Inherent genome instability underlies trisomy 21-associated myeloid malignancies.

Constitutional trisomy 21 (T21) is a state of aneuploidy associated with high incidence of childhood acute myeloid leukemia (AML). T21-associated AML is preceded by transient abnormal myelopoiesis (TAM), which is triggered by truncating mutations in GATA1 generating a short GATA1 isoform (GATA1s). T21-associated AML emerges due to secondary mutations in hematopoietic clones bearing GATA1s. Since aneuploidy generally impairs cellular fitness, the paradoxically elevated risk of myeloid malignancy in T21 is not fully understood. We hypothesized that individuals with T21 bear inherent genome instability in hematopoietic lineages that promotes leukemogenic mutations driving the genesis of TAM and AML. We found that individuals with T21 show increased chromosomal copy number variations (CNVs) compared to euploid individuals, suggesting that genome instability could be underlying predisposition to TAM and AML. Acquisition of GATA1s enforces myeloid skewing and maintenance of the hematopoietic progenitor state independently of T21; however, GATA1s in T21 hematopoietic progenitor cells (HPCs) further augments genome instability. Increased dosage of the chromosome 21 (chr21) gene DYRK1A impairs homology-directed DNA repair as a mechanism of elevated mutagenesis. These results posit a model wherein inherent genome instability in T21 drives myeloid malignancy in concert with GATA1s mutations.

The environmental stress response regulates ribosome content in cell cycle-arrested S. cerevisiae.

Prolonged cell cycle arrests occur naturally in differentiated cells and in response to various stresses such as nutrient deprivation or treatment with chemotherapeutic agents. Whether and how cells survive prolonged cell cycle arrests is not clear. Here, we used S. cerevisiae to compare physiological cell cycle arrests and genetically induced arrests in G1-, meta- and anaphase. Prolonged cell cycle arrest led to growth attenuation in all studied conditions, coincided with activation of the Environmental Stress Response (ESR) and with a reduced ribosome content as determined by whole ribosome purification and TMT mass spectrometry. Suppression of the ESR through hyperactivation of the Ras/PKA pathway reduced cell viability during prolonged arrests, demonstrating a cytoprotective role of the ESR. Attenuation of cell growth and activation of stress induced signaling pathways also occur in arrested human cell lines, raising the possibility that the response to prolonged cell cycle arrest is conserved.

Cell size is a determinant of stem cell potential during aging.

Stem cells are remarkably small. Whether small size is important for stem cell function is unknown. We find that hematopoietic stem cells (HSCs) enlarge under conditions known to decrease stem cell function. This decreased fitness of large HSCs is due to reduced proliferation and was accompanied by altered metabolism. Preventing HSC enlargement or reducing large HSCs in size averts the loss of stem cell potential under conditions causing stem cell exhaustion. Last, we show that murine and human HSCs enlarge during aging. Preventing this age-dependent enlargement improves HSC function. We conclude that small cell size is important for stem cell function in vivo and propose that stem cell enlargement contributes to their functional decline during aging.

Decreasing mitochondrial RNA polymerase activity reverses biased inheritance of hypersuppressive mtDNA.

Faithful inheritance of mitochondrial DNA (mtDNA) is crucial for cellular respiration/oxidative phosphorylation and mitochondrial membrane potential. However, how mtDNA is transmitted to progeny is not fully understood. We utilized hypersuppressive mtDNA, a class of respiratory deficient Saccharomyces cerevisiae mtDNA that is preferentially inherited over wild-type mtDNA (rho+), to uncover the factors governing mtDNA inheritance. We found that some regions of rho+ mtDNA persisted while others were lost after a specific hypersuppressive takeover indicating that hypersuppressive preferential inheritance may partially be due to active destruction of rho+ mtDNA. From a multicopy suppression screen, we found that overexpression of putative mitochondrial RNA exonuclease PET127 reduced biased inheritance of a subset of hypersuppressive genomes. This suppression required PET127 binding to the mitochondrial RNA polymerase RPO41 but not PET127 exonuclease activity. A temperature-sensitive allele of RPO41 improved rho+ mtDNA inheritance over a specific hypersuppressive mtDNA at semi-permissive temperatures revealing a previously unknown role for rho+ transcription in promoting hypersuppressive mtDNA inheritance.

