Loss of CREBBP and KMT2D cooperate to accelerate lymphomagenesis and shape the lymphoma immune microenvironment.

Despite regulating overlapping gene enhancers and pathways, CREBBP and KMT2D mutations recurrently co-occur in germinal center (GC) B cell-derived lymphomas, suggesting potential oncogenic cooperation. Herein, we report that combined haploinsufficiency of Crebbp and Kmt2d induces a more severe mouse lymphoma phenotype (vs either allele alone) and unexpectedly confers an immune evasive microenvironment manifesting as CD8+ T-cell exhaustion and reduced infiltration. This is linked to profound repression of immune synapse genes that mediate crosstalk with T-cells, resulting in aberrant GC B cell fate decisions. From the epigenetic perspective, we observe interaction and mutually dependent binding and function of CREBBP and KMT2D on chromatin. Their combined deficiency preferentially impairs activation of immune synapse-responsive super-enhancers, pointing to a particular dependency for both co-activators at these specialized regulatory elements. Together, our data provide an example where chromatin modifier mutations cooperatively shape and induce an immune-evasive microenvironment to facilitate lymphomagenesis.

ChromaFold predicts the 3D contact map from single-cell chromatin accessibility.

The identification of cell-type-specific 3D chromatin interactions between regulatory elements can help to decipher gene regulation and to interpret the function of disease-associated non-coding variants. However, current chromosome conformation capture (3C) technologies are unable to resolve interactions at this resolution when only small numbers of cells are available as input. We therefore present ChromaFold, a deep learning model that predicts 3D contact maps and regulatory interactions from single-cell ATAC sequencing (scATAC-seq) data alone. ChromaFold uses pseudobulk chromatin accessibility, co-accessibility profiles across metacells, and predicted CTCF motif tracks as input features and employs a lightweight architecture to enable training on standard GPUs. Once trained on paired scATAC-seq and Hi-C data in human cell lines and tissues, ChromaFold can accurately predict both the 3D contact map and peak-level interactions across diverse human and mouse test cell types. In benchmarking against a recent deep learning method that uses bulk ATAC-seq, DNA sequence, and CTCF ChIP-seq to make cell-type-specific predictions, ChromaFold yields superior prediction performance when including CTCF ChIP-seq data as an input and comparable performance without. Finally, fine-tuning ChromaFold on paired scATAC-seq and Hi-C in a complex tissue enables deconvolution of chromatin interactions across cell subpopulations. ChromaFold thus achieves state-of-the-art prediction of 3D contact maps and regulatory interactions using scATAC-seq alone as input data, enabling accurate inference of cell-type-specific interactions in settings where 3C-based assays are infeasible.

Cooperative super-enhancer inactivation caused by heterozygous loss of CREBBP and KMT2D skews B cell fate decisions and yields T cell-depleted lymphomas.

Mutations affecting enhancer chromatin regulators CREBBP and KMT2D are highly co-occurrent in germinal center (GC)-derived lymphomas and other tumors, even though regulating similar pathways. Herein, we report that combined haploinsufficiency of Crebbp and Kmt2d (C+K) indeed accelerated lymphomagenesis. C+K haploinsufficiency induced GC hyperplasia by altering cell fate decisions, skewing B cells away from memory and plasma cell differentiation. C+K deficiency particularly impaired enhancer activation for immune synapse genes involved in exiting the GC reaction. This effect was especially severe at super-enhancers for immunoregulatory and differentiation genes. Mechanistically, CREBBP and KMT2D formed a complex, were highly co-localized on chromatin, and were required for each-other's stable recruitment to enhancers. Notably, C+K lymphomas in mice and humans manifested significantly reduced CD8 + T-cell abundance. Hence, deficiency of C+K cooperatively induced an immune evasive phenotype due at least in part to failure to activate key immune synapse super-enhancers, associated with altered immune cell fate decisions. SIGNIFICANCE: Although CREBBP and KMT2D have similar enhancer regulatory functions, they are paradoxically co-mutated in lymphomas. We show that their combined loss causes specific disruption of super-enhancers driving immune synapse genes. Importantly, this leads to reduction of CD8 cells in lymphomas, linking super-enhancer function to immune surveillance, with implications for immunotherapy resistance.

