Early Hematopoietic Progenitor Cells Research Articles

Elucidation of the Unique Function of GATA1s in the Development of Transient Abnormal Myeloproliferative Disorders

Transient abnormal myeloproliferative disorder (TAM) is a pre-leukemic condition that occurs at birth in children with Down syndrome. The responsible gene of TAM, GATA1, is a hematopoietic transcription factor integral to hematopoiesis. The GATA1 gene has two translation start sites, giving rise to two protein variants: GATA1-fl, a full-length protein, and GATA1s, an isoform lacking the N-terminal domain. Notably, TAM blasts express only the GATA1s due to a mutation in the GATA1 gene, as opposed to normal cells which express both proteins. This implies that the precancerous state is not triggered by the production of an aberrant protein butrather by the absence of the full-length protein and the exclusive expression of the minor isoform protein also present in normal cells. This is in stark contrast with the functions of many other oncogenes. Thus, the prevailing view is that TAM development involves a “loss of function” due to deficiency of GATA1-fl. However, some studies suggest that GATA1s may have unique functions that actively drive the TAM phenotype. Because of these conflicting findings reported, the precise pathomechanism of TAM development remains elusive, despite the identification of the responsible gene. To address this, we analyzed the function of GATA1s using an induced pluripotent stem cell (iPSC) model of TAM. We previously examined the effect of increasing GATA1s expression levels during early hematopoietic differentiation stage by stage using the TAM-iPSC model. We found that, in the absence of GATA1-fl protein, overexpression of GATA1s in early hematopoietic progenitor cells (HPC) shifted megakaryocytic differentiation toward myeloid lineage, intensifying TAM severity by expanding progenitor cells. These findings suggest that GATA1s has a unique function distinct from that of GATA1-fl. In this study, we investigated the function of GATA1s proteins as transcription factors using chromatin immunoprecipitation sequencing (ChIP-seq). Our results demonstrated that genes involved in hematopoiesis or cell proliferation belong to the gene cluster to which overexpressed GATA1s (O/E GATA1s) binds solely in the absence of GATA1-fl at the early HPC stage. Gene Ontology (GO) analysis further validated the significant enrichment of terms relevant to the TAM phenotype, including regulation of myeloid cell differentiation and negative regulation of cell differentiation. Intriguingly, the genes to which O/E GATA1s binds when GATA1-fl is present were unrelated to hematopoiesis or cell proliferation. These observations suggest that GATA1s uniquely binds to genes, instigating TAM onset under GATA1-fl deficient conditions, thus confirming TAM as a disorder hallmarked by the absence of GATA1-fl due to the GATA1 gene mutation. Our RNA-sequencing data indicate that, at the early HPC stage, O/E GATA1s only induces changes in binding to gene regions involved in TAM pathogenesis, without altering gene expression levels. In contrast, gene expression analyses of other later stage HPC fractions (megakaryocyte-erythroid progenitor-like cells: MEP-like cells), revealed increased STAT3 expression and enrichment of the JAK-STAT pathway and myeloid cell differentiation pathway. These results suggest that TAM development may be influenced by shifts in the gene-binding region of GATA1s in the early HPC, which, in turn, induces TAM in more differentiated MEP-like cells by skewing their differentiation towards the myeloid lineage. In conclusion, our study provides important insights into the pathological mechanisms of TAM pathogenesis, particularly with respect to the role of GATA1 in driving the TAM phenotype. the unique function of GATA1s and their specific binding to genes involved in TAM pathogenesis shed light on the complex interplay between GATA1s and TAM pathogenesis shed light on the complex interplay between GATA1s and TAM pathogenesis. Understanding these mechanisms may pave the way for targeted therapies aimed at modulating GATA1s activity and reducing the severity of TAM.

Ras Activity Is Significantly Elevated in Patients with Primary Myelofibrosis and Is Agnostic to the Presence of RAS and JAK pathwaymutations

