A Novel AHI-1-BCR-ABL-JAK2 Interaction Complex Mediates Cellular Resistance to Tyrosine Kinase Inhibitors in CML .

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 38-38
Author(s):  
Donna DeGeer ◽  
Kathleen Newmarch ◽  
Leon Zhou ◽  
Min Chen ◽  
Kyi Min Saw ◽  
...  
Keyword(s):  
Stem Cells ◽  
Protein Expression ◽  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Total Protein ◽  
Kinase Inhibitors ◽  
K562 Cells ◽  
Cellular Resistance ◽  
Total Protein Expression

Abstract Abstract 38 The molecular signature of chronic myeloid leukemia (CML) is the BCR-ABL fusion gene originating in a multipotent hematopoietic stem cell. The BCR-ABL oncoprotein (p210BCR-ABL) has constitutively elevated tyrosine kinase activity that perturbs several signalling cascades, including the PI3K/AKT, JAK2/STAT5, NF-kB, and RAS/MAPK pathways. The current first line treatment for CML is the tyrosine kinase inhibitor imatinib mesylate (IM) that induces clinical remission in most chronic phase CML patients. However, early relapses and IM-resistant disease have emerged and are frequently associated with mutations in the BCR-ABL kinase domain. Our recent studies indicate that CML stem cells are less responsive to IM and other tyrosine kinase inhibitors and are critical target population for IM resistance. It is therefore critical to identify other therapies that target CML stem cells to prevent acquisition of resistance. One candidate target is AHI-1 (Abelson helper integration site 1), a recently discovered oncogene that is deregulated in primary leukemic stem cells from CML patients. AHI-1 contains several domains indicative of signalling functions, including an SH3 and a WD40-repeat domain. We have recently identified a novel AHI-1-BCR-ABL-JAK2 interaction complex that modulates BCR-ABL transforming activity both in vitro and in vivo and play a key role in the IM response/resistance of primary CML stem/progenitor cells. To investigate AHI-1's involvement in mediating this cellular resistance to IM and to test the comparative ability of new ABL and JAK2 inhibitors to inhibit this complex in CML cells, AHI-1 was either stably overexpressed in K562 cells by transduction of EF1a-AHI-1-IRES-YFP lentivirus or suppressed in K562 cells using RNA interference. Interestingly, an increase in cellular proliferation and colony formation and a decrease in apoptosis were observed in the presence of 1, 5 and 10 uM of IM when AHI-1 was overexpressed. Survival of these cells was similar to IM resistant K562 cells, which are highly resistant to IM in vitro and display higher AHI-1 protein expression than parental K562 cells. Suppression of AHI-1 had the opposite effect, with cells displaying heightened sensitivity to IM at concentrations as low as 1 uM. Phosphorylation and total protein expression levels of several proteins known to be involved in BCR-ABL signalling, including JAK2, STAT5, MAPK, SRC, AKT and NF-kB (P105, P50, and P65 subunits), were quantified by Western blot analysis. Elevated phosphorylation and total protein expression levels of several of these proteins were observed when AHI-1 was overexpresessed, in particular in the JAK2/STAT5 pathway and especially in the presence of Interleukin 3. Due to the strong effects AHI-1 had on this signalling cascade, we next inhibited JAK2 activity using a selective JAK2 inhibitor, TG101209, that is highly effective against the V617F mutation and inhibits JAK2 and STAT5 activities in polycythemia vera progenitor cells. AHI-1 overexpressing cells showed reduced proliferation and colony formation when treated with IM and TG101209 in combination compared to either IM or TG101209 alone. Interestingly, treatment with IM (5 uM) or dasatinib (150 nM, DA) in combination with TG101209 (100 nM) resulted in greater inhibition (81% and 85%) of CD34+ CML stem/progenitor cells from IM nonresponders (n=4), compared to the same cells treated with a combination of IM and DA (∼60%, p<0.05), as measured by colony-forming cell assays. CFSE tracking analysis of cell division in these cells further demonstrated additive antiproliferative activity as a result of combined ABL and JAK2 inhibitors. These results suggest that targeting both BCR-ABL and JAK2 activities may be a potential therapeutic option for IM resistant patients. Disclosures: No relevant conflicts of interest to declare.

