CDK4/6 inhibitors target SMARCA4-determined cyclin D1 deficiency in hypercalcemic small cell carcinoma of the ovary

Abstract

Inactivating mutations in SMARCA4 (BRG1), a key SWI/SNF chromatin remodelling gene, underlie small cell carcinoma of the ovary, hypercalcemic type (SCCOHT). To reveal its druggable vulnerabilities, we perform kinase-focused RNAi screens and uncover that SMARCA4-deficient SCCOHT cells are highly sensitive to the inhibition of cyclin-dependent kinase 4/6 (CDK4/6). SMARCA4 loss causes profound downregulation of cyclin D1, which limits CDK4/6 kinase activity in SCCOHT cells and leads to in vitro and in vivo susceptibility to CDK4/6 inhibitors. SCCOHT patient tumors are deficient in cyclin D1 yet retain the retinoblastoma-proficient/p16^INK4a-deficient profile associated with positive responses to CDK4/6 inhibitors. Thus, our findings indicate that CDK4/6 inhibitors, approved for a breast cancer subtype addicted to CDK4/6 activation, could be repurposed to treat SCCOHT. Moreover, our study suggests a novel paradigm whereby critically low oncogene levels, caused by loss of a driver tumor suppressor, may also be exploited therapeutically.

Fig. 1

SMARCA4-deficient SCCOHT cells are vulnerable to inhibition of CDK4/6 kinase activities. a Schematic outline of the shRNA screens for kinases whose inhibition is selectively lethal to SMARCA4-deficient SCCOHT cells (BIN-67) but not to SMARCA4-proficient control cells (IOSE80, OVCAR4). Cells were infected with the lentiviral shRNA library (T0) and cultured for selection for 14 days (T1). The relative abundance of shRNAs in the cell populations was determined by next-generation sequencing. b Analysis of the shRNA screens using the MAGeCK statistical software package. CDK6 (magenta) and CDK4 (blue) are the first two ranked genes that were negatively selected in BIN-67 cells. All genes were ranked based on their RRA (robust rank aggregation, top) or raw p values (bottom) generated from the MAGeCK analysis. c, d Validation of CDK6 and CDK4 in SCCOHT cells (BIN-67, SCCOHT-1, COV434) and SMARCA4-proficient controls (IOSE80, OVCAR4). c Colony-formation assay of the indicated cell lines expressing pLKO control or shRNAs targeting CDK6 or CDK4 after 10–15 days of culturing. For each cell line, all dishes were fixed at the same time, stained, and photographed. d Western blot analysis of CDK6 and CDK4 and phosphorylated RB at serine 795 (pRB-S795) in the cells described in c. HSP90 was used as a loading control. e–j SCCOHT cells are more vulnerable to inhibition of CDK4/6 kinase activities, compared to SMARCA4-proficient control cells. e BIN-67 cells stably expressing pLX304-GFP, pLX304-CDK6, or pLX304-CDK6^D163N were infected with viruses containing pLKO control or a shRNA targeting the 3’UTR of CDK6, selected for integration, and cultured for 14 days. All dishes were fixed at the same time. f Western blot analysis for CDK6, pRB-S795, and HSP90 in the cells described above. g, i BIN-67 (g) and OVCAR-4 (i) cells expressing pLX317-GFP, pLX317-CDK4, or pLX317-CDK4^D158N were infected with viruses containing pLKO control or a shRNA vector targeting the 3’UTR of CDK4, selected for integration, and cultured for 14 days. For each cell line, all dishes were fixed at the same time. h, j Western blot analysis for CDK4, pRB-S795, and HSP90 in the cells described above

Fig. 2

SCCOHT cells are highly sensitive to CDK4/6 inhibitors. a–c SMARCA4-deficient SCCOHT cells are highly sensitive to palbociclib treatment, similar to ER⁺ breast cancer cells. a Colony-formation assays in SCCOHT (BIN-67, SCCOHT-1, and COV434), SMARCA4-proficient ovarian (IOSE80, OVCAR4, and OVCAR8), and ER⁺ breast cancer (MCF7 and CAMA-1) cell lines. Cells were cultured in the absence or presence of palbociclib at the indicated concentrations for 10–21 days. For each cell line, all dishes were fixed at the same time. b Cell viability assay of the same cell line panel. Cells were treated with increasing concentrations of palbociclib for 5–10 days, and cell viability using CellTiter-Blue was determined by measuring the fluorescence (560/590 nm) in a microplate reader. Error bars: mean ± standard deviation (s.d.) of biological replicates (n = 4). c Palbociclib treatment suppresses RB phosphorylation in SCCOHT cells similar to ER⁺ breast cancer cells but not in IOSE80 and OVCAR4 cells. Levels of pRB-S795 in cells treated with 0, 100, and 300 nM of palbociclib for 24 h were documented by western blot analysis. d, e Transcriptome profiling in SCCOHT cells show that top ranked pathways affected upon palbociclib treatment are related to cell cycle regulation. RNA-Seq was performed in BIN-67 and SCCOHT-1 cells treated with 100 nM of palbociclib for 24 h. Common genes that significantly changed (p < 0.05) in both cell lines was analyzed using Gene Set Enrichment Analysis (GSEA). Top ten cellular processes by Gene Ontology (GO) term (d) and GSEA plots for the top three cellular processes (e) are shown. NES normalized enrichment score, FDR false discovery rate. f, g Transcriptome profiling in SCCOHT cells show that top ranked pathways affected upon CDK6 knockdown are related to cell cycle regulation. RNA-Seq was performed in BIN-67 and SCCOHT-1 cells expressing pLKO control or two independent shRNAs targeting CDK6. Common genes that significantly changed (p < 0.05) in both shRNAs and both cell lines were analyzed using GSEA. Top ten cellular processes by GO term (f) and GSEA plots for the top three cellular processes (g) are shown. NES normalized enrichment score, FDR false discovery rate

