Kurzfassung: Abstract 607 Developing erythrocytes acquire large amounts of iron through the transferrin (Tf) cycle for heme synthesis. The Tf cycle involves unidirectional transport of transferrin-transferrin receptor 1 (Tf-TfR1) complexes from the plasma membrane to the early and recycling endosomes (Figure). Besides the requirement for the basic trafficking machinery, specific sorting molecules exist to ensure the efficient re-cycling of Tf-TfR1 complexes and targeted iron delivery. The trafficking of TfR1 from recycling endosomes to the cell surface was shown to be mediated by Sec15L1, an exocyst component, as its mutation causes anemia in the hemoglobin deficit (hbd) mouse. The sorting mechanisms responsible for earlier trafficking steps in intracellular transferrin cycle, however, are poorly understood. Here we report that sorting nexin 3 (SNX3), a cargo-specific retromer component, facilitates the endocytic recycling of TfR1, and thus, is required for the proper delivery of iron to erythroid progenitors for heme synthesis (Figure). Snx3 is highly expressed in hematopoietic tissues of zebrafish and mouse. Morpholino-mediated knockdown of snx3 in zebrafish embryos leads to a profound anemia. shRNA silencing of Snx3 in mouse primary fetal liver cells and mouse Friend erythroleukemia (MEL) cells inhibits the production of hemoglobin. We demonstrate that these defects are due to impaired transferrin-mediated iron uptake and delivery to the mitochondria. The impaired iron assimilation can be complemented with non-transferrin bound iron chelates, such as Fe-SIH (salicylaldehyde isonicotinoyl hydrazone). Furthermore, we show that SNX3 may act through direct physical interaction with TfR1 to sort Tf-TfR1 complexes to the recycling endosomes. Our data from genetic, biochemical, and chemical biological studies collectively show that SNX3 regulates TfR1 trafficking and iron homeostasis in developing erythrocytes. The identification of SNX3 as an essential co-regulatory protein that regulates Tf-mediated iron delivery for heme synthesis provides a new genetic tool for exploring human disorders of iron metabolism, such as the hypochromic anemias, and erythropoiesis. * Cell Metabolism (in revision). Disclosures: No relevant conflicts of interest to declare.

Kurzfassung: Less-intensive induction therapies are increasingly used in older patients with acute myeloid leukemia (AML). Using an AML composite model (AML-CM) assigning higher scores to older age, increased comorbidity burdens, and adverse cytogenetic risks, we defined 3 distinct prognostic groups and compared outcomes after less-intensive vs intensive induction therapies in a multicenter retrospective cohort (n = 1292) treated at 6 institutions from 2008 to 2012 and a prospective cohort (n = 695) treated at 13 institutions from 2013 to 2017. Prospective study included impacts of Karnofsky performance status (KPS), quality of life (QOL), and physician perception of cure. In the retrospective cohort, recipients of less-intensive therapies were older and had more comorbidities, more adverse cytogenetics, and worse KPS. Less-intensive therapies were associated with higher risks of mortality in AML-CM scores of 4 to 6, 7 to 9, and ≥10. Results were independent of allogeneic transplantation and similar in those age 70 to 79 years. In the prospective cohort, the 2 groups were similar in baseline QOL, geriatric assessment, and patient outcome preferences. Higher mortality risks were seen after less-intensive therapies. However, in models adjusted for age, physician-assigned KPS, and chance of cure, mortality risks and QOL were similar. Less-intensive therapy recipients had shorter length of hospitalization (LOH). Our study questions the survival and QOL benefits (except LOH) of less-intensive therapies in patients with AML, including those age 70 to 79 years or with high comorbidity burdens. A randomized trial in older/medically infirm patients is required to better assess the value of less-intensive and intensive therapies or their combination. This trial was registered at www.clinicaltrials.gov as #NCT01929408.

