Bmi-1 Overexpression Synergizes with p210-BCR-ABL to Induce Stem Cell and Progenitor Transformation.

Abstract Abstract 3251 Poster Board III-1 Chronic myelogenous leukemia (CML) is a hematopoietic stem cell (HSC) malignancy induced by p210-BCR-ABL and characterized by myeloproliferation followed by poor-prognosis acute blastic transformation. Persistence of BCR-ABL+ HSCs in patients under tyrosine kinase inhibitor therapy suggests that inhibition of ABL-kinase alone is not sufficient to completely eliminate the leukemic stem cells and progenitor (LSC/P) population and a group of patients continue developing accelerated/blastic phase despite therapy. Expression of p210-BCR-ABL is necessary and sufficient to develop CML but the molecular mechanisms necessary for its blastic transformation remain elusive. The polycomb group gene Bmi1 plays an essential role in regulating the proliferative capacity of both normal and leukemic stem cells (Lessard J, et al. Nature 2003). Recently, expression of Bmi1 has been found significantly elevated in CD34+ cells of patients with advanced phases compared with patients in chronic-phase CML (Mohty M et al. Blood 2007). We therefore intend to determine whether Bmi1 expression in CML stem cells and progenitors, isolated from Scl/p210-BCR-ABL-expressing mice, is sufficient to accelerate significantly the development of blastic phase. Since simultaneous co-expression of Bmi1 and BCR-ABL in normal HSC/P may not faithfully recapitulate the progression events in CML transformation, we have over-expressed Bmi1 in a model of stem cell-dependent chronic phase CML. This model is based on the tetracycline-dependent expression of p210-BCR-ABL driven by the Scl promoter (Scl-tTA x TRE-BCR-ABL, Koschmieder S et al. Blood 2005), where expression of BCR-ABL is restricted to the HSC/P compartment. Scl-driven expression of BCR-ABL is predominantly active in HSC (Lin-/Sca1+/c-kit+; LSK) and progenitors (Lin-/c-kit+; LK) and is transplantable into secondary recipients (Sengupta A et al., ASH 2008). Expression of Bmi1 into Scl/p210-BCR-ABL-expressing cells significantly increases the HSC/P proliferation (2.5 fold) and also increases the frequency of colony forming cells (CFU-Cs) after serial propagation in liquid culture, compared to Bmi1 (4.6-fold) or BCR-ABL-expressing CML cells alone (517-fold). Furthermore, Bmi1 expression into Scl/p210 leukemic progenitors increases the hypermigratory phenotype of leukemic progenitors (3-fold increase over 5.5-fold increase induced by BCR/ABL alone; P<0.005) in response to CXCL12. Although, Bmi1 overexpression in Scl/p210 cells does not decrease further the reduced adhesion to fibronectin of BCR/ABL-transformed CML HSC/P, leukemic progenitors co-expressing Bmi1 and SCL/p210 have significantly reduced homing in bone marrow compared to Bmi1-expressing HSC/P (7.7 fold, P≤0.005). Altogether, these data indicate that Bmi-1 synergistically enhances the transformation phenotype of p210-BCR-ABL-expressing HSC/P and emphasize the role of epigenetic changes inducing overexpression of self-renewal genes in the pathogenesis of CML. These data suggest that Bmi-1 may represent a novel therapeutic target for CML LSC/P. Disclosures: Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

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Deletion of Rac2 inhibits Proliferation of Chronic Myelogenous Leukemia (CML) Stems Cells and Progenitors (HSC/P) In Vivo and Promotes Survival of Scl/p210-BCR-ABL Mice.

Blood ◽

10.1182/blood.v114.22.3253.3253 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3253-3253

Author(s):

Amitava Sengupta ◽

Jorden Arnett ◽

Susan Dunn ◽

Jose Cancelas

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Chronic Myelogenous Leukemia ◽

