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Loss of MAP3K7 Sensitizes Prostate Cancer Cells to CDK1/2 Inhibition and DNA Damage by Disrupting Homologous Recombination Satoshi Washino1, Leah C

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Loss of MAP3K7 Sensitizes Prostate Cancer Cells to CDK1/2 Inhibition and DNA Damage by Disrupting Homologous Recombination Satoshi Washino1, Leah C Published OnlineFirst July 12, 2019; DOI: 10.1158/1541-7786.MCR-18-1335 Cancer Genes and Networks Molecular Cancer Research Loss of MAP3K7 Sensitizes Prostate Cancer Cells to CDK1/2 Inhibition and DNA Damage by Disrupting Homologous Recombination Satoshi Washino1, Leah C. Rider1, Lina Romero1, Lauren K. Jillson1, Trisiani Affandi2, Angela M. Ohm2, Elaine T. Lam3, Mary E. Reyland2, James C. Costello1, and Scott D. Cramer1 Abstract The combined loss of CHD1 and MAP3K7 promotes aggres- without loss of Map3k7. Dinaciclib treatment reduced expres- sive prostate cancer by unknown mechanisms. Because both of sion of homologous recombination (HR) repair genes such as these genes are lost genetically in prostate cancer, they cannot ATM, ATR, BRCA2, and RAD51, blocked BRCA1 phosphory- be directly targeted. We applied an established computational lation, reduced RAD51 foci formation, and increased gH2AX systems pharmacology approach (TRAP) to identify altered foci selectively in prostate cells with suppression of Map3k7, signaling pathways and associated druggable targets. We com- thus inhibiting HR repair of chromosomal double-strand pared gene expression profiles of prostate cancer with coloss of breaks. Dinaciclib-induced HR disruption was also observed CHD1 and MAP3K7 with prostate cancer diploid for these in human prostate cells with knockdown of MAP3K7. Cotreat- genes using The Cancer Genome Atlas patient samples. This ment of dinaciclib with DNA-damaging agents or PARP inhib- analysis prioritized druggable target genes that included CDK1 itor resulted in a stronger cytotoxic effect on prostate cells with and CDK2. We validated that inhibitors of these druggable suppression of MAP3K7 compared with those without loss of target genes, including the CDK1/ CDK2 inhibitor dinaciclib, MAP3K7, or to each single agent. had antiproliferative and cytotoxic effects selectively on mouse prostate cells with knockdown of Chd1 and Map3k7. Dinaci- Implications: These findings demonstrate that loss of clib had stronger effects on prostate cells with suppression of MAP3K7 is a main contributing factor to drug response Map3k7 independent of Chd1 and also compared with cells through disruption of HR in prostate cancer. Introduction which encodes TGFb-activated kinase 1 (TAK1; ref. 4; P < 0.001; Fisher exact test). Homozygous or heterozygous deletion of Prostate cancer is characterized by large genomic rearrange- MAP3K7 in primary prostate cancer is significantly correlated ments including translocations, copy number losses, and with high Gleason grade tumors (2, 3). We previously demon- gains (1). MAP3K7 is deleted in 30%–40% of primary prostate strated that genomic deletion of CHD1 and MAP3K7 correlates cancer (homozygous and heterozygous deletion combined; with their respective mRNA expression in multiple patient cohorts refs. 2, 3). Loss of the Chromodomain Helicase DNA-binding and that low mRNA expression is correlated with poor disease- protein 1 (CHD1) gene (located on 5q21.1), which occurs in free survival (5). We recently demonstrated that MAP3K7 is a 20%–30% of prostate cancer (4, 5), is associated with an increased prostate tumor suppressor and that coloss with CHD1 promotes frequency of copy number alterations (CNA) and massive geno- aggressive prostate cancer (3, 5). The CHD1 gene encodes a mic rearrangements, termed chromothripsis (4). Not all of the histone-associated protein that is required for DNA double- CNAs associated with loss of CHD1 are random (4). CHD1 loss is strand break repair and CHD1 depletion enhances PARP inhibitor significantly associated with loss of MAP3K7 (located on 6q15.1), therapy (6). MAP3K7 encodes MAP kinase kinase kinase 7 that is immediately downstream of cytokine and stress response path- 1Department of Pharmacology, University of Colorado Anschutz Medical 2 ways. Loss of these two tumor suppressors represents a molecu- Campus, Aurora, Colorado. Department of Craniofacial Biology, University of fi Colorado Anschutz Medical Campus, Aurora, Colorado. 