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Chimeric antigen receptor (CAR) T cell therapy has shown remarkable success in the treatment of hematologic malignancies. Unfortunately, it has limited efficacy against solid tumors, even when the targeted antigens are well expressed. A better understanding of the underlying mechanisms of CAR T cell therapy resistance in solid tumors is necessary to develop strategies to improve efficacy. Here we report that solid tumors release small extracellular vesicles (sEVs) that carry both targeted tumor antigens and the immune checkpoint protein PD-L1. These sEVs acted as cell-free functional units to preferentially interact with cognate CAR T cells and efficiently inhibited their proliferation, migration, and function. In syngeneic mouse tumor models, blocking tumor sEV secretion not only boosted the infiltration and anti-tumor activity of CAR T cells but also improved endogenous anti-tumor immunity. These results suggest that solid tumors use sEVs as an active defense mechanism to resist CAR T cells and implicate tumor sEVs as a potential therapeutic target to optimize CAR T cell therapy against solid tumors.

Patient-derived xenografts (PDXs) model human intra-tumoral heterogeneity in the context of the intact tissue of immunocompromised mice. Histological imaging via hematoxylin and eosin (H&E) staining is performed on PDX samples for routine assessment and, in principle, captures the complex interplay between tumor and stromal cells. Deep learning (DL)-based analysis of large human H&E image repositories has extracted inter-cellular and morphological signals correlated with disease phenotype and therapeutic response. Here, we present an extensive, pan-cancer repository of nearly 1,000 PDX and paired human progenitor H&E images. These images, curated from the PDXNet consortium, are associated with genomic and transcriptomic data, clinical metadata, pathological assessment of cell composition, and, in several cases, detailed pathological annotation of tumor, stroma, and necrotic regions. We demonstrate that DL can be applied to these images to classify tumor regions with an accuracy of 0.87. Further, we show that DL can predict xenograft-transplant lymphoproliferative disorder, the unintended outgrowth of human lymphocytes at the transplantation site, with an accuracy of 0.97. This repository enables PDX-specific investigations of cancer biology through histopathological analysis and contributes important model system data that expand on existing human histology repositories. We expect the PDXNet Image Repository to be valuable for controlled digital pathology analysis, both for the evaluation of technical issues such as stain normalization and for development of novel computational methods based on spatial behaviors within cancer tissues. Citation Format: Brian S. White, Xing Yi Woo, Soner Koc, Todd Sheridan, Steven B. Neuhauser, Shidan Wang, Yvonne A. Evrard, John David Landua, R Jay Mashl, Sherri R. Davies, Bingliang Fang, Maria Gabriela Raso, Kurt W. Evans, Matthew H. Bailey, Yeqing Chen, Min Xiao, Jill Rubinstein, Ali Foroughi pour, Lacey Elizabeth Dobrolecki, Maihi Fujita, Junya Fujimoto, Guanghua Xiao, Ryan C. Fields, Jacqueline L. Mudd, Xiaowei Xu, Melinda G. Hollingshead, Shahanawaz Jiwani, PDXNet consortium, Tiffany A. Wallace, Jeffrey A. Moscow, James H. Doroshow, Nicholas Mitsiades, Salma Kaochar, Chong-xian Pan, Moon S. Chen, Luis G. Carvajal-Carmona, Alana L. Welm, Bryan E. Welm, Ramaswamy Govindan, Shunqiang Li, Michael A. Davies, Jack A. Roth, Funda Meric-Bernstam, Yang Xie, Meenhard Herlyn, Li Ding, Michael T. Lewis, Carol J. Bolt, Dennis A. Dean, Jeffrey H. Chuang. A pan-cancer PDX histology image repository with genomic and pathological annotations for deep learning analysis. [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 5407.

Metastatic melanoma is an aggressive disease. Despite recent improvements in therapy, drug resistant populations emerge even in drug-sensitive tumors. The ability to adapt to multiple types of stress contributes to tumor progression and therapy resistance. We have found a subset of melanoma cells with high Wnt5A and wild type p53 expression can transition to a slow-cycling state, rendering them resistant to most targeted therapy. Multiple types of stress, including DNA damage, targeted therapy, and aging increase Wnt5A and p53 in these metastatic cells. Inhibiting p53 blocks the slow cycling phenotype and promotes sensitivity to targeted therapy. A single dose of a p53 inhibitor at the commencement of BRAF/MEKi therapy was sufficient to prolong sensitivity to BRAF/MEKi therapy in Yumm1.7 derived tumors grown in aged mice (> 52 weeks). Upon analysis of PDX tumors with WTp53, treated with BRAF/MEKi combination or BRAFi therapy, we observed an increase in p53 positive cells as well as an increase in Wnt5A expression, further linking Wnt5A and p53 with resistance to targeted therapy. These data suggest that melanoma cells require p53 to survive multiple types of stress and that taking the paradoxical approach of inhibiting rather than activating WTp53 may sensitize previously resistant metastatic melanoma cells to therapy by temporarily driving them into a rapidly cycling state. WTp53 is typically thought of as a tumor suppressor due to its ability to regulate cell proliferation, stress response, and cell death. However, in metastatic melanoma cells, p53 promotes the expression of EDIL3, FAP, MMP3, and netrin G1, which are associated with poor prognosis, increased angiogenesis, and metastasis in multiple types of cancer. These data suggest that p53, typically thought of as a tumor suppressor, may be promoting the survival of a subset of highly resistant metastatic cells. Citation Format: Jasmin Giles, Maria Biancaniello, Mitchell Fane, Gretchen Alicea, Andrew Kossenkov, Amanpreet Kaur, Vito Rebecca, Xiaowei Xu, Meenhard Herlyn, Maureen Murphy, Ashani Weeraratna, Marie R. Webster. All stressed out: Phenoytpe plasticity and therapy resistance in melanoma [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 966.

Intratumoral heterogeneity and cellular plasticity enable tumors to alter phenotypes and adapt to foreign microenvironments and resist targeted inhibitors. While the ability to switch between phenotypic states has been broadly characterized, the key mechanisms that underlie tumor plasticity remain poorly understood. We studied the neural crest lineage transcription factor, SOX10, in the context of cutaneous melanoma and resistance to targeted therapies. SOX10 is heterogeneously expressed in melanoma samples. Using bio-informatics as well as in vivo and 3D in vitro melanoma models, SOX10 loss was sufficient to induce an invasive but slow proliferating phenotype in vitro and in vivo that was associated with expression of a mesenchymal gene set. Interestingly, while SOX10 knockout initially induced a targeted inhibitor tolerant state, longer exposure of co-mixed populations of SOX10 proficient and SOX10 deficient to targeted therapy drives the clonal selection of SOX10 knockout cells. Furthermore, cell lines generated from xenograft tumors that have acquired resistance to either vemurafenib, paradox-breaking BRAFi or the combination of BRAFi + MEKi showed dramatically reduced SOX10 expression compared to their parental counterparts. Altogether these data suggest that acquired resistant clones may arise from drug tolerant persister cells. As a strategy to selectively target this invasive, drug-tolerant SOX10-deficient sub-population, we screened a drug compound library and identified a class of cIAP1/2 inhibitors to be synthetically lethal for SOX10-deficient cells. Our preliminary data suggest that birinapant can delay or prevent resistance to BRAFi/MEKi in vivo. Together, these data suggest that SOX10 mediates phenotypic switching in cutaneous melanoma and enables tumor adaptation to altered microenvironments and drug treatments which could be targeted using cIAP1/2 inhibitors. Citation Format: Claudia Capparelli, Timothy J. Purwin, Manoela Tiago, Nicole Wilski, Danielle Pomante, McKenna Glasheen, Sheera Rosenbaum, Mai Q. Nguyen, Weijia Cai, Richard Zheng, Gaurav Kumar, Inna Chervoneva, Ayako Shimada, Adam E. Snook, Paolo Fortina, Xiaowei Xu, Kim Hookim, Edna Cukierman, Michael A. Davies, Meenhard Herlyn, Andrew E. Aplin. Targeting SOX10-deficient cells to reduce resistance to targeted therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 40.