Myelofibrosis (MF) is a myeloproliferative stem cell disorder arising from various clonal mutations in early myeloid-committed hematopoietic progenitor cells. The subsequent acquisition of additional mutations and cytokine signaling then heralds the recruitment of fibroblasts to the marrow space, leading to collagen deposition. MF disease behavior varies widely and hematopoietic stem cell transplantation remains the only curative therapeutic option. In non-transplant candidates, managing cytopenic disease is uniquely challenging as patients develop resistance to commonly-utilized Jak-inhibitors. Expanding the molecular landscape of the disease beyond Jak/Stat signaling with the goal of identifying new therapeutic targets has, accordingly, been an area of growing interest in MF research. Recently-published studies, including results from our own institution, have identified mutations that potentially activate Ras signaling as being associated with features of advanced MF (higher somatic mutation burden) as well as with shorter overall survival and progression to AML. The goals of our study is to expand on this prior data, characterize the activity of Ras signaling and determine its role in patients with MF. Peripheral blood mononuclear cells (PBMCs) from 27 patients with myelofibrosis (MF) at a single institution underwent integrated genomic-transcriptomic sequencing analyses using the University of Michigan MiOncoSeq platform. PBMCs were separated by Ficoll® followed by cell lysis and protein quantification. Ras activity was measured by the binding of effector Ras binding domain (RBD) via ELISA. Patients with MF were compared to healthy donors and to patients with either polycythemia vera (PV) or essential thrombocythemia (ET). Ras activity in MF was examined across different mutations and with respect to various clinical parameters. Multivariate analyses were performed to assess the impact of each individual mutation on Ras activity. Accompanying statistical analyses for all assessments were conducted using two-way unpaired t-tests. Overall, Ras activity in 21 assessable patients with MF was 1.64 fold higher than in PV/ET (12 patients, p=0.0462) and 1.74 fold higher than healthy donors (13 volunteers, p=0.0304). While the majority of patients in the MF cohort had driver mutations, including in JAK2 (73%) and CALR (15.4%), the individual types of driver mutations did not impact Ras activity. Within the Ras/MAP Kinase signaling pathway, mutations were present at a range of variant allele frequencies (VAF), including 2 patients with NRAS (one with VAF 42%, one <5%), 2 KRAS (both <5%), 2 NF1, 1 CBL and 1 RAF1 (VAFs not assessed). The individual impact of various driver mutations, epigenetic regulators and splicing factors as assessed by multivariate analysis identified a trend toward enhanced Ras activity with mutations in RAS, CALR, ASXL1, TET2 and TP53, suggesting that in addition to RAS, other mutations also activate Ras signaling in MF. With regards to clinical parameters, Ras activity was weakly correlated with spleen size and high molecular risk mutation (HMR) presence, although this did not reach statistical significance in this cohort due to a relatively small sample size. Next, we performed a differential enrichment of myeloid and lymphoid cells from PBMCs using EasySep™ and measured Ras activity in lymphoid and myeloid enriched cells. We observed that the ratio of myeloid:lymphoid activity in patients with MF with RAS/MAP Kinase pathway mutations is 3.8 times higher than in patients without such mutations (p=0.0012), suggesting that RAS pathway mutations have a predominant role in myeloid cells. Our results show that the majority of patients with MF have hyperactive Ras signaling compared to PV/ET or healthy donors, regardless of the presence of alterations in the Ras signaling pathway. This underscores the importance of functional assessment of Ras activity in patients with MF and provides evidence that targeting Ras signaling in MF may lead to therapeutic benefit. We are currently expanding our analysis to a larger cohort of MF patients and performing preclinical testing of Ras/MAP Kinase pathway inhibitors as a means of overcoming Jak-inhibitor resistance.

A Two-Part Study Evaluating the Combination of Tazemetostat and CPX-351 (Part 1) and Palbociclib with CPX-351 (Part 2) for the Treatment of Relapsed or Refractory Acute Myeloid Leukemia

BACKGROUND AND SIGNIFICANCE Acute myeloid leukemia (AML) is an aggressive cancer of early hematopoietic progenitor cells with only 25-30% of patients (pts) surviving 5 years from diagnosis. For fit pts, conventional chemotherapy with DNA-damaging agents remains the mainstay of treatment. Conventional chemotherapeutic agents cause DNA-damage by targeting cells during DNA replication. Chromatin structure is highly influential in the efficacy of DNA-damaging agents, as areas of compacted chromatin are more resistant to DNA-damaging effects. Areas of DNA marked with histone 3 lysine 27 trimethylation (H3K27me3) are transcriptionally repressed and exhibit reduced accessibility to DNA-damaging agents. Enhancer of Zeste Homolog 2 (EZH2) serves as the catalytic subunit of the polycomb repressive complex 2 which methylates H2K27 to produce a di- or trimethylated state (H3K27me2/3). In preclinical studies, treatment of AML cell lines with the combination of an EZH2 inhibitor (EZH2i) and doxorubicin resulted in an increase in DNA damage and apoptosis compared to those treated with doxorubicin alone. Further, a significant decrease in leukemia burden was seen in mice injected with THP-1 (AML) cells and treated with an EZH2i and doxorubicin and cytarabine compared to doxorubicin and cytarabine alone. This effect was further enhanced by pre-treatment of AML cells with palbociclib (PALB), a CDK4/6 inhibitor, by significantly increasing the number of cells in S phase when exposed to the EZH2i and doxorubicin combination, resulting in an increase in DNA damage and apoptosis compared to cells that were not pre-treated with PALB. These findings were reproduced in in vivo studies with mice injected with the primary AML cells (Porazzi P, et al. Cancer Res., 2021). STUDY DESIGN AND METHODS We will perform a 2-part phase I dose escalation study utilizing EZH2i tazemetostat (TAZ) in combination with CPX-351 induction chemotherapy and of PALB pre-treatment followed by CPX-351 in pts with relapsed or refractory (R/R) AML (NCT05627232). All pts will receive CPX-351 at standard dose on days 1, 3 and 5. Part 1 evaluate the addition of TAZ to CPX-351 via traditional 3+3 design. Pts will be treated with TAZ on days -1 to 6 at a starting dose of 600 mg twice daily to open chromatin concurrent with CPX-351 administration. Part 2 evaluate the pre-treatment with PALB via traditional 3+3 design. Pts will be treated with PALB starting dose of 100 mg daily on days -3 to -1 to induce cell cycle G1. PALB will be held on day 1 to allow cells to then enter S phase prior to administration of CPX-351. ( Figure 1). The study will enroll pts with R/R AML treated at Sidney Kimmel Cancer Center. Pts must be ≥18 years of age with R/R AML (non-acute promyelocytic leukemia) and have received at least 1 prior line of therapy and have adequate performance status and organ function. Pts whose participation would result in a total cumulative dose of daunorubicin greater than 550 mg/m 2 (450 mg/m 2 if they previously received mediastinal radiation) and those with active central nervous system involvement of AML, unstable cardiac disease or other severe concurrent disease will be excluded. The primary endpoints of Parts 1 and 2 will be to determine the maximum tolerated dose of TAZ and of PALB in combination with CPX-351, respectively. Secondary endpoints for Parts 1 and 2 include preliminary assessment of efficacy. Correlative studies to evaluate the on-target effect of TAZ on H3K27me3 and DNA-damage and of PALB on cell cycling will be performed at protocol-specified time points. If these two 2-drug combinations prove to be safe, we will amend the protocol to add Part 3 which will evaluate the safety of the 3-drug combination of pre-treatment with PALB followed by co-treatment with TAZ and CPX-351.

