BMS-214662 induces mitochondrial apoptosis in chronic myeloid leukemia (CML) stem/progenitor cells, including CD34+38− cells, through activation of protein kinase Cβ

Abstract Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder maintained by cancer stem cells. To target this population, we investigated the mechanism of action of BMS-214662, developed as a farnesyl transferase inhibitor (FTI) and unique in inducing apoptosis in these cells. By contrast, a related congener and equally effective FTI, BMS-225975 does not induce apoptosis, indicating a novel mechanism of action. BMS-214662 significantly and selectively induced apoptosis in primitive CD34+38− CML compared with normal cells. Apoptosis proceeded via the intrinsic pathway: Bax conformational changes, loss of mitochondrial membrane potential, generation of reactive oxygen species, release of cytochrome c, and caspase-9/3 activation were noted. Up-regulation of protein kinase Cβ (PKCβ), down-regulation of E2F1, and phosphorylation of cyclin A–associated cyclin-dependent kinase 2 preceded these changes. Cotreatment of CML CD34+ and CD34+38− cells with PKC modulators, bryostatin-1, or hispidin markedly decreased these early events and the subsequent apoptosis. None of these events was elicited by BMS-214662 in normal CD34+ cells or by BMS-225975 in CML CD34+ cells. These data suggest that BMS-214662 selectively elicits a latent apoptotic pathway in CML stem cells that is initiated by up-regulation of PKCβ and mediated by Bax activation, providing a molecular framework for development of novel therapeutics.

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Persistence of BCR-ABL-Expressing LEUKEMIC STEM CELLS IN Chronic MYELOID LEUKEMIA (CML) PATIENTS IN COMPLETE REMISSION with Undetectable MOLECULAR Disease

Blood ◽

10.1182/blood.v116.21.883.883 ◽

2010 ◽

Vol 116 (21) ◽

pp. 883-883 ◽

Cited By ~ 1

Author(s):

Jean-Claude Chomel ◽

Marie Laure Bonnet ◽

Nathalie Sorel ◽

Angelina Bertrand ◽

Marie Claude Meunier ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Leukemic Stem Cells ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Previously Treated ◽

Clonogenic Cells

Abstract Abstract 883 Currently Imatinib Mesylate (IM) represent the first line therapy for chronic myeloid leukemia (CML). Recent data suggest that despite unprecedented rates of complete cytogenetic responses (CCR) and major molecular responses (MMR) obtained, leukemic stem cells (LSC) persist in the majority of patients (pts). LSC have been shown to be resistant to in vitro treatments with tyrosine kinase inhibitors (TKI). Consequently, discontinuation of IM in pts with undetectable molecular residual leukemia (UMRL) attested by RQ-PCR, leads to molecular relapses in the majority of the cases. Although the persistence of CD34+ CD38- leukemic stem cells has been demonstrated in pts with complete cytogenetic remission (CCR), the persistence of BCR-ABL+ leukemic stem cells in UMRL pts with has not been studied so far. For this purpose, we have performed an extensive analysis of bone-marrow-derived clonogenic and primitive hematopoietic stem cells in 6 pts with RQ-PCR constantly negative in their blood samples. Concerning the treatments; 5 out of 6 pts were off therapy, 3 pts (UPN1, 2, 3) had been treated with interferon-a only (IFN) for 6–13 years and their therapy was discontinued for 11, 16 and 8 years, respectively and 2 pts (UPN4 and 5) had been treated successively with IFN and IM and their IM therapy was discontinued for 2 years. One patient (UPN6) had been treated with IM followed by dasatinib and was on dasatinib at the time of the study. UPN7 was previously treated with first IFN then IM (which induced a stable UMRL) and then she switched to dasatinib because of side effect with IM. Bone marrow cells were collected and CD34+ cells purified using immunomagnetic columns. After performing a clonogenic assay, CD34+ cells were used in long-term culture initiating cell (LTC-IC) assays with weekly half medium changes. At week+5, clonogenic assays were performed and LTC-IC-derived clonogenic cells activity was calculated. For each patient 20 individual and 20 pools of 10 clonogenic cells and 20 individual and 20 pools of 10 LTC-IC derived CFU-C were plucked in order to obtain information in at least 220 CFU-C. After RNA extraction, BCR-ABL was quantified by RQ-PCR and in each positive CFU-C a nested PCR was performed to confirm the results. In one patient (UPN7) a NOD/SCID mouse assay was performed. All 3 pts treated with IFN alone, had BCR-ABL+ clonogenic cells varying from 0.5% (UPN1, 2) to 45 % (UPN3). All 3 had LTC-IC derived CFU-C positive for BCR-ABL (UPN1: 20%; UPN2 5%; UPN3 3%). In two pts previously treated with IFN and IM, clonogenic CFU-C BCR-ABL positivity was 10% and 5% whereas LTC-IC-derived CFU-C was 5% in UPN4) and undetected on UPN5. In UPN6 treated with IM then dasatinib, 5% of CFU-C was BCR-ABL+ whereas 100% of LTC-IC derived CFU-C was positive. The analysis of SCID-NOD assays performed in CD34+ cells from patient UPN7 is ongoing. Overall, these data show, for the first time to our knowledge, that in pts in IFN and IFN/IM-induced long-term remissions, there is persistent clonogenic BCR-ABL+ output maintained by BCR-ABL-expressing stem cells in the absence of relapse. In the only patient with successively treated with IM and dasatinib, 100 % of primitive hematopoietic stem cells are BCR-ABL+, despite PCR-negativity in peripheral blood, suggesting their possible quiescence in vivo and highlighting a theoretical risk of relapse. It remains to be determined if in pts with TKI-induced remissions, the analysis of stem cell compartments could be of use for clinical decisions to discontinue therapy. Disclosures: No relevant conflicts of interest to declare.

