An atlas of cells in the human tonsil.

Palatine tonsils are secondary lymphoid organs (SLOs) representing the first line of immunological defense against inhaled or ingested pathogens. We generated an atlas of the human tonsil composed of >556,000 cells profiled across five different data modalities, including single-cell transcriptome, epigenome, proteome, and immune repertoire sequencing, as well as spatial transcriptomics. This census identified 121 cell types and states, defined developmental trajectories, and enabled an understanding of the functional units of the tonsil. Exemplarily, we stratified myeloid slan-like subtypes, established a BCL6 enhancer as locally active in follicle-associated T and B cells, and identified SIX5 as putative transcriptional regulator of plasma cell maturation. Analyses of a validation cohort confirmed the presence, annotation, and markers of tonsillar cell types and provided evidence of age-related compositional shifts. We demonstrate the value of this resource by annotating cells from B cell-derived mantle cell lymphomas, linking transcriptional heterogeneity to normal B cell differentiation states of the human tonsil.

Interleukin-10 receptor signaling promotes the maintenance of a PD-1int TCF-1+ CD8+ T cell population that sustains anti-tumor immunity.

T cell exhaustion limits anti-tumor immunity and responses to immunotherapy. Here, we explored the microenvironmental signals regulating T cell exhaustion using a model of chronic lymphocytic leukemia (CLL). Single-cell analyses identified a subset of PD-1hi, functionally impaired CD8+ T cells that accumulated in secondary lymphoid organs during disease progression and a functionally competent PD-1int subset. Frequencies of PD-1int TCF-1+ CD8+ T cells decreased upon Il10rb or Stat3 deletion, leading to accumulation of PD-1hi cells and accelerated tumor progression. Mechanistically, inhibition of IL-10R signaling altered chromatin accessibility and disrupted cooperativity between the transcription factors NFAT and AP-1, promoting a distinct NFAT-associated program. Low IL10 expression or loss of IL-10R-STAT3 signaling correlated with increased frequencies of exhausted CD8+ T cells and poor survival in CLL and in breast cancer patients. Thus, balance between PD-1hi, exhausted CD8+ T cells and functional PD-1int TCF-1+ CD8+ T cells is regulated by cell-intrinsic IL-10R signaling, with implications for immunotherapy.

Signatures of TOP1 transcription-associated mutagenesis in cancer and germline.

The mutational landscape is shaped by many processes. Genic regions are vulnerable to mutation but are preferentially protected by transcription-coupled repair1. In microorganisms, transcription has been demonstrated to be mutagenic2,3; however, the impact of transcription-associated mutagenesis remains to be established in higher eukaryotes4. Here we show that ID4-a cancer insertion-deletion (indel) mutation signature of unknown aetiology5 characterized by short (2 to 5 base pair) deletions -is due to a transcription-associated mutagenesis process. We demonstrate that defective ribonucleotide excision repair in mammals is associated with the ID4 signature, with mutations occurring at a TNT sequence motif, implicating topoisomerase 1 (TOP1) activity at sites of genome-embedded ribonucleotides as a mechanistic basis. Such TOP1-mediated deletions occur somatically in cancer, and the ID-TOP1 signature is also found in physiological settings, contributing to genic de novo indel mutations in the germline. Thus, although topoisomerases protect against genome instability by relieving topological stress6, their activity may also be an important source of mutations in the human genome.

SOX11 is a novel binding partner and endogenous inhibitor of SAMHD1 ara-CTPase activity in mantle cell lymphoma.

