Atypical Kinase RIOK2 Is a Master Regulator of Hematopoietic Cell Fate

Abstract Here we report the discovery of a new master regulator of cell fate during hematopoietic differentiation, one whose function has major implications for the treatment of blood disorders such as anemia. Anemia is a major comorbidity in aging, chronic diseases such as renal failure and inflammation, bone marrow failure disorders and in hematologic neoplasms such as myelodysplastic syndromes (MDS), affecting roughly one third of the world population. Anemia is also often diagnosed in patients treated with chemotherapy or other cytotoxic agents. The comorbidities of peripheral blood cytopenias especially in elderly patients with MDS often outweigh the treatment benefits from allogeneic stem cell transplants leaving only a handful of FDA-approved drugs/therapies for treatment of such disorders. There is thus a dire need to revisit the origins of hematopoietic differentiation defects underlying these hematologic disorders to identify additional targets for novel therapies in treating anemia. We present evidence establishing that right open reading frame kinase 2 (RIOK2), an understudied atypical kinase associated with pre-40S ribosome biogenesis (Ferreira-Cerca et al., Nat. Str. Biol. 2012), is also a master transcriptional regulator of hematopoietic lineage commitment that simultaneously drives erythroid differentiation and represses myeloid and megakaryocytic lineages. We show that ablation of RIOK2 expression leads to hematopoietic differentiation defects in primary human hematopoietic stem and progenitor cells, the cells of origin for hematologic neoplasms. We identity RIOK2 as an integral player in governing major blood cell differentiation processes: erythropoiesis, megakaryopoiesis and myelopoiesis. Analyses in primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) revealed that CRISPR/Cas9-mediated depletion of RIOK2 led to impaired erythropoiesis and a concomitant elevation in megakaryopoiesis and myelopoiesis. A more comprehensive analysis revealed that RIOK2 regulates the transcriptomic profiles of several key transcription factors that determine hematopoietic cell fate, including GATA1, GATA2, SPI1, RUNX3 and KLF1. Most importantly, we also observed a significant correlation between mRNA levels of RIOK2 and GATA1, GATA2, RUNX3 and KLF1 in MDS patient-derived bone marrow cells. We also demonstrate that loss of RIOK2 causes massive alterations in chromatin accessibility, both globally and specifically at the promoters of its putative target genes. This places RIOK2 at the apex of a transcriptional regulatory network controlling hematopoietic differentiation. We identify a previously unappreciated DNA-binding winged helix-turn-helix (wHTH) domain in RIOK2 conferring the protein with the properties and activities of a transcription factor. Transcriptomic profiling, structural modeling, chromatin immunoprecipitation-sequencing and a range of domain-deleted mutants reveal that RIOK2 functions as a bona-fide master transcription factor in hematopoiesis. We also identify two transactivation domains within the wHTH motif of RIOK2 that play integral roles in associating with the core transcriptional complex at promoter regions of genes. To the best of our knowledge, we present the first evidence of a protein that not only controls 40S ribosome biogenesis governing translation but also functions in the nucleus as a master transcription factor by regulating the expression of key transcription factors that determine hematopoietic cell fate. Our discovery of a novel master transcriptional regulator governing a multitude of hematopoietic lineages significantly advances our current understanding of the transcriptomic landscape underlying hematopoietic differentiation. We hope that our findings may lead to new approaches to target these newly identified regulatory networks in hematopoiesis that may be relevant not just for malignancies, but for other hematologic disorders as well, such as the anemia of aging, chronic and inflammatory diseases and aplastic anemias. We are hopeful that this study will also lay a foundation to discovering how proteins, like RIOK2, may integrate transcriptional processes with translational outcomes to drive cellular functions. Disclosures Raundhal: Jnana Therapeutics: Current Employment. Petsko: Amicus Therapeutics, MeiraGTx, Annovis Bio, Retromer Therapeutics, and Proclara Bioscience: Membership on an entity's Board of Directors or advisory committees; Denali Therapeutics, MeiraGTx, Annovis Bio, Retromer Therapeutics and Proclara Biosciences: Current equity holder in publicly-traded company. Glimcher: Kaleido Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Other: Former Director; Repare Therapeutics: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Abpro Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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The Role of Chromatin Factors in Hematopoietic Development

Blood ◽

10.1182/blood.v122.21.sci-48.sci-48 ◽

2013 ◽

Vol 122 (21) ◽

pp. SCI-48-SCI-48

Author(s):

Leonard I. Zon

Keyword(s):

Progenitor Cells ◽

Board Of Directors ◽

Large Scale ◽

Erythroid Progenitors ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Conditional Allele ◽

Stem And Progenitor Cells ◽

Equity Ownership

Abstract The initiation of blood-specific programs is orchestrated by key transcription factors. To generate a complete compendium of chromatin factors that establish the epigenetic code during developmental hematopoiesis, a large-scale reverse genetic screen was conducted targeting orthologs of 425 human chromatin factors in zebrafish. A set of chromatin regulators was identified that function at distinct steps of primitive and definitive blood formation, including factors not previously implicated in blood development. We identified 15 factors that regulate development of primitive erythroid progenitors and 29 factors that regulate development of definitive stem and progenitor cells. These chromatin factors are associated with SWI/SNF and ISWI chromatin remodeling, SET1/MLL methyltransferase, CBP/P300/HBO1/NuA4 acetyltransferase, Sin3A/NuRD deacetylase, and Polycomb repressive complexes. Knockdown of a class of chromatin factors led to an expansion of hematopoietic stem cells (HSCs). In collaboration with Nancy Speck’s laboratory, we have investigated the activity of one of these chromatin factors, CHD7, that led to an expansion of hematopoietic stem and progenitor cells in the aorta. Using a T-cell line, a CBFβ protein pulldown, and a mass spectrometric sequencing approach led to the finding that CBFβ immunoprecipitated RUNX1 and CHD7. By studying a conditional allele of murine Chd7, inactivation does not have an effect on peripheral blood counts, but Chd7-deleted HSCs purified based on phenotypic markers contain an increased number of functional HSCs. Our studies suggest that CHD7 suppresses hematopoiesis, and provides a novel control mechanism for the regulation of HSCs. Our work provides a comprehensive view of how specific chromatin factors and their associated complexes play a major role in the establishment of hematopoietic cells in vivo. Disclosures: Zon: Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

