Supp. Table 1 is an IC50 calculation of each individual HDACi (SAHA, VPA, ROMI) and PARPi (ABT-888) against the four TNBC cell lines MDA-MB-231, MDA-MB-436, HCC1937(MUT) and HCC1937(RECON). Supp. Table 2 displays the combination index of the HDACi at IC20 with 2.5uM PARPi for each of the 4 cell lines described in table 1. <1 synergy, >1 antagonism. Supp Fig 1. Is a DRGFP analysis of homologous recombination activity in to repair a GFP construct in the presence of the HDACi. We confirmed with immunofluorescence of RAD51 and marker of double strand breaks gammaH2Ax. Supp Fig 2. A) Is fluorescence microscopy displaying polyploidy in response to HDACi ROMI. B and C) Expression analysis of a set of DNA repair genes in response to HDACi (SAHA, VPA and ROMI). D) Protein expression analysis correlating with gene expression response. E) Validation of RAD51, BARD1 and FANCD2 depletion by siRNA. F-H) Cell cycle profile of MDA-MB-231 response to genetic depletion in the presence of HDACi (VPA)and PARPi (ABT-888). Supp. Fig 3. Optical and fluorescence microscopy of cells treated with combination ROMI and ABT-888.
ARTICLE ABSTRACT
The triple-negative breast cancer (TNBC) subtype represents a cancer that is highly aggressive with poor patient outcome. Current preclinical success has been gained through synthetic lethality, targeting genome instability with PARP inhibition in breast cancer cells that harbor silencing of the homologous recombination (HR) pathway. Histone deacetylase inhibitors (HDACi) are a class of drugs that mediate epigenetic changes in expression of HR pathway genes. Here, we compare the activity of the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA), the class I/IIa HDAC inhibitor valproic acid (VPA), and the HDAC1/2–specific inhibitor romidepsin (ROMI) for their capability to regulate DNA damage repair gene expression and in sensitizing TNBC to PARPi. We found that two of the HDACis tested, SAHA and ROMI, but not VPA, indeed inhibit HR repair and that RAD51, BARD1, and FANCD2 represent key proteins whose inhibition is required for HDACi-mediated therapy with PARP inhibition in TNBC. We also observed that restoration of BRCA1 function stabilizes the genome compared with mutant BRCA1 that results in enhanced polyploid population after combination treatment with HDACi and PARPi. Furthermore, we found that overexpression of the key HR protein RAD51 represents a mechanism for this resistance, promoting aberrant repair and the enhanced polyploidy observed. These findings highlight the key components of HR in guiding synthetic lethality with PARP inhibition and support the rationale for utilizing the novel combination of HDACi and PARPi against TNBC in the clinical setting. Mol Cancer Ther; 14(10); 2321–31. ©2015 AACR.