Senti-202, a Selective, Off-the-Shelf, Preclinical CAR-NK Cell Therapy with CD33 and/or FLT3 Activating CAR, Healthy Cell Protection from Endomucin (EMCN) Inhibitory CAR and Calibrated Release IL-15 for Hematologic Malignancies Including AML

Background: Patients with CD33 and/or FLT3 malignancies, which encompass most myeloid malignancies such as acute myeloid leukemia (AML), have a grim prognosis and a high unmet need. There are no approved cell therapies for patients with AML due to paucity of targets and an immunosuppressive bone marrow milieu. SENTI-202, a preclinical CAR-NK cell is engineered with a logic-gated gene circuit to overcome these challenges. Methods: SENTI-202 is engineered with a FLT3 OR CD33 NOT EMCN logic-gated gene circuit and a proprietary calibrated release IL-15 (crIL-15). These 3 chimeric proteins are engineered onto a natural killer (NK) cell (healthy adult peripheral blood-derived) to create an off-the-shelf chimeric antigen receptor (CAR) NK cell therapy. The bivalent CD33 OR FLT33 (OR GATE) activating CAR (aCAR) enables concurrent targeting of CD33+ and/or FLT3+ AML cells which kills both AML blasts and leukemic stem cells (LSCs) in vitro and in vivo. This concurrent targeting of two tumor antigens could potentially provide longer remission and less chance of relapse. The NOT EMCN (NOT GATE) inhibitory CAR (iCAR) protects healthy EMCN+ hematopoietic stem cells (HSCs) and early hematopoietic progenitor cells (HPCs) from off-tumor toxicity to potentially aid in post-treatment reconstitution of a healthy hematopoietic system. The crIL-15 provides both autocrine and paracrine IL-15 stimulation to the CAR-NK cells (and surrounding immune cells) to promote cell expansion, persistence, and tumor killing. While previous presentations (Garrison et al., 2021) focused on the performance of individual SENTI-202 components in a cell, this presentation focuses on functional data from the complete logic-gated gene circuit and the crIL-15 together in a cell (i.e., the final CAR-NK with all 3 genetic elements). Results: We demonstrated delivery of all circuit components using a single retroviral vector. We then investigated activity of the resulting CAR-NK cells using a traditional in vitro cytotoxicity assay (E:T=1:2; 20hrs) in which SENTI-202 showed >90% killing of leukemia cells (e.g. SEM; P≤0.001), maintained significant serial killing potential (3 rounds; P≤0.0001; P≤0.001; P≤0.001), as well as robust killing against primary AML blasts and LSC-enriched target cell populations. Importantly, SENTI-202 also exhibited significant tumor cell killing and improved survival (P≤0.001) in an MV4-11 xenograft AML mouse model. We investigated the ability of SENTI-202 to reduce off-tumor toxicity toward EMCN+ healthy cells, including primary HSCs. Within in vitro assays, SENTI-202 provided >50% protection to FLT3+ CD33+ model healthy cells that also expressed the EMCN safety antigen, and similar significant protection was maintained through serial protection assays (3 rounds; P≤0.0001; P≤0.001; P≤0.0001). As further evidence of the selectivity imparted using the OR/NOT logic gating approach, in vitro assays with SENTI-202 resulted in killing of leukemia cells but not primary healthy human HSCs and HPCs (~42% protection) (P≤0.01). Finally, in the presence of SENTI-202 in vivo, the NOT GATE protected and enabled significant (P≤0.0001) expansion of EMCN+ FLT3+ CD33+ model healthy cells. Conclusions: In summary, preclinical evaluation of SENTI-202 demonstrated selective killing of CD33 and/or FLT3 expressing AML cell lines while protecting healthy HSCs and HPCs. This selectivity is imparted by the novel logic-gated gene circuit engineered into healthy adult NK cells. Clinical evaluation of SENTI-202 is planned to evaluate the safety and efficacy in patients with hematologic malignancies with high unmet need, including AML.