Effectively Targeting Treatment-Naïve CML Stem/Progenitor Cells From Imatinib-Nonresponders With Combination Treatments Of JAK2 and ABL Inhibitors In Vitro and In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1474-1474
Author(s):  
Hanyang Lin ◽  
Min Chen ◽  
Katharina Rothe ◽  
Matthew V Lorenzi ◽  
Adrian Woolfson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Combination Treatment ◽  
K562 Cells ◽  
Hematopoietic Stem ◽  
Combination Treatments ◽  
Treatment Naïve

Abstract The hallmark of chronic myeloid leukemia (CML) is the presence of a BCR-ABL fusion gene that originates in hematopoietic stem cells. The BCR-ABL oncoprotein has constitutively elevated tyrosine kinase (TK) activity and drives CML pathogenesis. Introduction of Imatinib Mesylate (IM) and other tyrosine kinase inhibitor (TKI) therapies has had a major impact on the treatment of chronic phase CML, but early relapses and persistence of leukemic stem cells remain problematic. We have recently identified an AHI-1–BCR-ABL–JAK2 protein complex that contributes to the transforming activity of BCR-ABL and to IM-resistance of CML stem/progenitor cells. We therefore hypothesized that combined suppression of BCR-ABL and JAK2 activities might more effectively eliminate CML stem/progenitor cells in vitro and in vivo. Several JAK2 inhibitors are currently in various stages of clinical trials, but their off-target effects on normal primitive hematopoietic cells remain a concern. We have now examined the biological effects of an orally bioavailable, selective JAK2 inhibitor (BMS-911543) in combination with TKIs, including IM, dasatinib (DA) and nilotinib, both on CML cells lines and CD34+ treatment-naïve IM-nonresponder cells, which were obtained at diagnosis from CML patients who were classified subsequently, after initiation of IM therapy, as IM-nonresponders. In cell line studies, Western blot analysis showed that combination treatment was more effective at reducing pSTAT5 levels in K562 cells and IM-resistant K562 cells than single agents. In colony-forming cell (CFC) assays, combination treatment resulted in a greater reduction in colonies produced from these cells compared to single agents (2-3 fold, p<0.05). Similarly, intracellular staining analyses showed that combined exposure of CD34+ CML cells (n=4) to BMS-911543 and a TKI produced a deeper, more prolonged suppression of pSTAT5 and pCRKL activity than single agents (40-46% suppression for the combinations vs. 15-20% suppression for the single agents at 72 hrs, p<0.05). Combination treatments also resulted in greater inhibition of colony growth of CD34+ CML cells compared to single agents (n=7, 74-86% vs. 40-50%, p<0.05). Interestingly, the combination of BMS-911543 and a TKI almost completely inhibited BFU-E colony formation as compared to treatment with TKI alone (92-100% vs.63-66%, p<0.01). CFU-GM colonies were also more significantly reduced as a result of combination treatment, compared to single agents (49-71% vs. 30-39%, p<0.01). Long-term culture-initiating cell assays showed that more primitive cells were also more significantly eliminated by combination treatments (n=3, 2-3 fold, p<0.05). Importantly, our CFC data indicate that BMS-911543 is less toxic to normal bone marrow (BM) CD34+ cells (n=7) than the same cells from CML samples (n=7, 2-3 fold, p<0.05). To test the ability of combination treatments to eliminate primitive CML cells with in vivo leukemia propagating activity, we injected primitive CML cells intravenously into NSG mice and treated mice with inhibitors by oral gavage for two weeks. Mice undergoing combination treatment showed significantly reduced weight loss and engraftment levels in peripheral blood, BM , spleen, and liver compared to mice treated with single agents (0.14% vs. 6.14%, 1.17% vs. 26.3%, 2.46% vs. 51.5%, and 77.8% vs. 92.9%, respectively, p<0.05). H&E histology staining revealed that mice treated with DA and BMS-911543 had less infiltration of leukemic cells into their spleens and livers than mice treated with DA alone. Quantitative RT-PCR analysis further demonstrated a statistically significant reduction in BCR-ABL transcript levels in spleen, liver, and BM of mice treated with a combination of DA and BMS-911543 compared to mice treated with single agents (15-20 fold, p<0.05). Most importantly, combination treatments significantly enhanced survival of leukemic mice compared to mice treated with single agents (median survival of IM + BMS-911543 vs. IM: 70 days vs. 60.5 days, p<0.05; DA + BMS-911543 vs. DA: 96.5 days vs. 81 days, p<0.001). This study suggests that simultaneously targeting BCR-ABL and JAK2 activities in CML stem/progenitor cells may improve outcomes in patients, especially those destined to develop TKI resistance. Disclosures: No relevant conflicts of interest to declare.

