Jason Meyer Sheltzer

All Publications

• Paired CRISPR screens identify mitochondrial metabolism and UBE2H as aneuploid-specific dependencies in human cancer cell lines. bioRxiv : the preprint server for biology Schukken, K. M., Akalu, S. M., Zou, C., Kandikuppa, P. K., Hagenson, R. A., Keane, J. L., Lynch, M. P., Yoshimoto, T., Klingbeil, O., Sausville, E. L., Mishra, S., Vakoc, C. R., Storchova, Z., Aitken, S. J., Sheltzer, J. M. 2026

  Abstract

  Aneuploidy is a hallmark of cancer and imposes widespread cellular stress, including proteotoxicity, transcriptional dysregulation, and increased metabolic demand. Although these stresses are predicted to create therapeutic vulnerabilities, the genetic dependencies of aneuploid cells remain incompletely characterized. Here, we performed paired CRISPR loss-of-function screens in isogenic aneuploid and near-euploid cancer cell line models to systematically identify aneuploidy-specific dependencies. Seven genome-wide paired screens identified ribosomes, rRNA processing, spliceosome-mediated RNA processing, proteasome subunits, and mitochondrial metabolism as top aneuploid-specific dependency gene groups. To identify therapeutically targetable aneuploid dependencies, we performed 18 additional paired CRISPR screens using a focused druggable genome library. This analysis identified the ubiquitin-conjugating enzyme UBE2H as a top aneuploid-selective dependency. Functional validation confirmed aneuploid cell dependency on UBE2H, and mechanistic analyses linked UBE2H to mitochondrial protein abundance, suggesting a role in maintaining mitochondrial proteostasis under aneuploid stress. Together, these findings define core cellular systems that support the viability of aneuploid cells and identify UBE2H as a potential therapeutic vulnerability connecting ubiquitin signaling to mitochondrial homeostasis.

  View details for DOI 10.64898/2026.04.26.720636

  View details for PubMedID 42094535

  View details for PubMedCentralID PMC13142536

• Chromosome engineering to correct a complex rearrangement on Chromosome 8 reveals the effects of 8p syndrome on gene expression and neural differentiation. Genome research Lee, S. N., Banda, E. C., Qiao, L., Thompson, S. L., Singh, K., Hagenson, R. A., Davoli, T., Pinter, S. F., Sheltzer, J. M. 2026; 36 (3): 547-560

  Abstract

  Chromosomal rearrangements on the short arm of Chromosome 8 cause 8p syndrome, a rare developmental disorder characterized by neurodevelopmental delays, epilepsy, and cardiac abnormalities. Although significant progress has been made in managing the symptoms of 8p syndrome and other conditions caused by large-scale chromosomal aneuploidies, no therapeutic approach has yet been demonstrated to target the underlying disease-causing chromosome. Here, we establish a two-step approach to eliminate the abnormal copy of Chromosome 8 and restore euploidy in cells derived from an individual with a complex rearrangement of Chromosome 8p. Transcriptomic analysis revealed 361 differentially expressed genes between the proband and the euploid revertant, highlighting genes both within and outside the 8p region that may contribute to 8p syndrome pathology. Furthermore, we demonstrate that the proband exhibits a significant defect in neural differentiation that could be partially rescued by treatment with small-molecule inhibitors of cell death. Our work demonstrates the feasibility of using chromosome engineering to correct complex aneuploidies in vitro and establishes a platform to further dissect the pathophysiology of 8p syndrome and other conditions caused by chromosomal rearrangements.

  View details for DOI 10.1101/gr.280425.125

  View details for PubMedID 41526190

  View details for PubMedCentralID PMC12951946

• A clinical-stage oncology compound selectively targets drug-resistant cancers. bioRxiv : the preprint server for biology Long, K., Bhattacharjee, D., Newman-Stonebraker, S. H., Suhr, S., Mercado, B. Q., Scheib, E., Tighe, A., Romero, L., Thompson, S. L., Sausville, E. L., John, K. M., Julian, L., Mishra, S., Klingbeil, O., Gupta, P., Bhatt, U., Gao, A. C., Ricardo, S., Vakoc, C. R., Bornhauser, B. C., Corsello, S. M., Taylor, S. S., Chen, J., Holland, P. L., Sheltzer, J. M. 2026