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A therapeutically targetable mechanism of BCR-ABL–independent imatinib resistance in chronic myeloid leukemia

Science Translational Medicine ◽

10.1126/scitranslmed.3009073 ◽

2014 ◽

Vol 6 (252) ◽

pp. 252ra121-252ra121 ◽

Cited By ~ 63

Author(s):

Leyuan Ma ◽

Yi Shan ◽

Robert Bai ◽

Liting Xue ◽

Christopher A. Eide ◽

...

Keyword(s):

Stem Cells ◽

Chronic Myeloid Leukemia ◽

Protein Kinase ◽

Myeloid Leukemia ◽

Large Scale ◽

Combined Treatment ◽

Drug Binding ◽

Mitogen Activated Protein Kinase ◽

Imatinib Resistance ◽

Hematopoietic Stem

Resistance to the BCR-ABL inhibitor imatinib mesylate (IM) poses a major problem for the treatment of chronic myeloid leukemia (CML). IM resistance often results from a secondary mutation in BCR-ABL that interferes with drug binding. However, in many instances, there is no mutation in BCR-ABL, and the basis of such BCR-ABL–independent IM resistance remains to be elucidated. To gain insight into BCR-ABL–independent IM resistance mechanisms, we performed a large-scale RNA interference screen and identified IM-sensitizing genes (IMSGs) whose knockdown renders BCR-ABL+cells IM-resistant. In these IMSG knockdown cells, RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal–regulated kinase (ERK) signaling is sustained after IM treatment because of up-regulation ofPRKCH, which encodes the protein kinase C (PKC) family member PKCη, an activator of CRAF.PRKCHis also up-regulated in samples from CML patients with BCR-ABL–independent IM resistance. Combined treatment with IM and trametinib, a U.S. Food and Drug Administration–approved MEK inhibitor, synergistically kills BCR-ABL+IMSG knockdown cells and prolongs survival in mouse models of BCR-ABL–independent IM-resistant CML. Finally, we showed that CML stem cells contain high levels ofPRKCH, and this contributes to their intrinsic IM resistance. Combined treatment with IM and trametinib synergistically kills CML stem cells with negligible effect on normal hematopoietic stem cells. Collectively, our results identify a therapeutically targetable mechanism of BCR-ABL–independent IM resistance in CML and CML stem cells.

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Identification of BCR/ABL-negative primitive hematopoietic progenitor cells within chronic myeloid leukemia marrow

Blood ◽

10.1182/blood.v81.3.801.bloodjournal813801 ◽

1993 ◽

Vol 81 (3) ◽

pp. 801-807 ◽

Cited By ~ 1

Author(s):

T Leemhuis ◽

D Leibowitz ◽

G Cox ◽

R Silver ◽

EF Srour ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Chronic Phase ◽

Polymerase Chain Reaction Analysis ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem

Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell. It has been shown that normal stem cells coexist with malignant stem cells in the bone marrow of patients with chronic-phase CML. To characterize the primitive hematopoietic progenitor cells within CML marrow, CD34+DR- and CD34+DR+ cells were isolated using centrifugal elutriation, monoclonal antibody labeling, and flow cytometric cell sorting. Polymerase chain reaction analysis of RNA samples from these CD34+ subpopulations was used to detect the presence of the BCR/ABL translocation characteristic of CML. The CD34+DR+ subpopulation contained BCR/ABL(+) cells in 11 of 12 marrow samples studied, whereas the CD34+DR- subpopulation contained BCR/ABL(+) cells in 6 of 9 CML marrow specimens. These cell populations were assayed for hematopoietic progenitor cells, and individual hematopoietic colonies were analyzed by PCR for their BCR/ABL status. Results from six patients showed that nearly half of the myeloid colonies cloned from CD34+DR- cells were BCR/ABL(+), although the CD34+DR- subpopulation contained significantly fewer BCR/ABL(+) progenitor cells than either low-density bone marrow (LDBM) or the CD34+DR+ fraction. These CD34+ cells were also used to establish stromal cell-free long-term bone marrow cultures to assess the BCR/ABL status of hematopoietic stem cells within these CML marrow populations. After 28 days in culture, three of five cultures initiated with CD34+DR- cells produced BCR/ABL(-) cells. By contrast, only one of eight cultures initiated with CD34+DR+ cells were BCR/ABL(-) after 28 days. These results indicate that the CD34+DR- subpopulation of CML marrow still contains leukemic progenitor cells, although to a lesser extent than either LDBM or CD34+DR+ cells.

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Characterization of stage progression in chronic myeloid leukemia by DNA microarray with purified hematopoietic stem cells

Oncogene ◽

10.1038/sj.onc.1205029 ◽

2001 ◽

Vol 20 (57) ◽

pp. 8249-8257 ◽

Cited By ~ 56

Author(s):

Ken Ohmine ◽

Jun Ota ◽

Masuzu Ueda ◽

Shu-ichi Ueno ◽

Koji Yoshida ◽

...

Keyword(s):

Stem Cells ◽

Chronic Myeloid Leukemia ◽

Hematopoietic Stem Cells ◽

Dna Microarray ◽

Myeloid Leukemia ◽

Hematopoietic Stem ◽

Stage Progression

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Identification of BCR/ABL-negative primitive hematopoietic progenitor cells within chronic myeloid leukemia marrow

Blood ◽

10.1182/blood.v81.3.801.801 ◽

1993 ◽

Vol 81 (3) ◽

pp. 801-807 ◽

Cited By ~ 49

Author(s):

T Leemhuis ◽

D Leibowitz ◽

G Cox ◽

R Silver ◽

EF Srour ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Chronic Phase ◽

Polymerase Chain Reaction Analysis ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem

Abstract Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell. It has been shown that normal stem cells coexist with malignant stem cells in the bone marrow of patients with chronic-phase CML. To characterize the primitive hematopoietic progenitor cells within CML marrow, CD34+DR- and CD34+DR+ cells were isolated using centrifugal elutriation, monoclonal antibody labeling, and flow cytometric cell sorting. Polymerase chain reaction analysis of RNA samples from these CD34+ subpopulations was used to detect the presence of the BCR/ABL translocation characteristic of CML. The CD34+DR+ subpopulation contained BCR/ABL(+) cells in 11 of 12 marrow samples studied, whereas the CD34+DR- subpopulation contained BCR/ABL(+) cells in 6 of 9 CML marrow specimens. These cell populations were assayed for hematopoietic progenitor cells, and individual hematopoietic colonies were analyzed by PCR for their BCR/ABL status. Results from six patients showed that nearly half of the myeloid colonies cloned from CD34+DR- cells were BCR/ABL(+), although the CD34+DR- subpopulation contained significantly fewer BCR/ABL(+) progenitor cells than either low-density bone marrow (LDBM) or the CD34+DR+ fraction. These CD34+ cells were also used to establish stromal cell-free long-term bone marrow cultures to assess the BCR/ABL status of hematopoietic stem cells within these CML marrow populations. After 28 days in culture, three of five cultures initiated with CD34+DR- cells produced BCR/ABL(-) cells. By contrast, only one of eight cultures initiated with CD34+DR+ cells were BCR/ABL(-) after 28 days. These results indicate that the CD34+DR- subpopulation of CML marrow still contains leukemic progenitor cells, although to a lesser extent than either LDBM or CD34+DR+ cells.

