Ari Melnick

ARID1A, a subunit of the canonical BAF nucleosome remodeling complex, is commonly mutated in lymphomas. We show that ARID1A orchestrates B cell fate during the germinal center (GC) response, facilitating cooperative and sequential binding of PU.1 and NF-kB at crucial genes for cytokine and CD40 signaling. The absence of ARID1A tilts GC cell fate toward immature IgM+CD80−PD-L2− memory B cells, known for their potential to re-enter new GCs. When combined with BCL2 oncogene, ARID1A haploinsufficiency hastens the progression of aggressive follicular lymphomas (FLs) in mice. Patients with FL with ARID1A-inactivating mutations preferentially display an immature memory B cell-like state with increased transformation risk to aggressive disease. These observations offer mechanistic understanding into the emergence of both indolent and aggressive ARID1A-mutant lymphomas through the …

SMARCA4 encodes one of two mutually exclusive ATPase subunits in the BRG/BRM associated factor (BAF) complex that is recruited by transcription factors (TFs) to drive chromatin accessibility and transcriptional activation. SMARCA4 is among the most recurrently mutated genes in human cancer, including ∼30% of germinal center (GC)-derived Burkitt lymphomas. In mice, GC-specific Smarca4 haploinsufficiency cooperated with MYC over-expression to drive lymphomagenesis. Furthermore, monoallelic Smarca4 deletion drove GC hyperplasia with centroblast polarization via significantly increased rates of centrocyte recycling to the dark zone. Mechanistically, Smarca4 loss reduced the activity of TFs that are activated in centrocytes to drive GC-exit, including SPI1 (PU.1), IRF family, and NF-κB. Loss of activity for these factors phenocopied aberrant BCL6 activity within murine centrocytes and human Burkitt …

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 …

Angioimmunoblastic T cell lymphoma (AITL) is a peripheral T cell lymphoma that originates from T follicular helper (Tfh) cells and exhibits a prominent tumor microenvironment (TME). IDH2 and TET2 mutations co-occur frequently in AITL, but their contribution to tumorigenesis is poorly understood. We developed an AITL mouse model that is driven by Idh2 and Tet2 mutations. Malignant Tfh cells display aberrant transcriptomic and epigenetic programs that impair TCR signaling. Neoplastic Tfh cells bearing combined Idh2 and Tet2 mutations show altered cross-talk with germinal center B cells that promotes B cell clonal expansion while decreasing Fas-FasL interaction and reducing B cell apoptosis. The plasma cell count and angiogenesis are also increased in the Idh2-mutated tumors, implying a major relationship between Idh2 mutation and the characteristic AITL TME. Our mouse model recapitulates several …

Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapeutic regimens to fully eradicate disease-initiating leukemia stem cells (LSC). Previous studies have demonstrated that oxidative phosphorylation (OXPHOS) is an essential process that is targetable in LSC. Sirtuin 3 (SIRT3), a mitochondrial deacetylase with a multi-faceted role in metabolic regulation, has been shown to regulate OXPHOS in cancer models; however, it has not yet been studied in the context of LSC. Thus, we sought to identify if SIRT3 is important for LSC function. Using RNAi and a SIRT3 inhibitor (YC8-02), we demonstrate that SIRT3 is a critical target for the survival of primary human LSC but is not essential for normal human hematopoietic stem and progenitor cell function. In order to elucidate the molecular mechanisms by which SIRT3 is essential in LSC we combined transcriptomic …

Methods:In the current study, we collected progenitor cells from the kidney marrows of the adult tet2 m/m and tet2 wt/wt fish at 4 and 15 months of age and conducted enhanced reduced representation of bisulfite sequencing (ERRBS) and bulk RNA-seq to measure changes in DNA methylation and gene expression of hematopoietic stem and progenitor cells (HSPCs).Results and discussion:A global increase in DNA methylation of gene promoter regions and CpG islands was observed in tet2 m/m HSPCs at 4 months of age when compared with the wild type. Furthermore, hypermethylated genes were significantly enriched for targets of SUZ12 and the metal-response-element-binding transcription factor 2 (MTF2)—involved in the polycomb repressive complex 2 (PRC2). However, between 4 and 15 months of age, we observed a paradoxical global decrease in DNA methylation in tet2 m/m HSPCs. Gene expression …

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 …

Peripheral T-cell lymphomas (PTCL) with T-follicular helper phenotype (PTCL-TFH) has recurrent mutations affecting epigenetic regulators, which may contribute to aberrant DNA methylation and chemoresistance. This phase 2 study evaluated oral azacitidine (CC-486) plus cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) as initial treatment for PTCL. CC-486 at 300 mg daily was administered for 7 days before C1 of CHOP, and for 14 days before CHOP C2-6. The primary end point was end-of-treatment complete response (CR). Secondary end points included safety and survival. Correlative studies assessed mutations, gene expression, and methylation in tumor samples. Grade 3 to 4 hematologic toxicities were mostly neutropenia (71%), with febrile neutropenia uncommon (14%). Nonhematologic toxicities included fatigue (14%) and gastrointestinal symptoms (5%). In 20 evaluable …

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 …

Activated B-cell-like diffuse large B-cell lymphomas (ABC-DLBCLs) are characterized by constitutive activation of nuclear factor κB driven by the B-cell receptor (BCR) and Toll-like receptor (TLR) pathways. However, BCR-pathway-targeted therapies have limited impact on DLBCLs. Here we used >1,100 DLBCL patient samples to determine immune and extracellular matrix cues in the lymphoid tumour microenvironment (Ly-TME) and built representative synthetic-hydrogel-based B-cell-lymphoma organoids accordingly. We demonstrate that Ly-TME cellular and biophysical factors amplify the BCR–MYD88–TLR9 multiprotein supercomplex and induce cooperative signalling pathways in ABC-DLBCL cells, which reduce the efficacy of compounds targeting the BCR pathway members Bruton tyrosine kinase and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1). Combinatorial inhibition …

Multicellular life requires altruistic cooperation between cells. The adaptive immune system is a notable exception, wherein germinal center B cells compete vigorously for limiting positive selection signals. Studying primary human lymphomas and developing new mouse models, we found that mutations affecting BTG1 disrupt a critical immune gatekeeper mechanism that strictly limits B cell fitness during antibody affinity maturation. This mechanism converted germinal center B cells into supercompetitors that rapidly outstrip their normal counterparts. This effect was conferred by a small shift in MYC protein induction kinetics but resulted in aggressive invasive lymphomas, which in humans are linked to dire clinical outcomes. Our findings reveal a delicate evolutionary trade-off between natural selection of B cells to provide immunity and potentially dangerous features that recall the more competitive nature of …

The lymphoma field is undergoing a transformative moment, where research is revealing a comprehensive picture of how these tumors arise from the immune system. Epigenetic mechanisms are especially prominent given their critical role in driving immune cell phenotypes and cross‐talk with the immune microenvironment. Along these lines, there are exciting developments in the effort to develop novel precision epigenetic therapies. Although precision epigenetic targeted therapy is still extremely limited, there are many emerging targets and corresponding small molecules that have great promise for clinical translation. Herein we discuss these novel therapeutic agents, their potential application to lymphoma, their potential to serve as adjuvants to immunotherapy/cellular therapy agents, and how to deploy these agents in a precise and impactful manner.

