PROJECT SUMMARY WNT pathway hyperactivation is a dominant oncogenic driver in colorectal cancer (CRC), and directly linked to disease progression and drug resistance in many other cancer types, including lung (LUAD), breast and prostate cancer. Indeed, there is strong evidence from pre-clinical model systems that targeting hyperactive WNT signaling can provide significant therapeutic benefit in multiple cancer models. We and others have shown that blocking the function of redundant Tankyrase enzymes (TNKS and TNKS2) can suppress hyperactive WNT signaling, impede cancer cell proliferation and drive differentiation, particularly in CRC. However, WNT signaling is also critical for the homeostatic maintenance of multiple organ systems. Consequently, drugs that effectively block WNT, like TNKS inhibitors, have shown a range of on-target toxicities in essential tissues such as the intestine and bone. Defining a strategy for tumor-restricted WNT pathway suppression is a major goal in precision oncology. Advanced cancers often contain significant disruptions to their genomes, including gains and losses of large chromosome segments. These large-scale alterations encompassing many genes are presumed to support cancer cell growth. However, they also lead to loss of ‘passenger’ genes that do not drive cancer progression, but may unintentionally ‘rewire’ the signaling networks. Such cancer-specific collateral damage may provide opportunities for therapeutic intervention. We will test the hypothesis that a large chromosomal deletion on chromosome 8p, which induce loss of TNKS, create a tumor-specific dependency on TNKS2 that can be exploited to block WNT only in cancer cells. Because normal cells express both redundant family members, they should remain unaffected by selective targeting. In Aim 1, using a panel of CRC, LUAD and breast cancer cell lines and patient-derived organoids, in combination with CRISPR-based genome editing, we will determine how heterozygous and homozygous chromosome deletions impact the response to TNKS2 inhibition. Further, we will define downstream protein targets that are most sensitive to TNKS disruption and TNKS2 suppression. In Aim 2 we will exploit a unique transgenic shRNA system we developed, to define the safety of systemic and selective TNKS2 inhibition in vivo and determine the efficacy of selective TNKS2 inhibition in immunocompetent animal models of aggressive CRC and LUAD. Aim 3 will determine the efficacy of novel TNKS2-selective small molecules in cancer cell lines and organoid models and evaluate potential mechanisms of therapy resistance to TNKS2 inhibition. Identifying a safe and effective approach to block hyperactive WNT signaling in multiple tumor types could have a profound impact on the clinical management of advanced cancers. Thus, we believe our work will contribute substantially to the overall goal of developing safe and effective targeted therapies for WNT-driven cancers.