IM-resistant Chronic Myeloid Leukemia Cells Research Articles

Telomere loss is accompanied by decreased pool of shelterin proteins TRF2 and RAP1, elevated levels of TERRA and enhanced glycolysis in imatinib-resistant CML cells

Telomere length may be maintained by telomerase nucleoprotein complex and shelterin complex, namely TRF1, TRF2, TIN2, TPP1, POT1 and RAP1 proteins and modulated by TERRA expression. Telomere loss is observed during progression of chronic myeloid leukemia (CML) from the chronic phase (CML-CP) to the blastic phase (CML-BP). The introduction of tyrosine kinase inhibitors (TKIs), such as imatinib (IM), has changed outcome for majority of patients, however, a number of patients treated with TKIs may develop drug resistance. The molecular mechanisms underlying this phenomenon are not fully understood and require further investigation. In the present study, we demonstrate that IM-resistant BCR::ABL1 gene-positive CML K-562 and MEG-A2 cells are characterized by decreased telomere length, lowered protein levels of TRF2 and RAP1 and increased expression of TERRA in comparison to corresponding IM-sensitive CML cells and BCR::ABL1 gene-negative HL-60 cells. Furthermore, enhanced activity of glycolytic pathway was observed in IM-resistant CML cells. A negative correlation between a telomere length and advanced glycation end products (AGE) was also revealed in CD34+ cells isolated from CML patients. In conclusion, we suggest that affected expression of shelterin complex proteins, namely TRF2 and RAP1, TERRA levels, and glucose consumption rate may promote telomere dysfunction in IM-resistant CML cells.

Targeting MDC1 promotes apoptosis and sensitizes Imatinib resistance in CML cells by mainly disrupting non-homologous end-joining repair.

The first-line drug Imatinib (IM) has achieved a curative effect in most chronic myeloid leukemia (CML) patients, but drug resistance remains a problem. More alternative therapeutic strategies need to explore. In recent years, targeting dysregulated DNA repair mechanisms provided promising options for cancer treatment. Here, we discovered the versatile Mediator of DNA Damage Checkpoint 1 (MDC1) interacted with γ-H2AX and 53BP1 in the early stage of the DNA damage response of cells. MDC1 overexpressed in CML cell lines and patients' bone marrow mononuclear cells. By knocking down MDC1, non-homologous end-joining pathways were mainly inhibited, leading to an intense accumulation of unrepaired intracellular DNA damage and an apparent cell apoptosis promotion. Notably, targeting MDC1 further enhanced drug sensitivity in IM-resistant CML cells. Our work revealed that MDC1 is a prospective target for CML treatment through regulating DNA damage repair mechanism, and also an alternative option for IM resistance dilemma. This study extends the understanding of regulating dysfunctional DNA repair mechanisms for cancer treatment.

Identification of a Highly Deregulated eIF4F Translation Initiation Complex in Drug-Resistant BCR-ABL + Cells By a Phospho-Proteomic Antibody Microarray