RAS and its downstream cascades transmit cellular signals, resulting in increased transcription of genes involved in cell growth and division. Protein-coding gene targets of RAS signaling have been characterized extensively, but long noncoding RNAs (lncRNA) regulated by these processes have not. Using a custom-designed lncRNA microarray, we identified the lncRNA Orilnc1 as a genetic target of RAS that is critical for RAS oncogenicity. Orilnc1 expression was regulated by RAS–RAF–MEK–ERK signaling via the transcription factor AP1. Orilnc1 was highly expressed in BRAF-mutant cancers, such as melanoma. Silencing of Orilnc1 blocked tumor cell proliferation and growth in vitro and in vivo. In addition, Orilnc1 blockade reduced expression of cyclin E1 and induced G1–S cell-cycle arrest in tumor cells. Taken together, our results identify Orilnc1 as a novel, nonprotein mediator of RAS/RAF activation that may serve as a therapeutic target in RAS/RAF–driven cancers. Cancer Res; 77(14); 3745–57. ©2017 AACR.

Immune checkpoint inhibitor therapy (anti-CTLA4 or anti-PD1 antibodies) is rapidly emerging as a front-line treatment option for many solid tumors. However, only a third of melanoma patients respond to immune checkpoint blockade. Currently available mouse xenograft and transgenic models have many shortcomings and are unable to address the basis of therapy resistance and immune nonresponsiveness that are observed in patients. Thus, there is an urgent need to establish an in vivo model with a human immune microenvironment that can address issues of therapy resistance. Our laboratory has developed a novel humanized mouse melanoma model. Immunodeficient NSG mice were reconstituted with human CD34+ cells and after 8-12 weeks, mice are fully reconstituted with human innate (monocyte/myeloid lineage cells, dendritic cells and NK cells) and adaptive (T and B cells) immune cells. Humanized mice were then challenged with HLA-matched melanoma cells, and the functional ability of human immune cells to restrict tumor growth was monitored. Delayed tumor growth was observed in humanized mice, indicating in vivo sensitization of human immune cells to melanoma. This was confirmed by in vitro demonstration of human lymphocytes from tumor-bearing mice showing enhanced cytokine expression after stimulation with melanoma antigen peptides. In therapy studies, tumor-bearing humanized mice treated with anti-PD-1 showed restricted tumor growth. Anti-PD-1 therapy resulted in enhanced infiltration of T cells that correlated with tumor response. MassCyTOF studies were performed using a panel of immune markers to understand the mechanism of therapy nonresponsiveness in some tumors. Downmodulation of HLA-class I molecules and increased presence of FOXP3+ cells in the tumor region were seen. Our results suggest that humanized mouse melanoma model can be explored further to understand the causes of therapy resistance and immune nonresponsiveness. Citation Format: Rajasekharan Somasundaram, Anastasia Samarkina, Thomas Connelly, Robin Choi, Hedy Choi, Kar Muthumani, Xiaowei Xu, Klaus Kaestner, Meenhard Herlyn. Humanized mouse model: A model to understand mechanisms of immune non-responsiveness to immune checkpoint inhibitors in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A26.

High doses of ionizing radiation (IR) as delivered with hypofractionated radiotherapy (HFRT) can generate immunogenic cell death and elicit adaptive anti-tumor immunity through cross priming of tumor associated antigens. Resiquimod (R-848), a TLR7/8 agonist, generates a systemic antitumor immunity resulting in tumor regression and enhanced T-cell effector function in cutaneous T-cell lymphomas clinically. We hypothesized that combining HFRT with immunotherapy would boost the antitumorigenic effects of either monotherapies, and if so, could lead to a readily translated clinical modality. B16F10 (murine melanoma) and SCC7 (murine squamous cell carcinoma) cells were used as to grow flank subcutaneous syngeneic tumor models in C57BL/6J and C3H/HeJ mice respectively. Our studies indicate that intratumoral administration of resiquimod combined with HFRT to those tumors leads to a significantly stronger antitumor response than either single treatment in both tumor models. The combination therapy in the primary tumor led to complete tumor regression of the SCC7 tumors in 86% of the treated mice to just 14% rejection in the HFRT group alone. Tumor re-challenge of those mice led to tumor rejection in 83% in the combination group compared to 0% of the HFRT group indicating a potent systemic effect of the combined therapy. Furthermore, the combination therapy led to significant reduction of the index (treated) but moreover in the abscopal (untreated) tumors compared to either monotherapy confirming a systemic antitumor effect in this dual-tumor in vivo model. RT-qPCR analysis revealed an increase expression of Th-1 signature (IFNγ, IL-12, Prf1, GzmB) in both primary and abscopal tumors and flowcytometric analysis of the abscopal B16F10 tumors revealed an increase in CD8+ infiltration. A more thorough flowcytometric analysis following staining with the gp100 tetramer revealed an increased cross-priming against gp100 (PMEL) and enhanced gp100-specific T-cell infiltration within the tumor. Moreover, splenocytes that were re-stimulated in vitro with the gp100 peptide in an IFNγ ELISPOT assay, they increased in mice treated with the dual therapy. Finally, an in vitro study of B16F10 cells, which express TLR7/8 receptors, revealed a substantial increase in IFNβ and CCL5 expression with the dual treatment. Knocking down TLR7 and Tmem173 (STING) in those cells with siRNAs completely reversed the phenotypic IFN type I expression signature. In conclusion, the combination of HFRT with Resiquimod leads to very potent antitumor effects in both primary and abscopal melanoma and squamous cell carcinoma tumors driven by an enhanced systemic immunologic response. Our findings provide the rational for translating therapies combining radiotherapy and intratumoral resiquimod into early-phase clinical trials. Citation Format: Ilias V. Karagounis, Stefano Pierini, Noëlle François, Christoforos Thomas, Xiaowei Xu, Constantinos Koumenis, Andrea Facciabene, Amit Maity. Hypofractionated radiotherapy as a vaccine in combination with the TLR7/8 agonist resiquimod leads to significant abscopal tumor response and persistence antitumor immunity [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 6509.

