Tim Heffernan

Targeting YAP-Dependent MDSC Infiltration Impairs Tumor Progression.

UNLABELLEDnThe signaling mechanisms between prostate cancer cells and infiltrating immune cells may illuminate novel therapeutic approaches. Here, utilizing a prostate adenocarcinoma model driven by loss of Pten and Smad4, we identify polymorphonuclear myeloid-derived suppressor cells (MDSC) as the major infiltrating immune cell type, and depletion of MDSCs blocks progression. Employing a novel dual reporter prostate cancer model, epithelial and stromal transcriptomic profiling identified CXCL5 as a cancer-secreted chemokine to attract CXCR2-expressing MDSCs, and, correspondingly, pharmacologic inhibition of CXCR2 impeded tumor progression. Integrated analyses identified hyperactivated Hippo-YAP signaling in driving CXCL5 upregulation in cancer cells through the YAP-TEAD complex and promoting MDSC recruitment. Clinicopathologic studies reveal upregulation and activation of YAP1 in a subset of human prostate tumors, and the YAP1 signature is enriched in primary prostate tumor samples with stronger expression of MDSC-relevant genes. Together, YAP-driven MDSC recruitment via heterotypic CXCL5-CXCR2 signaling reveals an effective therapeutic strategy for advanced prostate cancer.nnnSIGNIFICANCEnWe demonstrate a critical role of MDSCs in prostate tumor progression and discover a cancer cell nonautonomous function of the Hippo-YAP pathway in regulation of CXCL5, a ligand for CXCR2-expressing MDSCs. Pharmacologic elimination of MDSCs or blocking the heterotypic CXCL5-CXCR2 signaling circuit elicits robust antitumor responses and prolongs survival.

Abstract A03: Perturbation of proteostasis is lethal in SMARCB1-deficient tumors

Alterations in chromatin remodeling genes have been increasingly implicated in human oncogenesis. The SWI/SNF complex, specifically, is involved in a plethora of biologic functions including cell cycle regulation, terminal differentiation, and regulation of cell metabolism. The crucial chromatin-remodeling function of the SWI/SNF complex during organogenesis and tissue specification is further supported by clinical data showing that the biallelic inactivation of the core subunits SMARCB1 and SMARCA4 results in the emergence of extremely aggressive pediatric malignancies characterized by a dramatic impairment of cell cycle regulation and cellular differentiation programs, resulting in highly lethal diseases characterized by early onset, widespread metastatic dissemination, and resistance to chemotherapy. So far the lack of conditional genetic models of malignant rhabdoid tumors (MRTs) made difficult to investigate the molecular bases of malignant transformation as well as the existence of dependencies associated with SMARCB1 loss. In order to identify the functional vulnerabilities of SMARCB1-deficient cancers, we developed an embryonic mosaic model of malignant rhabdoid tumors (MRTs) that faithfully recapitulates the clinicopathologic features of human disease. By using this novel experimental system we discovered that, upon SMARCB1 ablation, embryonic epithelial progenitors undergo a profound anabolic reprogramming resulting in a global increase in protein biosynthesis and in the adaptive activation of UPR and ER stress response. As a consequence, murine and human SMARCB1-deficient malignancies display an exquisite sensitivity to agents inducing proteotoxic stress and to the pharmacologic and genetic perturbation of autophagy. Our findings, therefore, have immediate clinical implications, paving the way for drug repositioning trials investigating combinations of agents with already known safety profiles targeting simultaneously UPR and the authopagic catabolic machinery in a class of orphan diseases affecting children with limited therapeutic options. Note: This abstract was not presented at the conference. Citation Format: Giannicola Genovese, Alessandro Carugo, Rosalba Minelli, Frederick, Scott Robinson, Pavlos Msaouel, Tim Heffernan, Andrea Viale, Nizar Tannir, Giulio Draetta. Perturbation of proteostasis is lethal in SMARCB1-deficient tumors [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A03.

