Chronic Myeloid Leukemia Cell Death Research Articles

Repurposing the Bis-Biguanide Alexidine in Combination with Tyrosine Kinase Inhibitors to Eliminate Leukemic Stem/Progenitor Cells in Chronic Myeloid Leukemia.

In CML, Leukemic Stem Cells (LSCs) that are insensitive to Tyrosine Kinase Inhibitors are responsible for leukemia maintenance and relapses upon TKI treatment arrest. We previously showed that downregulation of the BMI1 polycomb protein that is crucial for stem/progenitor cells self-renewal induced a CCNG2/dependent proliferation arrest leading to elimination of Chronic Myeloid Leukemia (CML) cells. Unfortunately, as of today, pharmacological inhibition of BMI1 has not made its way to the clinic. We used the Connectivity Map bioinformatic database to identify pharmacological molecules that could mimick BMI1 silencing, to induce CML cell death. We selected the bis-biguanide Alexidin (ALX) that produced a transcriptomic profile positively correlating with the one obtained after BMI silencing in K562 CML cells. We then evaluated the efficiency of ALX in combination with TKI on CML cells. Here we report that cell growth and clonogenic activity of K562 and LAMA-84 CML cell lines were strongly inhibited by ALX. ALX didn't modify BCR::ABL1 phosphorylation and didn't affect BMI1 expression but was able to increase CCNG2 expression leading to autophagic processes that preceed cell death. Besides, ALX could enhance the apoptotic response induced by any Tyrosine Kinase Inhibitors (TKI) of the three generations. We also noted a strong synergism between ALX and TKIs to increase expression of caspase-9 and caspase-3 and induce PARP cleavage, Bad expression and significantly decreased Bcl-xL family member expression. We also observed that the blockage of the mitochondrial respiratory chain by ALX can be associated with inhibition of glycolysis by 2-DG to achieve an enhanced inhibition of K562 proliferation and clonogenicity. ALX specifically affected the differentiation of BCR::ABL1-transduced healthy CD34+ cells but not of mock-infected healthy CD34+ control cells. Importantly, ALX strongly synergized with TKIs to inhibit clonogenicity of primary CML CD34+ cells from diagnosed patients. Long Term Culture of Initiating Cell (LTC-IC) and dilution of the fluorescent marker CFSE allowed us to observe that ALX and Imatinib (IM) partially reduced the number of LSCs by themselves but that the ALX/IM combination drastically reduced this cell compartment. Using an in vivo model of NSG mice intravenously injected with K562-Luciferase transduced CML cells, we showed that ALX combined with IM improved mice survival. Collectively, our results validate the use of ALX bis-biguanide to potentiate the action of conventional TKI treatment as a potential new therapeutic solution to eradicate CML LSCs.

A Central Contribution of TG2 Activity to the Antiproliferative and Pro-Apoptotic Effects of Caffeic Acid in K562 Cells of Human Chronic Myeloid Leukemia

Caffeic acid (CA) has shown antitumor activity in numerous solid and blood cancers. We have recently reported that CA is active in reducing proliferation and triggering apoptosis in both Imatinib-sensitive and resistant Chronic Myeloid Leukemia (CML) cells. Tissue transglutaminase type 2 (TG2) enzyme is involved in cell proliferation and apoptosis of numerous types of cancer. However, its activity has different effects depending on the type of tumor. This work investigated the possible involvement of TG2 activation in the triggering of CA-dependent anticancer effects on the K562 cell line, which was studied as a model of CML. CA-dependent changes in TG2 activity were compared with the effects on cell proliferation and apoptosis. The use of N-acetylcysteine (NAC), an antioxidant molecule, suggested that the antiproliferative effect of CA was due to the increase in reactive oxygen species (ROS). The use of a TG2 inhibitor showed that TG2 activity was responsible for the increase in ROS generated by CA and reduced both caspase activation and triggering of CA-dependent apoptosis. The knocking-down of TGM2 transcripts confirmed the crucial involvement of TG2 activation in CML cell death. In conclusion, the data reported, in addition to ascertaining the important role of TG2 activation in the antiproliferative and pro-apoptotic mechanism of CA allowed us to hypothesize a possible therapeutic utility of the molecules capable of triggering the activation pathways of TG2 in the treatment of CML.

