scholarly journals Biallelic TET2 Inactivation in Myeloid Neoplasia: From Clonal Hierarchy to Clinical Phenotypes

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1805-1805
Author(s):  
Hassan Awada ◽  
Yasunobu Nagata ◽  
Abhinav Goyal ◽  
Mohammad Fahad B. Asad ◽  
Bhumika J. Patel ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Normal Karyotype ◽  
Morphological Features ◽  
Survival Outcomes ◽  
Loss Of Function ◽  
Monocytic Differentiation ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Clinical Consequences ◽  
Myeloid Neoplasia

Abstract Genomic data has led to the identification of bio-markers of morphological features and disease sub-entities in myeloid neoplasia (MN). Somatic TET2 mutations (TET2MT) are frequently found in MN, particularly in chronic myelomonocytic leukemia (CMML). TET2MT are mostly loss-of-function and hypomorphic hits leading to inactivation of TET2 protein. In fact, impaired TET2 activity skews the differentiation of hematopoietic stem cells toward proliferating myeloid precursors favoring myeloid tumorigenesis. However, the contribution of TET2MT to clinico-hematological features in MN has been controversial, possibly due to studies containing too few patients relative to the combinatorial diversity of co-occurring lesions. We recently reported on the clonal architecture of TET2MT in patients with MN. Of these, 40% of the patients harbored biallelic TET2MT (biTET2MT). Further analysis showed a frequent occurrence of biallelic TET2 inactivation (biTET2i). To date, only a few studies have investigated the clinical consequences of biTET2i in MN. We hypothesized that the presence of biTET2i identifies a group of patho-morphological features that independently define a distinct MN subtype. To test our hypothesis, we studied correlations between mutational configuration, clinico-hematological/morphological features and survival outcomes in cases that were biTET2ivs. not (biTET2-), combining whole exome and targeted deep sequencing, SNP-arrays and conventional cytogenetics. Among 1,001 clinically annotated MN patients, 82 were biTET2i (66 biTET2MT, 13 hemizygous TET2MT and 3 homozygous TET2MT, i.e. UPD) and 919 were biTET2- (96 monoallelicTET2MT and 823 wild type). TET2 hits were ancestral lesions in 72% of biTET2ivs. 38% in biTET2- cases (P<.0001). When the 1stTET2 hit was ancestral in biTET2i, the most common subsequent hit was a 2ndTET2MT, followed by SRSF2MT, ASXL1MT, KRASMT/NRASMT and DNMT3AMT. Truncation mutations (frameshift or nonsense variants) were found in 83% of biTET2ivs. 65% of biTET2- cases (P=.02). A second TET2 hit in biTET2MT cases significantly increases the accrual of additional truncating changes. Furthermore, biTET2i were significantly enriched for additional hits in SRSF2MT (33%; P<.0001) and KRASMT/NRASMT (16%; P=.03) while biTET2- for TP53MT (11%; P=.03). SRSF2MT was also found to be significantly associated with biTET2i when compared to monoallelicTET2MT (P=.02). In contrast, biTET2i cases showed absence of SRSF2MT in the absence of monocytosis. We then assessed associations of biTET2i with specific genotype/phenotype. Clinical analyses revealed that cases with biTET2i compared to cases with biTET2- were older (91% ≥60 years vs. 74%, P=.0004) and more commonly had normal karyotype (65% vs. 45%; P=.0007). BiTET2i were enriched in patients with CMML1/2 (44% vs. 9%; P<.0001), and predominantly in lower-risk cases (62% vs. 47% in biTET2-; P=.003). While a second TET2 hit occurred frequently, biTET2i did not portend faster progression but rather associated with monocytic differentiation, consistent with its prevalence in CMML. In addition, among biTET2i with SRSF2MT or KRASMT/NRASMT, CMML was diagnosed in 70% (P=.001) and 77% (P=.01) of the cases, respectively, significantly higher than what was seen in the biTET2i population (44%). In biTET2- cases, leukopenia (81%; P<.0001), neutropenia (52%; P=.008), pancytopenia (27%; P=.008) and increased marrow blast percentages (≥5% in 33%; P=.01) were more prevalent than in biTET2i cases, which in return co-segregated with monocytosis (84%; P<.0001), marrow hypercellularity (cellularity >70% in 67%; P<.0001) and marked myeloid dysplasia (68%; P=.0003). Given our observation of a highly significant (P<.0001) relationship between biTET2i, CMML diagnosis and/or monocytosis, we also evaluated patients without frank diagnosis of CMML (CMML-) and compared biTET2ivs.biTET2- for associations with monocytosis and myeloid dysplasia, two hallmarks of CMML. Increased monocyte counts among CMML-cases were significantly overrepresented in biTET2i cases (72%; P=.03) vs.biTET2- (55%) as was myeloid dysplasia (72% vs. 46%; P=.0001). Lastly, biTET2i as a sole hit or in combination with other hits did not influence survival outcomes. In sum, biTET2i invariantly associates with distinct morphological and clinical phenotype. It may thus represent an early diagnostic marker of morphologic MN sub-entities. Disclosures Nazha: MEI: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy.