  Abstract

  Re-evaluating existing clinical compounds can uncover previously unrecognized mechanisms that reshape a drug's therapeutic potential. The small molecule Procaspase-Activating Compound 1 (PAC-1) entered oncology testing as a proposed activator of caspase-driven apoptosis. Here, we show that PAC-1-driven cytotoxicity occurs in the absence of executioner caspase expression, demonstrating that its anti-cancer activity occurs via an alternative mechanism. We provide genetic, biochemical, and biophysical evidence demonstrating that PAC-1 functions as a highly selective iron chelator that eliminates cancer cells by disrupting iron homeostasis. Unexpectedly, we discovered that expression of the key chemotherapy-resistance pump MDR1 confers marked hypersensitivity to PAC-1 treatment. While PAC-1 is only weakly effluxed by MDR1 under basal conditions, this process is potentiated when PAC-1 is bound to iron. Consequently, PAC-1 induces progressive iron depletion and selective cytotoxicity in otherwise drug-resistant MDR1-expressing cancer cells. Together, these findings redefine PAC-1's mechanism-of-action and establish a framework for exploiting multidrug resistance as a therapeutic vulnerability through targeted iron starvation.

  View details for DOI 10.1101/2025.11.26.690878

  View details for PubMedID 41394735

  View details for PubMedCentralID PMC12699294

• Protein buffering of aneuploidy is driven by coordinated factors identified through machine learning. Molecular systems biology Heller, E. M., Barthel, K., Räschle, M., Schukken, K. M., Sheltzer, J. M., Storchová, Z. 2026

  Abstract

  Aneuploidy, a hallmark of cancer, alters chromosome copy numbers and with that the abundance of hundreds of proteins. Evidence suggests that levels of proteins encoded on affected chromosomes are often buffered toward their abundances observed in diploids. Despite its prevalence, the molecular mechanisms driving this protein dosage compensation remain largely unknown. It is unclear whether all proteins are buffered similarly, what factors determine buffering, and whether dosage compensation varies across different cell lines or tumor types. Moreover, its potential adaptive advantage and therapeutic relevance remain unexplored. We established a novel approach to quantify protein dosage buffering in a gene copy number-dependent manner, showing that dosage compensation is widespread but variable in cancer samples. By developing multifactorial machine learning models, we identify gene dependency, protein complex participation, haploinsufficiency, and mRNA decay as key predictors of buffering. We show that dosage compensation affects oncogenic potential and that higher buffering correlates with reduced proteotoxic stress and increased drug resistance. These findings highlight protein dosage compensation as a crucial regulatory mechanism with therapeutic potential in aneuploid cancers.

  View details for DOI 10.1038/s44320-026-00187-9

  View details for PubMedID 41571838

  View details for PubMedCentralID 7771966

• Paralog Co-Targeting Identifies Selective Genetic Redundancies across Cancer Types CANCER DISCOVERY Gauthier-Coles, G., Sheltzer, J. M. 2024; 14 (12): 2312-2314

  Abstract

  In this issue, Klingbeil and colleagues deploy a paralog co-targeting strategy to reveal microtubule affinity-regulating kinases 2 and 3 as redundant negative regulators of the Hippo pathway and potentially actionable targets in YAP/TAZ-addicted tumors. See related article by Klingbeil et.al., p. 2471.

  View details for DOI 10.1158/2159-8290.CD-24-1349

  View details for Web of Science ID 001368753200004

  View details for PubMedID 39618283

• An elevated rate of whole-genome duplications in cancers from Black patients. Nature communications Brown, L. M., Hagenson, R. A., Koklič, T., Urbančič, I., Qiao, L., Strancar, J., Sheltzer, J. M. 2024; 15 (1): 8218

  Abstract

  In the United States, Black individuals have higher rates of cancer mortality than any other racial group. Here, we examine chromosome copy number changes in cancers from more than 1800 self-reported Black patients. We find that tumors from self-reported Black patients are significantly more likely to exhibit whole-genome duplications (WGDs), a genomic event that enhances metastasis and aggressive disease, compared to tumors from self-reported white patients. This increase in WGD frequency is observed across multiple cancer types, including breast, endometrial, and lung cancer, and is associated with shorter patient survival. We further demonstrate that combustion byproducts are capable of inducing WGDs in cell culture, and cancers from self-reported Black patients exhibit mutational signatures consistent with exposure to these carcinogens. In total, these findings identify a type of genomic alteration that is associated with environmental exposures and that may influence racial disparities in cancer outcomes.

