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The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q− syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle–arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors.

The rare PEL-negative phenotype is one of the last blood groups with an unknown genetic basis. By combining whole-exome sequencing and comparative global proteomic investigations, we found a large deletion in the ABCC4/MRP4 gene encoding an ATP-binding cassette (ABC) transporter in PEL-negative individuals. The loss of PEL expression on ABCC4-CRISPR-Cas9 K562 cells and its overexpression in ABCC4-transfected cells provided evidence that ABCC4 is the gene underlying the PEL blood group antigen. Although ABCC4 is an important cyclic nucleotide exporter, red blood cells from ABCC4null/PEL-negative individuals exhibited a normal guanosine 3′,5′-cyclic monophosphate level, suggesting a compensatory mechanism by other erythroid ABC transporters. Interestingly, PEL-negative individuals showed an impaired platelet aggregation, confirming a role for ABCC4 in platelet function. Finally, we showed that loss-of-function mutations in the ABCC4 gene, associated with leukemia outcome, altered the expression of the PEL antigen. In addition to ABCC4 genotyping, PEL phenotyping could open a new way toward drug dose adjustment for leukemia treatment.

The Plant Homeodomain 6 gene (PHF6) encodes a nucleolar and chromatin-associated leukemia tumor suppressor with proposed roles in transcription regulation. However, specific molecular mechanisms controlled by PHF6 remain rudimentarily understood. Here we show that PHF6 engages multiple nucleosome remodeling protein complexes, including nucleosome remodeling and deacetylase, SWI/SNF and ISWI factors, the replication machinery and DNA repair proteins. Moreover, after DNA damage, PHF6 localizes to sites of DNA injury, and its loss impairs the resolution of DNA breaks, with consequent accumulation of single- and double-strand DNA lesions. Native chromatin immunoprecipitation sequencing analyses show that PHF6 specifically associates with difficult-to-replicate heterochromatin at satellite DNA regions enriched in histone H3 lysine 9 trimethyl marks, and single-molecule locus-specific analyses identify PHF6 as an important regulator of genomic stability at fragile sites. These results extend our understanding of the molecular mechanisms controlling hematopoietic stem cell homeostasis and leukemia transformation by placing PHF6 at the crossroads of chromatin remodeling, replicative fork dynamics, and DNA repair.

Background: The treatment revolution of multiple myeloma (MM) with the advent of novel therapies, proteasome inhibitors, immunomodulators and newer drugs lead to increased survival. Clinical characteristics at diagnosis of Long-term survivor (>5 years after the start of treatment) were described; however data regarding the profile of lines of treatment is very limited. The aim of this study was to describe the profiles of response of this population. Methods: The Poitou‐Charentes cancer registry has exhaustively registered incident cases of MM from 2008 to 2010. The follow-up date was December 31st, 2018. Patients (pts) alive after 5 years from the start of first line treatment were considered long-term survivors and their data were collected from their medical files. Three pts profiles were studied, very long responder (VLR) treated with a single line; long responder (LR) treated with [2-3] lines; and advanced (AdMM) [4 lines +[. Smoldering Myeloma, AL amyloidosis, lost to follow-up pts were excluded from the analysis. Results: Among the 396 MM registered, 177 were alive after 5 years, and 158 were included in the study. The mean age was 62.3 +/-11, sex ratio 1.2, 51% IgG, 25% IgA and 20% Light Chain isotype. ISS1 was 42%, 34% ISS2 and 14% ISS3. The median follow-up after the 5-year landmark was 39.9 +/-13 months and 52% pts died during follow-up. Overall, the median number of lines was 2.9 +/-2, 44% had at least one ASCT, 83% received Bortezomib, 72% Lenalidomide, 48% Thalidomide, 22% Pomalidomide, 20% Daratumumab (usually 3 lines+). VLR represented 19%, 27% LR, and 54% AdMM. In VLR group, 43% received a Bortezomib-based regimen (2 or 3 drugs) followed by ASCT, 47% a Melphalan-based regimen plus Thalidomide (MPT) or Bortezomib (MPV). In LR group, first line comprised: 39% Bortezomib-based regimen and ASCT versus 24% MPT and 15% MPV. Second line for LR group: 75% had Lenalidomide single agent or combination, 22% had a Bortezomib-based regimen, 17% had an ASCT. In the LR group, 45% received a third line: 40% Lenalidomide, mostly single agent, 25% a Bortezomib-based regimen and 3% had an ASCT. The AdMM group had a mean of 5.9 +/-1.6 lines (range 4 -11) and 56% of RR patients had at least one ASCT. Conclusion: Approximately 40% of MM were long-term survivors at 5 years from start of therapy in 2010, mainly on the basis of proteasome inhibitors and immunomodulators-based regimens plus use of ASCT. The vast majority of pts reached the 5 years cut-off with [4;+[ lines, and very few pts were real long-term survivors with an early prolonged control of Myeloma. Future perspective will look into 10 years long-term survival, including novel drug developments with the advent of immunotherapy. Disclosures Sabirou: AbbVie: Research Funding. Leleu:Janssen: Honoraria; Amgen: Honoraria; Carsgen: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Sanofi: Honoraria; Takeda: Honoraria; Oncopeptide: Honoraria; Karyopharm: Honoraria; Merck: Honoraria; BMS: Honoraria.