scholarly journals Invariant phenotype and molecular association of biallelic TET2 mutant myeloid neoplasia

2019 ◽  
Vol 3 (3) ◽  
pp. 339-349 ◽  
Author(s):  
Hassan Awada ◽  
Yasunobu Nagata ◽  
Abhinav Goyal ◽  
Mohammad F. Asad ◽  
Bhumika Patel ◽  
...  
Keyword(s):  
Lower Risk ◽  
Assessment Tool ◽  
Uniparental Disomy ◽  
Chronic Myelomonocytic Leukemia ◽  
Loss Of Function ◽  
Wild Type ◽  
Monocytic Differentiation ◽  
Hematopoietic Stem ◽  
Myeloid Neoplasia ◽  
Myelomonocytic Leukemia

Abstract Somatic TET2 mutations (TET2MT) are frequent in myeloid neoplasia (MN), particularly chronic myelomonocytic leukemia (CMML). TET2MT includes mostly loss-of-function/hypomorphic hits. Impaired TET2 activity skews differentiation of hematopoietic stem cells toward proliferating myeloid precursors. This study was prompted by the observation of frequent biallelic TET2 gene inactivations (biTET2i) in CMML. We speculated that biTET2i might be associated with distinct clinicohematological features. We analyzed TET2MT in 1045 patients with MN. Of 82 biTET2i cases, 66 were biTET2MT, 13 were hemizygous TET2MT, and 3 were homozygous TET2MT (uniparental disomy); the remaining patients (denoted biTET2− hereafter) were either monoallelic TET2MT (n = 96) or wild-type TET2 (n = 823). Truncation mutations were found in 83% of biTET2i vs 65% of biTET2− cases (P = .02). TET2 hits were founder lesions in 72% of biTET2i vs 38% of biTET2− cases (P &lt; .0001). In biTET2i, significantly concurrent hits included SRSF2MT (33%; P &lt; .0001) and KRAS/NRASMT (16%; P = .03) as compared with biTET2−. When the first TET2 hit was ancestral in biTET2i, the most common subsequent hits affected a second TET2MT, followed by SRSF2MT, ASXL1MT, RASMT, and DNMT3AMT. BiTET2i patients without any monocytosis showed an absence of SRSF2MT. BiTET2i patients were older and had monocytosis, CMML, normal karyotypes, and lower-risk disease compared with biTET2− patients. Hence, while a second TET2 hit occurred frequently, biTET2i did not portend faster progression but rather determined monocytic differentiation, consistent with its prevalence in CMML. Additionally, biTET2i showed lower odds of cytopenias and marrow blasts (≥5%) and higher odds of myeloid dysplasia and marrow hypercellularity. Thus, biTET2i might represent an auxiliary assessment tool in MN.

scholarly journals Modeling the Development of SRSF2 Mutated Myeloid Malignancies By CRISPR/Cas9 Mediated Genome Engineering of Primary Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2160-2160
Author(s):  
Gabriel Pabst ◽  
Johannes Foßelteder ◽  
Angelika Schlacher ◽  
Lisa Auinger ◽  
Daniel Martinez-Krams ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Genome Engineering ◽  
Specific Effect ◽  
Monocytic Differentiation ◽  
Advisory Committees ◽  
Hematopoietic Stem

