Christopher McNair

Convergence of oncogenic and hormone receptor pathways promotes metastatic phenotypes.

Cyclin D1b is a splice variant of the cell cycle regulator cyclin D1 and is known to harbor divergent and highly oncogenic functions in human cancer. While cyclin D1b is induced during disease progression in many cancer types, the mechanisms underlying cyclin D1b function remain poorly understood. Herein, cell and human tumor xenograft models of prostate cancer were utilized to resolve the downstream pathways that are required for the protumorigenic functions of cyclin D1b. Specifically, cyclin D1b was found to modulate the expression of a large transcriptional network that cooperates with androgen receptor (AR) signaling to enhance tumor cell growth and invasive potential. Notably, cyclin D1b promoted AR-dependent activation of genes associated with metastatic phenotypes. Further exploration determined that transcriptional induction of SNAI2 (Slug) was essential for cyclin D1b-mediated proliferative and invasive properties, implicating Slug as a critical driver of disease progression. Importantly, cyclin D1b expression highly correlated with that of Slug in clinical samples of advanced disease. In vivo analyses provided strong evidence that Slug enhances both tumor growth and metastatic phenotypes. Collectively, these findings reveal the underpinning mechanisms behind the protumorigenic functions of cyclin D1b and demonstrate that the convergence of the cyclin D1b/AR and Slug pathways results in the activation of processes critical for the promotion of lethal tumor phenotypes.

Differential impact of RB status on E2F1 reprogramming in human cancer

The tumor suppressor protein retinoblastoma (RB) is mechanistically linked to suppression of transcription factor E2F1-mediated cell cycle regulation. For multiple tumor types, loss of RB function is associated with poor clinical outcome. RB action is abrogated either by direct depletion or through inactivation of RB function; however, the basis for this selectivity is unknown. Here, analysis of tumor samples and cell-free DNA from patients with advanced prostate cancer showed that direct RB loss was the preferred pathway of disruption in human disease. While RB loss was associated with lethal disease, RB-deficient tumors had no proliferative advantage and exhibited downstream effects distinct from cell cycle control. Mechanistically, RB loss led to E2F1 cistrome expansion and different binding specificity, alterations distinct from those observed after functional RB inactivation. Additionally, identification of protumorigenic transcriptional networks specific to RB loss that were validated in clinical samples demonstrated the ability of RB loss to differentially reprogram E2F1 in human cancers. Together, these findings not only identify tumor-suppressive functions of RB that are distinct from cell cycle control, but also demonstrate that the molecular consequence of RB loss is distinct from RB inactivation. Thus, these studies provide insight into how RB loss promotes disease progression, and identify new nodes for therapeutic intervention.

Therapeutic Challenge With a CDK 4/6 Inhibitor Induces an RB-dependent SMAC Mediated Apoptotic Response in Non-Small Cell Lung Cancer

Purpose: The retinoblastoma tumor suppressor (RB), a key regulator of cell-cycle progression and proliferation, is functionally suppressed in up to 50% of non–small cell lung cancer (NSCLC). RB function is exquisitely controlled by a series of proteins, including the CyclinD–CDK4/6 complex. In this study, we interrogated the capacity of a CDK4/6 inhibitor, palbociclib, to activate RB function. Experimental Design and Results: We employed multiple isogenic RB-proficient and -deficient NSCLC lines to interrogate the cytostatic and cytotoxic capacity of CDK 4/6 inhibition in vitro and in vivo. We demonstrate that while short-term exposure to palbociclib induces cellular senescence, prolonged exposure results in inhibition of tumor growth. Mechanistically, CDK 4/6 inhibition induces a proapoptotic transcriptional program through suppression of IAPs FOXM1 and Survivin, while simultaneously augmenting expression of SMAC and caspase-3 in an RB-dependent manner. Conclusions: This study uncovers a novel function of RB activation to induce cellular apoptosis through therapeutic administration of a palbociclib and provides a rationale for the clinical evaluation of CDK 4/6 inhibitors in the treatment of patients with NSCLC. Clin Cancer Res; 24(6); 1402–14. ©2018 AACR.

