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

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2546-2546
Author(s):  
Joseph Kim ◽  
Jung-Hyun Kim ◽  
Leslie Cope ◽  
Liping Li ◽  
Raajit Rampal ◽  
...  
Keyword(s):  
Antigen Presentation ◽  
Mhc Class I ◽  
Immune Evasion ◽  
Research Funding ◽  
Gene Networks ◽  
Immune Cell ◽  
High Mobility ◽  
Transcriptional Networks ◽  
Leukemic Transformation ◽  
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. Figure 1 Figure 1. Disclosures Rampal: 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.

Viral immune evasion: Lessons in MHC class I antigen presentation

2015 ◽  
Vol 27 (2) ◽  
pp. 125-137 ◽  
Author(s):  
Michael L. van de Weijer ◽  
Rutger D. Luteijn ◽  
Emmanuel J.H.J. Wiertz
Keyword(s):  
Antigen Presentation ◽  
Mhc Class I ◽  
Immune Evasion ◽  
Class I ◽  
Viral Immune Evasion ◽  
Mhc Class I Antigen

scholarly journals B2M Gene Expression Reflects an Immunologically Active Tumor Microenvironment in DLBCL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2778-2778
Author(s):  
Clare Gould ◽  
Colm Keane ◽  
Valentine Murigneux ◽  
Harald Oey ◽  
Jonathan Ellis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tumor Microenvironment ◽  
Antigen Presentation ◽  
Board Of Directors ◽  
Research Funding ◽  
Immune Cell ◽  
Immune Cell Infiltration ◽  
Protein Loss ◽  
Advisory Committees ◽  
Immune Effector

Introduction Interactions between tumor cells and the immune system play a critical role in regulating tumor development. Immune-based therapies have shown variable responses in diffuse large B-cell lymphoma (DLBCL) which suggests that the immune cell composition of the tumor microenvironment (TME) influences response to these agents. However, the key determinants of the immune TME are poorly understood. We have recently shown that indolent lymphomas can be stratified as immunologically 'hot' or 'cold' (Tobin et al J. Clin Oncol, in press) and sought to test for this in DLBCL. Also, given that beta-2-microglobulin (B2M) has a key role in antigen presentation and its loss is a frequent immune evasion mechanism in DLBCL, we determined the effect of B2M expression on the nature and magnitude of immune infiltration in the tumor microenvironment of DLBCL. Methods Ninety-seven de novo systemic DLBCL FFPE biopsies underwent targeted exon re-sequencing of B2M (Illumina) in addition to quantitative gene expression of Β2M, immune effector (CD4, CD8, CD56, CD137), immunosuppressive macrophage markers (CD68, CD163) and immune checkpoints (TIM3, LAG3, PD1 and PDL1) (Nanostring Technologies). B2M promoter methylation by mass array, immunohistochemistry for the above markers and high throughput T-cell receptor β sequencing (Adaptive Biotechnologies) were performed on a subset of cases. Genomic findings were validated in a whole exome and transcriptome cohort of approximately 1000 DLBCL samples (Reddy et al Cell 2017). Results Β2MMut were detected in 14/97 (14.4%) samples and had lower Β2M gene expression compared to Β2MWT (p = 0.0094) and all of these showed B2M protein loss. However, 29/40 (72%) of B2MWT samples tested also had B2M protein loss. There was no differential methylation of B2M promoter regions observed compared to lymph node controls. Results indicate that mechanisms other than mutation and methylation status contribute to loss of B2M surface expression and that a more comprehensive assessment of B2M expression within tumor tissues is achieved by B2M digital gene quantification. Consistent with this, there were no significant differences in expression of intra-tumoral immune markers between B2MMut and B2MWT tissues, whereas gene expression of B2M was significantly associated and positively correlated with the gene expression of CD4, CD8, CD56, CD137, CD68, CD163, PD1, PDL1, LAG3 and TIM3 (all p <0.01) and with the protein expression of CD8, CD56, CD137, PDL1 and LAG3 (all p <0.01), irrespective of their classification as an immune-effector, immune-checkpoint or macrophage markers. These observations are consistent with a co-ordinately regulated immune response, indicating an adaptive immune-checkpoint response to regulate immune-effector activation. In keeping with this, the housekeeper genes did not correlate with immune gene expression indicating that high or low co-ordinate expression of immune genes was not reflecting tissue RNA quality or quantity (Fig 1). Next, we tested the discovery cohort for relationships with the TCR repertoire. High B2M gene expression was significantly associated with reduced TCR diversity (p = 0.0101) compared to low B2M gene expression, suggesting that clonal T cell expansions are more likely with intact antigen presentation. The validation cohort also demonstrated that B2M gene expression correlated with immune cell infiltration and additionally showed that B2M positively correlated with the gene expression of HLA Class I//II molecules and a range of regulatory, transport and assembly molecules involved in the antigen presentation machinery pathway. However, no differential survival benefit was observed in patients with high versus low B2M. Conclusions In summary, digital gene expression is a robust measure of B2M quantification in the TME. Our data show that high B2M gene expression reflects an immunologically active or 'hot' tumor microenvironment in DLBCL characterised by higher levels of immune cell infiltration. These findings indicate that B2M gene expression level could be used as a biomarker of an active intra-tumoral immune response in DLBCL. Further studies are required to determine if B2M gene expression may have a role in stratifying the selection of patients in whom immune-based therapies are more likely to be effective. Disclosures Gould: NovoNordisk: Other: Travel funding - domestic flights to attend education, May 2018. Keane:MSD: Consultancy; BMS: Research Funding; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy, Other: Travel Grant. Hertzberg:Takeda: Consultancy, Honoraria; MSD: Consultancy; Roche: Consultancy, Honoraria. Gandhi:Gilead: Honoraria, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria.

