ALBERT

Description: Key Points Statement on the importance of climate change politics

Description: Despite advances in understanding of the biology of acute myeloid leukemia (AML), cure remains elusive for the majority of patients. Pro-survival molecules of BCL-2 family play critical roles in leukemia transformation and chemoresistance. The anti-leukemia potency of selective BCL-2 inhibitor venetoclax (ABT-199/GDC-0199) has been demonstrated in AML models (Pan et al. Cancer Discovery 2014). However, venetoclax is often associated with resistance due to its poor inhibition of MCL-1. RAF/MEK/ERK (MAPK) pathway is commonly activated in AML, and can stabilize anti-apoptotic MCL-1 and inactivate the pro-apoptotic BIM. In this study, we evaluated the anti-leukemia effects of concomitant BCL-2 and MAPK blockade by venetoclax in combination with MEK1/2 inhibitor GDC-0973 (cobimetinib). First, anti-leukemia activity of cobimetinib and venetoclax was examined in 18 primary AML samples with diverse genetic alterations. The combination significantly enhanced cell death, as compared to the single agent treatment (Fig 1A). Cobimetinib inhibited cell proliferation in the majority of AML cases (34.2 ± 23.7%) and the cell growth suppression was more profound in the combination group (60.2 ± 28.8%, p

Description: Background: Transfusion Services in Saudi Arabia are mainly hospital-based, where each major hospital recruit donors, test for Hepatitis B service antigen (HBs Ag), Hepatitis C antibody (Anti-HCV), Human Immune Deficiency Virus antibody (Anti-HIV), and Nucleic Acid Testing (NAT) for HBV-DNA, HCV-RNA, and HIV-RNA. In addition, Anti-HBc and Anti-HBs titer are tested for those who are negative for HBs Antigen and NAT, so that blood products from donors with high Anti-HBs titer (〉100 u/ml) can be used. The aim of this limited retrospective study is to find out the prevalence of Anti-HBc among blood donors in different blood donation centers in Riyadh city, Saudi Arabia and current policies regarding blood utilization from donors with positive Anti-HBc. Materials and Methods: We reviewed blood donor records in 4 large hospitals in Riyadh between 2011-2015. Hospital names are not disclosed for confidentiality reasons. Instead, they were named A, B, C, and D Results: (see Table) 1- The overall prevalence of Anti-HBc positive blood donors is 6.2%, however, there is a significant variation among institutions ranging from 2.8 % to 11.1 % (which needs to be investigated to explain reasons for this variation). 2- Some institutions defer all donors with positive anti-HBc (although they carry out all tests), while others carry out anti-HBs and NAT testing and use blood with high titer anti-HBs (〉 100 u/ml). Conclusion: There is a need for an expert consensus opinion regarding the cost-effectiveness of anti-HBc for either donor deferral or proceeding to NAT and anti-HBs titer testing. Such a consensus is practiced in many countries, based on the prevalence of anti-HBc and the availability of NAT testing Disclosures No relevant conflicts of interest to declare.

Description: RNA-binding proteins (RBPs) regulate many aspects of transcription and translation in a cell- and tissue-specific manner and are frequently dysregulated in malignancy. We systematically evaluated RBPs preferentially required in acute myeloid leukemia (AML) over other forms of cancer or normal hematopoietic precursors using a CRISPR/Cas9 domain-based, loss-of-function screen targeting 490 classical RBPs with 2,900 sgRNAs (Fig. A). This screen was performed in cells lines representing AML, T-cell acute lymphoblastic leukemia (T-ALL), and lung adenocarcinoma (LUAD) and revealed multiple RBPs preferentially required for AML survival, but not for T-ALL or LUAD survival. We identified genes encoding 21 RBPs that were 〉3-fold depleted in AML cells and significantly overexpressed in AML patient samples versus normal adult CD34+ precursors (p-value 〈 0.05; Fig. B). Amongst RBPs required and upregulated in AML was RBM39, an RBP described to be involved in a number of cellular processes and to interact with key splicing proteins SF3B1 and U2AF2. Genetic ablation of Rbm39 in mouse MLL-AF9 leukemia cells dramatically delayed AML development and progression (Fig. C). In parallel, it has recently been described that a class of clinically-validated anti-cancer sulfonamide compounds (including indisulam and E7820) mediate RBM39 degradation as their dominant