Introduction Most blood antigen-carrying proteins have important functions not only in red blood cells (RBCs) but also in other tissues. Hence, because of their polymorphisms and the existence of natural null phenotypes, blood groups also represent powerful tools to investigate human diseases. The PEL-negative rare blood type is one of the last with an unknown genetic basis. Family studies showed that the PEL-negative phenotype was inherited as an autosomal recessive trait, but the PEL locus remained undetermined since the first description of the corresponding antibody in French-Canadian individuals in 1996. Results Combining whole-exome sequencing and comparative global proteomic approaches using genomic DNA and red cell membrane proteins from four unrelated French-Canadian PEL-negative individuals and from controls, we identified i) the Multidrug Resistance-Associated Protein 4 (MRP4), also known as ABCC4, as the only missing protein in all PEL-negative RBCs and ii) a large deletion removing the last 10 exons of the ABCC4 gene. Western blot analyses confirmed that ABCC4 was present in the membrane of PEL-positive control RBCs but totally absent in the PEL-negative RBCs (Fig 1A). Western blot and flow cytometry analyses of transfected HEK293 cells revealed that over-expression of ABCC4 results in a significant increase in PEL antigen cell-surface expression (Fig 1B). Conversely, similar analyses of ABCC4-knockout K562 cells generated by the CRISPR-Cas9 strategy showed that the complete loss of ABCC4 abolished the expression of the PEL antigen (Fig 1C). PEL-negative individuals are therefore the first ever reported population lacking the complete expression of the ABCC4 protein. Surprisingly, despite the expected biological importance of ABCC4 as a cyclic nucleotide transporter, no apparent mutual symptoms or diseases were identified after investigation of the clinical history of the 12 PEL-negative members within the 4 families analyzed in this study. However, platelet investigation from one PEL-negative individual supported the role of this protein in the modulation of platelet aggregation, as previously observed in ABCC4-knockout mice studies To better understand the function of ABCC4 in mature RBCs, we measured the intracellular cGMP level in RBCs from 5 PEL-negative individuals and 5 controls. We showed that the cGMP level was not significantly impaired in the absence of ABCC4 suggesting that another cGMP transporter could compensate for the absence of ABCC4 on the RBC membrane. Interestingly, proteomic and flow cytometry analyses indicated that among the five other ABC transporters (ABCC1, ABCA7, ABCB6, ABCC5 and ABCG2) expressed on RBCs, only ABCG2 was increased in PEL-negative/ABCC4null RBCs compared to controls (1.3-fold; p=0.0007). This suggested that the absence of ABCC4 in PEL-negative individuals could be compensated by the over-expression of ABCG2. Our data, together with previous studies indicating that both ABCC4 and ABCG2 export cGMP, strongly suggest that the over-expression of ABCG2 in PEL-negative RBCs represents a compensatory mechanism for the lack of ABCC4, which could explain the absence of hematological abnormalities in PEL-negative individuals. Discussion Using genetic, molecular and cellular approaches, we demonstrated that ABCC4 is the gene underlying the novel PEL blood group system and that homozygosity for a large deletion in this gene is responsible for the rare PEL-negative phenotype. ABCC4 represents the third ABC transporter carrying a blood group system after ABCG2 (JR) and ABCB6 (LAN). The expression level of ABCC4 is strongly involved in drug resistance and toxicity, and in the clinical course of leukemia. As ABCC4 carries the PEL antigen, PEL phenotyping could be useful in the adjustment of an optimal drug dose and prevention of chemotherapy side effects in leukemia. Our findings are of immediate and important relevance in transfusion medicine and in a personalized medicine approach for chemotherapy treatment follow-up in leukemia. Furthermore, the exceptional natural "knockouts" of ABC transporters are highly valuable in understanding their function, not only in erythroid cells, but also in other cells or tissues. Disclosures Hermine: Novartis: Research Funding; Celgene: Research Funding; AB Science: Consultancy, Equity Ownership, Honoraria, Research Funding.

Introduction: Over the last decades, life expectancy of patients with hemophilia (PWH) has significantly improved and therefore, age-related comorbidities like cardiovascular disease (CVD) are now common features. Using antithrombotic and antiplatelet agents, as needed for managing CVD, proves complex in PWH given that these drugs, along with the often required invasive procedures, interfere with hemostasis; general recommendations are not applicable as such. Method: To collect data on current real-life practices in this patient population, the COCHE registry (COmorbidités Cardiovasculaires chez les patients HEmophiles) has been implemented back in July 2011. This French observational study sought to describe prospectively the management of CVD in PWH who require treatment with antiplatelet agents, antithrombotics, or both. Results: By January 2018, this registry had prospectively included 67 PWH: 59 hemophilia A and 8 hemophilia B patients (47 mild, 10 moderate, and 10 severe), with a median age of 65±12.5 years. Overall, 52 patients were included for coronary artery disease (CAD) and 16 for atrial fibrillation (AF), with one patient presenting both CAD and AF. Among the 67 patients enrolled, 49 were receiving on-demand replacement therapy. Identified cardiovascular risk factors were arterial hypertension (n=44), overweight or obesity (n= 35), dyslipidemia (n=30; four tested positive for human immune deficiency virus [HIV]), smoking (n=17), family coronary disease history (n=11), non-insulin dependent diabetes mellitus (NIDDM) (n=11), and insulin dependent diabetes mellitus (IDDM) (n=3). Conclusion: The proposals discussed herein are largely based on the experience we gained whilst managing PWH with CVD from the COCHE registry. To us, a multidisciplinary approach appears paramount. Disclosures Lebreton: Sobi: Consultancy, Research Funding.