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 …

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.SIGNIFICANCEAlthough 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.

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 …

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 …

B-cell non-Hodgkin lymphomas (B-NHLs) are highly heterogenous by genetic, phenotypic, and clinical appearance. Next-generation sequencing technologies and multi-dimensional data analyses have further refined the way these diseases can be more precisely classified by specific genomic, epigenomic, and transcriptomic characteristics. The molecular and genetic heterogeneity of B-NHLs may contribute to the poor outcome of some of these diseases, suggesting that more personalized precision-medicine approaches are needed for improved therapeutic efficacy. The germinal center (GC) B-cell like diffuse large B-cell lymphomas (GCB-DLBCLs) and follicular lymphomas (FLs) share specific epigenetic programs. These diseases often remain difficult to treat and surprisingly do not respond advanced immunotherapies, despite arising in secondary lymphoid organs at sites of antigen recognition. Epigenetic dysregulation is a hallmark of GCB-DLBCLs and FLs, with gain-of-function (GOF) mutations in the histone methyltransferase EZH2, loss-of-function (LOF) mutations in histone acetyl transferases CREBBP and EP300, and the histone methyltransferase KMT2D representing the most prevalent genetic lesions driving these diseases. These mutations have the common effect to disrupt the interactions between lymphoma cells and the immune microenvironment, via decreased antigen presentation and responsiveness to IFN- and CD40 signaling pathways. This indicates that immune evasion is a key step in GC B-cell lymphomagenesis. EZH2 inhibitors are now approved for the treatment of FL and selective HDAC3 inhibitors counteracting the …

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 …

During the early phase of primary humoral responses, activated B cells can differentiate into different types of effector cells, dependent on B cell receptor affinity for antigen. However, the pivotal transcription factors governing these processes remain to be elucidated. Here, we show that transcription factor Bach2 protein in activated B cells is transiently induced by affinity-related signals and mechanistic target of rapamycin complex 1 (mTORC1)-dependent translation to restrain their expansion and differentiation into plasma cells while promoting memory and germinal center (GC) B cell fates. Affinity-related signals also downregulate Bach2 mRNA expression in activated B cells and their descendant memory B cells. Sustained and higher concentrations of Bach2 antagonize the GC fate. Repression of Bach2 in memory B cells predisposes their cell-fate choices upon memory recall. Our study reveals that differential …

Dear Editor, Acute Myeloid Leukemia (AML) is a heterogeneous hematological malignancy that most commonly presents in patients over the age of 60 (aged AML or aAML). aAML is associated with worse prognosis compared to younger adult AML patients [1]. The current molecular criteria considered for risk stratification (somatic mutations and cytogenetic abnormalities) were largely derived from molecular profiles of patients younger than 60 years of age [2]. Risk classifiers focused on aAML patients have been proposed [3, 4], but they only assessed selected gene mutations and/or did not include uniformly treated patients. While recent clinical trials with newly developed AML therapeutics [5, 6] might offer further insight into prognostication, comprehensive genomics data were not generated for further analyses. Thus, specific molecular determinants of clinical outcomes in aAML patients who are uniformly treated …

Abstract 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 …

To the Editor: Multiple myeloma (MM) is a hematologic neoplasm characterized by a clonal expansion of malignant plasma cells (PCs) in the bone marrow, showing clinical, genetic, and epigenetic heterogeneity. Chromosomal translocations are one of the hallmarks of MM, and mainly involve the immunoglobulin heavy chain locus (IGH). These translocations usually result in the placement of various oncogenes under the control of IGH, leading to the up-regulation of genes that provide a selective growth advantage to MM cells. 1 Five recurrent IGH translocations have been described in MM; however, in many cases, the second gene involved is not defined in routine clinical analyses. Besides, recent studies have reported novel recurrent fusion partners and novel non-IGH fusions beyond well-known translocations. 2, 3 Nevertheless, these approaches did not consider the normal counterpart of B-cells, which may …

Enhancer of zeste homolog 2 (EZH2) is an epigenetic regulator that controls the normal biology of germinal B cells. Overexpression or mutation of EZH2 is associated with malignant transformation in a number of B-cell malignancies; thus, EZH2 inhibitors are an attractive therapeutic option for these targets. Several EZH2 inhibitors have entered clinical trials, but there remains an important question as to how EZH2 inhibitor mechanism of action differs in patients with mutant and wild-type EZH2. This review discusses the EZH2-driven mechanisms that lead to the development of B-cell lymphomas and act as therapeutic targets. Another key area of investigation is whether EZH2 inhibitors will work synergistically with existing immunomodulatory drugs and chemotherapy regimens. In summary, EZH2 inhibitors show potential as treatment for a range of B-cell lymphomas, and numerous clinical evaluations are currently …

In eukaryotic cells, the genome is three dimensionally (3D) organized with DNA interaction dynamics and topology changes that regulate gene expression and drive cell fate. Upon antigen stimulation, naive B cells are activated and form germinal centers (GC) for the generation of memory B cells and plasma cells. Thereby, terminal B-cell differentiation and associated humoral immune response require massive but rigorous 3D DNA reorganization. Here, we review the dynamics of genome reorganization during GC formation and the impact of its alterations on lymphomagenesis from the nucleosome structure to the higher order chromosome organization. We particularly discuss the identified architects of 3D DNA in GC B cells and the role of their mutations in B-cell lymphomas.

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 …

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 …

B cell translocation gene 1 (BTG1) protein belongs to the BTG/transducer of ERBB2 (TOB) family of antiproliferative proteins whose members regulate various key cellular processes such as cell cycle progression, apoptosis, and differentiation. Somatic missense mutations in BTG1 are found in ∼70% of a particularly malignant and disseminated subtype of diffuse large B cell lymphoma (DLBCL). Antiproliferative activity of BTG1 has been linked to its ability to associate with transcriptional cofactors and various enzymes. However, molecular mechanisms underlying these functional interactions and how the disease-linked mutations in BTG1 affect these mechanisms are currently unknown. To start filling these knowledge gaps, here, using atomistic molecular dynamics (MD) simulations, we explored structural, dynamic, and kinetic characteristics of BTG1 protein, and studied how various DLBCL mutations affect these …

© 2022 Takata et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/.

