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Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with 5-year survival of ∼50%. Genomic profiling studies have identified important somatic mutations in this disease which presents an opportunity for precision medicine. We demonstrate that KMT2D, a histone methyltransferase harbors somatic mutations in ∼17% of HNSCC and is associated with 2-year recurrence in TCGA data. Consistent with algorithmic prediction of bring a driver tumor-suppressor event, its loss results in larger oral tumors in immune-proficient orthotopic models. Mechanistically, we find that KMT2D knockdown or KMT2D mutation causes loss of H3K4me1-marked enhancers harboring IRF7/9 binding sites, which is known to regulate interferon signaling. Indeed, KMT2D loss in human and murine cell lines deregulated transcriptional levels of cytokine expression and impacted numerous immune signaling pathways, including interferon signaling. Consistently, Kmt2d knockdown in murine tumors exhibited decrease in IFNγ-producing effector T cells and an increase in T-cells with an exhausted phenotype. Epistasis experiments showed that exogenous treatment with IFNγ abrogated the increased tumor growth in Kmt2d-deficient oral tumors. Together, these results support the role of KMT2D as a tumor suppressor in HNSCC that regulates the tumor microenvironment by modulating H3K4me1-marked enhancers controlling interferon signaling.

Background The success of allogenic stem cell transplantation in curing AML suggests that the immune system can be harnessed to eradicate AML. In a phase 2 trial (NCT02397720) in relapsed/refractory (R/R) AML patients, we demonstrated that the azacitidine/nivolumab combination improved response rates and median overall survival compared with similar patients treated on other azacitidine-based studies (Daver et al Cancer Discovery 2019). The heterogenous response profiles and shorter duration of responses than seen in solid tumor patients suggested hitherto undefined tumor intrinsic, tumor microenvironment (TME) and T cell factors may impede PD-1 blockade therapy in AML. Methods We performed single cell RNA sequencing (scRNAseq) of 13,633 healthy bone marrow (BM) donor, and 113,394 BM cells from 22 aspirates (8 pre- and 14 post- treatment) from 8 R/R AML patients (median age 73 years; range 64-88 years) treated with azacitidine/nivolumab (Fig 1A). 3/8 patients were responders (2CR, 1 PR), while 2/8 and 3/8 had stable disease (SD), and no response (NR), respectively, allowing us to evaluate factors involved in response, relapse and resistance to azacitidine/nivolumab. Results A total of 60,753 AML and 52,641 TME cells passed scRNAseq quality check, with the proportion of identified AML cells correlating with clinical flow cytometry (r=0.87, p=1.5x10-7) and immunohistochemistry (r=0.73, p=0.0001). Pre- and post-treatment AML cells clustered by patient and had distinct cell cycle profiles regardless of response type, suggesting significant inter-tumor heterogeneity (Fig 1B). In an aggregate analysis of all cells at the pretreatment timepoint, the 3 responders had lower leukemia stemness (LSC17) scores compared with NR (p On paired single cell TCR analysis from 4,742 and 26,095 T cells from healthy and R/R AML BMs, respectively, T cells in the TME of AML patients had less clonal diversity and more oligoclonal dominance compared to healthy BMs (Fig 1G-H). Following treatment 76.9% and 72.4% of novel and expanded clones were contributed by responders, with non-responders contributing only 5% and 3.4% of the novel and expanded clones, respectively (Fig. 1I). Among responders, the majority of clones were either novel or expanded, whereas NR had mostly contracted clones. Conclusions: This is one of the first studies examining the effect of PD-1 blockade at single cell resolution in a hematologic malignancy. Further, this is the largest single study analyzing single AML cells longitudinally. AML cells harboring deletion 7/7q loss, enriched for LSC signature and metabolic/oxidative pathways, were features associated with resistance to azacitidine/nivolumab therapy. Azacitidine/nivolumab induced novel and expanded T cell clonotypes primarily in responders. Disentangling AML cells from their complex microenvironment revealed characteristics that shaped resistance to ICB-based therapy and could inform strategies to target AML vulnerabilities. Disclosures DiNardo: Takeda: Honoraria; Novartis: Consultancy; AbbVie: Consultancy, Honoraria, Research Funding; Syros: Honoraria; MedImmune: Honoraria; Jazz: Honoraria; Agios: Consultancy, Honoraria, Research Funding; ImmuneOnc: Honoraria; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; Notable Labs: Membership on an entity's Board of Directors or advisory committees. Kadia:Pfizer: Honoraria, Research Funding; Cyclacel: Research Funding; Incyte: Research Funding; Novartis: Honoraria; Abbvie: Honoraria, Research Funding; Astellas: Research Funding; Cellenkos: Research Funding; Ascentage: Research Funding; Amgen: Research Funding; JAZZ: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Astra Zeneca: Research Funding; Celgene: Research Funding; Genentech: Honoraria, Research Funding; Pulmotec: Research Funding. Ravandi:Celgene: Consultancy, Honoraria; Orsenix: Consultancy, Honoraria, Research Funding; Macrogenics: Research Funding; Astellas: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Xencor: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding. Borthakur:Xbiotech USA: Research Funding; BioLine Rx: Research Funding; PTC Therapeutics: Consultancy; BioLine Rx: Consultancy; Incyte: Research Funding; Novartis: Research Funding; Jannsen: Research Funding; Abbvie: Research Funding; Cyclacel: Research Funding; Argenx: Consultancy; FTC Therapeutics: Consultancy; Treadwell Therapeutics: Consultancy; PTC Therapeutics: Research Funding; Polaris: Research Funding; BMS: Research Funding; Oncoceutics: Research Funding; Nkarta Therapeutics: Consultancy; BioTherix: Consultancy; GSK: Research Funding; Curio Science LLC: Consultancy; AstraZeneca: Research Funding. Konopleva:Ascentage: Research Funding; Eli Lilly: Research Funding; Rafael Pharmaceutical: Research Funding; Forty-Seven: Consultancy, Research Funding; Ablynx: Research Funding; Amgen: Consultancy; F. Hoffmann La-Roche: Consultancy, Research Funding; AstraZeneca: Research Funding; AbbVie: Consultancy, Research Funding; Calithera: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Kisoji: Consultancy; Cellectis: Research Funding; Sanofi: Research Funding; Genentech: Consultancy, Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Agios: Research Funding. Garcia-Manero:Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Jazz Pharmaceuticals: Consultancy; Acceleron Pharmaceuticals: Consultancy, Honoraria; Helsinn Therapeutics: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria, Research Funding; Amphivena Therapeutics: Research Funding; Novartis: Research Funding; AbbVie: Honoraria, Research Funding; H3 Biomedicine: Research Funding; Onconova: Research Funding. Green:KDAc Therapeutics: Current equity holder in private company. 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Daver:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trillium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Research Funding; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Servier: Research Funding; Genentech: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novimmune: Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Trovagene: Research Funding; ImmunoGen: Research Funding; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