Distinct and opposite effects of leukemogenic Idh and Tet2 mutations in hematopoietic stem and progenitor cells.

Mutations in IDH1, IDH2, and TET2 are recurrently observed in myeloid neoplasms. IDH1 and IDH2 encode isocitrate dehydrogenase isoforms, which normally catalyze the conversion of isocitrate to α-ketoglutarate (α-KG). Oncogenic IDH1/2 mutations confer neomorphic activity, leading to the production of D-2-hydroxyglutarate (D-2-HG), a potent inhibitor of α-KG-dependent enzymes which include the TET methylcytosine dioxygenases. Given their mutual exclusivity in myeloid neoplasms, IDH1, IDH2, and TET2 mutations may converge on a common oncogenic mechanism. Contrary to this expectation, we observed that they have distinct, and even opposite, effects on hematopoietic stem and progenitor cells in genetically engineered mice. Epigenetic and single-cell transcriptomic analyses revealed that Idh2R172K and Tet2 loss-of-function have divergent consequences on the expression and activity of key hematopoietic and leukemogenic regulators. Notably, chromatin accessibility and transcriptional deregulation in Idh2R172K cells were partially disconnected from DNA methylation alterations. These results highlight unanticipated divergent effects of IDH1/2 and TET2 mutations, providing support for the optimization of genotype-specific therapies.

An Aged/Autoimmune B-cell Program Defines the Early Transformation of Extranodal Lymphomas.

A third of patients with diffuse large B-cell lymphoma (DLBCL) present with extranodal dissemination, which is associated with inferior clinical outcomes. MYD88L265P is a hallmark extranodal DLBCL mutation that supports lymphoma proliferation. Yet extranodal lymphomagenesis and the role of MYD88L265P in transformation remain mostly unknown. Here, we show that B cells expressing Myd88L252P (MYD88L265P murine equivalent) activate, proliferate, and differentiate with minimal T-cell costimulation. Additionally, Myd88L252P skewed B cells toward memory fate. Unexpectedly, the transcriptional and phenotypic profiles of B cells expressing Myd88L252P, or other extranodal lymphoma founder mutations, resembled those of CD11c+T-BET+ aged/autoimmune memory B cells (AiBC). AiBC-like cells progressively accumulated in animals prone to develop lymphomas, and ablation of T-BET, the AiBC master regulator, stripped mouse and human mutant B cells of their competitive fitness. By identifying a phenotypically defined prospective lymphoma precursor population and its dependencies, our findings pave the way for the early detection of premalignant states and targeted prophylactic interventions in high-risk patients. SIGNIFICANCE: Extranodal lymphomas feature a very poor prognosis. The identification of phenotypically distinguishable prospective precursor cells represents a milestone in the pursuit of earlier diagnosis, patient stratification, and prophylactic interventions. Conceptually, we found that extranodal lymphomas and autoimmune disorders harness overlapping pathogenic trajectories, suggesting these B-cell disorders develop and evolve within a spectrum. See related commentary by Leveille et al. (Blood Cancer Discov 2023;4:8-11). This article is highlighted in the In This Issue feature, p. 1.

Endothelial cell-leukemia interactions remodel drug responses, uncovering T-ALL vulnerabilities.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and often incurable disease. To uncover therapeutic vulnerabilities, we first developed T-ALL patient-derived tumor xenografts (PDXs) and exposed PDX cells to a library of 433 clinical-stage compounds in vitro. We identified 39 broadly active drugs with antileukemia activity. Because endothelial cells (ECs) can alter drug responses in T-ALL, we developed an EC/T-ALL coculture system. We found that ECs provide protumorigenic signals and mitigate drug responses in T-ALL PDXs. Whereas ECs broadly rescued several compounds in most models, for some drugs the rescue was restricted to individual PDXs, suggesting unique crosstalk interactions and/or intrinsic tumor features. Mechanistically, cocultured T-ALL cells and ECs underwent bidirectional transcriptomic changes at the single-cell level, highlighting distinct "education signatures." These changes were linked to bidirectional regulation of multiple pathways in T-ALL cells as well as in ECs. Remarkably, in vitro EC-educated T-ALL cells transcriptionally mirrored ex vivo splenic T-ALL at single-cell resolution. Last, 5 effective drugs from the 2 drug screenings were tested in vivo and shown to effectively delay tumor growth and dissemination thus prolonging overall survival. In sum, we developed a T-ALL/EC platform that elucidated leukemia-microenvironment interactions and identified effective compounds and therapeutic vulnerabilities.