ABSTRACT: Sterile alpha motif and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate triphosphohydrolase with ara-CTPase activity that confers cytarabine (ara-C) resistance in several hematological malignancies. Targeting SAMHD1's ara-CTPase activity has recently been demonstrated to enhance ara-C efficacy in acute myeloid leukemia. Here, we identify the transcription factor SRY-related HMG-box containing protein 11 (SOX11) as a novel direct binding partner and first known endogenous inhibitor of SAMHD1. SOX11 is aberrantly expressed not only in mantle cell lymphoma (MCL), but also in some Burkitt lymphomas. Coimmunoprecipitation of SOX11 followed by mass spectrometry in MCL cell lines identified SAMHD1 as the top SOX11 interaction partner, which was validated by proximity ligation assay. In vitro, SAMHD1 bound to the HMG box of SOX11 with low-micromolar affinity. In situ crosslinking studies further indicated that SOX11-SAMHD1 binding resulted in a reduced tetramerization of SAMHD1. Functionally, expression of SOX11 inhibited SAMHD1 ara-CTPase activity in a dose-dependent manner resulting in ara-C sensitization in cell lines and in a SOX11-inducible mouse model of MCL. In SOX11-negative MCL, SOX11-mediated ara-CTPase inhibition could be mimicked by adding the recently identified SAMHD1 inhibitor hydroxyurea. Taken together, our results identify SOX11 as a novel SAMHD1 interaction partner and its first known endogenous inhibitor with potentially important implications for clinical therapy stratification.

SOX11 is a novel binding partner and endogenous inhibitor of SAMHD1 ara-CTPase activity in mantle cell lymphoma.

The sterile alpha motif and histidine-aspartate (HD) domain containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate triphosphohydrolase with ara-CTPase activity that confers cytarabine (ara-C) resistance in several haematological malignancies. Targeting SAMHD1's ara-CTPase activity has recently been demonstrated to enhance ara-C efficacy in acute myeloid leukemia. Here, we identify the transcription factor SRY-related HMG-box containing protein 11 (SOX11) as a novel direct binding partner and first known endogenous inhibitor of SAMHD1. SOX11 is aberrantly expressed not only in mantle cell lymphoma (MCL), but also in some Burkitt lymphomas. Co-immunoprecipitation of SOX11 followed by mass spectrometry in MCL cell lines identified SAMHD1 as the top SOX11 interaction partner which was validated by proximity ligation assay. In vitro, SAMHD1 bound to the HMG box of SOX11 with low-micromolar affinity. In situ crosslinking studies further indicated that SOX11-SAMHD1 binding resulted in a reduced tetramerization of SAMHD1. Functionally, expression of SOX11 inhibited SAMHD1 ara-CTPase activity in a dose-dependent manner resulting in ara-C sensitization in cell lines and in a SOX11-inducible mouse model of MCL. In SOX11-negative MCL, SOX11-mediated ara-CTPase inhibition could be mimicked by adding the recently identified SAMHD1 inhibitor hydroxyurea. Taken together, our results identify SOX11 as a novel SAMHD1 interaction partner and its first known endogenous inhibitor with potentially important implications for clinical therapy stratification.

SOX11 expression is restricted to EBV-negative Burkitt lymphoma and associates with molecular genetic features.

SRY-related HMG-box gene 11 (SOX11) is a transcription factor overexpressed in mantle cell lymphoma (MCL), a subset of Burkitt lymphomas (BL) and precursor lymphoid cell neoplasms but is absent in normal B-cells and other B-cell lymphomas. SOX11 has an oncogenic role in MCL but its contribution to BL pathogenesis remains uncertain. Here, we observed that the presence of Epstein-Barr virus (EBV) and SOX11 expression were mutually exclusive in BL. SOX11 expression in EBV- BL was associated with an IG∷MYC translocation generated by aberrant class switch recombination, while in EBV-/SOX11- tumors the IG∷MYC translocation was mediated by mistaken somatic hypermutations. Interestingly, EBV- SOX11 expressing BL showed higher frequency of SMARCA4 and ID3 mutations compared to EBV-/SOX11- cases. By RNA-sequencing, we identified a SOX11-associated gene expression profile, with functional annotations showing partial overlap with the SOX11 transcriptional program of MCL. Contrary to MCL, no differences on cell migration or BCR signaling were found between SOX11- and SOX11+ BL cells. However, SOX11+ BL showed higher adhesion to VCAM-1 than SOX11- BL cell lines. Here we demonstrate that EBV- BL comprises two subsets of cases based on SOX11 expression. The mutual exclusion of SOX11 and EBV, and the association of SOX11 with a specific genetic landscape suggest a role of SOX11 in the early pathogenesis of BL.

