D-2HG comes out of its shell: Metabolic effects on the immune environment.

A recent study by Notarangelo et al.1 highlights the potential for tumor-derived D-2HG to inhibit neighboring T cell function through a novel mechanism.

Coordinated alterations in RNA splicing and epigenetic regulation drive leukaemogenesis.

Transcription and pre-mRNA splicing are key steps in the control of gene expression and mutations in genes regulating each of these processes are common in leukaemia1,2. Despite the frequent overlap of mutations affecting epigenetic regulation and splicing in leukaemia, how these processes influence one another to promote leukaemogenesis is not understood and, to our knowledge, there is no functional evidence that mutations in RNA splicing factors initiate leukaemia. Here, through analyses of transcriptomes from 982 patients with acute myeloid leukaemia, we identified frequent overlap of mutations in IDH2 and SRSF2 that together promote leukaemogenesis through coordinated effects on the epigenome and RNA splicing. Whereas mutations in either IDH2 or SRSF2 imparted distinct splicing changes, co-expression of mutant IDH2 altered the splicing effects of mutant SRSF2 and resulted in more profound splicing changes than either mutation alone. Consistent with this, co-expression of mutant IDH2 and SRSF2 resulted in lethal myelodysplasia with proliferative features in vivo and enhanced self-renewal in a manner not observed with either mutation alone. IDH2 and SRSF2 double-mutant cells exhibited aberrant splicing and reduced expression of INTS3, a member of the integrator complex3, concordant with increased stalling of RNA polymerase II (RNAPII). Aberrant INTS3 splicing contributed to leukaemogenesis in concert with mutant IDH2 and was dependent on mutant SRSF2 binding to cis elements in INTS3 mRNA and increased DNA methylation of INTS3. These data identify a pathogenic crosstalk between altered epigenetic state and splicing in a subset of leukaemias, provide functional evidence that mutations in splicing factors drive myeloid malignancy development, and identify spliceosomal changes as a mediator of IDH2-mutant leukaemogenesis.

Rituximab and lenalidomide for the treatment of relapsed or refractory indolent non-Hodgkin lymphoma: real-life experience.

The combination of rituximab and lenalidomide (R-len) stands as an established treatment for relapsed/refractory (R/R) indolent non-Hodgkin lymphoma (iNHL). However, the reproducibility of clinical trial results in routine clinical practice is unknown. To address this gap in knowledge, we reviewed our experience with patients diagnosed with R/R follicular lymphoma (FL) or marginal zone lymphoma (MZL) treated with this combination. Eighty-four patients underwent treatment with R-len, 69 (82%) affected by FL and 15 (18%) by MZL. The median age at the time of treatment initiation was 65 years (range, 39-94), 38 patients (45%) had a pre-treatment FLIPI score of 3-5, 19 (23%) had a bulky disease, 29 (37%) had a lymphoma refractory to the last treatment line, while in 20 (24%) cases the disease was refractory to rituximab. The best overall response rate (ORR) was 82%, and 52% achieved a complete response (CR). The best CR rates for FL and MZL patients were 55% and 40%, respectively. With a median follow-up of 22 months, the median progression-free survival (mPFS) was 22 months (95% CI 19-36) and the 2-year overall survival (OS) was 83% (95% CI 74-93). The median duration of CR (DoCR) was 46 months (95% CI 22-NR). Factors associated with shorter PFS in multivariate analysis were bulky disease and rituximab refractoriness. The most common adverse events (AE) included hematologic toxicity, fatigue and gastrointestinal disorders, such as diarrhea and constipation. Neutropenia and thrombocytopenia were the most common severe toxicities (grade ≥3 in 25% and 4%, respectively). No new safety signals were reported. Real-life results of R-len in patients with R/R iNHL appear consistent with those reported in prospective studies, and further support its use as comparator arm in controlled clinical trials.

GCN2 kinase activation by ATP-competitive kinase inhibitors.

