PTEN-null Tumors Research Articles

PTEN loss shapes macrophage dynamics in high grade serous ovarian carcinoma.

High-grade serous ovarian carcinoma (HGSC) remains a disease of poor prognosis that is unresponsive to current immune checkpoint inhibitors. Although PI3K pathway alterations, such as PTEN loss, are common in HGSC, attempts to target this pathway have been unsuccessful. We hypothesized that aberrant PI3K pathway activation may alter the HGSC immune microenvironment and present a targeting opportunity. Single-cell RNA sequencing identified populations of resident macrophages specific to Pten-null omental tumors in murine models, which were confirmed by flow cytometry. These macrophages derived from peritoneal fluid macrophages and had a unique gene expression program, marked by high expression of the enzyme heme oxygenase-1 (HMOX1). Targeting resident peritoneal macrophages prevented the appearance of HMOX1hi macrophages and reduced tumor growth. Furthermore, direct inhibition of HMOX1 extended survival in vivo. RNA sequencing identified IL33 in Pten-null tumor cells as a likely candidate driver leading to the appearance of HMOX1hi macrophages. Human HGSC tumors also contained HMOX1hi macrophages with a corresponding gene expression program. Moreover, the presence of these macrophages correlated with activated tumoral PI3K/mTOR signaling and poor overall survival in HGSC patients. In contrast, tumors with low numbers of HMOX1hi macrophages were marked by increased adaptive immune response gene expression. These data suggest targeting HMOX1hi macrophages as a potential therapeutic strategy for treating poor prognosis HGSC.

Chemotherapy-induced PTEN-L secretion promotes the selection of PTEN-deficient tumor cells

BackgroundPTEN loss has been identified in various tumor types and is linked to unfavorable clinical outcomes. In addition to PTEN mutation, multiple mechanisms contribute to PTEN loss during tumor development. However, the natural selection process of PTEN-deficient tumor cells remains unclear. Here, we aimed at further elucidating the role of PTEN-L in tumor progression.MethodsPTEN knockout cell lines were generated using CRISPR/Cas9 technology. Ni-NTA affinity column chromatography was employed for PTEN-L purification. Tumor cell metastasis was evaluated in murine models and observed using the IVIS Spectrum Imaging System. RNA-sequencing, western blotting, PCR, flow cytometry, and cell proliferation assays were employed to investigate tumor cell dormancy and related mechanisms.ResultsThe chemotherapeutic drugs, cisplatin, paclitaxel, and doxorubicin, induced tumor cells to secrete PTEN-long (PTEN-L), which shields PTEN-deficient tumor cells from chemotherapy-induced apoptosis better than it shields PTEN-intact cells. Further investigation revealed that PTEN-L treatment induced dormancy in PTEN-null tumor cells, characterized by an increase in p16 and p27 levels, cell-cycle arrest, reduced cell proliferation, and enhanced DNA repair. Furthermore, PTEN-L treatment selectively promoted the accumulation and growth of PTEN-null tumor cells in the lungs of C57BL/6J mice, while evading immune surveillance. Mechanistically, PTEN-L induced dormancy in PTEN-null tumor cells by activating the p38 signaling pathway. Addition of a p38 inhibitor effectively reversed dormancy and growth of PTEN-deficient tumor cells in the lungs. We also demonstrated that PTEN expression played a pivotal role in determining the outcome of PTEN-L-mediated antitumor therapy.ConclusionsIn summary, PTEN-L was identified as a potent inducer of dormancy in PTEN-deficient tumor cells, which increased their efficient selection within the tumor microenvironment.

PI3Kβ controls immune evasion in PTEN-deficient breast tumours.

Loss of the PTEN tumour suppressor is one of the most common oncogenic drivers across all cancer types1. PTEN is the major negative regulator of PI3K signalling. The PI3Kβ isoform has been shown to play an important role in PTEN-deficient tumours, but the mechanisms underlying the importance of PI3Kβ activity remain elusive. Here, using a syngeneic genetically engineered mouse model of invasive breast cancer driven by ablation of both Pten and Trp53 (which encodes p53), we show that genetic inactivation of PI3Kβ led to a robust anti-tumour immune response that abrogated tumour growth in syngeneic immunocompetent mice, but not in immunodeficient mice. Mechanistically, PI3Kβ inactivation in the PTEN-null setting led to reduced STAT3 signalling and increased the expression of immune stimulatory molecules, thereby promoting anti-tumour immune responses. Pharmacological PI3Kβ inhibition also elicited anti-tumour immunity and synergized with immunotherapy to inhibit tumour growth. Mice with complete responses to the combined treatment displayed immune memory and rejected tumours upon re-challenge. Our findings demonstrate a molecular mechanism linking PTEN loss and STAT3 activation in cancer and suggest that PI3Kβ controls immune escape in PTEN-null tumours, providing a rationale for combining PI3Kβ inhibitors with immunotherapy for the treatment of PTEN-deficient breast cancer.

Abstract 1124: PTEN loss confers resistance to anti-PD-1 therapy in NSCLC by increasing tumor infiltration of T regulatory cells

