CML Progenitor Cells Research Articles

Human Blast Crisis Leukemia Stem Cell Inhibition with a Novel Smoothened Antagonist.

AbstractAbstract 1223Sonic hedgehog (Shh) pathway activators, particularly smoothened (SMO), are implicated in driving mouse leukemia progression by promoting leukemia stem cell maintenance. Thus, we investigated the role of the Shh signaling pathway in a humanized mouse model of blast crisis (BC) CML as well as the effects of a novel SMO antagonist on leukemia stem cell (LSC) quiescence, survival and self-renewal both in vitro and in vivo. Methods:Human BC CML progenitor cells (n=3 patients) were treated for 7 days in vitro with vehicle (DMSO) or a clinical grade SMO antagonist (Pfizer; PF-04449913) and harvested for Shh target gene Q-PCR analysis. FACS-purified BC CML LSCs (CD34+CD38+Lin-PI-) were transplanted intrahepatically into newborn RAG2-/-gc-/- mice. When human progenitors were detectable in peripheral blood (8 weeks), mice were treated for 14 days daily by oral gavage with 200μl of drug or vehicle (50% PEG in HBSS). The treatment included dasatinib alone (50 mg/kg), PF-04449913 (100 mg/kg) with or without dasatinib (50mg/kg). After treatment the percentage of LSC (CD34+CD38+CD45RA+CD123+Lin-PI-) was determined by FACS analysis of the bone marrow, peripheral blood, spleen and liver and the number of myeloid sarcomas was evaluated. In addition, cell cycle analysis was also performed on the same hematopoietic tissues of vehicle compared with treated mice. To determine if self-renewal potential was abrogated by treatment, human leukemic progenitors were serially transplanted and myeloid sarcoma formation was assessed in secondary recipients after 8 weeks. Results:BC CML progenitors (n=3 patient samples) harbored increased expression of Gli and downregulation of Ptch1 transcripts compared to normal cord blood. Notably, Gli expression decreased following in vitro treatment with PF-04449913. Treatment with PF-04449913 alone in human BC CML LSC transplanted mice (n=12 exp., n=46 mice) significantly reduced spleen size (p ≤ 0.05). However, SMO inhibition alone did not significantly inhibit bone marrow engraftment compared with vehicle (n=12 exp., n= 47 mice). Although dasatinib alone decreases myeloid sarcoma formation (p ≤ 0.05), like the SMO antagonist alone it did not eradicate the LSC in the liver or bone marrow (n=12 exp., n=36 mice). Combination treatment (n=12 exp., n=34 mice) with PF-04449913 and dasatinib revealed a significant decrease in LSC hepatic engraftment (P≤0.05) compared with the PF-04449913 or dasatinib alone. Moreover, combination treatment completely eradicated the capacity of LSC to form myeloid sarcomas formation (0.0) in secondary transplant recipients (n=5) compared with dasatinib that had an average of 1.8 tumors (n=6), PF-04449913 that had an average of 4.9 (n=7) and vehicle group that had 3.8 tumors (n=16).Hematopoietic progenitor assays performed with normal cord blood CD34+ progenitors (n=3 samples) showed no reduction in colony survival following treatment with PF-04449913 (1μM) compared with vehicle. Furthermore, there was no significant inhibition of normal CD34+ colony replating following PF-04449913 compared with vehicle treatment. Cell cycle analysis demonstrated a reduction in quiescent human leukemic cells in the peripheral blood and marrow following SMO inhibitor treatment. Conclusions:Combination therapy with a potent BCR-ABL inhibitor, dasatinib, and a selective SMO inhibitor abrogates the self-renewal capacity of human imatinib resistant blast crisis leukemia stem cells. A phase I clinical trial with PF-04449913 is currently underway and a Phase 1b trial involving dasatinib and PF-04449913 for patients with advanced phase, imatinib resistant CML will begin in the near future. Disclosures:Jamieson:Pfizer: Research Funding; CIRM: Research Funding.

Abstract 5053: Depletion of PHLPP1 and 2 by Bcr-Abl promotes CML cell proliferation

Abstract [Objective] PHLPP1 and 2 (pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase 1 and 2) is identified as the dephosphorylation enzyme of Akt, the same as that of PP2A of the dephosphorylation enzyme of Akt, and regulate the cell-growth signal of Akt through a dephosphorylation of the phosphorylated Akt (p-Akt). Previously, we reported the expression of PHLPP1 and 2 were suppressed in CML cell lines. In this study, we investigated the the CML and its progenitor cell proliferation through the phosphorylation of Akt by depletion of PHLPP1 and 2. [Method] CML cell lines (K562, Meg01, SHG3) and the CML clinical specimen (n= 10) were used for this research. The changes in the expression of PHLPP1 and 2 by treatment with Abl kinase inhibitors (STI571, ANM107 and BMS354825) or knock down with Bcr-Abl gene were evaluated by RT-PCR method. The influence on the expression of PHLPP1 and 2 in AML cell line transfected with Bcr-Abl also evaluated. CML progenitor cells derived from CML patients separated by ALDH activity, and the expression of PHLPP1 and 2 were investigated by quantitative RT-PCR method. p-Ser473 Akt1, 2 and 3 by Abl kinase inhibitor or knock down with PHLPP1 and 2 were measured, and the influence on the effect of cell growth inhibition by MTT assay. The expression of PHLPP1 and 2 and colony formation in colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), colony forming unit-granulocyte, macrophage (CFU-GM) and burst forming unit-erythroid (BFU-E) derived from Bcr-Abl positive hematopoietic progenitor cells also evaluated. [Result] The expression of mRNA and protein of PHLPP1 and 2 were increased by treatment with Abl kinase inhibitor or Bcr-Abl knock down by siRNA in CML cell lines and AML cell line trasfected with Bcr-Abl gene. Moreover, p-Ser473 Akt 2 and 3 were decreased according to the expression of PHLPP1, and also p-Ser473 Akt1 and 3 according to the expression of PHLPP2. The Abl kinase inhibitors induced the expression of PHLPP1, 2 and the reduction of p-Ser473 Akt isoforms. The Abl kinase inhibitors inhibited the CML cell proliferation via the depletion of PHLPP1 and 2. In Bcr-Abl positive progenitor cells, the expression of PHLPP1 and 2 was increased, and colony formation was suppressed by Abl kinase inhibitor and by Bcr-Abl siRNA. The colony formation in progenitor cells knocked down PHLPP1 and 2 were decreased when treated with Abl kinase inhibitors. [Conclusion] These results suggest that Bcr-Abl might promote CML cell proliferarion through continuous phosphorylation of p-Ser473 Akt1, 2 and 3 by suppression of PHLPP1 and 2, and that the induction of PHLPP1 and 2 may be effective to regulate the cell proliferation in CML cells. It may be also that the induction of expression of PHLPPs has the potential possibility as the targets on the regulation of cell proliferation in Bcr-Abl positive progenitor cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5053.

