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 7

Plk1 knockdown inhibits growth of Bap1-deficient sarcomas.

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Plk1 knockdown inhibits growth of Bap1-deficient sarcomas. (A) High PLK...
(A) High PLK1 RNA expression correlates with poor prognosis in UPS (TCGA). Survival differences were analyzed using Kaplan-Meier curves and compared using the log-rank test. (B) IHC of a human sarcoma tissue array shows that PLK1 protein expression is significantly increased in UPS, MFS, fibrosarcoma, and dermatofibrosarcoma. 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) RNA sequencing and quantitative reverse transcription PCR confirm Plk1 upregulation in PB sarcomas. Statistical comparisons were performed using a 2-tailed Student’s t test. (D) Western blot confirms PLK1 knockdown; cell proliferation assays (CELLCYTE) demonstrate reduced growth following Plk1 knockdown. Statistical comparisons were performed using a 2-tailed Student’s t test. (E) Plk1 knockdown inhibits tumor growth and prolongs survival in a syngeneic mouse model. Survival was analyzed using Kaplan-Meier curves with significance assessed by the log-rank test. (F) CRISPR/Cas9–mediated mutation of Plk1 reduces the penetrance of Trp53/Bap1 loss–driven sarcomagenesis. Survival was analyzed using Kaplan-Meier curves with significance assessed by the log-rank test.