Abstract A52: Overcoming mitochondrial oxidative phosphorylation-mediated resistance to targeted therapies in melanoma

Abstracts: AACR Special Conference: Metabolism and Cancer; June 7-10, 2015; Bellevue, WAnnThere is a critical need to understand and overcome resistance to targeted therapy in cancer. While FDA-approved BRAF and MEK inhibitors achieve clinical responses in the majority of metastatic melanoma patients with a BRAFV600 mutation, a significant proportion of patients have minimal responses, and virtually all patients develop acquired resistance in a short period of time. We and others have shown that increased oxidative phosphorylation (OxPhos), which is critically dependent on the transcriptional co-activator PGC1α, can mediate de novo resistance to MAPK pathway inhibitors in melanomas with BRAFV600 mutations. We have also demonstrated that elevated OxPhos (a) mediates de novo resistance to MEK inhibitors in melanoma cell lines without BRAFV600 mutations; (b) characterizes 30-50% of BRAFV600-mutant cell lines and patients with acquired resistance to BRAF +/- MEK inhibitors; and (c) predicts sensitivity to dual inhibition of mTORC1/2 in vitro and in vivo . Inhibition of mTORC1/2 downregulates both PGC1α and OxPhos through a novel mechanism involving the subcellular localization of the transcription factor MITF. To further exploit the therapeutic potential of targeting OxPhos, we have evaluated the molecular and anti-melanoma activities of a novel OxPhos inhibitor, IACS-10759, developed at M.D. Anderson Cancer Center. IACS-10759 potently inhibits the electron transport chain Complex I of mitochondrial OxPhos at low nanomolar concentrations. Additionally, this molecule has favorable safety and pharmacokinetic profile in animal models. Normal melanocytes, skin fibroblasts and most melanoma cell lines were not sensitive to micromolar doses of IACS-10759. However, a subset of de novo and acquired resistant melanomas with high OxPhos were sensitive to low nanomolar concentrations of IACS-10759 and underwent apoptotic cell death with treatment. An additional subset of melanomas were sensitive to the combination of IACS-10759 with MEK or BRAF inhibitors. Early functional studies revealed AMPK-activation associated effects as mediators of sensitivity, in addition to the inability of the cells to use alternate metabolic pathways for energy generation. Our findings demonstrate that a high OxPhos metabolic phenotype is associated with de novo and acquired resistance to MAPK pathway targeted therapies in melanoma, and support that targeting mTORC1/2 and the OxPhos complex 1 are promising strategies for counteracting this resistance.nnCitation Format: Vashisht Gopal Yennu-Nanda, Zeping Hu, Victoria M. Thiele, Tim Heffernan, Maria DiFrancesco, Joe Marszalek, Ralph Deberadinis, Michael A. Davies. Overcoming mitochondrial oxidative phosphorylation-mediated resistance to targeted therapies in melanoma. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A52.

Abstract B105: HSP90 inhibitor, ganetespib, enhances responses to cancer immunotherapy through increased expression of interferon response genes

Recently, T cell based immunotherapies have moved to the forefront of cancer immunotherapy with the success of Adoptive T cell therapy (ACT) and Immune checkpoint blockade. ACT, where patients are treated with tumor infiltrating T cells (TILs), conferred a clinical response rate of ∼50%. Treatment with anti-CTLA4 therapy, Ipilimumab, conferred response rates of 10-20%, greatly improving the overall survival of patients with advanced melanoma. Despite the encouraging outcomes, there are relatively low response rates coupled with the delay of weeks to months before tumor shrinkage can be appreciated. Thus, understanding mechanisms of resistance to immune therapies, to improve response rates, shorten time to treatment effect and developing predictive biomarkers of response are vital to the care of melanoma patients. In order to identify possible resistance mechanisms to immunotherapy, a high-throughput in vitro screen with 850 different bio-active compounds (Selleckchem), was designed to search for agents that could either increase or decrease the resistance of melanoma tumor cells to T cell mediated killing. Paired patient derived human melanoma tumor samples and TILs were used to assess which compounds when used to treat the melanoma cell lines can enhance the cytotoxic activity of the TILs against the paired melanoma sample, using a flow cytometry based assay in which active caspase 3 was used as a read out of apoptosis. We identified heat shock protein 90 (HSP90) inhibitors amongst the top compounds that improved T cell mediated cytotoxicity of treated tumor cells. We show that treatment with the HSP90 inhibitor ganetespib (Synta) greatly improves T cell mediated cytotoxicity of human cancer cells lines in vitro. Furthermore, in vivo murine studies using the MC38/gp100 tumor model show that ganestespib in combination with anti-CTLA4, resulted in superior antitumor effect and survival compared to either treatment alone (Average tumor volume at day 21 of treatment: Vehicle 294.3mm3, α-CTLA4 193 mm3, Ganetespib 237.5 mm3 and Ganetespib + α-CTLA4 105.8 mm3, P Citation Format: Rina M. Mbofung, Jodi A. McKenzie, Shruti Malu, Chengwen Liu, Weiyi Peng, Isere Kuiatse, Leila Williams, Seram Devi, Zhe Wang, Trang Tieu, Tim Heffernan, Richard E. Davis, Rodabe Amaria, Patrick Hwu. HSP90 inhibitor, ganetespib, enhances responses to cancer immunotherapy through increased expression of interferon response genes [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B105.