Abstract Immune checkpoint inhibitors (ICIs) targeting PD-L1/PD-1 have dramatically improved the management of NSCLC patients. Nevertheless, more than 50% patients will present primary resistance or will develop secondary resistance within 12-25 months after treatment initiation. The mechanisms of resistance to ICIs may depend on tumor-intrinsic molecular/genetic alterations that generate an immunosuppressive tumor microenvironment (isTME). Using In silico analyses (TCGA and AACR-GENIE cohorts), and protein expression analysis in our institutional CIMA-CUN cohort, we have shown that >50% LUSC and ~28% LUAD tumors exhibited reduced PTEN mRNA/protein levels. Patients with PTEN-low tumors had higher levels of CXCL10, PD-L1 and PD-L2. In a cohort of stage IV NSCLC patients treated with ICIs, we found that patients with low tumor PTEN levels were significantly associated with worse OS (p=0.021). We have developed and characterized a Pten-null LUSC model using UN680 cells, which was also refractory to anti-PD-1 when implanted in syngeneic mice. Pten loss led to a deregulation of multiple pathways related to immunosuppression, with a positive enrichment in “IL-6/JAK/STAT3” and “TNF-α via NFkB” hallmarks, identified by GSEA. Tumors from the Pten-null LUSC model were highly metastatic and fibrotic, and secreted TGF-β/CXCL10, thus favoring conversion of CD4+ lymphocytes into Tregs. Human/mouse PTEN-low tumors were enriched in Tregs and immunosuppressive genes. Importantly, treatment of mice bearing Pten-null tumors with TLR agonists+anti-TGF-β, aimed to alter this isTME, led to tumor rejection and immunological memory in 100% of mice. Our results demonstrate that lack of PTEN causes immunotherapy resistance in LUSC by establishing an isTME that can be reversed therapeutically. Citation Format: Francisco Exposito, Miriam Redrado, Maeva Houry, Katherine Hastings, Magdalena Molero-Abraham, Teresa Lozano, Jose L. Solorzano, Julian Sanz Ortega, Vera Andradas, Ramon Amat, Esther Redin, Sergio Leon, Naroa Legarra, Javier García, Diego Serrano, Karmele Valencia, Camila Robles-Oteiza, Giorgia Foggetti, Enriqueta Felip, Juan J. Lasarte, Luis Paz-Ares, Jon Zugazagoitia, Katerina Politi, Luis M Montuenga, Alfonso Calvo. PTEN loss confers resistance to anti-PD-1 therapy in NSCLC by increasing tumor infiltration of T regulatory cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1124.

Abstract 5604: Targeting CD73 augments the efficacy of A2aR blockade in PTEN-deficient prostate cancer

Abstract Extracellular adenosine is an immunosuppressive molecule that is generated from ATP by the CD39 and CD73 ectoenzymes and exerts its antitumor effects through its interaction with the adenosine 2A receptor (A2AR) on immune cells. Approaches to curtail the immunosuppressive effects of extracellular adenosine primarily target CD73 and A2aR. In this study, we examined the immunomodulatory effects and antitumor effects of CD73 and A2aR blockade in the context of PTEN-deficient prostate cancer. The immunomodulatory effects of anti-CD73 antibody blockade (aCD73) or A2aR inhibition with the small molecule inhibitor AZD4635 were characterized in transgenic mice bearing Pten-null prostate tumors after one week of dosing. qPCR-based tumor immune profiling revealed distinct differences between the treatment groups that were characterized by the enrichment of gene signatures related to T cell, natural killer cell, and NK T cell in AZD4635 treated mice and macrophage and B cell in mice treated with aCD73. Immunohistochemical analysis showed significant 2.5- and 4.6-fold increases of granzyme B positive immune cells in the cancer glands of aCD73 and AZD4635 treated mice, respectively. Efficacy studies showed that tumors from mice after long-term treatment with AZD4635 were characterized with high adenosinergic signatures and particularly increased levels of Nt5e, the gene encoding CD73. Treatment with aCD73 and AZD4635 alone or in combination led to tumor burden reductions of 4.4%, 15.1% and 16.7%, respectively, after four weeks of treatment and -8.58%, 4.6% and 29.5%, respectively after eight weeks of treatment. Additional analyses showed that androgen deprivation therapy induced an adenosine high signature, and increased Nt5e expression levels and CD73-positive infiltrating MDSCs/neutrophils. The efficacy of AZD4635 versus aCD73 plus AZD4635 was evaluated in an in vivo castration-sensitive model of Pten-null prostate cancer treated with apalutamide plus androgen deprivation therapy via surgical castration. In this model, mice were treated for eight weeks to allow for the progression to castration-resistant prostate cancer (CRPC) and in this setting, the treatment combination of aCD73/AZD4635 improved tumor burden reduction to 34.3% from 23.1% in AZD4635 treated mice. Moreover 75% (6/8) of mice treated with aCD73/AZD4635 had tumor burden reductions greater than 30% relative to vehicle treated controls versus 28.6% (2/7) of mice in the AZD4635 only group. Our findings show that extracellular adenosine is a key immunosuppressive molecule in PTEN-deficient prostate cancer and provide evidence that implicates adenosinergic signaling via CD73 as a contributing factor to decreased efficacy of A2aR blockade and progression to CRPC. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Noriko Sako, Kazuko Sakai, Alwin Schuller, Kris Sachsenmeier, Kazutoshi Fujita, Eri Banno, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Targeting CD73 augments the efficacy of A2aR blockade in PTEN-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5604.

Abstract 5952: Curcumin monoglucuronide modulates the tumor microenvironment of Pten-null prostate tumors and exhibits antitumor activity

Abstract Curcumin is a naturally occurring compound found in turmeric and is well-known for its anticancer and anti-inflammatory properties, however, low solubility, poor absorption and fast clearance resulting in low systemic bioavailability has hindered therapeutic applications for free curcumin and has prompted the development of curcumin-based formulations with improved bioavailability. Curcumin monoglucuronide (CMG) is a water-soluble injectable prodrug that shows improved bioavailability over oral free curcumin and has demonstrated anticancer activity. In this study, we examine the therapeutic efficacy and immunomodulatory effects of CMG in mouse prostate cancer models driven by the conditional inactivation of Pten. The acute immunomodulatory effects of CMG were profiled in tumors from 28-week-old conditional Pten/Trp53 double knockout mice one-week after intraperitoneal dosing with CMG and were evaluated using a focused panel of qRT-PCR-based immune related genes, flow cytometry and immunohistochemistry (IHC). Overall, mice receiving CMG showed a higher inflammation profile compared to vehicle controls and was characterized with enrichments in macrophage, dendritic cell, and T cell gene signatures. CMG treated mice showed enhanced dendritic cell activity in tumors including an increased abundance of activated of migratory dendritic cells (CD11b+\CD11c+\XCR1+\MHC-II+\CD80+). Tumors from CMG-treated mice also contained increased T cell infiltration (including CD8+ and CD4+ T cells) as well as enrichment of genes related to T cell differentiation and activation and an increased abundance of CD4+\CD25+ regulatory T cells. While one week of dosing did not result in a significant reduction of tumor burden for CMG-treated mice, there was a 1.9-fold increase in cancer cell apoptosis as measures by cleaved caspase-3 IHC expression (P=0.004) and was correlated to leukocyte infiltration particularly in the ventral lobes of the prostate. The antitumor efficacy and long-term immunomodulatory effects of CMG were further evaluated in 16-week-old conditional Pten knockout after four weeks of dosing. In this model, tumor from mice treated with CMG showed a 10% reduction of tumor burden compared to vehicle controls (P=0.065). Tumors from CMG treated mice also had a 1.7-fold increase in apoptosis compared to vehicle treated mice (P=0.009) and tended to have reduced cancer cell proliferation compared to vehicle controls (P=0.148). Gene expression and flow cytometry analyses showed that tumors from mice treated with CMG for four weeks maintained improved tumor inflammation and showed an increased T cell infiltration over vehicle treated controls including effector cytotoxic T cells (CD8+\Granzyme B+). These findings provide preclinical evidence for the antitumor and immune modulatory activity of CMG in mouse hormone sensitive Pten-deficient prostate cancer. Citation Format: Yurie Kura, Marco A. De Velasco, Naomi Ando, Kazuko Sakai, Kazutoshi Fujita, Eri Banno, Yoshihiko Fujita, Mamoru Hashimoto, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Curcumin monoglucuronide modulates the tumor microenvironment of Pten-null prostate tumors and exhibits antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5952.

