Combinatorial effect of miR-486-5p & miR-22-3p mimics thrombopoietin's impact on hematopoietic stem & progenitor cells

Megakaryocytes shed and release submicron size microparticles (MkMPs), the most abundant microparticle in circulation. We have previously reported that MkMPs target peripheral-blood CD34+ hematopoietic stem/progenitor cells (HSPCs) to induce megakaryocytic differentiation and proliferation, and that small RNAs delivered to HSPCs via MkMPs play an important role in the development of this phenotype. Here, using single-molecule real-time (SMRT) RNA sequencing (RNAseq), we identify the top seven most abundant microRNAs (miRs) in MkMPs as potential candidates in mediating the effect of MkMPs on HSPCs. Using miR mimics, we demonstrate that among the seven most abundant miRs, two, miR-486-5p and miR-22-3p, are able to drive the Mk differentiation of HSPCs in the absence of thrombopoietin (TPO). The effect of these two miRs is comparable to the TPO- or MkMP-induced megakaryocytic differentiation of HSPCs, thus suggesting that these two miRs are responsible for this MkMP-induced phenotype. To probe the signaling through which MkMPs might enable this phenotype, we used kinase inhibitors of potential signaling pathways engaged in megakaryocytic differentiation. Our data suggest that MkMP-induced Mk differentiation of HSPCs is enabled through JNK and PI3K/Akt/mTOR signaling. Our data show that MkMPs activate Akt and mTOR phosphorylation. Furthermore, MkMPs downregulate PTEN expression, a direct target of miR-486-5p and a negative regulator of PI3K/Akt signaling, via JNK signaling. Taken together, our data provide a mechanistic understanding of the biological effect of MkMPs in inducing megakaryocytic differentiation of HSPCs, which, as was previously suggested, is a phenotype of potential physiological significance in stress megakaryopoiesis.

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ZNF16 (HZF1) promotes erythropoiesis and megakaryocytopoiesis via regulation of the c-KIT gene

Biochemical Journal ◽

10.1042/bj20130628 ◽

2014 ◽

Vol 458 (1) ◽

pp. 171-183 ◽

Cited By ~ 3

Author(s):

Jing Chen ◽

Xiao-Bo Li ◽

Rui Su ◽

Li Song ◽

Fang Wang ◽

...

Keyword(s):

Progenitor Cells ◽

Target Gene ◽

Megakaryocytic Differentiation ◽

Kit Gene ◽

Direct Target Gene ◽

Direct Target

The present study demonstrated that ZNF16 (HZF1) plays an important role in erythroid and megakaryocytic differentiation of human haematopoietic stem/progenitor cells, identified and validated c-KIT as a direct target gene of ZNF16, and demonstrated that ZNF16 functions via its regulation on the c-Kit/c-Raf/MEK/ERK/c-Jun/HEY1/GATA1 cascade.

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Combined Drug Targeting of p53-dependent and -independent Pathways Depletes Myelofibrosis Hematopoietic Stem/Progenitor Cells

Leukemia ◽

10.1038/s41375-021-01446-4 ◽

2021 ◽

Author(s):

Min Lu ◽

Lijuan Xia ◽

Nada Elmansy ◽

Cara Clementelli ◽

Douglas Tremblay ◽

...

Keyword(s):

Progenitor Cells ◽

Drug Targeting ◽

Patient Survival ◽

Death Receptor ◽

Negative Regulator ◽

Current Therapy ◽

Wild Type ◽

Hematopoietic Stem ◽

The Individual ◽

Trail Death Receptor

AbstractCurrent therapy for myelofibrosis (MF) results in a limited prolongation of patient survival. In order to improve treatment outcomes, we developed a strategy to effectively deplete MF hematopoietic stem/progenitor cells (HSPCs). In the present study, an imipridone, ONC201, was combined with RG7112, an antagonist of MDM2, a p53 negative regulator, to activate downstream events of the p53 and TNF-related apoptosis-inducing ligand (TRAIL)/death receptor (DR) pathways. As compared to treatment with the individual drugs, the combination of ONC201 and RG7112 promoted greater degrees of apoptosis of MF CD34+ cells through activation of both p53-dependent and -independent pathways. Importantly, treatment with ONC201-RG7112 not only decreased the number of JAK2V617F+ and calreticulin mutated colonies assayed from MF CD34+ cells, but allowed for the persistence or appearance of JAK2 wild type colonies. Treatment with ONC201 combined with RG7112 could be a potentially effective strategy for treating MF patients.