CREB/Sp1-mediated MCL1 expression and NFκB-mediated ABCB1 expression modulate the cytotoxicity of daunorubicin in chronic myeloid leukemia cells

Although some studies have hinted at the therapeutic potential of daunorubicin (DNR) in chronic myeloid leukemia (CML), the mechanism by which DNR induces CML cell death is unclear. Therefore, this study aimed to investigate DNR-induced cell death signaling pathways in CML cell lines K562 and KU812. DNR-triggered apoptosis in K562 cells was characterized by inhibition of MCL1 expression, while restoration of MCL1 expression protected K562 cells from DNR-mediated cytotoxicity. In addition, DNR induced NOX4-dependent ROS production, leading to the activation of p38 MAPK and inactivation of Akt and ERK. Activated p38 MAPK stimulated protein phosphatase 2A-dependent dephosphorylation of CREB. Since Akt-mediated activation of ERK reduced β-TrCP mRNA stability, the inactivation of Akt-ERK axis increased β-TrCP expression, which in turn promoted proteasomal degradation of Sp1. Inhibition of CREB phosphorylation and Sp1 expression simultaneously reduced MCL1 transcription and protein expression. DNR-induced MCL1 suppression was not reliant on its ability to induce DNA damage. In addition, DNR induced the expression of drug exporter ABCB1 in K562 cells through the p38 MAPK/NFκB-mediated pathway, while imatinib or ABT-199 inhibited the DNR-induced effect. The combination of imatinib or ABT-199 with DNR showed synergistic cytotoxicity in K562 cells by increasing intracellular DNR retention. Cumulatively, our data indicate that DNR induces MCL1 downregulation in K562 cells by promoting p38 MAPK-mediated dephosphorylation of CREB and inhibiting the Akt-ERK axis-mediated Sp1 protein stabilization. Furthermore, experimental evidence indicates that DNR-induced death of KU812 cells occurs through a similar pathway.

Antimetabolic cooperativity with the clinically approved l-asparaginase and tyrosine kinase inhibitors to eradicate CML stem cells

ObjectiveLong-term treatment with tyrosine kinase inhibitors (TKI) represents an effective cure for chronic myeloid leukemia (CML) patients and discontinuation of TKI therapy is now proposed to patient with deep molecular responses. However, evidence demonstrating that TKI are unable to fully eradicate dormant leukemic stem cells (LSC) indicate that new therapeutic strategies are needed to control LSC and to prevent relapse. In this study we investigated the metabolic pathways responsible for CML surviving to imatinib exposure and its potential therapeutic utility to improve the efficacy of TKI against stem-like CML cells. MethodsUsing complementary cell-based techniques, metabolism was characterized in a large panel of BCR-ABL+ cell lines as well as primary CD34+ stem-like cells from CML patients exposed to TKI and L-Asparaginases. Colony forming cell (CFC) assay and flow cytometry were used to identify CML progenitor and stem like-cells. Preclinical models of leukemia dormancy were used to test the effect of treatments. ResultsAlthough TKI suppressed glycolysis, compensatory glutamine-dependent mitochondrial oxidation supported ATP synthesis and CML cell survival. Glutamine metabolism was inhibited by L-asparaginases such as Kidrolase or Erwinase without inducing predominant CML cell death. However, clinically relevant concentrations of TKI render CML cells susceptible to Kidrolase. The combination of TKI with Lasparaginase reactivates the intinsic apoptotic pathway leading to efficient CML cell death. ConclusionTargeting glutamine metabolism with the FDA-approved drug, Kidrolase in combination with TKI that suppress glycolysis represents an effective and widely applicable therapeutic strategy for eradicating stem-like CML cells.

Prolonged unfolded protein reaction is involved in the induction of chronic myeloid leukemia cell death upon oprozomib treatment.