Protein-tyrosine kinase inhibitors (TKIs) have been effective for treatment of early stages of chronic myeloid leukemia (CML). However, BCR-ABL-dependent resistance mechanisms and TKI-unresponsive quiescent leukemic stem cells (LSCs) can result in drug resistance and disease relapse. We have demonstrated that Abelson helper integration site-1 (AHI-1) is a highly deregulated protein in CML LSCs. It interacts with BCR-ABL through the AHI-1 WD40 domain and with other proteins, like dynamin-2 (DNM2), through its SH3 domain, to enhance leukemic-initiating activities and TKI resistance. To uncover downstream effects of the AHI-1-BCR-ABL-DNM2 complex and its biological role in mediating TKI resistance in CML stem/progenitor cells, an advanced antibody microarray analysis was then used to investigate differences in the proteome and phosphorylation landscape of BCR-ABL + cells co-transduced with wild-type Ahi-1 or Ahi-1 SH3 Δ mutant in the presence or absence of imatinib (IM). The microarray simultaneously quantified the differences in expression and phosphorylation sites of proteins in multiple signaling pathways using 1325 antibodies in duplicate measurements, and each microarray analysis was performed in duplicate. Significant changes in antibody signals for protein expression or phosphorylation were determined using limma. This analysis revealed that the overexpression of wild-type Ahi-1 (WT Ahi-1) has a profound differential effect, compared to the SH3 domain deleted Ahi-1 (Ahi-1 SH3 Δ), on the expression and phosphorylation status of proteins in BCR-ABL + cells with and without IM treatment. BCR-ABL + cells co-expressing WT Ahi-1 had a greater number of significantly differential antibody signals (7 increases, 42 decreases) when compared to BCR-ABL + cells, while Ahi-1 SH3 Δ expressing cells resulted in fewer significantly differential antibody signals (12 increases, 2 decreases) compared to BCR-ABL + cells. IM treatment resulted in WT Ahi-1 expressing cells having the greatest number of significantly differential antibody signals (7 increases, 56 decreases) compared to BCR-ABL + cells (5 decreases) and those co-expressing Ahi-1 SH3 Δ (2 increases, 9 decreases). Pathway enrichment analysis, using gProfiler, identified that the targets with significantly increased differential antibody signal after IM treatment in WT Ahi-1 cells were related to the regulation of translation initiation complex (p>0.0001). Interestingly, our RNA-seq dataset analysis further identified several members of the eukaryotic initiation factor 4F (eIF4F) complex to be significantly upregulated in CD34 + CML patient cells compared to normal bone marrow, particularly eIF4G1, the scaffold protein of the eIF4F complex involved in translation initiation (2-fold, p=0.001), as well as mTOR, a key regulator that controls the assembly of the eIF4F complex. This finding prompted us to further explore the regulation of translation initiation and the members of the eIF4F complex in BCR-ABL + cells. Immunoblotting demonstrated that BCR-ABL + cells co-transduced with WT Ahi-1 showed increased expression of eIF4G1 (3-fold) and eIF4B (2-fold), a cofactor that regulates the helicase activity of the eIF4F complex, compared to BCR-ABL + cells. Additionally, Cyclin D3, a gene reported to be sensitive to eIF4F translational activity, was found to have slightly increased expression (1.4-fold) in WT Ahi-1 cells compared to BCR-ABL + cells. Most interestingly, these results were also demonstrated in IM-resistant CML cells as compared to IM-sensitive cells, with an increase in eIF4G1 expression (2-fold), phosphorylation of eIF4B (5-fold, p=0.003), and Cyclin D3 expression (4-fold, p<0.05). Mechanistically, eIF4G1 knockdown by shRNA impaired survival (5-fold, p<0.0001) and increases TKI sensitivity in IM-resistant cells (3-fold, p<0.05). A new protein interaction between eIF4G1 and the mRNA cap-binding protein eIF4E was further identified in these cells using a proximity ligation assay. Thus, we have uncovered that the eIF4F complex, the key regulator of the mRNA-ribosome recruitment phase of translation initiation, has increased activity in IM-resistant cells, which may contribute to the regulation of TKI resistance in CML. DisclosuresNo relevant conflicts of interest to declare.

Circ_0080145 Enhances Imatinib Resistance of Chronic Myeloid Leukemia by Regulating miR-326/PPFIA1 Axis.

Background: Acquired multidrug resistance is often blamed for the failure of chemotherapy in patients with malignant tumors, including chronic myeloid leukemia (CML). In this study, the authors investigated the role of circular RNA 0080145 (circ_0080145) in imatinib (IM) resistance of CML. Materials and Methods: Quantitative real-time polymerase chain reaction was applied to measure the expression of circ_0080145, microRNA-326 (miR-326), and PTPRF interacting protein alpha 1 (PPFIA1) mRNA. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to determine the half maximal inhibitory concentration (IC50) of IM and cell proliferation. Flow cytometry analysis was utilized to assess cell apoptosis. The levels of glucose uptake and lactate production were examined using specific kits. Protein levels were detected through Western blot assay. The targeting relationship between miR-326 and circ_0080145 or PPFIA1 was verified by dual-luciferase reporter assay. The murine xenograft model was constructed to investigate the effect of circ_0080145 in vivo. Results: circ_0080145 was upregulated in IM-resistant CML patients and cells. circ_0080145 silencing suppressed IM resistance, cell growth, and glycolysis and induced apoptosis in IM-resistant CML cells in vitro. Moreover, circ_0080145 knockdown blocked tumor growth and IM resistance in vivo. miR-326 was a target of circ_0080145, and miR-326 inhibition restored the effects of circ_0080145 silencing on cell progression and IM resistance. In addition, PPFIA1 was a target gene of miR-326. The suppressive roles in IM resistance, cell growth and glycolysis, and the promotional role in apoptosis mediated by miR-326 were abolished by PPFIA1 overexpression in IM-resistant CML cells. Conclusion: circ_0080145 contributes to IM resistance via modulating miR-326/PPFIA1 axis, which might provide a novel avenue for CML therapy.

