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Loss of BOP1 Confers Resistance to BRAF Kinase Inhibitors in Melanoma by Activating MAP Kinase Pathway

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Loss of BOP1 Confers Resistance to BRAF Kinase Inhibitors in Melanoma by Activating MAP Kinase Pathway Loss of BOP1 confers resistance to BRAF kinase inhibitors in melanoma by activating MAP kinase pathway Romi Guptaa, Suresh Bugidea, Biao Wanga, Michael R. Greenb,1, Douglas B. Johnsonc, and Narendra Wajapeyeeb,1 aDepartment of Pathology, Yale University School of Medicine, New Haven, CT 06510; bDepartment of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605; and cDepartment of Medicine, Vanderbilt University Medical Center, Nashville, TN 37240 Contributed by Michael R. Green, January 14, 2019 (sent for review December 24, 2018; reviewed by Min Li and Pankaj K. Singh) Acquired resistance to BRAF kinase inhibitors (BRAFi) is the Furthermore, analysis of matched patient-derived BRAFi or primary cause for their limited clinical benefit. Although several BRAFi+MEKi pre- and progressed melanoma samples revealed mechanisms of acquired BRAFi resistance have been identified, the reduced BOP1 protein expression in progressed samples. basis for acquired resistance remains unknown in over 40% of melanomas. We performed a large-scale short-hairpin RNA screen, Results targeting 363 epigenetic regulators and identified Block of Pro- A Large-Scale Epigenome-Wide Human shRNA Screen Identifies liferation 1 (BOP1) as a factor the loss of which results in resistance Candidates That Confer Resistance to BRAF Inhibitors. Epigenetic to BRAFi both in cell culture and in mice. BOP1 knockdown pro- alterations are shown to play an important role in the regulation moted down-regulation of the MAPK phosphatases DUSP4 and of cancer cell growth and their response to targeted therapies DUSP6 via a transcription-based mechanism, leading to increased (15–17). Therefore, to determine the role of epigenetic regula- MAPK signaling and BRAFi resistance. Finally, analysis of matched tors in conferring resistance to BRAFi, we performed a large- + patient-derived BRAFi or BRAFi MEKi pre- and progressed mela- scale, unbiased, epigenome-wide shRNA screen by targeting 363 noma samples revealed reduced BOP1 protein expression in pro- known and predicted epigenetic regulators with 1862 shRNAs gressed samples. Collectively, our results demonstrate that loss of (SI Appendix, Table S1). For this screen, we infected the BRAF- BOP1 and the resulting activation of the MAPK pathway is a MEDICAL SCIENCES mutant melanoma line A375 with the epigenetic regulator clinically relevant mechanism for acquired resistance to BRAFi shRNA library at a multiplicity of infection (MOI) of 0.2 and in melanoma. selected with puromycin to enrich for shRNA-expressing cells. μ BOP1 | drug resistance | melanoma | BRAF inhibitor | RNAi After selection, shRNA-containing cells were treated with 2 M of vemurafenib for 4 wk. Surviving colonies were then harvested, and genomic DNA was isolated and sequenced for shRNA iden- elanoma is an aggressive cancer that frequently metasta- tification (Fig. 1A). From this analysis, we identified shRNAs sizes to various distal organs (1, 2). Although treatment of M corresponding to six different epigenetic regulators in vemurafenib- melanoma at early stages is generally effective, even with several resistant A375 colonies (SI Appendix,TableS2). improvements in current therapeutic approaches the median survival of patients with metastatic melanoma is only 4.5– 12.5 mo (1, 3). Genomic sequencing of melanoma has identified Significance oncogenic mutations in the BRAF gene in over 50% of tumors (4, 5). Acquiring oncogenic mutations in the BRAF gene causes Oncogenic mutations in the BRAF gene are found in ∼50% of constitutive activation of the BRAF → MEK → ERK pathway melanomas and drive melanoma growth. Thus, BRAF kinase and is necessary for melanoma growth and progression (4, 6). inhibitors (BRAFi), such as vemurafenib and dabrafenib, have These findings have led to the development and approval of been developed and used for the treatment of BRAF-mutant several BRAF and MEK kinase inhibitors by the Food and Drug metastatic melanoma in clinic. However, the clinical benefits of Administration for treating unresectable metastatic melanoma BRAFi are temporary and short-lived due to the emergence of (7, 8). However, although melanoma patients initially respond drug resistance. Although several mechanisms of acquired robustly to BRAF kinase targeted therapy, they show acquired BRAFi resistance have been identified, the basis for acquired resistance within a matter of a few months, resulting in disease resistance remains unknown in over 40% of melanomas. progression. Due to the high prevalence of this problem, in- Identifying a