Joe R. Delaney

Screening Methods to Discover the FDA-Approved Cancer Drug Encorafenib as Optimally Selective for Metallothionein Gene Loss Ovarian Cancer

Background/Objectives: All 11 metallothionein protein-coding genes are located on human chromosome 16q13. It is unique among human genetics to have an entire pathway’s genes clustered in a short chromosomal region. Since solid tumors, particularly high-grade serous ovarian cancer (HGSC), exhibit high rates of monoallelic aneuploidy, this region is commonly lost. Studies have not yet been performed to determine what vulnerability may be created in cancer cells with low metallothionein expression. Here, a screen of FDA-approved cancer small molecule drugs for those best targeting low metallothionein ovarian cancer was completed. Methods: Screening methods were tested and compared using vehicle-treated negative controls and cadmium chloride, a positive control for cell loss selective for low metallothionein cells. CAOV3 cells, which are unique in their expression of only two metallothionein isoforms, were used, with or without shRNA knockdown of the predominantly expressed MT2A gene. A library of FDA-approved molecules was then screened. Results: The optimal assay utilized Hoechst 33342 nuclear staining and mechanized fluorescent microscope counting of cell content. Encorafenib, an RAF inhibitor, was identified as the most selective for enhanced cytotoxicity in MT2A knockdown cells compared to scrambled controls. Conclusions: The nuclear stain Hoechst 33342, assessed by fluorescence microscopy, provides a low variance, moderate throughput platform for cancer cell loss screens. Low metallothionein ovarian cancer cells exhibit a vulnerability to the RAF inhibitor encorafenib.

Autophagy unrelated transcriptional mechanisms of hydroxychloroquine resistance revealed by integrated multi-omics of evolved cancer cells

MYC is Sufficient to Generate Mid-Life High-Grade Serous Ovarian and Uterine Serous Carcinomas in a p53-R270H Mouse Model

Abstract Genetically engineered mouse models (GEMM) have fundamentally changed how ovarian cancer etiology, early detection, and treatment are understood. MYC, an oncogene, is amongst the most amplified genes in high-grade serous ovarian cancer (HGSOC), but it has not previously been utilized to drive HGSOC GEMMs. We coupled Myc and dominant-negative mutant p53-R270H with a fallopian tube epithelium (FTE)-specific promoter Ovgp1 to generate a new GEMM of HGSOC. Female mice developed lethal cancer at an average of 14.5 months. Histopathologic examination of mice revealed HGSOC characteristics, including nuclear p53 and nuclear MYC in clusters of cells within the FTE and ovarian surface epithelium. Unexpectedly, nuclear p53 and MYC clustered cell expression was also identified in the uterine luminal epithelium, possibly from intraepithelial metastasis from the FTE. Extracted tumor cells exhibited strong loss of heterozygosity at the p53 locus, leaving the mutant allele. Copy-number alterations in these cancer cells were prevalent, disrupting a large fraction of genes. Transcriptome profiles most closely matched human HGSOC and serous endometrial cancer. Taken together, these results demonstrate that the Myc and Trp53-R270H transgenes were able to recapitulate many phenotypic hallmarks of HGSOC through the utilization of strictly human-mimetic genetic hallmarks of HGSOC. This new mouse model enables further exploration of ovarian cancer pathogenesis, particularly in the 50% of HGSOC which lack homology-directed repair mutations. Histologic and transcriptomic findings are consistent with the hypothesis that uterine serous cancer may originate from the FTE. Significance: Mouse models using transgenes which generate spontaneous cancers are essential tools to examine the etiology of human diseases. Here, the first Myc-driven spontaneous model is described as a valid HGSOC model. Surprisingly, aspects of uterine serous carcinoma were also observed in this model.

MYC and HSF1 Cooperate to Drive Sensitivity to Polo-like Kinase 1 Inhibitor Volasertib in High-grade Serous Ovarian Cancer

Abstract Ovarian cancer is a deadly gynecologic disease with frequent recurrence. Current treatments for patients include platinum-based therapy regimens with PARP inhibitors specific for homologous recombination–deficient high-grade serous ovarian cancers (HGSOC). Despite initial effectiveness, patients inevitably develop disease progression as tumor cells acquire resistance. Toward the development of new therapeutic avenues, we describe a gene amplification involving both heat shock factor 1 (HSF1) and MYC, wherein these two genes are co-amplified in more than 30% of patients with HGSOC. We further found that HSF1 and MYC transcriptional activities were highly correlated with human HGSOC tumors and cell lines, suggesting that they may cooperate in the disease. CUT&RUN sequencing for HSF1 and MYC revealed overlapping HSF1 and MYC binding throughout the genome. Moreover, the binding peaks of both transcription factors in HGSOC cells were nearly identical, and a protein–protein interaction between HSF1 and MYC was detected, supporting molecular cooperation. Supporting a functional cooperation of these two transcription factors, the growth of HGSOC cells with the co-amplification was dependent on both HSF1 and MYC. To identify a therapeutic target that could take advantage of this unique HSF1 and MYC dependency, polo-like kinase 1 (PLK1) was correlated with HSF1 and MYC in HGSOC specimens. Targeting PLK1 with volasertib revealed a greater than 200-fold increased potency in HSF1–MYC co-amplified HGSOC cells compared with those with wild-type HSF1 and MYC copy numbers. Although the success of volasertib and other PLK1 inhibitors in clinical trials has been modest, the current study suggests that targeting PLK1 using a precision medicine approach based on HSF1–MYC co-amplification as a biomarker in HGSOC would improve therapy response and patient outcomes. Significance: We show that HSF1 and MYC genes are co-amplified in more than 30% of HGSOC and demonstrate that HSF1 and MYC functionally cooperate to drive the growth of HGSOC cells. This work provides the foundation for HSF1 and MYC co-amplification as a biomarker for treatment efficacy of the polo-like kinase 1 inhibitor volasertib in HGSOC.

Single-cell analysis of copy-number alterations in serous ovarian cancer reveals substantial heterogeneity in both low- and high-grade tumors

Unusually high aneuploidy is a hallmark of epithelial serous ovarian cancer (SOC). Previous analyses have focused on aneuploidy on average across all tumor cells. With the expansion of single-cell sequencing technologies, however, an analysis of copy number heterogeneity cell-to-cell is now technically feasible. Here, we describe an analysis of single-cell RNA sequencing (scRNA-seq) data to infer arm-level aneuploidy in individual serous ovarian cancer cells. By first clustering high-quality sequenced epithelial versus non-epithelial cells, high-confidence tumor cell populations were identified. InferCNV was used to predict segmented copy-number alterations (CNAs), which were then used to determine arm-level aneuploidy at the single-cell level. Control comparisons of normal cells to normal cells showed zero arm-level aneuploidy, whereas a median of four aneuploid events were detectable in cancer cells. A heterogeneity analysis of high-grade tumor cells compared to low-grade tumor cells showed similar levels of cell-to-cell variation between cancer grades. Metastatic tumors potentially showed selection pressure with reduced cell-to-cell variation compared to cells from primary tumors. Minor cell populations with CNAs similar to metastatic cells were identified within the matched primary tumors. Taken together, these results provide a minimum estimate for single-cell aneuploidy in serous ovarian cancer and demonstrate the utility of single-cell sequencing for CNA analysis.