Jason M. Sheltzer

Transcriptional consequences of aneuploidy

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.

The aneuploidy paradox: costs and benefits of an incorrect karyotype

Aneuploidy has a paradoxical effect on cell proliferation. In all normal cells analyzed to date, aneuploidy has been found to decrease the rate of cell proliferation. Yet, aneuploidy is also a hallmark of cancer, a disease of enhanced proliferative capacity, and aneuploid cells are frequently recovered following the experimental evolution of microorganisms. Thus, in certain contexts, aneuploidy might also have growth-advantageous properties. New models of aneuploidy and chromosomal instability have shed light on the diverse effects that karyotypic imbalances have on cellular phenotypes, and suggest novel ways of understanding the role of aneuploidy in development and disease.

Elite male faculty in the life sciences employ fewer women

Significance Despite decades of progress, men still greatly outnumber women among biology faculty in the United States. Here, we show that high-achieving faculty members who are male train 10–40% fewer women in their laboratories relative to the number of women trained by other investigators. These skewed employment patterns may result from self-selection among female scientists or they may result from conscious or unconscious bias on the part of some faculty members. The dearth of women who are trained in these laboratories likely limits the number of female candidates who are most competitive for faculty job searches. Women make up over one-half of all doctoral recipients in biology-related fields but are vastly underrepresented at the faculty level in the life sciences. To explore the current causes of women’s underrepresentation in biology, we collected publicly accessible data from university directories and faculty websites about the composition of biology laboratories at leading academic institutions in the United States. We found that male faculty members tended to employ fewer female graduate students and postdoctoral researchers (postdocs) than female faculty members did. Furthermore, elite male faculty—those whose research was funded by the Howard Hughes Medical Institute, who had been elected to the National Academy of Sciences, or who had won a major career award—trained significantly fewer women than other male faculty members. In contrast, elite female faculty did not exhibit a gender bias in employment patterns. New assistant professors at the institutions that we surveyed were largely comprised of postdoctoral researchers from these prominent laboratories, and correspondingly, the laboratories that produced assistant professors had an overabundance of male postdocs. Thus, one cause of the leaky pipeline in biomedical research may be the exclusion of women, or their self-selected absence, from certain high-achieving laboratories.

A transcriptional and metabolic signature of primary aneuploidy is present in chromosomally-unstable cancer cells and informs clinical prognosis

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.

Mitotic entry in the presence of DNA damage is a widespread property of aneuploidy in yeast

Genetic instability is a hallmark of aneuploidy in budding and fission yeast. All aneuploid yeast strains analyzed to date harbor elevated levels of Rad52-GFP foci, a sign of DNA damage. Here we investigate how continuously elevated levels of DNA damage affect aneuploid cells. We show that Rad52-GFP foci form during S phase, consistent with the observation that DNA replication initiation and elongation are impaired in some aneuploid yeast strains. We furthermore find that although DNA damage is low in aneuploid cells, it nevertheless has dramatic consequences. Many aneuploid yeast strains adapt to DNA damage and undergo mitosis despite the presence of unrepaired DNA leading to cell death. Wild-type cells exposed to low levels of DNA damage exhibit a similar phenotype, indicating that adaptation to low levels of unrepaired DNA is a general property of the cells response to DNA damage. Our results indicate that by causing low levels of DNA damage, whole-chromosome aneuploidies lead to DNA breaks that persist into mitosis. Such breaks provide the substrate for translocations and deletions that are a hallmark of cancer.

The Class V Myosin Myo2p Is Required for Fus2p Transport and Actin Polarization during the Yeast Mating Response

Mating yeast cells remove their cell walls and fuse their plasma membranes in a spatially restricted cell contact region. Cell wall removal is dependent on Fus2p, an amphiphysin-associated Rho-GEF homolog. As mating cells polarize, Fus2p-GFP localizes to the tip of the mating projection, where cell fusion will occur, and to cytoplasmic puncta, which show rapid movement toward the tip. Movement requires polymerized actin, whereas tip localization is dependent on both actin and a membrane protein, Fus1p. Here, we show that Fus2p-GFP movement is specifically dependent on Myo2p, a type V myosin, and not on Myo4p, another type V myosin, or Myo3p and Myo5p, type I myosins. Fus2p-GFP tip localization and actin polarization in shmoos are also dependent on Myo2p. A temperature-sensitive tropomyosin mutation and Myo2p alleles that specifically disrupt vesicle binding caused rapid loss of actin patch organization, indicating that transport is required to maintain actin polarity. Mutant shmoos lost actin polarity more rapidly than mitotic cells, suggesting that the maintenance of cell polarity in shmoos is more sensitive to perturbation. The different velocities, differential sensitivity to mutation and lack of colocalization suggest that Fus2p and Sec4p, another Myo2p cargo associated with exocytotic vesicles, reside predominantly on different cellular organelles.

CRISPR/Cas9 mutagenesis invalidates a putative cancer dependency targeted in on-going clinical trials

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. DOI: http://dx.doi.org/10.7554/eLife.24179.001

MELK expression correlates with tumor mitotic activity but is not required for cancer growth

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.

Single-chromosome aneuploidy commonly functions as a tumor suppressor

Whole-chromosome aneuploidy is a hallmark of human malignancies. The prevalence of chromosome segregation errors in cancer – first noted more than 100 years ago – has led to the widespread belief that aneuploidy plays a crucial role in tumor development. Here, we set out to test this hypothesis. We transduced congenic euploid and trisomic fibroblasts with 14 different oncogenes or oncogene combinations, thereby creating genetically-matched cancer cell lines that differ only in karyotype. Surprisingly, nearly all aneuploid cell lines divided slowly in vitro, formed few colonies in soft agar, and grew poorly as xenografts, relative to matched euploid lines. Similar results were obtained when comparing a near-diploid human colorectal cancer cell line with derivatives of that line that harbored extra chromosomes. Only a few aneuploid lines grew at close to wild-type levels, and no aneuploid line exhibited greater tumorigenic capabilities than its euploid counterpart. These results demonstrate that rather than promoting tumorigenesis, aneuploidy, particularly single chromosome gains, can very often function as a tumor suppressor. Moreover, our results suggest one potential way that cancers can overcome the tumor suppressive effects of aneuploidy: rapidly-growing aneuploid cell lines that had evolved in vitro or in vivo demonstrated recurrent karyotype changes that were absent from their euploid counterparts. Thus, the genome-destabilizing effects of single-chromosome aneuploidy may facilitate the development of balanced, high-complexity karyotypes that are frequently found in advanced malignancies.

An integrated analysis of the epigenetic, genetic, and transcriptional patterns associated with outcome across cancer types

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 genomic profiles from 33,036 solid tumors with known patient outcomes. Contrary to expectations, we find that mutations in cancer driver genes are almost never associated with patient survival time. In contrast, copy number changes in these same genes are broadly prognostic. Analysis of methylation, microRNA, mRNA, and protein expression patterns in primary tumors define several additional prognostic patterns, including signatures of tumor mitotic activity and tissue de-differentiation. Co-expression analysis with a cell cycle meta-gene distinguished proliferation-dependent and ‐independent prognostic features, allowing us to construct multivariate survival models with improved stratification power. In total, our analysis provides a comprehensive resource for biomarker and therapeutic target identification, and suggests that copy number and methylation profiling should complement tumor sequencing efforts to improve patient risk assessment.