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).

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.

BCOR is a component of a variant Polycomb repressive complex 1 (PRC1.1). PRC1 and PRC2 complexes together constitute a major gene regulatory system critical for appropriate cellular differentiation. The gene is upregulated in germinal center (GC) B cells and mutated in a number of hematologic malignancies. We report BCOR inactivating alterations in 4/7 classic Hodgkin lymphoma (cHL) cell lines, subclonal somatic mutations in Hodgkin and Reed–Sternberg (HRS) cells of 4/10 cHL cases, and deletions in HRS cells of 7/17 primary cHL cases. In mice, conditional loss of Bcor driven by AID-Cre in GC B cells resulted in gene expression changes of 46 genes (>2-fold) including upregulated Lef1 that encodes a transcription factor responsible for establishing T-cell identity and Il9r (interleukin-9 receptor), an important member of the cytokine network in cHL. Our findings suggest a role for BCOR loss in cHL …

High-order three-dimensional (3D) interactions between more than two genomic loci are common in human chromatin, but their role in gene regulation is unclear. Previous high-order 3D chromatin assays either measure distant interactions across the genome or proximal interactions at selected targets. To address this gap, we developed Pore-C, which combines chromatin conformation capture with nanopore sequencing of concatemers to profile proximal high-order chromatin contacts at the genome scale. We also developed the statistical method Chromunity to identify sets of genomic loci with frequencies of high-order contacts significantly higher than background (‘synergies’). Applying these methods to human cell lines, we found that synergies were enriched in enhancers and promoters in active chromatin and in highly transcribed and lineage-defining genes. In prostate cancer cells, these included binding …

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 …

Intravital confocal microscopy and two-photon microscopy are powerful tools to explore the dynamic behavior of immune cells in mouse lymph nodes (LNs), with penetration depth of ~100 and ~300 μm, respectively. Here, we used intravital three-photon microscopy to visualize the popliteal LN through its entire depth (600–900 μm). We determined the laser average power and pulse energy that caused measurable perturbation in lymphocyte migration. Long-wavelength three-photon imaging within permissible parameters was able to image the entire LN vasculature in vivo and measure CD8+ T cells and CD4+ T cell motility in the T cell zone over the entire depth of the LN. We observed that the motility of naive CD4+ T cells in the T cell zone during lipopolysaccharide-induced inflammation was dependent on depth. As such, intravital three-photon microscopy had the potential to examine immune cell behavior in …

The activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) has an aggressive course and is associated with poor prognosis in the relapsed or refractory setting. ABC-DLBCL is characterized by chronic active signaling of NF-κB, which is dependent on the CARD11-BCL10-MALT1 (CBM) complex. MALT1 is a key effector of the CBM complex and activates canonical NF-κB and AP-1 among other transcription factors via distinct protease and scaffold functions. There is therefore growing interest in therapeutic targeting of MALT1 for B-cell malignancies. Here, we review recent advances in therapeutic targeting of MALT1 for ABC-DLBCL. Covalent and allosteric MALT1 protease inhibitors have been developed which inhibit growth of ABC-DLBCL in preclinical models, and two clinical MALT1 protease inhibitors are being developed in phase I clinical trials. Importantly, these compounds can overcome …

Clonal hematopoiesis (CH) occurs when blood cells harboring an advantageous mutation propagate faster than others. These mutations confer a risk for hematological cancers and cardiovascular disease. Here, we analyze CH in blood samples from a pair of twin astronauts over 4 years in bulk and fractionated cell populations using a targeted CH panel, linked-read whole-genome sequencing, and deep RNA sequencing. We show CH with distinct mutational profiles and increasing allelic fraction that includes a high-risk, TET2 clone in one subject and two DNMT3A mutations on distinct alleles in the other twin. These astronauts exhibit CH almost two decades prior to the mean age at which it is typically detected and show larger shifts in clone size than age-matched controls or radiotherapy patients, based on a longitudinal cohort of 157 cancer patients. As such, longitudinal monitoring of CH may serve as an …

Cell fate decisions during development are governed by multi-factorial regulatory mechanisms including chromatin remodeling, DNA methylation, binding of transcription factors to specific loci, RNA transcription and protein synthesis. However, the mechanisms by which such regulatory 'dimensions' coordinate cell fate decisions are currently poorly understood. Here we quantified the multi-dimensional molecular changes that occur in mouse embryonic stem cells (mESCs) upon depletion of Estrogen related receptor beta (Esrrb), a key pluripotency regulator. Comparative analyses of expression changes subsequent to depletion of Esrrb or Nanog, indicated that a system of interlocked feed-forward loops involving both factors, plays a central part in regulating the timing of mESC fate decisions. Taken together, our meta-analyses support a hierarchical model in which pluripotency is maintained by an Oct4-Sox2 regulatory module, while the timing of differentiation is regulated by a Nanog-Esrrb module.

Relapse in acute myeloid leukemia (AML) patients remains a clinical challenge. The majority of AML patients who receive induction treatment with combination chemotherapy achieve clinicopathologic remission. However, a significant proportion of these patients will relapse and succumb to chemoresistant disease [1]. The biological mechanisms that contribute to relapsed AML are yet to be fully deciphered. Previous studies investigating genetic contributions to AML disease relapse included small numbers of patient samples and/or focused on a small number of AML subtypes. These studies have suggested that disease relapse is associated with founder clone recurrence, subclonal expansion and/or the occurrence of relapse-specific events (reviewed in [2]). To better understand the somatic genomic changes that drive AML relapse, we analyzed specimens (n= 120) from a clinically annotated adult relapsed AML …

Patients with acute myeloid leukemia (AML) frequently relapse after chemotherapy, yet the mechanism by which AML reemerges is not fully understood. Herein, we show that primary AML cells enter a senescence-like phenotype following chemotherapy in vitro and in vivo. This is accompanied by induction of senescence/inflammatory and embryonic diapause transcriptional programs, with downregulation of MYC and leukemia stem cell genes. Single-cell RNA sequencing suggested depletion of leukemia stem cells in vitro and in vivo, and enrichment for subpopulations with distinct senescence-like cells. This senescence effect was transient and conferred superior colony-forming and engraftment potential. Entry into this senescence-like phenotype was dependent on ATR, and persistence of AML cells was severely impaired by ATR inhibitors. Altogether, we propose that AML relapse is facilitated …