Motive and Opportunity: MYC rearrangements in high-grade B-cell lymphoma with MYC and BCL2 rearrangements-an LLMPP study.

Rearrangements that place the oncogenes MYC, BCL2, or BCL6 adjacent to superenhancers are common in mature B-cell lymphomas. Lymphomas with diffuse large B-cell lymphoma (DLBCL) or high-grade morphology with both MYC and BCL2 rearrangements are classified as high-grade B-cell lymphoma with MYC and BCL2 rearrangements ("double hit": HGBCL-DH-BCL2) and are associated with aggressive disease and poor outcomes. Although it is established that MYC rearrangements involving immunoglobulin (IG) loci are associated with inferior outcomes relative to those involving other non-IG superenhancers, the frequency of, and mechanisms driving, IG vs non-IG MYC rearrangements have not been elucidated. Here we used custom targeted capture and/or whole genome sequencing to characterize oncogene rearrangements across 883 mature B-cell lymphomas including Burkitt lymphoma, follicular lymphoma, DLBCL, and HGBCL-DH-BCL2 tumors. We demonstrate that, while BCL2 rearrangement topology is consistent across entities, HGBCL-DH-BCL2 have distinct MYC rearrangement architecture relative to tumors with single MYC rearrangements or with both MYC and BCL6 rearrangements (HGBCL-DH-BCL6), including both a higher frequency of non-IG rearrangements and different architecture of MYC::IGH rearrangements. The distinct MYC rearrangement patterns in HGBCL-DH-BCL2 occur on the background of high levels of somatic hypermutation across MYC partner loci in HGBCL-DH-BCL2, creating more opportunity to form these rearrangements. Furthermore, because one IGH allele is already disrupted by the existing BCL2 rearrangement, the MYC rearrangement architecture in HGBCL-DH-BCL2 likely reflects selective pressure to preserve both BCL2 and B cell receptor expression. These data provide new mechanistic explanations for the distinct patterns of MYC rearrangements observed across different lymphoma entities.

B-cell non-Hodgkin lymphomas.

B-cell lymphomas occur with an incidence of 20 new cases per 100 000 people per year in high-income countries. They can affect any organ and are characterised by heterogeneous clinical presentations and courses, varying from asymptomatic, to indolent, to very aggressive cases. Since the topic of B-cell non-Hodgkin lymphomas was last reviewed in The Lancet in 2017, a deeper understanding of the biological background of this heterogeneous group of malignancies, the availability of new diagnostic methods, and the development and implementation of new targeted and immunotherapeutic approaches have improved our ability to treat patients. This Seminar provides an overview of the pathobiology, classification, and prognostication of B-cell non-Hodgkin lymphomas and summarises the current knowledge and standard of care regarding biology and clinical management of the most common subtypes of mature B-cell non-Hodgkin lymphomas. It also highlights new findings in deciphering the molecular background of disease development and the implementation of new therapeutic approaches, particularly those targeting the immune system.

Chromatin activation profiling of stereotyped chronic lymphocytic leukemias reveals a subset 8-specific signature.

The chromatin activation landscape of chronic lymphocytic leukemia (CLL) with stereotyped B-cell receptor immunoglobulin is currently unknown. In this study, we report the results of a whole-genome chromatin profiling of histone 3 lysine 27 acetylation of 22 CLLs from major subsets, which were compared against nonstereotyped CLLs and normal B-cell subpopulations. Although subsets 1, 2, and 4 did not differ much from their nonstereotyped CLL counterparts, subset 8 displayed a remarkably distinct chromatin activation profile. In particular, we identified 209 de novo active regulatory elements in this subset, which showed similar patterns with U-CLLs undergoing Richter transformation. These regions were enriched for binding sites of 9 overexpressed transcription factors. In 78 of 209 regions, we identified 113 candidate overexpressed target genes, 11 regions being associated with more than 2 adjacent genes. These included blocks of up to 7 genes, suggesting local coupregulation within the same genome compartment. Our findings further underscore the uniqueness of subset 8 CLL, notable for the highest risk of Richter's transformation among all CLLs and provide additional clues to decipher the molecular basis of its clinical behavior.

