Hematopoietic Growth Factor Receptor Research Articles

Discovery of an unconventional lamprey lymphocyte lineage highlights divergent features in vertebrate adaptive immune system evolution

Lymphocyte receptors independently evolved in both jawed and jawless vertebrates with similar adaptive immune responses. However, the diversity of functional subtypes and molecular architecture in jawless vertebrate lymphocytes, comparable to jawed species, is not well defined. Here, we profile the gills, intestines, and blood of the lamprey, Lampetra morii, with single-cell RNA sequencing, using a full-length transcriptome as a reference. Our findings reveal higher tissue-specific heterogeneity among T-like cells in contrast to B-like cells. Notably, we identify a unique T-like cell subtype expressing a homolog of the nonlymphoid hematopoietic growth factor receptor, MPL-like (MPL-L). These MPL-L+ T-like cells exhibit features distinct from T cells of jawed vertebrates, particularly in their elevated expression of hematopoietic genes. We further discovered that MPL-L+ VLRA+ T-like cells are widely present in the typhlosole, gill, liver, kidney, and skin of lamprey and they proliferate in response to both a T cell mitogen and recombinant human thrombopoietin. These findings provide new insights into the adaptive immune response in jawless vertebrates, shedding new light on the evolution of adaptive immunity.

A c- MPL Splice Variant up-Regulated in Myeloproliferative Neoplasms Modulates Thrombopoietin Signaling and Induces Cycling of Hematopoietic Stem Cells and Inflammatory Progenitors

Introduction: Myeloproliferative neoplasms (MPN) are clonal disorders caused by mutations that activate JAK/STAT signaling through the thrombopoietin receptor, MPL, leading to overproduction of myeloid lineages. MPL is the only hematopoietic growth factor receptor expressed on the surface of hematopoietic stem cells (HSC) where it maintains their quiescence by tethering HSCs to osteoblasts in the endosteal niche. MPL is also expressed on megakaryocytes and platelets. Individuals born with loss of function mutations in the distal MPL cytokine receptor homology domain (CRHD) develop thrombocytopenia and progressive bone marrow failure. How MPL functions in MPN remains unclear. Here, we describe a novel MPL splice variant ( MPL-SV) that is present in CD34 + cells and platelets from healthy controls, but overexpressed in the MPN. We therefore sought to: 1) define MPL-SV function in hematopoiesis and MPN, and, 2) elucidate mechanisms underlying MPL-SV. Methods: After identifying a novel MPL-SV transcript from JAK2 V617F MPNplatelets, we compared its expression to MPL transcripts lacking the deletion in MPN patients and healthy controls. To assess its function, we generated transgenic (Tg) mice expressing varied levels of MPL-SV in HSC. Next, we examined responses of the Tg mice to thrombopoietin (THPO), hematopoietic stem progenitor cell (HSPC) frequency by flow cytometry, and HSPC function by colony-forming assays (CFA). To elucidate mechanisms, we performed single cell RNA sequencing (scRNAseq) of bone marrow (BM)-derived Lin -, Sca +, c-kit + (LSK) cells followed by pathway analysis. Results: Using primers to amplify the MPL CRHD in platelets from patients with JAK2 V617F MPN, we discovered an MPL- SV lacking 7 amino acids in the MPL amino-terminal domain, a common hotspot for activating MPN mutations. MPL-SV is present in CD34 + cells and platelets of healthy controls, but overexpressed in MPN relative to MPL transcripts lacking the deletion. To examine its function, we generated transgenic (Tg) mice expressing MPL-SV in HSC. Tg miceexpressing a single copy of MPL-SV have unperturbed hematopoiesis, whereas Tg mice expressing 6 copies of the transgene ( MPL-SV-6) develop marked thrombocytopenia, mild splenomegaly, and a modest increase in red cells by 9-12 weeks of age. Baseline serum THPO levels are elevated in Tg MPL-SV-6 mice and they fail to increase platelet counts following exogenous THPO administration, suggesting defective function of MPL-SV in binding and/or internalizing THPO. To further assess MPL-SV function, we compared CFA from HSPC of Tg MPL-SV-6 and wildtype mice, which show a decrease in total colony forming units (CFU) from BM or spleen-derived MPL-SV-6 HSPC. Accordingly, there was a decrease in LSK cells, multi-potent progenitors (MPPs), and megakaryocytes (Mk) in BM by flow cytometry. By contrast, spleens show increases in LK cells and short-term HSC. Further analysis by scRNAseq of BM-derived LSK cells reveal a marked decrease in both quiescent HSC and HSC with a Mk-bias associated with a relative expansion in more proliferative HSC, including those with a bias towards erythroid and mature myeloid differentiation. Pathway analysis reveal that MPL-SV activates transcriptional networks involved in TNF-α signaling via NF-κB and oxidative phosphorylation. Analysis of cell-cell interactions by CellChat show decreased signaling through intercellular adhesion molecule 1 (ICAM-1) in MPL-SV-6 HSC compared to controls. Importantly, ICAM1 maintains quiescent HSC within their BM niche. Similarly, cell cycle analysis from scRNAseq indicate that quiescent HSC are stimulated to cycle and generate HSC primed to differentiate into erythroid and myeloid progeny. Conclusions: We discovered a novel MPL-SV that is up-regulated in MPN. In Tg mice, MPL-SV impairs responses to THPO and disrupts hematopoiesis by depleting quiescent HSC and BM megakaryocytes. Single cell transcriptomics suggest that MPL-SV drives expansion of cycling HSC and HSC primed to differentiate into progenitors involved in inflammation, including myeloid cells. Mechanistically, HSPC expressing MPL-SV-6 activate transcriptional networks involved in TNFα signaling via NF-κB pathways. Our results highlight the central role for MPL in hematopoiesis and suggest that aberrant MPL splicing contributes to MPN pathogenesis.

