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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 883-883 ◽  
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.

scholarly journals BCR-ABL enhances differentiation of long-term repopulating hematopoietic stem cells

Blood ◽  
2010 ◽  
Vol 115 (16) ◽  
pp. 3185-3195 ◽  
Author(s):  
Mirle Schemionek ◽  
Christian Elling ◽  
Ulrich Steidl ◽  
Nicole Bäumer ◽  
Ashley Hamilton ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Multipotent Progenitor Cells

Abstract In a previously developed inducible transgenic mouse model of chronic myeloid leukemia, we now demonstrate that the disease is transplantable using BCR-ABL+ Lin−Sca-1+c-kit+ (LSK) cells. Interestingly, the phenotype is more severe when unfractionated bone marrow cells are transplanted, yet neither progenitor cells (Lin−Sca-1−c-kit+), nor mature granulocytes (CD11b+Gr-1+), nor potential stem cell niche cells (CD45−Ter119−) are able to transmit the disease or alter the phenotype. The phenotype is largely independent of BCR-ABL priming before transplantation. However, prolonged BCR-ABL expression abrogates the potential of LSK cells to induce full-blown disease in secondary recipients and increases the fraction of multipotent progenitor cells at the expense of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow. BCR-ABL alters the expression of genes involved in proliferation, survival, and hematopoietic development, probably contributing to the reduced LT-HSC frequency within BCR-ABL+ LSK cells. Reversion of BCR-ABL, or treatment with imatinib, eradicates mature cells, whereas leukemic stem cells persist, giving rise to relapsed chronic myeloid leukemia on reinduction of BCR-ABL, or imatinib withdrawal. Our results suggest that BCR-ABL induces differentiation of LT-HSCs and decreases their self-renewal capacity.

scholarly journals DYRK2 Inhibits the Self-Renewal of Leukemic Stem Cells in Chronic Myeloid Leukemia By Inducing Degradation of c-Myc Downstream of the Reprogramming Factor KLF4

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1879-1879
Author(s):  
Chun Shik Park ◽  
Ye Shen ◽  
Koramit Suppipat ◽  
Andrew Lewis ◽  
Julie Tomolonis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Parp Cleavage ◽  
Leukemic Stem Cells ◽  
Vitamin K3 ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Chronic myeloid leukemia (CML) is a blood cancer originated by expression of BCR-ABL, a constitutively activated kinase product of the chromosomal translocation t(9;22), in hematopoietic stem cells (HSC). Although tyrosine kinase inhibitors (TKI) can efficiently induce molecular remission in CML patients, drug discontinuation often leads to relapse caused by reactivation of leukemic stem cells (LSC) spared from TKI therapy via BCR-ABL-independent mechanisms of self-renewal and survival. Thus, there is a need for alternative drugs for relapse patients to prevent expansion of BCR-ABL-positive LSC during discontinuation of chemotherapy or emergence of chemoresistance. We found that somatic deletion of the reprogramming factor Krüppel-like factor 4 (KLF4) in BCR-ABL(p210)-induced CML severely impaired disease maintenance. This inability to sustain CML in the absence of KLF4 was caused by a progressive attrition of LSCs in bone marrow and the spleen and impaired ability of LSCs to recapitulate leukemia in secondary recipients. Analyses of global gene expression and genome-wide binding of KLF4 revealed that the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) is repressed by KLF4 in CML LSCs. Immunoblots revealed elevated levels of DYRK2 protein that were associated with a reduction of c-Myc protein and increased levels of p53 (S46) phosphorylation and PARP cleavage in KLF4-deficient LSCs purified from the bone marrow of CML mice. Genomic silencing of KLF4 in the murine CML cell line 32D-BCR-ABL resulted in increased levels of DYRK2 and phosphorylated c-Myc (S62) leading to diminished levels of c-Myc protein, which was reverted by treatment with a proteasome inhibitor, suggesting that KLF4 prevents c-Myc degradation triggered by DYRK2-mediated priming phosphorylation. Consistent with an inhibitory role in leukemia, DYRK2 levels are significantly reduced both in CD34+CD38+ and CD34+CD38− cells from CML patients compared to normal stem/progenitor cells. Aiming at pharmacological activation of DYRK2 to abrogate self-renewal and survival of CML cells, we treated CML cells with vitamin K3 that inhibits Siah2, an ubiquitin E3 ligase involved in Dyrk2 proteolysis. Vitamin K3, and not Vitamin K1 and K2, induces dose-dependent cytotoxicity in a panel of human-derived CML cell lines by stabilizing Dyrk2 protein and consequently promoting c-Myc degradation. Interestingly, combination of vitamin K3 with Imatinib exhibit additive effect inducing cytotoxicity in CML cells. Collectively, the identification of Dyrk2 as a critical mediator of LSC downfall is a novel paradigm poised to support the development of LSC-specific therapy to induce treatment-free remission in conjunction with Imitinib in CML patients. Disclosures No relevant conflicts of interest to declare.