  View details for DOI 10.1038/s41467-024-52554-5

  View details for PubMedID 39300140

  View details for PubMedCentralID PMC11413164

• Evolving copy number gains promote tumor expansion and bolster mutational diversification NATURE COMMUNICATIONS Wang, Z., Xia, Y., Mills, L., Nikolakopoulos, A. N., Maeser, N., Dehm, S. M., Sheltzer, J. M., Sun, R. 2024; 15 (1): 2025

  Abstract

  The timing and fitness effect of somatic copy number alterations (SCNA) in cancer evolution remains poorly understood. Here we present a framework to determine the timing of a clonal SCNA that encompasses multiple gains. This involves calculating the proportion of time from its last gain to the onset of population expansion (lead time) as well as the proportion of time prior to its first gain (initiation time). Our method capitalizes on the observation that a genomic segment, while in a specific copy number (CN) state, accumulates point mutations proportionally to its CN. Analyzing 184 whole genome sequenced samples from 75 patients across five tumor types, we commonly observe late gains following early initiating events, occurring just before the clonal expansion relevant to the sampling. These include gains acquired after genome doubling in more than 60% of cases. Notably, mathematical modeling suggests that late clonal gains may contain final-expansion drivers. Lastly, SCNAs bolster mutational diversification between subpopulations, exacerbating the circle of proliferation and increasing heterogeneity.

  View details for DOI 10.1038/s41467-024-46414-5

  View details for Web of Science ID 001180826600011

  View details for PubMedID 38448455

  View details for PubMedCentralID PMC10918155

• Inhibition of a lower potency target drives the anticancer activity of a clinical p38 inhibitor. Cell chemical biology Bhattacharjee, D., Bakar, J., Chitnis, S. P., Sausville, E. L., Ashtekar, K. D., Mendelson, B. E., Long, K., Smith, J. C., Heppner, D. E., Sheltzer, J. M. 2023; 30 (10): 1211-1222.e5

  Abstract

  The small-molecule drug ralimetinib was developed as an inhibitor of the p38α mitogen-activated protein kinase, and it has advanced to phase 2 clinical trials in oncology. Here, we demonstrate that ralimetinib resembles EGFR-targeting drugs in pharmacogenomic profiling experiments and that ralimetinib inhibits EGFR kinase activity in vitro and in cellulo. While ralimetinib sensitivity is unaffected by deletion of the genes encoding p38α and p38β, its effects are blocked by expression of the EGFR-T790M gatekeeper mutation. Finally, we solved the cocrystal structure of ralimetinib bound to EGFR, providing further evidence that this drug functions as an ATP-competitive EGFR inhibitor. We conclude that, though ralimetinib is >30-fold less potent against EGFR compared to p38α, its ability to inhibit EGFR drives its primary anticancer effects. Our results call into question the value of p38α as an anticancer target, and we describe a multi-modal approach that can be used to uncover a drug's mechanism-of-action.

  View details for DOI 10.1016/j.chembiol.2023.09.013

  View details for PubMedID 37827156

  View details for PubMedCentralID PMC10715717

• Oncogene-like addiction to aneuploidy in human cancers. Science (New York, N.Y.) Girish, V., Lakhani, A. A., Thompson, S. L., Scaduto, C. M., Brown, L. M., Hagenson, R. A., Sausville, E. L., Mendelson, B. E., Kandikuppa, P. K., Lukow, D. A., Yuan, M. L., Stevens, E. C., Lee, S. N., Schukken, K. M., Akalu, S. M., Vasudevan, A., Zou, C., Salovska, B., Li, W., Smith, J. C., Taylor, A. M., Martienssen, R. A., Liu, Y., Sun, R., Sheltzer, J. M. 2023; 381 (6660): eadg4521

  Abstract

  Most cancers exhibit aneuploidy, but its functional significance in tumor development is controversial. Here, we describe ReDACT (Restoring Disomy in Aneuploid cells using CRISPR Targeting), a set of chromosome engineering tools that allow us to eliminate specific aneuploidies from cancer genomes. Using ReDACT, we created a panel of isogenic cells that have or lack common aneuploidies, and we demonstrate that trisomy of chromosome 1q is required for malignant growth in cancers harboring this alteration. Mechanistically, gaining chromosome 1q increases the expression of MDM4 and suppresses p53 signaling, and we show that TP53 mutations are mutually exclusive with 1q aneuploidy in human cancers. Thus, tumor cells can be dependent on specific aneuploidies, raising the possibility that these "aneuploidy addictions" could be targeted as a therapeutic strategy.