3Department of Internal larly de ned subtype of aggressive prostate cancer with unknown Medicine, Division of Medical Oncology, University of Colorado Anschutz therapeutic sensitivities. Because both of these genes are lost Medical Campus, Aurora, Colorado. genetically in prostate cancer, they cannot be directly targeted. Note: Supplementary data for this article are available at Molecular Cancer Therefore, the pathways that are altered must be characterized to Research Online (http://mcr.aacrjournals.org/). identify therapeutic vulnerabilities. To identify potential signaling pathways altered in prostate J.C. Costello and S.D. Cramer contributed equally to this article. cancer with loss of CHD1 and/or MAP3K7, we applied an estab- Corresponding Author: Scott D. Cramer, University of Colorado, Anschutz lished computational systems pharmacology approach, Tran- Medical Center, 12801 E 17th Ave, L18-6117, Mail Stop 8303, PO Box 6511, Aurora, scriptional Regulatory Association with Pathways (TRAP; ref. 7), CO 80045. Phone: 303-724-6276; Fax: 303-724-3663; E-mail: [email protected] to gene expression data in The Cancer Genome Atlas (TCGA; ref. 8). Through this approach, we identified a number of altered Mol Cancer Res 2019;XX:XX–XX signaling pathways and a set of predicted druggable targets. We doi: 10.1158/1541-7786.MCR-18-1335 investigated several targets in prostate cells with suppression of Ó2019 American Association for Cancer Research. CHD1 and MAP3K7 expression and identified several promising www.aacrjournals.org OF1 Downloaded from mcr.aacrjournals.org on October 2, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst July 12, 2019; DOI: 10.1158/1541-7786.MCR-18-1335 Washino et al. drugs, including dinaciclib, a multiple cyclin-dependent kinase luminescence was read using a Modulus Microplate reader. For (CDK) inhibitor. We showed that loss of MAP3K7 was the main WFU3 and LNCaP, cell proliferation was determined by the contributing alteration driving increased sensitivity to these percent confluence of each well using Incucyte Zoom scanning. drugs, and that treatment with a CDK inhibitor paired with either The proportion of cells per treatment group was normalized to PARP inhibitor or DNA-damaging agents resulted in potent cell control wells when indicated. Annexin V- and PI-positive cell death in cells with loss of MAP3K7. counts were normalized to percent confluence in each well. Cell-cycle analysis and cell sorting Materials and Methods Staining of cells for cell-cycle analysis was performed as Agents described previously (11). Briefly, WFU3 (2–5 Â 105) and All drugs were purchased from Selleckchem except for doce- BHPrE1 cells (1 Â 105) were seeded in a 6-well plate and taxel that was purchased from Sigma-Aldrich. incubated overnight. WFU3 cells were exposed to vehicle, ola- parib 10 mmol/L, dinaciclib 60 nmol/L, or the combination of Cell culture these drugs for 24 hours prior to collection. BHPrE1 cells were All cell lines were routinely tested for Mycoplasma contamina- exposed to vehicle, docetaxel 1 nmol/L, dinaciclib 4 nmol/L, or tion by DAPI staining and PCR. LNCaP cells were obtained from the combination of these drugs for 24 hours prior to collection. ATCC and used below passage 50. LNCaP cells were validated by Cells were washed once with PBS, pelleted, and vortexed before spectral karyotyping. Generation and maintenance of WFU3 adding 1 mL Krishan stain solution (11). Cells were vortexed mouse prostate epithelial progenitor/stem cells were described again and incubated overnight at 4C before analysis with a previously (9). Wild-type WFU3 cells generate benign ductal Gallios instrument using CXP software (Beckman Coulter). A structures and represent a clean model to dissect the contributions total of 10,000 events were acquired for each sample and the of specific genetic alterations to therapeutics and signaling (5, 9). percent of cells in G1, S, and G2–M phases was determined with LNCaP cells were maintained as described previously (5). Gen- ModFit LT v4.0.5 software (Verity Software House) eration of shControl, shMAP3K7, shCHD1, and shDouble cell Cell sorting for the cell-cycle phases was performed as lines in WFU3 and LNCaP was described previously (5). BHPrE1 follows: WFU3 cells (0.3–2 Â 106) were seeded in a 15-cm cells, a spontaneously immortalized benign prostate cell line dish and incubated overnight. Cells were exposed to vehicle, or that generates benign ductal structures in vivo, were a generous the combination of dinaciclib and olaparib for 36 hours prior gift from Simon Hayward NorthShore University HealthSystem to collection. Cells were washed once with PBS, pelleted, and Research Institute, Evanston, IL (10). As there is no record of prior vortexed before adding Hoechst33342 (Thermo Fisher Scien- spectral karyotyping, we validated that BHPrE1 cells had a unique tific, #H3570). Cells were incubated in a medium containing karyotype. BHPrE1 cells were maintained and cultured in DMEM/ 60 mg/mL of Hoecht33342 for 45 minutes with rocking in an F12 with 1% insulin–transferrin selenium (Gibco, #41400045), incubator. Around 4 Â 106 cells in each condition were sorted 1% bovine pituitary extract (Hammond Cell Technology, #1078- on the basis of G1,S,andG2–M phases, respectively, using UV NZ), 10 mg/mL EGF (Corning, #354001), 1% penicillin/strepto- laser with MoFlo XDP70 (Beckman Coulter). Collected cells mycin (Thermo Fisher Scientific, #15140122), and 5% FBS were harvested and proteins were isolated. (Gemini Bio-Products,
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