Germline risk of clonal haematopoiesis.

Clonal haematopoiesis (CH) is a common, age-related expansion of blood cells with somatic mutations that is associated with an increased risk of haematological malignancies, cardiovascular disease and all-cause mortality. CH may be caused by point mutations in genes associated with myeloid neoplasms, chromosomal copy number changes and loss of heterozygosity events. How inherited and environmental factors shape the incidence of CH is incompletely understood. Even though the several varieties of CH may have distinct phenotypic consequences, recent research points to an underlying genetic architecture that is highly overlapping. Moreover, there are numerous commonalities between the inherited variation associated with CH and that which has been linked to age-associated biomarkers and diseases. In this Review, we synthesize what is currently known about how inherited variation shapes the risk of CH and how this genetic architecture intersects with the biology of diseases that occur with ageing.

Molecular and clinical presentation of UBA1-mutated myelodysplastic syndromes.

Mutations in UBA1, which are disease-defining for VEXAS syndrome, have been reported in patients diagnosed with myelodysplastic syndromes (MDS). Here, we define the prevalence and clinical associations of UBA1 mutations in a representative cohort of patients with MDS. Digital droplet PCR profiling of a selected cohort of 375 male patients lacking MDS disease-defining mutations or established WHO disease classification identified 28 patients (7%) with UBA1 p.M41T/V/L mutations. Using targeted sequencing of UBA1 in a representative MDS cohort (n=2,027), we identified an additional 27 variants in 26 patients (1%), which we classified as likely/pathogenic (n=12) and unknown significance (n=15). Among the total 40 patients with likely/pathogenic variants (2%), all were male and 63% were classified by WHO2016 as MDS-MLD/SLD. Patients had a median of one additional myeloid gene mutation, often in TET2 (n=12), DNMT3A (n=10), ASXL1 (n=3), or SF3B1 (n=3). Retrospective clinical review where possible showed that 83% (28/34) UBA1-mutant cases had VEXAS-associated diagnoses or inflammatory clinical presentation. The prevalence of UBA1-mutations in MDS patients argues for systematic screening for UBA1 in the management of MDS.

Molecular Taxonomy of Myelodysplastic Syndromes and its Clinical Implications.

Myelodysplastic syndromes/neoplasms (MDS) are clonal hematologic disorders characterized by morphologic abnormalities of myeloid cells and peripheral cytopenias. While genetic abnormalities underlie the pathogenesis of these disorders and their heterogeneity, current classifications of MDS rely predominantly on morphology. We performed genomic profiling of 3,233 patients with MDS or related disorders to delineate molecular subtypes and define their clinical implications. Gene mutations, copy-number alterations (CNAs), and copy-neutral loss of heterozygosity (cnLOH) were derived from targeted sequencing of a 152-gene panel, with abnormalities identified in 91, 43, and 11% of patients, respectively. We characterized 16 molecular groups, encompassing 86% of patients, using information from 21 genes, 6 cytogenetic events, and LOH at the TP53 and TET2 loci. Two residual groups defined by negative findings (molecularly not-otherwise specified, absence of recurrent drivers) comprised 14% of patients. The groups varied in size from 0.5% to 14% of patients and were associated with distinct clinical phenotypes and outcomes. The median bone marrow blast percentage across groups ranged from 1.5 to 10%, and the median overall survival from 0.9 to 8.2 years. We validated 5 well-characterized entities, added further evidence to support 3 previously reported subsets, and described 8 novel groups. The prognostic influence of bone marrow blasts depended on the genetic subtypes. Within genetic subgroups, therapy-related MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) had comparable clinical and outcome profiles to primary MDS. In conclusion, genetically-derived subgroups of MDS are clinically relevant and may inform future classification schemas and translational therapeutic research.

