A tailored in vivo CRISPR screen identifies BAP1 as a potent tumor suppressor of sarcoma
Jianguo Huang, Xingliang Liu, Warren Floyd, William Haugh, Zhaoyu Sun, Melissa J. Kasiewicz, Yaping Wu, Brian Piening, John T. Welle, Wesley K. Rosales, Venkatesh Rajamanickam, So Young Kim, Eric S. Xu, Lixia Luo, Yan Ma, Rutulkumar Patel, Ziqiang Zhang, Brady Bernard, William L. Redmond, Walter J. Urba, R. Bryan Bell, David G. Kirsch
Jianguo Huang, Xingliang Liu, Warren Floyd, William Haugh, Zhaoyu Sun, Melissa J. Kasiewicz, Yaping Wu, Brian Piening, John T. Welle, Wesley K. Rosales, Venkatesh Rajamanickam, So Young Kim, Eric S. Xu, Lixia Luo, Yan Ma, Rutulkumar Patel, Ziqiang Zhang, Brady Bernard, William L. Redmond, Walter J. Urba, R. Bryan Bell, David G. Kirsch
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Research Article Genetics Oncology

A tailored in vivo CRISPR screen identifies BAP1 as a potent tumor suppressor of sarcoma

Abstract

Undifferentiated pleomorphic sarcoma (UPS) is one of the most common adult soft-tissue sarcomas (STSs), yet therapeutic progress remains limited because of the absence of recurrent oncogenic driver mutations. To identify tumor suppressors contributing to UPS pathogenesis, we performed a customized in vivo CRISPR/Cas9 screen in mice. This approach identified BRCA1-associated protein 1 (BAP1) as a potent tumor suppressor in STS. Integrative analyses using RNA sequencing, multiplex immunohistochemistry, and flow cytometry revealed that Bap1-deficient sarcomas exhibited a markedly immunosuppressive tumor microenvironment. Consistent with these findings, BAP1 protein expression was reduced in human UPS, whereas polo-like kinase 1 (PLK1) expression was elevated. Functional studies demonstrated that PLK1 was required for the growth and survival of Bap1-deficient sarcomas. Pharmacologic inhibition of PLK1 with volasertib significantly suppressed tumor growth in both syngeneic and autochthonous mouse models. Moreover, combining PLK1 inhibition with anti–PD-1 therapy enhanced tumor control and improved survival compared with either treatment alone. Together, these results identify PLK1 as a potential therapeutic vulnerability in BAP1-deficient sarcomas and support further evaluation of combined PLK1 inhibition and immune checkpoint blockade as a treatment strategy for a subset of STSs.

Authors

Jianguo Huang, Xingliang Liu, Warren Floyd, William Haugh, Zhaoyu Sun, Melissa J. Kasiewicz, Yaping Wu, Brian Piening, John T. Welle, Wesley K. Rosales, Venkatesh Rajamanickam, So Young Kim, Eric S. Xu, Lixia Luo, Yan Ma, Rutulkumar Patel, Ziqiang Zhang, Brady Bernard, William L. Redmond, Walter J. Urba, R. Bryan Bell, David G. Kirsch

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Figure 3

Validation of Bap1 as a potent tumor suppressor in UPS.

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Validation of Bap1 as a potent tumor suppressor in UPS. (A) Mutation rat...
(A) Mutation rates of 3 genes (TP53, RB1, and BAP1) in human UPS/MFS according to TCGA database. (B) IHC of a human sarcoma tissue array shows that BAP1 expression is significantly reduced in UPS, MFS, fibrosarcoma, and dermatofibrosarcoma compared with normal muscle (NM). Scale bars: 50 μm. Quantification was performed on independent tissue cores, and statistical significance was assessed using a 2-tailed Student’s t test. (C) IVE of plasmids expressing sgRNAs targeting Trp53 and Bap1 or Trp53 and Rb1 induced tumors in Rosa26loxP-Cas9/loxP-Cas9 mice. Tumor-free survival (latency to tumor formation) was analyzed using Kaplan-Meier curves and compared using the log-rank test. (D) Tumor quadrupling times were similar between PR CRISPR and PB CRISPR tumors. Statistical comparisons were performed using a 2-tailed Student’s t test. (E) Growth curves of representative PR and PB CRISPR tumors. (F) IVE of Cre recombinase and sgRNA targeting Bap1 (n = 11) induced significantly higher tumor penetrance compared with Cre alone or Cre plus sgRNA targeting Crebbp (3 tumors in 18 mice). Tumor-free survival (latency to tumor formation) was analyzed using Kaplan-Meier curves and compared using the log-rank test. (G) PRB Cre/CRISPR tumors grew significantly faster than PR Cre tumors. Statistical comparisons were performed using a 2-tailed Student’s t test. (H) Growth curves of representative PR Cre and PRB Cre/CRISPR tumors. For all experiments, n denotes biologically independent mice. Data are presented as mean ± SEM unless otherwise indicated. Representative images and growth curves are from experiments repeated at least twice with similar results.