scholarly journals C/EBPβ is a critical mediator of IFN-α–induced exhaustion of chronic myeloid leukemia stem cells

2019 ◽  
Vol 3 (3) ◽  
pp. 476-488 ◽  
Author(s):  
Asumi Yokota ◽  
Hideyo Hirai ◽  
Ryuichi Sato ◽  
Hiroko Adachi ◽  
Fumiko Sato ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Interferon Α ◽  
Dependent Manner ◽  
Distal Enhancer ◽  
Induced Differentiation ◽  
Ifn Γ

Abstract Even in the era of ABL tyrosine kinase inhibitors, eradication of chronic myeloid leukemia (CML) stem cells is necessary for complete cure of the disease. Interferon-α (IFN-α) has long been used for the treatment of chronic-phase CML, but its mechanisms of action against CML stem cells remain unclear. We found that IFN-α upregulated CCAAT/enhancer binding protein β (C/EBPβ) in BCR-ABL–expressing mouse cells by activating STAT1 and STAT5, which were recruited to a newly identified 3′ distal enhancer of Cebpb that contains tandemly aligned IFN-γ–activated site elements. Suppression or deletion of the IFN-γ–activated site elements abrogated IFN-α–dependent upregulation of C/EBPβ. IFN-α induced differentiation and exhaustion of CML stem cells, both in vitro and in vivo, in a C/EBPβ-dependent manner. In addition, IFN-α upregulated C/EBPβ and induced exhaustion of lineage− CD34+ cells from CML patients. Collectively, these results clearly indicate that C/EBPβ is a critical mediator of IFN-α–induced differentiation and exhaustion of CML stem cells.

Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 319-325 ◽  
Author(s):  
Susan M. Graham ◽  
Heather G. Jørgensen ◽  
Elaine Allan ◽  
Charlie Pearson ◽  
Michael J. Alcorn ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Growth Factors ◽  
Chronic Myeloid Leukemia ◽  
Antiproliferative Activity ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Chronic Phase ◽  
Quiescent State

In clinical trials, the tyrosine kinase inhibitor STI571 has proven highly effective in reducing leukemic cell burden in chronic myeloid leukemia (CML). The overall sensitivity of CML CD34+ progenitor cells to STI571 and the degree to which cell death was dependent on cell cycle status were determined. Stem cells (Lin−CD34+) from the peripheral blood of patients with CML in chronic phase and from granulocyte–colony-stimulating factor–mobilized healthy donors were labeled with carboxy-fluorescein diacetate succinimidyl diester dye to enable high-resolution tracking of cell division. Then they were cultured for 3 days with and without growth factors ± STI571. After culture, the cells were separated by fluorescence-activated cell sorting into populations of viable quiescent versus cycling cells for genotyping. For healthy controls, in the presence of growth factors, STI571 affected neither cell cycle kinetics nor recovery of viable cells. In the absence of growth factors, normal cells were unable to divide. For CML samples, in the presence or absence of growth factors, the response to STI571 was variable. In the most sensitive cases, STI571 killed almost all dividing cells; however, a significant population of viable CD34+ cells was recovered in the undivided peak and confirmed to be part of the leukemic clone. STI571 also appeared to exhibit antiproliferative activity on the quiescent population. These studies confirm that CML stem cells remain viable in a quiescent state even in the presence of growth factors and STI571. Despite dramatic short-term responses in vivo, such in vitro insensitivity to STI571, in combination with its demonstrated antiproliferative activity, could translate into disease relapse after prolonged therapy.

scholarly journals Cytotoxic T cells induce proliferation of chronic myeloid leukemia stem cells by secreting interferon-γ

2013 ◽  
Vol 210 (3) ◽  
pp. 605-621 ◽  
Author(s):  
Christian Schürch ◽  
Carsten Riether ◽  
Michael A. Amrein ◽  
Adrian F. Ochsenbein
Keyword(s):  
Stem Cells ◽  
Immune System ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Cytotoxic T Cells ◽  
Treatment Strategies ◽  
Hematopoietic Stem ◽  
Ifn Γ

Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia arising from the oncogenic break point cluster region/Abelson murine leukemia viral oncogene homolog 1 translocation in hematopoietic stem cells (HSCs), resulting in a leukemia stem cell (LSC). Curing CML depends on the eradication of LSCs. Unfortunately, LSCs are resistant to current treatment strategies. The host’s immune system is thought to contribute to disease control, and several immunotherapy strategies are under investigation. However, the interaction of the immune system with LSCs is poorly defined. In the present study, we use a murine CML model to show that LSCs express major histocompatibility complex (MHC) and co-stimulatory molecules and are recognized and killed by leukemia-specific CD8+ effector CTLs in vitro. In contrast, therapeutic infusions of effector CTLs into CML mice in vivo failed to eradicate LSCs but, paradoxically, increased LSC numbers. LSC proliferation and differentiation was induced by CTL-secreted IFN-γ. Effector CTLs were only able to eliminate LSCs in a situation with minimal leukemia load where CTL-secreted IFN-γ levels were low. In addition, IFN-γ increased proliferation and colony formation of CD34+ stem/progenitor cells from CML patients in vitro. Our study reveals a novel mechanism by which the immune system contributes to leukemia progression and may be important to improve T cell–based immunotherapy against leukemia.

scholarly journals Multicolor Immunophenotyping of Candidate Leukemic Stem Cell Markers in CD34+CD38- Chronic Myeloid Leukemia Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2922-2922
Author(s):  
Martin Culen ◽  
Marianna Romzova ◽  
Dagmar Smitalova ◽  
Tomas Loja ◽  
Jiri Mayer
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Correlation Coefficient ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Surface Expression ◽  
Specific Marker ◽  
Stem Cell Markers ◽  
Similar Frequency ◽  
Median Difference

Introduction: Detection of leukemic stem cells (LSCs) may represent a new potential prognostic parameter in chronic myeloid leukemia (CML) and a tool for minimal residual disease monitoring in combination with standard qPCR method. To date CD26 is studied as a most specific marker for CML LSC detection. Several other candidate LSC markers have been reported, such as IL1-RAP, CD25 and CD93, however a side-by-side testing of their specificity is lacking. Recently, we have identified CD69 molecule to be overexpressed in CD34+CD38-CD26+ cells, which makes this antigen another candidate marker for LSC. Aim: To compare the surface expression of LSC markers CD69, CD26, CD25, CD56, IL1-RAP, CD56, CD93 in bulk CD34+CD38- population at diagnosis using a multicolor phenotypization assay Methods: In total, 44 patients were analyzed at diagnosis of chronic phase CML before administration of any treatment. Fresh (n=38) or cryopreserved (n=6) leukocytes obtained by erythrolysis were stained with CD45, CD34, CD38, CD25, CD26, CD56, CD69, CD93, IL1-RAP antibodies and 7-AAD for selection of live cells. Analysis was performed on FACSAria Fusion (BD Biosciences). Acquisition of live mononuclear cells ranged from 2×104 to 2.5×106. Results: Expression of candidate LSC markers CD26, CD25, CD56, IL1-RAP, CD56, CD93 was analyzed in BM of 35 patients who carried at least 30 CD34+CD38- cells. Median percentage of marker positive cells was 34% for IL1-RAP, 31% for CD25, 23% for CD26, 16% for CD56 and 2% for CD93, from the parent CD34+CD38- population. Next we analyzed the overlap and combination for the three best markers - IL1-RAP, CD25 and CD26. The 3-combination (defined as IL1-RAP or CD25 or CD26 expression) identified 40% of CD34+CD38- positive cells, which was more than any of the markers alone. Expression of the three markers showed good overlap and ruled out mutually exclusive expression of the markers. This was demonstrated by median difference of 0.4% of CD34+CD38- cells (range: 0-18%) and a correlation coefficient r2=0.9914, when comparing the 3-combination and the best performing marker in each patient. In contrast, in 12/35 (34%) of patients, one of the three markers failed to identify at least half of the cells positive for another marker. In 21/35 patients, we also analyzed the expression of CD69 in the CD34+CD38- compartment. The CD69 showed similar performance as the 3-combination of CD26, CD25, and IL1-RAP, 59% vs 54% positive cells, respectively. We observed excellent overlap between CD69 and 3-combination expression in individual patients with median difference of 0% of CD34+CD38- cells (range 0-15%) and correlation coefficient r2=0.9890. Furthermore, we compared marker positivity in BM vs PB in 19 paired samples. Both, sample types showed similar frequency of CD34+CD38- cells (5×10-3 in BM, and 2×10-3 for PB), but PB carried higher percentage of LSCs identified by the 3-combination - median 76 vs 52% cells. Conclusions: We show an overlap in surface expression of three previously reported CML LSC markers - IL1-RAP, CD25 and CD26. Nevertheless, a combination of these markers can detect more positive cells than any of the markers alone. Moreover, we demonstrate that CD69 identifies the same cells within the CD34+CD38- compartment as the combination of three above mentioned markers, which makes CD69 the best candidate for routine CML LSC quantification. Disclosures Mayer: AOP Orphan Pharmaceuticals AG: Research Funding.