Decoding the molecular heterogeneity of pediatric monomorphic post-solid organ transplant lymphoproliferative disorders.

Posttransplant lymphoproliferative disorders (PTLDs) represent a broad spectrum of lymphoid proliferations, frequently associated with Epstein-Barr virus (EBV) infection. The molecular profile of pediatric monomorphic PTLDs (mPTLDs) has not been elucidated, and it is unknown whether they display similar genetic features as their counterpart in adult and immunocompetent (IMC) pediatric patients. In this study, we investigated 31 cases of pediatric mPTLD after solid organ transplantation, including 24 diffuse large B-cell lymphomas (DLBCLs), mostly classified as activated B cell, and 7 cases of Burkitt lymphoma (BL), 93% of which were EBV positive. We performed an integrated molecular approach, including fluorescence in situ hybridization, targeted gene sequencing, and copy number (CN) arrays. Overall, PTLD-BL carried mutations in MYC, ID3, DDX3X, ARID1A, or CCND3 resembling IMC-BL, higher mutational burden than PTLD-DLBCL, and lesser CN alterations than IMC-BL. PTLD-DLBCL showed a very heterogeneous genomic profile with fewer mutations and CN alterations than IMC-DLBCL. Epigenetic modifiers and genes of the Notch pathway were the most recurrently mutated in PTLD-DLBCL (both 28%). Mutations in cell cycle and Notch pathways correlated with a worse outcome. All 7 patients with PTLD-BL were alive after treatment with pediatric B-cell non-Hodgkin lymphoma protocols, whereas 54% of patients with DLBCL were cured with immunosuppression reduction, rituximab, and/or low-dose chemotherapy. These findings highlight the low complexity of pediatric PTLD-DLBCL, their good response to low-intensity treatment, and the shared pathogenesis between PTLD-BL and EBV-positive IMC-BL. We also suggest new potential parameters that could help in the diagnosis and the design of better therapeutic strategies for these patients.

The U1 spliceosomal RNA is recurrently mutated in multiple cancers.

Cancers are caused by genomic alterations known as drivers. Hundreds of drivers in coding genes are known but, to date, only a handful of noncoding drivers have been discovered-despite intensive searching1,2. Attention has recently shifted to the role of altered RNA splicing in cancer; driver mutations that lead to transcriptome-wide aberrant splicing have been identified in multiple types of cancer, although these mutations have only been found in protein-coding splicing factors such as splicing factor 3b subunit 1 (SF3B1)3-6. By contrast, cancer-related alterations in the noncoding component of the spliceosome-a series of small nuclear RNAs (snRNAs)-have barely been studied, owing to the combined challenges of characterizing noncoding cancer drivers and the repetitive nature of snRNA genes1,7,8. Here we report a highly recurrent A>C somatic mutation at the third base of U1 snRNA in several types of tumour. The primary function of U1 snRNA is to recognize the 5' splice site via base-pairing. This mutation changes the preferential A-U base-pairing between U1 snRNA and the 5' splice site to C-G base-pairing, and thus creates novel splice junctions and alters the splicing pattern of multiple genes-including known drivers of cancer. Clinically, the A>C mutation is associated with heavy alcohol use in patients with hepatocellular carcinoma, and with the aggressive subtype of chronic lymphocytic leukaemia with unmutated immunoglobulin heavy-chain variable regions. The mutation in U1 snRNA also independently confers an adverse prognosis to patients with chronic lymphocytic leukaemia. Our study demonstrates a noncoding driver in spliceosomal RNAs, reveals a mechanism of aberrant splicing in cancer and may represent a new target for treatment. Our findings also suggest that driver discovery should be extended to a wider range of genomic regions.