To select the most efficient chemical to induce apoptosis in leukemia cells, a multidrug screen was applied on bone marrow mononuclear cells from chronic myeloid leukemia (CML) patients. Oprozomib (Cpd 21) was chosen for the subsequent experiments. The isobaric tags for relative and absolute quantitation (iTRAQ) was then performed to identify the responsible pathway relative to apoptosis and the results showed that endoplasmic reticulum (ER) chaperones were upregulated. Apoptosis was attributed to a joint effect of calcium leakage andPERK and IRE1α phosphorylation. The PERK branch was responsible for the first wave of cell death that occurred within 24 hours. The later wave of apoptosis was mediated by IRE1α, which transmit apoptotic signals through the ASK‐JNK‐BIM axis. Release of Ca2+ from ER into cytosol resulted in activation of calpain, which, in turn, cleaved caspase‐12. Our data also explained the selective killing effects of oprozomib on CML cells, which relied on proteasome activity. The present study demonstrated that prolonged inhibition of proteasome to trigger unfolded protein response could be an alternative strategy for treating CML in light of tyrosine kinase inhibitors resistance.

Parthenolide and DMAPT Induce Cell Death in Chronic Myeloid Leukemia Cells Via Generation of Reactive Oxygen Species

Chronic Myeloid Leukemia (CML) is a malignant hematologic disease characterized by the presence of a chromosomal alteration known as the Philadelphia Chromosome (Ph). Ph originates in Leukemia Stem Cells (LSC) and is caused by the reciprocal translocation between chromosome 9 and 22, giving rise to the Bcr-Abl oncogene, which produces a constitutively active tyrosine kinase -the BCR-ABL oncoprotein. This CML feature has made it possible the development of Tyrosine Kinase Inhibitors (TKI's) Imatinib, Dasatinib, and Nilotinib, which are able to inhibit BCR-ABL and take patients into molecular remission. However TKI's are not able to eliminate the population of LSC, which cause patients to relapse if treatment is discontinued. This has made it necessary search for alternative ways to eliminate LSC in CML patients.Parthenolide (PTL), a sesquiterpene lactone extracted from the feverfew plant (Thanacethum partenium), which has been used in folk medicine to treat inflammatory symptoms, has shown activity against LSC in Acute Myeloid Leukemia (AML). Recently Guzman and collaborators have developed an analog of PTL, named DMAPT, which is orally bioavailable. Both molecules have shown to be active inhibitors of NFkB and induce high levels of Reactive Oxygen Species (ROS) in LSC of AML. In the present study we asked whether CML cells (LSC, Progenitors and CML cell lines) are sensitive to PTL and DMAPT.Cell lines K562, Kcl-22 and HL60 (as an AML control) were cultured in liquid suspension cultures with increasing concentrations of PTL or DMAPT at a cell density of 300,000 cells per ml. At 24 hours of exposure, PTL and DMAPT showed significant induction of apoptosis at 7.5 µM and 10 µM, respectively. To determine whether NFkB activity is inhibited by PTL and DMAPT, K562, Kcl-22, HL60 cell lines, as well as CD34+ cells from a CML patient in Chronic phase where exposed to 7.5 µM PTL or 10 µM DMAPT. After 6 hours, protein lysis was induced and Western blots were performed with protein extracts from 250,000 cells per lane. Membranes where probed for Phosphorilated-p65, total p65 and b-actin as a loading control. We observed a decrease in the levels of phosphorylated p65 in cells treated with PTL and DMAPT, compared with untreated cells, while levels of total p65 remained constant.We had previosly used DCFDA stain and HMOX expression, to observe ROS induction in CML cells exposed to PTL. To asses if ROS induction is necessary for CML cell death we pretreated cells with N-acetyl cysteine (NAC), precursor of glutathione, for 1 hour before treating cells with 7.5 µM PTL or 10 µM DMAPT, and evaluated ROS levels by staining with Cell rox deep red, Mitosox-PE, and also assessing cell viability with YoPro-1 and 7AAD stain. After 6 hours of PTL treatment, we found that NAC pretreatment diminished ROS levels in cytoplasm and mitochondrial dysfunction when cells were treated with PTL. Pretreatment with NAC was able to rescue the majority of cells from apoptosis induced by PTL. Similar results were observed in cultures treated with DMAPT.Taken together, our results indicate that PTL and DMAPT were able to induce cell death via apoptosis in CML cells. This process seems to involve inhibition of p65 phosphorylation and an increase in ROS levels, which we found to be necessary for cell death to occur. DisclosuresNo relevant conflicts of interest to declare.

