Proliferation Of Hematopoietic Progenitors Research Articles

Dysregulated myeloid differentiation in colitis is induced by inflammatory osteoclasts in a TNFα-dependent manner

Inflammatory bowel disease (IBD) is characterized by very severe intestinal inflammation associated with extra-intestinal manifestations. One of the most critical ones is bone destruction, which remains a major cause of morbidity and a risk factor for osteopenia and osteoporosis in IBD patients. In various mouse models of IBD, we and other have demonstrated concomitant bone loss due to a significant increase in osteoclast activity. Besides bone resorption, osteoclasts are known to control hematopoietic niches in vivo and modulate inflammatory responses in vitro, suggesting they may participate in chronic inflammation in vivo. Here, using different models of colitis, we showed that osteoclast inhibition significantly reduced disease severity and that induction of osteoclast differentiation by RANKL contributed to disease worsening. Our results demonstrate a direct link between osteoclast activity and myeloid cell accumulation in the intestine during colitis. RNAseq analysis of osteoclasts from colitic mice revealed overexpression of genes involved in the remodeling of hematopoietic stem cell niches. We also demonstrated that osteoclasts induced hematopoietic progenitor proliferation accompanied by a myeloid skewing in the early phases of colitis, which was confirmed in a model of RANKL-induced osteoclastogenesis. Mechanistically, inhibition of TNF-α reduced the induction of myeloid skewing by OCL both in vitro and in vivo. Lastly, we observed that osteoclastic activity and the proportion of myeloid cells in the blood are positively correlated in patients with Crohn’s disease. Collectively, our results shed light on a new role of osteoclasts in colitis in vivo, demonstrating they exert their colitogenic activity through an early action on hematopoiesis, leading to an increase in myelopoiesis sustaining gut inflammation.

Exploring the role of GHRH antagonist MIA-602 in overcoming Doxorubicin-resistance in acute myeloid leukemia.

Acute myeloid leukemia (AML) is characterized by the rapid proliferation of mutagenic hematopoietic progenitors in the bone marrow. Conventional therapies include chemotherapy and bone marrow stem cell transplantation; however, they are often associated with poor prognosis. Notably, growth hormone-releasing hormone (GHRH) receptor antagonist MIA-602 has been shown to impede the growth of various human cancer cell lines, including AML. This investigation examined the impact of MIA-602 as monotherapy and in combination with Doxorubicin on three Doxorubicin-resistant AML cell lines, KG-1A, U-937, and K-562. The in vitro results revealed a significant reduction in cell viability for all treated wild-type cells. Doxorubicin-resistant clones were similarly susceptible to MIA-602 as the wild-type counterpart. Our in vivo experiment of xenografted nude mice with Doxorubicin-resistant K-562 revealed a reduction in tumor volume with MIA-602 treatment compared to control. Our study demonstrates that these three AML cell lines, and their Doxorubicin-resistant clones, are susceptible to GHRH antagonist MIA-602.

Abstract 4620: Exploring the role of GHRH antagonist MIA-602 in overcoming doxorubicin resistance in acute myeloid leukemia

Abstract Acute Myeloid Leukemia (AML) is characterized by the uncontrolled proliferation of immature hematopoietic progenitors, often associated with a poor prognosis. Conventional therapies involve chemotherapy and bone marrow stem cell transplantation. The growth hormone releasing hormone receptor (GHRH-R) antagonist MIA-602 has exhibited inhibitory effects on the growth of various human cancer cell lines, including AML cells. This study aimed to investigate the impact of MIA-602 alone and in combination with doxorubicin on three doxorubicin-resistant AML cell lines, K-562, U-937, and KG-1A. Initial examinations confirmed the presence of GHRH receptors in both wild-type and doxorubicin-resistant cell lines through western blot analysis. Doxorubicin was administered at concentrations of 0.005, 0.01, and 0.05 μg/ml, while MIA-602 was used at 5 μmol/L. Subsequent experiments involved culturing both wild-type and doxorubicin-resistant clones with MIA-602 at concentrations ranging from 0.05 μmol/L to 5 μmol/L for 24 and 48 hours. The results revealed a significant, dose- and time-dependent reduction in cell proliferation across all six cell lines. Both wild-type and doxorubicin-resistant clones exhibited a comparable decrease in cell proliferation when exposed to MIA-602 at concentrations greater than 0.05 μmol/L (p < 0.05) after 24 and 48 hours. In conclusion, our study demonstrates that these three AML cell lines and their doxorubicin-resistant clones remain susceptible to GHRH antagonist MIA-602. These findings may pave the way for the development of novel therapies or drug combinations using GHRH antagonists such as MIA-602 for treating AML. This is of particular interest for those who are resistant to conventional chemotherapy, further broadening the spectrum of treatment options available. Additional investigations are warranted to progress these findings to in vivo xenograft models. Citation Format: Simonetta I. Gaumond, Joel Costoya, Andrew V. Schally, Joaquin J. Jimenez. Exploring the role of GHRH antagonist MIA-602 in overcoming doxorubicin resistance in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4620.