In contrast to the curative effect of allogenic stem cell transplantation in acute myeloid leukemia via T cell activity, only modest responses are achieved with checkpoint-blockade therapy, which might be explained by T cell phenotypes and T cell receptor (TCR) repertoires. Here, we show by paired single-cell RNA analysis and TCR repertoire profiling of bone marrow cells in relapsed/refractory acute myeloid leukemia patients pre/post azacytidine+nivolumab treatment that the disease-related T cell subsets are highly heterogeneous, and their abundance changes following PD-1 blockade-based treatment. TCR repertoires expand and primarily emerge from CD8+ cells in patients responding to treatment or having a stable disease, while TCR repertoires contract in therapy-resistant patients. Trajectory analysis reveals a continuum of CD8+ T cell phenotypes, characterized by differential expression of granzyme B and a bone marrow-residing memory CD8+ T cell subset, in which a population with stem-like properties expressing granzyme K is enriched in responders. Chromosome 7/7q loss, on the other hand, is a cancer-intrinsic genomic marker of PD-1 blockade resistance in AML. In summary, our study reveals that adaptive T cell plasticity and genomic alterations determine responses to PD-1 blockade in acute myeloid leukemia., The response rate of relapsed/refractory acute myeloid leukemia patients to PD-1 checkpoint blockade is low and unpredictable. Authors here show by single cell RNA sequencing, T cell receptor profiling and genomic analysis that the phenotypes and repertoire of CD8 + T cells and loss of chromosome 7/7q are important determinants of response.