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Adenylate Kinase 2 Links Energy Metabolism and Cell Fate in Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood-2019-129424 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3705-3705

Author(s):

Wenqing Wang ◽

Avni Awani ◽

Andrew Devilbiss ◽

Thomas Mathews ◽

Daniel Thomas ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Fate ◽

Rna Seq ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Knock Out ◽

Stem And Progenitor Cells ◽

Equity Ownership ◽

And Control

While hematopoietic stem and progenitor cells (HSPCs) were thought to rely mainly on glycolysis for energy supply, emerging evidence suggests that defects in mitochondrial functions can impact HSPC development with respect to self-renewal, differentiation and aging. The exact mechanisms underlying metabolic reprogramming and cell fate decisions during human hematopoiesis, however, remain elusive. Biallelic mutations in the mitochondrial enzyme adenylate kinase 2 (AK2), cause reticular dysgenesis (RD), one of the most profound forms of severe combined immunodeficiency (SCID). AK2 catalyzes the interconversion between adenine nucleotides and thereby controls the availability of ADP for oxidative phosphorylation. Clinically, RD patients not only present with profound lymphopenia, typical for classic SCID, but also suffer from severe congenital neutropenia. The developmental arrest across the T, NK and granulocytic lineages suggests that AK2 deficiency causes a metabolic defect with global impact on hematopoiesis. Our prior work in induced pluripotent stem cells (iPSCs) from RD patients has shown that maturation-arrested iPSC-derived HSPCs exhibit increased oxidative stress and an energy-depleted adenine nucleotide profile, suggesting that AK2-regulated mitochondrial bioenergetics play an integral role in HSPC differentiation. Therefore, RD serves as an excellent model to study the impact of mitochondrial metabolism during human HSPC development. Methods: Since iPSCs do not recapitulate definitive hematopoiesis, we developed an AK2 biallelic knock-out model in primary human HSPCs using CRISPR/Cas9 gene editing. Employing a homologous recombination-mediated dual color reporter strategy, we were able to select for HSPCs with biallelic AK2 knock-out. HSPCs edited at the safe harbor AAVS1 site were used as a control. FACS purified AK2-/- and AAVS1-/- HSPCs were in vitro differentiated along the granulocytic lineage, and cells at various differentiation stages were sorted for RNA-seq and metabolomics analysis. Results: We analyzed the myeloid differentiation potential of AK2-/- HSPCs in vitro. Compared to AAVS1-/- controls, AK2-/- HSPCs displayed a severely decreased colony forming potential of both myeloid and erythroid lineages. In addition, AK2-/- HSPCs showed a granulocytic maturation arrest at the HLA-DR-, CD117+ promyelocyte stage, consistent with the characteristic phenotype observed in RD patients. We then performed RNA-seq studies on in vitro differentiated promyelocyte and neutrophil subpopulations derived from AK2-/- and control HSPCs. The RNA-seq analysis showed differential gene expression in glutathione metabolism and IL-10 signaling pathways, suggesting an increase in oxidative stress and inflammation, respectively, caused by AK2 deficiency. In addition, genes implicated in antimicrobial function and granule synthesis were downregulated in AK2-/- neutrophils, suggesting a functional defect. Liquid chromatography-mass spectrometry (LC-MS/MS) studies to delineate differences in metabolite profile conferred by AK2 deficiency at different stages of HSPC development are currently in progress. Conclusions: We have established the first cell-traceable biallelic AK2 CRISPR knock-out model in primary human HSPCs that recapitulates the myeloid phenotype of RD patients. This model allows us to profile AK2 knock-out cells at different developmental stages. AK2-/- granulocyte precursors showed a transcriptional signature suggestive of worsening oxidative stress, inflammation and defective effector cell functions during maturation. To understand the mechanistic underpinnings for these observations we are now using a global metabolomics approach to profile the changes in energy metabolites that occur during development in AK2-deficient and control HSPC subpopulations. Understanding how metabolism governs differentiation and self-renewal of human HSPCs has important translational implications to improve hematopoietic stem cell products and transplantation outcomes. Disclosures Morrison: Frequency Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; OncoMed Pharmaceuticals: Equity Ownership; GI Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Kolon Gene Therapeutics: Consultancy; Protein Fluidics: Other: Stock Options.

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Stag2 Regulates Hematopoietic Differentiation and Self-Renewal through Alterations in Gene Expression and Topological Control

Blood ◽

10.1182/blood-2019-123464 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 279-279

Author(s):

Aaron D Viny ◽

Robert L Bowman ◽

Yu Liu ◽

Vincent-Philippe Lavallee ◽

Shira Eisman ◽

...