Modulation of human thrombopoietin receptor conformations uncouples JAK2 V617F-driven activation from cytokine-induced stimulation

AbstractThe thrombopoietin receptor (TpoR) plays a central role in myeloproliferative neoplasms (MPNs). Mutations in JAK2, calreticulin, or TpoR itself drive the constitutive activation of TpoR and uncontrolled proliferation and differentiation of hematopoietic stem cells and progenitors. The JAK2 V617F mutation is responsible for most MPNs, and all driver mutants induce pathologic TpoR activation. Existing therapeutic strategies have focused on JAK2 kinase inhibitors that are unable to differentiate between the mutated MPN clone and healthy cells. Surprisingly, the targeting of TpoR itself has remained poorly explored despite its central role in pathology. Here, we performed a comprehensive characterization of human TpoR activation under physiological and pathological conditions, focusing on the JAK2 V617F mutant. Using a system of controlled dimerization of the transmembrane and cytosolic domains of TpoR, we discovered that human TpoR (hTpoR) adopts different dimeric conformations upon Tpo-induced vs JAK2 V617F–mediated activation. We identified the amino acids and specific dimeric conformation of hTpoR responsible for activation in complex with JAK2 V617F and confirmed our findings in the full-length receptor context in hematopoietic cell lines and primary bone marrow cells. Remarkably, we found that the modulation of hTpoR conformations by point mutations allowed for specific inhibition of JAK2 V617F–driven activation without affecting Tpo-induced signaling. Our results demonstrate that modulation of the hTpoR conformation is a viable therapeutic strategy for JAK2 V617F–positive MPNs and set the path for novel drug development by identifying precise residues of hTpoR involved in JAK2 V617F–specific activation.

Impact of Double-Stranded RNA Internalization on Hematopoietic Progenitors and Krebs-2 Cells and Mechanism

It is well-established that double-stranded RNA (dsRNA) exhibits noticeable radioprotective and radiotherapeutic effects. The experiments conducted in this study directly demonstrated that dsRNA was delivered into the cell in its native form and that it induced hematopoietic progenitor proliferation. The 68 bp synthetic dsRNA labeled with 6-carboxyfluorescein (FAM) was internalized into mouse hematopoietic progenitors, c-Kit+ (a marker of long-term hematopoietic stem cells) cells and CD34+ (a marker of short-term hematopoietic stem cells and multipotent progenitors) cells. Treating bone marrow cells with dsRNA stimulated the growth of colonies, mainly cells of the granulocyte-macrophage lineage. A total of 0.8% of Krebs-2 cells internalized FAM-dsRNA and were simultaneously CD34+ cells. dsRNA in its native state was delivered into the cell, where it was present without any signs of processing. dsRNA binding to a cell was independent of cell charge. dsRNA internalization was related to the receptor-mediated process that requires energy from ATP. Synthetic dsRNA did not degrade in the bloodstream for at least 2 h. Hematopoietic precursors that had captured dsRNA reinfused into the bloodstream and populated the bone marrow and spleen. This study, for the first time, directly proved that synthetic dsRNA is internalized into a eukaryotic cell via a natural mechanism.

221P The role of TNF-alpha in thymus dysfunction during acute myeloid leukemia

Acute myeloid leukemia (AML) is characterized by an increased proliferation of hematopoietic progenitors or precursors (blasts) of the different myeloid lineages. Studies performed in AML-affected patients revealed a T-cell immunodeficiency, characterized by a decreased number of peripheral T lymphocytes' TRECs and a restricted repertoire.

TLR9-Selective Therapeutic Targeting of MDS Clones with a CpG Payload-Delivery Strategy

Inflammatory molecules and their receptors serve as regulatory cues driving the proliferation of hematopoietic stem and progenitors (HSPC) in myelodysplastic syndromes (MDS). Recent studies indicate that inflammaging (inflammation in aging) in the bone marrow (BM) microenvironment contributes to hematopoietic impairment and MDS clone expansion. However, attempts to develop therapeutics targeting these processes have been hampered by potential off target effects due to the lack of identification of selective biomarkers to specifically target the MDS malignant clone. Therefore, a better understanding of the mechanisms of MDS inflammaging processes for selection and target of malignant clones remains a key goal. Several groups, including ours, have found that innate immune receptors are over expressed in MDS BM HSPC, including Toll-like receptors (TLRs). Overexpression of these receptors was associated with secretion of inflammatory cytokines and mediators including S100A9. S100A9, a Damage-Associated Molecular Pattern (DAMPs) molecule, causes pyroptosis of HSPC and induces genomic instability leading to MDS clonal expansion in an autocrine amplification loop. However, most of the TLRs and associated factors are hard to target due to their wide expression on healthy immune cells. We found that TLR9, usually an intracellular receptor, is upregulated and translocated to the surface of MDS progenitors, which thereby presents an opportunity for disease-specific targeting through its natural ligand, CpG oligonucleotides. TLR9 is one of the main pathogen recognition receptors expressed intracellularly in innate immune cells and recognizes bacterial nucleic acid moieties. Its gene expression has been previously found to be upregulated in MDS and now our work demonstrates a functional upregulation and translocation in low-risk disease. Flow cytometric sorting of low risk MDS BM cells with labeled CpG separated cells based on mutational burden suggesting that surface TLR9 is specific to malignant clones. Furthermore, pyroptotic release of nucleic acid DAMPs, such as RNA:DNA hybrids and our recently published biomarker oxidized mitochondrial DNA, is linked to the translocation of TLR9 from intracellular endosomes to the surface of these cells. This overexpression was validated in our unique inflammaging animal model, the S100A9 transgenic (Tg) mice, that phenocopies human MDS. Moreover, treatment of human BM mononuclear cells (MNC) with recombinant human S100A9 induced surface expression of TLR9, through the accumulation of S100A9-induced RNA:DNA hybrids and oxidized mitochondrial DNA. Therefore, this paradoxical surface expression of TLR9 in MDS BM HSPCs provides an opportunity to selectively target malignant cells through the targeted delivery of cytotoxins using our newly developed CpG-linked payload deliver strategy. CpG has been used safely in phase I and Phase II clinical trials where it has been shown to be well tolerated, although not effective as a stand-alone therapy. Therefore, we linked CpG to dendrimer particles (CpG-Den) providing a cationic lipid surface structure capable of disrupting cells only after internalization through TLR9. The CpG-Den linkage ratio can be manipulated to modify cytotoxicity strength and the particle size avoids leukocyte engulfment. Using both primary MDS BM MNC and BM MNC from S100A9Tg mice we demonstrate that treatment in vitro and in vivo with CpG-Den selectively targets surface TLR9+ HSPC, increasing healthy hematopoiesis in colony forming assays, and BM expression of HSPC, as well ameliorating anemia in vivo in the S100A9Tg mice. Use of this therapy also led to a decrease in suppressive myeloid cells likely aiding in the reduction of the inflammaging cycle. Localized targeting was confirmed in vivo by labeling CpG-Den with IR800 revealing specific accumulation in the mice BM. Our data supports TLR9-targeting potential in a proof-of-concept strategy to selectively target primary MDS malignant cells to restore and enhance hematopoiesis while avoiding toxicities.