Identification of AHI-1 Involvement in Mediating Cellular Resistance to Imatinib Mesylate in Chronic Myeloid Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3201-3201
Author(s):  
Donna DeGeer ◽  
Leon Zhou ◽  
Min Chen ◽  
Yun Zhao ◽  
Ali G Turhan ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Protein Expression ◽  
Cell Line ◽  
Myeloid Leukemia ◽  
K562 Cells ◽  
K562 Cell Line ◽  
Cellular Resistance ◽  
Expression Levels ◽  
Total Protein Expression

Abstract Chronic myeloid leukemia (CML) is a clonal multilineage myeloproliferative disorder arising from the neoplastic transformation of a pluripotent hematopoietic stem cell that acquires a unique BCR-ABL fusion gene. The BCR-ABL oncoprotein displays constitutively elevated tyrosine kinase activity that deregulates cellular proliferation and apoptosis control through effects on several common signal transduction cascades, including the PI3K/AKT, JAK2/STAT5, and NF-kB pathways. The current first line treatment for CML involves administration of the tyrosine kinase inhibitor imatinib mesylate (IM) that has shown promise in treating chronic phase CML patients. However, early relapses and IM-resistant disease have emerged and are frequently associated with mutations in the BCR-ABL kinase domain that affect inhibitor binding. AHI-1 (Abelson helper integration site 1) is a recently discovered oncogene that has been demonstrated to be highly deregulated in a CML cell line (K562) and in primary leukemic stem/progenitor cells from CML patients. AHI-1 contains several unique domains that are indicative of signalling functions, including both an SH3 and a WD40-repeat domain. We have recently demonstrated that overexpression of murine Ahi-1 is able to transform IL-3 dependent Baf3, resulting in cells able to grow in the absence of growth factors. When these transduced cells were injected into sublethally irradiated NOD/SCID immunodeficient mice, the mice developed leukemia, demonstrating the oncogenic properties of Ahi-1. Interestingly, these in vitro and in vivo effects can be enhanced by co-transduction of BCR-ABL in these cells. In addition, a direct interaction between AHI-1 and BCR-ABL at endogenous levels was identified in K562 cells and this protein interaction complex further mediated IM response/resistance in CML stem/progenitor cells. To further investigate AHI-1’s involvement in mediating this cellular resistance to IM, AHI-1 was either stably overexpressed in K562 cells by transduction of EF1a-AHI-1-IRES-YFP lentivirus or was suppressed in K562 cells using a lentiviral-mediated RNA interference approach. Interestingly, overexpression of AHI-1 in K562 cells significantly increased cellular survival in the presence of 1, 5 and 10 uM of IM as measured by a viability assay; survival of these cells was similar to that observed in an IM resistant K562 cell line reported to be highly resistant to IM in vitro. Furthermore, suppression of AHI-1 had the opposite effect, with cells displaying heightened sensitivity to IM at concentrations as low as 1 uM. Phosphorylation and protein expression levels of several proteins known to be involved in BCR-ABL signalling, including JAK2, STAT5, AKT and NF-kB (P105, P50, and P65 subunits), were then quantified by Western blot analysis. Interestingly, elevated phosphorylation and protein expression levels of JAK2, and STAT5 and total protein expression levels NF-kB p105/p55 subunits were observed in both the AHI-1 overexpressing K562 cells and IM resistant K562 cells, while reduced phosphorylation and protein expression of these same proteins was observed in AHI-1 suppressed K562 cells. Differential expression of phosphorylated NF-kB p65 subunit at serine 536 was observed, while total protein expression levels did not significantly differ. Phosphorylated AKT expression levels were only affected in AHI-1 suppressed K562 cells, and total AKT protein expression was not affected in AHI-1 overexpressed or suppressed cells. Interestingly, AHI-1 protein expression was highly elevated at endogenous levels in the IM resistant K562 cells relative to a parental K562 cell line. These findings suggest that AHI-1 may play an important role in mediating cellular resistance to IM through activation of several signalling proteins involved in BCR-ABL signalling pathway, including JAK2 and STAT5.

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.