Research Funding ◽

Myelogenous Leukemia ◽

Leukemic Stem Cells ◽

Hematopoietic Stem ◽

Rac Gtpases ◽

And Migration

Abstract Abstract 3253 Poster Board III-1 Chronic myelogenous leukemia (CML) is a hematopoietic stem cell (HSC) malignancy induced by p210-BCR-ABL and is characterized by myeloproliferation in the bone marrow (BM) and egress of leukemic stem cells and progenitors (LSC/P) to extramedullary sites. Persistence of BCR-ABL+ HSCs in patients under imatinib suggests that inhibition of ABL-kinase alone is not sufficient to completely eliminate the LSC/P population. Rac GTPases represent integrative molecular switches for p210-BCR-ABL-induced HSC transformation and combined pharmacological and genetic attenuation of Rac GTPases significantly prolong survival in vivo, as reported in a retroviral transduction/transplantation model (Thomas EK & Cancelas JA et al, Cancer Cell 2008). Here, we analyzed the role of Rac2 GTPase in the leukemic maintenance and in the interaction of LSC/P with the leukemic microenvironment in vivo. We used a stem cell leukemia (Scl) promoter-driven, tet-off, Scl-tTA x TRE-BCR-ABL (Scl/p210-BCR-ABL) binary transgenic mouse model (Koschmieder S et al., Blood 2005), where expression of BCR-ABL is restricted to the HSC/P compartment, allowing the study of the intrinsic molecular changes in LSC/P during leukemogenesis. In these mice, Scl-driven expression of BCR-ABL is active in HSC (Lin-/Sca1+/c-kit+; LSK) and progenitors (Lin-/c-kit+/Sca-1-; LK), and CML development is associated with the activation of downstream signaling effectors CrkL, p38-MAPK and JNK. Additionally, Scl/p210-BCR-ABL mice had increased cycling of LSK cells and expansion of circulating and splenic, but not BM, LSC/P, suggesting egress of LSC/Ps from the marrow. These mice share all the characteristics of HSC/P transformation in CML, including increased HSC/P proliferation and survival, severely reduced adhesion to fibronectin, increased migration towards CXCL12, increased cell surface expression of CD44 and decreased expression of L-selectin. Myeloproliferative disease (MPD) in these mice is transplantable into recipient mice, and CML splenocytes have a 10-fold increase in homing to the spleen than towards BM (P<0.05). Leukemic splenocytes are also enriched in endosteal lodging progenitors, compared to the BM-derived progenitors (1.9-fold, P≤0.05). In order to determine the contribution of Rac2 GTPase in the transformation phenotype of leukemic stem cells and progenitors, Scl/p210 mice were intercrossed with Rac2-/- mice. Interestingly loss of Rac2 GTPase alone significantly prolongs survival of the leukemic mice (P≤0.001). Prolonged survival, as observed in Scl/p210 x Rac2-/-, is associated with significantly reduced proliferation of leukemic LK (3-fold, P<0.05) and LSK (6-fold P<0.005) cells, both in BM as well as in spleen, in vivo. Scl/p210 x Rac2-/- mice are also characterized by increased apoptosis (1.7-fold) and lower frequency of LSK cells (2-fold) compared to the Scl/p210 mice in vivo. However, deletion of Rac2 does not significantly reverse the adhesion and migration transformation phenotype of LSC/P. In summary, Rac2 deficiency induces a significant survival of CML mice in a HSC-initiated model of disease through decrease proliferation and survival but does not reverse the transformation phenotype affecting adhesion and migration. This murine model may represent an adequate in vivo system to dissect out the specific signaling pathways involved in p210-BCR-ABL-induced stem cell transformation. Disclosures: Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

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The quiescent fraction of chronic myeloid leukemic stem cells depends on BMPR1B, Stat3 and BMP4-niche signals to persist in patients in remission

Haematologica ◽

10.3324/haematol.2019.232793 ◽

2020 ◽

Vol 106 (1) ◽

pp. 111-122 ◽

Cited By ~ 4

Author(s):

Sandrine Jeanpierre ◽

Kawtar Arizkane ◽

Supat Thongjuea ◽

Elodie Grockowiak ◽

Kevin Geistlich ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Tyrosine Kinase ◽

Stromal Cells ◽

Kinase Inhibitor ◽

Bmp Signaling ◽

Myelogenous Leukemia ◽

Leukemic Stem Cells ◽

Hematopoietic Stem

Chronic myelogenous leukemia arises from the transformation of hematopoietic stem cells by the BCR-ABL oncogene. Though transformed cells are predominantly BCR-ABL-dependent and sensitive to tyrosine kinase inhibitor treatment, some BMPR1B+ leukemic stem cells are treatment-insensitive and rely, among others, on the bone morphogenetic protein (BMP) pathway for their survival via a BMP4 autocrine loop. Here, we further studied the involvement of BMP signaling in favoring residual leukemic stem cell persistence in the bone marrow of patients having achieved remission under treatment. We demonstrate by single-cell RNA-Seq analysis that a sub-fraction of surviving BMPR1B+ leukemic stem cells are co-enriched in BMP signaling, quiescence and stem cell signatures, without modulation of the canonical BMP target genes, but enrichment in actors of the Jak2/Stat3 signaling pathway. Indeed, based on a new model of persisting CD34+CD38- leukemic stem cells, we show that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways. Interestingly, we reveal that only the BMPR1B+ cells adhering to stromal cells display a quiescent status. Surprisingly, this quiescence is induced by treatment, while non-adherent BMPR1B+ cells treated with tyrosine kinase inhibitors continued to proliferate. The subsequent targeting of BMPR1B and Jak2 pathways decreased quiescent leukemic stem cells by promoting their cell cycle re-entry and differentiation. Moreover, while Jak2-inhibitors alone increased BMP4 production by mesenchymal cells, the addition of the newly described BMPR1B inhibitor (E6201) impaired BMP4-mediated production by stromal cells. Altogether, our data demonstrate that targeting both BMPR1B and Jak2/Stat3 efficiently impacts persisting and dormant leukemic stem cells hidden in their bone marrow microenvironment.