MAPK reliance via acquired CDK4/6 inhibitor resistance in cancer

Purpose: Loss of cell-cycle control is a hallmark of cancer, which can be targeted with agents, including cyclin-dependent kinase-4/6 (CDK4/6) kinase inhibitors that impinge upon the G1–S cell-cycle checkpoint via maintaining activity of the retinoblastoma tumor suppressor (RB). This class of drugs is under clinical investigation for various solid tumor types and has recently been FDA-approved for treatment of breast cancer. However, development of therapeutic resistance is not uncommon. Experimental Design: In this study, palbociclib (a CDK4/6 inhibitor) resistance was established in models of early stage, RB-positive cancer. Results: This study demonstrates that acquired palbociclib resistance renders cancer cells broadly resistant to CDK4/6 inhibitors. Acquired resistance was associated with aggressive in vitro and in vivo phenotypes, including proliferation, migration, and invasion. Integration of RNA sequencing analysis and phosphoproteomics profiling revealed rewiring of the kinome, with a strong enrichment for enhanced MAPK signaling across all resistance models, which resulted in aggressive in vitro and in vivo phenotypes and prometastatic signaling. However, CDK4/6 inhibitor–resistant models were sensitized to MEK inhibitors, revealing reliance on active MAPK signaling to promote tumor cell growth and invasion. Conclusions: In sum, these studies identify MAPK reliance in acquired CDK4/6 inhibitor resistance that promotes aggressive disease, while nominating MEK inhibition as putative novel therapeutic strategy to treat or prevent CDK4/6 inhibitor resistance in cancer. Clin Cancer Res; 24(17); 4201–14. ©2018 AACR.

Abstract IA03: Differential impact of RB pathway status on E2F1 reprogramming and disease progression in human prostate cancer

The retinoblastoma tumor suppressor (RB) is mechanistically linked to suppression of E2F1-mediated cell cycle regulation. Abrogation of RB function is associated with poor clinical outcome across various tumor types, which frequently elicit a preference for either RB depletion or functional inactivation, yet the basis for selectivity is unknown. Here, examination of RB pathway alterations in advanced prostate cancer revealed that cyclin dependent kinase (CDK)/cyclin/CDKi alterations are infrequent, and identify RB loss as the major mechanism of pathway disruption in human disease. Furthermore, RB status was readily traced through cell-free DNA analyses in human specimens, thus identifying new ways to assign RB status in the clinical setting. Strikingly, RB depletion in human disease was not associated with a higher Ki67 index, indicating a role for the RB/E2F1 pathway in regulating processes distinct from cell cycle control and associated with lethal-stage disease. Subsequent mechanistic investigation utilized isogenic prostate cancer models, wherein RB could be differentially inactivated through depletion or through hormone-induced, CDK-mediated phosphorylation. Unbiased molecular interrogation uncovered a novel E2F1 cistrome and downstream engagement of transcriptional networks exclusively observed after RB loss, with binding specificity divergent from canonically described E2F1 binding patterns. Additionally, E2F1 cistrome alterations elicited by RB depletion were seen to be distinct from those after phosphorylation-induced RB functional inactivation, providing needed insight into the basis of selectivity for RB loss versus CDK-mediated inactivation observed in human disease. Analyses of human CRPC tumor samples further underscored the clinical relevance of RB loss-induced gene expression programs, which were significantly correlated with reprogrammed E2F1 binding identified herein. Taken together, the studies presented are the first to identify the consequences of RB loss, demonstrating molecular distinction from RB inactivation and illustrating the clinical relevance of RB loss-induced E2F rewiring. Citation Format: Christopher McNair, Kexin Xu, Amy C. Mandigo, Matteo Benelli, Benjamin Leiby, Daniel Rodrigues, Johan Lindberg, Henrik Gronberg, Bram De Laere, Luc Dirix, Tapio Visakorpi, Fugen Li, Felix Y. Feng, Johann de Bono, Francesca Demichelis, Mark A Rubin, Myles Brown, Karen E. Knudsen. Differential impact of RB pathway status on E2F1 reprogramming and disease progression in human prostate cancer [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 IA03.

Abstract B040: Differential impact of RB status on E2F1 reprogramming in human cancer