Inhibition of Bcl-2/Bcl-XL Promotes Apoptosis in Blast Crisis CML Including Quiescent Primitive Progenitor Cells Regardless of Cellular Responses to Tyrosine Kinase Inhibitors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 646-646
Author(s):  
Duncan H. Mak ◽  
Wendy D. Schober ◽  
Marina Konopleva ◽  
Jorge Cortes ◽  
Hagop M. Kantarjian ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Progenitor Cells ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
Antiapoptotic Proteins ◽  
Cell Compartments ◽  
Cd34 Cells

Abstract Abstract 646 The advent of imatinib, a Bcr-Abl tyrosine kinase inhibitor revolutionized the treatment for patients with CML. Development of resistance, limited activity in blast crisis CML, and more importantly, insensitivity of quiescent primitive CD34+ CML progenitor cells are evolving problems facing this therapy. Antiapoptotic Bcl-2 proteins were known to be highly expressed in Bcr-Abl expressing cells and inhibition of Bcl-2/Bcl-XL by the selective inhibitor ABT-737 was reported to augment the killing of tyrosine kinase inhibitors in CML cells. However, its effect on quiescent primitive CD34+ CML progenitor cells is unknown. To investigate the effect of activating the apoptotic machinery in quiescent primitive CD34+CML progenitor cells, which are resistant to current therapies, we first compared the expression of antiapoptotic proteins in proliferating and quiescent primitive CD34+CML progenitor cells. Cells obtained from patients with blast crisis CML were stained with the fluorescent 5-(and 6-) carboxy-fluorescein diacetate succinimidyl ester, a cell proliferation tracking dye, and cultured in vitro for 4-6 days. Cells were then stained with CD34 antibody and FACS sorted into proliferating and quiescent CD34+/PI- CML progenitor cells. RNA levels of antiapoptotic proteins in these two cell populations (n=8) were determined by real-time RT-PCR: quiescent and proliferating primitive CD34+ CML progenitor cells expressed similar levels of Bcl-2, Bcl-XL, Mcl-1, and XIAP implying that like total blast cells, quiescent primitive CD34+CML progenitor cells may also be sensitive to agents targeting these proteins. We next treated 5 samples obtained from patients with blast crisis CML with ABT-737 and measured apoptosis in total CD34+ cells, proliferating CD34+ cells, and quiescent CD34+ cells. All 5 patients were resistant to or relapsed from imatinib and nilotinib and/or dasatinib treatments and they were insensitive to imatinib in vitro as expected. However, cells from 4 patients were sensitive to ABT-737, in bulk blasts and in both proliferating and quiescent CD34+ CML cell compartments: % specific apoptosis with 100 nM of ABT-737=40.8±7.7, 38.4±8.5, 40.0±5.1, respectively at 24 hours. Interestingly, when ABT-737 was combined with imatinib, cell death was greatly enhanced in cells from all 5 patients in all cell compartments (combination index=0.059±0.032, 0.041±0.025, 0.111±0.042, respectively). Furthermore, we showed previously, that triptolide, an antitumor agent from a Chinese herb, induces apoptosis in both proliferating and quiescent primitive CD34+CML progenitor cells by decreasing Mcl-1 which is a resistant factor for ABT-737, XIAP, and Bcr-Abl protein levels (Mak D. et al., MCT in press). When ABT-737 was combined with triptolide, a significant increase of cell death was found in total CD34+ and proliferating as well as quiescent primitive CD34+CML cells with combination index at EC50=0.57, 0.55, and 0.56, respectively in cells from the 5 patients suggesting a high degree of synergism. In summary, Bcl-2, Bcl-XL, Mcl-1, and XIAP are equally expressed in proliferating and quiescent primitive CML cells and targeting Bcl-2/Bcl-XL promotes death of blast crisis CML cells, tyrosine kinase inhibitor resistant CML cells, and quiescent primitive CD34+ CML progenitor cells. Researches suggest that the combination of apoptosis inducing agents and tyrosine kinase inhibitor is a novel strategy to overcome tyrosine kinase resistance, eradicate quiescent primitive CML progenitor cells, and improve current therapy for patients with CML. Disclosures: No relevant conflicts of interest to declare.