A novel HDAC inhibitor chidamide combined with imatinib synergistically targets tyrosine kinase inhibitor resistant chronic myeloid leukemia cells

Chidamide is a novel selective histone deacetylase inhibitor (HDACi) with promising activity in hematological malignancies, but its role in chronic myeloid leukemia (CML) was marginally addressed. In this study, we firstly demonstrated that chidamide alone inhibited CML cells proliferation, induced apoptosis and cell cycle arrest. Further, chidamide combined with imatinib (IM) induced synergistic lethality in CML cell line KBM5, as well as IM-resistant CML cells KBM5T315I, associated with a marked reduction of Bcr-Abl kinase activity and acetyl-histone H3 expression. The combination treatment markedly inhibited constitutive activity of β-catenin signaling in IM-resistant cells and abolished the protective effects of mesenchymal stromal cells (MSCs) to CML cells. In addition, the co-treatment significantly reduced Bcr-Abl and β-catenin transcript levels and induced apoptosis of primary CD34+ stem/progenitor cells derived from blast crisis (BC)-CML patients, but exhibited minimal toxicity to normal CD34+ progenitors. Collectively, our data show that combination of chidamide and imatinib synergistically targets tyrosine kinase inhibitor (TKI) -resistant BC-CML cells via inhibition of Bcr-Abl and β-catenin signaling, suggesting that this combination has the potential for treating TKI-resistant CML and improving clinical outcomes of BC-CML patients.

MicroRNA-153-3p enhances the sensitivity of chronic myeloid leukemia cells to imatinib by inhibiting B-cell lymphoma-2-mediated autophagy.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease caused by abnormal DNA replication of bone marrow stem cells and chemotherapy resistance is a major obstacle to the effective treatment of patients with CML. Imatinib (IM), a tyrosine kinase inhibitor (TKI), is a first-line drug clinically used for CML. Mounting evidence has indicated that the dysregulation of microRNAs (miRNAs) is associated with the chemoresistance of CML. In this study, miR-153-3p, which had been implicated with numerous types of tumors, was identified to be downregulated in IM-resistant CML cells. Upregulation of miR-153-3p significantly increased IM sensitivity and decreased the survival rate of IM-resistant CML cells, whereas downregulation of miR-153-3p attenuated these effects in IM-resistant CML cells. Upregulated miR-153-3p could decrease the autophagy caused by IM in IM-resistant CML cells. Dual-luciferase reporter assays confirmed that Bcl-2 is a direct target of miR-153-3p. Bcl-2 restoration reversed the increased sensitivity to IM induced by miR-153-3p-mimic transfection in IM-resistant CML cells. The results of the present study showed that dysregulated miR-153-3p may target Bcl-2 to promote the development of IM resistance and attenuate IM-induced apoptosis in CML. Therefore, miR-153-3p upregulation combined with IM treatment may serve as a promising therapeutic strategy for patients with low sensitivity.

Heme oxygenase-1 reduces the sensitivity to imatinib through nonselective activation of histone deacetylases in chronic myeloid leukemia.

Resistance towards imatinib (IM) remains troublesome in treating many chronic myeloid leukemia (CML) patients. Heme oxygenase-1 (HO-1) is a key enzyme of antioxidative metabolism in association with cell resistance to apoptosis. Our previous studies have shown that overexpression of HO-1 resulted in resistance development to IM in CML cells, while the mechanism remains unclear. In the current study, the IM-resistant CML cells K562R indicated upregulation of some of the histone deacetylases (HDACs) compared with K562 cells. Therefore, we herein postulated HO-1 was associated with HDACs. Silencing HO-1 expression in K562R cells inhibited the expression of some HDACs, and the sensitivity to IM was increased. K562 cells transfected with HO-1 resisted IM and underwent obvious some HDACs. These findings related to the inhibitory effects of high HO-1 expression on the reactive oxygen species (ROS) signaling pathway that negatively regulated HDACs. Increased expression of HO-1 activated HDACs by inhibiting ROS production. In summary, HO-1, which is involved in the development of drug resistance in CML cells by regulating the expression of HDACs, is probably a novel target for improving CML therapy.