scholarly journals An Evolutionarily Conserved Function of Polycomb Silences the MHC Class I Antigen Presentation Pathway and Enables Immune Evasion in Cancer

Cancer Cell ◽  
2019 ◽  
Vol 36 (4) ◽  
pp. 385-401.e8 ◽  
Author(s):  
Marian L. Burr ◽  
Christina E. Sparbier ◽  
Kah Lok Chan ◽  
Yih-Chih Chan ◽  
Ariena Kersbergen ◽  
...  
Keyword(s):  
Antigen Presentation ◽  
Mhc Class I ◽  
Immune Evasion ◽  
Class I ◽  
Antigen Presentation Pathway ◽  
Presentation Pathway ◽  
Evolutionarily Conserved ◽  
Mhc Class I Antigen

scholarly journals Comparative Genomic Analyses Defines Shared and Unique Features of cHL and PMBL and New Mechanisms of Sensitivity to PD-1 Blockade

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1493-1493
Author(s):  
Kirsty Wienand ◽  
Bjoern Chapuy ◽  
Chip Stewart ◽  
Andrew Dunford ◽  
David Wu ◽  
...  
Keyword(s):  
Antigen Presentation ◽  
Board Of Directors ◽  
Signaling Pathway ◽  
Mhc Class I ◽  
Research Funding ◽  
Class I ◽  
Newly Diagnosed ◽  
Advisory Committees ◽  
Biopsy Specimens ◽  
Genetic Bases