Gene expression profiling in normal cytogenetic acute myeloid leukaemia of Malay patient

Background: Insight into recent molecular analyses of patients with acute myeloid leukaemia (AML) and a normal cytogenetic have revealed a striking heterogeneity with regard to the presence of acquired changes in gene expression. Nano String technology which is aimed to identify descriptive profiling of normal cytogenetic AML in differentially expressed genes involved in the different pathways in normal cytogenetic AML patients.
 Materials and method: Blood samples were obtained from eight at diagnosis patients with normal cytogenetics AML, two follow-up patients and two normal healthy controls prior to RNA extractions. RNA gene expression assay was performed using Nano Stringn Counter® Pan Cancer Pathway Panel (Nanostring Technologies, Seattle, Washington). Briefly, 100 ng of each total RNA sample was used as input into then Counter Pan Cancer Pathway Panel sample preparation. Data was extracted using then Counter RCC Collector and raw data output was imported into nSolver v2.6 analysis software for data analysis.
 Results: The age range was from 13-69 years, with a median age-range of 41 years. We found the most enriched up regulated genes in newly diagnosed normal cytogenetic AML enlisted MPO (7.25 log FC), FLT3 (5.02 log FC), MYCN (4.99 log FC), MYB (4.74 log FC), ITGA9 (4.48 log FC), KIT (4.41 log FC), MCM2 (3.47 log FC), RAD51 (3.40 log FC), CCNA2 (3.37 log FC) and PROM1 (3.24 log FC) which those commonly be found in AML cases. For highly expressed down regulated genes were GZMB (-5.80 log FC), IL8 (-5.79 log FC), TNFRSFSF10C (-5.03 log FC), LEF1 (-4.82 log FC), IL2RB (-4.70 log FC), IL7R (-4.44 log FC), BCL2A1 (-4.15 log FC), RASGRP1 (-4.13 log FC), IL1R2 (-4.00 log FC) and HSPA6 (-3.99 log FC).
 Discussion and conclusion: Nano String required smaller amounts of starting material, and can perform mRNA expression profiling with digital precision, therefore the results do not require further validation by another method. MPO expression was significantly associated with disease-free survival (DFS). Previous report demonstrated that the groups by the MPO expression in the intermediate cytogenetic risk group showed a significant difference in DFS (p<0.001). A study by Valk et al., (2004) showed FLT3, a hematopoietic growth factor receptor, is the most common molecular abnormality in AML. The presence of such mutations in FLT3 and elevated expression of the transcription factor EVI1 confer a poor prognosis. N-MYC expression levels in AML samples from patients with favorable, intermediate, or unfavorable prognosis were compared with that in CD34+ cells from four healthy bone marrow donors. The most highly down regulated gene in newly diagnosed AML goes to Granzyme B (GZMB) which involved in cytolytic activity that showed high correlation with other transcripts expressed in activated cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells as well as lymphocyte activation-related gene validating it as a robust and specific metric of active cellular immunity. Increased expression of IL-8 and/or its receptors has been characterized in cancer cells, endothelial cells, infiltrating neutrophils, and tumor-associated macrophages, suggesting that IL-8 may function as a significant regulatory factor within the tumor microenvironment.
 Bangladesh Journal of Medical Science Vol.20(3) 2021 p.556-562

The Thrombopoietin Receptor, MPL, Is a Therapeutic Target of Opportunity in the MPN.

The myeloproliferative neoplasms, polycythemia vera, essential thrombocytosis and primary myelofibrosis share driver mutations that either activate the thrombopoietin receptor, MPL, or indirectly activate it through mutations in the gene for JAK2, its cognate tyrosine kinase. Paradoxically, although the myeloproliferative neoplasms are classified as neoplasms because they are clonal hematopoietic stem cell disorders, the mutations affecting MPL or JAK2 are gain-of-function, resulting in increased production of normal erythrocytes, myeloid cells and platelets. Constitutive JAK2 activation provides the basis for the shared clinical features of the myeloproliferative neoplasms. A second molecular abnormality, impaired posttranslational processing of MPL is also shared by these disorders but has not received the recognition it deserves. This abnormality is important because MPL is the only hematopoietic growth factor receptor expressed in hematopoietic stem cells; because MPL is a proto-oncogene; because impaired MPL processing results in chronic elevation of plasma thrombopoietin, and since these diseases involve normal hematopoietic stem cells, they have proven resistant to therapies used in other myeloid neoplasms. We hypothesize that MPL offers a selective therapeutic target in the myeloproliferative neoplasms since impaired MPL processing is unique to the involved stem cells, while MPL is required for hematopoietic stem cell survival and quiescent in their bone marrow niches. In this review, we will discuss myeloproliferative neoplasm hematopoietic stem cell pathophysiology in the context of the behavior of MPL and its ligand thrombopoietin and the ability of thrombopoietin gene deletion to abrogate the disease phenotype in vivo in a JAK2 V617 transgenic mouse model of PV.

The MPL mutation.

Myeloproliferative neoplasms (MPN) patients share driver mutations in JAK2, MPL or CALR genes leading to the activation of the thrombopoietin receptor (TPOR) and downstream signaling pathways. JAK2 mutation drives all the three major entities of MPN (Polycythemia Vera, Essential Thrombocythemia and Primary Myelofibrosis) through the constitutive activation of TPOR, erythropoietin (EPOR) and colony stimulating factor 3 receptor (CSF3R) signaling. MPL is a proto-oncogene encoding for TPOR, the hematopoietic growth factor receptor of myeloid stem cells. MPL mutants induce the stable dimerization of TPOR that in turn activate JAK2 and the thrombopoietin pathway. The thrombopoietin pathway plays an important role in the development of megakaryocytes and platelets as well as the self-renewal of hematopoietic stem cells. Little wonder therefore that mutations of MPL result in thrombocytosis, leading to an abnormal MPL trafficking or receptor activation. Finally, some extremely rare germline genetic variants in MPL can induce MPN-like hereditary disease. Against this molecular background, TPOR is a key actor in the MPN development and MPL mutations are of major relevance to fully elucidate the molecular mechanisms underlying the clinical manifestations of MPN and to arrange novel therapeutic strategies aiming to disrupt the dysegulated signaling cascade. This chapter will focus on the role MPL in the pathogenesis of MPN and in familial thrombocytosis and will review these different subtypes of somatic and germline genetic variants by dissecting how they impact clinical phenotype.