Distinct Genetically Determined Origins of Myd88/BCL2-Driven Aggressive Lymphoma Rationalize Targeted Therapeutic Intervention Strategies.

Genomic profiling revealed the identity of at least 5 subtypes of diffuse large B-cell lymphoma (DLBCL), including the MCD/C5 cluster characterized by aberrations in MYD88, BCL2, PRDM1, and/or SPIB. We generated mouse models harboring B cell-specific Prdm1 or Spib aberrations on the background of oncogenic Myd88 and Bcl2 lesions. We deployed whole-exome sequencing, transcriptome, flow-cytometry, and mass cytometry analyses to demonstrate that Prdm1- or Spib-altered lymphomas display molecular features consistent with prememory B cells and light-zone B cells, whereas lymphomas lacking these alterations were enriched for late light-zone and plasmablast-associated gene sets. Consistent with the phenotypic evidence for increased B cell receptor signaling activity in Prdm1-altered lymphomas, we demonstrate that combined BTK/BCL2 inhibition displays therapeutic activity in mice and in five of six relapsed/refractory DLBCL patients. Moreover, Prdm1-altered lymphomas were immunogenic upon transplantation into immuno-competent hosts, displayed an actionable PD-L1 surface expression, and were sensitive to antimurine-CD19-CAR-T cell therapy, in vivo. SIGNIFICANCE: Relapsed/refractory DLBCL remains a major medical challenge, and most of these patients succumb to their disease. Here, we generated mouse models, faithfully recapitulating the biology of MYD88-driven human DLBCL. These models revealed robust preclinical activity of combined BTK/BCL2 inhibition. We confirmed activity of this regimen in pretreated non-GCB-DLBCL patients. See related commentary by Leveille et al., p. 8. This article is highlighted in the In This Issue feature, p. 1.

TP53 mutations and RNA-binding protein MUSASHI-2 drive resistance to PRMT5-targeted therapy in B-cell lymphoma.

To identify drivers of sensitivity and resistance to Protein Arginine Methyltransferase 5 (PRMT5) inhibition, we perform a genome-wide CRISPR/Cas9 screen. We identify TP53 and RNA-binding protein MUSASHI2 (MSI2) as the top-ranked sensitizer and driver of resistance to specific PRMT5i, GSK-591, respectively. TP53 deletion and TP53R248W mutation are biomarkers of resistance to GSK-591. PRMT5 expression correlates with MSI2 expression in lymphoma patients. MSI2 depletion and pharmacological inhibition using Ro 08-2750 (Ro) both synergize with GSK-591 to reduce cell growth. Ro reduces MSI2 binding to its global targets and dual treatment of Ro and PRMT5 inhibitors result in synergistic gene expression changes including cell cycle, P53 and MYC signatures. Dual MSI2 and PRMT5 inhibition further blocks c-MYC and BCL-2 translation. BCL-2 depletion or inhibition with venetoclax synergizes with a PRMT5 inhibitor by inducing reduced cell growth and apoptosis. Thus, we propose a therapeutic strategy in lymphoma that combines PRMT5 with MSI2 or BCL-2 inhibition.

Tee-ing up a New Follicular Lymphoma Classification System.

In this issue of Blood Cancer Discovery, Han and colleagues find that follicular lymphomas (FL) can be stratified into distinct classes with clinical and functional relevance based on their T-cell subset composition. Their findings further indicate that pairing of FL cell MHCII expression with specific T-cell markers may represent a useful diagnostic approach to select patients for particular immunotherapies or immune augmentation therapies independent of genetic profiling. See related article by Han et al., p. 428 (4).

