Supplemental Figures S1-S7 from An Effective Epigenetic-PARP Inhibitor Combination Therapy for Breast and Ovarian Cancers Independent of BRCA Mutations
Authors: Pulliam Nicholas , Fang Fang , Ozes Ali R. , Tang Jessica , Adewuyi Adeoluwa , Keer Harold , Lyons John , Baylin Stephen B. , Matei Daniela , Nakshatri Harikrishna , Rassool Feyruz V. , Miller Kathy D. , Nephew Kenneth P. 
Published: April 2023

<p>Supplemental Figure S1: PEO1 cells ectopically expressing vector control or BRCA2 were subjected to (A) RAD51 foci formation assay or (B) treated with increasing concentrations of Cisplatin (CDDP) and subjected to MTT assay for IC50 determination. DNMT1 expression was measured in PEO1 and PEO4 cells treated with or without 1nM Talaz by (C) qRT-PCR or (D) Western blot analysis (bottom: Images were quantified using ImageJ software and quantification is representative of three independent experiments {plus minus} SEM; two-tailed student's t-test). (E) PEO1 (Top) and PEO4 (Bottom) cells were treated with 20nM guadecitabine (Guad) and 1nM talazoparib (Talaz), alone or in combination for 72hrs. 24hrs post treatment caspase 3/7 activity assay was performed. Results are representative of at least three independent experiments. (F) Ovarian cancer cells were treated with the indicated dose of 5- azacytidine (AZA) and talazoparib (Talaz), alone or in combination for 72hrs. Supplemental Figure S2: A panel of OC cell lines (see table for description: Supplemental Table 1) was treated with (A-G) 20nM guadecitabine (Guad), (H-I) 5-azacytidine (AZA), or 1nM talazoparib (Talaz), alone and in combination. Following treatment cells were subjected to clonogenic survival assays and drug synergism analysis; x-axis is indicative of Fraction affected (FA); y-axis is indicative of combination index (CI). Combinations beneath dashed black line are synergistic. Combination treatment schemas are as followed: Priming ('Prime') daily treatment with 20nM Guad for 3 days, 24hrs recovery, followed by one treatment with 1nM Talaz or Co-administration ('Co-ad') treatment with both 20nM guadecitabine (Guad) and 1nM talazoparib (Talaz) on day 1, and 20nM Guad on days 2 and 3. Results are representative of at least three independent experiments {plus minus} SEM. * p<0.01, ** p<0.001, *** p<0.0001 compared to control, # p<0.01, ## p<0.001 relative to bracketed treatment. F. G. C. D. A. B. E. Supplemental S3 Supplemental Figure S3: (A-G) A panel of breast cancer cell lines (see table: Supplemental Table 1) was treated with either 20nM guadecitabine (Guad) or 1nM talazoparib (Talaz), alone and in combination. Following treatment cells were subjected to clonogenic survival assays and drug synergism analysis; x-axis is indicative of Fraction affected (FA); y-axis is indicative of combination index (CI). Combinations beneath dashed black line are synergistic. Combination treatment schemas are as followed: Priming ('Prime') daily treatment with 20nM Guad for 3 days, 24hrs recovery, followed by one treatment with 1nM Talaz or Co-administration ('Co-ad') treatment with both 20nM Guad and 1nM Talaz on day 1, and 20nM Guad on days 2 and 3. Results are representative of at least three independent experiments {plus minus} SEM. * p<0.01, ** p<0.001, *** p<0.0001 compared to control, # p<0.01, ## p<0.001 relative to bracketed treatment. Supplemental Figure S3: (A-G) A panel of breast cancer cell lines (see table: Supplemental Table 1) was treated with either 20nM guadecitabine (Guad) or 1nM talazoparib (Talaz), alone and in combination. Following treatment cells were subjected to clonogenic survival assays and drug synergism analysis; x-axis is indicative of Fraction affected (FA); y-axis is indicative of combination index (CI). Combinations beneath dashed black line are synergistic. Combination treatment schemas are as followed: Priming ('Prime') daily treatment with 20nM Guad for 3 days, 24hrs recovery, followed by one treatment with 1nM Talaz or Co-administration ('Co-ad') treatment with both 20nM Guad and 1nM Talaz on day 1, and 20nM Guad on days 2 and 3. Results are representative of at least three independent experiments {plus minus} SEM. * p<0.01, ** p<0.001, *** p<0.0001 compared to control, # p<0.01, ## p<0.001 relative to bracketed treatment. Supplemental Figure S4: (A) Western blot analysis of PEO4 cells following BRCA2 knock down, compared to vector control knock down. (B) PEO4 cells were treated with 20nM guadecitabine (Guad) and 1nM talazoparib (Talaz), alone and in combination, following knock down of BRCA2 or control. Treated cells were subjected to clonogenic survival assay. Results are representative of three independent experiments. (C) Western blot analysis of PEO4 cells against indicated antibodies ectopically expressing BRCA2, compared to vector control. Low exposure