Primitive Hematopoietic Progenitor Cells Research Articles

A DESCRIPTIVE STUDY TO ASSESS KNOWLEDGE REGARDING UMBILICAL CORD BLOOD BANKING AMONG WOMEN IN THE REPRODUCTIVE AGE

Umbilical cord blood stem cells are the name given to the blood remaining in the umbilical cord and placenta after childbirth. Umbilical cord blood has the potential to be a huge source of primitive hematopoietic stem and progenitor cells that can be used to reconstruct the hematopoietic system and restore immunological function in individuals who require treatment. A descriptive research methodology was adopted in this study and 100 reproductive age group women in Sharda Hospital were selected in the present study. Convenience sampling technique was adopted for the data collection. The information was gathered via a knowledge questionnaire, which was used to assess knowledge regarding umbilical cord blood banking. The result revealed that 30 women had poor knowledge, 63 women had moderate knowledge and 7 had knowledge about umbilical cord blood banking. The importance of umbilical cord blood banking information was given among selected 100 participating women via the help of a pamphlet to raise awareness of uses of umbilical cord bloodbanking.

A germline point mutation in the MYC-FBW7 phosphodegron initiates hematopoietic malignancies.

Oncogenic activation of MYC in cancers predominantly involves increased transcription rather than coding region mutations. However, MYC-dependent lymphomas frequently acquire point mutations in the MYC phosphodegron, including at threonine 58 (T58), where phosphorylation permits binding via the FBW7 ubiquitin ligase triggering MYC degradation. To understand how T58 phosphorylation functions in normal cell physiology, we introduced an alanine mutation at T58 (T58A) into the endogenous c-Myc locus in the mouse germline. While MYC-T58A mice develop normally, lymphomas and myeloid leukemias emerge in ∼60% of adult homozygous T58A mice. We found that primitive hematopoietic progenitor cells from MYC-T58A mice exhibit aberrant self-renewal normally associated with hematopoietic stem cells (HSCs) and up-regulate a subset of MYC target genes important in maintaining stem/progenitor cell balance. In lymphocytes, genomic occupancy by MYC-T58A was increased at all promoters compared with WT MYC, while genes differentially expressed in a T58A-dependent manner were significantly more proximal to MYC-bound enhancers. MYC-T58A lymphocyte progenitors exhibited metabolic alterations and decreased activation of inflammatory and apoptotic pathways. Our data demonstrate that a single point mutation stabilizing MYC is sufficient to skew target gene expression, producing a profound gain of function in multipotential hematopoietic progenitors associated with self-renewal and initiation of lymphomas and leukemias.

792-P: GLP-1 RA Therapy Increases Circulating Vascular Regenerative Cell Content and Decreases Inflammatory Burden in People with T2D

CV outcome trials have established that long-term use of GLP-1 receptor agonists (GLP-1RAs) yields important CV benefits in people with T2D. Similar to SGLT2i, GLP-1RAs are recommended for people with T2D who have established CVD and heart failure regardless of their A1C. In a randomized trial of people with T2D and coronary artery disease, use of the SGLT2i empagliflozin for 6 months increased the frequency of vascular regenerative (VR) cells in the circulation. To determine if GLP-1RA therapy has a similar impact on the same VR cell populations, we retrospectively analyzed data from an ongoing research program with adults >40 years of age with T2D. Peripheral blood mononuclear cells were isolated and profiled with an established flow cytometry assay that was founded on the activity of the anti-oxidative enzyme aldehyde dehydrogenase (ALDH) that is highly expressed in progenitor (ALDHhi) versus mature (ALDHlow) cells. At baseline, 17 participants were on a GLP-1RA (9 of whom were also on an SGLT2i), 33 were on an SGLT2i but not a GLP-1RA, and 28 were on neither drug class. Mean age, weight, BMI, A1C, and duration of T2D was balanced across the groups. The frequency of ALDHhiSSClow cells (primitive hematopoietic and endothelial progenitor cells that coordinate angiogenesis) was higher in participants who were on a GLP-1RA (0.064%) or an SGLT2i (0.048%) relative to those not on either drug class (0.037%; P<0.05 for both). The frequencies of VR ALDHhiSSCmid monocytes in the three groups were comparable. The frequency of pro-inflammatory ALDHhiSSChi granulocyte precursor cells in participants on a GLP-1RA (2.9%) was lower than that in individuals on an SGLT2i (4.3%; P=0.05) or not on either drug class (5.2%; P<0.01). GLP-1RA therapy in people with T2D was associated with greater VR cell content and lower inflammatory burden. Augmented vessel repair mediated by higher VR cell content may account in part for the CV benefits observed with GLP-1RA in the setting of T2D. Disclosure A.Krishnaraj: None. B.Park: None. E.Bakbak: None. Y.Pan: None. A.Quan: None. H.Teoh: None. S.Verma: Advisory Panel; Amgen Canada, AstraZeneca, Bayer Inc., Boehringer Ingelheim and Eli Lilly Alliance, HLS Therapeutics Inc., Janssen Pharmaceuticals, Inc., Novartis Canada, Novartis, Novo Nordisk, Novo Nordisk Canada Inc., Consultant; AstraZeneca, Other Relationship; Amarin Corporation, AstraZeneca, Bayer Inc., Boehringer Ingelheim and Eli Lilly Alliance, Boehringer Ingelheim International GmbH, Canadian Medical and Surgical Knowledge Translation Research Group, EOCI Pharmacomm, HLS Therapeutics Inc., Janssen Pharmaceuticals, Inc., Novartis Canada, Novo Nordisk, Novo Nordisk Canada Inc., Pfizer Inc., PhaseBio Pharmaceuticals, Inc., S & L Solutions Event Management Inc, Sanofi, Sun Pharmaceutical Industries Ltd., Toronto Knowledge Translation Working Group, Research Support; Amarin Corporation, Amgen Canada, AstraZeneca, Bayer Inc., Boehringer Ingelheim International GmbH, HLS Therapeutics Inc., Novartis, Novo Nordisk, Pfizer Inc., PhaseBio Pharmaceuticals, Inc. D.A.Hess: None.

