Unlocking mechanisms of resistance to targeted therapy in thyroid cancer

Division Of Basic Sciences - Nci

Investigators

Abstract

Aim 1: Identify novel genes or essential pathways required for resistance to BRAFV600E inhibition utilizing genome wide and focused CRISPR-based screens. First, using BRAFV600E-ATC cell line 8505c, we will harness a human CRISPR knockout library of 19,050 genes to identify genes whose loss enhances cell death by the BRAF selective inhibitor Dabrafenib. Dabrafenib has a better toxicity profile compared to the canonical inhibitor vemurafenib and was recently approved by the FDA for the treatment of advanced and metastatic thyroid cancer. BRAF activity will be inhibited using 400 nM of Dabrafenib, a dose which yielded about 20% of cell death and a complete inhibition of phospho-MEK and phospho-ERK1/2 in a 14-days treatment survival assay. Mechanisms of resistance to targeted therapy including those involving BRAFV600E inhibitors is a complex process which involve various alterations that enable cancer cells to escape death. To more specifically unlock these mechanisms of resistance, we engineered a focused CRISPR/cas9 and CRISPR/dCas9 transcriptional activation (SAM system) libraries targeting genes sets selected based on strong correlations with BRAFV600E activation and dependency, in collaboration with the Genome modification Core of the NCI-Frederick. We will utilize single sgRNA CRISPR guides targeting top genes identified in our CRISPR screens output to validate the putative synthetic lethality with BRAFV600E inhibition. To further substantiate the significance of essential gene (s) in vivo, we will use two models 1) ATC cells 8505c will be depleted for the validated gene(s) and used in an immunocompromised xenograft model for ATC. 2) The murine BRAFV600E-driven ATC cell line will be depleted for the validated gene(s) and implanted orthotopically into immunocompetent mice. In both mouse models tumor growth and metastasis of wild type and target-depleted cells will be evaluated in response to dabrafenib. This study will identify novel genes involved in resistance to BRAFV600E inhibition and will unfold new avenues of therapeutic strategies in patients with anaplastic thyroid cancer. Aim2: Targeting ATM and BRAFV600E in anaplastic thyroid cancer for precision medicine. Preclinical studies in cancer therapy have shown that targeting major genome maintenance genes such as ATM, ATR, or harnessing deficiency in DNA damage response genes such as BRCA1, may help to overcome drug resistance in multiple solid malignancies. These findings provide insights into targeting DNA repair proteins to sensitize cancer cells to targeted therapies. In line with these observations, we found that ATM expression is strongly associated with adverse clinical features and aggressive thyroid cancer histology, thereby suggesting that ATM may play a key role in mediating thyroid cancer metastasis, particularly in BRAF-driven thyroid tumors. ATM is a major genome maintenance gene involved in the repair of double-stranded DNA breaks. While several reports have demonstrated links between genetic alterations in ATM and DNA repair response genes, little is known about how changes in ATM expression level and kinase activity affect cancer progression. We aim to determine the extent to which ATM expression level is associated with key features of thyroid cancer aggressiveness, and whether ATM could serve as a druggable therapeutic target in advanced and metastatic thyroid cancer. To elucidate the role of ATM in thyroid cancer progression we performed functional studies and found that ATM is a key regulator of VEGF secretion and angiogenesis in ATC. Furthermore, ATM depletion or its inhibition with a highly selective ATM inhibitor reduced tumor metastasis in a model of ATC xenografts. On the other hand, little is known about the impact of BRAFV600E inhibition on DNA repair pathways in solid tumors. Our preliminary data show that increasing doses of BRAFV600E inhibitors dabrafenib (FDA approved) and PLX4720 (modified vemurafenib) lead to a robust phosphorylation of ATM on its Serine1981 residue in BRAFV600E driven ATC cells. These observations are consistent with two major recent findings demonstrating an increased phosphorylation of ATM in EGFR and BRAFV600E inhibitors treated NSCLC and melanoma cells respectively. Together, these data suggest that therapy targeting oncogenes such as BRAFV600E induces dependency on ATM activation in cancer cells, and combination of ATM inhibitor AZD1390 (currently in clinical trial) with specific BRAFV600E inhibitors such as dabrafrenib will unlock acquired resistance to BRAFV600E inhibitors in ATC. Remarkably, we found that AZD1390 sensitizes ATC and PDTC BRAFV00E driven cells to dabrafenib. The validation of the clinical benefit of this combination therapy in a genetically engineered ATC mouse model (BRAFV600E; Trp53R270H) with a similar phenotype and disease aggressiveness as in human patients, will provide a valuable new therapeutic strategy that may significantly improve the clinical management of patients with undifferentiated thyroid cancer.