Augmentation of NK Cell Proliferation and Anti-tumor Immunity by Transgenic Expression of Receptors for EPO or TPO

Many investigational adoptive immunotherapy regimens utilizing natural killer (NK) cells require the administration of interleukin-2 (IL-2) or IL-15, but these cytokines cause serious dose-dependent toxicities. To reduce or preclude the necessity for IL-2 use, we investigated whether genetic engineering of NK cells to express the erythropoietin (EPO) receptor (EPOR) or thrombopoietin (TPO) receptor (c-MPL) could be used as a method to improve NK cell survival and function. Viral transduction of NK-92 cells to express EPOR or c-MPL receptors conveyed signaling via appropriate pathways, protected cells from apoptosis, augmented cellular proliferation, and increased cell cytotoxic function in response to EPO or TPO ligands invitro. In the presence of TPO, viral transduction of primary human NK cells to express c-MPL enhanced cellular proliferation and increased degranulation and cytokine production toward target cells invitro. In contrast, transgenic expression of EPOR did not augment the proliferation of primary NK cells. In immunodeficient mice receiving TPO, invivo persistence of primary human NK cells genetically modified to express c-MPL was higher compared with control NK cells. These data support the concept that genetic manipulation of NK cells to express hematopoietic growth factor receptors could be used as a strategy to augment NK cell proliferation and antitumor immunity.

Thrombopoietin is required for full phenotype expression in a JAK2V617F transgenic mouse model of polycythemia vera.

The myeloproliferative neoplasms, polycythemia vera, essential thrombocytosis and primary myelofibrosis are hematopoietic stem cell disorders and share driver mutations that either directly activate the thrombopoietin receptor, MPL, or activate it indirectly through gain-of-function mutations in the gene for JAK2, its cognate tyrosine kinase. Paradoxically, MPL surface expression in hematopoietic stem cells is also reduced in the myeloproliferative neoplasms due to abnormal post-translational glycosylation and premature destruction of JAK2, suggesting that the myeloproliferative neoplasms are disorders of MPL processing since MPL is the only hematopoietic growth factor receptor in hematopoietic stem cells. To examine this possibility, we genetically manipulated MPL expression and maturation in a JAK2V617F transgenic mouse model of polycythemia vera. Elimination of MPL expression completely abrogated the polycythemia vera phenotype in this JAK2V617F transgenic mouse model, which could only be partially restored by expression of one MPL allele. Most importantly, elimination of thrombopoietin gene expression abrogated the polycythemia vera phenotype in this JAK2V617F transgenic mouse model, which could be completely restored by expression of a single thrombopoietin allele. These data indicate that polycythemia vera is in part a thrombopoietin-dependent disorder and that targeting the MPL-thrombopoietin axis could be an effective, nonmyelotoxic therapeutic strategy in this disorder.

C-MPL provides tumor-targeted T-cell receptor-transgenic T cells with costimulation and cytokine signals

AbstractAdoptively transferred T-cell receptor (TCR)-engineered T cells depend on host-derived costimulation and cytokine signals for their full and sustained activation. However, in patients with cancer, both signals are frequently impaired. Hence, we developed a novel strategy that combines both essential signals in 1 transgene by expressing the nonlymphoid hematopoietic growth factor receptor c-MPL (myeloproliferative leukemia), the receptor for thrombopoietin (TPO), in T cells. c-MPL signaling activates pathways shared with conventional costimulatory and cytokine receptor signaling. Thus, we hypothesized that host-derived TPO, present in the tumor microenvironment, or pharmacological c-MPL agonists approved by the US Food and Drug Administration could deliver both signals to c-MPL-engineered TCR-transgenic T cells. We found that c-MPL+ polyclonal T cells expand and proliferate in response to TPO, and persist longer after adoptive transfer in immunodeficient human TPO-transgenic mice. In TCR-transgenic T cells, c-MPL activation enhances antitumor function, T-cell expansion, and cytokine production and preserves a central memory phenotype. c-MPL signaling also enables sequential tumor cell killing, enhances the formation of effective immune synapses, and improves antileukemic activity in vivo in a leukemia xenograft model. We identify the type 1 interferon pathway as a molecular mechanism by which c-MPL mediates immune stimulation in T cells. In conclusion, we present a novel immunotherapeutic strategy using c-MPL-enhanced transgenic T cells responding to either endogenously produced TPO (a microenvironment factor in hematologic malignancies) or c-MPL-targeted pharmacological agents.

Structural Determinants of the Gain-of-Function Phenotype of Human Leukemia-associated Mutant CBL Oncogene

Mutations of the tyrosine kinase-directed ubiquitin ligase CBL cause myeloid leukemias, but the molecular determinants of the dominant leukemogenic activity of mutant CBL oncogenes are unclear. Here, we first define a gain-of-function attribute of the most common leukemia-associated CBL mutant, Y371H, by demonstrating its ability to increase proliferation of hematopoietic stem/progenitor cells (HSPCs) derived from CBL-null and CBL/CBL-B-null mice. Next, we express second-site point/deletion mutants of CBL-Y371H in CBL/CBL-B-null HSPCs or the cytokine-dependent human leukemic cell line TF-1 to show that individual or combined Tyr → Phe mutations of established phosphotyrosine residues (Tyr-700, Tyr-731, and Tyr-774) had little impact on the activity of the CBL-Y371H mutant in HSPCs, and the triple Tyr → Phe mutant was only modestly impaired in TF-1 cells. In contrast, intact tyrosine kinase-binding (TKB) domain and proline-rich region (PRR) were critical in both cell models. PRR deletion reduced the stem cell factor (SCF)-induced hyper-phosphorylation of the CBL-Y371H mutant and the c-KIT receptor and eliminated the sustained p-ERK1/2 and p-AKT induction by SCF. GST fusion protein pulldowns followed by phospho-specific antibody array analysis identified distinct CBL TKB domains or PRR-binding proteins that are phosphorylated in CBL-Y371H-expressing TF-1 cells. Our results support a model of mutant CBL gain-of-function in which mutant CBL proteins effectively compete with the remaining wild type CBL-B and juxtapose TKB domain-associated PTKs with PRR-associated signaling proteins to hyper-activate signaling downstream of hematopoietic growth factor receptors. Elucidation of mutant CBL domains required for leukemogenesis should facilitate targeted therapy approaches for patients with mutant CBL-driven leukemias.

