Supplementary Figures S1-S11 from The Novel, Small-Molecule DNA Methylation Inhibitor SGI-110 as an Ovarian Cancer Chemosensitizer
Authors: Fang Fang , Munck Joanne , Tang Jessica , Taverna Pietro , Wang Yinu , Miller David F.B. , Pilrose Jay , Choy Gavin , Azab Mohammad , Pawelczak Katherine S. , VanderVere-Carozza Pamela , Wagner Michael , Lyons John , Matei Daniela , Turchi John J. , Nephew Kenneth P. 
Published: April 2023

<p>Supplementary Figure S1. Diagrams of biweekly and QD5 drug (SGI-110 and CDDP) treatment schedules for mice. (A). For non-tumor bearing mice study, mice (3 per group) were injected with different doses of SGI-110 (2 mg/kg, 5 mg/kg, s.c.), CDDP (2 mg/kg, 4 mg/kg, i.p.) or in combination of the 2 drugs. The treatment was performed in 2 schedules as shown. (B). For A2780 tumor bearing mice study, mice (5 per group) were injected with OC cells subcutaneously on the right flanks, and followed by different doses of SGI-110 (2 mg/kg, s.c.), CDDP (2 mg/kg, 4 mg/kg, i.p.) or in combination of the 2 drugs after a palpable tumor was formed on each mouse. The treatment was performed in 2 schedules as shown. Blood samples were collected on the indicated days. Supplementary Figure S2. 5-aza-dC treatment modulates CDDP sensitization and alters DNA methylation and gene expression in vitro. Comparison of cell growth rates of parental A2780 cells (A), A2780-CDDP resistant cells (B), and CP70 CDDP resistant cells (C) treated with 5Î¼M 5-aza-dC, or vehicle (DMSO 1:2000) for 48 hours followed by CDDP ranging from 0-50Î¼M CDDP. Mean values {plus minus} S.E.M. of 8 independent experiments in duplicate are reported. All treatments were significantly different, at P < 0.05, than vehicle control cells. IC50 values are listed in Supplementary Table S2. Data shown represent means {plus minus} S.E.M. from 8 experiments. Supplementary Figure S3. SGI-110 treatment alters DNA methylation in vitro. Parental A2780 cells, A2780-CDDP resistant cells, and CP70-CDDP resistant cells were treated with 5 Î¼M SGI-110 for 48 hours, and DNAs were extracted for pyrosequencing analysis for specific genes (AKT1S1: subunit of mTORC1; HOXA10: embryonic development; IFNAR1 and IL2RG: receptor subunit in Jak/STAT pathway; AKT1: serine/threonine kinase in apoptosis; BRCA1: DNA repair; STAT5B: transcription factor; LRP6; AXIN1: cytoplasmic G-protein signaling regulator; CTNNB1: Î²-catenin; and CSKN1D: casein kinase). Supplementary Figure S4. 5-aza-dC and SGI-110 treatment modulated CDDP sensitization. Cells were treated with 0.1 Î¼M SGI-110, 0.3 Î¼M SGI-110 or DMSO (control) for 3-consecutive days, prior to treatment with a range of CDDP and cell viability determined on day 8 using the alamar blue assay. IC50s are shown in the boxes under each graph. Data shown represent means {plus minus} S.E.M. from three independent experiments. Supplementary Figure S5. Effects of SGI-110 on MLH1 expression levels in OC cell lines. Cells were treated for 3-consecutive days with 0.1 Î¼M SGI-110, 0.3 Î¼M SGI-110, 1 Î¼M SGI-110 or DMSO (control) and expression levels of MLH1 determined. (A). Western blot analysis of MLH1 expression in A2780 cells. (B). Western blot analysis of MLH1 expression in OAW28 and OVCAR8 cells. Supplementary Figure S6. SGI-110 induces the expression of potential CDDP-resistance biomarkers in OAW28. OAW28 cells were treated for 3-consecutive days with 0.1 Î¼M SGI-110, 0.3 Î¼M SGI-110, 1 Î¼M SGI-110 or DMSO (control) and mRNA levels of 19 genes determined by RT-PCRFold change in mRNA levels were calculated as 2(-Î”Î”CT) relative to DMSO-treated cells. Of the 19 genes analyzed (Supplementary Table S4), only genes for which there was a significant difference (P< 0.05) in expression levels between SGI-110 and DMSO-treated cells are included in the figure. Data shown represents means {plus minus} S.E.M. from triplicate experiments. Supplementary Figure S7. SGI-110 is well tolerated in non-tumor bearing mice. Mice were treated as Supplemental Figure S1A and were measured for BW twice a week. (A). BW curves for QD5 schedule: 8-day treatment followed by a 4-week recovery. 5 mice were sacrificed on Day 8 to Day 10 (2 mice in SGI-110 5 mg/kg; 2 mice in SGI-110 5 mg/kg + cisplatin 2 mg/kg; 1 mouse in SGI-110 5 mg/kg + cisplatin 4 mg/kg). (B). BW curves for biweekly schedule: 4 treatment cycles (7 days a cycle) followed by a 3-week recovery. Data shown represent means {plus minus} S.E.M. from 5 mice. Supplementary Figure S8. Decreased tumor growth from parental-derived A2780 xenografts treated with SGI-110 and CDDP in the QD5 and biweekly regimens. CDDP-resistant A2780 xenograft tumor volume was compared among single agent and combination treatment to vehicle control in two treatment schedules. (*: P<0.05, **: P<0.01, ***: P<0.001). (A). QD5 treatment. (D). biweekly treatment. Data shown represent means {plus minus} S.E.M. from 5 tumors. Supplementary Figure S9. Decrease of tumor growth of parental and resistant-derived A2780 xenografts treated by SGI-110 and CDDP in the QD5 and biweekly regimens. Area under the curve (AUC) of A2780 xenograft tumor volume was compared among single agent and combination treatment to vehicle control in two treatment schedules. (*: P<0.05, **: P<0.01). (A). Parental A2780 xenograft tumor growth, QD5 treatment. (B). Parental A2780 xenograft tumor growh, biweekly treatment. (C). A2780-CDDP resistant xenograft tumor growth, QD5 treatment. (D). A2780-CDDP resistant xenograft tumor growth, biweekly treatment. Data shown represent means {plus minus} S.E.M. from 5 tumors. Supplementary Figure S10. SGI-110 is well tolerated in tumor bearing mice Mice were injected with CDDP-resistant A2780 OC cells, treated as Supplemental Figure S1B, and were measured for BW twice a week (*: P<0.05). (A). BW curves for QD5 schedule: 8-day treatment followed by a 2-week recovery. (B). BW curves for biweekly schedule: 2 treatment cycles (7 days a cycle) followed by a 2-week recovery. Data shown represent means {plus minus} S.E.M. from 5 mice. Supplementary Figure S11. SGI-110 increases platinum DNA adducts in vitro. (A). Numbers of platinum adducts were increased by SGI-110 pretreatment in both A2780 parental (A) and CDDP resistant (B) cells. A t-test was performed between SGI-110 (+) and SGI-110 (-) at each time point (*: P<0.05). Data shown represent means {plus minus} S.E.M. from 3 experiments.</p>