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Imatinib mesylate therapy for relapse after allogeneic stem cell transplantation for chronic myelogenous leukemia

Blood ◽

10.1182/blood.v100.5.1590.h81702001590_1590_1595 ◽

2002 ◽

Vol 100 (5) ◽

pp. 1590-1595 ◽

Cited By ~ 120

Author(s):

Hagop M. Kantarjian ◽

Susan O'Brien ◽

Jorge E. Cortes ◽

Sergio A. Giralt ◽

Mary Beth Rios ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Transplantation ◽

Imatinib Mesylate ◽

Allogeneic Stem Cell Transplantation ◽

Response Rate ◽

Chronic Phase ◽

Complete Response ◽

Cytogenetic Response ◽

Myelogenous Leukemia ◽

Blastic Phase

Twenty-eight adults with chronic myelogenous leukemia (CML) that had relapsed after allogeneic stem cell transplantation (SCT) received imatinib mesylate (400-1000 mg/d). Disease was in chronic phase in 5 patients, accelerated in 15, and blastic in 8 (7 medullary, 1 extramedullary); median time from transplantation to relapse was 9 months (range, 1-137 months). Thirteen patients had undergone salvage donor lymphocyte infusion (DLI) (median time from DLI to imatinib mesylate therapy, 4 months [range, 2-39 months]). The overall response rate was 79% (22 of 28 patients); the complete hematologic response (CHR) rate was 74% (17 of 23 patients), and the cytogenetic response rate was 58% (15 of 26 patients; complete response in 9 [35%] patients). CHR rates were 100% for chronic phase, 83% for accelerated phase, and 43% for blastic phase. The patient with extramedullary blastic disease achieved complete response. Cytogenetic response rates were 63% (12 of 19 patients) for chronic or accelerated phases (complete cytogenetic response in 8) and 43% for blastic phase (3 of 7 patients). At median follow-up of 15 months, 19 patients were alive, 9 with no evidence of disease. The 1-year estimated survival rate was 74%. Five patients had recurrence of grade 3 (3 patients) or grades 1 to 2 (2 patients) graft-versus-host disease (GVHD). Severe granulocytopenia developed in 43% of patients and thrombocytopenia in 27%; both conditions reversed with dose adjustments of imatinib mesylate. We conclude that imatinib mesylate effectively controlled CML that recurred after allogeneic SCT, but it was associated with side effects including myelosuppression and recurrence of severe GVHD.

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Bone Marrow (BM) Microenviroment Factors As Early Markers of Response in Patients with Newly Diagnosed Chronic Phase Chronic Myelogenous Leukemia (CML-CP) Treated with Nilotinib

Blood ◽

10.1182/blood.v126.23.1570.1570 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1570-1570

Author(s):

Santa Errichiello ◽

Simona Caruso ◽

Concetta Quintarelli ◽

Biagio De Angelis ◽

Novella Pugliese ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Research Funding ◽