Despite novel therapies for melanoma, drug resistance remains a significant hurdle to achieving optimal responses. NRAS mutant melanoma is an archetype of therapeutic challenges in the field, which we used to test drug combinations to avert drug resistance. We show that the BET family member BRD4 is expressed at high levels in NRAS-mutant melanoma, and that high levels of BRD4 are associated with poor patient survival. Combining BET and MEK inhibitors synergistically curbed the growth of NRAS mutant melanoma and prolonged the survival of mice bearing tumors refractory to MAPK inhibitors and immunotherapy. Transcriptomic analysis revealed that combining BET and MEK inhibitors mitigated a MAPK- and checkpoint-inhibitor resistance transcriptional signature, downregulated the transcription factor TCF19, and induced apoptosis. Notably, TCF19 levels were downregulated in post-treatment biopsies from patients that responded to targeted or immunotherapies. In contrast, TCF19 levels were upregulated in tumor samples from non-responder patients. Furthermore, global proteomic analysis of PDX derived from patients resistant to immune checkpoint inhibitor indicate that the major downstream effects of concurrent BET and MEK inhibition is lipid metabolism. We are investigating the impact and mechanism whereby lipids modulate therapy response. Together our studies demonstrate that co-targeting MEK and BET can offset therapy resistance, offering a potential salvage strategy for melanomas with no other therapeutic options, and possibly other treatment-resistant tumor types. Citation Format: Jessie Villanueva, Ileabett M. Echevarria-Vargas, Patricia I. Reyes-Uribe, Adam Guterres, Amruta Ronghe, Delaine M. Zayas-Bazan Burgos, Qin Liu, Xiaowei Xu, David W. Speicher. Co-targeting BET and MEK as salvage therapy for MAPKi and checkpoint inhibitor resistant melanoma [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 3842.

Background: The HER2 pathway can be inhibited by potent targeting agents such as lapatinib (L), trastuzumab (T), or their combination (LT), but acquired and de novo resistance still occur. Resistance to these drugs remains a major hurdle in the management of HER2+ breast cancer. Consequently, elucidation of mechanisms of acquired therapeutic resistance to HER2-directed therapies is of critical importance. Methods: To obtain clues to the mechanisms for resistance we developed a panel of HER2+ breast cancer cell lines resistant to L, T, or LT. Parental cells and resistant derivatives of the HER2+ BT474 cell line were characterized by RNA-seq. Genes that were overexpressed in resistant compared to parental cells were confirmed by RT-PCR, Western blotting, and immunohistochemistry (IHC). Cell growth and cell signaling were assessed in parental and resistant cell lines after down-regulation (by siRNA) or overexpression (via an inducible cDNA) of IRS4 in the presence or absence of treatment. The effect of IRS4 overexpression on L resistance was assessed in a BT474 xenograft model. The proteins that interact with IRS4 were identified by co-immunoprecipitation with IRS4 followed by separation of the associated proteins by SDS-PAGE and microsequencing by mass spectrometry. Results: RNA-seq analysis revealed that IRS4 was the most up-regulated gene in BT474 L or LT resistant derivatives in which HER2 signaling is effectively inhibited, but not T alone, where HER2 signaling is reactivated. Western blotting and IHC validated this result and identified membrane localization of IRS4. Knockdown of IRS4 in L- or LT-resistant cells reversed resistance and restored growth inhibition. IRS4 knockdown also inhibited downstream signaling, with a reduction in pAKT but not in pMAPK. Induction of the cell cycle regulator p27 and down-regulation of survivin were observed after IRS4 knockdown. Overexpression of IRS4 cDNA in parental BT474 and SKBR3 cells led to resistance to L/LT, increased pAkt, and decreased the apoptotic marker cleaved PARP in the presence of L or the LT combination. The BT474 xenograft model showed that IRS4 overexpression in the absence of treatment had no effect on tumor growth but it significantly reduced the inhibitory effect of lapatinib (p=0.002). A group of proteins that interact with IRS4 in BT474 L-resistant cells were identified by mass spectrometry. The roles of these proteins in IRS4-mediated resistance to lapatinb-containing regimens are under investigation. Conclusion: IRS4 overexpression is a critical factor in causing resistance to lapatinib-containing regimens in BT474 cells. Investigation of IRS4 and its signaling partners in HER2+ human tumors resistant to lapatinib will be important to determine if this mechanism is also operative in patients. Citation Format: Lanfang Qin, Maria B Hahn, Xiaoyong Fu, Martin J Shea, Mario Giuliano, Sarmistha Nanda, Xiaowei Xu, Huizhong Hu, Sung Yun Jung, Laura M Heiser, Nicholas Wang, Joe W Gray, Susan G Hilsenbeck, Chad Creighton, Chad A Shaw, Gary C Chamness, Dean P Edwards, Sabrina Herrera, Carolina Gutierrez, C Kent Osborne, Rachel Schiff. Overexpression of insulin receptor substrate 4 can mediate acquired resistance to lapatinib-containing regimens in HER2+ breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-13.

Mutations in the BRAF serine/threonine kinase gene are frequently found in cutaneous melanomas. Activation of hypoxia inducible factor-1α (HIF-1α) in response to both hypoxic stress and oncogenic signals has important implications in cancer development and progression. Here, we report that mutant BRAFV600E increases HIF-1α expression in melanoma cells. Our microarray profiling data in 35 melanoma and melanocyte cell lines showed that HIF-1α gene expression was significantly increased in melanomas harboring BRAFV600E mutation. Stable suppression of mutant BRAFV600E or both wild-type and mutant BRAFV600E by RNA interference in melanoma cells resulted in significantly decreased HIF-1α expression. Knockdown of mutant BRAFV600E induced significant reduction of cell survival and proliferation under hypoxic conditions, whereas knockdown of both wild-type and mutant BRAFV600E resulted in further reduction. The effects of BRAF knockdown can be rescued by reintroducing BRAFV600E into tumor cells. Transfection of BRAFV600E into melanoma cells with wild-type BRAF induced significantly more hypoxic tolerance. Knockdown of HIF-1α in melanoma cells resulted in decreased cell survival under hypoxic conditions. Pharmacologic inhibition of BRAF by BAY 43-9006 also resulted in decreased HIF-1α expression. Although HIF-1α translational rate was not changed, the protein was less stable in BRAF knockdown cells. In additional, von Hippel-Lindau protein expression was significantly increased in BRAF knockdown cells. Our data show for the first time that BRAFV600E mutation increases HIF-1α expression and melanoma cell survival under hypoxic conditions and suggest that effects of the oncogenic V600E BRAF mutation may be partially mediated through the HIF-1α pathway. [Cancer Res 2007;67(7):3177–84]

Recent studies suggest that cancer can arise from a cancer stem cell (CSC), a tumor-initiating cell that has properties similar to those of stem cells. CSCs have been identified in several malignancies, including those of blood, brain, and breast. Here, we test whether stem cell–like populations exist in human melanomas. In ∼20% of the metastatic melanomas cultured in growth medium suitable for human embryonic stem cells, we found a subpopulation of cells propagating as nonadherent spheres, whereas in standard medium, adherent monolayer cultures were established. Individual cells from melanoma spheres (melanoma spheroid cells) could differentiate under appropriate conditions into multiple cell lineages, such as melanocytic, adipocytic, osteocytic, and chondrocytic lineages, which recapitulates the plasticity of neural crest stem cells. Multipotent melanoma spheroid cells persisted after serial cloning in vitro and transplantation in vivo, indicating their ability to self-renew. Furthermore, they were more tumorigenic than adherent cells when grafted to mice. We identified similar multipotent spheroid cells in melanoma cell lines and found that the stem cell population was enriched in a CD20+ fraction of melanoma cells. Based on these findings, we propose that melanomas can contain a subpopulation of stem cells that contribute to heterogeneity and tumorigenesis. Targeting this population may lead to effective treatments for melanomas.