Classical Hodgkin lymphoma (cHL) and primary mediastinal large B-cell lymphoma (PMBL) are aggressive tumors with distinct cells of origin and pathomorphological features. However, these lymphomas share certain transcriptional signatures and aberrant signaling pathways. CHLs and PMBLs both exhibit constitutive activation of NF-κB and JAK/STAT signaling and genetic bases of PD-1 mediated immune evasion including frequent 9p24.1/PD-L1/PD-L2 copy gains. In both lymphomas, PD-1 blockade is a FDA-approved therapy for relapsed/refractory disease. To characterize genetic bases of response to PD-1 blockade and identify complementary treatment targets in cHL and PMBL, we defined the comprehensive genetic signatures of both diseases. First, we obtained flow cytometry-sorted Hodgkin Reed Sternberg (HRS) cells from 23 biopsies of newly diagnosed cHLs and intact tumor biopsy specimens from 37 newly diagnosed PMBLs. The isolated HRS cells and paired normal DNAs and PMBL biopsy specimens were subjected to whole exome sequencing using an optimized workflow for low input samples and an expanded bait set to capture structural variants (SVs), including translocations. We used newly developed and established analytical pipelines to analyze tumor samples without paired normals (PMBLs) and identify significantly mutated genes (candidate cancer genes [CCGs], MutSig2CV, CLUMPS), SCNAs (GISTIC2.0) and SVs(4 algorithms) in both cHL and PMBL. In cHL, we identified 15 CCGs, 13 recurrent SCNAs, SVs in ETV6 and CIITA, complementary alterations of JAK/STAT, NF-κB and PI3K signaling pathway components and a median number of 11 genetic drivers per tumor. Previously unappreciated aspects of the cHL genetic signature included the increased incidence of driver mutational events in cHLs with ARID1A alterations (p=0.012). Analyses of co-occurring genetic events in EBV+ and EBV- cHLs confirmed that EBV- cHLs were significantly more likely to exhibit alterations of specific NF-κB signaling intermediaries (such as TNFAIP3 mutation and/or focal copy loss, p=0.006) and perturbations of MHC class I antigen presentation pathway components (inactivating B2M mutations, HLA-B mutations or focal copy loss of 6p21.32/HLA-B, p=0.008). The latter findings provide genetic bases for the reported differences in cell surface expression of MHC class I in EBV+ and EBV- cHLs. In PMBL, we defined 15 CCGs and more selective perturbations of specific epigenetic modifiers (ZNF217 and EZH2), transcription factors (PAX5 and IRF2BP2) and TP53, in comparison with cHL. The majority of these alterations were clonal supporting their role as early drivers. We identified 18 SCNAs and additional SVs in CIITA and PD-1 ligands, recurrent alterations of JAK/STAT and NF-κB signaling pathway components and a median of 9 genetic drivers per PMBL. Antigen presentation pathways in PMBL were perturbed by multiple recurrent alterations, including B2M mutations, focal copy losses of B2M and the MHCI/II loci, SVs of CTIIA and EZH2 mutations. There was a significant correlation between genetic perturbations of MHC class I pathway components and absence of MHC class I expression in PMBL, as previously described in cHL. Recurrent cHL alterations including B2M, TNFAIP3, STAT6, GNA13 and XPO1 CCGs and 2p/2p15/2p16.1, 6p21.32, 6q23.2 and 9p/9p24.1 SCNAs were also identified in &gt;20% of PMBLs, highlighting shared pathogenetic mechanisms in these diseases. These tumors of predominantly young adults (median age: cHL 26 yrs; PMBL 34 yrs) both had a high rate of spontaneous deamination of CpGs, a clock-like mutational signature that is typically associated with aging. CHLs and PMBLs both exhibited previously uncharacterized molecular features that may increase sensitivity to PD-1 blockade, including high mutational burdens, in comparison with other lymphoid and solid tumors. In particular, the mutational burden in EBV- cHLs was among the highest reported, similar to that in carcinogen-induced cancers (melanoma and NSCLC). Additionally, both cHLs and PMBLs had an increased incidence of microsatellite instability and APOBEC mutational signatures, features associated with a more favorable response to PD-1 blockade. Taken together, these data define genetic similarities and differences in cHL and PMBL and establish a framework to comprehensively assess molecular bases of response to PD-1 blockade and develop rational combination therapies in these diseases. Disclosures Armand: Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Otsuka: Research Funding; Sigma Tau: Research Funding; Adaptive: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Affimed: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Research Funding; Pfizer: Consultancy; ADC Therapeutics: Consultancy; Infinity: Consultancy; Genentech: Research Funding; Tensha: Research Funding. Rodig:Merck: Research Funding; Affirmed: Research Funding; Kite, a Gilead Company: Research Funding; Bristol Myers Squib: Consultancy, Honoraria, Other: Travel Expenses, Speakers Bureau. Fromm:Merck, Inc.: Research Funding. Getz:Pharmacyclics: Research Funding; IBM: Research Funding; MuTect, ABSOLTUE, MutSig and POLYSOLVER: Patents & Royalties: MuTect, ABSOLTUE, MutSig and POLYSOLVER. Shipp:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Merck & Co.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Frequent Loss of IRF2 in Cancers Leads to Immune Evasion through Decreased MHC Class I Antigen Presentation and Increased PD-L1 Expression