The JAK2V617F mutation is present in the majority of patients with polycythemia vera and one-half of those with essential thrombocythemia and primary myelofibrosis. JAK2V617F is a gain-of-function mutation resulting in constitutive JAK2 signaling involved in the pathogenesis of these diseases. JAK2V617F has been shown to promote S-phase entry. Here, we demonstrate that the CDC25A phosphatase, a key regulator of the G1/S cell-cycle transition, is constitutively overexpressed in JAK2V617F-positive cell lines, JAK2-mutated patient CD36+ progenitors, and in vitro–differentiated proerythroblasts. Accordingly, CDC25A is overexpressed in BM and spleen of Jak2V617F knock-in mice compared with wild-type littermates. By using murine FDC-P1–EPOR and human HEL and SET-2 cell lines, we found that JAK2V617F-induced CDC25A up-regulation was caused neither by increased CDC25A transcription or stability nor by the involvement of its upstream regulators Akt and MAPK. Instead, our results suggest that CDC25A is regulated at the translational level through STAT5 and the translational initiation factor eIF2α. CDC25A inhibition reduces the clonogenic and proliferative potential of JAK2V617F-expressing cell lines and erythroid progenitors while moderately affecting normal erythroid differentiation. These results suggest that CDC25A deregulation may be involved in hematopoietic cells expansion in JAK2V617F patients, making this protein an attracting potential therapeutic target.

M-CSF–driven differentiation of peripheral blood monocytes is one of the sources of tissue macrophages. In humans and mice, the differentiation process involves the activation of caspases that cleave a limited number of proteins. One of these proteins is nucleophosmin (NPM1), a multifunctional and ubiquitous protein. Here, we show that caspases activated in monocytes exposed to M-CSF cleave NPM1 at D213 to generate a 30-kDa N-terminal fragment. The protein is further cleaved into a 20-kDa fragment, which involves cathepsin B. NPM1 fragments contribute to the limited motility, migration, and phagocytosis capabilities of resting macrophages. Their activation with lipopolysaccharides inhibits proteolytic processes and restores expression of the full-length protein that negatively regulates the transcription of genes encoding inflammatory cytokines (eg, NPM1 is recruited with NF-κB on the MCP1 gene promoter to decrease its transcription). In mice with heterozygous npm gene deletion, cytokine production in response to lipopolysaccharides, including CXCL1 (KC), MCP1, and MIP2, is dramatically enhanced. These results indicate a dual function of NPM1 in M-CSF–differentiated macrophages. Proteolysis of the protein participates in the establishment of a mature macrophage phenotype. In response to inflammatory stimuli, the full-length protein negatively regulates inflammatory cytokine production.