Abstract Introduction: Acute Myeloid Leukemia (AML) is a malignant disease of the bone marrow that can arise from a premalignant condition called clonal hematopoiesis of indeterminate potential (CHIP). Mutations in Serine and Arginine-rich Splicing Factor 2 (SRSF2) are detected in CHIP and mediate a high risk for AML development. Here we used CRISPR/Cas9-mediated genome engineering to introduce a heterozygous SRSF2P95H mutation into primary human hematopoietic stem and progenitor cells (HSPCs) and investigated its functional consequences using both in vitro and in vivo assays. Methods: We used CRISPR/Cas9 technology to introduce a heterozygous mutant (mut) SRSF2P95H into the endogenous SRSF2 gene locus of healthy cord blood HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6) (Figure A). This allows for targeted in-frame integration of mut and/or wildtype (WT) SRSF2 cDNA under the control of the endogenous SRSF2 promoter. Notably, an integrated fluorescent reporter enables the isolation and tracking of heterozygously mutated HSPCs (Figure B). Methylcellulose colony and long-term competition assays of SRSF2 mut and WT HSPCs were performed in vitro. Cells were analyzed by flow cytometry and characterized cytomorphologically. In addition, bulk RNA-seq analyses were performed to characterize differential gene expression and abnormal splicing events. Xenotransplantation into NSG-SGM3 mice was performed in order to assess stem cell characteristics and the in vivo leukemogenic potential of SRSF2 mut HSPCs. Finally, we investigated the mutation-specific effect of the splicing inhibitor Indisulam to determine if SRSF2 mut cells are particularly vulnerable to splicing inhibition. Results: Colony assays (n=9) revealed impaired erythroid and increased monocytic differentiation of SRSF2 mut HSPCs. Quantification of colonies showed a lower frequency of erythroid BFU-E in SRSF2 mut compared to SRSF2 WT HSPCs (mean ± SD; 33.3 ± 12.5% vs. 17.4 ± 10.8%, p=0.00002). In contrast, the frequency of myeloid CFU-M colonies was higher in SRSF2 mut HSPCs compared to SRSF2 WT HSPCs (38.3 ± 7.3% vs. 22.6 ± 6.8%, p = 0.0003) (Figure C). Long-term in vitro competition assays revealed an outgrowth of SRSF2 mut over WT cells in 2 out of 7 donors. Strikingly, after three months of in vitro culture, in one donor, the SRSF2 mut cells developed a blast-like morphology with strong CD34 expression (Figure D). To assess stem cell characteristics and the leukemogenic potential in vivo, we transplanted SRSF2 mut HSPCs from 4 different donors into immunodeficient NSG-SGM3 mice (n=11). SRSF2 mut cells showed a myeloid-skewed engraftment. Cytomorphologic analysis of long-term engrafted SRSF2 mut myeloid cells revealed dysplastic changes such as nuclear abnormalities and extensive cytoplasmic vacuolization. In 4 out of 11 xenografts, human engraftment substantially increased over time with a parallel outgrowth of the SRSF2 mut clone and the appearance of blast-like cells resembling transformation into myeloid leukemia (Figure E). Comparative RNA-seq analysis identified 138 differentially spliced genes, with exon skipping being the dominant altered splicing type. Gene ontology (GO) analysis on differentially expressed genes revealed "Acute Myeloid Leukemia" among the most enriched terms (p-val = 8.2E-07, min FDR = 1.486E-04). When testing the SRSF2-mutation specific effect of the splicing inhibitor Indisulam, SRSF2 mut HSPCs show a significantly lower IC-50 than WT cells (977nM vs. 3574 nM). Strikingly, in competition- and CFU-assays, Indisulam preferentially eradicates SRSF2 mut hematopoietic cells, while sparing WT cells. Conclusion: Using our CRISPR/Cas9 approach, we can successfully introduce heterozygous SRSF2P95H mutants in primary human HSPCs. Mutant SRSF2P95H leads to increased monocytic differentiation, impaired erythroid differentiation, and phenocopy SRSF2P95H driven diseases in patients. Importantly, we show for the first time that the SRSF2 mutation alone is sufficient to induce dysplastic features and even transform healthy human HSPCs into AML-like blasts. Our model allows the identification and therapeutic investigation of specific cellular vulnerabilities caused by SRSF2 mutations and highlights Indisulam as a potential compound to specifically treat individuals carrying a SRSF2 mutation. Figure 1 Figure 1. Disclosures Ediriwickrema: Nanosive SAS: Patents & Royalties. Greinix: Novartis: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Therakos: 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: Celgene: Consultancy, Honoraria; AbbVie: Consultancy; Novartis: Consultancy. 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 Therapeutic 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: Patents & Royalties: inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences, Inc.. Reinisch: Pfizer: Consultancy; Celgene: Research Funding.

scholarly journals Eltrombopag Creates a Transient Chemical Phenocopy of TET2 Loss of Function That Contributes to Hematopoietic Precursor Expansion