Recent examination of advanced prostate cancer (PCa) has suggested a major mechanism of progression to castration-resistant disease (CRPC) to be loss of the retinoblastoma (RB) protein. Along with its critical role in controlling cell cycle progression, RB is known to have important tumor-suppressor functions, and has been shown in PCa to be lost exclusively in late-stage disease. Additionally, loss of RB has been shown to correlate with increased E2F1 transcript and protein expression, via E2F-dependent mechanisms. Despite the vital role RB loss has been shown to play in this fatal stage of disease, the molecular underpinnings remain undefined. Thus, in order to elucidate these CRPC specific alterations, the current study utilizes isogenic models of RB loss in combination with genome-wide binding and transcriptional studies. Data presented herein demonstrate that loss of RB is frequent in CRPC, and represents the main mechanism of RB pathways disruption in PCa as detected through analyses of tumor samples and cell-free DNA. However, this phenomenon is not correlated with changes in proliferative indices, suggesting a role for RB loss outside of canonical cell cycle control. Further, RB loss induces significant genome-wide transcriptional alterations, including upregulation in Myc, E2F, and DNA-repair related pathways. Additionally, loss of RB significantly expands E2F1 binding capacity in castrate conditions, while largely maintaining the RB-intact E2F1 cistrome. Strikingly, while the current RB/E2F1 paradigm suggests that E2F1 exclusively occupies promoter regions of DNA in order to regulate transcriptional changes, RB loss induces marked reprogramming of E2F1 occupied regions, with a distinct increase in enhancer-bound E2F1. Further, motif analyses suggest divergence away from canonical E2F1 binding motifs after RB loss, specifically in regions of expanded E2F1 binding, and additionally suggest likely interaction of novel E2F1 cofactors under RB loss conditions. Interestingly, changes in E2F1 binding capacity after RB loss were seen to be distinct from those detected after androgen-induced RB inactivation, suggesting that the molecular alterations underlying RB loss are discrete from those resulting from functional inactivation. With respect to putative mechanism, it is of note that chromatin accessibility was not significantly altered to sufficiently explain the widespread changes in E2F1 cistrome, regardless of RB status, suggesting a mechanism outside simple opportunistic E2F1 binding after RB loss. Finally, interrogation of a CRPC patient tumor cohort showed predictive capacity for an “Expanded E2F1 Signature,” resulting from genes exhibiting gained E2F1 binding and differential expression after RB loss, in predicting loss of RB in patient samples, and indicating a novel E2F1-driven set of targets vital for CRPC transition in human disease. Together, these data present the first insight into E2F1 activity resulting from RB loss, and the role these changes play in progression to CRPC. Citation Format: Christopher McNair, Kexin Xu, Amy C. Mandigo, Matteo Benelli, Benjamin Leiby, Daniel Rodrigues, Johan Lindberg, Henrik Gronberg, Mateus Crespo, Bram De Laere, Luc Dirix, Tapio Visakorpi, Fugen Li, Felix Y. Feng, Johann de Bono, Francesca Demichelis, Mark A. Rubin, Myles Brown, Karen E. Knudsen. Differential impact of RB status on E2F1 reprogramming in human cancer [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 B040.

Abstract LB-264: Preclinical evaluation of DNA-PK as a therapeutic target in prostate cancer

Prostatic adenocarcinoma (PCa) is dependent on androgen receptor (AR) signaling at all stages of disease, as AR activation induces both cell proliferation and survival. Though organ–confined disease can be treated, the response is transient. Reactivation of AR leads to castration resistant prostate cancer (CRPC), which remains fatal. Previously published studies demonstrate that AR activation promotes tumor cell survival and proliferation through a feed-forward loop involving the DNA repair factor DNA-dependent protein kinase (DNA-PK). Recent data from our lab has highlighted the role of DNA-PK in DNA damage repair as well as transcriptional regulation of pro-metastatic signaling. Strikingly, DNA-PK is the most deregulated kinase in metastatic CRPC. DNAPK is highly elevated and hyperactivated, and is an independent predictor of metastasis and overall survival in patients with high-risk disease. Concordantly, DNAPK suppression attenuated DNA-PK functions in NHEJ and transcriptional regulation. DNA-PK inhibition is sufficient to prevent proliferation in vitro and ex vivo, as well as tumor metastasis in vivo. Combined these findings highlight importance of DNA-PK functions as a transcriptional regulator and a mediator of NHEJ DNA repair and nominate it as a therapeutic target in PCa. Data presented here will interrogate the translational capacity of a specific DNA-PK inhibitor, a dual DNA-PK/TOR kinase inhibitor and a specific TOR kinase inhibitor in CRPC models. Currently, CC-115, a dual kinase inhibitor, is the only DNA-PK inhibitor in Phase1/2 trial in combination with Enzalutamide in PCa. Unbiased transcriptomic analyses were performed and will be used to identify mechanisms of action that lead to suppression of PCa growth in vitro and ex vivo. These findings will provide insight in the effectiveness of DNA-PK inhibitors as a single agent or combinatorial treatments and provide rational for its placement in the correct clinical space. Citation Format: Emanuela Dylgjeri, Jonathan F. Goodwin, Christopher M. McNair, Ayesha A. Shafi, Vishal Kothari, Felix Feng, Dana Rathkop, Karen Knudsen. Preclinical evaluation of DNA-PK as a therapeutic target in prostate cancer [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 LB-264. doi:10.1158/1538-7445.AM2017-LB-264