Clonal selection of stable aneuploidies in progenitor cells drives high-prevalence tumorigenesis.

Chromosome gains and losses are a frequent feature of human cancers. However, how these aberrations can outweigh the detrimental effects of aneuploidy remains unclear. An initial comparison of existing chromosomal instability (CIN) mouse models suggests that aneuploidy accumulates to low levels in these animals. We therefore developed a novel mouse model that enables unprecedented levels of chromosome missegregation in the adult animal. At the earliest stages of T-cell development, cells with random chromosome gains and/or losses are selected against, but CIN eventually results in the expansion of progenitors with clonal chromosomal imbalances. Clonal selection leads to the development of T-cell lymphomas with stereotypic karyotypes in which chromosome 15, containing the Myc oncogene, is gained with high prevalence. Expressing human MYC from chromosome 6 (MYCChr6) is sufficient to change the karyotype of these lymphomas to include universal chromosome 6 gains. Interestingly, while chromosome 15 is still gained in MYCChr6 tumors after genetic ablation of the endogenous Myc locus, this chromosome is not efficiently gained after deletion of one copy of Rad21, suggesting a synergistic effect of both MYC and RAD21 in driving chromosome 15 gains. Our results show that the initial detrimental effects of random missegregation are outbalanced by clonal selection, which is dictated by the chromosomal location and nature of certain genes and is sufficient to drive cancer with high prevalence.

Opportunities, barriers, and recommendations in down syndrome research.

BACKGROUND: Recent advances in medical care have increased life expectancy and improved the quality of life for people with Down syndrome (DS). These advances are the result of both pre-clinical and clinical research but much about DS is still poorly understood. In 2020, the NIH announced their plan to update their DS research plan and requested input from the scientific and advocacy community. OBJECTIVE: The National Down Syndrome Society (NDSS) and the LuMind IDSC Foundation worked together with scientific and medical experts to develop recommendations for the NIH research plan. METHODS: NDSS and LuMind IDSC assembled over 50 experts across multiple disciplines and organized them in eleven working groups focused on specific issues for people with DS. RESULTS: This review article summarizes the research gaps and recommendations that have the potential to improve the health and quality of life for people with DS within the next decade. CONCLUSIONS: This review highlights many of the scientific gaps that exist in DS research. Based on these gaps, a multidisciplinary group of DS experts has made recommendations to advance DS research. This paper may also aid policymakers and the DS community to build a comprehensive national DS research strategy.

Cell adaptation to aneuploidy by the environmental stress response dampens induction of the cytosolic unfolded-protein response.

Aneuploid yeast cells are in a chronic state of proteotoxicity, yet do not constitutively induce the cytosolic unfolded protein response, or heat shock response (HSR) by heat shock factor 1 (Hsf1). Here, we demonstrate that an active environmental stress response (ESR), a hallmark of aneuploidy across different models, suppresses Hsf1 induction in models of single-chromosome gain. Furthermore, engineered activation of the ESR in the absence of stress was sufficient to suppress Hsf1 activation in euploid cells by subsequent heat shock while increasing thermotolerance and blocking formation of heat-induced protein aggregates. Suppression of the ESR in aneuploid cells resulted in longer cell doubling times and decreased viability in the presence of additional proteotoxicity. Last, we show that in euploids, Hsf1 induction by heat shock is curbed by the ESR. Strikingly, we found a similar relationship between the ESR and the HSR using an inducible model of aneuploidy. Our work explains a long-standing paradox in the field and provides new insights into conserved mechanisms of proteostasis with potential relevance to cancers associated with aneuploidy.

Aneuploid senescent cells activate NF-κB to promote their immune clearance by NK cells.