Targeting Treatment-Naïve CML Stem/Progenitor Cells From Imatinib-Nonresponders with JAK2 (BMS-911543)/ABL Inhibitor Combination Therapy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1339-1339
Author(s):  
Hanyang Lin ◽  
Katharina Rothe ◽  
Matthew V Lorenzi ◽  
Adrian Woolfson ◽  
Xiaoyan Jiang
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Combination Treatment ◽  
Kinase Activity ◽  
K562 Cells ◽  
Tyrosine Kinase Activity ◽  
Employment Equity ◽  
Combination Treatments ◽  
Equity Ownership

Abstract Abstract 1339 Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder characterized by a BCR-ABL fusion gene with constitutive tyrosine kinase activity that drives the pathogenesis of the disease. Deregulated tyrosine kinase activity of BCR-ABL perturbs several intracellular signaling pathways, including the JAK2/STAT5 pathway. In particular, JAK2 physically interacts with the C-terminal region of BCR-ABL and is one of the most prominent targets of BCR-ABL. Recent introduction of imatinib mesylate (IM) and other tyrosine kinase inhibitor (TKI) therapies have had a major impact on treatment of chronic phase CML. However, early relapse, acquired drug resistance and ineffectiveness in eliminating leukemic stem cells remain impediments to successful treatment for many patients. Improved treatment approaches to prevent development of resistant subclones, by combined targeting of other key molecular elements active in CML stem cells, are thus clearly needed. One candidate is a complex between the oncoproteins encoded by Abelson helper integration site-1 (AHI-1), BCR-ABL and JAK2 that we recently identified. This complex contributes to the transforming activity of BCR-ABL and also plays a key role in the IM response/resistance of primary CML stem/progenitor cells. We have therefore hypothesized that combined suppression of BCR-ABL and JAK2 activities might be more effective in eliminating treatment-naïve CML stem/progenitor cells in patients destined to develop resistance to TKI monotherapy. Several JAK2 inhibitors are currently in various stages of clinical trials, but their nonspecific, off-target effects on normal primitive hematopoietic cells remain a concern. We have now examined the biological effects of a new, highly specific JAK2 inhibitor (BMS-911543) in combination with a number of TKIs, including IM, dasatinib and nilotinib. First, Western blot analysis showed that combination treatment was more effective at reducing pSTAT5 levels in K562 cells and IM-resistant K562 cells than single agents. Also, in colony-forming cell (CFC) assays, combination treatment resulted in a greater reduction in colonies produced from these cells compared to single agents, especially in medium-sized colonies (50–500 cells per colony, 2–3 fold, p<0.05). To determine whether the results obtained following the simultaneous targeting of BCR-ABL and JAK2 in CML cell lines would extend to primary primitive CML cells and whether this combined BCR-ABL-JAK2 targeting approach could also be therapeutically effective for CML patients who do not respond adequately to treatment with a single TKI, we investigated primitive CML cells obtained at diagnosis from seven CML patients who were classified retrospectively as IM-nonresponders, after initiation of IM therapy. Similar to our cell line studies, intracellular staining analysis showed that combined exposure of CD34+CML cells (n=3) to BMS-911543 and a TKI produced a deeper and more prolonged suppression of pSTAT5 activity than a single agent (40–46% suppression for the combinations vs. 15–20% suppression for the single agents at 72 hrs, p<0.05). Combination treatments also caused greater inhibition in colony growth of these cells compared to single treatments (74–86% vs. 40–50%, p<0.05). Interestingly, the combination of BMS-911543 and a TKI almost completely inhibits BFU-E colony formation as compared to treatment with TKI alone (92–100% vs.63–66%, p<0.01). CFU-GM colonies were also more significantly reduced as a result of combination treatment, compared to single agents (49–71% vs. 30–39%, p<0.01). Furthermore, long-term culture-initiating cell (LTC-IC) assays showed that the more primitive cells were also more significantly eliminated by combination treatments than by single agents (2–5 fold, p<0.05), indicating the merit of combination therapy in targeting very primitive cells. Most importantly, our CFC data indicate that BMS-911543 has far less toxicity on normal bone marrow CD34+ cells (n=4) than CML samples (2–3 fold, p<0.05). This study suggests a rational strategy for improving therapeutic outcomes in CML IM-nonresponders by simultaneously targeting both BCR-ABL and JAK2 activities in primary CML stem/progenitor cells. Disclosures: Lorenzi: Bristol-Myers Squibb: Employment, Equity Ownership. Woolfson:Bristol-Myers Squibb: Employment, Equity Ownership.