cellular mechanism of action. This occurs via novel interactions with the DCAF15 adapter protein of the CUL4/Ddb1 ubiquitin ligase complex with RBM39 as a neo-substrate. Treatment of MOLM-13 cells xenografted into mice with indisulam conferred significant anti-leukemic effects and improved overall survival (Fig. D). To explore the mechanism of RBM39 dependence in AML, we performed proteomic analyses of RBM39 interacting proteins in MOLM-13 cells as well as transcriptome-wide analysis of RBM39 RNA binding by enhanced UV cross-linking and immunoprecipitation (eCLIP) in the same cells. RBM39 physically interacted with an entire network of RBPs identified by our CRISPR screen as crucial for AML cell survival in addition to interacting with the core SF3b splicing complex. Further, anti-RBM39 eCLIP revealed RBM39 binding to exonic regions and most enriched at exon/intron borders at 5' and 3' splice sites of pre-mRNA (Fig. E), suggesting a prominent role of RBM39 in regulating splicing. Consistent with this, RNA-sequencing of AML cells following RBM39 deletion revealed significant effects of RBM39 loss on RNA splicing, most prominently causing increased cassette exon skipping (Fig. F). Recent studies suggest that myeloid leukemias with mutations in RNA splicing factors are sensitized to pharmacologic perturbation of RNA splicing. Analysis of the effects of RBM39 degrading compounds over a panel of 18 AML cells revealed that leukemia cells bearing splicing factor mutations or with high DCAF15 expression were the most sensitive to treatment (Fig. G). Genetic introduction of SF3B1, SRSF2, or U2AF1 hotspot mutations in K562 or NALM6 cells resulted in a 20-50% reduction in IC50 in response to sulfonamides. We next performed RNA sequencing of isogenic K562 cells with or without knockin of SF3B1K700E and SRSF2P95H mutations into the endogenous loci, and treated at the IC50 of E7820 or E7107, a small molecule that inhibits the SF3b core spliceosome complex. Treatment with either drug dramatically increased cassette exon skipping events and intron retention relative to DMSO control, with greater effects in splicing mutant cells. However, at equipotent doses, E7820 markedly increased mis-splicing compared with E7107. Furthermore, E7820 treatment resulted in mis-splicing of a number of RBP targets identified in our CRISPR screen as being required for AML survival, including SUPT6H, hnRNPH, and SRSF10, as well as RBM3 and U2AF2, consistent with previous observations (Fig. H). Here through systematic evaluation of RBPs across several cancers, we identify RBPs specifically required in AML. In so doing we identify a network of functionally and physically interacting RBPs upregulated in AML over normal precursors. Genetic or pharmacologic elimination one such RBP, RBM39, led to aberrant splicing of multiple members of this RBP network as well as of transcriptional regulators required for AML survival. These data suggest important clinical potential for anti-cancer sulfonamide treatment in splicing mutant myeloid leukemias. Disclosures Uehara: Eisai: Employment. Owa:Eisai: Employment.

Description: Recent studies of the spectrum of somatic genetic alterations in acute myeloid leukemia (AML) have identified frequent somatic mutations in genes that encode proteins important in the epigenetic regulation of gene transcription. This includes proteins involved in the modification of DNA cytosine residues and enzymes which catalyze posttranslational modifications of histones. Here we describe the clinical, biological, and therapeutic relevance of mutations in epigenetic regulators in AML. In particular, we focus on the role of loss-of-function mutations in TET2, gain-of-function mutations in IDH1 and IDH2, and loss-of-function mutations in ASXL1 and mutations of unclear impact in DNMT3A in AML pathogenesis and therapy. Multiple studies have consistently identified that mutations in these genes have prognostic relevance, particularly in intermediate-risk AML patients, arguing for inclusion of mutational testing of these genetic abnormalities in routine clinical practice. Moreover, biochemical, biological, and epigenomic analyses of the effects of these mutations have informed the development of novel therapies which target pathways deregulated by these mutations. Our understanding of the effects of these mutations on hematopoiesis and potential for therapeutic targeting of specific AML subsets is also reviewed here.

