Carly J. King

Src and STAT3 inhibitors synergize to promote tumor inhibition in renal cell carcinoma

The intracytoplasmic tyrosine kinase Src serves both as a conduit and a regulator for multiple processes required for the proliferation and survival cancer cells. In some cancers, Src engages with receptor tyrosine kinases to mediate downstream signaling and in other cancers, it regulates gene expression. Src therefore represents a viable oncologic target. However, clinical responses to Src inhibitors, such as dasatinib have been disappointing to date. We identified Stat3 signaling as a potential bypass mechanism that enables renal cell carcinoma (RCC) cells to escape dasatinib treatment. Combined Src-Stat3 inhibition using dasatinib and CYT387 (a JAK/STAT inhibitor) synergistically reduced cell proliferation and increased apoptosis in RCC cells. Moreover, dasatinib and CYT387 combine to suppress YAP1, a transcriptional co-activator that promotes cell proliferation, survival and organ size. Importantly, this combination was well tolerated, and caused marked tumor inhibition in RCC xenografts. These results suggest that combination therapy with inhibitors of Stat3 signaling may be a useful therapeutic approach to increase the efficacy of Src inhibitors.

LSD1 activates a lethal prostate cancer gene network independently of its demethylase function

Significance Medical castration or interference with androgen receptor (AR) function is the principal treatment for advanced prostate cancer. However, progression is universal, and therapies following the emergence of castration resistance do not offer durable control of the disease. Lysine-specific demethylase 1 (LSD1) is an important regulator of gene expression, including in cancer. Here, we show that LSD1 is highly expressed in tumors of patients with lethal castration-resistant prostate cancer (CRPC) and that LSD1 promotes AR-independent survival in CRPC cells in a noncanonical, demethylase-independent manner. We determined that the drug SP-2509 acts as an allosteric inhibitor of LSD1–blocking demethylase-independent functions. Our demonstration of tumor suppression with this inhibitor in CRPC preclinical models provides the rationale for clinical trials with LSD1 inhibitors. Medical castration that interferes with androgen receptor (AR) function is the principal treatment for advanced prostate cancer. However, clinical progression is universal, and tumors with AR-independent resistance mechanisms appear to be increasing in frequency. Consequently, there is an urgent need to develop new treatments targeting molecular pathways enriched in lethal prostate cancer. Lysine-specific demethylase 1 (LSD1) is a histone demethylase and an important regulator of gene expression. Here, we show that LSD1 promotes the survival of prostate cancer cells, including those that are castration-resistant, independently of its demethylase function and of the AR. Importantly, this effect is explained in part by activation of a lethal prostate cancer gene network in collaboration with LSD1’s binding protein, ZNF217. Finally, that a small-molecule LSD1 inhibitor―SP-2509―blocks important demethylase-independent functions and suppresses castration-resistant prostate cancer cell viability demonstrates the potential of LSD1 inhibition in this disease.

Cellular androgen content influences enzalutamide agonism of F877L mutant androgen receptor.

Prostate cancer is the most commonly diagnosed and second-most lethal cancer among men in the United States. The vast majority of prostate cancer deaths are due to castration-resistant prostate cancer (CRPC) – the lethal form of the disease that has progressed despite therapies that interfere with activation of androgen receptor (AR) signaling. One emergent resistance mechanism to medical castration is synthesis of intratumoral androgens that activate the AR. This insight led to the development of the AR antagonist enzalutamide. However, resistance to enzalutamide invariably develops, and disease progression is nearly universal. One mechanism of resistance to enzalutamide is an F877L mutation in the AR ligand-binding domain that can convert enzalutamide to an agonist of AR activity. However, mechanisms that contribute to the agonist switch had not been fully clarified, and there were no therapies to block AR F877L. Using cell line models of castration-resistant prostate cancer (CRPC), we determined that cellular androgen content influences enzalutamide agonism of mutant F877L AR. Further, enzalutamide treatment of AR F877L-expressing cell lines recapitulated the effects of androgen activation of F877L AR or wild-type AR. Because the BET bromodomain inhibitor JQ-1 was previously shown to block androgen activation of wild-type AR, we tested JQ-1 in AR F877L-expressing CRPC models. We determined that JQ-1 suppressed androgen or enzalutamide activation of mutant F877L AR and suppressed growth of mutant F877L AR CRPC tumors in vivo, demonstrating a new strategy to treat tumors harboring this mutation.