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.

Role of Osteoclasts in Regulation of Human Hematopoietic Stem Cell Niche and Fate.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4253-4253
Author(s):  
Shmuel Yaccoby ◽  
Kenichiro Yata ◽  
Yun Ge ◽  
Bart Barlogie ◽  
Joshua Epstein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Scid Mice ◽  
Common Mechanism ◽  
Hematopoietic Stem ◽  
Variable Expression ◽  
Cd34 Cells ◽  
Rabbit Bone

Abstract Recent studies indicate that osteoblasts play an important role in maintaining hematopoietic stem cells (HSCs) niche in the bone marrow microenvironment. The aim of study was to test the effect of osteoclasts on the fate of HSCs in a long term co-culture assay. To generate osteoclasts, peripheral blood mononuclear cells from mobilized donors were cultured for 6–10 days in αMEM media supplemented with 10% FCS, M-CSF and RANKL. After removal of non-adherent cells, the cultures contained 95% multinucleated osteoclasts and their precursors. These osteoclasts expressed TRAP and formed resorption pits on bone slices (Yaccoby et al., Cancer Res., 2004). CD34+ cells were purified from donor PBSCs and cord blood using immunomagnetic beads separation (>95% purity). Adult and cord blood HSCs were co-cultured with osteoclasts for up to 3 and 10 months, respectively, in media lacking any cytokines. Because osteoclasts do not survive long without M-CSF and RANKL, the HSCs were transferred to fresh osteoclast cultures every 6–10 days. Unlike their tight adherence to stromal cells, HSCs did not adhere to the osteoclasts and were easily recovered from co-cultures by gentle pipetting. Following 1 to 3 weeks of co-culture, committed HSCs rapidly differentiated into various hematopoietic cell lineage, followed by phagocytosis of terminal differentiated hematopoietic cells by the osteoclasts. The remaining HSCs were highly viable (>90% by trypan blue exclusion) and gradually lost their CD34 expression, so that the cultures contained subpopulations of HSCs expressing CD34−/lowCD38+ and CD34−/lowCD38−. Quantitive real time RT-PCR (qRT-PCR) revealed loss of expression of CD34 and reduced expression of CD45 by HSCs co-cultured with osteoclasts longer than 6 weeks. Variable expression of CD34 on HSCs was previously reported in murine but not human HSCs (Tajima et al., Blood, 2001). The co-cultured HSCs showed reduced capacity of generating in vitro hematopoietic colonies, and did not differentiate into osteoclasts upon stimulation with M-CSF and RANKL. We next tested the long term engraftment of these co-cultured HSCs in 2 animal models. In the first model, cord blood and adult HSCs from 2 donors recovered after >6 weeks in co-culture were injected I.V. into irradiated NOD/SCID mice. In the second novel model, co-cultured cord blood and adult HSCs from 2 donors were injected directly into rabbit bones implanted subcutaneously in SCID mice (SCID-rab model), 6–8 weeks after rabbit bone implantation. After 2–4 months, 10%±3% human CD45-expressing cells were identified in the NOD/SCID mice femora and 8%±4% in the SCID-rab mice rabbit bone. Our study suggests that osteoclasts promote rapid differentiation of committed HSCs and induce conversion of CD34+ cells to CD34− stem cells with self renewal potential. Intriguingly, long term co-culture of primary CD138-selected myeloma plasma cells (n=16) with osteoclasts resulted in dedifferentiation of tumor cells from a mature CD45− phenotype to an immature, CD45-expressing cells, suggesting a common mechanism of osteoclast-induced HSC and myeloma cell plasticity. This indicates that osteoclasts are important bone marrow component regulating human HSC niche, plasticity and fate.

Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 112-118 ◽  
Author(s):  
Mo A. Dao ◽  
Jesusa Arevalo ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cd34 Expression

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

scholarly journals The Role of Notch in Endothelial Cell-Mediated Protection of AML Precursors from Targeted Therapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3947-3947
Author(s):  
Quy Le ◽  
Brandon Hadland ◽  
Soheil Meshinchi ◽  
Irwin D. Bernstein
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Notch Signaling ◽  
Liquid Culture ◽  
Critical Role ◽  
Common Mutation ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Inhibitory Antibodies

Abstract Background: AML is an aggressive hematologic malignancy that remains difficult to treat. A common mutation found in AML is FLT3-ITD, occurring in 15% of childhood AML. Although chemotherapy has successfully induced remission, patients with a high FLT3 ITD:WT allelic ratio (FLT3-AR) exhibit a high relapse rate, requiring hematopoietic stem cell transplantation to increase the chance of long-term remission. In this study, we demonstrate the requirement of ECs for survival of FLT3-ITD progenitors from primary pediatric AML specimens in the presence of AC220, a potent and selective inhibitor of FLT3. We further show that the Notch pathway plays a role in EC-mediated protection amongst patient samples with high FLT3-AR, suggesting the potential therapeutic use of Notch blockade in the treatment of this high-risk subset. Results: To determine whether ECs confer protection to FLT3-ITD progenitors, we quantified the number of CFC present after 2 weeks of liquid culture or EC co-culture with AC220 (added at days 0, 3 and 7) from four AML specimens with high FLT3-AR (≥1). We used PCR to determine the presence of FLT3-ITD in individual CFC. We found that the numbers of FLT3-ITD CFC (p=0.007) and FLT3-WT CFC (p=0.044) were reduced in liquid culture compared to EC co-culture, suggesting that ECs mediate the survival of FLT3-ITD hematopoietic progenitors against the therapeutic treatment of AC220. Previously, we demonstrated that ECs are critical for the growth and expansion of hematopoietic stem cells, which is dependent on the activation of Notch signaling. We asked whether Notch plays a role in EC-mediated protection of AML progenitors against AC220, using RNA-seq analysis on three FLT3-ITD-harboring AML. Among the significantly altered genes (FDR<0.05), we found an enrichment of Notch target genes that were expressed at significantly higher levels in AC220-treated cells compared to DMSO-treated cells, including HES1, HES4, NRARP, CDKN1A, CCND1, andGATA3, suggesting that Notch signaling may facilitate EC-mediated protection against AC220. Next, we assessed the effect of inhibiting Notch signaling on AML progenitor survival during AC220 treatment in EC co-culture, using inhibitory antibodies specific to the Negative Regulatory Region (NRR) of both Notch1 and Notch2 receptors (anti-NRR1 and anti-NRR2; kindly provided by Chris Siebel, Genentech). We co-cultured bone marrow cells from eight patient specimens with low FLT3-AR (<1) and five patient specimens with high FLT3-AR (≥1), with ECs and briefly treated the co-cultures with Notch inhibitory antibodies or IgG1 antibody for 3 days. AC220 was added to the cultures at days 0, 3 and 7. We assessed CFC numbers present after 2 weeks of culture. Patient samples with low FLT3-AR did not exhibit changes in the numbers of FLT3-ITD CFC (p = 0.735) and FLT3-WT CFC (p = 0.489) in response to Notch inhibition relative to IgG1 control. In contrast, patient samples with high FLT3-AR showed reduction in the number of FLT3-ITD CFC (p=0.019) but the number of FLT3-WT CFC remained unaffected (p=0.874). These results suggest a critical role for Notch in EC-mediated protection in AML with high FLT3-AR. Conclusion: Our studies suggest that inhibiting Notch signaling may have therapeutic potential for overcoming drug resistance induced by the tumor microenvironment in a subset of AML with high FLT3-AR. We have previously shown that a high FLT3-AR is associated with the presence of FLT3-ITD in the least mature hematopoietic subset (CD34+ CD33- precursors), which is thought to contain leukemic stem cells, and this association is correlated with poorer outcome. Additionally, AML cells that give rise to CFC after long-term co-culture with bone marrow stroma or ECs are derived from the CD34+CD33- AML precursors. Ongoing studies aim to determine whether Notch signaling plays a role in the survival of AML CD34+CD33- cells with the goal of eliminating leukemic stem cells responsible for relapse. Disclosures No relevant conflicts of interest to declare.