During the germinal center (GC) reaction, B cells undergo profound transcriptional, epigenetic and genomic architectural changes. How such changes are established remains unknown. Mapping chromatin accessibility during the humoral immune response, we show that OCT2 was the dominant transcription factor linked to differential accessibility of GC regulatory elements. Silent chromatin regions destined to become GC-specific super-enhancers (SEs) contained pre-positioned OCT2-binding sites in naive B cells (NBs). These preloaded SE ‘seeds’ featured spatial clustering of regulatory elements enriched in OCT2 DNA-binding motifs that became heavily loaded with OCT2 and its GC-specific coactivator OCAB in GC B cells (GCBs). SEs with high abundance of pre-positioned OCT2 binding preferentially formed long-range chromatin contacts in GCs, to support expression of GC-specifying factors. Gain in …

Treatment-persistent residual tumors impede curative cancer therapy. To understand this cancer cell state we generated models of treatment persistence that simulate the residual tumors. We observe that treatment-persistent tumor cells in organoids, xenografts, and cancer patients adopt a distinct and reversible transcriptional program resembling that of embryonic diapause, a dormant stage of suspended development triggered by stress and associated with suppressed Myc activity and overall biosynthesis. In cancer cells, depleting Myc or inhibiting Brd4, a Myc transcriptional co-activator, attenuates drug cytotoxicity through a dormant diapause-like adaptation with reduced apoptotic priming. Conversely, inducible Myc upregulation enhances acute chemotherapeutic activity. Maintaining residual cells in dormancy after chemotherapy by inhibiting Myc activity or interfering with the diapause-like adaptation by …

In less than nine months, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) killed over a million people, including >25,000 in New York City (NYC) alone. The COVID-19 pandemic caused by SARS-CoV-2 highlights clinical needs to detect infection, track strain evolution, and identify biomarkers of disease course. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs and a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, viral, and microbial profiling. We applied these methods to clinical specimens gathered from 669 patients in New York City during the first two months of the outbreak, yielding a broad molecular portrait of the emerging COVID-19 disease. We find significant enrichment of a NYC-distinctive clade of the virus (20C), as well as host responses in interferon, ACE …

Summary In this issue of Blood Cancer Discovery, Yan and colleagues discovered that mitochondrial deacylase, SIRT5, is required in AML cells to support mitochondrial oxidative phosphorylation, maintain redox homeostasis, and drive glutaminolysis. The new SIRT5 inhibitor, NRD167, can efficiently target SIRT5 in AMLs at micromolar range and may constitute a novel therapeutic approach to improve clinical outcomes of patients with AML. See related article by Yan et al., p. 266.

Loss-of-function mutations in KMT2D are a striking feature of germinal center (GC) lymphomas, resulting in decreased histone 3 lysine 4 (H3K4) methylation and altered gene expression. We hypothesized that inhibition of the KDM5 family, which demethylates H3K4me3/me2, would reestablish H3K4 methylation and restore the expression of genes repressed on loss of KMT2D. KDM5 inhibition increased H3K4me3 levels and caused an antiproliferative response in vitro, which was markedly greater in both endogenous and gene-edited KMT2D mutant diffuse large B-cell lymphoma cell lines, whereas tumor growth was inhibited in KMT2D mutant xenografts in vivo. KDM5 inhibition reactivated both KMT2D-dependent and -independent genes, resulting in diminished B-cell signaling and altered expression of B-cell lymphoma 2 (BCL2) family members, including BCL2 itself. KDM5 inhibition may offer an effective …

Clinical and genetic risk factors are currently used in multiple myeloma (MM) to stratify patients and to design specific therapies. However, these systems do not capture the heterogeneity of the disease supporting the development of new prognostic factors. In this study, we identified active promoters and alternative active promoters in 6 different B cell subpopulations, including bone-marrow plasma cells, and 32 MM patient samples, using RNA-seq data. We find that expression initiated at both regular and alternative promoters was specific of each B cell subpopulation or MM plasma cells, showing a remarkable level of consistency with chromatin-based promoter definition. Interestingly, using 595 MM patient samples from the CoMMpass dataset, we observed that the expression derived from some alternative promoters was associated with lower progression-free and overall survival in MM patients independently of …

During the germinal center (GC) reaction, B cells undergo extensive redistribution of cohesin complex and three-dimensional reorganization of their genomes. Yet, the significance of cohesin and architectural programming in the humoral immune response is unknown. Herein we report that homozygous deletion of Smc3, encoding the cohesin ATPase subunit, abrogated GC formation, while, in marked contrast, Smc3 haploinsufficiency resulted in GC hyperplasia, skewing of GC polarity and impaired plasma cell (PC) differentiation. Genome-wide chromosomal conformation and transcriptional profiling revealed defects in GC B cell terminal differentiation programs controlled by the lymphoma epigenetic tumor suppressors Tet2 and Kmt2d and failure of Smc3-haploinsufficient GC B cells to switch from B cell- to PC-defining transcription factors. Smc3 haploinsufficiency preferentially impaired the connectivity of …

The capacity of the adaptive immune system to respond to antigenic challenges relies on rapid diversification, expansion, and functional specialization of mature B cells. To accomplish this, activated B cells are transiently endowed with phenotypes that would normally be suppressed in somatic cells, such as enhanced proliferative potential and tolerance for genomic instability. Acquisition of these traits, directed by immune signaling cues and orchestrated by transcriptional and epigenetic programs, sets the stage for malignant transformation, often due to somatic mutations targeting epigenetic machinery in B cell lymphomas. This review examines how such mutations hack the epigenome to reprogram the immune response in such a way as to facilitate the emergence of malignant traits, suppress immune surveillance, and ultimately drive transformation and progression of a diverse spectrum of B cell lymphomas …

Multiple myeloma (MM) is an incurable disease, whose clinical heterogeneity makes its management challenging, highlighting the need for biological features to guide improved therapies. Deregulation of specific long non-coding RNAs (lncRNAs) has been shown in MM, nevertheless, the complete lncRNA transcriptome has not yet been elucidated. In this work, we identified 40,511 novel lncRNAs in MM samples. lncRNAs accounted for 82% of the MM transcriptome and were more heterogeneously expressed than coding genes. A total of 10,351 overexpressed and 9,535 downregulated lncRNAs were identified in MM patients when compared with normal bone-marrow plasma cells. Transcriptional dynamics study of lncRNAs in the context of normal B-cell maturation revealed 989 lncRNAs with exclusive expression in MM, among which 89 showed de novo epigenomic activation. Knockdown studies on one of …

Transcriptional deregulation is a central event in the development of acute myeloid leukemia (AML). To identify potential disturbances in gene regulation, we conducted an unbiased screen of allele-specific expression (ASE) in 209 AML cases. The gene encoding GATA binding protein 2 (GATA2) displayed ASE more often than any other myeloid- or cancer-related gene. GATA2 ASE was strongly associated with CEBPA double mutations (DMs), with 95% of cases presenting GATA2 ASE. In CEBPA DM AML with GATA2 mutations, the mutated allele was preferentially expressed. We found that GATA2 ASE was a somatic event lost in complete remission, supporting the notion that it plays a role in CEBPA DM AML. Acquisition of GATA2 ASE involved silencing of 1 allele via promoter methylation and concurrent overactivation of the other allele, thereby preserving expression levels. Notably, promoter methylation …