KLF1 is an essential erythroid transcription factor (EKLF) involved both in the ß-globin switch from fetal to adult and in definitive erythropoiesis. KLF1 also activates genes encoding heme biosynthetic enzymes, as well as genes involved in the cell cycle and the synthesis of membrane proteins. Heterozygosity for one or several nucleotide changes in KLF1 may be responsible for the so-called dominant Lu(a-b-) phenotype, also known as In(Lu) for "Inhibitor Lutheran" and characterized by a dramatic reduction in the expression levels of the Lutheran blood group antigens and a slight elevation in hemoglobin F (HbF). Several other blood group antigens are also under the direct control of KLF1, but their number and expression level in In(Lu) red blood cells (RBCs) remain a matter of debate. In addition, mutations in KLF1 in humans lead to anemia, acantocytosis and some of these variants, such as CDA type IV, are characterized by ineffective terminal differentiation and defects in enucleation. Given the major role of KLF1 in erythropoiesis, it would not be surprising that In(Lu) RBCs show abnormal properties. In this work, we performed proteomic studies to quantify membrane proteins in In(Lu) vs control RBCs. Results: using label-free mass spectrometry, we analyzed the expression levels of membrane proteins in 5 In(Lu) and 4 control RBCs. Hierarchical clustering allowed to identify two modulation profiles of protein expression (Figure 1). The first profile (red color) is composed of 49 proteins over-expressed in In(Lu) RBCs, with the majority of them corresponding to the "26S proteasome complex" (PSMA, PSMB, PSMD, and PSME). In addition, we confirmed the overexpression of the 26S proteasome complex in In(Lu) RBCs by western blot analyses. The second profile (green color) includes 23 membrane proteins with a lower expression level in In(Lu) RBCs; these proteins correspond to blood group antigens (e.g. Lu/BCAM, CD44), and to cytoskeletal proteins (e.g. dematin, flotillin). Five proteins carrying blood group systems show a decrease of expression in In(Lu) RBCs over 40%: Chido/Rodgers, Xg, Indian, Duffy and Lutheran: antigens. While our results indicating of decreased expression of Indian and Chido/Rodgers systems are consistent with previous reports, the Duffy expression was unexpectedly found to be decreased by 86%. Since we evidenced an important increase in the proteasome complex, and taking into account that KLF1 is involved in the regulation or erythropoiesis, we, investigated the expression profile of the 26 S proteasome expression during erythropoiesis. We observed an important decrease of all proteins of the proteasome complex during erythropoiesis. Conclusion: from our preliminary results reported in this study, we hypothesized that the increase of the 26S proteasome in the In(Lu) individuals may result from defects in organelle loss and could explain the profound dyserytropoiesis observed in the "nan" mice null for KLF1. Figure 1: Comparative proteomics of red blood cell membrane proteins in In(Lu) blood group type. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: Sapacitabine is a novel oral nucleoside analogue with a unique ability to induce single-strand DNA breaks after incorporation into DNA, leading to production of double-strand DNA breaks and/or G2 cell cycle arrest. In AML cell lines, the active metabolite of sapacitabine, CNDAC, is synergistic with hypomethylating agents (HMAs) particularly when treated with HMAs first. In a pilot study, there were 6 CRs and 2 PRs in 25 patients treated with sapacitabine in alternating cycles with decitabine. This global randomized phase 3 study evaluated the survival benefit of this treatment strategy vs. decitabine monotherapy. Methods: Decitabine 20 mg/m2 was administered intravenously daily x 5 days of a 4-week cycle (for the control arm and odd cycles of the study arm) alternating with sapacitabine 300 mg p.o. b.i.d. x 3 days/week x 2 weeks of a 4-week cycle (even cycles of the study arm). The safety of these doses was further evaluated in the lead-in phase of the phase 3 study to confirm the findings from the pilot study. Eligible patients were ≥70 years with untreated AML and unsuitable for or unwilling to receive standard induction chemotherapy. Patients who had received HMAs for prior MDS or MPD were excluded. Results: For 482 patients randomized to receive decitabine/sapacitabine (n=241) vs. decitabine only (n=241), randomization was stratified by the presence of antecedent MDS or MPN, peripheral white blood cell count (WBC 40,000 in 59 patients (12%); 194 patients (40%) had unfavorable cytogenetic risk by SWOG criteria. Disease characteristics were well balanced in both arms. In total, 13.7% of patients achieved CR, more on the study arm vs. control (16.6% vs. 10.8%). A total of 37.3% treated patients received ≥5 cycles of treatment, similar on both arms, as were 30- and 60-day death rates. Median overall survival was 5.9 months on the study arm vs. 5.7 months on control arm, which did not reach a statistically significant difference. In the subgroup of patients with 10% patients were thrombocytopenia, anemia, neutropenia, pneumonia, febrile neutropenia, sepsis, and disease progression. Conclusion: The regimen of sapacitabine administered in alternating cycles with decitabine was active and well tolerated but it did not significantly improve overall survival as compared to decitabine monotherapy. Further analyses are being conducted to characterize the subgroups of patients who appeared to have benefited from this treatment regimen and the potential cost savings associated with the use of an oral drug. Disclosures Kantarjian: Amgen: Research Funding; Pfizer: Research Funding; Delta-Fly Pharma: Research Funding; ARIAD: Research Funding; Bristol-Meyers Squibb: Research Funding; Novartis: Research Funding. Goldberg: Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; Celgene: Speakers Bureau; COTA: Employment, Equity Ownership; Pfizer: Honoraria; Ariad: Speakers Bureau; Jazz: Speakers Bureau. Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees. Strickland: Boehringer Ingelheim: Consultancy; Daiichi Sankyo: Consultancy; CTI BioPharma: Consultancy; Tolero Pharmaceuticals: Consultancy; Sunesis Pharamaceuticals: Consultancy, Research Funding; Baxalta: Consultancy; Alexion Pharmaceuticals: Consultancy; Astellas Pharma: Honoraria. Rubenstein: Alexion Pharmaceuticals: Speakers Bureau. Arellano: Cephalon Oncology: Research Funding. Schiller: bluebird bio: Research Funding; mateon therapeutics: Research Funding. Gaidano: Roche: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Montesinos: Celgene Corporation: Honoraria, Research Funding. Rizzieri: Shire: Research Funding; Erytech: Research Funding. Subramanian: Boehringer-Ingelheim, Lilly, BMS, Astra-Zeneca, Pfizer, Biocept: Consultancy, Speakers Bureau. Buyse: IDDI: Employment, Equity Ownership. Chiao: Cyclacel LTD: Employment, Equity Ownership.

Haploinsufficiency in genes encoding ribosomal proteins (RP) or ribosome-associated proteins either by mutation or deletion leads to a predominant erythroid phenotype. In acquired 5q- myelodysplasic syndrome (MDS), the macrocytic anemia has been linked to the monoallelic deletion of RPS14 gene which results in altered ribosome biogenesis. Because of the defective maturation of the small 40S ribosome subunit, the RPL5/RPL11/5SrARN complex, normally involved in the assembly of the large 60S subunit assembly, accumulates and inhibits E3-ligase-HDM2 leading to the stabilization and activation of p53 resultig in cell cycle arrest, increased apoptosis and defective differentiation of maturing erythroblasts. In the present work, we hypothesized that p53 could play a key role in the control of normal erythroid differentiation by ribosome biogenesis and we further investigated the involvement of the decreased pool of ribosome on erythroid defects in the 5q- syndrome. To investigate the first hypothesis, we studied the kinetics of ribosome biogenesis in human primary erythroblasts by mass spectrometry after pulse-SILAC. We noted that ribosome renewal collapses begining at the polychromatophilic erythroblast stage. We subsequently used the pharmacological agent CX-5461 to inhibit RNA polymerase I. When ribosome biogenesis in proerythroblasts is blocked by CX-5461, p53 is activated and proerythroblasts enter the terminal differentiation by expressing GATA1-erythroid target genes without apoptosis. By ChIP-seq in primary erythroblasts, we demonstrated that p53 binds to 1289 genes including 263 genes specifically activated by CX-5461, 6 of them being upregulated during CX-5461-induced erythroid differentiation and 3 of them being known GATA1 targets. We further used an shRNA strategy to demonstrate that one of these genes is required to permit entry into terminal erythroid differentiation when ribosome biogenesis is abrogated. We thus showed that normal erythroid differentiation is controlled by ribosome biogenesis through a p53-dependent checkpoint. In 5q- syndrome, ribosome biogenesis is continuously decreased along all stages of erythropoiesis and in contrast to the normal conditions, erythroid differentiation is defective with an excess of apoptosis affecting mature erythroblasts. We previously reported that GATA1 is targeted by a caspase-dependent cleavage since GATA1 is not protected by its chaperone HSP70 in the nucleus of MDS erythroblasts. We confirmed that GATA1 protein is decreased in 5q- primary erythroblasts and in RPS14 shRNA-expressing normal erythroblasts. To obtain further insights into the defective erythroid maturation of RPS14-deleted erythroblasts, we developed an inducible shRNA to RPS14 in the UT7/Epo cell line. Polysome profiling confirmed the decrease of 40S subunit and absolute quantification of RP by deep proteomics demonstrated a 50% decrease of RPS in conjunction with 50% reduction of ribosome content in these cells. GATA1 expression was decreased and was only partially rescued by treatment with either caspase inhibitor qVD or proteasome inhibitor, bortezomib. We then tested the hypothesis of a decrease in GATA1 translation, as previously shown in a shRNA RPS19 Diamond-Blackfan model, by analyzing and comparing the global transcriptome and the translatome corresponding to transcripts present in high molecular weight polysomes using Affymetrix HTA 2.0 microarrays. We observed a decoupling between transcriptome and translatome suggesting a selectivity of translational defects. Thermodynamic characteristics i.e. the fold energy, energy per base and length of the 3'UTR and the energy per base of the 5'UTR (Vienna RNA Package, UCSC genome browser) were the determinants of transcript selection on the polysome. The shortest transcripts with a highly structured 3'UTR including GATA1 were the transcripts which were less effectively translated. Consistently, the diminution of GATA1 protein was associated with a decrease of its target genes. Our results suggest that GATA1 is a potentially interesting therapeutic target. In summary, our results show that ribosome biogenesis controls erythroid differentiation via a p53-dependent transcriptional regulation and that a reduction of the ribosome pool leads to a selective translation at the expense of erythroid master gene GATA1. Disclosures Fontenay: Celgene: Research Funding.