2019 ◽  
Vol 203 (7) ◽  
pp. 1999-2010 ◽  
Author(s):  
Barry A. Kriegsman ◽  
Pranitha Vangala ◽  
Benjamin J. Chen ◽  
Paul Meraner ◽  
Abraham L. Brass ◽  
...  
Keyword(s):  
Antigen Presentation ◽  
Mhc Class I ◽  
Immune Evasion ◽  
Class I ◽  
Mhc Class I Antigen

scholarly journals High Mobility Group A1 Chromatin Regulators: Key Epigenetic Switches and Therapeutic Targets Required for Leukemic Transformation in JAK2 Mutant MPN

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1680-1680
Author(s):  
Liping Li ◽  
Wenyan Lu ◽  
Alison R. Moliterno ◽  
Lingling Xian ◽  
Joseph Kim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
High Mobility ◽  
Transcriptional Networks ◽  
Murine Models ◽  
Rna Seq ◽  
Leukemic Transformation ◽  
Cycle Progression ◽  
Cd34 Cells

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by hyperactive JAK/STAT signaling and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML). However, mechanisms driving progression remain elusive and therapies are ineffective after leukemia develops. The High Mobility Group A1/2 (HMGA1/2) genes encode oncogenic chromatin remodeling proteins which are overexpressed in aggressive solid tumors where they portend adverse outcomes. HMGA1/2 genes are also up-regulated in hematologic malignancies and MPN with disease progression. In murine models, Hmga1/2 overexpression drives clonal expansion and deregulated proliferation while Hmga1 overexpression is sufficient for lymphoid leukemic transformation. We therefore sought to: 1) test the hypothesis that HMGA1/2 proteins are rational therapeutic targets required for leukemic transformation in MPN, 2) elucidate mechanisms mediated by HMGA1/2 during disease progression, and, 3) identify therapeutic approaches to disrupt HMGA function and intercept the transition from chronic disease to aggressive leukemia. Methods: We compared HMGA1/2 in JAK2V617F mutant AML cell lines from MPN patients (DAMI, SET-2), CD34+ cells from PV patients during chronic and transformation phases, and JAK2V617F murine models of PV (transgenic JAK2V617F) and PV-AML (transgenic JAK2V617F/MPLSV). To elucidate HMGA1/2 function, we silenced HMGA1 or HMGA2 via short hairpin RNA in human MPN-AML cells and generated murine models of PV-AML with heterozygous Hmga1 or Hmga2 deficiency. To dissect molecular mechanisms underlying HMGA, we compared RNA-Seq from MPN-AML cell lines after gene silencing. Finally, to identify therapies to target HMGA pathways, we integrated the RNA-Seq data with the Broad Connectivity Map (cMAP). Results: There is a marked up-regulation in HMGA1/2 in CD34+ cells from PV patients after transformation to AML and in leukemic blasts from our PV-AML mouse model. Conversely, silencing HMGA1 or HMGA2 in human MPN-AML cell lines (DAMI, SET-2) dramatically halts proliferation, disrupts clonogenicity, and prevents leukemia development in mice. Further, heterozygous Hmga1 deficiency prolongs survival in the transgenic PV-AML murine model with fulminant leukemia and early mortality, although Hmga2 deficiency has no effect. RNA-Seq analyses from human MPN-AML cell lines revealed that HMGA1 up-regulates transcriptional networks involved in cell cycle progressions (E2F targets, mitotic spindle, G2M checkpoint, MYC targets) while repressing immune pathways (inflammation, interferon gamma) and oxidative phosphorylation. HMGA2 up-regulates similar pathways, but represses TNFalpha signaling. cMAP identified inhibitors of histone deacetylation and cell cycle progression as potential agents to target HMGA1 pathways; DNA synthesis inhibitors were predicted to target HMGA2 pathways. Cytotoxicity assays demonstrate that epigenetic therapy with HDAC inhibitors synergizes with Ruxolitinib in JAK2 mutant MPN cells after transformation to leukemia. Conclusions: HMGA1/2 genes are overexpressed in MPN with highest levels after leukemic transformation. Further, silencing HMGA1/2 disrupts leukemogenic phenotypes in vitro and prevents the development of leukemia in mice. In addition, heterozygous deficiency of Hmga1 prolongs survival in a fulminant MPN-AML model. Mechanistically, RNA-Seq analyses revealed that HMGA amplifies transcriptional networks involved cell cycle progression, which can be targeted with epigenetic therapies. Our findings further underscore the key role for HMGA as an epigenetic switch required for leukemic transformation in MPN and opens the door to novel therapeutic approaches to intercept the transition from chronic indolent disease to aggressive leukemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals HMGA1 Chromatin Regulators Drive Progression in Myeloproliferative Neoplasms through Epigenetic Rewiring to Induce Networks Involved in GATA2 and Proliferation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 625-625
Author(s):  
Liping Li ◽  
Jung-Hyun Kim ◽  
Wenyan Lu ◽  
Leslie Cope ◽  
Donna M Williams ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Chromatin Accessibility ◽  
Epigenetic Therapy ◽  
Transcriptional Networks ◽  
Leukemic Transformation ◽  
Histone Marks ◽  
Chromatin Regulators