Imetelstat in patients with lower-risk myelodysplastic syndromes who have relapsed or are refractory to erythropoiesis-stimulating agents (IMerge): a multinational, randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Unmet medical needs remain in patients with red blood cell transfusion-dependent (RBC-TD) lower-risk myelodysplastic syndromes (LR-MDS) who are not responding to or are ineligible for erythropoiesis-stimulating agents (ESAs). Imetelstat, a competitive telomerase inhibitor, showed promising results in a phase 2 trial. We aimed to compare the RBC transfusion independence (RBC-TI) rate with imetelstat versus placebo in patients with RBC-TD LR-MDS. METHODS: In phase 3 of IMerge, a double-blind, placebo-controlled trial conducted in 118 sites including university hospitals, cancer centres, and outpatient clinics in 17 countries, patients (aged ≥18 years) with ESA-relapsed, ESA-refractory, or ESA-ineligible LR-MDS (low or intermediate-1 risk disease as per International Prognostic Scoring System [IPSS] criteria) were randomly assigned via a computer-generated schedule (2:1) to receive imetelstat 7·5 mg/kg or placebo, administered as a 2-h intravenous infusion, every 4 weeks until disease progression, unacceptable toxic effects, or withdrawal of consent. Randomisation was stratified by previous RBC transfusion burden and IPSS risk group. Patients, investigators, and those analysing the data were masked to group assignment. The primary endpoint was 8-week RBC-TI, defined as the proportion of patients without RBC transfusions for at least 8 consecutive weeks starting on the day of randomisation until subsequent anti-cancer therapy, if any. Primary efficacy analyses were performed in the intention-to-treat population, and safety analyses were conducted in patients who received at least one dose of trial medication or placebo. This trial is registered with ClinicalTrials.gov (NCT02598661; substudy active and recruiting). FINDINGS: Between Sept 11, 2019, and Oct 13, 2021, 178 patients were enrolled and randomly assigned (118 to imetelstat and 60 to placebo). 111 (62%) were male and 67 (38%) were female. 91 (77%) of 118 patients had discontinued treatment by data cutoff in the imetelstat group versus 45 (75%) in the placebo group; a further one patient in the placebo group did not receive treatment. Median follow-up was 19·5 months (IQR 12·0-23·4) in the imetelstat group and 17·5 months (12·1-22·7) in the placebo group. In the imetelstat group, 47 (40% [95% CI 30·9-49·3]) patients had an RBC-TI of at least 8 weeks versus nine (15% [7·1-26·6]) in the placebo group (rate difference 25% [9·9 to 36·9]; p=0·0008). Overall, 107 (91%) of 118 patients receiving imetelstat and 28 (47%) of 59 patients receiving placebo had grade 3-4 treatment-emergent adverse events. The most common treatment-emergent grade 3-4 adverse events in patients taking imetelstat were neutropenia (80 [68%] patients who received imetelstat vs two [3%] who received placebo) and thrombocytopenia (73 [62%] vs five [8%]). No treatment-related deaths were reported. INTERPRETATION: Imetelstat offers a novel mechanism of action with durable transfusion independence (approximately 1 year) and disease-modifying activity for heavily transfused patients with LR-MDS who are not responding to or are ineligible for ESAs. FUNDING: Janssen Research & Development before April 18, 2019, and Geron Corporation thereafter.

Interactive network-based clustering and investigation of multimorbidity association matrices with associationSubgraphs.

MOTIVATION: Making sense of networked multivariate association patterns is vitally important to many areas of high-dimensional analysis. Unfortunately, as the data-space dimensions grow, the number of association pairs increases in O(n2); this means that traditional visualizations such as heatmaps quickly become too complicated to parse effectively. RESULTS: Here, we present associationSubgraphs: a new interactive visualization method to quickly and intuitively explore high-dimensional association datasets using network percolation and clustering. The goal is to provide an efficient investigation of association subgraphs, each containing a subset of variables with stronger and more frequent associations among themselves than the remaining variables outside the subset, by showing the entire clustering dynamics and providing subgraphs under all possible cutoff values at once. Particularly, we apply associationSubgraphs to a phenome-wide multimorbidity association matrix generated from an electronic health record and provide an online, interactive demonstration for exploring multimorbidity subgraphs. AVAILABILITY AND IMPLEMENTATION: An R package implementing both the algorithm and visualization components of associationSubgraphs is available at https://github.com/tbilab/associationsubgraphs. Online documentation is available at https://prod.tbilab.org/associationsubgraphs_info/. A demo using a multimorbidity association matrix is available at https://prod.tbilab.org/associationsubgraphs-example/.

International Consensus Classification of Myeloid Neoplasms and Acute Leukemias: integrating morphologic, clinical, and genomic data.