Integrative molecular network analysis identifies emergent enzalutamide resistance mechanisms in prostate cancer

Recent work demonstrates that castration-resistant prostate cancer (CRPC) tumors harbor countless genomic aberrations that control many hallmarks of cancer. While some specific mutations in CRPC may be actionable, many others are not. We hypothesized that genomic aberrations in cancer may operate in concert to promote drug resistance and tumor progression, and that organization of these genomic aberrations into therapeutically targetable pathways may improve our ability to treat CRPC. To identify the molecular underpinnings of enzalutamide-resistant CRPC, we performed transcriptional and copy number profiling studies using paired enzalutamide-sensitive and resistant LNCaP prostate cancer cell lines. Gene networks associated with enzalutamide resistance were revealed by performing an integrative genomic analysis with the PAthway Representation and Analysis by Direct Reference on Graphical Models (PARADIGM) tool. Amongst the pathways enriched in the enzalutamide-resistant cells were those associated with MEK, EGFR, RAS, and NFKB. Functional validation studies of 64 genes identified 10 candidate genes whose suppression led to greater effects on cell viability in enzalutamide-resistant cells as compared to sensitive parental cells. Examination of a patient cohort demonstrated that several of our functionally-validated gene hits are deregulated in metastatic CRPC tumor samples, suggesting that they may be clinically relevant therapeutic targets for patients with enzalutamide-resistant CRPC. Altogether, our approach demonstrates the potential of integrative genomic analyses to clarify determinants of drug resistance and rational co-targeting strategies to overcome resistance.

Abstract 2406: LSD1 promotes castration-resistant prostate cancer cell survival independently of the androgen receptor and of histone demethylation

Background: Androgen deprivation therapy (ADT) or interference with androgen receptor (AR) function is the principal treatment for advanced prostate cancer. However, progression is universal, and therapies following the emergence of castration resistance do not offer durable control of the disease. Lysine specific demethylase 1 (LSD1) is a histone demethylase and a key regulator of gene expression in cancer. Prior work demonstrates that LSD1 may act as a cofactor of the AR in androgen-dependent prostate cancer cells. In this report, we describe a distinct role of LSD1 as a driver of proliferation and survival of castration-resistant prostate cancer (CRPC) cells independently of the AR and independently of histone demethylation. Methods: We used gain and loss of function studies to determine the importance of LSD1 for survival of prostate cancer cells. To identify transcriptional networks that contribute to cell survival, we suppressed LSD1 with RNAi and measured gene expression changes with microarrays. To determine the importance of histone demethylation in regulation of these gene networks, we suppressed LSD1 and measured levels of LSD1 canonical histone substrates (H3K4me2 and H3K9me2) genome-wide with chromatin immunoprecipitation-sequencing. Results: Cell viability assays demonstrated that LSD1 is important for proliferation and survival of CRPC cells independently of the AR. Microarray studies demonstrated that LSD1 activates androgen-independent genes that comprise cell cycle and embryonic stem cell maintenance gene sets that are enriched in lethal human tumors. Importantly, our global epigenomic studies after LSD1 suppression demonstrated that LSD1 activates these gene sets independently of demethylation of its canonical histone substrates. Conclusions: Our results demonstrate that LSD1 promotes CRPC cell survival independently of the AR and suggest that LSD1 regulates key pathways in CRPC through demethylation of non-histone substrates or via a scaffold function―mechanisms we are currently investigating. In summary, LSD1 contributes to CRPC cell survival through non-canonical mechanisms and represents an attractive therapeutic target in lethal prostate cancer. Citation Format: Archana Sehrawat, Lina Gao, Junior Tayou, Armand Bankhead, Laura M. Heiser, Carly J. King, Yuliang Wang, Jacob Schwartzman, Joshua Urrutia, Daniel J. Coleman, Sheila Weinmann, Bhaskar V. Kallakury, Deborah L. Berry, Reina Haque, Stephen K. Van Den Eeden, Tomasz M. Beer, George V. Thomas, Shannon McWeeney, Joshi J. Alumkal. LSD1 promotes castration-resistant prostate cancer cell survival independently of the androgen receptor and of histone demethylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2406. doi:10.1158/1538-7445.AM2017-2406