Small-molecule kinase inhibitors represent a major group of cancer therapeutics, but tumor responses are often incomplete. To identify pathways that modulate kinase inhibitor response, we conducted a genome-wide knockout (KO) screen in glioblastoma cells treated with the pan-ErbB inhibitor neratinib. Loss of general control nonderepressible 2 (GCN2) kinase rendered cells resistant to neratinib, whereas depletion of the GADD34 phosphatase increased neratinib sensitivity. Loss of GCN2 conferred neratinib resistance by preventing binding and activation of GCN2 by neratinib. Several other Food and Drug Administration (FDA)-approved inhibitors, such erlotinib and sunitinib, also bound and activated GCN2. Our results highlight the utility of genome-wide functional screens to uncover novel mechanisms of drug action and document the role of the integrated stress response (ISR) in modulating the response to inhibitors of oncogenic kinases.

Acquired resistance to IDH inhibition through trans or cis dimer-interface mutations.

Somatic mutations in the isocitrate dehydrogenase 2 gene (IDH2) contribute to the pathogenesis of acute myeloid leukaemia (AML) through the production of the oncometabolite 2-hydroxyglutarate (2HG)1-8. Enasidenib (AG-221) is an allosteric inhibitor that binds to the IDH2 dimer interface and blocks the production of 2HG by IDH2 mutants9,10. In a phase I/II clinical trial, enasidenib inhibited the production of 2HG and induced clinical responses in relapsed or refractory IDH2-mutant AML11. Here we describe two patients with IDH2-mutant AML who had a clinical response to enasidenib followed by clinical resistance, disease progression, and a recurrent increase in circulating levels of 2HG. We show that therapeutic resistance is associated with the emergence of second-site IDH2 mutations in trans, such that the resistance mutations occurred in the IDH2 allele without the neomorphic R140Q mutation. The in trans mutations occurred at glutamine 316 (Q316E) and isoleucine 319 (I319M), which are at the interface where enasidenib binds to the IDH2 dimer. The expression of either of these mutant disease alleles alone did not induce the production of 2HG; however, the expression of the Q316E or I319M mutation together with the R140Q mutation in trans allowed 2HG production that was resistant to inhibition by enasidenib. Biochemical studies predicted that resistance to allosteric IDH inhibitors could also occur via IDH dimer-interface mutations in cis, which was confirmed in a patient with acquired resistance to the IDH1 inhibitor ivosidenib (AG-120). Our observations uncover a mechanism of acquired resistance to a targeted therapy and underscore the importance of 2HG production in the pathogenesis of IDH-mutant malignancies.

Transcription factor T-bet represses expression of the inhibitory receptor PD-1 and sustains virus-specific CD8+ T cell responses during chronic infection.

T cell exhaustion has a major role in failure to control chronic infection. High expression of inhibitory receptors, including PD-1, and the inability to sustain functional T cell responses contribute to exhaustion. However, the transcriptional control of these processes remains unclear. Here we demonstrate that the transcription factor T-bet regulated the exhaustion of CD8(+) T cells and the expression of inhibitory receptors. T-bet directly repressed transcription of the gene encoding PD-1 and resulted in lower expression of other inhibitory receptors. Although a greater abundance of T-bet promoted terminal differentiation after acute infection, high T-bet expression sustained exhausted CD8(+) T cells and repressed the expression of inhibitory receptors during chronic viral infection. Persistent antigenic stimulation caused downregulation of T-bet, which resulted in more severe exhaustion of CD8(+) T cells. Our observations suggest therapeutic opportunities involving higher T-bet expression during chronic infection.

FXR mediates T cell-intrinsic responses to reduced feeding during infection.

Reduced nutrient intake is a widely conserved manifestation of sickness behavior with poorly characterized effects on adaptive immune responses. During infectious challenges, naive T cells encountering their cognate antigen become activated and differentiate into highly proliferative effector T cells. Despite their evident metabolic shift upon activation, it remains unclear how effector T cells respond to changes in nutrient availability in vivo. Here, we show that spontaneous or imposed feeding reduction during infection decreases the numbers of splenic lymphocytes. Effector T cells showed cell-intrinsic responses dependent on the nuclear receptor Farnesoid X Receptor (FXR). Deletion of FXR in T cells prevented starvation-induced loss of lymphocytes and increased effector T cell fitness in nutrient-limiting conditions, but imparted greater weight loss to the host. FXR deficiency increased the contribution of glutamine and fatty acids toward respiration and enhanced cell survival under low-glucose conditions. Provision of glucose during anorexia of infection rescued effector T cells, suggesting that this sugar is a limiting nutrient for activated lymphocytes and that alternative fuel usage may affect cell survival in starved animals. Altogether, we identified a mechanism by which the host scales immune responses according to food intake, featuring FXR as a T cell-intrinsic sensor.