  View details for DOI 10.1126/science.adg4521

  View details for PubMedID 37410869

  View details for PubMedCentralID PMC10753973

• Ricolinostat is not a highly selective HDAC6 inhibitor. Nature cancer Médard, G., Sheltzer, J. M. 2023; 4 (6): 807-808

  View details for DOI 10.1038/s43018-023-00582-3

  View details for PubMedID 37322365

  View details for PubMedCentralID 7717492

• Extensive protein dosage compensation in aneuploid human cancers. Genome research Schukken, K. M., Sheltzer, J. M. 2022; 32 (7): 1254-1270

  Abstract

  Aneuploidy is a hallmark of human cancers, but the effects of aneuploidy on protein expression remain poorly understood. To uncover how chromosome copy number changes influence the cancer proteome, we conducted an analysis of hundreds of human cancer cell lines and tumors with matched copy number, RNA expression, and protein expression data. We found that a majority of proteins show dosage compensation and fail to change by the degree expected based on chromosome copy number alone. We uncovered a variety of gene groups that were recurrently buffered upon both chromosome gain and loss, including protein complex subunits and cell cycle genes. Several genetic and biophysical factors were predictive of protein buffering, highlighting complex post-translational regulatory mechanisms that maintain appropriate gene product dosage. Finally, we established that chromosomal aneuploidy has a moderate effect on the expression of oncogenes and tumor suppressors, showing that these key cancer drivers can be subject to dosage compensation as well. In total, our comprehensive analysis of aneuploidy and dosage compensation across cancers will help identify the key driver genes encoded on altered chromosomes and will shed light on the overall consequences of aneuploidy during tumor development.

  View details for DOI 10.1101/gr.276378.121

  View details for PubMedID 35701073

  View details for PubMedCentralID PMC9341510

• 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

  Abstract

  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

• Genome-wide identification and analysis of prognostic features in human cancers. Cell reports Smith, J. C., Sheltzer, J. M. 2022; 38 (13): 110569

  Abstract

  Clinical decisions in cancer rely on precisely assessing patient risk. To improve our ability to identify the most aggressive malignancies, we constructed genome-wide survival models using gene expression, copy number, methylation, and mutation data from 10,884 patients. We identified more than 100,000 significant prognostic biomarkers and demonstrate that these genomic features can predict patient outcomes in clinically ambiguous situations. While adverse biomarkers are commonly believed to represent cancer driver genes and promising therapeutic targets, we show that cancer features associated with shorter survival times are not enriched for either oncogenes or for successful drug targets. Instead, the strongest adverse biomarkers represent widely expressed cell-cycle and housekeeping genes, and, correspondingly, nearly all therapies directed against these features have failed in clinical trials. In total, our analysis establishes a rich resource for prognostic biomarker analysis and clarifies the use of patient survival data in preclinical cancer research and therapeutic development.

  View details for DOI 10.1016/j.celrep.2022.110569

  View details for PubMedID 35354049

  View details for PubMedCentralID PMC9042322

• Chromosomal instability and aneuploidy as causes of cancer drug resistance. Trends in cancer Lukow, D. A., Sheltzer, J. M. 2022; 8 (1): 43-53

  Abstract

  High levels of aneuploidy and chromosomal instability (CIN) are correlated with poor patient outcomes, though the mechanism(s) underlying this relationship have not been established. Recent evidence has demonstrated that chromosome copy number changes can function as point mutation-independent sources of drug resistance in cancer, which may partially explain this clinical association. CIN generates intratumoral heterogeneity in the form of gene dosage alterations, upon which the selective pressures induced by drug treatments can act. Thus, although CIN and aneuploidy impair cell fitness under most conditions, CIN can augment cellular adaptability, establishing CIN as a bet-hedging mechanism in tumor evolution. CIN may also endow cancers with unique vulnerabilities, which could be exploited therapeutically to achieve better patient outcomes.