Arsenic impairs the lineage commitment of hematopoietic progenitor cells through the attenuation of GATA-2 DNA binding activity

Arsenic exposure produces significant hematotoxicity in vitro and in vivo. Our previous work shows that arsenic (in the form of arsenite, AsIII) interacts with the zinc finger domains of GATA-1, inhibiting the function of this critical transcription factor, and resulting in the suppression of erythropoiesis. In addition to GATA-1, GATA-2 also plays a key role in the regulation of hematopoiesis. GATA-1 and GATA-2 have similar zinc finger domains (C4-type) that are structurally favorable for AsIII interactions. Taking this into consideration, we hypothesized that early stages of hematopoietic differentiation that are dependent on the function of GATA-2 may also be disrupted by AsIII exposure. We found that in vitro AsIII exposures disrupt the erythromegakaryocytic lineage commitment and differentiation of erythropoietin-stimulated primary mouse bone marrow hematopoietic progenitor cells (HPCs), producing an aberrant accumulation of cells in early stages of hematopoiesis and subsequent reduction of committed erythro-megakaryocyte progenitor cells. Arsenic significantly accumulated in the GATA-2 protein, causing the loss of zinc, and disruption of GATA-2 function, as measured by chromatin immunoprecipitation and the expression of GATA-2 responsive genes. Our results show that the attenuation of GATA-2 function is an important mechanism contributing to the aberrant lineage commitment and differentiation of early HPCs. Collectively, findings from the present study suggest that the AsIII-induced disruption of erythro-megakaryopoiesis may contribute to the onset and/or exacerbation of hematological disorders, such as anemia.

A review of the mutagenic potential of N-ethyl-N-nitrosourea (ENU) to induce hematological malignancies.

This review is intended to summarize the existing literature on the mutagenicity of N-ethyl-N-nitrosourea (ENU) in inducing hematological malignancies, including acute myeloid leukemia (AML) in mice. Blood or hematological malignancies are the most common malignant disorders seen in people of all age groups. Driven by a number of genetic alterations, leukemia rule out the normal proliferation and differentiation of hematopoietic stem cells (HSCs) and their progenitors in the bone marrow (BM) and severely affects blood functions. Out of all hematological malignancies, AML is the most aggressive type, with a high incidence and mortality rate. AML is found as either de novo or secondary therapeutic AML (t-AML). t-AML is a serious adverse consequence of alkylator chemotherapy to the cancer patient and alone constitutes about 10%-20% of all reported AML cases. Cancer patients who received alkylator chemotherapy are at an elevated risk of developing t-AML. ENU has a long history of use as a potent carcinogen that induces blood malignancies in mice and rats that are pathologically similar to human AML and t-AML. ENU, once entered into the body, circulates all over the body tissues and reaches BM. It creates an overall state of suppression within the BM by damaging the marrow cells, alkylating the DNA, and forming DNA adducts within the early and late hematopoietic stem and progenitor cells. The BM holds a weak DNA repair mechanism due to low alkyltransferase, and poly [ADP-ribose] polymerase (PARP) enzyme content often fails to obliterate those adducts, acting as a catalyst to bring genetic abnormalities, including point gene mutations as well as chromosomal alterations, for example, translocation and inversion. Taking advantage of ENU-induced immune-suppressed state and weak immune surveillance, these mutations remain viable and slowly give rise to transformed HSCs. This review also highlights the carcinogenic nature of ENU and the complex relation between the ENU's overall toxicity in the induction of hematological malignancies.

Globin vector regulatory elements are active in early hematopoietic progenitor cells

The globin genes are archetypal tissue-specific genes that are silent in most tissues but for late-stage erythroblasts upon terminal erythroid differentiation. The transcriptional activation of the β-globin gene is under the control of proximal and distal regulatory elements located on chromosome 11p15.4, including the β-globin locus control region (LCR). The incorporation of selected LCR elements in lentiviral vectors encoding β and β-like globin genes has enabled successful genetic treatment of the β-thalassemias and sickle cell disease. However, recent occurrences of benign clonal expansions in thalassemic patients and myelodysplastic syndrome in patients with sickle cell disease call attention to the non-erythroid functions of these powerful vectors. Here we demonstrate that lentivirally encoded LCR elements, in particular HS1 and HS2, can be activated in early hematopoietic cells including hematopoietic stem cells and myeloid progenitors. This activity is position-dependent and results in the transcriptional activation of a nearby reporter gene in these progenitor cell populations. We further show that flanking a globin vector with an insulator can effectively restrain this non-erythroid activity without impairing therapeutic globin expression. Globin lentiviral vectors harboring powerful LCR HS elements may thus expose to the risk of trans-activating cancer-related genes, which can be mitigated by a suitable insulator.

Spectrum of Molecular Modes of Immune Escape in Idiopathic Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria

The pathogenesis of idiopathic aplastic anemia (IAA) involves a human leukocyte antigen (HLA)-restricted T-cell autoreactivity against unknown antigens preferentially distributed on early hematopoietic stem and progenitor cells (HSPCs). Genetically acquired GPI-anchor and HLA deficiency have been both linked to clonal immune evasion from T-cell pressure. We hypothesized that, in analogy to anti-tumor adaptive immune evasion, pathophysiology of immune escape in IAA originates together with a broader dysfunction of antigen presentation/processing machinery and immune regulatory proteins, beyond HLA molecules, as an effect of immune pressure under T-cell attack. This initial immune reaction would produce up-modulation of these pathways, ultimately promoting the acquisition of mutations and expansion of immune resistant clones.To test this hypothesis, we first performed single-cell RNAseq analysis in HSPCs in IAA patients at disease manifestation, 1 which showed signatures of dysfunction of antigen presentation machinery, with up-regulation of most of the HLA molecules, proteasome subunits and endoplasmic reticulum related organelle transporters. Strikingly, DRB1 was among the top 3 genes upregulated in IAA patients compared to controls (q-values 1.23E-35; Fig.1A), underscoring the etiological impact that antigen presentation via this locus has in the initiation of autoimmune process. Mild upregulation was also seen in DQB1 and B loci (q-values 4.7E-07 and 2.1E-10, respectively).We then studied molecular escape mechanisms by genotyping 204 IAA and PNH patients, with either a targeted or whole genome sequencing (WGS) platform. By application of a newly in-house developed bioinformatic pipeline, we detected somatic aberrations in HLA region involving both class I and II alleles in 36% of IAA patients including point mutations, frameshift insertions or deletions and copy number variations inducing allelic loss. B*14:02 and A*02:01 emerged as the most commonly mutated class I alleles with a few hotspot mutations identified, particularly in exon 1 (c.19C>T, p.R7X, Fig.1B,C), confirming previous reports. 2,3 In class II, DQB1 and DPA1 loci were frequently targeted by fine mutational events, while more complex allelic loss phenomena interested prevalently DRB1 and DQB1 loci. Those aberrations were identified at diagnosis (35%), during disease follow-up (33%) or at the time of malignant evolution (27%), with higher clonal size in specimens collected during the course of the disease (median VAF 3% [2-27%] at diagnosis, 8% [2-98%] at follow-up, and 2.2% [2.0-6.1%] at evolution). Of 41 patients with at least one HLA aberration and characterized with an extended genotypic study, only 6 harbored also >1 somatic myeloid mutation (14%), versus 30/90 (33%) not affected by somatic hits in HLA (p=.026; Fig.1D). HLA aberrant cases also showed lower number of somatic myeloid mutations (OR=0.44; p=.0262) with driver hits rarely present (Fig.1E). In terms of PIGA mutations, an increased PIGA mosaicism was observed in the HLA mutant group, underlying that both processes have similar pathophysiologic origin as a product of the immune selection pressure (OR: 1.55 [95%CI 1.1-4.2], p=.0201). We then investigated, through WGS of 53 patients, the presence of somatic mutations in other immune genes which could be triggered by immune pressure. Hence, in 47% of the cases we were able to find pathogenic or likely pathogenic hits in genes encompassing proteasome complex, vesicle trafficking, transactivators and interferon regulatory factors, including CREBBP, TAP1, CIITA, PSMC5, PSMB4 and IRF9 (Fig.1F), whose pathogenicity was computationally assessed through recently implemented somatic classifiers. 4 Those hits were not mutually exclusive neither with HLA nor with PIGA mutations, however their VAF was significantly lower compared to concurrent HLA and PIGA lesions, underscoring their lower driver potential within the immune escape environment compared to PNH and HLA-lacking clones.Altogether our results describe the diversity of molecular and immune events taking place in IAA and PNH. Our study suggests that following initial immune insult, clonal architecture of residual hematopoiesis can be dominated by multiple modes of immune escape, agonistically participating to a mechanism of “adaptive” clonal recovery, likely in opposition to the “maladaptive” malignant progression. [Display omitted] DisclosuresMaciejewski: Alexion: Consultancy; Regeneron: Consultancy; Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy.

High-volume endurance exercise training stimulates hematopoiesis by increasing ACE NH2-terminal activity.

One of the health benefits of endurance exercise training (ET) is the stimulation of hematopoiesis. However, the mechanisms underlying ET-induced hematopoietic adaptations are understudied. N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) inhibits proliferation of early hematopoietic progenitor cells. The angiotensin I-converting enzyme (ACE) NH2-terminal promotes hematopoiesis by inhibiting the anti-hematopoietic effect of Ac-SDKP. Here we demonstrate for the first time the role of ACE NH2-terminal in ET-induced hematopoietic adaptations. Wistar rats were subjected to 10 weeks of moderate-(T1) and high-(T2) volume swimming-training. Although both protocols induced classical ET-associated adaptations, only T2 increased plasma ACE NH2-domain activity (by 40%, P=0.0003) and reduced Ac-SDKP levels (by 50%, P<0.0001). T2 increased the number of hematopoietic stem cells (HSCs; ∼200%, P=0.0008), early erythroid progenitor colonies (∼300%, P<0.0001) and reticulocytes (∼500%, P=0.0007), and reduced erythrocyte lifespan (∼50%, P=0.022). Following, Wistar rats were subjected to T2 or T2 combined with ACE NH2-terminal inhibition (captopril (Cap) treatment: 10 mg.kg-1.day-1). T2 combined with ACE NH2-terminal inhibition prevented Ac-SDKP decrease and attenuated ET-induced hematopoietic adaptations. Altogether, our findings show that ET-induced hematopoiesis was at least partially associated with increased ACE NH2-terminal activity and reduction in the hematopoietic inhibitor Ac-SDKP.

Lineage-Selective Disturbance of Early Human Hematopoietic Progenitor Cell Differentiation by the Commonly Used Plasticizer Di-2-ethylhexyl Phthalate via Reactive Oxygen Species: Fatty Acid Oxidation Makes the Difference.