Immune checkpoint inhibitors result in impressive clinical responses but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here, we report major tumor regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation (RT) on a phase one clinical trial and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumors, resistance was common. Computational analysis of genome-wide and immune profiles of mice revealed resistance was due to T cell exhaustion driven by adaptive resistance and prolonged interferon-gamma exposure, resulting in STAT1-mediated upregulation of PD-L1 on melanoma cells and tumor macrophages. Accordingly, optimal response in melanoma and other cancer types requires RT, anti-CTLA4, and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T regulatory cells, and macrophage depletion and/or PD-L1 blockade reverses T cell exhaustion. RT promotes the infiltration of intratumoral antigen-specific CD8 T cells and enhances the diversity of the T cell receptor (TCR) repertoire. RT with dual checkpoint blockade shapes the TCR repertoire of the expanded peripheral clones in a manner consistent with antigen-driven selection. Similar to results from mice, patients on our clinical trial with tumors showing high PD-L1 did not respond to RT + anti-CTLA4, demonstrated persistent T cell exhaustion, and rapidly progressed. In contrast, patients with low PD-L1 on melanoma cells or macrophages had markedly improved survival, with the best survival observed among those patients with low PD-L1 on both cell types. Thus, our results suggest that 1) RT can enhance response to anti-CTLA4 when the TCR and/or antigen repertoire are sub-optimal, 2) upregulation of PD-L1 through STAT1-mediated adaptive resistance mechanisms inhibits response to anti-CTLA4-based therapy unless PD-L1/PD-1 is blocked, and 3) the combination of RT, anti-CTLA4, and anti-PD-L1 promotes response and immunity through distinct mechanisms. Finally, although PD-L1 was a dominant resistance mechanism in our models, PD-L1-independent resistance mechanisms were also evident. The next generation of clinical trials based on these findings are underway. Citation Format: Christina Twyman-Saint Victor, Andrew Rech, Joseph Benci, Amit Maity, Ramesh Rengan, Kristen Pauken, Erietta Stelekati, Bihui Xu, Hannah Dada, Pamela Odorizzi, Ramin Herati, Ravi Amaravadi, Lynn Schuchter, Hemant Ishwaran, Rosemarie Mick, Daniel Pryma, Xiaowei Xu, Michael Feldman, Tara Gangadhar, Steve Hahn, John Wherry, Robert Vonderheide, Andy Minn. Mechanisms of tumor response and resistance to radiation and dual checkpoint blockade in mice and patients. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR05.

Current treatments against malignant melanoma, including the use of small molecule inhibitors, are displaying encouraging results in the clinic. However, tumor resistance develops even for patients who initially respond favorably. To identify novel drug targets and compounds with anti-melanoma activity, we screened a panel of genetically distinct human-derived metastatic melanoma cell lines against structurally diverse organometallic kinase inhibitors. We observed a compound that preferentially inhibits melanoma cell proliferation compared to normal primary fibroblasts and melanocytes in adherent and three-dimensional (3D) cultures. We further showed that this compound (SM200) is pro-apoptotic and anti-invasive in 3D melanoma cell cultures. A kinome screen to identify the targets responsible for the anti-melanoma effects of SM200 revealed PIM kinases to be highly inhibited. PIM1 knockdown studies to validate this target in human metastatic melanoma indicate that PIM1 contributes to melanoma growth in 3D culture and in a xenograft model. We then evaluated the clinically available PIM kinase inhibitor SGI-1776 in our melanoma models and observed reduced cell proliferation in 2D, 3D, and in vivo melanoma models (as seen for SM200) suggesting an important role for PIM kinases in melanoma pathobiology. Taken together, our findings suggest that PIM kinases are valid targets for melanoma and PIM kinase inhibitors could enhance the effects of current therapies in the clinic. Citation Format: Adina M. Vultur, Batool Shannan, Quan Chen, Andrea Watters, Stefan Mollin, Eric Meggers, Clemens Krepler, Michela Perego, Ling Li, Phyllis A. Gimotty, Xiaowei Xu, Meenhard Herlyn. Compound screen identifies PIM kinases as therapeutic targets for melanoma. [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 687. doi:10.1158/1538-7445.AM2015-687

Optimal results with immune checkpoint inhibitors such as CTLA4 and PD-1 will likely require combination therapy. This raises important questions about tumor resistance and non-redundant mechanisms of action. Pre-clinical and clinical data indicate that radiation (RT) may augment responses to immune checkpoint inhibition. We therefore evaluated this combination for metastatic melanoma using parallel studies in mice and humans. In a phase I clinical trial with 19 patients with multiple melanoma metastases, a single index lesion was irradiated with hypofractionated RT, delivered over two or three fractions, followed by four cycles of the anti-CTLA4 antibody ipilimumab. We reproduced this therapy in mice using the melanoma cell line B16-F10. For this, each flank of C57BL/6 mice was implanted with tumors to model multiple metastases. Mice received anti-CTLA4 (on days 5, 8, and 11), irradiation of one tumor using an image-guided micro-irradiator (20 Gy x 1 on day 8), or both treatments. Mechanistic studies were performed on material obtained from patients and mice at baseline and thereafter. Overall, treatment in the phase I study was well tolerated and toxicity was similar to that reported for anti-CTLA4. Major tumor regressions were observed in a subset of patients with metastatic melanoma treated with anti-CTLA4 + RT. In mice, although combined treatment enhanced the CD8 T cell to Treg ratios and improved responses in irradiated and unirradiated tumors, resistance was common. Genome-wide and unbiased analyses revealed that resistant tumors have increased PD-L1, interferon-stimulated genes, and exhausted T cells that depress the CD8/Treg ratio. Patients and mice with high PD-L1 tumors that were treated with RT + anti-CTLA4 poorly reinvigorated exhausted T cells, did not respond, and rapidly progressed. In mice, adding anti-PD-L1/PD-1 to RT + anti-CTLA4 reinvigorated exhausted T cells, leading to complete responses and immunity across multiple cancer types. The extent of T cell exhaustion/reinvigoration predicts response and can be assessed through peripheral blood. Resistance to RT + anti-CTLA4 results from depression in the CD8/Treg ratio due to elevated tumor PD-L1 and persistent T cell exhaustion. Both clinical and pre-clinical data suggest that the combination of RT with CTLA4 and PD-1 checkpoint blockade is a rational, non-redundant approach to overcoming tumor resistance and improving immunity in multiple cancer types. Citation Format: Andrew J. Rech, Christina Twyman-Saint Victor, Amit Maity, Ramesh Rengan, Kristen E. Pauken, Erietta Stelekati, Joseph Benci, Bihui Xu, Hannah Dada, Pamela M. Odorizzi, Ramin S. Herati, Kathleen D. Mansfield, Dana Patsch, Ravi K. Amaravadi, Lynn M. Schuchter, Hemant Ishwaran, Rosemarie Mick, Daniel Pryma, Xiaowei Xu, Michael D. Feldman, Tara C. Gangadhar, Stephen M. Hahn, E. John Wherry, Andy J. Minn, Robert H. Vonderheide. Radiation and dual immune checkpoint blockade overcome tumor resistance and distinctly improve immunity. [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 2858. doi:10.1158/1538-7445.AM2015-2858