Abstract Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by hyperactive JAK/STAT signaling and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML). However, mechanisms driving progression remain elusive and therapies are ineffective after leukemic transformation. The High Mobility Group A1 (HMGA1) gene encodes oncogenic chromatin regulators which are overexpressed in diverse tumors where they portend adverse outcomes (Resar Cancer Res 2010; Xian et al Nature Commun 2017). Hmga1 induces leukemic transformation in transgenic mice and HMGA1 is overexpressed in refractory myeloid malignancies (Resar et al Cancer Res 2018). Further, germline lesions within the HMGA1 loci increase the risk for developing MPN (Bao et al Nature 2020). We therefore sought to: 1) test the hypothesis that HMGA1 drives MPN progression by rewiring transcriptional networks to foster leukemogenesis, and, 2) identify mechanisms underlying HMGA1 that could be targeted with therapy. Methods: To elucidate the function of HMGA1, we disrupted HMGA1 expression via CRISPR/Cas9 or short hairpin RNA (shRNA) targeting 2 different sequences per gene and assessed proliferation, colony formation, apoptosis, and leukemogenesis. We also generated JAK2 V617F transgenic mouse models of MF with Hmga1 deficiency. To dissect molecular mechanisms underlying HMGA1, we integrated RNAseq, ATACseq, and chromatin immunoprecipitation (ChIP) from MPN-AML cell lines (DAMI, SET-2). Next, we tested whether HMGA1 depletion synergizes with ruxolitinib in preventing leukemic engraftment in mice. To identify drugs to target HMGA1 networks, we applied the Broad Institute Connectivity Map (CMAP). Results: HMGA1 is overexpressed in CD34 + cells from patients with JAK2 V617F MPN with highest levels after transformation to MF or AML in 3 independent cohorts. CRISPR/Cas9 inactivation or shRNA-mediated HMGA1 silencing disrupts proliferation, decreases the frequency of cells in S phase, increases apoptosis, and impairs clonogenicity in human MPN-AML cell lines. HMGA1 depletion also prevents leukemic engraftment in mice. Surprisingly, loss of just a single Hmga1 allele prevents progression to MF in JAK2 V617Fmurine models of MPN, decreasing erythrocytosis, thrombocytosis, and preventing splenomegaly and fibrosis of the spleen and bone marrow. Further, Hmga1 deficiency preferentially prevents expansion in long-term HSC, granulocyte-macrophage progenitors, and megakaryocyte-erythroid progenitors in JAK2 V617F mice. RNAseq revealed genes induced by HMGA1 that govern cell cycle progression (E2F targets, mitotic spindle, G2M checkpoint, MYC targets) and cell fate decisions (GATA2 networks), including the GATA2 master regulator gene. Silencing GATA2 recapitulates anti-leukemia phenotypes observed with HMGA1 deficiency whereas restoring GATA2 in MPN-AML cells with HMGA1 silencing partially rescues leukemia phenotypes, increasing clonogenicity and leukemic engraftment. Mechanistically, HMGA1 binds directly to AT-rich sequences near the GATA2 developmental enhancer (+9.5), enhances chromatin accessibility, and recruits active histone marks (H3K4me1/3) to induce GATA2 expression. HMGA1 depletion enhances responses to the JAK/STAT Inhibitor, ruxolitinib, delaying leukemic engraftment and prolonging survival in murine models of JAK2 V617F MPN-AML. Further, epigenetic drugs predicted to target HMGA1 transcriptional networks using CMAP synergize with JAK inhibitors to disrupt proliferation in human MPN-AML cells. HMGA1 and GATA2 are co-expressed and up-regulated with progression from MF to AML in matched patient samples. Moreover, HMGA1 transcriptional networks are activated in leukemic blasts, thus underscoring the role of HMGA1 in human MPN progression. Conclusions: We uncovered a previously unknown epigenetic program whereby HMGA1 enhances chromatin accessibility and recruits activating histone marks to induce transcriptional networks required for progression in MPN, including direct transactivation of GATA2. Further, HMGA1 networks can be targeted with epigenetic therapy and synergize with ruxolitinib. Together, our studies reveal a new paradigm whereby HMGA1 up-regulates GATA2 and proliferation networks to drive disease progression and illuminate HMGA1 as a novel therapeutic target in MPN. Figure 1 Figure 1. Disclosures Rampal: Jazz Pharmaceuticals: Consultancy; Incyte: Consultancy, Research Funding; Kartos: Consultancy; Constellation: Research Funding; Pharmaessentia: Consultancy; Blueprint: Consultancy; Disc Medicine: Consultancy; Stemline: Consultancy, Research Funding; BMS/Celgene: Consultancy; Novartis: Consultancy; Sierra Oncology: Consultancy; CTI: Consultancy; Abbvie: Consultancy; Memorial Sloan Kettering: Current Employment. Stubbs: Incyte Research Institute: Current Employment, Current holder of individual stocks in a privately-held company.

