RAF1 amplification drives a subset of bladder tumors and confers sensitivity to MAPK-directed therapeutics
Raie T. Bekele, Amruta S. Samant, Amin H. Nassar, Jonathan So, Elizabeth P. Garcia, Catherine R. Curran, Justin H. Hwang, David L. Mayhew, Anwesha Nag, Aaron R. Thorner, Judit Börcsök, Zsofia Sztupinszki, Chong-Xian Pan, Joaquim Bellmunt, David J. Kwiatkowski, Guru P. Sonpavde, Eliezer M. Van Allen, Kent W. Mouw
Raie T. Bekele, Amruta S. Samant, Amin H. Nassar, Jonathan So, Elizabeth P. Garcia, Catherine R. Curran, Justin H. Hwang, David L. Mayhew, Anwesha Nag, Aaron R. Thorner, Judit Börcsök, Zsofia Sztupinszki, Chong-Xian Pan, Joaquim Bellmunt, David J. Kwiatkowski, Guru P. Sonpavde, Eliezer M. Van Allen, Kent W. Mouw
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Research Article Oncology

RAF1 amplification drives a subset of bladder tumors and confers sensitivity to MAPK-directed therapeutics

Abstract

Bladder cancer is a genetically heterogeneous disease, and novel therapeutic strategies are needed to expand treatment options and improve clinical outcomes. Here, we identified a unique subset of urothelial tumors with focal amplification of the RAF1 (CRAF) kinase gene. RAF1-amplified tumors had activation of the RAF/MEK/ERK signaling pathway and exhibited a luminal gene expression pattern. Genetic studies demonstrated that RAF1-amplified tumors were dependent upon RAF1 activity for survival, and RAF1-activated cell lines and patient-derived models were sensitive to available and emerging RAF inhibitors as well as combined RAF plus MEK inhibition. Furthermore, we found that bladder tumors with HRAS- or NRAS-activating mutations were dependent on RAF1-mediated signaling and were sensitive to RAF1-targeted therapy. Together, these data identified RAF1 activation as a dependency in a subset making up nearly 20% of urothelial tumors and suggested that targeting RAF1-mediated signaling represents a rational therapeutic strategy.

Authors

Raie T. Bekele, Amruta S. Samant, Amin H. Nassar, Jonathan So, Elizabeth P. Garcia, Catherine R. Curran, Justin H. Hwang, David L. Mayhew, Anwesha Nag, Aaron R. Thorner, Judit Börcsök, Zsofia Sztupinszki, Chong-Xian Pan, Joaquim Bellmunt, David J. Kwiatkowski, Guru P. Sonpavde, Eliezer M. Van Allen, Kent W. Mouw

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Figure 3

RAF1-amplified cell lines are dependent on RAF1 signaling.

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RAF1-amplified cell lines are dependent on RAF1 signaling. (A) RAF1 mRN...
(A) RAF1 mRNA expression (x axis) and DNA copy number (y axis) across 36 bladder cancer cell lines from the DepMap identify 2 cell lines (5637 and UMUC9) with high RAF1 levels. (B) RAF1 immunoblot in bladder epithelial and tumor cell lines confirms high levels of RAF1 protein expression in 5637 and UMUC9. (C) RAF1 depletion by siRNA kills RAF1-amplified cell lines, but has minimal effect on bladder cancer cell lines without RAF1 amplification. Unmagnified. NTC, nontargeting control siRNA. (D) Quantification of the relative signal intensity from the viability assay in C. ***P < 0.0001, ANOVA with Bonferroni’s post hoc test. (E) RAF1 depletion by siRNA abrogates RAF/MEK/ERK signaling in RAF1-amplified bladder cancer cell lines, as shown by immunoblot (blots were run in parallel from the same sample). (F) RAF1 gene-dependency scores for bladder cancer cell lines from CRISPR-Cas9 essentiality screens from DepMap (33). A low score indicates a higher likelihood that the gene is essential in a given cell line. RAF1-amplified cell lines (UMUC and 5637) are shown in red, an HRAS mutant cell line (T24) in green, NRAS mutant cell lines (Ku-19-19 and BFTC905) in orange, and a MEK2 mutant cell line (JMSU1) in yellow. Cell lines without alterations in any of these 4 genes are shown in blue. The bottom panel shows the distribution of RAF1 dependency scores across the 29 bladder cancer cell lines analyzed.