Impact of the Aryl Hydrocarbon Receptor on Aurora A Kinase and the G2/M Phase Pathway in Hematopoietic Stem and Progenitor Cells

Recent evidence suggests that the environment-sensing transcription factor aryl hydrocarbon receptor (AHR) is an important regulator of hematopoiesis. Yet, the mechanisms and extent of AHR-mediated regulation within the most primitive hematopoietic cells, hematopoietic stem and progenitor cells (HSPCs), are poorly understood. Through a combination of transcriptomic and flow cytometric approaches, this study provides new insight into how the AHR influences hematopoietic stem and progenitor cells. Comparative analysis of intraphenotypic transcriptomes of hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells from AHR knockout (AHR KO) and wild type mice revealed significant differences in gene expression patterns. Notable among these were differences in expression of cell cycle regulators, specifically an enrichment of G2/M checkpoint genes when Ahr was absent. This included the regulator Aurora A kinase (Aurka, AurA). Analysis of AurA protein levels in HSPC subsets using flow cytometry, in combination with inducible AHR KO or in vivo AHR antagonism, showed that attenuation of AHR increased levels of AurA in HSCs and lineage-biased MPP cells. Overall, these data highlight a potential novel mechanism by which AHR controls HSC homeostasis and HSPC differentiation. These findings advance the understanding of how AHR influences and regulates primitive hematopoiesis.

Effects of Rigosertib (RIGO) Alone or in Combination with Azacitidine or Vorinostat on Epigenetic Reprogramming of CD34+ Cells in the Myelodysplastic Syndrome

Rigosertib (RIG) which functions as a RAS-mimetic, binds to the RAS-binding domain (RBD) impacting many pathways that involve RBD-mediated interactions, including the RAS-Raf and AKT/PI3-kinase pathways (Divakar et al., Cell 2016). Recently, we demonstrated that RIG also functions as an epigenetic modifier in MDS. RIG alone or in combination with Azacitidine (AZA) exerted epigenetic effects on the global histone post-translational modifications (PTMs), chromatin remodelers, HDACs, and DNMTs and caused enhanced apoptosis in MDS (MDS-L) and AML (BW-90) cell lines as well as in bone marrow cells (BM) derived from pts with MDS (Chaurasia et al ASCO 2016). In clinical trials, the combination of RIG and AZA demonstrated an overall response rate of 76% in all MDS patients; 62% response in pts who had failed prior hypomethyating agent (HMA) (Navada et al ASH 2016) and 85% in HMA naive pts. The growing evidence suggests that combined therapy may be more effective than either agent alone. To further study these effects, we explored activity of RIG in combination with Vorinostat (VOR), a histone deacetylase inhibitor, on the cell lines. Combined treatment in sequential order VOR/RIG and RIG/VOR potentiates far greater effects on the global PTMs, HDACs, DNMTs, chromatin remodelers, cell cycle checkpoint proteins and PI3/AKT pathway than RIG/AZA or AZA/RIG.RIG in combination with AZA or VOR (AZA/RIG, RIG/AZA, VOR/RIG or RIG/VOR) demonstrated differential effects on the association of RNA polymerase II (Pol II) with active histone marks (H3K4me3 and H3K4me2) in MDS-L and BW-90 cells. An overall decrease in association of Pol II/H3K4me2 was observed with the combinations (AZA/RIG, VOR/RIG or vice versa) compared to control in MDS-L and BW-90, 10-33% (ANOVA, p=0.0006), 9-20% (ANOVA, p=0.0004), respectively. Significant differences were observed in association of H3K4me3/Pol II in BW-90 cells (7-30%; ANOVA, p<0.0001). Similarly, in BM from a pt with MDS after 1 cycle of RIG and AZA treatment demonstrated a decrease in association of Pol II with H3K4me2 (67%) and H3K4me3 (28%).Treatment of MDS-L cells with RIG alone or in combination with AZA failed to induce expansion of CD34+ cells, but the RIG/VOR combination induced 1.9-fold expansion of CD34+ cells (ANOVA p=0.002). Individually AZA or VOR treatment led to an enhanced expansion of CD34+ cells, 3.8 and 4.0 fold, respectively (ANOVA, p=0.004). RIG alone or with AZA yielded maximum aldehyde dehydrogenase (ALDH) activity, a marker of primitive hematopoietic stem and progenitor cells (HSPCs) (ANOVA, p=0.006). However, a marked decrease in ALDH activity was observed in AZA or VOR or RIG/VOR, that was inversely proportion to the expansion of CD34+ cells.In a single MDS pt treated with RIG/AZA, expansion of primitive HPSCs expressing low levels of CD34 was observed. A highly expressed subpopulation of HPSCs, which co-existed prior to treatment of the pt and disappeared upon treatment, suggests either an induced cell death or commitment of HSPCs towards improved lineage differentiation in conjunction with clinical response.In order to evaluate effects of RIG on expansion of CD34+ cells, we examined the pluripotent genes (SOX2, OCT4 NANOG and ZIC3) in the BM from MDS pts, prior and after RIG/AZA treatment. RNA expression levels of SOX2, OCT4, NANOG and ZIC3 were elevated post treatment (≥2 fold). In MDS-L cells the pluripotency genes were highly upregulated in the presence of RIG or RIG/AZA or RIG/VOR (1.7-34 folds). These findings indicate that expression of pluripotency genes is a consequence of epigenetic reprogramming that favors expansion of more primitive HSPCs in a pt, while increase in ALDH activity was inversely proportional to the enhanced expansion of CD34+ cells in MDS-L suggesting a more primitive state of HSPC.The epigenetic events modulated by RIG in combination with either AZA or VOR led to global histone PTMs, differential Pol II association with active histone marks, epigenetic reprogramming of pluripotency genes and expansion of primitive HSPC with downregulation of the PI3K/AKT pathway and cell cycle checkpoint proteins. Collectively, these studies indicate that epigenetic effects of RIG on chromatin alterations lead to HSPC reprogramming that may lead to improved hematopoietic function and response in the clinical setting. These preclinical models suggest potential novel clinical strategies to improve outcomes for patients with higher-risk MDS. DisclosuresReddy:Onconova Therapeutics: Equity Ownership.