Kurzfassung: Red cells synthesize large amounts of heme during terminal differentiation. Central to this process is the transport and trafficking of heme synthesis intermediates within the cell. Despite the importance of transport during heme synthesis, the molecules involved in this process are largely unknown. In a screen for genes that are upregulated during erythroid terminal differentiation, we identified Tmem14c, a predicted multi-pass transmembrane protein as an essential component of the porphyrin metabolism pathway. Here, we report that Tmem14c facilitates the synthesis of mitochondrial protoporphyrin IX from coproporphyrinogen III and is thus required for heme synthesis. Tmem14c is a mitochondrial inner-membrane protein enriched in vertebrate hematopoietic tissues and is required for terminal erythropoiesis. Tmem14c gene-trap mouse embryos are severely anemic and mostly die by E13.5 (Fig. A). Fetal liver erythroid cells derived from gene-trap embryos experience maturation arrest. shRNA silencing of Tmem14c in Friend murine erythroleukemia (MEL) cells results in a significant decrease in de-novo heme synthesis. The biochemical defect is due to a decrease in mitochondrial protoporphyrin IX synthesis, while cytoplasmic porphyrin levels remain normal (Fig. B). The heme synthesis defect in Tmem14c-silenced MEL cells is complemented with a protoporphyrin IX analog. These data show the role of Tmem14c in regulating the terminal steps in mitochondrial porphyrin trafficking. Our findings collectively demonstrate that Tmem14c is required for the transport of mitochondrial porphyrins in developing erythroid cells. Due to its inner-mitochondrial localization and its relative proximity to heme synthetic enzymes coproporphyrinogen oxidase and protoporphyrinogen oxidase (Rhee et al., 2013 Science), Tmem14c can function as a molecular adaptor that facilitates the interaction of proteins involved in porphyrin transport, or as a protoporphyrinogen IX transporter (Fig. C). The identification of Tmem14c as an essential regulator of porphyrin transport and heme synthesis provides a novel genetic tool for exploring erythropoiesis and disorders of heme synthesis such as porphyria and anemia. Disclosures: No relevant conflicts of interest to declare.

Kurzfassung: Protamine sulfate is a positively charged polypeptide widely used to reverse heparin-induced anticoagulation. Paradoxically, prospective randomized trials have shown that protamine administration for heparin neutralization is associated with increased bleeding, particularly after cardiothoracic surgery with cardiopulmonary bypass. The molecular mechanism(s) through which protamine mediates this anticoagulant effect has not been defined. In vivo administration of pharmacologic doses of protamine to BALB/c mice significantly reduced plasma thrombin generation and prolonged tail-bleeding time (from 120 to 199 seconds). Similarly, in pooled normal human plasma, protamine caused significant dose-dependent prolongations of both prothrombin time and activated partial thromboplastin time. Protamine also markedly attenuated tissue factor-initiated thrombin generation in human plasma, causing a significant decrease in endogenous thrombin potential (41% ± 7%). As expected, low-dose protamine effectively reversed the anticoagulant activity of unfractionated heparin in plasma. However, elevated protamine concentrations were associated with progressive dose-dependent reduction in thrombin generation. To assess the mechanism by which protamine mediates down-regulation of thrombin generation, the effect of protamine on factor V activation was assessed. Protamine was found to significantly reduce the rate of factor V activation by both thrombin and factor Xa. Protamine mediates its anticoagulant activity in plasma by down-regulation of thrombin generation via a novel mechanism, specifically inhibition of factor V activation.

Kurzfassung: We designed a prospective, observational study enrolling patients presenting for treatment of acute myeloid leukemia (AML) at 13 institutions to analyze associations between hematopoietic cell transplantation (HCT) and survival, quality of life (QOL), and function in: the entire cohort, those aged ≥65 years, those with high comorbidity burden, intermediate cytogenetic risk, adverse cytogenetic risk, and first complete remission with or without measurable residual disease. Patient were assessed 8 times over 2 years. Time-dependent regression models were used. Among 692 patients that were evaluable, 46% received HCT with a 2-year survival of 58%. In unadjusted models, HCT was associated with reduced risks of mortality most of the subgroups. However, after accounting for covariates associated with increased mortality (age, comorbidity burden, disease risks, frailty, impaired QOL, depression, and impaired function), the associations between HCT and longer survival disappeared in most subgroups. Although function, social life, performance status, and depressive symptoms were better for those selected for HCT, these health advantages were lost after receiving HCT. Recipients and nonrecipients of HCT similarly ranked and expected cure as main goal of therapy, whereas physicians had greater expectations for cure than the former. Accounting for health impairments negates survival benefits from HCT for AML, suggesting that the unadjusted observed benefit is mostly owing to selection of the healthier candidates. Considering patients’ overall expectations of cure but also the QOL burdens of HCT motivate the need for randomized trials to identify the best candidates for HCT. This trial was registered at www.clinicaltrials.gov as #NCT01929408.