The classification of myeloid neoplasms and acute leukemias was last updated in 2016 within a collaboration between the World Health Organization (WHO), the Society for Hematopathology, and the European Association for Haematopathology. This collaboration was primarily based on input from a clinical advisory committees (CACs) composed of pathologists, hematologists, oncologists, geneticists, and bioinformaticians from around the world. The recent advances in our understanding of the biology of hematologic malignancies, the experience with the use of the 2016 WHO classification in clinical practice, and the results of clinical trials have indicated the need for further revising and updating the classification. As a continuation of this CAC-based process, the authors, a group with expertise in the clinical, pathologic, and genetic aspects of these disorders, developed the International Consensus Classification (ICC) of myeloid neoplasms and acute leukemias. Using a multiparameter approach, the main objective of the consensus process was the definition of real disease entities, including the introduction of new entities and refined criteria for existing diagnostic categories, based on accumulated data. The ICC is aimed at facilitating diagnosis and prognostication of these neoplasms, improving treatment of affected patients, and allowing the design of innovative clinical trials.

TLR3 agonism augments CD47 inhibition in acute myeloid leukemia.

CD47-SIRPa is a myeloid check point pathway that promotes phagocytosis of cells lacking markers for self-recognition. Tumor cells can overexpress CD47 and bind to SIRPa on macrophages, preventing phagocytosis. CD47 expression is enhanced and correlated with a negative prognosis in acute myeloid leukemia (AML), with its blockade leading to cell clearance. ALX90 is an engineered fusion protein with high affinity for CD47. Composed of the N-terminal D1 domain of SIRPα genetically linked to an inactive Fc domain from human immunoglobulin (Ig) G, ALX90 is designed to avoid potential toxicity of CD47-expressing red blood cells. Venetoclax (VEN) is a specific B-cell lymphoma-2 (BCL-2) inhibitor that can restore apoptosis in malignant cells. In AML, VEN is combined with azanucleosides to induce superior remission rates, however treatment for refractory/relapse is an unmet need. We questioned whether the anti-tumor activity of a VENbased regimen can be augmented through CD47 inhibition (CD47i) in AML and how this triplet may be enhanced. Human AML cell lines were sensitive to ALX90 and its addition increased efficacy of a VEN plus azacitidin (VEN+AZA) regimen in vivo. However, CD47i failed to clear bone marrow tumor burden in PDX models. We hypothesized that the loss of resident macrophages in the bone marrow in AML reduced efficiency of CD47i. Therefore, we attempted to enhance this medullary macrophage population with agonism of TLR3 via polyinosinic:polycytidylic acid (poly(I:C)), which led to expansion and activation of medullary macrophages in in vivo AML PDX models and potentiated CD47i. In summary, the addition of poly(I:C) can enhance medullary macrophage populations to potentiate the phagocytosis merited by therapeutic inhibition of CD47.

Apoptolidin family glycomacrolides target leukemia through inhibition of ATP synthase.

Cancer cells have long been recognized to exhibit unique bioenergetic requirements. The apoptolidin family of glycomacrolides are distinguished by their selective cytotoxicity towards oncogene-transformed cells, yet their molecular mechanism remains uncertain. We used photoaffinity analogs of the apoptolidins to identify the F1 subcomplex of mitochondrial ATP synthase as the target of apoptolidin A. Cryogenic electron microscopy (cryo-EM) of apoptolidin and ammocidin-ATP synthase complexes revealed a novel shared mode of inhibition that was confirmed by deep mutational scanning of the binding interface to reveal resistance mutations which were confirmed using CRISPR-Cas9. Ammocidin A was found to suppress leukemia progression in vivo at doses that were tolerated with minimal toxicity. The combination of cellular, structural, mutagenesis, and in vivo evidence defines the mechanism of action of apoptolidin family glycomacrolides and establishes a path to address oxidative phosphorylation-dependent cancers.

Luspatercept in Patients with Lower-Risk Myelodysplastic Syndromes.