HOTAIR expression and prognostic impact in acute myeloid leukemia patients

BackgroundAcute myeloid leukemia (AML) is a disorder characterized by a rapid onset of symptoms attributable to bone marrow failure due to clonal proliferation of primitive hematopoietic stem cells or progenitor cells. Epigenetic abnormalities play an important role in the development and progression of acute leukemia. Long non-coding ribonucleic acid (lncRNA) plays an important role in epigenetic regulation. Homeobox (Hox) transcript antisense intergenic RNA (HOTAIR) is a lncRNA which has been determined to be a negative prognostic indicator in various solid-tumor patients. However, its role in hematopoietic tumors as AML is to be assessed. This study aimed at measuring lncRNA HOTAIR expression level on bone marrow (BM) mononuclear cells in newly diagnosed AML patients and correlating its expression with their outcome and different prognostic variables. This provides new prospective for a novel marker involved in development and progression of AML which can be used as a diagnostic marker and a target of therapy. The current study included 65 subjects divided into 35 newly diagnosed AML adult patients (before initiation of chemotherapy) and 30 non-leukemic adult patients who are candidates for BM aspiration for causes other than hematological malignancies as immune thrombocytopenic purpura and hypersplenism as controls. HOTAIR expression was measured on BM mononuclear cells by quantitative reverse transcription polymerase chain reaction (qRT-PCR).ResultsHOTAIR expression was found to be significantly upregulated in AML patients (probability (p) value = 0.000) and it can be used as a diagnostic biomarker of AML as confirmed by a significant difference between cases and controls using receiver operating characteristic curve (ROC) analysis. However, it was not significantly correlated with event free survival (EFS) or prognostic variables.ConclusionThis study showed that the expression of HOTAIR is upregulated in de novo AML patients and can be used as a diagnostic marker. However, highly expressed HOTAIR is not associated with poor prognosis.

TET2 and DNMT3A Mutations Exert Divergent Effects on DNA Repair and Sensitivity of Leukemia Cells to PARP Inhibitors

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that both affect the epigenetic regulation of DNA methylation. Although the proteins antagonistically regulate the epigenetic mark of 5-methylcytosine (5-mC), where DNMT3A catalyzes addition of 5-mC while TET2 oxidizes 5-mC as a first step in DNA demethylation, mutations in both genes appear in a similar spectrum of human hematopoietic malignancies. Mutations in both genes often co-occur with activating mutations in oncogenic tyrosine kinases (OTKs) such as FLT3ITD, BCR-ABL1, JAK2V617F, MPLW515L or mutations affecting related signaling pathways such as NRASG12D and CALRdel52. Moreover, while the mutations exert divergent effect on primitive hematopoietic progenitor cells, they lead to similar disease phenotypes, suggesting the roles of these mutations in hematopoietic malignancies may relate to mechanisms outside of DNA methylation. OTK-positive malignant cells accumulate high numbers of spontaneous and drug-induced DNA double-strand breaks (DSBs) in comparison to normal cells, but they manage to survive because of their enhanced/altered ability to repair these breaks. DSBs, the most lethal DNA lesions, are repaired by two major mechanisms, BRCA1/2-dependent homologous recombination (HR) and DNA-PK -mediated non-homologous end-joining (D-NHEJ). Both HR and D-NHEJ repair DSBs in proliferating cells, while D-NHEJ plays a major role in quiescent cells. PARP1 -dependent alternative NHEJ (Alt-NHEJ) serves as back-up in both proliferating and quiescent cells. The existence of these redundant pathways creates the opportunity to employ a phenomenon called "synthetic lethality", which was originally applied to eliminate cancer cells with mutations in BRCA1 and BRCA2 by PARP inhibitor (PARPi). Our previous report [M. Nieborowska-Skorska et al., Gene expression and mutation-guided synthetic lethality eradicates proliferating and quiescent leukemia cells. J Clin Invest 127, 2392-2406 (2017)] suggested that certain leukemias are sensitive to PARPi-triggered synthetic lethality. Here we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing OTKs. Malignant TET2-deficient cells display downregulation of BRCA1 and LIG4 resulting in reduced activity of HR and D-NHEJ, respectively, and rely on Alt-NHEJ to protect them from the toxic effects of replication stress and drug-induced DSBs. Conversely, DNMT3A-deficient cells favor HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells are sensitive to PARPi treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity and/or TET2 - Wilms tumor 1 (WT1) binding ability were responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Our findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. Disclosures Valent: Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding. Tallman:Abbvie: Research Funding; Cellerant: Research Funding; Orsenix: Research Funding; ADC Therapeutics: Research Funding; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; Glycomimetics: Research Funding; Rafael: Research Funding; Amgen: Research Funding; Bioline rx: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; KAHR: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents &amp; Royalties. Martinelli:Roche: Consultancy; Pfizer: Consultancy, Research Funding, Speakers Bureau; Incyte: Consultancy; Jazz: Consultancy; Janssen: Consultancy; Daichii Sankyo: Consultancy, Research Funding; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy; AbbVie: Consultancy, Research Funding. Vassiliou:Kymab Ltd - Monoclonal antibody company. Currently not working in myeloid cancers or clonal haematopoiesis.: Consultancy.