Purpose: Combining immunostimulatory monoclonal (mAb) and checkpoint inhibition may improve response rates and overall survival in patients with metastatic melanoma. CP-870,893 is an agonistic fully human IgG2 mAb targeting the immune stimulatory molecule CD40 (αCD40). Tremelimumab (treme) is a fully human IgG2 mAb targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA4), a negative co-stimulatory molecule found on T cells. Methods: Patients with metastatic melanoma were enrolled on a single-arm open-label phase I study. αCD40 and treme were dosed i.v. every 3 weeks and 12 weeks, respectively. Dose escalation followed a 3 + 3 strategy with alternating increases of αCD40 and treme in each cohort. Outcomes were scored using the Common Terminology Criteria for Adverse Events v3.0 and RECIST 1.0. Flow cytometric analysis of baseline blood samples was compared to samples obtained at least 3 weeks after the last dose of αCD40 and treme. Results: Toxicity: Twenty-four patients were enrolled and treated at 4 different dose levels. Two patients were replaced per protocol due to rapid, symptomatic progression of disease; these patients were not evaluated for response but were included in toxicity analysis. Dose-limiting toxicities (DLT), considered to be likely due to treatment, were colitis (n = 1) and hypophysitis (n = 1) and uveitis (n = 1). Cytokine release syndrome (CRS), grade 1-2, occurring within 24 hours of CP administration, was the most common treatment-related toxicity occurring in 19 (79.2%) patients. CRS symptoms were controlled with standard supportive care. All episodes of CRS resolved within 24 hours of αCD40 administration. The estimated maximum tolerated dose was 0.2 mg/kg of αCD40 and 10 mg/kg of treme. Outcomes: Overall objective response rate (ORR) was 27.3% (best response): two patients (9.1%) had complete responses to treatment and four (18.2%) patients had partial responses. The median follow-up was 22 months with a median progression free survival of 2.5 months and a median overall survival (OS) of 26.1 months. Correlative studies: Flow cytometric analysis of peripheral blood lymphocytes revealed changes in CD8+ T cell phenotypes. Double positive granzyme B+ and Ki-67+ cells increased from 0.56 ± 0.17% (mean ± standard error) for all CD8+ T cells at baseline to 1.01 ± 0.23% post-treatment (p = 0.03). CD8+ T cells expressing both programmed cell death 1 and eomesodermin were also higher after treatment, increasing from 7.7 ± 1.1% to 10.4 ±1.8% (p = 0.04) of all CD8+ T cells. Conclusion: Combination therapy with αCD40 and treme was well-tolerated in patients with metastatic melanoma, with rates of CRS and other toxicity similar to previously reported rates for αCD40 or treme treatments alone. Overall ORR was 27.3%, with median OS of 26.1 months. Given the tolerability, antitumor activity, and biomarker evidence of immune activation, expanded study of this combination should be pursued. Citation Format: David L. Bajor, Rosemarie Mick, Matthew J. Riese, Lee P. Richman, Xiaowei Xu, Drew A. Torigian, Erietta Stelekati, Martha Sweeney, Brendan Sullivan, Lynn M. Schuchter, Ravi Amaravadi, E. John Wherry, Robert H. Vonderheide. Combination of agonistic CD40 monoclonal antibody CP-870,893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma. [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 CT137. doi:10.1158/1538-7445.AM2015-CT137

Hepatocellular carcinoma (HCC) is the fifth most common type of cancer worldwide. However, current therapeutic approaches for this epidemic disease are limited and its 5-year survival rate has not been improved in the past decades. Patient-derived xenograft (PDX) tumor models have become an excellent in vivo system for understanding of disease biology and drug discovery. In order to identify new therapeutic targets for HCC, whole-exome sequencing (WES) was performed on more than 60 HCC PDX models. Among them, four models exhibited point mutations in JAK1 (Janus Kinase) gene, which were confirmed by Sanger sequencing. To explore the transforming capability, these mutations were then introduced into 293T and Ba/F3 cells by transient transfection and retroviral infection, respectively. The results demonstrated that JAK1S703I mutation was able to activate JAK-STAT (Signal Transducer and Activator of Transcription) signaling pathway and drive cell proliferation in the absence of cytokine stimulation in vitro. Furthermore, a modest sensitivity to the treatment of a JAK1/2 inhibitor, Ruxolitinib, was observed in JAK1S703I mutant PDX model, but not in other mutant or wild type models. Pharmacodynamic analysis showed that phosphorylation of STAT3 in the Ruxolitinib-treated tumor tissues was significantly inhibited. Collectively, our results suggested that JAK1S703I is an activating mutation for JAK-STAT signaling pathway in vitro and in vivo. JAK-STAT pathway might represent a new therapeutic opportunity for HCC. Monotherapy using a more potent inhibitor and combinatory therapy may be further explored in JAK1S703I mutant PDX model. Citation Format: Shuqun Yang, Chonglin Luo, Qingyang Gu, Qiang Xu, Hongye Sun, Ziliang Qian, Yexiong Tan, Hao Wu, Yuxin Qin, Yuhong Shen, Xiaowei Xu, Shu-Hui Chen, Chi-Chung Chan, Hongyang Wang, Mao Mao, Douglas D. Fang. Activating JAK1-S703I mutation may predict the sensitivity of JAK-STAT inhibition in hepatocellular carcinoma patient-derived xenograft tumor models. [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 688. doi:10.1158/1538-7445.AM2015-688