Genomic profiling for clinical decision making in lymphoid neoplasms.

With the introduction of large-scale molecular profiling methods and high-throughput sequencing technologies, the genomic features of most lymphoid neoplasms have been characterized at an unprecedented scale. Although the principles for the classification and diagnosis of these disorders, founded on a multidimensional definition of disease entities, have been consolidated over the past 25 years, novel genomic data have markedly enhanced our understanding of lymphomagenesis and enriched the description of disease entities at the molecular level. Yet, the current diagnosis of lymphoid tumors is largely based on morphological assessment and immunophenotyping, with only few entities being defined by genomic criteria. This paper, which accompanies the International Consensus Classification of mature lymphoid neoplasms, will address how established assays and newly developed technologies for molecular testing already complement clinical diagnoses and provide a novel lens on disease classification. More specifically, their contributions to diagnosis refinement, risk stratification, and therapy prediction will be considered for the main categories of lymphoid neoplasms. The potential of whole-genome sequencing, circulating tumor DNA analyses, single-cell analyses, and epigenetic profiling will be discussed because these will likely become important future tools for implementing precision medicine approaches in clinical decision making for patients with lymphoid malignancies.

BCL10 Mutations Define Distinct Dependencies Guiding Precision Therapy for DLBCL.

Activated B cell-like diffuse large B-cell lymphomas (ABC-DLBCL) have unfavorable outcomes and chronic activation of CARD11-BCL10-MALT1 (CBM) signal amplification complexes that form due to polymerization of BCL10 subunits, which is affected by recurrent somatic mutations in ABC-DLBCLs. Herein, we show that BCL10 mutants fall into at least two functionally distinct classes: missense mutations of the BCL10 CARD domain and truncation of its C-terminal tail. Truncating mutations abrogated a motif through which MALT1 inhibits BCL10 polymerization, trapping MALT1 in its activated filament-bound state. CARD missense mutations enhanced BCL10 filament formation, forming glutamine network structures that stabilize BCL10 filaments. Mutant forms of BCL10 were less dependent on upstream CARD11 activation and thus manifested resistance to BTK inhibitors, whereas BCL10 truncating but not CARD mutants were hypersensitive to MALT1 inhibitors. Therefore, BCL10 mutations are potential biomarkers for BTK inhibitor resistance in ABC-DLBCL, and further precision can be achieved by selecting therapy based on specific biochemical effects of distinct mutation classes. SIGNIFICANCE: ABC-DLBCLs feature frequent mutations of signaling mediators that converge on the CBM complex. We use structure-function approaches to reveal that BCL10 mutations fall into two distinct biochemical classes. Both classes confer resistance to BTK inhibitors, whereas BCL10 truncations confer hyperresponsiveness to MALT1 inhibitors, providing a road map for precision therapies in ABC-DLBCLs. See related commentary by Phelan and Oellerich, p. 1844. This article is highlighted in the In This Issue feature, p. 1825.

The International Consensus Classification of Mature Lymphoid Neoplasms: a report from the Clinical Advisory Committee.

Since the publication of the Revised European-American Classification of Lymphoid Neoplasms in 1994, subsequent updates of the classification of lymphoid neoplasms have been generated through iterative international efforts to achieve broad consensus among hematopathologists, geneticists, molecular scientists, and clinicians. Significant progress has recently been made in the characterization of malignancies of the immune system, with many new insights provided by genomic studies. They have led to this proposal. We have followed the same process that was successfully used for the third and fourth editions of the World Health Organization Classification of Hematologic Neoplasms. The definition, recommended studies, and criteria for the diagnosis of many entities have been extensively refined. Some categories considered provisional have now been upgraded to definite entities. Terminology for some diseases has been revised to adapt nomenclature to the current knowledge of their biology, but these modifications have been restricted to well-justified situations. Major findings from recent genomic studies have impacted the conceptual framework and diagnostic criteria for many disease entities. These changes will have an impact on optimal clinical management. The conclusions of this work are summarized in this report as the proposed International Consensus Classification of mature lymphoid, histiocytic, and dendritic cell tumors.