Description: The Blood and Marrow Transplant Clinical Trials Network conducted 2 parallel multicenter phase 2 trials for individuals with leukemia or lymphoma and no suitable related donor. Reduced intensity conditioning (RIC) was used with either unrelated double umbilical cord blood (dUCB) or HLA-haploidentical related donor bone marrow (Haplo-marrow) transplantation. For both trials, the transplantation conditioning regimen incorporated cyclophosphamide, fludarabine, and 200 cGy of total body irradiation. The 1-year probabilities of overall and progression-free survival were 54% and 46%, respectively, after dUCB transplantation (n = 50) and 62% and 48%, respectively, after Haplo-marrow transplantation (n = 50). The day +56 cumulative incidence of neutrophil recovery was 94% after dUCB and 96% after Haplo-marrow transplantation. The 100-day cumulative incidence of grade II-IV acute GVHD was 40% after dUCB and 32% after Haplo-marrow transplantation. The 1-year cumulative incidences of nonrelapse mortality and relapse after dUCB transplantation were 24% and 31%, respectively, with corresponding results of 7% and 45%, respectively, after Haplo-marrow transplantation. These multicenter studies confirm the utility of dUCB and Haplo-marrow as alternative donor sources and set the stage for a multicenter randomized clinical trial to assess the relative efficacy of these 2 strategies. The trials are registered at www.clinicaltrials.gov under NCT00864227 (BMT CTN 0604) and NCT00849147 (BMT CTN 0603).

Description: Mutations in the RNA splicing factor SF3B1 are common in MDS and other myeloid malignancies. SF3B1 mutations promote expression of mRNAs that use an aberrant, intron proximal 3' splice site (ss). Despite the consistency of this finding, linking aberrant splicing changes to disease pathogenesis has been a challenge. Here we identify aberrant splicing and downregulated expression of BRD9, a member of the recently described ATP-dependent non-canonical BAF (ncBAF) chromatin remodeling complex, across SF3B1 mutant leukemias. In so doing, we identify a novel role for altered ncBAF function in hematopoiesis and MDS. To systematically identify functionally important aberrant splicing events created by mutant SF3B1, we integrated differential splicing events in SF3B1 mutant versus wild-type MDS with a positive enrichment CRISPR screen mimicking splicing changes induced by mutant SF3B1 that promote NMD (non-sense mediated mRNA decay). We tested whether loss of any gene functionally inactivated by SF3B1 mutations promoted transformation of Ba/F3 and 32D cells. This identified a specific NMD-inducing aberrant splicing event in BRD9 which promoted cytokine independence (Fig. A) and exhibited striking aberrant splicing across CLL and MDS and across all mutational hotspots in SF3B1 (Fig. B). SF3B1 mutations cause exonization of a normally intronic sequence in BRD9, resulting in inclusion of a poison exon that interrupts BRD9's reading frame (Fig. C) and reduced BRD9 mRNA and protein expression through NMD (Fig. D). We confirmed that mutant SF3B1 suppressed full-length BRD9 levels without generating truncated BRD9 protein. Loss of BRD9 impaired ncBAF complex formation as indicated by abolished interaction between the ncBAF specific component GLTSCR1 and the ATPase subunit BRG1 upon chemical or spliceosomal BRD9 ablation (Fig. D). Given that prior work has linked mutant SF3B1 to use of aberrant 3' ss, we sought to understand the molecular basis for aberrant exon inclusion in BRD9 by mutant SF3B1. Lariat sequencing of SF3B1 mutant versus WT K562 cells and BRD9 minigene analyses identified use of a deep intronic branchpoint adenosine by mutant SF3B1 to promote BRD9 poison exon inclusion (Fig. E). The data above suggest a role for BRD9 downregulation in SF3B1 mutant leukemia. While prior work indicated that BRD9 is required in MLL-rearranged AML (Hohmman et al. Nature Chemical Biology 2016), the role of BRD9 in normal hematopoiesis or other subtypes of myeloid neoplasms has not been evaluated. Genetic downregulation of BRD9 in normal human hematopoietic progenitors from cord blood promoted myelopoiesis while impairing megakaryopoiesis. Interestingly and unexpectedly, BRD9 loss in CD34+ cells promoted terminal erythroid differentiation in vitro. To further evaluate BRD9's role in hematopoiesis in vivo, we also generated mice with inducible knockout of the bromodomain of BRD9 (required for BRD9 function) and generation of a frameshift transcript resulting in