Pellicano F, Cilloni D, Helgason GV, Messa F, Panuzzo C, Arruga F, Bracco E, Allan E, Huntly BJ, Holyoake TL, Saglio G. FOXO transcription factor activity is partially retained in quiescent CML stem cells and induced by tyrosine kinase inhibitors in CML progenitor cells [published online ahead of print December 1, 2009]. Blood. doi 10.1182/blood-2009-06-226621.

Pellicano F, Cilloni D, Helgason GV, Messa F, Panuzzo C, Arruga F, Bracco E, Allan E, Huntly BJ, Holyoake TL, Saglio G. FOXO transcription factor activity is partially retained in quiescent CML stem cells and induced by tyrosine kinase inhibitors in CML progenitor cells [published online ahead of print December 1, 2009]. Blood. doi 10.1182/blood-2009-06-226621.

Bortezomib induces apoptosis in primitive chronic myeloid leukemia cells including LTC-IC and NOD/SCID repopulating cells

AbstractChronic myeloid leukemia (CML) is treated effectively with tyrosine kinase inhibitors (TKIs); however, 2 key problems remain—the insensitivity of CML stem and progenitor cells to TKIs and the emergence of TKI-resistant BCR-ABL mutations. BCR-ABL activity is associated with increased proteasome activity and proteasome inhibitors (PIs) are cytotoxic against CML cell lines. We demonstrate that bortezomib is antiproliferative and induces apoptosis in chronic phase (CP) CD34+ CML cells at clinically achievable concentrations. We also show that bortezomib targets primitive CML cells, with effects on CD34+38−, long-term culture-initiating (LTC-IC) and nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells. Bortezomib is not selective for CML cells and induces apoptosis in normal CD34+38− cells. The effects against CML cells are seen when bortezomib is used alone and in combination with dasatinib. Bortezomib causes proteasome but not BCR-ABL inhibition and is also effective in inhibiting proteasome activity and inducing apoptosis in cell lines expressing BCR-ABL mutations, including T315I. By targeting both TKI-insensitive stem and progenitor cells and TKI-resistant BCR-ABL mutations, we believe that bortezomib offers a potential therapeutic option in CML. Because of known toxicities, including myelosuppression, the likely initial clinical application of bortezomib in CML would be in resistant and advanced disease.

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.

Abstract 646The 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.

Effective Targeting of Quiescent CML Stem Cells by Histone Deacetylase Inhibitors in Combination with Imatinib Mesylate.

Abstract 190▪▪This icon denotes an abstract that is clinically relevant.The BCR-ABL tyrosine kinase inhibitor imatinib mesylate (IM) is highly effective in inducing remissions and improving survival in CML patients but fails to eliminate leukemia stem cells, which remain a potential source of relapse. Quiescent leukemia stem cells resist apoptosis following BCR-ABL kinase inhibition, and other strategies are required for their elimination. Histone deacetylase inhibitors (HDACi) have shown promise in the treatment of several cancers, and it is of particular interest that reports suggest that they are also capable of inducing apoptosis in non-proliferating cells. It is known that the potent pan-HDACi LAQ824 (LAQ) and LBH589 (LBH) can induce apoptosis in CML cell lines and blast crisis CML cells. However, the effect of HDACi on quiescent leukemia stem cells from chronic phase CML patients is not known. Here we investigated the effects of LAQ and LBH, alone and in combination with IM, on CML stem and progenitor cells. CML and normal CD34+ cells were cultured with LAQ (10–100nM) or LBH (25–50nM) alone, IM (1mM) alone, and LAQ or LBH with IM for 96 hours. HDACi treatment effectively enhanced Histone H3 and H4 acetylation in CML CD34+ cells. HDACi treatment by itself induced less apoptosis in CML compared to normal CD34+CD38- primitive and CD34+CD38+ committed progenitors, but was highly effective in inducing apoptosis in CML progenitors when combined with IM. In addition, the combination induced significantly higher apoptosis in CML compared with normal CD34+ cells, and unlike IM, also induced apoptosis in non-dividing cells. Combination treatment also inhibited the proliferation of CML progenitor cells as measured by CFSE and growth of CML CFC in methylcellulose progenitor assays. Treatment of CML CD34+ cells with IM resulted in modest reduction in levels of engraftment in NSG mice. In contrast treatment with LAQ824 plus IM resulted in abrogation of engraftment of BCR-ABL+ CML cells (p<0.001). Significantly less inhibition of normal compared to CML cell engraftment was seen following LAQ824 and IM treatment (p=0.006). We used a transgenic Scl-tTa-BCR-ABL mouse model to investigate the effect of HDACi treatment on CML stem cells in vivo. SCLtTA/BCR-ABL transgenic mice were crossed with GFP transgenic mice to allow tracking of transplanted cells. Recipient mice developed CML-like disease 3–4 weeks after transplantation. Mice were treated with IM (200mg/kg daily by gavage), LBH (30 mg/kg IP 3 times a week), LBH with IM, or vehicle alone (control) for 4 weeks. LBH combined with IM resulted in greater reduction in WBC, neutrophils and GFP+ cells than LBH or IM alone. Significantly increased apoptosis and a profound reduction of Lin- Sca-1+ Kit+ (LSK) stem cells were seen in mice treated with IM plus LBH (p<0.001) but not IM or LBH alone. LBH plus IM treatment did not significantly inhibit LSK cells in normal mice. BCR-ABL-transgenic mice treated with IM plus LBH demonstrated prolonged leukemia-free survival after discontinuing treatment compared with mice treated with IM or LBH alone (p<0.05). Combined IM and LAQ824 treatment resulted in marked inhibition of the anti-apoptotic protein MCL-1 in CML CD34+ cells (p<0.001). RNAi mediated inhibition of MCL-1 expression resulted in increased apoptosis in CML compared with normal CD34+ cells that was further enhanced by IM treatment. Our results indicate that treatment with HDACi plus IM effectively and selectively targets leukemia stem cells in CML, suggest a potential role for MCL-1 inhibition in HDACi induced apoptosis, and support ongoing clinical trials of LBH combined with IM to eliminate residual leukemia stem cells in IM-treated CML patients. Disclosures:Bhatia:Novartis: Consultancy.