Evaluation of clinical parameters and biomarkers in older, untreated mantle cell lymphoma patients receiving bendamustine-rituximab.

Mantle cell lymphoma (MCL) is clinically and biologically heterogeneous. While various prognostic features have been proposed, none currently impact therapy selection, particularly in older patients, for whom treatment is primarily dictated by age and comorbidities. Herein, we undertook a comprehensive comparison of clinicopathological features in a cohort of patients 60 years and older, uniformly treated with bendamustine and rituximab, with a median survival of >8 years. The strongest prognostic indicators in this cohort were a high-risk call by a simplified MCL international prognostic index (s-MIPI) (HR: 3.32, 95% CI: 1.65-6.68 compared to low risk), a high-risk call by MCL35 (HR: 10.34, 95% CI: 2.37-45.20 compared to low risk) and blastoid cytology (HR: 4.21, 95% CR: 1.92-9.22 compared to classic). Patients called high risk by both the s-MIPI and MCL35 had the most dismal prognosis (HR: 11.58, 95% CI: 4.10-32.72), while those with high risk by either had a moderate but clinically relevant prognosis (HR: 2.95, 95% CI: 1.49-5.82). A robust assay to assess proliferation, such as MCL35, along with stringent guidelines for cytological evaluation of MCL, in combination with MIPI, may be a strong path to risk-stratify older MCL patients in future clinical trials.

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.

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.

BCL3 rearrangements in B-cell lymphoid neoplasms occur in two breakpoint clusters associated with different diseases.

The t(14;19)(q32;q13) often juxtaposes BCL3 with immunoglobulin heavy chain (IGH) resulting in overexpression of the gene. In contrast to other oncogenic translocations, BCL3 rearrangement (BCL3-R) has been associated with a broad spectrum of lymphoid neoplasms. Here we report an integrative whole-genome sequence, transcriptomic, and DNA methylation analysis of 13 lymphoid neoplasms with BCL3-R. The resolution of the breakpoints at single base-pair revealed that they occur in two clusters at 5' (n=9) and 3' (n=4) regions of BCL3 associated with two different biological and clinical entities. Both breakpoints were mediated by aberrant class switch recombination of the IGH locus. However, the 5' breakpoints (upstream) juxtaposed BCL3 next to an IGH enhancer leading to overexpression of the gene whereas the 3' breakpoints (downstream) positioned BCL3 outside the influence of the IGH and were not associated with its expression. Upstream BCL3-R tumors had unmutated IGHV, trisomy 12, and mutated genes frequently seen in chronic lymphocytic leukemia (CLL) but had an atypical CLL morphology, immunophenotype, DNA methylome, and expression profile that differ from conventional CLL. In contrast, downstream BCL3-R neoplasms were atypical splenic or nodal marginal zone lymphomas (MZL) with mutated IGHV, complex karyotypes and mutated genes typical of MZL. Two of the latter four tumors transformed to a large B-cell lymphoma. We designed a novel fluorescence in situ hybridization assay that recognizes the two different breakpoints and validated these findings in 17 independent tumors. Overall, upstream or downstream breakpoints of BCL3-R are mainly associated with two subtypes of lymphoid neoplasms with different (epi)genomic, expression, and clinicopathological features resembling atypical CLL and MZL, respectively.

A low total metabolic tumor volume independently predicts for a longer time to first treatment in initially observed, low tumor burden follicular lymphoma.