Exposure to ubiquitous endocrine-disrupting chemicals (EDCs) is a major public health concern. We analyzed the physiological impact of the EDC, di-2-ethylhexyl phthalate (DEHP), and found that its metabolite, mono-2-ethylhexyl phthalate (MEHP), had significant adverse effects on myeloid hematopoiesis at environmentally relevant concentrations. An analysis of the underlying mechanism revealed that MEHP promotes increases in reactive oxygen species (ROS) by reducing the activity of superoxide dismutase in all lineages, possibly via its actions at the aryl hydrocarbon receptor. This leads to a metabolic shift away from glycolysis toward the pentose phosphate pathway and ultimately results in the death of hematopoietic cells that rely on glycolysis for energy production. By contrast, cells that utilize fatty acid oxidation for energy production are not susceptible to this outcome due to their capacity to uncouple ATP production. These responses were also detected in non-hematopoietic cells exposed to alternate inducers of ROS.

BAALC Is a Key Mediator of Leukemia Development in Congenital Neutropenia

We recently reported high frequency of cooperating acquired CSF3R and RUNX1 mutations in severe congenital neutropenia (CN) patients that developed AML or MDS. CN is a pre-leukemic bone marrow failure syndrome (BMFS) and longitudinal retrospective analysis of CN bone marrow samples allows identification of the pathological intracellular events that lead to leukemogenic transformation.In the present study, we report the identification of a key mechanism of leukemogenesis in CN. We made use of the induced pluripotent stem cell (iPSC) model and embryoid body (EB)-based in vitro hematopoietic differentiation of iPSCs generated from a CN patient harboring ELANE mutation before (CN-iPSCs) and after (CN/AML-iPSCs) overt AML. CN/AML-iPSCs clones revealed CSF3R and RUNX1 mutations and trisomy 21. EB-based hematopoietic differentiation revealed that CN-iPSCs produced similar amounts of CD34+ cells, but approximately 2-fold reduction of mature granulocytes and CFU-G colonies in CFU-assay, as compared to healthy donor-derived iPSCs. CN/AML-iPSCs produced CD34+ cells but did not differentiate into mature granulocytes at all.Analysis of the gene expression profiles of five CN/AML patients including our index patient, revealed that genes upregulated in early hematopoietic stem/progenitor cells (HSPCs), such as Brain And Acute Leukemia, Cytoplasmic (BAALC), Hematopoietic Prostaglandin D Synthase (HPGDS), Natriuretic Peptide Receptor 3 (NPR3) and CD109 were markedly increased in CN/AML blasts harboring both RUNX1 and CSF3R mutations, as compared to hematopoietic cells prior to leukemia. This genetic signature suggests transformation of hematopoietic cells into more primitive hematopoietic progenitors after acquisition of CSF3R and RUNX1 mutations. We were able to confirm strong increase of BAALC, HPGDS, NPR3 and CD109 mRNA levels in CD34+ cells derived from CN/AML-iPSCs, as compared to CN-iPSCs, suggesting that our iPSC-model is reliable to study leukemogenesis.In order to evaluate which of these candidate genes (BAALC, HPGDS, NPR3 and CD109)is responsible for the leukemogenic transformation in CN, we generated CN/AML-iPSCs clones deficient for one selected genes. We established CRISPR-Cas9-mediated gene knockout using transfection of CN/AML-iPSCs with pSpCas9(BB)-2A-GFP vector expressing Cas9 and sgRNA, that were designed to specifically target first exon of each candidate gene. CN/AML-iPSCs transfected with specific sgRNAs were sorted and single cell clones were analyzed further.Knockout of the selected genes did not affect survival and pluripotency of CN/AML-iPSCs and we were able to study EB-based hematopoietic differentiation of gene-edited CN/AML-iPSCs clones. Interestingly, knockout of HPGDS, NPR3 or CD109 did not correct defective granulocytic differentiation of CN/AML-iPSCs, whereas knockout of BAALC in CN/AML-iPSCs resulted in the tremendous increase of the granulocytic differentiation, as assessed by FACS, CFU assay, morphological analysis of cytospin preparations and nitroblue tetrazolium (NBT) assay. Granulocytic differentiation of BAALC -deficient CN/AML-iPSCs was comparable to CN-iPSCs, but was still reduced in comparison to healthy donor-derived iPSCs.These data clearly showed that knockout of BAALC in CN/AML-iPSCs overcomes differentiation block at the early hematopoietic progenitor cell stage, demonstrating a fundamental role of BAALC in leukemia development in CN. BAALC, HPGDS and CD109 are known to be markedly up-regulated in RUNX1 -mutated de novo AML and qRT-PCR of CD34+ cells generated from BAALC -knockout CN/AML-iPSCs revealed a decrease of mRNA expression levels of BAALC, HPGDS and CD109 . At the same time, knockout of RUNX1 in CN/AML-iPSCs using CRISPR/Cas9-editing revealed even higher decrease of mRNA levels of BAALC, NPR3 and CD109 . These data suggest that HPGDS and CD109 up-regulation is downstream of RUNX1 -mediated BAALC induction in AML.Taken together, we established a reliable in vitro model to study leukemogenesis in the pre-leukemic BMFS, using CN as an example. With the help of this model, we identified BAALC up-regulation as a key “leukemogenic” factor downstream of RUNX1 and CSF3R mutations. Based on these observations, we conclude that inhibition of BAALC in hematopoietic cells of CN patients may prevent leukemogenic transformation of hematopoiesis or eliminate AML blasts in CN-AML. DisclosuresNo relevant conflicts of interest to declare.

Developing the right tools for the job: Lin28 regulation of early life T-cell development and function.

