Nathanael Gray is the Krishnan-Shah Family Professor of Chemical and Systems Biology at Stanford, Co-Director of Cancer Drug Discovery Co-Leader of the Cancer Therapeutics Research Program, Member of Chem-H, and Program Leader for Small Molecule Drug Discovery for the Innovative Medicines Accelerator (IMA). His research utilizes the tools of synthetic chemistry, protein biochemistry, and cancer biology to discover and validate new strategies for the inhibition of anti-cancer targets. Dr. Gray’s research has had broad impact in the areas of kinase inhibitor design and in circumventing drug resistance.
Dr. Gray received his PhD in organic chemistry from the University of California at Berkeley in 1999 after receiving his BS degree with the highest honor award from the same institution in 1995. After completing his PhD, Dr. Gray was recruited to the newly established Genomics Institute of the Novartis Research Foundation (GNF) in San Diego, California. During his six year stay at GNF, Dr. Gray became the director of biological chemistry where he supervised a group of over fifty researchers integrating chemical, biological and pharmacological approaches towards the development of new experimental drugs. Some of the notable accomplishments of Dr. Gray’s team at GNF include: discovery of the first allosteric inhibitors of wild-type and mutant forms of BCR-ABL which resulted in clinical development of ABL001; discovery of the first selective inhibitors of the Anaplastic Lymphoma Kinase (ALK), an achievement that led to the development of now FDA-approved drugs such as ceritinib (LDK378) for the treatment of EML4-ALK expressing non-small cell lung cancer (NSCLC); and discovery that sphingosine-1-phosphate receptor-1 (S1P1) is the pharmacologically relevant target of the immunosuppressant drug Fingomilod (FTY720) followed by the development of Siponimod (BAF312), which is currently used for the treatment of multiple sclerosis.
In 2006, Dr. Gray returned to academia as a faculty member at the Dana Farber Cancer Institute and Harvard Medical School in Boston. There, he has established a discovery chemistry group that focuses on developing first-in-class inhibitors for newly emerging biological targets, including resistant alleles of existing targets, as well as inhibitors of well-validated targets, such as Her3 and RAS, that have previously been considered recalcitrant to small molecule drug development. Dr. Gray’s team developed covalent inhibitors of the T790M mutant of EGFR inspired the development of Osimertinib (AZD9291), now FDA approved for treatment of patients with relapsed lung cancer due to resistance to first generation EGFR inhibitors. Dr. Gray has also developed structure-based, generalized approaches for designing drugs to overcome one of the most common mechanisms of resistance observed against most kinase inhibitor drugs, mutation of the so-called "gatekeeper" residue, which has been observed in resistance to drugs targeting BCR-ABL, c-KIT and PDGFR.
In 2021, Dr. Gray joined Stanford University where he has joined the Stanford Cancer Institute, Chem-H and the Innovative Medicines Accelerator (IMA) to spur the development of prototype drugs.
These contributions have been recognized through numerous awards including the National Science Foundation’s Career award in 2007, the Damon Runyon Foundation Innovator award in 2008, the American Association for Cancer Research for Team Science in 2010 and for Outstanding Achievement in 2011 and the American Chemical Society award for Biological Chemistry in 2011, and the Nancy Lurie Marks endowed professorship in 2015 and the Paul Marks Prize in 2019, and the Hope Funds for Cancer Research in 2023.

Academic Appointments

2023-24 Courses

Stanford Advisees

All Publications

  • Broad-spectrum activity against mosquito-borne flaviviruses achieved by a targeted protein degradation mechanism. Nature communications Liu, H. Y., Li, Z., Reindl, T., He, Z., Qiu, X., Golden, R. P., Donovan, K. A., Bailey, A., Fischer, E. S., Zhang, T., Gray, N. S., Yang, P. L. 2024; 15 (1): 5179


    Viral genetic diversity presents significant challenges in developing antivirals with broad-spectrum activity and high barriers to resistance. Here we report development of proteolysis targeting chimeras (PROTACs) targeting the dengue virus envelope (E) protein through coupling of known E fusion inhibitors to ligands of the CRL4CRBN E3 ubiquitin ligase. The resulting small molecules block viral entry through inhibition of E-mediated membrane fusion and interfere with viral particle production by depleting intracellular E in infected Huh 7.5 cells. This activity is retained in the presence of point mutations previously shown to confer partial resistance to the parental inhibitors due to decreased inhibitor-binding. The E PROTACs also exhibit broadened spectrum of activity compared to the parental E inhibitors against a panel of mosquito-borne flaviviruses. These findings encourage further exploration of targeted protein degradation as a differentiated and potentially advantageous modality for development of broad-spectrum direct-acting antivirals.

    View details for DOI 10.1038/s41467-024-49161-9

    View details for PubMedID 38898037

    View details for PubMedCentralID PMC11187112

  • Discovery of Potent Degraders of the Dengue Virus Envelope Protein. bioRxiv : the preprint server for biology Li, Z., Liu, H. Y., He, Z., Chakravarty, A., Golden, R. P., Jiang, Z., You, I., Yue, H., Donovan, K. A., Du, G., Che, J., Tse, J., Che, I., Lu, W., Fischer, E. S., Zhang, T., Gray, N. S., Yang, P. L. 2024


    Targeted protein degradation has been widely adopted as a new approach to eliminate both established and previously recalcitrant therapeutic targets. Here we report the development of small molecule degraders of the envelope (E) protein of dengue virus. We developed two classes of bivalent E-degraders, linking two previously reported E-binding small molecules, GNF-2 and CVM-2-12-2, to a glutarimide-based recruiter of the CRL4CRBN ligase to effect proteosome-mediated degradation of the E protein. ZXH-2-107 (based on GNF-2) is an E degrader with ABL inhibition while ZXH-8-004 (based on CVM-2-12-2) is a selective and potent E-degrader. These two compounds provide proof-of-concept that difficult-to-drug targets such as a viral envelope protein can be effectively eliminated using a bivalent degrader and provide starting points for the future development of a new class antiviral drugs.

    View details for DOI 10.1101/2024.06.01.596987

    View details for PubMedID 38854003

    View details for PubMedCentralID PMC11160776

  • Down-regulation of AKT proteins slows the growth of mutant-KRAS pancreatic tumors. bioRxiv : the preprint server for biology Chen, C., Jiang, Y. P., You, I., Gray, N. S., Lin, R. Z. 2024


    Serine/threonine kinase AKT isoforms play a well-established role in cell metabolism and growth. Most pancreatic adenocarcinoma (PDAC) harbors activation mutations of KRAS, which activates the PI3K/AKT signaling pathway. However, AKT inhibitors are not effective in the treatment of pancreatic cancer. To better understand the role of AKT signaling in mutant-KRAS pancreatic tumors, this study utilizes proteolysis-targeting chimeras (PROTACs) and CRISPR-Cas9-genome editing to investigate AKT proteins. PROTAC down-regulation of AKT proteins markedly slowed the growth of three pancreatic tumor cell lines harboring mutant KRAS. In contrast, inhibition of AKT kinase activity alone had very little effect on the growth of these cell lines. Concurrent genetic deletion of all AKT isoforms (AKT1, AKT2, and AKT3) in the KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cell line also dramatically slowed its growth in vitro and when orthotopically implanted in syngeneic mice. Surprisingly, insulin-like growth factor-1 (IGF-1), but not epidermal growth factor (EGF), restored KPC cell growth in serum-deprived conditions and the IGF-1 growth stimulation effect was AKT dependent. RNA-seq analysis of AKT1/2/3-deficient KPC cells suggested that reduced cholesterol synthesis may be responsible for the decreased response to IGF-1 stimulation. These results indicate that the presence of all three AKT isoforms supports pancreatic tumor cell growth and pharmacological degradation of AKT proteins may be more effective than AKT catalytic inhibitors for treating pancreatic cancer.

    View details for DOI 10.1101/2024.05.03.592345

    View details for PubMedID 38746217

    View details for PubMedCentralID PMC11092743

  • Author Correction: Targeting DCAF5 suppresses SMARCB1-mutant cancer by stabilizing SWI/SNF. Nature Radko-Juettner, S., Yue, H., Myers, J. A., Carter, R. D., Robertson, A. N., Mittal, P., Zhu, Z., Hansen, B. S., Donovan, K. A., Hunkeler, M., Rosikiewicz, W., Wu, Z., McReynolds, M. G., Roy Burman, S. S., Schmoker, A. M., Mageed, N., Brown, S. A., Mobley, R. J., Partridge, J. F., Stewart, E. A., Pruett-Miller, S. M., Nabet, B., Peng, J., Gray, N. S., Fischer, E. S., Roberts, C. W. 2024

    View details for DOI 10.1038/s41586-024-07402-3

    View details for PubMedID 38684813

  • Reciprocal antagonism of PIN1-APC/CCDH1 governs mitotic protein stability and cell cycle entry. Nature communications Ke, S., Dang, F., Wang, L., Chen, J. Y., Naik, M. T., Li, W., Thavamani, A., Kim, N., Naik, N. M., Sui, H., Tang, W., Qiu, C., Koikawa, K., Batalini, F., Stern Gatof, E., Isaza, D. A., Patel, J. M., Wang, X., Clohessy, J. G., Heng, Y. J., Lahav, G., Liu, Y., Gray, N. S., Zhou, X. Z., Wei, W., Wulf, G. M., Lu, K. P. 2024; 15 (1): 3220


    Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/CCDH1) prevents cell-cycle entry by targeting crucial mitotic proteins for degradation. Phosphorylation of its co-activator CDH1 modulates the E3 ligase activity, but little is known about its regulation after phosphorylation and how to effectively harness APC/CCDH1 activity to treat cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1)-catalyzed phosphorylation-dependent cis-trans prolyl isomerization drives tumor malignancy. However, the mechanisms controlling its protein turnover remain elusive. Through proteomic screens and structural characterizations, we identify a reciprocal antagonism of PIN1-APC/CCDH1 mediated by domain-oriented phosphorylation-dependent dual interactions as a fundamental mechanism governing mitotic protein stability and cell-cycle entry. Remarkably, combined PIN1 and cyclin-dependent protein kinases (CDKs) inhibition creates a positive feedback loop of PIN1 inhibition and APC/CCDH1 activation to irreversibly degrade PIN1 and other crucial mitotic proteins, which force permanent cell-cycle exit and trigger anti-tumor immunity, translating into synergistic efficacy against triple-negative breast cancer.

    View details for DOI 10.1038/s41467-024-47427-w

    View details for PubMedID 38622115

    View details for PubMedCentralID PMC11018817

  • Targeting DCAF5 suppresses SMARCB1-mutant cancer by stabilizing SWI/SNF. Nature Radko-Juettner, S., Yue, H., Myers, J. A., Carter, R. D., Robertson, A. N., Mittal, P., Zhu, Z., Hansen, B. S., Donovan, K. A., Hunkeler, M., Rosikiewicz, W., Wu, Z., McReynolds, M. G., Roy Burman, S. S., Schmoker, A. M., Mageed, N., Brown, S. A., Mobley, R. J., Partridge, J. F., Stewart, E. A., Pruett-Miller, S. M., Nabet, B., Peng, J., Gray, N. S., Fischer, E. S., Roberts, C. W. 2024


    Whereas oncogenes can potentially be inhibited with small molecules, the loss of tumour suppressors is more common and is problematic because the tumour-suppressor proteins are no longer present to be targeted. Notable examples include SMARCB1-mutant cancers, which are highly lethal malignancies driven by the inactivation of a subunit of SWI/SNF (also known as BAF) chromatin-remodelling complexes. Here, to generate mechanistic insights into the consequences of SMARCB1 mutation and to identify vulnerabilities, we contributed 14 SMARCB1-mutant cell lines to a near genome-wide CRISPR screen as part of the Cancer Dependency Map Project1-3. We report that the little-studied gene DDB1-CUL4-associated factor 5 (DCAF5) is required for the survival of SMARCB1-mutant cancers. We show that DCAF5 has a quality-control function for SWI/SNF complexes and promotes the degradation of incompletely assembled SWI/SNF complexes in the absence of SMARCB1. After depletion of DCAF5, SMARCB1-deficient SWI/SNF complexes reaccumulate, bind to target loci and restore SWI/SNF-mediated gene expression to levels that are sufficient to reverse the cancer state, including in vivo. Consequently, cancer results not from the loss of SMARCB1 function per se, but rather from DCAF5-mediated degradation of SWI/SNF complexes. These data indicate that therapeutic targeting of ubiquitin-mediated quality-control factors may effectively reverse the malignant state of some cancers driven by disruption of tumour suppressor complexes.

    View details for DOI 10.1038/s41586-024-07250-1

    View details for PubMedID 38538798

  • The dual HCK/BTK inhibitor KIN-8194 impairs growth and integrin-mediated adhesion of BTKi-resistant mantle cell lymphoma. Leukemia Lantermans, H. C., Ma, F., Kuil, A., van Kesteren, S., Yasinoglu, S., Yang, G., Buhrlage, S. J., Wang, J., Gray, N. S., Kersten, M. J., Treon, S. P., Pals, S. T., Spaargaren, M. 2024


    Although Bruton's tyrosine kinase (BTK) inhibitors (BTKi) have significantly improved patient prognosis, mantle cell lymphoma (MCL) is still considered incurable due to primary and acquired resistance. We have recently shown that aberrant expression of the Src-family tyrosine kinase hematopoietic cell kinase (HCK) in MCL correlates with poor prognosis, and that genetic HCK perturbation impairs growth and integrin-mediated adhesion of MCL cells. Here, we show that KIN-8194, a dual inhibitor of BTK and HCK with in vivo activity against Myd88-L265P-driven diffuse large B-cell lymphoma and Waldenström Macroglobulinemia, has a potent growth inhibitory effect in MCL cell lines and primary MCL cells, irrespective of their sensitivity to BTKi (ibrutinib and acalabrutinib). In BTKi-resistant cells this is mediated by inhibition of HCK, which results in repression of AKT-S6 signaling. In addition, KIN-8194 inhibits integrin-mediated adhesion of BTKi-sensitive and insensitive MCL cells to fibronectin and stromal cells in an HCK-dependent manner. Finally, we show that MCL cells with acquired BTKi resistance retain their sensitivity to KIN-8194. Taken together, our data demonstrate that KIN-8194 inhibits growth and integrin-mediated adhesion of BTKi-sensitive MCL cells, as well as MCL cells with primary or acquired BTKi resistance. This renders KIN-8194 a promising novel treatment for MCL patients.

    View details for DOI 10.1038/s41375-024-02207-9

    View details for PubMedID 38454120

    View details for PubMedCentralID 3790509

  • Multiomic profiling of breast cancer cells uncovers stress MAPK-associated sensitivity to AKT degradation. Science signaling Erickson, E. C., You, I., Perry, G., Dugourd, A., Donovan, K. A., Crafter, C., Johannes, J. W., Williamson, S., Moss, J. I., Ros, S., Ziegler, R. E., Barry, S. T., Fischer, E. S., Gray, N. S., Madsen, R. R., Toker, A. 2024; 17 (825): eadf2670


    More than 50% of human tumors display hyperactivation of the serine/threonine kinase AKT. Despite evidence of clinical efficacy, the therapeutic window of the current generation of AKT inhibitors could be improved. Here, we report the development of a second-generation AKT degrader, INY-05-040, which outperformed catalytic AKT inhibition with respect to cellular suppression of AKT-dependent phenotypes in breast cancer cell lines. A growth inhibition screen with 288 cancer cell lines confirmed that INY-05-040 had a substantially higher potency than our first-generation AKT degrader (INY-03-041), with both compounds outperforming catalytic AKT inhibition by GDC-0068. Using multiomic profiling and causal network integration in breast cancer cells, we demonstrated that the enhanced efficacy of INY-05-040 was associated with sustained suppression of AKT signaling, which was followed by induction of the stress mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK). Further integration of growth inhibition assays with publicly available transcriptomic, proteomic, and reverse phase protein array (RPPA) measurements established low basal JNK signaling as a biomarker for breast cancer sensitivity to AKT degradation. Together, our study presents a framework for mapping the network-wide signaling effects of therapeutically relevant compounds and identifies INY-05-040 as a potent pharmacological suppressor of AKT signaling.