Watchful waiting is an acceptable management strategy for advanced-stage, low tumor burden (LTB) patients with follicular lymphoma (FL). However, the prediction of how long this treatment-free observation period will last remains imperfect. We explored whether total metabolic tumor volume (TMTV) and other positron emission tomography parameters were predictive of time to first treatment (TTFT). We analyzed 97 grade 1-3A advanced-stage LTB FL patients and found that a high TMTV was associated with other tumor burden features at diagnosis. Patients with a TMTV above our established cutoff of 50 mL had a significantly shorter median duration of observation (2.6 vs. 8.8 years; p = 0.001). At 5 years, 77% of patients with a high TMTV and 46% of patients with a low TMTV required treatment. In the multivariable analysis, a high TMTV was the only independent factor predicting TTFT (hazard ratio = 2.09; p = 0.017). Overall, TMTV is a strong predictor of the duration of observation in LTB FL patients. Upon validation of our cutoff in external series and standardization of the methodology, the TMTV could become an additional factor to consider deferring or initiating treatment in otherwise LTB patients.

A multiprotein supercomplex controlling oncogenic signalling in lymphoma.

B cell receptor (BCR) signalling has emerged as a therapeutic target in B cell lymphomas, but inhibiting this pathway in diffuse large B cell lymphoma (DLBCL) has benefited only a subset of patients1. Gene expression profiling identified two major subtypes of DLBCL, known as germinal centre B cell-like and activated B cell-like (ABC)2,3, that show poor outcomes after immunochemotherapy in ABC. Autoantigens drive BCR-dependent activation of NF-κB in ABC DLBCL through a kinase signalling cascade of SYK, BTK and PKCß to promote the assembly of the CARD11-BCL10-MALT1 adaptor complex, which recruits and activates IκB kinase4-6. Genome sequencing revealed gain-of-function mutations that target the CD79A and CD79B BCR subunits and the Toll-like receptor signalling adaptor MYD885,7, with MYD88(L265P) being the most prevalent isoform. In a clinical trial, the BTK inhibitor ibrutinib produced responses in 37% of cases of ABC1. The most striking response rate (80%) was observed in tumours with both CD79B and MYD88(L265P) mutations, but how these mutations cooperate to promote dependence on BCR signalling remains unclear. Here we used genome-wide CRISPR-Cas9 screening and functional proteomics to determine the molecular basis of exceptional clinical responses to ibrutinib. We discovered a new mode of oncogenic BCR signalling in ibrutinib-responsive cell lines and biopsies, coordinated by a multiprotein supercomplex formed by MYD88, TLR9 and the BCR (hereafter termed the My-T-BCR supercomplex). The My-T-BCR supercomplex co-localizes with mTOR on endolysosomes, where it drives pro-survival NF-κB and mTOR signalling. Inhibitors of BCR and mTOR signalling cooperatively decreased the formation and function of the My-T-BCR supercomplex, providing mechanistic insight into their synergistic toxicity for My-T-BCR+ DLBCL cells. My-T-BCR supercomplexes characterized ibrutinib-responsive malignancies and distinguished ibrutinib responders from non-responders. Our data provide a framework for the rational design of oncogenic signalling inhibitors in molecularly defined subsets of DLBCL.

Chromatin activation as a unifying principle underlying pathogenic mechanisms in multiple myeloma.

Multiple myeloma (MM) is a plasma cell neoplasm associated with a broad variety of genetic lesions. In spite of this genetic heterogeneity, MMs share a characteristic malignant phenotype whose underlying molecular basis remains poorly characterized. In the present study, we examined plasma cells from MM using a multi-epigenomics approach and demonstrated that, when compared to normal B cells, malignant plasma cells showed an extensive activation of regulatory elements, in part affecting coregulated adjacent genes. Among target genes up-regulated by this process, we found members of the NOTCH, NF-kB, MTOR signaling, and TP53 signaling pathways. Other activated genes included sets involved in osteoblast differentiation and response to oxidative stress, all of which have been shown to be associated with the MM phenotype and clinical behavior. We functionally characterized MM-specific active distant enhancers controlling the expression of thioredoxin (TXN), a major regulator of cellular redox status and, in addition, identified PRDM5 as a novel essential gene for MM. Collectively, our data indicate that aberrant chromatin activation is a unifying feature underlying the malignant plasma cell phenotype.