scholarly journals Leukemia Stem Cells as a Potential Target to Achieve Therapy-Free Remission in Chronic Myeloid Leukemia

Cancers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 5822
Author(s):  
Kyoko Ito ◽  
Keisuke Ito
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Drug Binding ◽  
Leukemia Stem Cells ◽  
Phase Point ◽  
Hematopoietic Stem ◽  
Wide Range ◽  
Tki Resistance

Leukemia stem cells (LSCs, also known as leukemia-initiating cells) not only drive leukemia initiation and progression, but also contribute to drug resistance and/or disease relapse. Therefore, eradication of every last LSC is critical for a patient’s long-term cure. Chronic myeloid leukemia (CML) is a myeloproliferative disorder that arises from multipotent hematopoietic stem and progenitor cells. Tyrosine kinase inhibitors (TKIs) have dramatically improved long-term outcomes and quality of life for patients with CML in the chronic phase. Point mutations of the kinase domain of BCR-ABL1 lead to TKI resistance through a reduction in drug binding, and as a result, several new generations of TKIs have been introduced to the clinic. Some patients develop TKI resistance without known mutations, however, and the presence of LSCs is believed to be at least partially associated with resistance development and CML relapse. We previously proposed targeting quiescent LSCs as a therapeutic approach to CML, and a number of potential strategies for targeting insensitive LSCs have been presented over the last decade. The identification of specific markers distinguishing CML-LSCs from healthy HSCs, and the potential contributions of the bone marrow microenvironment to CML pathogenesis, have also been explored. Nonetheless, 25% of CML patients are still expected to switch TKIs at least once, and various TKI discontinuation studies have shown a wide range in the incidence of molecular relapse (from 30% to 60%). In this review, we revisit the current knowledge regarding the role(s) of LSCs in CML leukemogenesis and response to pharmacological treatment and explore how durable treatment-free remission may be achieved and maintained after discontinuing TKI treatment.

scholarly journals KLF4 Controls Leukemic Stem Cell Self-Renewal in MLL-AF9-Induced Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1231-1231
Author(s):  
Andrew Lewis ◽  
Chun Shik Park ◽  
Monica Puppi ◽  
H. Daniel Lacorazza
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Epigenetic Mechanisms ◽  
Leukemic Stem Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Limiting Dilution