The immune system plays a major role in the protection against cancer. Identifying and characterizing the pathways mediating this immune surveillance are thus critical for understanding how cancer cells are recognized and eliminated. Aneuploidy is a hallmark of cancer, and we previously found that untransformed cells that had undergone senescence due to highly abnormal karyotypes are eliminated by natural killer (NK) cells in vitro. However, the mechanisms underlying this process remained elusive. Here, using an in vitro NK cell killing system, we show that non-cell-autonomous mechanisms in aneuploid cells predominantly mediate their clearance by NK cells. Our data indicate that in untransformed aneuploid cells, NF-κB signaling upregulation is central to elicit this immune response. Inactivating NF-κB abolishes NK cell-mediated clearance of untransformed aneuploid cells. In cancer cell lines, NF-κB upregulation also correlates with the degree of aneuploidy. However, such upregulation in cancer cells is not sufficient to trigger NK cell-mediated clearance, suggesting that additional mechanisms might be at play during cancer evolution to counteract NF-κB-mediated immunogenicity.

RAD21 is a driver of chromosome 8 gain in Ewing sarcoma to mitigate replication stress.

Aneuploidy, defined as whole-chromosome gain or loss, causes cellular stress but, paradoxically, is a frequent occurrence in cancers. Here, we investigate why ∼50% of Ewing sarcomas, driven by the EWS-FLI1 fusion oncogene, harbor chromosome 8 gains. Expression of the EWS-FLI1 fusion in primary cells causes replication stress that can result in cellular senescence. Using an evolution approach, we show that trisomy 8 mitigates EWS-FLI1-induced replication stress through gain of a copy of RAD21. Low-level ectopic expression of RAD21 is sufficient to dampen replication stress and improve proliferation in EWS-FLI1-expressing cells. Conversely, deleting one copy in trisomy 8 cells largely neutralizes the fitness benefit of chromosome 8 gain and reduces tumorgenicity of a Ewing sarcoma cancer cell line in soft agar assays. We propose that RAD21 promotes tumorigenesis through single gene copy gain. Such genes may explain some recurrent aneuploidies in cancer.

Cross-compartment signal propagation in the mitotic exit network.

In budding yeast, the mitotic exit network (MEN), a GTPase signaling cascade, integrates spatial and temporal cues to promote exit from mitosis. This signal integration requires transmission of a signal generated on the cytoplasmic face of spindle pole bodies (SPBs; yeast equivalent of centrosomes) to the nucleolus, where the MEN effector protein Cdc14 resides. Here, we show that the MEN activating signal at SPBs is relayed to Cdc14 in the nucleolus through the dynamic localization of its terminal kinase complex Dbf2-Mob1. Cdc15, the protein kinase that activates Dbf2-Mob1 at SPBs, also regulates its nuclear access. Once in the nucleus, priming phosphorylation of Cfi1/Net1, the nucleolar anchor of Cdc14, by the Polo-like kinase Cdc5 targets Dbf2-Mob1 to the nucleolus. Nucleolar Dbf2-Mob1 then phosphorylates Cfi1/Net1 and Cdc14, activating Cdc14. The kinase-primed transmission of the MEN signal from the cytoplasm to the nucleolus exemplifies how signaling cascades can bridge distant inputs and responses.

Aneuploidy increases resistance to chemotherapeutics by antagonizing cell division.

Aneuploidy, defined as whole chromosome gains and losses, is associated with poor patient prognosis in many cancer types. However, the condition causes cellular stress and cell cycle delays, foremost in G1 and S phase. Here, we investigate how aneuploidy causes both slow proliferation and poor disease outcome. We test the hypothesis that aneuploidy brings about resistance to chemotherapies because of a general feature of the aneuploid condition-G1 delays. We show that single chromosome gains lead to increased resistance to the frontline chemotherapeutics cisplatin and paclitaxel. Furthermore, G1 cell cycle delays are sufficient to increase chemotherapeutic resistance in euploid cells. Mechanistically, G1 delays increase drug resistance to cisplatin and paclitaxel by reducing their ability to damage DNA and microtubules, respectively. Finally, we show that our findings are clinically relevant. Aneuploidy correlates with slowed proliferation and drug resistance in the Cancer Cell Line Encyclopedia (CCLE) dataset. We conclude that a general and seemingly detrimental effect of aneuploidy, slowed proliferation, provides a selective benefit to cancer cells during chemotherapy treatment.

A somatic evolutionary model of the dynamics of aneuploid cells during hematopoietic reconstitution.