    View details for DOI 10.1126/scisignal.adf2670

    View details for PubMedID 38412255

  • Molecular Bidents with Two Electrophilic Warheads as a New Pharmacological Modality ACS CENTRAL SCIENCE Li, Z., Jiang, J., Ficarro, S. B., Beyett, T. S., To, C., Tavares, I., Zhu, Y., Li, J., Eck, M. J., Janne, P. A., Marto, J. A., Zhang, T., Che, J., Gray, N. S. 2024
  • ZNL0325, a Pyrazolopyrimidine-Based Covalent Probe, Demonstrates an Alternative Binding Mode for Kinases. Journal of medicinal chemistry Li, Z., Lu, W., Beyett, T. S., Ficarro, S. B., Jiang, J., Tse, J., Kim, A. Y., Marto, J. A., Che, J., Jänne, P. A., Eck, M. J., Zhang, T., Gray, N. S. 2024


    The pyrazolopyrimidine (PP) heterocycle is a versatile and widely deployed core scaffold for the development of kinase inhibitors. Typically, a 4-amino-substituted pyrazolopyrimidine binds in the ATP-binding pocket in a conformation analogous to the 6-aminopurine of ATP. Here, we report the discovery of ZNL0325 which exhibits a flipped binding mode where the C3 position is oriented toward the ribose binding pocket. ZNL0325 and its analogues feature an acrylamide side chain at the C3 position which is capable of forming a covalent bond with multiple kinases that possess a cysteine at the αD-1 position including BTK, EGFR, BLK, and JAK3. These findings suggest that the ability to form a covalent bond can override the preferred noncovalent binding conformation of the heterocyclic core and provides an opportunity to create structurally distinct covalent kinase inhibitors.

    View details for DOI 10.1021/acs.jmedchem.3c01891

    View details for PubMedID 38300264

  • Discovery of Potent Antimalarial Type II Kinase Inhibitors with Selectivity over Human Kinases. Journal of medicinal chemistry Wang, L., Bohmer, M. J., Wang, J., Nardella, F., Calla, J., Laureano De Souza, M., Schindler, K. A., Montejo, L., Mittal, N., Rocamora, F., Treat, M., Charlton, J. T., Tumwebaze, P. K., Rosenthal, P. J., Cooper, R. A., Chakrabarti, R., Winzeler, E. A., Chakrabarti, D., Gray, N. S. 2024


    While progress has been made in the effort to eradicate malaria, the disease remains a significant threat to global health. Acquired resistance to frontline treatments is emerging in Africa, urging a need for the development of novel antimalarial agents. Repurposing human kinase inhibitors provides a potential expedited route given the availability of a diverse array of kinase-targeting drugs that are approved or in clinical trials. Phenotypic screening of a library of type II human kinase inhibitors identified compound 1 as a lead antimalarial, which was initially developed to target human ephrin type A receptor 2 (EphA2). Here, we report a structure-activity relationship study and lead optimization of compound 1, which led to compound 33, with improved antimalarial activity and selectivity.

    View details for DOI 10.1021/acs.jmedchem.3c02046

    View details for PubMedID 38214254

  • Functional Dissection of Cellular Programs to Uncover Novel Gene Dependencies in AML Ellegast, J. M., Alexe, G., Baniya, S., Hamze, A., Taillon, A., Adane, B., Conway, A., Zhang, T., Gray, N. S., Armstrong, S. A., Stegmaier, K. AMER SOC HEMATOLOGY. 2023
  • Exploration of the Tunability of BRD4 Degradation by DCAF16 Trans-labelling Covalent Glues. bioRxiv : the preprint server for biology Hassan, M. M., Li, Y. D., Ma, M. W., Teng, M., Byun, W. S., Puvar, K., Lumpkin, R., Sandoval, B., Rutter, J. C., Jin, C. Y., Wang, M. Y., Xu, S., Schmoker, A. M., Cheong, H., Groendyke, B. J., Qi, J., Fischer, E. S., Ebert, B. L., Gray, N. S. 2023


    Small molecules that can induce protein degradation by inducing proximity between a desired target and an E3 ligase have the potential to greatly expand the number of proteins that can be manipulated pharmacologically. Current strategies for targeted protein degradation are mostly limited in their target scope to proteins with preexisting ligands. Alternate modalities such as molecular glues, as exemplified by the glutarimide class of ligands for the CUL4CRBN ligase, have been mostly discovered serendipitously. We recently reported a trans-labelling covalent glue mechanism which we named 'Template-assisted covalent modification', where an electrophile decorated small molecule binder of BRD4 was effectively delivered to a cysteine residue on an E3 ligase DCAF16 as a consequence of a BRD4-DCAF16 protein-protein interaction. Herein, we report our medicinal chemistry efforts to evaluate how various electrophilic modifications to the BRD4 binder, JQ1, affect DCAF16 trans-labeling and subsequent BRD4 degradation efficiency. We discovered a decent correlation between the ability of the electrophilic small molecule to induce ternary complex formation between BRD4 and DCAF16 with its ability to induce BRD4 degradation. Moreover, we show that a more solvent-exposed warhead presentation is optimal for DCAF16 recruitment and subsequent BRD4 degradation. Unlike the sensitivity of CUL4CRBN glue degraders to chemical modifications, the diversity of covalent attachments in this class of BRD4 glue degraders suggests a high tolerance and tunability for the BRD4-DCAF16 interaction. This offers a potential new avenue for a rational design of covalent glue degraders by introducing covalent warheads to known binders.

    View details for DOI 10.1101/2023.10.07.561308

    View details for PubMedID 37873358

    View details for PubMedCentralID PMC10592706

  • Chemical Specification of E3 Ubiquitin Ligase Engagement by Cysteine-Reactive Chemistry. Journal of the American Chemical Society Sarott, R. C., You, I., Li, Y. D., Toenjes, S. T., Donovan, K. A., Seo, P., Ordonez, M., Byun, W. S., Hassan, M. M., Wachter, F., Chouchani, E. T., Słabicki, M., Fischer, E. S., Ebert, B. L., Hinshaw, S. M., Gray, N. S. 2023


    Targeted protein degradation relies on small molecules that induce new protein-protein interactions between targets and the cellular protein degradation machinery. Most of these small molecules feature specific ligands for ubiquitin ligases. Recently, the attachment of cysteine-reactive chemical groups to pre-existing small molecule inhibitors has been shown to drive specific target degradation. We demonstrate here that different cysteine-reactive groups can specify target degradation via distinct ubiquitin ligases. By focusing on the bromodomain ligand JQ1, we identify cysteine-reactive functional groups that drive BRD4 degradation by either DCAF16 or DCAF11. Unlike proteolysis-targeting chimeric molecules (PROTACs), the new compounds use a single small molecule ligand with a well-positioned cysteine-reactive group to induce protein degradation. The finding that nearly identical compounds can engage multiple ubiquitination pathways suggests that targeting cellular pathways that search for and eliminate chemically reactive proteins is a feasible avenue for converting existing small molecule drugs into protein degrader molecules.

    View details for DOI 10.1021/jacs.3c06622

    View details for PubMedID 37767920

  • Genome-Wide CRISPR Screens Identify Multiple Synthetic Lethal Targets That Enhance KRASG12C Inhibitor Efficacy. Cancer research Mukhopadhyay, S., Huang, H. Y., Lin, Z., Ranieri, M., Li, S., Sahu, S., Liu, Y., Ban, Y., Guidry, K., Hu, H., Lopez, A., Sherman, F., Tan, Y. J., Lee, Y. T., Armstrong, A. P., Dolgalev, I., Sahu, P., Zhang, T., Lu, W., Gray, N. S., Christensen, J. G., Tang, T. T., Velcheti, V., Khodadadi-Jamayran, A., Wong, K. K., Neel, B. G. 2023


    Non-small lung cancers (NSCLCs) frequently (~30%) harbor KRAS driver mutations, half of which are KRASG12C. KRAS-mutant NSCLC with co-mutated STK11 and/or KEAP1 is particularly refractory to conventional, targeted, and immune therapy. Development of KRASG12C inhibitors (G12Cis) provided a major therapeutic advance, but resistance still limits their efficacy. To identify genes whose deletion augments efficacy of the G12Cis adagrasib (MRTX-849) or adagrasib plus TNO155 (SHP2i), we performed genome-wide CRISPR/Cas9 screens on KRAS/STK11-mutant NSCLC lines. Recurrent, potentially targetable, synthetic lethal (SL) genes were identified, including serine-threonine kinases, tRNA-modifying and proteoglycan synthesis enzymes, and YAP/TAZ/TEAD pathway components. Several SL genes were confirmed by siRNA/shRNA experiments, and the YAP/TAZ/TEAD pathway was extensively validated in vitro and in mice. Mechanistic studies showed that G12Ci treatment induced gene expression of RHO paralogs and activators, increased RHOA activation, and evoked ROCK-dependent nuclear translocation of YAP. Mice and patients with acquired G12Ci- or G12Ci/SHP2i-resistant tumors showed strong overlap with SL pathways, arguing for the relevance of the screen results. These findings provide a landscape of potential targets for future combination strategies, some of which can be tested rapidly in the clinic.

    View details for DOI 10.1158/0008-5472.CAN-23-2729

    View details for PubMedID 37729426

  • Proteomics-Based Discovery of First-in-Class Chemical Probes for Programmed Cell Death Protein 2 (PDCD2). Angewandte Chemie (International ed. in English) Ji, W., Byun, W. S., Lu, W., Zhu, X., Donovan, K. A., Dwyer, B., Che, J., Yuan, L., Abulaiti, X., Corsello, S. M., Fischer, E. S., Zhang, T., Gray, N. S. 2023: e202308292


    Chemical probes are essential tools for understanding biological systems and for credentialing potential biomedical targets. Programmed cell death 2 (PDCD2) is a member of the B-cell lymphoma 2 (Bcl-2) family of proteins, which are critical regulators of apoptosis. Here we report the discovery and characterization of 10e, a first-in-class small molecule degrader of PDCD2. We discovered PDCD2 degrader by serendipity using a chemical proteomics approach in contrast to the conventional approach for making bivalent degraders starting from a known binding ligand targeting the protein of interest. Using 10e as a pharmacological probe, we demonstrate that PDCD2 functions as a critical regulator of cell growth by modulating the progression of the cell cycle in T lymphoblasts. Our work provides a useful pharmacological probe for investigating PDCD2 function and highlights using chemical proteomics to discover selective small molecule degraders of unanticipated targets.

    View details for DOI 10.1002/anie.202308292

    View details for PubMedID 37658265

  • Author Correction: Rewiring cancer drivers to activate apoptosis. Nature Gourisankar, S., Krokhotin, A., Ji, W., Liu, X., Chang, C. Y., Kim, S. H., Li, Z., Wenderski, W., Simanauskaite, J. M., Yang, H., Vogel, H., Zhang, T., Green, M. R., Gray, N. S., Crabtree, G. R. 2023

    View details for DOI 10.1038/s41586-023-06543-1

    View details for PubMedID 37596490

  • Development of a Highly Potent and Selective Degrader of LRRK2. Bioorganic & medicinal chemistry letters Hatcher, J. M., Zwirek, M., Sarhan, A. R., Vatsan, P. S., Tonelli, F., Alessi, D. R., Davies, P., Gray, N. S. 2023: 129449


    The discovery of disease-modifying therapies for Parkinson's Disease (PD) represents a critical need in neurodegenerative medicine. Genetic mutations in leucine-rich repeat kinase 2 (LRRK2) are risk factors for the development of PD, and some of these mutations have been linked to increased LRRK2 kinase activity and neuronal toxicity in cellular and animal models. Furthermore, LRRK2 function as a scaffolding protein in several pathways has been implicated as a plausible mechanism underlying neurodegeneration caused by LRRK2 mutations. Given that both the kinase activity and scaffolding function of LRRK2 have been linked to neurodegeneration, we developed proteolysis-targeting chimeras (PROTACs) targeting LRRK2. The degrader molecule JH-XII-03-02 (6) displayed high potency and remarkable selectivity for LRKK2 when assessed in a of 468 panel kinases and serves the dual purpose of eliminating both the kinase activity as well as the scaffolding function of LRRK2.

    View details for DOI 10.1016/j.bmcl.2023.129449

    View details for PubMedID 37591317

  • Targeted kinase degradation via the KLHDC2 ubiquitin E3 ligase. Cell chemical biology Kim, Y., Seo, P., Jeon, E., You, I., Hwang, K., Kim, N., Tse, J., Bae, J., Choi, H., Hinshaw, S. M., Gray, N. S., Sim, T. 2023


    Chemically induced protein degradation is a powerful strategy for perturbing cellular biochemistry. The predominant mechanism of action for protein degrader drugs involves an induced proximity between the cellular ubiquitin-conjugation machinery and a target. Unlike traditional small molecule enzyme inhibition, targeted protein degradation can clear an undesired protein from cells. We demonstrate here the use of peptide ligands for Kelch-like homology domain-containing protein 2 (KLHDC2), a substrate adapter protein and member of the cullin-2 (CUL2) ubiquitin ligase complex, for targeted protein degradation. Peptide-based bivalent compounds that can induce proximity between KLHDC2 and target proteins cause degradation of the targeted factors. The cellular activity of these compounds depends on KLHDC2 binding. This work demonstrates the utility of KLHDC2 for targeted protein degradation and exemplifies a strategy for the rational design of peptide-based ligands useful for this purpose.

    View details for DOI 10.1016/j.chembiol.2023.07.008

    View details for PubMedID 37567174

  • Rewiring cancer drivers to activate apoptosis. Nature Gourisankar, S., Krokhotin, A., Ji, W., Liu, X., Chang, C., Kim, S. H., Li, Z., Wenderski, W., Simanauskaite, J. M., Yang, H., Vogel, H., Zhang, T., Green, M. R., Gray, N. S., Crabtree, G. R. 2023


    Genes that drive the proliferation, survival, invasion and metastasis of malignant cells have been identified for many human cancers1-4. Independent studies have identified cell death pathways that eliminate cells for the good of the organism5,6. The coexistence of cell death pathways with driver mutations suggests that the cancer driver could be rewired to activate cell death using chemical inducers of proximity (CIPs). Here we describe a new class of molecules called transcriptional/epigenetic CIPs (TCIPs) that recruit the endogenous cancer driver, or a downstream transcription factor, to the promoters of cell death genes, thereby activating their expression. We focused on diffuse large B cell lymphoma, in which the transcription factor B cell lymphoma 6 (BCL6) is deregulated7. BCL6 binds to the promoters of cell death genes and epigenetically suppresses their expression8. We produced TCIPs by covalently linking small molecules that bind BCL6 to those that bind to transcriptional activators that contribute to the oncogenic program, such as BRD4. The most potent molecule, TCIP1, increases binding of BRD4 by 50% over genomic BCL6-binding sites to produce transcriptional elongation at pro-apoptotic target genes within 15min, while reducing binding of BRD4 over enhancers by only 10%, reflecting a gain-of-function mechanism. TCIP1 kills diffuse large B cell lymphoma cell lines, including chemotherapy-resistant, TP53-mutant lines, at EC50 of 1-10nM in 72h and exhibits cell-specific and tissue-specific effects, capturing the combinatorial specificity inherent to transcription. The TCIP concept also has therapeutic applications in regulating the expression of genes for regenerative medicine and developmental disorders.

    View details for DOI 10.1038/s41586-023-06348-2

    View details for PubMedID 37495688

  • The rise of degrader drugs. Cell chemical biology Teng, M., Gray, N. S. 2023


    The cancer genomics revolution has served up a plethora of promising and challenging targets for the drug discovery community. The field of targeted protein degradation (TPD) uses small molecules to reprogram the protein homeostasis system to destroy desired target proteins. In the last decade, remarkable progress has enabled the rational development of degraders for a large number of target proteins, with over 20 molecules targeting more than 12 proteins entering clinical development. While TPD has been fully credentialed by the prior development of immunomodulatory drug (IMiD) class for the treatment of multiple myeloma, the field is poised for a "Gleevec moment" in which robust clinical efficacy of a rationally developed novel degrader against a preselected target is firmly established. Here, we endeavor to provide a high-level evaluation of exciting developments in the field and comment on steps that may realize the full potential of this new therapeutic modality.

    View details for DOI 10.1016/j.chembiol.2023.06.020

    View details for PubMedID 37494935

  • New scaffolds for type II JAK2 inhibitors overcome the acquired G993A resistance mutation. Cell chemical biology Arwood, M. L., Liu, Y., Harkins, S. K., Weinstock, D. M., Yang, L., Stevenson, K. E., Plana, O. D., Dong, J., Cirka, H., Jones, K. L., Virtanen, A. T., Gupta, D. G., Ceas, A., Lawney, B., Yoda, A., Leahy, C., Hao, M., He, Z., Choi, H. G., Wang, Y., Silvennoinen, O., Hubbard, S. R., Zhang, T., Gray, N. S., Li, L. S. 2023


    Recurrent JAK2 alterations are observed in myeloproliferative neoplasms, B-cell acute lymphoblastic leukemia, and other hematologic malignancies. Currently available type I JAK2 inhibitors have limited activity in these diseases. Preclinical data support the improved efficacy of type II JAK2 inhibitors, which lock the kinase in the inactive conformation. By screening small molecule libraries, we identified a lead compound with JAK2 selectivity. We highlight analogs with on-target biochemical and cellular activity and demonstrate in vivo activity using a mouse model of polycythemia vera. We present a co-crystal structure that confirms the type II binding mode of our compounds with the "DFG-out" conformation of the JAK2 activation loop. Finally, we identify a JAK2 G993A mutation that confers resistance to the type II JAK2 inhibitor CHZ868 but not to our analogs. These data provide a template for identifying novel type II kinase inhibitors and inform further development of agents targeting JAK2 that overcome resistance.

