Tumor suppressor vulnerability conferred by aneuploid loss of haploinsufficient metallothionein genes

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Abstract Cancer will rarely be cured through pharmacologic targeting of single genes. Tumors evolve in response to selection pressure. Precision oncology often targets a single gene product, and that gene simply becomes mutated once the drug is administered. Although individual genes may mutate, tumor biology is nonetheless constrained to dysregulating specific pathways for each cancer type. We have previously created cutting-edge bioinformatic tools to better understand which constrained pathways are acting as tumor suppressors and oncogenes, due to collaborative gene dysregulation at the molecular pathway level. Aneuploidy is a major cause of molecular pathway changes in cancer and unfortunately each aneuploid event alters both predicted driver genes and unknown passenger genes. Investigation of causal aneuploid changes in a tumor remains difficult, if not impossible, to study with current cell biology and genetic tools. However, we have discovered a unique, commonly suppressed (70% of high-grade serous ovarian cancers have a monoallelic loss, correlating with average reduced expression), pathway which is amenable to well-controlled basic science experimentation: the cadmium response pathway. It is composed of 11 highly homologous metallothionein genes arrayed on a single chromosomal locus. Metallothioneins sequester the bulk of intracellular Zn2+ and environmental genotoxic Cd2+ ions. The loss of the metallothionein locus is associated with chromosome instability and occurs early in tumor formation. Our in vitro assays show controlled metallothionein suppression results in elevated DNA damage. However, the role of metallothioneins as tumor suppressors and as regulators of cancer cellular and molecular biology is largely unknown. This project will establish specific tumor suppressor phenotypes of metallothioneins in ovarian cancer and determine if this aneuploid pathway will serve as a representative example of how multi- genic vulnerabilities can better enable next-generation cancer therapies. We will (1) characterize in vivo the effects of metallothionein gene loss in spontaneous tumor formation in ovarian cancer, (2) develop controlled models of gene suppression enabling suppression of all 11 genes, including by a synthetic dead-Cas9-based transcription factor, (3) determine which cadmium-dependent and cadmium-independent metallothionein- regulated molecular pathways convey tumor suppressor functions, and (4) discover drug classes which best selectively kill low-metallothionein cells. Genetic tools created by this project will enable causal investigation of entire molecular pathways for future projects. Taken together, this innovative research program will directly test how an uncharacterized aneuploid-suppressed pathway contributes to oncogenesis and remains a pharmacologically targetable vulnerability throughout tumor development.