Abstract Melanoma is an aggressive neoplasm with increasing incidence that bears the infamous distinction of being a recalcitrant cancer, i.e. a cancer with poor prognosis, lacking progress in diagnosis and treatment. In addition to conventional therapy, melanoma treatment is currently based on targeting the BRAF/MEK/ERK signal transduction pathway and immune checkpoints; however, advanced therapeutic approaches based on novel targets are urgently needed. We reasoned that the base excision repair enzyme Thymine DNA Glycosylase (TDG) could be such a target for its dual role in safeguarding genome stability and in effecting active DNA demethylation downstream the Ten-Eleven Translocation (TET) dioxygenases. TDG knockdown in melanoma cell lines causes cell cycle arrest, senescence and death by mitotic alterations, and impairs xenograft formation. Importantly, untransformed melanocytes are not affected by TDG knockdown, and adult mice with conditional knockout of TDG are viable. Candidate TDG inhibitors, identified through a high-throughput screen, reduced viability and clonogenic capacity of melanoma cell lines. Candidate TDG inhibitors increased cellular levels of 5-carboxylcytosine, the last intermediate in DNA demethylation, which is specifically removed by TDG, indicating successful targeting. These findings suggest that TDG may provide critical functions in cancer cells, but not in normal cells, that make it a highly suitable anti-melanoma drug target. By potentially disrupting both DNA repair and the epigenetic state, targeting TDG may represent a completely new approach to melanoma therapy. Citation Format: Rossella Tricarico, Pietro Mancuso, Vikram Bhattacharjee, Laura Cosentino, Emmanuelle Nicolas, Margret Einarson, Neil Beeharry, Karthik Devarajan, Rich Katz, Dorjbal G. Dorjsuren, Anton Simeonov, Yuwaraj Kadariya, Guillaume Davidson, Joseph R. Testa, Irwin Davidson, Lionel Larue, Robert W. Sobol, Timothy Yen, Alfonso Bellacosa. Thymine DNA glycosylase (TDG) as a novel target for melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1940.
Acquired resistance of metastatic melanoma (MM) tumors to BRAF V600E inhibitors (BRAFi's) is commonplace in the clinic. Habitual relapse of patients contributes to <20% 5-year survival rates in MM. We previously identified serine synthesis as a critical detrminant of late-stage cancer cell resistance to BRAFi's. Pre-treatment with DNA damaging agent gemcitabine (a nucleoside analog) re-sensitized drug-resistant cancer cells to BRAFi's dabrafenib and vemurafenib. Importantly, the combination treatments were effective against BRAF wild type cancer cells potentially expanding the clinical reach of BRAFi's. In this study, we identify the antifolate methotrexate (MTX) as a sensitizer of acquired- and intrinsically-resistant MM cells to BRAFi's dabrafenib and encorafenib. We identify a novel, positive correlation between dabrafenib treatments and repair delay of MTX induced single-strand DNA (ssDNA) breaks. Cells arrest in G1 phase following simultaneous MTX + dabrafenib treatments and eventually die via apoptosis. Importantly, we identify RAS codon 12 activating mutations as prognostic markers for MTX + BRAFi treatment efficacy. We describe a method of killing drug-resistant MM cells that if translated has the potential to improve MM patient survival.
Abstract Melanoma is an aggressive neoplasm with increasing incidence that is classified by the NCI as a recalcitrant cancer, i.e., a cancer with poor prognosis, lacking progress in diagnosis and treatment. In addition to conventional therapy, melanoma treatment is currently based on targeting the BRAF/MEK/ERK signaling pathway and immune checkpoints. As drug resistance remains a major obstacle to treatment success, advanced therapeutic approaches based on novel targets are still urgently needed. We reasoned that the base excision repair enzyme thymine DNA glycosylase (TDG) could be such a target for its dual role in safeguarding the genome and the epigenome, by performing the last of the multiple steps in DNA demethylation. Here we show that TDG knockdown in melanoma cell lines causes cell cycle arrest, senescence, and death by mitotic alterations; alters the transcriptome and methylome; and impairs xenograft tumor formation. Importantly, untransformed melanocytes are minimally affected by TDG knockdown, and adult mice with conditional knockout of Tdg are viable. Candidate TDG inhibitors, identified through a high-throughput fluorescence-based screen, reduced viability and clonogenic capacity of melanoma cell lines and increased cellular levels of 5-carboxylcytosine, the last intermediate in DNA demethylation, indicating successful on-target activity. These findings suggest that TDG may provide critical functions specific to cancer cells that make it a highly suitable anti-melanoma drug target. By potentially disrupting both DNA repair and the epigenetic state, targeting TDG may represent a completely new approach to melanoma therapy.
Abstract Acquired resistance of metastatic melanoma (MM) tumors to BRAF V600E inhibitors (BRAFis) is commonplace in the clinic. Habitual relapse of patients contributes to <20% 5-year survival rates in MM. We previously identified serine synthesis as a critical mechanism of late-stage cancer cell resistance to BRAFis. Pretreatment with DNA-damaging agent and nucleoside analog gemcitabine resensitized drug-resistant cancer cells to BRAFis dabrafenib (Tafinlar) and vemurafenib (Zelboraf). The combination treatments were effective against BRAF wild type cancer cells, potentially expanding the clinical reach of BRAFis. In this study, we identify two additional DNA-damaging agents that resensitized drug-resistant MM and pancreatic cancer cells to BRAFis dabrafenib and encorafenib. We identify the antifolate and DNA-damaging agent methotrexate (MTX), and the topoisomerase 1 inhibitor and DNA-damaging agent camptothecin (Camp) as sensitizers of late-stage, drug-resistant cancer cells to BRAFis dabrafenib and encorafenib (LGX818). Importantly, we show a novel positive correlation between DNA repair delay and and allosteric MAPK activation via BRAFi treatment in BRAF wild type cells. Cells arrest in G1/S following DNA-damaging agent + BRAFi combination treatments and ultimately, cell death is triggered via apoptosis, presumably due to DNA damage repair delays. Additionally, we have identified activating KRAS codon 12 mutations as critical to the efficacy of combination treatments. In summary, we have elucidated a novel method of cell death induction via DNA-damaging agent + BRAFi combination treatments that has the potential to significantly impact late-stage MM and pancreatic cancer therapy in the clinic. Citation Format: Vikram Bhattacharjee. Killing late-stage cancer cells by coupling DNA damage induction with MAPK pathway activation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A074.