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Combination of tyrosine kinase inhibitors and the MCL1 inhibitor S63845 exerts synergistic antitumorigenic effects on CML cells

Cell Death and Disease ◽

10.1038/s41419-021-04154-0 ◽

2021 ◽

Vol 12 (10) ◽

Author(s):

Alena Malyukova ◽

Dorina Ujvari ◽

Elham Yektaei-Karin ◽

Ana Zovko ◽

Harsha S. Madapura ◽

...

Keyword(s):

Tyrosine Kinase ◽

Progenitor Cells ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Residual Disease ◽

Chronic Phase ◽

Myeloid Cell ◽

Hematologic Malignancies ◽

Hematopoietic Stem ◽

Tki Treatment

AbstractTyrosine kinase inhibitor (TKI) treatment has dramatically improved the survival of chronic myeloid leukemia (CML) patients, but measurable residual disease typically persists. To more effectively eradicate leukemia cells, simultaneous targeting of BCR-ABL1 and additional CML-related survival proteins has been proposed. Notably, several highly specific myeloid cell leukemia 1 (MCL1) inhibitors have recently entered clinical trials for various hematologic malignancies, although not for CML, reflecting the insensitivity of CML cell lines to single MCL1 inhibition. Here, we show that combining TKI (imatinib, nilotinib, dasatinib, or asciminib) treatment with the small-molecule MCL1 inhibitor S63845 exerted strong synergistic antiviability and proapoptotic effects on CML lines and CD34+ stem/progenitor cells isolated from untreated CML patients in chronic phase. Using wild-type BCR-ABL1-harboring CML lines and their T315I-mutated sublines (generated by CRISPR/Cas9-mediated homologous recombination), we prove that the synergistic proapoptotic effect of the drug combination depended on TKI-mediated BCR-ABL1 inhibition, but not on TKI-related off-target mechanisms. Moreover, we demonstrate that colony formation of CML but not normal hematopoietic stem/progenitor cells became markedly reduced upon combination treatment compared to imatinib monotherapy. Our results suggest that dual targeting of MCL1 and BCR-ABL1 activity may efficiently eradicate residual CML cells without affecting normal hematopoietic stem/progenitors.

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CD1d expression on and regulation of murine hematopoietic stem and progenitor cells

Blood ◽

10.1182/blood-2012-01-404012 ◽

2012 ◽

Vol 119 (24) ◽

pp. 5731-5741 ◽

Cited By ~ 5

Author(s):

Hal E. Broxmeyer ◽

Kent Christopherson ◽

Giao Hangoc ◽

Scott Cooper ◽

Charlie Mantel ◽

...

Keyword(s):

Progenitor Cells ◽

Colony Formation ◽

Immune Cell ◽

Cytoplasmic Tail ◽

Negative Regulator ◽

Hematopoietic Stem ◽

Synergistic Enhancement ◽

Stem And Progenitor Cells ◽

Different Strains

Abstract In the present study, surface CD1d, which is involved in immune cell interactions, was assessed for effects on hematopoiesis. Mouse BM hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) express CD1d. The numbers and cycling status of HPCs in the BM and spleen of different strains of cd1d−/− mice were enhanced significantly, suggesting that CD1d is a negative regulator of HPCs. In support of this, CD1d was required for the SCF and Flt3 ligand synergistic enhancement of CSF induction of HPC colony formation and for HPC response to myelosuppressive chemokines. Colony formation by immature subsets of HPCs was greatly enhanced when normal, but not cd1d−/−, BM cells were pretreated with CD1d Abs in vitro. These effects required the full CD1d cytoplasmic tail. In contrast, long-term, but not short-term, repopulating HSC engraftment was impaired significantly, an effect that was minimally influenced by the presence of a truncated CD1d cytoplasmic tail. Pretreatment of normal BM cells with CD1d Abs greatly enhanced their engraftment of HSCs. The results of the present study implicate CD1d in a previously unrecognized regulatory role of normal and stressed hematopoiesis.

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HOXA10 Expression Induces by Abl Kinase Inhibitors Enhanced Apoptosis through PI3K Pathway in CML Cells.

Blood ◽

10.1182/blood.v110.11.4529.4529 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4529-4529

Author(s):

Isao Hirano ◽

Yuya Sugimoto ◽

Keiji Okinaka ◽

Takaaki Ono ◽

Kaori Shinjo ◽

...

