Abstract 4034: ALKBH3 deficiency and liability to alkylating agents in CRC models

Abstract

Abstract Colorectal cancer (CRC) is the third most frequent malignancy and the second leading cause of cancer death worldwide. Despite the increase in therapeutic options for the treatment of metastatic CRC (mCRC), cytotoxic chemotherapy using fluoropyrimidines, oxaliplatin, and irinotecan still constitutes the mainstay of the treatment. These genotoxic drugs function by inducing direct or indirect DNA damage, which leads to cell cycle block and cell death. We previously showed that the presence of transcriptional repression of O-6-methylguanine-DNA methyltransferase (MGMT), a gene involved in direct repair of methylated guanines, confers increased sensitivity to temozolomide treatment -a DNA alkylating agent- in mCRC patients. We conducted a screening for down-regulation of DNA repair genes in a large panel of 245 molecularly annotated human colorectal cancer cell lines with the aim to identify other pharmacological liabilities to alkylating agents. 5 cell lines (2%) were identified to be significantly downregulated for alpha-ketoglutarate-dependent dioxygenase alkB homolog 3 (ALKBH3). ALKBH3 encodes a DNA dioxygenase that counteracts toxic alkylation damage inflicted by SN2 agents such as methyl methane sulfonate (MMS), and is particularly active at repairing 3-methyl-Cytosine (3meC) residues on single stranded DNA (ssDNA). Previous reports have shown that epigenetic loss of ALKBH3 has prognostic implication in lung cancer, Hodgkin lymphoma, Hepatocellular carcinoma (HCC) and breast cancer. However, no predictive value is currently known for ALKBH3 downregulation with respect to alkylating agents treatment. In order to evaluate the pharmacogenomics of ALKBH3-deficient CRC we firstly validated the absence of ALKBH3 protein by WB analysis in our CRC cell panel, then we confirmed ALKBH3 CpG promoter methylation, as previously described in literature for breast cancer models. Subsequently, we evaluated the sensitivity to several alkylating agents, including temozolomide, cisplatin, MMS, dacarbazine, lomustine, and carmustine in our panel of ALKBH3-null cell lines in comparison with a panel of ALKBH3-proficient cell lines. One MGMT-methylated CRC cell line was used as positive control for high sensitivity to temozolomide. We performed both short term (96 hours) and long term (up to 14 days) cell viability assay, using as readout for cell viability CellTitre Glow (CTG) and crystal violet staining, respectively. No statistically significant differences in sensitivity to alkylating agents was found in ALKBH3-null cell lines compared to ALKBH3-proficient cell lines, irrespectively from other genetic biomarkers such as microsatellite status or RAS/BRAF mutations. Taken together, these data demonstrate that ALKBH3 expression is not a candidate predictive biomarker for sensitivity to alkylating agents in CRC, most likely because of a high degree of redundancy of the systems involved in direct repair of alkylated DNA. Citation Format: Pietro Paolo Vitiello, Elisa Mariella, Pietro Andrei, Vito Amodio, Giovanni Germano, Annalisa Lorenzato, Carlotta Cancelliere, Alberto Bardelli. ALKBH3 deficiency and liability to alkylating agents in CRC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4034.