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 453-453
Author(s):  
Yihong Guan ◽  
Bhumika J. Patel ◽  
Metis Hasipek ◽  
Dale Grabowski ◽  
Hassan Awada ◽  
...  
Keyword(s):  
Board Of Directors ◽  
In Silico ◽  
Structural Model ◽  
Data Monitoring Committee ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Free System ◽  
Dose Dependent

Eltrombopag (Epag) is FDA approved for immune thrombocytopenic purpura (ITP) and aplastic anemia (AA), in which it induces tri-lineage responses in primary and refractory settings. These biologic effects suggest that Epag helps to regenerate not only committed megakaryocytic progenitors, but also hematopoietic stem and progenitor cells (HSPCs). Epag is a small molecule thrombopoietin receptor (TpoR) agonist that activates the JAK-STAT pathway to increase platelet counts similar to the polypeptide based TpoR agonist Nplate. In addition, some of Epag's activity may, unlike that of Nplate, be independent of TpoR. Epag increases HSC self-renewal in mice despite the lack of binding to murine TpoR and showed efficacy in a TpoR-deficient strain. Here we show that Epag binds and inhibits TET2 in an iron-chelation independent manner, to in this way increase precursor expansion. Since iron is a key prosthetic component of the TET2 enzyme, we determined if Epag sequestration of iron in HSPCs inhibits TET2 function. In silico modeling indicated that Epag can form a tripartate complex with Fe2+, αKG and TET2 (Fig.A). Epag interacted with TET2 via N1387 and H1984 forming a two-way H-bond and also coordinating Fe2+ sandwiched between N-Oxalylglycine a surrogate for aKG and H1381 residues of TET2 (Fig.A). To experimentally confirm the computational structural model and study the effect of Epag on TET2, we used an ELISA-based TET2 activity assay in a cell-free system. We found that Epag inhibits TET2 in a dose-dependent manner with an IC50 of 1.6±0.1 µM in the presence of 25 µM each of aKG and Fe2+ (Fig.B). Interestingly, this observed IC50 of Epag for TET2 inhibition is 10-fold lower than the plasma Cmax of Epag that is produced in humans at standard clinical doses. Therefore, we performed a dose dependent TET2 rescue experiment by increasing aKG and Fe2+. There was no proportional effect on the TET2 inhibitory IC50 of Epag upon increasing either Fe2+ or αKG suggesting the inhibition of TET2 is independent of both these co-factors (Fig.B). This was consistent with in silico structural model data indicating that Epag specifically binds and traps TET2 in an inactive state, explaining why increasing concentration of Fe2+ or Fe3+ failed to rescue TET2 activity (Fig.C). Also consistent with this model of how Epag inhibits TET2, we did not experimentally observe any significant effect of ascorbic acid (100 µM), known to activate TET2 through maintenance of Fe3+↔Fe2+ homeostasis during TET2 catalysis. Underscoring likely relevance of TpoR independent actions of Epag, Epag treatment of K562 cells displaying undetectable levels of TpoR mRNA as well as protein, significantly reduced levels of 5hmC, while Tpo had no effects on 5hmC (Fig.D). We are currently measuring, after written informed consent on an IRB approved protocol, 5hmc levels serially in patients who are receiving Epag. In summary, we demonstrate TpoR-independent actions of Epag, its direct inhibition of TET2 activity, most likely by locking TET2 in an inactive configuration. Given the fundamental role of TET2 in promoting differentiation, this mechanism-of-action of Epag could be one pathway by which it expands HSPCs, independent of TpoR. In short, Epag creates a transient chemical phenocopy of TET2 loss of function, simultaneously having the capacity to activate JAK-STAT signaling via TpoR. These actions together can explain the clinical potency of Epag. Figure Disclosures Nazha: Abbvie: Consultancy; Tolero, Karyopharma: Honoraria; Daiichi Sankyo: Consultancy; Incyte: Speakers Bureau; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau; Jazz Pharmacutical: Research Funding. Saunthararajah:Novo Nordisk: Consultancy; EpiDestiny: Consultancy, Equity Ownership, Patents & Royalties. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

scholarly journals Outcome of Allogeneic Hematopoietic Stem Cell Transplantation in Children and Adolescents with GATA2-Related Myelodysplastic Syndrome

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2033-2033
Author(s):  
Rachel Bortnick ◽  
Marcin Wlodarski ◽  
Valerie de Haas ◽  
Barbara De Moerloose ◽  
Michael Dworzak ◽  
...  
Keyword(s):  
Stem Cell ◽  
Children And Adolescents ◽  
Board Of Directors ◽  
Conditioning Regimen ◽  
Normal Karyotype ◽  
Trisomy 8 ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Monosomy 7 ◽  
Gata2 Deficiency