Keyword(s):

Progenitor Cells ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Pi3k Pathway ◽

Hematopoietic Progenitor ◽

Leukemic Transformation ◽

Hematopoietic Stem ◽

Abl Kinase ◽

Abl Kinase Inhibitors

Abstract [Background] Homeobox (Hox) genes are grouped together in 4 clusters, A to D. Recent studies has shown that the Hox proteins are important in the control of differentiation and proliferation in hematopoietic cells. We found that the Abl kinase inhibitors increased the expression of HoxA10 gene in CML cells. In this study, we analyzed the role of HoxA10 in CML cell lines and the hematopoietic progenitor cells derived from CML patients by inhibiting the expression of HoxA10. Moreover, we investigated whether the regulation of HoxA10 eradicate Ph+ hematopoietic stem/progenitor cells, which are the targets for leukemic transformation in CML. [Methods] We used AMN107 and BMS354825 for the Abl kinase inhibitors, LY294002 for a PI3K inhibitor, PP2 for a Src kinase inhibitor, and SB203580 for a p38 MAP kinase inhibitor. For analysis of HoxA10 mRNA and protein, RT-PCR and western blot were performed in K562, Meg-01 and U937 cells, which untreated or treated with AMN107, BMS354825, LY294002, PP2, or SB203580 respectively. We then attempted to localize the intracellular locations of HoxA10 in K562 and Meg01, which untreated or treated with AMN107, BMS354825, or LY294002 by using conforcal fluorescence microscopy. For analysis of proliferation in K562, Meg-01 and U937 transfected with siRNA HoxA10, MTT assays were performed in untransfected or transfected K562, Meg-01 and U937 treated with or without AMN107, BMS354825, or LY294002. Finally, we counted the colony numbers of CFU-GEMM, CFU-GM, and BFU-E in K562 and Meg-01 treated with the Abl kinase inhibitors or LY294002. Results Both K562 and Meg01 cells expressed HoxA10 mRNA and protein at lower level compared to U937 cells. Interestingly, treatment with AMN107, BMS354825, or LY294002 increased the expression of HoxA10 mRNA and protein in both K562 and Meg01 cells. The fluorescence of HoxA10 was more strongly observed in the area corresponding to the cell’s cytoplasm than nucleus, and the treatment with AMN107, BMS354825, or LY294002 increased the fluorescence in nucleus of K562 and Meg01 cells in a time-dependent manner. In K562 and Meg01 cells transfected with the siRNA HoxA10, treatment with AMN107 or BMS354825 slightly inhibited the proliferation compared to K562 and Meg01 transfected with control siRNA. Finally, we showed that the inhibition of HoxA10 expression by siRNA increased the numbers of CFU-GEMM, BFU-E, and CFU-GM when the cells were treated with the combination of BMS354825 and LY294002 compared to control cells. [Conclusions] In this study, we showed that the Abl kinase inhibitors induced the expression of HoxA10, and HoxA10 was regulated by PI3K pathway in CML cells. This finding indicates a new insight in the regulation of cell proliferation via the PI3K signal pathway in CML cells. Moreover, we found the role of HoxA10 in CML cell lines and the hematopoietic progenitor cells derived from CML patients by inhibiting the expression of HoxA10. We showed that the regulation of HoxA10 eradicated Bcr-Abl+ hematopoietic stem/progenitor cells, which are the targets for leukemic transformation in CML.

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LNK (SH2B3) Inhibition Expands Healthy and Fanconi Anemia Human Hematopoietic Stem and Progenitor Cells

Blood Advances ◽

10.1182/bloodadvances.2021004205 ◽

2021 ◽

Author(s):

Nicholas Holdreith ◽

Grace Y Lee ◽

Vemika Chandra ◽

Carlo Salas Salinas ◽

Peter Nicholas ◽

...

Keyword(s):

Gene Therapy ◽

Bone Marrow ◽

Progenitor Cells ◽

Fanconi Anemia ◽

Adaptor Protein ◽

Negative Regulator ◽

Hematopoietic Stem ◽

Monogenic Diseases ◽

Stem And Progenitor Cells ◽

Human Hscs

Hematopoietic stem cell transplantation (HSCT) remains the only curative treatment for a variety of hematological diseases. Allogenic HSCT requires hematopoietic stem cells (HSCs) from matched donors and comes with cytotoxicity and mortality. Recent advances in genome modification of HSCs have demonstrated the possibility of using autologous HSCT-based gene therapy to cure monogenic diseases, such as the inherited bone marrow failure (BMF) syndrome Fanconi Anemia (FA). However, for FA and other BMF syndromes insufficient HSC numbers with functional defects results in delayed hematopoietic recovery and increased risk of graft failure. We and others previously identified the adaptor protein Lnk (Sh2b3) as a critical negative regulator of murine HSC homeostasis. However, whether LNK (SH2B3) controls human HSCs has not been studied. Here, we demonstrate that depletion of LNK via lentiviral expression of miR30-based short hairpin RNAs (shRNAs) resulted in robust expansion of transplantable human HSCs that provided balanced multilineage reconstitution in primary and secondary mouse recipients. Importantly, LNK depletion enhanced cytokine mediated JAK/STAT activation in CD34+ hematopoietic stem and progenitor cells (HSPCs). Moreover, we demonstrate that LNK depletion expands primary HSPCs associated with FA. In xenotransplant, engraftment defects of FANCD2-depleted FA-like HSCs were markedly improved by LNK inhibition. Finally, targeting LNK in primary bone marrow HSPCs from FA patients enhanced their colony forming potential in vitro. Together, these results demonstrate the potential of targeting LNK to expand HSCs to improve HSCT and HSCT-based gene therapy.