Identification and interrogation of the gene regulatory network of CEBPA-double mutant acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy caused by mutations in genes encoding transcriptional and epigenetic regulators together with signaling genes. It is characterized by a disturbance of differentiation and abnormal proliferation of hematopoietic progenitors. We have previously shown that each AML subtype establishes its own core gene regulatory network (GRN), consisting of transcription factors binding to their target genes and imposing a specific gene expression pattern that is required for AML maintenance. In this study, we integrate gene expression, open chromatin and ChIP data with promoter-capture Hi-C data to define a refined core GRN common to all patients with CEBPA-double mutant (CEBPAN/C) AML. These mutations disrupt the structure of a major regulator of myelopoiesis. We identify the binding sites of mutated C/EBPα proteins in primary cells, we show that C/EBPα, AP-1 factors and RUNX1 colocalize and are required for AML maintenance, and we employ single cell experiments to link important network nodes to the specific differentiation trajectory from leukemic stem to blast cells. Taken together, our study provides an important resource which predicts the specific therapeutic vulnerabilities of this AML subtype in human cells.

Flt3 Signaling in B Lymphocyte Development and Humoral Immunity

The Class III receptor tyrosine kinase Flt3 and its ligand, the Flt3-ligand (FL), play an integral role in regulating the proliferation, differentiation, and survival of multipotent hematopoietic and lymphoid progenitors from which B cell precursors derive in bone marrow (BM). More recently, essential roles for Flt3 signaling in the regulation of peripheral B cell development and affinity maturation have come to light. Experimental findings derived from a multitude of mouse models have reinforced the importance of molecular and cellular regulation of Flt3 and FL in lymphohematopoiesis and adaptive immunity. Here, we provide a comprehensive review of the current state of the knowledge regarding molecular and cellular regulation of Flt3/FL and the roles of Flt3 signaling in hematopoietic stem cell (HSC) activation, lymphoid development, BM B lymphopoiesis, and peripheral B cell development. Cumulatively, the literature has reinforced the importance of Flt3 signaling in B cell development and function. However, it has also identified gaps in the knowledge regarding Flt3-dependent developmental-stage specific gene regulatory circuits essential for steady-state B lymphopoiesis that will be the focus of future studies.

Granulocyte-colony stimulating factor (G-CSF): an emerging therapeutic approach for amyotrophic lateral sclerosis (ALS).

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by neuronal degeneration and inflammation in the nerves. G-CSF is a 19.6-kDa hematopoietic growth factor which is essential for the proliferation and differentiation of granulocyte hematopoietic progenitors. G-CSF exerts neuroprotective activities by induction of neuronal regeneration, inhibition of neuronal apoptosis, mobilization of Hematopoietic stem cells (HSCs), regulation of pro and anti-inflammatory cytokines, and activation of angiogenesis. Pre-clinical studies have shown significant efficacy of G-CSF therapy in mSOD1G93A mice models. G-CSF treatments were able to increase the survival of mice. However, clinical studies on ALS patients failed to clone pre-clinical results. Considering the potential role of G-CSF in nervous system regeneration, this study aimed to comprehensively review the clinical and pre-clinical studies addressing G-CSF in ALS treatment.

VEGF Receptor 1 Promotes Hypoxia-Induced Hematopoietic Progenitor Proliferation and Differentiation.

Although it is well known that hypoxia incites unleashed cellular inflammation, the mechanisms of exaggerated cellular inflammation in hypoxic conditions are not known. We observed augmented proliferation of hematopoietic stem and progenitor cells (HSPC), precursors of inflammatory leukocytes, in mice under hypoxia. Consistently, a transcriptomic analysis of human HSPC exposed to hypoxic conditions revealed elevated expression of genes involved in progenitor proliferation and differentiation. Additionally, bone marrow cells in mice expressed high amount of vascular endothelial growth factor (VEGF), and HSPC elevated VEGF receptor 1 (VEGFr1) and its target genes in hypoxic conditions. In line with this, VEGFr1 blockade in vivo and in vitro decreased HSPC proliferation and attenuated inflammation. In silico and ChIP experiments demonstrated that HIF-1α binds to the promoter region of VEGFR1. Correspondingly, HIF1a silencing decreased VEGFr1 expression in HSPC and diminished their proliferation. These results indicate that VEGF signaling in HSPC is an important mediator of their proliferation and differentiation in hypoxia-induced inflammation and represents a potential therapeutic target to prevent aberrant inflammation in hypoxia-associated diseases.