Downregulation of Mir-181a Restores Chemosensitivity to Imatinib Mesylate in Chronic Myeloid Leukemia Cells Resistant to Tyrosine Kinase Inhibitors

Abelson Interactor protein 1 (Abi-1) is a negative regulator of Abelson kinase and interacts with its oncogenic form Bcr-Abl1 (Brehme et al., PNAS 2009:106;7414). We have recently shown that Abi-1 also modulates interactions between chronic myeloid leukemia (CML) stem/progenitor cells and their microenvironment, and has a role in acquired drug resistance. Our data indicated that abnormally low levels of Abi-1 in CML cells correlate with increased expression of integrin α4, deregulation of Fak/Akt/Erk signaling, enhanced adhesive properties, quiescence, and resistance to imatinib mesylate (IM, Chorzalska et al., Leukemia 2014:28;2165). We have found miR-181a to be significantly upregulated in IM-resistant CML cells, and miRNA expression profiling identified miR-181a as a potential regulator of Abi-1 expression. Furthermore, a bioinformatics analysis matching the miR-181a seed region to conserved ABI1 sequences using TargetScan 6.2 (Lewis, Burge, & Bartel, Cell 2005;120.15) determined that a 3'-untranslated region of ABI1 (position 131-137) is a target for miR-181a. This suggested that miR-181a may regulate Abi-1 levels in IM-resistant CML cells. We hypothesized that decreasing the levels of miR-181a in those cells would restore the intracellular levels of Abi-1, and consequently reduce the expression of integrin α4, decrease the adhesion and quiescence, and restore their sensitivity to IM.To test this hypothesis, we designed and constructed a miRNA sponge (Figure 1) using the methodology established by Kluvier et al. (Methods, 2012:58;113). The sponge can endogenously sequester and functionally reduce levels of miR-181a in CML IM-resistant cells (K562-STI-R cell line), thus allowing to measure the effect of miR-181a loss on Abi-1, integrin α4 levels and chemoresistance. K562-STI-R CML cell line was electroporated with pcDNA3-SanDI vector encoding the miR-181a sponge or a control mismatched miRNA sponge.Using qPCR, we confirmed a 35% percent decrease in miR-181a transcript levels in IM-resistant K562-STI-R CML cell line expressing the design miR-181a sponge, compared with controls (p=0.0015). This was paralleled by a 38% increase in ABI1 mRNA levels (p=0.046) and about 40% increase in Abi-1 protein levels (p=0.03) as evaluated by immunoblotting followed by densitometry, indicating that functionally decreased intracellular levels of miR-181a resulted in increased expression of ABI1. Because we previously demonstrated that down-regulation of ABI1 increases expression of integrin α4 and adhesion of IM-resistant CML cells while decreasing their proliferative potential, we then evaluated ITGA4 transcript levels and expression of integrin α4 in CML cells transfected with the miR-181a sponge. We observed a 20% decrease in ITGA4 transcript and integrin α4 protein levels compared with controls (p=0.051). Phenotypic evaluation of the chemoresistant K562-STI-R cells expressing the miR-181a sponge showed a 36% (p=0.04) increase in the cell number. Decreased number of Annexin V-positive cells suggested that miR-181a sequestration had a modest inhibitory effect on apoptosis. We then evaluated chemosensitivity of the miR-181a sponge-expressing K562-STI-R cells by culturing them in increased concentrations of IM (10, 100 and 200 µM), and measuring apoptosis by Annexin V staining using flow cytometry. We observed 30% (p=0.01), 46% (p=0.023) and 68% (p=0.018) increase in the number of apoptotic cells in miR-181a sponge-expressing cells compared with control cultured at 10, 100 and 200 µM of IM, respectively.In summary, this study provides the first piece of evidence that miR-181a is an epigenetic regulator of ABI1, and that downregulation of miR-181a by a miRNA sponge can effectively increase the intracellular levels of Abi-1 in IM-resistant CML cells, consequently increasing their sensitivity to Bcr-Abl inhibition. Utilization of targeted miRNA inhibitors may represent a new approach to restore chemosensitivity of IM-resistant CML cells. [Display omitted] DisclosuresOlszewski:Bristol-Myers Squibb, Inc.: Consultancy; Genentech, Inc.: Research Funding.