  View details for DOI 10.1016/j.trecan.2021.09.002

  View details for PubMedID 34593353

• Chromosomal instability accelerates the evolution of resistance to anti-cancer therapies. Developmental cell Lukow, D. A., Sausville, E. L., Suri, P., Chunduri, N. K., Wieland, A., Leu, J., Smith, J. C., Girish, V., Kumar, A. A., Kendall, J., Wang, Z., Storchova, Z., Sheltzer, J. M. 2021; 56 (17): 2427-2439.e4

  Abstract

  Aneuploidy is a ubiquitous feature of human tumors, but the acquisition of aneuploidy typically antagonizes cellular fitness. To investigate how aneuploidy could contribute to tumor growth, we triggered periods of chromosomal instability (CIN) in human cells and then exposed them to different culture environments. We discovered that transient CIN reproducibly accelerates the acquisition of resistance to anti-cancer therapies. Single-cell sequencing revealed that these resistant populations develop recurrent aneuploidies, and independently deriving one chromosome-loss event that was frequently observed in paclitaxel-resistant cells was sufficient to decrease paclitaxel sensitivity. Finally, we demonstrated that intrinsic levels of CIN correlate with poor responses to numerous therapies in human tumors. Our results show that, although CIN generally decreases cancer cell fitness, it also provides phenotypic plasticity to cancer cells that can allow them to adapt to diverse stressful environments. Moreover, our findings suggest that aneuploidy may function as an under-explored cause of therapy failure.

  View details for DOI 10.1016/j.devcel.2021.07.009

  View details for PubMedID 34352222

  View details for PubMedCentralID PMC8933054

• A CRISPR Competition Assay to Identify Cancer Genetic Dependencies. Bio-protocol Girish, V., Sheltzer, J. M. 2020; 10 (14): e3682

  Abstract

  The CRISPR/Cas9 system is a powerful tool for genome editing, wherein the RNA-guided nuclease Cas9 can be directed to introduce double-stranded breaks (DSBs) at a targeted locus. In mammalian cells, these DSBs are typically repaired through error-prone processes, resulting in insertions or deletions (indels) at the targeted locus. Researchers can use these Cas9-mediated lesions to probe the consequences of loss-of-function perturbations in genes of interest. Here, we describe an optimized protocol to identify specific genes required for cancer cell fitness through a CRISPR-mediated cellular competition assay. Identifying these genetic dependencies is of utmost importance, as they provide potential targets for anti-cancer drug development. This protocol provides researchers with a robust and scalable approach to investigate gene dependencies in a variety of cell lines and cancer types and to validate the results of high-throughput or whole-genome screens.

  View details for DOI 10.21769/BioProtoc.3682

  View details for PubMedID 33659353

  View details for PubMedCentralID PMC7842800

• Single-Chromosomal Gains Can Function as Metastasis Suppressors and Promoters in Colon Cancer. Developmental cell Vasudevan, A., Baruah, P. S., Smith, J. C., Wang, Z., Sayles, N. M., Andrews, P., Kendall, J., Leu, J., Chunduri, N. K., Levy, D., Wigler, M., Storchová, Z., Sheltzer, J. M. 2020; 52 (4): 413-428.e6

  Abstract

  High levels of cancer aneuploidy are frequently associated with poor prognosis. To examine the relationship between aneuploidy and cancer progression, we analyzed a series of congenic cell lines that harbor single extra chromosomes. We found that across 13 different trisomic cell lines, 12 trisomies suppressed invasiveness or were largely neutral, while a single trisomy increased metastatic behavior by triggering a partial epithelial-mesenchymal transition. In contrast, we discovered that chromosomal instability activates cGAS/STING signaling but strongly suppresses invasiveness. By analyzing patient copy-number data, we demonstrate that specific aneuploidies are associated with distinct outcomes, and the acquisition of certain aneuploidies is in fact linked with a favorable prognosis. Thus, aneuploidy is not a uniform driver of malignancy, and different aneuploidies can uniquely influence tumor progression. At the same time, the gain of a single chromosome is capable of inducing a profound cell state transition, thereby linking genomic plasticity, phenotypic plasticity, and metastasis.

  View details for DOI 10.1016/j.devcel.2020.01.034

  View details for PubMedID 32097652

  View details for PubMedCentralID PMC7354079

• Discovering and validating cancer genetic dependencies: approaches and pitfalls. Nature reviews. Genetics Lin, A. n., Sheltzer, J. M. 2020

  Abstract

  Cancer 'genetic dependencies' - genes whose products are essential for cancer cell fitness - are promising targets for therapeutic development. However, recent evidence has cast doubt on the validity of several putative dependencies that are currently being targeted in cancer clinical trials, underscoring the challenges inherent in correctly identifying cancer-essential genes. Here we review several common techniques and platforms for discovering and characterizing cancer dependencies. We discuss the strengths and drawbacks of different gene-perturbation approaches, and we highlight the use of poorly validated genetic and pharmacological agents as a common cause of target misidentification. A careful consideration of the limitations of current technologies and cancer models will improve our ability to correctly uncover cancer genetic dependencies and will facilitate the development of improved therapeutic agents.