Abstract 3354: A gene expression signature of high serum cholesterol and prostate cancer outcomes

Abstract Statin use is associated with reduced risk of lethal prostate cancer potentially through effects on serum cholesterol. Using data from a prospective study with measured serum cholesterol among men subsequently diagnosed with prostate cancer, we developed a tumour gene expression score to reflect the pre-diagnosis serum cholesterol environment these tumours developed in, and applied this score to a separate study to examine the relationship with prostate cancer-specific outcomes. We identified 127 men with prostate cancer in the prospective Health Professionals Follow-up Study, whose prostate tumors underwent whole genome gene expression profiling. Pre-diagnosis serum cholesterol was measured a median of 3.3 years (IQR 1.5-4.8 years) prior to cancer diagnosis. We excluded men using statins either at time of blood draw for serum cholesterol measurement or at cancer diagnosis. Using tumor biopsy whole genome transcriptomic data from 232 prostate cancer patients treated with radiotherapy in Northern Ireland (NI), we generated the serum cholesterol score by summing z-score-transformed expression for each gene. PTEN status was measured using a published 50-gene transcriptional signature. Both signatures were dichotomized using the median cut-point. Age-adjusted and multivariable Cox regression analysis was used to compute HRs and 95% CI for associations of the cholesterol gene score with biochemical recurrence and metastasis risk. We identified in HPFS a set of 30 genes associated with pre-diagnostic serum cholesterol with a fold change ≥4 which included PTEN. In the NI Cohort, tumors with above median cholesterol scores had higher expression of the PTEN-null signature (p<0.001). Increased serum cholesterol tumor score was associated with higher Gleason grade (p<0.001); this relationship was stronger in PTEN-null tumours relative to PTEN-intact. Over a median follow-up of 11 years, 68 men experienced biochemical recurrence, and 28 developed metastasis. In age-adjusted analysis, patients with high serum cholesterol score had increased risk of recurrence (HR 1.84; 95% CI 1.13-2.99) and metastasis (HR 1.98; 95% CI 0.91-4.29), though the latter was not statistically significant. Associations with recurrence and metastasis were attenuated following further adjustment for Gleason score (HR 1.65; 95% CI, 0.98-2.77 and HR 1.49; 95% CI 0.66-3.35, respectively). Men with increased tumor gene expression of a serum cholesterol score, derived to reflect tumor development within a high serum cholesterol environment, were more likely to be diagnosed with a PTEN-null prostate cancer. These men had more aggressive prostate cancer at diagnosis and increased risk of recurrence, particularly men with PTEN-null disease. Our results support recent findings showing a reduced risk of PTEN-null prostate cancer in statin users, and inform our understanding of the potential role of serum cholesterol in prostate cancer etiology. Citation Format: Sarah Jane Winter, Sophia R. Halliday, Travis A. Gerke, Lorelei Ann Mucci, Konrad H. Stopsack, Gillian Prue, Suneil Jain, Emma H. Allott. A gene expression signature of high serum cholesterol and prostate cancer outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3354.

Correlation of ABI1 and PTEN expression during prostate tumor progression.

172 Background: Mechanisms of tumor invasion are not well defined. PTEN, a key tumor suppressor frequently inactivated in epithelial cancers, acts as a central node that controls tumor invasion. Despite PI-3 kinase-phospho-Akt pathway activation resulting in enhanced tumor growth, prostate tumors with PTEN loss undergo p53-mediated senescence that restricts tumor invasion. Methods: ABI1 downregulation is associated with epithelial-mesenchymal transition in highly invasive prostate tumors; these tumors frequently loose PTEN; therefore we set to examine genetic interaction of ABI1 and PTEN using novel mouse model of prostate cancer. We analyzed the correlation of ABI1 and PTEN expression in human PCa tumor tissue. Results: Here, using Abi1/Pten KO mouse model we identified a novel mechanism that guards tumor invasion. In Pten-null tumors upregulation of Abi1 leads to sequestration of activated Src kinase. In the absence of Abi1, this regulation is lost leading to activation of non-canonical WNT-SRC-STAT3 axis and enhanced invasion through activation of MMP2 activity. This molecular mechanism explains progression of tumors with Pten loss from PIN to invasive carcinoma upon concomitant Abi1 inactivation. In human tumors with low Abi1 and Pten are associated with aggressive phenotype, biochemical recurrence and metastasis. Conclusions: ABI1 acts as failsafe mechanism in PTEN null tumors by restricting SRC-mediated tumor invasion. ABI1 might have a predictive value in clinical setting in context of PTEN levels.