Myelogenous Leukemia ◽

Hematopoietic Progenitor Cell ◽

Newly Diagnosed ◽

Hematopoietic Stem ◽

Optimal Response

Abstract Introduction Tyrosine Kinase Inhibitors (TKI) have completely changed the scenario of CML and dramatically improved the outcomes. Thus, early identification of patients expecting poor outcome is crucial to offer alternative TKI regimens or in some selected cases stem cell transplantation before disease progression may occur. The Evaluating Nilotinib Efficacy and Safety in Trial as First-Line Treatment (ENEST1st) is a phase 3b is an open-label study of nilotinib 300 mg twice daily (BID) in adults with newly diagnosed BCR-ABL positive CP-CML. Aim of the ENEST1st sub-study N10 was to investigate BM microenvironment markers that regulate leukemic stem cells in the bone marrow (BM) niche of Nilotinib-treated patients. Methods The study enrolled patients in 21 Italian ENEST1st participating centers. Response was based on ELN recommendations (Baccarani M, et al. Blood 2013 122:872-884). In an interim analysis, molecular and cytogenetic response by 24 months was assessed. Mononuclear cells were collected from BM and PB samples at the screening visit (V0) and after 3 months of treatment (V4). RT-qPCR for the expression of 10 genes (ARF, KIT, CXCR4, FLT3, LIF, NANOg, PML, PRAME, SET and TIE), involved in the stemness and hematopoietic stem cells survival signaling regulation was conducted. RT-qPCR data were normalized by the expression of GUS mRNA (normalized copy number, NCN). Plasma samples were collected at different time points from both BM or PB samples. Concentrations of 20 different analytes, including IL-1a, IL-3, M-CSF, SCF, SDF1-a, TRAIL, HGF, PDGF-bb, IL1b, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, G-CSF, GM-CSF, MIP-1a, TNF-a, and VEGF, were simultaneously evaluated using commercially available multiplex bead-based sandwich immunoassay kits. Results 33 out of 37 patients enrolled were available for an interim molecular analysis at 24 months: an optimal response was achieved in 25 patients, a warning response in 5 patients and a failure response in 3 patients. We observed a significant correlation between the expression of two genes involved in the regulation of stem cell pluripotency (NANOg) or cytokine signaling (SET) and patient outcome. Indeed, NANOg and SET mRNA were significantly down-regulated in PB samples at diagnosis of patients with optimal response compared to patients with warning/failure response (NANOg mRNA: 0.3±0.25 NCN vs 0.6±0.7 NCN, respectively; p=0.05; SET mRNA: 0.2±0.3 NCN vs 2.3±4.2 NCN, respectively; p=0.03). We also investigated the plasma level of several factors involved in the hematopoietic stem cells (HSCs). Some of these markers showed a significant correlation with patient's outcome when evaluated at diagnosis in either PB or BM samples. Indeed, high level of IL12 (in the BM samples), or HGF, mCSF and SCF (in the PB samples) were associated to a worst prognosis markers, since significantly correlating with no MMR@12months (IL12, p=0.03), intermediate/high Socal score (mCSF, p=0.03; SCF, p=0.03), no reduction of MMR below to 1 at 3 month (SCF, p=0.04) or warning/failure response to Nilotinib treatment (HGF, p=0.03; SCF, p=0.04). Indeed, we find a lower levels of PDGFb, SDF1, TNFa, TRAIL (in the BM samples), and HGF, SDF1, TRAIL (in the PB samples) in those patients with intermediate/high Hasford or Sokal score (PDGFb, p=0.0007; SDF1, p=0.02), warning/failure response to Nilotinib treatment (HGF, p=0.03) or lacking of MMR4.0 (SDF1, p=0.01; TNFa, p=0.02; TRAIL, p=0.05). Conclusion/Summary Taken together, our results suggest that the expression analysis of genes involved in cell pluripotency (NANOg) and/or cell signaling (SET) at baseline, may indicate early achievement of deep molecular response in shown CML-CP patients treated with nilotinib. In addition, in patients with optimal response to Nilotinib, high concentration of SDF-1, TRAIL (inversely correlated with BCR-ABL, and associated to an higher susceptibility to apoptosis in the leukemic blasts) were observed as well as BM TNF (cell-extrinsic and potent endogenous suppressor of HSC activity). A lower concentration of several factors associated to hematopoietic progenitor cell growth and survival (including HGF, SCF and IL12) were observed compared to patients failing to achieve response to Nilotinib. These data strongly suggest that stromal microenvironment supports the viability of BCR-ABL cells in BM niches through direct feeding, or environment releasing of survival factors. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:MSD: Consultancy; BMS: Speakers Bureau; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy. Saglio:Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Novartis Pharmaceutical Corporation: Consultancy, Honoraria. Galimberti:Novartis: Employment. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

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The role of stem cell transplantation for chronic myelogenous leukemia in the 21st century

Blood ◽

10.1182/blood-2014-10-567784 ◽

2015 ◽

Vol 125 (21) ◽

pp. 3230-3235 ◽

Cited By ~ 23

Author(s):

A. John Barrett ◽

Sawa Ito

Keyword(s):

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Chronic Myelogenous Leukemia ◽

Kinase Inhibitors ◽

Conditioning Regimen ◽

Chronic Phase ◽

Myelogenous Leukemia ◽

Blastic Phase

Abstract The introduction of tyrosine kinase inhibitors (TKIs), a treatment of chronic myelogenous leukemia (CML), has largely replaced curative strategies based on allogeneic stem cell transplantation (SCT). Nevertheless, SCT still remains an option for accelerated/blastic-phase and selected chronic-phase CML. Transplant outcomes can be optimized by peritransplant TKIs, conditioning regimen, BCR-ABL monitoring, and relapse management. Controversies exist in transplant timing, pediatric CML, alternative donors, and economics. SCT continues to serve as a platform of “operational cure” for CML with TKIs and immunotherapies.