Targeting Hedgehog signaling pathway and autophagy overcomes drug resistance of BCR-ABL-positive chronic myeloid leukemia

The frontline tyrosine kinase inhibitor (TKI) imatinib has revolutionized the treatment of patients with chronic myeloid leukemia (CML). However, drug resistance is the major clinical challenge in the treatment of CML. The Hedgehog (Hh) signaling pathway and autophagy are both related to tumorigenesis, cancer therapy, and drug resistance. This study was conducted to explore whether the Hh pathway could regulate autophagy in CML cells and whether simultaneously regulating the Hh pathway and autophagy could induce cell death of drug-sensitive or -resistant BCR-ABL+ CML cells. Our results indicated that pharmacological or genetic inhibition of Hh pathway could markedly induce autophagy in BCR-ABL+ CML cells. Autophagic inhibitors or ATG5 and ATG7 silencing could significantly enhance CML cell death induced by Hh pathway suppression. Based on the above findings, our study demonstrated that simultaneously inhibiting the Hh pathway and autophagy could markedly reduce cell viability and induce apoptosis of imatinib-sensitive or -resistant BCR-ABL+ cells. Moreover, this combination had little cytotoxicity in human peripheral blood mononuclear cells (PBMCs). Furthermore, this combined strategy was related to PARP cleavage, CASP3 and CASP9 cleavage, and inhibition of the BCR-ABL oncoprotein. In conclusion, this study indicated that simultaneously inhibiting the Hh pathway and autophagy could potently kill imatinib-sensitive or -resistant BCR-ABL+ cells, providing a novel concept that simultaneously inhibiting the Hh pathway and autophagy might be a potent new strategy to overcome CML drug resistance.

Kinase Inhibitor Therapy in CML: It's What's Inside That Counts

Kinase Inhibitor Therapy in CML: It's What's Inside That Counts

Combination of Imatinib with CXCR4 Antagonist BKT140 Overcomes the Protective Effect of Stroma and Targets CML in Vitro and in Vivo

AbstractAbstract 3735 Background:Chronic myeloid leukemia (CML) is myeloproliferative disease of hematopoietic stem cells that is driven by the constitutively active oncogenic tyrosine kinase BCR-ABL. Inhibition of BCR-ABL activity by tyrosine kinase inhibitors (TKIs) such as imatinib mesylate, revolutionized the treatment of CML and remains a major therapeutic strategy for CML patients. However, existence of quiescent leukemic stem cells (LSCs) that are resistant to TKIs and may be responsible for CML resistance and recurrence continues to pose challenges for CML cure. Thus, novel therapies that eradicate LSCs are in need. The bone marrow (BM) microenvironment is believed to have a role in protecting LSCs and CML cells from TKIs-induced apoptosis. Chemokine receptor CXCR4 and its ligand CXCL12 have a key role in the trafficking and retention of normal and leukemic SCs in the BM niche. Although, CXCR4 signaling is impaired in CML cells, it can be elevated by the imatinib treatment and may induce stroma-promoted chemo-resistance. Therefore, CXCR4 inhibition may antagonize the survival and spread of CML and LSC cells and may restore their sensitivity to TKIs in the BM microenvironment context. Methods:In order to study the role of CXCR4 in CML progression, we over-expressed CXCR4 in K562 cells using a lentiviral vector and generated a cell line with high and stable CXCR4 expression. Results:CXCL12 stimulation did not significantly affect the migration of K562 cells in vitro. In contrast, elevated CXCR4 increased the in vitro survival and proliferation of K562 cells in response to CXCL12 treatment, suggesting an important role of CXCR4/CXCL12 axis in CML growth. In accordance, we found that in vitro treatment with CXCR4 antagonist BKT140 (8 μM) directly inhibited the cell growth by 40–60% and induced cell death of three CML cell lines tested (K562, KCL22 and LAMA84). Combination of BKT140 (8 μM) with IC50 concentrations of imatinib (0.2–0.3 μM) significantly increased the anti-CML apoptotic effect in achieving 90–95% reduction in cell viability in vitro (p<0.01). Furthermore, to investigate the interaction between CML cells and BM microenvironment, we evaluated the effect of BM stromal cells on CML growth and response to imatinib treatment in vitro in co-culture experiments. We found that murine BM stromal cells 14F1.1 and MS5, as well as primary human BM stromal cells (BMSCs) significantly increased the proliferation of CML cell lines K562 and LAMA84 (p<0.01) and protected them from imatinib-induced apoptosis. We next wanted to assess whether BCL6 is involved in the stroma-mediated TKI resistance as it was recently reported that CML cells up-regulate proto-oncogene BCL6 in response to TKI treatment. Indeed, the BM stromal cells remarkably elevated BCL6 mRNA expression in the CML cells in response to imatinib treatment, suggesting the possible role of BCL6 in stroma-mediated TKI resistance. Addition of BKT140 to the co-culture system reversed the protective effect of the stroma, re-sensitizing CML cells to imatinib and effectively promoting CML cell death. Moreover, combination of imatinib with BKT140 decreased BCL6 mRNA levels in CML cells co-cultured with BMSCS. To further explore the role of CXCR4 inhibition in vivo, we established a xenograft bioluminescent model of CML. Luciferase-transduced K562 cells were intra-peritoneally injected into NOD/SCID mice and tumor burden was quantified using bioluminescence imaging. Both imatinib (0.6 mg/injection) and BKT140 (100 μg/injection), administered intra-peritoneally, effectively reduced disease burden. However, combined treatment demonstrated increased potency in vivo. Conclusions:Taken together, our data indicate the importance of CXCR4/CXCL12 axis in CML growth and CML-BM stroma interaction. CXCR4 inhibition with BKT140 antagonist efficiently synergized with imatinib both in vitro as well as in vivo, over-coming the protective effect of the BM stroma. These results provide the rational basis for CXCR4-targeted therapy in combination with TKI to override drug resistance and suppress residual disease. Disclosures:No relevant conflicts of interest to declare.

Involvement of ROS in chlorogenic acid-induced apoptosis of Bcr-Abl + CML cells

Chlorogenic acid (Chl) has been reported to possess a wide range of biological and pharmacological properties including induction of apoptosis of Bcr-Abl + chronic myeloid leukemia (CML) cell lines and clinical leukemia samples via inhibition of Bcr-Abl phosphorylation. Here we studied the mechanisms of action of Chl in greater detail. Chl treatment induced an early accumulation of intracellular reactive oxygen species (ROS) in Bcr-Abl + cells leading to downregulation of Bcr-Abl phosphorylation and apoptosis. Chl treatment upregulated death receptor DR5 and induced loss of mitochondrial membrane potential accompanied by release of cytochrome c from the mitochondria to the cytosol. Pharmacological inhibition of caspase-8 partially inhibited apoptosis, whereas caspase-9 and pan-caspase inhibitor almost completely blocked the killing. Knocking down DR5 using siRNA completely attenuated Chl-induced caspase-8 cleavage but partially inhibited apoptosis. Antioxidant NAC attenuated Chl-induced oxidative stress-mediated inhibition of Bcr-Abl phosphorylation, DR5 upregulation, caspase activation and CML cell death. Our data suggested the involvement of parallel death pathways that converged in mitochondria. The role of ROS in Chl-induced death was confirmed with primary leukemia cells from CML patients in vitro as well as in vivo in nude mice bearing K562 xenografts. Collectively, our results establish the role of ROS for Chl-mediated preferential killing of Bcr-Abl + cells.

Coordination of Intrinsic, Extrinsic and Endoplasmic Reticulum-Mediated Apoptosis by Imatinib Mesylate Combined with Arsenic Trioxide in Chronic Myeloid Leukemia.

Abstract A treatment strategy that combines arsenic trioxide (ATO) with the tyrosine kinase inhibitor imatinib mesylate (STI571, Gleevec) appears to induce more cell apoptosis than STI571 alone in chronic myeloid leukemia (CML). To understand the mechanisms underlying the synergistic action of these agents, we applied cDNA microarrays, component plane presentation integrated self-organizing maps (CPP-SOM) and methods of protein biochemistry to study proapoptotic and apoptotic activities in programmed cell death induced by STI571, ATO and the combination of the two agents in detail. Numerous features with temporospatial relationship were revealed, indicating the coordinated regulation of molecular networks from various aspects of proapoptotic and apoptotic activities in CML. In consequence, STI571 induces the intrinsic pathway of cell apoptosis, whereas ATO induces the ER stress-mediated pathway of cell apoptosis, and the combination of the two agents induces the intrinsic, extrinsic and ER stress mediated pathways of cell apoptosis, which results in a more effective and efficient induction of programmed cell death in CML. The potential impact of this study on the development of more sophisticated therapy in human malignancy can be substantial.