Kurzfassung: This dose-escalation study was performed to evaluate the hematologic activity, biological effects, immunogenicity, and toxicity of PIXY321 (an interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein) administered after high-dose carboplatin (CBDCA) treatment. Patients with advanced cancers received CBDCA at 800 mg/m2 intravenously on day 0 of repeated 28-day cycles. In part A of the study, patients were treated with CBDCA alone during cycle 1 and then received PIXY321 on days 1 through 18 of cycle 2 and later cycles. In part B, patients received 18 days of PIXY321 beginning on day 1 of all CBDCA cycles, including cycle 1. PIXY321 was administered subcutaneously in 2 divided doses. Total doses of 135, 250, 500, 750, and 1,000 μg/m2/d were administered to successive cohorts of 3 to 6 patients in part A. In part B, patient groups received PIXY321 doses of 750, 1,000, and 1,250 μg/m2/d. The hematologic effects of PIXY321 were assessed in the first 2 cycles of therapy. Anti-PIXY321 antibody formation was assessed by enzyme-linked immunosorbent assay (ELISA) and neutralization assay. Of the 49 patients enrolled, 31 were fully evaluable for hematologic efficacy. When comparing the first B cycle (cycle B-1; with PIXY321) with the first A cycle (cycle A-1; without PIXY321), the fusion protein had no significant effect on platelet nadirs or duration of platelets less than 20,000/μL but was able to speed the time of recovery of platelet counts to 100,000/μL (15v 20 days; P = .01). Significant improvements in neutrophil nadir and duration of ANC less than 500 were observed in cycles A-2 and B-1 (with PIXY321) as compared with cycle A-1 (without PIXY321). Initial PIXY321 prophylaxis (cycle A-2 and cycle B-1), enhanced the recovery of ANC to greater than 1,500/μL by an average of at least 8 days as compared with cycle A-1 (without PIXY321;P ≤ .004). However, positive PIXY321 hematologic effects were lost in the second course of PIXY321 among patients treated in part B. ELISA analysis showed that 92% of patients had developed neutralizing anti-PIXY321 antibodies by the completion of 2 PIXY321-containing cycles. The incidental action of PIXY321 to depress serum cholesterol levels was also abrogated during cycle B-2. We conclude that PIXY321 was active in speeding hematologic recovery but that neutralizing anti-PIXY321 antibody formation suppressed the hematologic and biochemical effects by the second cycle of PIXY321 administration. The immunogenicity of this fusion protein provides a cautionary warning that clinical development of bioengineered human molecules requires thorough testing for immune neutralization.

Kurzfassung: Congenital dyserythropoietic anemia (CDA) type II is the most frequent type of congenital dyserythropoietic anemia; it is transmitted in an autosomal recessive fashion and is characterized by ineffective erythropoiesis, peripheral hemolysis, bi-multinuclearity in the erythroblasts, and hypoglycosylation of red blood cell (RBC) membrane proteins such as band 3. The disease is generally caused by biallelic mutations in the SEC23B gene. However, there are a small portion of patients with clinical and hematologic features of CDA II that are negative for mutations in SEC23B, suggesting that alternative etiologies for such disturbed erythropoiesis exist. We identified two siblings of Italian origin who had dyserythropoiesis with a chronic macrocytic anemia. Their parents were healthy with normal hematologic parameters. No history of consanguinity for at least three generations was noted. The affected siblings had anisopoikylocytosis on peripheral blood smear with stomatocytes (8-9%), spherocytes (4-5%), rare ovalocytes, and dacryocytes. RBCs osmotic fragility was increased but the red cells had normal eosin-5-maleimide (EMA)-binding. Serum ferritin and transferrin saturation were increased in only one sibling. Bone marrow morphology revealed erythroid hyperplasia (myeloid: erythroid ratio = 0.6) with binuclearity and megaloblastic changes, as well as occasional cytoplasmic bridging between cells at different stage of maturation; electron microscopy of bone marrow erythroblasts showed multiple membranes that ran parallel to the plasma membrane or that were grouped in stacked segments, possibly attributable to residual endoplasmic reticulum (ER) cisternae. SDS-PAGE analysis of RBC ghosts from both siblings demonstrated hypoglycosylation of band 3 and GLUT1, as well as residual residual Protein Disulphide Isomerase (PDI) positive ER remnants, as observed in classical CDA II cases. However, in contrast to CDAII, the Ham's