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Identification of Heme Oxygenase 1 (HO-1) as a Novel Negative Regulator of Mobilization of Hematopoietic Stem/Progenitor Cells

Stem Cell Reviews and Reports ◽

10.1007/s12015-014-9547-7 ◽

2014 ◽

Vol 11 (1) ◽

pp. 110-118 ◽

Cited By ~ 36

Author(s):

Marcin Wysoczynski ◽

Janina Ratajczak ◽

Daniel Pedziwiatr ◽

Gregg Rokosh ◽

Roberto Bolli ◽

...

Keyword(s):

Progenitor Cells ◽

Heme Oxygenase ◽

Negative Regulator ◽

Heme Oxygenase 1 ◽

Hematopoietic Stem

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The Function of AML1 (RUNX1) C-Deletion Mutant in Hematopoietic Stem/Progenitor Cells.

Blood ◽

10.1182/blood.v106.11.1738.1738 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1738-1738

Author(s):

Yusuke Satoh ◽

Itaru Matsumura ◽

Sachiko Ezoe ◽

Hirokazu Tanaka ◽

Takafumi Yokota ◽

...

Keyword(s):

Progenitor Cells ◽

Es Cells ◽

Hematopoietic Cells ◽

Embryonic Stem ◽

Dominant Negative ◽

Negative Regulator ◽

Receptor System ◽

Hematopoietic Stem ◽

Platelet Disorder ◽

Aml1 Gene

Abstract AML1 (RUNX1) is a family member of heterodimeric transcription factors named core binding factors (CBFs). AML1 binds to its target DNA through the RUNT domain and is frequently involved in chromosomal translocations associated with human leukemias. Also, AML1 was found to be mutated in a substantial fraction of myelodysplastic syndrome (MDS) patients. Although most of AML1 mutations identified in AML patients were located in the N-terminal region, the C-terminal mutaions yielding mutant AML lacking the C-terminal domain (AML1dC) were frequently observed in MDS patients. Based on the fact that the haploinsufficiency of AML1, which results from heterozygous missense mutation of the AML1 gene, causes familial platelet disorder (FPD) with predisposition to AML, and that the conditional deletion of the AML1 gene using the Cre-LoxP system in adult mice results in the failure of megakaryopoiesis and subsequent platelet production, we speculate that AML1 might be involved in the thrombopoietin (TPO)/c-Mpl (its receptor) system, which is a major regulator of this process. Also, we examined the mechanism through which AML1dC affects the function of wtAML1. At first, we examined the role of AML1 in the regulation of the c-mpl promoter with luciferase assays. In 293T cells, we found that wild-type AML1 (wtAML1) activated the c-mpl promoter by 3.5 fold. This effect was dose-dependently inhibited by a dominant-negative form of AML1, AML1-MTG8. In addition, we found that AML1dC inhibited the function of wtAML1 with efficiencies similar to AML1-MTG8 (or with lesser efficiencies than AML1-MTG8). Next, we tried to determine the element responsive to AML1 in the c-mpl promoter. Using various mutants of the c-mpl promoter, we found that wtAML1 activates the element between −135 and −116, which contains the typical AML1-binding sequence. Furthermore, we confirmed that wtAML1 bound to this element in electrophoretic mobility shift assays using nuclear extract of 293T cells transfected with wtAML1. Also, in this assay, we found that AML1dC bound to the consensus DNA sequence more strongly than wtAML1 and inhibited the DNA-binding of wtAML1 competitively. Next, we examined the roles of AML1 in the c-Mpl expression in hematopoietic cells by expressing AML1dC in the OP-9 system, in which hematopoietic cells develop from embryonic stem (ES) cells during the coculture with OP-9 cells. In contrast to the results obtained from 293T cells, when AML1dC was inducibly expressed with the Tet-off system, c-Mpl was expressed more intensely in Lin−Sca1+ hematopoietic stem/progenitor cells than mock-transfected Lin−Sca1+ cells, indicating that AML1 is a negative regulator of the c-Mpl expression in hematopoietic stem/progenitor cells. In contrast, the surface expression of c-Mpl in mature megakaryocytes was hardly affected by AMLdC in the OP-9 system. We also examined the effects of AML1 on the c-Mpl expression in normal murine bone marrow Lin−Sca-1+ cells by expressing AML1dC with the retrovirus system. As a result, we again found that AML1dC enhanced the c-Mpl expression in murine Lin−Sca-1+ cells. Together, our results indicate that AML1 regulates the c-Mpl expression both positively and negatively according to cell types (cf, 293T cells, hematopoietic stem/progenitor cells, and megakaroycytes). Particularly, although AML1 was previously reported to positively regulate of the c-Mpl expression in megakaryocytes, it was supposed be a negative regulator in hematopoietic stem/progenitor cells.