  View details for DOI 10.1038/s41576-020-0247-7

  View details for PubMedID 32561862

• Increasing gender diversity in the STEM research workforce SCIENCE Greider, C. W., Sheltzer, J. M., Cantalupo, N. C., Copeland, W. B., Dasgupta, N., Hopkins, N., Jansen, J. M., Joshua-Tor, L., McDowell, G. S., Metcalf, J. L., McLaughlin, B., Olivarius, A., O'Shea, E. K., Raymond, J. L., Ruebain, D., Steitz, J. A., Stillman, B., Tilghman, S. M., Valian, V., Villa-Komaroff, L., Wong, J. Y. 2019; 366 (6466): 692-+

  View details for DOI 10.1126/science.aaz0649

  View details for Web of Science ID 000496500400032

  View details for PubMedID 31699926

• Off-target toxicity is a common mechanism of action of cancer drugs undergoing clinical trials. Science translational medicine Lin, A., Giuliano, C. J., Palladino, A., John, K. M., Abramowicz, C., Yuan, M. L., Sausville, E. L., Lukow, D. A., Liu, L., Chait, A. R., Galluzzo, Z. C., Tucker, C., Sheltzer, J. M. 2019; 11 (509)

  Abstract

  Ninety-seven percent of drug-indication pairs that are tested in clinical trials in oncology never advance to receive U.S. Food and Drug Administration approval. While lack of efficacy and dose-limiting toxicities are the most common causes of trial failure, the reason(s) why so many new drugs encounter these problems is not well understood. Using CRISPR-Cas9 mutagenesis, we investigated a set of cancer drugs and drug targets in various stages of clinical testing. We show that-contrary to previous reports obtained predominantly with RNA interference and small-molecule inhibitors-the proteins ostensibly targeted by these drugs are nonessential for cancer cell proliferation. Moreover, the efficacy of each drug that we tested was unaffected by the loss of its putative target, indicating that these compounds kill cells via off-target effects. By applying a genetic target-deconvolution strategy, we found that the mischaracterized anticancer agent OTS964 is actually a potent inhibitor of the cyclin-dependent kinase CDK11 and that multiple cancer types are addicted to CDK11 expression. We suggest that stringent genetic validation of the mechanism of action of cancer drugs in the preclinical setting may decrease the number of therapies tested in human patients that fail to provide any clinical benefit.

  View details for DOI 10.1126/scitranslmed.aaw8412

  View details for PubMedID 31511426

  View details for PubMedCentralID PMC7717492

• Generating Single Cell-Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9. Current protocols in molecular biology Giuliano, C. J., Lin, A., Girish, V., Sheltzer, J. M. 2019; 128 (1): e100

  Abstract

  CRISPR/Cas9 technology enables the rapid generation of loss-of-function mutations in a targeted gene in mammalian cells. A single cell harboring those mutations can be used to establish a new cell line, thereby creating a CRISPR-induced knockout clone. These clonal cell lines serve as crucial tools for exploring protein function, analyzing the consequences of gene loss, and investigating the specificity of biological reagents. However, the successful derivation of knockout clones can be technically challenging and may be complicated by multiple factors, including incomplete target ablation and interclonal heterogeneity. Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. We provide strategies for guide RNA design, CRISPR delivery, and knockout validation that facilitate the derivation of true knockout clones and are amenable to multiplexed gene targeting. These protocols will be broadly useful for researchers seeking to apply CRISPR to study gene function in mammalian cells. © 2019 The Authors.

  View details for DOI 10.1002/cpmb.100

  View details for PubMedID 31503414

  View details for PubMedCentralID PMC6741428

• Systematic identification of mutations and copy number alterations associated with cancer patient prognosis. eLife Smith, J. C., Sheltzer, J. M. 2018; 7

  Abstract

  Successful treatment decisions in cancer depend on the accurate assessment of patient risk. To improve our understanding of the molecular alterations that underlie deadly malignancies, we analyzed the genomic profiles of 17,879 tumors from patients with known outcomes. We find that mutations in almost all cancer driver genes contain remarkably little information on patient prognosis. However, CNAs in these same driver genes harbor significant prognostic power. Focal CNAs are associated with worse outcomes than broad alterations, and CNAs in many driver genes remain prognostic when controlling for stage, grade, TP53 status, and total aneuploidy. By performing a meta-analysis across independent patient cohorts, we identify robust prognostic biomarkers in specific cancer types, and we demonstrate that a subset of these alterations also confer specific therapeutic vulnerabilities. In total, our analysis establishes a comprehensive resource for cancer biomarker identification and underscores the importance of gene copy number profiling in assessing clinical risk.