Acquired hemophilia A is a rare bleeding disorder caused by autoantibodies to coagulation FVIII. Bleeding episodes at presentation are spontaneous and severe in most cases. Optimal hemostatic therapy is controversial, and available data are from observational and retrospective studies only. The EACH2 registry, a multicenter, pan-European, Web-based database, reports current patient management. The aim was to assess the control of first bleeding episodes treated with a bypassing agent (rFVIIa or aPCC), FVIII, or DDAVP among 501 registered patients. Of 482 patients with one or more bleeding episodes, 144 (30%) received no treatment for bleeding; 31 were treated with symptomatic therapy only. Among 307 patients treated with a first-line hemostatic agent, 174 (56.7%) received rFVIIa, 63 (20.5%) aPCC, 56 (18.2%) FVIII, and 14 (4.6%) DDAVP. Bleeding was controlled in 269 of 338 (79.6%) patients treated with a first-line hemostatic agent or ancillary therapy alone. Propensity score matching was applied to allow unbiased comparison between treatment groups. Bleeding control was significantly higher in patients treated with bypassing agents versus FVIII/DDAVP (93.3% vs 68.3%; P = .003). Bleeding control was similar between rFVIIa and aPCC (93.0%; P = 1). Thrombotic events were reported in 3.6% of treated patients with a similar incidence between rFVIIa (2.9%) and aPCC (4.8%).

Background We and others have shown that normal human erythroid cell maturation requires a transient activation of caspase-3 at late stages of maturation (Zermati et al, J Exp Med 2001). We further documented that, in human erythroblasts, the chaperone HSP70 is constitutively expressed and, at late stages of maturation, translocates into the nucleus and protects GATA-1, the master transcriptional factor critical for erythropoiesis, from caspase-3 cleavage (Ribeil et al, Nature 2007). During the maturation of human β-TM erythroblasts, HSP70 is sequestrated by excess of α-globin chains in the cytoplasm and as a consequence, GATA-1 is no longer protected from caspase-3 cleavage resulting in end-stage maturation arrest and apoptosis (Arlet et al, Nature 2013). Understanding the molecular mechanisms that regulate the localization of HSP70 during erythroid differentiation may help to find new therapeutic targets to reduce ineffective erythropoiesis in beta-thalassemia. Methods CD34 positive cells from normal and thalassemic peripheral blood were cultured in IMDM/BIT media in the presence of SCF, IL3, IL6 for seven days and subsequently cultured for additional 7 to 9 days in media containing SCF, IL3 and Epo. Erythroblasts differentiation, HSP70 localization were analysed by FACS, AMNIS stream, confocal microscopy and western blot analysis. RNAseq and proteomic analysis of highly purified erythroid cells at all distinct stages of differentiation were used to assess expression levels of various exportins. Duolink and Octet analyses were used to assess protein proximity and affinity of interactions, respectively. Results During erythroid differentiation, Hikeshi, the cognate nuclear importin of HSP70, is constitutively expressed and enables HSP70 nucleus entry as assessed by siRNA experiments. However, its expression was not regulated during erythroid differentiation. In contrast, exportin expression analysis showed marked differences in expression levels of XPO1 and XPO7 during erythroid differentiation. XPO1 expression being reduced at the time of c-kit down-regulation and caspase 3 activation while there was a marked increase in XPO7 expression at the late stages of terminal erythroid differentiation. XPO1 interacted in vivo (Duolink analysis) and in vitro with HSP70 (Octet analysis). Likewise, the previously described HSP70 S400A mutant (in the Leucine-rich Nuclear Export Sequence), which is constitutively located in the nucleus interacted with XPO1 with lower affinity compared to HSP70 WT. Stem Cell Factor (SCF) starvation and Pi3k inhibition led to decreased in vivo HSP70/XPO1 interactions. However, neither phosphorylation of HSP70 nor XPO1 were detected by Nanopro and proteomic analysis, and XPO1 expression was not regulated by Pi3K pathway. Expression of RanGTP Activating Protein (RanGAP), a protein critical for XPO1/cargo interaction, was down-regulated at the moment of caspase 3 activation during erythroid maturation, which may explain the decrease in HSP70/XPO-1 interactions. Inhibitors of XPO1 (leptomycin B and KPT 251) were able to induce HSP70 nuclear localization at early stages of differentiation (proE). In erythroid progenitors from β-TM patients, treatment with the Selective Inhibitor of Nuclear Export compound KPT-251 rescued nuclear HSP70 localization and GATA1 expression, and resulted in improved of erythroid terminal differentiation, without cytotoxicity, of thalassemic erythroid progenitors. Conclusion XPO1 is a major regulator of erythropoiesis through the regulation of HSP70 nuclear localization and is a potential new target to decrease ineffective erythropoiesis of thalassemia. Specific XPO-1 inhibitors currently in clinical development are being tested for potential therapy in thalassemic erythroid progenitors. Disclosures No relevant conflicts of interest to declare.