Abstract 4700: BET bromodomain inhibition is a promising treatment strategy for distinct subsets of lethal castration-resistant prostate cancer

Background: While treatment options for patients with castration-resistant prostate cancer (CRPC) are expanding, one American man is still predicted to die every 19 minutes from this disease this year. Moreover, more widespread use of novel and more potent AR-targeting agents has led to increased clinical frequency of virulent androgen and AR-independent CRPC subsets(Small, Huang et al. 2015). Currently, there are limited treatment options for men with CRPC who are resistant to these AR-targeting agents, clearly demonstrating an urgent need to develop more effective therapies for CRPC patients. Recent published reports demonstrate an important role for BET bromodomain chromatin reader proteins in prostate cancer models that are androgen or AR-dependent (Gao, Schwartzman et al. 2013; Wyce, Degenhardt et al. 2013; Asangani, Dommeti et al. 2014; Chan, Selth et al. 2015). However, there was limited information about the anti-tumor activity of this class of drugs in androgen-independent, enzalutamide-resistant, or AR-independent CRPC models. The studies reported herein were designed to address that deficit. Methods: We treated a panel of CRPC cell lines with dose escalation of the BET bromodomain inhibitor JQ1 and measured cell viability. To clarify molecular mechanisms that contribute to the anti-tumor effect, we treated CRPC cell lines with JQ1 and measured gene expression changes with RNA-sequencing. Finally, to determine the anti-tumor activity of JQ1 in vivo, we implanted enzalutamide resistant or AR-null CRPC xenografts in immunocompromised mice and treated them with JQ1. Results: All cell lines were sensitive to JQ1 with similar GI50 values (all Conclusions: BET bromodomain inhibition is a promising treatment strategy for distinct subsets of lethal CRPC. Our work using a broad panel of CRPC models sheds further light on pharmacodynamic markers of response and target proteins whose function is impacted by BET bromodomain inhibition. These findings may have implications for the design of BET bromodomain inhibitor clinical trials in men with CRPC and interpretation of on-target effects in those trials. Citation Format: Lina Gao, Daniel J. Coleman, Carly J. King, Jacob Schwartzman, Nicholas Wang, Amanda Esch, Joshua Urrutia, Archana Sehrawat, Laura M. Heiser, Joshi J. Alumkal. BET bromodomain inhibition is a promising treatment strategy for distinct subsets of lethal castration-resistant prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4700.

Abstract 3592: Androgens interfere with enzalutamide agonism of mutant F876L androgen receptor