A dimeric peptide with erythropoiesis-stimulating activity uniquely affects erythropoietin receptor ligation and cell surface expression.

Erythropoiesis-stimulating agents (ESAs) that exert long-acting antianemia effects have been developed recently, but their mechanisms are poorly understood. Analyses reveal unique erythropoietin receptor (EPOR)-binding properties for one such ESA, the synthetic EPOR agonist peginesatide. Compared with recombinant human EPO and darbepoietin, peginesatide exhibited a slow on rate, but sustained EPOR residency and resistant displacement. In EPO-dependent human erythroid progenitor UT7epo cells, culture in peginesatide unexpectedly upmodulated endogenous cell surface EPOR levels with parallel increases in full-length EPOR-68K levels. These unique properties are suggested to contribute to the durable activity of this (and perhaps additional) dimeric peptide hematopoietic growth factor receptor agonist.

MPL SV, a Unique MPN MPL Splice Variant, Provokes a Fulminant Myeloid Leukemia in a JAK2 V617F Transgenic Mouse Model of Polycythemia Vera

Introduction: The MPN, polycythemia vera (PV), essential thrombocytosis (ET) and primary myelofibrosis (PMF) are clonal stem cell disorders, which share mutations constitutively activating the physiologic signal transduction pathways for hematopoiesis. Although the MPN have different natural histories, they share in common transformation to myelofibrosis and acute leukemia at differing frequencies and not explainable by a specific mutation. Impaired MPL expression, due to incomplete glycosylation is another common denominator amongst the MPN. Significantly, MPL is the only hematopoietic growth factor receptor expressed in MPN HSC, and we have demonstrated that the PV phenotype in a JAK2 V617F transgenic (V617tg) mouse could be abrogated by elimination of MPL or its ligand, TPO. Impaired MPL expression in the MPN cannot be completely explained by receptor-activated down regulation because not all MPN driver mutations directly activate MPL. However, we have discovered another common denominator in the MPN, variant MPL splicing, eliminating 7 amino acids in the MPL N-terminal domain, a common hotspot for both MPL driver and inactivating mutations, which impairs MPL glycosylation and expression.Methods: To determine the role of the MPL splice variant (MPL SV) in MPN pathophysiology, we cloned the full length MPLSV cDNA and created a transgenic mouse (MPLSV tg) using the same VAV promoter as the V617Ftg mouse to ensure hematopoietic cell-specific transcription. The MPLSV tg mice were produced by pronuclear injection of purified MPLSV cDNA into B6SJLF1 mice. Founders were crossed into a C57Bl/6 background, and then were crossed with V617Ftg mice in either an MPL knockout or wild type background. Mice were phenotyped by blood counts and necropsy with morphologic and immunophenotyping of bone marrow and tumor masses.Results: Amongst 19 founder B6SJLF1 mice, 6 expressed the MPLSV transgene with copy numbers ranging from one to 30; single copy number mice had no hematopoietic phenotype but all mice with higher MPLSV tg copy numbers had thrombocytopenia (median platelet count 500,000/µL; range 200- 600,000; wild type, 750,000/µL; 550-950,000). MPLSV tg mice from several different founders bred into a C57Bl/6 background for 6 generations maintained consistent copy numbers, Mendelian ratios and hematopoietic phenotypes with 100% penetrance and an inverse correlation between MPLSV copy number and the platelet count. The number of double transgenic (V617Ftg/MPLSV tg) offspring observed in the MPLSV tg to V617Ftg crosses were slightly lower than expected (20% vs 25%), indicating reduced embryo viability. Four week old V617Ftg/MPLSV tg mice displayed tumorous abnormalities of the head and hips as well as small size and failure to thrive (median weight 8.5 gms; range 8-9; wild type, 13 gms; 10-14), and enlarged spleens (median weight 0.29 gms; range 0.15-0.64; wild type 0.086 gms; 0.05-0.10). Histologically, the V617Ftg/MPLSV tg-associated head and hip abnormalities represented monomorphic myeloid sarcomas extending through the calvarium both extra and intracranially and from the femur into surrounding muscle. Both the marrow and spleen were diffusely infiltrated by large blasts with abundant basophilic cytoplasm, expressing CD34, CD61 and CD117 but lacking CD127, consistent with a myeloid origin. All V617Ftg/MPLSV tg mice either died or required humane sacrifice by 6 weeks. The extramedullary tumor was transplantable in secondary recipients, and flow cytometry-based phenotyping showed that the tumors (both primary and secondary) were CD34, CD117, CD61 and CD71- positive. Penetrance of the leukemia phenotype was 100% in V617Ftg/MPLSV tg from all founder lines with multiple copies of the MPLSV tg, whether in an MPL knockout or wild type background. The leukemia phenotype was never observed with the MPLSV tg alone or with V617Ftg alone despite observation of over 300 V617Ftg mice up to 50 weeks of age.Conclusion: We identified an MPL SV in human MPN that was functional in mice with a dominant-negative effect with respect to platelet production and at the same time synergized with JAK2 V617F to create a fulminant myeloid malignancy. We have recently shown that knockout of MPL or TPO alone abrogates the PV phenotype in the V617Ftg mouse, whereas the MPLSV uniquely drives a highly penetrant and fulminant leukemia, establishing MPL and TPO as targets for mitigation of malignant transformation in the MPN. DisclosuresSpivak:Incyte: Membership on an entity's Board of Directors or advisory committees. Moliterno:incyte: Membership on an entity's Board of Directors or advisory committees.