scholarly journals The Multiple Immune-Evasion Genes of Murine Cytomegalovirus Are Not Redundant

2001 ◽  
Vol 194 (7) ◽  
pp. 967-978 ◽  
Author(s):  
Daniel G. Kavanagh ◽  
Marielle C. Gold ◽  
Markus Wagner ◽  
Ulrich H. Koszinowski ◽  
Ann B. Hill
Keyword(s):  
Antigen Presentation ◽  
Mhc Class I ◽  
Cytotoxic T Lymphocytes ◽  
Immune Evasion ◽  
Surface Expression ◽  
Class I ◽  
Murine Cytomegalovirus ◽  
Histocompatibility Complex ◽  
Golgi Compartment ◽  
Partial Retention

Both human cytomegaloviruses (HCMVs) and murine cytomegaloviruses (MCMVs) encode multiple genes that interfere with antigen presentation by major histocompatibility complex (MHC) class I, and thus protect infected targets from lysis by virus-specific cytotoxic T lymphocytes (CTLs). HCMV has been shown to encode four such genes and MCMV to encode two. MCMV m152 blocks the export of class I from a pre-Golgi compartment, and MCMV m6 directs class I to the lysosome for degradation. A third MCMV gene, m4, encodes a glycoprotein which is expressed at the cell surface in association with class I. Here we here show that m4 is a CTL-evasion gene which, unlike previously described immune-evasion genes, inhibited CTLs without blocking class I surface expression. m152 was necessary to block antigen presentation to both Kb- and Db-restricted CTL clones, while m4 was necessary to block presentation only to Kb-restricted clones. m152 caused complete retention of Db, but only partial retention of Kb, in a pre-Golgi compartment. Thus, while m152 effectively inhibited Db-restricted CTLs, m4 was required to completely inhibit Kb-restricted CTLs. We propose that cytomegaloviruses encode multiple immune-evasion genes in order to cope with the diversity of class I molecules in outbred host populations.

scholarly journals Comprehensive Genomic Analysis of Flow-Sorted Hodgkin Reed Sternberg Cells Reveals Additional Genetic Bases of Immune Evasion

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1559-1559 ◽  
Author(s):  
Kirsty Wienand ◽  
Bjoern Chapuy ◽  
Chip Stewart ◽  
Andrew Dunford ◽  
David Wu ◽  
...  
Keyword(s):  
B Cells ◽  
Mhc Class I ◽  
Immune Evasion ◽  
Research Funding ◽  
Class I ◽  
Stat Signaling ◽  
Genetic Mechanisms ◽  
Cell Expression ◽  
Inactivating Mutations ◽  
Genetic Bases