Aneuploidy is a feature of many cancers. Recent studies demonstrate that in the hematopoietic stem and progenitor cell (HSPC) compartment aneuploid cells have reduced fitness and are efficiently purged from the bone marrow. However, early phases of hematopoietic reconstitution following bone marrow transplantation provide a window of opportunity whereby aneuploid cells rise in frequency, only to decline to basal levels thereafter. Here we demonstrate by Monte Carlo modeling that two mechanisms could underlie this aneuploidy peak: rapid expansion of the engrafted HSPC population and bone marrow microenvironment degradation caused by pre-transplantation radiation treatment. Both mechanisms reduce the strength of purifying selection acting in early post-transplantation bone marrow. We explore the contribution of other factors such as alterations in cell division rates that affect the strength of purifying selection, the balance of drift and selection imposed by the HSPC population size, and the mutation-selection balance dependent on the rate of aneuploidy generation per cell division. We propose a somatic evolutionary model for the dynamics of cells with aneuploidy or other fitness-reducing mutations during hematopoietic reconstitution following bone marrow transplantation. Similar alterations in the strength of purifying selection during cancer development could help explain the paradox of aneuploidy abundance in tumors despite somatic fitness costs.

Emergence of a High-Plasticity Cell State during Lung Cancer Evolution.

Tumor evolution from a single cell into a malignant, heterogeneous tissue remains poorly understood. Here, we profile single-cell transcriptomes of genetically engineered mouse lung tumors at seven stages, from pre-neoplastic hyperplasia to adenocarcinoma. The diversity of transcriptional states increases over time and is reproducible across tumors and mice. Cancer cells progressively adopt alternate lineage identities, computationally predicted to be mediated through a common transitional, high-plasticity cell state (HPCS). Accordingly, HPCS cells prospectively isolated from mouse tumors and human patient-derived xenografts display high capacity for differentiation and proliferation. The HPCS program is associated with poor survival across human cancers and demonstrates chemoresistance in mice. Our study reveals a central principle underpinning intra-tumoral heterogeneity and motivates therapeutic targeting of the HPCS.

The environmental stress response causes ribosome loss in aneuploid yeast cells.

Aneuploidy, a condition characterized by whole chromosome gains and losses, is often associated with significant cellular stress and decreased fitness. However, how cells respond to the aneuploid state has remained controversial. In aneuploid budding yeast, two opposing gene-expression patterns have been reported: the "environmental stress response" (ESR) and the "common aneuploidy gene-expression" (CAGE) signature, in which many ESR genes are oppositely regulated. Here, we investigate this controversy. We show that the CAGE signature is not an aneuploidy-specific gene-expression signature but the result of normalizing the gene-expression profile of actively proliferating aneuploid cells to that of euploid cells grown into stationary phase. Because growth into stationary phase is among the strongest inducers of the ESR, the ESR in aneuploid cells was masked when stationary phase euploid cells were used for normalization in transcriptomic studies. When exponentially growing euploid cells are used in gene-expression comparisons with aneuploid cells, the CAGE signature is no longer evident in aneuploid cells. Instead, aneuploid cells exhibit the ESR. We further show that the ESR causes selective ribosome loss in aneuploid cells, providing an explanation for the decreased cellular density of aneuploid cells. We conclude that aneuploid budding yeast cells mount the ESR, rather than the CAGE signature, in response to aneuploidy-induced cellular stresses, resulting in selective ribosome loss. We propose that the ESR serves two purposes in aneuploid cells: protecting cells from aneuploidy-induced cellular stresses and preventing excessive cellular enlargement during slowed cell cycles by down-regulating translation capacity.

Aneuploidy and a deregulated DNA damage response suggest haploinsufficiency in breast tissues of BRCA2 mutation carriers.