Discovery of Protein Modifications Using Differential Tandem Mass Spectrometry Proteomics.

Recent studies have revealed diverse amino acid, post-translational, and noncanonical modifications of proteins in diverse organisms and tissues. However, their unbiased detection and analysis remain hindered by technical limitations. Here, we present a spectral alignment method for the identification of protein modifications using high-resolution mass spectrometry proteomics. Termed SAMPEI for spectral alignment-based modified peptide identification, this open-source algorithm is designed for the discovery of functional protein and peptide signaling modifications, without prior knowledge of their identities. Using synthetic standards and controlled chemical labeling experiments, we demonstrate its high specificity and sensitivity for the discovery of substoichiometric protein modifications in complex cellular extracts. SAMPEI mapping of mouse macrophage differentiation revealed diverse post-translational protein modifications, including distinct forms of cysteine itaconatylation. SAMPEI's robust parametrization and versatility are expected to facilitate the discovery of biological modifications of diverse macromolecules. SAMPEI is implemented as a Python package and is available open-source from BioConda and GitHub (https://github.com/FenyoLab/SAMPEI).

Induction of sarcomas by mutant IDH2.

More than 50% of patients with chondrosarcomas exhibit gain-of-function mutations in either isocitrate dehydrogenase 1 (IDH1) or IDH2. In this study, we performed genome-wide CpG methylation sequencing of chondrosarcoma biopsies and found that IDH mutations were associated with DNA hypermethylation at CpG islands but not other genomic regions. Regions of CpG island hypermethylation were enriched for genes implicated in stem cell maintenance/differentiation and lineage specification. In murine 10T1/2 mesenchymal progenitor cells, expression of mutant IDH2 led to DNA hypermethylation and an impairment in differentiation that could be reversed by treatment with DNA-hypomethylating agents. Introduction of mutant IDH2 also induced loss of contact inhibition and generated undifferentiated sarcomas in vivo. The oncogenic potential of mutant IDH2 correlated with the ability to produce 2-hydroxyglutarate. Together, these data demonstrate that neomorphic IDH2 mutations can be oncogenic in mesenchymal cells.

Using genomic data for selecting the treatment of lymphoma patients.

PURPOSE OF REVIEW: Genomic profiling platforms provide unprecedented genetic information of lymphoma biology, yet information has yet to be readily integrated into clinical medicine. This review summarizes the important concepts of utilizing genomics to aide disease management. RECENT FINDINGS: A wide range of clinical grade genetic sequencing platforms are available, therefore the selection of sequencing platform should ideally be based on biological and clinical questions, as well as the strength and weaknesses of individual platform. Different evidence-based guidelines exist to aide clinical judgment; however, few have well curated, easy to search platforms. Using one guideline proposed by several regulatory groups, our review summarizes genetic alterations with diagnostic, prognostic and therapeutic potential in the major subtypes of lymphoma. SUMMARY: A comprehensive database of genetic alterations that contribute to clinical care in lymphoma is needed. Ideally, a database which accounts for single and pathway-based genetic alterations may be developed to guide development and interventions for management of lymphoma.

L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH.

The metabolite 2-hydroxyglutarate (2HG) can be produced as either a D-R- or L-S- enantiomer, each of which inhibits α-ketoglutarate (αKG)-dependent enzymes involved in diverse biologic processes. Oncogenic mutations in isocitrate dehydrogenase (IDH) produce D-2HG, which causes a pathologic blockade in cell differentiation. On the other hand, oxygen limitation leads to accumulation of L-2HG, which can facilitate physiologic adaptation to hypoxic stress in both normal and malignant cells. Here we demonstrate that purified lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) catalyze stereospecific production of L-2HG via 'promiscuous' reduction of the alternative substrate αKG. Acidic pH enhances production of L-2HG by promoting a protonated form of αKG that binds to a key residue in the substrate-binding pocket of LDHA. Acid-enhanced production of L-2HG leads to stabilization of hypoxia-inducible factor 1 alpha (HIF-1α) in normoxia. These findings offer insights into mechanisms whereby microenvironmental factors influence production of metabolites that alter cell fate and function.

Integrated genomic DNA/RNA profiling of hematologic malignancies in the clinical setting.