*the first two authors are co-first authors Introduction. Erythropoiesis is a complex process starting from pluripotent medullary progenitors and leading to the production of highly specialized and enucleated erythrocytes. Two successive phases are generally distinguished: an amplification phase with intense proliferation of morphologically similar progenitors and a terminal differentiation phase with few cell divisions and strong cellular modifications. Although erythropoiesis is a continuous process, these modifications allow the identification of specific maturation stages and the passage from one stage to the following one seems to correlate with a cell division. Several transcriptomic analyses of erythroid differentiation have been published but only few and very limited proteomic studies have been reported. Since post transcriptomic modifications are responsible for a large part of the proteome variations, a direct proteomic analysis of the erythroid differentiation is required to accurately assess the modifications that occur during this process. Results. For this study, we used CD34+ cord blood progenitors and an optimized three step cell culture method allowing the production of highly synchronized cell populations of erythroid cells at various differentiation stages. Several cellular populations from erythroid progenitors up to reticulocytes were analyzed by a label-free analysis and mass spectrometry that led to the absolute quantification of more than 6000 proteins with a false discovery rate of less than 1% (n=3). Moreover, the relative expression of well-known stage-specific erythroid markers such as TFRC, BAND3 or GLUT1, transcription factors, heme biosynthesis enzymes followed the expected pattern. To complete this study, we performed a quantitative analysis of the repartition of proteins between the generated reticulocytes and the expelled nucleus (pyrenocyte). To do that, pyrenocytes and reticulocytes were sorted by FACS according to size, Hoechst 33342 and glycophorin A labelling. Equal numbers of reticulocytes and pyrenocytes were used to prepare peptides that were analyzed by mass spectrometry after iTRAQ labelling. These experiments allowed the quantitative repartition of 1153 proteins including most erythrocyte-specific membrane proteins. Conclusion. All these results significantly increase our knowledge of the protein expression pattern during erythropoiesis and should constitute a valuable data base for subsequent studies regarding both physiological and disordered erythropoiesis. Disclosures No relevant conflicts of interest to declare.

Interleukin-7 (IL-7), the principal cytokine implicated in thymopoiesis and peripheral T-cell homeostasis, is presently under evaluation in human diseases characterized by persistent lymphopenia. Unexpectedly, before the eventual IL-7–driven T-cell expansion, all treated patients showed a profound T-cell depletion 24 hours after injection. The current study uses the rhesus macaque model to investigate the mechanisms involved in this IL-7–induced T-cell depletion. We identify a new critical function of IL-7 that induces massive and rapid T-cell migration from the blood into various organs, including lymph nodes, parts of the intestine, and the skin. This homing process was initiated after the induction of chemokine receptor expression by circulating T cells and the production of corresponding chemokines in target organs. Finally, we demonstrate that the IL-7–induced cell cycling is initiated within these organs before T cells migrate back into the bloodstream, indicating that T-cell homing is required for in vivo IL-7 function.

We studied T-cell reconstitution in 31 primary T-cell–immunodeficient patients who had undergone hematopoietic stem-cell transplantation (HSCT) over 10 years previously. In 19 patients, there was no evidence of myeloid chimerism because little or no myeloablation had been performed. Given this context, we sought factors associated with good long-term T-cell reconstitution. We found that all patients having undergone full myeloablation had donor myeloid cells and persistent thymopoiesis, as evidenced by the presence of naive T cells carrying T-cell receptor excision circles (TRECs). In 9 patients with host myeloid chimerism, sustained thymic output was also observed and appeared to be associated with γc deficiency. It is therefore possible that the complete absence of thymic progenitors characterizing this condition created a more favorable environment for thymic seeding by a population of early progenitor cells with the potential for self-renewal, thus enabling long-term (> 10 years) T-cell production.