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.

Abstract 187First 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.

Depletion of PHLPP1 and 2 by Bcr-Abl Promotes CML Cell Proliferation through the Continuous Phosphorylation of Akt.

Abstract 2182Poster Board II-159 ObjectivePHLPP1 and 2 (pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase 1 and 2) is identified as the dephosphorylation enzyme of Akt, the same as that of PP2A of the dephosphorylation enzyme of Akt, and regulate the cell-growth signal of Akt through a dephosphorylation of the phosphorylated Akt (p-Akt). Previously, we reported the expression of PHLPP1 and 2 were suppressed in CML cell lines. In this study, we investigated the the CML and its progenitor cell proliferation through the phosphorylation of Akt by depletion of PHLPP1 and 2. MethodCML cell lines (K562, Meg01, SHG3) and the CML clinical specimen (n= 10) were used for this research. The changes in the expression of PHLPP1 and 2 by treatment with Abl kinase inhibitors (STI571, AMN107 and BMS354825) or knock down with Bcr-Abl gene were evaluated by RT-PCR method. The influence on the expression of PHLPP1 and 2 in AML cell line transfected with Bcr-Abl also evaluated. CML progenitor cells derived from CML patients separated by ALDH activity, and the expression of PHLPP1 and 2 were investigated by quantitative RT-PCR method. p-Ser473 Akt1, 2 and 3 by Abl kinase inhibitor or knock down with PHLPP1 and 2 were measured, and the influence on the effect of cell growth inhibition by MTT assay. The expression of PHLPP1 and 2 and colony formation in colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), colony forming unit-granulocyte, macrophage (CFU-GM) and burst forming unit-erythroid (BFU-E) derived from Bcr-Abl positive hematopoietic progenitor cells also evaluated. ResultThe expression of mRNA and protein of PHLPP1 and 2 were increased by treatment with Abl kinase inhibitor or Bcr-Abl knock down by siRNA in CML cell lines and AML cell line trasfected with Bcr-Abl gene. Moreover, p-Ser473 Akt 2 and 3 were decreased according to the expression of PHLPP1, and also p-Ser473 Akt1 and 3 according to the expression of PHLPP2. The Abl kinase inhibitors induced the expression of PHLPP1, 2 and the reduction of p-Ser473 Akt isoforms. The Abl kinase inhibitors inhibited the CML cell proliferation via the depletion of PHLPP1 and 2. In Bcr-Abl positive progenitor cells, the expression of PHLPP1 and 2 was increased, and colony formation was suppressed by Abl kinase inhibitor and by Bcr-Abl siRNA. The colony formation in progenitor cells knocked down PHLPP1 and 2 were decreased when treated with Abl kinase inhibitors. ConclusionThese results suggest that Bcr-Abl might promote CML cell proliferarion through continuous phosphorylation of p-Ser473 Akt1, 2 and 3 by suppression of PHLPP1 and 2, and that the induction of PHLPP1 and 2 may be effective to regulate the cell proliferation in CML cells. It may be also that the induction of expression of PHLPPs has the potential possibility as the targets on the regulation of cell proliferation in Bcr-Abl positive progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Imatinib Induced Re-Activation of FoxO3 Transcription Factor in CML Is Responsible for the Induction of a Quiescent Status of CD34 Leukaemic Progenitor Cells.

The FoxO family of transcription factors is regulated by PI3K/Akt induced phosphorylation resulting in nuclear exclusion and degradation. Nuclear FoxO transcribes proapoptotic molecules and cell cycle inhibitors. In CML cells the TK activity of Bcr-Abl leads to the abnormal activation of downstream effectors including PI3K/Akt. The aim of this study was to investigate the role of FoxO3 in Bcr-Abl induced apoptotic arrest and cell growth and the effect of imatinib (IM) induced re-activation of FoxO3 activity in CML progenitor cells. BM cells were collected from 52 CML patients and 20 healthy donors. The expression level of FoxO3 was tested by RQ-PCR. The protein amount and localization was analyzed by Western blot and immunofluorescence, DNA binding activity was measured by EMSA. In addition, FoxO3 was analyzed in CML primary cells and CD34+ cells after IM incubation. Cell cycle and the expression levels of CD47, which has been demonstrated to increased during progression through the cell cycle and stem cell mobilization, was measured by FACS in CD34+ cell population. In addition K562 cells was transfected with pECE-FoxO3 to clarify FoxO3 effects on cell growth and apoptosis. Finally we used our already set up model of Drosophila melanogaster (Dm) transgenic for human Bcr-Abl to study the pathway leading to FoxO3 inactivation. We found that, despite either FoxO3 mRNA levels or protein amount are similar in CML cells compared to controls, FoxO3 protein is equally distributed in the nucleus and cytoplasm in controls but it is completely cytoplasmatic in CML cells and it enters the nucleus during in vivo IM treatment or in vitro IM incubation. Additionally, FoxO3 DNA binding activity in CML patients is completely absent at diagnosis and reappears after IM treatment. Moreover FoxO3 overexpression in transfected cells results into a 49±9 % reduction of proliferation which was further reduced of 75±5 % after IM incubation. Furthermore, we demonstrated that IM incubation results into the reactivation of FoxO3 in Ph+ CD34+ cells inducing quiescence into this population as demonstrated by the comparison of cell cycle kinetics and by a decreased expression of CD47. Finally, the progeny obtained from the crossbreeding of Bcr-Abl flies and flies transgenic for FoxO showed a rescue of FoxO phenotype demonstrating that FoxO inactivation is Bcr-Abl mediated. Overall, these in vitro and in vivo experiments suggest that FoxO3 is inactivated in CML cells and its delocalization is mainly dependant from Bcr-Abl activity. The antiproliferative activity of IM may be mediated by FoxO3 re-localization. On the other side, FoxO3 re-activation induced by IM results into a quiescence of Bcr-Abl CD34+ progenitor cells, which raises a hypothesis that FoxO3 could play a role in IM resistance. This investigation was conducted by CML Correlative Studies Network (CCSN), TOPS, which is sponsored by Novartis Oncology