Mutational screening of RTK-BRAF genes in de novo adult acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is derived from primitive hematopoietic stem cells or progenitor cells due to genetic variations such as chromosomal abnormalities and molecular alterations. Mutations in the RTK-BRAF genes are involved in leukaemogenesis. Few reports simultaneously concerning the mutation alteration of the RTK-BRAF genes have been publicized in AML subjects. The current study aimed to screen the mutations of RTK-BRAF genes in Iranian de novo adult AML subjects. MethodsMutations of RTK-BRAF genes were investigated in bone marrow specimens from 58 patients with de novo adult AML. High-Resolution Melt (HRM) curve analysis was performed for the identification of FLT3-ITD and D835 mutations. Mutations in the NPM1 (exon12), BRAF (exons11 and 15), PTPN11(exons 3 and 13) and c-FMS (exon22) were detected using Sanger sequencing method. ResultsTotally, FLT3 and NPM1 mutations were found in 34.48% and 25.86% of AML patients, respectively. Moreover, mutations in the FLT3 and NPM1 genes were significantly higher in normal karyotype AML subjects (52.38% and 38%, respectively) than patients with abnormal karyotype (p-value = 0.001 and p-value = 0.002, respectively). No mutations were detected in other RTK-BRAF genes. ConclusionAccording to the findings of this research, in addition to the chromosomal abnormalities in the patients with acute myeloid leukemia, FLT3 and NPM1 mutations were the most recurrent genetic alterations. Identification of these alterations may help to choose appropriate treatment and improve the patients' outcome.

Inflammatory bone marrow microenvironment.

Self-renewing hematopoietic stem cells and their progeny, lineage-specific downstream progenitors, maintain steady-state hematopoiesis in the bone marrow (BM). Accumulating evidence over the last few years indicates that not only primitive hematopoietic stem and progenitor cells (HSPCs), but also cells defining the microenvironment of the BM (BM niche), sense hematopoietic stress signals. They respond by directing and orchestrating hematopoiesis via not only cell-intrinsic but also cell-extrinsic mechanisms. Inflammation has many beneficial roles by activating the immune system in tissue repair and as a defense mechanism. However, chronic inflammation can have detrimental effects by stressing HSPCs, leading to cell (DNA) damage resulting in BM failure or even to leukemia. Emerging data have demonstrated that the BM microenvironment plays a significant role in the pathogenesis of hematopoietic malignancies, in particular, through disrupted inflammatory signaling, specifically in niche (microenvironmental) cells. Clonal selection in the context of microenvironmental alterations can occur in the context of toxic insults (eg, chemotherapy), not only aging but also inflammation. In this review, we summarize mechanisms that lead to an inflammatory BM microenvironment and discuss how this affects normal hematopoiesis. We pay particular attention to the process of aging, which is known to involve low-grade inflammation and is also associated with age-related clonal hematopoiesis and potentially malignant transformation.

Differential Expression of CD49f Discriminates the Independently Emerged Hematopoietic Stem Cells and Erythroid-Biased Progenitors

While hematopoietic stem cells (HSCs) can sustain the production of all types of mature blood cells throughout the life, there also exists HSC-independent hematopoiesis, which partially supports embryonic hematopoiesis and generation of specific types of adult hematopoietic cells (e.g., macrophages). Examples of the HSC-independent hematopoiesis include (i) the primitive wave of hematopoiesis that produces unipotent progenitors for erythrocytes, megakaryocytes or macrophages, and (ii) the "pro-definitive" hematopoiesis that produces multipotent erythro-myeloid progenitors (EMPs). Given that HSCs and HSC-independent progenitors are both derived from endothelial cells in distinct or overlapping hematopoietic sites, tracing their developmental origins and clarifying the regulatory mechanism will enhance our understanding of the profound difference between them and may improve in vitro generation of HSCs. Human HSCs have been refined based on the expression of CD49f (ITGA6). In combination with other HSC markers (CD34+CD38-CD45RA-CD43+CD90+), high expression of CD49f identifies long-term multilineage engrafting HSCs, whereas the cells with low CD49f represent a subtype of hematopoietic progenitor cells (HPCs) that possess transient engrafting activity. Meanwhile, CD49f has also been shown to be heterogeneously expressed in hemogenic endothelial cells (HECs), which give rise to both HSCs and EMPs via endothelial-to-hematopoietic transition (EHT). Thus, determining the changes (i.e., persistence, gain or loss) of CD49f expression during EHT is a key step in tracing the origins of HSCs and HSC-independent HPCs. In this study, using an in vitro system of HSC differentiation from human embryonic stem cells (hESCs), we observed that, while CD49f is highly expressed in all hESCs, only a portion of HECs express CD49f. Importantly, live cell imaging analysis revealed that CD49f expression persists during EHT, which is accompanied by initiating CD43 expression. To test whether the differential CD49f expression is associated with HSC versus HPC functions, we sorted the CD49fhigh and CD49flow cells and performed colony forming assay and gene expression profiling. The results showed that the CD49fhigh cells have multilineage potential, whereas the CD49flow cells lack lymphoid potential but show a strong erythroid preference. Gene expression analysis confirmed that the CD49fhigh and CD49flow cells represent HSCs and erythroid-biased HPCs, respectively, and that the Wnt and Notch signaling pathways may play a role in their functions. Collectively, these observations suggest that the CD49fhigh and the CD49flow cells are concurrently derived from the CD49fhigh and CD49flow HECs, thus modeling the in vivo generation of HSCs and HSC-independent HPCs. Based on the in vitro observations, we proposed that CD49f in vivo may also specify the distinct HSPCs emerged at different developmental stages/sites. To test this hypothesis, we isolated mouse primitive HPCs, EMPs and definitive HSCs, as well as their parental HECs, from yolk sac, embryo, and aorta-gonad-mesonephros (AGM) of different embryonic stages and determined their CD49f expression. The results showed that the primitive erythroid progenitors have lowest, whereas the definitive AGM HSCs have highest, CD49f levels; this trend was also observed in the related HECs isolated from various stages/sites. Thus, it is likely that the embryonic hematopoiesis is recapitulated, at least partially, by the in vitro system in terms of the sequential emergence of HSPCs ranging from unipotent erythroid progenitors to multipotent definitive HSCs, and this may also underlie the situation that EMPs and HSCs can be produced at the same stage/site but independently from different HECs. In summary, using the in vitro HSC differentiation system, we found that the differential expression of CD49f discriminates HSCs and HSC-independent progenitors, which are concurrently emerged from HECs. The persistent CD49f expression during EHT suggests that the fates of HSCs and HSC-independent HPCs are pre-defined in their parental HECs. Combining our in vivo data, the differential expression of CD49f also provide a possible regulatory mechanism for the multi-wave hematopoiesis. Further exploring the function and mechanism of CD49f in these regulations should be important for fully understanding the precisely regulated HSC generation and activities. Disclosures No relevant conflicts of interest to declare.