Establishment of a cost-effective method to detect FLT-ITD and D835 mutations in acute myeloid leukemia patients in the Taiwanese population

ObjectiveThe FMS-related tyrosine kinase 3 (FLT-3) gene is a hematopoietic growth factor receptor gene, an independent negative prognostic factor, which affects the proliferation and differentiation of stem cells or hematopoietic progenitor cells. Patients with FLT-3 gene mutations have a worse prognosis and responsiveness to chemotherapy than those without these mutations. Our study aims to establish a conventional detection method for FLT3-ITD and D835 mutations in patients with acute myeloid leukemia (AML). Materials and methodsIn this study, we recruited 100 patients with AML. Primers were designed to distinguish between wild-type FLT-3, FLT3-ITD, and D835 variants. Methods using a polymerase chain reaction (PCR)-Agilent 2100 Bioanalyzer, PCR-ABI PRISM 3100 Genetic Analyzer, and PCR–agarose gel electrophoresis were compared. ResultsA high-accuracy, easily operated, low-cost technique to detect the FLT-3 variation with 99.9% specificity was established in this study. The PCR platform, the Agilent 2100 Bioanalyzer (plus DNA 1000 LabChip kit) chip analysis platform, and the ABI PRISM 3100 Genetic Analyzer (plus GeneScan-500 size standard) short tandem repeat (STR) fluorescence analysis platform were used in different experimental comparisons. The ABI PRISM 3100 Genetic Analyzer (plus GeneScan-500 size standard) STR fluorescence analysis platform was the most suitable method to detect FLT3 variants. This method has a high degree of sensitivity, accuracy, and a specificity of 99.9%. ConclusionAML with the homozygous mutated FLT-3 may have a worse cure rate than AML with heterozygous mutation. This mutation is not related to drug resistance, but is a factor in a high risk of relapse; it is also related to unfavorable overall survival. Our designed detection methods should provide key information to develop personalized medicine for AML patients.

“RHEX-26”, a Novel EPO/EPOR-Regulated Mediator of Human Erythroid Progenitor Cell Expansion

Abstract 682Erythropoietin (EPO) and its receptor (EPOR) serve well as an informative paradigm for understanding hematopoietic growth factor effects and receptor action mechanisms. Recently, interest in EPO/EPOR investigations has been heightened based on the clinical emergence of novel EPO mimetics, and by reported EPO/EPOR activities in modulating cellular immunity, diabetic susceptibility, retinopathy and the cytoprotection of select non-hematopoietic tissues. To better understand EPOR action modes, we presently have applied a global phosphoproteomics approach to specifically discover new targets of EPO/EPOR-regulated tyrosine phosphorylation. Using an EPO-dependent erythroid progenitor cell model, our LC-MS/MS approach was first validated for known EPO/EPOR-targets (e.g., JAK2, STATs, SHIPs, PLCgamma, GABs). Second, among certain known targets, new phosphorylation sites were identified. Third, several novel major downstream targets of EPO/EPOR were discovered. One (a previously uncharacterized open reading frame here designated as “RHEX26”) was induced ∼20-fold in tyrosine-phosphorylation by EPO treatment. Quantitative RT-PCR analyses of tissues and primary hematopoietic cells indicate RHEX26 expression to be erythroid- restricted. Antibodies prepared to RHEX26 and tyrosine phosphorylated-RHEX26 confirmed strong EPO-induced phosphorylation; cell fractionation and Western blot studies demonstrated membrane residence of this 26 kDa phosphoprotein; and immunofluorescence microscopy showed sharp RHEX26 sub-localization as an integral plasma membrane protein. In functional analyses, lentiviral shRNA knockdown of RHEX26 proved to markedly inhibit the EPO-dependent expansion of erythroid progenitor cells (clonal colony-forming analyses demonstrated >80% specific inhibition), while effects on cell viability were limited. Mechanistically, RHEX26 is coupled (in part) to GRB2, with co-immunoprecipitation observed specifically upon EPO exposure, but without dependency on RHEX26 tyrosine phosphorylation. Finally, genealogy analyses intriguingly indicate RHEX26 to be well-conserved only in H. sapiens and primates, while no ortholog is present in the rat, mouse, or zebrafish. RHEX26, as a novel EPO/EPOR 26 kDa target, therefore is proposed to comprise an important new Regulator of Human Erythroid eXpansion. Disclosures:Leu:Affymax, Inc.: Employment, own stock in Affymax, Inc. Young:Affymax, Inc.: Employment, own stock in Affymax, Inc. Schatz:Affymax, Inc.: Employment, own stock in Affymax, Inc. Green:Affymax, Inc.: Employment, own stock in Affymax, Inc.