Angiotensin-converting Enzyme Insertion/Deletion Polymorphism (rs4646994) and Susceptibility to Acute Lymphoblastic Leukemia: A Case–control Study

BACKGROUND: Angiotensin-converting enzyme (ACE) stimulates the proliferation of bone marrow hematopoietic progenitors and thought to be involved in pathological neoplastic hematopoiesis and leukemogenesis. AIM: This study aimed to investigate the association between ACE gene I/D polymorphism and the risk of acute lymphoblastic leukemia (ALL). MATERIALS AND METHODS: A total of 96 subjects were recruited for the study, 48 patients with ALL, and 48 apparently healthy volunteers as a control group. Genomic DNA was extracted from peripheral leukocytes and ACE I/D polymorphism was analysed using allele-specific polymerase chain reaction. RESULTS: In both study groups, the ACE D/D polymorphic genotype was the most frequent (52.1% and 54.2%, respectively), followed by the ID genotype (47.9% and 45.8% respectively), while the II genotype was completely absent in both study groups. The distribution of the polymorphic genotypes among the study groups was not significantly different (p = 0. 0.398). The frequency of the D allele was 0.76 in the patients and 0.77 in the control group, while the frequency of I allele was 0.24 in the patients and 0.23 in the control group. No deviation from Hardy–Weinberg equilibrium was observed (χ2 = 4.24, df = 1, p = 0.12). CONCLUSION: ACE I/D polymorphism is not associated with susceptibility to ALL among the Sudanese population.

2013 – A LABEL-FREE, HIGH-THROUGHPUT PHENOTYPIC SCREEN OF ADIPOCYTIC DIFFERENTIATION MODULATORS TO ACCELERATE HEMATOPOIETIC RECOVERY

Bone marrow (BM) preadipocytes and adipocytes are part of the same differentiation axis and co-exist within this organ with hematopoietic cells. Mature adipocytes can slow down hematopoietic progenitor proliferation whereas pre-adipocytes efficiently support their growth, suggesting that modulating the adipocytic differentiation axis could steer hematopoietic function. We used digital holographic microscopy to perform a label-free, high-throughput in vitro phenotypic screen of more than 4000 compounds to identify modulators of adipocytic differentiation. Candidate compounds were counter-screened for niche-mediated effects on hematopoietic proliferation. The screen identified calcipotriol, a vitamin D analogue. Treatment with calcipotriol remodeled adipocytes in a dose-dependent manner and increased hematopoietic progenitors in mice undergoing lethal irradiation followed by BM transplantation. Furthermore, treatment with calcipotriol rescued mortality in CXCL12 haplo-insufficient mice undergoing limiting-dose BM transplantation. Our approach provides a useful tool to study adipocyte biology, demonstrates the feasibility of modulating the adipocytic differentiation axis in the BM and indicates its potential role in clinically relevant conditions of high hematopoietic demand. Bone marrow (BM) preadipocytes and adipocytes are part of the same differentiation axis and co-exist within this organ with hematopoietic cells. Mature adipocytes can slow down hematopoietic progenitor proliferation whereas pre-adipocytes efficiently support their growth, suggesting that modulating the adipocytic differentiation axis could steer hematopoietic function. We used digital holographic microscopy to perform a label-free, high-throughput in vitro phenotypic screen of more than 4000 compounds to identify modulators of adipocytic differentiation. Candidate compounds were counter-screened for niche-mediated effects on hematopoietic proliferation. The screen identified calcipotriol, a vitamin D analogue. Treatment with calcipotriol remodeled adipocytes in a dose-dependent manner and increased hematopoietic progenitors in mice undergoing lethal irradiation followed by BM transplantation. Furthermore, treatment with calcipotriol rescued mortality in CXCL12 haplo-insufficient mice undergoing limiting-dose BM transplantation. Our approach provides a useful tool to study adipocyte biology, demonstrates the feasibility of modulating the adipocytic differentiation axis in the BM and indicates its potential role in clinically relevant conditions of high hematopoietic demand.

3082 – NONALCOHOLIC STEATOHEPATITIS PROMOTES EMERGENCY MYELOPOIESIS AND IMMUNE EFFECTOR DYSFUNCTION

Neutrophils and macrophages act as sentinels and help protect against injury and infection. Emergency myelopoiesis is triggered during this process and increases production of myeloid cells from hematopoietic stem and progenitor cells (HSPCs). This process is largely studied under acute stressors such as infection, but the impact of organ-specific inflammation on emergency myelopoiesis is poorly explored. Metabolic disorders induce metainflammation, a low-grade systemic chronic inflammation that results in elevated myeloid cells in circulation. The link of metainflammation to emergency myelopoiesis is not understood. We examined emergency myelopoiesis and immune cell function in a zebrafish model for Nonalcoholic steatohepatitis (NASH), an aggressive, pro-inflammatory hepatic manifestation of metabolic syndrome. To induce NASH, zebrafish larvae were fed a high-cholesterol diet for 1 week leading to liver steatohepatitis and chronic systemic inflammation. We detected increased proliferation of hematopoietic progenitors, as evidenced by increased 5-ethynyl-2’deoxyuridine (5-EdU) incorporation in hematopoietic tissues. Consistent with these findings, cd41:GFP-positive HSPCs as well as the total number of neutrophils and macrophages were increased in NASH larvae when compared to larvae fed with normal diet. In addition, neutrophils from NASH larvae displayed a hyperactive and dysfunctional response, as evidenced by exacerbated recruitment of lyz:H2B-mCherry-positive neutrophils to a tailfin injury. Neutrophils in NASH zebrafish also had increased speed, delayed resolution, and increased neutrophil reactive oxygen species production at the injury site. These data suggest that metabolic disorders, such as NASH, can promote emergency myelopoiesis and immune effector dysfunction leading to a host of inflammatory deregulation in these individuals. Neutrophils and macrophages act as sentinels and help protect against injury and infection. Emergency myelopoiesis is triggered during this process and increases production of myeloid cells from hematopoietic stem and progenitor cells (HSPCs). This process is largely studied under acute stressors such as infection, but the impact of organ-specific inflammation on emergency myelopoiesis is poorly explored. Metabolic disorders induce metainflammation, a low-grade systemic chronic inflammation that results in elevated myeloid cells in circulation. The link of metainflammation to emergency myelopoiesis is not understood. We examined emergency myelopoiesis and immune cell function in a zebrafish model for Nonalcoholic steatohepatitis (NASH), an aggressive, pro-inflammatory hepatic manifestation of metabolic syndrome. To induce NASH, zebrafish larvae were fed a high-cholesterol diet for 1 week leading to liver steatohepatitis and chronic systemic inflammation. We detected increased proliferation of hematopoietic progenitors, as evidenced by increased 5-ethynyl-2’deoxyuridine (5-EdU) incorporation in hematopoietic tissues. Consistent with these findings, cd41:GFP-positive HSPCs as well as the total number of neutrophils and macrophages were increased in NASH larvae when compared to larvae fed with normal diet. In addition, neutrophils from NASH larvae displayed a hyperactive and dysfunctional response, as evidenced by exacerbated recruitment of lyz:H2B-mCherry-positive neutrophils to a tailfin injury. Neutrophils in NASH zebrafish also had increased speed, delayed resolution, and increased neutrophil reactive oxygen species production at the injury site. These data suggest that metabolic disorders, such as NASH, can promote emergency myelopoiesis and immune effector dysfunction leading to a host of inflammatory deregulation in these individuals.