is 10 seconds and high exposure is 30 seconds. Western blot is representative of three individual experiments. (D) Vector control and BRCA2 overexpressing PEO4 cells were treated with 20nM guadecitabine (Guad) and increasing concentrations of talazoparib (Talaz), as indicated by the x-axis, and subjected to cell viability assays. Results are representative of three independent experiments, performed in triplicate {plus minus} SEM. * p<0.01, ** p<0.001, *** p<0.0001 compared to control, # p<0.01, ## p<0.001 relative to bracketed treatment. Supplemental Figure 5: (A) Basal PAR concentration was measured in PEO1 and PEO4 cells by PAR-capture ELISA. Results are representative of three independent experiments {plus minus} SEM (B) Luciferase assay measuring basal ATP levels between PEO1 and PEO4 cells. Results are normalized to PEO1 cell line luciferase activity. Results are representative of two individual (biological) experiments. (C) Basal expression of ROS-associated genes in PEO1 and PEO4 cells was measured by qRT-PCR. Results are normalized to PEO1 cell line expression. (D) PEO1 cells were treated with 20nM guadecitabine (Guad) and 1nM talazoparib (Talaz), alone or in combination, and expression of ROS-associated genes was measured by qRT-PCR. Results are normalized to untreated control. Results are representative of three independent experiments. (E) PEO4 cells were treated with 20nM guadecitabine (Guad) and 1nM talazoparib (Talaz), alone or in combination, and expression of ROS-associated genes was measured by qRTPCR. Results are normalized to untreated control and are representative of three independent experiments. PEO1 and PEO4 cells were treated with 5μM RG108 (non-nucleoside analog DNMTi), 24hr post treatment cells were subjected to (F) western blot analysis against the indicated antibodies and (G) ROS levels measured. Quantification is representative of three individual experiments (Mean {plus minus} SEM; two-tailed student's t-test). * p<0.01, ** p<0.001, *** p<0.0001 compared to control, # p<0.01, ## p<0.001 relative to bracketed treatment. Supplemental Figure 6: (A) PEO4 cells were treated with 1mM H2O2 in the presence and absence of 5μM PKA inhibitor H89, (left) western blot analysis against the indicated antibodies and (right) PAR capture ELISA was performed. (B) Basal cAMP concentrations were measured between PEO1 and PEO4 cells. Results are representative of three independent experiments. (C) OC and breast cancer cell lines were treated with 1nM talazoparib (Talaz) for 24hrs and cAMP levels measured. Quantification is representative of three separate experiments. (D) PEO1 and PEO4 cells were treated with 10μM FSK (adenylate cyclase activator) in the presence and absence of 5μM PKA inhibitor H89 or 1nM talazoparib (Talaz). Western blot analysis of cell lysates against indicated antibodies was performed. Western blot is representative of three individual blots. (E) PEO4 cells were treated with 20nm Guad and the indicated concentrations of Talaz, with or without 5μM PKA inhibitor H89 pre-treatment for 24hrs. Treated cells were subjected to colony formation assays (F) PEO1 cells were treated with 1nM talazoparib (Talaz) in the presence and absence of 5μM H89 (PKA inhibitor), and cell viability assay was performed. Results are representative of three individual experiments, performed in duplicate (Mean {plus minus} SEM). * p<0.01, ** p<0.001, *** p<0.0001 compared to control, # p<0.01, ## p<0.001 relative to bracketed treatment. Supplemental Figure 7: (A) A2780 cells were treated with 500nM AZA, 1nM Talaz or the combination, and subjected to (Top) western blot analysis, following chromatin isolation. (Bottom) Quantification of PARP1 chromatin localization is representative of three independent experiments, and performed using ImageJ software. (B) PEO1 and PEO4 cells were treated with 20nM guadecitabine (Guad) or 1μM veliparib (Velip), alone and in combination for 72hrs, and clonogenic survival assay performed. Quantification is representative three individual experiments (Mean {plus minus} SEM). (C) PEO1 and PEO4 cells were treated with 1μM veliparib (Velip) and cAMP levels measured. Quantification is representative of three separate experiments (Mean {plus minus} SEM; two-tailed student's t-test). (D) Proposed model: (Left; "Priming") DNMTi increases intracellular ROS accumulation. Increased ROS promotes rapid PARP activation by stimulation of the cAMP/PKA signal transduction pathway, priming the cell towards PARPi sensitivity. (Right, "Trapping") DNMTi-PARPi co-administration induces DNMT1 and PARP1 localization to DNA, increasing DNA damage. Both contribute to PARPi response, regardless of BRCA status and function. * p<0.05, ** p<0.01, *** p<0.001 compared to control, # p<0.01, ## p<0.001 relative to bracketed treatment.</p>