The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs), and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded blood and bone marrow samples with high accuracy in a clinically relevant time frame, which is performed in our Clinical Laboratory Improvement Amendments-certified College of American Pathologists-accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs, and gene fusions, with similar accuracy to lower-throughput assays that focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis, and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically relevant genomic alterations with therapeutic relevance.

A role for the transcriptional repressor Blimp-1 in CD8(+) T cell exhaustion during chronic viral infection.

T cell exhaustion is common during chronic infections and can prevent optimal immunity. Although recent studies have demonstrated the importance of inhibitory receptors and other pathways in T cell exhaustion, the underlying transcriptional mechanisms are unknown. Here, we define a role for the transcription factor Blimp-1 in CD8(+) T cell exhaustion during chronic viral infection. Blimp-1 repressed key aspects of normal memory CD8(+) T cell differentiation and promoted high expression of inhibitory receptors during chronic infection. These cardinal features of CD8(+) T cell exhaustion were corrected by conditionally deleting Blimp-1. Although high expression of Blimp-1 fostered aspects of CD8(+) T cell exhaustion, haploinsufficiency indicated that moderate Blimp-1 expression sustained some effector function during chronic viral infection. Thus, we identify Blimp-1 as a transcriptional regulator of CD8(+) T cell exhaustion during chronic viral infection and propose that Blimp-1 acts as a transcriptional rheostat balancing effector function and T cell exhaustion.

Genomic and functional analysis of leukemic transformation of myeloproliferative neoplasms.

Patients with myeloproliferative neoplasms (MPNs) are at significant, cumulative risk of leukemic transformation to acute myeloid leukemia (AML), which is associated with adverse clinical outcome and resistance to standard AML therapies. We performed genomic profiling of post-MPN AML samples; these studies demonstrate somatic tumor protein 53 (TP53) mutations are common in JAK2V617F-mutant, post-MPN AML but not in chronic-phase MPN and lead to clonal dominance of JAK2V617F/TP53-mutant leukemic cells. Consistent with these data, expression of JAK2V617F combined with Tp53 loss led to fully penetrant AML in vivo. JAK2V617F-mutant, Tp53-deficient AML was characterized by an expanded megakaryocyte erythroid progenitor population that was able to propagate the disease in secondary recipients. In vitro studies revealed that post-MPN AML cells were sensitive to decitabine, the JAK1/2 inhibitor ruxolitinib, or the heat shock protein 90 inhibitor 8-(6-iodobenzo[d][1.3]dioxol-5-ylthio)-9-(3-(isopropylamino)propyl)-9H-purine-6-amine (PU-H71). Treatment with ruxolitinib or PU-H71 improved survival of mice engrafted with JAK2V617F-mutant, Tp53-deficient AML, demonstrating therapeutic efficacy for these targeted therapies and providing a rationale for testing these therapies in post-MPN AML.

Pre-TCR signaling inactivates Notch1 transcription by antagonizing E2A.

Precise control of the timing and magnitude of Notch signaling is essential for the normal development of many tissues, but the feedback loops that regulate Notch are poorly understood. Developing T cells provide an excellent context to address this issue. Notch1 signals initiate T-cell development and increase in intensity during maturation of early T-cell progenitors (ETP) to the DN3 stage. As DN3 cells undergo beta-selection, during which cells expressing functionally rearranged TCRbeta proliferate and differentiate into CD4(+)CD8(+) progeny, Notch1 signaling is abruptly down-regulated. In this report, we investigate the mechanisms that control Notch1 expression during thymopoiesis. We show that Notch1 and E2A directly regulate Notch1 transcription in pre-beta-selected thymocytes. Following successful beta-selection, pre-TCR signaling rapidly inhibits Notch1 transcription via signals that up-regulate Id3, an E2A inhibitor. Consistent with a regulatory role for Id3 in Notch1 down-regulation, post-beta-selected Id3-deficient thymocytes maintain Notch1 transcription, whereas enforced Id3 expression decreases Notch1 expression and abrogates Notch1-dependent T-cell survival. These data provide new insights into Notch1 regulation in T-cell progenitors and reveal a direct link between pre-TCR signaling and Notch1 expression during thymocyte development. Our findings also suggest new strategies for inhibiting Notch1 signaling in pathologic conditions.