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

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 801-807 ◽  
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.

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

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 801-807 ◽  
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.

scholarly journals Autografting with cultured marrow in chronic myeloid leukemia: results of a pilot study [see comments]

Blood ◽  
1994 ◽  
Vol 84 (3) ◽  
pp. 724-732 ◽  
Author(s):  
MJ Barnett ◽  
CJ Eaves ◽  
GL Phillips ◽  
RD Gascoyne ◽  
DE Hogge ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Intensive Therapy ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Phase Group ◽  
Cytogenetic Remission ◽  
Group 2 ◽  
Group 1

Abstract Incubation of chronic myeloid leukemia (CML) marrow for 10 days in vitro causes a marked and selective loss of very primitive Philadelphia chromosome (Ph)+ as compared with Ph- progenitors. We have autografted 22 patients with CML (16 in first chronic phase [group 1] and 6 with more advanced disease [group 2]) with marrow treated in this way to facilitate restoration of Ph- hematopoiesis after intensive therapy. Hematologic recovery to greater than 0.5 x 10(9)/L neutrophils occurred in 16 patients, and to greater than 20 x 10(9)/L platelets in 15 of 21 evaluable patients at a median of 29 and 48 days postautograft, respectively. Regenerating marrow cells were 100% Ph- in 13 patients and 75% to 94% Ph- in 3. Between 4 and 36 months (median 12) postautograft, Ph+ cells became detectable in all but 1 (who died in remission) of the 13 patients who achieved complete cytogenetic remission. Four of 7 evaluable patients treated with low-dose interferon alpha were returned to complete cytogenetic remission. Thirteen group 1 patients (81%) are alive 1.0 to 5.7 years (median 2.6) after autografting: 4 in complete cytogenetic remission, 2 in hematologic remission, 6 in chronic phase, and 1 in myeloid blast phase. Three group 2 patients (50%) are alive at 2.6, 3.8, and 4.3 years after autografting: 1 in partial cytogenetic remission, 1 in chronic phase, and 1 in accelerated phase. Thus, autografts of cultured marrow can result in prolonged restoration of Ph- hematopoiesis for some patients with CML.

scholarly journals Phenotypic heterogeneity of primitive leukemic hematopoietic cells in patients with chronic myeloid leukemia

Blood ◽  
1992 ◽  
Vol 80 (10) ◽  
pp. 2522-2530 ◽  
Author(s):  
C Udomsakdi ◽  
CJ Eaves ◽  
PM Lansdorp ◽  
AC Eaves
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Philadelphia Chromosome ◽  
Significant Proportion ◽  
Hematopoietic Cells ◽  
Chronic Phase ◽  
Leukemic Cells ◽  
Clonogenic Cell ◽  
Clonogenic Cells

Abstract The peripheral blood of chronic myeloid leukemia (CML) patients with chronic-phase disease and elevated white blood cell (WBC) counts typically contains markedly increased numbers of a variety of neoplastic pluripotent and lineage-restricted hematopoietic progenitors. These include cells detected in standard colony assays as well as their more primitive precursors. The latter are referred to as long-term culture-initiating cells (LTC-IC) because of their ability to generate clonogenic cell progeny detectable after a minimum of 5 weeks incubation on competent fibroblast feeder layers. In this study, we have investigated a number of the properties of the LTC-IC and clonogenic cells present in the blood of such CML patients with high WBC counts. This included an analysis of the light scattering properties of these progenitors, as well as their expression of CD34 and HLA-DR, Rhodamine-123 staining, and in vitro sensitivity to 4- hydroperoxycyclophosphamide. In the case of LTC-IC, the production of different types of lineage-restricted and multipotent progeny was also analyzed. Most of the circulating LTC-IC and clonogenic cells in the CML patients studied (on average approximately 70% and approximately 90%, respectively) showed features of proliferating or activated cells. This is in marked contrast to the majority of progenitors in the blood of normal individuals and most of the LTC-IC in normal marrow, all of which exhibit a phenotype expected of quiescent cells. Interestingly, a significant proportion of the circulating clonogenic cells and LTC-IC in the CML samples studied (on average approximately 10% and approximately 30%, respectively) appeared to be phenotypically similar to normal circulating progenitors, although their absolute numbers were indicative of a neoplastic origin. Both phenotypes of circulating CML clonogenic cells and LTC-IC could be obtained at approximately 10% to 20% purity by differential multiparameter sorting. These findings suggest that expansion of the Philadelphia chromosome-positive clone at the level of the earliest types of hematopoietic cells results from the activation of mechanisms that enable some, but not all, signals that block the cycling of normal stem cells to be bypassed or overcome. In addition, they provide strategies for purifying these primitive leukemic cells that should facilitate further analysis of the mechanisms underlying their abnormal proliferative behavior.

