All Publications

  • Targeted Polymersome Delivery of a Stapled Peptide for Drugging the Tumor Protein p53:BCL-2-Family Axis in Diffuse Large B-Cell Lymphoma ACS NANO Schnorenberg, M. R., Hawley, K. M., Thomas-Toth, A. T., Watkins, E. A., Tian, Y., Ting, J. M., Leak, L. B., Kucera, I. M., Raczy, M. M., Kung, A. L., Hubbell, J. A., Tirrell, M. V., LaBelle, J. L. 2023


    Diffuse large B-cell lymphoma (DLBCL) remains a formidable diagnosis in need of new treatment paradigms. In this work, we elucidated an opportunity for therapeutic synergy in DLBCL by reactivating tumor protein p53 with a stapled peptide, ATSP-7041, thereby priming cells for apoptosis and enhancing their sensitivity to BCL-2 family modulation with a BH3-mimetic, ABT-263 (navitoclax). While this combination was highly effective at activating apoptosis in DLBCL in vitro, it was highly toxic in vivo, resulting in a prohibitively narrow therapeutic window. We, therefore, developed a targeted nanomedicine delivery platform to maintain the therapeutic potency of this combination while minimizing its toxicity via packaging and targeted delivery of a stapled peptide. We developed a CD19-targeted polymersome using block copolymers of poly(ethylene glycol) disulfide linked to poly(propylene sulfide) (PEG-SS-PPS) for ATSP-7041 delivery into DLBCL cells. Intracellular delivery was optimized in vitro and validated in vivo by using an aggressive human DLBCL xenograft model. Targeted delivery of ATSP-7041 unlocked the ability to systemically cotreat with ABT-263, resulting in delayed tumor growth, prolonged survival, and no overt toxicity. This work demonstrates a proof-of-concept for antigen-specific targeting of polymersome nanomedicines, targeted delivery of a stapled peptide in vivo, and synergistic dual intrinsic apoptotic therapy against DLBCL via direct p53 reactivation and BCL-2 family modulation.

    View details for DOI 10.1021/acsnano.3c04112

    View details for Web of Science ID 001065436200001

    View details for PubMedID 37688780

  • Protocol for detection of ferroptosis in cultured cells. STAR protocols Murray, M. B., Leak, L. B., Lee, W. C., Dixon, S. J. 2023; 4 (3): 102457


    Mammalian cells can die by apoptosis or by one of several non-apoptotic mechanisms, such as ferroptosis. Here, we present a protocol to distinguish ferroptosis from other cell death mechanisms in cultured cells. We describe steps for seeding cells, administering mechanism-specific cell death inducers and inhibitors, and measuring cell death and viability. We then detail the use of molecular markers to verify mechanisms of cell death. This protocol can be used to identify and distinguish ferroptosis in 2D and 3D cultures. For complete details on the use and execution of this protocol, please refer to Ko, et al. (2019),1 Magtanong, et al. (2022),2 and Armenta, et al. (2022).3.

    View details for DOI 10.1016/j.xpro.2023.102457

    View details for PubMedID 37556320

  • Surveying the landscape of emerging and understudied cell death mechanisms. Biochimica et biophysica acta. Molecular cell research Leak, L., Dixon, S. J. 2023: 119432


    Cell death can be a highly regulated process. A large and growing number of mammalian cell death mechanisms have been described over the past few decades. Major pathways with established roles in normal or disease biology include apoptosis, necroptosis, pyroptosis and ferroptosis. However, additional non-apoptotic cell death mechanisms with unique morphological, genetic, and biochemical features have also been described. These mechanisms may play highly specialized physiological roles or only become activated in response to specific lethal stimuli or conditions. Understanding the nature of these emerging and understudied mechanisms may provide new insight into cell death biology and suggest new treatments for diseases such as cancer and neurodegeneration.

    View details for DOI 10.1016/j.bbamcr.2023.119432

    View details for PubMedID 36690038

  • Forging a path toward a more sustainable laboratory. Trends in biochemical sciences Leak, L. B., Tamborski, J., Commissaris, A., Brophy, J. A. 2023; 48 (1): 5-8


    Scientific discovery has advanced human society in countless ways, but research requires the expenditure of energy and resources. This Scientific Life article details one laboratory's efforts to reduce the environmental impact of wet-lab research and provides a series of resources to improve lab sustainability.