    View details for DOI 10.1016/j.chembiol.2023.05.007

    View details for PubMedID 37290440

  • Development of Mutant-Selective Allosteric EGFR Inhibitors for Drug-Resistant Lung Cancer Beyett, T., To, C., Heppner, D. E., Gero, T. W., Gray, N. S., Scott, D. A., Janne, P. A., Eck, M. J. AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS. 2023
  • Genome-scale functional genomics identify genes preferentially essential for multiple myeloma cells compared to other neoplasias NATURE CANCER Simoes, R., Shirasaki, R., Downey-Kopyscinski, S. L., Matthews, G. M., Barwick, B. G., Gupta, V. A., Dupere-Richer, D., Yamano, S., Hu, Y., Sheffer, M., Dhimolea, E., Dashevsky, O., Gandolfi, S., Ishiguro, K., Meyers, R. M., Bryan, J. G., Dharia, N. V., Hengeveld, P. J., Bruggenthies, J. B., Tang, H., Aguirre, A. J., Sievers, Q. L., Ebert, B. L., Glassner, B. J., Ott, C. J., Bradner, J. E., Kwiatkowski, N. P., Auclair, D., Levy, J., Keats, J. J., Groen, R. J., Gray, N. S., Culhane, A. C., McFarland, J. M., Dempster, J. M., Licht, J. D., Boise, L. H., Hahn, W. C., Vazquez, F., Tsherniak, A., Mitsiades, C. S. 2023; 4 (5): 754-+


    Clinical progress in multiple myeloma (MM), an incurable plasma cell (PC) neoplasia, has been driven by therapies that have limited applications beyond MM/PC neoplasias and do not target specific oncogenic mutations in MM. Instead, these agents target pathways critical for PC biology yet largely dispensable for malignant or normal cells of most other lineages. Here we systematically characterized the lineage-preferential molecular dependencies of MM through genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) studies in 19 MM versus hundreds of non-MM lines and identified 116 genes whose disruption more significantly affects MM cell fitness compared with other malignancies. These genes, some known, others not previously linked to MM, encode transcription factors, chromatin modifiers, endoplasmic reticulum components, metabolic regulators or signaling molecules. Most of these genes are not among the top amplified, overexpressed or mutated in MM. Functional genomics approaches thus define new therapeutic targets in MM not readily identifiable by standard genomic, transcriptional or epigenetic profiling analyses.

    View details for DOI 10.1038/s43018-023-00550-x

    View details for Web of Science ID 000995948000003

    View details for PubMedID 37237081

    View details for PubMedCentralID 4468029

  • Development and characterization of selective FAK inhibitors and PROTACs with in vivo activity. Chembiochem : a European journal of chemical biology Koide, E., Mohardt, M. L., Doctor, Z. M., Yang, A., Hao, M., Donovan, K. A., Kuismi, C. C., Nelson, A. J., Abell, K., Aguiar, M., Che, J., Stokes, M. P., Zhang, T., Aguirre, A. J., Fischer, E. S., Gray, N. S., Jiang, B., Nabet, B. 2023: e202300141


    Focal adhesion kinase (FAK) is an attractive drug target due to its overexpression in cancer. FAK functions as a non-receptor tyrosine kinase and scaffolding protein, coordinating several downstream signaling effectors and cellular processes. While drug discovery efforts have largely focused on targeting FAK kinase activity, FAK inhibitors have failed to show efficacy as single agents in clinical trials. Here, using structure-guided design, we report the development of a selective FAK inhibitor (BSJ-04-175) and degrader (BSJ-04-146) to evaluate the consequences and advantages of abolishing all FAK activity in cancer models. BSJ-04-146 achieves rapid and potent FAK degradation with high proteome-wide specificity in cancer cells and induces durable degradation in mice. Compared to kinase inhibition, targeted degradation of FAK exhibits pronounced improved activity on downstream signaling and cancer cell viability and migration. Together, BSJ-04-175 and BSJ-04-146 are valuable chemical tools to dissect the specific consequences of targeting FAK through small molecule inhibition or degradation.

    View details for DOI 10.1002/cbic.202300141

    View details for PubMedID 37088717

  • Catalytic Degraders Effectively Address Kinase Site Mutations in EML4-ALK Oncogenic Fusions. Journal of medicinal chemistry Gao, Y., Jiang, B., Kim, H., Berberich, M. J., Che, J., Donovan, K. A., Hatcher, J. M., Huerta, F., Kwiatkowski, N. P., Liu, Y., Liuni, P. P., Metivier, R. J., Murali, V. K., Nowak, R. P., Zhang, T., Fischer, E. S., Gray, N. S., Jones, L. H. 2023


    Heterobifunctional degraders, known as proteolysis targeting chimeras (PROTACs), theoretically possess a catalytic mode-of-action, yet few studies have either confirmed or exploited this potential advantage of event-driven pharmacology. Degraders of oncogenic EML4-ALK fusions were developed by conjugating ALK inhibitors to cereblon ligands. Simultaneous optimization of pharmacology and compound properties using ternary complex modeling and physicochemical considerations yielded multiple catalytic degraders that were more resilient to clinically relevant ATP-binding site mutations than kinase inhibitor drugs. Our strategy culminated in the design of the orally bioavailable derivative CPD-1224 that avoided hemolysis (a feature of detergent-like PROTACs), degraded the otherwise recalcitrant mutant L1196M/G1202R in vivo, and commensurately slowed tumor growth, while the third generation ALK inhibitor drug lorlatinib had no effect. These results validate our original therapeutic hypothesis by exemplifying opportunities for catalytic degraders to proactively address binding site resistant mutations in cancer.

    View details for DOI 10.1021/acs.jmedchem.2c01864

    View details for PubMedID 37036171

  • USP9X mediates an acute adaptive response to MAPK suppression in pancreatic cancer but creates multiple actionable therapeutic vulnerabilities. Cell reports. Medicine Perurena, N., Lock, R., Davis, R. A., Raghavan, S., Pilla, N. F., Ng, R., Loi, P., Guild, C. J., Miller, A. L., Sicinska, E., Cleary, J. M., Rubinson, D. A., Wolpin, B. M., Gray, N. S., Santagata, S., Hahn, W. C., Morton, J. P., Sansom, O. J., Aguirre, A. J., Cichowski, K. 2023: 101007


    Pancreatic ductal adenocarcinomas (PDACs) frequently harbor KRAS mutations. Although MEK inhibitors represent a plausible therapeutic option, most PDACs are innately resistant to these agents. Here, we identify a critical adaptive response that mediates resistance. Specifically, we show that MEK inhibitors upregulate the anti-apoptotic protein Mcl-1 by triggering an association with its deubiquitinase, USP9X, resulting in acute Mcl-1 stabilization and protection from apoptosis. Notably, these findings contrast the canonical positive regulation of Mcl-1 by RAS/ERK. We further show that Mcl-1 inhibitors and cyclin-dependent kinase (CDK) inhibitors, which suppress Mcl-1 transcription, prevent this protective response and induce tumor regression when combined with MEK inhibitors. Finally, we identify USP9X as an additional potential therapeutic target. Together, these studies (1) demonstrate that USP9X regulates a critical mechanism of resistance in PDAC, (2) reveal an unexpected mechanism of Mcl-1 regulation in response to RAS pathway suppression, and (3) provide multiple distinct promising therapeutic strategies for this deadly malignancy.

    View details for DOI 10.1016/j.xcrm.2023.101007

    View details for PubMedID 37030295

  • ITK degradation to block T cell receptor signaling and overcome therapeutic resistance in T cell lymphomas. Cell chemical biology Jiang, B., Weinstock, D. M., Donovan, K. A., Sun, H. W., Wolfe, A., Amaka, S., Donaldson, N. L., Wu, G., Jiang, Y., Wilcox, R. A., Fischer, E. S., Gray, N. S., Wu, W. 2023


    Interleukin (IL)-2-inducible T cell kinase (ITK) is essential for T cell receptor (TCR) signaling and plays an integral role in T cell proliferation and differentiation. Unlike the ITK homolog BTK, no inhibitors of ITK are currently US Food and Drug Administration (FDA) approved. In addition, recent studies have identified mutations within BTK that confer resistance to both covalent and non-covalent inhibitors. Here, as an alternative strategy, we report the development of BSJ-05-037, a potent and selective heterobifunctional degrader of ITK. BSJ-05-037 displayed enhanced anti-proliferative effects relative to its parent inhibitor BMS-509744, blocked the activation of NF-kB/GATA-3 signaling, and increased the sensitivity of T cell lymphoma cells to cytotoxic chemotherapy both in vitro and in vivo. In summary, targeted degradation of ITK is a novel approach to modulate TCR signal strength that could have broad application for the investigation and treatment of T cell-mediated diseases.

    View details for DOI 10.1016/j.chembiol.2023.03.007

    View details for PubMedID 37015223

  • Structure-Based Design of Y-Shaped Covalent TEAD Inhibitors. Journal of medicinal chemistry Lu, W., Fan, M., Ji, W., Tse, J., You, I., Ficarro, S. B., Tavares, I., Che, J., Kim, A. Y., Zhu, X., Boghossian, A., Rees, M. G., Ronan, M. M., Roth, J. A., Hinshaw, S. M., Nabet, B., Corsello, S. M., Kwiatkowski, N., Marto, J. A., Zhang, T., Gray, N. S. 2023


    Transcriptional enhanced associate domain (TEAD) proteins together with their transcriptional coactivator yes-associated protein (YAP) and transcriptional coactivator with the PDZ-binding motif (TAZ) are important transcription factors and cofactors that regulate gene expression in the Hippo pathway. In mammals, the TEAD families have four homologues: TEAD1 (TEF-1), TEAD2 (TEF-4), TEAD3 (TEF-5), and TEAD4 (TEF-3). Aberrant expression and hyperactivation of TEAD/YAP signaling have been implicated in a variety of malignancies. Recently, TEADs were recognized as being palmitoylated in cells, and the lipophilic palmitate pocket has been successfully targeted by both covalent and noncovalent ligands. In this report, we present the medicinal chemistry effort to develop MYF-03-176 (compound 22) as a selective, cysteine-covalent TEAD inhibitor. MYF-03-176 (compound 22) significantly inhibits TEAD-regulated gene expression and proliferation of the cell lines with TEAD dependence including those derived from mesothelioma and liposarcoma.

    View details for DOI 10.1021/acs.jmedchem.2c01548

    View details for PubMedID 36946421

  • Shining light on reprogramming Tregs for cancer therapy. Cell chemical biology Wang, E. S., Gray, N. S. 2023; 30 (3): 231-233


    In this issue of Cell Chemical Biology, Bonazzi etal. demonstrate that pharmacologically degrading the transcription factor Helios (IKZF2) results in destabilization of regulatory Tcells, which normally restrain anti-tumor immunity. These results highlight how molecular glue degraders can selectively target previously undruggable proteins with potential applications in the clinic.

    View details for DOI 10.1016/j.chembiol.2023.02.009

    View details for PubMedID 36931248

  • Human Polo-like Kinase Inhibitors as Antiplasmodials. ACS infectious diseases Bohmer, M. J., Wang, J., Istvan, E. S., Luth, M. R., Collins, J. E., Huttlin, E. L., Wang, L., Mittal, N., Hao, M., Kwiatkowski, N. P., Gygi, S. P., Chakrabarti, R., Deng, X., Goldberg, D. E., Winzeler, E. A., Gray, N. S., Chakrabarti, D. 2023


    Protein kinases have proven to be a very productive class of therapeutic targets, and over 90 inhibitors are currently in clinical use primarily for the treatment of cancer. Repurposing these inhibitors as antimalarials could provide an accelerated path to drug development. In this study, we identified BI-2536, a known potent human polo-like kinase 1 inhibitor, with low nanomolar antiplasmodial activity. Screening of additional PLK1 inhibitors revealed further antiplasmodial candidates despite the lack of an obvious orthologue of PLKs in Plasmodium. A subset of these inhibitors was profiled for their in vitro killing profile, and commonalities between the killing rate and inhibition of nuclear replication were noted. A kinase panel screen identified PfNEK3 as a shared target of these PLK1 inhibitors; however, phosphoproteome analysis confirmed distinct signaling pathways were disrupted by two structurally distinct inhibitors, suggesting PfNEK3 may not be the sole target. Genomic analysis of BI-2536-resistant parasites revealed mutations in genes associated with the starvation-induced stress response, suggesting BI-2536 may also inhibit an aminoacyl-tRNA synthetase.

    View details for DOI 10.1021/acsinfecdis.3c00025

    View details for PubMedID 36919909

  • Targeting the Dark Lipid Kinase PIP4K2C with aPotent and Selective Binder and Degrader. Angewandte Chemie (International ed. in English) Teng, M., Jiang, J., Wang, E. S., Geng, Q., Toenjes, S. T., Donovan, K. A., Mageed, N., Yue, H., Nowak, R. P., Wang, J., Manz, T. D., Fischer, E. S., Cantley, L. C., Gray, N. S. 2023: e202302364


    Phosphatidylinositol 5-phosphate 4-kinase, type II, gamma (PIP4K2C) remains a poorly understood lipid kinase with minimal enzymatic activity but potential scaffolding roles in immune modulation and autophagy-dependent catabolism. Achieving potent and selective agents for PIP4K2C while sparing other lipid and non-lipid kinases has been challenging. Here, we report the discovery of the highly potent PIP4K2C binder TMX-4102, which shows exclusive binding selectivity for PIP4K2C. Furthermore, we elaborated this molecule into TMX-4153, a bivalent degrader capable of rapidly and selectively degrading endogenous PIP4K2C. Collectively, our work demonstrates that PIP4K2C is a tractable and a degradable target, and that TMX-4102 and TMX-4153 are useful leads to further interrogate the biological roles and therapeutic potential of PIP4K2C.

    View details for DOI 10.1002/anie.202302364

    View details for PubMedID 36898968

  • CDK7 controls E2F- and MYC-driven proliferative and metabolic vulnerabilities in multiple myeloma. Blood Yao, Y., Fong Ng, J., Park, W. D., Samur, M. K., Morelli, E., Encinas, J., Chyra, Z., Xu, Y., Derebail, S., Epstein, C. B., Nabet, B., Chesi, M., Gray, N. S., Young, R., Kwiatkowski, N., Mitsiades, C. S., Anderson, K. C., Lin, C. Y., Munshi, N. C., Fulciniti, M. 2023


    Therapeutic targeting of CDK7 has proven beneficial in pre-clinical studies, yet the off-target effects of currently available CDK7 inhibitors make it difficult to pinpoint the exact mechanisms behind MM cell death mediated by CDK7 inhibition. Here, we show that CDK7 expression positively correlates with E2F and MYC transcriptional programs in multiple myeloma (MM) patient cells; and its selective targeting counteracts E2F activity via perturbation of the CDKs/Rb axis and impairs MYC-regulated metabolic gene signatures translating into defects in glycolysis and reduced levels of lactate production in MM cells. CDK7 inhibition using the covalent small molecule inhibitor YKL-5-124 elicits a strong therapeutic response with minimal effects on normal cells, and causes in vivo tumor regression increasing survival in several MM mouse models including a genetically engineered mouse model of MYC-dependent MM. Through its role as a critical cofactor and regulator of MYC and E2F activity, CDK7 is therefore a master regulator of oncogenic cellular programs supporting MM growth and survival, and a valuable therapeutic target providing rationale for development of YKL-5-124 for clinical use.

    View details for DOI 10.1182/blood.2022018885

    View details for PubMedID 36877894

  • Development of a Covalent Inhibitor of c-Jun N-Terminal Protein Kinase (JNK) 2/3 with Selectivity over JNK1. Journal of medicinal chemistry Lu, W., Liu, Y., Gao, Y., Geng, Q., Gurbani, D., Li, L., Ficarro, S. B., Meyer, C. J., Sinha, D., You, I., Tse, J., He, Z., Ji, W., Che, J., Kim, A. Y., Yu, T., Wen, K., Anderson, K. C., Marto, J. A., Westover, K. D., Zhang, T., Gray, N. S. 2023


    The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family, which includes JNK1-JNK3. Interestingly, JNK1 and JNK2 show opposing functions, with JNK2 activity favoring cell survival and JNK1 stimulating apoptosis. Isoform-selective small molecule inhibitors of JNK1 or JNK2 would be useful as pharmacological probes but have been difficult to develop due to the similarity of their ATP binding pockets. Here, we describe the discovery of a covalent inhibitor YL5084, the first such inhibitor that displays selectivity for JNK2 over JNK1. We demonstrated that YL5084 forms a covalent bond with Cys116 of JNK2, exhibits a 20-fold higher Kinact/KI compared to that of JNK1, and engages JNK2 in cells. However, YL5084 exhibited JNK2-independent antiproliferative effects in multiple myeloma cells, suggesting the existence of additional targets relevant in this context. Thus, although not fully optimized, YL5084 represents a useful chemical starting point for the future development of JNK2-selective chemical probes.

