Cooperative Targeting of Bcl-2 Family Proteins By ABT-199 (GDC-0199) and Tyrosine Kinase Inhibitors to Eradicate Blast Crisis CML and CML Stem/Progenitor Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 512-512 ◽  
Author(s):  
Bing Z Carter ◽  
Po Yee Mak ◽  
Hong Mu ◽  
Hongsheng Zhou ◽  
Duncan H Mak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Blast Crisis ◽  
Single Agent ◽  
Specific Inhibitor ◽  
Chronic Phase

Abstract Bcr-Abl tyrosine kinase supports CML cell survival in part by regulating antiapoptotic Bcl-2 proteins such as Bcl-xL and Mcl-1. Tyrosine kinase inhibition, the front-line therapy for patients with chronic phase CML, is less effective in blast crisis (BC) patients and inactive against quiescent CML stem/progenitor cells. We reported that ABT-737, a dual Bcl-2/Bcl-xL inhibitor, induces apoptosis in BC CML cells including CD34+quiescent CML cells. ABT-199, a potent Bcl-2 specific inhibitor, has entered clinical trials for various hematological malignancies. We hypothesized that cooperative targeting of antiapoptotic Bcl-2 proteins using a combination of ABT-199 and tyrosine kinase inhibitors (TKIs) would exert enhanced activity against BC CML and CML stem/progenitor cells. Cells from patients (n=4) with TKI-resistant BC CML were treated with ABT-199, TKIs, and combinations. Although exerting low activity by itself, ABT-199 in combination with TKIs synergistically induced apoptosis (CI<0.1) in bulk and CD34+38- cells from these patients regardless of their previous clinical responses to TKIs. The combinations had minimal activity against normal CD34+cells (n=3). Mechanistic studies demonstrated that nilotinib inhibited the expression of Bcl-xL and Mcl-1 mRNA and protein, even in cells from TKI (including nilotinib) resistant patients. Individual inhibition of Bcl-xL or Mcl-1, and even more so inhibition of both, by siRNAs increased the sensitivity of cells to ABT-199, suggesting that cooperative inhibition of Bcl-2 by ABT-199 and Bcl-xL/Mcl-1 by TKIs contributes to the synergy. To evaluate the effect of these combinations on TKI-insensitive quiescent stem/progenitor CML cells, BC CML patient cells were stained with the cell division-tracking dye carboxyfluorescein succinimidyl ester (CFSE) and then co-cultured with human bone marrow (BM)-derived mesenchymal stromal cells (MSCs). Once proliferating and quiescent cells were distinguishable by flow cytometry, cells were treated with ABT-199, TKIs, and their combinations for 48 hours with or without MSC co-culture. Apoptosis was measured in proliferating and quiescent progenitor cells, defined as the percentage of annexin V positivity in CD34+CFSEdim and CD34+CFSEbright cells, respectively. ABT-199 as a single agent decreased viability of CML cells cultured alone or co-cultured with MSCs in both proliferating (IC50=191±103nM and 194±64nM, respectively) and quiescent (IC50=221±75nM and 205±123nM, respectively) CD34+ CML cells. Combinations of ABT-199 with TKIs, including imatinib, nilotinib, dasatinib, or ponatinib, synergistically induced death (CI<0.2) and decreased the number of viable cells in proliferating as well as quiescent CD34+progenitor cell populations (n=6). All 6 patients were resistant to TKIs, and 4 had mutations in the BCR-ABL gene, including three with the T315I mutation. To further test the ability of ABT-199 and TKI combinations to eradicate CML stem cells, we used an inducible transgenic CML mouse model in which the BCR-ABL gene is expressed under control of a tet-regulated enhancer of the murine stem cell leukemia (Scl) gene, allowing targeted BCR-ABL expression in stem/progenitor cells. Once BM cells from transgenic Scl-tTa-BCR-ABL/GFP mice were engrafted in wild type recipient mice, the mice were treated with ABT-199, nilotinib, or both. At the end of a 3-week treatment period, each single agent alone, and even more so with the combinations, significantly decreased blood total GFP+ WBC (12.9±1.4, 5.2±0.3, 6.1±0.4, and 1.6±0.3 x106/ml in controls, ABT-199, nilotinib, and combination, respectively) and neutrophils (1.43±0.03, 0.49±0.06, 0.32±0.03, and 0.25±0.05 x106/ml in the respective groups). ABT-199 (P=0.02), and more so with the combination (P<0.01) but not nilotinib alone (P=0.29), significantly decreased BM GFP+ LSK cells (12.0±1.2, 6.8±0.6, 9.5±1.6, and 2.2±0.2 x103 cells in the respective groups). The in vivo experiments are ongoing. Conclusions: ABT-199 and TKIs cooperatively target antiapoptotic Bcl-2 family proteins. This combination is highly effective in killing bulk and CD34+38- CML cells and quiescent CD34+ CML stem/progenitor cells from BC CML patients in vitro and in suppressing leukemia and leukemia stem cells in vivo. This strategy has the potential to eradicate BC CML cells and CML stem/progenitor cells, neither of which are effectively targeted by TKIs alone. Disclosures Carter: AbbVie, Inc.: Research Funding. Leverson:AbbVie, Inc.: Employment. Konopleva:AbbVie, Inc: clinic trial Other.