Abstract Classical Hodgkin lymphoma (cHL) is composed of rare malignant Hodgkin Reed Sternberg (HRS) cells within an extensive, but ineffective, inflammatory/immune cell infiltrate. Emerging data suggests that cHLs use multiple genetic mechanisms to evade immune recognition. We previously found that HRS cells exhibit near-universal somatic copy number alterations (SCNAs) involving chromosome 9p24.1/PD-1-L1/PD-L2 and rare chromosomal rearrangements of PD-L1 or PD-L2. The 9p24.1 amplicon also includes JAK2, which increases JAK2 copy numbers, augments JAK2/STAT signaling and further induces PD-1 ligand expression. However, HRS cells also have inactivating mutations of B2M and decreased or absent MHC class I expression. In cHL, clinical responses to PD-1 blockade are unrelated to HRS cell expression of MHC class I but closely associated with HRS cell expression of MHC class II, highlighting the potential role of CD4+ T-cell effectors (J Clin Oncol 2018;36:942-50). To define genetic bases of response and resistance to PD-1 blockade and identify complementary treatment targets, we performed whole exome sequencing (WES) of HRS cells. We first used a previously described multi-color flow cytometric sorting protocol (Methods 2012; 57:368-75) to obtain highly purified CD30+ HRS cells and normal B cells from the excisional biopsies of 25 newly diagnosed cHLs. The isolated HRS cells and paired normal B cells were then subjected to WES using an optimized workflow for low input samples and an expanded bait set to capture structural variants (SVs). We used established analytical pipelines to identify significantly mutated genes (candidate cancer genes [CCGs], MutSig2CV), SCNAs (GISTIC2.0) and SVs (4 algorithms). With improved methodology and purity (median of 80%) of the isolated HRS cells, we defined 15 significantly mutated CCGs, 21 recurrent SCNAs, including 6 CN gains (4 focal and 2 arm level) and 15 CN losses (14 focal and 1 arm level), and low frequency SVs. We identified 2 cHLs as hypermutators with MSI signatures due to splice site mutations in MSH2 or missense mutations in POLE. Excluding the 2 hypermutators, the analyzed cHLs had a median mutational density of 6.4 mutations/Mb, that falls within the top quartile of reported cancer mutational frequencies (Nature 2013 499:214). We also identified a previously unappreciated high incidence of ARID1A mutations (24%) in cHL. This is noteworthy because ARID1A deficiency increases mutational load and augments the efficacy of PD-1 blockade in murine models (Nature Med 2018;24:556). Together, the observed MSI signatures, relatively high mutational burden and newly identified ARID1A mutations in cHL represent additional potential genetic bases for the efficacy of PD-1 blockade. Notably, these cHLs also exhibited recurrent 9p24.1 copy gain (80%) and multiple genetic bases of enhanced JAK/STAT signaling including JAK2 copy gain (80%), STAT6 mutations (32%) involving known hotspots (D419 and N421) in the DNA-binding domain and frequent inactivating SOCS1 mutations (68%). We also identified multiple genetic bases for immune evasion, including B2M inactivating mutations (36%), HLA-B mutations (16%) and 6p21.32/HLA-B copy loss (28%), copy loss of the larger 6p21.32 region and inactivating CIITA SVs (8%). Additional signaling pathways were perturbed by multiple genetic mechanisms in these cHLs. For example, NF-κB pathway alterations included: TNFAIP3 mutations (24%) and 6q23.2/TNFAIP3 copy loss (56%), 12% biallelic; NFKBIE mutations (24%) and 6q21.32/NFKBIE copy loss (12%); and NFKBIA mutations (16%). The gene encoding the nuclear export protein, XPO1, was perturbed by E571K mutations (24%) and frequent 2p15/XPO1 copy gain (72%). Additionally, GNA13, an activator of RHOA and modifier of PI3K signaling, was mutated in 24% of cases. Of interest, cHL recurrent alterations including B2M, TNFAIP3, STAT6, and GNA13 mutations and 6q23.2 and 9p24.1 SCNAs were also identified in > 20% of examined primary mediastinal B-cell lymphomas, highlighting shared pathogenetic mechanisms in these diseases. In summary, comprehensive genomic analyses of purified HRS cells reveal new genetic bases of immune evasion, potential mechanisms of response and resistance to PD-1 blockade and additional targetable alterations. KW, BC, CS, AD and DW contributed equally. JF, GG and MS contributed equally. Disclosures Rodig: Affimed: Research Funding; KITE: Research Funding; Merck: Research Funding; Bristol Myers Squibb: Research Funding. Shipp:Merck: Research Funding; Bayer: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria.

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