    View details for DOI 10.1021/acs.jmedchem.2c01834

    View details for PubMedID 36826833

  • Depletion of creatine phosphagen energetics with a covalent creatine kinase inhibitor. Nature chemical biology Darabedian, N., Ji, W., Fan, M., Lin, S., Seo, H. S., Vinogradova, E. V., Yaron, T. M., Mills, E. L., Xiao, H., Senkane, K., Huntsman, E. M., Johnson, J. L., Che, J., Cantley, L. C., Cravatt, B. F., Dhe-Paganon, S., Stegmaier, K., Zhang, T., Gray, N. S., Chouchani, E. T. 2023


    Creatine kinases (CKs) provide local ATP production in periods of elevated energetic demand, such as during rapid anabolism and growth. Thus, creatine energetics has emerged as a major metabolic liability in many rapidly proliferating cancers. Whether CKs can be targeted therapeutically is unknown because no potent or selective CK inhibitors have been developed. Here we leverage an active site cysteine present in all CK isoforms to develop a selective covalent inhibitor of creatine phosphagen energetics, CKi. Using deep chemoproteomics, we discover that CKi selectively engages the active site cysteine of CKs in cells. A co-crystal structure of CKi with creatine kinase B indicates active site inhibition that prevents bidirectional phosphotransfer. In cells, CKi and its analogs rapidly and selectively deplete creatine phosphate, and drive toxicity selectively in CK-dependent acute myeloid leukemia. Finally, we use CKi to uncover an essential role for CKs in the regulation of proinflammatory cytokine production in macrophages.

    View details for DOI 10.1038/s41589-023-01273-x

    View details for PubMedID 36823351

    View details for PubMedCentralID 5540325

  • Reciprocal inhibition of PIN1 and APC/CCDH1 controls timely G1/S transition and creates therapeutic vulnerability. Research square Ke, S., Dang, F., Wang, L., Chen, J. Y., Naik, M. T., Thavamani, A., Liu, Y., Li, W., Kim, N., Naik, N. M., Sui, H., Tang, W., Qiu, C., Koikawa, K., Batalini, F., Wang, X., Clohessy, J. G., Heng, Y. J., Lahav, G., Gray, N. S., Zho, X. Z., Wei, W., Wulf, G. M., Lu, K. P. 2023


    Cyclin-dependent kinases (CDKs) mediated phosphorylation inactivates the anaphase-promoting complex (APC/CCDH1), an E3 ubiquitin ligase that contains the co-activator CDH1, to promote G1/S transition. PIN1 is a phosphorylation-directed proline isomerase and a master cancer signaling regulator. However, little are known about APC/CCDH1 regulation after phosphorylation and about PIN1 ubiquitin ligases. Here we uncover a domain-oriented reciprocal inhibition that controls the timely G1/S transition: The non-phosphorylated APC/CCDH1 E3 ligase targets PIN1 for degradation in G1 phase, restraining G1/S transition; APC/CCDH1 itself, after phosphorylation by CDKs, is inactivated by PIN1-catalyzed isomerization, promoting G1/S transition. In cancer, PIN1 overexpression and APC/CCDH1 inactivation reinforce each other to promote uncontrolled proliferation and tumorigenesis. Importantly, combined PIN1- and CDK4/6-inhibition reactivates APC/CCDH1 resulting in PIN1 degradation and an insurmountable G1 arrest that translates into synergistic anti-tumor activity against triple-negative breast cancer in vivo. Reciprocal inhibition of PIN1 and APC/CCDH1 is a novel mechanism to control timely G1/S transition that can be harnessed for synergistic anti-cancer therapy.

    View details for DOI 10.21203/

    View details for PubMedID 36711754

    View details for PubMedCentralID PMC9882653

  • The ETS transcription factor ETV6 constrains the transcriptional activity of EWS-FLI to promote Ewing sarcoma NATURE CELL BIOLOGY Lu, D. Y., Ellegast, J. M., Ross, K. N., Malone, C. F., Lin, S., Mabe, N. W., Dharia, N. V., Meyer, A., Conway, A., Su, A. H., Selich-Anderson, J., Taslim, C., Byrum, A. K., Seong, B. A., Adane, B., Gray, N. S., Rivera, M. N., Lessnick, S. L., Stegmaier, K. 2023: 285-297


    Transcription factors (TFs) are frequently mutated in cancer. Paediatric cancers exhibit few mutations genome-wide but frequently harbour sentinel mutations that affect TFs, which provides a context to precisely study the transcriptional circuits that support mutant TF-driven oncogenesis. A broadly relevant mechanism that has garnered intense focus involves the ability of mutant TFs to hijack wild-type lineage-specific TFs in self-reinforcing transcriptional circuits. However, it is not known whether this specific type of circuitry is equally crucial in all mutant TF-driven cancers. Here we describe an alternative yet central transcriptional mechanism that promotes Ewing sarcoma, wherein constraint, rather than reinforcement, of the activity of the fusion TF EWS-FLI supports cancer growth. We discover that ETV6 is a crucial TF dependency that is specific to this disease because it, counter-intuitively, represses the transcriptional output of EWS-FLI. This work discovers a previously undescribed transcriptional mechanism that promotes cancer.

    View details for DOI 10.1038/s41556-022-01059-8

    View details for Web of Science ID 000916317800006

    View details for PubMedID 36658220

    View details for PubMedCentralID PMC9928584

  • Advancing targeted protein degrader discovery by measuring cereblon engagement in cells. Methods in enzymology Zerfas, B. L., Huerta, F., Liu, H., Du, G., Gray, N. S., Jones, L. H., Nowak, R. P. 2023; 681: 169-188


    Measurement of target engagement in cells is critical to understand the molecular pharmacology of drugs and chemical probes. Many targeted protein degraders engage the E3 ligase CRL4CRBN and induce proximity with target neosubstrates resulting in their polyubiquitination and subsequent proteasomal degradation. Here we describe the development of a sensitive and robust cellular NanoBRET-based assay that measures occupancy of the CRBN ligand binding site. The assay is based on a bioluminescence resonance energy transfer (BRET) between NanoLuc luciferase tagged CRBN and a BODIPY-lenalidomide tracer which can be competed out by CRBN ligands, including PROTACs and molecular glues. The assay is compatible with a 384-well plate setup, does not require transfections and can be performed in a single day with only 3-4h of laboratory time. The protocols can be used to design other NanoLuc fusion engagement assays based on BODIPY tracers.

    View details for DOI 10.1016/bs.mie.2022.08.013

    View details for PubMedID 36764756

  • Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders BioRxiv - preprint Li, Y., Ma, M. W., Hassan, M. M., et al 2023
  • Development of potent and selective degraders of PI5P4Kgamma. European journal of medicinal chemistry Ji, W., Wang, E. S., Manz, T. D., Jiang, J., Donovan, K. A., Abulaiti, X., Fischer, E. S., Cantley, L. C., Zhang, T., Gray, N. S. 2022; 247: 115027


    Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks), a family of three members in mammals (alpha, beta and gamma), have emerged as potential therapeutic targets due to their role in regulating many important cellular signaling pathways. In comparison to the PI5P4Kalpha and PI5P4Kbeta, which usually have similar expression profiles across cancer cells, PI5P4Kgamma exhibits distinct expression patterns, and pathological functions for PI5P4Kgamma have been proposed in the context of cancer and neurodegenerative diseases. PI5P4Kgamma has very low kinase activity and has been proposed to inhibit the PI4P5Ks through scaffolding function, providing a rationale for developing a selective PI5P4Kgamma degrader. Here, we report the development and characterization of JWZ-1-80, a first-in-class PI5P4Kgamma degrader. JWZ-1-80 potently degrades PI5P4Kgamma via the ubiquitin-proteasome system and exhibits proteome-wide selectivity and is therefore a useful tool compound for further dissecting the biological functions of PI5P4Kgamma.

    View details for DOI 10.1016/j.ejmech.2022.115027

    View details for PubMedID 36584631

  • Development and Utility of a PAK1-Selective Degrader. Journal of medicinal chemistry Chow, H., Karchugina, S., Groendyke, B. J., Toenjes, S., Hatcher, J., Donovan, K. A., Fischer, E. S., Abalakov, G., Faezov, B., Dunbrack, R., Gray, N. S., Chernoff, J. 2022


    Overexpression of PAK1, a druggable kinase, is common in several malignancies, and inhibition of PAK1 by small molecules has been shown to impede the growth and survival of such cells. Potent inhibitors of PAKs 1-3 have been described, but clinical development has been hindered by recent findings that PAK2 function is required for normal cardiovascular function in adult mice. A unique allosteric PAK1-selective inhibitor, NVS-PAK1-1, provides a potential path forward, but has modest potency. Here, we report the development of BJG-05-039, a PAK1-selective degrader consisting of NVS-PAK1-1 conjugated to lenalidomide, a recruiter of the E3 ubiquitin ligase substrate adaptor Cereblon. BJG-05-039 induced selective degradation of PAK1 and displayed enhanced anti-proliferative effects relative to its parent compound in PAK1-dependent, but not PAK2-dependent, cell lines. Our findings suggest that selective PAK1 degradation may confer more potent pharmacological effects compared with catalytic inhibition and highlight the potential advantages of PAK1-targeted degradation.

    View details for DOI 10.1021/acs.jmedchem.2c00756

    View details for PubMedID 36416208

  • Transcriptional Antagonism by CDK8 Inhibition Improves Therapeutic Efficacy of MEK Inhibitors. Cancer research Malone, C. F., Kim, M., Alexe, G., Engel, K., Forman, A. B., Robichaud, A., Saur Conway, A., Goodale, A., Meyer, A., Khalid, D., Thayakumar, A., Hatcher, J. M., Gray, N. S., Piccioni, F., Stegmaier, K. 2022


    Aberrant RAS/MAPK signaling is a common driver of oncogenesis that can be therapeutically targeted with clinically approved MEK inhibitors. Disease progression on single agent MEK inhibitors is common, however, and combination therapies are typically required to achieve significant clinical benefit in advanced cancers. Here we focused on identifying MEK inhibitor-based combination therapies in neuroblastoma with mutations that activate the RAS/MAPK signaling pathway, which are rare at diagnosis but frequent in relapsed neuroblastoma. A genome-scale CRISPR-Cas9 functional genomic screen was deployed to identify genes that when knocked out sensitize RAS-mutant neuroblastoma to MEK inhibition. Loss of either CCNC or CDK8, two members of the mediator kinase module, sensitized neuroblastoma to MEK inhibition. Furthermore, small molecule kinase inhibitors of CDK8 improved response to MEK inhibitors in vitro and in vivo in RAS-mutant neuroblastoma and other adult solid tumors. Transcriptional profiling revealed that loss of CDK8 or CCNC antagonized the transcriptional signature induced by MEK inhibition. When combined, loss of CDK8 or CCNC prevented the compensatory upregulation of pro-growth gene expression induced by MEK inhibition. These findings propose a new therapeutic combination for RAS-mutant neuroblastoma and may have clinical relevance for other RAS-driven malignancies.

    View details for DOI 10.1158/0008-5472.CAN-21-4309

    View details for PubMedID 36398965

  • Therapeutic efficacy of selective CDK7 inhibition in pancreatic cancer mediated by induction of R-loop formation, DNA replication stress and genomic instability Yang, A., Jiang, J., Li, Z., Kapner, K. S., Feng, H., Lowder, K. E., Kuljanin, M., Johnson, W., Uribe, G., Neggers, J. E., Liu, S., Zhang, T., Decaprio, J., Sicinska, E., Wolpin, B. M., Kwiatkowski, N. P., Dougan, S. K., Mancias, J. D., Gray, N. S., Aguirre, A. J. AMER ASSOC CANCER RESEARCH. 2022: 38
  • Covalent disruptor of YAP-TEAD association suppresses defective hippo signaling. eLife Fan, M., Lu, W., Che, J., Kwiatkowski, N. P., Gao, Y., Seo, H., Ficarro, S. B., Gokhale, P. C., Liu, Y., Geffken, E. A., Lakhani, J., Song, K., Kuljanin, M., Ji, W., Jiang, J., He, Z., Tse, J., Boghossian, A. S., Rees, M. G., Ronan, M. M., Roth, J. A., Mancias, J. D., Marto, J. A., Dhe-Paganon, S., Zhang, T., Gray, N. S. 2022; 11


    The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03-69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03-69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03-69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03-69 led to an in vivo compatible compound MYF-03-176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration.

    View details for DOI 10.7554/eLife.78810

    View details for PubMedID 36300789

  • Acute pharmacological degradation of ERK5 does not inhibit cellular immune response or proliferation. Cell chemical biology You, I., Donovan, K. A., Krupnick, N. M., Boghossian, A. S., Rees, M. G., Ronan, M. M., Roth, J. A., Fischer, E. S., Wang, E. S., Gray, N. S. 2022


    Recent interest in the role that extracellular signal-regulated kinase 5 (ERK5) plays in various diseases, particularly cancer and inflammation, has grown. Phenotypes observed from genetic knockdown or deletion of ERK5 suggested that targeting ERK5 could have therapeutic potential in various disease settings, motivating the development ATP-competitive ERK5 inhibitors. However, these inhibitors were unable to recapitulate the effects of genetic loss of ERK5, suggesting that ERK5 may have key kinase-independent roles. To investigate potential non-catalytic functions of ERK5, we report the development of INY-06-061, a potent and selective heterobifunctional degrader of ERK5. In contrast to results reported through genetic knockdown of ERK5, INY-06-061-induced ERK5 degradation did not induce anti-proliferative effects in multiple cancer cell lines or suppress inflammatory responses in primary endothelial cells. Thus, we developed and characterized a chemical tool useful for validating phenotypes reported to be associated with genetic ERK5 ablation and for guiding future ERK5-directed drug discovery efforts.

    View details for DOI 10.1016/j.chembiol.2022.09.004

    View details for PubMedID 36220104

  • Anti-SARS-CoV-2 Activity of Targeted Kinase Inhibitors: Repurposing Clinically Available Drugs for COVID-19 Therapy. Journal of medical virology Boytz, R., Slabicki, M., Ramaswamy, S., Patten, J. J., Zou, C., Meng, C., Hurst, B. L., Wang, J., Nowak, R. P., Yang, P. L., Sattler, M., Stone, R. M., Griffin, J. D., Gray, N. S., Gummuluru, S., Davey, R. A., Weisberg, E. 2022


    PURPOSE: Coronavirus disease 2019 (COVID-19) remains a major public health concern, and vaccine unavailability, hesitancy, or failure underscore the need for discovery of efficacious antiviral drug therapies. Numerous approved drugs target protein kinases associated with viral life cycle and symptoms of infection. Repurposing of kinase inhibitors is appealing as they have been vetted for safety and are more accessible for COVID-19 treatment. However, an understanding of drug mechanism is needed to improve our understanding of the factors involved in pathogenesis.METHODS: We tested the in vitro activity of three kinase inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including inhibitors of AXL kinase, a host cell factor that contributes to successful SARS-CoV-2 infection. Using multiple cell-based assays and approaches, gilteritinib, nintedanib, and imatinib were thoroughly evaluated for activity against SARS-CoV-2 variants.RESULTS: Each drug exhibited antiviral activity, but with stark differences in potency, suggesting differences in host dependency for kinase targets. Importantly, for gilteritinib, the amount of compound needed to achieve 90% infection inhibition, at least in part involving blockade of spike protein-mediated viral entry and at concentrations not inducing phospholipidosis (PLD), approached a clinically achievable concentration. Knockout of AXL, a target of gilteritinib and nintedanib, impaired SARS-CoV-2 variant infectivity, supporting a role for AXL in SARS-CoV-2 infection and supporting further investigation of drug-mediated AXL inhibition as a COVID-19 treatment.CONCLUSIONS: This work supports further evaluation of AXL-targeting kinase inhibitors as potential antiviral agents and treatments for COVID-19. Additional mechanistic studies are needed to determine underlying differences in virus response. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/jmv.28157

    View details for PubMedID 36117402

  • Cereblon covalent modulation through structure-based design of histidine targeting chemical probes. RSC chemical biology Cruite, J. T., Dann, G. P., Che, J., Donovan, K. A., Ferrao, S., Ficarro, S. B., Fischer, E. S., Gray, N. S., Huerta, F., Kong, N. R., Liu, H., Marto, J. A., Metivier, R. J., Nowak, R. P., Zerfas, B. L., Jones, L. H. 2022; 3 (9): 1105-1110


    Electrophilic biocompatible warheads, particularly cysteine-reactive acrylamides, have enabled the development of covalent inhibitor drugs and chemical biology probes, but cysteine is rarely present in protein binding sites. Therefore, expansion of the list of targetable amino acid residues is required to augment the synthetic bology toolkit of site-selective protein modifications. This work describes the first rational targeting of a specific histidine residue in a protein binding site using sulfonyl exchange chemistry. Structure-based drug design was used to incorporate sulfonyl fluoride and triazole reactive groups into the isoindolinone thalidomide congener EM12 to yield potent covalent inhibitors of the cereblon E3 ubiquitin ligase complex through engagement of His353. Conversely, the fluorosulfate derivative EM12-FS labels His353, but degrades a novel neosubstrate, the protein N-terminal glutamine amidohydrolase NTAQ1, which is involved in the N-end rule pathway and DNA damage response. Targeted protein degradation using cereblon ligands has become an important new drug discovery modality and the chemical probes and covalent labeling strategy described here will broadly impact this exciting area of therapeutic research.