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The Risk of Residual Molecular and Cytogenetic Disease in Patients With Philadelphia-Chromosome Positive First Chronic Phase Chronic Myelogenous Leukemia Is Reduced After Transplantation of Allogeneic Peripheral Blood Stem Cells Compared With Bone Marrow

Blood ◽

10.1182/blood.v94.2.384 ◽

1999 ◽

Vol 94 (2) ◽

pp. 384-389 ◽

Cited By ~ 84

Author(s):

Ahmet H. Elmaagacli ◽

Dietrich W. Beelen ◽

Bertram Opalka ◽

Siegfried Seeber ◽

Ulrich W. Schaefer

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Peripheral Blood ◽

Philadelphia Chromosome ◽

Chronic Phase ◽

Disease Recurrence ◽

Myelogenous Leukemia ◽

Peripheral Blood Stem Cells ◽

Blood Stem Cells

Abstract The detection of residual molecular and cytogenetic disease was prospectively compared in patients with Philadelphia-chromosome (Ph1) positive first chronic phase chronic myelogenous leukemia (CML) who underwent allogeneic transplantation of unmanipulated peripheral blood stem cells (PBSCT) (n = 29) or bone marrow (BM) (n = 62) using genotypically HLA-identical sibling donors or partially HLA-matched extended family donors. A molecular relapse (MR), as defined by two consecutive positive polymerase chain reaction (PCR) assays for the detection of M-bcr-abl transcripts in a 4-week interval, was found in two of 29 (7%) patients after PBSCT compared with 20 of 62 (32%) patients after bone marrow transplantation (BMT). This corresponds to a 4-year molecular relapse estimate (± standard error) of 7% ± 5% after PBSCT and of 44% ± 8% after BMT (P < .009). With identical follow-up periods of survivors in both patient subsets between 6 and 55 months (median, 28 months), 14 of the 20 patients with MR after BMT progressed to an isolated cytogenetic (n = 10) or a hematologic (n = 4) disease recurrence, resulting in a 4-year cytogenetic relapse estimate of 47% ± 11%, while none of the patients after PBSCT has so far relapsed (P < .006). Multivariate analysis including all potential influencial factors of posttransplant disease recurrence identified the source of stem cells (P < .02) as the only independent predictor of molecular relapse. In conclusion, this prospective comparison of molecular and cytogenetic residual disease demonstrates that peripheral blood stem cell transplants have a more pronounced activity against residual CML cells than bone marrow transplants. Prospective randomized trials comparing PBSCT and BMT in patients with first chronic phase Ph1-positive CML are strictly required to further substantiate differences in the antileukemic activity of the two stem cell sources.

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BMP Pathway Alterations in Chronic Myelogenous Leukemia Lead to the Amplification of Primitive Progenitors Output in Chronic Phase

Blood ◽

10.1182/blood.v120.21.1663.1663 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1663-1663

Author(s):

Franck E. Nicolini ◽

Bastien Laperoussaz ◽

Sandrine Jeanpierre ◽

Karen Sagorny ◽

Bastien Kaniewski ◽

...

Keyword(s):

Stem Cell ◽

Research Funding ◽

Stem Cell Niche ◽

Chronic Phase ◽

Myelogenous Leukemia ◽

Disease Phenotype ◽

Serum Free ◽

Bmp Pathway ◽

Cell Niche ◽

Serum Free Conditions

Abstract Abstract 1663 The presence of inherently resistant Ph+ leukemic stem cells (LSC) to tyrosine kinase inhibitors (TKI) in chronic phase chronic myelogenous leukemia (CP CML) might be responsible for the long-term disease persistence, requiring the indefinite prolongation of TKI, and represent a reservoir for the initiation of an overt resistance. Despite quiescence, these Ph+ LSC maintain high level interactions with their microenvironment and might display some alterations to different pathways to survive, responsible for disease persistence. In this perspective, we studied some actors involved in the maintenance of the stem cell niche and normal hematopoiesis (Jeanpierre S. et al. Blood 2008), the bone morphogenetic proteins (BMP-2 and BMP-4) in a series of 45 CP CML patients harvested at diagnosis and compared the results to that of 15 normal bone marrow (BM) counterparts from healthy donors. BMPs are members of the TGF-b family, highly secreted by the normal BM stem cell niche, that regulate numerous cellular events such as proliferation, differentiation, migration, adhesion and apoptosis in various cellular systems including hematopiesis. The deregulation of these events is the hallmark of CP CML and they are all involved in the CML disease phenotype in CP. All CML and BM samples were CD34+ purified. RQ-PCRs on total cells, CD34− and CD34+ progenitors for the different members of the BMP pathway show specific alterations in CML: in the CD34+ primitive compartment, BMP-4 itself is downregulated as well as Bambi, Smad1, Smad5, and Runx2 while Smad6 is specifically upregulated. Interestingly, while analyzing the CD34+ and CD34− compartments from individual patients, we observed a differential expression of the signaling pathway inhibitors Smad6 and Follistatin (low levels in leukemic CD34−, high levels in leukemic CD34+ cells when compared to their normal purified counterparts), and BMP-2, BMP-4, and three of the BMP specific receptors (BMP-RIa, BMP-RIb and BMP-RII) are significantly and selectively downregulated in CD34+ CML progenitors. We further characterized the impact of 7-days in vitro exposure to BMP-2 and BMP-4 in 4GF (IL-6, SCF, IL-11, IL-3) serum-free conditions of CD34+ CML and normal BM progenitors using immunophenotypic, CFC and LTC-IC assays. The functional assays results suggest in responding patients, that BMP-4 is able to selectively amplify the genotyped Ph+ LTC-IC compartment (while BMP-2 partly maintains it, whereas TPO amplifies normal Ph− LTC-IC as well as Ph+ LTC-IC) as mentioned in figure 1. Figure 1: Total LTC-IC-derived week 5 colonies per 1000 seeded cells within the responding CML samples. Black bars are CML samples and white bars are normal BM counterparts, (*: p<0.05). Figure 1:. Total LTC-IC-derived week 5 colonies per 1000 seeded cells within the responding CML samples. Black bars are CML samples and white bars are normal BM counterparts, (*: p<0.05). The CFC compartment was highly amplified when exposed to either BMP-2 or BMP-4 (but not TPO) when compared to normal BM progenitors as controls. In addition, within responding patient samples, only BMP-4 significantly amplified the number of immature cells as compared to TPO or BMP-2 treated cells. Single cell experiments in serum-free conditions with sorted CD34+38− chronic phase CML cells from diagnosis, in parallel with normal BM cells, in the presence or not of BMP-2 & −4 have been performed. A high cloning efficiency was observed with CML cells (∼17%), compared to that with normal BM counterparts (3%) as usual, and in addition, BMP-2 seemed to promote erythroid commitment of primitive CML cells, while BMP-4 increased CFU-GM and CFU-GEMM output. In conclusion, these data provide some evidences that the BMP pathway is selectively altered in primitive CP CML progenitors at diagnosis, and its molecular and functional alterations might be involved in the disease phenotype. Whether this is a cause or a consequence of the disease and its involvement in stem cell persistence on TKI is currently under examination. Disclosures: Nicolini: Novartis: Research Funding. Sagorny:BMS: Research Funding. maguer-Satta:Novartis: Research Funding.

