The High Mobility Group A1 Chromatin Regulator Drives Immune Evasion during MPN Progression By Repressing Genes Involved in Antigen Presentation and Immune Attack

Abstract

Introduction: Myeloproliferative Neoplasms (MPN) are blood diseases caused by mutations in hematopoietic stem cells (HSCs) which lead to clonal expansion and overproduction of myeloid lineages. Individuals with MPN are at increased risk for transformation to bone marrow fibrosis (myelofibrosis, MF) and acute myeloid leukemia (AML), both of which are associated with poor clinical outcomes. However, targetable mechanisms underlying progression remain elusive. The High Mobility Group A1 (HMGA1) gene encodes chromatin regulators which are enriched in stem cells and aberrantly overexpressed in aggressive tumors (Xian et al Nature Commun 2017;8:15008, Resar et al Cancer Res 2018;78:1890). Transgenic mice misexpressing Hmga1 in lymphoid cells develop lethal leukemia by dysregulating gene networks associated with aberrant proliferation and inflammation (Hillion et al Cancer Res 2008;68:10121, Schuldenfrei et al BMC Genomics, 2011;12:549). We discovered that HMGA1 is overexpressed in MPN with progression and required for leukemic transformation in preclinical models (Resar et al Blood 2018;132 Suppl 1:102). We therefore sought to: 1) test the hypothesis that HMGA1 drives MPN progression by dysregulating gene networks involved in immune evasion, and, 2) identify mediators of immune escape that could be disrupted in therapy.Methods: To elucidate transcriptional networks regulated by HMGA1 during MPN progression to AML, we integrated multi-omics sequencing (seq) analyses, including RNAseq, chromatin immunoprecipitation seq (ChIPseq), and ATACseq in AML cell lines from JAK2 V617Fmutant MPN after leukemic transformation (DAMI, SET-2) + HMGA1 depletion. HMGA1 gene expression was inactivated using CRISPR/Cas9 or short hairpin RNA (shRNA)-mediated gene silencing. Gene set enrichment analysis was used to dissect molecular mechanisms underlying immune invasion by HMGA1. To validate results in human MPN, RNAseq was performed in peripheral blood mononuclear cells (PBMCs) from matched MF patients who transformed to AML. To reconstruct the immune cell composition of primary MPN samples, we applied xCell, a robust computational method that converts gene expression profiles to immune cell types. Transcriptional networks were validated at the level of mRNA and protein via quantitative RT-PCR and flow cytometry. To identify drugs to disrupt HMGA1 immune evasion networks, we applied the Broad Institute Connectivity Map (CMAP) and cytotoxicity assays.Results: Integration of RNAseq, ChIPseq, and ATACseq in MPN AML cells (DAMI, SET-2) revealed that HMGA1 represses genes involved in immune activation (inflammatory response, TNFa signaling, NF-κB networks) and antigen presentation [Interferon gamma (IFNγ) response networks], including genes encoding the major histocompatibility complex (MHC) class I and II antigens. Inhibiting HMGA1 results in up-regulation of MHC class I and II antigen genes, with greatest induction of HLA-DRA (the alpha paralog for HLA Class II antigens). Similarly, HMGA1 depletion increases cell surface expression of HLA-DRA antigens. Strikingly, RNAseq from MPN patients with MF after transformation to AML reveal that HMGA1 is up-regulated in MPN AML concurrent with repression in gene networks of immune activation and antigen presentation. Immune cell transcriptomes are also depleted in MPN cells after leukemic transformation. To determine if immune evasion by HMGA1 could be modulated in therapy, we applied CMAP which identified histone deacetylase inhibitors (HDACi) as drugs targeting the HMGA1 transcriptome. The HDACi, entinostat, is cytotoxic and synergizes with the JAK inhibitor, ruxolitinib, in MPN AML cell lines. Further, entinostat induces expression of MHC class II genes and antigens. Moreover, HMGA1 depletion enhances sensitivity of MPN AML cells to entinostat.Conclusions: We discovered an epigenetic program whereby HMGA1 drives immune evasion during MPN progression by binding to chromatin and enhancing chromatin accessibility to activate transcriptional networks that repress antigen presentation and immune attack. Most importantly, HMGA1 immune evasion networks are dysregulated in human MPN and can be targeted by HDACi therapy. Together, our studies reveal a new paradigm whereby HMGA1 down-regulates MHC antigens during MPN progression, suggesting that targeting HMGA1 networks could activate an immune attack and prevent MPN progression. [Display omitted] DisclosuresRampal: Novartis: Consultancy; Pharmaessentia: Consultancy; Kartos: Consultancy; Blueprint: Consultancy; Disc Medicine: Consultancy; BMS/Celgene: Consultancy; Stemline: Consultancy, Research Funding; Constellation: Research Funding; Jazz Pharmaceuticals: Consultancy; Sierra Oncology: Consultancy; Abbvie: Consultancy; CTI: Consultancy; Incyte: Consultancy, Research Funding; Memorial Sloan Kettering: Current Employment.