HOXA10 expression induced by Abl kinase inhibitors enhanced apoptosis through PI3K pathway in CML cells

Chronic myelogenous leukemia is characterized by the reciprocal chromosomal translocation (9;22), which generates a novel fusion gene, BCR-ABL. Bcr-Abl-expressing leukemia cells are highly resistant to apoptosis. Imatinib an Abl kinase inhibitor, is a highly effective agent for patients with CML. However, a small percentage of these patients and most advanced-phase patients relapse on imatinib therapy. It is poorly understood whether the Abl kinase inhibitors are able to eradicate CML progenitor or stem cells. In this study, we investigated the role of HOXA10 in CML cell lines and the hematopoietic progenitor cells derived from CML patients, and whether the regulation of HOXA10 eradicates Bcr-Abl + hematopoietic stem/progenitor cells. The Abl kinase inhibitors and PI3K inhibitor, LY294002, induced the expression of HOXA10, and it enhanced apoptosis in CML cells. Moreover, the reduction of HOXA10 expression by siRNA in CML cells inhibited apoptosis by treatment with the Abl kinase inhibitors and LY294002. These results revealed that HOXA10 had an important role in induction of apoptosis by the Abl kinase inhibitors in CML cells. Finally, we showed that the inhibition of HOXA10 expression by siRNA increased the numbers of CFU-GEMM, BFU-E, and CFU-GM when the cells were treated with the combination of BMS354825 and LY294002 compared to control cells, and HOXA10 played a critical role in the committed colony-formation in CML. This study shows for the first time that the Abl kinase inhibitor and LY294002 induced HOXA10, and HOXA10 had an important role in apoptosis or cell growth inhibition in CML cells in vitro.

Enhanced Phosphorylation and Altered Localization Lead to Impairment of p27kip Activity in CML Progenitor Cells Despite Enhanced Protein Translation and Expression.

The cyclin-dependent kinase inhibitor p27kip is a key regulator of hematopoietic progenitor proliferation and pool size. The activity of p27 can be regulated by modulation of its expression and localization in different cellular compartments. The levels of p27 protein expression are reported to be reduced in BCR-ABL expressing cell lines. In contrast p27 levels have been reported to be elevated in BCR/ABL expressing CML progenitor cells. However, CML progenitors paradoxically demonstrate enhanced proliferation despite having elevated levels of p27. Therefore the regulation of p27 protein expression and activity in primary CML progenitor cells is not well understood and requires further investigation. We have therefore performed a careful assessment of p27 expression, phosphorylation and localization in CD34+ cells from CML patients as well as in cord blood CD34+ cells transduced with retroviral vectors to ectopically express the BCR-ABL gene. We observed that p27 protein levels were markedly increased in both primary CML CD34+ cells and BCR/ABL transduced cells. CML CD34+ cells and BCR/ABL transduced CD34+ cells also demonstrated increased phosphorylation of p27 on T157 (an Akt-dependent phosphorylation site) and T187 (a cdk2-dependent site) on western blotting as well as increased tyrosine phosphorylation of p27 in immunoprecipitation studies. Immunofluorescence microscopy analysis of cells labeled with anti-p27 antibodies demonstrated primarily nuclear localization of p27 in normal CD34+ cells, but markedly reduced nuclear and increased cytoplasmic localization of p27 in CML CD34+ cells and BCR/ABL transduced CD34+ cells. Cdk2 activity was maintained, cdk4 activity was increased and phosphorylated RB levels were increased in BCR-ABL expressing hematopoietic cells, indicating CDKI activity was reduced in these cells despite increased total p27 levels. Consistent with this BCR-ABL expressing CD34+ cells demonstrated enhanced cell cycling compared to controls. Mutation of the Y177 residue in BCR-ABL (BCR-ABL-Y177F), which abrogates Grb2 binding and reduces Ras and Akt activation by BCR-ABL, reversed BCR-ABL induced abnormalities in p27 expression, phosphorylation and localization. Treatment of BCR-ABL transduced CD34+ cells with the PI-3K inhibitor LY294002 resulted in reduced phosphorylation of p27 on T157 but not T187, and restored nuclear localization of p27, further indicating an important role for AKT signaling in abnormal cytoplasmic localization of p27. Quantitative reverse transcription PCR analysis indicated that p27 mRNA levels were similar in BCR/ABL expressing and control CD34+ cells, suggesting that p27 expression is regulated mainly at the posttranscriptional level. Metabolic labeling of cells with S35-methionine showed that p27 translation was increased in BCR/ABL expressing cells, explaining the observed increase in total p27 protein levels. We conclude that p27 CDKI activity is reduced in BCR-ABL expressing progenitors as a result of altered p27 phosphorylation at important regulatory sites and a shift in cellular localization of p27 from the nucleus to the cytoplasm. The enhanced level of total p27 in primary CML progenitors reflects enhanced protein translation, which may be a compensatory response to reduced p27 CDKI activity. These results provide important insights into p27 deregulation in CML progenitors and its potential role in the expansion of the hematopoietic progenitor pool size in CML.

In Vivo Eradication of Human Clonigenic Acute Lymphoblastic Leukemic Cells Expressing the Wilms Tumor Protein, WT-1, by HLA Restricted WT-1 Specific T-Cells in NOD/SCID Mice Monitored by Bioluminescent Imaging.