The Pan-Bcl-2 Family Inhibitor 97C1 Targets Blast Crisis Chronic Myeloid Leukemia Stem Cells but Spares Normal Cord Blood Progenitor Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 516-516 ◽  
Author(s):  
Daniel Goff ◽  
Alice Shih ◽  
Angela Court Recart ◽  
Larisa Balaian ◽  
Ryan Chuang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Blast Crisis ◽  
Chemotherapy Resistance ◽  
Chronic Phase ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 516 Introduction: Several studies have demonstrated the role of leukemia stem cells (LSC) in the development and maintenance of human chronic myeloid leukemia (CML). These cells, which first develop in chronic phase CML (CP CML) with acquisition of the BCR-ABL fusion protein, are often quiescent and can be highly resistant to apoptosis induced by drugs and radiotherapy that target rapidly dividing cells. Data has also shown that CML LSC become increasingly resistant to BCR-ABL inhibition with progression to blast crisis CML (BC CML). Bcl-2 family proteins are key regulators of apoptosis and have been shown by numerous studies to regulate cancer resistance to chemotherapy. This family of proteins has also been implicated in the development of BC CML, however most studies have focused on CML cell lines and their expression of Bcl-2 family proteins in vitro. Thus, there is relatively little data on expression of Bcl-2 family proteins in primary CML LSC and on the role of these proteins in regulating chemotherapy resistance in CML LSC in vivo. As Bcl-2 family proteins are known regulators of chemotherapy resistance we hypothesized that human BC CML LSC may overexpress these proteins compared to normal hematopoietic stem cells. We analyzed Bcl-2 family mRNA and protein expression in CP CML and BC CML LSC and compared this expression to normal cord blood stem and progenitor cells. We also analyzed whether these cells were sensitive to chemotherapy treatment in vitro. Finally, we tested whether a high potency pan-Bcl-2 inhibitor, 97C1, could effectively kill CML LSC in vitro and in vivo. Methods: Bcl-2 and Mcl-1 protein expression was measured in primary CP CML, BC CML, and normal cord blood cells using intracellular FACS. We also measured Bcl-2, Mcl-1, Bcl-X, and Bfl-1 mRNA expression in FACS sorted CD34+CD38+lin− cells (LSC) from these samples. For all drug studies we used either serially transplanted CD34+ cells derived from primary BC CML patient samples or primary CD34+ normal cord blood cells. In vitro drug responses were tested by culturing CD34+ cells either alone or in co-culture with a mouse bone marrow stromal cell line (SL/M2). Effects on colony formation and replating were also tested by culturing sorted CD34+CD38+lin− cells in methylcellulose in the presence and absence of drug. For in vivo testing of 97C1 we transplanted neonatal RAG2-/-yc-/- mice with CD34+ cells from 3 different BC CML and cord blood samples. Transplanted mice were screened for peripheral blood engraftment at 6–8 weeks post-transplant and engrafted mice were then treated for 2 weeks with 97C1 by IP injection. Following the treatment period the mice were sacrificed and hemotapoietic organs were analyzed for human engraftment by FACS. Results: BC CML progenitors expressed higher levels of Bcl-2 and Mcl-1 protein compared to normal cord blood and chronic phase CML cells. mRNA expression of Mcl-1, Bcl-X, and Bfl-1 was also increased in BC CML progenitors compared to CP CML progenitors. While BC CML LSC cultured in vitro were resistant to etoposide and dasatinib-induced cell death, 97C1 treatment led to a dose-dependent increase in cell death along with a dose-dependent decrease in the frequency of CD34+CD38+lin− cells compared to vehicle treated controls. While cord blood progenitor cells were also sensitive to 97C1 treatment they had an IC50 around 10 times higher than that for the BC CML cells (100nM versus 10nM). Importantly, 97C1 treatment did not inhibit cord blood colony formation or colony replating in vitro. Mice transplanted with BC CML LSC developed CML in 6–8 weeks post-transplant with diffuse myeloid sarcomas and engraftment of human CD34+CD38+lin− cells in the peripheral blood, liver, spleen, and bone marrow. In vivo treatment with 97C1 led to a significant reduction in both total human engraftment and engraftment of CD34+CD38+lin− cells in all hematopoietic organs analyzed. Conclusion: Our results demonstrate that BC CML LSC are resistant to conventional chemotherapy but are sensitive to 97C1 in vitro and in vivo. Broad-spectrum inhibition of Bcl-2 family proteins may help to eliminate CML LSC while sparing normal hematopoietic stem and progenitor cells. Disclosures: Jamieson: CoronadoBiosciences: Research Funding; CIRM: Research Funding.

