Many chemotherapeutic drugs are differentially effective from one patient to the next. Understanding the causes of this variability is a critical step towards the development of personalized treatments and improvements to existing medications. Here, we investigate sensitivity to a group of anti-neoplastic drugs that target topoisomerase II using the model organism Caenorhabditis elegans. We show that wild strains of C. elegans vary in their sensitivity to these drugs, and we use an unbiased genetic approach to demonstrate that this natural variation is explained by a methionine-to-glutamine substitution in topoisomerase II (TOP-2). The presence of a non-polar methionine at this residue increases hydrophobic interactions between TOP-2 and its poison etoposide, as compared to a polar glutamine. We hypothesize that this stabilizing interaction results in increased genomic instability in strains that contain a methionine residue. The residue affected by this substitution is conserved from yeast to humans and is one of the few differences between the two human topoisomerase II isoforms (methionine in hTOPIIα and glutamine in hTOPIIβ). We go on to show that this amino acid difference between the two human topoisomerase isoforms influences cytotoxicity of topoisomerase II poisons in human cell lines. These results explain why hTOPIIα and hTOPIIβ are differentially affected by various poisons and demonstrate the utility of C. elegans in understanding the genetics of drug responses.

Mosaic Variegated Aneuploidy (MVA) syndrome is a rare autosomal recessive disorder characterized by inaccurate chromosome segregation and high rates of near-diploid aneuploidy. Children with MVA syndrome die at an early age, are cancer prone, and have progeroid features like facial dysmorphisms, short stature, and cataracts. The majority of MVA cases are linked to mutations in BUBR1, a mitotic checkpoint gene required for proper chromosome segregation. Affected patients either have bi-allelic BUBR1 mutations, with one allele harboring a missense mutation and the other a nonsense mutation, or mono-allelic BUBR1 mutations combined with allelic variants that yield low amounts of wild-type BubR1 protein. Parents of MVA patients that carry single allele mutations have mild mitotic defects, but whether they are at risk for any of the pathologies associated with MVA syndrome is unknown. To address this, we engineered a mouse model for the nonsense mutation 2211insGTTA (referred to as GTTA) found in MVA patients with bi-allelic BUBR1 mutations. Here we report that both the median and maximum lifespans of the resulting BubR1(+/GTTA) mice are significantly reduced. Furthermore, BubR1(+/GTTA) mice develop several aging-related phenotypes at an accelerated rate, including cataract formation, lordokyphosis, skeletal muscle wasting, impaired exercise ability, and fat loss. BubR1(+/GTTA) mice develop mild aneuploidies and show enhanced growth of carcinogen-induced tumors. Collectively, these data demonstrate that the BUBR1 GTTA mutation compromises longevity and healthspan, raising the interesting possibility that mono-allelic changes in BUBR1 might contribute to differences in aging rates in the general population.

Renal angiomyolipoma is a kidney tumor in the perivascular epithelioid (PEComa) family that is common in patients with Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM) but occurs rarely sporadically. Though histologically benign, renal angiomyolipoma can cause life-threatening hemorrhage and kidney failure. Both angiomyolipoma and LAM have mutations in TSC2 or TSC1. However, the frequency and contribution of other somatic events in tumor development is unknown. We performed whole exome sequencing in 32 resected tumor samples (n = 30 angiomyolipoma, n = 2 LAM) from 15 subjects, including three with TSC. Two germline and 22 somatic inactivating mutations in TSC2 were identified, and one germline TSC1 mutation. Twenty of 32 (62%) samples showed copy neutral LOH (CN-LOH) in TSC2 or TSC1 with at least 8 different LOH regions, and 30 of 32 (94%) had biallelic loss of either TSC2 or TSC1. Whole exome sequencing identified a median of 4 somatic non-synonymous coding region mutations (other than in TSC2/TSC1), a mutation rate lower than nearly all other cancer types. Three genes with mutations were known cancer associated genes (BAP1, ARHGAP35 and SPEN), but they were mutated in a single sample each, and were missense variants with uncertain functional effects. Analysis of sixteen angiomyolipomas from a TSC subject showed both second hit point mutations and CN-LOH in TSC2, many of which were distinct, indicating that they were of independent clonal origin. However, three tumors had two shared mutations in addition to private somatic mutations, suggesting a branching evolutionary pattern of tumor development following initiating loss of TSC2. Our results indicate that TSC2 and less commonly TSC1 alterations are the primary essential driver event in angiomyolipoma/LAM, whereas other somatic mutations are rare and likely do not contribute to tumor development.

The discovery that somatic cells are reprogrammable to pluripotency by ectopic expression of a small subset of transcription factors has created great potential for the development of broadly applicable stem-cell-based therapies. One of the concerns regarding the safe use of induced pluripotent stem cells (iPSCs) in therapeutic applications is loss of genomic integrity, a hallmark of various human conditions and diseases, including cancer. Structural chromosome defects such as short telomeres and double-strand breaks are known to limit reprogramming of somatic cells into iPSCs, but whether defects that cause whole-chromosome instability (W-CIN) preclude reprogramming is unknown. Here we demonstrate, using aneuploidy-prone mouse embryonic fibroblasts (MEFs) in which chromosome missegregation is driven by BubR1 or RanBP2 insufficiency, that W-CIN is not a barrier to reprogramming. Unexpectedly, the two W-CIN defects had contrasting effects on iPSC genomic integrity, with BubR1 hypomorphic MEFs almost exclusively yielding aneuploid iPSC clones and RanBP2 hypomorphic MEFs karyotypically normal iPSC clones. Moreover, BubR1-insufficient iPSC clones were karyotypically unstable, whereas RanBP2-insufficient iPSC clones were rather stable. These findings suggest that aneuploid cells can be selected for or against during reprogramming depending on the W-CIN gene defect and present the novel concept that somatic cell W-CIN can be concealed in the pluripotent state. Thus, karyotypic analysis of somatic cells of origin in addition to iPSC lines is necessary for safe application of reprogramming technology.