PB1702 EXPRESSION OF ANGIOTENSIN‐CONVERTING ENZYME IN PATIENTS WITH ACUTE LEUKEMIAS

Background:Blast cells are characterized by aberrant expression normal markers of early hematopoietic progenitor cells (HPC). Angiotensin‐converting enzyme (ACE/CD143), a key regulator of renin‐angiotensin system, was shown to be expressed in blood‐forming tissues of the human embryo and primitive human HPC. Recent studies indicated that CD143 is over‐expressed in leukemic myeloid blast cells.Aims:To evaluate the surface expression of CD143 in blast cells in patients with newly diagnosed acute leukemia (AL).Methods:The expression of CD143 was estimated in patients with newly diagnosed untreated acute leukemia, using eight‐color flow cytometry. The blast gate was defined on the basis of CD45dim expression and side‐scatter characteristics. Anti‐CD143 monoclonal antibody (clone BB9, BD) conjugated with phycoerythrin was used for analysis of CD143 expression. The percentage of positive cells (PPC) and the mean fluorescence intensity (MFI) was applied to describe expression. The expression of CD143 was devided into three groups according range of PPC ≤1%, 1 – 20 %, and ≥20 %.Results:A total of untreated 68 patients with newly diagnosed acute leukemia were included in the study group. They were diagnosed with non‐promyelocytic acute myeloid leukemia (non‐APL AML) – 35 (51.5 %), of them 3 (8.5 %) patients were related to favorable genetic group, 9 (25.7 %) – intermediate, 23 (65.8 %) – adverse; promyelocytic leukemia (APL) – 10 (14.7 %); B acute lymphoblastic leukemia/lymphoma (B‐ALL) – 13 (19.1 %), including 2 patients with Ph‐positive B‐ALL; T acute lymphoblastic leukemia/lymphoma (T‐ALL) – 10 (14.7 %), including 2 cases of early T‐precursor ALL (ETP). Median age of patients was 35.5 years, 29 male, 39 female.In total group with CD143 expression with PPC≥20% was observed in 7% (5/68) of all cases AL. CD143 expression in non‐APL AML with a range of PPC 1 – 20 % was detected in 12/35 cases (34.2 %), PPC ≥20 % was observed in 2/35 cases with complex karyotype (5.7 %). In group with APL 2/10 (20 %) patients had 1 – 20%, and 2/10 (20 %) patients had &gt;20 %. Among T‐ALL only 1/10 (10 %) had a CD143 expression. ACE expression in B‐ALL was absent (defined as ≤1 %). Median MFI of ACE expression was higher in patients with APL – 477 (range 159 – 4796) than in non‐APL AML ‐ 175 (range 43 – 3253). CD143 expression with PPC≥1% was significantly higher in patients with AML (non‐APL AML and APL), than in ALL (B‐ and T‐ALL), 40 % (18/45) vs. 4.3 % (1/23) (p = 0.0016).Summary/Conclusion:Thus, CD143 expression with PPC≥20% was observed only in 7% (5/68) of all cases AL, 2 was observed in AML with PML‐RARA, 2 ‐ AML with complex karyotype and 1 in ETP‐ALL. No expression of CD143 was detected in B‐ALL. CD143 expression was significantly higher in myeloid blast cells than lymphoid. No correlation was found between the expression of ACE and age, sex, neuroleukemia, leucocytes count, bone marrow blasts, lactic acid dehydrogenase activity and cytogenetic abnormalities in different subgroups. The significance of CD143 expression in AL is not completely clear and has to be studied.

Enumeration of primitive haematopoietic progenitor cells to improve the quality of cord blood product testing

Background & Aim Human cord blood-derived primitive CD133+CD34− cells have been shown to engraft in animal models of transplantation. Using a modified ISHAGE method, we have developed a flow cytometry-based assay to enumerate CD133+CD34− haematopoietic progenitor cells (HPCs) in cord blood (CB) samples that meet regulatory requirements. Methods, Results & Conclusion A non-linear regression with one phase decay model was fitted between CD133+CD34− and CD34+ HPCs compartments. The proportion of CD133+CD34− HPCs was found to inversely correlate with CD34+ HPCs (n=42, R2=0.76). CD133+CD34− cells were significantly smaller than CD34+ cells (p We analysed survival using a threshold of 0.45% CD34+ of total nucleated cells (TNC) in cohorts of transplant recipients who received a single CBU from the Sydney Cord Blood Bank. Patients that died within 3 months of transplant were censored from the analysis. The proportion of CD34+ HPCs in the CBU had no effect on survival when TNC dose was more than 7 × 107/kg (n=44; graph on left). However, in patients who received TNC dose of 3-6 × 107/kg a significant improvement in survival (76% vs 52% at 36 months) was observed when the proportion of CD34+ cells was With the advancements in flow cytometric technology and clinical grade reagents, and with access to high quality CBU final product, we were able to demonstrate that primitive CD133+CD34− cells can be enumerated. These data suggest that CD133+CD34− HPCs may contribute to long-term engraftment after cord blood transplantation. We believe that the role of CD133+CD34− HPCs in long-term engraftment should be further evaluated and that prospective studies to determine the infused CD133+CD34− HPC dose are required to elucidate the potential role of these primitive cells in engraftment and survival.

EZH2 Inhibitor GSK126 Suppresses Antitumor Immunity by Driving Production of Myeloid-Derived Suppressor Cells.