Long Term Observation of CML Patients after Imatinib Resistance Associated with BCR-ABL Mutations.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1086-1086 ◽  
Author(s):  
Andreas Hochhaus ◽  
T. Ernst ◽  
P. Erben ◽  
M. C. Mueller ◽  
M. Emig ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Median Survival ◽  
Kinase Inhibitors ◽  
Clonal Selection ◽  
Direct Sequencing ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Activation Loop ◽  
Imatinib Resistance

Abstract The major cause of imatinib resistance in CML patients (pts) is clonal selection of cells harboring mutations of the BCR-ABL tyrosine kinase associated with various degrees of insensitivity to the drug. Management and outcome of these pts have been a matter of debate. In particular, retrospective studies suggested a poor prognosis of pts with P-loop mutations, whilst others did not confirm this observation. We sought to determine the outcome of 121 CML pts (73 m, 48 f; median age 61 yrs, range 28–80) with BCR-ABL mutations detected by direct sequencing and associated with hematologic (n=105), cytogenetic (n=14), or molecular (n=2) resistance. Imatinib was commenced between 8/99 and 3/05 after a median of 33.1 mo (range 0–312.3) after diagnosis, the median exposure to the drug was 17.0 mo (0.9–55.4). Resistance occurred after 18.1 mo (0.9–55.4) in chronic phase (CP, n=63), 18.3 mo (5.1–52.8) in accelerated phase (AP, n=33), 11.4 mo (0.9–47.7) in myeloid blast crisis (BC, n=20), and 9.1 mo (1.8–45.6) in lymphoid BC (n=5). Resistance was associated with 41 different mutations leading to a change of 32 amino acids (aa) of the P-loop area (n=38), T315I (n=15), the activation loop (n=11), and other sites (n=74). 15 pts showed 2, 2 pts 3, and one patient 4 mutations in parallel. In addition, resistance was associated with a loss of 27 aa including the P-loop in 2 pts. ABL polymorphisms were detected in 3 pts (T315T, n=1; K247R, n=2). Median time until resistance was 12.1 mo (0.9–52.8) for pts with P-loop mutations, 11.5 mo (2.8–47.7) for T315I, 17.2 mo (5.0–35.5) for activation loop mutations, and 20.4 (2.0–55.4) for others. After hematologic resistance, imatinib therapy was continued for 3.5 mo (0–54.1). Dose was increased in 42 pts up to 800mg/d, which was associated with disease stabilization in 7, hematologic improvement in 4, and cytogenetic or molecular improvement in 2 cases. In 46 pts, imatinib was combined with other drugs, 33 received low dose cytarabine. After imatinib withdrawal, treatment was continued with hydroxyurea, n=53, cytarabine, n=32, and/or other cytostatic drugs. 10 pts underwent allogeneic stem cell transplantation (SCT), 5 of them died. Recently, 2nd generation tyrosine kinase inhibitors were administered to 35 pts (dasatinib, n=19, AMN107, n=15, both drugs, n=1). Censoring pts at the time of SCT or start of alternative tyrosine kinase inhibitors, overall survival after resistance was 29.7 mo (0–52.3). 