Keyword(s):

Gene Regulation ◽

Board Of Directors ◽

Research Funding ◽

Chromatin Accessibility ◽

Hematopoietic Differentiation ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Chromatin Architecture ◽

Transcriptional Output

Cell-type specific transcriptional programs are regulated by the activity of tissue-specific transcription factors and enhancer elements within structurally defined topologically associating domains (TADs). The coordinated and dynamic changes in chromatin architecture are highly regulated as transcriptional output is influenced by chromatin accessibility, histone modification, promoter enhance interactions, and recruitment of transcriptional co-activator complexes. The genes which contribute to transcriptional regulation, including members of the cohesin complex, are frequently mutated in human cancers, including leukemias, Ewing sarcoma, and glioblastoma multiforme. Despite this, the mechanistic role of STAG2 in gene regulation, hematopoietic function, and tumor suppression has not been delineated. We show that somatic Stag2 deletion in hematopoietic stem/progenitor cells (HSPC) results in altered hematopoietic function, increased self-renewal, and impaired differentiation across all three lineages consistent with myelodysplasia. Chromatin immunoprecipitation sequencing of Stag2-deficient HSPCs revealed that Stag2 and Stag1 have both shared and non-redundant cistromes with Stag1/2 common binding sites enriching at TAD boundaries with CTCF occupancy. This maintains TAD integrity in the setting of Stag2 loss of function which we confirmed with Hi-C chromosome capture. High resolution of the Hi-C data at 10kB resolution identified a specific role for Stag2, but not Stag1, in maintaining short-range chromatin interactions, specifically at genes with PU.1 and IRF8 motifs. While co-deletion of Stag2 and Stag1 resulted in synthetic lethality, Stag2 loss alone resulted in decreased chromatin accessibility and reduced transcriptional output at key PU.1 target loci involved in lineage-specification, including reduced Ebf1 and Pax5 expression resulting in impaired B-lineage differentiation. We investigated whether expression of PU.1 could overcome the non-permissive chromatin state at these key downstream targets and rescue hematopoietic differentiation; however, PU.1 expression could not restore Ebf1 expression or B cell differentiation and did not attenuate the serial replating capacity of Stag2 deficient hematopoietic stem/progenitor cells (HSPCs). Given this transcriptional "choke-point" we investigated whether expression of Ebf1 would have a more significant impact on Stag2 deficient cells. Indeed Ebf1 rescue restored B cell colony formation/differentiation in vitro and in vivo and abrogated serial replating of Stag2 deficient HSPCs. These data highlight the non-hierarchical and non-redundant relationship between transcription factors and chromatin architecture and demonstrate a key role for Stag2-regulated local interactions in transcription factor output and hematopoietic differentiation. Nonetheless, the mechanistic underpinnings of the structural basis for transcriptional regulation remain associative. We have recently been able to reduce the cell input for Hi-C assays such that we can now analyze the chromatin architecture of purified populations and model the structural transition from Lin- Sca-1+ Kit+ hematopoietic stem cells to committed granulocyte macrophage precursor cells both in normal hematopoiesis and in the Stag2 deficient setting. Previous studies using in vitro systems have shown that complete cohesin depletion results in the loss of structural loop components; however, cohesin levels are reduced, but not absent, in cancer cells. As such, our studies highlight a key role for locus-specific alterations in gene regulation and DNA interactions in Stag2 deficient cells, which results in altered gene expression and contributes to transformation. Taken together, these data identify a key role for Stag2 loss in transcriptional dysregulation distinct from its shared role with Stag1 in chromosomal segregation. Moreover, we illustrate a critical link between cohesin, chromosomal contacts, and gene regulation that contributes to hematopoietic transformation. Disclosures Viny: Mission Bio: Other: Sponsored travel; Hematology News: Membership on an entity's Board of Directors or advisory committees. Dekker:Arima Genomics: Membership on an entity's Board of Directors or advisory committees. Levine:Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Lilly: Honoraria; Gilead: Consultancy; Novartis: Consultancy; Prelude Therapeutics: Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; Loxo: Membership on an entity's Board of Directors or advisory committees.

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Single Cell Analysis of Adult Human Hematopoietic Stem and Progenitor Cells Identifies a Novel Lymphoid Primed Multipotent Progenitor That Expands in Relapsed Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2021-148993 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3259-3259

Author(s):

Asiri Ediriwickrema ◽

Bogdan A. Luca ◽

Aaron M Newman ◽

Andrew J Gentles ◽

Ravindra Majeti

Keyword(s):

Single Cell ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Surface Marker ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Multipotent Progenitors ◽

