DNA Demethylating Agents Generate a Brcaness Effect in Multiple Sporadic Tumor Types: Prediction for Sensitivity to PARP Inhibitors in AML
Poly (ADP ribose) polymerase inhibitors (PARPis) have shown clinical efficacy in breast and ovarian cancers with inherited mutations in BRCA genes that abrogate homologous recombination (HR) DNA double strand break (DSB) repair activity. However, to date, PARPishave not had success in sporadic breast and ovarian cancers, or other cancers, such as acute myeloid leukemia (AML) with intact BRCA genes. We have recently reported that triple negative breast cancer (TNBC) and AML are sensitive to low doses of the potent PARPiTalazoparib in combination with DNA methyl transferase inhibitors (DNMTi) decitabine (DAC) or azacitidine (AZA) in vitro and in vivo. In these cancer models, the DNMTi/PARPicombination increases amplitude and retention of PARP1 directly at laser induced DNA damage sites, increasing DNA DSBs, promoting synergistic tumor cytotoxicity, blunting cell self renewal, and leading to strong anti tumor responses. These results have led to a Phase I/II Clinical trial of DNMTi/PARPi combination therapy in patients with relapsed/refractory AML that is underway. In identifying the molecular pathways that may underlie efficacy of this inhibitor combination in sporadic TNBC, ovarian cancer and AML, we find that low doses of DNMTisreprogram the cancer genome, in part by changing gene expression patterns of DSB repair genes, potentially creating a HR defect (HRD) or "BRCAness" effect. Expression array (Agilent Human 44K v2) analysis was performed on RNA extracted from multiple BRCA intact sporadic cell lines from AML (including, Kasumi 1, KG 1 and MOLM 14) TNBC (including, MDA MB 231, SUM159) and ovarian cancer (including, A2780) on days 1, 3, 5 and 7 and 10 post DNMTi(DAC 10nM or AZA 500nM) treatment. Hallmark, KEGG and reactome gene ontologies were utilized. Only gene sets with log fold change (FDR) .250 were considered significant and genes demonstrating 2 fold or greater change with drug treatment were selected. While multiple DNA repair pathway genes were found both up and down regulated post DNMTi treatment, one or more HR genes, were consistently downregulated in all cell lines examined. QPCR of mRNA and/or immunoblotting of HR proteins, in particular RAD51, BRCA1, BRCA2, or HR related genes, FEN1 or FANCD2, showed significantly decreased expression with DNMTi treatment (p The above HRD like or BRCAness phenotype with DNMTi treatment translated to sensitivity to PARPis in primary bone marrow mononuclear cells (BMMNC) from AML patients. Thus, in 6 of 8 (75% AML samples, HR gene expression by QPCR of RNA and HR functional activity by immunostaining for RAD51 foci, following DAC treatment (10nM, 72 hrs) DNMTi treatment resulted in a significant decrease in HR or HR related gene expression, including RAD51, BRCA1, BRCA2, and decreased levels of RAD51 foci (p Disclosures Civin: ConverGene LLC: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees, Research Funding; 3DBioWork Inc: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; BD (Becton Dickinson) Honoraria; GPB Scientific LLC: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees. Kingsbury: 3DBioWorks: Consultancy, Equity Ownership.