ABSTRACT: Effective Targeting of Quiescent Chronic Myelogenous Leukemia Stem Cells by Histone Deacetylase Inhibitors in Combination with Imatinib Mesylate
Project description:ABL1 kinase inhibitors such as imatinib mesylate (IM) are effective in managing chronic myelogenous leukemia (CML) but incapable of eliminating leukemia stem cells (LSCs), suggesting that kinaseM-bM-^HM-^Rindependent pathways support LSC survival. Given that the bone marrow hypoxic microenvironment supports hematopoietic stem cells, we investigated if hypoxia similarly contributes to LSC persistence. Importantly, we found that while BCRM-bM-^HM-^RABL1 kinase remained effectively inhibited by IM under hypoxia, apoptosis became partially suppressed. Furthermore, hypoxia enhanced the clonogenicity of CML cells, as well as their efficiency in repopulating immunodeficient mice, both in the presence and absence of IM. HIF1M-bM-^HM-^RM-NM-1, which is the master regulator of the hypoxia transcriptional response is expressed in the bone marrow specimens of CML individuals. In vitro, HIF1M-bM-^HM-^RM-NM-1 is stabilized during hypoxia and its expression and transcriptional activity can be partially attenuated by concurrent IM treatment. Expression analysis demonstrates at the whole transcriptome level that hypoxia and IM regulate distinct subsets of genes. Functionally, knockdown of HIF1M-bM-^HM-^RM-NM-1 abolished the enhanced clonogenicity during hypoxia. Taken together, our results suggest that in the hypoxic microenvironment, HIF1M-bM-^HM-^RM-NM-1 signaling supports LSC persistence independently of BCRM-bM-^HM-^RABL1 kinase activity. Thus targeting HIF1M-bM-^HM-^RM-NM-1 and its pathway components may be therapeutically important for the complete eradication of LSCs. 24 samples consisting CD34+ bone marrow aspirates of 3 chronic phase patients that were subjected to 24h or 96h of DMSO/Normoxia (21% oxygen, 5% carbon dioxide) control, 2 M-BM-5M Imatinib, hypoxia (0.5% oxygen, 5% carbon dioxide) or combined Imatinib/hypoxia treatments in triplicate cultures.
Project description:Although Bcr-Abl kinase inhibitors have proven effective in the treatment of chronic myeloid leukemia (CML), they generally fail to completely eradicate Bcr-Abl+ leukemia cells. To identify genes whose inhibition sensitizes Bcr-Abl+ leukemias to killing by Bcr-Abl inhibitors, we performed an RNAi-based synthetic lethal screen with imatinib in CML cells. This screen identified numerous components of a Wnt/Ca2+/NFAT signaling pathway. Antagonism of this pathway led to impaired NFAT activity, decreased cytokine production and enhanced sensitivity to Bcr-Abl inhibition. Furthermore, NFAT inhibition with cyclosporin A facilitated leukemia cell elimination by the Bcr-Abl inhibitor dasatinib and markedly improved survival in a mouse model of Bcr-Abl+ acute lymphoblastic leukemia (ALL). Targeting this pathway in combination with Bcr-Abl inhibition could improve treatment of Bcr-Abl+ leukemias. We utilized a genome-wide shRNA library in combination with microarray analysis to screen for gene targets in chronic myeloid leukemia cells that cooperate with imatinib.
Project description:Chronic myelogenous leukemia (CML) is a malignant stem cell disease characterized by a reciprocal translocation between chromosome 9 and 22. The selective bcr-abl tyrosine-kinase inhibitor Imatinib has become the therapy of choice for patients with newly diagnosed CML including those previously considered candidates for allogeneic haematopoietic stem cell transplantation. The tyrosine-kinase inhibitor Nilotinib is a derivate of Imatinib with higher potency. To examine the molecular and functional effects of Nilotinib and Imatinib in chronic myelogenous leukemia, we performed gene expression and functional analyses in K562 cells following treatment with the two tyrosine kinase inhibitors.