38 pts died; of these, mutations were located in the P-loop, n=16, at T315, n=4, or at other sites, n=18. Median survival after hematologic resistance was 29.7 mo for pts with P-loop mutations, 13.0 mo for pts with T315I, and 33.5 mo for others (n.s.). Considering CP pts only, median survival in case of P-loop mutations was 35.8 mo and has not been reached for pts with T315I and others (n.s.). In advanced disease, median survival with P-loop mutations was 17.0 mo, for T315I 13.0 mo, and for others 29.5 mo (n.s.). In conclusion, this prospective analysis failed to reveal a significantly impaired survival of pts with P-loop mutations compared to others. There is, however, a trend for a survival disadvantage in pts harboring T315I. Outcome of CML pts with mutations associated with insensitivity to imatinib, in particular P-loop and T315I mutations can be improved by early detection of the mutation and rapid withdrawal of the drug.

scholarly journals Exosomes from Bone Marrow Microenvironment-Derived Mesenchymal Stem Cells Affect CML Cells Growth and Promote Drug Resistance to Tyrosine Kinase Inhibitors

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Xiaoyan Zhang ◽  
Yazhi Yang ◽  
Yang Yang ◽  
Huijun Chen ◽  
Huaijun Tu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Drug Resistance ◽  
Mesenchymal Stem Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Bone Marrow Microenvironment

Although major advances have been achieved in the treatment of chronic myeloid leukemia (CML) by using tyrosine kinase inhibitors, patients relapse after withdrawal and need long-term medication. This reflects the CML clones have not been eliminated completely. The precise mechanisms for the maintenance of CML cells are not yet fully understood. The bone marrow microenvironment constitutes the sanctuary for leukemic cells. Mesenchymal stem cells (MSC) are an important component of the bone marrow microenvironment (BM). It plays an important role in the development and drug resistance of CML. Accumulating evidence indicates that exosomes play a vital role in cell-to-cell communication. We successfully isolated and purified exosomes from human bone marrow microenvironment-derived mesenchymal stem cells (hBMMSC-Exo) by serial centrifugation. In the present study, we investigated the effect of hBMMSC-Exo on the proliferation, apoptosis, and drug resistance of CML cells. The results demonstrated that hBMMSC-Exo had the ability to inhibit the proliferation of CML cells in vitro via miR-15a and arrest cell cycle in the G0/G1 phase. However, the results obtained from BALB/c nu/nu mice studies apparently contradicted the in vitro results. In fact, hBMMSC-Exo increased tumor incidence and promoted tumor growth in vivo. Further study showed the antiapoptotic protein Bcl-2 expression increased, whereas the Caspase3 expression decreased. Moreover, the in vivo study in the xenograft tumor model showed that hBMMSC-Exo promoted the proliferation and decreased the sensitivity of CML cells to tyrosine kinase inhibitors, resulting in drug resistance. These results demonstrated that hBMMSC-Exo supported the maintenance of CML cells and drug resistance in BM by cell-extrinsic protective mechanisms. They also suggested that hBMMSC-Exo might be a potential target to overcome the microenvironment-mediated drug resistance.