Background Allogeneic stem cell transplantation can cure relapsed/refractor (R/R) AML via grafted T cells versus leukemia effect, but not a viable option to many patients. By combining azacitidine with nivolumab, we harnessed T cell activity and demonstrated 33% response rates. The tumor microenvironment (TME) factors impacting response and resistance to PD-1 blockade-based treatment in AML are unknown. Methods We performed single cell RNA sequencing (scRNAseq) on 113,394 bone marrow (BM) cells, paired with >30,000 single cell T cell receptor (scTCR) repertoires, from 8 pre- and 14 post- azacitidine/nivolumab treatment aspirates of 8 R/R AML patients (median age 73 years; 3 responders; 3 non-responders; 2 stable disease) (figure 1). Results Inferred copy number loss of chromosome 7/7q (chr7/7q) by scRNAseq was associated with resistance to azacitidine/nivolumab (figure 2A), which was validated in a larger cohort based on clinical karyotyping (figure 2B). There was significant enrichment (q Conclusions Chr7/7q loss is associated with resistance to PD-1 blockade. CD8 cells exist in a continuum in BMs of patients with AML and GZMK expression identifies a stem-like, memory T cell subset. The subverted T cells can be reinvigorated via PD-1 blockade and induce responses in AML driven via novel and expanded clones demonstrating AML T cell plasticity and adaptability. Further functional characterization of GZMK expressing lymphocytes in mediating antileukemic responses is underway. Ethics Approval The study was approved by IRB at MD Anderson Cancer Center

Acute myeloid leukemia (AML) and effector cells of immune checkpoint blockade (ICB) therapy co-reside in a complex bone marrow (BM) milieu. The interplay of tumor intrinsic and microenvironment (TME) mechanisms that influences the response to ICB-based therapies in AML have not been elucidated. Here we report our analyses of single cell RNA profiling of more than 127,000 BM cells from healthy donors and relapsed/refractory (R/R) AML patients at pre/post treatment with azacitidine/nivolumab, paired with single cell T cell receptor (TCR) repertoire profiles, to uncover factors impacting response and resistance. Loss of chromosome 7/7q conferred an immunosuppressive TME and was associated with resistance to ICB-based therapy in R/R AML. Our trajectory analysis revealed a continuum of CD8+ T cell phenotypes, characterized by differential expression of granzyme B (GZMB) and GZMK. GZMK expression defined a BM residing memory CD8+ T cell subset with stem-like properties likely an intermediary between naïve and cytotoxic lymphocytes. Responses to ICB-based therapy were primarily driven by novel and expanded T cell clonotypes. Our findings support an adaptable T cell plasticity in response to PD-1 blockade in AML. Disentangling AML cells from their complex, immune-rich microenvironment revealed characteristics that shaped resistance to ICB-based therapy and could inform strategies to target AML vulnerabilities.SignificanceDetermining the cellular and molecular underpinnings of response and resistance to PD-1 blockade based therapy in AML can guide immune-based therapeutic strategies. Our results reveal AML intrinsic characteristics (chromosome 7/7q status and oxidative stressors) and tumor microenvironment to modulate responses to checkpoint blockers. CD8 cells exist in the bone marrow in a continuum with GZMK expression defining a memory, stem-like T cell population that could play a role in response to therapy.

β-hydroxybutyrate (β-OHB) provides an essential metabolic energy source during fasting and functions as an epigenetic regulator by lysine β-hydroxybutyrylation (Kbhb) modification of core histones H3 and H4. We report that H3K9bhb, but not H4K12bhb, becomes highly enriched at proximal promoters of genes regulating lipolytic and ketogenic metabolic programs in small intestinal (SI) crypts during fasting and these pathways are transcriptionally activated in Lgr5 + SI stem cells. Similar Kbhb enrichment is observed in Lgr5 + stem cell-enriched epithelial spheroids treated with β-OHB in vitro. Combinatorial chromatin state analysis reveals that H3K9bhb enrichment is associated with active chromatin states and that fasting enriches for an H3K9bhb–H3K27ac signature at active metabolic gene promoters and distal DNA enhancer elements. Intestinal knockout of Hmgcs2 results in a marked loss of H3K9bhb associated fasting-induced gene programs suggesting that local production of β-OHB is responsible for epigenetic reprogramming. We conclude that specific modulation of H3K9bhb in SI crypts is a key epigenetic event in response to fasting.