WT-1 is expressed in 60–80% of acute leukemias, CML and high risk forms of MDS. Its expression has been hypothesized to be critical to the growth or survival of leukemic stem cells. Previously, alloreactive HLAA0201− T-cells recognizing a complex of WT-1 peptide and HLA A0201 were reported to prevent growth of leukemic HLA A0201+ CD34+ Ph+CML progenitor cells in NOD/ SCID mice (Transplantation, vol 75, No9, 2003). In this study, we have assessed the capacity of HLA-restricted, WT-1 peptide specific CTL (WT1-CTL) lacking alloreactivity to prevent the outgrowth of a human acute preB-lymphocytic leukemia (B-ALL)in NOD/SCID mice. This leukemia contained 65% of the blasts expressed WT-1 as determined by FACS analysis. For these studies the leukemic cells were transduced to express a luciferase reporter gene, permitting sequential monitoring of growth in vivo by bioluminescent imaging. WT-1 specific T-cells were generated from normal HLA A0201+ donor PBMC by in vitro sensitization with autologous dendritic cells loaded with the immunogenic HLA A0201 binding WT-1 peptide, RMFPNAPYL, and shown to be selectively cytotoxic against HLA A0201+WT-1+ leukemias and peptide loaded PHA blasts. T-cells from the same donor sensitized with autologous EBV BLCL and exhibiting HLA A0201 restricted EBV-specific cytotoxic activity served as controls. WT-1-CTL or EBV CTL were co-incubated in vitro with the WT-1+ HLA A0201+ BALL-LUC at a 4:1 effector target ratio for 7 hours at 37°C. Thereafter, separate groups of 5 NOD/SCID mice received intravenous infusions of cells from each of the co-cultures, at doses providing 12 × 106 WT1 CTL or EBVCTL and 3 × 106 BALL-LUC cells/mouse. A third group received 3×106 BALL-LUC alone. Leukemia growth was monitored at 2–3 day intervals from day 1–45 post infusion. In all 3 groups, BALL-LUC could be detected in the thorax by imaging at day 1. In mice treated with BALL-LUC alone or together with EBV-CTL, signal accumulation in the thorax increased steadily through 45 days of observation. By day 17, BALL-LUC were also detected throughout the head, abdomen and pelvis, and thereafter also increased until sacrifice at day 45. Autopsy confirmed presence of leukemic nodules in the lung and leukemic cells in blood, spleen and marrow as well as other organs. In contrast, in mice treated with WT1-CTL+ BALL LUC, signal intensity in lung decreased by day 4. In 4/5 of these mice, BALL-LUC could not be detected thereafter. In one mouse from this group, BALL-LUC were first detected in the head 31 days post infusion. At autopsy on day 45, this mouse had detectable BALL in the skull but in no other sites. WT-1 expression of residual leukemic cells is being analyzed. The other mice treated with WT-1 CTL had no detectable residual disease. These results suggest that clonogenic BALL cells express WT-1 and are susceptible to eradication in vivo by WT-1 peptide specific cytotoxic T-cells. The elimination of such clonogenic leukemic cells is sufficient to prevent subsequent development of leukemia.

Immune Effects of Imatinib in Chronic Myelogenous Leukemia In Vitro.

Imatinib mesylate, a potent and selective inhibitor of the BCR-ABL tyrosine kinase, has been shown to induce durable haematological and major cytogenetic responses in a high percentage of CML patients. However in most patients the disease recurs, when imatinib is discontinued. In contrast, allogeneic stem cell transplantation (ASCT) is considered to be curative by the immune effect of donor T cells against CML progenitor cells. In this context, the role of imatinib is controversial; it may improve the results of ASCT by reducing the tumour load, it may also reduce the effect of donor lymphocyte transfusions (DLT) by impairing the function of T cells and the capacity of myeloid cells to present antigen. Patient derived CML-cells were studied for the stimulation of allogeneic HLA-matched and mismatched T-cells in the presence and absence of imatinib. In a 5 day culture the proliferative response of HLA-mismatched T cells was evaluated in presence of different concentrations of imatinib (0, 1, 2, or 5 micro M) and various responder-to-stimulator ratios. Thereby, proliferation was detected via a CFDA based assays and the activation profile (CD25, CD69) of the T-cells was determined by FACS. Cr51-release assays were performed after a 7 day culture of CML cells with HLA-matched T cells to test cytotoxicity of CD8+ T-cells. In addition, we characterized the antigen-presenting profile (CD14, CD33, HLA-DR, CD40, CD80, CD86, CD54, CD58) of the CML cells over a 5 day culture with and without imatinib. The presence of imatinib inhibited the proliferative capacity of allogeneic T-cells in a dose-dependent manner. Also, the expression of T cell activation markers was reduced in the presence of the different imatinib concentrations. Preincubation of CML cells with imatinib for 48 hours strengthened the effect on proliferation and activation of T cells. Moreover, imatinib impaired the cytotoxic function of T-cell (HLA-matched setting; CR51-release assay) also in a dose-dependent manner. Finally, the antigen-presenting profile of the myeloid leukemia cells was down regulated by increasing concentrations of imatinib. In summary, imatinib may interfere with the T cellular immune response and the antigen presenting profile on the CML cells in vitro. These results may have an impact on new strategies of treatment of CML with immunotherapy.

Bortezomib Has Anti-Proliferative and Apoptotic Effects Against CML Stem Cells, Including the Quiescent Population.