Abstract 4002: Enhancing the antitumor efficacy of immunotherapy by using the topoisomerase I inhibitor MM398

Melanoma is a highly aggressive form of skin cancer, whose rates of morbidity and mortality are increasing. The development of immunotherapies like anti-PDL1 and anti-CTLA4 antibodies has resulted in fundamental advances in the treatment of some cancers. However, long lasting responses are only observed in a subset of immunotherapy-treated patients. This shortfall highlights the need for a better understanding of the molecular mechanisms that govern tumor response to immunotherapy. To address this need, autologous patient-derived tumor cell lines and tumor infiltrating lymphocytes (TILs) were utilized in an in vitro high throughput screen, to identify compounds that increase the sensitivity of melanoma cells to T cell mediated cytotoxicity. The screen consisted of an 850 compound library. One group of compounds that was most able to enhance T cell killing of melanoma cells was topoisomerase I (Top1) inhibitors such as topotecan and irinotecan. Our results indicate that treatment of melanoma cells with a Top1 inhibitor prior to exposure to autologous T cells produced a synergistic increase in tumor cell death, as measured by intracellular staining of activated caspase 3. We have also recapitulated this finding in an in vivo model, where a better anti-tumor effect was observed in tumor bearing mice treated with an antibody against the co-inhibitory molecule PDL1 in combination with MM398 (nanoliposomal irinotecan), than in cohorts treated with either α-PDL1 or Top1 inhibitor alone. These findings suggest synergism between Top1 inhibitors and immune-based therapies in the treatment of melanoma. Molecular changes elicited by inhibition of Top1 are now being investigated to identify the factors that mediate the effect of Top1 inhibitors on T cell-mediated killing of melanoma. We have identified a p53-driven gene signature in Top1 inhibitor-treated melanoma cell lines and are investigating the functional relevance of Tumor Protein p53 Inducible Nuclear Protein 1 (TP53INP1) in mediating increased T cell killing of Top1 inhibitor-treated melanoma cells. Our results indicate that TP53INP1 is a critical component of this apoptotic response, as overexpression of TP53INP1 in melanoma cells increased their susceptibility to T cell mediated cytotoxicity. Complementary to this observation, we have also found that knockdown of TP53INP1 by shRNA, impedes the sensitivity of Top1 inhibitor-treated melanoma cells to T cell mediated killing. Understanding how Top1 inhibitors enhance melanoma killing by immunotherapy will allow for the development of predictive biomarkers, and also augment immune-based therapeutic strategies to ensure durable responses in a larger population of melanoma patients. By using melanoma as a model disease system, we can gain valuable insights into the dynamics of cancer immune response that may be applied to other cancers where effective treatment strategies are also lacking. Citation Format: Jodi A. McKenzie, Rina M. Mbofung, Shruti Malu, Rodabe N. Amaria, Richard E. Davis, Li Zhang, Trang N. Tieu, Tim P. Heffernan, Patrick Hwu. Enhancing the antitumor efficacy of immunotherapy by using the topoisomerase I inhibitor MM398. [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 4002.