test performed with 15 normal serum samples was negative, and no mutations were detected in the SEC23B gene. To uncover the underlying etiologies, whole-exome sequencing was conducted on all available family members. After filtering for common variants, only a single gene had biallelic mutations in the affected siblings, which were transmitted from the unaffected heterozygous parents. The identified mutations resided in the PARP4 gene, which encodes a poly-ADP ribose polymerase enzyme, and were predicted to be deleterious. We demonstrate that knockdown of PARP4 using shRNA in primary human erythroid progenitors results in impaired erythroid differentiation and increased apoptosis. In addition, morpholino-mediated knockdown of the PARP4 orthologue in the zebrafish resulted in dyserythropoiesis and anemia in developing embryos. Sequencing of PARP4 in additional rare cases of CDA II without an identified molecular basis will help to uncover the frequency and spectrum of PARP4 mutations leading to dyserythropoiesis. The finding of a new gene implicated in a similar type of CDA with features such as redundant ER membranes offers the potential for more mechanistic dissection of the role of both SEC23B and PARP4 in erythroid development and suggests that new insight can be gained into the underlying pathophysiology of both normal and disordered erythropoiesis through the study of such rare cases. Disclosures No relevant conflicts of interest to declare.

Kurzfassung: Introduction: Survival rates continue to improve after allogeneic HCT (Gooley et al, NEJM, 2013). Population-based studies also indicate overall improvement in survival of older (60-80 years old) AML patients (pts) (Bower, Blood Cancer Journal, 2016). Yet, only a small minority (6%-8%) of them receive HCT (Medeiros, Ann Hematol. 2015). Given these potentially incongruent findings and the changing face of survival in AML, we designed the first prospective multi-center longitudinal study dating from first presentation of adults with AML to be treated at one of 13 different referral centers that provide both AML treatment and HCT. We compared survival according to whether or not pts received HCT at later time points. Methods: We enrolled 695 pts (Table 1). Data on demographics, AML status, cytogenetic risks per European Leukemia Network (ELN), and response; age; comorbidities per the HCT-comorbidity index (CI); function including activities of daily living (ADL); frailty; geriatric assessment including cognition; QOL including the Functional Assessment of Cancer Therapy-Bone Marrow Transplant Scale (FACT-BMT), Euro-QOL 5-Dimension scale, ENRICHD Social Support Instrument, Social Activity Log, and Patient Health Questionnaire 9-item Depression Scale (PHQ-9) were collected at enrollment and at 1, 3, 6, 9, 12, 18, and 24 months thereafter. We used time-dependent Cox regression analyses to identify baseline and time-dependent risk factors associated with mortality in the overall population. The factors identified as significantly associated with mortality (p 〈 0.05) were used to develop multivariate models examining the association between HCT and mortality within 1) the general population as well as those with 2) intermediate vs 3) unfavorable ELN risk, and 4) vulnerable pts (age ≥60 years or HCT-CI scores ≥4). The latter group constituted the majority (76%). In these analyses, all pts were considered to be in the non-HCT group until receipt of HCT at which time they enter the HCT group. The contribution of deaths to the hazard ratio (HR) for HCT reflects the relative number and characteristics of pts remaining at risk in the two groups at the time a death occurs. Results: Median follow-up was 16.8 months (range 0.1-52.4). In the initial multivariate analyses, the following were identified as significantly associated with an increased risk of mortality (Table 2): HCT-CI scores ≥5 (p 〈 0.0001), age ≥70 years (p 〈 0.0001), intermediate (p=0.03) and high ELN risk (p 〈 0.0001), relapsed/refractory AML at enrollment (p=0.0005), relapse or refractory response to initial treatment after enrollment (p 〈 0.0001), frailty per walk test (p=0.004), impaired QOL per FACT-G scores (p=0.02), increased depression per PHQ-9 (p=0.03), and dependent status per ADL scores 〈 14 (p=0.05). Survival after HCT was 58% at 2-years. Initial unadjusted analyses showed significantly lower risks of mortality in association with receiving allogeneic HCT (p=0.0003). These findings were similar in pts with intermediate (p=0.0005) or unfavorable (p 〈 0.0001) ELN risk and in vulnerable pts (p 〈 0.0001) (Table 3). However, in