Abstract 5874: Cdk4/6 kinase inhibitor resistance in prostate cancer

Non-organ confined prostate cancer (PCa) is often effectively, but only transiently treated by targeting the androgen receptor (AR) signaling axis through androgen depletion strategies, often coupled with AR antagonists. Unfortunately, disease recurs within a median of 3-4 years, presenting as castration resistant PCa (CRPC), for which there are limited therapeutic options. This emphasizes the need for more efficacious drugs and a patient-tailored approach towards cancer therapy to improve disease outcome. One class of drugs currently tested clinically, Cdk4/6 kinase inhibitors, blocks phosphorylation of the retinoblastoma (RB) tumor suppressor, thereby boosting its function, and likely preventing castration resistance. As Cdk4/6 inhibitor resistance has already been reported in other cancers, some PCa patients are anticipated to develop drug resistance. Here, we created palbociclib-resistant PCa cell models by continuously culturing them in presence of the drug to unravel mechanisms of acquired resistance, and assessed them for cross-resistance to ribociclib and response to other therapeutics. While the parental PCa cell models, Cdk4/6 inhibitors efficiently induce a G1 cell cycle arrest, the resistant cell lines bypass this cell cycle checkpoint. Although loss of RB is a known mechanism for Cdk4/6i resistance, none of the models lost RB expression. Strikingly, these originally hormone-sensitive cell lines, upon developing Cdk4/6 inhibitor resistance display altered response to selected therapeutic regimens. Mechanisms of resistance, as informed by Whole Exome Sequencing and RNASeq, will be discussed. Citation Format: Renee de Leeuw, Matthew J. Schiewer, Christopher McNair, Michael A. Augello, Akihiro Yoshida, Edward S. Hazard, Sean Courtney, Gerard T. Hardiman, Justin Drake, Felix Y. Feng, Scott Tomlins, Maha H. Hussain, J Alan Diehl, William K. Kelly, Karen E. Knudsen. Cdk4/6 kinase inhibitor resistance in prostate cancer [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 5874. doi:10.1158/1538-7445.AM2017-5874

Abstract 1844: Genome wide analysis of AR-cell cycle interplay reveals novel functions in cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The androgen receptor (AR) plays a vital role in prostate cancer (PCa) due in part to its ability to interact with cell cycle components in order to drive cell cycle transition. Numerous points of cross-talk have been identified, wherein specific components of the cell cycle machinery “feed back” to modulate AR function, and these interactions are thought to be altered in human malignancies. Despite these observations, the majority of genome wide analyses for AR have been performed in cells that have exited cell cycle (G0). Here, the cell cycle dependent AR transcriptome and cistrome was identified, revealing new and unexpected functions for AR in cycling tumor cells. In studies to be discussed, cells were arrested in 5 distinct phases of the cell cycle, stimulated with androgen, and AR activity assessed through gene expression and ChIP-Seq analyses. In AR binding analyses, significant overlap was seen with previously identified sites, but were accompanied by novel binding events that could be segregated into those that are specific to cycling cells and occur in all phases (“cell cycle common”) or show cell cycle stage specific binding (“phase exclusive”). Over 50% of the cell cycle common sites, and up to 95% of the phases exclusive sites were novel AR occupied sites. Additionally, using a “guilty by association” approach to determine potentially AR regulated genes from this novel cistromic data, it was determined that close to 50% of cell cycle common, and 70% of phase exclusive binding uncover novel candidates for AR regulation. Cistrome data was therefore overlaid with microarray data, to prioritize discovery of meaningful, cell cycle specific AR binding events. Analyses to be discussed reveal striking new insight into disease relevant AR function. In sum, these data rigorously demonstrate that AR acts in a cell cycle dependent manner, and that these functions of AR have a major impact on tumor cell phenotypes. Citation Format: Christopher McNair, Jonathan Goodwin, Michael Augello, Alfonso Urbanucci, Matthew Schiewer, Clay Comstock, Adam Ertel, Liguo Wang, Qianben Wang, Ian Mills, Wei Li, Jason Carroll, Karen Knudsen. Genome wide analysis of AR-cell cycle interplay reveals novel functions in 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 1844. doi:10.1158/1538-7445.AM2015-1844