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Patient Characteristics Associated with Successful Mobilization of Peripheral Blood Stem and Progenitor Cells Following Cytotoxic Chemotherapy and Peg-G-CSF Stimulation.

Blood ◽

10.1182/blood.v110.11.4920.4920 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4920-4920

Author(s):

Ingmar Bruns ◽

Johannes C. Fischer ◽

Akos G. Czibere ◽

Cangül Kilic ◽

Roland Fenk ◽

...

Keyword(s):

Cell Count ◽

Progenitor Cells ◽

Peripheral Blood ◽

Serum Levels ◽

Cytotoxic Chemotherapy ◽

Cell Yield ◽

Hematopoietic Stem ◽

Peripheral Blood Cd34 ◽

Stem And Progenitor Cells ◽

Cell Mobilization

Abstract Mobilized peripheral blood stem and progenitor cells are nowadays widely used for transplantation of hematopoietic stem and progenitor cells (PBSCT). These cells can be mobilized into the peripheral blood with cytotoxic chemotherapy, cytokines or both. Currently, G-CSF is most frequently used due to its high efficacy and lack of serious toxicity. However, a serious patient-to-patient variation in the yield of peripheral blood stem and progenitor cells is a feature common of all mobilizations schemes. Therefore, factors determining the collection efficacy have been identified for G-CSF mobilization. Recently a polyethylenglycole-conjugated G-CSF (Peg-G-CSF) has been introduced which has a 12-fold longer half-life than the original compound and therefore leads to long-lasting G-CSF serum-levels after a single injection. Studies on Peg-G-CSF included only small cohorts and no attempts have been made to identify factors influencing the mobilization of blood stem and progenitor cells. Therefore, we retrospectively analyzed 101 unselected patients (66 with multiple myeloma, 26 with non-Hodgkin-lymphoma, 7 with Hodgkin’s disease, 1 with Ewing sarcoma, 1 with malignant germ cell tumor). 27% of patients had active disease, while all others where at least in partial remission after conventional chemotherapy. Patients were treated with a broad range of chemotherapy regimens. The number of cytotoxic chemotherapy cycles administered prior to the mobilization therapy ranged from 1 to 11 (median 4). Mobilization chemotherapy was followed by 6 mg or 12 mg Peg-G-CSF (median 6 mg). Median peripheral blood CD34+ cell maximum in all patients was 65.3/μl (range 0.2–1084 per μl). 12 mg Peg-G-CSF led to a significantly earlier CD34+ cell maximum in the peripheral blood compared to 6 mg Peg-G-CSF (median 13 days vs 15 days, respectively; p=0.01). Overall, a median yield of 8.5 x 10^6 CD34+ cells/kg bodyweight (range 0.2–72.4 x 10^6) was reached with a single apheresis (median, range 1–4). To search for predictors of hematopoietic stem and progenitor cell mobilization, multiple regression analysis was used and revealed CD34+ cell count/μl peripheral blood at the day of apheresis and the processed blood volume during apheresis as predictors for the CD34+ cell yield per kilogram bodyweight. Age, sex, disease type and status were not significantly related to the CD34+ cell count/μl peripheral blood nor the CD34+ cell yield. Interestingly, the number of previous chemotherapy cycles was correlated with the CD34+ cell maximum (p=0.027) with fewer chemotherapy cycles leading to a higher peripheral blood CD34+ cell count and vice versa. In contrast, radiation therapy prior to CD34+ cell mobilization led to a significantly later occurrence of the CD34+ cell maximum in the peripheral blood. Our results confirm the feasibility and efficacy of PBPC mobilization with single dose Peg-G-CSF after cytotoxic chemotherapy shown in previous clinical trials analyzing the largest patient cohort to date and predictors for successful stem cell mobilization with Peg-G-CSF could be identified.

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