FGFR1 and FGFR2 are amplified in many tumor types, yet what determines response to FGFR inhibition in amplified cancers is unknown. In a translational clinical trial, we show that gastric cancers with high-level clonal FGFR2 amplification have a high response rate to the selective FGFR inhibitor AZD4547, whereas cancers with subclonal or low-level amplification did not respond. Using cell lines and patient-derived xenograft models, we show that high-level FGFR2 amplification initiates a distinct oncogene addiction phenotype, characterized by FGFR2-mediated transactivation of alternative receptor kinases, bringing PI3K/mTOR signaling under FGFR control. Signaling in low-level FGFR1-amplified cancers is more restricted to MAPK signaling, limiting sensitivity to FGFR inhibition. Finally, we show that circulating tumor DNA screening can identify high-level clonally amplified cancers. Our data provide a mechanistic understanding of the distinct pattern of oncogene addiction seen in highly amplified cancers and demonstrate the importance of clonality in predicting response to targeted therapy. Significance Robust single-agent response to FGFR inhibition is seen only in high-level FGFR-amplified cancers, with copy-number level dictating response to FGFR inhibition in vitro, in vivo, and in the clinic. High-level amplification of FGFR2 is relatively rare in gastric and breast cancers, and we show that screening for amplification in circulating tumor DNA may present a viable strategy to screen patients.

Resistance to androgen receptor (AR) blockade in castration-resistant prostate cancer (CRPC) is associated with sustained AR signaling, including through alternative splicing of AR (AR-SV). Inhibitors of transcriptional coactivators that regulate AR activity, including the paralog histone acetyltransferase proteins p300 and CBP, are attractive therapeutic targets for lethal prostate cancer. Herein, we validate targeting p300/CBP as a therapeutic strategy for lethal prostate cancer and describe CCS1477, a novel small-molecule inhibitor of the p300/CBP conserved bromodomain. We show that CCS1477 inhibits cell proliferation in prostate cancer cell lines and decreases AR- and C-MYC–regulated gene expression. In AR-SV–driven models, CCS1477 has antitumor activity, regulating AR and C-MYC signaling. Early clinical studies suggest that CCS1477 modulates KLK3 blood levels and regulates CRPC biopsy biomarker expression. Overall, CCS1477 shows promise for the treatment of patients with advanced prostate cancer. Significance: Treating CRPC remains challenging due to persistent AR signaling. Inhibiting transcriptional AR coactivators is an attractive therapeutic strategy. CCS1477, an inhibitor of p300/CBP, inhibits growth and AR activity in CRPC models, and can affect metastatic CRPC target expression in serial clinical biopsies. See related commentary by Rasool et al., p. 1011. This article is highlighted in the In This Issue feature, p. 995

Welti et al; Targeting BET family proteins in CRPC 2 Statement of Translational RelevanceAdvanced prostate cancer invariably progresses to lethal castration resistant prostate cancer (CRPC). Resistance to current androgen receptor (AR) targeting therapies is associated with the development of AR aberrations including the constitutively active AR splice variant 7 (AR-V7). Currently, no clinically available therapies effectively inhibit aberrant AR signaling. BRD4, a bromodomain and extraterminal (BET) family protein, is a critical AR coregulator. We show that BRD4 expression associates with patient outcome and AR driven transcription in lethal prostate cancer. Moreover, BET inhibitors (BETi) reduce AR splicing and AR-V7 expression by regulating alternative splicing, abrogating AR signaling and inhibiting growth of CRPC patient derived models. Clinical studies with BETi in CRPC should pursue pharmacodynamics studies evaluating abrogation of AR splicing and persistent AR signaling to optimize the development of these drugs for the treatment of CRPC.Research. Experimental Design: We determined associations between BET expression, AR driven transcription and patient outcome; and the effect and mechanism by which chemical BETi (JQ1 and GSK1210151A; I-BET151) and BET family protein knockdown regulates AR-V7 expression and AR signaling in prostate cancer (PC) models.Results: Nuclear BRD4 protein expression increases significantly (p=<0.01) with castration resistance in same patient treatment naïve (median H-score; interquartile range: 100; 100-170) and CRPC (150; 110-200) biopsies, with higher expression at diagnosis associating with worse outcome (HR 3.25, 95% CI 1.50-7.01; p=<0.001). BRD2, BRD3 and BRD4 RNA expression in CRPC biopsies correlates with AR driven transcription (all p=<0.001). Chemical BETi, and combined BET family protein knockdown, reduce AR-V7 expression and AR signaling. This was not recapitulated by C-MYC knockdown. In addition, we show that BETi regulates RNA processing thereby reducing alternative splicing and AR-V7 expression. Furthermore, BETi reduce growth of PC cells and patient derived organoids with known AR mutations, AR amplification and AR-V7 expression. Finally, BETi, unlike enzalutamide, decreases persistent AR signaling and growth (p=<0.001) of a patient derived xenograft model of CRPC with AR amplification and AR-V7 expression. Conclusion:BETi merit clinical evaluation as inhibitors of AR splicing and function, with trials demonstrating their blockade in proof of mechanism pharmacodynamic studies.

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PARP inhibitors (PARPis) have clinical efficacy in BRCA-deficient cancers, but not BRCA-intact tumors, including glioblastoma (GBM). We show that MYC or MYCN amplification in patient-derived glioblastoma stem-like cells (GSCs) generates sensitivity to PARPi via Myc-mediated transcriptional repression of CDK18 , while most tumors without amplification are not sensitive. In response to PARPi, CDK18 facilitates ATR activation by interacting with ATR and regulating ATR-Rad9/ATR-ETAA1 interactions; thereby promoting homologous recombination (HR) and PARPi resistance. CDK18 knockdown or ATR inhibition in GSCs suppressed HR and conferred PARPi sensitivity, with ATR inhibitors synergizing with PARPis or sensitizing GSCs. ATR inhibitor VE822 combined with PARPi extended survival of mice bearing GSC-derived orthotopic tumors, irrespective of PARPi-sensitivity. These studies identify a role of CDK18 in ATR-regulated HR. We propose that combined blockade of ATR and PARP is an effective strategy for GBM, even for low-Myc GSCs that do not respond to PARPi alone, and potentially other PARPi-refractory tumors.