SETD2 Haploinsufficiency Enhances Germinal Center-Associated AICDA Somatic Hypermutation to Drive B-cell Lymphomagenesis.

SETD2 is the sole histone methyltransferase responsible for H3K36me3, with roles in splicing, transcription initiation, and DNA damage response. Homozygous disruption of SETD2 yields a tumor suppressor effect in various cancers. However, SETD2 mutation is typically heterozygous in diffuse large B-cell lymphomas. Here we show that heterozygous Setd2 deficiency results in germinal center (GC) hyperplasia and increased competitive fitness, with reduced DNA damage checkpoint activity and apoptosis, resulting in accelerated lymphomagenesis. Impaired DNA damage sensing in Setd2-haploinsufficient germinal center B (GCB) and lymphoma cells associated with increased AICDA-induced somatic hypermutation, complex structural variants, and increased translocations including those activating MYC. DNA damage was selectively increased on the nontemplate strand, and H3K36me3 loss was associated with greater RNAPII processivity and mutational burden, suggesting that SETD2-mediated H3K36me3 is required for proper sensing of cytosine deamination. Hence, Setd2 haploinsufficiency delineates a novel GCB context-specific oncogenic pathway involving defective epigenetic surveillance of AICDA-mediated effects on transcribed genes. SIGNIFICANCE: Our findings define a B cell-specific oncogenic effect of SETD2 heterozygous mutation, which unleashes AICDA mutagenesis of nontemplate strand DNA in the GC reaction, resulting in lymphomas with heavy mutational burden. GC-derived lymphomas did not tolerate SETD2 homozygous deletion, pointing to a novel context-specific therapeutic vulnerability. This article is highlighted in the In This Issue feature, p. 1599.

Tumor-associated antigen PRAME exhibits dualistic functions that are targetable in diffuse large B cell lymphoma.

PRAME is a prominent member of the cancer testis antigen family of proteins, which triggers autologous T cell-mediated immune responses. Integrative genomic analysis in diffuse large B cell lymphoma (DLBCL) uncovered recurrent and highly focal deletions of 22q11.22, including the PRAME gene, which were associated with poor outcome. PRAME-deleted tumors showed cytotoxic T cell immune escape and were associated with cold tumor microenvironments. In addition, PRAME downmodulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. In turn, PRC2-regulated genes were repressed in isogenic PRAME-KO lymphoma cell lines, and PRAME was found to directly interact with EZH2 as a negative regulator. EZH2 inhibition with EPZ-6438 abrogated these extrinsic and intrinsic effects, leading to PRAME expression and microenvironment restoration in vivo. Our data highlight multiple functions of PRAME during lymphomagenesis and provide a preclinical rationale for synergistic therapies combining epigenetic reprogramming with PRAME-targeted therapies.

Blocking UBE2N abrogates oncogenic immune signaling in acute myeloid leukemia.

Dysregulation of innate immune signaling pathways is implicated in various hematologic malignancies. However, these pathways have not been systematically examined in acute myeloid leukemia (AML). We report that AML hematopoietic stem and progenitor cells (HSPCs) exhibit a high frequency of dysregulated innate immune-related and inflammatory pathways, referred to as oncogenic immune signaling states. Through gene expression analyses and functional studies in human AML cell lines and patient-derived samples, we found that the ubiquitin-conjugating enzyme UBE2N is required for leukemic cell function in vitro and in vivo by maintaining oncogenic immune signaling states. It is known that the enzyme function of UBE2N can be inhibited by interfering with thioester formation between ubiquitin and the active site. We performed in silico structure-based and cellular-based screens and identified two related small-molecule inhibitors UC-764864/65 that targeted UBE2N at its active site. Using these small-molecule inhibitors as chemical probes, we further revealed the therapeutic efficacy of interfering with UBE2N function. This resulted in the blocking of ubiquitination of innate immune- and inflammatory-related substrates in human AML cell lines. Inhibition of UBE2N function disrupted oncogenic immune signaling by promoting cell death of leukemic HSPCs while sparing normal HSPCs in vitro. Moreover, baseline oncogenic immune signaling states in leukemic cells derived from discrete subsets of patients with AML exhibited a selective dependency on UBE2N function in vitro and in vivo. Our study reveals that interfering with UBE2N abrogates leukemic HSPC function and underscores the dependency of AML cells on UBE2N-dependent oncogenic immune signaling states.

