Fig 1: BCL2L1 is frequently gained in OvCa. Reproduced from TCGA OvCa dataset, shown were (A) copy number alteration of Cytoband 20q11.21 that encompassed BCL2L1; (B) frequency of gain (pink) and amplification (red) of BCL2L1 across all cancers in TCGA dataset; (C) differential expression of BCL2L1 across TCGA cancers; (D) Reproduced from CPTAC dataset, shown were protein level of BCL2L1 in different clinicopathological parameters; (E) representative IHC images of BCL2L1 in normal ovary tissue and ovarian cancer tissue; (F) overall and progression-free survival of OvCa cases with or without BCL2L1 gain/amplification in TCGA cohort; (G) overall survival of OvCa cases with top 30% or bottom 30% of BCL2L1 expression in TCGA cohort (*P < .05; **P < .01; ***P < .001; ****P < .0001).
Fig 2: BCL2L1 gain is associated with multi-resistance. Reproduced from TCGA OvCa dataset, shown were (A) BCL2L1 expression in different copy number alteration in OvCa; Reproduced from GDSC dataset, shown were (B) volcano plot revealing drug sensitivity of cancer cells with 20q11.21 gain; (C) cancer cells with BCL2L1 gain showing resistance to 2 BCL2-targeted compounds; (D) western blotting showing BCL-xL level in 3 OvCa cell lines with different BCL2L1 status treated or untreated with BCL2L1 knockdown (KD) by shRNA; (E) cell viability detected by MTT assay in 3 OvCa cell lines over 10-day course with or without BCL2L1-KD plus cisplatin (Pt, 1 mg/mL in 0.9% NaCl); Gene enrichment analysis using (F) NET-GE platform and (G) GSEA method showing genes enriched in BCL2L1-gained OvCa cases in TCGA cohort; (H) shown was PCR validation of sgRNAs using 3 pairs of primers with Cruser method in SK-OV-3 cells prepared for CRISPR/Cas9 knockout (KO) of BCL2L1; (I) western blotting showing BCL-xL level in SK-OV-3 cells with or without BCL2L1-KO; (J) cell viability of SK-OV-3 cells with or without BCL2L1-KO plus cisplatin over 5-day course; Western blotting showing (K) levels of FGFR/NCAM family members in SK-OV-3 cells with or without BCL2L1-KO and (L) BCL2L1 (BCL-xL) level in SK-OV-3 cells with both KD of BCL2L1 and one of FGFR/NCAM family members (N = 5 in all assays; *P < .05; **P < .01; ***P < .001; ****P < .0001).
Fig 3: Dual inhibition of FGFR/BCL-xL is feasible in vivo. (A) weight monitoring of SK-OV-3 cell-implanted xenograft mice treated with 25 mg/kg/d of A-1331852, 100-mg/kg of BLU9931, Cisplatin equivalent of 75 mg/m2 of dose in humans; (B) tumor growth of SK-OV-3 cell-implanted xenograft mice treated with A-1331852 (A) and BLU9931 (B) or vehicle control with representative tumor image at endpoint, discontinued curve in control group indicating individuals reaching endpoint of tumor size; (C) Kaplan-Meier survival curve of control and treatment group compared by Log-rank test; (D) representative immunohistochemical staining of BCL-xL in extracted tumors; (E) schematic cartoon showing association between FGFR4/NCAM and BCL2L1 with drug sensitivity profile in the setting of current study. (N = 10 in all assays; Scale bar = 1cm; *P < .05; **P < .01; ***P < .001; ****P < .0001).
Fig 4: Combined inhibition is needed for BCL2L1-gained OvCa. Shown were sigmoidal curve fitting for IC50s of (A) A-1331852 and BLU-9931 in SK-OV-3 cells with or without BCL2L1-KO and (B) A-1331852 in SK-OV-3 cells treated with set dose of BLU-9931 at 5 µM; (C) Cell viability of SK-OV-3 cells treated with A-1331852 (A) at 3 µM and/or BLU-9931 (B) at 5 µM and/or cisplatin (Pt) at 1 mg/mL in the 5-day course detected by MTT; (D) colony formation assay; (E) EDU-stained proliferation assay; (F) cell cycle profiling and (G) apoptosis analysis examined by flow cytometry; Transwell-based (H) invasion and (I) migration; and (J) wound-healing assay in SK-OV-3 cells treated with A-1331852, BLU-9931 and cisplatin all at 72 h of treatment with doses shown in panel (C) (N = 5 in all assays; *P < .05; **P < .01; ***P < .001; ****P < .0001).
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