Genomic Abnormalities as Biomarkers and Therapeutic Targets in Acute Myeloid Leukemia.

Simple SummaryAML is a heterogenous malignancy with a variety of underlying genomic abnormalities. Some of the genetic aberrations in AML have led to the development of specific inhibitors which were approved by the Food and Drug Administration (FDA) and are currently used to treat eligible patients. In this review, we describe five gene mutations for which approved inhibitors have been developed, the response of AML patients to these inhibitors, and the known mechanism(s) of resistance. This review also highlights the significance of developing function-based screens for target discovery in the era of personalized medicine.Acute myeloid leukemia (AML) is a highly heterogeneous malignancy characterized by the clonal expansion of myeloid stem and progenitor cells in the bone marrow, peripheral blood, and other tissues. AML results from the acquisition of gene mutations or chromosomal abnormalities that induce proliferation or block differentiation of hematopoietic progenitors. A combination of cytogenetic profiling and gene mutation analyses are essential for the proper diagnosis, classification, prognosis, and treatment of AML. In the present review, we provide a summary of genomic abnormalities in AML that have emerged as both markers of disease and therapeutic targets. We discuss the abnormalities of RARA, FLT3, BCL2, IDH1, and IDH2, their significance as therapeutic targets in AML, and how various mechanisms cause resistance to the currently FDA-approved inhibitors. We also discuss the limitations of current genomic approaches for producing a comprehensive picture of the activated signaling pathways at diagnosis or at relapse in AML patients, and how innovative technologies combining genomic and functional methods will improve the discovery of novel therapeutic targets in AML. The ultimate goal is to optimize a personalized medicine approach for AML patients and possibly those with other types of cancers.

Cytotoxic Marine Alkaloid 3,10-Dibromofascaplysin Induces Apoptosis and Synergizes with Cytarabine Resulting in Leukemia Cell Death.

Myeloid leukemia is a hematologic neoplasia characterized by a clonal proliferation of hematopoietic stem cell progenitors. Patient prognosis varies depending on the subtype of leukemia as well as eligibility for intensive treatment regimens and allogeneic stem cell transplantation. Although significant progress has been made in the therapy of patients including novel targeted treatment approaches, there is still an urgent need to optimize treatment outcome. The most common therapy is based on the use of chemotherapeutics cytarabine and anthrayclines. Here, we studied the effect of the recently synthesized marine alkaloid 3,10-dibromofascaplysin (DBF) in myeloid leukemia cells. Unsubstituted fascaplysin was early found to affect cell cycle via inhibiting CDK4/6, thus we compared the activity of DBF and other brominated derivatives with known CDK4/6 inhibitor palbociclib, which was earlier shown to be a promising candidate to treat leukemia. Unexpectedly, the effect DBF on cell cycle differs from palbociclib. In fact, DBF induced leukemic cells apoptosis and decreased the expression of genes responsible for cancer cell survival. Simultaneously, DBF was found to activate the E2F1 transcription factor. Using bioinformatical approaches we evaluated the possible molecular mechanisms, which may be associated with DBF-induced activation of E2F1. Finally, we found that DBF synergistically increase the cytotoxic effect of cytarabine in different myeloid leukemia cell lines. In conclusion, DBF is a promising drug candidate, which may be used in combinational therapeutics approaches to reduce leukemia cell growth.