SO-10 An open-label, multi-centre, phase Ib/II study of PI3Kβ selective inhibitor GSK2636771 administered in combination with paclitaxel in patients with advanced gastric cancer having alterations in PI3K/Akt pathway

Loss of PTEN expression accompanied with activation of PI3K/AKT pathway commonly occurs in gastric cancer. GSK2636771 is a potent, orally bioavailable, adenosine triphosphate-competitive selective inhibitor of PI3Kβ with minimal off-target effects. Here, we conducted a dose-finding study with safety and efficacy analysis of GSK2636771 combined with paclitaxel in patients with advanced gastric cancer (AGC) who progressed from first-line chemotherapy. In this multi-center phase Ib/II study, patients with PTEN-deficient AGC who failed to respond to first-line chemotherapy with fluoropyrimidine and platinum were eligible. PTEN expression was evaluated by immunohistochemistry and the PTEN-deficient was defined by < 100 of PTEN histoscore (PTEN H-score). A standard 3+3 design included two dose levels of GSK2636771 (300mg once daily and 200mg once daily) in combination with paclitaxel (80mg/m2 on days 1, 8, and 15) during phase Ib study to determine the recommended phase II dose (RP2D). Phase II study was subsequently conducted based on the RP2D derived from phase Ib study to assess safety and antitumor activity. Primary objectives included defining RP2D and assessment of progression-free survival (PFS) in patients administered with RP2D. Secondary objectives included overall survival (OS), objective response rate (ORR), disease control rate (DCR), safety, and biomarker analysis incorporating investigation of PTEN H-score (0 vs. 1-99) and somatic alteration of cancer by next-generation sequencing (NGS). This trial is registered with ClinicalTrials.gov, number NCT02615730. A total of 42 patients were enrolled, median age was 59 years old (range: 32-88 years old) and most patients were male (34/42). During phase Ib study, grade 3 hypocalcemia was observed in 2 out of 5 patients with GSK2636771 300mg once daily combined with paclitaxel. The RP2D was GSK2636771 200mg once daily combined with paclitaxel (80mg/m2 on days 1, 8, and 15). The median follow-up duration of 29.9 months [95% confidential interval (CI), 21.5-38.3 weeks]. Among 37 patients treated with RP2D with and 33.4 weeks (95% CI, 26.2-40.7 weeks), median PFS and OS were 12.1 (95% CI, 11.1-13.2 weeks) and 33.4 weeks (95% CI, 26.2-40.7 weeks), respectively. Among 28 patients with measurable disease and administered with RP2D, confirmed response and disease control was observed in 5 patients (ORR=17.9%) and 19 patients (DCR=67.9%), respectively. Patients with PTENnull tumors (H-score 0) exhibited favorable PFS (18.9 vs. 11.6 weeks, P=0.026) compared to those with PTENpartial loss tumors (H-score 1-99). Most common any grade of adverse events (AEs) were neutropenia (38.1%), peripheral neuropathy (28.8%), anorexia (21.4%), diarrhea (16.7%), and myalgia (14.3%). No treatment related death or unexpected AEs resulting in treatment cessation were observed with the RP2D. GSK2636771 combined with paclitaxel revealed manageable toxicities and antitumor activity in patients with PTEN-deficient AGC who progressed after first-line chemotherapy. Complete loss of PTEN expression may be associated with clinical benefit from GSK2636771 and paclitaxel. Further analysis of the association with biomarkers and clinical outcomes is underway.

Abstract PS18-13: Men1611 is a pi3k α/β selective and δ sparing inhibitor with long-lasting antitumor activity in different genetic backgrounds of pik3ca mutant breast cancer models

Abstract Dysregulation of the PI3K/AKT/mTOR pathway has the potential to trigger the activation of class I phosphoinositide 3-kinases (PI3Ks) and eventually lead to cancer. Class I PI3Ks are heterodimers composed of the regulatory subunit p85 and the catalytic subunit p110. There are four isoforms of p110, alpha and beta, important in solid tumors, and delta and gamma, preferentially expressed in leukocytes, and drivers in hematological malignancies. While activation of PI3K p110alpha is the most frequent oncogenic event in solid tumors, p110beta plays an important role in PTEN deficient cancers and in mediating resistance to p110alpha selective PI3K inhibitors.MEN1611 is a PI3K inhibitor active on p110alpha (both mutants and wt), beta and gamma (8.6- and 2.2- fold less potent compared to the alpha, respectively), while sparing the delta isoform (36-fold less potent compared to the alpha). Thus, MEN1611 differs from other selective PI3K inhibitors such as alpelisib (p110alpha selective) and taselisib (p110beta sparing). MEN1611 is currently in clinical development for patients with HER2 positive advanced or metastatic breast cancer in combination with trastuzumab with/without fulvestrant (B-PRECISE-01). Patient-derived xenograft (PDX) models and breast cancer cell lines with different genetic background were used to assess the antitumor activity of MEN1611 in p110 alpha- and beta- dependent tumors. Healthy-donors derived CD69+ B-cells, which are primarily dependent on p110 delta for proliferation and survival were used to test the activity of the compound against the p110delta. According to the biochemical selectivity, MEN1611 demonstrated a lower cytotoxic potential in a p110delta-driven cellular model (CD19+ B-cells), when compared to taselisib (&amp;gt;150-fold) and an improved cytotoxic activity in the p110beta-driven cellular model (PTEN-null breast cancer cells) when compared to alpelisib. Moreover, MEN1611 selectively decreases the p110alpha protein levels in PIK3CA mutated breast cancer cells in a concentration-dependent manner, with a corresponding reduction in pAKT. In vivo, MEN1611 monotherapy showed significant and long-lasting anti-tumor activity, reflected in tumor-stasis or tumor regression in several trastuzumab-resistant PIK3CA-mutant HER2 positive PDXs with different concomitant genomic aberrations. The combination of trastuzumab and MEN1611 significantly improved the efficacy compared to single agent treatment. Interestingly, MEN1611 monotherapy showed a significant tumor volume inhibition in a PTEN-null breast cancer PDX model. Overall, our data provide evidence that MEN1611 is active against HER2 positive and PTEN-null breast tumors. Moreover, its delta-sparing profile may have potential implications on MEN1611 tolerability and ability to overcome resistance to p110alpha selective inhibitors. Citation Format: Alessio Fiascarelli, Corrado Carrisi, Giuseppe Merlino, Stefania Capano, Diego Bisignano, Simone Talucci, Alessandro Bressan, Daniele Bellarosa, Mario Bigioni, Maurizio Scaltriti, Joaquin Arribas, Andrea Pellacani, Massimiliano Salerno, Monica Binaschi. Men1611 is a pi3k α/β selective and δ sparing inhibitor with long-lasting antitumor activity in different genetic backgrounds of pik3ca mutant breast cancer models [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-13.