Stem And Progenitor Cells ◽

Surface Marker Analysis

Abstract Background: Hematopoietic stem and progenitor cells (HSPCs) are capable of generating all human blood cells. Although these cells have been extensively evaluated using both sorted (Corces et al. Nat. Genet. 2016) and single cell assays (Pellin et al. Nat. Commun. 2019), there remains significant uncertainty as to the degree of heterogeneity within HSPC subpopulations and their relevance to disease. The phenotypic diversity within HSPCs needs to be better characterized in order to understand the pathogenesis of numerous blood disorders including hematologic malignancies. To address this need, we extensively characterized healthy bone marrow mononuclear cells (BMMCs) with both whole transcriptome analysis (WTA) and surface marker evaluation. We hypothesized that by utilizing concurrent RNA and multiplexed (n=35) surface marker analysis, we could not only improve HSPC clustering but also characterize specific phenotypic states along unique hematopoietic differentiation trajectories. This analysis allowed us to identify new subpopulations within currently defined hematopoietic stem cells (HSCs), multipotent progenitors (MPPs) and lymphoid primed multipotent progenitors (LMPPs). Methods: Three healthy adult BMMC samples were stained with antibody conjugated oligonucleotides (BD Abseq) and analyzed using a nanowell cell capture system (BD Rhapsody). We filtered 8,070 high quality cells for 2,508 HSPCs, myeloid cells, and lymphocyte precursors. These cells were first clustered using matrix factorization in order to identify unique HSPC states. Novel clusters within the HSC, MPP, and LMPP subpopulations were purified using flow cytometry, and functionally evaluated with both in vitro and in vivo assays. Results: The antibody-derived tags (ADTs) obtained from BD Abseq comprised 33 of the most informative features (n=2000) and resulted in more stable clustering as determined by within sum of squares (WSS = 898 versus 934 for mRNA alone for 20 clusters). Additionally, we were able to design a targeted HSPC panel (n=500 genes) with Abseq which identified similar cell clusters compared to the WTA alone and WTA plus ADT data (Rand index = 0.88). HSPC clustering identified the putative hematopoietic stem cell (HSC), a multipotent progenitor (MPP), and a new lymphoid primed multipotent progenitor (LMPP). A new computational sorting strategy was derived to purify these primitive HSPCs, and subsequently validated with flow cytometry. The functional evaluation of these sorted populations revealed that HSC and MPP cells were capable of increased serial replating ability in vitro suggesting enhanced self-renewal capabilities compared to LMPP cells. All three HSPC subpopulations produced gradients of erythroid and myeloid colonies in methylcellulose, and T-cells in T-cell expansion assays in vitro. The HSCs were the only cells that were able to produce long term engraftment in immunodeficient mice after serial transplantations. Additionally, common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP) clusters that expressed canonical surface markers were identified. The primitive HSPC clusters were converted into a signature matrix using CIBERSORTx (Newman et al. Nat. Biotechnol. 2019), and bulk acute myeloid leukemia (AML) and healthy samples were deconvolved into respective healthy cell clusters. Using multivariate Cox proportional hazard analysis, we found that high levels of MPPs at diagnosis was the strongest feature associated with worse overall survival (HR = 78.46, 95% confidence interval 7.32-828). Interestingly, after performing paired analysis of diagnostic and relapse cases, the HSC and LMPP expanded significantly in relapsed AML after chemotherapy (p < 0.05) whereas the MPP expanded considerably after stem cell transplant but did not reach significance (p = 0.09). Conclusions: In summary, we identified new cell type clusters within previously defined HSC, MPP, and LMPP subpopulations, and unique surface marker associations using combined single cell WTA and surface marker analysis. We were able to purify these primitive HSPCs using these new markers, in addition to classical markers like CD34, CD90, and CD45RA. Importantly, deconvolution analysis provided preliminary insights into their clinical relevance in AML. Disclosures Ediriwickrema: Nanosive SAS: Patents & Royalties. Majeti: BeyondSpring Inc: Membership on an entity's Board of Directors or advisory committees; Circbio inc: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutics Solutions Inc: Membership on an entity's Board of Directors or advisory committees; Coherus Biosciences: Membership on an entity's Board of Directors or advisory committees; Acuta Capital Partners: Consultancy; Gilead Sciences, Inc: Patents & Royalties.

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Introduction and Genetic Correction of Calreticulin Mutations in Human Hematopoietic Stem and Progenitor Cells Sheds Light on MPN Pathogenesis

Blood ◽

10.1182/blood-2021-147919 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2541-2541

Author(s):

Johannes Foßelteder ◽

Angelika Schlacher ◽

Gabriel Pabst ◽

Bettina Amtmann ◽

Wolfgang Schöll ◽

...

Keyword(s):

Board Of Directors ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Dna And Rna ◽

Stem And Progenitor Cells ◽

Calr Mutations

Abstract Introduction: Recurrent mutations in calreticulin (CALR) are present in 70% to 80% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients without a JAK2 or MPL mutation. Despite recent advances in understanding mutant CALR, the detailed mechanisms are not fully elucidated, and current knowledge is mainly based on transgenic mouse models or human cancer cell lines. Thus, to more faithfully model MPN pathogenesis, we first aimed to introduce heterozygous type-1 and type-2 CALR mutations into healthy human hematopoietic stem and progenitor cells (HSPCs) via targeted CRISPR/Cas9-mediated gene knock-in (KI) and investigate its impact on HSPC function in vitro and in vivo. Second, we aimed to correct CALR mutations in patient-derived HSPCs to study their dependence on the initial driver event to exert an MPN phenotype. Methods: We used CRISPR/Cas9 to introduce heterozygous CALR mutations into the endogenous gene locus of healthy cord blood-derived HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6). Briefly, Cas9-sgRNA ribonucleoprotein (RNP) was used to cut the DNA. Simultaneously AAV6, carrying either a mutation-bearing or a wildtype control cDNA, was co-delivered to allow for targeted in-frame integration. This way, mutant CALR remains under the control of the endogenous promoter. Concurrent integration of a fluorescent reporter downstream of the mutated exon, enabled purification and tracking of modified cells via flow cytometry. Purified CRISPR-modified HSPCs were used for in vitro collagen-based colony-forming assays, proliferation and differentiation assays in liquid culture, and intrafemoral transplantation into immunodeficient NSG mice to assess their pathogenic potential. Results: Our CRISPR/Cas9 KI strategy enabled us to efficiently generate and enrich for heterozygous CALR mutant human HSPCs. Modified cells harbor the mutation at the endogenous CALR locus with intact gene regulatory regions. Correct integration and transcript expression were confirmed on DNA and RNA level by sanger sequencing. Additionally, CALR mutant protein expression was confirmed via immunohistochemistry using a diagnostically approved mutant-specific antibody. Type-1 and type-2 CALR mutations led to TPO-independent growth of CD34 + HSPC-derived cells and a two-fold (p<0.01) increase of megakaryocyte colonies in collagen-based media compared to wildtype control KI. These findings were corroborated by significantly enhanced CD41 + CD42b + megakaryocyte formation of CALR mutant HSPCs upon liquid culture differentiation. When transplanted into sublethally irradiated immunodeficient NSG mice, CALR mutant HSPCs showed robust engraftment in the bone marrow with a myeloid lineage skewing, outcompetition of wildtype cells and increased formation of CALR mutant CD41 + megakaryocyte progenitors. To investigate, if removal of type-1 and type-2 CALR mutations can ameliorate MPNs, we utilized our KI strategy to correct both CALR mutations in MPN patient-derived HSPCs by replacing them with wildtype sequences. A successful correction was confirmed on DNA and RNA level and by the absence of mutant CALR protein. Opposite to the results from introducing CALR mutations, correcting the mutations led to a two-fold decrease in megakaryocyte colony formation. Interestingly this was only seen in ET and post-ET MF samples, whereas primary MF samples were unaffected, underscoring the importance of other secondary genetic driver events in the pathogenesis of primary MF. Conclusion: Our system allows us to investigate human MPN pathogenesis prospectively and shed light on the transforming mechanisms of mutant CALR in primary HSPCs. We could show that CALR mutations prime HSPCs toward the formation of platelet-producing megakaryocytes. Genetic correction of CALR mutations in MPN patient-derived HSPCs revealed a dependence on the oncogenic mutant CALR driver event in ET and post-ET MF patients, opening the possibility of an ex vivo gene correction approach to remove mutant CALR in patient-derived HSPCs . Lastly, since MPN patient-derived cells have notoriously low engraftment potential in mice, our CRISPR/Cas9-engineered CALR mutant model also provides a powerful new strategy to generate MPN xenotransplants with defined genotypes for the evaluation of novel therapies. Disclosures Greinix: Celgene: Consultancy; Therakos: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Novartis: Consultancy. Sill: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zebisch: Novartis: Consultancy; AbbVie: Consultancy; Celgene: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.