The cohesin complex plays critical roles in genomic stability and gene expression through effects on 3D architecture. Cohesin core subunit genes are mutated across a wide cross-section of cancers, but not in germinal center (GC) derived lymphomas. In spite of this, haploinsufficiency of cohesin ATPase subunit Smc3 was shown to contribute to malignant transformation of GC B-cells in mice. Herein we explored potential mechanisms and clinical relevance of Smc3 deficiency in GC lymphomagenesis. Transcriptional profiling of Smc3 haploinsufficient murine lymphomas revealed downregulation of genes repressed by loss of epigenetic tumor suppressors Tet2 and Kmt2d. Profiling 3D chromosomal interactions in lymphomas revealed impaired enhancer-promoter interactions affecting genes like Tet2, which was aberrantly downregulated in Smc3 deficient lymphomas. Tet2 plays important roles in B-cell exit from the GC reaction, and single cell RNA-seq profiles and phenotypic trajectory analysis in Smc3 mutant mice revealed a specific defect in commitment to the final steps of plasma cell differentiation. Although Smc3 deficiency resulted in structural abnormalities in GC B-cells, there was no increase of somatic mutations or structural variants in Smc3 haploinsufficient lymphomas, suggesting that cohesin deficiency largely induces lymphomas through disruption of enhancer-promoter interactions of terminal differentiation and tumor suppressor genes. Strikingly, the presence of the Smc3 haploinsufficient GC B-cell transcriptional signature in human patients with GC-derived diffuse large B-cell lymphoma (DLBCL) was linked to inferior clinical …

Aberrant cell fate decisions due to transcriptional misregulation are central to malignant transformation. Histones are the major constituents of chromatin, and mutations in histone-encoding genes are increasingly recognized as drivers of oncogenic transformation. Mutations in linker histone H1 genes were recently identified as drivers of peripheral lymphoid malignancy. Loss of H1 in germinal center B cells results in widespread chromatin decompaction, redistribution of core histone modifications, and reactivation of stem cell–specific transcriptional programs. This review explores how linker histones and mutations therein regulate chromatin structure, highlighting reciprocal relationships between epigenetic circuits, and discusses the emerging role of aberrant three-dimensional chromatin architecture in malignancy.

H1 linker histones are the most abundant chromatin-binding proteins. In vitro studies indicate that their association with chromatin determines nucleosome spacing and enables arrays of nucleosomes to fold into more compact chromatin structures. However, the in vivo roles of H1 are poorly understood. Here we show that the local density of H1 controls the balance of repressive and active chromatin domains by promoting genomic compaction. We generated a conditional triple-H1-knockout mouse strain and depleted H1 in haematopoietic cells. H1 depletion in T cells leads to de-repression of T cell activation genes, a process that mimics normal T cell activation. Comparison of chromatin structure in normal and H1-depleted CD8+ T cells reveals that H1-mediated chromatin compaction occurs primarily in regions of the genome containing higher than average levels of H1: the chromosome conformation capture (Hi …

MALT1 inhibitors are promising therapeutic agents for B-cell lymphomas that are dependent on constitutive or aberrant signaling pathways. However, a potential limitation for signal transduction–targeted therapies is the occurrence of feedback mechanisms that enable escape from the full impact of such drugs. Here, we used a functional genomics screen in activated B-cell–like (ABC) diffuse large B-cell lymphoma (DLBCL) cells treated with a small molecule irreversible inhibitor of MALT1 to identify genes that might confer resistance or enhance the activity of MALT1 inhibition (MALT1i). We find that loss of B-cell receptor (BCR)- and phosphatidylinositol 3-kinase (PI3K)-activating proteins enhanced sensitivity, whereas loss of negative regulators of these pathways (eg, TRAF2, TNFAIP3) promoted resistance. These findings were validated by knockdown of individual genes and a combinatorial drug screen …

B-cell lymphoma 6 (BCL6) is a master regulator of germinal center formation that produce antibody-secreting plasma cells and memory B-cells for sustained immune responses. The BTB domain of BCL6 (BCL6BTB) forms a homodimer that mediates transcriptional repression by recruiting its corepressor proteins to form a biologically functional transcriptional complex. The protein–protein interaction (PPI) between the BCL6BTB and its corepressors has emerged as a therapeutic target for the treatment of DLBCL and a number of other human cancers. This Perspective provides an overview of recent advances in the development of BCL6BTB inhibitors from reversible inhibitors, irreversible inhibitors, to BCL6 degraders. Inhibitor design and medicinal chemistry strategies for the development of novel compounds will be provided. The binding mode of new inhibitors to BCL6BTB are highlighted. Also, the in vitro and in …

A hallmark of acute myeloid leukemia (AML) is the inability of self-renewing malignant cells to mature into a non-dividing terminally differentiated state. This differentiation block has been linked to dysregulation of multiple cellular processes, including transcriptional, chromatin, and metabolic regulation. The transcription factor HOXA9 and the histone demethylase LSD1 are examples of such regulators that promote differentiation blockade in AML. To identify metabolic targets that interact with LSD1 inhibition to promote myeloid maturation, we screened a small molecule library to identify druggable substrates. We found that differentiation caused by LSD1 inhibition is enhanced by combined perturbation of purine nucleotide salvage and de novo lipogenesis pathways, and identified multiple lines of evidence to support the specificity of these pathways and suggest a potential basis of how perturbation of these …

Genomic classification has improved risk assignment of pediatric, but not adult B-lineage acute lymphoblastic leukemia (B-ALL). The international UKALLXII/ECOG-ACRIN E2993 (#NCT00002514) trial accrued 1229 adolescent/adult patients with BCR-ABL1− B-ALL (aged 14 to 65 years). Although 93% of patients achieved remission, 41% relapsed at a median of 13 months (range, 28 days to 12 years). Five-year overall survival (OS) was 42% (95% confidence interval, 39, 44). Transcriptome sequencing, gene expression profiling, cytogenetics, and fusion polymerase chain reaction enabled genomic subtyping of 282 patient samples, of which 264 were eligible for trial, accounting for 64.5% of E2993 patients. Among patients with outcome data, 29.5% with favorable outcomes (5-year OS 65% to 80%) were deemed standard risk (DUX4-rearranged [9.2%], ETV6-RUNX1/-like [2.3%], TCF3-PBX1 [6.9%], PAX5 …

Background Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA’s Epigenomics Quality Control Group. Results Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing …

Linker histone H1 proteins bind to nucleosomes and facilitate chromatin compaction, although their biological functions are poorly understood. Mutations in the genes that encode H1 isoforms B–E (H1B, H1C, H1D and H1E; also known as H1-5, H1-2, H1-3 and H1-4, respectively) are highly recurrent in B cell lymphomas, but the pathogenic relevance of these mutations to cancer and the mechanisms that are involved are unknown. Here we show that lymphoma-associated H1 alleles are genetic driver mutations in lymphomas. Disruption of H1 function results in a profound architectural remodelling of the genome, which is characterized by large-scale yet focal shifts of chromatin from a compacted to a relaxed state. This decompaction drives distinct changes in epigenetic states, primarily owing to a gain of histone H3 dimethylation at lysine 36 (H3K36me2) and/or loss of repressive H3 trimethylation at lysine 27 …

B-cell lymphoma 6 (BCL6) is a transcription repressor and proto-oncogene that plays a crucial role in the innate and adaptive immune system and lymphoid neoplasms. However, its role in myeloid malignancies remains unclear. Here, we explored the role of BCL6 in acute myeloid leukemia (AML). BCL6 was expressed at variable and often high levels in AML cell lines and primary AML samples. AMLs with higher levels of BCL6 were generally sensitive to treatment with BCL6 inhibitors, with the exception of those with monocytic differentiation. Gene expression profiling of AML cells treated with a BCL6 inhibitor revealed induction of BCL6-repressed target genes and transcriptional programs linked to DNA damage checkpoints and downregulation of stem cell genes. Ex vivo treatment of primary AML cells with BCL6 inhibitors induced apoptosis and decreased colony-forming capacity, which correlated with the …

Diffuse large B cell lymphoma (DLBCL) is the most common histological subtype of non-Hodgkin B cell lymphoma (NHL), and manifests highly heterogeneous genetic/phenotypic characteristics as well as variable responses to conventional immunochemotherapy. Genetic profiling of DLBCL patients has revealed highly recurrent mutations of epigenetic regulator genes such as CREBBP, KMT2D, EZH2 and TET2. These mutations drive malignant transformation through aberrant epigenetic programming of B-cells and may influence clinical outcomes. These and other chromatin modifier genes also play critical roles in normal B-cells, as they undergo the various phenotypic transitions characteristic of the humoral immune response. Many of these functions have to do with impairing immune surveillance and may critically mediate resistance to immunotherapies. In this review, we describe how epigenetic dysfunction …

DNA methylation is aberrant in cancer, but the dynamics, regulatory role and clinical implications of such epigenetic changes are still poorly understood. Here, reduced representation bisulfite sequencing (RRBS) profiles of 1538 breast tumors and 244 normal breast tissues from the METABRIC cohort are reported, facilitating detailed analysis of DNA methylation within a rich context of genomic, transcriptional, and clinical data. Tumor methylation from immune and stromal signatures are deconvoluted leading to the discovery of a tumor replication-linked clock with genome-wide methylation loss in non-CpG island sites. Unexpectedly, methylation in most tumor CpG islands follows two replication-independent processes of gain (MG) or loss (ML) that we term epigenomic instability. Epigenomic instability is correlated with tumor grade and stage, TP53 mutations and poorer prognosis. After controlling for these global …

Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease. Transcriptomic and genetic characterization of DLBCL has increased the understanding of its intrinsic pathogenesis and provided potential therapeutic targets. However, the role of the microenvironment in DLBCL biology remains less understood. Here, we performed a transcriptomic analysis of the microenvironment of 4,655 DLBCLs from multiple independent cohorts and described four major lymphoma microenvironment categories that associate with distinct biological aberrations and clinical behavior. We also found evidence of genetic and epigenetic mechanisms deployed by cancer cells to evade microenvironmental constraints of lymphoma growth, supporting the rationale for implementing DNA hypomethylating agents in selected patients with DLBCL. In addition, our work uncovered new …

Diffuse large B cell lymphoma (DLBCL) is a markedly phenotypically heterogenous disease, frequently assessed using bulk genomic techniques that blur the intrinsic heterogeneity of each tumor. In this issue of Cancer Cell, Steen et al. have utilized a computational framework called EcoTyper to skillfully dissect bulk transcriptomic tumor profiles into different cell type components in an unsupervised manner.

Based on gene expression profiles, diffuse large B-cell lymphoma (DLBCL) is subdivided into germinal center B-cell–like (GCB) and activated B-cell–like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88 as well as BCL2 copy-number gains. Here, we employ immune phenotyping, RNA sequencing, and whole-exome sequencing to characterize a Myd88- and BCL2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression compared with GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and PD-1 blockade …

The prognosis for people with the high-grade brain tumor glioblastoma is very poor, due largely to low cell death in response to genotoxic therapy. The transcription factor BCL6, a protein that normally suppresses the DNA damage response during immune cell maturation, and a known driver of B-cell lymphoma, was shown to mediate the survival of glioblastoma cells. Expression was observed in glioblastoma tumor specimens and cell lines. When BCL6 expression or activity was reduced in these lines, increased apoptosis and a profound loss of proliferation was observed, consistent with gene expression signatures suggestive of anti-apoptotic and pro-survival signaling role for BCL6 in glioblastoma. Further, treatment with the standard therapies for glioblastoma—ionizing radiation and temozolomide—both induced BCL6 expression in vitro, and an in vivo orthotopic animal model of glioblastoma. Importantly, inhibition of BCL6 in combination with genotoxic therapies enhanced the therapeutic effect. Together these data demonstrate that BCL6 is an active transcription factor in glioblastoma, that it drives survival of cells, and that it increased with DNA damage, which increased the survival rate of therapy-treated cells. This makes BCL6 an excellent therapeutic target in glioblastoma—by increasing sensitivity to standard DNA damaging therapy, BCL6 inhibitors have real potential to improve the outcome for people with this disease.

