Ponatinib Shows Potent Antitumor Activity in Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) through Multikinase Inhibition

Abstract

Purpose: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by loss of the SWI/SNF ATPase subunits SMARCA4 and SMARCA2. A great need exists for effective targeted therapies for SCCOHT.Experimental Design: To identify underlying therapeutic vulnerabilities in SCCOHT, we conducted high-throughput siRNA and drug screens. Complementary proteomics approaches profiled kinases inhibited by ponatinib. Ponatinib was tested for efficacy in two patient-derived xenograft (PDX) models and one cell-line xenograft model of SCCOHT.Results: The receptor tyrosine kinase (RTK) family was enriched in siRNA screen hits, with FGFRs and PDGFRs being overlapping hits between drug and siRNA screens. Of multiple potent drug classes in SCCOHT cell lines, RTK inhibitors were only one of two classes with selectivity in SCCOHT relative to three SWI/SNF wild-type ovarian cancer cell lines. We further identified ponatinib as the most effective clinically approved RTK inhibitor. Reexpression of SMARCA4 was shown to confer a 1.7-fold increase in resistance to ponatinib. Subsequent proteomic assessment of ponatinib target modulation in SCCOHT cell models confirmed inhibition of nine known ponatinib target kinases alongside 77 noncanonical ponatinib targets in SCCOHT. Finally, ponatinib delayed tumor doubling time 4-fold in SCCOHT-1 xenografts while reducing final tumor volumes in SCCOHT PDX models by 58.6% and 42.5%.Conclusions: Ponatinib is an effective agent for SMARCA4-mutant SCCOHT in both in vitro and in vivo preclinical models through its inhibition of multiple kinases. Clinical investigation of this FDA-approved oncology drug in SCCOHT is warranted. Clin Cancer Res; 24(8); 1932-43. ©2018 AACR.

Figure 1. High-throughput screen network analysis.

(A) Protein interaction network constructed for RNAi validated screen hits using ReactomeFI network resources, as further described in the supplemental materials and methods. Curated interactions are represented by solid edges with arrows for interactions involved in catalysis or activation and edges with “T” bar for inhibitory connections. Dotted edges are those interactions that are computationally predicted. Nodes in the network represent validate RNAi hits. Nodes with no edges are not pictured here (full validated hit list in Supplemental Table 3). Genes that were identified as PDGFR pathway members by ReactomeFI are highlighted in yellow, all other genes colored in blue. (B) Chemical hit entity association network for verified chemical hits and their targets and associated biological concepts in SCCOHT cell line, BIN67. RTK associated hits are highlighted in box.

Figure 2. Sensitivity of SCCOHT and other SWI/SNF mutant cell lines to RTK inhibition.

DDR curves for various RTKi in BIN67 cells. Cell viability was assayed after 72 hours treatment. (B) Summary of IC₅₀ values for RTKi tested in SCCOHT cell lines, along with selected agents tested in SWI/SNF-mutant MRT cell lines (G401 and G402) and lung cancer cell lines (A427 and H522) vs SWI/SNF-wildtype immortalized granulosa cells (SVOG3e). Conditions not tested are in gray. (C) Comparison of IC₅₀ values for ponatinib in SCCOHT cell lines (BIN67, SCCOHT-1, and COV434) versus ovarian cancer cell lines selected for their wildtype SWI/SNF status (OVCAR-3, OVCAR-5, and OVCAR-8). (D) Fold change in ponatinib IC₅₀ value following doxycycline treatment in parental COV434 cells and two COV434 pIND20 BRG1 clones (inducible SMARCA4). (E) Cell viability of BIN67 cells following knockdown of ponatinib target genes. Known ponatinib target genes that were initial hits in the siRNA screen (FGFR4, PDGFRA, and PDGFRB) were validated in a secondary siRNA screen. Data shown is mean cell viability of 3 independent replicates of 4 distinct siRNAs relative to internal GFP-targeting siRNA control. All three target genes were considered validated hits based on criteria described in methods.

Figure 3. RTK expression in SCCOHT primary tumors and downstream RTK signaling inhibited by ponatinib in SCCOHT cells.

(A) Expression of all RTK genes in four SCCOHT primary tumors from RNA-Seq. Expression is displayed in FPKM, and genes are hierarchically clustered. The six most highly expressed RTKs are in inset. (B) Phosphorylation of signaling components downstream of RTKs are inhibited by ponatinib within 30 min treatment in BIN67 cells.

Figure 4. RTK signaling inhibited by ponatinib in SCCOHT cells.

(A) Phospho-RTK array performed on lysates from BIN67 cells treated with ponatinib for 1 hour. Background-normalized spot intensity relative to vehicle-treated control is represented as a heatmap, where blue indicates an increase in phosphorylation and red indicates a decrease. RTKs are arranged from top to bottom based on mean basal dot intensity. Known ponatinib targets are highlighted in yellow, kinases inhibited in both cell lines are outlined in orange and kinases inhibited in one cell line only are outlined in green. (B and C) Kinases identified from MIB-MS assay (B) and (C) ABPP assay in SCCOHT-1 cells (blue circles, 2μM, biological triplicates) and BIN67 cells (red circles, IC₅₀, technical replicates) or in both cell lines (yellow circles) mapped to kinase dendrogram. The gene lists are color-coded based on corresponding kinase family in the dendrogram. Atypical protein kinases are listed in black. Differentially expressed kinases are shown in lighter shaded circles. Uniquely expressed kinases are shown in darker shaded circles and are marked with an asterisk in the gene list.

Figure 5. Efficacy of ponatinib in animal models of SCCOHT.

(A) SCCOHT-1 xenograft growth with treatment with vehicle or ponatinib. (B) Survival of SCCOHT-1 xenograft-bearing mice following treatment with vehicle or ponatinib. (C and D) Tumor volumes of SCCOHT PDX model ‘465 (C) or ‘040 (D) growth with treatment with vehicle or ponatinib. Insets in (C) and (D) show tumor volumes at day 30. (E) Phospho-RTK array performed on lysates from flash-frozen PDX tumor tissues following treatment with ponatinib for six hours or at the experimental endpoint for the efficacy studies in Figure 5C and 5D and analyzed as in Figure 4A. Data represents the average of two tumors per group.