    View details for DOI 10.1039/d2cb00078d

    View details for PubMedID 36128501

    View details for PubMedCentralID PMC9428674

  • Exploring the target scope of KEAP1 E3 ligase-based PROTACs. Cell chemical biology Du, G., Jiang, J., Henning, N. J., Safaee, N., Koide, E., Nowak, R. P., Donovan, K. A., Yoon, H., You, I., Yue, H., Eleuteri, N. A., He, Z., Li, Z., Huang, H. T., Che, J., Nabet, B., Zhang, T., Fischer, E. S., Gray, N. S. 2022


    Targeted protein degradation (TPD) uses small molecules to recruit E3 ubiquitin ligases into the proximity of proteins of interest, inducing ubiquitination-dependent degradation. A major bottleneck in the TPD field is the lack of accessible E3 ligase ligands for developing degraders. To expand the E3 ligase toolbox, we sought to convert the Kelch-like ECH-associated protein 1 (KEAP1) inhibitor KI696 into a recruitment handle for several targets. While we were able to generate KEAP1-recruiting degraders of BET family and murine focal adhesion kinase (FAK), we discovered that the target scope of KEAP1 was narrow, as targets easily degraded using a cereblon (CRBN)-recruiting degrader were refractory to KEAP1-mediated degradation. Linking the KEAP1-binding ligand to a CRBN-binding ligand resulted in a molecule that induced degradation of KEAP1 but not CRBN. In sum, we characterize tool compounds to explore KEAP1-mediated ubiquitination and delineate the challenges of exploiting new E3 ligases for generating bivalent degraders.

    View details for DOI 10.1016/j.chembiol.2022.08.003

    View details for PubMedID 36070758

  • Redirecting the Neo-Substrate Specificity of Cereblon-Targeting PROTACs to Helios. ACS chemical biology Verano, A. L., You, I., Donovan, K. A., Mageed, N., Yue, H., Nowak, R. P., Fischer, E. S., Wang, E. S., Gray, N. S. 2022


    Immunomodulatory imide drugs (IMiDs), such as thalidomide and its analogues, are some of the most commonly utilized E3 ligase ligands for the development of proteolysis targeting chimeras (PROTACs). While the canonical neo-substrates of IMiDs (i.e., Ikaros and Aiolos) are often considered to be unwanted targets of PROTACs, maintaining the degradation of these neo-substrates also provides the opportunity to synergistically degrade multiple proteins with a single compound. Here, we report the development of ALV-07-082-03, a CDK4/CDK6/Helios triple degrader that consists of palbociclib, an FDA-approved CDK4/6 inhibitor, conjugated to DKY709, a novel IMiD-based Helios degrader. Pharmacological codegradation of CDK4/6 and Helios resulted in potent suppression of downstream signaling and proliferation in cancer cells, as well as enhanced derepression of IL-2 secretion. Thus, not only do we demonstrate the possibility of rationally redirecting the neo-substrate specificity of PROTACs by incorporating alternative molecular glue molecules as E3 ligase ligands but our findings also suggest that cotargeting CDK4/6 and Helios may have synergistic effects.

    View details for DOI 10.1021/acschembio.2c00439

    View details for PubMedID 36007246

  • PRM-LIVE Accelerates Target Class-based Selectivity Profiling of Small Molecule Inhibitors Zhu, H., Ficarro, S. B., Alexander, W. M., Fleming, L. E., Adelmant, G., Zhang, T., Willetts, M., Decker, J., Brehmer, S., Krause, M., East, M. P., Gray, N. S., Johnson, G. L., Kruppa, G., Mellors, J., Chan, W., Thompson, J., Tavares, I., Bratt, A. S., Buhrlage, S. J., Marto, J. A. ELSEVIER. 2022: S80
  • Targeting transcription in heart failure via CDK7/12/13 inhibition. Nature communications Hsu, A., Duan, Q., Day, D. S., Luo, X., McMahon, S., Huang, Y., Feldman, Z. B., Jiang, Z., Zhang, T., Liang, Y., Alexanian, M., Padmanabhan, A., Brown, J. D., Lin, C. Y., Gray, N. S., Young, R. A., Bruneau, B. G., Haldar, S. M. 2022; 13 (1): 4345


    Heart failure with reduced ejection fraction (HFrEF) is associated with high mortality, highlighting an urgent need for new therapeutic strategies. As stress-activated cardiac signaling cascades converge on the nucleus to drive maladaptive gene programs, interdicting pathological transcription is a conceptually attractive approach for HFrEF therapy. Here, we demonstrate that CDK7/12/13 are critical regulators of transcription activation in the heart that can be pharmacologically inhibited to improve HFrEF. CDK7/12/13 inhibition using the first-in-class inhibitor THZ1 or RNAi blocks stress-induced transcription and pathologic hypertrophy in cultured rodent cardiomyocytes. THZ1 potently attenuates adverse cardiac remodeling and HFrEF pathogenesis in mice and blocks cardinal features of disease in human iPSC-derived cardiomyocytes. THZ1 suppresses Pol II enrichment at stress-transactivated cardiac genes and inhibits a specific pathologic gene program in the failing mouse heart. These data identify CDK7/12/13 as druggable regulators of cardiac gene transactivation during disease-related stress, suggesting that HFrEF features a critical dependency on transcription that can be therapeutically exploited.

    View details for DOI 10.1038/s41467-022-31541-8

    View details for PubMedID 35896549

  • Quinazolinones as allosteric fourth-generation EGFR inhibitors for the treatment of NSCLC BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Gero, T. W., Heppner, D. E., Beyett, T. S., To, C., Azevedo, S. C., Jang, J., Bunnell, T., Feru, F., Li, Z., Shin, B., Soroko, K. M., Gokhale, P. C., Gray, N. S., Janne, P. A., Eck, M. J., Scott, D. A. 2022; 68: 128718


    The C797S mutation confers resistance to covalent EGFR inhibitors used in the treatment of lung tumors with the activating L858R mutation. Isoindolinones such as JBJ-4-125-02 bind in an allosteric pocket and are active against this mutation, with high selectivity over wild-type EGFR. The most potent examples we developed from that series have a potential chemical instability risk from the combination of the amide and phenol groups. We explored a scaffold hopping approach to identify new series of allosteric EGFR inhibitors that retained good potency in the absence of the phenol group. The 5-F quinazolinone 34 demonstrated tumor regression in an H1975 efficacy model upon once daily oral dosing at 25 mg/kg.

    View details for DOI 10.1016/j.bmcl.2022.128718

    View details for Web of Science ID 000800009100001

    View details for PubMedID 35378251

  • Temporal resolution of gene derepression and proteome changes upon PROTAC-mediated degradation of BCL11A protein in erythroid cells. Cell chemical biology Mehta, S., Buyanbat, A., Kai, Y., Karayel, O., Goldman, S. R., Seruggia, D., Zhang, K., Fujiwara, Y., Donovan, K. A., Zhu, Q., Yang, H., Nabet, B., Gray, N. S., Mann, M., Fischer, E. S., Adelman, K., Orkin, S. H. 2022


    Reactivation of fetal hemoglobin expression by the downregulation of BCL11A is a promising treatment for beta-hemoglobinopathies. A detailed understanding of BCL11A-mediated repression of gamma-globin gene (HBG1/2) transcription is lacking, as studies to date used perturbations by shRNA or CRISPR-Cas9 gene editing. We leveraged the dTAG PROTAC degradation platform to acutely deplete BCL11A protein in erythroid cells and examined consequences by nascent transcriptomics, proteomics, chromatin accessibility, and histone profiling. Among 31 genes repressed by BCL11A, HBG1/2 and HBZ show the most abundant and progressive changes in transcription and chromatin accessibility upon BCL11A loss. Transcriptional changes at HBG1/2 were detected in <2 h. Robust HBG1/2 reactivation upon acute BCL11A depletion occurred without the loss of promoter 5-methylcytosine (5mC). Using targeted protein degradation, we establish a hierarchy of gene reactivation at BCL11A targets, in which nascent transcription is followed by increased chromatin accessibility, and both are uncoupled from promoter DNA methylation at the HBG1/2 loci.

    View details for DOI 10.1016/j.chembiol.2022.06.007

    View details for PubMedID 35839780

  • Cereblon covalent modulation through structure-based design of histidine targeting chemical probes RSC CHEMICAL BIOLOGY Cruite, J. T., Dann, G. P., Che, J., Donovan, K. A., Ferrao, S., Ficarro, S. B., Fischer, E. S., Gray, N. S., Huerta, F., Kong, N. R., Liu, H., Marto, J. A., Metivier, R. J., Zerfas, B. L., Jones, L. H. 2022

    View details for DOI 10.1039/d2cb00078d

    View details for Web of Science ID 000824800100001

  • Transcriptional antagonism by CDK8 inhibition improves therapeutic efficacy of MEK inhibitors Malone, C. F., Kim, M., Alexe, G., Forman, A. B., Robichaud, A., Conway, A., Goodale, A., Hatcher, J. M., Gray, N. S., Piccioni, F., Stegmaier, K. AMER ASSOC CANCER RESEARCH. 2022
  • Tuning microtubule dynamics to enhance cancer therapy by modulating FER-mediated CRMP2 phosphorylation (vol 9, 476, 2018) NATURE COMMUNICATIONS Zheng, Y., Sethi, R., Mangala, L. S., Taylor, C., Goldsmith, J., Wang, M., Masuda, K., Carrami, E. M., Mannion, D., Miranda, F., Herrero-Gonzalez, S., Hellner, K., Chen, F., Alsaadi, A., Albukhari, A., Fotso, D., Yau, C., Jiang, D., Pradeep, S., Rodriguez-Aguayo, C., Lopez-Berestein, G., Knapp, S., Gray, N. S., Campo, L., Myers, K. A., Dhar, S., Ferguson, D., Bast, R. C., Sood, A. K., von Delft, F., Ahmed, A. 2022; 13 (1): 3352

    View details for DOI 10.1038/s41467-022-31011-1

    View details for Web of Science ID 000810123400001

    View details for PubMedID 35688808

  • Publication Criteria and Requirements for Studies on Protein Kinase Inhibitors-What Is Expected? ("It is pretty easy to make a bad kinase inhibitor") JOURNAL OF MEDICINAL CHEMISTRY Laufer, S., Bajorath, J., Gehringer, M., Gray, N., Frye, S., Lindsley, C. W. 2022; 65 (10): 6973-6974

    View details for DOI 10.1021/acs.jmedchem.2c00623

    View details for Web of Science ID 000808007900002

    View details for PubMedID 35512193

  • The Dawn of Allosteric BCR-ABL1 Drugs: From a Phenotypic Screening Hit to an Approved Drug. Journal of medicinal chemistry Teng, M., Luskin, M. R., Cowan-Jacob, S. W., Ding, Q., Fabbro, D., Gray, N. S. 2022


    Chronic myeloid leukemia (CML) is driven by the constitutive activity of the BCR-ABL1 fusion oncoprotein. Despite the great success of drugs that target the BCR-ABL1 ATP-binding site in transforming CML into a manageable disease, emerging resistance point mutations impair inhibitor binding, thereby limiting the effectiveness of these drugs. Recently, allosteric inhibitors that interact with the ABL1 myristate-binding site have been shown to awaken an endogenous regulatory mechanism and reset full-length BCR-ABL1 into an inactive assembled state. The discovery and development of these allosteric inhibitors demonstrates an in-depth understanding of the fundamental regulatory mechanisms of kinases. In this review, we illustrate the structural basis of c-ABL1's dynamic regulation of autoinhibition and activation, discuss the discovery of allosteric inhibitors and the characterization of their mechanism of action, present the therapeutic potential of dual binding to delay the development of mutation-driven acquired resistance, and suggest key lessons learned from this program.

    View details for DOI 10.1021/acs.jmedchem.2c00373

    View details for PubMedID 35609336

  • Molecular basis for cooperative binding and synergy of ATP-site and allosteric EGFR inhibitors. Nature communications Beyett, T. S., To, C., Heppner, D. E., Rana, J. K., Schmoker, A. M., Jang, J., De Clercq, D. J., Gomez, G., Scott, D. A., Gray, N. S., Janne, P. A., Eck, M. J. 2022; 13 (1): 2530


    Lung cancer is frequently caused by activating mutations in the epidermal growth factor receptor (EGFR). Allosteric EGFR inhibitors offer promise as the next generation of therapeutics, as they are unaffected by common ATP-site resistance mutations and synergize with the drug osimertinib. Here, we examine combinations of ATP-competitive and allosteric inhibitors to better understand the molecular basis for synergy. We identify a subset of irreversible EGFR inhibitors that display positive binding cooperativity and synergy with the allosteric inhibitor JBJ-04-125-02 in several EGFR variants. Structural analysis of these complexes reveals conformational changes occur mainly in the phosphate-binding loop (P-loop). Mutation of F723 in the P-loop reduces cooperative binding and synergy, supporting a mechanism in which F723-mediated contacts between the P-loop and the allosteric inhibitor are critical for synergy. These structural and mechanistic insights will aid in the identification and development of additional inhibitor combinations with potential clinical value.

    View details for DOI 10.1038/s41467-022-30258-y

    View details for PubMedID 35534503

  • Synthesis and Structure-Activity relationships of cyclin-dependent kinase 11 inhibitors based on a diaminothiazole scaffold. European journal of medicinal chemistry Li, Z., Ishida, R., Liu, Y., Wang, J., Li, Y., Gao, Y., Jiang, J., Che, J., Sheltzer, J. M., Robers, M. B., Zhang, T., Westover, K. D., Nabet, B., Gray, N. S. 2022; 238: 114433


    Cyclin-dependent kinases (CDK) are attractive targets for drug discovery due to their wide range of cellular functions. CDK11 is an understudied CDK with roles in transcription and splicing, cell cycle regulation, neuronal function, and apoptosis. In this study, we describe a medicinal chemistry campaign to identify a CDK11 inhibitor. Employing a promising but nonselective CDK11-targeting scaffold (JWD-047), extensive structure-guided medicinal chemistry modifications led to the identification of ZNL-05-044. A combination of biochemical evaluations and NanoBRET cellular assays for target engagement guided the SAR towards a 2,4-diaminothiazoles CDK11 probe with significantly improved kinome-wide selectivity over JWD-047. CDK11 inhibition with ZNL-05-044 leads to G2/M cell cycle arrest, consistent with prior work evaluating OTS964, and impacts CDK11-dependent mRNA splicing in cells. Together, ZNL-05-044 serves as a tool compound for further optimization and interrogation of the consequences of CDK11 inhibition.

    View details for DOI 10.1016/j.ejmech.2022.114433

    View details for PubMedID 35597007

  • A preclinical platform for assessing antitumor effects and systemic toxicities of cancer drug targets. Proceedings of the National Academy of Sciences of the United States of America Li, X., Huang, C. H., Sánchez-Rivera, F. J., Kennedy, M. C., Tschaharganeh, D. F., Morris, J. P., Montinaro, A., O'Rourke, K. P., Banito, A., Wilkinson, J. E., Chen, C. C., Ho, Y. J., Dow, L. E., Tian, S., Luan, W., de Stanchina, E., Zhang, T., Gray, N. S., Walczak, H., Lowe, S. W. 2022; 119 (17): e2110557119


    SignificanceMany new cancer drugs fail at the clinical stage owing to poor efficacy and/or excessive toxicity, though whether this reflects shortcomings of the target or the drug is often unclear. To gain earlier insights into factors that can influence the therapeutic index of target inhibition in vivo, we combine inducible RNA interference and somatic engineering technologies to produce a cost-effective platform that enables systemic and inducible suppression of candidate target in normal tissues and tumor cells in the same mouse. By comparing the consequences of genetic and pharmacological CDK9 inhibition, we establish the utility of this platform to predict factors influencing the therapeutic index. Additionally, our studies provide support, and some cautionary notes, for the clinical development of CDK9 inhibitors.