T cells comprise a functionally heterogeneous cell population that has important roles in the immune system. While T cells are broadly considered to be a component of the antigen-specific adaptive immune response, certain T-cell subsets display innate-like effector characteristics whereas others perform immunosuppressive functions. These functionally diverse T-cell populations preferentially arise at different stages of ontogeny and are tailored to the immunological priorities of the organism over time. Many differences in early life versus adult T-cell phenotypes can be attributed to the cell-intrinsic properties of the distinct progenitors that seed the thymus throughout development. It is becoming clear that Lin28, an evolutionarily conserved, heterochronic RNA-binding protein that is differentially expressed among early life and adult hematopoietic progenitor cells, plays a substantial role in influencing early T-cell development and function. Here, we discuss the mechanisms by which Lin28 shapes the T-cell landscape to protect the developing fetus and newborn. Manipulation of the Lin28 gene regulatory network is being considered as one means of improving hematopoietic stem cell transplant outcomes; as such, understanding the impact of Lin28 on T-cell function is of clinical relevance.

Pluripotent stem cell model of early hematopoiesis in Down syndrome reveals quantitative effects of short-form GATA1 protein on lineage specification.

Children with Down syndrome (DS) are susceptible to two blood disorders, transient abnormal myelopoiesis (TAM) and Down syndrome-associated acute megakaryocytic leukemia (DS-AMKL). Mutations in GATA binding protein 1 (GATA1) have been identified as the cause of these diseases, and the expression levels of the resulting protein, short-form GATA1 (GATA1s), are known to correlate with the severity of TAM. On the other hand, despite the presence of GATA1 mutations in almost all cases of DS-AMKL, the incidence of DS-AMKL in TAM patients is inversely correlated with the expression of GATA1s. This discovery has required the need to clarify the role of GATA1s in generating the cells of origin linked to the risk of both diseases. Focusing on this point, we examined the characteristics of GATA1 mutant trisomy-21 pluripotent stem cells transfected with a doxycycline (Dox)-inducible GATA1s expression cassette in a stepwise hematopoietic differentiation protocol. We found that higher GATA1s expression significantly reduced commitment into the megakaryocytic lineage at the early hematopoietic progenitor cell (HPC) stage, but once committed, the effect was reversed in progenitor cells and acted to maintain the progenitors. These differentiation stage-dependent reversal effects were in contrast to the results of myeloid lineage, where GATA1s simply sustained and increased the number of immature myeloid cells. These results suggest that although GATA1 mutant cells cause the increase in myeloid and megakaryocytic progenitors regardless of the intensity of GATA1s expression, the pathways vary with the expression level. This study provides experimental support for the paradoxical clinical features of GATA1 mutations in the two diseases.

CD34 is not Expressed by Blasts in a Third of B-ALL Patients and its Negativity is associated with Aberrant Marker Expression: A Retrospective Analysis

Background: CD34 antigen is expressed by early hematopoietic progenitor cells and acute leukemia cells. Its expression is associated with good prognosis in acute myeloid leukemia. Literature is sparse on its prognostic significance in B- acute lymphoblastic leukemia (B-ALL) especially from India. Hence the present study was undertaken to analyse the frequency of CD34 expression in B-ALL in Indian patients and determine its prognostic significance by associating with other prognostic markers and aberrant antigen expression. Methods:Seventy-five B-ALL patients diagnosed by flow cytometry over a period of 3½ year were studied. Correlation of CD34 expression was studied with gender, age, total leucocyte count (TLC), French-American-British (FAB) morphological type, immuno-phenotypic markers, cytogenetics and minimal residual disease. Differences between groups were evaluated using Student’s T-test for quantitative data and Chi-square test/Fishers exact T-test for qualitative variables. P value <0.05 was considered significant. Results:CD34 was positive in 66.66% (50/75) cases while it was negative in rest (33.33%; 25/75) cases. CD13, CD33, CD5, CD7 and CD11b were more frequently expressed in CD34 negative B-ALL (p=0.025). No association of CD34 expression was found with gender, age, TLC, FAB morphological type, other immune-phenotypic markers, MRD and cytogenetics studied.Conclusions:The expression of CD34 does not associate with known prognostic markers in B-ALL. However, absence of CD34 is associated with aberrant immunophenotypic expression (myeloid + T-cell antigens) in these patients. Larger studies with larger sample size and more extensive immunophenotypic panel needs to be done in Indian setup to confirm these findings.

Leukemogenic pathway of infant leukemia with MLL fusion

Acute lymphoblastic leukemia (ALL) in infants remains an intractable and difficult-to-treat leukemia as compared to other pediatric ALLs, for which considerable progress has been achieved in terms of treatment outcomes in recent years. The leukemic cells in infants with ALL frequently carry chromosome translocations involving 11q23, resulting in the rearrangement and fusion of the MLL (KMT2A) gene. Among many MLL fusion genes, MLL-AF4 (KMT2A-AFF1) fusion is characteristically observed in infants with ALL, representing a hallmark of poor prognosis. In MLL-AF4-positive infants with ALL, first leukemic cells with MLL-AF4 were generated in utero. Analysis of several murine and human leukemia models revealed that the target cells for tumorigenesis by MLL-AF4 were not the hematopoietic progenitor cells of the bone marrow, but the early hematopoietic progenitor cells present in the fetal liver during the embryonic period and possibly the undifferentiated cells prior to the commitment to hematopoietic cells in the fetus. Elucidation of the leukemogenic process of infant ALL with MLL-AF4 may lead to early, pre-symptomatic diagnosis of leukemia, resulting in the improvement of prognosis and prevention of the onset of ALL in infants.