reduced Brd9 expression (Fig. F). Loss of Brd9 resulted in macrocytosis with bone marrow erythroid dysplasia in a dosage-dependent manner, along with impaired lymphopoiesis and myeloid skewing. Moreover, competitive transplantation of hematopoietic precursors from these mice revealed that ablation of Brd9 function impaired lymphoid reconstitution while promoting advantage of myeloid cells and hematopoietic precursors (Fig. G-I). In myeloid leukemia cells, introduction of SF3B1K700E or downregulation of BRD9 resulted in increased chromatin accessibility at promoters with a significant overlap in commonly upregulated genes. This finding suggests shared epigenetic effects of SF3B1K700E mutations and BRD9 loss (Fig. J). These data identify aberrant splicing of BRD9 across the spectrum of SF3B1 mutant cancers and identify a novel role for downregulation of ncBAF function in MDS pathogenesis. Consistent with human genetic data, genetic ablation of BRD9 function in mouse and human hematopoietic cells resulted in myeloid skewing and dyserythropoiesis. These data suggest that targeted correction of aberrant BRD9 splicing might serve as a novel therapeutic approach for SF3B1-mutant leukemias. Of note, treatment with drugs impairing the binding of mutant SF3B1 to RNA resulted in a dose-dependent rescue of aberrant BRD9 splicing in vitro (Fig. K) and in treatment of an SF3B1 mutant AML patient-derived xenograft in vivo. Figure Disclosures Kadoch: Foghorn Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Background Hypomethylating agent (HMA) therapy represents the standard of care for patients with higher risk myelodysplastic syndromes (MDS) although only 50% of patients respond to treatment. Recent evidence from molecular profiling through next-generation sequencing (NGS) in myeloid diseases has been conflicting as to the value of somatic mutations as a biomarker for response to HMA. In particular, there have been conflicting data on response rates and outcomes in TP53 mutant (MT) MDS and acute myeloid leukemia (AML) based on azacitidine versus decitabine (Welch et al., NEJM 2016; Garcia-Manero et al., NEJM 2017). However, the TP53 mutant cohorts in these studies were small (median 23 patients, range 13-39) and heterogeneous based on treatment status (treatment naïve versus relapse/refractory). Therefore, our goal was to characterize outcomes of TP53 mutant MDS patients who received frontline HMA therapy. Patients and Methods TP53 MT MDS and oligoblastic AML (20-30% blasts) cases were retrospectively identified from the Moffitt Cancer Center MDS database. All patients had NGS of TP53 and up to 53 additional genes performed prior to the initiation of HMA. The lower limit of VAF detection was set at 5% and the minimum depth of coverage at each position was 500X. Clinical variables and outcomes of MDS patients were characterized at the time of sample procurement. Fisher's exact tests were used for comparative analyses. Kaplan-Meier curves were used to estimate overall survival (OS) and analyzed from the date of HMA initiation. Response rates and outcomes of TP53 MT patients were compared to a cohort of wildtype (WT) patients (n=63). Results From May 2013 to May 2018, a total of 71 patients with TP53 mutant MDS were identified with a median age of 68 years (39-82) and male predominance (66%). Fourteen patients (20%) had multiple mutations in TP53. Of the cohort, 82% of patients (n=58) were treated with azacitidine (88% (n=51) with AZA monotherapy; 12% (n=7) with AZA in combination (2 pts with lenalidomide and 5 pts with investigational agents)) with 18% (n=13) receiving decitabine. The median # of HMA cycles was 4 (range 1-33). Thirteen pts (18%) proceeded to allogeneic hematopoietic stem cell transplant (HSCT). Of the cohort, 18% (n=13) obtained complete remission (CR) with 39% (n=28) overall response rate (ORR). There was no difference in CR or ORR in pts treated with AZA vs DAC (P=0.24 and P=0.2, respectively). At a median follow up 20 months, the median OS of the entire cohort was 9.7 months. There was no difference in median OS between AZA vs AZA combo vs DAC (7.6 vs 15.2 vs 12.5 months; P = 0.44; Figure 1A). TP53 variant allele frequency (VAF 〉 20% vs ≤ 20%) was not predictive of outcomes to HMA (7.8 vs 10.4 months, P = 0.36). However, TP53 MT patients who had clonal response to HMA (i.e. VAF 〈 5%; n=19 (27%)) had improved OS (14.5 vs 7.5 months; HR 0.33, 95% CI 0.18 to 0.59; P = 0.001; Figure 1B). In multivariable analysis incorporating age, revised