Abstract 3521: Transcriptome analysis of TCGA prostate cancer samples identifies an association of poorer survival and aggressive disease biology with CDC-like kinase (CLK) expression and spliceosome regulation

Abstract In prostate cancer, alternative splicing of mRNA and spliceosome activity are implicated in several areas of disease pathogenesis. This is exemplified by the strong association of androgen receptor splice variants with treatment resistance and poor clinical outcome in castration-resistant disease. Therefore, pharmacologic targeting of spliceosome-regulating proteins such as CLKs and serine/arginine-rich splicing factors (SRSFs) represents a novel treatment approach for prostate cancer. To evaluate the therapeutic potential of inhibiting CLK activity in prostate cancer, the association between splicing-related gene expression and survival was investigated in The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) data collection (N=495). Survival analysis of RNA-seq data assessed 17,879 genes to measure their association with progression-free interval (PFI). Using transcript per million as the metric for normalized gene expression, age-adjusted Cox proportional hazards regression models were performed for each gene (R v3.6.0, coxph v2.43-3). A total of 3,145 genes significantly correlated with worse prognosis (P-adj&amp;lt;0.10, Cox coefficient &amp;gt;0). CLK1 (P-adj=0.0218, HR=1.5939), CLK2 (P-adj=0.001298, HR=2.1393), and SRSF2 (P-adj=0.00167, HR=3.2917) were found to be positively associated with poorer PFI, ranking 1202, 400, and 437, respectively. Reactome pathway analysis of the significant gene set showed that mRNA splicing and processing accounted for 5 of the 19 pathways that were strongly associated with poorer PFI. An additional pathway analysis (GSEA v.3.0, MSigDB v6.2) of tumors categorized by PTEN status to assess relationship with disease severity showed that mRNA splicing (P-adj=0.0243, NES=1.7714) was enriched in PTEN-null vs. PTEN-wt tumors. Other pathways of interest, including Wnt signaling (P-adj=0.0187, NES=1.846), cell cycle (P-adj=0.0124, NES=1.974), chromatin remodeling (P-adj=0.0135, NES=1.901), DNA damage repair (P-adj=0.013974, NES=1.8934), and PTEN regulation (P-adj=0.0230, NES=1.7861), were also enriched in PTEN-null tumors. Lastly, a survival analysis within all TCGA-PRAD patients showed that low CLK1 (P=0.03) and CLK2 (P=0.0004) expression, individually, were associated with a better prognosis vs. their high-expressing counterparts. Analysis of CLK3 and CLK4 expression did not reach statistical significance. Collectively, these findings revealed an association of spliceosome activity and CLK1/2 expression with aggressive disease biology in prostate cancer. A Phase 1 study of SM08502, a novel, small-molecule pan-CLK inhibitor, in subjects with advanced solid tumors is ongoing (NCT03355066). This analysis nominates prostate cancer as a tumor type worth further exploring for the clinical activity of SM08502. Citation Format: Shawn Cho, Atish D. Choudhury, Catherine Fleener, Long Do, Carine Bossard, Heekyung Chung, Timothy J. Phalen, Steven Cha. Transcriptome analysis of TCGA prostate cancer samples identifies an association of poorer survival and aggressive disease biology with CDC-like kinase (CLK) expression and spliceosome regulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3521.

Abstract 2535: Screening PTEN-loss associated lncRNAs in prostate cancer

Abstract Phosphatase and tensin homologue (PTEN) is a tumor suppressor gene that is frequently inactivated by deletion in prostate cancer (PCa). Occurring in around 20% of primary PCa tumors, and up to 50% in castration resistant tumors, it is the most frequent genomic aberration in PCa. Loss of PTEN activates the phosphoinositide 3-kinase-RAC-alpha serine/threonine-protein kinase (PI3K-AKT) pathway, which is associated with poor clinical outcomes. Despite the consequences of PTEN loss being well studied, most of what is known is restricted to protein-coding genes, with relatively little information about the role of non-coding genes. Using our recently created resource - the FC-R2 expression atlas, which encompasses expression levels for thousands of lncRNAs recently unveiled by the FANTOM consortium - we analyzed differential gene expression of PTEN-null vs PTEN-intact tumors with the goal of characterizing the molecular landscape of PTEN loss. First, we generated a consensus signature using two large PCa cohorts with experimentally validated PTEN status by Immunohistochemistry (IHC), applying a meta-analysis approach. This signature encompassed mainly protein coding genes due it being microarray based. In order to expand this signature beyond the coding genes, we relied on FC-R2-based TCGA-PRAD data. Since PTEN status was not available by IHC, we opted to call the status based on CNV. Then, we proceed to generate a PTEN-null signature using a generalized linear model approach. Both signatures were compared for concordance using correspondence-at-the-top plots and hypergeometric confidence intervals. Gene set enrichment analysis was performed in both signatures using a collection of obtained from the MSigDB database in order to characterize pathways involved in this event. Our results showed that the signature based on IHC validated samples agreed significantly with the CNV-based signature from TCGA for the genes in common. In the differential gene expression analysis on the TCGA cohort we observed 203 significant coding genes and 171 significant non-coding genes (FDR ≤ 0.01, LogFC ≥ 1). Notably, we identified several lncRNAs that have not been associated with PCa or PTEN loss, these include many classes of non-coding RNAs characterized by the FANTOM consortium such as: enhancers and promoters genes. Gene set enrichment analysis revealed that PTEN-null tumors are associated with epithelial-mesenchymal transition suggesting a possible role for these lncRNAs. In conclusion, by leveraging our resources, we were able to obtain comprehensive landscape of the PTEN loss in PCa for both the coding and non-coding counterpart. Furthermore, the association of many lncRNAs with PTEN loss was observed, many recently annotated by the FANTOM consortium, which can help us understand how genes are regulated in this event. In this work we show that despite being widely studied, there are still many components of PTEN loss in the form of lncRNAs highlighting potential markers for PTEN loss and clinical outcomes. Citation Format: Eddie Luidy Imada, Diego Fernando Sanchez, Wikum Dinalankara, Tamara Lotan, Luigi Marchionni. Screening PTEN-loss associated lncRNAs in prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2535.

The Mechanisms Underlying PTEN Loss in Human Tumors Suggest Potential Therapeutic Opportunities.

In this review, we will first briefly describe the diverse molecular mechanisms associated with PTEN loss of function in cancer. We will then proceed to discuss the molecular mechanisms linking PTEN loss to PI3K activation and demonstrate how these mechanisms suggest possible therapeutic approaches for patients with PTEN-null tumors.