BACKGROUND: Patients with anemia and lower-risk myelodysplastic syndromes in whom erythropoiesis-stimulating agent therapy is not effective generally become dependent on red-cell transfusions. Luspatercept, a recombinant fusion protein that binds transforming growth factor ß superfamily ligands to reduce SMAD2 and SMAD3 signaling, showed promising results in a phase 2 study. METHODS: In a double-blind, placebo-controlled, phase 3 trial, we randomly assigned patients with very-low-risk, low-risk, or intermediate-risk myelodysplastic syndromes (defined according to the Revised International Prognostic Scoring System) with ring sideroblasts who had been receiving regular red-cell transfusions to receive either luspatercept (at a dose of 1.0 up to 1.75 mg per kilogram of body weight) or placebo, administered subcutaneously every 3 weeks. The primary end point was transfusion independence for 8 weeks or longer during weeks 1 through 24, and the key secondary end point was transfusion independence for 12 weeks or longer, assessed during both weeks 1 through 24 and weeks 1 through 48. RESULTS: Of the 229 patients enrolled, 153 were randomly assigned to receive luspatercept and 76 to receive placebo; the baseline characteristics of the patients were balanced. Transfusion independence for 8 weeks or longer was observed in 38% of the patients in the luspatercept group, as compared with 13% of those in the placebo group (P<0.001). A higher percentage of patients in the luspatercept group than in the placebo group met the key secondary end point (28% vs. 8% for weeks 1 through 24, and 33% vs. 12% for weeks 1 through 48; P<0.001 for both comparisons). The most common luspatercept-associated adverse events (of any grade) included fatigue, diarrhea, asthenia, nausea, and dizziness. The incidence of adverse events decreased over time. CONCLUSIONS: Luspatercept reduced the severity of anemia in patients with lower-risk myelodysplastic syndromes with ring sideroblasts who had been receiving regular red-cell transfusions and who had disease that was refractory to or unlikely to respond to erythropoiesis-stimulating agents or who had discontinued such agents owing to an adverse event. (Funded by Celgene and Acceleron Pharma; MEDALIST ClinicalTrials.gov number, NCT02631070; EudraCT number, 2015-003454-41.).

Ivosidenib or enasidenib combined with intensive chemotherapy in patients with newly diagnosed AML: a phase 1 study.

Ivosidenib (AG-120) and enasidenib (AG-221) are targeted oral inhibitors of the mutant isocitrate dehydrogenase (mIDH) 1 and 2 enzymes, respectively. Given their effectiveness as single agents in mIDH1/2 relapsed or refractory acute myeloid leukemia (AML), this phase 1 study evaluated the safety and efficacy of ivosidenib or enasidenib combined with intensive chemotherapy in patients with newly diagnosed mIDH1/2 AML. Ivosidenib 500 mg once daily and enasidenib 100 mg once daily were well tolerated in this setting, with safety profiles generally consistent with those of induction and consolidation chemotherapy alone. The frequency of IDH differentiation syndrome was low, as expected given the concurrent administration of cytotoxic chemotherapy. In patients receiving ivosidenib, the frequency and grades of QT interval prolongation were similar to those observed with ivosidenib monotherapy. Increases in total bilirubin were more frequently observed in patients treated with enasidenib, consistent with this inhibitor's known potential to inhibit UGT1A1, but did not appear to have significant clinical consequences. In patients receiving ivosidenib (n = 60) or enasidenib (n = 91), end-of-induction complete remission (CR) rates were 55% and 47%, respectively, and CR/CR with incomplete neutrophil or platelet recovery (CR/CRi/CRp) rates were 72% and 63%, respectively. In patients with a best overall response of CR/CRi/CRp, 16/41 (39%) receiving ivosidenib had IDH1 mutation clearance and 15/64 (23%) receiving enasidenib had IDH2 mutation clearance by digital polymerase chain reaction; furthermore, 16/20 (80%) and 10/16 (63%), respectively, became negative for measurable residual disease by multiparameter flow cytometry. This trial was registered at www.clinicaltrials.gov as #NCT02632708.

Selective inhibition of MCL1 overcomes venetoclax resistance in a murine model of myelodysplastic syndromes.

Treatment for myelodysplastic syndromes (MDS) remains insufficient due to clonal heterogeneity and lack of effective clinical therapies. Dysregulation of apoptosis is observed across MDS subtypes regardless of mutations and represents an attractive therapeutic opportunity. Venetoclax (VEN), a selective inhibitor of anti-apoptotic protein B-cell lymphoma- 2 (BCL2), has yielded impressive responses in older patients with acute myeloid leukemia (AML) and high risk MDS. BCL2 family anti-apoptotic proteins BCL-XL and induced myeloid cell leukemia 1 (MCL1) are implicated in leukemia survival, and upregulation of MCL1 is seen in VEN-resistant AML and MDS. We determined in vitro sensitivity of MDS patient samples to selective inhibitors of BCL2, BCL-XL and MCL1. While VEN response positively correlated with MDS with excess blasts, all MDS subtypes responded to MCL1 inhibition. Treatment with combined VEN + MCL1 inhibtion was synergistic in all MDS subtypes without significant injury to normal hematopoiesis and reduced MDS engraftment in MISTRG6 mice, supporting the pursuit of clinical trials with combined BCL2 + MCL1 inhibition in MDS.