Stathmin 1 Is Highly Expressed in Acute Promyelocytic Leukemia and Microtubule Dynamics Is a Potential Target for ATRA-Resistant APL Cells

Introduction : Stathmin 1 is a microtubule destabilizer protein involved in the proliferation and differentiation of early hematopoietic progenitors. Aberrant Stathmin 1 expression has been reported in hematological malignancies. Stathmin 1 silencing reduces cell proliferation and clonogenicity of leukemia cell lines. A previous study identified that the PML-RARα fusion protein, which is necessary to acute promyelocytic leukemia (APL) genesis, positively regulates Stathmin 1 at transcription and protein activity levels. However, Stathmin 1 expression and its impact on prognosis and leukemia cell biology have rarely been studied in APL. Objectives : To evaluate Stathmin 1 expression and its association with laboratory and clinical parameters in a cohort of APL patients. To investigate Stathmin 1 expression and activity upon all-trans retinoic acid (ATRA) treatment and during cell cycle in the PML-RARα cell lines. Finally, to phenocopy the effects of Stathmin 1 inhibition using the pharmacological microtubule stabilizing agent paclitaxel. Methods : Bone marrow samples from 121 APL patients enrolled in the International Consortium on Acute Promyelocytic Leukemia (IC-APL) at diagnosis and 22 healthy donors were included. Patients were treated with ATRA and chemotherapy as described elsewhere. Patients were dichotomized according to Stathmin 1 expression using the median as cut-off. The PML-RARα cell lines, NB4 (ATRA-sensible) and NB4-R2 (ATRA-resistant) were used for functional assays. Cell cycle was synchronized by double thymidine block. For dose-response curves (MTT), PML-RARα cells were treated with graded concentrations of paclitaxel and IC50 values were calculated using a nonlinear regression analysis. For apoptosis assay (annexin V/PI), PML-RARα cells were treated with ATRA (1 µM) and/or IC50 paclitaxel dose for 72 hours. Gene and protein expressions were evaluated by qPCR and Western blot, and microtubule networks by confocal microscopy. For statistical analyses, Fisher's exact test or Chi-square test were used to compare categorical variables. Mann-Whitney test, t Student test or ANOVA test and Bonferroni post-test were used to compare continuous variables. Log-rank test and Cox regression were used for survival analyzes. P -value <0.05 was considerate statistically significant. Results : Stathmin 1 transcripts were significantly increased in bone marrow samples from APL patients compared with healthy donors (median 1.21 [range 0.02-4.62] versus (vs.) 0.66 [0.01-1.71]; p <0.01). Stathmin 1 levels did not impact on disease-free survival, overall survival and complete remission rates of APL patients. No relevant association between Stathmin 1 expression and clinical and laboratory parameters were observed. In NB4 cells, but not in NB4-R2, ATRA induced granulocytic differentiation, associated with a prominent reduction of Stathmin 1 gene and protein expression (p <0.05). Paradoxically, ATRA-induced granulocytic differentiation strongly reduced α-tubulin acetylation (microtubule stability marker) in NB4 cells. Using thymidine-synchronized cells, we observed that Stathmin 1 phosphorylation at serine 16 (an inhibitory site) was predominant in pool of mitosis-enriched cells, and its phosphorylation was marked reduced in pool of G1-enriched cells. NB4 and NB4-R2 cells presented high sensibility to paclitaxel treatment (IC50 of 3.6 nM and 1.8 nM, respectively). In NB4-R2 cells, but not in NB4 cells, paclitaxel increased the number of apoptotic cells after 72 hours of ATRA treatment; increased annexin V and caspase 3 cleavage (p <0.05). Paclitaxel treatment increased acetyl-α-tubulin, y-H2AX and CDKN1A levels. Conclusions : Stathmin 1 is highly expressed, but does not impact clinical outcomes in APL patients. Stathmin 1 reduces during ATRA-induced cell differentiation. Stathmin 1 activity is reduced during mitosis (as demonstrate by increased p-Stathmin 1S16) and increases post-mitosis (reduced p-Stathmin 1S16), indicating that this protein participates in microtubule dynamics and cell cycle progression in PML-RARα cells. Paclitaxel was an efficient pharmacological agent in inducing microtubule stabilization, reducing cell viability, increasing mitotic catastrophe as monotherapy or in combination with ATRA, in PML-RARα cells, including ATRA-resistant cells. Our study provides new insights on Stathmin 1 and microtubule dynamics in APL. DisclosuresBittencourt:Janssen, Takeda: Honoraria.

Effects of CSF3R mutations on Myeloid Differentiation and Proliferation of Hematopoietic Cells of Congenital Neutropenia Patients