A basal-enriched microRNA is required for prostate tumorigenesis in a Pten knockout mouse model.

MicroRNAs (miRNAs) play important roles in prostate cancer development. However, it remains unclear how individual miRNAs contribute to the initiation and progression of prostate cancer. Here we show that a basal layer-enriched miRNA is required for prostate tumorigenesis. We identify miR-205 as the most highly expressed miRNA and enriched in the basal cells of the prostate. Although miR-205 is not required for normal prostate development and homeostasis, genetic deletion of miR-205 in a Pten null tumor model significantly compromises tumor progression and does not promote metastasis. In Pten null basal cells, loss of miR-205 attenuates pAkt levels and promotes cellular senescence. Furthermore, although overexpression of miR-205 in prostate cancer cells with luminal phenotypes inhibits cell growth in both human and mouse, miR-205 has a minimal effect on the growth of a normal human prostate cell line. Taken together, we have provided genetic evidence for a requirement of miR-205 in the progression of Pten null-induced prostate cancer.

Phosphatase and tensin homolog deleted on chromosome ten deficiency sensitizes tumor cells to lithium chloride treatment

Objective: To identify the therapeutic efficacy of lithium chloride (LiCl) on phosphatase and tensin homolog deleted on chromosome ten (PTEN)-deficient tumors. Methods: First, the Catalogue of Somatic Mutations in Cancer for mutation spectrum of human endometrial carcinoma samples was analyzed. Second, the relationship between PTEN abundance and LiCl inhibition of endometrial cancer cell lines using Pten(+/+) and Pten(-/-) mouse embryonic fibroblast (MEF) lines was investigated. Moreover, potential alterations of mammalian target of rapamycin (mTOR) signaling pathway after treatment with LiCl were checked.Last,LiCl's efficacy on PTEN null tumors was studied. Results: PTEN was mutated in 39% of endometrial carcinomas. LiCl preferentially inhibited the proliferation of PTEN-deficient endometrial carcinoma cells and MEFs. Furthermore, LiCl blocked PTEN-deficient tumor development. Mechanistically, LiCl down-regulated mTOR signaling. Conclusions: PTEN is the most frequently mutated gene in endometrial carcinoma.By targeting mTOR signaling pathway,LiCl is a promising regimen for the treatment of tumors with PTEN deficiency.

Abstract 4621: Developing genetically modified homograft models of mouse prostate cancer for efficacy evaluation of combinatory immunotherapies

Abstract Despite the early approval of Sipuleucel-T for metastatic castration-resistant prostate cancer, which often perceived as a milestone achievement in cancer immunotherapy, subsequent progress in prostate cancer immunotherapy development has been limited by disappointing results with tumor vaccines and its resistance to immune checkpoint inhibitors, such as PD1 &amp; PD-L1. It is now generally accepted that we need to tackle prostate cancer by combinatorial approaches of chemo-, targeted- and immuno-therapies. Highly relevant preclinical models are very much needed for proof of principle efficacy evaluation. Genetically engineered mouse models (GEMM) recapitulate key aspects of human prostate cancer in both histo- &amp; molecular- pathology. Among those, PTEN loss of function (LOF) in prostate epithelium is one of the central events in human prostate cancer. PTEN haploinsufficiency in mice is sufficient to drive mouse prostatic intraepithelial neoplasia (PIN) formation, while loss of both alleles of PTEN in mouse prostate leads to hyperplasia at 4 weeks, PIN at 6 weeks, and fully invasive adenocarcinoma from 12 weeks of age. PTEN null tumors are also resistant to androgen depletion. In the meantime, although KRAS mutation are not often seen in human prostate cancer, activation of MAPK pathway often happens in advanced tumor. Mutant KRAS or BRAF can robustly promote mouse prostate cancer progression. Prostate specific Pten null and Kras G12D; Pten null mouse prostate cancer models have been well characterized by a number of labs. However, parental GEMM models are difficult to be used for pharmacological studies due to the spontaneous nature of tumor onset and progression. The compound mutant mice are also costly to breed. Here we report generation of transplantable mouse prostate cancer homograft models by passaging the primary tumor subcutaneously in the C57BL/6 mice. These mouse tumors featuring Pbsn-Cre;LSL-KrasG12D/+;Ptenflox/flox or Pbsn-Cre;Ptenflox/flox retain morphological similarity to moderate to poorly differentiated human prostate cancer. These homograft tumors show high levels of M2 macrophage infiltration. Growth of these mouse prostate tumors are resistant to androgen depletion. They are also resistant to the treatment of AR inhibitor enzalutamide, only marginally responsive to docetaxel, but sensitive to treatments with mTOR inhibitor everolimus. When treated with immune checkpoint antibodies, i.e. PD1 and CTLA4, these tumors showed increased T-cell infiltration, but very modest growth inhibition. We are now testing a variety of combinatory therapies with chemotherapies, immune modulators and immune checkpoint antibodies. The results will be presented at the meeting. Citation Format: Jessie Jingjing Wang, Yanrui Song, Annie Xiaoyu An, Likun Zhang, Henry Qixiang Li, Davy Xuesong Ouyang. Developing genetically modified homograft models of mouse prostate cancer for efficacy evaluation of combinatory immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4621.