Foxo Transcription Factor Activity Is Retained in Quiescent Chronic Myeloid Leukaemia Stem Cells and Activated by Tyrosine Kinase Inhibitors to Mediate “induced-quiescence” in More Mature progenitors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 187-187 ◽  
Author(s):  
Daniela Cilloni ◽  
Francesca Pellicano ◽  
Vignir G Helgason ◽  
Cristina Panuzzo ◽  
Francesca Messa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Tyrosine Kinase ◽  
Cell Cycle Arrest ◽  
Progenitor Cells ◽  
Transcriptional Activity ◽  
Kinase Inhibitors ◽  
Akt Pathway ◽  
Cycle Arrest

Abstract Abstract 187 First line therapy for CML involves tyrosine kinase inhibitors (TKIs) which can induce rapid cytogenetic responses in the majority of patients in chronic phase (CP), but do not eliminate BCR-ABL transcripts in the majority, suggesting persistence of residual disease. These findings, together with the rapid kinetics of relapse in patients who discontinue TKIs, suggest the presence of a reservoir of TKI-resistant leukaemic stem cells, although the mechanism for TKI-insensitivity of CML stem cells remains unclear. The FoxO family of transcription factors is mainly regulated by PI3K/Akt induced phosphorylation, resulting in nuclear exclusion and degradation. FOXO activity is implicated in maintaining haemopoietic stem cell (HSC) quiescence. Its transcriptional activity in normal HSC results in cell cycle arrest by expression of p27, p130, p21, down-regulation of Cyclin D and protection from oxidative stress. Cell line studies suggest that FOXOs may play a central role in the anti-proliferative effects of TKIs, but their role in primary CML stem cells has not been previously investigated. Methods: Quiescent CML stem cells were isolated phenotypically (Lin−CD34+38−) by FACS from the total CD34+ stem/progenitor cell population and D-FISH analysis performed to determine the percentage of Ph+ cells. The expression levels of Spred1, FOXO1, FOXO3a, FOXO4 and Cyclin D1 were evaluated by Real-Time-PCR. Protein levels and localization were studied by Western blot, immunofluorescence and FACS. EMSA assay was used to evaluate FOXO3a DNA binding activity. K562 cells were transfected with wt FOXO3a and the constitutively active triple mutant (TM) form of FOXO. After transfection, proliferation and apoptosis were tested using incorporation of H3 thymidine and annexin V detection by FACS, respectively. Results: We found that BCR-ABL, through activation of the PI3K/Akt pathway, induces phosphorylation and cytoplasmic localization of FOXO in CD34+ CML cells, thereby blocking its transcriptional activity as demonstrated by EMSA and by the inhibition of FOXO target genes, including Spred1. Incubation with TKIs decreased phosphorylation and induced re-localization of FOXO to the nucleus in CD34+ CML cells, thus restoring FOXO transcriptional activity. This resulted in decreased levels of Cyclin D1 and reduced ROS. Similar effects and a dramatic reduction of cell proliferation, accompanied by significant apoptosis, were observed by forcing the expression of FOXO3a in K562 through transfection of the TM form, confirming the role of FOXO in inducing cell cycle arrest. Incubation experiments using LY294002, rapamycin and TKIs established that the reactivation of FOXO by TKI is mediated by the PI3K/Akt pathway. Interestingly, we found that phosphorylation of FOXO1, 3a and 4 was higher (i.e. cytoplasmic and inactive) in proliferating CD34+38+ CML cells, as compared to more primitive and quiescent CD34+38−90+ CML cells, although the activity of BCR-ABL, measured by analysis of p-CrKL was found to be similar in both populations, indicating that although present, BCR-ABL was not inducing phosphorylation of FOXO in the quiescent population. In this sense the quiescent CML stem cells resembled normal HSC in terms of FOXO regulation. In conclusion our data indicate that TKIs initiate a process in CML stem and progenitor cells that maintains their quiescence and therefore potential resistance to TKIs themselves. The anti-proliferative activity of TKIs against primary CML CD34+ cells is mediated, at least in part, by the re-activation of FOXO1, 3a and 4. BCR-ABL appears to play a different role in more mature progenitor cells compared to primitive quiescent stem cells, suggesting the possibility of an incomplete activity of BCR-ABL at the stem cell level or alternatively, the possibility that FOXO activity at this level provides the dominant signal responsible for intrinsic quiescence. Disclosures: No relevant conflicts of interest to declare.