Acute myeloid leukemia (AML) develops from sequential mutations which transform hematopoietic stem and progenitor cells (HSPCs) in the bone marrow into leukemic stem cells (LSCs) which drive the progression of frank leukemia. Especially poor outcomes in elderly patients coupled with frequent relapse have led to a dismal 28.3% 5-year survival, warranting the need for innovative therapeutic approaches. Successful targeted therapy will selectively eliminate LSCs, which possess distinct characteristics enabling self-renewal and chemotherapeutic resistance, while sparing normal HSPCs. We theorized that KLF4, a zinc finger transcription factor, maintains key self-renewal pathways in LSCs due to its known importance in preserving stemness in embryonic and cancer stem cells. KLF4 alters gene transcription through its activating and repressing domains as well as remodeling chromatin through various epigenetic mechanisms, and work from our lab has demonstrated that loss of KLF4 in leukemia driven by the BCR-ABL fusion oncogene results in depletion of LSCs (Park et. al in revision) while enhancing self-renewal of hematopoietic stem cells. To address this hypothesis, mice featuring floxed Klf4 gene (Klf4fl/fl) were crossed with transgenic Vav-iCre mice to produce mice with hematopoietic-specific deletion of Klf4 (Klf4Δ/Δ). The murine t(9;11)(p21;q23) translocation (MLL-AF9 or MA9) transduction model has previously been shown to reflect clinical disease attributes, and represents the MLL-rearranged human patient subset with particularly poor prognosis and relatively higher levels of KLF4. Lin−Sca-1+c-Kit+ (LSK) cells from Klf4fl/fl and Klf4Δ/Δ mice were transduced with retrovirus containing MA9 and GFP reporter and transplanted into lethally-irradiated wild-type (WT) mice to generate trackable Klf4fl/fl and Klf4Δ/ΔAMLs. Recipients of both MA9Klf4fl/fl and Klf4Δ/Δ cells developed a rapid expansion of leukemic cells with myeloid immunophenotype by flow cytometric analysis (CD11b+Gr-1+; 68-91%), characterized as AML with latency of approximately 44.5 days. To quantify the defect induced by loss of KLF4 in the leukemic stem cell population, we performed secondary transplant of multiple limiting-dilution cell doses of primary transformed leukemic bone marrow from moribund mice. Klf4Δ/Δ AML mice exhibited significantly improved survival in all dose-cohorts, in some cases presenting no detectable leukemic cells at completion of monitoring (225 days). Limiting dilution analysis using the ELDA online software tool demonstrated a 7-fold reduction from 1 in 513 in Klf4fl/fl to 1 in 3836 in Klf4Δ/Δ AML bone marrow cells capable of leukemic initiation function (p<0.001), a hallmark of LSCs. Using the ERCre-tamoxifen inducible deletion system, Klf4 deletion 15 days post-transplant of AML significantly improved survival of Klf4Δ/Δ mice compared to controls, demonstrating KLF4 promotes maintenance of disease. Plating of leukemic bone marrow from Klf4Δ/Δ mice in methylcellulose medium revealed a reduction in serial colony-forming ability, further supporting a defect in self-renewal. To further determine the mechanisms connected to this reduction in functional LSCs, we isolated leukemic granulocyte-macrophage progenitors (L-GMPs), a population previously reported to be highly enriched for functional LSCs and representing a comparable cellular subset in human clinical samples, from Klf4fl/fl and Klf4Δ/Δ AMLs and conducted RNA-Seq to identify potential transcriptional targets of KLF4 with therapeutic promise. Taken together, these data suggest a novel function of the stemness transcription factor KLF4 in the preservation of leukemic stem cells in AML. Whereas prior models based on KLF4 expression in human cell lines and bulk AML samples have proposed a tumor suppressive role, our work suggests KLF4 supports expansion of leukemic cells with a stem cell phenotype and serial assays suggest an effect on LSC self-renewal. Further studies are being conducted to define the transcriptional and epigenetic mechanisms governing these findings. Understanding the molecular changes induced by loss of KLF4 presents promise for development of new therapies selectively targeting LSCs. Disclosures No relevant conflicts of interest to declare.

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