Background GATA2 deficiency is an inherited immunodeficiency and predisposition syndrome with a high risk of developing myelodysplastic syndrome (MDS) early in life. Allogeneic hematopoietic stem cell transplantation (HSCT) is presently the only curative therapy for affected patients (pts), but to date there has been no larger study examining in detail outcomes after HSCT for GATA2-related pediatric MDS. Here we report the results of an analysis of pts with a germline GATA2 mutation undergoing HSCT for a diagnosis of MDS enrolled in the registry of the European Working Group of MDS in Childhood (EWOG-MDS). Patients and transplantation procedure Of the 87 pts with GATA2 deficiency registered before the age of 18 years, 66 underwent HSCT between 01/1997 and 11/2018. One pt had to be excluded from the analysis due to lack of data. The 65 remaining pts (34 males/31 females) were transplanted at a median age of 13.5 (4.6-19.9) years. Twenty-seven pts were transplanted for refractory cytopenia of childhood (RCC), while 38 pts had advanced disease. The highest bone marrow (BM) blast percentage prior to HSCT was 5-19% (n=23), 20-29% (n= 9) or >30% blasts (n=5); in one pt with myelofibrotic MDS a blast count was not attainable. Karyotypes included monosomy 7 (n=44), der (1;7) (n=4), trisomy 8 (n=4), random aberration (n=1) or a normal karyotype (n=12). Five of the 38 pts with an increased blast percentage had received intensive AML-type therapy prior to HSCT. Pts were grafted from a matched sibling donor (MSD; n=17), unrelated donor (UD; n=40) or a mismatched family donor (MMFD; n=8). The stem cell source was BM (n=37), peripheral blood (n=27) or cord blood (n=1). Pts were prepared with a busulfan-based (n=35), treosulfan-based (n=21), total body irradiation-based (n=5) or an alternative conditioning regimen (n=4). Results At 5 years the probability of overall survival (pOS) and disease-free survival (DFS) was 0.74 [0.62-0.86] and 0.69 [0.57-0.81], respectively, non-relapse mortality was 0.15 [0.08-0.27] and the cumulative incidence of relapse was 0.16 [0.09-0.29]. All pts engrafted initially. The cumulative incidence of acute graft versus host disease (GVHD) grade II-IV and III-IV was 0.34 [0.24-0.48] and 0.12 [0.06-0.24], respectively, and of overall and extensive chronic GVHD 0.25 [0.16-0.39] and 0.08 [0.03-0.20]. The most common post-transplant infections were viral (39 of the 43 pts with infections) with one pt each with EBV-related post-transplant lymphoproliferative disease and primary CMV disease. There were no mycobacterial infections. The most common non-infectious complications were hepatobiliary (13 pts, including 3 with veno-occlusive disease) and pulmonary (10 pts, 5 of whom had been prepared with a busulfan-based conditioning regimen). Pts with >20% BM blasts showed a trend towards a poorer DFS (0.52 [0.24-0.80]) compared to pts with 5-19% blasts (0.72 [0.53-0.91]) or pts with RCC (0.80 [0.64-0.96]; p=0.15). Examining the influence of karyotype in pts with RCC, there were a total of 2 relapses and 3 deaths (1 after relapse) among the 12 pts with monosomy 7, while there was one event among the 15 RCC pts with a normal karyotype (n=10, 1 death), trisomy 8 (n=3), der (1;7) (n=1) or random aberration (n=1). Limiting the analysis to 9/10 or 10/10 HLA matched-donors, DFS was comparable for pts transplanted from an UD (0.73 [0.55-0.91]) versus a MSD (0.82 [0.64-1.00]). Of the 8 pts transplanted from a MMFD, one patient died after secondary graft failure. No major difference in outcome was seen according to age at HSCT, gender, time from diagnosis to HSCT or stem cell source. Of the five pts who had received AML-type therapy prior to HSCT, three died of a transplant-related cause or relapse. Conclusions and perspectives In summary, HSCT resulted in a pOS of 0.74 in this cohort of children and adolescents with GATA2 deficiency and MDS. Pts with increased blasts had a tendency towards poorer outcomes. The high risk of developing advanced MDS and the better outcome in early stages of the disease indicates that HSCT should be performed early in the clinical course of children diagnosed with GATA2 deficiency and MDS. Of note, there was no indication of excessive toxicity, disease-associated comorbidities or an increased risk of GVHD. The HSCT outcomes of children and adolescents with MDS and GATA2 deficiency are similar to what has been previously published for pts transplanted for MDS in the absence of GATA2 germline disease. Disclosures Bader: Amgen (Brasil), Novartis: Consultancy, Speakers Bureau; Celgene: Consultancy; Medac: Patents & Royalties, Research Funding; Riemser, Neovii: Research Funding. Locatelli:Miltenyi: Honoraria; bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees. Niemeyer:Celgene: Consultancy.