Transcriptional Changes Induced by Imatinib and Nilotinib in the Chronic Myelogenous Leukemia (CML) Cell Line K562.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4250-4250
Author(s):  
Daniela Bruennert ◽  
Ingmar Bruns ◽  
Norbert Gattermann ◽  
Ralf Kronenwett ◽  
Rainer Haas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
K562 Cells ◽  
Significant Differential Expression ◽  
Number Of Genes ◽  
In Vitro Treatment

Abstract Abstract 4250 Nilotinib is a selective bcr-abl tyrosine kinase inhibitor that is 30-fold more potent than Imatinib in vitro. To examine the molecular and functional effects of Nilotinib and Imatinib we performed gene expression and functional analyses in K562 cells following in vitro treatment with the two tyrosine kinase inhibitors. Affymetrix U133A 2.0 microarrays covering 21.722 probe sets were used to analyse the gene expression profile of 5×10 7 K562 cells after 24h in vitro treatment with Imatinib (0.5 μM) or Nilotinib (0.05 μM) (half maximal inhibitory concentration). Gene expression data of the treated cells were compared with data of untreated cells. In addition, proliferation (MTS Assay, Promega), apoptosis (Cell Death Detection ELISAPLUS, Roche) and cell cycle (FITC BrdU Flow Kit, BD Pharmingen) assays were performed. Protein levels of STAT5, pSTAT5(Thr694), PIM-1 and PIM-2 were detected with Western blots. Particular emphasis was put on 303 genes which we found to be differentially expressed in primary CD34+ cells from patients with CML in vivo during treatment with Imatinib (Bruennert et al, Leukemia, 2008). In K562 cells, we found that Imatinib led to a significant differential expression of 45 of those genes. In general, the effect of Nilotinib with regard to the number of genes affected and degree of suppression was more pronounced resulting in a significant differential expression of 120 genes of the aforementioned genes. Of note, genes affected by Nilotinib included all genes altered by Imatinib. Downregulation of genes involved in cell cycle was observed in 17 genes following Nilotinib exposure, but only in the PIM-1 gene following Imatinib exposure. This effect of Nilotinib is in line with the results of cell cycle experiments showing that Nilotinib exposed cells had the lowest proportion of actively cycling cells. The proportion of apoptotic K562 cells was 5.5-fold greater following treatment with Nilotinib in comparison to treatment with DMSO, whereas Imatinib treated K562 cells had a 1.3-fold higher apoptosis rate compared to DMSO treated 562 cells after 24 hours. The superiority of Nilotinib is also reflected by the results of protein analysis of STAT5, pSTAT5(Thr694), PIM-1 and PIM-2: Both tyrosine kinase inhibitors completely inhibit the phosphorylation of STAT5, but Nilotinib inhibits PIM-1 and PIM-2 stronger than Imatinib (by 85% vs. 70%) in K562 cells. In summary, on a molecular level Nilotinib is apparently more potent than Imatinib with regard to the number of genes affected and the degree of their suppression. Among the 45 genes that were significant differentially expressed with both drugs, genes of Imatinib treated K562 cells were downregulated 1.68fold (mean) whereas genes of Nilotinib treated K562 cells were downregulated 2.41fold (mean). The genes altered are mainly associated with cell cycle regulation. In addition to this Nilotinib has a stronger inhibitory effect on PIM 1 and 2. Disclosures: Gattermann: Novartis: Honoraria, Participation in Advisory Boards on deferasirox clinical trials.

MJ05, a Novel p53 Activating Compound, Effectively and Selectively Eliminates Human CML Stem/Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1464-1464
Author(s):  
Su Chu ◽  
Catherine Drummond ◽  
Ling Li ◽  
YinWei Ho ◽  
Sonia Lain ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Kinase Inhibitors ◽  
Conflicts Of Interest ◽  
P53 Protein ◽  
Wild Type ◽  
Inhibition Of Proliferation ◽  
Protein Levels ◽  
Cd34 Cells