Background: Poly [ADP-ribose] polymerase (PARP) is an important mediator of DNA damage repair. Preclinical and clinical studies have indicated PARP inhibitors (i) as promising agents in DNA-repair-defective cancers, especially in the presence of BRCA1/2 alterations. Recent evidence also suggests a role for additional DNA damage-independent functions of PARP, involving transcriptional and epigenetic regulation. Particularly, PARP-1 can interact with the estrogen receptor (ER) and modulate its transcriptional activity in vascular smooth muscle cells, and PARPi can circumvent endocrine resistance in prostate cancer cells. However, the effect of PARPi on the efficacy of endocrine therapy (EndoTx) in ER+ breast cancer is unknown. Here we aimed to establish these effects using the PARPi olaparib (AZD2281, Olap) in our preclinical models. Methods: The effects of Olap (0.01-1μM) alone or in combination with EndoTx [estrogen deprivation (ED) or tamoxifen (Tam)] were tested in vitro in three ER+ parental cell lines (MCF7, ZR75-1, and T47D; BRCAwt) and their endocrine resistant (R) derivatives. Clonogenic assays were used to assess Olap-induced changes in cell growth. In vivo, ovariectomized nude mice bearing 200 mm3 tumors growing in the presence of estrogen supplementation (E2 pellets), were randomized to either continued E2 (control), ED, or ED+Tam; all ± oral Olap (100mg/kg daily). Results: In MCF7 parental cells in vitro, Olap, in a dose dependent manner, inhibited E2-stimulated growth and increased the growth inhibitory effect of EndoTx. In addition, at a clinically relevant dose (1μM), Olap significantly inhibited the growth of MCF7 EndoR cell derivatives. Similar results were also observed in the other 2 ER+ cell models. In vivo, long-term Olap-treatment in the presence of EndoTx (Tam ∼200days, and ED ∼300days) was well tolerated and no apparent drug-related toxicity was observed. Olap did not affect the growth of E2-stimulated MCF7 xenografts. In contrast, Olap enhanced sensitivity to Tam by delaying time to tumor progression (TTP; size doubling) from a median of 95 days to 155 days (p = 0.03), and numerically but not significantly reduced time to tumor regression (TTR; size halving) from 138 to 51 days (p = 0.2). Olap also numerically accelerated median time to tumor regression in the presence of ED (34 ED vs. 25 days ED+Olap; p = 0.23), but had no effect on TTP (P = 0.36). Conclusion: Our results suggest a potential role for Olap in enhancing EndoTx efficacy and circumventing resistance in the absence of BRCA mutations. The absence of any toxicity and negative interaction by adding Olap to EndoTx in the in vivo experiment supports the inclusion of ER+ breast cancer patients in clinical trials using PARPi. Further studies are warranted to better understand the biology of PARP and the efficacy of PARPi in ER+ breast cancer, especially in the presence of EndoTx and resistance. Citation Format: Agostina Nardone, Amit Goldstein, Martin J. Shea, Tamika Mithchell, Xiaoyong Fu, Carmine De Angelis, Huizhong Hu, Xiaowei Xu, Mahitha Rajendran, Mark O'Connor, Gershon Locker, Susan Hilsenbeck, Kent Osborne, Rachel Schiff. PARP inhibition effects on endocrine therapy and resistance in estrogen receptor positive (ER+) breast cancer models. [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 LB-166. doi:10.1158/1538-7445.AM2015-LB-166

Background: Mortality rates due to melanoma are continuously increasing. Specifically, in older patients, melanoma is very aggressive and has a poor prognosis, compared to the young. Therefore, there is an urgent need to understand the underlying molecular mechanisms behind this age-related aggression. The expression level of klotho, an emerging tumor suppressor, declines with the progression of age and is associated with several pathologies related to human aging. A previous study from our group has demonstrated that loss of klotho in melanoma cells is associated with enhanced melanoma cell motility by decreasing the ability of melanoma cells to internalize Wnt5A, a non-canonical Wnt protein, known to be one of the drivers of melanoma metastasis. Therefore, we hypothesize that the loss of Klotho in aging microenvironment contributes to enhanced Wnt5A signaling and ultimately results in age-related melanoma aggression. Methods: In order to study the effects of aged microenvironment on melanoma cells, we have obtained and cultured normal skin fibroblasts from aged (55-65 years old) and young donors (25-35 years old). The expression levels of klotho and Wnt5A in these fibroblasts were analyzed by immunoblot, immunofluorescence and real time-PCR assays. We examined melanoma cell/ fibroblast co-cultures and artificial skin reconstructs made with these fibroblasts to understand the age-related effects of tumor microenvironment on the disease progression. We also used immunohistochemistry technique to examine the expression levels of Klotho and Wnt5A in these artificial skin reconstructs and in the paraffin-embedded skin sections obtained from young and old melanoma patients. Results: Our western blot and real-time PCR results of the young vs. old fibroblasts show that klotho expression level is lost with progression of age. Analysis of artificial skin reconstructs made using skin fibroblasts isolated from aged donors demonstrate induced invasion of melanoma cells to a much greater extent than those cultured from young donors. We also analyzed these skin reconstructs for the expression levels of Klotho and Wnt5A, using immunohistochemistry technique and the results confirm that Klotho is decreased in aged fibroblasts. Furthermore, we also found that the loss of Klotho in the aged microenvironment translated to a loss of Klotho in melanoma cells. Our western blot results also suggest that loss of Klotho in the aging microenvironment enhances Wnt5A internalization and signaling and this promotes tumor metastasis. Our immunohistochemistry results using patient samples further extend our in vitro findings and support our hypothesis that Klotho plays pivotal role in age-related melanoma progression. Significance: These results for the first time demonstrate a correlation between age-related melanoma progression and the loss of klotho level. In addition, these findings provide a molecular explanation of age-related melanoma aggression. Citation Format: Reeti Behera, Amanpreet Kaur, Katie Marchbank, Vanessa Dang, Marie Webster, Xiaowei Xu, Ashani T. Weeraratna. Role of Klotho in age-related melanoma progression. [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 1556. doi:10.1158/1538-7445.AM2015-1556

Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12-week neoadjuvant trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall pathologic complete response (pCR) rate was 27%. To investigate resistance mechanisms, we developed 10 HER2+ BC cell line models resistant (R) to one or both drugs (LR/TR/LTR). To discover potential predictive markers/therapeutic targets to circumvent resistance, we completed genomic profiling of the cell lines and a subset of pre-treatment specimens from TBCRC006. Methods: Parental (P) and LR/TR/LTR lines of 10 cell line models were profiled with whole exome/RNA sequencing. Mutations detected in R lines but not in P lines of the same model were identified. Mutation-specific Q-PCR was designed for sensitive quantification. Resistant cell and xenograft tumor growth were measured in response to drugs. Whole exome sequencing (>100X) and Ampliseq of 17 baseline tumor/normal pairs from TBCRC006 were performed. Results: We found and validated the HER2 L755S mutation in the BT474/ATCC-LTR line and BT474/AZ-LR line (in ∼30% of DNA/RNA), in which the HER pathway was reactivated for resistance. Overexpression of this mutation was previously shown to induce LR in HER2-negative BC cell lines, and resistant growth of BT474/AZ-LR line is significantly inhibited by HER2-L755S-specific siRNA knock-down, suggesting its role as an acquired L/LT resistance driver in HER2+ BC. Sequencing of BT474/AZ-LR single cell clones found the mutation in ∼30% of HER2 copies in every cell. Using mutation-specific Q-PCR, we found statistically higher HER2 L755S levels in two BT474 parentals compared to P lines of SKBR3, AU565, and UACC812. These data suggest that HER2 L755S resistant subclones preexist in the BT474 parentals and were selected by L treatment to become the major clone in the two R lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of BT474/AZ-LR and BT474/ATCC-LTR cells (IC50: Afa 0.02μM vs. L 3 μM) and BT474/AZ-LR xenografts. Whole exome sequencing/Ampliseq of TBCRC006 found the HER2 L755S mutation in 1/17 primaries. This patient did not achieve pCR. The variant was present in 2% of DNA on both platforms, indicating a subclonal event of the resistance mutation. Conclusion: Acquired L/LT resistance in the two BT474 R lines is due to selection of HER2 L755S subclones present in parental cells. The higher HER2 L755S levels in BT474 parentals compared with other parentals, and detection of its subclonal presence in a pre-treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group with an irreversible TKI like Afa may prevent resistance and improve clinical outcome of this subset of HER2+ BC. Citation Format: Xiaowei Xu, Agostina Nardone, Huizhong Hu, Lanfang Qin, Sarmistha Nanda, Laura Heiser, Nicholas Wang, Kyle Covington, Edward Chen, Alexander Renwick, Tamika Mitchell, Marty Shea, Tao Wang, Carmine De Angelis, Alejandro Contreras, Carolina Gutierrez, Suzanne Fuqua, Gary Chamness, Chad Shaw, Marilyn Li, David Wheeler, Susan Hilsenbeck, Mothaffar Fahed Rimawi, Joe Gray, C.Kent Osborne, Rachel Schiff. Clonal evolution of the HER2 L755S mutation as a mechanism of acquired HER-targeted therapy resistance. [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 737. doi:10.1158/1538-7445.AM2015-737