CML is a disease of the haemopoietic stem cell (HSC) caused by the constitutively active tyrosine kinase Bcr-Abl. The current first-line therapy for CML is imatinib (IM), a tyrosine kinase inhibitor (TKI) effective in establishing durable disease control in the majority of patients in chronic phase. However, the quiescent leukaemic HSC (Ph+HSC) population is insensitive to the apoptotic effects of this and more potent TKIs - such as dasatinib or nilotinib. These TKIs exert potent anti-proliferative effects against CML stem and progenitor cells, resulting in accumulation of quiescent cells in vitro. This Ph+HSC reservoir of disease in treated patients is now recognised to be a critical target if we wish to achieve complete eradication of residual disease. Bcr-Abl+ cells have abnormally high levels of proteasome activity and expression and in common with other cancer cells have been shown to be relatively more sensitive to the effects of proteasome inhibitors (PI) than their non-malignant counterparts. We have assessed the potential role for PI in CML using Bcr-Abl+ cell lines (K562 and Ba/F3 with mutant/wild type Bcr-Abl) and Ph+HSC obtained from leukapheresis products of patients, taken with consent, at time of diagnosis with CML. We have demonstrated apoptotic and anti-proliferative effects of PI (MG132 and bortezomib) in wild type and mutated Bcr-Abl+ cell lines (including T315I) and in Ph+HSC (n=7) by viable cell counts, thymidine incorporation and flow cytometry for annexin V and 7AAD. The apoptotic effects also occurred in the quiescent Ph+HSC fraction with an IC50 of 5–7.5nM (bortezomib)- similar to that seen in the total CD34+ cell population and lower than that previously reported for cells from multiple myeloma patients. These results were compared to parallel FACS-sorted cell populations from non-CML Ph- leukapheresis products and a therapeutic window was seen. CFSE tracking of cell division in CML samples in vitro indicated depletion of the quiescent fraction, in contrast to the accumulation of these cells demonstrated with IM, nilotinib or dasatinib, either alone or in combination. The intracellular effects of exposure of these cell populations to bortezomib were assessed by quantitatively measuring the intracellular accumulation of polyubiquitinated proteins seen following PI treatment. Interestingly no clear effect on Bcr-Abl activity was demonstrated with PI treatment of Ph+HSC (measured by immunoblotting with antibody to phosphorylated Crkl), though in Bcr-Abl+ cell lines detectable activity was reduced. Drug combination experiments (Calcusyn) were performed with bortezomib and IM or dasatinib to assess the effect of simultaneous inhibition of Bcr-Abl and the proteasome - the combination of a TKI with a PI resulted in additive effects, however no clear synergistic effects of significance were seen. Our results indicate that PI are capable of inducing CML stem cell specific apoptosis, suggesting the proteasome may be a relevant target for eradication of residual disease in CML following TKI therapy.

Tyrosine Kinase Inhibitors Dasatinib, Nilotinib and Imatinib Have an Impact on Both CD8+ T Lymphocytes and CD4+CD25+FoxP3+ Regulatory T Cells by Downregulation of the NF-κB Pathway.

Background: For CML/ALL patients with refractory disease or at relapse after allogeneic stem cell transplantation (allo-PBSCT), one might administer tyrosine kinase inhibitors (TKIs) like imatinib (Glivec), nilotinib (Tasigna) and dasatinib (Sprycel), or donor lymphocyte infusions (DLIs). TKIs will inhibit the proliferation of CML progenitor cells, but might also hamper the graft-versus-leukemia (GVL) effect considered to be crucial for the eradication of the disease. Moreover, CD8+ T cells specific for cytomegalovirus (CMVpp65) might be impaired.Methods: Imatinib, nilotinib and dasatinib were added at concentrations of 0–20 μM, 0–4 μM and 0–50 nM respectively to proliferation assays of Tregs and CD8+ T cells. Mixed lymphocyte peptide cultures (MLPCs) were performed with peptides derived froma.influenza matrix protein (IMP),b.CMVpp65 as a viral antigen andc.the receptor for hyaluronic acid mediated motility (RHAMM-R3) as a leukemia-associated antigen.CD8+ T cells from these MLPCs from healthy donors and patients with CML after allo-PSCT by tetramer staining/multi-color flow cytometry and enzyme linked immunosorbent spot (ELISPOT) assays, as well as Tregs after 3 days of culture with anti-CD3 and anti-CD28. Activity of T cells from the peripheral blood of patients under dasatinib and after withdrawal of dasatinib. Western blots (WBs) for T cell receptor (TCR) related molecules and NFkB were performed.Results: The release of interferon gamma and granzyme B by CD8+ HLA-A2/tetramer+effector T cells specific for IMP, CMVpp65 and RHAMM-R3 was inhibited by all TKIs in a dose-dependent fashion and correlating to the time of TKI exposure. The inhibition was reversible after removal of the drugs from the MLPC. The proliferation and function of Tregs were also significantly inhibited by TKIs in a dose-related fashion. In WBs, TKIs decreased the expression of ZAP70, Lck and Akt, as well as NFκB p65/p100/p105 and c-Rel. T cell activity was impaired when TKIs were clinically administered. The potency of T cell inhibition was imatinib: nilotinib: dasatinib = 1:2:40 under therapeutical serum respectively culture medium levels (2 μM:1 μM: 25 nM).Conclusion: When administering TKIs to patients after allo-PBSCT, the inhibition of both CD8+ and Tregs must be taken into consideration with respect to GVL and anti-viral T cell response. Inhibition of CD8+ T lymphocytes was partially reversible after removal of TKIs from the cultures. Using western blotting analysis, we found that these effects are mediated at least in part by down-regulating the levels of phosphorylation of TCR and NF-κB family members.

Increased eIF4E Expression and Phosphorylation in Late Phase Chronic Myelogenous Leukemia Occurs in a Bcr-Abl-Dependent Manner, and Can Be Targeted by a Novel Mnk Kinase Inhibitor, CGP57380, To Overcome Imatinib-Resistance.