Fig. 3

Cyclin D1 deficiency in SCCOHT cells results in vulnerability to CDK4/6 inhibition. a SMARCA4 loss in SCCOHT is associated with cyclin D1 deficiency and reduced CDK4 expression. Western blot analysis of key cell cycle regulators in a cell line panel: non-transformed ovarian epithelial (IOSE80), ovarian carcinoma (OVCAR4, OVCAR8), SCCOHT (BIN-67, SCCOHT-1, COV434), and ER⁺ breast cancer (CAMA-1, MCF7). l.e. long exposure. b SCCOHT cells express low levels of CCND1 mRNA. Relative CCND1 expression (normalized to GAPDH) in the cell panel described above were measured by qRT-PCR. Error bars: mean ± s.d. of biological replicates (n = 3). c SCCOHT cells have lower total CDK4 kinase activities compared to SMARCA4-proficient control cells. Normalized amount of immunoprecipitated CDK4 kinase complexes from IOSE80 and SCCOHT cell lines were subjected to in vitro kinase assays using recombinant RB. Upper, western blot analysis of immunoprecipitated CDK4 input; lower, kinase assay radiography. d–i Ectopic cyclin D1 expression increases RB phosphorylation and confers resistance to palbociclib in SCCOHT cells. d, e Western blot analysis of immunoprecipitations using an antibody against CDK4 or IgG in SCCOHT-1 (d) and BIN-67 (e) cells stably expressing GFP or CCND1. f, g Western blot analysis of SCCOHT-1 (f) and BIN-67 (g) cells with stable ectopic expression of GFP, CDK4, or CCND1. h, i Colony-formation assay of SCCOHT-1 (h) and BIN-67 (i) cells (described in f, g) treated with palbociclib. j, k Spontaneously palbociclib-resistant clones of SCCOHT-1 expressed elevated cyclin D1 and RB phosphorylation. j Cell viability assay of SCCOHT-1 parental cells and resistant clones (R1 and R2) treated with palbociclib for 9 days. Error bars: mean ± standard deviation (s.d.) of biological replicates (n = 4). k Western blot analysis for the indicated proteins in the cells described above. l, m Cyclin D1 knockdown in palbociclib-resistant SCCOHT-1 cells resensitizes them to palbociclib. l Cell viability assay of SCCOHT-1 R1 cells expressing pLKO control or two independent shRNAs targeting cyclin D1 treated with palbociclib for 9 days. Error bars: mean ± standard deviation (s.d.) of biological replicates (n = 4). m Western blot analysis for the indicated proteins in the cells described above