  View details for DOI 10.7554/eLife.39217

  View details for PubMedID 30526857

  View details for PubMedCentralID PMC6289580

• MELK expression correlates with tumor mitotic activity but is not required for cancer growth. eLife Giuliano, C. J., Lin, A., Smith, J. C., Palladino, A. C., Sheltzer, J. M. 2018; 7

  Abstract

  The Maternal Embryonic Leucine Zipper Kinase (MELK) has been identified as a promising therapeutic target in multiple cancer types. MELK over-expression is associated with aggressive disease, and MELK has been implicated in numerous cancer-related processes, including chemotherapy resistance, stem cell renewal, and tumor growth. Previously, we established that triple-negative breast cancer cell lines harboring CRISPR/Cas9-induced null mutations in MELK proliferate at wild-type levels in vitro (Lin et al., 2017). Here, we generate several additional knockout clones of MELK and demonstrate that across cancer types, cells lacking MELK exhibit wild-type growth in vitro, under environmental stress, in the presence of cytotoxic chemotherapies, and in vivo. By combining our MELK-knockout clones with a recently described, highly specific MELK inhibitor, we further demonstrate that the acute inhibition of MELK results in no specific anti-proliferative phenotype. Analysis of gene expression data from cohorts of cancer patients identifies MELK expression as a correlate of tumor mitotic activity, explaining its association with poor clinical prognosis. In total, our results demonstrate the power of CRISPR/Cas9-based genetic approaches to investigate cancer drug targets, and call into question the rationale for treating patients with anti-MELK monotherapies.

  View details for DOI 10.7554/eLife.32838

  View details for PubMedID 29417930

  View details for PubMedCentralID PMC5805410

• CRISPR/Cas9 mutagenesis invalidates a putative cancer dependency targeted in on-going clinical trials. eLife Lin, A., Giuliano, C. J., Sayles, N. M., Sheltzer, J. M. 2017; 6

  Abstract

  The Maternal Embryonic Leucine Zipper Kinase (MELK) has been reported to be a genetic dependency in several cancer types. MELK RNAi and small-molecule inhibitors of MELK block the proliferation of various cancer cell lines, and MELK knockdown has been described as particularly effective against the highly-aggressive basal/triple-negative subtype of breast cancer. Based on these preclinical results, the MELK inhibitor OTS167 is currently being tested as a novel chemotherapy agent in several clinical trials. Here, we report that mutagenizing MELK with CRISPR/Cas9 has no effect on the fitness of basal breast cancer cell lines or cell lines from six other cancer types. Cells that harbor null mutations in MELK exhibit wild-type doubling times, cytokinesis, and anchorage-independent growth. Furthermore, MELK-knockout lines remain sensitive to OTS167, suggesting that this drug blocks cell division through an off-target mechanism. In total, our results undermine the rationale for a series of current clinical trials and provide an experimental approach for the use of CRISPR/Cas9 in preclinical target validation that can be broadly applied.

  View details for DOI 10.7554/eLife.24179

  View details for PubMedID 28337968

  View details for PubMedCentralID PMC5365317

• Single-chromosome Gains Commonly Function as Tumor Suppressors. Cancer cell Sheltzer, J. M., Ko, J. H., Replogle, J. M., Habibe Burgos, N. C., Chung, E. S., Meehl, C. M., Sayles, N. M., Passerini, V., Storchova, Z., Amon, A. 2017; 31 (2): 240-255

  Abstract

  Aneuploidy is a hallmark of cancer, although its effects on tumorigenesis are unclear. Here, we investigated the relationship between aneuploidy and cancer development using cells engineered to harbor single extra chromosomes. We found that nearly all trisomic cell lines grew poorly in vitro and as xenografts, relative to genetically matched euploid cells. Moreover, the activation of several oncogenic pathways failed to alleviate the fitness defect induced by aneuploidy. However, following prolonged growth, trisomic cells acquired additional chromosomal alterations that were largely absent from their euploid counterparts and that correlated with improved fitness. Thus, while single-chromosome gains can suppress transformation, the genome-destabilizing effects of aneuploidy confer an evolutionary flexibility that may contribute to the aggressive growth of advanced malignancies with complex karyotypes.