Oral cedazuridine/decitabine for MDS and CMML: a phase 2 pharmacokinetic/pharmacodynamic randomized crossover study.

This phase 2 study was designed to compare systemic decitabine exposure, demethylation activity, and safety in the first 2 cycles with cedazuridine 100 mg/decitabine 35 mg vs standard decitabine 20 mg/m2 IV. Adults with International Prognostic Scoring System intermediate-1/2- or high-risk myelodysplastic syndromes (MDS) or chronic myelomonocytic leukemia (CMML) were randomized 1:1 to receive oral cedazuridine/decitabine or IV decitabine in cycle 1, followed by crossover to the other treatment in cycle 2. All patients received oral cedazuridine/decitabine in subsequent cycles. Cedazuridine and decitabine were given initially as separate capsules in a dose-confirmation stage and then as a single fixed-dose combination (FDC) tablet. Primary end points: mean decitabine systemic exposure (geometric least-squares mean [LSM]) of oral/IV 5-day area under curve from time 0 to last measurable concentration (AUClast), percentage long interspersed nuclear element 1 (LINE-1) DNA demethylation for oral cedazuridine/decitabine vs IV decitabine, and clinical response. Eighty patients were randomized and treated. Oral/IV ratios of geometric LSM 5-day AUClast (80% confidence interval) were 93.5% (82.1-106.5) and 97.6% (80.5-118.3) for the dose-confirmation and FDC stages, respectively. Differences in mean %LINE-1 demethylation between oral and IV were ≤1%. Clinical responses were observed in 48 patients (60%), including 17 (21%) with complete response. The most common grade ≥3 adverse events regardless of causality were neutropenia (46%), thrombocytopenia (38%), and febrile neutropenia (29%). Oral cedazuridine/decitabine (100/35 mg) produced similar systemic decitabine exposure, DNA demethylation, and safety vs decitabine 20 mg/m2 IV in the first 2 cycles, with similar efficacy. This study is registered at www.clinicaltrials.gov as #NCT02103478.

SF3B1-mutant MDS as a distinct disease subtype: a proposal from the International Working Group for the Prognosis of MDS.

The 2016 revision of the World Health Organization classification of tumors of hematopoietic and lymphoid tissues is characterized by a closer integration of morphology and molecular genetics. Notwithstanding, the myelodysplastic syndrome (MDS) with isolated del(5q) remains so far the only MDS subtype defined by a genetic abnormality. Approximately half of MDS patients carry somatic mutations in spliceosome genes, with SF3B1 being the most commonly mutated one. SF3B1 mutation identifies a condition characterized by ring sideroblasts (RS), ineffective erythropoiesis, and indolent clinical course. A large body of evidence supports recognition of SF3B1-mutant MDS as a distinct nosologic entity. To further validate this notion, we interrogated the data set of the International Working Group for the Prognosis of MDS (IWG-PM). Based on the findings of our analyses, we propose the following diagnostic criteria for SF3B1-mutant MDS: (1) cytopenia as defined by standard hematologic values, (2) somatic SF3B1 mutation, (3) morphologic dysplasia (with or without RS), and (4) bone marrow blasts <5% and peripheral blood blasts <1%. Selected concomitant genetic lesions represent exclusion criteria for the proposed entity. In patients with clonal cytopenia of undetermined significance, SF3B1 mutation is almost invariably associated with subsequent development of overt MDS with RS, suggesting that this genetic lesion might provide presumptive evidence of MDS in the setting of persistent unexplained cytopenia. Diagnosis of SF3B1-mutant MDS has considerable clinical implications in terms of risk stratification and therapeutic decision making. In fact, this condition has a relatively good prognosis and may respond to luspatercept with abolishment of the transfusion requirement.

Pevonedistat and azacitidine upregulate NOXA (PMAIP1) to increase sensitivity to venetoclax in preclinical models of acute myeloid leukemia.