Abstract 3431: PI3Kbeta regulates macropinocytosis in PTEN-null tumor cells

Abstract Class I phosphoinositide 3-kinases (PI3Ks) have been previously implicated in the regulation of vesicular trafficking, including receptor-mediated endocytosis and macropinocytosis. Using NIH3T3 cells in which the genes for the PI3Kbeta or PI3Kalpha catalytic subunits were deleted by CRISPR/Cas9, we were unable to detect any defects in receptor-mediated endocytosis of [125I]-PDGF or in pinocytosis of Lucifer yellow. However, we observed a marked inhibition of macropinocytosis in PI3Kbeta knockout cells; deletion of the other major PI3K isoform in these cells, PI3Kalpha, had no effect. These studies were confirmed using isoform-selective inhibitors: only inhibition of PI3Kbeta blocked macropinocytosis. Similarly, in MDA-MB-231 breast cancer cells, HGF-stimulated macropinocytosis was markedly reduced by inhibition of PI3Kbeta, but not the other PI3K isoforms. Notably, expression of a mutant PI3Kbeta that cannot be activated by Gbeta-gamma, or treatment of WT cells with pertussis toxin, also inhibited macropinocytosis in MDA-MB-231 cells. A requirement for Gbeta-gamma binding to PI3Kbeta was also seen in primary macrophages from mice expressing WT or Gbeta-gamma-uncoupled PI3Kbeta, as both CSF-1 and C5a-stimulated macropinocytosis was inhibited by mutant PI3Kbeta. To define the mechanism by which PI3Kbeta regulates macropinocytosis, we measured circular dorsal ruffle (CDR) formation. Genetic ablation or inhibition of PI3Kbeta completely suppressed CDR formation in PDGF-stimulated NIH3T3 cells, whereas inhibition or ablation of other PI3K isoforms had no effect. While previous studies have suggested that Class I PI3Ks act late in macropinosome formation, just prior to macropinosome sealing, our data suggest that PI3Kbeta functions early in macropinocytosis, as it mediates CDR formation in a Gbeta-gamma-dependent manner. Given previous work showing a positive feedback loop involving PI3Kbeta and Rac1, we tested whether macropinosome formation was rescued by expression of CA-Rac1. CA-Rac1 expression potently stimulated macropinocytosis in MDA-MB-231 cells. Strikingly, this was completely blocked by expression of the Gbeta-gamma-uncoupled PI3Kbeta, and partially blocked by expression of a mutant PI3Kbeta that cannot bind Rac1. Finally, given the well established role of PI3Kbeta in the growth of tumor cells lacking the PTEN tumor suppressor, as well as recent studies showing that high rates of basal macropinocytosis can serve as a nutrient a uptake mechanism in PTEN null cells, we measured macropinocytosis in two PTEN-deficient tumor lines, BT549 (breast) and PC3 (prostate). Both lines showed elevated basal levels of macropinocytosis, which were specifically blocked by inhibition of PI3Kbeta. Our data support a highly specific role for PI3Kbeta in the regulation of macropinocytosis, which may contribute the role of PI3Kbeta in the growth of PTEN-null tumor cells. Citation Format: Gilbert Salloum, Charles Jakubik, Anne R. Bresnick, Jonathan M. Backer. PI3Kbeta regulates macropinocytosis in PTEN-null tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3431.

Combined Inhibition of PI3Kβ and mTOR Inhibits Growth of PTEN-null Tumors

Loss of the tumor suppressor PTEN confers a tumor cell dependency on the PI3Kβ isoform. Achieving maximal inhibition of tumor growth through PI3K pathway inhibition requires sustained inhibition of PI3K signaling; however, efficacy is often limited by suboptimal inhibition or reactivation of the pathway. To select combinations that deliver comprehensive suppression of PI3K signaling in PTEN-null tumors, the PI3Kβ inhibitor AZD8186 was combined with inhibitors of kinases implicated in pathway reactivation in an extended cell proliferation assay. Inhibiting PI3Kβ and mTOR gave the most effective antiproliferative effects across a panel of PTEN-null tumor cell lines. The combination of AZD8186 and the mTOR inhibitor vistusertib was also effective in vivo controlling growth of PTEN-null tumor models of TNBC, prostate, and renal cancers. In vitro, the combination resulted in increased suppression of pNDRG1, p4EBP1, as well as HMGCS1 with reduced pNDRG1 and p4EBP1 more closely associated with effective suppression of proliferation. In vivo biomarker analysis revealed that the monotherapy and combination treatment consistently reduced similar biomarkers, while combination increased nuclear translocation of the transcription factor FOXO3 and reduction in glucose uptake. These data suggest that combining the PI3Kβ inhibitor AZD8186 and vistusertib has potential to be an effective combination treatment for PTEN-null tumors. Mol Cancer Ther; 17(11); 2309-19. ©2018 AACR.

833P - AZD8186, a potent and selective inhibitor of PI3Kβ/δ, as monotherapy and in combination with abiraterone acetate plus prednisone (AAP), in patients (pts) with metastatic castrate-resistant prostate cancer (mCRPC)

Background: The PI3K pathway plays an important role in cell growth and survival of PTEN-null tumours. An ongoing phase 1/2 study (NCT01884285) previously reported that AZD8186, a potent and selective inhibitor of PI3Kβ (minimal PI3Kδ activity), can be well tolerated in pts with solid tumours; here we present preliminary data in heavily pretreated mCRPC. Methods: Pts with mCRPC received escalating doses of AZD8186 (5 days on, 2 days off; 2 days on, 5 days off; or continuous schedules) as monotherapy (study Part A) or in combination with AAP (1000 mg qd + prednisone 10 mg qd, study Part C1) until progressive disease (PD) or dose-limiting toxicities. Analyses included tolerability, RECIST tumour response, prostate-specific antigen response, circulating tumour cell counts and cell-free DNA. Results: Fifty-two pts with mCRPC were treated with AZD8186 as monotherapy (n = 39) or in combination with AAP (n = 13). Prior treatment status: AAP (n = 14), enzalutamide (enza, n = 10), both (n = 21) or AAP/enza-naive (n = 7). Diarrhoea (39%) and nausea (27%) were the most frequently reported adverse events (AEs, all grades) related to AZD8186. Grade 3 AEs of interest included diarrhoea/colitis (10%), which was fully reversible with dose interruption/SoC treatment, and rash (7%). Two (4%) pts had grade 4 AEs (thrombocytopenia, hypokalaemia); no grade 5 AEs. AZD8186 did not appear to alter tolerability of AAP. Among pts with RECIST measurable disease, one had a confirmed partial response (Part C1), 10 had stable disease, nine had PD. Nine (17%) pts had reduction in PSA >30%. Twelve pts completed >16 weeks of treatment, five pts >24 weeks (PTEN-proficient [n = 0], PTEN-deficient [n = 3], PTEN-unknown [n = 2]). Recruitment of pts with PTEN-deficient mCRPC into an expansion phase in combination with AAP is ongoing. Conclusions: Data from this phase 1/2 study indicates that the tolerability of AZD8186 supports combination treatment with AAP in pts with metastatic prostate cancer. Preliminary evidence of antitumour activity has been observed. Updated results will be presented. Clinical trial identification: NCT01884285. Editorial acknowledgement: Medical writing and editorial assistance was provided by Tyrone Daniel from Bioscript Medical (Macclesfield, UK) and funded by AstraZeneca. Legal entity responsible for the study: AstraZeneca. Funding: AstraZeneca. Disclosure: J.S. de Bono: Advisory board member: AZ, GSK, Merck Serono, MSD, Sanofi Aventis, Taiho, Genentech-Roche, Seattle Genetics, Bayer, Endocyte, Shattuck, Harpoon, Pfizer Oncology, Janssen, Menarini, Daiichi; Research support: GSK, Sanofi, Genentech, Daiichi, Taiho, Merck Serono, MSD, Bayer, AZ. A. Hansen: Research support: Genentech/Roche, Merck, GlaxoSmithKline, Bristol Myers Squibb, Novartis, Boston Biomedical, Boehringer-Ingelheim, Karyopharm. A.D. Choudhury: Honoraria: Bayer, Astellas, Janssen. C. Higano: Advisory boards and/or steering committees: Astellas, Bayer, Blue Earth Diagnostics, Clovis Oncology, Ferring, Janssen, Myriad, and Tolmar; Institutional research funding: Aptevo, Aragon, Astellas, AstraZeneca, Bayer, Hoffman LaRoche, Janssen, Medivation, Pfizer; Spouse is a co-founder of, and has a leadership role at, CTI Biopharma. D.E. Rathkopf: Advisory/consulting fees: Janssen; Research funding for institution: AstraZeneca, Astellas Pharma, Celgene, Genentech, Janssen, Medivation, Millenium, Novartis, Pfizer, Roche, Taiho, Tracon. K.B. Wisinski: Advisory board: AstraZeneca. S. Barry, E. de Bruin, W. Brugger, S. Colebrook, T. Klinowska, P.G.S. Mortimer: Employment and stock ownership: AstraZeneca. M. Moschetta: Employee: AstraZeneca. L.L. Siu: Honoraria: AstraZeneca/MedImmune. G. Shapiro: Consulting/advisory relationship: Eli Lilly, Pfizer, G1 Therapeutics, Merck/EMD Serono, Roche, Almac; Research funding: Lilly, Pfizer, Merck/EMD Serono. All other authors have declared no conflicts of interest.