Production of Murine Macrophages from Hoxb8-Immortalized Myeloblasts: Utility and Use in the Context of Salmonella Infection.

Salmonella enterica is a Gram-negative intracellular pathogen that causes a range of life-threatening diseases in humans and animals worldwide. In a systemic infection, the ability of Salmonella to survive/replicate in macrophages, particularly in the liver and spleen, is crucial for virulence. Transformed macrophage cell lines and primary macrophages prepared from mouse bone marrow are commonly used models for the study of Salmonella infection. However, these models raise technical or ethical issues that highlight the need for alternative methods. This chapter describes a technique for immortalizing early hematopoietic progenitor cells derived from wild-type or transgenic mice and using them to produce macrophages. It validates, through a specific example, the interest of this cellular approach for the study of Salmonella infection.

Design of New Therapeutic Agents Targeting FLT3 Receptor Tyrosine Kinase Using Molecular Docking and 3D-QSAR Approach

Background: FMS-like tyrosine kinase-3 (FLT3) belongs to the class III Receptor Tyrosine Kinase (RTK) family. FLT3 is involved in normal hematopoiesis and is generally expressed in early hematopoietic progenitor cells. Mutations either with an internal tandem duplication of FMS-like tyrosine kinase-3 (FLT3-ITD) or point mutation at the activation loop leads to the Acute Myeloid Leukemia (AML), a highly heterogeneous disease. Thus, FLT3 is an important therapeutic target for AML. Method: In the present work, docking and 3D-QSAR techniques were performed on a series of diaminopyrimidine derivatives as FLT3 kinase antagonists. Results: Docking study recognized important active site residues such as Leu616, Gly617, Val624, Ala642, Phe830, Tyr693, Cys694, Cys695, Tyr696 and Gly697 that participate in the inhibition of FLT3 kinase. Receptor-based CoMFA, RF-CoMFA and CoMSIA models were developed. RFCoMFA model revealed relatively better statistical results compared to other models. Furthermore, the selected RF-CoMFA model was evaluated using various validation techniques. Contour maps of the RF-CoMFA illustrated that steric and electronegative substitutions were favored at R1 position whereas steric and electropositive substitutions were favored at R2 position to enhance the potency. Conclusion: Based on the designed strategy, we derived from the contour map analysis, 14 novel FLT3 inhibitors were designed and their activities were predicted. These designed inhibitors exhibited more potent activity than the most active compounds of the dataset.

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.

Single-cell analysis reveals the KIT D816V mutation in haematopoietic stem and progenitor cells in systemic mastocytosis.

BackgroundSystemic mastocytosis (SM) is a haematological disease characterised by organ infiltration by neoplastic mast cells. Almost all SM patients have a mutation in the gene encoding the tyrosine kinase receptor KIT causing a D816V substitution and autoactivation of the receptor. Mast cells and CD34+ haematopoietic progenitors can carry the mutation; however, in which progenitor cell subset the mutation arises is unknown. We aimed to investigate the distribution of the D816V mutation in single mast cells and single haematopoietic stem and progenitor cells.MethodsFluorescence-activated single-cell index sorting and KIT D816V mutation assessment were applied to analyse mast cells and >10,000 CD34+ bone marrow progenitors across 10 haematopoietic progenitor subsets. In vitro assays verified cell-forming potential.FindingsWe found that in SM 60–99% of the mast cells harboured the KIT D816V mutation. Despite increased frequencies of mast cells in SM patients compared with control subjects, the haematopoietic progenitor subset frequencies were comparable. Nevertheless, the mutation could be detected throughout the haematopoietic landscape of SM patients, from haematopoietic stem cells to more lineage-primed progenitors. In addition, we demonstrate that FcεRI+ bone marrow progenitors exhibit mast cell-forming potential, and we describe aberrant CD45RA expression on SM mast cells for the first time.InterpretationThe KIT D816V mutation arises in early haematopoietic stem and progenitor cells and the mutation frequency is approaching 100% in mature mast cells, which express the aberrant marker CD45RA.

Midostaurin for the management of FLT3-mutated acute myeloid leukemia and advanced systemic mastocytosis.

This article reviews the pharmacology, efficacy, safety, cost, and future directions of midostaurin for the treatment of acute myeloid leukemia (AML), aggressive systemic mastocytosis, systemic mastocytosis with associated hematological neoplasm, and mast cell leukemia, collectively known as advanced systemic mastocytosis (SM). Midostaurin was approved by the U.S. Food and Drug Administration for the treatment of FMS-like tyrosine kinase-3 (FLT3)- mutated AML. FLT3 is a tyrosine kinase which plays a key role in proliferation of early hematopoietic progenitor cells. In addition to having activity against FLT3, midostaurin inhibits several other tyrosine kinases which led to its approval for the treatment of advanced SM. Midostaurin offers a novel strategy to treat both FLT3-mutated AML and advanced SM. With a comparable adverse effect profile to other agents and substantial antiproliferative activity, midostaurin offers a therapeutic option for patients who have historically been difficult to treat.