BMS-214662 potently induces apoptosis of chronic myeloid leukemia stem and progenitor cells and synergizes with tyrosine kinase inhibitors

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2843-2853 ◽  
Author(s):  
Mhairi Copland ◽  
Francesca Pellicano ◽  
Linda Richmond ◽  
Elaine K. Allan ◽  
Ashley Hamilton ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Hematopoietic Stem

Chronic myeloid leukemia (CML), a hematopoietic stem-cell disorder, cannot be eradicated by conventional chemotherapy or the tyrosine kinase inhibitor imatinib mesylate (IM). To target CML stem/progenitor cells, we investigated BMS-214662, a cytotoxic farnesyltransferase inhibitor, previously reported to kill nonproliferating tumor cells. IM or dasatinib alone reversibly arrested proliferation of CML stem/progenitor cells without inducing apoptosis. In contrast, BMS-214662, alone or in combination with IM or dasatinib, potently induced apoptosis of both proliferating and quiescent CML stem/progenitor cells with less than 1% recovery of Philadelphia-positive long-term culture-initiating cells. Normal stem/progenitor cells were relatively spared by BMS-214662, suggesting selectivity for leukemic stem/progenitor cells. The ability to induce selective apoptosis of leukemic stem/progenitor cells was unique to BMS-214662 and not seen with a structurally similar agent BMS-225975. BMS-214662 was cytotoxic against CML blast crisis stem/progenitor cells, particularly in combination with a tyrosine kinase inhibitor and equally effective in cell lines harboring wild-type vs mutant BCR-ABL, including the T315I mutation. This is the first report of an agent with activity in resistant and blast crisis CML that selectively kills CML stem/progenitor cells through apoptosis and offers potential for eradication of chronic phase CML.

Effective and Selective Elimination of CML Stem Cells Using Novel Ethacrynic Acid Derivatives

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4508-4508
Author(s):  
Su Chu ◽  
YinWei Ho ◽  
Guisen Zhao ◽  
Tessa L. Holyoake ◽  
Samuel Waxman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Protein Expression ◽  
Progenitor Cells ◽  
Ethacrynic Acid ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Cd34 Cells ◽  
Induced Apoptosis

