ACQUIRED RESISTANCE TO BRAF INHIBITOR SENSITIZES MELANOMA CELLS TO CHK1 INHIBITION-INDUCED REPLICATION STRESS

Vol 2, 2021 - 142474
DR - Doctoral Student
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Abstract

INTRODUCTION AND OBJECTIVES: Melanomas frequently harbor BRAF mutations, which deregulate the MAPK pathway and contribute to tumor development and progression. Treatment with BRAF inhibitor induces rapid regression of metastasis, but most melanoma cells eventually acquire drug resistance. These BRAF inhibitor-resistant (BRAFiR) cells accumulate a range of genetic and functional alterations that enable them to escape alternative pharmacological agents. The search for these modifications is critical to develop new treatment approaches for BRAFiR patients. Here, we investigated the potential of Chk1, a kinase of the DNA Damage Response (DDR) pathway, as a therapeutic target in BRAFiR melanomas. MATERIAL AND METHODS: To address Chk1 inhibitor (Chk1i) sensitivity, we used two different Chk1i (GDC0575 and AZD7762) and compared BRAFiR cells to their respective treatment-naïve cells. We analyzed in vitro cell death by caspase cleavage and in vivo tumor growth in a human melanoma xenograft model. Due to the role of Chk1 in cell cycle regulation, we monitored cell cycle progression using a fluorescent system and time-lapse microscopy. To estimate Chk1i-induced replication stress, we evaluated nucleotide incorporation by flow cytometry and DNA fibers assay, and the levels of phospho-RPA and yH2AX were quantified by western blotting. To investigate the factors responsible for Chk1i sensitivity, we performed exome and transcriptome sequencing and explored the differences between BRAFiR and treatment-naïve cells. RESULTS AND CONCLUSION: We demonstrated that most BRAFiR cells are hypersensitive to Chk1i compared to their respective treatment-naïve cells both in vitro and in vivo. We showed that S phase is crucial for Chk1i-induced cytotoxicity in BRAFiR cells, in which they partially fail to incorporate nucleotides. Upon Chk1 inhibition, BRAFiR cells hypersensitive to Chk1i showed a greater increase in phospho-RPA and yH2AX, indicating that DNA replication stress is exacerbated in these cells. Interestingly, DNA fibers assay demonstrated that, in the absence of any inhibitor, BRAFiR cells show an increase in replication origin firing. In agreement, the transcriptome profiling showed increased expression of G1/S transition regulators in BRAFiR cells. Exome sequencing revealed mutations in genes belonging to the RAS family of GTPases in all BRAFiR cells classified as hypersensitive to Chk1i. Using an independent melanoma cell line panel, we confirmed that the presence of RAS mutations is associated with higher Chk1i sensitivity, in line with the literature showing a synthetic interaction between RAS mutations and inhibition of DDR pathway members in several tumors. In summary, we show here that enhanced sensitivity to Chk1i-induced replication stress is common among BRAFiR melanoma cells that harbor RAS mutations. Further work is ongoing to investigate the effect of different RAS mutations on Chk1i sensitivity in melanoma cells.

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Institutions
  • 1 Instituto Nacional de Câncer (INCA)
  • 2 Netherlands Cancer Institute (NKI)
Track
  • 2. Cellular Biology
Keywords
Melanoma
Chk1 inhibitor
RAS mutation