Background How a quiescent, terminally differentiated plasma cell transforms into an aggressive and lethal disease such as multiple myeloma (MM) remains a scientific puzzle. The abnormal genomic landscape of MM patients has been previously characterized but has not been fully capable of explaining the pathogenesis of the disease. Furthermore, MM is a remarkably heterogeneous disease with no unifying transforming mechanism. Recently, breakthrough discoveries in the epigenetics field have transformed our understanding of neoplastic transformation. However, the chromatin regulatory network underlying MM onset and progression has just started to be characterized. Aims To identify the core epigenetic mechanisms underlying myelomagenesis by integrating a multi‐omics characterization of MM patients in the context of normal B cell differentiation. Methods We have generated and integrated genome …

Epigenetic allele diversity is linked to inferior prognosis in acute myeloid leukemia (AML). However, the source of epiallele heterogeneity in AML is unknown. Herein we analyzed epiallele diversity in a genetically and clinically annotated AML cohort. Notably, AML driver mutations linked to transcription factors and favorable outcome are associated with epigenetic destabilization in a defined set of susceptible loci. In contrast, AML subtypes linked to inferior prognosis manifest greater abundance and highly stochastic epiallele patterning. We report an epiallele outcome classifier supporting the link between epigenetic diversity and treatment failure. Mouse models with TET2 or IDH2 mutations show that epiallele diversity is especially strongly induced by IDH mutations, precedes transformation to AML, and is enhanced by cooperation between somatic mutations. Furthermore, epiallele complexity was …

Liquid biopsies based on cell-free DNA (cfDNA) or exosomes provide a noninvasive approach to monitor human health and disease but have not been utilized for astronauts. Here, we profile cfDNA characteristics, including fragment size, cellular deconvolution, and nucleosome positioning, in an astronaut during a year-long mission on the International Space Station, compared to his identical twin on Earth and healthy donors. We observed a significant increase in the proportion of cell-free mitochondrial DNA (cf-mtDNA) inflight, and analysis of post-flight exosomes in plasma revealed a 30-fold increase in circulating exosomes and patient-specific protein cargo (including brain-derived peptides) after the year-long mission. This longitudinal analysis of astronaut cfDNA during spaceflight and the exosome profiles highlights their utility for astronaut health monitoring, as well as cf-mtDNA levels as a potential biomarker …

CREBBP mutations are highly recurrent in B-cell lymphomas and either inactivate its histone acetyltransferase (HAT) domain or truncate the protein. Herein, we show that these two classes of mutations yield different degrees of disruption of the epigenome, with HAT mutations being more severe and associated with inferior clinical outcome. Genes perturbed by CREBBP mutation are direct targets of the BCL6–HDAC3 onco-repressor complex. Accordingly, we show that HDAC3-selective inhibitors reverse CREBBP-mutant aberrant epigenetic programming, resulting in: (i) growth inhibition of lymphoma cells through induction of BCL6 target genes such as CDKN1A and (ii) restoration of immune surveillance due to induction of BCL6-repressed IFN pathway and antigen-presenting genes. By reactivating these genes, exposure to HDAC3 inhibitors restored the ability of tumor-infiltrating …

The most aggressive B cell lymphomas frequently manifest extranodal distribution and carry somatic mutations in the poorly characterized gene TBL1XR1. Here, we show that TBL1XR1 mutations skew the humoral immune response toward generating abnormal immature memory B cells (MB), while impairing plasma cell differentiation. At the molecular level, TBL1XR1 mutants co-opt SMRT/HDAC3 repressor complexes toward binding the MB cell transcription factor (TF) BACH2 at the expense of the germinal center (GC) TF BCL6, leading to pre-memory transcriptional reprogramming and cell-fate bias. Upon antigen recall, TBL1XR1 mutant MB cells fail to differentiate into plasma cells and instead preferentially reenter new GC reactions, providing evidence for a cyclic reentry lymphomagenesis mechanism. Ultimately, TBL1XR1 alterations lead to a striking extranodal immunoblastic lymphoma phenotype that mimics …

Although we are just beginning to understand the mechanisms that regulate the epigenome, aberrant epigenetic programming has already emerged as a hallmark of hematologic malignancies including acute myeloid leukemia (AML) and B-cell lymphomas. Although these diseases arise from the hematopoietic system, the epigenetic mechanisms that drive these malignancies are quite different. Yet, in all of these tumors, somatic mutations in transcription factors and epigenetic modifiers are the most commonly mutated set of genes and result in multilayered disruption of the epigenome. Myeloid and lymphoid neoplasms generally manifest epigenetic allele diversity, which contributes to tumor cell population fitness regardless of the underlying genetics. Epigenetic therapies are emerging as one of the most promising new approaches for these patients. However, effective targeting of the epigenome must consider the …

During affinity maturation, germinal center (GC) B cells alternate between proliferation and somatic hypermutation in the dark zone (DZ) and affinity-dependent selection in the light zone (LZ). This anatomical segregation imposes that the vigorous proliferation that allows clonal expansion of positively selected GC B cells takes place ostensibly in the absence of the signals that triggered selection in the LZ, as if by “inertia.” We find that such inertial cycles specifically require the cell cycle regulator cyclin D3. Cyclin D3 dose-dependently controls the extent to which B cells proliferate in the DZ and is essential for effective clonal expansion of GC B cells in response to strong T follicular helper (Tfh) cell help. Introduction into the Ccnd3 gene of a Burkitt lymphoma–associated gain-of-function mutation (T283A) leads to larger GCs with increased DZ proliferation and, in older mice, clonal B cell lymphoproliferation, suggesting …

Despite the global rapid increase in the number of clinical trials employing chimeric antigen receptors (CARs), no comprehensive survey of their scope, targets and design exists. In this study, we present an interactive CAR clinical trial database, spanning 64 targets deployed in T cells (CAR-T), natural killer cells (CAR-NK) or mixtures (CAR-NK/T) from over 500 clinical trials in 20 countries, encompassing >20,000 patients. By combining these data with transcriptional and proteomic data, we create a ‘targetable landscape’ for CAR cell therapies based on 13,206 proteins and RNAs across 78 tissues, 124 cell types and 20 cancer types. These data suggest a landscape of over 100 single targets and over 100,000 target pairs using logical switches for CAR cell engineering. Our analysis of the CAR cellular therapeutic landscape may aid the design of future therapies, improve target-to-patient matching, and ultimately …

The TET2 DNA hydroxymethyltransferase is frequently disrupted by somatic mutations in diffuse large B cell lymphomas (DLBCLs), a tumor that originates from germinal center (GC) B cells. Here, we show that TET2 deficiency leads to DNA hypermethylation of regulatory elements in GC B cells, associated with silencing of the respective genes. This hypermethylation affects the binding of transcription factors including those involved in exit from the GC reaction and involves pathways such as B cell receptor, antigen presentation, CD40, and others. Normal GC B cells manifest a typical hypomethylation signature, which is caused by AID, the enzyme that mediates somatic hypermutation. However, AID-induced demethylation is markedly impaired in TET2-deficient GC B cells, suggesting that AID epigenetic effects are partially dependent on TET2. Last, we find that TET2 mutant DLBCLs also manifest the aberrant TET2 …