Abstract Tyrosine kinase inhibitors (TKI) are the mainstay of CML treatment but fail to eliminate leukemia stem cells (LSC), leading to high risk of disease recurrence when treatment is stopped. There is considerable interest in developing new strategies to target CML LSC. We have previously shown that p53 activation following SIRT1 deacetylase inhibition can inhibit growth and survival of TKI-treated CML LSC (Cancer Cell 2012, 21:266). We are therefore interested in investigating other strategies to activate p53 as potential approaches to target CML LSC. While conducting a screen of 20,000 small molecules for ability to activate p53-dependent transcription in TP53 wild-type ARN8 human melanoma cells we identified MJ05 amongst the top-ranking compounds. Importantly MJ05 did not activate p53-dependent transcription in T22 fibroblast cells. Increased p53 protein levels in ARN8 cells were seen within 6 hours and were accompanied by an increase in p21, pig3 and mdm2 mRNA and protein levels. Dependency on p53 was confirmed using p53-null and wild-type H1299 cells. Activation of p53 occurred without concurrent increase in DNA damage evidence by g-H2AX labeling, increased p53 Ser 15-phosphorylation, or inhibition of p53-HDM2 interaction. MJ05 treatment inhibited S-Phase progression of ARN8 cells without inhibition of ATM, ATR or DNA-PK phosphorylation, suggesting a unique mechanism of action. We tested the effect of MJ05 on primary normal or CML CD34+ cell by itself and in combination with TKI inhibitor Nilotinib, and compared with effects on Nutlin, a well studied inhibitor of p53-HDM2 interactions. Apoptosis was assessed by Annexin V labeling, proliferation by CFSE labeling, and colony forming cell (CFC) frequency, in methylcellulose progenitor assays. Treatment with MJ05 (5 and 10μM), with or without Nilotinib (1μM), for 72 hours in the presence of low concentrations of growth factor significantly and selectively increased apoptosis, inhibited proliferation and reduced colony CFC frequency in CML CD34+ cells compared to normal CD34+ cells. Combination of MJ05 with Nilotinib (1μM) resulted in significant increase in apoptosis of CML but not normal CD34+ cells. In contrast treatment with Nutlin (2 and 5 μM) resulted in similar increase in apoptosis in CML and normal CD34+ cells. We next evaluated the effect of treatment with MJ05 (10μM), Nilotinib and the combination for 72 hours on purified CML and normal CD34+CD38- stem/primitive progenitor cells and CD34+CD38+ committed progenitor cells. MJ05 significantly enhanced apoptosis of CML but not normal CD34+CD38- cells and CD34+CD38+ cells. Apoptosis was further enhanced by combination with Nilotinib (Table). MJ05 also resulted in significant reduction of proliferation in CML CD34+38+ and CD34+38- cells, with significantly less inhibition of proliferation of normal cells. MJ05 treatment markedly reduced CFU-GM and BFU-E generation from CML compared to normal CD34+CD38- and CD34+CD38+ cells. The combination with Nilotinib resulted in almost complete abrogation of CML CFC (Table). MJ05 resulted in significantly less inhibition of normal CFC, with greater effect on normal BFU-E compared to CFU-GM. Ongoing xenograft experiments are testing the effect of in vitro treatment with MJ05, Nilotinib or the combination on engraftment of CML and normal stem cells in NSG mice. Our studies indicate that MJ05, a unique, potent and selective p53 activating compound, is remarkably effective in inducing apoptosis and inhibiting growth of primitive CML stem/progenitor cells by itself and to an even greater extent in combination with Nilotinib. MJ05 treatment has significantly lesser effects on normal stem cells, and may offer a promising approach to selectively target CML LSC in combination with Nilotinib.Table 1NormalCMLUntreatedMj05+NilMj05+NilUntreatedMj05NilMj05+NilCD34+38+% Apoptosis4.5±0.19.0±1.14.13±018.8±7.17.5±0.815.9±4.412.3±1.723±7.4CFU-GM86±757.3±4.191±3.852±278.7±19.95±336±141±0BFU-E166±15.315±3.2155.7±9.314±1.595±19.30.7±0.721.3±5.50.3±03CD34+38-% Apoptosis4.2±0.26.1±0.34.2±0.15.3±0.423.0±1.230±4.114.6±3.850.1±7.2CFU-GM67.3±15.941.7±5.549±4.730.3±4.9192.7±50.66±2.372.67±48.70.7±0.7BFU-E58.7±20.211±2.526.3±2.97.6±0.9150.3±58.71±0.672±440.3±0.3 Disclosures: No relevant conflicts of interest to declare.

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.

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