Differential Regulation of MicroRNA Expression In Polycythemia Vera CD34+ Cells

AbstractAbstract 4785 Introduction:Polycythemia vera (PV) is a clonal stem cell disorder characterized by unregulated red cell, white cell and platelet production, a phenotype attributable to an activating point mutation (V617F) in JAK2, a tyrosine kinase utilized for signaling by Type 1 hematopoietic growth factor receptors. However, it is generally conceded that JAK2 V617F is not the initiating mutation in PV, nor are any of the other gene mutations, deletions and amplifications discovered to date. Deregulated gene transcription is also a feature of PV but a unified mechanism for this also cannot be ascribed to the currently known genetic defects. MicroRNAs (miRs) are non-coding 18–22nt RNAs that function as posttranscriptional gene repressors by binding to complementary target sequences in the mRNA 3′UTR to inhibit protein translation. There is growing evidence that miRs regulate hematopoiesis not only at the committed hematopoietic progenitor cell level but also at the level of the hematopoietic stem cell (HSC). Studies of miR behavior in PV have documented differential expression during erythroid differentiation as well as differential expression in granulocytes and platelets. However, PV is a stem cell disorder and to date there has been no analysis of miR behavior in PV HSC. We report here differential miR expression in PV peripheral blood (pb) CD34+ cells. Methods:We studied pb CD34+ cells from eight PV patients (four females and four males, all JAK2 V617F-positive) and six normal donors (three females and three males) in conjunction with a study of mRNA expression in PV pb CD34+ cells by oligonucleotide microarray. Using total cell RNA, microRNA expression was analyzed with the Agilent Human microRNA Microarray Kit Version 3, which contains probes for 866 human miRs. The TaqMan MicroRNA Assay (Applied Biosystems) was used for quantitative reverse-transcription PCR to verify differential miR expression. The Student unpaired t test was used for statistical analysis and P values £ 0.05 were considered significant. Results:Overall, in the 8 PV patients as a group, there were 130 positive calls with differential regulation of 81 miRs (p <0.05) (23 up regulated and 58 down regulated) and there was no correlation between miR expression and the JAK2 V617F allele burden. Given the phenotypic and genotypic differences between men and women PV patients, we analyzed differential miR expression separately by gender to identify gender-specific effects. 75 miRs (26 up and 49 down regulated) were differentially expressed in the male PV patients and 32 (7 up and 25 down regulated) in the women, confirming similar observations of gender-related differences in global mRNA expression in PV pb CD34+ cells. Amongst the differentially regulated miRs in the group as a whole were the miR-H1 group, miR-125a-3p and 196b. The miR-H1 group, also known as miR-17-92, is an oncomir with a unique gene structure from which one primary transcript yields six distinct miRs. This cluster was only up regulated in male PV patients. Likewise, miR-125a-3p was also up regulated only in males. Its expression is dependent on DICER, a gene also only up regulated in male patients. MiR-125a-3p has been identified as being preferentially expressed in HSC, acts to expand HSCs by preventing apoptosis and favors a myeloid fate. MiR-196b, which was concordantly down regulated in both men and women, is normally up regulated in HSC. Altogether, consistent with their different phenotypes, 31 miRs were differently regulated solely in the male patients (16 up regulated and 15 down), while only 12 miRs were differently regulated solely in the women (2 up and 10 down regulated) Seven miRs were concordantly up (miR-575) or down regulated (miR-150; miR-181c; miR-196b; miR-551b; miR-769-5p and miR-874) by both the men and women patients. Notably, down regulation of miR-150 at the HSC level is consistent with its role in lineage-specific progenitor cell commitment. Since there appears to be little overlap with respect to target genes for this core group of miRs, multiple venues for translational control are likely to be involved in the pathogenesis of PV. Conclusion:These data establish the presence of differential miR expression in PV HSC and provide insight into potential mechanisms for PV HSC expansion, confirm a genetic basis for the gender-specific differences that characterize PV and identify novel therapeutic targets for drug discovery. Disclosures:No relevant conflicts of interest to declare.

Most purported antibodies to the human granulocyte colony-stimulating factor receptor are not specific

Antibodies to human granulocyte colony-stimulating factor receptor (HuG-CSFR) are widely available and have been used in numerous studies to evaluate the expression of this protein on normal and malignant cells of hematopoietic and nonhematopoietic origin. Spurred by recent studies that demonstrated that two commonly used antibodies against the erythropoietin and thrombopoietin receptors can in fact bind to completely unrelated and more broadly expressed proteins, we screened 27 commercially available monoclonal and polyclonal antibodies with claimed specificity to HuG-CSFR to determine if they are specific to this receptor. Antibodies were evaluated by Western blotting, flow cytometry, and immunohistochemistry using 293T cells engineered to overexpress HuG-CSFR protein, immortalized human hematopoietic cell lines expressing endogenous G-CSFR, and purified human neutrophils. Only two monoclonal antibodies and one polyclonal antibody could be employed using defined Western blotting or flow cytometry protocols to detect G-CSFR protein in cell lysates or on the surface of cells that express G-CSFR messenger RNA with no binding to cells that did not express the gene. None of the antibodies were suitable for immunohistochemistry. Competitive inhibition with soluble G-CSFR extracellular domain and small inhibitory RNA-mediated knock-down of G-CSFR messenger RNA further demonstrated the limited specificity of these antibodies for HuG-CSFR expressed on the cell surface. Most commercially available anti-HuG-CSFR antibodies do not bind specifically to this protein. These studies highlight the need for investigators to validate antibodies in their own systems to avoid the inadvertent use of nonspecifically binding antibodies that could lead, as exemplified in this case with a hematopoietic growth factor receptor, to erroneous conclusions about protein expression.

The p110α Catalytic Isoform of PI3 Kinase Is Important for Erythropoiesis, but Has a Minimal Role in Hematopoietic Stem Cell Self-Renewal.

Abstract 3620Poster Board III-556PIK3CA, which encodes the p110α catalytic isoform of PI3 kinase (PI3K), is mutated in many human cancers, and is an attractive therapeutic target. However, PI3K may also be important during hematopoiesis, as it is activated by hematopoietic growth factor receptors which control hematopoietic stem cell (HSC) and progenitor proliferation, differentiation, and self-renewal, such as erythropoietin receptor (epoR), c-kit receptor, and fms-like tyrosine kinase 3 (FLT3). In hematopoietic cells, receptor tyrosine kinases signal through the catalytic p110 subunit of PI3K, which has 3 isoforms (α, β, δ). However, the roles of PI3K and its specific catalytic isoforms in normal HSC function are poorly understood. We hypothesized that signaling through the p110α isoform is important for hematopoiesis and HSC self-renewal. We have used the Cre-loxP system to delete p110α in the HSCs of adult mice by breeding p110αF/F mice to Mx1-Cre transgenic mice. p110αF/F;Mx1-Cre+ (Cre+) mice and their p110αF/F (Cre-) littermates were injected with PolyI-PolyC (pIpC) at 4-6 weeks of age to induce Cre-mediated excision at the PIK3CA locus specifically in hematopoietic cells. Deletion of p110α in the bone marrow (BM) was verified by PCR and by immunoblot. We observed that, by four weeks after pIpC treatment, Cre+ mice developed microcytic anemia compared with Cre- littermates, characterized by a decreased mean hemoglobin (p<0.0001) and decreased mean corpuscular volume, while white blood cell counts and platelet counts were unaffected. Cre+ mice also had significantly decreased spleen, liver, and thymus weights. Flow cytometry analyses of bone marrow and spleen cells revealed a relative block in erythropoiesis in the spleens of Cre+ mice, with expansion of the basophilic erythroblast population, and a decrease in the most mature nucleated erythroblast population. Colony assays of splenocytes in erythropoietin-containing methylcellulose medium revealed a 52% decrease in BFU-E colony formation by p110α-deleted cells in response to erythropoietin, suggesting that loss of p110α may lead to blunted epoR signaling (p=0.009). Multiparameter flow cytometry revealed that the overall number of Lin-Sca1+ckit+ (LSK) cells, which contains the HSC population, was increased two-fold in the BM of Cre+ mice compared with Cre- littermates (p=0.01). To determine whether loss of p110α affects long-term HSC self-renewal in vivo, we performed competitive repopulation assays, in which CD45.2+ BM cells from PIPC-treated Cre+ mice or Cre- controls were transplanted together with CD45.1+CD45.2+ competitor BM cells into lethally irradiated CD45.1+ recipient mice. Donor BM chimerism (%CD45.2+ cells) at 16 weeks was mildly reduced in the absence of p110α, but Cre+ cells were still capable of long-term reconstitution. In summary, we have found that the p110α catalytic isoform is specifically required for erythropoiesis, but has only a small role in HSC homeostasis and in differentiation of the other hematopoietic lineages. This suggests that pharmacologic targeting of p110α in cancer therapy may result in mild anemia, but should otherwise have minimal hematologic toxicity. Disclosures:Gilliland:Merck Research Laboratories: Employment. Roberts:Novartis Pharmaceuticals, Inc.: Consultancy. Zhao:Novartis Pharmaceuticals, Inc.: Consultancy.