  View details for DOI 10.1016/j.ccell.2016.12.004

  View details for PubMedID 28089890

  View details for PubMedCentralID PMC5713901

• A transcriptional and metabolic signature of primary aneuploidy is present in chromosomally unstable cancer cells and informs clinical prognosis. Cancer research Sheltzer, J. M. 2013; 73 (21): 6401-12

  Abstract

  Aneuploidy is invariably associated with poor proliferation of primary cells, but the specific contributions of abnormal karyotypes to cancer, a disease characterized by aneuploidy and dysregulated proliferation, remain unclear. In this study, I demonstrate that the transcriptional alterations caused by aneuploidy in primary cells are also present in chromosomally unstable cancer cell lines, but the same alterations are not common to all aneuploid cancers. Chromosomally unstable cancer lines and aneuploid primary cells also share an increase in glycolytic and TCA cycle flux. The biological response to aneuploidy is associated with cellular stress and slow proliferation, and a 70-gene signature derived from primary aneuploid cells was defined as a strong predictor of increased survival in several cancers. Inversely, a transcriptional signature derived from clonal aneuploidy in tumors correlated with high mitotic activity and poor prognosis. Together, these findings suggested that there are two types of aneuploidy in cancer: one is clonal aneuploidy, which is selected during tumor evolution and associated with robust growth, and the other is subclonal aneuploidy caused by chromosomal instability (CIN). Subclonal aneuploidy more closely resembles the stressed state of primary aneuploid cells, yet CIN is not benign; a subset of genes upregulated in high-CIN cancers predict aggressive disease in human patients in a proliferation-independent manner.

  View details for DOI 10.1158/0008-5472.CAN-13-0749

  View details for PubMedID 24041940

  View details for PubMedCentralID PMC3901577

• Transcriptional consequences of aneuploidy. Proceedings of the National Academy of Sciences of the United States of America Sheltzer, J. M., Torres, E. M., Dunham, M. J., Amon, A. 2012; 109 (31): 12644-9

  Abstract

  Aneuploidy, or an aberrant karyotype, results in developmental disabilities and has been implicated in tumorigenesis. However, the causes of aneuploidy-induced phenotypes and the consequences of aneuploidy on cell physiology remain poorly understood. We have performed a metaanalysis on gene expression data from aneuploid cells in diverse organisms, including yeast, plants, mice, and humans. We found highly related gene expression patterns that are conserved between species: genes that were involved in the response to stress were consistently upregulated, and genes associated with the cell cycle and cell proliferation were downregulated in aneuploid cells. Within species, different aneuploidies induced similar changes in gene expression, independent of the specific chromosomal aberrations. Taken together, our results demonstrate that aneuploidies of different chromosomes and in different organisms impact similar cellular pathways and cause a stereotypical antiproliferative response that must be overcome before transformation.

  View details for DOI 10.1073/pnas.1209227109

  View details for PubMedID 22802626

  View details for PubMedCentralID PMC3411958

• Aneuploidy drives genomic instability in yeast. Science (New York, N.Y.) Sheltzer, J. M., Blank, H. M., Pfau, S. J., Tange, Y., George, B. M., Humpton, T. J., Brito, I. L., Hiraoka, Y., Niwa, O., Amon, A. 2011; 333 (6045): 1026-30

  Abstract

  Aneuploidy decreases cellular fitness, yet it is also associated with cancer, a disease of enhanced proliferative capacity. To investigate one mechanism by which aneuploidy could contribute to tumorigenesis, we examined the effects of aneuploidy on genomic stability. We analyzed 13 budding yeast strains that carry extra copies of single chromosomes and found that all aneuploid strains exhibited one or more forms of genomic instability. Most strains displayed increased chromosome loss and mitotic recombination, as well as defective DNA damage repair. Aneuploid fission yeast strains also exhibited defects in mitotic recombination. Aneuploidy-induced genomic instability could facilitate the development of genetic alterations that drive malignant growth in cancer.

  View details for DOI 10.1126/science.1206412

  View details for PubMedID 21852501

  View details for PubMedCentralID PMC3278960