    View details for DOI 10.1073/pnas.2110557119

    View details for PubMedID 35442775

  • Selective Macrocyclic Inhibitors of DYRK1A/B. ACS medicinal chemistry letters Powell, C. E., Hatcher, J. M., Jiang, J., Vatsan, P. S., Che, J., Gray, N. S. 2022; 13 (4): 577-585


    Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) is a therapeutic target of interest due to the roles it plays in both neurological diseases and cancer. We present the development of the first macrocyclic inhibitors of DYRK1A. Initial lead inhibitor JH-XIV-68-3 (3) displayed selectivity for DYRK1A and close family member DYRK1B in biochemical and cellular assays, and demonstrated antitumor efficacy in head and neck squamous cell carcinoma (HNSCC) cell lines. However, we noted that it suffered from rapid aldehyde oxidase (AO)-mediated metabolism. To overcome this liability, we generated a derivative (JH-XVII-10 (10)), where fluorine was introduced to block the 2-position of the azaindole and render the molecule resistant to AO activity. We showed that 10 maintains remarkable potency and selectivity in biochemical and cellular assays as well as antitumor efficacy in HNSCC cell lines and improved metabolic stability. Therefore, 10 represents a promising new scaffold for developing DYRK1A-targeting chemical probes and therapeutics.

    View details for DOI 10.1021/acsmedchemlett.1c00630

    View details for PubMedID 35450378

  • An allosteric inhibitor against the therapy-resistant mutant forms of EGFR in non-small cell lung cancer. Nature cancer To, C., Beyett, T. S., Jang, J., Feng, W. W., Bahcall, M., Haikala, H. M., Shin, B. H., Heppner, D. E., Rana, J. K., Leeper, B. A., Soroko, K. M., Poitras, M. J., Gokhale, P. C., Kobayashi, Y., Wahid, K., Kurppa, K. J., Gero, T. W., Cameron, M. D., Ogino, A., Mushajiang, M., Xu, C., Zhang, Y., Scott, D. A., Eck, M. J., Gray, N. S., Janne, P. A. 2022


    Epidermal growth factor receptor (EGFR) therapy using small-molecule tyrosine kinase inhibitors (TKIs) is initially efficacious in patients with EGFR-mutant lung cancer, although drug resistance eventually develops. Allosteric EGFR inhibitors, which bind to a different EGFR site than existing ATP-competitive EGFR TKIs, have been developed as a strategy to overcome therapy-resistant EGFR mutations. Here we identify and characterize JBJ-09-063, a mutant-selective allosteric EGFR inhibitor that is effective across EGFR TKI-sensitive and resistant models, including those with EGFR T790M and C797S mutations. We further uncover that EGFR homo- or heterodimerization with other ERBB family members, as well as the EGFR L747S mutation, confers resistance to JBJ-09-063, but not to ATP-competitive EGFR TKIs. Overall, our studies highlight the potential clinical utility of JBJ-09-063 as a single agent or in combination with EGFR TKIs to define more effective strategies to treat EGFR-mutant lung cancer.

    View details for DOI 10.1038/s43018-022-00351-8

    View details for PubMedID 35422503

  • Unleashing cell-intrinsic inflammation as a strategy to kill AML blasts. Cancer discovery Ellegast, J. M., Alexe, G., Hamze, A., Lin, S., Uckelmann, H. J., Rauch, P. J., Pimkin, M., Ross, L. S., Dharia, N. V., Robichaud, A. L., Saur Conway, A., Khalid, D., Perry, J. A., Wunderlich, M., Benajiba, L., Pikman, Y., Nabet, B., Gray, N. S., Orkin, S. H., Stegmaier, K. 2022


    Leukemic blasts are immune cells gone awry. We hypothesized that dysregulation of inflammatory pathways contributes to the maintenance of their leukemic state and can be exploited as a cell-intrinsic, self-directed immunotherapy. To this end, we applied genome-wide screens to discover genetic vulnerabilities in acute myeloid leukemia (AML) cells implicated in inflammatory pathways. We identified the immune modulator interferon regulatory factor 2 binding protein 2 (IRF2BP2) as a selective AML dependency. We validated AML cell dependency on IRF2BP2 with genetic and protein degradation approaches in vitro and genetically in vivo. Chromatin and global gene expression studies demonstrated that IRF2BP2 represses IL-1B/TNFa signaling via NF-kappaB, and IRF2BP2 perturbation results in an acute inflammatory state leading to AML cell death. These findings elucidate a hitherto unexplored AML dependency, reveal cell-intrinsic inflammatory signaling as a mechanism priming leukemic blasts for regulated cell death, and establish IRF2BP2-mediated transcriptional repression as a mechanism for blast survival.

    View details for DOI 10.1158/2159-8290.CD-21-0956

    View details for PubMedID 35405016

  • A new role for the SRC family kinase HCK as a driver of SYK activation in MYD88 mutated lymphomas. Blood advances Munshi, M., Liu, X., Kofides, A., Tsakmaklis, N., Guerrera, M. L., Hunter, Z. R., Palomba, M. L., Argyropoulos, K. V., Patterson, C. J., Canning, A. G., Meid, K. E., Gustine, J., Branagan, A. R., Flynn, C., Sarosiek, S., Castillo, J. J., Wang, J., Buhrlage, S., Gray, N. S., Munshi, N. C., Anderson, K. C., Treon, S. P., Yang, G. 2022


    The SRC family kinase (SFK) HCK is transcriptionally upregulated and activated by mutated MYD88 (MYD88Mut), a key adaptor for Toll-receptor signaling. HCK activates BTK, AKT and ERK in MYD88Mut lymphomas. SYK, a BCR component is activated in MYD88Mut lymphoma cells. While the SFK LYN serves as a trigger for SYK activation in MYD88Mut ABC DLBCL cells, LYN activity is muted in MYD88Mut WM cells. We therefore investigated a role for HCK in mediating SYK activation. Over-expression of wild-type (HCKWT) or gatekeeper mutated (HCKThr333Met) HCK in MYD88Mut lymphoma cells triggered SYK activation. Conversely, HCK knockdown reduced p-SYK in MYD88Mut lymphoma cells. Co-immunoprecipitation experiments showed that HCK was complexed with p-SYK in MYD88Mut BCWM.1 and TMD8 cells, but not in MYD88 wild-type (WT) Ramos cells. Rescue experiments in MYD88Mut lymphoma cells expressing HCKThr333Met led to persistent HCK and SYK activation and resistance to the HCK inhibitor A419259. Treatment of primary MYD88Mut WM cells with A419259 reduced p-HCK and p-SYK expression. Taken together, our findings show that SYK is activated by HCK in MYD88Mut B-cell lymphomas cells; broaden the pro-survival signaling generated by aberrant HCK expression in response to MYD88Mut; and help define HCK as an important therapeutic target in MYD88Mut B-cell lymphomas.

    View details for DOI 10.1182/bloodadvances.2021006147

    View details for PubMedID 35255496

  • Discovery and Optimization of Tau Targeted Protein Degraders Enabled by Patient Induced Pluripotent Stem Cells-Derived Neuronal Models of Tauopathy FRONTIERS IN CELLULAR NEUROSCIENCE Silva, M., Nandi, G., Donovan, K. A., Cai, Q., Berry, B. C., Nowak, R. P., Fischer, E. S., Gray, N. S., Ferguson, F. M., Haggarty, S. J. 2022; 16: 801179


    Accumulation of misfolded, aggregating proteins concurrent with disease onset and progression is a hallmark of neurodegenerative proteinopathies. An important class of these are tauopathies, such as frontotemporal dementia (FTD) and Alzheimer's disease (AD), associated with accumulation of aberrant forms of tau protein in the brain. Pathological tau undergoes abnormal post-translational modifications, misfolding, oligomerization and changes in solubility, cellular redistribution, and spreading. Development and testing of experimental therapeutics that target these pathological tau conformers requires use of cellular models that recapitulate neuronal endogenous, non-heterologous tau expression under genomic and physiological contexts relevant to disease. In this study, we employed FTD-patient induced pluripotent stem cells (iPSC)-derived neurons, expressing a tau variant or mutation, as primary models for driving a medicinal chemistry campaign around tau targeting degrader series. Our screening goal was to establish structure-activity relationships (SAR) for the different chemical series to identify the molecular composition that most efficiently led to tau degradation in human FTD ex vivo neurons. We describe the identification of the lead compound QC-01-175 and follow-up optimization strategies for this molecule. We present three final lead molecules with tau degradation activity in mutant neurons, which establishes potential disease relevance and will drive future studies on specificity and pharmacological properties.

    View details for DOI 10.3389/fncel.2022.801179

    View details for Web of Science ID 000775600200001

    View details for PubMedID 35317195

    View details for PubMedCentralID PMC8934437

  • Novel Macrocyclic Peptidomimetics Targeting the Polo-Box Domain of Polo-Like Kinase 1. Journal of medicinal chemistry Ryu, S., Park, J., Ham, Y. J., Lim, D. C., Kwiatkowski, N. P., Kim, D., Bhunia, D., Kim, N. D., Yaffe, M. B., Son, W., Kim, N., Choi, T., Swain, P., Kim, C., Lee, J., Gray, N. S., Lee, K. S., Sim, T. 1800


    The polo-box domain (PBD) of Plk1 is a promising target for cancer therapeutics. We designed and synthesized novel phosphorylated macrocyclic peptidomimetics targeting PBD based on acyclic phosphopeptide PMQSpTPL. The inhibitory activities of 16e on Plk1-PBD is >30-fold higher than those of PMQSpTPL. Both 16a and 16e possess excellent selectivity for Plk1-PBD over Plk2/3-PBD. Analysis of the cocrystal structure of Plk1-PBD in complex with 16a reveals that the 3-(trifluoromethyl)benzoyl group in 16a interacts with Arg516 through a pi-stacking interaction. This pi-stacking interaction, which has not been reported previously, provides insight into the design of novel and potent Plk1-PBD inhibitors. Furthermore, 16h, a PEGlyated macrocyclic phosphopeptide derivative, induces Plk1 delocalization and mitotic failure in HeLa cells. Also, the number of phospho-H3-positive cells in a zebrafish embryo increases in proportion to the amount of 16a. Collectively, the novel macrocyclic peptidomimetics should serve as valuable templates for the design of potent and novel Plk1-PBD inhibitors.

    View details for DOI 10.1021/acs.jmedchem.1c01359

    View details for PubMedID 35029981

  • A novel HER2-selective kinase inhibitor is effective in HER2 mutant and amplified non-small cell lung cancer. Cancer research Son, J., Jang, J., Beyett, T. S., Eum, Y., Haikala, H. M., Verano, A., Lin, M., Hatcher, J. M., Kwiatkowski, N. P., Eser, P. Ö., Poitras, M. J., Wang, S., Xu, M., Gokhale, P. C., Cameron, M. D., Eck, M. J., Gray, N. S., Janne, P. A. 2022


    In-frame insertions in exon 20 of human epidermal growth factor receptor-2 (HER2) are the most common HER2 mutations in non-small cell lung cancer (NSCLC) patients, a disease in which approved EGFR/HER2 tyrosine kinase inhibitors (TKIs) display poor efficiency and undesirable side effects due to their strong inhibition of wild-type EGFR. Here, we report a HER2-selective covalent TKI, JBJ-08-178-01, that targets multiple HER2 activating mutations including exon 20 insertions as well as amplification. JBJ-08-178-01 displayed strong selectivity towards HER2 mutants over wild-type EGFR compared with other EGFR/HER2 TKIs. Determination of the crystal structure of HER2 in complex with JBJ-08-178-01 suggests that an interaction between the inhibitor and Ser783 may be responsible for HER2 selectivity. The compound showed strong antitumoral activity in HER2-mutant or -amplified in vitro and in vivo. Treatment with JBJ-08-178-01 also led to a reduction in total HER2 by promoting proteasomal degradation of the receptor. Taken together, the dual activity of JBJ-08-178-01 as a selective inhibitor and destabilizer of HER2 represents a combination which may lead to better efficacy and tolerance in NSCLC patients harboring HER2 genetic alterations or amplification.

    View details for DOI 10.1158/0008-5472.CAN-21-2693

    View details for PubMedID 35149586

  • Development of PDE6D and CK1alpha Degraders through Chemical Derivatization of FPFT-2216. Journal of medicinal chemistry Teng, M., Lu, W., Donovan, K. A., Sun, J., Krupnick, N. M., Nowak, R. P., Li, Y., Sperling, A. S., Zhang, T., Ebert, B. L., Fischer, E. S., Gray, N. S. 1800


    Immunomodulatory drugs are a class of drugs approved for the treatment of multiple myeloma. These compounds exert their clinical effects by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif, resulting in degradation of degron-containing targets. However, although many cellular proteins feature the degron motif, only a subset of those are degradable via this strategy. Here, we demonstrated that FPFT-2216, a previously reported "molecular glue" compound, degrades PDE6D, in addition to IKZF1, IKZF3, and CK1alpha. We used FPFT-2216 as a starting point for a focused medicinal chemistry campaign and developed TMX-4100 and TMX-4116, which exhibit greater selectivity for degrading PDE6D and CK1alpha, respectively. We also showed that the region in PDE6D that interacts with the FPFT-2216 derivatives is not the previously pursued prenyl-binding pocket. Moreover, we found that PDE6D depletion by FPFT-2216 does not impede the growth of KRASG12C-dependent MIA PaCa-2 cells, highlighting the challenges of drugging PDE6D-KRAS. Taken together, the approach we described here represents a general scheme to rapidly develop selective degraders by reprogramming E3 ubiquitin ligase substrate specificity.

    View details for DOI 10.1021/acs.jmedchem.1c01832

    View details for PubMedID 34965125

  • Prospects for Antibacterial Discovery and Development JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Privalsky, T. M., Soohoo, A. M., Wang, J., Walsh, C. T., Wright, G. D., Gordon, E. M., Gray, N. S., Khosla, C. 2021; 143 (50): 21127-21142


    The rising prevalence of multidrug-resistant bacteria is an urgent health crisis that can only be countered through renewed investment in the discovery and development of antibiotics. There is no panacea for the antibacterial resistance crisis; instead, a multifaceted approach is called for. In this Perspective we make the case that, in the face of evolving clinical needs and enabling technologies, numerous validated antibacterial targets and associated lead molecules deserve a second look. At the same time, many worthy targets lack good leads despite harboring druggable active sites. Creative and inspired techniques buoy discovery efforts; while soil screening efforts frequently lead to antibiotic rediscovery, researchers have found success searching for new antibiotic leads by studying underexplored ecological niches or by leveraging the abundance of available data from genome mining efforts. The judicious use of "polypharmacology" (i.e., the ability of a drug to alter the activities of multiple targets) can also provide new opportunities, as can the continued search for inhibitors of resistance enzymes with the capacity to breathe new life into old antibiotics. We conclude by highlighting available pharmacoeconomic models for antibacterial discovery and development while making the case for new ones.

    View details for DOI 10.1021/jacs.1c10200

    View details for Web of Science ID 000750819800018

    View details for PubMedID 34860516

  • A New Role for the SRC Family Member HCK As a Driver of BCR/SYK Signaling in MYD88 Mutated Lymphomas Munshi, M., Liu, X., Kofides, A., Tsakmaklis, N., Demos, M. G., Guerrera, M., Hunter, Z. R., Palomba, M., Argyropoulos, K. V., Patterson, C. J., Meid, K., Gustine, J., Castillo, J. J., Sarosiek, S., Flynn, C. A., Wang, J., Buhrlage, S. J., Gray, N. S., Munshi, N. C., Anderson, K. C., Yang, G., Treon, S. P. AMER SOC HEMATOLOGY. 2021
  • Abemaciclib is a potent inhibitor of DYRK1A and HIP kinases involved in transcriptional regulation. Nature communications Kaltheuner, I. H., Anand, K., Moecking, J., Duster, R., Wang, J., Gray, N. S., Geyer, M. 2021; 12 (1): 6607


    Homeodomain-interacting protein kinases (HIPKs) belong to the CMGC kinase family and are closely related to dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). HIPKs are regulators of various signaling pathways and involved in the pathology of cancer, chronic fibrosis, diabetes, and multiple neurodegenerative diseases. Here, we report the crystal structure of HIPK3 in its apo form at 2.5A resolution. Recombinant HIPKs and DYRK1A are auto-activated and phosphorylate the negative elongation factor SPT5, the transcription factor c-Myc, and the C-terminal domain of RNA polymerase II, suggesting a direct function in transcriptional regulation. Based on a database search, we identified abemaciclib, an FDA-approved Cdk4/Cdk6 inhibitor used for the treatment of metastatic breast cancer, as potent inhibitor of HIPK2, HIPK3, and DYRK1A. We determined the crystal structures of HIPK3 and DYRK1A bound to abemaciclib, showing a similar binding mode to the hinge region of the kinase as observed for Cdk6. Remarkably, DYRK1A is inhibited by abemaciclib to the same extent as Cdk4/Cdk6 in vitro, raising the question of whether targeting of DYRK1A contributes to the transcriptional inhibition and therapeutic activity of abemaciclib.