The potential of hematopoietic growth factors for treatment of Alzheimer's disease: a mini-review

There are no effective interventions that significantly forestall or reverse neurodegeneration and cognitive decline in Alzheimer's disease. In the past decade, the generation of new neurons has been recognized to continue throughout adult life in the brain's neurogenic zones. A major challenge has been to find ways to harness the potential of the brain's own neural stem cells to repair or replace injured and dying neurons. The administration of hematopoietic growth factors or cytokines has been shown to promote brain repair by a number of mechanisms, including increased neurogenesis, anti-apoptosis and increased mobilization of bone marrow-derived microglia into brain. In this light, cytokine treatments may provide a new therapeutic approach for many brain disorders, including neurodegenerative diseases like Alzheimer's disease. In addition, neuronal hematopoietic growth factor receptors provide novel targets for the discovery of peptide-mimetic drugs that can forestall or reverse the pathological progression of Alzheimer's disease.

Ectopic Expression of the Extracellular Domain of Mpl Is Sufficient to Induce a Hematopoietic Population Crisis

The thrombopoietin receptor Mpl is required for regeneration of hematopoietic stem cells and governs megakaryocytic differentiation. Patients with inherited MPL deficiency suffer from severe thrombocytopenia which progresses to aplastic anemia, a disorder called congenital amegakaryocytic thrombocytopenia (CAMT). As a first step towards a potential gene therapy for MPL deficiency, we retrovirally expressed the receptor in a murine bone marrow transplantation model. An initial series of vectors used a strong enhancer-promoter derived from the spleen-focus forming virus (SFFV). Mice transplanted with hematopoietic cells modified by these constructs developed a profound yet transient elevation of multi-lineage hematopoiesis due to increased signaling of the Thpo receptor on target and non-target cells. Mice developed increased leukocyte, erythrocyte and platelet counts (2–3 times elevated) compared to GFP control animals. Histopathology revealed an elevated number of mature megakaryocytes with atypical features like numerous giant forms in the bone marrow and the spleen and atypical neoformation of bone in the spleen leading to the diagnosis of chronic myeloproliferative disorder (CMPD). A minority of mice (3/27) developed erythroleukemia with almost 100% CD71 and TER119 double positive cells as detected by flow cytometry. Histopathology presented infiltration of erythroblasts in all hematopoietic tissues like the BM, spleen and liver. Molecular analysis revealed retroviral vector insertions in sfpi1, fli1 and klf3 that seem to be the major driving force for the development of leukemia in these animals. Somewhat unexpectedly, in the majority of mice the CMPD converted into a progressive, potentially lethal pancytopenia. Animals had severely reduced blood cell counts with only 50% of leukocyte, 20% of erythrocyte and 10% of platelet counts compared to GFP control animals. This population crisis affected all major blood lineages and also involved co-existing unmodified hematopoiesis. Histopathology presented a dysmegakaryopoiesis with an increased number of atypical micro-megakaryocytes, histiocytes with erythrocytophagocytosis and atypical mast cell proliferation diagnosed as a myelodysplastic syndrome (MDS)-like disorder. In the bone marrow, pancytopenic mice had reduced cell numbers of the primitive cell fraction (LSK cells). To address the mechanism of pancytopenia, we expressed a dominant negative form of Mpl (dnMpl) consisting of the extracellular and transmembrane domain and lacking the intracellular signal transduction domain. Animals transplanted with dnMpl-modified cells failed to show the initial CMPD but developed the same pancytopenic, MDS-like end stage. A vector expressing Mpl under control of the PGK promoter or a fragment of the Mpl-promoter reduced or completely avoided the side effects (CMPD, MDS-like disorder) observed with vectors using stronger promoters. The induction of a hematopoietic population homeostasis thus depends upon Mpl expression levels, indicating the need for strictly regulated transgene expression in gene therapy for MPL deficiency. As ectopic expression of the extracellular domain is sufficient to cause MDS, sequestration of crucial niche factors like Thpo may contribute to the pathogenesis of this disorder. This study demonstrates that ectopic expression of a hematopoietic growth factor receptor may disturb organ homeostasis through interference with intra- and extracellular mechanisms of cell communication.

