George Xu

The state of melanoma: challenges and opportunities

The Melanoma Research Foundation (MRF) has charted a comprehensive assessment of the current state of melanoma research and care. Intensive discussions among members of the MRF Scientific Advisory Council and Breakthrough Consortium, a group that included clinicians and scientists, focused on four thematic areas – diagnosis/early detection, prevention, tumor cell dormancy (including metastasis), and therapy (response and resistance). These discussions extended over the course of 2015 and culminated at the Society of Melanoma Research 2015 International Congress in November. Each of the four groups has outlined their thoughts as per the current status, challenges, and opportunities in the four respective areas. The current state and immediate and long‐term needs of the melanoma field, from basic research to clinical management, are presented in the following report.

TBX2 expression is regulated by PAX3 in the melanocyte lineage

The paired box homeotic gene 3 (PAX3) is a crucial regulator for the maintenance of melanocytic progenitor cells and has a poorly defined role in melanoma. To understand how PAX3 affects melanocyte and melanoma proliferation, we identified potential PAX3 downstream targets through gene expression profiling. Here, we identify T‐box 2 (TBX2), a key developmental regulator of cell identity and an antisenescence factor in melanoma, as a directly regulated PAX3 target. We also found that TBX2 is involved in the survival of melanoma cells and is overexpressed in some melanoma specimens. The identification of TBX2 as a target for PAX3 provides a key insight into how PAX3 may contribute to melanoma evolution and may provide opportunities for prosenescence therapeutic intervention aimed at disrupting the ability of PAX3 to regulate TBX2.

Analysis of HIF-1a and its regulator, PHD2, in retroperitoneal sarcomas: clinico-pathologic implications.

Hypoxia is known to play important role in cancer biology. In sarcomas, hypoxia-induced protein biomarkers such as Hypoxia Inducible Factor-1a (HIF-1a), vascular endothelial growth factor (VEGF), and Erythropoietin (Epo) have been previously reported in only a few studies. Moreover, the biologic significance and relationship to tumorigenesis of these hypoxia-induced biomarkers is not well understood in the context of sarcoma. The HIF negative regulator, Prolyl Hydroxylase Domain protein 2 (PHD2) has not been evaluated in sarcomas. We examined the expression of PHD2, HIF-1a, and several other hypoxia induced biomarkers in a series of clinically characterized, retroperitoneal sarcomas with immunohistochemical methods. Expression of these proteins was analyzed and correlated with clinical outcome. Increased HIF-1a expression was associated with shorter overall and disease free survival. PHD2 expression was detected in the majority of sarcoma cases, with increased expression correlating with high tumor grade but not with survival. Though changes in PHD2 expression alone did not correlate with overall and disease free survival, reduced/absent PHD2 expression in the presence of HIF-1a expression was associated with shorter overall and disease-free survival than that of other HIF-1a/PHD2 expression profiles. These observations suggest that regulation and expression of both PHD2 and HIF-1a are important to the biology of sarcomas, and that loss of PHD2 function has an additional adverse effect in the prognosis of sarcomas in tumors expressing HIF-1a. The biologic and therapeutic implications of HIF-1a and PHD2 expression in retroperitoneal sarcomas warrant further investigation.

Abstract A52: Radiation and dual PD-L1 and CTLA4 checkpoint blockade non-redundantly improves tumor resistance, response, and immunity

Background: Immune checkpoint inhibitors such as CTLA4 and PD-1 result in impressive clinical responses, but optimal results will require combination therapy. This raises fundamental questions about non-redundancy and mechanisms of resistance. Emerging data indicate that combining immune checkpoint inhibitors with radiation (RT) may hold promise. We therefore evaluated this combination for metastatic melanoma using parallel studies in mice and humans. Methods: 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. Results: 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. Conclusions: 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, anti-CTLA4, and anti-PDL1 is a rational and non-redundant approach to improving resistance, response, and immunity to melanoma and other cancer types. Citation Format: Andrew J. Rech, Christina Twyman, Amit Maity, Ramesh Rengan, Kristen E. Pauken, Erietta Stelekati, Bihui Xu, Hannah Dada, Pamela M. Odorizzi, Ramin D. Herati, Kathleen D. Mansfield, Dana Patsch, Ravi Amaravadi, Lynn Schuchter, Hemant Ishwaran, Rosemarie Mick, Daniel Pryma, George Xu, Michael Feldman, Tara C. Gangadhar, Stephen Hahn, E. John Wherry, Andy J. Minn, Robert H. Vonderheide. Radiation and dual PD-L1 and CTLA4 checkpoint blockade non-redundantly improves tumor resistance, response, and immunity. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A52.

Prediction of late-onset Alzheimer’s disease-associated enhancer elements

Background: Genome-wide association studies (GWAS) have added substantially to our understanding of the genetic factors implicated in late-onset Alzheimer’s disease (LOAD). However, GWAS alone lacks the granularity to determine causal variants from among thousands of candidates in each significant region. Determination of true causal variants must therefore be conducted through additional analyses following GWAS. This is further complicated by the fact that an estimated 93% of GWAS signals implicate variants in non-protein-coding regions where mechanisms involve indirect gene regulation (Maurano et al. 2012). Furthermore, regulatory elements do not necessarily regulate the closest genes. Therefore two key questions arise: (1) How do we distinguish between the multitudes of non-coding variants with significant P-values to determine causal variants? (2) How do we determine which target genes these causal variants regulate?Methods:We developed a pipeline integrating annotation data from ENCODE, NIH Roadmap Epigenomics, and FANTOM5 data among others for the purpose of detecting cell-type-specific enhancer-promoter relationships affected by LOAD-associated non-coding SNPs. From the 21 genome-wide significant LOAD-associated regions identified by the IGAP GWAS, 1,980 candidate SNPs were identified. Candidates were then prioritized for enhancer potential based on consensus among cell-type-specific and non-cell-type-specific enhancer markings. Target genes were predicted for top candidates based on gene expression QTL and chromatin conformation (3C and Hi-C) results, as well as through enhancer-promoter correlation of both open chromatin and RNA expression levels. Results:Using our pipeline we identified a set of five top candidate LOAD-associated enhancer SNPs, which are supported by multiple independent monocyte-specific enhancer marks. These SNPs were linked to the genes PTK2B, CASS4, MS4A4A, MS4A6A, and TAS2R41 by both FANTOM5 enhancer-promoter RNA expression correlation as well as monocyte-specific eQTL studies. We are currently in the process of performing luciferase assays in the THP-1 monocyte cell line in order to validate the enhancers’ activities. Conclusions: Through utilization of genomic annotation data, we were able to identify candidate causal SNPs within LOAD-associated non-protein coding regions. Further wet-lab validations are underway; we will report the findings during the AAIC 2015 meeting.