Targeted therapy represents a class of cancer treatments that interferes with specific cancer-associated molecules and aims to specifically attack reprogrammed cancer cells, leaving normal cells unaffected. In this dissertation, we investigate novel approaches for targeted therapy in Clear Cell-Renal Cell Carcinoma (CC-RCC) and Triple Negative Breast Cancer (TNBC).

In CC-RCC, we uncovered an interaction of the tumor suppressor gene VHL with the Rho/ROCK signaling pathway, identifying this pathway as a potential therapeutic target. We found that ROCK inhibition preferentially induces massive mitotic errors in VHL-deficient cells and suggest that the induction of mitotic catastrophe represents a novel therapeutic strategy for VHL-deficient cancers. We further demonstrate that Cyclin-dependent kinase (CDK) inhibitors show significant cytotoxic activity in VHL-deficient CC-RCC in vitro and in a patient-derived xenograft (PDX) orthotopic mouse model. Importantly, we show that these inhibitors do not induce apoptosis in non-transformed cells, or CC-RCC cells engineered to re-express VHL. In TNBC, we obtained clinical evidence that the oncogene CUB Domain Containing Protein 1 (CDCP1) is co-activated with the oncogenic kinase c-Src and its downstream target Protein Kinase C δ (PKC-δ) in a subset of patient specimens, and that this class of patients may benefit from the emerging therapeutics targeting this pathway.

Together, these studies contribute to expansion of the targeted therapy library in CC-RCC and TNBC and help to set the stage for more focused preclinical studies to evaluate the efficacy of targeting these pathways.

Clear Cell Renal Cell Carcinoma (CC-RCC) is a devastating disease in its metastatic manifestation with a 5-year survival rate of 11.7%. The loss of the tumor suppressor von Hippel-Lindau (VHL) has been shown to drive the initiation and progression of CC-RCC. Since most of the currently approved FDA therapies act on a patient’s endothelial cells to reduce angiogenesis, instead of directly on the tumor, new targeted therapies are needed to treat this disease. One method for identifying targeted and tumor specific therapies is by identifying synthetic lethal interactions with the most common mutations in the cancer.

We conducted an annotated chemical library screen in a CC-RCC cell line with and without VHL re-expressed and identified seven potential synthetic lethal interactions. Validation of these potential hits confirmed that inhibition of Rho Kinase (ROCK) 1 is synthetically lethal with VHL loss in CC-RCC. We then confirmed the interaction both genetically via siRNA knockdown and with multiple ROCK inhibitors. The synthetic lethality interaction effect between ROCK1 inhibition and VHL loss is dependent on the overactivation of HIFs that occurs upon VHL loss. Furthermore, treatment with the ROCK inhibitor Y-27632 inhibited tumor growth in vivo in a subcutaneous xenograft model using 786-O CC-RCC cells.

While ROCK inhibitors have great potential to become CC-RCC therapeutics, with the exception of Fasudil approved in Japan and China for treating cerebral vasospasm and pulmonary hypertension, the existing inhibitors are currently limited to topical applications for glaucoma. Statins, HMG CoA Reductase inhibitors, can disrupt the Rho/ROCK pathway at doses administered for treating hypercholesterolemia. We have confirmed the synthetic lethal effect of statin treatment in VHL-deficient CC-RCC. The effect is cytostatic at low nanomolar doses and becomes cytotoxic as the dose is increased into the low micromolar. The addition of both Mevalonate and Geranylgeranyl pyrophosphate (GGPP) can fully rescue the effect. Increasing ROCK activity with arachidonic acid only partially rescues the effect suggesting that statins act through more synthetic lethal partners beyond the Rho/ROCK pathway. In vivo, treatment with Fluvastatin decreased tumor initiation and caused tumor regression in established tumors in subcutaneous xenograft models using 786-O CC-RCC cells.