Abstract B043: Cancer stem cell selection drives adverse response to androgen-deprivation therapy

Abstract Purpose: The dramatic responses tumors display to targeted therapies are frequently limited by acquired or pre-existing mechanisms of therapy resistance. In prostate cancer (PCa), we found that targeted androgen receptor signaling blockade by the androgen antagonist enzalutamide (Enz) paradoxically enhanced metastasis. In mouse models, these prometastatic effects are mediated by the chemoattractant CCL2. We have previously demonstrated that androgen blockade-dependent induction of CCL2 is regulated by TNF signaling. Thus, we asked whether TNF mediates the prometastatic effects of Enz. Further, since androgen-deprivation therapy (ADT) selectively increases stem/progenitor (S/P) cell populations in PCa, we asked whether the increase in TNF expression following ADT is due to selection for a TNF-expressing prostate cancer S/P cell population. Methods: PCa and microenvironment-derived cell lines were employed as in vitro model systems. These include the human (LNCaP, C4-2) and murine (P8, CaP8) PCa cell lines and their castration-resistant prostate cancer (CRPC) and immortalized macrophage-like (THP1) and the stromal myofibroblast (WPMY1) cell lines. Following Enz and anti-TNF treatment, cytokine mRNA levels and protein secretion from CRPC and microenvironment cells was measured by Q-RT-PCR and ELISA, respectively. PCa expression of S/P cell markers following Enz treatment was analyzed by immunofluorescence. S/P cells were sorted by fluorescence-activated cell sorting (FACS) and captured. Cytokine secretion from S/P and “bulk” (more differentiated) tumor cell populations were measured by ELISA. S/P cell phenotype was validated by soft agar colony assay. CRPC and macrophage migration/invasion were measured in transwells. Cytokines in PTEN-null tumors at various times post-castration were measured by ELISA. Macrophage infiltration into these tumors was analyzed by immunohistochemistry using an F4/80 antibody. Results: Androgen withdrawal or Enz induced TNF mRNA and protein secretion in human CRPC (C4-2) cells. Enz induced autocrine CCL2 expression in C4-2 as well as a murine CRPC cell line PTEN-CaP8, while exogenous TNF induced CCL2 in THP1 and WPMY1 cells. TNF was most potent in myofibroblast cultures, suggesting that ADT induces CCL2 via paracrine interactions within the tumor microenvironment. A soluble TNF receptor (etanercept) blocked Enz-induced CCL2 secretion and mRNA expression in CRPC cells, implying dependence on secreted TNF. Etanercept inhibited Enz-induced migration and invasion of C4-2. Oncomine and GEO analysis of gene expression in primary PCa and metastatic CRPC datasets showed coordinate upregulation of both TNF and an S/P cell marker (integrin a6) in CRPC, relative to castration-sensitive primary PCa. Consistent with the Oncomine data, we found Enz increased the fraction of S/P marker (CD166 and integrin a6) expressing cells in C4-2 and LNCaP, with time. In parallel, TNF and CCL2 were also induced in these cultures, suggesting that the increase in TNF expression that occurs following ADT may be due to selection for a TNF-expressing S/P cells. Indeed, TNF and CCL2 secretion from sorted CD166+ C4-2 cells was significantly higher than from CD166- cells. Coculturing CD166+ cells with WPMY1 cells led to higher CCL2 secretion and more C4-2 and THP1 cell migration relative to CD166- cells. Consistent with this in vitro data, TNF and CCL2 expression, along with macrophage infiltration, was significantly elevated in PTEN-null tumors 35-day post-castration. Conclusions: ADT induces TNF production by selecting for a TNF-expressing S/P cell-like population. TNF signaling then mediates ADT induction of CRPC metastasis via CCL2. Since anti-TNF therapies are safe and FDA approved, combining ADT with anti-TNF therapy might reduce resistance (metastasis) while allowing continued tumor control. Citation Format: Kai Sha, Lingxiang Jiang, Chawnshang Chang, Dean Tang, Kent L. Nastiuk, John J. Krolewski. Cancer stem cell selection drives adverse response to androgen-deprivation therapy [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B043.