Women harboring heterozygous germline mutations of BRCA2 have a 50 to 80% risk of developing breast cancer, yet the pathogenesis of these cancers is poorly understood. To reveal early steps in BRCA2-associated carcinogenesis, we analyzed sorted cell populations from freshly-isolated, non-cancerous breast tissues of BRCA2 mutation carriers and matched controls. Single-cell whole-genome sequencing demonstrates that >25% of BRCA2 carrier (BRCA2mut/+ ) luminal progenitor (LP) cells exhibit sub-chromosomal copy number variations, which are rarely observed in non-carriers. Correspondingly, primary BRCA2mut/+ breast epithelia exhibit DNA damage together with attenuated replication checkpoint and apoptotic responses, and an age-associated expansion of the LP compartment. We provide evidence that these phenotypes do not require loss of the wild-type BRCA2 allele. Collectively, our findings suggest that BRCA2 haploinsufficiency and associated DNA damage precede histologic abnormalities in vivo. Using these hallmarks of cancer predisposition will yield unanticipated opportunities for improved risk assessment and prevention strategies in high-risk patients.

Spindle pole bodies function as signal amplifiers in the Mitotic Exit Network.

The Mitotic Exit Network (MEN), a budding yeast Ras-like signal transduction cascade, translates nuclear position into a signal to exit from mitosis. Here we describe how scaffolding the MEN onto spindle pole bodies (SPB-centrosome equivalent) allows the MEN to couple the final stages of mitosis to spindle position. Through the quantitative analysis of the localization of MEN components, we determined the relative importance of MEN signaling from the SPB that is delivered into the daughter cell (dSPB) during anaphase and the SPB that remains in the mother cell. Movement of half of the nucleus into the bud during anaphase causes the active form of the MEN GTPase Tem1 to accumulate at the dSPB. In response to Tem1's activity at the dSPB, the MEN kinase cascade, which functions downstream of Tem1, accumulates at both SPBs. This localization to both SPBs serves an important role in promoting efficient exit from mitosis. Cells that harbor only one SPB delay exit from mitosis. We propose that MEN signaling is initiated by Tem1 at the dSPB and that association of the downstream MEN kinases with both SPBs serves to amplify MEN signaling, enabling the timely exit from mitosis.

Relevance and Regulation of Cell Density.

Cell density shows very little variation within a given cell type. For example, in humans variability in cell density among cells of a given cell type is 100 times smaller than variation in cell mass. This tight control indicates that maintenance of a cell type-specific cell density is important for cell function. Indeed, pathological conditions such as cellular senescence are accompanied by changes in cell density. Despite the apparent importance of cell-type-specific density, we know little about how cell density affects cell function, how it is controlled, and how it sometimes changes as part of a developmental process or in response to changes in the environment. The recent development of new technologies to accurately measure the cell density of single cells in suspension and in tissues is likely to provide answers to these important questions.

Context is everything: aneuploidy in cancer.

Cancer is driven by multiple types of genetic alterations, which range in size from point mutations to whole-chromosome gains and losses, known as aneuploidy. Chromosome instability, the process that gives rise to aneuploidy, can promote tumorigenesis by increasing genetic heterogeneity and promoting tumour evolution. However, much less is known about how aneuploidy itself contributes to tumour formation and progression. Unlike some pan-cancer oncogenes and tumour suppressor genes that drive transformation in virtually all cell types and cellular contexts, aneuploidy is not a universal promoter of tumorigenesis. Instead, recent studies suggest that aneuploidy is a context-dependent, cancer-type-specific oncogenic event that may have clinical relevance as a prognostic marker and as a potential therapeutic target.

Evaluation of Chen et al.: Overexpression of Protein Complexes and Aneuploidy.

One snapshot of the peer review process for "Overdosage of Balanced Protein Complexes Reduces Proliferation Rate in Aneuploid Cells" (Chen et al., 2019).

Protein aggregation mediates stoichiometry of protein complexes in aneuploid cells.

Aneuploidy, a condition characterized by chromosome gains and losses, causes reduced fitness and numerous cellular stresses, including increased protein aggregation. Here, we identify protein complex stoichiometry imbalances as a major cause of protein aggregation in aneuploid cells. Subunits of protein complexes encoded on excess chromosomes aggregate in aneuploid cells, which is suppressed when expression of other subunits is coordinately altered. We further show that excess subunits are either degraded or aggregate and that protein aggregation is nearly as effective as protein degradation at lowering levels of excess proteins. Our study explains why proteotoxic stress is a universal feature of the aneuploid state and reveals protein aggregation as a form of dosage compensation to cope with disproportionate expression of protein complex subunits.