Combined, these studies identify ROCK inhibitors and HMG CoA Reductase inhibitors as promising new therapies for treating VHL-deficient CC-RCC and the biomarkers (VHL/HIF pathway) by which patients can be stratified for clinical trials.

CUB Domain Containing Protein 1 (CDCP1) is a transmembrane glycoprotein that has mainly been implicated in driving migration and metastasis of carcinoma cells. We focus on its role in triple-negative breast cancer (TNBC) metastasis, liver metabolic disorders (metabolic syndrome, non-alcoholic fatty liver disease, and type-2 diabetes mellitus), and wound healing.

In TNBC cells, we uncovered a novel mechanism of CDCP1 activation via cleavage and homo-dimerization to promote trans-phosphorylation of its downstream target, PKCδ, by Src kinase. Furthermore, we uncovered a novel role of CDCP1 as a regulator of lipid metabolism in TNBC. We found that CDCP1 reduces cytoplasmic lipid droplet (LD) abundance and promotes fatty acid β-oxidation (FAO) in TNBC cells. We found that CDCP1 regulates lipid metabolism partially by reducing Acyl-CoA Synthetase Ligases (ACSLs). All of this leads to CDCP1-induced TNBC metastasis, which can be inhibited by blocking CDCP1 homo-dimerization via overexpression of the extracellular portion of cleaved CDCP1 (ECC). ECC reduces phosphorylation of PKCδ and increases ACSL activity and LD abundance in TNBC cells to reduce metastasis of TNBC tumors to the lungs.

Physiologically, we found that CDCP1 regulates LD abundance in the livers of mice; CDCP1 knockout (KO) mice had higher LD abundance than CDCP1 heterozygous (Het) mice. We also found that CDCP1 KO mice had reduced wound healing capacity of dermal punch biopsy wounds compared to CDCP1 Het mice.

Further, we present preliminary findings in the quest to identify important residues in CDCP1 dimerization and the development of a surrogate biomarker of CDCP1 activity in breast cancer.

Combined, these studies give a comprehensive look at the broad range of pathological and physiological effects CDCP1 has and contributes groundwork for future investigation into these diverse and potentially organ-type specific roles of CDCP1.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer, with von Hippel-Lindau (VHL) gene mutations prevalent in approximately 90% of cases. We have previously demonstrated synthetic lethality between VHL loss and the inhibition of Cyclin-Dependent Kinases (CDKs) in ccRCC, using the multi-CDK inhibitor Dinaciclib. Here, we extend these findings by investigating the specific role of CDK9 in synthetic lethality with VHL loss. CDK9 is a transcriptional CDK involved in the process of transcription elongation and splicing. Using three distinct and specific CDK9 inhibitors (AZD45753, KB0742 and NVP-2), we confirmed preferential toxicity towards VHL-deficient ccRCC cells that acted independently of the hypoxia-inducible factor (HIF) pathway, the main pathway regulated by VHL. An analysis of the current literature connecting inhibition of CDK9 to cancer cell death suggests that the prime mechanism of action is the reduction of CDK9’s target pro-survival proteins, MCL-1 or BFL-1. Interestingly, we found that BFL-1, not MCL-1, is the critical anti-apoptotic protein involved in the mechanism of synthetic lethality. The literature further suggested that CDK9 inhibition might cause immunogenic cell death (ICD). In vivo, immunocompetent and immunodeficient mouse studies using AZD4573 showed that CDK9 inhibition suppressed ccRCC tumor growth, although we did not observe significant changes in immune cell populations upon treatment in an immunocompetent mouse model. At the same time, CDK9 inhibition did not compromise immune system. Our results offer novel insights into the mechanism of CDK9 inhibition / VHL loss synthetic lethality in ccRCC and provide a compelling rationale for further development of CDK9 inhibitors as potential therapeutics in VHL-deficient ccRCC. The BFL-1 pathway, which is a part of the mechanism of AZD4573 action, represents a biomarker for drug sensitivity, paving the way for improved patient stratification and treatment outcomes.