Dysregulation of apoptotic machinery is one mechanism by which acute myeloid leukemia (AML) acquires a clonal survival advantage. B-cell lymphoma protein-2 (BCL2) overexpression is a common feature in hematologic malignancies. The selective BCL2 inhibitor, venetoclax (VEN) is used in combination with azacitidine (AZA), a DNAmethyltransferase inhibitor (DNMTi), to treat patients with AML. Despite promising response rates to VEN/AZA, resistance to the agent is common. One identified mechanism of resistance is the upregulation of myeloid cell leukemia-1 protein (MCL1). Pevonedistat (PEV), a novel agent that inhibits NEDD8-activating enzyme, and AZA both upregulate NOXA (PMAIP1), a BCL2 family protein that competes with effector molecules at the BH3 binding site of MCL1. We demonstrate that PEV/AZA combination induces NOXA to a greater degree than either PEV or AZA alone, which enhances VEN-mediated apoptosis. Herein, using AML cell lines and primary AML patient samples ex vivo, including in cells with genetic alterations linked to treatment resistance, we demonstrate robust activity of the PEV/VEN/AZA triplet. These findings were corroborated in preclinical systemic engrafted models of AML. Collectively, these results provide rational for combining PEV/VEN/AZA as a novel therapeutic approach in overcoming AML resistance in current therapies.

The ABNL-MARRO 001 study: a phase 1-2 study of randomly allocated active myeloid target compound combinations in MDS/MPN overlap syndromes.

BACKGROUND: Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) comprise several rare hematologic malignancies with shared concomitant dysplastic and proliferative clinicopathologic features of bone marrow failure and propensity of acute leukemic transformation, and have significant impact on patient quality of life. The only approved disease-modifying therapies for any of the MDS/MPN are DNA methyltransferase inhibitors (DNMTi) for patients with dysplastic CMML, and still, outcomes are generally poor, making this an important area of unmet clinical need. Due to both the rarity and the heterogeneous nature of MDS/MPN, they have been challenging to study in dedicated prospective studies. Thus, refining first-line treatment strategies has been difficult, and optimal salvage treatments following DNMTi failure have also not been rigorously studied. ABNL-MARRO (A Basket study of Novel therapy for untreated MDS/MPN and Relapsed/Refractory Overlap Syndromes) is an international cooperation that leverages the expertise of the MDS/MPN International Working Group (IWG) and provides the framework for collaborative studies to advance treatment of MDS/MPN and to explore clinical and pathologic markers of disease severity, prognosis, and treatment response. METHODS: ABNL MARRO 001 (AM-001) is an open label, randomly allocated phase 1/2 study that will test novel treatment combinations in MDS/MPNs, beginning with the novel targeted agent itacitinib, a selective JAK1 inhibitor, combined with ASTX727, a fixed dose oral combination of the DNMTi decitabine and the cytidine deaminase inhibitor cedazuridine to improve decitabine bioavailability. DISCUSSION: Beyond the primary objectives of the study to evaluate the safety and efficacy of novel treatment combinations in MDS/MPN, the study will (i) Establish the ABNL MARRO infrastructure for future prospective studies, (ii) Forge innovative scientific research that will improve our understanding of pathogenetic mechanisms of disease, and (iii) Inform the clinical application of diagnostic criteria, risk stratification and prognostication tools, as well as response assessments in this heterogeneous patient population. TRIAL REGISTRATION: This trial was registered with ClinicalTrials.gov on August 19, 2019 (Registration No. NCT04061421).

Increased Ripk1-mediated bone marrow necroptosis leads to myelodysplasia and bone marrow failure in mice.

Hematopoiesis is a dynamic system that requires balanced cell division, differentiation, and death. The 2 major modes of programmed cell death, apoptosis and necroptosis, share molecular machinery but diverge in outcome with important implications for the microenvironment; apoptotic cells are removed in an immune silent process, whereas necroptotic cells leak cellular contents that incite inflammation. Given the importance of cytokine-directed cues for hematopoietic cell survival and differentiation, the impact on hematopoietic homeostasis of biasing cell death fate to necroptosis is substantial and poorly understood. Here, we present a mouse model with increased bone marrow necroptosis. Deletion of the proapoptotic Bcl-2 family members Bax and Bak inhibits bone marrow apoptosis. Further deletion of the BH3-only member Bid (to generate Vav CreBaxBakBid triple-knockout [TKO] mice) leads to unrestrained bone marrow necroptosis driven by increased Rip1 kinase (Ripk1). TKO mice display loss of progenitor cells, leading to increased cytokine production and increased stem cell proliferation and exhaustion and culminating in bone marrow failure. Genetically restoring Ripk1 to wild-type levels restores peripheral red cell counts as well as normal cytokine production. TKO bone marrow is hypercellular with abnormal differentiation, resembling the human disorder myelodysplastic syndrome (MDS), and we demonstrate increased necroptosis in MDS bone marrow. Finally, we show that Bid impacts necroptotic signaling through modulation of caspase-8-mediated Ripk1 degradation. Thus, we demonstrate that dysregulated necroptosis in hematopoiesis promotes bone marrow progenitor cell death that incites inflammation, impairs hematopoietic stem cells, and recapitulates the salient features of the bone marrow failure disorder MDS.

The CDK7 inhibitor THZ1 alters RNA polymerase dynamics at the 5' and 3' ends of genes.

The t(8;21) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML). We found that t(8;21) AML were extremely sensitive to THZ1, which triggered apoptosis after only 4 h. We used precision nuclear run-on transcription sequencing (PROseq) to define the global effects of THZ1 and other CDK inhibitors on RNA polymerase II dynamics. Inhibition of CDK7 using THZ1 caused wide-spread loss of promoter-proximal paused RNA polymerase. This loss of 5' pausing was associated with accumulation of polymerases in the body of a large number of genes. However, there were modest effects on genes regulated by 'super-enhancers'. At the 3' ends of genes, treatment with THZ1 suppressed RNA polymerase 'read through' at the end of the last exon, which resembled a phenotype associated with a mutant RNA polymerase with slower elongation rates. Consistent with this hypothesis, polyA site-sequencing (PolyA-seq) did not detect differences in poly A sites after THZ1 treatment. PROseq analysis after short treatments with THZ1 suggested that these 3' effects were due to altered CDK7 activity at the 5' end of long genes, and were likely to be due to slower rates of elongation.

The evolving role of next generation sequencing in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are clonal haematological disorders characterized by haematopoietic cell dysplasia, peripheral blood cytopenias, and a predisposition for developing acute myeloid leukaemia (AML). Cytogenetics have historically been important in diagnosis and prognosis in MDS, but the growing accessibility of next generation sequencing (NGS) has led to growing research in the roles of molecular genetic variation on clinical decision-making in these disorders. Multiple genes have been previously studied and found to be associated with specific outcomes or disease types within MDS and knowledge of mutations in these genes provides insight into previously defined MDS subtypes. Knowledge of these mutations also informs development of novel therapies in the treatment of MDS. The precise role of NGS in the diagnosis, prognosis and monitoring of MDS remains unclear but the improvements in NGS technology and accessibility affords clinicians an additional practice tool to provide the best care for patients.

Pevonedistat, a first-in-class NEDD8-activating enzyme inhibitor, combined with azacitidine in patients with AML.

Pevonedistat (TAK-924/MLN4924) is a novel inhibitor of NEDD8-activating enzyme (NAE) with single-agent activity in relapsed/refractory acute myeloid leukemia (AML). We performed a phase 1b study of pevonedistat (PEV) with azacitidine (AZA) based on synergistic activity seen preclinically. Primary objectives included safety and tolerability, and secondary objectives included pharmacokinetics (PK) and disease response. Patients ≥60 years with treatment-naive AML (unfit for standard induction therapy) received PEV 20 or 30 mg/m2 IV on days 1, 3, and 5 combined with fixed-dose AZA (75 mg/m2 IV/subcutaneously) on days 1 to 5, 8, and 9, every 28 days. The most common treatment-emergent adverse events were constipation (48%), nausea (42%), fatigue (42%), and anemia (39%). In total, 11 deaths were observed and considered unrelated to study therapy by the investigators. Transient elevations in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were dose limiting. The recommended phase 2 dose (RP2D) of PEV in this combination is 20 mg/m2 PEV PK was not altered by the addition of AZA. Overall response rate (ORR) based on an intent-to-treat analysis was 50% (20 complete remissions [CRs], 5 complete remission with incomplete peripheral count recovery, 7 partial remissions [PRs]), with an 8.3-month median duration of remission. In patients receiving ≥6 cycles of therapy (n = 23, 44%), ORR was 83%. In patients with TP53 mutations, the composite CR/PR rate was 80% (4/5). Two of these patients stayed on study for >10 cycles. Baseline bone marrow blast percentage or cytogenetic/molecular risk did not influence ORR. This study was registered at www.clinicaltrials.gov as #NCT01814826.