Abstract Tyrosine kinase inhibitors (TKI) are highly effective in the treatment of CML but do not eliminate primitive, quiescent leukemia stem cells (LSC), which persist as a potential source of leukemia relapse. Additional strategies to enhance eradication of LSC are required to increase the possibility of treatment free remissions for CML patients. Glutathione S-transferase P1-1 (GSTP1-1) is a Phase II detoxifying enzyme which is overexpressed in several cancers and causes drug resistance. The diuretic Ethacrynic acid (EA) is a GSTP1-1 activity inhibitor and also induces celldeath in malignant cells at high concentrations. We have synthesized two EAoxadiazole analogs, 6U and 6S, which demonstrate enhancedpro-apoptotic effects in CML K562 cells expressing high levels of GSTP1-1. Previously we found that 6U and 6S induced apoptosis through downregulation of anti-apoptotic protein MCL-1 in addition to their GSTP1-1 activity inhibition. We extended these observations to primary CML stem/progenitor cells. We observed increased expression of GSTP1-1 mRNA and protein, associated with increased expression of MCL-1, BCL2 and BCL-xL, in chronic phase (CP) and blast crisis (BC) CML compared to normal CD34+ cells. Treatment of CP CML CD34+ cells with 6U or 6S (1 to 6µM) for 24 to 48 hours resulted in a significant dose-dependent induction of apoptosis, inhibition of cell growth, and reduction in colony forming cell (CFC) numbers, with 6U demonstrating greater efficacy than 6S. Treatment with 6U did not induce significant apoptosis of normal (NL) CD34+ cells at doses below 4µM. 6U induced significantly less apoptosis in NL compared with CML CD34+ cells (2µM, p<0.05). We further tested the activity of 6U against purified CML and normal CD34+CD38- stem/primitive progenitors and CD34+CD38+ committed progenitors with or without the BCR-ABL TKI Dasatinib (DAS). 6U treatment induced apoptosis of CML, but not normal, CD34+CD38- and CD34+CD38+ cells (Table). Combination of 6U with DAS (50nM) selectively enhanced apoptosis of CML compared to normal cells, including quiescent, slowly dividing CML LSC that are resistant to TKI-induced apoptosis (p≤0.01). Treatment with 6U alone or with DAS, significantly increased G1, and decreased S/G2/M phase of CML, but not in normal CD34+ cells, and reduced CFC growth from CML CD34+CD38+ cells (Table). CML, but not normal CD34+ cells, treated with 6U, with or without DAS, prior to transplant, showed significantly reduced engraftment in NSG mice, indicating selective inhibition of in vivo repopulating CML LSC (Table). Treatment with 6U was also effective in inducing apoptosis and inhibiting CFC growth in BC CML progenitor cells (Table). 6U treatment resulted in down-regulation of GSTPI1-1 and MCL-1 protein expression in CP and BC CML, but not in normal CD34+ cells. Interestingly 6U treatment also reduced BCR-ABL protein expression in CP and BC CML CD34+ cells. We conclude that CML CP and BC LSC express high levels of GSTP1-1 and anti-apoptotic proteins, which can be targeted by the novel EA derivative 6U through a new mechanism. Since 6U has significantly lesser effects on normal stem cells, it may offer a promising and innovative approach to selectively target CP and BC CML LSC in combination with TKI inhibitors. Abstract 4508. Table CML CP Normal CML BC Ctrl 6U DAS DAS+ 6U Ctrl 6U DAS DAS+ 6U Ctrl 6U DAS DAS+ 6U Apoptosis (normal, CP CML: CD34+CD38-; CML BC CD34+) 3.4± 0.9 15.9±6.7 9.4± 2.6 47.4±13.6 ** 3.3± 0.9 5.1± 1.0 1.6± 0.2 7.0± 1.2 * 3.4± 0.7 30±12.7 10.6±1.8 43.3±14.1 ** CFU-GM (normal, CP CML: CD34+CD38+; CML BC CD34+) 71.3± 7.8 7± 3.2 ** 21± 7.3 ** 5 ± 2.3 ** 121±19.3 102.7±6.2 134.3±15.9 103±5.1 288.5±89.4 26.5±11.3 *** 82.7±33.1 ** 8 ± 3.6 *** NSG engraftment (CD34+) 1.8± 0.3 0.4± 0.1 *** 0.8± 0.3 ** 0.4± 0.04 *** 68.2± 4.9 61± 2.2 68.1± 2.9 64.2± 3.9 Data shown are mean ± SEM of 3-6 samples. Significance, compared to controls. *p≤0.05,**p≤0.01, ***p≤0.001 Disclosures No relevant conflicts of interest to declare.

scholarly journals Activation of apoptosis signaling eliminates CD34+ progenitor cells in blast crisis CML independent of response to tyrosine kinase inhibitors

Leukemia ◽  
2011 ◽  
Vol 26 (4) ◽  
pp. 788-794 ◽  
Author(s):  
D H Mak ◽  
R-Y Wang ◽  
W D Schober ◽  
M Konopleva ◽  
J Cortes ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Blast Crisis

In Vivo Analyses of Leukemia Stem Cells in Genetically Defined Murine Models of Chronic and Blast Crisis CML.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 236-236
Author(s):  
Craig T. Jordan ◽  
Sarah J. Neering ◽  
Pin-Yi Wang ◽  
Randall M. Rossi ◽  
Timothy Bushnell
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Leukemic Cells ◽  
Leukemia Stem Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Normal Controls