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Mir-99 Is a Regulator of Hematopoietic and Leukemic Stem Cell Differentiation

Blood ◽

10.1182/blood.v126.23.3063.3063 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3063-3063 ◽

Cited By ~ 1

Author(s):

Mona Khalaj ◽

Wenhuo Hu ◽

Christopher Y. Park

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Growth ◽

Fold Increase ◽

Negative Regulator ◽

Myeloid Differentiation ◽

P Value ◽

Leukemic Stem Cells ◽

Hematopoietic Stem ◽

Fold Reduction

Abstract MicroRNAs play an important role in the regulation of normal and malignant stem cells of the hematopoietic system. Analysis of microRNA-Seq data from 168 acute myeloid leukemia (AML) patients in the TCGA database reveals widespread dysregulation of miRNA expression, including miR-99b. Interestingly, miR-99b expression inversely correlates with differentiation status of the AMLs as determined by the French-American-British classification scheme, suggesting miR-99 is a negative regulator of differentiation. We also have shown that miR-99a and miR-99b are highly expressed in mouse hematopoietic stem cells (HSCs) compared to their more differentiated progeny. Thus, we hypothesized that miR-99 is a regulator of differentiation in normal and leukemic stem cells. To test the function of miR-99 in normal hematopoiesis, we knocked down miR-99a or miR-99b in mouse HSCs (Lin-cKit+Sca1+CD34-SLAM+) using lentiviral vectors, resulting in ~3.5 fold reduction in the number of colonies formed in methylcellulose assays upon secondary plating (P=0.01), with the average colony being approximately 2 times smaller as measured by the number of cells (P=0.03). Consistently, transplantation assays demonstrated >10-fold reduction in long-term engraftment capacity of HSCs upon miR-99 knockdown (KD) at 16 weeks (P=0.0004). Analysis of peripheral blood of the transplanted mice revealed ~3-fold increase in the proportion of donor-derived myeloid cells, suggesting that miR-99 may regulate the self-renewal and/or limit differentiation of HSPCs (P=0.01). Consistent with these observations, gene set enrichment analysis (GSEA) using the RNA-sequencing data generated from hematopoietic stem and progenitor cells (HSPCs) with miR-99 KD revealed significant enrichment for the myeloid differentiation gene signature (Lindstedt-dendritic-cell-maturation, Nominal p value =0.0, FDR q value=0.02). Moreover, analysis of GFP+ miR-99 KD cells after second plating revealed a 3-fold increase in apoptosis as measured by caspase 3 activation. To better understand the mechanism mediating the reduced reconstitution capacity of miR-99 KD HSCs, we analyzed the composition of progenitors in the bone marrow of transplanted mice. miR-99 KD resulted in a highly significant decrease in the percentage of myeloid progenitors of donor-derived cells 16 weeks post transplantation (60% decrease, P=0.01), consistent with miR-99 KD inducing HSPC differentiation. Overall, these data suggest that miR-99 is a negative regulator of HSPC differentiation. Consistent with a role in maintaining LSCs, miR-99 KD in the AML cell lines MonoMac6 and U937 induced expression of myeloid differentiation markers including CD15, CD14 and CD13. This was accompanied by a 2-50 fold reduction in cell growth depending on the AML cell line tested and a significant increase in apoptosis; the levels of miR-99 expression correlated with the degree of the growth defect. In addition, concomitant expression of miR-99b KD and MLL-AF9 overexpression vectors in HSPCs followed by serial methocult assays lead to decreased colony formation in third platings. We also observed a 50% reduction in the percentage of Lin-Sca-1-c-Kit+ myeloid progenitors in secondarily plated cells (P value=0.01). These data suggest in MLL-AF9+ leukemia, miR-99 acts to restrain cell growth and maintain the undifferentiated state of leukemic blasts. Together, these data indicate that miR-99a/b regulates the proliferation of normal and leukemic stem cells by negatively regulating apoptosis and maintaining the stem cell state. Disclosures No relevant conflicts of interest to declare.