The molecular mechanisms which mediate progression of chronic phase (CP) CML to accelerated and blast phase (BP) disease remain unclear, although one feature that correlates with progression is increased expression of the Bcr-Abl protein itself (Barnes et al., Can. Res. 2005). Increased Bcr-Abl expression is likely to contribute to the more aggressive behavior of BP disease, but the downstream factors that are dysregulated by the increased amounts of Bcr-Abl protein remain to be determined. In these studies we turned our attention to eIF4E since forced expression of eIF4E is transforming, and because increased levels of eIF4E have been found in BP but not CP CML (Topisirovic et al., Mol. Cell. Bio. 2003). eIF4E plays a critical role in cap-dependent translation and allows recruitment of the translation machinery to mRNA. eIF4E is phosphorylated at Ser209, and phosphorylation correlates with exposure to growth factors and increased cap-dependent translation. Using a panel of primary CML cells representing patients at various stages of disease, we confirmed that both Bcr-Abl and eIF4E protein levels were elevated in BP samples compared to those in CP, and furthermore that phosphorylation at Ser209 was dependent on Bcr-Abl kinase activity in BP but not CP samples. We next went on to explore the role of eIF4E phosphorylation in BP CML. Because eIF4E is exclusively phosphorylated at Ser209 by the MAPK signal-integrating kinases (Mnk1/2), we used a small molecule inhibitor of Mnk1/2, CGP57380, to inhibit eIF4E phosphorylation (kind gift of Dr. H. Gram, Novartis). Using MTS assays, we found that CGP57380 exhibited synergistic activity with imatinib mesyalte (IM) against Ba/F3-Bcr-Abl and K562 cells, and that this was associated with increased caspase-3 activation. Consistent with a role for eIF4E phosphorylation in cap-dependent translation, we found that CGP57380 augmented the IM-mediated inhibition of cap-binding complex (eIF4F) formation, as well as loading of mRNA onto polysomes. Interestingly, we also uncovered the existence of a novel negative-feedback loop regulating Mnk kinase. Here, treatment with CGP57380 resulted in increased phosphorylation of Mnk1 as well as its upstream activator, ERK, in a time- and dose-dependent manner. Because activation of the MEK/ERK pathway is essential to Bcr-Abl-mediated transformation, this finding suggested that the full activity of CGP57380 might be obscured by this feedback loop. In support of this, the addition of the MEK inhibitor, U0126, to the IM/CGP57380combination resulted in increased activity against CML cells. The triple combination was also effective against Ba/F3-Bcr-Abl cells harboring the E255K and T315I mutations, but not parental Ba/F3 cells (reduced by 50, 23, and 15% respectively of DMSO-treated controls by MTS assay). Colony forming assays also demonstrated the activity of the IM/CGP57380 combination against CML progenitor cells. In conclusion, our data demonstrate that:eIF4E protein expression and phosphorylation are upregulated in a Bcr-Abl-dependent manner in BP CML;Inhibition of eIF4E phosphorylation by the novel Mnk kinase inhibitor, CGP57380, synergizes with IM in killing CML cells, as well as overcomes certain forms of IM-resistance;The addition of CGP57380 to IM results in inhibition of key steps in cap-dependent mRNA translation, and may provide a mechanistic explanation for the activity of this agent in CML.

Pre-Stimulation of CML CD34+ Cells with High Concentrations of Growth Factors Counters Imatinib-Mediated Proliferation Suppression and Enhances Apoptosis of Non-Dividing Cells.

While imatinib mesylate is a highly effective treatment for CML, there is accumulating evidence that it may not adequately target quiescent malignant HSCs. In vitro exposure to imatinib inhibits CML progenitor growth primarily through suppression of abnormally enhanced proliferation. Apoptosis appears to be restricted to dividing cells while quiescent progenitors are resistant to apoptosis. One approach to more effectively enhance the sensitivity of HSCs to imatinib may be to induce them to cycle using hematopoietic growth factors (GF). We have shown that exposure of CML CD34+ progenitors to imatinib (1μM) in high GF conditions (100ng/ml SCF and FL3, 20ng/ml IL6, G-CSF and IL3) reduced the total number of viable, undivided cells compared to control cells cultured in 100-fold lower GF conditions (low GF). High GF treated cells were more proliferative but less sensitive to imatinib-mediated apoptosis (Blood 2004, 104:2967). We hypothesized that pre-stimulation with high GF prior to imatinib exposure would further reduce viable, non-dividing CML progenitors. CML CD34+ cells were cultured in high GF for 48 hours and then exposed to imatinib (1μM) for 48 hours in either high or low GF conditions. Compared to cells exposed to imatinib without any pre-stimulation, high GF pre-stimulation significantly reduced imatinib-mediated inhibition of proliferation in both low GF (22±5%, p=0.0009) and high GF (18±3%; p=0.0003). Pre-stimulation decreased imatinib-mediated apoptosis when compared to the same conditions with no pre-stimulation [19±2% for imatinib treatment in low GF (p<0.0001) and 7±3% in high GF (p=0.064)]. However, although overall apoptosis decreased, pre-stimulation resulted in increased apoptosis of undivided cells exposed to imatinib in either low GF (14±5%; p=0.022) or high GF (13±6%, p=0.065). These results are notable since increased apoptosis of undivided cells was not previously observed in any other condition. Importantly, the percent of input cells remaining viable and undivided decreased significantly for pre-stimulated cells exposed to imatinib in low GF (19±3%; p<0.0001) or high GF (7±2%; p=0.016). These results highlight the potential use of GF stimulation to enhance targeting of CML HSC. Additional studies examined whether GF readily available for clinical use (G-CSF and/or GM-CSF) could also enhance imatinib targeting of quiescent CML progenitors. CML CD34+ cells were exposed to 1mM imatinib for 96 hours in a basal low GF cocktail (250pg/ml G-CSF, 10pg/ml GM-CSF, 200pg/ml SCF, 1ng/ml IL6, 200pg/ml MIP1α, 50pg/ml LIF) alone or with the addition of G-CSF (50ng/ml) and/or GM-CSF (10ng/ml). While overall apoptosis decreased, apoptosis of undivided cells significantly increased for cells exposed to imatinib in high concentrations of G-CSF + GM-CSF compared to those in basal GF alone (8.7±1.3%; p=0.007). The percent of input cells remaining viable and undivided in the presence of imatinib significantly decreased with high G-CSF + GM-CSF compared to basal GF alone (7.2%±1.1; p=0.007). In conclusion, pre-stimulation with high concentrations of GF can lead to increased proliferation and enhance reduction of non-dividing CML CD34+ cells by imatinib. These results are of significance because non-dividing primitive cells have previously proven highly resistant to elimination by imatinib and support translational clinical studies to investigate whether intermittent GF administration can enhance elimination of residual CML stem and progenitor cells in patients in remission on imatinib treatment.