Activated B-cell (ABC)-diffuse large B-cell lymphomas (DLBCLs) are clinically aggressive and phenotypically complex malignancies, whose transformation mechanisms remain unclear. Partially differentiated antigen-secreting cells (plasmablasts) have long been regarded as cells-of-origin for these tumors, despite lack of definitive experimental evidence. Recent DLBCL reclassification based on mutational landscapes identified MCD/C5 tumors as specific ABC-DLBCLs with unfavorable clinical outcome, activating mutations in the signaling adaptors MYD88 and CD79B, and immune evasion through mutation of antigen-presenting genes. MCD/C5s manifest prominent extranodal dissemination and similarities with primary extranodal lymphomas (PENLs). In this regard, recent studies on TBL1XR1, a gene recurrently mutated in MCD/C5s and PENLs, suggest that aberrant memory B cells (MBs), and not …

The National Aeronautics and Space Administration (NASA) Twins Study created an integrative molecular profile of an astronaut during NASA's first 1-year mission on the International Space Station (ISS) and included comparisons to an identical Earth-bound twin. The unique biochemical profiles observed when landing on Earth after such a long mission (e.g., spikes in interleukin-1 [IL-1]/6/10, c-reactive protein [CRP], C-C motif chemokine ligand 2 [CCL2], IL-1 receptor antagonist [IL-1ra], and tumor necrosis factor alpha [TNF-α]) opened new questions about the human body's response to gravity and how to plan for future astronauts, particularly around initiation or resolution of inflammation. Here, single-cell, multi-omic (100-plex epitope profile and gene expression) profiling of peripheral blood mononuclear cells (PBMCs) showed changes to blood cell composition and gene expression post-flight, specifically for …

Cancer cells adapt their metabolic activities to support growth and proliferation. However, increased activity of metabolic enzymes is not usually considered an initiating event in the malignant process. Here, we investigate the possible role of the enzyme serine hydroxymethyltransferase-2 (SHMT2) in lymphoma initiation. SHMT2 localizes to the most frequent region of copy number gains at chromosome 12q14.1 in lymphoma. Elevated expression of SHMT2 cooperates with BCL2 in lymphoma development; loss or inhibition of SHMT2 impairs lymphoma cell survival. SHMT2 catalyzes the conversion of serine to glycine and produces an activated one-carbon unit that can be used to support S-adenosyl methionine synthesis. SHMT2 induces changes in DNA and histone methylation patterns leading to promoter silencing of previously uncharacterized mutational genes, such as SASH1 and PTPRM. Together, our …

Molecular alterations in the histone methyltransferase EZH2 and the antiapoptotic protein Bcl-2 frequently co-occur in diffuse large B-cell lymphoma (DLBCL). Because DLBCL tumors with these characteristics are likely dependent on both oncogenes, dual targeting of EZH2 and Bcl-2 is a rational therapeutic approach. We hypothesized that EZH2 and Bcl-2 inhibition would be synergistic in DLBCL. To test this, we evaluated the EZH2 inhibitor tazemetostat and the Bcl-2 inhibitor venetoclax in DLBCL cells, 3-dimensional lymphoma organoids, and patient-derived xenografts (PDXs). We found that tazemetostat and venetoclax are synergistic in DLBCL cells and 3-dimensional lymphoma organoids that harbor an EZH2 mutation and an IGH/BCL2 translocation but not in wild-type cells. Tazemetostat treatment results in upregulation of proapoptotic Bcl-2 family members and priming of mitochondria to BH3-mediated …

We have identified and validated a spaceflight-associated microRNA (miRNA) signature that is shared by rodents and humans in response to simulated, short-duration and long-duration spaceflight. Previous studies have identified miRNAs that regulate rodent responses to spaceflight in low-Earth orbit, and we have confirmed the expression of these proposed spaceflight-associated miRNAs in rodents reacting to simulated spaceflight conditions. Moreover, astronaut samples from the NASA Twins Study confirmed these expression signatures in miRNA sequencing, single-cell RNA sequencing (scRNA-seq), and single-cell assay for transposase accessible chromatin (scATAC-seq) data. Additionally, a subset of these miRNAs (miR-125, miR-16, and let-7a) was found to regulate vascular damage caused by simulated deep space radiation. To demonstrate the physiological relevance of key spaceflight-associated …

Follicular lymphomas (FLs) are slow-growing, indolent tumors containing extensive follicular dendritic cell (FDC) networks and recurrent EZH2 gain-of-function mutations. Paradoxically, FLs originate from highly proliferative germinal center (GC) B cells with proliferation strictly dependent on interactions with T follicular helper cells. Herein, we show that EZH2 mutations initiate FL by attenuating GC B cell requirement for T cell help and driving slow expansion of GC centrocytes that become enmeshed with and dependent on FDCs. By impairing T cell help, mutant EZH2 prevents induction of proliferative MYC programs. Thus, EZH2 mutation fosters malignant transformation by epigenetically reprograming B cells to form an aberrant immunological niche that reflects characteristic features of human FLs, explaining how indolent tumors arise from GC B cells.

Histone post-translational modifications (PTMs) create a powerful regulatory mechanism for maintaining chromosomal integrity in cells. Histone acetylation and methylation, the most widely studied histone PTMs, act in concert with chromatin-associated proteins to control access to genetic information during transcription. Alterations in cellular histone PTMs have been linked to disease states and have crucial biomarker and therapeutic potential. Traditional bottom-up mass spectrometry of histones requires large numbers of cells, typically one million or more. However, for some cell subtype-specific studies, it is difficult or impossible to obtain such large numbers of cells and quantification of rare histone PTMs is often unachievable. An established targeted LC-MS/MS method was used to quantify the abundance of histone PTMs from cell lines and primary human specimens. Sample preparation was modified by omitting nuclear isolation and reducing the rounds of histone derivatization to improve detection of histone peptides down to 1,000 cells. In the current study, we developed and validated a quantitative LC-MS/MS approach tailored for a targeted histone assay of 75 histone peptides with as few as 10,000 cells. Furthermore, we were able to detect and quantify 61 histone peptides from just 1,000 primary human stem cells. Detection of 37 histone peptides was possible from 1,000 acute myeloid leukemia patient cells. We anticipate that this revised method can be used in many applications where achieving large cell numbers is challenging, including rare human cell populations.

Locus control region (LCR) functions define cellular identity and have critical roles in diseases such as cancer, although the hierarchy of structural components and associated factors that drive functionality are incompletely understood. Here we show that OCA-B, a B cell-specific coactivator essential for germinal center (GC) formation, forms a ternary complex with the lymphoid-enriched OCT2 and GC-specific MEF2B transcription factors and that this complex occupies and activates an LCR that regulates the BCL6 proto-oncogene and is uniquely required by normal and malignant GC B cells. Mechanistically, through OCA-B-MED1 interactions, this complex is required for Mediator association with the BCL6 promoter. Densely tiled CRISPRi screening indicates that only LCR segments heavily bound by this ternary complex are essential for its function. Our results demonstrate how an intimately linked complex of …