Enhancer of zeste homolog (EZH2) is a key epigenetic regulator of gene expression and is frequently overexpressed in various cancer types, suggesting a role in oncogenesis. The therapeutic potential of EZH2 inhibitors is currently being explored, but their effect on antitumor immunity is largely unknown. Here we report that suppressing EZH2 activity using EZH2 inhibitor GSK126 resulted in increased numbers of myeloid-derived suppressor cells (MDSC) and fewer CD4+ and IFNγ+CD8+ T cells, which are involved in antitumor immunity. Addition of a neutralizing antibody against the myeloid differentiation antigen GR-1 or gemcitabine/5-fluorouracil-depleted MDSCs alleviated MDSC-mediated immunosuppression and increased CD4+ and CD8+ T-cell tumor infiltration and GSK126 therapeutic efficacy. Mechanistically, we identified a novel pathway of MDSC production in cancer in which EZH2 inhibition directs myeloid differentiation from primitive hematopoietic progenitor cells. These findings suggest that modulating the tumor immune microenvironment may improve the efficacy of EZH2 inhibitors. SIGNIFICANCE: This study uncovers a potential mechanism behind disappointing results of a phase I clinical trial of EZH2 inhibitor GSK126 and identifies a translatable combinational strategy to overcome it.

Anesthesia consideration for cesarean section in chronic myeloid leukemia diagnosed during pregnancy: An interesting case report and brief review of literature

Chronic myeloid leukemia (CML) is a myeloproliferative disorder with clonal expansion of transformed primitive hematopoietic progenitor cells without loss of their capacity to differentiate. Annual incidence of CML in females ranges from 0.6 to 1.6 per 100,000 populations. In developing countries like India where onset of hematological malignancy occurs in early age, thereby increases chances of concomitant occurrence of CML and pregnancy together. Although the incidence is rare, anesthesia management of pregnant female presenting more so for an emergency cesarean section (C/S) is challenging because of the physiological changes during pregnancy, presence of anemia, coagulopathy, immunosuppressant drugs, leukocytosis, and rarely blast cells in circulation. We report the successful management of a 19-year-old primigravida, recently diagnosed with CML, planned for emergency C/S under general anesthesia in view of meconium-stained liquor with fetal distress.

Role of RasGRP3 in EPO/EPOR Signaling and Transmigration of Human Hematopoietic CD34+ Cells

Introduction: Umbilical cord blood (UCB) is a salient source of primitive hematopoietic stem progenitor cells (HSPCs) for bone marrow (BM) reconstitution in patients with hematologic and non-hematologic malignancies. However, a relatively low number of HSPCs in UCB units and poor BM homing efficiency greatly hinders the clinical application of UCB CD34+ cells for transplantation. To overcome these hurdles, we developed two independent strategies that increase CD34+ cell numbers and improve BM homing efficiency of UCB HSPCs. First, we expanded UCB HSPCs by culturing them in decellularized Wharton's jelly matrix (DWJM), a biometric scaffold mimicking the 3-dimenstional (3D) microenvironment of BM. Second, we enhanced the in vitro transmigration and in vivo BM homing efficiency of UCB CD34+ cells by blocking EPO/EPOR signaling. Both approaches enhance UCB CD34+ cell migration toward stromal cell-derived factor 1 (SDF1). In this study, we employed RNA-Seq and RT-PCR approaches to analyze UCB HSPCs treated with EPO and co-cultured with DWJM, aiming to identify molecules that regulate UCB HSPC transmigration via EPO/EPOR signaling.Methods: CD34+ cells from highly enriched UCB units (>90% purity) were treated with EPO for 24 hours and separately co-cultured with DWJM for 1 week. UCB CD34+ cells were collected and subjected to RNA-Seq and real-time PCR (RT-PCR) analyses. In vitro transmigration toward SDF-1 was assessed by transwell assay. To assess the involvement of RasGRP3 in UCB CD34+ cell mobility, cells were treated with 100 nM ingenol-3-angelate (I3A), a diacylglycerol (DAG) analog that specifically targets RAS Guanyl Releasing-Protein 3 (RasGRP3), for 16 hours followed by transwell assay. Anti-EPOR antibody-treated or EPO-treated cells were used as controls. In addition, RasGRP3 gene expression was examined in CD34+ cells from peripheral blood (PB) and BM samples collected from the same donor, and compared to RasGRP3 expression in UCB CD34+ cells. Unpaired, 2-tailed t-test was used to analyze results.Results: RasGRP3 was identified by RNA-Seq from the two independent approaches, EPO treatment and DWJM co-culture. EPO downregulated and DWJM upregulated RasGRP3 gene expression in UCB CD34+ cells. RasGRP3 expression was confirmed by qPCR. UCB CD34+ cells that migrated to the bottom chamber of the transwell assays, a population that has a higher mobility, showed an elevated RasGRP3 gene expression and a decreased EPOR cell surface expression. Activation of RasGRP3 by DAG analog I3A induced a significant increase in RasGRP3 gene expression (control: I3A treatment = 1: 202 ± 58, p=0.00012) that was associated with an enhanced transmigration capability (control: I3A = 41%+/-5: 54%+/- 3, p=0.032). Knocking-down of RasGRP3 in K562 cells, a known EPOR expressing cell line, impaired the transmigration capability of K562. CD34+ cells in peripheral blood (PB) showed a higher level of RasGRP3 gene expression compared to CD34+ cells in BM samples from the same healthy donors. RasGRP3 expression in PB CD34+ cells was significantly higher than BM and UCB CD34+ cells (qPCR signals relative to BM, BM: PB: UCB = 1: 431±65: 21±8, p=0.0012, 0.0023, and <0.0001 for BM vs. PB, BM vs. UCB and PB vs. UCB, respectively).Conclusions: By employing transwell assays, flow cytometry and molecular analyses, we demonstrate for the first time that RasGRP3, a protein responsible for GDP/GTP exchange of Ras, regulates the transmigration ability of human CD34+ cells. In addition, our findings connect RasGRP3 expression to the EPOR-mediated signaling pathway in CD34+ cells. A significantly higher level of RasGRP3 expression in PB CD34+ cells than its counterparts in BM might provide an explanation for why PB HSPCs show relatively faster BM engraftment than BM HSPCs during transplantation. Ongoing follow-up studies will elucidate the molecular mechanism(s) underlying EPOR signaling, which holds clinical potential to improve the BM homing deficiency of UCB CD34+ cells via modulating EPOR and RasGRP3 expression (Figure 1). [Display omitted] DisclosuresLiesveld:Onconova: Other: DSMB; Abbvie: Honoraria. Aljitawi:Medpace: Consultancy; The University of Rochester Medical Center: Patents & Royalties: Pending patent related to decellularized Wharton's jelly matrix.