Selective Inhibition of Lysophosphatidic Acid Acyltransferase-β by CT32228 Inhibits MAPK and Enhances the Antileukemic Activity of Imatinib in BCR-ABL Expressing Cell Lines Resistant to Imatinib.

Background: Lysophosphatidic acid acyltransferase-β (LPAAT-β) catalyzes the conversion of lysophosphatidic acid (LPA) to phosphatidic acid (PA), an essential component of the Ras pathway. In vascular smooth muscle cells, specific inhibitors of LPAAT-β interfere with the ras and PI3-kinase dependent signaling pathways thereby inducing growth arrest and apoptosis. We have previously shown that CT32228, a specific inhibitor of LPAAT-β, selectively inhibits the growth of CML progenitor cells in cultures without cytokines [ASH 2003, #2415]. Since Bcr-Abl is usually reactivated in cells resistant to the Abl kinase inhibitor imatinib, and Ras and PI3 kinase are downstream targets of Bcr-Abl, we hypothesized that a specific inhibitor of LPAAT-β may have activity against Bcr-Abl-positive CML with resistance to imatinib.Methods: Antiproliferative activity was assessed using the MTS-proliferation assay. Statistical analysis of combination studies (CT32228 + imatinib) was performed by applying the median effect method. Flow cytometry after staining with propidium iodide (PI) was performed to assess the cell cycle, co-staining with annexin allowed the detection of apoptotic cells. Signaling studies were performed by immunoblotting.Results: CT32228 inhibited the proliferation of Bcr-Abl expressing cell lines (AR230-s, K562, KCL22, Baf/BCR-ABL-s, 32DBCR-ABL, MO7p210) with IC50 concentrations between 23 and 105 nM. Similar IC50 values were obtained in lines expressing imatinib resistant BCR-ABL-mutants (Y253F/M351T/T315I/H396R) or amplified wild-type-BCR-ABL (Baf/BCR-ABL-r1, AR230-r), demonstrating the absence of significant cross-resistance. Combination studies with imatinib demonstrated additive to synergistic antiproliferative activity only in cell lines with residual sensitivity towards imatinib. FACS analysis of K562 cells treated with CT32228 showed accumulation of cells in G2/M as early as 6 h after start of treatment. After 24 h, 50% of cells were in G2/M and apoptosis was demonstrable with annexin/PI staining. Consistent with the G2/M arrest, multinucleated cells were evident microscopically at 24 h. Further analysis of apoptotic signalling pathways revealed activation of caspase-3 and PARP cleavage, while activation of caspase-8 and -9 was not demonstrated. In K562 cells treated with CT32228, Erk1/2 phosphorylation was abrogated, consistent with inhibition of mitogen activated kinase signalling as a result of decreased Ras signalling. In contrast phosphorylation of Akt was unaffected suggesting that inhibition of LPAAT-β does not interfere with the PI3K pathway in BCR-ABL-positive cells.Conclusion: CT32228 exerts antileukemic activity in imatinib-resistant cells without cross-resistance to imatinib and is synergistic in combination with imatinib. Cytotoxicity is mediated by cell cycle arrest in G2/M and induction of apoptosis. Interference with the Ras-pathway through MAPK-inhibition may play a key role in the antileukemic effect of CT32228. This data provides the framework for evaluation of CT32228 and related compounds to improve imatinib-based treatment of CML.

The progenitor cell inhibition assay to measure the anti-leukemic reactivity of T cell clones against acute and chronic myeloid leukemia

Adoptive immunotherapy with donor T lymphocytes may be used as a treatment for relapsed leukemia after allogeneic hematopoietic stem cell transplantation (SCT). In vitro selected and expanded anti-leukemic T cells may be more effective in inducing a response in vivo. To identify the anti-leukemic reactivity of in vitro generated T cells, standard target cell read-out assays like the 51Cr-release assay are not always appropriate. We developed an assay in which the ability of T cells to antigen specifically inhibit the in vitro growth of leukemic progenitor cells in the presence of cytokines can be measured. This assay allows the evaluation of the cytolytic or suppressive potential of leukemia reactive T cells for prolonged periods of time. The assay is based on inhibition of [ 3H]thymidine incorporation by the leukemic progenitor cells induced by multiple hematopoietic growth factors. T cell clones with a known specificity were used to compare the analytic potential of the new assay with those of other cytotoxicity assays. Based on the results of the T cell clones, a modification of a limiting dilution assay was developed to identify anti-leukemic allogeneic T cells in HLA identical donor–recipient combinations selected on their ability to inhibit the in vitro growth of CML or AML progenitor cells, to be used for the generation of leukemia-reactive CTL lines for clinical use.

Imatinib (STI571) Provides Only Limited Selectivity for CML Cells and Treatment Might Be Complicated by Silent BCR-ABL Genes

Very promising results have been obtained in clinical trials on chronic-phase chronic myeloid leukemia (CP-CML) patients treated with imatinib mesylate (IM; Gleevec®, STI571), a BCR-ABL tyrosine kinase inhibitor. However, we found that IM caused considerable inhibition of normal hematopoietic progenitor cells upon treating control bone marrow (BM) cultures. In vitro IM treatment gave a decrease in the yield and size of colonies from BM of untreated CP-CML patients that was only two to three times that from the normal samples. Moreover, about 30% of myeloid progenitors (CFU-GM) from CML BM still formed colonies in the presence of IM, most of which had BCR-ABL RNA. About half of these treated colonies also displayed methylation of the internal ABL Pa promoter, a CML-specific epigenetic alteration, which was used in this study as a marker for BCR-ABL translocation-containing cells. However, ~5-8% of the treated or the untreated CML BM-derived colonies had no detectable BCR-ABL RNA by two or three rounds of RT-PCR despite being positive for the internal standard RNA and displaying hallmarks of CML, either t(9;22)(q34;q11) or ABL Pa methylation. Our results indicate that IM is only partially specific for CML progenitor cells compared to normal hematopoietic progenitor cells and suggest that some CML cells may have a silent BCR-ABL oncogene that could interfere with therapy.