Abstract 1441: Systems-level interrogation of resistance mechanisms to immunotherapy through pooled shRNA screens

Despite the impressive clinical efficacy of immunotherapy in some patients, many still do not respond or progress following an initial response. The molecular mechanisms underlying the tumor resistance in those non-responders remain largely undefined. To address this issue, we set out to perform high-throughput unbiased pooled shRNA screens to identify critical genes that confer immune resistance. Patient-derived melanoma cells were transduced with barcoded pooled lentiviral shRNA libraries that targeted the human kinome, followed by exposure to cytotoxicity mediated by autologous tumor infiltrating lymphocytes. Tumor cells were then subject to genomic deep sequencing and integrated analysis to identify depleted barcodes and corresponding genes. One identified candidate of particular interest is Aurora Kinase A (AURKA), a cell cycle regulator that has previously been shown to contribute to tumorigenesis and correlate with poor prognosis for cancer patients. Importantly, our independent screening with a bioactive compound library also implicated Aurora Kinase as an immune resistance candidate against T cell immunotherapy. Further studies showed that suppression of Aurora kinase activity with a pan inhibitor - AMG900, exhibited a synergistic cytotoxic effect with tumor infiltrating lymphocyte-mediated killing on autologous tumor cells from patients. Furthermore, our Nanostring analysis of tumor biopsies from patients that received adoptive T cell therapy revealed significantly increased expression of AURKA in tumors from non-responding patient when compared with responder counterparts. These results have substantiated the validity of our pooled shRNA screening platform. Further investigations are ongoing to elucidate the underpinnings of Aurora kinase-mediated tumor resistance to immunotherapy and to functionally and physiologically validate other putative targets we have identified. Citation Format: Zhe Wang, Shruti Malu, Weiyi Peng, Jodi McKenzie, Rina Mbofung, Leila Williams, Sahil Seth, Tim Heffernan, Patrick Hwu. Systems-level interrogation of resistance mechanisms to immunotherapy through pooled shRNA screens. [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 1441.

Abstract 4795: A novel gene fusion in glioblastoma and a radiation response methylation signature identified by genomic characterization of glioma sphere-forming cells

Purpose: High fidelity models of the lethal primary brain tumor glioblastoma (GBM) are essential to develop new therapies. Glioma sphere-forming cells (GSCs) are derived from surgical specimens and are thought to play important roles in tumor maintenance and treatment resistance. We performed genomic characterization of the largest reported panel of GSCs. We hypothesized that GSCs would recapitulate the genomic alterations of their GBMs of origin while identifying novel changes identifiable only in a pure tumor cell population. Methods: All GSCs were obtained at the time of surgical resection and all analyses were conducted at early passage. We performed exome and transcriptome sequencing, DNA methylation profiling (Illumina Infinium 450K Bead Arrays) and DNA copy number determination (Affymetrix OncoScan). Radiation (RT) sensitivity was determined by clonogenic survival and in vivo survival by orthotopic xenograft. Results: We analyzed 43 GSCs, 40 of which had tissue available from their tumors of origin. Somatically mutated genes previously described in GBM, such as TP53, EGFR, PTEN, NF1, PIK3CA and RB1, were found at similar mutation frequencies. Likewise, DNA copy number variations were similar to their matched tumor and those reported by the TCGA, with novel or more pronounced alterations, such as MYC application and QKI deletion, identified in the GSCs. GSCs were classified into TCGA GBM subtypes by expression signatures, identifying a subset of GSCs with a subtype differing from their matched tumors that correlated to decreased stromal enrichment. GSCs exhibited upregulation of self-renewal pathways, such as MYC, WNT, and NOTCH, and of stem-cell factors, such as MSI1, NESTIN, OLIG2, and SOX2, consistent with the stem-like phenotype attributed to GSCs. Transcript analyses identified the previously reported FGFR3-TACC3 and EGFR-SEPT14 gene fusions as well as a novel KIF1B-KMT2A (MLL) fusion, which was found to have been retained in the matching recurrent GBM as well as the GSC derived from the recurrence. A signature derived by the differential methylation pattern of RT sensitive vs. resistant GSCs was applied to the subset of TCGA cases that received upfront RT. Survival by methylation class in this subset was significantly different (median survival 84 vs. 61 weeks; HR 1.64 adjusting for patient age, p-value Conclusions: Based on genomic analyses, GSCs are robust models of GBM which can be used for therapeutic development. We have identified a novel gene fusion involving MLL with a predicted driving role suggesting a new mode of gliomagenesis. A methylation signature predictive of RT response may have potential for personalizing RT treatment of GBM patients and provides insights into RT sensitivity phenotypes. Citation Format: Qianghu Wang, Ravesanker Ezhilarasan, Lindsey D. Goodman, Joy Gumin, Siyuan Zheng, Kosuke Yoshihara, Peng Sun, Jie Yang, Tim Heffernan, Giulio Draetta, Kenneth D. Aldape, Frederick F. Lang, Roel G.W. Verhaak, Erik P. Sulman. A novel gene fusion in glioblastoma and a radiation response methylation signature identified by genomic characterization of glioma sphere-forming cells. [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 4795. doi:10.1158/1538-7445.AM2015-4795

