ALBERT

Description: Key Points Inhibition of calcineurin-NFAT signaling increases megakaryocyte and platelet counts. Inhibition of calcineurin-NFAT signaling increases proliferation of megakaryocyte progenitors.

Description: Abstract 2287 High throughput genomic testing for blood groups allows large scale antigen typing and assessment of donor pool compatibility with chronic transfusion dependent populations, particularly thalassemia or sickle cell disease (SCD) patients, in order to decrease RBC alloimmunization. Thus, it is crucial to determine if the quantity and antigen diversity of the donor pool meets the demands to sustain these patients on phenotype/genotype extended matched chronic transfusion protocols. We calculated the most common extended RBC predicted phenotypes for the 12 major clinically significant blood group antigens, D, CcEe, K, Jka/b, Fya/b, and Ss, in patients undergoing chronic transfusion for sickle cell disease (n=203) or thalassemia (n=98). The most common phenotypes in each diagnostic group were used to determine the prevalence of these phenotypes in a single day donor inventory (n=5,000) and stratified by ethnic group (70% Caucasian, 10% African-American, 13% Hispanic, 5% Asian, and 2% Other). All patient samples were tested for the presence of the GATA mutation which disrupts erythroid expression of Fy(b), and if present deemed not at risk for Fy(b) alloimmunization. The majority of patients with SCD and thalassemia are RhD positive (97% and 90% respectively), but differ in extended Rh phenotype, with Ro (Dce) prevalent in SCD pa tients (61%), and R1 (DCe) in thalassemia patients (79%). For patients with SCD, the most prevalent antigen-negative phenotypes were 17% C-E-K-,Fy(a-),Jk(b-), S-; 9% C-E-K-, Fy(a-),S-; 5% E-K-,Fy(a-),S-; and 4% C-K-,Fy(a-), Jk(b-), S-. In patients with thalassemia, no minor antigen profile exceeded 5% of individuals. The most prevalent antigen-negative profiles were 5% E-c-K-, Fy(b-),Jk(b-),S-, and 4% E-K-. Comparison of the most prevalent antigen-negative phenotype in patients with SCD with the donors revealed only 0.06% Caucasian (n=2), but 20% of the African-American donors (n=90) were antigen-negative matches. For the second most prevalent phenotype, 0.08% Caucasian (n=3) and 33% of African-American (n=167), 2% of Hispanic (n=13), and 5% other (n=5) were antigen-negative matches. For the third and fourth prevalent phenotypes, 47% of African-American (n=233) and 23% (n=115) respectively, were antigen-negative matches, while only 0.14% (n=5) and 0.06% (n=2) of Caucasians, but 5% (n=31) and 2% (n=13) of Hispanic donors were appropriate matches, respectively. For the thalassemia patients, antigen matches for the most common phenotype were found most often in Asian (13%) and donors identifying as “other” (6%). Matches were present in only 2% of the Caucasians, 2% of Hispanics, and 1.8% of African-American donors for thalassemia patients. These results confirmed the importance and impact of African-American donors for extended antigen-matching for patients with SCD. Less than 1% of our Caucasian donors could serve as extended matching for SCD. Nearly 20% of patients with SCD are negative for a common group of antigens, which allows future donor recruitment efforts focused on extended antigen profiles of the donor. Patients with thalassemia do not have a common antigen-negative profile, but extended matching for these patients can be improved by increase recruitment of Asian donors. High throughput genotyping enables typing of large numbers of donors, and potentially the majority of the donor inventory. Analysis of antigen-negative phenotypes in the donor pool with analysis of patient groups is important for inventory management, focused donor recruitment, and improved transfusion practice by avoiding alloimmunization. Disclosures: Stassinopoulos: Cerus: Employment, Equity Ownership, Patents & Royalties.

Description: Abstract 3450 Our goal is to generate sufficient PLTs from ex vivo-generated MKs for clinical utility in PLT transfusions. A critical step in this process begins with ex vivo-generated hMKs and deriving clinically relevant PLTs. We reported that infused mature, high-ploidy murine (m) MKs derived from fetal liver (FL) cells increased PLT counts in recipient mice in a clinically relevant fashion, thus avoiding the need to generate ex vivo functional PLTs. We examined whether this strategy applies to hMKs derived from FL cells (gestational age, 17–21 weeks) and bone marrow (BM) cells in a xeno-transfusion model using immunodeficient NOD/SCID/IL-2Rγcnull (NSG) mice. Infused hPLTs isolated from blood had a half-life (T1/2) of 10 hours (hrs), compared to 24 hrs for infused murine PLTs. The hPLTs were functional in NSG mice as demonstrated by their incorporation into growing thrombi in situ. Both hFL hematopoietic mononuclear cells and hBM-CD34+ cells were cultured in serum-free media supplemented with optimized cytokine cocktails to generate hMKs. In contrast to the murine studies where the FL cell-derived mMKs were the most efficient source of derived mPLTs, FL cell-derived hMKs had low ploidy (0% ≥ 8N ploidy), gave rise to ∼16 PLTs/infused hMK, and had a short T1/2 (6 hrs). In contrast, 17% of hBM cell-derived MKs had a ploidy of ≥ 8N, and after infusion into NSG mice, resulted in a wave of MKs transiently entrapped in the pulmonary microvasculature and then over ∼0.5–3 hrs released PLTs with a T1/2 of 10 hrs, comparable to infused hPLTs. Maximally, we achieved a level of 5% of circulating total PLTs being derived from human cells with ∼32 PLTs/infused hMK. These hPLTs were normal in size, displayed normal levels of surface markers, were functional, and incorporated into growing thrombi. One strategy to increase hPLT yield is to expose developing MKs to drugs reported to increase MK maturation, thrombopoiesis, and/or facilitate hematopoietic progenitor cell expansion. Such drugs include dimethylfasudil (diMF) (an inhibitor of several kinases involved in polyploidization), UNC0638 (a G9a histone methyltransferase inhibitor), SR1 (an AhR antagonist), and nicotinamide (a sirtuin histone/protein deacetylases inhibitor). Although diMF promoted size and polyploidization of hMKs, diMF markedly worsened yield of PLTs/infused hMK and decreased PLTs T1/2 in vivo. UNC0638 led to significant cell expansion, but lowered hMKs ploidy and PLTs/infused hMK yield. Nicotinamide increased maturation, size and polyploidization of hMKs, but PLT release following MK infusion needs further study. Of note, SR1 that has been reported to promote the expansion of human HSC, not only increased size and ploidy of hMKs, but also hPLT release in vitro and in vivo. SR1-treated hMKs resulted in a 3-fold increased yield of normal size, T1/2 and functional PLTs/infused hMK compared to a DMSO-treated control. In summary, like mMKs, infused hMKs into mice release PLTs in the pulmonary vasculature though at a lower efficiency. Released hPLTs were functional and T1/2 was as expected. diMF enhanced MK ploidy, but worsened PLT yield and T1/2, while an AhR antagonist SR1 that also improved MK ploidy appears to markedly enhance yield of PLT/infused hMK, while maintaining T1/2. The ability of SR1 to enhance PLT release from induced pluripotent stem cells (iPSCs)-derived MKs remains to be tested, but this drug appears to be a strong candidate for a therapeutic strategy to take ex vivo-grown hMKs and generate PLTs in clinical relevant numbers. Disclosures: No relevant conflicts of interest to declare.

Description: Commitment of multipotential hematopoietic progenitors to unique cell fates is determined by the induction and interplay of specific nuclear factors that reinforce unilineage transcriptional programs. Previously, we reported that loss of transcription factor GATA-1 promotes the expansion of bipotential megakaryocyte erythroid progenitors (MEPs) from embryonic stem cell or fetal liver derived hematopoietic progenitors. These mutant cells, termed G1ME (for Gata-1− Megakaryocyte-Erythroid), proliferate in culture and differentiate into committed megakaryocytes and erythroblasts when GATA-1 activity is restored. To evaluate gene regulation at the MEP stage of hematopoiesis, we performed microarray analysis of G1ME cells before and after GATA-1-induced differentiation. Expression of GATA-1 in G1ME cells induced numerous erythroid and megakaryocytic target genes. In addition, undifferentiated G1ME cells expressed numerous granulocyte and macrophage genes, suggesting that loss of GATA-1 derepresses a myeloid program in MEPs. Myeloid genes were rapidly inhibited upon expression of GATA-1. In particular, mRNA encoding the myeloid transcription factor PU.1 and more than thirty of its downstream targets were repressed by GATA-1. Chromatin immunoprecipitation (ChIP) showed that GATA-1 bound directly to the PU.1 gene (Sfpi1) at the promoter and at a −18kb upstream region coincident with repression. At the same regions, GATA-1 triggered release of both GATA-2, a related transcription factor expressed in multipotential progenitors, and PU.1, a positive autoregulator of its own gene. While GATA-1 is known to inhibit PU.1 functions through direct protein interactions, this work shows that antagonism also occurs at the level of Sfpi1 gene transcription. Together, our findings indicate that GATA-1 not only positively regulates an erythro-megakaryocytic program of gene expression, but also actively restrains myelopoiesis by inhibiting Sfpi1 expression directly. More generally, our work reveals a new mechanism through which cross-antagonism between transcription factors reinforces lineage commitment decisions in hematopoiesis.

Description: Abstract 911 Infants with Down syndrome (DS, trisomy 21, T21) frequently exhibit hematological abnormalities including polycythemia and/or thrombocytopenia. About 10% of DS infants develop transient myeloproliferative disease (TMD), which usually self-resolves. However, approximately 30% of affected patients develop acute megakaryoblastic leukemia (AMKL) by age 4 years. Both TMD and AMKL are accompanied by somatic GATA1 gene mutations that give rise to GATA-1s (for “short”), an amino-truncated protein lacking amino acids 1–81. Thus, DS-associated AMKL requires at least three sequentially occurring genetic abnormalities in hematopoietic cells: germline T21, somatic GATA1 mutations in fetal progenitors, and postnatal mutations in additional, currently unidentified genes. To analyze this malignant progression step by step and better understand T21-associated hematopoietic abnormalities, we created induced pluripotent stem cells (iPSCs) from DS subjects (n=3), TMD blasts (n=5) and controls (n=3). All iPSC lines exhibited signature features of pluripotency and retained their relevant genotypes: T21, T21+GATA1s and normal euploid. We compared the blood-forming capacities of iPSC lines by generating embryoid bodies in defined medium containing hematopoietic cytokines. Stage-matched embryoid bodies of each genotype produced similar numbers CD41+/235+/43+ hematopoietic progenitors capable of erythroid, myeloid and megakaryocytic differentiation. However, in methylcellulose colony assays, progenitors from DS iPSCs contained 13.5-fold increased numbers of burst forming unit erythroid (BFU-E) progenitors compared to control iPSCs (p=.009) (Table). While the absolute numbers of colony forming unit-megakaryocytes (CFU-MK) were similar between DS and wild type iPSC-derived progenitors (p=0.21), the CFU-MK:CFU-myeloid ratio was increased in progenitors from DS iPSCs (p=0.014). Thus, DS iPSC-derived hematopoietic progenitors exhibit increased propensity for erythro-megakaryocytic differentiation, similar to what occurs in DS fetal liver (Chou et al; Tunstall-Pedoe et al, Blood v112, 2008). In contrast, CD41+/235+/43+ progenitors from all 5 DS TMD iPSC lines studied (T21/GATA1s) exhibited complete absence of erythroid developmental potential in liquid culture and methylcellulose colony assays (p

Description: Key Points RH genotyping of red cells may improve matching of patients and donors and reduce Rh alloimmunization. RH genotype matching may improve use of an African American blood donor inventory.

Description: Key Points DNA methylation changes during the development of DS-AMKL occur in sequential waves of opposing losses and gains of methylation. Each wave of DNA methylation abnormalities targets specific gene networks that contribute to distinct biological features of the disease.

Description: Red blood cell alloimmunization can be a life-threatening complication for patients with sickle cell disease (SCD) receiving therapeutic transfusions. However, it remains unknown why only some (20-60%) SCD patients develop alloantibodies whereas others do not. Because of ongoing hemolysis in SCD, we have investigated the effects of toxic hemin on T cell responses of patients with SCD on chronic transfusion therapy. We found impaired monocyte control of proinflammatory T cells in response to hemin in alloimmunized SCD patients, partly due to defective levels of monocyte heme-oxygenase-1 (HO-1), an anti-inflammatory heme degrading enzyme that catabolizes heme into iron, biliverdin and carbon monoxide (CO). Monocytes are precursors of dendritic cells (DCs), which represent the antigen presenting cells most able to initiate and regulate immune responses. However, little is known about the functional properties of DCs in SCD patients. We therefore hypothesized that DCs of alloimmunized SCD patients would also have impaired response to hemin. Monocyte-derived DCs (moDCs) from healthy donor controls (n=11) and a cohort of SCD patients (aged 15-30 on chronic transfusion therapy every 3-4 weeks for at least two years using C,E,K phenotyped-matched, leukodepleted units) grouped either as "non-alloimmunized" (no history of antibody production, n=6), versus "alloimmunized" (with a history of having produced at least one alloantibody, n=7) were matured in the absence or presence of hemin (5uM and 20uM) with TLR agonists: LPS/IFNg (TLR4 agonist+STAT1 activation) or R848 (TLR7/8 agonist). Hemin-exposed mature and immature (no TLR agonist) moDCs were then assayed for HO-1 expression, cytokine production, co-stimulation, and T cell priming. Interestingly, upon hemin exposure, immature moDCs from healthy donors and non-alloimmunized patients upregulated more HO-1 (1056±206 fold; mean fluorescent intensity (MFI): 12283±1818 at 20uM hemin) than alloimmunized patients (fold increase 494±49; MFI: 7422±959, p

Description: Introduction RBC alloimmunization is common in patients with sickle cell disease (SCD). Despite serological matching RBCs for major Rh antigens, Rh alloimmunization remains problematic. The Rh blood group is encoded by two genes RHD and RHCE, which exhibit extensive nucleotide polymorphism and chromosome structural changes, resulting in the formation of Rh variant antigens. Rh variants can result in loss of protein epitopes or expression of neo-epitopes, and are common in SCD patients. Hence SCD patients harboring Rh variants can be predisposed to Rh alloimmunization. Given the limitation of traditional serologic antigen typing for detection of Rh variants, molecular genotyping has become required. A DNA microarray-based platform, BioArray RHCE and RHD BeadChip (Immuncor) is available for RH genotyping. However, it detects the most common, but not all, variants. Whole exome sequence data have been used for prediction of Rh variants (Chou, et. al, Blood Adv., 2017), offer some advantages, including detection of rare variants, structural rearrangements and copy number variation. However, whole genome sequence (WGS) analysis of RHD/RHCE is challenging due to difficulties in mapping next generation sequencing (NGS) reads to this duplicated gene family. We developed a computational algorithm to identify RH variants using WGS data. Methods The pipeline included three major components, RH allele database construction, RH variant calling, and classification of Rh blood group according the identified variants. The RH allele database was built based on NCBI Blood Group Antigen Gene Mutation (BGMUT) and International Society of Blood Transfusion (ISBT) database. Since the alleles in the BGMUT and ISBT databases were specified according to conventional RH genes (RHD, L08429; RHCE, DQ322275) that are different from those on reference human genome, we first called the variations based on the reference human genome. The positions of the identified variations were subsequently corrected to match with the BGMUT and ISBT annotation system. Next, the NGS reads with low base quality and/or mapping quality were discarded during the variation calling step. Synonymous and non-synonymous amino acid changes were characterized for each polymorphism. Haplotypes were constructed for the segments with NGS read support. Gene sequencing coverage was calculated to determine gene deletions or amplifications. Lastly, we implemented an algorithm to predict RH genotypes based on a selection of candidate alleles by read-mapping profile which considers both sequence variations and sequence consistency followed by a likelihood-based ranking of all pairwise combinations of the selected alleles. The allele combination with the highest likelihood is considered the most likely pair of alleles at a given locus. Patient specimens used in this study were from participants of the Sickle Cell Clinical Research and Intervention Program (SCCRIP, Hankins et al. Pediatr Blood Cancer. 2018). Results We validated our method in a cohort of 58 SCD patients whose RH genotypes had been determined by BioArray RhCE and RhD BeadChip and supplementary molecular tests that identify the most common variants among individuals of African descent. In this validation cohort including a total of 11 RHD and 13 RHCE alleles, our approach achieved a concordance rate of 85.85% (91 of 106 alleles) for RHD and 83.02% (88 of 106 alleles) for RHCE genotyping. WGS was highly sensitive in distinguishing homozygosity from heterozygosity of genes. By comparing the numbers of NGS reads on RH regions and whole genome average coverage, heterozygous deletion can be determined. Since WGS provides comprehensive genotyping, our analysis identified single nucleotide polymorphisms that were not identified by the BeadChip and supplemental molecular testing. The final source of discordance was likely due to the short read length of NGS such that haplotype phases cannot be correctly predicted if the variations are separated by thousands of base pairs, for which long read DNA sequencing or RNA/cDNA sequencing are required. Evaluation of the identified discrepancies is ongoing. Conclusions We developed and validated a diagnostic method for RH genotyping that leveraged the accuracy and flexibility of RH genotyping based on WGS data. With further optimization of our method, this may be useful for RBC genotype matching sickle cell patients to blood donors in the future. Disclosures Hankins: Novartis: Research Funding; Global Blood Therapeutics: Research Funding; NCQA: Consultancy; bluebird bio: Consultancy.