the adjusted models, the advantage of HCT in reducing mortality rates was lost both in the overall population (p=0.21, see figure) as well as in the other groups (p 〉 0.54, 0.40, and 0.51, respectively, Table 3). Formal tests of interactions (Table 3) showed no statistically compelling evidence that the association of HCT and mortality varies with respect to the timing of mortality or to the underlying ELN risk. Conclusions: In a prospective observational study, adjusting for key AML-specific and pt-specific variables negated the observed benefit of HCT over non-HCT therapies in reducing mortality rates among AML pts. Our results might reflect 1) improvement in supportive care and non-HCT therapies, 2) a relatively high non-relapse mortality early after HCT and the need for longer follow-up to demonstrate an adjusted benefit of HCT, and 3) the high selectivity of the transplant eligibility process, as we accounted here for variables that are often ignored in "genetic assignment" randomized studies (i.e. comorbidities and function). New randomized trials are needed; however, these trials have to be more inclusive of vulnerable pts and measure pt-specific variables. Trials focusing on reducing burden of comorbidities, frailty and poor function are needed alongside trials to treat AML with or without HCT. Disclosures Gerds: Celgene: Consultancy; Apexx Oncology: Consultancy; CTI Biopharma: Consultancy; Incyte: Consultancy. Shami:JSK Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Baston Biologics Company: Membership on an entity's Board of Directors or advisory committees; Lone Star Biotherapies: Equity Ownership; Pfizer: Consultancy. Rizzieri:Teva: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Arog: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Wang:Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Novartis: Speakers Bureau; Novartis: Speakers Bureau; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Faderl:Jazz Pharmaceuticals: Employment, Equity Ownership. Koprivnikar:Alexion: Consultancy, Speakers Bureau; Amgen: Speakers Bureau; Otsuka: Consultancy. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Becker:GlycoMimetics: Research Funding.

Kurzfassung: Megaloblastic anemia characterizes molecularly heterogeneous disorders, most commonly marked by macrocytosis, red blood cell enlargement, and abnormal nuclear maturation. Aside from vitamin B12 and folate deficiencies, genetic defects may be responsible for hereditary forms of megaloblastic anemia, though some remain unknown (Ghersim C, et al. 2013 J. Biol. Chem.). Loss of function of the gene Sideroflexin-4 (SFXN4) has been implicated as a cause for combined macrocytic anemia and mitochondriopathy. We have previously shown a defect in SFXN4 in humans and successfully modeled the erythroid and mitochondrial defects in the zebrafish (Hildick-Smith GJ, et al. 2013 AJHG). Here, we expand on prior studies involving SFXN4 in zebrafish and human fibroblasts by generating a murine cultured cell model deficient in SFXN4. Clustered regularly interspaced short palindromic repeats (CRISPR)-cas targeting technology (Cong L, et al. 2013 Science) was utilized to generate SFXN4 knockout Friend mouse erythroid leukemia (MEL) clones. We conducted a screen of clones using qRT-PCR to select MEL clones with the lowest expression of Sfxn4 mRNA. The CRISPR knockout clone shows significantly reduced Sfxn4 mRNA relative to wild type (Panel A). Furthermore, the CRISPR knockout's reduced Sfxn4 mRNA is comparable to Sfxn4 mRNA levels in clones silenced by shRNA hairpin constructs (data now shown). We also investigated the differentiation and maturation potential of the CRISPR-derived SFXN4 knockout clone. Initially, the MEL clone was differentiated for 3-5 days with DMSO treatment. o-dianisidine stains revealed that the CRISPR knockout clone had a reduction in hemoglobinization. Furthermore, Wright-Giemsa staining demonstrated that the same clone possessed megaloblastic features with immature maturation and a high nuclear to cytoplasmic ratio (Panel B). Both sets of results from experiments support the hypothesis for an essential role of SFXN4 in vertebrate red cell development. In summary, our data: (1) supports the hypothesis of SFXN4's involvement in erythropoiesis and candidacy as a gene for combined macrocytic anemia and mitochondriopathy, and (2) demonstrates the promise of shRNA knockdown and now CRISPR SFXN4 knockout clones as tools for further studying the biochemical relationship between SFXN4 and megaloblastic anemia. Disclosures: No relevant conflicts of interest to declare.