Project description:Imatinib is highly effective in the treatment of chronic myelogenous leukemia (CML), but the primary and acquired imatinib resistance remains the big hurdle. Molecular mechanisms for CML resistance to tyrosine kinase inhibitors, beyond point mutations in BCR-ABL kinase domain, still need to be addressed. Here, we demonstrated that TXNIP is a novel BCR-ABL target gene. Suppression of TXNIP is responsible for BCR-ABL triggered glucose metabolic reprogramming and mitochondrial homeostasis. Mechanistically, Miz-1/P300 complex transactivates TXNIP through the recognition of TXNIP core promoter region, responding to the c-Myc suppression by either imatinib or BCR-ABL knockdown. TXNIP restoration sensitizes CML cells to imatinib treatment and compromises imatinib resistant CML cell survival, predominantly through the blockage of both glycolysis and glucose oxidation which results in the mitochondrial dysfunction and ATP production. In particular, TXNIP suppresses expressions of the key glycolytic enzyme, HK2 and LDHA, potentially through Fbw7-dependent c-Myc degradation. In accordance, BCR-ABL suppression of TXNIP provides a novel survival pathway for the transformation of mouse BM cells. Combination of drug inducing TXNIP expression with imatinib synergistically kills CML cells from patients and further extends the survival of CML mice. Thus, the activation of TXNIP represents an effective strategy for CML treatment to overcome resistance.
Project description:Chronic myelogenous leukemia (CML) is a malignant stem cell disease characterized by a reciprocal translocation between chromosome 9 and 22. The selective bcr-abl tyrosine-kinase inhibitor Imatinib has become the therapy of choice for patients with newly diagnosed CML including those previously considered candidates for allogeneic haematopoietic stem cell transplantation. The tyrosine-kinase inhibitor Nilotinib is a derivate of Imatinib with higher potency. To examine the molecular and functional effects of Nilotinib and Imatinib in chronic myelogenous leukemia, we performed gene expression and functional analyses in K562 cells following treatment with the two tyrosine kinase inhibitors. Experiment Overall Design: Affymetrix U133A 2.0 microarrays were used to examine the gene expression profile of K562 cells after in vitro treatment with Imatinib (0.5 µM) or Nilotinib (0.05 µM) for 24 hours. Gene expression data of the treated cells were compared with data of untreated cells.
Project description:ABL1 kinase inhibitors such as imatinib mesylate (IM) are effective in managing chronic myelogenous leukemia (CML) but incapable of eliminating leukemia stem cells (LSCs), suggesting that kinase−independent pathways support LSC survival. Given that the bone marrow hypoxic microenvironment supports hematopoietic stem cells, we investigated if hypoxia similarly contributes to LSC persistence. Importantly, we found that while BCR−ABL1 kinase remained effectively inhibited by IM under hypoxia, apoptosis became partially suppressed. Furthermore, hypoxia enhanced the clonogenicity of CML cells, as well as their efficiency in repopulating immunodeficient mice, both in the presence and absence of IM. HIF1−α, which is the master regulator of the hypoxia transcriptional response is expressed in the bone marrow specimens of CML individuals. In vitro, HIF1−α is stabilized during hypoxia and its expression and transcriptional activity can be partially attenuated by concurrent IM treatment. Expression analysis demonstrates at the whole transcriptome level that hypoxia and IM regulate distinct subsets of genes. Functionally, knockdown of HIF1−α abolished the enhanced clonogenicity during hypoxia. Taken together, our results suggest that in the hypoxic microenvironment, HIF1−α signaling supports LSC persistence independently of BCR−ABL1 kinase activity. Thus targeting HIF1−α and its pathway components may be therapeutically important for the complete eradication of LSCs.
Project description:A comparison between parental K562 cells (CML) and two clones derived from this cell line : ImaR and PDR which are resistant against Imatinib mesylate and PD166326 respectively , two inhibitors of BCR-ABL. Keywords: gene expression, comparison
Project description:Expression profile of 14 Chronic Myeloid Leukemia Philadelphia chromossome negative patients after allo haematopoietc stem cell transplantation and Chronic Myeloid Leukemia treated with imatinib mesylate. We tested 754 microRNAs by reverse transcription quantitative polymerase chain reaction (RT-qPCR) array for each patient.
Project description:We generated iPSCs from imatinib-sensitive chronic myelogenous leukemia (CML) patient samples. We used microarrays tc ompare the gene expression pattern among CML-iPSCs and normal cord blood (CB) iPSCs.