    View details for DOI 10.1038/s41467-021-26935-z

    View details for PubMedID 34785661

  • Development of Highly Potent and Selective Pyrazolopyridine Inhibitor of CDK8/19. ACS medicinal chemistry letters Hatcher, J. M., Vatsan, P. S., Wang, E., Jiang, J., Gray, N. S. 2021; 12 (11): 1689-1693


    CDK8 and its paralog CDK19 are cyclin-dependent kinases that are core components of the so-called Mediator complex that has essential roles as a positive and negative regulator of gene expression. Several efforts to develop inhibitors have yielded natural and synthetic ATP-competitive compounds including cortistatin A, Sel120, BCD-115, CCT251921 (1), and MSC2530818 (2). Here, we used a hybridization approach starting from CCT251921 and MSC2530818 to derive new inhibitors with the aim of developing highly potent and selective inhibitors of CDK8/19. Initial compounds suffered from rapid aldehyde oxidase-mediated metabolism. This liability was overcome by utilizing a pyrazolopyridine hinge binder with a chlorine at the C-3 position. These efforts resulted in JH-XVI-178 (compound 15), a highly potent and selective inhibitor of CDK8/19 that displays low clearance and moderate oral pharmacokinetic properties.

    View details for DOI 10.1021/acsmedchemlett.1c00300

    View details for PubMedID 34795857

  • Targeting transcription cycles in cancer. Nature reviews. Cancer Vervoort, S. J., Devlin, J. R., Kwiatkowski, N., Teng, M., Gray, N. S., Johnstone, R. W. 2021


    Accurate control of gene expression is essential for normal development and dysregulation of transcription underpins cancer onset and progression. Similar to cell cycle regulation, RNA polymerase II-driven transcription can be considered as a unidirectional multistep cycle, with thousands of unique transcription cycles occurring in concert within each cell. Each transcription cycle comprises recruitment, initiation, pausing, elongation, termination and recycling stages that are tightly controlled by the coordinated action of transcriptional cyclin-dependent kinases and their cognate cyclins as well as the opposing activity of transcriptional phosphatases. Oncogenic dysregulation of transcription can entail defective control of gene expression, either at select loci or more globally, impacting a large proportion of the genome. The resultant dependency on the core-transcriptional machinery is believed to render 'transcriptionally addicted' cancers sensitive to perturbation of transcription. Based on these findings, small molecules targeting transcriptional cyclin-dependent kinases and associated proteins hold promise for the treatment of cancer. Here, we utilize the transcription cycles concept to explain how dysregulation of these finely tuned gene expression processes may drive tumorigenesis and how therapeutically beneficial responses may arise from global or selective transcriptional perturbation. This conceptual framework helps to explain tumour-selective transcriptional dependencies and facilitates the rational design of combination therapies.

    View details for DOI 10.1038/s41568-021-00411-8

    View details for PubMedID 34675395

  • INK4 tumor suppressor proteins mediate resistance to CDK4/6 kinase inhibitors. Cancer discovery Li, Q., Jiang, B., Guo, J., Shao, H., Del Priore, I. S., Chang, Q., Kudo, R., Li, Z., Razavi, P., Liu, B., Boghossian, A. S., Rees, M. G., Ronan, M. M., Roth, J. A., Donovan, K. A., Palafox, M., Reis-Filho, J. S., de Stanchina, E., Fischer, E. S., Rosen, N., Serra, V., Koff, A., Chodera, J. D., Gray, N. S., Chandarlapaty, S. 2021


    Cyclin-dependent kinases 4 and 6 (CDK4/6), represent a major therapeutic vulnerability for breast cancer. The kinases are clinically targeted via ATP competitive inhibitors (CDK4/6i); however, drug resistance commonly emerges over time. To understand CDK4/6i resistance, we surveyed over 1,300 breast cancers and identify several genetic alterations (e.g. FAT1, PTEN or ARID1A loss) converging on upregulation of CDK6. Mechanistically, we demonstrate CDK6 causes resistance by inducing and binding CDK inhibitor INK4 proteins (e.g. p18INK4C). In vitro binding and kinase assays together with physical modeling reveal that the p18INK4C/D-cyclin/CDK6 complex occludes CDK4/6i binding while only weakly suppressing ATP binding. Suppression of INK4 expression or its binding to CDK6 restores CDK4/6i sensitivity. To overcome this constraint, we developed bifunctional degraders conjugating palbociclib with E3 ligands. Two resulting lead compounds potently degraded CDK4/6, leading to substantial antitumor effects in vivo, demonstrating the promising therapeutic potential for retargeting CDK4/6 despite CDK4/6i resistance.

    View details for DOI 10.1158/2159-8290.CD-20-1726

    View details for PubMedID 34544752

  • TRIM8 modulates the EWS/FLI oncoprotein to promote survival in Ewing sarcoma CANCER CELL Seong, B. A., Dharia, N., Lin, S., Donovan, K. A., Chong, S., Robichaud, A., Conway, A., Hamze, A., Ross, L., Alexe, G., Adane, B., Nabet, B., Ferguson, F. M., Stolte, B., Wang, E., Sun, J., Darzacq, X., Piccioni, F., Gray, N. S., Fischer, E. S., Stegmaier, K. 2021; 39 (9): 1262-+


    Fusion-transcription factors (fusion-TFs) represent a class of driver oncoproteins that are difficult to therapeutically target. Recently, protein degradation has emerged as a strategy to target these challenging oncoproteins. The mechanisms that regulate fusion-TF stability, however, are generally unknown. Using CRISPR-Cas9 screening, we discovered tripartite motif-containing 8 (TRIM8) as an E3 ubiquitin ligase that ubiquitinates and degrades EWS/FLI, a driver fusion-TF in Ewing sarcoma. Moreover, we identified TRIM8 as a selective dependency in Ewing sarcoma compared with >700 other cancer cell lines. Mechanistically, TRIM8 knockout led to an increase in EWS/FLI protein levels that was not tolerated. EWS/FLI acts as a neomorphic substrate for TRIM8, defining the selective nature of the dependency. Our results demonstrate that fusion-TF protein stability is tightly regulated and highlight fusion oncoprotein-specific regulators as selective therapeutic targets. This study provides a tractable strategy to therapeutically exploit oncogene overdose in Ewing sarcoma and potentially other fusion-TF-driven cancers.

    View details for DOI 10.1016/j.ccell.2021.07.003

    View details for Web of Science ID 000695626600016

    View details for PubMedID 34329586

    View details for PubMedCentralID PMC8443273

  • Exploring Ligand-Directed N-Acyl-N-alkylsulfonamide-Based Acylation Chemistry for Potential Targeted Degrader Development. ACS medicinal chemistry letters Teng, M., Jiang, J., Ficarro, S. B., Seo, H., Bae, J. H., Donovan, K. A., Fischer, E. S., Zhang, T., Dhe-Paganon, S., Marto, J. A., Gray, N. S. 2021; 12 (8): 1302-1307


    Ligand-directed bioconjugation strategies have been used for selective protein labeling in live cells or tissue samples in applications such as live-cell imaging. Here we hypothesized that a similar strategy could be used for targeted protein degradation. To test this possibility, we developed a series of CDK2-targeting N-acyl-N-alkylsulfonamide (NASA)-containing acylation probes. The probes featured three components: a CDK2 homing ligand, a CRL4CRBN E3 ligase recruiting ligand, and a NASA functionality. We determined that upon target binding, NASA-mediated reaction resulted in selective functionalization of Lys89 on purified or native CDK2. However, we were unable to observe CDK2 degradation, which is in contrast to the efficient degradation achieved by the use of a structurally similar reversible bivalent degrader. Our analysis suggests that the lack of degradation is due to the failure to form a productive CDK2:CRBN complex. Therefore, although this work demonstrates that NASA chemistry can be used for protein labeling, whether this strategy could enable efficient protein degradation remains an open question.

    View details for DOI 10.1021/acsmedchemlett.1c00285

    View details for PubMedID 34413960

  • Targeting Pin1 renders pancreatic cancer eradicable by synergizing with immunochemotherapy. Cell Koikawa, K., Kibe, S., Suizu, F., Sekino, N., Kim, N., Manz, T. D., Pinch, B. J., Akshinthala, D., Verma, A., Gaglia, G., Nezu, Y., Ke, S., Qiu, C., Ohuchida, K., Oda, Y., Lee, T. H., Wegiel, B., Clohessy, J. G., London, N., Santagata, S., Wulf, G. M., Hidalgo, M., Muthuswamy, S. K., Nakamura, M., Gray, N. S., Zhou, X. Z., Lu, K. P. 2021


    Pancreatic ductal adenocarcinoma (PDAC) is characterized by notorious resistance to current therapies attributed to inherent tumor heterogeneity and highly desmoplastic and immunosuppressive tumor microenvironment (TME). Unique proline isomerase Pin1 regulates multiple cancer pathways, but its role in the TME and cancer immunotherapy is unknown. Here, we find that Pin1 is overexpressed both in cancer cells and cancer-associated fibroblasts (CAFs) and correlates with poor survival in PDAC patients. Targeting Pin1 using clinically available drugs induces complete elimination or sustained remissions of aggressive PDAC by synergizing with anti-PD-1 and gemcitabine in diverse model systems. Mechanistically, Pin1 drives the desmoplastic and immunosuppressive TME by acting on CAFs and induces lysosomal degradation of the PD-1 ligand PD-L1 and the gemcitabine transporter ENT1 in cancer cells, besides activating multiple cancer pathways. Thus, Pin1 inhibition simultaneously blocks multiple cancer pathways, disrupts the desmoplastic and immunosuppressive TME, and upregulates PD-L1 and ENT1, rendering PDAC eradicable by immunochemotherapy.

    View details for DOI 10.1016/j.cell.2021.07.020

    View details for PubMedID 34388391

  • Dual targeting of salt inducible kinases and CSF1R uncouples bone formation and bone resorption ELIFE Tang, C., Andrade, C., O'Meara, M. J., Yoon, S., Sato, T., Brooks, D. J., Bouxsein, M. L., Martins, J., Wang, J., Gray, N. S., Misof, B., Roschger, P., Boulin, S., Klaushofer, K., Velduis-Vlug, A., Vegting, Y., Rosen, C. J., O'Connell, D., Sundberg, T. B., Xavier, R. J., Ung, P., Schlessinger, A., Kronenberg, H. M., Berdeaux, R., Foretz, M., Wein, M. N. 2021; 10


    Bone formation and resorption are typically coupled, such that the efficacy of anabolic osteoporosis treatments may be limited by bone destruction. The multi-kinase inhibitor YKL-05-099 potently inhibits salt inducible kinases (SIKs) and may represent a promising new class of bone anabolic agents. Here, we report that YKL-05-099 increases bone formation in hypogonadal female mice without increasing bone resorption. Postnatal mice with inducible, global deletion of SIK2 and SIK3 show increased bone mass, increased bone formation, and, distinct from the effects of YKL-05-099, increased bone resorption. No cell-intrinsic role of SIKs in osteoclasts was noted. In addition to blocking SIKs, YKL-05-099 also binds and inhibits CSF1R, the receptor for the osteoclastogenic cytokine M-CSF. Modeling reveals that YKL-05-099 binds to SIK2 and CSF1R in a similar manner. Dual targeting of SIK2/3 and CSF1R induces bone formation without concomitantly increasing bone resorption and thereby may overcome limitations of most current anabolic osteoporosis therapies.

    View details for DOI 10.7554/eLife.67772

    View details for Web of Science ID 000765167000001

    View details for PubMedID 34160349

    View details for PubMedCentralID PMC8238509

  • ULK1 inhibition overcomes compromised antigen presentation and restores antitumor immunity in LKB1-mutant lung cancer NATURE CANCER Deng, J., Thennavan, A., Dolgalev, I., Chen, T., Li, J., Marzio, A., Poirier, J. T., Peng, D. H., Bulatovic, M., Mukhopadhyay, S., Silver, H., Papadopoulos, E., Pyon, V., Thakurdin, C., Han, H., Li, F., Li, S., Ding, H., Hu, H., Pan, Y., Weerasekara, V., Jiang, B., Wang, E. S., Ahearn, I., Philips, M., Papagiannakopoulos, T., Tsirigos, A., Rothenberg, E., Gainor, J., Freeman, G. J., Rudin, C. M., Gray, N. S., Hammerman, P. S., Pagano, M., Heymach, J. V., Perou, C. M., Bardeesy, N., Wong, K. 2021
  • Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo. Nature chemical biology Dubiella, C., Pinch, B. J., Koikawa, K., Zaidman, D., Poon, E., Manz, T. D., Nabet, B., He, S., Resnick, E., Rogel, A., Langer, E. M., Daniel, C. J., Seo, H., Chen, Y., Adelmant, G., Sharifzadeh, S., Ficarro, S. B., Jamin, Y., Martins da Costa, B., Zimmerman, M. W., Lian, X., Kibe, S., Kozono, S., Doctor, Z. M., Browne, C. M., Yang, A., Stoler-Barak, L., Shah, R. B., Vangos, N. E., Geffken, E. A., Oren, R., Koide, E., Sidi, S., Shulman, Z., Wang, C., Marto, J. A., Dhe-Paganon, S., Look, T., Zhou, X. Z., Lu, K. P., Sears, R. C., Chesler, L., Gray, N. S., London, N. 2021


    The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.

    View details for DOI 10.1038/s41589-021-00786-7

    View details for PubMedID 33972797

  • ULK1 inhibition overcomes compromised antigen presentation and restores antitumor immunity in LKB1 mutant lung cancer. Nature cancer Deng, J., Thennavan, A., Dolgalev, I., Chen, T., Li, J., Marzio, A., Poirier, J. T., Peng, D. H., Bulatovic, M., Mukhopadhyay, S., Silver, H., Papadopoulos, E., Pyon, V., Thakurdin, C., Han, H., Li, F., Li, S., Ding, H., Hu, H., Pan, Y., Weerasekara, V., Jiang, B., Wang, E. S., Ahearn, I., Philips, M., Papagiannakopoulos, T., Tsirigos, A., Rothenberg, E., Gainor, J., Freeman, G. J., Rudin, C. M., Gray, N. S., Hammerman, P. S., Pagano, M., Heymach, J. V., Perou, C. M., Bardeesy, N., Wong, K. K. 2021; 2 (5): 503-514

    View details for DOI 10.1038/s43018-021-00208-6

    View details for PubMedID 34142094

    View details for PubMedCentralID PMC8205437

  • Discovery of a Potent Degrader for Fibroblast Growth Factor Receptor 1/2. Angewandte Chemie (International ed. in English) Du, G., Jiang, J., Wu, Q., Henning, N. J., Donovan, K. A., Yue, H., Che, J., Lu, W., Fischer, E. S., Bardeesy, N., Zhang, T., Gray, N. S. 2021


    Aberrant activation of FGFR signaling occurs in many cancers, and ATP-competitive FGFR inhibitors have received regulatory approval. Despite demonstrating clinical efficacy, these inhibitors exhibit dose-limiting toxicity, potentially due to a lack of selectivity amongst the FGFR family and are poorly tolerated. Here, we report the discovery and characterization of DGY-09-192, a bivalent degrader that couples the pan-FGFR inhibitor BGJ398 to a CRL2VHL E3 ligase recruiting ligand, which preferentially induces FGFR1&2 degradation while largely sparing FGFR3&4. DGY-09-192 exhibited two-digit nanomolar DC50 s for both wildtype FGFR2 and several FGFR2-fusions, resulting in degradation-dependent antiproliferative activity in representative gastric cancer and cholangiocarcinoma cells. Importantly, DGY-09-192 induced degradation of a clinically relevant FGFR2 fusion protein in a xenograft model. Taken together, we demonstrate that DGY-09-192 has potential as a prototype FGFR degrader.

    View details for DOI 10.1002/anie.202101328

    View details for PubMedID 33915015

  • PRM-LIVE with Trapped Ion Mobility Spectrometry and Its Application in Selectivity Profiling of Kinase Inhibitors. Analytical chemistry Zhu, H., Ficarro, S. B., Alexander, W. M., Fleming, L. E., Adelmant, G., Zhang, T., Willetts, M., Decker, J., Brehmer, S., Krause, M., East, M. P., Gray, N. S., Johnson, G. L., Kruppa, G., Marto, J. A. 2021


    Parallel reaction monitoring (PRM) has emerged as a popular approach for targeted protein quantification. With high ion utilization efficiency and first-in-class acquisition speed, the timsTOF Pro provides a powerful platform for PRM analysis. However, sporadic chromatographic drift in peptide retention time represents a fundamental limitation for the reproducible multiplexing of targets across PRM acquisitions. Here, we present PRM-LIVE, an extensible, Python-based acquisition engine for the timsTOF Pro, which dynamically adjusts detection windows for reproducible target scheduling. In this initial implementation, we used iRT peptides as retention time standards and demonstrated reproducible detection and quantification of 1857 tryptic peptides from the cell lysate in a 60 min PRM-LIVE acquisition. As an application in functional proteomics, we use PRM-LIVE in an activity-based protein profiling platform to assess binding selectivity of small-molecule inhibitors against 220 endogenous human kinases.