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Long-Term Experience with Large Granular Lymphocytic Leukemia Evolving after Solid Organ and Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood-2019-130167 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1226-1226

Author(s):

Hassan Awada ◽

Reda Z. Mahfouz ◽

Jibran Durrani ◽

Ashwin Kishtagari ◽

Deepa Jagadeesh ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Viral Infections ◽

De Novo ◽

Post Transplantation ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Post Transplant ◽

Stat3 Mutations

T-cell large granular lymphocyte leukemia (T-LGLL) is a clonal proliferation of cytotoxic T lymphocytes (CTL). T-LGLL mainly manifest in elderly and is associated with autoimmune diseases including rheumatoid arthritis (RA), B cell dyscrasias, non-hematologic cancers and immunodeficiency (e.g., hypogammaglobulinemia). LGL manifestations often resemble reactive immune processes leading to the dilemmas that LGLs act like CTL expansion during viral infections (for example EBV associated infectious mononucleosis). While studying a cohort of 246 adult patients with T-LGLL seen at Cleveland Clinic over the past 10 years, we encountered 15 cases of overt T-LGLL following transplantation of solid organs (SOT; n=8) and hematopoietic stem cell transplantation (HSCT; n=7). Although early studies reported on the occurrence of LGL post-transplant, these studies focused on the analysis of oligoclonality skewed reactive CTL responses rather than frank T-LGLL. We aimed to characterize post-transplantation T-LGLL in SOT and HSCT simultaneously and compare them to a control group of 231 de novo T-LGLL (cases with no history of SOT or HSCT). To characterize an unambiguous "WHO-defined T-LGLL" we applied stringent and uniform criteria. All cases were diagnosed if 3 out of 4 criteria were fulfilled, including: 1) LGL count >500/µL in blood for more than 6 months; 2) abnormal CTLs expressing CD3, CD8 and CD57 by flow cytometry; 3) preferential usage of a TCR Vβ family by flow cytometry; 4) TCR gene rearrangement by PCR. In addition, targeted deep sequencing for STAT3 mutations was performed and charts of bone marrow biopsies were reviewed to exclude other possible conditions. Diagnosis was made 0.2-27 yrs post-transplantation (median: 4 yrs). At the time of T-LGLL diagnosis, relative lymphocytosis (15-91%), T lymphocytosis (49-99%) and elevated absolute LGL counts (>500 /µL; 93%) were also seen. Post-transplantation T-LGLL were significantly younger than de novo T-LGLL, (median age: 48 vs. 61 yr; P<.0001). Sixty% of post-transplantation T-LGLL patients were males. Fifteen% of patients had more cytogenetic abnormalities compared to de novo T-LGLL, had a lower absolute LGL count (median: 4.5 vs. 8.5 k/µL) and had less frequent neutropenia, thrombocytopenia and anemia (27 vs. 43%, 33 vs. 35% and 20% vs. 55%; P=.01). TCR Vb analysis identified clonal expansion of ≥1 of the Vb proteins in 60% (n=9) of the patients; the remaining 40% (n=6) of the cases had either a clonal process involving a Vb protein not tested in the panel (20%; n=3) or no clear expansion (20%; n=3). Signs of rejection were observed in 20% (n=3/15) and GvHD in 13% (n=2/15) of the patients. Post-transplantation, 27% of cases presented with neutropenia (absolute neutrophil count <1.5 x109/L; n=4), 33% with thrombocytopenia (platelet count <150 x109/L; n=5) and 25% with anemia (hemoglobin <10 g/dL; n=3). T-LGLL evolved in 10 patients (67%; 10/15) despite IST including cyclosporine (n=5), tacrolimus (n=4), mycophenolate mofetil (n=5), cyclophosphamide (n=1), anti-thymocyte globulin (n=1), and corticosteroids (n=6). Lymphadenopathy and splenomegaly were seen in 13% (n=2) and 33% (n=5) of the patients. Other conditions observed were MGUS (20%; n=3) and RA (7%; n=1). Conventional cytogenetic showed normal karyotype in 89% (n=11, tested individuals 13/15). Somatic STAT3 mutations were identified in 2 patients. Sixty% of cases (n=9) were seropositive for EBV when tested at different time points after transplant. Similarly, 53% (n=8) were seropositive for CMV, of which, 5 were positive post-transplantation and 3 pre-/post-transplantation. The complexity of T-LGLL expansion post-transplantation might be due to several mechanisms including active viral infections, latent oncogenic viral reactivation and graft allo-antigenic stimulation. However, in our cohort graft rejection or GvHD was encountered in a few patients (2 allo-HSCT recipients). Autoimmune conditions were present in 50% of SOT recipients (n=4/ 8, including RA, ulcerative colitis, systemic lupus erythematosus). Some of our patients also had low immunoglobulin levels. Overt EBV (post-transplant lymphoproliferative disorder) and CMV reactivation was diagnosed in only 27% (4/15) of the patients. In sum we report the long term follow up of a cohort of T-LGLL and emphasize the expansion of T-LGLL post-transplant highlighting the difficulty in assigning one unique origin of LGLL. Disclosures Hill: Genentech: Consultancy, Research Funding; Takeda: Research Funding; Celegene: Consultancy, Honoraria, Research Funding; Kite: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Consultancy, Honoraria; Amgen: Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; TG therapeutics: Research Funding; AstraZeneca: Consultancy, Honoraria. Majhail:Atara Bio: Consultancy; Mallinckrodt: Honoraria; Nkarta: Consultancy; Anthem, Inc.: Consultancy; Incyte: Consultancy. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

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Older Age and Increased Neutrophil-to-Lymphocyte Ratio (NLR) Are Predictors of Mortality in a Multiethnic Urban Cohort of Hematologic Neoplasms and COVID-19 Patients

Blood ◽

10.1182/blood-2020-138842 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 34-35

Author(s):

Angelica D'Aiello ◽

Sumaira Zareef ◽

Kith Pradhan ◽

Amanda Lombardo ◽

Fariha Khatun ◽

...

Keyword(s):

Cancer Treatment ◽

Board Of Directors ◽

Median Time ◽

Lymphocyte Count ◽

Research Funding ◽

Hematologic Neoplasms ◽

Private Company ◽

Advisory Committees ◽

Treatment Status ◽

Race Ethnicity

Introduction: We sought to compare outcomes among patients with hematologic neoplasms diagnosed with COVID-19 infection in a multiethnic urban academic medical center. Methods: A retrospective analysis of patients with hematologic neoplasms diagnosed with COVID-19 from March 17th to June 8th2020 was conducted. Subjects included were censored at last point of contact. Variables collected included age, gender, race/ethnicity, hematologic diagnosis, cancer treatment status, baseline and follow-up COVID-19 testing, neutrophil count, and lymphocyte count at time of diagnosis. Associations between hematologic diagnosis, cancer treatment status, age, gender, race/ethnicity, neutrophil-to-lymphocyte ratio (NLR), and overall survival (OS) were assessed using the Kaplan-Meier method with logrank test. Results: A total of 102 subjects with hematologic neoplasms and COVID-19 infection treated in Montefiore Health system were identified (Table 1). Thirty-nine (38%) subjects were undergoing active treatment, including 17 (16%) receiving conventional chemotherapy agents, 12 (12%) targeted therapy, and 10 (10%) combination therapy. Of those subjects, twenty (50%) experienced delay or discontinuation of treatment due to COVID-19 infection. Four subjects (4%) showed persistent infection by PCR at median duration of 25.1 days after initial diagnosis. Ten subjects (9.8%) showed clearance of the virus by PCR with median time-to-clearance of 51.8 days. Of 9 subjects with serologic testing, 8 tested positive for COVID-19 IgG antibody at median time of 62 days after initial COVID-19 diagnosis. Forty-seven (47%) subjects expired as a result of COVID-19 disease at the time of analysis. Disease type, treatment status, race/ethnicity, age, and gender showed no significant association with mortality. Patients older than 70 had worse outcomes than the younger population (p = 0.0082). Median neutrophil and lymphocyte count at time of diagnosis was 4500 and 900, respectively. NLR greater than 9 was associated with worse survival when compared to NLR less than 9 (p=0.0067). Conclusions: COVID-19 infection has adverse effects on patients with hematological neoplasms. Subjects older than 70 years had a significantly worse prognosis. Notably, subjects actively being treated with chemotherapy did not have worse outcomes than those not being treated in our cohort, supporting the notion than active COVID-19 infection per se should not result in treatment delays. In addition, high NLR correlates with worsened survival, suggesting that this could be a potential prognostic factor for COVID-19 mortality in the hematologic neoplasms population. Disclosures Steidl: Stelexis Therapeutics: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Research Funding; Pieris Pharmaceuticals: Consultancy; Aileron Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Verma:stelexis: Current equity holder in private company; BMS: Consultancy, Research Funding; Medpacto: Research Funding; Janssen: Research Funding; acceleron: Consultancy, Honoraria.

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Pegaspargase Can Safely be Administered in Adults Age 40 and Older with Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2019-124727 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3816-3816 ◽

Cited By ~ 2

Author(s):

Ryan J. Daley ◽

Sridevi Rajeeve ◽

Charlene C. Kabel ◽

Jeremy J. Pappacena ◽

Sarah E. Stump ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Board Of Directors ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Philadelphia Chromosome ◽

Hypersensitivity Reactions ◽

Newly Diagnosed ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Frontline Treatment