Background The blood filtering organ in zebrafish embryos is the pronephros, which consists of two functional nephrons. Segmentation of a nephron into different domains is essential for its function and is well conserved among vertebrates. Zebrafish has been extensively used as a model to understand nephron segmentation during development. Here, we have identified EF-hand domain containing 2 (Efhc2) as a novel component of genetic programme regulating nephron segmentation in zebrafish. Human EFHC2 is a protein with one predicted calcium-binding EF-hand motif and three DM10 domains, whose function is unknown. EFHC2 has been implicated in several brain-related genetic diseases like Turner syndrome and juvenile myoclonic epilepsy. However, there is limited information on its normal physiological function. Results efhc2 mRNA is primarily expressed in the pronephros of zebrafish embryos. Other sites of expression include olfactory placode, notochord, otic vesicle, epiphysis and neuromast cells. Morpholino antisense oligonucleotide-mediated knock-down of Efhc2 resulted in defects in pronephros development and function in zebrafish embryos. Efhc2 knock-down leads to expansion of distal early segment of pronephros, whereas, the corpuscle of stannius and distal late segments were reduced. The number of multi-ciliated cells (MCC) that are present in a salt-and-pepper fashion throughout the middle of each nephron and vital for fluid flow were also reduced. It is known that retinoic acid (RA) signaling regulates pronephros segmentation in vertebrates and we show that Efhc2 function is crucial for nephron segmentation in zebrafish. Our data suggests that RA and Efhc2 function independent of each other in pronephros segmentation. However, Efhc2 and RA synergistically regulate MCC development. Conclusion In this study, we have identified Efhc2 as a regulator of segmentation of the distal part of nephron and pronephros function during zebrafish development. Electronic supplementary material The online version of this article (10.1186/s13578-018-0253-z) contains supplementary material, which is available to authorized users.

SUMMARY β-hydroxybutyrate (β-OHB) is an essential metabolic energy source during fasting and functions as a chromatin regulator by lysine β-hydroxybutyrylation (Kbhb) modification of the core histones H3 and H4. We report that Kbhb on histone H3 (H3K9bhb) is enriched at proximal promoters of critical gene subsets associated with lipolytic and ketogenic metabolic pathways in small intestine (SI) crypts during fasting. Similar Kbhb enrichment is observed in Lgr5+ stem cell-enriched epithelial spheroids treated with β-OHB in vitro. Combinatorial chromatin state analysis reveals that H3K9bhb is associated with active chromatin states and that fasting enriches for an H3K9bhb-H3K27ac signature at active metabolic gene promoters and distal enhancer elements. Intestinal knockout of Hmgcs2 results in marked loss of H3K9bhb-associated loci, suggesting that local production of β-OHB is responsible for chromatin reprogramming within the SI crypt. We conclude that modulation of H3K9bhb in SI crypts is a key gene regulatory event in response to fasting., Graphical Abstract, In brief Terranova et al. demonstrate that fasting induces production of HMGCS2 and β-hydroxybutyrate in small intestine (SI) crypt cells. This causes enrichment of H3K9bhb within regulatory regions of critical metabolic genes in crypt epithelial cells. Loss of intestinal Hmgcs2 impairs H3K9bhb enrichment and affects expression of H3K9bhb-associated metabolic gene programs.

SUMMARY The dynamic evolution of chromatin state patterns during metastasis, their relationship with bona fide genetic drivers, and their therapeutic vulnerabilities are not completely understood. Combinatorial chromatin state profiling of 46 melanoma samples reveals an association of NRAS mutants with bivalent histone H3 lysine 27 trimethylation (H3K27me3) and Polycomb repressive complex 2. Reprogramming of bivalent domains during metastasis occurs on master transcription factors of a mesenchymal phenotype, including ZEB1, TWIST1, and CDH1. Resolution of bivalency using pharmacological inhibition of EZH2 decreases invasive capacity of melanoma cells and markedly reduces tumor burden in vivo, specifically in NRAS mutants. Coincident with bivalent reprogramming, the increased expression of pro-metastatic and melanocyte-specific cell-identity genes is associated with exceptionally wide H3K4me3 domains, suggesting a role for this epigenetic element. Overall, we demonstrate that reprogramming of bivalent and broad domains represents key epigenetic alterations in metastatic melanoma and that EZH2 plus MEK inhibition may provide a promising therapeutic strategy for NRAS mutant melanoma patients., Graphical abstract, In brief Terranova et al. provide a comprehensive epigenome resource for melanoma encompassing 284 chromatin maps. They find key regulatory roles for bivalent and broad domains in expression of pro-metastatic genes and identify EZH2 plus MEK inhibition as a therapeutic strategy for NRAS mutant melanomas.