Multiple lines of evidence demonstrate that castration-resistant prostate cancers (CRPCs) remain reliant on androgens that activate the androgen receptor (AR). Treatment with the novel anti-androgen enzalutamide improves both progression-free and overall survival in CRPC patients (Beer, 2014, Scher, 2012). However, progression is universal. Recently, F876L mutations in the AR ligand binding domain have been described (Balbas, 2013, Korpal, 2013, Joseph, 2013). This mutation confers resistance to enzalutamide treatment and in some cases converts enzalutamide to an AR agonist in pre-clinical studies (Balbas, 2013, Korpal, 2013, Joseph, 2013). Clinical studies demonstrate that ∼10% of patients harbor F876L mutations after treatment with novel anti-androgens (Joseph, 2013). However, anti-androgen withdrawal effects (PSA responses) after discontinuing enzalutamide are rarely seen clinically (Rodriguez-Vida, 2014). An enzalutamide-resistant cell line was developed after chronic treatment of LNCaP cells in vivo. We found that this resistant cell line was dependent on AR expression for survival and that this cell line harbored an AR F876L mutation. When these resistant cells were cultured in complete serum, enzalutamide treatment did not lead to agonistic effects. However, enzalutamide treatment of these resistant cells or cell lines with ectopic expression of AR F876L cultured in androgen-depleted serum led to a significant agonistic effect - an effect that was attenuated by the addition of androgens to culture. Finally, prior work and our own demonstrated that F876L mutant and wild-type AR activate similar pathways (Joseph, 2013). Therefore, we determined if suppression of previously described transcriptional co-activators of wild-type AR also blocked AR F876L function. We found several targetable AR co-activators that met that standard. Our data demonstrate that androgens interfere with enzalutamide-induced agonism of F876L mutant AR. Because androgens persist in enzalutamide-resistant CRPC, AR activation by androgens, rather than enzalutamide, may explain why enzalutamide discontinuation does not lead to anti-androgen withdrawal effects clinically. Further, our results provide a cautionary note on therapeutic efforts to deplete androgens concomitantly with enzalutamide treatment as this may accentuate AR agonism by enzalutamide in tumors harboring AR F876L mutations. Finally, targeting critical AR transcriptional co-activators is a promising strategy to suppress mutant AR F876L function irrespective of whether the AR agonist is androgens or enzalutamide. Citation Format: Daniel Coleman, Katy Van Hook, Robert Lisac, Carly King, Nicholas Wang, Jacob Schwartzman, Martin Gleave, Lina Gao, Joshua Urrutia, Laura Heiser, Joshi J. Alumkal. Androgens interfere with enzalutamide agonism of mutant F876L androgen receptor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3592. doi:10.1158/1538-7445.AM2015-3592

Abstract 731: Integrative genomic analysis to identify emergent enzalutamide resistance mechanisms in castration-resistant prostate cancer

Background: Multiple lines of evidence demonstrate that castration-resistant prostate cancers (CRPCs) remain reliant on androgens that activate the androgen receptor. Treatment with the novel anti-androgen enzalutamide improves progression-free survival and overall survival in CRPC patients; however, nearly 50% of patients never respond, and progression is universal (Beer, 2014, Scher, 2012). Mechanisms of enzalutamide resistance are largely unknown and few treatments exist for enzalutamide-resistant CRPC. Recent work demonstrates that CRPC tumors harbor countless genomic aberrations that control many hallmarks of cancer (Grasso, 2012, Hanahan and Weinberg, 2011). Based on our prior work (Heiser, 2012, Vaske, 2010), we hypothesize that these aberrations operate in concert to drive enzalutamide resistance and influence specific cancer hallmarks. Methods: We performed genomic studies using paired enzalutamide-sensitive and resistant LNCaP cell models. After transcriptional and copy number profiling, we performed an integrative pathway-informed PARADIGM analysis to identify differentially regulated cellular networks (Heiser, 2012, Vaske, 2010). These large-scale networks underwent regression analysis to identify sub-networks associated with acquired resistance. Genes residing within significant sub-networks were nominated for functional validation studies with RNAi or existing therapeutic compounds that impinge upon significant sub-networks in resistant models. Results: We used PARADIGM to compare the genomic alterations between the parental and enzalutamide-resistant cell line models and identified critical deregulated networks that may be targeted therapeutically. Currently, we are applying this same PARADIGM analysis to additional model systems and metastatic patient tumors obtained prior to treatment and at the time of disease progression through a West Coast Dream Team prospective enzalutamide clinical trial. Conclusions: PARADIGM integrative genomic analysis identifies specific sub-networks that contribute to enzalutamide resistance. A predicted outcome of our efforts is the development of rationally designed clinical trials with specific enzalutamide drug combinations in distinct molecular subsets of CRPC patients in the near-term. Citation Format: Josha Woodward, Carly King, Daniel Coleman, Robert Lisac, Jacob Schwartzman, Nicholas Wang, Martin Gleave, Joe Gray, George Thomas, Tomasz M. Beer, Katy Van Hook, Robert Baertsch, Ted Goldstein, Josh Stuart, Lina Gao, Joshua Urrutia, Laura Heiser, Joshi J. Alumkal. Integrative genomic analysis to identify emergent enzalutamide resistance mechanisms in castration-resistant prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 731. doi:10.1158/1538-7445.AM2015-731