Over the last 25 years, with the clinical approval of recombinant human granulocyte colony-stimulating factor (rhG-CSF) remarkable progress has been achieved in the therapy of severe congenital neutropenia (CN). Nevertheless, CN patients have a high rate of transformation to myelodysplasia (MDS) or acute myeloid leukemia (AML). This risk is especially high in the group of patients who harbor acquired G-CSFR mutations, suggesting that these mutations are involved in the leukemogenesis. Deep sequencing of the intracellular part of G-CSFR allowed us to identify a subset of CN patients with a high mutant allele frequency (MAF) of G-CSFR mutations in the granulocytic compartment. We performed CSF3R mutation analysis of myeloid colonies from colony forming unit (CFU) assay of BM and CD34+ samples of 3 ELANE -CN patients, in which the percentage of granulocytes with G-CSFR mutation varied between 22.6% and 81%. Average number of sequenced colonies per CFU sample was 18. Intriguingly, we found that only cell clones with WT G-CSFR were grown in CFU assay. We hypothesized that lack of G-CSFR mutant clones in CFU assay is due to abnormal signaling downstream of truncated G-CSFR. To compare myeloid differentiation of G-CSFR mutant and WT G-CSFR hematopoietic cells, we generated ELANE -CN patient-derived induced pluripotent stem cells (CN-iPSCs) and performed CRISPR/Cas9 genome editing of CSF3R . Using 2 different sgRNAs specific for the intracellular region of G-CSFR, we introduced nucleotide indels at amino acid positions 740 and 735 (NP_000751.1) in CN-iPSCs. Subsequently, homozygous and heterozygous CN-iPSCs clones with truncated distal part of cytoplasmic domain of G-CSF receptor and subsequent loss of 3 out of 4 conserved tyrosine residues were generated.We compared myeloid differentiation of healthy donor iPSCs (HD-iPSCs), CN-iPSCs and CN-iPSCs with heterozygous and homozygous truncated G-CSF receptor (G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs, respectively). Absolute cell count during myeloid differentiation (18-32 day) of HD-iPSCs, G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs didn't differ significantly (p>0.05). In contrast, absolute cell count was found to be elevated from day 25 to day 32 of differentiation when G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs hematopoietic progenitors were compared with CN-iPSCs-derived hematopoietic cells (p<0.05). FACS analysis of CN-iPSCs derived hematopoietic progenitors with and without truncated G-CSF receptor using a panel of hematopoietic markers (KDR, CD34, CD235a, CD41a, CD45 and CD33) showed no difference in percentage of early progenitors (CD34+/KDR+), E/MK precursors (CD41a+CD235a+/ CD45-), CD34+/CD45+ and CD34+/CD43+ cells. Intriguingly, we observed increase in percentage of CD34+/CD43+ (p<0.01), CD33+/CD45+ (p<0.05) and CD41a+CD235a+/ CD45- (p<0.05) cells at day 14 of myeloid differentiation of G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs versus HD-iPSc. As expected, relative number CD14+ and CD16+ cells were not changed upon myeloid differentiation of hematopoietic progenitors with mutant G-CSFR but relative levels of CD15+ and CD15+/CD16+ were negatively affected by homozygous and heterozygous G-CSFR mutations. Next, CFU assay of hematopoietic progenitors derived from G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs produced markedly less colonies than HD- and CN-iPSCs derived hematopoietic cells. Cell morphology analysis also confirmed the reduced number of mature granulocytes seen after myeloid differentiation of G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs. Finally, reprogramming of BM cells from CN patients into iPSCs followed by mutation analysis of single cell derived iPSCs clones allowed us to identify iPSCs with naturally acquired G-CSFR mutations. Preliminary results revealed reduced myeloid differentiation of these cells, which is in line with data generated from G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs.Using a combination of patient CN-iPSCs and CRISPR/Cas9 gene editing we were able to recapitulate main features of G-CSFR mutant versus G-CSFR WT CN cells - defective neutrophil differentiation and increased proliferation of human hematopoietic progenitors. Since virtually all studies of truncated G-CSFR receptor signaling have been performed in murine cytokine-responsive myeloid cell lines, our approach is more suitable to explore human aberrant G-CSF receptor signaling in depth. DisclosuresNo relevant conflicts of interest to declare.

Results of a Non-Interventional Clinical Trial of Apograft — a Novel Technology for GvHD Prevention

IntroductionGvHD is a threat to the wellbeing and survival of patients following hematopoietic stem cell transplantation (HSCT). In-vivo and ex-vivo T cell depletion are the most effective methods for GvHD prevention, but attendant risks of infection, graft failure and leukemic recurrence offset the advantages of these manipulations. Less extensive T cell depletion leaves many patients at risk for the ravages of treatment-resistant GvHD. Alternative approaches to eliminate GvHD-causing cells are needed. Fas ligand (FasL), a member of the tumor necrosis factor (TNF) family, can selectively induce apoptosis of mature T cells while sparing stem and progenitor cells. We have developed a novel FasL-mediated selection process which eliminates mature T cell subsets in hematopoietic stem cell grafts and effectively prevents GvHD in pre-clinical models without impairing engraftment and immune system recovery. We report here data from ex-vivo and in-vivo studies supporting the use of FasL-mediated depletion of mature T-cells, a process we have named ApoGraft, as a novel effective method for GvHD prophylaxis in the context of allogeneic HSCT.Methods:Aliquots of mobilized peripheral blood cells (MPBC) were collected from 104 consenting healthy donors and were used ex-vivo for optimization of the ApoGraft manufacturing process. ApoGraft is manufactured under GMP guidelines, and includes two washing steps, a 2 hr incubation of the graft at 37oC with human recombinant different concentrations of FasL (MegaFasL, Adipogen), and 2 additional washing steps to remove unbound FasL. Aliquots from 25 MPBC apheresis that had been subjected to the complete ApoGraft process were analyzed ex-vivo for cell yield, viability and cellular constitution by FACS and for clonogenicity potential by colony forming unit (CFU) assays. Additionally, engraftment and GvHD prevention of ApoGraft were evaluated with 7 aliquots of MPBC apheresis in NOD-SCID IL2-Rgamma-null (NSG) mice (n=349). Here we report results from representative studies were unfractionated ApoGraft (2.5x10^6 cells) or CD34+ purified cells (1.0x10^5 cells) from ApoGraft were transplanted into irradiated (2-2.75Gy) NSG mice. The mice were monitored clinically for GVHD occurrence post transplantation; engraftment and differentiation were evaluated using FACS analysis.Results:Cellular viability was 98% (±1%) in the ApoGraft samples analyzed. Comparison of ApoGraft and Starting Material (SM) showed a mean 19.9% (±4.3%) reduction of total CD3+ T cells; early apoptosis of CD3+ cell increased by 5-fold as compared to Starting Material. Importantly, the percentage of viable CD34+ stem and progenitor cells was not affected by ApoGraft processing. Proliferation and differentiation of hematopoietic progenitors in colony forming unit (CFU) assay was not affected. In NSG mice infused with unfractionated ApoGraft or CD34+ cells purified from it, chimerism level and differentiation were similar to those of mice infused with control MPBC (figure 1A-1B). Most importantly, ApoGraft infused mice had lower GvHD clinical score and prolonged survival as compared to mice infused with control MPBC (maximal score 1.7±0.7 vs. 8.8±0.8) (Figure 1C).Conclusion:ApoGraft is an apoptosis-based selection process that eliminates mature T cells from stem cells grafts, while preserving CD34+ cells quantity, quality and functionality. In our preclinical murine model, ApoGraft reduced GvHD without impairing engraftment. Ongoing animal studies are evaluating the graft vs. leukemic capabilities of ApoGraft and the preservation of anti-viral immunity after ApoGraft processing. Additionally, ApoGraft is currently being evaluated in a Phase I/IIa study (NCT02828878) in subjects with hematological malignancies undergoing matched related allogeneic HSCT. [Display omitted] DisclosuresZuckerman:BioSight Ltd.: Consultancy. Yarkoni:Cellect Biotechnology Ltd.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Nechushtan:Cellect Biotechnology Ltd.: Employment. Rodionov:Cellect Biotechnology Ltd.: Employment. Gez:Cellect Biotechnology Ltd.: Employment. Rosenzwaig:Cellect Biotechnology Ltd.: Employment. Rodin:Cellect Biotechnology Ltd.: Employment. Pereg:Cellect Biotechnology Ltd.: Employment.