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Targeting Acute Myeloid Leukemia Stem Cells by MUC1-C Subunit Inhibition

Blood ◽

10.1182/blood.v116.21.848.848 ◽

2010 ◽

Vol 116 (21) ◽

pp. 848-848 ◽

Cited By ~ 1

Author(s):

Dina Stroopinsky ◽

Jacalyn Rosenblatt ◽

Keisuke Ito ◽

Li Yin ◽

Hasan Rajabi ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Colony Formation ◽

Myeloid Leukemia ◽

Muc1 Expression ◽

Hematopoietic Stem ◽

Flow Cytometric ◽

Control Peptide ◽

Cd34 Cells ◽

Acute Myeloid

Abstract Abstract 848 Introduction: Acute myeloid leukemia (AML) arises from a malignant stem cell population that is resistant to cytotoxic therapy and represents a critical reservoir of conferring disease recurrence. A major focus of investigation is the identification of unique markers on leukemia stem cells (LSCs) that differentiate them from normal hematopoietic stem cells and thereby serve as potential therapeutic targets. MUC1 is a high molecular weight transmembrane glycoprotein that is aberrantly expressed in many epithelial tumors and confers cell growth and survival. We have developed an inhibitor of the MUC1-C receptor subunit that blocks oligomer formation and nuclear localization. In the present study, we have examined expression of MUC1 on LSCs as compared to normal hematopoietic stem cells and studied the effect of MUC1-C inhibition on the functional properties of LSCs. Methods and Results: Using multichannel flow cytometric analysis, we isolated the LSC compartment as defined by CD34+/CD38-/lineage- cells from bone marrow specimens obtained from patients with active AML. The majority of LSCs strongly expressed MUC1 with a mean percentage of 77% (n=6). These findings were confirmed by immunocytochemical staining of LSCs isolated by flow cytometric sorting. MUC1 expression was not detectable on the CD34- fraction of AML cells, but was present on the granulocyte-macrophage progenitor (GMP) fraction (CD34+/CD38+ cells) (mean=83%; n=6). In contrast, MUC1 expression was not observed on CD34+ progenitors isolated from normal donors (18%, n=6). In concert with these findings, RT-PCR analysis for MUC1 RNA demonstrated expression in CD34+ cells isolated from AML patients, but not normal volunteers. Notably, we also found that MUC1 expression selectively identifies malignant hematopoietic progenitors in a patient with chimerism between normal and leukemia derived stem cells. The presence of MUC1+CD34+ cells was detected in a patient with AML who achieved a morphologic complete remission following sex mismatched allogeneic transplantation. Using Bioview technology, we found that MUC1 is expressed only in the recipient (XX) CD34+ cells, representing residual malignant cells, whereas the donor (XY) derived CD34+ cells, representing the majority of the progenitors, lacked MUC1 expression. We subsequently examined the effects of MUC1-C inhibition on the capacity of leukemic progenitors to proliferate and support colony formation. MUC1-C inhibition with the GO-203 cell-penetrating peptide resulted in downregulation of the β-catenin pathway, an important modulator of cell division and survival, which is known to support the LSC phenotype. No significant change was detected with a control peptide, or with MUC1-C inhibition of progenitors isolated from a normal control. Furthermore, MUC1-C inhibition resulted in apoptosis, as demonstrated by flow cytometric staining for AnnexinV in AML CD34+ cells, but not in CD34+ progenitors isolated from normal volunteers (mean Annexin positive cells 53% and 5%, respectively, n=4). Consistent with these findings, the MUC1-C inhibitor, but not the control, peptide resulted in cell death of CD34+ cells isolated from AML patients, but not normal controls. Most significantly, exposure of CD34+ AML cells to the MUC1-C inhibitor resulted in loss of their capacity for colony formation in vitro with mean colonies of 4 and 40 for those cells exposed to the MUC1 inhibitor and a control peptide (n=2). In contrast, colony formation by normal hematopoietic stem cells was unaffected. Conclusions: MUC1 is selectively expressed by leukemic progenitors and may be used to differentiate malignant from normal hematopoietic stem cell populations. MUC1-C receptor subunit inhibition results in (i) downregulation of b-catenin signaling, (ii) induction of apoptosis and cell death, and (iii) disruption of the capacity to induce leukemia colony formation. Disclosures: Stone: genzyme: Consultancy; celgene: Consultancy; novartis: Research Funding. Kufe:Genus Oncology: Consultancy, Equity Ownership.