CD8+ T cell metabolic flexibility elicited by CD28-ARS2 axis-driven alternative splicing of PKM supports antitumor immunity.

Metabolic flexibility has emerged as a critical determinant of CD8+ T-cell antitumor activity, yet the mechanisms driving the metabolic flexibility of T cells have not been determined. In this study, we investigated the influence of the nuclear cap-binding complex (CBC) adaptor protein ARS2 on mature T cells. In doing so, we discovered a novel signaling axis that endows activated CD8+ T cells with flexibility of glucose catabolism. ARS2 upregulation driven by CD28 signaling reinforced splicing factor recruitment to pre-mRNAs and affected approximately one-third of T-cell activation-induced alternative splicing events. Among these effects, the CD28-ARS2 axis suppressed the expression of the M1 isoform of pyruvate kinase in favor of PKM2, a key determinant of CD8+ T-cell glucose utilization, interferon gamma production, and antitumor effector function. Importantly, PKM alternative splicing occurred independently of CD28-driven PI3K pathway activation, revealing a novel means by which costimulation reprograms glucose metabolism in CD8+ T cells.

Expression of the transcription factor cKrox in peripheral CD8 T cells reveals substantial postthymic plasticity in CD4-CD8 lineage differentiation.

Most T cells belong to either of two lineages defined by the mutually exclusive expression of CD4 and CD8 coreceptors: CD4 T cells are major histocompatibility complex (MHC) II restricted and have helper function, whereas CD8 T cells are MHC I restricted and have cytotoxic function. The divergence between these two lineages occurs during intrathymic selection and is thought to be irreversible in mature T cells. It is, however, unclear whether the CD4-CD8 differentiation of postthymic T cells retains some level of plasticity or is stably maintained by mechanisms distinct from those that set lineage choice in the thymus. To address this issue, we examined if coreceptor or effector gene expression in mature CD8 T cells remains sensitive to the zinc finger transcription factor cKrox, which promotes CD4 and inhibits CD8 differentiation when expressed in thymocytes. We show that cKrox transduction into CD8 T cells inhibits their expression of CD8 and cytotoxic effector genes and impairs their cytotoxic activity, and that it promotes expression of helper-specific genes, although not of CD4 itself. These observations reveal a persistent degree of plasticity in CD4-CD8 differentiation in mature T cells.

Requirement for T-bet in the aberrant differentiation of unhelped memory CD8+ T cells.

Immunity to intracellular pathogens requires dynamic balance between terminal differentiation of short-lived, cytotoxic effector CD8+ T cells and self-renewal of central-memory CD8+ T cells. We now show that T-bet represses transcription of IL-7Ralpha and drives differentiation of effector and effector-memory CD8+ T cells at the expense of central-memory cells. We also found T-bet to be overexpressed in CD8+ T cells that differentiated in the absence of CD4+ T cell help, a condition that is associated with defective central-memory formation. Finally, deletion of T-bet corrected the abnormal phenotypic and functional properties of "unhelped" memory CD8+ T cells. T-bet, thus, appears to function as a molecular switch between central- and effector-memory cell differentiation. Antagonism of T-bet may, therefore, represent a novel strategy to offset dysfunctional programming of memory CD8+ T cells.

A multimodal atlas of tumour metabolism reveals the architecture of gene-metabolite covariation.

Tumour metabolism is controlled by coordinated changes in metabolite abundance and gene expression, but simultaneous quantification of metabolites and transcripts in primary tissue is rare. To overcome this limitation and to study gene-metabolite covariation in cancer, we assemble the Cancer Atlas of Metabolic Profiles of metabolomic and transcriptomic data from 988 tumour and control specimens spanning 11 cancer types in published and newly generated datasets. Meta-analysis of the Cancer Atlas of Metabolic Profiles reveals two classes of gene-metabolite covariation that transcend cancer types. The first corresponds to gene-metabolite pairs engaged in direct enzyme-substrate interactions, identifying putative genes controlling metabolite pool sizes. A second class of gene-metabolite covariation represents a small number of hub metabolites, including quinolinate and nicotinamide adenine dinucleotide, which correlate to many genes specifically expressed in immune cell populations. These results provide evidence that gene-metabolite covariation in cellularly heterogeneous tissue arises, in part, from both mechanistic interactions between genes and metabolites, and from remodelling of the bulk metabolome in specific immune microenvironments.