scholarly journals The Biological Inferences from the Ranking of SF3B1 Mutations in the Clonal Hierarchy of Myeloid Neoplasia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5411-5411
Author(s):  
Hassan Awada ◽  
Jibran Durrani ◽  
Ashwin Kishtagari ◽  
Vera Adema ◽  
Cassandra M Kerr ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Chromosomal Abnormalities ◽  
Clonal Evolution ◽  
Data Monitoring Committee ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Clonal Architecture ◽  
Clonal Nature ◽  
Myeloid Neoplasia

Chromosomal abnormalities can be founder lesions (e.g., t (8; 21), inv (16), inv (3)), initiate or advance disease progression (both founder and secondary hits e.g., ASXL1, TP53, RUNX1) or can be obligatory secondary hits (FLT3, NPM1). Hence, the rank of these mutations may determine the biological properties and clinical outcomes. However, while many mechanistic studies have been undertaken without identifying the key pathogenetic factors resulting from SF3B1 mutations, important biological clues can be derived from the consequences of SF3B1 alterations in the context of the clonal architecture of myeloid neoplasia (MN). SF3B1 mutant patients often have a homogeneous phenotype with isolated erythroid dysplasia, ring sideroblasts (RS) and favorable prognoses. Studies in primary MDS cells have suggested that SF3B1 mutations are initiating lesions and provide a marked clonal advantage to MDS-RS cells by propagating from rare lympho-myeloid hematopoietic stem cells. However, there is significant diversity of clinical phenotypes and outcomes including the observation that the disappearance of RS can be observed during the disease course of clonal MN and might suggest cellular shifts due to acquisition of additional hits. In such scenarios, the cell's fate in the context of SF3B1 mutations is pre-defined by the predominance of expanded hits. We took advantage of our detailed database of molecularly and clinical annotated cases with MN to study the SF3B1 mutatome and describe whether the clonal nature (ancestral vs. secondary) might change the clinical and phenotypic trajectories of MDS cells and whether the concatenation of mutations decreases the competitiveness of SF3B1 clones, leading to the dominance of other driver genes and subsequently to clonal evolution. The clonal hierarchy was resolved using our in-house designed VAF-based bioanalytic method and confirmed by the PyClone pipeline, which showed a high level of concordance. We first assigned clonal hierarchy to SF3B1 mutations by using VAFs (adjusted for copy number and zygosity) and classifying the mutations into dominant (if a cutoff of at least 5% difference between VAFs existed), secondary (any subsequent sub-clonal hit) and co-dominant hits (if the difference of VAFs between two mutations was <5%). In total, we identified 140 dominant (SF3B1DOM), 121 secondary (SF3B1SEC) and 74 co-dominant SF3B1 mutations. For the purpose of this study, we set aside co-dominant SF3B1 mutations. Focusing on SF3B1DOM and SF3B1SEC, SF3B1DOM were often associated with a normocellular bone marrow compared to SF3B1SEC (n=42 vs. 26; P=0.02) and were less likely enriched in multi-dysplastic myeloid cells (29% vs. 53%; P=0.01). As such, SF3B1DOM tended to be more frequently detected in lower-risk MDS (P=0.05) in the subtypes of MDS-RS and MLD-RS (RS≥15%: 67% vs. 41%; P=0.01) compared to other disease subtypes. Twenty-three percent of patients with SF3B1SEC had secondary acute myeloid leukemia (sAML) (P=0.03). SF3B1SEC patients tended to have a lower median platelet count than patients with SF3B1DOM (97 vs. 130 x 109/L; P=0.05). SF3B1SEC was also more associated with bi-cytopenia compared to SF3B1DOM (52% vs. 36%; P=0.01). No specific association was found between SF3B1 clonal nature and cytogenetic abnormalities, suggesting that additional mutations might be the main contributors in the evolution of MDS to AML. Of note, patients with SF3B1SEC had half OS compared to patients with SF3B1DOM (SF3B1SECvs. SF3B1DOM: 15.9 mo. vs. 39.7 mo., P= 0.0001), suggesting that in cases evolving to AML, expanding hits might have dramatically skewed the favorable nature of SF3B1 mutations. Indeed, mutations preceding SF3B1 mainly affected lineage-restricted genes associated with repression of erythroid programs (RUNX1, 23%), terminal monocytic differentiation (TET2, 9%), transcriptional corepressors (BCOR/L1, 8%) and development of leukemia (DNMT3A, 8%). In conclusion, our study of the clonal architecture of SF3B1 mutations highlights that clonal progression of cases with MN harboring SF3B1 mutations might be inferred by the rank of additional genetic lesions cooperating with SF3B1. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Advani:Abbvie: Research Funding; Macrogenics: Research Funding; Pfizer: Honoraria, Research Funding; Amgen: Research Funding; Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy. Nazha:Tolero, Karyopharma: Honoraria; Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Abbvie: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. 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.