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Targeting MUC1 as a Marker for Myeloid Leukemia Stem Cells by DC/AML Fusions.

Blood ◽

10.1182/blood.v110.11.1794.1794 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1794-1794

Author(s):

Jacalyn Rosenblatt ◽

Zekui Wu ◽

Corrine Lenahan ◽

Adam Bissonnette ◽

Baldev Vasir ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Stem Cell ◽

Myeloid Leukemia ◽

Chronic Phase ◽

Fold Increase ◽

Leukemia Cells ◽

Leukemia Stem Cells ◽

Cell Compartment ◽

Stem Cell Compartment

Abstract The epithelial mucin antigen (MUC1) is aberrantly expressed in many epithelial tumors and hematologic malignancies and promotes oncogenesis and tumor progression. MUC1 is recognized by the T cell repertoire and has served as a target for cellular immunotherapy. In the present study, we examined MUC1 as a marker for myeloid leukemia cells and their progenitors and its capacity to serve as a target for leukemia stem cells. Myeloid leukemia cells were isolated from bone marrow aspirates or peripheral blood in patients with high levels of circulating disease. MUC1 was not expressed on unselected leukemia samples (mean expression 3%, n=12). Similarly, low levels of MUC1 expression were seen in leukemic blasts with monocytoid differentiation (mean expression 2.7%, n=5). A subset of leukemia specimens underwent CD34 selection by magnetic bead separation. In contrast to unselected cells, 38% of CD34+ leukemia cells expressed MUC1 (n=5). The leukemia stem cell compartment was isolated by separating CD34+/CD38−/ lineage- fractions by flow cytometric sorting. Leukemia stem cells demonstrated strong expression of MUC-1 by immunohistochemical staining and FACS analysis. Similarly, we examined MUC1 expression on progenitor cells derived from chronic phase chronic myeloid leukemia and following blast transformation. MUC1 was seen in only 4% of CD34+ cells obtained from chronic phase CML samples (n=4) while uniform expression was observed in samples derived from patients with accelerated/blastic phase disease. These data suggest that MUC1 serves as a marker for early leukemia progenitors and is associated with blastic transformation. We assessed the capacity of a cancer vaccine consisting of dendritic cell (DC)/myeloid leukemia fusions to stimulate immune responses that target MUC1 and other antigens expressed by the stem cell compartment. DCs were generated from adherent mononuclear cells that were cultured with GM-CSF and IL-4 and matured with TNFa. DCs were fused with patient derived myeloid leukemia cells using polyethylene glycol as previously described. Fusion cells were quantified by determining the percentage of cells that expressed unique DC and leukemia antigens. DC/AML fusions induced the expansion of MUC1 specific T cells. Stimulation of autologous T cells with DC/AML fusions resulted in a mean 3 fold increase in CD8+ cells binding the MUC-1 tetramer (N=4). DC/AML fusions stimulated anti-tumor immune responses that targeted leukemia stem cells. Fusion stimulated T cells demonstrated increased expression of IFNγ following exposure to lysate generated from unselected leukemia cells (29 fold) and leukemia stem cells (28 fold). In contrast, exposure to renal carcinoma lysate generated only a 5 fold increase in IFNγ. In summary, these findings suggest that leukemic progenitors in AML and accelerated/blast phase CML express MUC-1. DC/tumor fusion vaccines target the MUC-1 protein and the stem cell compartment, and may be a potent immunotherapeutic strategy to eliminate the malignant stem cell clone in AML.