    View details for DOI 10.1016/j.tibs.2022.09.001

    View details for PubMedID 36563657

  • A Novel Cell Death Mechanism Involving the Sphingosine-to-Glycerophospholipid Pathway. FASEB journal : official publication of the Federation of American Societies for Experimental Biology Leak, L., Dixon, S. 2022; 36 Suppl 1


    Apoptosis is the most well-studied form of cell death, but there exist other cell death pathways that also have roles in normal physiology and disease. It is unclear what other forms of cell death remain to be discovered and how these pathways could inform our understanding of cell and molecular biology. Caspase-independent lethal 56 (CIL56) is a compound that kills cells via a novel cell death mechanism. We conducted a genome-wide CRISPR screen to identify novel regulators of this death pathway. From the screen, we identified trans-2-enoyl-CoA reductase (TECR) as a key regulator of CIL56-induced death. TECR is localized to the endoplasmic reticulum and is required for very long-chain fatty acid (VLCFA) synthesis. When the gene encoding TECR is disrupted, cells become resistant to CIL56-induced death. We find that VLCFA synthesis is dispensable for CIL56-induced death. On the contrary, TECR has a non-canonical role in the conversion of sphingosine to palmitate in the sphingosine-to-glycerophospholipid pathway. Through chemical complementation analyses, we find that this pathway is important for death induction by CIL56. It remains unclear how this pathway could be acting to execute cell death. It is known that palmitate produced in the endoplasmic reticulum can be incorporated into ceramide species, which have long been associated with cell death. Preliminary lipidomic analyses reveal a dramatic increase in palmitoyl-ceramide upon CIL56 treatment. We are investigating the role of this species in CIL56-induced cell death to further characterize the mechanism of death and its importance.

    View details for DOI 10.1096/fasebj.2022.36.S1.R2783

    View details for PubMedID 35556625

  • Clonal architecture predicts clinical outcomes and drug sensitivity in acute myeloid leukemia. Nature communications Benard, B. A., Leak, L. B., Azizi, A., Thomas, D., Gentles, A. J., Majeti, R. 1800; 12 (1): 7244


    The impact of clonal heterogeneity on disease behavior or drug response in acute myeloid leukemia remains poorly understood. Using a cohort of 2,829 patients, we identify features of clonality associated with clinical features and drug sensitivities. High variant allele frequency for 7 mutations (including NRAS and TET2) associate with dismal prognosis; elevated GATA2 variant allele frequency correlates with better outcomes. Clinical features such as white blood cell count and blast percentage correlate with the subclonal abundance of mutations such as TP53 and IDH1. Furthermore, patients with cohesin mutations occurring before NPM1, or transcription factor mutations occurring before splicing factor mutations, show shorter survival. Surprisingly, a branched pattern of clonal evolution is associated with superior clinical outcomes. Finally, several mutations (including NRAS and IDH1) predict drug sensitivity based on their subclonal abundance. Together, these results demonstrate the importance of assessing clonal heterogeneity with implications for prognosis and actionable biomarkers for therapy.

    View details for DOI 10.1038/s41467-021-27472-5

    View details for PubMedID 34903734

  • Preferential targeting of MCL-1 by a hydrocarbon-stapled BIM BH3 peptide. Oncotarget Hadji, A., Schmitt, G. K., Schnorenberg, M. R., Roach, L., Hickey, C. M., Leak, L. B., Tirrell, M. V., LaBelle, J. L. 2019; 10 (58): 6219-6233


    BCL-2 family proteins are central regulators of apoptosis and represent prime therapeutic targets for overcoming cell death resistance in malignancies. However, plasticity of anti-apoptotic members, such as MCL-1, often allows for a switch in cell death dependency patterns that lie outside the binding profile of targeted BH3-mimetics. Therefore discovery of therapeutics that effectively inactivate all anti-apoptotic members is a high priority. To address this we tested the potency of a hydrocarbon stapled BIM BH3 peptide (BIM SAHB A ) to overcome both BCL-2 and MCL-1 apoptotic resistance given BIM's naturally wide ranging affinity for all BCL-2 family multidomain members. BIM SAHB A effectively killed diffuse large B-cell lymphoma (DLBCL) cell lines regardless of their anti-apoptotic dependence. Despite BIM BH3's ability to bind all BCL-2 anti-apoptotic proteins, BIM SAHB A 's dominant intracellular target was MCL-1 and this specificity was exploited in sequenced combination BH3-mimetic treatments targeting BCL-2, BCL-XL, and BCL-W. Extending this MCL-1 functional dependence, mouse embryonic fibroblasts (MEFs) deficient in MCL-1 were resistant to mitochondrial changes induced by BIM SAHB A . This study demonstrates the importance of understanding BH3 mimetic functional intracellular affinities for optimized use and highlights the diagnostic and therapeutic promise of a BIM BH3 peptide mimetic as a potential MCL-1 inhibitor.

    View details for DOI 10.18632/oncotarget.27262

    View details for PubMedID 31692812

    View details for PubMedCentralID PMC6817437