Introduction: Asparaginase (ASP) has demonstrated a survival benefit in pediatric patients (pts) with acute lymphoblastic leukemia (ALL) and is now part of standard-of-care frontline treatment. As a result, asparaginase preparations have been incorporated into the treatment of adult ALL to improve outcomes. Pegaspargase (PEG-ASP), a modified version of asparaginase with prolonged asparagine depletion, appears to be safe in adults up to age 40 (Stock, et al., Blood, 2019), but is associated with a unique spectrum of toxicities, the risks of which appear to increase with age. Therefore, the safety of PEG-ASP remains a significant concern in older adults w/ ALL. Methods: We conducted a single center retrospective chart review of pts age ≥40 years who received PEG-ASP as part of frontline induction/consolidation or reinduction, between March 2008 and June 2018 at Memorial Sloan Kettering Cancer Center. The primary objective was to evaluate the tolerability and toxicity of PEG-ASP based on the incidence and severity of ASP-related toxicities (hypersensitivity reactions, hypertriglyceridemia, hyperbilirubinemia, transaminitis, pancreatitis, hypofibrinogenemia, etc) according to the Common Terminology Criteria for Adverse Events, version 4.03. Laboratory values recorded were either the peak or the nadir, the more appropriate for toxicity assessment, within a 4-week period following PEG-ASP administration. Secondary objectives were to determine the total number of doses of PEG-ASP administered in comparison to the number of doses intended, and to characterize the rationale for PEG-ASP discontinuation when applicable. Fisher's exact test was used to compare the incidence of PEG-ASP toxicities with respect to pt and treatment characteristics (regimen, age, BMI, gender, Philadelphia chromosome positive (Ph+) vs. Ph-, presence of extramedullary disease, PEG-ASP dose). P values were not adjusted for multiple comparisons. Results: We identified 60 pts with ALL (40 B-ALL and 20 T-ALL) who received at least one dose of PEG-ASP. Nine pts were Ph+. The median pt age at initiation of the treatment was 53, (range, 40 to 80), and 19 pts had a BMI ≥30 kg/m2. Forty-four pts received treatment for newly diagnosed ALL, and 16 pts for relapsed disease. Table 1 lists pt baseline characteristics. Among the 44 pts with newly diagnosed ALL, 27 pts received PEG-ASP as part of pediatric or pediatric-inspired regimens at doses of 2000 - 2500 units/m2, and 1 pt received a modified dose of 1000 units/m2 due to age. The remaining 16 pts received PEG-ASP at doses of 1000 - 2000 units/m2 for consolidation, per established adult regimens (ALL-2 and L-20; Lamanna, et al., Cancer, 2013). Grade 3/4 ASP-related toxicities with a >10% incidence included: hyperbilirubinemia, transaminitis, hypoalbuminemia, hyperglycemia, hypofibrinogenemia, and hypertriglyceridemia. Frontline treatment regimens in which PEG-ASP was used in consolidation cycles only (ALL-2, L-20) were associated w/ a lower incidence of hyperbilirubinemia (p=0.009) and hypertriglyceridemia (p<0.001) compared to those regimens that included PEG-ASP during induction (pediatric/pediatric-inspired regimens) (Table 2). Younger age (40-59 vs. ≥60 years) was associated with a greater risk of hypertriglyceridemia (p<0.001) and higher PEG-ASP dose (≥2000 vs. <2000 units/m2) was associated with a greater risk of hypertriglyceridemia and hypofibrinogenemia (p=0.002 and p=0.025, respectively). Thirty-eight pts (63%) received all intended doses of PEG-ASP. Six pts stopped PEG-ASP to proceed to allogeneic hematopoietic stem cell transplantation (5 in CR1, 1 in CR2), and 7 pts stopped for hypersensitivity reactions. Hepatotoxicity was the only ASP-related toxicity that led to PEG-ASP discontinuation occurring in 5 pts (hyperbilirubinemia, N=4; transaminitis, N=1). The total number of intended doses of PEG-ASP based on regimens used was 186, and 112 were administered. Conclusion: PEG-ASP was incorporated into the treatment of 60 adult ALL pts age ≥40, with manageable toxicity. Seven pts discontinued PEG-ASP due to hypersensitivity reactions and 5 discontinued due to hepatotoxicity, but other reported toxicities did not lead to PEG-ASP discontinuation and the majority of the pts completed all intended doses of PEG-ASP. This study suggests that with careful monitoring, PEG-ASP can safely be administered in adults ≥40 years of age. Disclosures Rajeeve: ASH-HONORS Grant: Research Funding. Tallman:UpToDate: Patents & Royalties; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Research Funding; Biosight: Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees. Geyer:Dava Oncology: Honoraria; Amgen: Research Funding. Park:Takeda: Consultancy; Allogene: Consultancy; Amgen: Consultancy; AstraZeneca: Consultancy; Autolus: Consultancy; GSK: Consultancy; Incyte: Consultancy; Kite Pharma: Consultancy; Novartis: Consultancy.

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Lower Hematopoietic Progenitor Cell Counts and Yields at Subsequent Donations Is Influenced By a Shorter Inter-Donation Interval between the First and Subsequent Mobilizations

Blood ◽

10.1182/blood-2019-123492 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1962-1962

Author(s):

Sandhya R. Panch ◽

Brent R. Logan ◽

Jennifer A. Sees ◽

Bipin N. Savani ◽

Nirali N. Shah ◽

...

Keyword(s):

Stem Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Time Interval ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Peripheral Blood Cd34 ◽