Background: Klotho, an emerging tumor suppressor, was originally identified as an anti-aging gene. The klotho expression level declines with the progression of age and is associated with several pathologies related to human aging. Mortality rates due to melanoma are continuously increasing. Specifically, in older patients, melanoma is very aggressive and has a very poor prognosis, compared to the young. Therefore, there is an urgent need to understand the underlying molecular mechanisms behind this age-related aggression. A previous study from our group has demonstrated that loss of klotho in melanoma cells is associated with enhanced melanoma cell motility by decreasing the ability of melanoma cells to internalize Wnt5A, a non-canonical Wnt protein, known to be a driver of melanoma metastasis. Therefore, we hypothesize that the loss of Klotho in aging microenvironment contributes to enhanced Wnt5A signaling, ultimately resulting in age-related melanoma aggression. Methods: In order to study the effects of aged microenvironment on melanoma cells, we have obtained and cultured normal skin fibroblasts from aged (55-65 years old) and young donors (25-35 years old) from the Baltimore Longitudinal Study of Aging (BLSA). The expression levels of klotho and Wnt5A in these fibroblasts were analyzed by immunoblot, immunofluorescence and real time-PCR assays. We examined melanoma cell/ fibroblast co-cultures and artificial skin reconstructs made with these fibroblasts to understand the age-related effects of tumor microenvironment on the disease progression. We also used immunohistochemistry technique to examine the expression levels of Klotho and Wnt5A in these artificial skin reconstructs and in the paraffin-embedded skin sections obtained from young and old melanoma patients. Results: Our western blot and real-time PCR results of the young vs. old fibroblasts show that klotho expression level is lost and Wnt5A expression is gained with progression of age. Analysis of artificial skin reconstructs made using skin fibroblasts isolated from aged donors demonstrate induced invasion of melanoma cells to a much greater extent than those cultured from young donors. We also analyzed these skin reconstructs for the expression levels of Klotho and Wnt5A, using immunohistochemistry technique and our results confirm that Klotho is decreased in aged fibroblasts, furthermore, we also found that the loss of Klotho in the aged microenvironment also gets translated to a loss of Klotho in melanoma cells, and a gain of Wnt5A. Our immunohistochemistry results with the patient samples further extend our in vitro findings and support our hypothesis that Klotho-Wnt5A cross talk plays pivotal role in age-related melanoma progression. Significance: These results for the first time demonstrate a correlation between age-related melanoma progression and the loss of klotho and gain in Wnt5A expression levels in the aging tumor microenvironment. In addition, these findings provide a molecular explanation of age-related melanoma aggression. Citation Format: Reeti Behera, Katie Marchbank, Amanpreet Kaur, Vanessa Dang, Marie Webster, Michael O'Connell, Xiaowei Xu, Ashani Weeraratna. Crosstalk between klotho and wnt5A drives age-related melanoma progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A11.

The incidence of melanoma rises dramatically after the age of 55. Due to an increase in aging population, it is important to study the change in molecular mechanisms due to aging that would allow development of therapies that are tailored to the age of the patients. The role of the tumor microenvironment in modulating cancer characteristics is widely recognized and it also provides targets for therapeutic intervention. Due to this, we hypothesized that changes in tumor microenvironment due to aging could affect the progression of the melanoma. We obtained skin fibroblasts from healthy donors aged 25-35, as well as skin fibroblasts from healthy donors aged 55-65. We cultured these fibroblasts and used conditioned media from them to affect the invasion of melanoma cells in 3D spheroid invasion assays, where aged fibroblasts promoted invasion of melanoma cells into collagen. We also built artificial skin (reconstructs) using young and aged fibroblasts and demonstrated that skin built with aged fibroblasts promoted melanoma cell invasion. Finally using a transgenic mouse model of melanoma (Yumm1.7, BRAFV600E/PTEN-/-) we observed that the injection of melanoma cells into the tail vein of aged mice (52 weeks) formed metastastic colonies much more rapidly than those injected into the tail vein of young mice (8 weeks). To study the factors involved in the aging microenvironment, we performed a proteomics study of the secretome from young and aged fibroblasts. From this study, we observed that aged fibroblasts secreted inhibitors of canonical Wnt signaling, as well as increased deposition of extracellular matrix components in the aging microenvironment. Since inhibition of canonical Wnt signaling has been linked to decreased sensitivity towards BRAF inhibitors in melanoma, we injected Yumm 1.7 cells subcutaneously into aged and young mice. These mice were then treated with BRAF inhibitors. We observed an increased resistance in response to therapy in the aged mice. We also prepared skin reconstruct from fibroblasts with knockdown of the proteins that we identified from secretome and treated them with PLX4720. These results indicated the role of these extracellular matrix proteins in melanoma. We are exploring the mechanisms of how these extracellular matrix proteins affect the sensitivity towards chemotherapeutics. We concluded that aging could alter tumor microenvironment thus resulting in increased metastasis and therapy resistance. It is important that the studies in cancer therapies take into account the age of the patient to achieve better response in patients. Citation Format: Amanpreet Kaur, Katie Marchbank, Vanessa Dang, Michael O'Connell, Marie Webster, Jessica Appleton, Phil Cheng, Alexander Valiga, Rachel Morissette, Nazli McDonnell, Luigi Ferrucci, Andrew Kossenkov, Katrina Meeth, Marcus Bosenberg, Hsin-Yao Tang, Xiangfan Yin, William Wood, III, Elin Lehrmann, Kevin Becker, Keith Flaherty, Dennie Frederick, Jennifer Wargo, Katherine Aird, Rugang Zhang, Xiaowei Xu, Qin Liu, David Speicher, Ashani Weeraratna. Aging microenvironment modulates melanoma invasion and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A04.

Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12-week neoadjuvant trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall pathologic complete response rate (pCR) was 27%. To investigate resistance mechanisms our lab developed 10 HER2+ BC cell lines resistant (R) to these drugs (LR/TR/LTR). To discover potential predictive markers/therapeutic targets to circumvent resistance, we completed genomic profiling of the cell line panel and a subset of pre-treatment baseline specimens from TBCRC006. Methods: Parental (P) lines and LR/TR/LTR derivatives of 9 HER2+ BC cell line models were profiled with whole exome and RNA sequencing. Mutations detected in R lines but not in same-model P lines were identified. cDNAs were assessed by targeted Sanger sequencing. Single cells of the BT474AZ-LR line were cloned and their cDNAs were sequenced. Mutant-specific Q-PCR was designed to sensitively quantify mutations. Whole exome sequencing (minimum depth 100X) of 17 baseline tumor/normal pairs from TBCRC006 were performed on Illumina HiSeq. Results: We found and validated the HER2 L755S mutation in the BT474ATCC-LTR line and the BT474AZ-LR line (∼30% of DNA/RNA/cDNA in BT474AZ-LR), in which the HER pathway was reactivated to cause resistance. Overexpression of this mutation was previously shown to induce L resistance in HER2-negative BC cell lines, suggesting a role as an acquired L/LT resistance driver in HER2+ BC. Sanger sequencing of BT474AZ-LR single cell clones found the HER2 L755S mutation in every clone but only in ∼30% of the HER2 copies. Using sensitive mutant-specific Q-PCR, we found statistically higher levels of HER2 L755S expression in BT474ATCC-P and BT474AZ-P compared to parentals of other HER2+ BC cell lines (UACC812/AU565/SKBR3/SUM190). These data suggest that this mutation exists subclonally within BT474 parental lines and was selected to become the more dominant population in the two resistant lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of both BT474ATCC-LTR/AZ-LR cells (IC50: Afa 0.02µM vs. L 3 µM). Western blots confirmed inhibition of the HER and downstream Akt and MAPK signaling in the LR cells by Afa. Sequencing of TBCRC006 baseline samples found the HER2 L755S mutation in 1/17 subjects. This patient did not achieve pCR after neoadjuvant LT treatment. The variant was present in 2% of the reads, indicating it as a subclonal event in this patient’s baseline tumor. Conclusion: Acquired resistance in two of our BT474 LR/LTR lines is due to selection of HER2 L755S subclones present in the parental cell population. The higher HER2 L755S levels detected in BT474 parentals compared with other HER2+ BC parental lines, and detection of its subclonal presence in a pre-treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group with an irreversible TKI like Afa may prevent resistance and improve clinical outcome of this subset of HER2+ BC. Citation Format: Xiaowei Xu, Agostina Nardone, Huizhong Hu, Lanfang Qin, Sarmistha Nanda, Laura M Heiser, Nicholas Wang, Kyle R Covington, Edward S Chen, Alexander Renwick, Tao Wang, Carmine De Angelis, Alejandro Contreras, Carolina Gutierrez, Suzanne AW Fuqua, Gary C Chamness, Chad Shaw, David A Wheeler, Joe W Gray, Susan G Hilsenbeck, Mothaffar F Rimawi, C Kent Osborne, Rachel Schiff. Clonal evolution of the HER2 L755S mutation leads to acquired HER-targeted therapy resistance that can be reversed by the irreversible HER1/2 inhibitor afatinib [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-03.

Autophagy, a catabolic process that maintains cellular homeostasis, is a primary therapy resistance mechanism in cancer. Emerging studies indicate that autophagy is involved in secretion through non-classical mechanisms. However, the effects of autophagy-mediated secretion on the tumor microenvironment are poorly understood. Further, while autophagy is a potential cancer therapeutic target, efforts to inhibit autophagy in clinical trials have been hampered by sub-optimal methods to quantitatively monitor tumor autophagy levels. Here, we leveraged the recent findings that autophagy is implicated in non-classical secretion to identify secreted proteins associated with autophagy in melanoma. The conditioned media of low autophagy WM793 melanoma cells was compared to its highly autophagic metastatic derivative, 1205Lu, using quantitative proteomics. These comparisons identified secreted proteins associated with high autophagy, such as IL-1β, IL-8, LIF, FAM3C, and DKK3, which have previously been implicated in tumorigenesis. These proteins were shown to be elevated in supernatants of an independent panel of high autophagy melanoma cell lines compared with low autophagy cells, suggesting a mechanistic link between autophagy level and secretion of these proteins. In addition, secretion of these proteins increased when melanoma cells with intrinsically low autophagy levels were treated with an autophagy-inducing tat-Beclin 1 peptide. Further supporting a mechanistic link, Atg7 silencing in an intrinsically high autophagy cell line resulted in significantly decreased secretion. Finally, serum collected from untreated metastatic melanoma patients with high tumor autophagy levels exhibited higher levels of these proteins than serum from low autophagy patients. These results suggest that autophagy-related secretion affects the tumor microenvironment and that measurement of extracellular autophagy-associated secreted proteins in tumors and in plasma can serve as surrogates for intracellular autophagy dynamics in tumor cells. Citation Format: Adam Akl, Xiaowei Xu, Shengfu Piao, Ravi K. Amaravadi, David W. Speicher. Identification of secreted proteins that reflect intracellular autophagy dynamics in melanoma. [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 310. doi:10.1158/1538-7445.AM2014-310

Norcantharidin (NCTD) is the demethylated analog of cantharidin, a naturally occurring toxin isolated from Mylabris. NCTD has been used to treat hepatocellular carcinoma with clinical efficacy and limited side effects. However, the underlying mechanism of its antitumor effects remains elusive. We have previously shown that TR3 expression is significantly decreased in metastatic melanomas and it is implicated in regulation of melanoma cell apoptosis. In this study, we showed that NCTD inhibited melanoma cell viability and induced apoptosis in vitro. NCTD induced translocation of TR3 from nucleus to mitochondria where it colocalized with Bcl-2 in melanoma cells. NCTD also increased Cytochome c release from mitochondria to the cytoplasm. There was also increased expression of Bax and cleaved caspase-3, accompanied with decreased expression of Bcl2 and Nf-kB2. The effects of NCTD were inhibited by knockdown TR3 expression in melanoma cells. To study its function in vivo, we treated Tyr::cre;BRAFCa/+;Ptenlox/lox mice with NCTD. Temozolomide and PBS were used as controls, and ten mice were used in each group. Temozolomide modestly increased mice survival, whereas NCTD significantly improved the survival of treated mice. Our data indicates that NCTD inhibits melanoma growth by inducing cell apoptosis via activation of a TR3 dependent pathway. These results suggest that NCTD is a potential therapeutic agent for melanoma. Financial support was provided by CA-093372, CA-116103 to XX. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5043. doi:10.1158/1538-7445.AM2011-5043

There is enormous interest to target cancer stem cells (CSCs) for clinical treatment. CSCs are highly tumorigenic and resistant to chemotherapy. Nevertheless, there is still controversial surrounding CSCs and the origin of CSCs remains elusive. In this study, we showed that expression of Oct4 gene or transmembrane delivery of Oct4 protein promoted dedifferentiation of melanoma cells to CSC-like cells. The dedifferentiated melanoma cells displayed dramatic morphological changes and acquired the ability to form tumor spheroids. They showed markedly increased resistance to chemotherapeutic agents and hypoxic injury. In the subcutaneous xenograft or tail vein injection assays, these cells had significantly increased tumorigenic capacity. The dedifferentiated melanoma cells showed significantly decreased expression of melanocytic markers and acquired many features associated with CSCs such as multipotent differentiation capacity and expression of melanoma CSC markers such as ABCB5 and CD271. Oct4 induced dedifferentiation was associated with endogenous Oct4 promoter activity, expression of Nanog and Klf4, and global gene expression changes that enriched for transcription factors. Furthermore, ShRNA mediated knockdown of Oct4 in dedifferentiated cells led to diminished CSC phenotypes. Oct4 expression was detectable in a subpopulation of tumor cells in clinical melanoma tissues. Our results indicate that CSC phenotype is dynamic and may be acquired through dedifferentiation and Oct4 is a positive regulator of tumor dedifferentiation. These data suggest Oct4 mediated differentiation may play an important role during tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4376. doi:10.1158/1538-7445.AM2011-4376