Introduction: Allogeneic hematopoietic stem cell transplantation (ASCT) remains the only curative option for patients with relapsed/refractory (R/R) AML, achieved via the grafted T cells versus leukemia effect. Since ASCT is not viable option to many AML patients, our group investigated (NCT02397720) whether T cells can be harnessed to eradicate R/R AML by combining azacitidine, a hypomethylating agent, and nivolumab, a PD-1 blocker. The tumor microenvironment (TME) factors impacting response and resistance to PD-1 blockade-based treatment in AML is unknown. Methods: We performed single cell RNA sequencing (scRNAseq) of 13,633 healthy bone marrow (BM) donor, and 113,394 BM cells, paired with >30,000 single cell T cell receptor (scTCR) repertoires, from 22 aspirates (8 pre- and 14 post- treatment) from 8 R/R AML patients (median age 73 years) treated with azacitidine/nivolumab. 3/8 patients were responders, while 2/8 and 3/8 had stable disease, and no response, respectively. Results: We identified marked variation in the T cell components across AML patients at pre- and post- treatment, demonstrating significant dynamic changes in CD4, CD8 and non-classical T cells populations, including mucosal associated invariant T (MAIT) cells. Among CD8 cells, we identified a unique GZMK-enriched population that was higher at pretreatment in responders compared to non-responders. Pseudotemporal trajectory analysis revealed a continuum of CD8 cell states that is intermediated by the less exhausted, GZMK-enriched CD8 population. We similarly identified GZMK to discriminate between 2 MAIT populations. Further characterization of GZMK-enriched cells revealed increased expression of the stem-like T cell transcription factor TCF7, and the T cell memory transcription factor EOMES. Pathway enrichment of the 33 overlapping genes in the GZMK-enriched CD8 and MAIT versus cytotoxic T lymphocyte (CD8 CTL) signatures demonstrated highest enrichment for pathways involved in leukocyte differentiation, calcium signaling, and cytokine production. scTCR clonotype assessment revealed more shared clonotypes with the terminally effector CD8 CTL cells following PD-1 blockade. Following treatment, novel clones represented 38.7% (39/101) of total clones, followed by contracted clones (32.6%) and expanded (28.7%) clones. However, 76.9% and 72.4% of novel and expanded clones were contributed by the responders. On the other hand, non-responders contributed only 5% and 3.4% of the novel and expanded clones, respectively. Conclusion: We identified a CD8 continuum in BMs of patients with AML. The response to combined azacitidine/nivolumab is driven by emergence of novel clones and expansion of prior clones demonstrating T cell plasticity and adaptability. Our results demonstrate that the subverted T cells can be reinvigorated via PD-1 blockade and elicit responses in AML and warrants further functional characterization of GZMK expressing lymphocytes in mediating antileukemic responses. This abstract is also being presented as PO059. Citation Format: Hussein A. Abbas, Dapeng Hao, Katarzyna Tomczak, Praveen Barrodia, Jin Seon Im, Patrick K. Reville, Gheath Al-Atrash, Jing Ning, Maomao Ding, Jairo T. Mathews, Latasha Little, Jianhua Zhang, Sreyashi Basu, Marina Konopleva, Guillermo Garcia-Manero, Michael R. Green, Padmanee Sharma, James P. Allison, Steven M. Kornblau, Kunal Rai, Linghua Wang, Naval Daver, Andrew Futreal. Single-cell profiling of acute myeloid leukemia (AML) and its microenvironment reveals a CD8 continuum and adaptable T cell plasticity in response to PD-1 blockade-based therapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR003.

The nucleolar protein 4-like (NOL4L) gene is present on chromosome 20 (20q11.21) in humans. Parts of this gene have been shown to fuse with RUNX1 and PAX5 in acute myeloid leukemia and acute lymphoblastic leukemia, respectively. The normal function of NOL4L in humans and other organisms is not well understood. The expression patterns and functions of NOL4L homologs during vertebrate development have not been reported. We sought to address these questions by studying the expression pattern of zebrafish nol4l during embryogenesis. Our data show that Znol4l mRNA is expressed in multiple organs in zebrafish embryos. The sites of expression include parts of the brain, spinal cord, pronephros, hematopoietic cells and gut.