Cell Counts ◽

Cd34 Cells

Introduction: Approximately 7% of unrelated hematopoietic stem cell (HSC) donors are asked to donate a subsequent time to the same or different recipient. In a recent large CIBMTR study of second time donors, Stroncek et al. incidentally found that second peripheral blood stem cell (PBSC) collections had lower total CD34+ cells, CD34+ cells per liter of whole blood processed, and CD34+ cells per kg donor weight. Based on smaller studies, the time between the two independent PBSC donations (inter-donation interval) as well as donor sex, race and baseline lymphocyte counts appear to influence CD34+ cell yields at subsequent donations. Our objective was to retrospectively evaluate factors contributory to CD34+ cell yields at subsequent PBSC donation amongst NMDP donors. Methods. The study population consisted of filgrastim (G-CSF) mobilized PBSC donors through the NMDP/CIBMTR between 2006 and 2017, with a subsequent donation of the same product. evaluated the impact of inter-donation interval, donor demographics (age, BMI, race, sex, G-CSF dose, year of procedure, need for central line) and changes in complete blood counts (CBC), on the CD34+ cell yields/liter (x106/L) of blood processed at second donation and pre-apheresis (Day 5) peripheral blood CD34+ cell counts/liter (x106/L) at second donation. Linear regression was used to model log cell yields as a function of donor and collection related variables, time between donations, and changes in baseline values from first to second donation. Stepwise model building, along with interactions among significant variables were assessed. The Pearson chi-square test or the Kruskal-Wallis test compared discrete variables or continuous variables, respectively. For multivariate analysis, a significance level of 0.01 was used due to the large number of variables considered. Results: Among 513 PBSC donors who subsequently donated a second PBSC product, clinically relevant decreases in values at the second donation were observed in pre-apheresis CD34+ cells (73.9 vs. 68.6; p=0.03), CD34+cells/L blood processed (32.2 vs. 30.1; p=0.06), and total final CD34+ cell count (x106) (608 vs. 556; p=0.02). Median time interval between first and second PBSC donations was 11.7 months (range: 0.3-128.1). Using the median pre-apheresis peripheral blood CD34+ cell counts from donation 1 as the cut-off for high versus low mobilizers, we found that individuals who were likely to be high or low mobilizers at first donation were also likely to be high or low mobilizers at second donation, respectively (Table 1). This was independent of the inter-donation interval. In multivariate analyses, those with an inter-donation interval of >12 months, demonstrated higher CD34+cells/L blood processed compared to donors donating within a year (mean ratio 1.15, p<0.0001). Change in donor BMI was also a predictor for PBSC yields. If donor BMI decreased at second donation, so did the CD34+cells/L blood processed (0.74, p <0.0001). An average G-CSF dose above 960mcg was also associated with an increase in CD34+cells/L blood processed compared to donors who received less than 960mcg (1.04, p=0.005). (Table 2A). Pre-apheresis peripheral blood CD34+ cells on Day 5 of second donation were also affected by the inter-donation interval, with higher cell counts associated with a longer time interval (>12 months) between donations (1.23, p<0.0001). Further, independent of the inter-donation interval, GCSF doses greater than 960mcg per day associated with higher pre-apheresis CD34+ cells at second donation (1.26, p<0.0001); as was a higher baseline WBC count (>6.9) (1.3, p<0.0001) (Table 2B). Conclusions: In this large retrospective study of second time unrelated PBSC donors, a longer inter-donation interval was confirmed to be associated with better PBSC mobilization and collection. Given hematopoietic stem cell cycling times of 9-12 months in humans, where possible, repeat donors may be chosen based on these intervals to optimize PBSC yields. Changes in BMI are also to be considered while recruiting repeat donors. Some of these parameters may be improved marginally by increasing G-CSF dose within permissible limits. In most instances, however, sub-optimal mobilizers at first donation appear to donate suboptimal numbers of HSC at their subsequent donation. Disclosures Pulsipher: CSL Behring: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Research Funding; Bellicum: Consultancy; Amgen: Other: Lecture; Jazz: Other: Education for employees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Medac: Honoraria. Shaw:Therakos: Other: Speaker Engagement.

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Poor Outcomes of HLA-Mismatched Allogeneic Hematopoietic Cell Transplantation and High Ferritin Levels after a Reduced-Intensity Conditioning Regimen in Patients with Advanced Myelofibrosis

Blood ◽

10.1182/blood-2019-130338 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5742-5742

Author(s):

Han Bi Lee ◽

Jae-Ho Yoon ◽

Gi June Min ◽

Sung-Soo Park ◽

Silvia Park ◽

...

Keyword(s):

Board Of Directors ◽

Cell Transplantation ◽

Research Funding ◽

Acute Gvhd ◽

Hematopoietic Cell Transplantation ◽

Hematopoietic Cell ◽

Allogeneic Hematopoietic Cell Transplantation ◽

Reduced Intensity Conditioning ◽

Advisory Committees ◽

Reduced Intensity

Allogeneic hematopoietic cell transplantation (allo-HCT) preconditioning intensity, donor choice, and graft-versus-host disease (GVHD) prophylaxis for advanced myelofibrosis (MF) have not been fully elucidated. Thirty-five patients with advanced MF were treated with reduced-intensity conditioning (RIC) allo-HCT. We searched for matched sibling (n=16) followed by matched (n=10) or mismatched (n=5) unrelated and familial mismatched donors (n=4). Preconditioning regimen consisted of fludarabine (total 150 mg/m2) and busulfan (total 6.4 mg/kg) with total body irradiation≤ 400cGy. All showed engraftments, but four (11.4%) showed either leukemic relapse (n=3) or delayed graft failure (n=1). Two-year overall survival (OS) and non-relapse mortality (NRM) was 60.0% and 29.9%, respectively. Acute GVHD was observed in 19 patients, and grade III-IV acute GVHD was higher with HLA-mismatch (70% vs. 20%, p=0.008). Significant hepatic GVHD was observed in nine patients (5 acute, 4 chronic), and six of them died. Multivariate analysis revealed inferior OS with HLA-mismatch (HR=6.40, 95%CI 1.6-25.7, p=0.009) and in patients with high ferritin level at post-HCT D+21 (HR=7.22, 95%CI 1.9-27.5, p=0.004), which were related to hepatic GVHD and high NRM. RIC allo-HCT can be a valid choice for advanced MF. However, HLA-mismatch and high post-HCT ferritin levels related to significant hepatic GVHD should be regarded as poor-risk parameters. Disclosures Kim: Handok: Honoraria; Amgen: Honoraria; Celgene: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Hanmi: Consultancy, Honoraria; AGP: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; SL VaxiGen: Consultancy, Honoraria; Novartis: Consultancy; Janssen: Honoraria; Daiichi Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Otsuka: Honoraria; BL & H: Research Funding; Chugai: Honoraria; Yuhan: Honoraria; Sanofi-Genzyme: Honoraria, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Lee:Alexion: Consultancy, Honoraria, Research Funding; Achillion: Research Funding.

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