Fig. 4

Cyclin D1 deficiency in SCCOHT cells is caused by SMARCA4 loss. a–c RNA-Seq analysis in BIN-67 and SCCOHT-1 cells stably expressing pReceiver control or pReceiver-SMARCA4 identified CCND1 as the top ranked cell cycle regulator upregulated upon SMARCA4 restoration (n = 3). a, b Venn diagrams showing the genes upregulated (a) or downregulated (b) upon SMARCA4 restoration (fold change >3, adjusted p < 0.05). c Heatmap of the top 50 genes upregulated upon SMARCA4 restoration in both BIN-67 and SCCOHT-1 cells. Red arrow points to CCND1. d, e SMARCA4 restoration in SCCOHT cells upregulated CCND1 mRNA (d) and cyclin D1 protein (e) levels. d Relative expression levels of CCND1 mRNA (normalized to GAPDH) in SCCOHT cells were measured by qRT-PCR. Error bars: mean ± s.d. of biological replicates (n = 3; two-tailed t test, **p < 0.01). e Western blot analysis for the indicated proteins in the cells described above. f, g SMARCA4 knockdown in SMARCA4-proficient cells suppressed CCND1 mRNA (f) and cyclin D1 protein (g) levels. f Relative expression levels of CCND1 mRNA (normalized to GAPDH) in IOSE80 and OVCAR4 cells were measured by qRT-PCR. Error bars: mean ± s.d. of biological replicates (n = 3; two-tailed t test, ****p < 0.0001, **p < 0.01). g Western blot analysis for the indicated proteins in the cells described above. h, i SMARCA4 occupancy in the CCND1 promoter region. Chromatin immunoprecipitation experiments were performed in SCCOHT cells (SCCOHT-1, BIN-67) expressing pReceiver or pReceiver-SMARCA4 (h) and in SMARCA4-proficient cells (IOSE80, OVCAR4) expressing pLKO or shRNA targeting SMARCA4 (i), using an antibody against SMARCA4 or IgG. qPCR was used to analyze SMARCA4 occupancy using the primer sets for CCND1, CCND3, and CCNE1 locus as indicated. p promoter, TSS transcription start site, error bars: mean ± s.d. of measurement replicates of a representative experiment (n = 3; two-tailed t test, *p < 0.05, **p < 0.01)

Fig. 5

Palbociclib is effective in suppressing SCCOHT tumor growth in vivo. Palbociclib significantly suppresses tumor growth in xenograft models of BIN-67 (a, b, e, f) and SCCOHT-1 (c, d, g, h), as well as a patient-derived xenograft (PDX) model of SCCOHT (i). After tumor establishment (3–4 weeks for BIN-67 and SCCOHT-1; 3 months for the PDX), mice were treated daily with vehicle (ctrl) or palbociclib (palbo) for the indicated days. a, c Tumor volume fold change from Day 1 of treatment in BIN-67 (a, n = 5 per group) and SCCOHT-1 (d, n = 4 for vehicle, n = 5 for palbociclib; 150 mg kg⁻¹) models (two-way ANOVA, ****p < 0.0001). b, d The final tumor volume and weight measured at necropsy in BIN-67 (b, n = 8 per group) and SCCOHT-1 (d, n = 4 for vehicle, n = 5 for palbociclib) models (two-tailed t test, ****p < 0.0001, **p < 0.01). e–h Palbociclib treatment resulted in suppression of RB phosphorylation, Ki67 expression, and mitotic index in BIN-67 and SCCOHT-1 xenograft tumors of the trial end points. Representative images of IHC (p-RB, Ki67) and hematoxylin and eosin (H&E) analysis of BIN-67 (e) and SCCOHT-1 (g) xenograft tumor tissues. Quantification results of p-RB, Ki67, and mitotic count of BIN-67 (f, n = 3) and SCCOHT-1 (h, n = 4). In the H&E images, black arrows point to mitotic active cells as an example. Bar 50 µm; two-tailed t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. i Tumor volume fold change from Day 1 of treatment in the SCCOHT PDX model (n = 6 for vehicle, n = 3 for palbociclib). Mice were treated with the initial dose of 150 mg kg⁻¹ of palbociclib. After first signs of any animal discomfort, we reduced the dose to 100 mg kg⁻¹. Mice that showed weight loss were not included for the analysis. Two-way ANOVA, **p < 0.01. Error bars: mean ± standard error of mean (s.e.m.)

Fig. 6

SCCOHT patient tumors expressed reduced levels of cyclin D1 mRNA and protein. a, b SCCOHT tumors expressed significantly lower CCND1 mRNA levels compared to ovarian high-grade serous carcinomas (HGSCs). Heatmaps (a) and boxplots (b) showing CCND1 and CDK4 mRNA levels obtained from a NanoString gene expression study in SCCOHT patient tumors (n = 17) relative to HGSCs (n = 6). Two-tailed t test, *p < 0.05. c qRT–PCR analysis of an independent cohort of fresh-frozen patient tumor samples show that SCCOHT (n = 5) expressed significantly low levels of CCND1 mRNA (normalized to GAPDH) compared to HGSCs (n = 7). Two-tailed t test, **p < 0.01; ns not significant. d, e SCCOHT patient tumors express low levels of cyclin D1 and retain the RB-proficient/p16-deficient profile associated with positive responses to palbociclib. Immunohistochemistry (IHC) analysis coupled with unbiased automated quantification were performed on formalin-fixed paraffin-embedded HGSC (n = 52) and SCCOHT (n = 32; 4 of which were also analyzed by qRT-PCR in Fig. 6c) patient tumors for the expression of indicated key regulators of G1- to S-phase cell cycle. Representative images of the IHC analysis (d) and quantification results (e) are shown. Bar 50 µm; non-parametric Mann–Whitney test, ***p < 0.001, ****p < 0.0001; Error bars: mean ± standard error of mean (s.e.m.)