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

Blood ◽  
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.

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

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3816-3816 ◽  
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.

scholarly journals 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 ◽  
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.

scholarly journals Epigenetic Activation of the pH Regulator MCT4 in Acute Myeloid Leukemia Exploits a Fundamental Metabolic Process of Enhancing Cell Growth through Proton Shifting

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3765-3765
Author(s):  
Cheuk-Him Man ◽  
David T. Scadden ◽  
Francois Mercier ◽  
Nian Liu ◽  
Wentao Dong ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Growth ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Monocarboxylate Transporter ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Equity Ownership ◽  
Acute Myeloid

Acute myeloid leukemia (AML) cells exhibit metabolic alterations that may provide therapeutic targets not necessarily evident in the cancer cell genome. Among the metabolic features we noted in AML compared with normal hematopoietic stem and progenitors (HSPC) was a strikingly consistent alkaline intracellular pH (pHi). Among candidate proton regulators, monocarboxylate transporter 4 (MCT4) mRNA and protein were differentially increased in multiple human and mouse AML cell lines and primary AML cells. MCT4 is a plasma membrane H+and lactate co-transporter whose activity necessarily shifts protons extracellularly as intracellular lactate is extruded. MCT4 activity is increased when overexpressed or with increased intracellular lactate generated by glycolysis in the setting of nutrient abundance. With increased MCT4 activity, extracellular lactate and protons will increase causing extracellular acidification while alkalinizing the intracellular compartment. MCT4-knockout (MCT4-KO) of mouse and human AMLdid not induce compensatory MCT1 expression, reduced pHi, suppressed proliferation and improved animal survival. Growth reduction was experimentally defined to be due to intracellular acidification rather than lactate accumulation by independent modulation of those parameters. MCT4-KOmetabolic profiling demonstrated decreased ATP/ADP and increased NADP+/NADPH suggesting suppression of glycolysis and the pentose phosphate pathway (PPP) that was confirmed by stable isotopic carbon flux analyses. Notably,the enzymatic activity of purified gatekeeper enzymes, hexokinase 1 (HK1), pyruvate kinase M2 isoform (PKM2) and glucose-6-phosphate dehydrogenase (G6PDH) was sensitive to pH with increased activity at the leukemic pHi (pH 7.6) compared to normal pHi (pH 7.3). Evaluating MCT4 transcriptional regulation, we defined that activating histonemarks, H3K27ac and H3K4me3, were enriched at the MCT4 promoter region as were transcriptional regulators MLL1 and Brd4 by ChIP in AML compared with normal cells. Pharmacologic inhibition of Brd4 suppressed Brd4 and H3K27ac enrichment and MCT4 expression in AML and reduced leukemic cell growth. To determine whether MCT4 based pHi changes were sufficient to increase cell proliferation, we overexpressed MCT4 in normal HSPC and demonstrated in vivo increases in growth in conjunction with pHi alkalization. Some other cell types also were increased in their growth kinetics by MCT4 overexpression and pHi increase. Therefore, proton shifting may be a means by which cells respond to nutrient abundance, co-transporting lactate and protons out of the cell, increasing the activity of enzymes that enhance PPP and glycolysis for biomass generation. Epigenetic changes in AML appear to exploit that process by increasing MCT4 expression to enforce proton exclusion thereby gaining a growth advantage without dependence on signaling pathways. Inhibiting MCT4 and intracellular alkalization may diminish the ability of AML to outcompete normal hematopoiesis. Figure Disclosures Scadden: Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Sponsored research; Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; Fog Pharma: Consultancy; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Equity Ownership.