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A Bio-Integrative Approach Identifies an Inflammatory Signature in Chronic Myeloid Leukemia (CML) Stem Cells That Is Highly Perturbed in CML Blast Crisis and Involves REL transcription Factor

Blood ◽

10.1182/blood.v126.23.4017.4017 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4017-4017

Author(s):

Christophe Desterke ◽

Ludovic Marie-Sainte ◽

Amine Sbitti ◽

Ali Naama ◽

Annelise Bennaceur-Griscelli ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Stem Cell ◽

Chronic Myeloid Leukemia ◽

Disease Progression ◽

Myeloid Leukemia ◽

Blast Crisis ◽

Integrative Approach ◽

Hematopoietic Stem ◽

Gene Set

Abstract Chronic myeloid leukemia is a clonal myeloproliferative neoplasm defined by the presence of BCR-ABL fusion gene. This oncogenic event occurs in a hematopoietic stem cell (HSC) involved in CML initiation, maintenance, relapse and progression. Several evidences suggest that inflammatory pathways may participate to the pathophysiology of CML as well as disease progression to blast crisis. It has been shown that NFKB/REL pathway is constitutively activated both in BCR-ABL positive leukemic cell lines as well as in primary blast cells from CML-BC patients. More recent works identified IL6 as key cytokine acting on CML multipotent progenitors and their normal bystander counterpart to favor their differentiation toward the myeloid lineage. In addition, high levels of autocrine TNFα secretion by quiescent CML stem/progenitor cells activate NFKB pathway and promote their survival. Although all of these observations are linked to inflammatory processes, a focused analysis of inflammatory pathways in primary CML stem cells has not been performed so far. In the present study we undertook a text-mining strategy using pubmed e-querying to generate an exhaustive set of genes linked to inflammation. Then we integrated transcriptome analysis of highly purified CML stem cells to evaluate the contribution of these genes in CML development and progression. Methods : We queried 6 key words (Inflammation, macrophages, inflammatory response, chemokines, leukocytes and interleukins) that returned a total of 332000 hits in Pubmed. A raw gene set of 918 genes was found significantly associated (p<0.05) with these hits. Using R-package, we applied a false discovery rate correction that decreased the set to 588 relevant genes. The expression level of this gene set was then analyzed in previously reported microarray data (GEO accession: GSE47927) of highly purified normal cord blood CD34+CD38-CD90+ HSCs (CB; n=3), chronic phase (CP; n= 6), accelerated phase (AP; n =4) and Blast crisis (BC; n=2) CML cells. Results: Among the 588 genes related to inflammation we found 70 genes differentially expressed between the four groups (normal, CP, AP and BC, p<0.01; ANOVA test). Enrichment analysis confirmed 29 up regulated genes (NES = 2.12; p<0.0001) among which IL-6, PARP1, IL1R2, IRF5, IRF8, IL20. 39 genes such as STAT3, STAT4, CD47, CXCR4 IL-11, IL15, TLR-1, were down-regulated in CML CD34+CD38-CD90+ (all phases) as compared with normal HSCs (NES = -2,58; p<0.0001). Using principal component analysis on the 70 inflammatory deregulated genes we identified 10 genes such as IRAK1, IL1R2, VEGF and ESAM that discriminate "all phase" CML samples from normal HSCs (Dim 2 = 22.7%). Another inflammatory gene subset (n=26 genes) comprising IL6, REL, CXCR4, CXCL2, IL11, TLR1, IL1R2, PPARA highly separated CML stem cells according to the disease phase. The later gene set highly separates CP and AP-CML stem cells from BC-CML stem cell (Dim 1 = 50.3%). We next performed a random forest analysis with machine learning (1000 trees) and found that the inflammatory transcript level that best predicted CML phase was REL transcription factor. The expression of 413 genes were found positively correlated with REL expression in CP, AP and BC-CML CD34+CD38-CD90+ cells (r>0.75 and p-value <0.001). A search using JASPAR and TRANSFAC database identified a significant enrichment of NFKB1 and RELA binding motif in the promoter regions of these 413 genes (p<0.00001) among which several regulatory factors of hematopoietic stem cell biology. Conclusion : Using a bio-integrative approach we identified a specific inflammatory signature in CD34+CD38-CD90+ CML stem cells. This inflammatory network is highly altered in blast crisis suggesting its contribution to disease evolution. We identified REL overexpression as a good predictor for disease progression to blast crisis and found NFKB1and RELA (p=3.2x10-13) as the best REL target candidates. RELA/NFKB1 was previously shown to be constitutively activated in CML and Ph+ ALL and this analysis suggests that this may also take place in the most primitive subset of CML cells although REL may be the main partner of NFKB in CML stem cells. These results which are currently validated using functional assays, could lead to identification of novel therapeutic strategies. Disclosures Turhan: Bristol Myers Squibb: Consultancy; Novartis: Research Funding.

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