Abstract Studies to date have shown that primary human leukemia stem cells (LSC) are resistant to standard chemotherapy agents and are likely to be a major cause of drug refractory disease and relapse. Therefore, elucidating the in vivo biology of LSC is critical in order to develop more effective therapeutic regimens. To this end, we report the first genetically defined model of LSC, using syngeneic murine systems in which the biological features of human LSC are recapitulated. The approach employs retroviral vectors to transduce normal murine hematopoietic stem cells with either BCR/ABL-GFP alone, or in combination with Nup98/HoxA9-YFP. Expression of BCR/ABL creates a well-described model of chronic phase CML, whereas expression of BCR/ABL in combination with Nup98/HoxA9 induces acute disease that mimics blast crisis CML. Analysis of the normal cell competent to generate LSC indicates that the BCR/ABL mutation must occur in primitive HSC in order to manifest disease, however, subsequent progression to blast crisis can occur through mutation in cells at the myeloid progenitor stage. Characterization of stem cells in these models revealed several striking features. First, chronic phase stem cells are1 phenotypically identical to normal hematopoietic stem cells (lin−, Sca-1+, c-kit+) and display cell cycle rates (percentage of cells in S or G2 phase) that are nearly double normal controls. However, the overall frequency of such cells is not elevated. In contrast, blast crisis stem cells show a distinct immunophenotype (lin−, Sca-1+, c-kit-lo, Flt3+, CD150−) and cycle rates nearly identical to normal controls, but are approximately 10-fold increased numbers. These data indicate that BCR/ABL alone functions as a stem cell mitogen, but does not enhance self-renewal, whereas added expression of Nup98/HoxA9 is sufficient to increase self-renewal, but return cell cycle regulation to normal levels. Furthermore, analysis of co-resident non-leukemic cells in each model shows that while the cycle activity of normal stem cells (HSC) was not affected, the cycle rates of normal progenitors (lin−, c-kit+) were substantially reduced. Thus, in either disease, active suppression of normal progenitors is evident and thereby increases the growth advantage of malignant populations. To test methods for modulation of normal vs. leukemic cells in vivo, we challenged blast crisis animals with ara-C (single dose, 100mg/kg) or imatinib mesylate (200mg/kg/day for 3 consecutive days) and assessed the consequences in primitive populations. The data indicate that ara-C reduced frequency and cycle rate of progenitor cells in vivo, but that the effects were identical between normal and malignant populations. Thus, at least for short-term studies there was no therapeutic index for ara-C at the level of primitive cells. In contrast, treatment with imatinib induced a 50% increase in the cycle rate and a 2–4 fold increase in numbers of progenitor cells. These findings imply a homeostatic mechanism in blast crisis leukemia, where pressure towards the malignant population may induce increased activity of stem and progenitor cells. In summary, this model provides a novel means by which the biology of LSCs may be directly characterized and the consequences of candidate treatment regimens can be assessed with regard to normal vs. leukemia stem cells in vivo.

scholarly journals Targeting USP47 overcomes tyrosine kinase inhibitor resistance and eradicates leukemia stem/progenitor cells in chronic myelogenous leukemia

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hu Lei ◽  
Han-Zhang Xu ◽  
Hui-Zhuang Shan ◽  
Meng Liu ◽  
Ying Lu ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Chronic Myelogenous Leukemia ◽  
Drug Targets ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Myelogenous Leukemia ◽  
Tki Resistance

AbstractIdentifying novel drug targets to overcome resistance to tyrosine kinase inhibitors (TKIs) and eradicating leukemia stem/progenitor cells are required for the treatment of chronic myelogenous leukemia (CML). Here, we show that ubiquitin-specific peptidase 47 (USP47) is a potential target to overcome TKI resistance. Functional analysis shows that USP47 knockdown represses proliferation of CML cells sensitive or resistant to imatinib in vitro and in vivo. The knockout of Usp47 significantly inhibits BCR-ABL and BCR-ABLT315I-induced CML in mice with the reduction of Lin−Sca1+c-Kit+ CML stem/progenitor cells. Mechanistic studies show that stabilizing Y-box binding protein 1 contributes to USP47-mediated DNA damage repair in CML cells. Inhibiting USP47 by P22077 exerts cytotoxicity to CML cells with or without TKI resistance in vitro and in vivo. Moreover, P22077 eliminates leukemia stem/progenitor cells in CML mice. Together, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate leukemia stem/progenitor cells in CML.

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