Abstract 1191: Translational Proof-of-Concept (TransPoC), a not-for-profit research organization enabling access to large-scale translational oncology platforms: The Patient-Derived Xenograft network

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnTransPoC is a not-for-profit research organization that will deliver open-access, large-scale translational oncology platforms to enable greater clinical proof-of-concept success for new cancer therapies. TransPoC will comprise three platforms: 1. CPN - Cancer Cell “PoC” Network for screening compounds against 1000+ genomically-characterized cell lines; 2. MPN- Mouse “PoC” Network - a multi-site platform for mouse preclinical trials using genomically-characterized Patient-Derived Xenograft (PDX) models; 3. BioIT - analysis and integration of genomic information and pharmacological profiling data. Here we present an overview of the Mouse “PoC” Network, define a path to implementation of multi-center pre-clinical trials in mice and describe a pilot study to demonstrate the feasibility of implementing such a network. PDX models are increasingly used in pre-clinical studies as they capture and retain the histological, molecular, and genetic heterogeneity of the original tumor compared to cell line derived xenografts and are therefore a closer representation of a patients tumor in situ. To enable transformative preclinical studies, models need to be characterized in a manner similar to tumor samples in The Cancer Genome Atlas and the International Cancer Genome Consortium, and must be assembled in sufficient quantity to capture clinically relevant major cancer (sub)types. To achieve this, TransPoC is building a global network of mouse PDX “hospitals” with genomic and metabolomic profiles characterized in a consistent manner. In addition, each mouse hospital will utilize common SOPs to generate comparable pharmacology data sets across sites that will include testing standard of care agents. BioIT will enable deep interrogation of data sets and provide pipelines for pharmacogenomics correlates of response to both standard and novel agents. To date, the network has collated over 2,000 PDX models and will enable sponsors to execute multi-center pre-clinical trials in a manner similar to those used in multi-institutional cooperative clinical trials. To demonstrate the viability of MPN, a pilot study has been initiated at 6 sites located in Canada, Italy, China and USA to evaluate the activity of MEK and RAF inhibitors against a panel of BRAF/KRAS mutant melanoma and colorectal cancer PDX models. An update on the initial tolerability, PK/PD/efficacy studies and molecular characterization of PDX models in the network will be presented. TransPoC continues to recruit new sites and characterize their PDX models for incorporation into MPN for use by TransPoC sponsors. Through this effort TransPoC enables rapid assessment of standard and novel investigational therapies to determine their therapeutic potential for translation to clinical trials with a mission to improve the chance of observing clinical proof-of-concept.nnCitation Format: Peter G. Smith, David Sutton, Andrea Bertotti, Livio Trusolino, Susan Airhart, Ming S. Tsao, Bradly G. Wouters, S. Gail Eckhardt, Lai Wang, Tim Heffernan, David Verbel, Andrea Gerken, Peter Fekkes, Lihua Yu, Lihua Yu, Markus Warmuth. Translational Proof-of-Concept (TransPoC), a not-for-profit research organization enabling access to large-scale translational oncology platforms: The Patient-Derived Xenograft network. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1191. doi:10.1158/1538-7445.AM2014-1191