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Leukemic Stem Cells and Progenitors Demonstrate Impaired Interaction with the Hematopoietic Microenvironment in Vivo in An Inducible Murine Model of Chronic Myelogenous Leukemia

Blood ◽

10.1182/blood.v112.11.191.191 ◽

2008 ◽

Vol 112 (11) ◽

pp. 191-191

Author(s):

Amitava Sengupta ◽

Jose Cancelas

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Chronic Myelogenous Leukemia ◽

Murine Model ◽

Chronic Phase ◽

Myelogenous Leukemia ◽

Cell Surface Expression ◽

Leukemic Stem Cells ◽

Hematopoietic Microenvironment

Abstract Chronic myelogenous leukemia (CML) is a stem cell malignancy induced by p210 BCR-ABL and characterized by myeloproliferation in BM and egression of leukemic stem cells and progenitors (LSC/P) to extramedullary sites. Persistence of BCR-ABL+ HSC in patients under Imatinib suggests inhibition of ABL-kinase alone is not sufficient to eliminate the LSC/P. One of the major hallmarks of CML induced by signaling downstream BCR-ABL is the loss of control of the hematopoietic microenvironment on LSC/P. Expression of p210 BCR-ABL has been associated with loss of adhesion to the bone marrow, impaired migration in response to CXCL12 and decreased retention in the BM. In order to study the putative LSC/P niches in steady-state chronic-phase leukemia, we have analyzed the ability of LSC/P to proliferate and get retained in the bone marrow (BM) in an inducible model of CML. Binary transgenic SCL-tTA/TRE-BCR-ABL mice (Koschmieder S et al., Blood 2005) express p210 BCR-ABL in LSC/P upon doxycycline withdrawal (CML mice). Induced myeloproliferation was associated with activation of the downstream signaling effectors CrkL and p38-MAPK and expansion of circulating (Table 1) and splenic LSC/P but not in BM, suggesting massive LSC/P egression from the marrow (Table 2). Proliferation analysis showed that myeloid expansion in the spleen was secondary to increased cycling of Lin−Sca1+c-Kit+ (LSK) cells (3.1-fold increase in S-phase cells, P<0.05), but not in Lin−/c-Kit+ (LK) population, compared with the control spleens. In agreement with the LSC/P BM content data, the frequency of BM-derived LSK and LK cells incorporating BrdU in CML and in control mice remained similar, suggesting a specific egression of LSC/P from the BM to extramedullary sites. To test whether this model truly represented a model of BM LSC/P egression, we compared the splenic and BM LSC/P compared with their controls regarding their adhesion molecule expression, interaction with the hematopoietic microenvironment (HM) and homing to the overall marrow cavity and endosteal space. Splenic, but not BM-derived, LSK and LSK CD34+ ST-HSCs had increased cell surface expression of CD44 compared to controls (1.35 fold, P=0.006 and 1.23 fold, P<0.05 respectively) and decreased expression of L-selectin (8.7 fold, P<0.05) while expression of CXCR4, α4β1 and α5β1 integrins remain similar in bone marrow and splenocytes from CML and control mice. CML BM progenitors also showed 18-fold reduced adhesion to fibronectin and 1.4-fold increased migration towards CXCL12 compared to control BM progenitors. Myeloproliferative disease was transplantable into non-transgenic littermates and homing of CML BM progenitors was increased (4.3 fold, P<0.005) in myeloablated littermate recipient BM. However, lineage-negative leukemic BM-derived cells which had increased homing in BM of recipient mice had an impaired ability to migrate to the BM endosteal space compared with their littermate controls (control: 31 ± 18% vs CML mice: 17.6 ± 17%), suggesting an specific impairment to lodge in specialized anatomically-defined hematopoietic “niches”. Altogether, this murine model may represent an adequate in vivo system to analyze the ability of p210 BCR-ABL-expressing LSC/P to interact with BM niches and study the control of the hematopoietic microenvironment on LSC/P survival, proliferation and retention. Table 1 Increase in circulating LSC/P in the CML mice after withdrawal of doxycyclin Peripheral Blood LSK (×103)Cells/mL Blood P<0.05 LT-HSC(×103)Cells/mL Blood P<0.05 CFU-GM+BFU-E/mL Blood P<0.05 Control 1.56 ± 0.25 0.459 ± 0.29 60.86 ± 51.09 CML mice 3.56 ± 1.52 2.159 ± 2.03 869.6 ± 628.4 Table 2. Immunophenotypic analysis of BM and splenocytes in control and CML mice Population BM (Cells ×104) (Control) BM (Cells x104) (CML) SP (Cells ×104) (Control) SP (Cells x104) (CML) C-Kit + Sca1 + 24.3 ± 9.9 21.3 ± 11 6.8 ± 4.5 30.1 ± 12.3 (P<0.05) Mac1 + Gr1 + 1779 ± 307 1583 ± 265 78.4 ± 32 608 ± 377 (P<0.05) CFU-C/10 5 Cells 342 ± 66 334 ± 99 63.3 ± 7.09 79 ± 6.54 (P<0.05)

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