scholarly journals Inducible SBDS Deficiency Impairs Bone Marrow Niche Function to Engraft Donor Hematopoietic Stem Cell after Transplantation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3199-3199
Author(s):  
Ji Zha ◽  
Lori Kunselman ◽  
Hongbo Michael Xie ◽  
Brian Ennis ◽  
Jian-Meng Fan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mouse Model ◽  
Board Of Directors ◽  
Hematopoietic Stem Cell ◽  
Graft Failure ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Bm Niche ◽  
Deficient Mice

Hematopoietic stem cell (HSC) transplantation (HSCT) is required for curative therapy for patients with high-risk hematologic malignancies, and a number of non-malignant disorders including inherited bone marrow failure syndromes (iBMFS). Strategies to enhance bone marrow (BM) niche capacity to engraft donor HSC have the potential to improve HSCT outcome by decreasing graft failure rates and enabling reduction in conditioning intensity and regimen-associated complications. Several studies in animal models of iBMFS have demonstrated that BM niche dysfunction contributes to both the pathogenesis of iBMFS, as well as impaired graft function after HSCT. We hypothesize that such iBMFS mouse models are useful tools for discovering targetable niche elements critical for donor engraftment after HSCT. Here, we report the development of a novel mouse model of Shwachman-Diamond Syndrome (SDS) driven by conditional Sbds deletion, which demonstrates profound impairment of healthy donor hematopoietic engraftment after HSCT due to pathway-specific dysfunctional signaling within SBDS-deficient recipient niches. We first attempted to delete Sbds specifically in mature osteoblasts by crossing Sbdsfl/flmice with Col1a1Cre+mice. However, the Col1a1CreSbdsExc progenies are embryonic lethal at E12-E15 stage due to developmental musculoskeletal abnormalities. Alternatively, we generated an inducible SDS mouse model by crossing Sbdsfl/flmice with Mx1Cre+ mice, and inducing Sbds deletion in Mx1-inducible BM hematopoietic and osteolineage niche cells by polyinosinic-polycytidilic acid (pIpC) administration. Compared with Sbdsfl/flcontrols, Mx1CreSbdsExc mice develop significantly decreased platelet counts, an inverted peripheral blood myeloid/lymphoid cell ratio, and reduced long-term HSC within BM, consistent with stress hematopoiesis seen in BMF and myelodysplastic syndromes. To assess whether inducible SBDS deficiency impacts niche function to engraft donor HSC, we transplanted GFP+ wildtype donor BM into pIpC-treated Mx1CreSbdsExc mice and Sbdsfl/flcontrols after 1100 cGy of total body irradiation (TBI). Following transplantation, Mx1CreSbdsExc recipient mice exhibit significantly higher mortality than controls (Figure 1). The decreased survival was related to primary graft failure, as Mx1CreSbdsExc mice exhibit persistent BM aplasia after HSCT and decreased GFP+ reconstitution in competitive secondary transplantation assays. We next sought to identify the molecular and cellular defects within BM niche cells that contribute to the engraftment deficits in SBDS-deficient mice. We performed RNA-seq analysis on the BM stromal cells from irradiated Mx1CreSbdsExc mice versus controls, and the results revealed that SBDS deficiency in BM niche cells caused disrupted gene expression within osteoclast differentiation, FcγR-mediated phagocytosis, and VEGF signaling pathways. Multiplex ELISA assays showed that the BM niche of irradiated Mx1CreSbdsExc mice expresses lower levels of CXCL12, P-selectin and IGF-1, along with higher levels of G-CSF, CCL3, osteopontin and CCL9 than controls. Together, these results suggest that poor donor HSC engraftment in SBDS-deficient mice is likely caused by alterations in niche-mediated donor HSC homing/retention, bone metabolism, host monocyte survival, signaling within IGF-1 and VEGF pathways, and an increased inflammatory state within BM niches. Moreover, flow cytometry analysis showed that compared to controls, the BM niche of irradiated Mx1CreSbdsExc mice contained far fewer megakaryocytes, a hematopoietic cell component of BM niches that we previously demonstrated to be critical in promoting osteoblastic niche expansion and donor HSC engraftment. Taken together, our data demonstrated that SBDS deficiency in BM niches results in reduced capacity to engraft donor HSC. We have identified multiple molecular and cellular defects in the SBDS-deficient niche contributing to this phenotype. Such niche signaling pathway-specific deficits implicate these pathways as critical for donor engraftment during HSCT, and suggest their potential role as targets of therapeutic approaches to enhance donor engraftment and improve HSCT outcome in any condition for which HSCT is required for cure. Disclosures Olson: Merck: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

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