    View details for DOI 10.1021/acs.analchem.1c02349

    View details for PubMedID 34606255

  • Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition SCIENCE ADVANCES Fassl, A., Brain, C., Abu-Remaileh, M., Stukan, I., Butter, D., Stepien, P., Feit, A. S., Bergholz, J., Michowski, W., Otto, T., Sheng, Q., Loo, A., Michael, W., Tiedt, R., DeAngelis, C., Schiff, R., Jiang, B., Jovanovic, B., Nowak, K., Ericsson, M., Cameron, M., Gray, N., Dillon, D., Zhao, J. J., Sabatini, D. M., Jeselsohn, R., Brown, M., Polyak, K., Sicinski, P. 2020; 6 (25)
  • Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition. Science advances Fassl, A., Brain, C., Abu-Remaileh, M., Stukan, I., Butter, D., Stepien, P., Feit, A. S., Bergholz, J., Michowski, W., Otto, T., Sheng, Q., Loo, A., Michael, W., Tiedt, R., DeAngelis, C., Schiff, R., Jiang, B., Jovanovic, B., Nowak, K., Ericsson, M., Cameron, M., Gray, N., Dillon, D., Zhao, J. J., Sabatini, D. M., Jeselsohn, R., Brown, M., Polyak, K., Sicinski, P. 2020; 6 (25)


    Inhibitors of cyclin-dependent kinases CDK4 and CDK6 have been approved for treatment of hormone receptor-positive breast cancers. In contrast, triple-negative breast cancers (TNBCs) are resistant to CDK4/6 inhibition. Here, we demonstrate that a subset of TNBC critically requires CDK4/6 for proliferation, and yet, these TNBC are resistant to CDK4/6 inhibition due to sequestration of CDK4/6 inhibitors into tumor cell lysosomes. This sequestration is caused by enhanced lysosomal biogenesis and increased lysosomal numbers in TNBC cells. We developed new CDK4/6 inhibitor compounds that evade the lysosomal sequestration and are efficacious against resistant TNBC. We also show that coadministration of lysosomotropic or lysosome-destabilizing compounds (an antibiotic azithromycin, an antidepressant siramesine, an antimalaria compound chloroquine) renders resistant tumor cells sensitive to currently used CDK4/6 inhibitors. Lastly, coinhibition of CDK2 arrested proliferation of CDK4/6 inhibitor-resistant cells. These observations may extend the use of CDK4/6 inhibitors to TNBCs that are refractory to current anti-CDK4/6 therapies.

    View details for DOI 10.1126/sciadv.abb2210

    View details for PubMedID 32937480

  • Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition. Science advances Fassl, A. n., Brain, C. n., Abu-Remaileh, M. n., Stukan, I. n., Butter, D. n., Stepien, P. n., Feit, A. S., Bergholz, J. n., Michowski, W. n., Otto, T. n., Sheng, Q. n., Loo, A. n., Michael, W. n., Tiedt, R. n., DeAngelis, C. n., Schiff, R. n., Jiang, B. n., Jovanovic, B. n., Nowak, K. n., Ericsson, M. n., Cameron, M. n., Gray, N. n., Dillon, D. n., Zhao, J. J., Sabatini, D. M., Jeselsohn, R. n., Brown, M. n., Polyak, K. n., Sicinski, P. n. 2020; 6 (25): eabb2210


    Inhibitors of cyclin-dependent kinases CDK4 and CDK6 have been approved for treatment of hormone receptor-positive breast cancers. In contrast, triple-negative breast cancers (TNBCs) are resistant to CDK4/6 inhibition. Here, we demonstrate that a subset of TNBC critically requires CDK4/6 for proliferation, and yet, these TNBC are resistant to CDK4/6 inhibition due to sequestration of CDK4/6 inhibitors into tumor cell lysosomes. This sequestration is caused by enhanced lysosomal biogenesis and increased lysosomal numbers in TNBC cells. We developed new CDK4/6 inhibitor compounds that evade the lysosomal sequestration and are efficacious against resistant TNBC. We also show that coadministration of lysosomotropic or lysosome-destabilizing compounds (an antibiotic azithromycin, an antidepressant siramesine, an antimalaria compound chloroquine) renders resistant tumor cells sensitive to currently used CDK4/6 inhibitors. Lastly, coinhibition of CDK2 arrested proliferation of CDK4/6 inhibitor-resistant cells. These observations may extend the use of CDK4/6 inhibitors to TNBCs that are refractory to current anti-CDK4/6 therapies.

    View details for DOI 10.1126/sciadv.abb2210

    View details for PubMedID 32704543

    View details for PubMedCentralID PMC7360435

  • Coordinating Tissue Regeneration Through Transforming Growth Factor-beta Activated Kinase 1 Inactivation and Reactivation STEM CELLS Hsieh, H., Agarwal, S., Cholok, D. J., Loder, S. J., Kaneko, K., Huber, A., Chung, M. T., Ranganathan, K., Habbouche, J., Li, J., Butts, J., Reimer, J., Kaura, A., Drake, J., Breuler, C., Priest, C. R., Nguyen, J., Brownley, C., Peterson, J., Ozgurel, S., Niknafs, Y. S., Li, S., Inagaki, M., Scott, G., Krebsbach, P. H., Longaker, M. T., Westover, K., Gray, N., Ninomiya-Tsuji, J., Mishina, Y., Levi, B. 2019; 37 (6): 766–78

    View details for DOI 10.1002/stem.2991

    View details for Web of Science ID 000474038500008

  • Coordinating Tissue Regeneration through TGF-beta Activated Kinase 1 (TAK1) In-activation and Re-activation. Stem cells (Dayton, Ohio) Sung Hsieh, H. H., Agarwal, S., Cholok, D. J., Loder, S. J., Kaneko, K., Huber, A., Chung, M. T., Ranganathan, K., Habbouche, J., Li, J., Butts, J., Reimer, J., Kaura, A., Drake, J., Breuler, C., Priest, C. R., Nguyen, J., Brownley, C., Peterson, J., Ozgurel, S. U., Niknafs, Y. S., Li, S., Inagaki, M., Scott, G., Krebsbach, P., Longaker, M. T., Westover, K., Gray, N., Ninomiya-Tsuji, J., Mishina, Y., Levi, B. 2019


    Aberrant wound healing presents as inappropriate or insufficient tissue formation. Using a model of musculoskeletal injury, we demonstrate that loss of TGF-beta activated kinase 1 (TAK1) signaling reduces inappropriate tissue formation (heterotopic ossification) through reduced cellular differentiation. Upon identifying increased proliferation with loss of TAK1 signaling, we considered a regenerative approach to address insufficient tissue production through coordinated inactivation of TAK1 to promote cellular proliferation, followed by re-activation to elicit differentiation and extracellular matrix (ECM) production. While the current regenerative medicine paradigm is centered on the effects of drug treatment ("drug on"), the impact of drug withdrawal ("drug off") implicit in these regimens are unknown. Because current TAK1 inhibitors are unable to phenocopy genetic Tak1 loss, we introduce the dual-inducible COmbinational Sequential Inversion ENgineering (COSIEN) mouse model. The COSIEN mouse model, which allows us to study the response to targeted drug treatment ("drug on") and subsequent withdrawal ("drug off") through genetic modification, was used here to inactivate and re-activate Tak1 with the purpose of augmenting tissue regeneration in a calvarial defect model. Our study reveals the importance of both the "drug on" (Cre-mediated inactivation) and "drug off" (Flp-mediated re-activation) states during regenerative therapy using a mouse model with broad utility to study targeted therapies for disease. SIGNIFICANCE STATEMENT: We target the TAK1 pathway to reduce heterotopic ossification, a pathologic condition in which bone develops within muscle or soft tissues. We show that Tak1 knockout leads to cellular proliferation; this can be harnessed to increase the number of cells present at the injury site. Using a mouse model, we inactivate and reactivate the Tak1 gene. We show that inactivation and reactivation of Tak1 can improve bony healing through the coordination of increased proliferation (inactivation) followed by differentiation (reactivation). This approach elucidates a new paradigm in regenerative medicine in which coordination between treatment and withdrawal of treatment can augment healing. © AlphaMed Press 2019.

    View details for PubMedID 30786091

  • A Next Generation Connectivity Map: L1000 Platform and the First 1,000,000 Profiles CELL Subramanian, A., Narayan, R., Corsello, S. M., Peck, D. D., Natoli, T. E., Lu, X., Gould, J., Davis, J. F., Tubelli, A. A., Asiedu, J. K., Lahr, D. L., Hirschman, J. E., Liu, Z., Donahue, M., Julian, B., Khan, M., Wadden, D., Smith, I. C., Lam, D., Liberzon, A., Toder, C., Bagul, M., Orzechowski, M., Enache, O. M., Piccioni, F., Johnson, S. A., Lyons, N. J., Berger, A. H., Shamji, A. F., Brooks, A. N., Vrcic, A., Flynn, C., Rosains, J., Takeda, D. Y., Hu, R., Davison, D., Lamb, J., Ardlie, K., Hogstrom, L., Greenside, P., Gray, N. S., Clemons, P. A., Silver, S., Wu, X., Zhao, W., Read-Button, W., Wu, X., Haggarty, S. J., Ronco, L. V., Boehm, J. S., Schreiber, S. L., Doench, J. G., Bittker, J. A., Root, D. E., Wong, B., Golub, T. R. 2017; 171 (6): 1437-+


    We previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs, and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high-throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMap can be used to discover mechanism of action of small molecules, functionally annotate genetic variants of disease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at

    View details for DOI 10.1016/j.cell.2017.10.049

    View details for Web of Science ID 000417362700023

    View details for PubMedID 29195078

  • SIKs control osteocyte responses to parathyroid hormone NATURE COMMUNICATIONS Wein, M. N., Liang, Y., Goransson, O., Sundberg, T. B., Wang, J., Williams, E. A., O'Meara, M. J., Govea, N., Beqo, B., Nishimori, S., Nagano, K., Brooks, D. J., Martins, J. S., Corbin, B., Anselmo, A., Sadreyev, R., Wu, J. Y., Sakamoto, K., Foretz, M., Xavier, R. J., Baron, R., Bouxsein, M. L., Gardella, T. J., Divieti-Pajevic, P., Gray, N. S., Kronenberg, H. M. 2016; 7


    Parathyroid hormone (PTH) activates receptors on osteocytes to orchestrate bone formation and resorption. Here we show that PTH inhibition of SOST (sclerostin), a WNT antagonist, requires HDAC4 and HDAC5, whereas PTH stimulation of RANKL, a stimulator of bone resorption, requires CRTC2. Salt inducible kinases (SIKs) control subcellular localization of HDAC4/5 and CRTC2. PTH regulates both HDAC4/5 and CRTC2 localization via phosphorylation and inhibition of SIK2. Like PTH, new small molecule SIK inhibitors cause decreased phosphorylation and increased nuclear translocation of HDAC4/5 and CRTC2. SIK inhibition mimics many of the effects of PTH in osteocytes as assessed by RNA-seq in cultured osteocytes and following in vivo administration. Once daily treatment with the small molecule SIK inhibitor YKL-05-099 increases bone formation and bone mass. Therefore, a major arm of PTH signalling in osteocytes involves SIK inhibition, and small molecule SIK inhibitors may be applied therapeutically to mimic skeletal effects of PTH.

    View details for DOI 10.1038/ncomms13176

    View details for PubMedID 27759007

  • Pathophysiological significance and therapeutic targeting of germinal center kinase in diffuse large B-cell lymphoma. Blood Matthews, J. M., Bhatt, S., Patricelli, M. P., Nomanbhoy, T. K., Jiang, X., Natkunam, Y., Gentles, A. J., Martinez, E., Zhu, D., Chapman, J. R., Cortizas, E., Shyam, R., Chinichian, S., Advani, R., Tan, L., Zhang, J., Choi, H. G., Tibshirani, R., Buhrlage, S. J., Gratzinger, D., Verdun, R., Gray, N. S., Lossos, I. S. 2016; 128 (2): 239-248


    Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), yet 40-50% of patients will eventually succumb to their disease demonstrating a pressing need for novel therapeutic options. Gene expression profiling has identified messenger RNA's that lead to transformation, but critical events transforming cells are normally executed by kinases. Therefore, we hypothesized that previously unrecognized kinases may contribute to DLBCL pathogenesis. We performed the first comprehensive analysis of global kinase activity in DLBCL, to identify novel therapeutic targets, and discovered that Germinal Center Kinase (GCK) was extensively activated. GCK RNA interference and small molecule inhibition induced cell cycle arrest and apoptosis in DLBCL cell lines and primary tumors in vitro and decreased the tumor growth rate in vivo, resulting in a significantly extended lifespan of mice bearing DLBCL xenografts. GCK expression was also linked to adverse clinical outcome in a cohort of 151 primary DLBCL patients. These studies demonstrate, for the first time, that GCK is a molecular therapeutic target in DLBCL tumors and that inhibiting GCK may significantly extend DLBCL patient survival. Since the majority of DLBCL tumors (~80%) exhibit activation of GCK, this therapy may be applicable to most patients.

    View details for DOI 10.1182/blood-2016-02-696856

    View details for PubMedID 27151888

  • Activation of HIPK2 Promotes ER Stress-Mediated Neurodegeneration in Amyotrophic Lateral Sclerosis. Neuron Lee, S., Shang, Y., Redmond, S. A., Urisman, A., Tang, A. A., Li, K. H., Burlingame, A. L., Pak, R. A., Jovicic, A., Gitler, A. D., Wang, J., Gray, N. S., Seeley, W. W., Siddique, T., Bigio, E. H., Lee, V. M., Trojanowski, J. Q., Chan, J. R., Huang, E. J. 2016; 91 (1): 41-55


    Persistent accumulation of misfolded proteins causes endoplasmic reticulum (ER) stress, a prominent feature in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Here we report the identification of homeodomain interacting protein kinase 2 (HIPK2) as the essential link that promotes ER-stress-induced cell death via the IRE1α-ASK1-JNK pathway. ER stress, induced by tunicamycin or SOD1(G93A), activates HIPK2 by phosphorylating highly conserved serine and threonine residues (S359/T360) within the activation loop of the HIPK2 kinase domain. In SOD1(G93A) mice, loss of HIPK2 delays disease onset, reduces cell death in spinal motor neurons, mitigates glial pathology, and improves survival. Remarkably, HIPK2 activation positively correlates with TDP-43 proteinopathy in NEFH-tTA/tetO-hTDP-43ΔNLS mice, sporadic ALS and C9ORF72 ALS, and blocking HIPK2 kinase activity protects motor neurons from TDP-43 cytotoxicity. These results reveal a previously unrecognized role of HIPK2 activation in ER-stress-mediated neurodegeneration and its potential role as a biomarker and therapeutic target for ALS. VIDEO ABSTRACT.

    View details for DOI 10.1016/j.neuron.2016.05.021

    View details for PubMedID 27321923

    View details for PubMedCentralID PMC4938715

  • The immunological evolution of catalysis SCIENCE Patten, P. A., Gray, N. S., Yang, P. L., Marks, C. B., Wedemayer, G. J., Boniface, J. J., Stevens, R. C., Schultz, P. G. 1996; 271 (5252): 1086–91


    The germline genes used by the mouse to generate the esterolytic antibody 48G7 were cloned and expressed in an effort to increase our understanding of the detailed molecular mechanisms by which the immune system evolves catalytic function. The nine replacement mutations that were fixed during affinity maturation increased affinity for the transition state analogue by a factor of 10(4), primarily the result of a decrease in the dissociation rate of the hapten-antibody complex. There was a corresponding increase in the rate of reaction of antibody with substrate, k(cat)/k(m), from 1.7 x 10(2)M(-1) min(-1) to 1.4 x 10(4)M(-1) min(-1). The three-dimensional crystal structure of the 48G7-transition state analogue complex at 2.0 angstroms resolution indicates that one of the nine residues in which somatic mutations have been fixed directly contact the hapten. Thus, in the case of 48G7, affinity maturation appears to play a conformational role, either in reorganizing the active site geometry of limiting side-chain and backbone flexibility of the germline antibody. The crystal structure and analysis of somatic and directed active site mutants underscore the role of transition state stabilization in the evolution of this catalytic antibody.

    View details for DOI 10.1126/science.271.5252.1086

    View details for Web of Science ID A1996TW70100029

    View details for PubMedID 8599084