System-wide transcriptome damage and tissue identity loss in COVID-19 patients.

The molecular mechanisms underlying the clinical manifestations of coronavirus disease 2019 (COVID-19), and what distinguishes them from common seasonal influenza virus and other lung injury states such as acute respiratory distress syndrome, remain poorly understood. To address these challenges, we combine transcriptional profiling of 646 clinical nasopharyngeal swabs and 39 patient autopsy tissues to define body-wide transcriptome changes in response to COVID-19. We then match these data with spatial protein and expression profiling across 357 tissue sections from 16 representative patient lung samples and identify tissue-compartment-specific damage wrought by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, evident as a function of varying viral loads during the clinical course of infection and tissue-type-specific expression states. Overall, our findings reveal a systemic disruption of canonical cellular and transcriptional pathways across all tissues, which can inform subsequent studies to combat the mortality of COVID-19 and to better understand the molecular dynamics of lethal SARS-CoV-2 and other respiratory infections.

Histone 3 Methyltransferases Alter Melanoma Initiation and Progression Through Discrete Mechanisms.

Perturbations to the epigenome are known drivers of tumorigenesis. In melanoma, alterations in histone methyltransferases that catalyze methylation at histone 3 lysine 9 and histone 3 lysine 27-two sites of critical post-translational modification-have been reported. To study the function of these methyltransferases in melanoma, we engineered melanocytes to express histone 3 lysine-to-methionine mutations at lysine 9 and lysine 27, which are known to inhibit the activity of histone methyltransferases, in a zebrafish melanoma model. Using this system, we found that loss of histone 3 lysine 9 methylation dramatically suppressed melanoma formation and that inhibition of histone 3 lysine 9 methyltransferases in human melanoma cells increased innate immune response signatures. In contrast, loss of histone 3 lysine 27 methylation significantly accelerated melanoma formation. We identified FOXD1 as a top target of PRC2 that is silenced in melanocytes and found that aberrant overexpression of FOXD1 accelerated melanoma onset. Collectively, these data demonstrate how histone 3 lysine-to-methionine mutations can be used to uncover critical roles for methyltransferases.

Translational Activation of ATF4 through Mitochondrial Anaplerotic Metabolic Pathways Is Required for DLBCL Growth and Survival.

Diffuse large B-cell lymphomas (DLBCL) are broadly dependent on anaplerotic metabolism regulated by mitochondrial SIRT3. Herein we find that translational upregulation of ATF4 is coupled with anaplerotic metabolism in DLBCLs due to nutrient deprivation caused by SIRT3 driving rapid flux of glutamine into the tricarboxylic acid (TCA) cycle. SIRT3 depletion led to ATF4 downregulation and cell death, which was rescued by ectopic ATF4 expression. Mechanistically, ATF4 translation is inhibited in SIRT3-deficient cells due to the increased pools of amino acids derived from compensatory autophagy and decreased glutamine consumption by the TCA cycle. Absence of ATF4 further aggravates this state through downregulation of its target genes, including genes for amino acid biosynthesis and import. Collectively, we identify a SIRT3-ATF4 axis required to maintain survival of DLBCL cells by enabling them to optimize amino acid uptake and utilization. Targeting ATF4 translation can potentiate the cytotoxic effect of SIRT3 inhibitor to DLBCL cells. SIGNIFICANCE: We discovered the link between SIRT3 and ATF4 in DLBCL cells, which connected lymphoma amino acid metabolism with ATF4 translation via metabolic stress signals. SIRT3-ATF4 axis is required in DLBCL cells regardless of subtype, which indicates a common metabolic vulnerability in DLBCLs and can serve as a therapeutic target.This article is highlighted in the In This Issue feature, p. 1.