international prognostic scoring system (IPSS-R) category, HSCT status, or type of HMA, TP53 clonal clearance remained an independent covariate for improved OS (HR 0.34, 95% CI 0.16 to 0.72; P = 0.005). Pts who underwent HSCT (n=13) had a trend for improved OS (14.5 months vs 7.9 months; P = 0.09). Notably in transplanted pts who had serial TP53 NGS (n=7), pts who achieved a VAF 〈 5% had significant improved OS (16.3 months vs 8.9 months; P=0.03). Compared to higher risk MDS/AML TP53 WT patients treated with HMA, there was no difference in CR (18% vs 14% (P = 0.64) or ORR rates (39% vs 40%). In contrast, TP53 MT patients had significantly inferior OS with HMA therapy (9.7 vs 15.4 months; HR 2.14, 95% CI 1.32. to 3.27; P = 0.001; Figure 1C). Conclusion In this large cohort of higher risk MDS and oligoblastic AML pts who received frontline HMA therapy, TP53 MT patients have significantly inferior OS with no significant differences in response rates or outcomes by HMA. TP53 MT patients who achieve maximum clonal suppression with HMA treatment (i.e. VAF 〈 5%) have improved OS as well as improved outcome with HSCT. Novel therapy targeting TP53 mutation is needed to improve outcomes. Figure 1. Figure 1. Disclosures Sallman: Celgene: Research Funding, Speakers Bureau. Sweet:Agios: Consultancy; Jazz: Speakers Bureau; Astellas: Consultancy; Phizer: Consultancy; Phizer: Consultancy; Astellas: Consultancy; Jazz: Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Agios: Consultancy; BMS: Honoraria; Celgene: Honoraria, Speakers Bureau; BMS: Honoraria. List:Celgene: Research Funding. Komrokji:Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau.

Description: Background.This study aimed to determine the progression-free survival and response rate using early therapeutic intervention in patients with high-risk smoldering multiple myeloma (SMM) using the combination of ixazomib, lenalidomide, and dexamethasone. Methods.Patients enrolled on study met eligibility for high-risk SMM based on the newly defined criteria proposed by Rajkumar et al. (Blood 2014). The treatment plan was designed to be administered on an outpatient basis where patients receive 9 cycles of induction therapy of ixazomib (4mg) at days 1, 8, and 15, in combination with lenalidomide (25mg) at days 1-21 and dexamethasone at days 1, 8, 15, and 22. The induction phase was followed by ixazomib (4mg) and lenalidomide (15mg) maintenance for another 15 cycles. A treatment cycle was defined as 28 consecutive days for a total of 24 months period. Bone marrow samples of all patients were obtained before starting therapy for baseline assessment for minimal residual disease (MRD) testing, whole-exome sequencing (WES), and RNA sequencing of plasma and bone marrow microenvironment cells. Moreover, blood samples were obtained at screening and before each cycle for isolating cell-free DNA (cfDNA) and circulating tumor cells (CTCs). Results.In total, 53 of the planned 62 patients have been enrolled in this study from February 2017 to May 2019. The median age of the patients enrolled was 61 years (range, 41 to 84) with 22 male (41.5%). The analysis was conducted on patients who have completed at least 1 cycle of therapy (n=45). The median follow-up for the trial is 14.4 months (range: 2- 27.6). Interphase fluorescence in situ hybridization (iFISH) was successful in 37 patients (82.2%). High-risk cytogenetics (defined as the presence of t(4;14), 17p deletion, and 1q gain) were found in 20 patients (54%). The median number of cycles completed was 14 cycles (range: 1-24). According to the study's inclusion criteria, baseline markers showed that 15, 14, and 13 patients had 3, 4, and 5 high-risk features, respectively. Moreover, 24 patients (53.3%) met the criteria of high-risk SMM, according to the Mayo 2018 model. The most common grade 3 adverse events were hypertension (6.3%), hypophosphatemia (4.2%), and rash (4.2%). Grade 4 thrombocytopenia and neutropenia were each reported in 4.4% of patients, and hyperglycemia was reported in 2.2%. Stem cells were collected in all eligible patients by the end of the induction phase. As of the abstract date, the overall response rate (partial response or better) in participants who completed at least 1 cycle of treatment was 91.1% (41/45), with 14 Complete Responses (CR, 31.1%), 9 very good partial responses (VGPR, 20%), 18 partial responses (40%), and 4 minimal Responses (MR, 10%). ORR in patients who completed the induction phase (≥9 cycles) was 97% (n= 32/33), with 