Plerixafor Overcomes Lenalidomide-Associated Stem Cell Collection Failure and Affects Collected Myeloid and Erythroid Progenitors

▪High-dose chemotherapy followed by autologous hematopoietic cell transplantation (auto-HCT) remains standard of care for transplant-eligible multiple myeloma (MM) patients (pts). MM frontline therapy typically includes lenalidomide (LEN). However, studies have indicated that LEN-containing regimens are associated with an increase in peripheral blood stem cell (PBSC) collection failure. Since most health insurance carriers do not reimburse for PBSC collection if there is no intent to proceed with auto-HCT soon after collection, a common current practice is early auto-HCT after a few cycles of LEN-containing regimen. This may prevent a subset of pts from achieving the deepest possible response prior to transplant. Recently, plerixafor has been used sporadically to ameliorate the suppressive effect of LEN on PBSC collection, however its effect on collected graft composition is unknown. Here, we examine the influence of plerixafor on PBSC mobilization, graft composition, and auto-HCT outcomes.MethodsPts who underwent auto-HCT between 2007 and 2016 at our institution were included. After excluding pts without complete progenitor cell assays, 94 pts formed the final analysis set. The primary end point was PBSC collection failure, defined as the inability to collect at least 5×10^6 CD34+ cells/kg on first apheresis attempt. Secondary endpoints were frequency, in-vitro proliferation, and differentiation capability of hematopoietic progenitors. Granulocyte-Monocyte Colony-Forming Units (CFU-GM) and Burst-Forming Erythroid Units (BFU-E) colonies were scored after 14 days of incubation. Effects of baseline and treatment variables on PBSC collection failure were examined by logistic regression. Significant variables were entered in a multivariate regression model.ResultsPBSC mobilization was achieved with G-CSF and cyclophosphamide (21 pts), G-CSF alone (8 pts), or with G-CSF and plerixafor (65 pts). Pts that received LEN-containing regimens as the immediate line of therapy before collection (LENpos, N=51) were compared to those who did not (LENneg, N=43). Median number of LEN cycles was 4 (range 1-13). 15 pts (16%) had day 1 collection failure with no difference between LENpos and LENneg groups (17% vs. 14%, respectively; P=0.392). Only 1 pts (1.1%) failed final collection. Collection days and median number of collected CD34+ cells were similar in both groups. Older age (OR: 1.85, 95% CI: 1.11-2.54, P=0.001), lower platelets on day of stem cell collection (OR: 1.87, 95% CI: 1.09-2.98, P=0.021), and lack of plerixafor use for PBSC mobilization (OR: 1.96, 95% CI: 1.11-3.52, P<0.001) were risk factors associated with worse stem cell collection in univariate analysis (Table 1). All three parameters detected by univariate analysis retained statistical negative predictive significance. LEN exposure did not affect the collection failure rate (OR: 1.15, 95% CI: 0.81-1.36, P=0.356). There was no difference between LENpog and LENneg groups in median ratio of CFU-GM/total nucleated cells (TNC) (10.5 vs. 11.7 10^-4, P=0.381) neither BFU-E/TNC (7.2 vs. 7.9 10^-4, P= 0.253), indicating similar LEN effect on collected myeloid and erythroid progenitors. The number of collected CFU-GM and BFU-E correlated with collected TNC in both groups (LENpos: R2:0.400, P=0.031, LENneg: R2: 0.405, P=0.001; and LENpos: R2:0.357, P=0.009, LENneg: R2: 0.292, P=0.001; respectively, Figure 1). However there were no difference between LENpos and LENneg group. LENpos pts that did not use plerixafor had significantly lower collected CFU-GM and BGU-E compared to the remainder of pts (P=0.003 and P=0.021, respectively; Figure 3). Mean time to neutrophil and platelet engraftments were similar in both groups (Figure-2). There was one platelet engraftment failure in the LENneg group. There was no detected difference in progression-free survival between the two groups (3-year PFS rate: 71% vs. 74%, HR: 0.91, CI: 0.72-1.16, p=0.27).ConclusionsIn our cohort of heavily pre-treated pts, the length of LEN usage prior to PBSC collection did not affect PBSC collection, graft quality or engraftment when plerixafor was added to the mobilization regimen, suggesting plerixafor may overcome potential deleterious effects of LEN on PBSC collection. The impact of LEN on PBSC collection and transplantation needs further study in prospective clinical trials. [Display omitted] DisclosuresDe Lima:Celgene Corporation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees. Malek:Sanofi: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.