Kurzfassung: Congenital sideroblastic anemias (CSAs) are uncommon inherited diseases resulting from defects in heme biosynthesis, mitochondrial translation or mitochondrial iron-sulfur cluster (ISC) assembly. CSAs are characterized by pathological mitochondrial iron deposits in bone marrow erythroblasts. Recently, mutations in mitochondrialheat shock protein 70 (HSPA9), a critical chaperone involved in mitochondrial ISC assembly, have been reported as a cause of non-syndromic CSA. Human heat shock cognate protein 20 (HSCB), a highly conserved mitochondrial co-chaperone, is the primary binding partner of HSPA9. HSCB allows the transfer of nascent ISC to HSPA9 and stimulates its ATPase activity, promoting ISC transfer to target proteins. To identify novel genes responsible for CSA, we performed whole exome sequencing on more than 75 CSA probands and their family members. In one patient, a young woman, with pancytopenia characterized by a normocytic anemia with numerous bone marrow ringed sideroblasts, we identified two variants in HSCB : a paternally-inherited promoter variant (c.-134C & gt;A) predicted to disrupt a conserved ETS transcription factor binding site, and a maternally-inherited frameshift (c.259dup, p.T87fs*27). A fibroblast cell-line derived from the proband showed a decrease in HSCB expression, but normal HSPA9 expression compared to healthy, unrelated controls. Impairment of ETS1-dependent transcriptional activation of the promoter variant was demonstrated in K562 cells transfected with an HSCB-luciferase reporter construct. K562 cells were also employed to determine if reduced expression of HSCB could result in impaired erythroid metabolism, maturation, or proliferation. K562 cells infected with shRNA directed against HSCB were deficient in multiple mitochondrial respiratory complexes, had abnormal iron metabolism and a defect of protein lipoylation, all consistent with defective ISC metabolism. In addition, both IRP1 and IRP2 expression were decreased and cell surface transferrin receptor 1 (TFR1) expression was enhanced, suggesting disturbed cellular iron metabolism. Nevertheless, cells lacking HSCB partially retained an ability to respond to iron chelation and iron overload. Cells lacking HSCB lose their ability to hemoglobinize in response to sodium butyrate treatment (Figure 1A). This defect was confirmed in vivo using a morpholino strategy in zebrafish, as fish lacking HSCB are also unable to hemoglobize (Fig 1B). We generated an Hscb conditional mouse to better elucidate the underlying pathophysiology of the disease. Heterozygous (Hscb+/-) animals have no discernable phenotype; however, null animals die prior to embryonic day E7.5. Thus, to avoid this lethality, we employed Vav-cre animals (Tg(Vav1-cre)1Graf) to evaluate the loss of HSCB specifically in the hematopoietic compartment. Hscbc/- Vav-cre+ pups are pale and growth retarded compared to control littermates and die at approximately p10 with severe pancytopenia. To assess the loss of HSCB specifically in the erythroid lineage, we bred conditional animals to EpoR-cre (Eportm1(EGFP/cre)Uk) mice. Hscbc/- EpoR-cre+ mice die at approximately E12.5 due to a complete failure of erythropoiesis (Figure 1C). Finally, temporally inducible, hematopoietic-specific deletion animals were generated by transplantation of fetal livers from Mx-Cre (Tg(Mx1-cre)1Cgn) positive Hscbc/- animals. After polyinosinic:polycytidylic acid (pIpC) induction, global defects of hematopoiesis were observed in Mx-Cre+ animals, leading to their death 3-weeks post-induction from profound pancytopenia. A transient siderocytosis was seen in the peripheral blood between days 6-8 post-pIpC. Flow cytometry using FSC-TER119-CD44 gating strategy confirmed the defect in erythropoiesis. Taken together, these data demonstrate that HSCB is essential for hematopoiesis; both whole animal and in vitro cell culture models recapitulate the patient's phenotype, suggesting that the two patient mutations are likely disease-causing. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.