Introduction. Whereas von Willebrand disease is the most common constitutional bleeding disorder, acquired von Willebrand syndrome (AVWS) is rare with an estimated prevalence between 0.04 to 0.5%(1). AVWS has been related in various pathophysiological conditions including cardiovascular diseases such as aortic stenosis, autoimmune and lymphoproliferative disorders. To our knowledge, the prevalence of AVWS in lymphoproliferative disorders has never been investigated. Methods. We conducted an observational monocentric prospective study in Caen university hospital to evaluate the incidence of AVWS in B cell chronic lymphoproliferative disorders (B-CLPD) and characterize its phenotype. Inclusion criteria were the presence of a BCLPD in patients with no personal or family history of bleeding. Every enrolled patients was tested for Biological parameters were measured including closure time, von Willebrand Antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo) and factor VIII procoagulant activity (FVIII:C). The bleeding phenotype was evaluated using Tossetto bleeding score(2). AVWS was suspected when patients presented a decrease of VWF:Ag and VWF:RCo and/or a VWF:RCo/VWF:Ag below 0.7. Results. A total of 147 patients were included with the following diagnosis: fifty five patients (37%) with chronic lymphocytic leukemia, 48 patients (33%) with monoclonal gammopathies of undetermined significance (MGUS), 22 patients (15 %) with non hodgkin B cell lymphoma, 9 patients (6 %) with multiple myeloma, 4 patients (3 %) with Waldenstrom macroglobulinemia and 9 patients with other B-CLPD. Closure times, with epinephrine and ADP, were prolonged for six patients (6/147, 4.1%) with median levels of VWF:Ag, VWF:RCo, FVIII:C and VWF:RCo/VWF:Ag ratio at 29.5 IU/dL [9-284], 11.4 IU/dL [1-140] , 42.5 [6-204] and 0.3 [0.04-0.84], respectively. Five of these 6 patients had MGUS and 1 patient presented a follicular lymphoma. Serum protein electrophoresis revealed a monoclonal component in 5 patients with a median concentration at 8.45 g/L [4.4-9.1]. Four out of these 6 patients presented mucocutaneous bleedings including menorragia, ecchymoses, epistaxis, gingival bleedings, post-operative bleedings and gastro intestinal bleedings. The median bleeding score of these six patients was 4.5 [-1 – 12]. In four patients, the biological phenotype was a type 2 von Willebrand disease, with decreased VWF:RCo/VWF:Ag ratio ( Table 1. Different proposed cutoffs with their prospective sensitivity, specificity, and likelihood ratios (LR). Cutoff > Sensitivity % 95% CI Specificity % 95% CI LR + LR - 503.50 100.00 66.37% to 100.0% 0.30 0.007557% to 1.652% 1.00 0.00 2514.00 88.89 51.75% to 99.72% 75.22 70.24% to 79.76% 3.59 0.15 5020.00 77.78 39.99% to 97.19% 85.67 81.46% to 89.24% 5.43 0.26 Conclusion: In this original prospective study, we observed AVWS for 4.1% of patients with B-CLPD. Monoclonal gammopathies of undetermined significance (MGUS) were associated with AVWS in 5 of 6 patients with AVWS. For patients with type 2 phenotypes, an increased clearance of VWF was the main mechanism of AVWS. The annual follow-up of these patients will give informations regarding the time of appearance and clinical informations for AVWS. Tiede A et al. Blood 2011. Tosetto A et al. Blood Rev 2007. Disclosures No relevant conflicts of interest to declare.

Background Nilotinib is approved for use in pts with CML after failure of imatinib and in newly diagnosed CP-CML. In the ENESTnd trial, 1st line nilotinib was associated with a higher frequency of hypercholesterolemia than imatinib. Moreover, several studies reported a nilotinib-associated risk of artery occlusive disease (AOD). In ENESTnd, pre-existing treatment for dyslipidemia did not preclude pts from enrolment. To avoid this pitfall and to determine which cholesterol fractions are affected and whether triglycerides (TG) are modified, we prospectively studied the plasma lipid profile of nilotinib-treated pts without drug treatment for dyslipidemia. Methods Pts (n=25) in our center with CP-CML either newly diagnosed or after failure of imatinib were proposed 1st or 2nd line nilotinib provided that neither very high risk for fatal cardiovascular disease (CVD) nor nilotinib-resistant ABL kinase domain mutation were present at baseline. Pts with drug treatment for dyslipidemia at baseline were excluded from this study. Total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C) and TG were measured prior to the start of nilotinib and at 3, 6, 9 and 12 months. Fasting glucose (FG) and glycosylated hemoglobin (HbA1C) were also measured. Results are shown as medians (min-max). ESC/EAS 2012 guidelines were used to determine CVD risk categories and for the management of dyslipidemias. Results Median age at nilotinib initiation was 52 years (19-69), 13 were males (52%). Sokal risk group was low in 11 pts (44%), intermediate in 11 (44%) and high in 3 (12%). Nilotinib was given upfront in 20 pts (80%) and after 1st line imatinib in 5 pts (20%). Median time from diagnosis was 0.8 month (0.4-4.3) in the former and 15.7 months (2.5-47.9) in the latter. Initial nilotinib dosing regimen was 300mg BID in 22 pts (88%) and 400mg BID in 3 pts (12%). Baseline CVD risk factors included age ≥ 45 years in men and ≥ 55 years in women in 33 pts (52%), active smoking or ceased during the previous year in 6 pts (24%), body mass index (BMI) ≥ 25 kg/m2in 14 pts (56%), hypertension in 1 pt (4%), type 2 diabetes mellitus (DM) in 1 pt (4%). Two pts were discovered pre DM. The CVD risk was low in 12 pts (48%), moderate in 11 pts (44%) and high in 2 pts (8%). At baseline, no pt required drug intervention by lipid lowering agents based on LDL-C, 11 pts (44%) had low HDL-C (≤0.4g/L) and 7 (28%) had high TG (≥1.5g/L). Median FG and HBA1C were 0.88g/L (0.63-1.26) and 5.5% (4.7-6.5), respectively. All pts were given lifestyle medical advice. At M3, TC, LDL-C and HDL-C had increased by a mean of 26.9%, 31.8% and 36.3%, respectively. Median TC at baseline and M3 was 1.84g/L (1.17-2.74) and 2.27g/L (1.08-3.62) (p Three pts (12%) required statins during the first year of nilotinib therapy: 2 pts with a moderate CVD risk at baseline and in whom LDL-C rose above 1.9g/L and 1 pt with a moderate CVD risk at baseline who developed peripheral artery disease (PAD) at M12, leading to nilotinib discontinuation. By M12, the CVD risk changed from low to high in 1 pt due do to the onset of DM and from moderate to very high in the pt with PAD. Conclusions Nilotinib is associated with a significant and early increase in TC, LDL-C and HDL-C. In contrast, a decrease in TG occurs but this may be due to lifestyle medical advice given to our patients. All patients considered for nilotinib therapy should have an evaluation of blood lipids at baseline and early after the start of therapy. Requirement for lifestyle modifications and lipid lowering treatment by statins should be determined at baseline and throughout therapy according to TC, LDL-C and CVD risk category. The role of nilotinib-induced high LDL-C in the onset of AOD needs to be investigated. Disclosures: Rea: Novartis: Honoraria; BMS: Honoraria; Teva: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Gautier:Novartis: Honoraria. Messas:Novartis: Honoraria. Dombret:Novartis: Research Funding.