Sequential Treatment with Rigosertib Followed By Azacitidine Maximizes the Effects on the Interferon Signaling Pathway in Hematopoietic Cells in Myelodysplastic Syndrome (MDS)

Background: MDS is a clonal stem cell disorder characterized by abnormal maturation and differentiation of hematopoietic cells. Azacitidine (AZA), a hypomethylating agent (HMA), was approved by the FDA for patients (pts) with MDS and is the standard of care as 1st line therapy for higher- risk disease. About 50% of MDS pts respond to AZA, with a median duration of response of 14 - 24 month. For those pts responding to an HMA, most either relapse or progress with worsening bone marrow failure (BMF) and have a median survival of 4 to 6 months after treatment failure. Both primary and secondary resistance remains significant clinical problems and result in poor survival. A recent study reveals that AZA activates several immunomodulatory pathways leading to activation of the antiviral defense pathway and upregulation of interferon signaling (IFN) (Chiappinelli et al, 2017). INF has multiple effects on hematopoiesis and appears highly regulated. Interferon can stimulate hematopoietic stem cells (HSC) which may be countered through activation of p38 MAPK; inhibition of the latter may improve hematopoiesis. Short-term effects of IFN stimulate myeloid and erythroid hematopoiesis.Rigosertib (RIG) is a Ras-mimetic that blocks the activation of Ras effector proteins. It has been identified as a novel anti-cancer drug which inhibits cell cycle progression and induces apoptosis of cancer cells (Athuluri-Divakar et al, 2016). It activates apoptosis related pathways by ameliorating several signaling pathways like Akt, p38, MAPK/ JNK, STAT3, β-catenin, GSK3α/β and PLK1 that can be dysregulated in MDS pts (Xu et al, 2014). In vitro, the combination of RIG with AZA (Skidan et al, AACR 2006) showed synergistic effect in inducing cell death in a sequence dependent manner requiring RIG priming. We have reported that it acts as a histone deacetylase inhibitor with chromatin modifying activity (Chaurasia et al, ASCO 2016). In a Phase I/II study the combination of RIG and AZA (NCT 01926587) produced an ORR of 85% in pts who were HMA naïve and importantly 62% in HMA failures (Navada et al, EHA 2017). The ability to reverse the clinical resistance phenotype is a novel observation with important clinical implications and understanding the mechanisms is critical to develop ways of reversing resistance.Methods: We investigated the in vitro effects of RIG combined with AZA on two cell lines one resistant and one sensitive to AZA: BW90, an AZA resistant line and MDS-L, AZA sensitive and in specimens from 2 pts treated on a clinical trial of RIG and AZA in combination, to determine effects on hematopoietic cells and IN signaling.Results: QT-PCR studies demonstrated that individual treatment of MDS-L and BW90 with RIG or combined with AZA in sequential treatment (AZA/RIG or RIG/AZA) altered chromatin remodeler (KDM2a, SET1, JMJD3 and LRWD1) transcript levels in a cell line specific context. Exposure of cells from a bone marrow from a patient prior to treatment with RIG alone or RIG/AZA failed to induce expansion of CD34+ cells and yielded maximum aldehyde dehydrogenase (ALDH) activity, a marker of primitive hematopoietic stem and progenitor cells (HSCs) (ANOVA, p=0.006). AZA alone yielded a 3.8 fold expansion of CD34+, with marked decrease in ALDH activity that was inversely proportional to the expansion of CD34+ cells. Expansion of CD34+ cells led to ≥2 fold increase in pluripotent genes (SOX2, OCT4, NANOG and ZIC3) expression levels. QT PCR studies of the effects of AZA or RIG alone or in combination on INF yielded differential effects on INFa and INFb expression as follows compared to control reported as percent fold increase for INFa - AZA 2x; RIG 0.75x, AZA/RIG 1.5x; RIG/AZA 2.5x; INFb AZA 1.3x; RIG 0.6x, AZA/RIG 1.2x; RIG/AZA 0.7x. There was a significant increase in INFa (p=<0.0001) but not INFb, with RIG priming compared to AZA alone or AZA/RIG.Conclusion: Treatments with RIG either alone or combined with AZA results in epigenetic reprogramming of pluripotency genes, and expansion of primitive HSPC. This may serve to regulate HSPC pluripotency and expression of a maturation program. A significant increase in expression of IFNa seen after exposure to RIG/AZA may lead to enhanced hematopoietic function and may explain the differences in the alternative sequence of AZA/RIG. Further studies are underway to define more specific effects on these pathways to explain the impact on the resistance phenotype. DisclosuresNavada:Onconova: Research Funding. Silverman:Mount Sinai School of Medicine: Employment; Onconova Therapeutics Inc.: Patents & Royalties, Research Funding; Celgene: Research Funding; Medimmune: Research Funding; Johnson and Johnson: Research Funding; Bayer: Research Funding.

Serum Stem Cell Growth Factor Beta for the Prediction of Therapy Response in Hepatocellular Carcinoma.

Introduction Chronic inflammatory response is one of major contributors in the development of hepatocellular carcinoma (HCC). Inflammatory molecules, such as cytokines and growth factors in the circulation, can be useful in the diagnosis and prognosis of the patients. The stem cell growth factor beta (SCGFβ), a newly found protein, is a secreted sulfated glycoprotein and it functions as a growth factor for primitive hematopoietic progenitor cells. The level of SCGFβ had been reported to be elevated in several cancer types. However, there is very few or even no information on this protein in the study of HCC, even more in clinical studies. Methods A multiplex immunoassay panel of 48 cytokines and growth factors were utilized to screen 68 sera from 29 HCC patients at pretreatment (T0), 1 month (T1), and 6 months (T6) after treatment by either radiofrequency ablation (RF) or transarterial chemoembolization (TACE). Treatment response was evaluated according to mRECIST criteria. Results Immunoassay screening showed that the levels of IL-17, CTACK, TNFα, IL-2Rα, IL-8, and SCGFβ were different in Complete Responders (CR) and Nonresponders (NR) groups. At T0 and T1, the SCGFβ level was significantly the highest in NR (23.8 and 40.7 ng/mL, respectively), followed by early recurrence (25.4 and 25.0 ng/mL), and CR (6.7 and 5.3 ng/mL), independently from HCV, stages, and treatment type. Low basal SCGFβ level was associated with longer disease-free survival compared to high SCGFβ. Conclusion In this study, for the first time, we demonstrate that the high level of serum SCGFβ at pre- and posttreatment is associated with HCC nonresponsiveness.

InVitro Differentiation of Gata2 and Ly6a Reporter Embryonic Stem Cells Corresponds to InVivo Waves of Hematopoietic Cell Generation.

SummaryIn vivo hematopoietic generation occurs in waves of primitive and definitive cell emergence. Differentiation cultures of pluripotent embryonic stem cells (ESCs) offer an accessible source of hematopoietic cells for blood-related research and therapeutic strategies. However, despite many approaches, it remains a goal to robustly generate hematopoietic progenitor and stem cells (HP/SCs) in vitro from ESCs. This is partly due to the inability to efficiently promote, enrich, and/or molecularly direct hematopoietic emergence. Here, we use Gata2Venus (G2V) and Ly6a(SCA1)GFP (LG) reporter ESCs, derived from well-characterized mouse models of HP/SC emergence, to show that during in vitro differentiation they report emergent waves of primitive hematopoietic progenitor cells (HPCs), definitive HPCs, and B-lymphoid cell potential. These results, facilitated by enrichment of single and double reporter cells with HPC properties, demonstrate that in vitro ESC differentiation approximates the waves of hematopoietic cell generation found in vivo, thus raising possibilities for enrichment of rare ESC-derived HP/SCs.

Exposure to Low Levels of Lead in Utero and Umbilical Cord Blood DNA Methylation in Project Viva: An Epigenome-Wide Association Study.

Background:Early-life exposure to lead is associated with deficits in neurodevelopment and with hematopoietic system toxicity. DNA methylation may be one of the underlying mechanisms for the adverse effects of prenatal lead on the offspring, but epigenome-wide methylation data for low levels of prenatal lead exposure are lacking.Objectives:We investigated the association between prenatal maternal lead exposure and epigenome-wide DNA methylation in umbilical cord blood nucleated cells in Project Viva, a prospective U.S.-based prebirth cohort with relatively low levels of lead exposure.Methods:Among 268 mother–infant pairs, we measured lead concentrations in red blood cells (RBC) from prenatal maternal blood samples, and using HumanMethylation450 Bead Chips, we measured genome-wide methylation levels at 482,397 CpG loci in umbilical cord blood and retained 394,460 loci after quality control. After adjustment for batch effects, cell types, and covariates, we used robust linear regression models to examine associations of prenatal lead exposure with DNA methylation in cord blood at epigenome-wide significance level [false discovery rate ].Results:The mean [standard deviation (SD)] maternal RBC lead level was 1.22 (0.63) . CpG cg10773601 showed an epigenome-wide significant negative association with prenatal lead exposure ( per doubling increase in lead exposure; ) and was annotated to C-Type Lectin Domain Family 11, Member A (CLEC11A), which functions as a growth factor for primitive hematopoietic progenitor cells. In sex-specific analyses, we identified more CpGs with among female infants () than among male infants (). One CpG (cg24637308), which showed a strong negative association with prenatal lead exposure among female infants ( per doubling increase in lead exposure; ), was annotated to Dynein Heavy Chain Domain 1 gene (DNHD1) which is highly expressed in human brain. Interestingly, there were strong correlations between blood and brain methylation for CpG (cg24637308) based on another independent set of samples with a high proportion of female participants.Conclusion:Prenatal low-level lead exposure was associated with newborn DNA methylation, particularly in female infants. https://doi.org/10.1289/EHP1246

CBP/Catenin antagonists: Targeting LSCs' Achilles heel.

Cancer stem cells (CSCs), including leukemia stem cells (LSCs), exhibit self-renewal capacity and differentiation potential and have the capacity to maintain or renew and propagate a tumor/leukemia. The initial isolation of CSCs/LSCs was in adult myelogenous leukemia, although more recently, the existence of CSCs in a wide variety of other cancers has been reported. CSCs, in general, and LSCs, specifically with respect to this review, are responsible for initiation of disease, therapeutic resistance and ultimately disease relapse. One key focus in cancer research over the past decade has been the development of therapies that safely eliminate the LSC/CSC population. One major obstacle to this goal is the identification of key mechanisms that distinguish LSCs from normal endogenous hematopoietic stem cells. An additional daunting feature that has recently come to light with advances in next-generation sequencing and single-cell sequencing is the heterogeneity within leukemias/tumors, with multiple combinations of mutations, gain and loss of function of genes, and so on being capable of driving disease, even within the CSC/LSC population. The focus of this review/perspective is on our work in identifying and validating, in both chronic myelogenous leukemia and acute lymphoblastic leukemia, a safe and efficacious mechanism to target an evolutionarily conserved signaling nexus, which constitutes a common "Achilles heel" for LSCs/CSCs, using small molecule-specific CBP/catenin antagonists.