IGLV3-21R110 identifies an aggressive biological subtype of chronic lymphocytic leukemia with intermediate epigenetics.

B-cell receptor (BCR) signaling is crucial for chronic lymphocytic leukemia (CLL) biology. IGLV3-21-expressing B cells may acquire a single point mutation (R110) that triggers autonomous BCR signaling, conferring aggressive behavior. Epigenetic studies have defined 3 CLL subtypes based on methylation signatures reminiscent of naïve-like (n-CLL), intermediate (i-CLL), and memory-like (m-CLL) B cells with different biological features. i-CLL carries a borderline IGHV mutational load and significantly higher use of IGHV3-21/IGLV3-21. To determine the clinical and biological features of IGLV3-21R110 CLL and its relationship to these epigenetic subtypes, we characterized the immunoglobulin gene of 584 CLL cases using whole-genome/exome and RNA sequencing. IGLV3-21R110 was detected in 6.5% of cases: 30 (38%) of 79 i-CLLs, 5 (1.7%) of 291 m-CLLs, and 1 (0.5%) of 189 n-CLLs. All stereotype subset 2 cases carried IGLV3-21R110, whereas 62% of IGLV3-21R110 i-CLL cases had nonstereotyped BCR immunoglobulins. IGLV3-21R110 i-CLL had a significantly higher number of SF3B1 and ATM mutations and total number of driver alterations. However, the R110 mutation was the sole alteration in 1 i-CLL and was accompanied only by del(13q) in 3. Although IGHV mutational status varied, IGLV3-21R110 i-CLL transcriptomically resembled n-CLL/unmutated IGHV CLL with a specific signature including WNT5A/B overexpression. In contrast, i-CLL lacking IGLV3-21R110 mirrored m-CLL/mutated IGHV. Patients with IGLV3-21R110 i-CLL had a short time to first treatment and overall survival similar to those of n-CLL/unmutated IGHV patients, whereas patients with non-IGLV3-21R110 i-CLL had a good prognosis similar to that of patients with m-CLL/mutated IGHV. IGLV3-21R110 defines a CLL subgroup with specific biological features and an unfavorable prognosis independent of IGHV mutational status and epigenetic subtype.

The protein landscape of chronic lymphocytic leukemia.

Many functional consequences of mutations on tumor phenotypes in chronic lymphocytic leukemia (CLL) are unknown. This may be in part due to a scarcity of information on the proteome of CLL. We profiled the proteome of 117 CLL patient samples with data-independent acquisition mass spectrometry and integrated the results with genomic, transcriptomic, ex vivo drug response, and clinical outcome data. We found trisomy 12, IGHV mutational status, mutated SF3B1, trisomy 19, del(17)(p13), del(11)(q22.3), mutated DDX3X and MED12 to influence protein expression (false discovery rate [FDR] = 5%). Trisomy 12 and IGHV status were the major determinants of protein expression variation in CLL as shown by principal-component analysis (1055 and 542 differentially expressed proteins, FDR = 5%). Gene set enrichment analyses of CLL with trisomy 12 implicated B-cell receptor (BCR)/phosphatidylinositol 3-kinase (PI3K)/AKT signaling as a tumor driver. These findings were supported by analyses of protein abundance buffering and protein complex formation, which identified limited protein abundance buffering and an upregulated protein complex involved in BCR, AKT, MAPK, and PI3K signaling in trisomy 12 CLL. A survey of proteins associated with trisomy 12/IGHV-independent drug response linked STAT2 protein expression with response to kinase inhibitors, including Bruton tyrosine kinase and mitogen-activated protein kinase kinase (MEK) inhibitors. STAT2 was upregulated in unmutated IGHV CLL and trisomy 12 CLL and required for chemokine/cytokine signaling (interferon response). This study highlights the importance of protein abundance data as a nonredundant layer of information in tumor biology and provides a protein expression reference map for CLL.