Abstract 1686 Background: ENESTnd established the superior efficacy of frontline NIL vs IM in pts with CML-CP. An association between NIL treatment and hyperglycemia has been previously reported. NIL-mediated increases in fasting plasma glucose levels (FGLs) have been shown to be reversible, with levels returning to normal on treatment discontinuation; in contrast, IM has been shown to decrease FGLs in pts with diabetes (Breccia M, Alimena G. Leuk Res. 2009;33:871–875). Here, we report the incidence of hyperglycemia and related glycemic outcomes with NIL and IM by 3 y in ENESTnd. Methods: Pts were randomized to receive NIL 300 mg twice daily (BID; n = 282), NIL 400 mg BID (n = 281), or IM 400 mg once daily (QD; n = 283). This analysis included randomized pts who had ≥1 dose of study drug and no drug treatment for diabetes mellitus /prediabetes at baseline (BL; 264, 262, and 267 pts in the NIL 300 mg BID, NIL 400 mg BID, and IM arms, respectively); pts with treated diabetes at BL (15, 15, and 13 pts, respectively) were excluded. Hyperglycemia was defined as FGL ≥1.1 g/L, prediabetes as FGL ≥1.1 g/L to < 1.26 g/L or glycated hemoglobin (HbA1C) ≥5.7% to < 6.5%, and diabetes as FGL ≥1.26 g/L or HbA1C ≥6.5%. These criteria represent a combination of World Health Organization and American Diabetes Association guidelines. Results: At BL, median age and mean body mass index (BMI) were similar across the 3 arms (Table). In the NIL 300 mg BID, NIL 400 mg BID, and IM arms, respectively, 34.9%, 35.5%, and 37.8% of pts had untreated prediabetes at BL, and 6.1%, 6.1%, and 3.4% of pts had untreated diabetes at BL. Median time on treatment in the 3 arms was similar (≈ 3 y). Cumulative incidence of hyperglycemia by 3 y was higher with NIL vs IM (Table). By 3 y, grade 3/4 hyperglycemia was reported in 6 (2.3%), 7 (2.7%), and 0 pts on NIL 300 mg BID, NIL 400 mg BID, and IM, respectively; no pt discontinued due to hyperglycemia. Median time to hyperglycemia was longer with NIL 300 mg BID than with NIL 400 mg BID. From BL to 3 y, there were minimal changes in mean BMI and mean HbA1C in all 3 arms; greater increases in mean fasting insulin were observed with NIL vs IM. Excluding pts with diabetes at BL, 20.1%, 22.8%, and 8.9% of pts developed diabetes by 3 y, and 48.8%, 51.6%, and 50.0% developed prediabetes by 3 y (NIL 300 mg BID, NIL 400 mg BID, and IM, respectively). Relatively few pts required antidiabetic medication by 3 y. Based on a multivariate analysis, prediabetes at BL was a risk factor for development of hyperglycemia in all 3 treatment arms (P =.0252.0004, and.0296 [NIL 300 mg BID, NIL 400 mg BID, and IM arms, respectively]). Age > 60 y at BL was a risk factor for NIL 300 mg BID (P =.0183) and IM (P =.0010) but was not a risk factor for NIL 400 mg BID (P =.6001). Dyslipidemia (P =.0455) at BL was identified as an additional risk factor for development of hyperglycemia on IM in the multivariate analysis. Conclusions: In ENESTnd, the incidence of hyperglycemia by 3 y was higher with NIL vs IM. However, most events were low grade, HbA1C levels remained stable over time, and few pts required antidiabetic medication. The underlying mechanism of action driving NIL-mediated hyperglycemia is unknown, but the increases in fasting insulin observed suggest NIL therapy is associated with insulin resistance. NIL-treated pts with CML-CP and BL risk factors for diabetes should be monitored closely. Disclosures: Delphine: Novartis: Honoraria; BMS: Honoraria; Teva: Honoraria. Gautier:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Breccia:Novartis: Honoraria; BMS: Honoraria; Celgene: Honoraria. Saglio:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy. Hughes:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria; CSL: Research Funding. Kantarjian:Novartis Pharmaceuticals Corp: Consultancy, Research Funding; BMS: Research Funding; Pfizer: Research Funding. Kemp:Novartis Pharmaceuticals Corp: Employment. Gao:Novartis: Employment. Piccolo:Novartis Pharma AG: Employment. Larson:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy; Ariad: Consultancy, Research Funding. Hochhaus:BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding.