Disease-Causing Mutations in SF3B1 Alter Splicing by Disrupting Interaction with SUGP1.

SF3B1, which encodes an essential spliceosomal protein, is frequently mutated in myelodysplastic syndromes (MDS) and many cancers. However, the defect of mutant SF3B1 is unknown. Here, we analyzed RNA sequencing data from MDS patients and confirmed that SF3B1 mutants use aberrant 3' splice sites. To elucidate the underlying mechanism, we purified complexes containing either wild-type or the hotspot K700E mutant SF3B1 and found that levels of a poorly studied spliceosomal protein, SUGP1, were reduced in mutant spliceosomes. Strikingly, SUGP1 knockdown completely recapitulated the splicing errors, whereas SUGP1 overexpression drove the protein, which our data suggest plays an important role in branchsite recognition, into the mutant spliceosome and partially rescued splicing. Other hotspot SF3B1 mutants showed similar altered splicing and diminished interaction with SUGP1. Our study demonstrates that SUGP1 loss is a common defect of spliceosomes with disease-causing SF3B1 mutations and, because this defect can be rescued, suggests possibilities for therapeutic intervention.

SF3B1 mutant-induced missplicing of MAP3K7 causes anemia in myelodysplastic syndromes.

SF3B1 is the most frequently mutated RNA splicing factor in cancer, including in ∼25% of myelodysplastic syndromes (MDS) patients. SF3B1-mutated MDS, which is strongly associated with ringed sideroblast morphology, is characterized by ineffective erythropoiesis, leading to severe, often fatal anemia. However, functional evidence linking SF3B1 mutations to the anemia described in MDS patients harboring this genetic aberration is weak, and the underlying mechanism is completely unknown. Using isogenic SF3B1 WT and mutant cell lines, normal human CD34 cells, and MDS patient cells, we define a previously unrecognized role of the kinase MAP3K7, encoded by a known mutant SF3B1-targeted transcript, in controlling proper terminal erythroid differentiation, and show how MAP3K7 missplicing leads to the anemia characteristic of SF3B1-mutated MDS, although not to ringed sideroblast formation. We found that p38 MAPK is deactivated in SF3B1 mutant isogenic and patient cells and that MAP3K7 is an upstream positive effector of p38 MAPK. We demonstrate that disruption of this MAP3K7-p38 MAPK pathway leads to premature down-regulation of GATA1, a master regulator of erythroid differentiation, and that this is sufficient to trigger accelerated differentiation, erythroid hyperplasia, and ultimately apoptosis. Our findings thus define the mechanism leading to the severe anemia found in MDS patients harboring SF3B1 mutations.

U2AF35(S34F) Promotes Transformation by Directing Aberrant ATG7 Pre-mRNA 3' End Formation.

Recurrent mutations in the splicing factor U2AF35 are found in several cancers and myelodysplastic syndrome (MDS). How oncogenic U2AF35 mutants promote transformation remains to be determined. Here we derive cell lines transformed by the oncogenic U2AF35(S34F) mutant and identify aberrantly processed pre-mRNAs by deep sequencing. We find that in U2AF35(S34F)-transformed cells the autophagy-related factor 7 (Atg7) pre-mRNA is abnormally processed, which unexpectedly is not due to altered splicing but rather selection of a distal cleavage and polyadenylation (CP) site. This longer Atg7 mRNA is translated inefficiently, leading to decreased ATG7 levels and an autophagy defect that predisposes cells to secondary mutations, resulting in transformation. MDS and acute myeloid leukemia patient samples harboring U2AF35(S34F) have a similar increased use of the ATG7 distal CP site, and previous studies have shown that mice with hematopoietic cells lacking Atg7 develop an MDS-like syndrome. Collectively, our results reveal a basis for U2AF35(S34F) oncogenic activity.

ASXL1 mutations are associated with a response to alvocidib and 5-azacytidine combination in myelodysplastic neoplasms.

Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMAs) are promising therapeutic approaches in acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS). Alvocidib, a cyclin-dependent kinase 9 (CDK9) inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has previously shown clinical activity in AML. Availability of biomarkers for response to the alvocidib + 5- AZA could also extend the rationale of this treatment concept to high-risk MDS. In this study, we performed a comprehensive in vitro assessment of alvocidib and 5-AZA effects in n=45 high-risk MDS patients. Our data revealed additive cytotoxic effects of the combination treatment. Mutational profiling of MDS samples identified ASXL1 mutations as predictors of response. Further, increased response rates were associated with higher gene-expression of the pro-apoptotic factor NOXA in ASXL1 mutated samples. The higher sensitivity of ASXL1 mutant cells to the combination treatment was confirmed in vivo in ASXL1Y588X transgenic mice. Overall, our study demonstrated augmented activity for the alvocidib + 5-AZA combination in higher-risk MDS and identified ASXL1 mutations as a biomarker of response for potential stratification studies.

Gene-edited stem cells enable CD33-directed immune therapy for myeloid malignancies.

Antigen-directed immunotherapies for acute myeloid leukemia (AML), such as chimeric antigen receptor T cells (CAR-Ts) or antibody-drug conjugates (ADCs), are associated with severe toxicities due to the lack of unique targetable antigens that can distinguish leukemic cells from normal myeloid cells or myeloid progenitors. Here, we present an approach to treat AML by targeting the lineage-specific myeloid antigen CD33. Our approach combines CD33-targeted CAR-T cells, or the ADC Gemtuzumab Ozogamicin with the transplantation of hematopoietic stem cells that have been engineered to ablate CD33 expression using genomic engineering methods. We show highly efficient genetic ablation of CD33 antigen using CRISPR/Cas9 technology in human stem/progenitor cells (HSPC) and provide evidence that the deletion of CD33 in HSPC doesn't impair their ability to engraft and to repopulate a functional multilineage hematopoietic system in vivo. Whole-genome sequencing and RNA sequencing analysis revealed no detectable off-target mutagenesis and no loss of functional p53 pathways. Using a human AML cell line (HL-60), we modeled a postremission marrow with minimal residual disease and showed that the transplantation of CD33-ablated HSPCs with CD33-targeted immunotherapy leads to leukemia clearance, without myelosuppression, as demonstrated by the engraftment and recovery of multilineage descendants of CD33-ablated HSPCs. Our study thus contributes to the advancement of targeted immunotherapy and could be replicated in other malignancies.

Comprehensive phenotyping of erythropoiesis in human bone marrow: Evaluation of normal and ineffective erythropoiesis.

Identification of stage-specific erythroid cells is critical for studies of normal and disordered human erythropoiesis. While immunophenotypic strategies have previously been developed to identify cells at each stage of terminal erythroid differentiation, erythroid progenitors are currently defined very broadly. Refined strategies to identify and characterize BFU-E and CFU-E subsets are critically needed. To address this unmet need, a flow cytometry-based technique was developed that combines the established surface markers CD34 and CD36 with CD117, CD71, and CD105. This combination allowed for the separation of erythroid progenitor cells into four discrete populations along a continuum of progressive maturation, with increasing cell size and decreasing nuclear/cytoplasmic ratio, proliferative capacity and stem cell factor responsiveness. This strategy was validated in uncultured, primary erythroid cells isolated from bone marrow of healthy individuals. Functional colony assays of these progenitor populations revealed enrichment of BFU-E only in the earliest population, transitioning to cells yielding BFU-E and CFU-E, then CFU-E only. Utilizing CD34/CD105 and GPA/CD105 profiles, all four progenitor stages and all five stages of terminal erythroid differentiation could be identified. Applying this immunophenotyping strategy to primary bone marrow cells from patients with myelodysplastic syndrome, identified defects in erythroid progenitors and in terminal erythroid differentiation. This novel immunophenotyping technique will be a valuable tool for studies of normal and perturbed human erythropoiesis. It will allow for the discovery of stage-specific molecular and functional insights into normal erythropoiesis as well as for identification and characterization of stage-specific defects in inherited and acquired disorders of erythropoiesis.

Leukaemogenesis induced by an activating ß-catenin mutation in osteoblasts.

Cells of the osteoblast lineage affect the homing and the number of long-term repopulating haematopoietic stem cells, haematopoietic stem cell mobilization and lineage determination and B cell lymphopoiesis. Osteoblasts were recently implicated in pre-leukaemic conditions in mice. However, a single genetic change in osteoblasts that can induce leukaemogenesis has not been shown. Here we show that an activating mutation of ß-catenin in mouse osteoblasts alters the differentiation potential of myeloid and lymphoid progenitors leading to development of acute myeloid leukaemia with common chromosomal aberrations and cell autonomous progression. Activated ß-catenin stimulates expression of the Notch ligand jagged 1 in osteoblasts. Subsequent activation of Notch signalling in haematopoietic stem cell progenitors induces the malignant changes. Genetic or pharmacological inhibition of Notch signalling ameliorates acute myeloid leukaemia and demonstrates the pathogenic role of the Notch pathway. In 38% of patients with myelodysplastic syndromes or acute myeloid leukaemia, increased ß-catenin signalling and nuclear accumulation was identified in osteoblasts and these patients showed increased Notch signalling in haematopoietic cells. These findings demonstrate that genetic alterations in osteoblasts can induce acute myeloid leukaemia, identify molecular signals leading to this transformation and suggest a potential novel pharmacotherapeutic approach to acute myeloid leukaemia.

Improving Treatment for Myelodysplastic Syndromes Patients.

OPINION STATEMENT: Aging is the most potent of carcinogens, especially for the bone marrow stem cell clonal disorders called myelodysplastic syndromes (MDS). Age-associated changes in the microenvironment or the soil of the bone marrow (BM) as well as in the cell or the seed provide a growth advantage for clonal myeloid cells. Slowly accumulating senescent cells which can no longer divide because they have reached the end of their proliferative life cycle, but which continue to produce metabolic debris, overwhelm the natural autophagy mechanisms resulting in pro-inflammatory changes in the BM soil. In addition, the seed or stem cells acquire passenger mutations with each round of proliferation resulting from DNA copying errors. Some mutations commonly associated with MDS can be found in older, otherwise healthy individuals; however, when combined with other passenger mutations or in the setting of a noxious soil, the result could be a proliferative advantage for one stem cell over others, leading to its clonal expansion and development of the clinical syndrome. When considering therapeutic options for MDS patients, the important considerations are related to both the common co-morbidities of an elderly population along with the heterogeneous passenger mutations and the inflammatory changes in the soil. At present, allogeneic stem cell transplant is the only potentially curative option in MDS. Palliative strategies are directed at improving the quality of life and prolonging survival. Only three drugs are FDA approved, two being the hypomethylating agents azacytidine and decitabine while the third is lenalidomide which is restricted to lower risk MDS patients with deletion 5q. Promising future therapies are directed at reversing the pro-inflammatory changes in the microenvironment (luspatercept) or targeting specific mutations isocitrate dehydrogenase (IDH)1, IDH2, p53, EZH2. More durable responses are to be expected when the seed and soil are targeted simultaneously through a combination of drugs.

Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities.

Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (<1% of total). Of these events, 374 involved the inclusion of cassette exons, and the inclusion was either increased (206) or decreased (168). We detected a specific motif (UCCA/UG) enriched in the more-included exons and a distinct motif (UGGA/UG) in the more-excluded exons. RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG but bound less tightly to UGGAG sites. Thus in most cases the pattern of exon inclusion or exclusion correlated with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein-protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation and also reveal a group of misspliced mRNA isoforms for potential therapeutic targeting.

Activity of the oral mitogen-activated protein kinase kinase inhibitor trametinib in RAS-mutant relapsed or refractory myeloid malignancies.

BACKGROUND: RAS/RAF/mitogen-activated protein kinase activation is common in myeloid malignancies. Trametinib, a mitogen-activated protein kinase kinase 1 (MEK1)/MEK2 inhibitor with activity against multiple myeloid cell lines at low nanomolar concentrations, was evaluated for safety and clinical activity in patients with relapsed/refractory leukemias. METHODS: This phase 1/2 study accrued patients with any relapsed/refractory leukemia in phase 1. In phase 2, this study accrued patients with relapsed/refractory acute myeloid leukemia (AML) or high-risk myelodysplastic syndromes (MDS) with NRAS or KRAS mutations (cohort 1); patients with AML, MDS, or chronic myelomonocytic leukemia (CMML) with a RAS wild-type mutation or an unknown mutation status (cohort 2); and patients with CMML with an NRAS or KRAS mutation (cohorts 3). RESULTS: The most commonly reported treatment-related adverse events were diarrhea, rash, nausea, and increased alanine aminotransferase levels. The phase 2 recommended dose for Trametinib was 2 mg orally daily. The overall response rates were 20%, 3%, and 27% for cohorts 1, 2, and 3, respectively, and this indicated preferential activity among RAS-mutated myeloid malignancies. Repeated cycles of trametinib were well tolerated with manageable or reversible toxicities; these results were similar to those of other trametinib studies. CONCLUSIONS: The selective, single-agent activity of trametinib against RAS-mutated myeloid malignancies validates its therapeutic potential. Combination strategies based on a better understanding of the hierarchical role of mutations and signaling in myeloid malignancies are likely to improve the response rate and duration. Cancer 2016;122:1871-9. © 2016 American Cancer Society.

Inhibition of leukemia cell engraftment and disease progression in mice by osteoblasts.

The bone marrow niche is thought to act as a permissive microenvironment required for emergence or progression of hematologic cancers. We hypothesized that osteoblasts, components of the niche involved in hematopoietic stem cell (HSC) function, influence the fate of leukemic blasts. We show that osteoblast numbers decrease by 55% in myelodysplasia and acute myeloid leukemia patients. Further, genetic depletion of osteoblasts in mouse models of acute leukemia increased circulating blasts and tumor engraftment in the marrow and spleen leading to higher tumor burden and shorter survival. Myelopoiesis increased and was coupled with a reduction in B lymphopoiesis and compromised erythropoiesis, suggesting that hematopoietic lineage/progression was altered. Treatment of mice with acute myeloid or lymphoblastic leukemia with a pharmacologic inhibitor of the synthesis of duodenal serotonin, a hormone suppressing osteoblast numbers, inhibited loss of osteoblasts. Maintenance of the osteoblast pool restored normal marrow function, reduced tumor burden, and prolonged survival. Leukemia prevention was attributable to maintenance of osteoblast numbers because inhibition of serotonin receptors alone in leukemic blasts did not affect leukemia progression. These results suggest that osteoblasts play a fundamental role in propagating leukemia in the marrow and may be a therapeutic target to induce hostility of the niche to leukemia blasts.

Prospective international validation of the Quality of Life in Myelodysplasia Scale (QUALMS).

Disease-specific measures of quality of life can improve assessment of disease-related symptoms and psychosocial sequelae. We report on the development and validation of the Quality of Life in Myelodysplasia Scale (QUALMS), a 38-item assessment tool for patients with myelodysplastic syndromes (MDS). In 2014-2015, a multi-center cohort of patients with myelodysplasia completed the QUALMS, as well as the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (QLQ-C30) and the Functional Assessment of Cancer Therapy Anemia Scale (FACT-An); a second administration was undertaken three to six months later. A total of 255 patients from the United States, Canada and Italy participated. Median age was 72 years, 56.1% were men, and the International Prognostic Scoring System distribution was 40.4% low, 42.0% intermediate-1, 13.3% intermediate-2 and 2.3% high. QUALMS scores ranged from 24 to 99 (higher scores are better), with a mean of 67.2 [standard deviation (SD)=15.2]. The measure was internally consistent (α=0.92), and moderately correlated with the multi-item QLQ-C30 scales and the FACT-An (r=-0.65 to 0.79; all P<0.001). Patients with hemoglobin of 8 g/dL or under scored lower than those with hemoglobin over 10 g/dL (61.8 vs 71.1; P<0.001), and transfusion-dependent patients scored lower than transfusion-independent patients (62.4 vs 69.7; P<0.01). Principal components analysis revealed "physical burden", "benefit-finding", and "emotional burden" subscales. There was good overall test-retest reliability among those with stable hemoglobin (r=0.81), and significant changes for patients hospitalized or with infections between administrations (both P<0.01). These data suggest the QUALMS is a valuable tool for assessing MDS-specific quality of life in the modern treatment era.

A double-blind, placebo-controlled trial of ruxolitinib for myelofibrosis.

BACKGROUND: Ruxolitinib, a selective inhibitor of Janus kinase (JAK) 1 and 2, has clinically significant activity in myelofibrosis. METHODS: In this double-blind trial, we randomly assigned patients with intermediate-2 or high-risk myelofibrosis to twice-daily oral ruxolitinib (155 patients) or placebo (154 patients). The primary end point was the proportion of patients with a reduction in spleen volume of 35% or more at 24 weeks, assessed by means of magnetic resonance imaging. Secondary end points included the durability of response, changes in symptom burden (assessed by the total symptom score), and overall survival. RESULTS: The primary end point was reached in 41.9% of patients in the ruxolitinib group as compared with 0.7% in the placebo group (P<0.001). A reduction in spleen volume was maintained in patients who received ruxolitinib; 67.0% of the patients with a response had the response for 48 weeks or more. There was an improvement of 50% or more in the total symptom score at 24 weeks in 45.9% of patients who received ruxolitinib as compared with 5.3% of patients who received placebo (P<0.001). Thirteen deaths occurred in the ruxolitinib group as compared with 24 deaths in the placebo group (hazard ratio, 0.50; 95% confidence interval, 0.25 to 0.98; P=0.04). The rate of discontinuation of the study drug because of adverse events was 11.0% in the ruxolitinib group and 10.6% in the placebo group. Among patients who received ruxolitinib, anemia and thrombocytopenia were the most common adverse events, but they rarely led to discontinuation of the drug (in one patient for each event). Two patients had transformation to acute myeloid leukemia; both were in the ruxolitinib group. CONCLUSIONS: Ruxolitinib, as compared with placebo, provided significant clinical benefits in patients with myelofibrosis by reducing spleen size, ameliorating debilitating myelofibrosis-related symptoms, and improving overall survival. These benefits came at the cost of more frequent anemia and thrombocytopenia in the early part of the treatment period. (Funded by Incyte; COMFORT-I ClinicalTrials.gov number, NCT00952289.).

Isolation and functional characterization of human erythroblasts at distinct stages: implications for understanding of normal and disordered erythropoiesis in vivo.

Terminal erythroid differentiation starts from morphologically recognizable proerythroblasts that proliferate and differentiate to generate red cells. Although this process has been extensively studied in mice, its characterization in humans is limited. By examining the dynamic changes of expression of membrane proteins during in vitro human terminal erythroid differentiation, we identified band 3 and α4 integrin as optimal surface markers for isolating 5 morphologically distinct populations at successive developmental stages. Functional analysis revealed that these purified cell populations have distinct mitotic capacity. Use of band 3 and α4 integrin enabled us to isolate erythroblasts at specific developmental stages from primary human bone marrow. The ratio of erythroblasts at successive stages followed the predicted 1:2:4:8:16 pattern. In contrast, bone marrows from myelodysplastic syndrome patients exhibited altered terminal erythroid differentiation profiles. Thus, our findings not only provide new insights into the genesis of the red cell membrane during human terminal erythroid differentiation but also offer a means of isolating and quantifying each developmental stage during terminal erythropoiesis in vivo. Our findings should facilitate a comprehensive cellular and molecular characterization of each specific developmental stage of human erythroblasts and should provide a powerful means of identifying stage-specific defects in diseases associated with pathological erythropoiesis.

Efficacy, safety, and survival with ruxolitinib in patients with myelofibrosis: results of a median 3-year follow-up of COMFORT-I.

In the phase III COMFORT-I study, the Janus kinase 1 (JAK1)/JAK2 inhibitor ruxolitinib provided significant improvements in splenomegaly, key symptoms, and quality-of-life measures and was associated with an overall survival benefit relative to placebo in patients with intermediate-2 or high-risk myelofibrosis. This planned analysis assessed the long-term efficacy and safety of ruxolitinib at a median follow-up of 149 weeks. At data cutoff, approximately 50% of patients originally randomized to ruxolitinib remained on treatment whereas all patients originally assigned to placebo had discontinued or crossed over to ruxolitinib. At week 144, mean spleen volume reduction was 34% with ruxolitinib. Previously observed improvements in quality-of-life measures were sustained with longer-term ruxolitinib therapy. Overall survival continued to favor ruxolitinib despite the majority of placebo patients crossing over to ruxolitinib [hazard ratio 0.69 (95% confidence interval: 0.46-1.03); P = 0.067]. Exploratory analyses suggest that crossover may have contributed to an underestimation of the true survival difference between the treatment groups. Ruxolitinib continued to be generally well tolerated; there was no pattern of worsening grade ≥ 3 anemia or thrombocytopenia with longer-term ruxolitinib exposure. These longer-term data continue to support the efficacy and safety of ruxolitinib in patients with myelofibrosis. The study is registered at clinicaltrials.gov: NCT00952289.

Clinical activity and safety of the dual pathway inhibitor rigosertib for higher risk myelodysplastic syndromes following DNA methyltransferase inhibitor therapy.

Rigosertib (ON 01910.Na) is an inhibitor of the phosphoinositide 3-kinase and polo-like kinase pathways that induces mitotic arrest and apoptosis in neoplastic cells, while sparing normal cells. Our purpose is to summarize the clinical activity and safety of intravenous (IV) rigosertib delivered by an external ambulatory infusion pump in patients with refractory anemia with excess blasts-1, -2, or, -t myelodysplastic syndromes (MDS) following prior treatment with DNA methyltransferase (DNMT) inhibitors. A total of 39 patients with MDS who fulfilled these criteria were enrolled in four phase 1-2 clinical trials of IV rigosertib. Thirty five (88%) had higher risk disease according to the Revised International Prognostic Scoring System. Median overall survival for this group of 39 patients was 35 weeks. Of 30 evaluable patients with follow-up bone marrow biopsies, 12 (40%) achieved complete (n = 5) or partial (n = 7) bone marrow blast responses. In addition, 15 patients achieved stabilization of bone marrow blasts. One patient with a complete bone marrow response also achieved a complete cytogenetic response. A second patient with stable bone marrow blasts achieved a partial cytogenetic response. Two of the responding patients and three patients with stable disease had hematological improvements. Rigosertib-induced bone marrow blast decreases and stability appeared to be predictive of prolonged survival. IV rigosertib had a favorable safety profile without significant myelosuppression. Most common drug-related toxicities included fatigue, diarrhea, nausea, dysuria, and hematuria. In summary, IV rigosertib is well tolerated and has clinical activity in patients with higher risk MDS following DNMT inhibitor treatment. A multinational pivotal phase 3 randomized clinical trial of rigosertib versus best supportive care for patients with MDS with excess blasts following prior treatment with DNMT inhibitors (ONTIME: ON 01910.Na Trial In Myelodysplastic SyndromE) has recently completed enrollment.

Clinical effect of point mutations in myelodysplastic syndromes.

BACKGROUND: Myelodysplastic syndromes are clinically heterogeneous disorders characterized by clonal hematopoiesis, impaired differentiation, peripheral-blood cytopenias, and a risk of progression to acute myeloid leukemia. Somatic mutations may influence the clinical phenotype but are not included in current prognostic scoring systems. METHODS: We used a combination of genomic approaches, including next-generation sequencing and mass spectrometry-based genotyping, to identify mutations in samples of bone marrow aspirate from 439 patients with myelodysplastic syndromes. We then examined whether the mutation status for each gene was associated with clinical variables, including specific cytopenias, the proportion of blasts, and overall survival. RESULTS: We identified somatic mutations in 18 genes, including two, ETV6 and GNAS, that have not been reported to be mutated in patients with myelodysplastic syndromes. A total of 51% of all patients had at least one point mutation, including 52% of the patients with normal cytogenetics. Mutations in RUNX1, TP53, and NRAS were most strongly associated with severe thrombocytopenia (P<0.001 for all comparisons) and an increased proportion of bone marrow blasts (P<0.006 for all comparisons). In a multivariable Cox regression model, the presence of mutations in five genes retained independent prognostic significance: TP53 (hazard ratio for death from any cause, 2.48; 95% confidence interval [CI], 1.60 to 3.84), EZH2 (hazard ratio, 2.13; 95% CI, 1.36 to 3.33), ETV6 (hazard ratio, 2.04; 95% CI, 1.08 to 3.86), RUNX1 (hazard ratio, 1.47; 95% CI, 1.01 to 2.15), and ASXL1 (hazard ratio, 1.38; 95% CI, 1.00 to 1.89). CONCLUSIONS: Somatic point mutations are common in myelodysplastic syndromes and are associated with specific clinical features. Mutations in TP53, EZH2, ETV6, RUNX1, and ASXL1 are predictors of poor overall survival in patients with myelodysplastic syndromes, independently of established risk factors. (Funded by the National Institutes of Health and others.).

Comparison of placebo and best available therapy for the treatment of myelofibrosis in the phase 3 COMFORT studies.

Prior to Janus kinase inhibitors, available therapies for myelofibrosis were generally supportive and did not improve survival. This analysis compares efficacy outcomes of patients with myelofibrosis in the control arms (placebo [n=154] and best available therapy [n=73]) from the two phase 3 COntrolled MyeloFibrosis study with ORal JAK inhibitor Treatment (COMFORT) studies. Spleen volume was assessed by magnetic resonance imaging/computed tomography at baseline and every 12 weeks through week 72; spleen length was assessed by palpation at each study visit. Health-related quality of life and symptoms were assessed using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 Items at baseline and in weeks 4, 8, 12, 16 and 24 in COMFORT-I and in weeks 8, 16, 24 and 48 in COMFORT-II. The demographic and baseline characteristics were similar between the control arms of the two studies. One patient who received placebo and no patients who received best available therapy had a ≥35% reduction in spleen volume from baseline at week 24. At 24 weeks, neither placebo nor best available therapy had produced clinically meaningful changes in global quality of life or symptom scales. Non-hematologic adverse events were mostly grade 1/2; the most frequently reported adverse events in each group were abdominal pain, fatigue, peripheral edema and diarrhea. These data suggest that non-Janus kinase inhibitor therapies provide little improvement in splenomegaly, symptoms or quality of life as compared with placebo. Both COMFORT-I (NCT00952289) and COMFORT-II (NCT00934544) studies have been appropriately registered with clinicaltrials.gov.

Identification of RPS14 as a 5q- syndrome gene by RNA interference screen.

Somatic chromosomal deletions in cancer are thought to indicate the location of tumour suppressor genes, by which a complete loss of gene function occurs through biallelic deletion, point mutation or epigenetic silencing, thus fulfilling Knudson's two-hit hypothesis. In many recurrent deletions, however, such biallelic inactivation has not been found. One prominent example is the 5q- syndrome, a subtype of myelodysplastic syndrome characterized by a defect in erythroid differentiation. Here we describe an RNA-mediated interference (RNAi)-based approach to discovery of the 5q- disease gene. We found that partial loss of function of the ribosomal subunit protein RPS14 phenocopies the disease in normal haematopoietic progenitor cells, and also that forced expression of RPS14 rescues the disease phenotype in patient-derived bone marrow cells. In addition, we identified a block in the processing of pre-ribosomal RNA in RPS14-deficient cells that is functionally equivalent to the defect in Diamond-Blackfan anaemia, linking the molecular pathophysiology of the 5q- syndrome to a congenital syndrome causing bone marrow failure. These results indicate that the 5q- syndrome is caused by a defect in ribosomal protein function and suggest that RNAi screening is an effective strategy for identifying causal haploinsufficiency disease genes.

scRNAseq and High-Throughput Spatial Analysis of Tumor and Normal Microenvironment in Solid Tumors Reveal a Possible Origin of Circulating Tumor Hybrid Cells.

Metastatic cancer is a leading cause of death in cancer patients worldwide. While circulating hybrid cells (CHCs) are implicated in metastatic spread, studies documenting their tissue origin remain sparse, with limited candidate approaches using one-two markers. Utilizing high-throughput single-cell and spatial transcriptomics, we identified tumor hybrid cells (THCs) co-expressing epithelial and macrophage markers and expressing a distinct transcriptome. Rarely, normal tissue showed these cells (NHCs), but their transcriptome was easily distinguishable from THCs. THCs with unique transcriptomes were observed in breast and colon cancers, suggesting this to be a generalizable phenomenon across cancer types. This study establishes a framework for HC identification in large datasets, providing compelling evidence for their tissue residence and offering comprehensive transcriptomic characterization. Furthermore, it sheds light on their differential function and identifies pathways that could explain their newly acquired invasive capabilities. THCs should be considered as potential therapeutic targets.