14(42.4%) and 9 (27.2%) having CR and VGPR, respectively. All patients who had a CR have also achieved a stringent CR. Six patients have completed the treatment protocol and are currently on follow-up. As of July 2019, none of the patients have progressed to overt MM. MRD testing by next-generation sequencing is ongoing for patients who achieved CR or VGPR and will be presented at the meeting. Conclusion.The combination of ixazomib, lenalidomide, and dexamethasone is an effective and well-tolerated intervention in high-risk smoldering myeloma with 91% ORR and 54.7% CR and VGPR to date. The high response rate, convenient schedule and manageable toxicity build on prior studies which have shown efficacy of lenalidomide and dexamethasone in high risk smoldering myeloma. Longer follow-up for disease outcome is ongoing. Disclosures Bustoros, MD: Takeda: Honoraria. Nadeem:Celgene: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Sanofi: Consultancy. Prescott:Janssen: Equity Ownership. Munshi:Takeda: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Adaptive: Consultancy; Abbvie: Consultancy; Abbvie: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Amgen: Consultancy; Janssen: Consultancy. Anderson:OncoPep: Other: Scientific founder ; C4 Therapeutics: Other: Scientific founder ; Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; Sanofi-Aventis: Other: Advisory Board. Richardson:Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Ghobrial:Amgen: Consultancy; Celgene: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; Takeda: Consultancy. OffLabel Disclosure: Ixazomib, Lenalidomide and Dexamethasone is an investigational combination in high-risk smoldering multiple myeloma and has not been approved by the US Food and Drug Administration or any other regulatory agency worldwide for the use under investigation.

Description: The Congenital Dyserythropoietic Anemia Registry (CDAR, ClinicalTrials.gov Identifier: NCT02964494) was created to investigate the natural history, biology, and molecular pathogenetic mechanisms of CDA. To date, there are 6 genes known to cause CDA (CDAN1, C15orf41, SEC23B, KIF23, KLF1, GATA1). However, 57% of patients registered in CDAR so far (17 out of 33 patients) have an unidentified genetic cause. We have utilized whole exome sequencing (WES) in family-trio design to search for novel candidate gene mutations that may be responsible for the disease. Three unrelated patients with dyserythropoiesis, hemolytic anemia, and neurodevelopmental delay were found to have missense mutations in the gene VPS4A which encodes an ATPase that participates with the ESCRT III machinery in endosomal vesicle trafficking, centrosome localization, and the abscission step of cytokinesis. It has been shown to play an essential role in division of HeLa cells in vitro where it concentrates at the spindle poles during mitosis and at the midbody during cytokinesis. The aim of this work is to validate the pathogenetic role of these VPS4A variants in CDA and further investigate the role of VPS4A in erythropoiesis. Patients 1 and 3 had de novo mutations (R284W and G203A) and transfusion-dependent anemia with presence of binucleated erythroblasts in the bone marrow resembling CDA type I. Of note, the patients' erythroblasts exhibited cytoplasmic bridges (Figure 1A) rather than the nuclear chromatin bridges observed in CDA-I. Patient 2, offspring of consanguineous parents, presented with hemolytic anemia and was found to have a homozygous mutation (A28V) in a highly conserved alanine residue in the microtubule-interacting domain (MIT) of VPS4A. She had rare evidence of dyserythropoiesis with fewer than 3% binucleated erythroblasts in bone marrow studies. All three patients had significant neurodevelopmental delay with axial hypotonia and appendicular hypertonia. Flow cytometry analysis of peripheral blood from each of these patients revealed a unique cell population which is negative for RNA (by thiazole orange) but still CD71 positive suggesting that loss of VPS4A function also impacts reticulocyte maturation, likely because of defective endosomal vesicle trafficking. Using CD34+ cells in ex vivo erythropoiesis cultures, we first confirmed that VPS4A is expressed in human erythroblasts and localizes at the spindle poles and midbody during mitosis and cytokinesis in these cells. RNA isolated from reticulocytes from patients 1 and 2 was assessed for expression of VPS4A and the paralogous VPS4B. Samples from patient 1 had reduced expression of VPS4A (