Favorable long-term follow-up results over 6 years for response, survival, and safety with imatinib mesylate therapy in chronic-phase chronic myeloid leukemia after failure of interferon-α treatment

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1039-1043 ◽  
Author(s):  
Andreas Hochhaus ◽  
Brian Druker ◽  
Charles Sawyers ◽  
Francois Guilhot ◽  
Charles A. Schiffer ◽  
...  
Keyword(s):  
Overall Survival ◽  
Chronic Myeloid Leukemia ◽  
Imatinib Mesylate ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Cytogenetic Response ◽  
Interferon Α ◽  
Imatinib Treatment ◽  
Serious Adverse Events ◽  
Response Level

Abstract Imatinib mesylate, a targeted inhibitor of BCR-ABL tyrosine kinase, is the standard of care for chronic myeloid leukemia (CML). A phase 2 trial of imatinib in late chronic-phase (CP) CML after interferon-α (IFNα) failure enrolled 532 patients, 454 with a confirmed diagnosis of CP CML. Median time from diagnosis was 34 months; median duration of imatinib treatment was 65 months. Cumulative best rates of major cytogenetic response (MCyR) and complete cytogenetic response (CCyR) were 67% and 57%, respectively. At the 5-year landmark, 184 (41%) of the 454 patients are in CCyR. At more than 6 years, 199 (44%) of the 454 patients remain on imatinib. Most responses occurred within 12 months of starting imatinib; however, some patients achieved initial MCyR and CCyR more than 5 years after imatinib initiation. Estimated rates of freedom from progression to accelerated phase (AP) and blastic phase (BP) and overall survival at 6 years were 61% and 76%, respectively. Both freedom from progression to AP/BP and overall survival (OS) were associated with cytogenetic response level at 12 months. No increase in rates of serious adverse events was observed with continuous use of imatinib for up to 6.5 years, compared with earlier time points. Imatinib continues to be an effective and safe therapy for patients with CP CML after failure of IFN.

scholarly journals OCCURRENCE OF SECONDARY MALIGNANCIES IN CHRONIC MYELOID LEUKEMIA DURING THERAPY WITH IMATINIB MESYLATE-SINGLE INSTITUTION EXPERIENCE

2015 ◽  
Vol 7 ◽  
pp. e2015003 ◽  
Author(s):  
Grzegorz Helbig ◽  
Grazyna Bober ◽  
Marek Seweryn ◽  
Ryszard Wichary ◽  
Andrzej Tukiendorf ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Imatinib Mesylate ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Disease Free Survival ◽  
Interferon Α ◽  
Secondary Malignancies ◽  
Single Institution ◽  
Chi Squared ◽  
Disseminated Disease

Introduction. Imatinib mesylate (IM) remains a treatment of choice for chronic myeloid leukemia (CML) showing a remarkable efficacy and providing a perspective for a long disease-free survival. Due to the long-term administration of IM, the questions about the possible impact on the development of secondary malignancies (SM) are raised.Objective. To investigate the frequency and clinical outcome of secondary malignancies during IM therapy for CML.Material and Methods. The records of 221 CML patients treated with IM between 2003-2013 in single institution were reviewed. The Chi-squared test was used for statistic analysis.Results. Secondary malignancies developed in eight out of the 221 patients (3.6%) receiving IM for a median of 61 months (range, 10-137 months). Female/male ratio was 5/3. Two patients were diagnosed with their CML at accelerated phase whereas 6 had chronic phase. The median age at IM initiation was 58 years (range, 31-72 years).  Five of these 8 SM patients received IM after other treatments failure: interferon α (n=5), hydroxyurea (n=4) and cytarabine (n=1). Three patients received IM as a frontline therapy. All patients were on IM at 400mg daily at SM occurrence. The therapy for SM included surgery (n=3); chemotherapy only (n=3); and chemotherapy followed by radiotherapy (n=1). One patient did not receive treatment due to disseminated disease. At the time of SM development all patients were in hematologic and cytogenetic remission (CCR) of their CML and all patients continued their IM while receiving treatment for their SM.  Among 8 patients with SM, five patients are alive and remain in CCR on IM whereas 3 patients died due to SM. The observed incidence of SM was found to be comparable with that expected in the age-adjusted population (chi-squared=0.4; p=0.52).Conclusions. The association between IM therapy for CML and SM development has not been found. 

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