Transplanted CD34+ Cells Stimulated with Flt3 Ligand and Granulocyte-Colony Stimulating Factor Display An Impaired Early Reconstitution Capacity

Flt3/flk2 belongs to the class III hematopoietic growth factor receptors with intrinsic kinase activity, ligand-activated tyrosine phosphorylation and a central role in early stages of hematopoiesis. Flt3-ligand (FL) has been shown to enhance the effects of several growth factors, including Granulocyte-colony stimulating factor (G-CSF). In this study the reconstitution ability of FL+G-CSF primed CD34+ cells was evaluated in a nonhuman primate model of radiation-induced myeloablative treatment followed by transplantation of stimulated autologous bone marrow (BM) and/or peripheral blood (PB) cells. Rhesus monkeys (n=9) were subjected to subcutaneous injections with FL (100 μg/kg/day) and G-CSF (100 μg/kg/day) for 7 to 14 days and subsequently irradiated with a lethal dose of total body irradiation (TBI) followed by transplantation with primed stem cells or with a midlethal dose of TBI (n=2) without transplantation. White blood cell counts (WBC) in PB showed a steep increase and reached a mean level of 98.9×109/L (71.5–131.7×109/L) after 7 days (P<0.01). BM cellularity increased from 35.2×109/L before treatment to 193.8×109/L after priming (95.0–348.2×109/L) (P<0.01). CD34+ cells displayed a 1-log increase (P<0.01) in BM and a 2-log increase in PB (P<0.01). Both total nucleated and CD34+ cell numbers reached after 7 days a steady state level, which was maintained until cessation of growth factor treatment. Treatment with FL+G-CSF resulted in a moderate increase (2-fold) of CFU-GM in BM after 7 days (P<0.05) but did not affect BFU-E, consistent with the predilection of FL for myeloid and lymphoid cells, in contrast to SCF, which predominantly influences the erythroid and thrombopoietic lineage. The effect of treatment with FL+G-CSF on WBC was significantly greater than treatment with either G-CSF at day 7 (P<0.05) or FL at day 7 and 14 (P<0.01). Lethally irradiated monkeys, transplanted with FL+G-CSF stimulated cells displayed a much slower regeneration of WBC, platelets and reticulocytes relative to the cell number transplanted and were consequently in need of prolonged antimicrobial prophylaxis and transfusions. Furthermore, these monkeys displayed regeneration patterns consistent with a 1-log depletion of repopulating cells relative to non-stimulated BM cells. Mid-lethal irradiated FL+G-CSF treated monkeys were not transplanted to study the intrinsic regeneration capacity of primed BM. These monkeys displayed normal regeneration of reticulocytes, platelets and WBC. Thus, stimulation with FL+G-CSF leads to a steep and synergistic increase of nucleated and CD34+ cells in both BM and PB. Priming with FL+G-CSF does not affect the intrinsic regeneration capacity of BM after radiation; but when transplanted, primed CD34+ cells do not per se contribute to accelerated short-term engraftment after transplantation, rather the reconstitution patterns were consistent with on average one order of magnitude stem cell depletion relative to the numbers of CD34+ cells transplanted.

YRRL Motifs in the Cytoplasmic Domain of the Thrombopoietin Receptor Mpl Regulate Receptor Internalization and Degradation.

Thrombopoietin (TPO), acting through its receptor Mpl, promotes survival and proliferation of hematopoietic progenitor cells and also drives megakaryocyte differentiation. The pro-proliferation and survival signals activated by TPO must therefore be tightly regulated to prevent uncontrolled cell growth. Several mechanisms to down-regulate hematopoietic growth factor signaling have been identified, including increased expression of suppressors of cytokine signaling (SOCS) proteins, activation of protein phosphatases, and endocytosis and degradation of activated growth factor receptors. In this work we determined the mechanisms that control TPO-stimulated Mpl internalization and defined the processes leading to Mpl degradation using IL-3-dependent BaF3 cells engineered to express wild type (WT) and mutant forms of human Mpl. Stimulation of BaF-Mpl cells with TPO lead to rapid endocytosis of Mpl which was blocked by pre-treatment with the clathrin-mediated endocytosis inhibitor monodansylcadaverine, indicating that Mpl internalization is clathrin-mediated. Additionally, we found that inhibition of Janus kinase 2 (Jak2) and Src-family kinases greatly reduced Mpl internalization. Sites of clathrin assembly on the plasma membrane contain the adaptor protein complex AP2, which associates with transmembrane proteins enabling targeted endocytosis. Association of AP2 and transmembrane proteins relies on an interaction between AP2μ2 subunit and internalization motifs YXXΦ (where X=any amino acid and Φ=bulky hydrophobic) expressed by target proteins. Mpl contains two YXXΦ motifs, at cytoplasmic Y8 (Y8RRL) and Y78 (Y78RRL). BaF-Mpl cells transfected with siRNA targeted to AP2 displayed greatly reduced TPO-mediated internalization of Mpl, demonstrating that AP2-Mpl association is critical for Mpl endocytosis. Next, we introduced Y to F point mutations at Mpl cytoplasmic Y8 and Y78 individually, and in combination (Y8+78F). Internalization was greatly reduced in Mpl Y78F and Mpl Y8+78F after TPO stimulation, whereas Mpl Y8F internalization was only slightly attenuated. Furthermore, we found that Mpl Y78F and Y8+78F exhibited increased proliferation and increased strength and duration of activated Jak2 and AKT in response to TPO, compared to Mpl WT and Y8F. In addition to mediating endocytosis, cytoplasmic YXXΦ motifs located between 6 and 9 residues from the transmembrane domain have been shown to mediate lysosomal targeting of proteins following endocytosis. Consequently, we studied the role of Mpl Y8RRL motif in Mpl lysosome targeting. BaF-Mpl WT and Y8F cells were pretreated with cyclohexamide and stimulated with TPO for up to 2 hours. Mpl Y8F displayed greatly reduced Mpl degradation in response to TPO compared to WT. Furthermore, Mpl Y8F recycled back to the plasma membrane following TPO starvation quicker than WT Mpl, suggesting that the Mpl Y8F remains in endosomes, rather than being targeted to lysosomes. Our data shows that Mpl cytoplasmic YRRL motifs are responsible for both TPO-mediated internalization via interactions with AP2 and lysosomal targeting following endocytosis. These findings significantly advance our understanding of normal Mpl function. Further study of internalization motifs, which are present in receptors for other hematopoietic growth factors including; erythropoietin, granulocyte colony stimulating factor, leukemia inhibitory factor and interleukin, may highlight the importance of these sequences in mediating hematopoiesis and potentially aid identification of novel targets in hematological disease.