new mechanism of acquired resistance to BRAFi tensive efforts have focused on identifying the causes of re- may provide new opportunities to effectively treat BRAF- sistance to BRAF and MEK kinase inhibitors, and several mutant melanoma. In this study, we have identified Block of mechanisms have been identified (9, 10). These mechanisms can Proliferation 1 as a new factor the loss of which results in re- be broadly categorized as either dependent or independent of sistance to BRAFi. the MAPK pathway (11, 12). Block of proliferation 1 (BOP1) contains WD40 repeats and Author contributions: R.G., S.B., M.R.G., D.B.J., and N.W. designed research; R.G., S.B., B.W., M.R.G., and N.W. performed research; R.G., S.B., B.W., M.R.G., D.B.J., and N.W. has been shown to be involved in 28S and 5.8S ribosomal RNA contributed new reagents/analytic tools; R.G., S.B., M.R.G., D.B.J., and N.W. analyzed data; (rRNA) processing and 60S ribosome biogenesis (13). BOP1 is and R.G., S.B., D.B.J., and N.W. wrote the paper. also part of the PES1-BOP1-WDR12 (PeBoW) complex, and Reviewers: M.L., University of Oklahoma Health Sciences Center; and P.K.S., University of inactivation of subunits from this complex inhibits rRNA pro- Nebraska Medical Center. cessing and ribosome biogenesis (13, 14). Here, using a large- The authors declare no conflict of interest. scale short-hairpin RNA (shRNA) screen, we have identified Published under the PNAS license. that loss of BOP1 causes resistance to BRAF kinase inhibitor 1To whom correspondence may be addressed. Email: [email protected] or (BRAFi). We show that loss of BOP1 results in reduced ex- [email protected]. pression of Dual specificity phosphatase 4 (DUSP4) and Dual This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. specificity phosphatase 6 (DUSP6), which results in activation of 1073/pnas.1821889116/-/DCSupplemental. the MAP kinase pathway, resulting in resistance to BRAFi. Published online February 19, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1821889116 PNAS | March 5, 2019 | vol. 116 | no. 10 | 4583–4591 Downloaded by guest on September 29, 2021 A375 NS shRNA A B NS shRNA #1 NS shRNA 150 HAT1 shRNA #1 150 #2 150 #1 BOP1 shRNA HMGB1 shRNA #2 #2 al (%) al Infect cells with human epigenetic regulators 100 100 viv 100 shRNA library sur (363 genes, targeted by 1862 shRNAs) cell 50 50 50 ve cell survival (%) i ve i ive cell survival (%) Puromycin select t **** **** ns Relat Relat Vemurafenib treatment 0 Rela 0 0 (2 μM, 4 weeks) 0.00 0.05 0.10 0.25 0.50 1.00 0.00 0.05 0.10 0.25 0.50 1.00 0.00 0.05 0.10 0.25 0.50 1.00 Vemurafenib (μM) Vemurafenib (μM) Vemurafenib (μM) NS shRNA NS shRNA NS shRNA 150 #1 150 #1 150 #1 HMGN1 shRNA ING5 shRNA KDM4C shRNA #2 #2 #2 Isolate surviving colonies l (%) ival (%) ival 100 viva 100 100 urv Purify genomic DNA ur s s cell 50 50 ecell 50 PCR amplify, sequence, identify ive tiv shRNAs causing resistance *** **** ns Relat Rela to vemurafenib 0 0 Relative cell survival (%) 0 0.00 0.05 0.10 0.25 0.50 1.00 0.00 0.05 0.10 0.25 0.50 1.00 0.00 0.05 0.10 0.25 0.50 1.00 Vemurafenib (μM) Vemurafenib (μM) Vemurafenib (μM) C D SKMEL-239 SKMEL-28 SKMEL-239 SKMEL-28 M249 M229 SKMEL-239 NS shRNA P 150 NS shRNA 150 150 NS shRNA 150 NS shRNA R PR PR ) #1 #1 BOP1 #1 BOP1 shRNA shRNA HAT1 #1 p-ERK1/2 #2 #2 shRNA HAT1 shRNA #2 #2 total-ERK1/2 100 100 100 100 BOP1 50 50 50 50 HAT1 **** **** **** Relative cell survival (%) Relative cell survival (%) ING5 Relative cell survival (% 0 0 0 Relative cell survival (%) 0 KDM4C 0.00 0.05 0.10 0.25 0.50 1.00 0.00 0.05 0.10 0.25 0.50 1.00 0.00 0.05 0.10 0.25 0.50 1.00 0.00 0.05 0.10 0.25 0.50 1.00 μ μ μ ACTINB Vemurafenib ( M) Vemurafenib ( M) Vemurafenib ( M) Vemurafenib (μM) Fig. 1. Large-scale shRNA screen to identify epigenetic regulators of vemurafenib resistance in BRAF-mutant melanoma. (A) Schematic for large-scale epigenetic regulator shRNA screen. (B) Relative cell survival (%) as measured by MTT assay of A375 cells expressing shRNAs targeting the indicated genes or NS control shRNAs that were treated with the indicated concentrations of vemurafenib for 3 d. (C) Immunoblotting for the indicated proteins in three pairs of BRAFi-sensitive (P) or resistant (R) BRAF-mutant melanoma cell lines. (D) Relative cell survival (%) as measured by MTT assay of SKMEL-239 or SKMEL-28 cells expressing shRNAs targeting the indicated genes or NS control shRNAs that were treated with indicated concentrations of vemurafenib for 3 d. Data are presented as the mean ± SEM. ns, not significant. ***P < 0.001 and ****P < 0.0001. Next, we individually knocked down expression of all six genes sults, vemurafenib-treated melanoma cells expressing BOP1 and identified from our primary screen in A375 cells (SI Appendix, HAT1 shRNAs showed significantly larger colonies compared Fig.
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