PKC Pathways Mediate BCR-ABL-Independent Imatinib Resistance in Chronic Myeloid Leukemia

Abstract Resistance to imatinib (IM) has posed a great obstacle in treating Chronic Myeloid Leukemia(CML). New generations of tyrosine kinase inhibitors (TKIs) have been developed for BCR-ABL dependent IM resistance, which is commonly due to BCR-ABL kinase-domain mutations. However, in 50% or more of IM-resistant CML patients there is no mutation in BCR-ABL, and intrinsic resistance of CML stem cells often causes disease relapse. Understanding the mechanism of BCR-ABL independent IM resistance is therefore essential for devising strategies to eradicate resistant and residual leukemia. Through a genome-wide shRNA screen in a IM-sensitive human CML cell line, K562 cells, we identified 16 imatinib-sensitizing genes (IMSGs), knockdown of which increased IM resistance of multiple human CML cell lines (e.g., K562, KYO-1 cells. &gt;2 folds increment), and primary CML mice bone marrow cells (&gt; 2 folds of CFUs). Knocking down 11 IMSGs increased IC50IM by more than 5 folds, and also conferred equivalent level of resistance to the second generation TKI, Dasatinib. Extensive signaling pathway analysis by immune-blotting for characteristic protein phosphorylation markers revealed co-activation of PKC and MEK/ERK pathway in majority of IMSG knockdown K562 cell lines. qRT-PCR analysis in IMSG knockdown cells identified PRKCH as the predominant deregulated PKC family member. Mild overexpression of PRKCH increased IC50IM by 10-20 folds, and in vitro phosphorylation confirmed that PRKCH could directly phosphorylate and activate CRAF, therefore MEK/ERK pathway. PRKCH is highly expressed in IM-resistant samples with wildtype BCR-ABL compared to those with mutant BCR-ABL (p&lt;0.05, Mann Whitney test) or IM-sensitive samples (p&lt;0.01, Mann Whitney test). Accordingly, multiple IMSGs were also significantly down-regulated (i.e.,CLEC5A, ELF5, WNT7B, p&lt;0.05), supporting a suppressive role of diverse IMSGs on PRKCH expression. Specifically, we found ELF5 acted as a transcription repressor of PRKCH. To investigate whether simultaneous inhibition of BCR-ABL and MEK/ERK signaling could more efficiently kill BCR-ABL-independent IM-resistant CML cells, we analyzed the effect of combining IM treatment with a FDA-approved MEK inhibitor, Trametinib, in multiple BCR-ABL independent IM-resistant CML mouse models, and primary resistant CML cells. Treatment with both drugs showed a substantially greater effect than either drug alone, and in many instances, the effect of combined drug treatment was synergistic. qRT-PCR analysis showed that PRKCH expression in IM-resistant CML stem cells is higher than that in IM-sensitive CML progenitors (4~25 folds, p&lt;0.01, Student’s t test), implying an important role of PRKCH in CML stem cell resistance. We established a PRKCH knockdown CML mouse model by co-expressing BCR-ABL and a PRKCH shRNA and performed stem cells analysis with or without IM treatment. Although both PRKCH knockdown and IM treatment abolishes MEK/ERK signaling in CML progenitors, only PRKCH controls MEK/ERK pathway in CML stem cells while IM barely has any effect. Combined IM treatment with PRKCH knockdown or MEK inhibition substantially increased the apoptosis rate of CML stem cells (p&lt;0.01, Student’s t test), and prolonged CML mice survival(n=5, p&lt;0.05, Log-rank test). In addition to MEK/ERK pathway, we found PRKCH could activate PKD2, a downstream effector of novel PKCs. PRKCH expression increases upon IM treatment, along with increased PKD2 activity (S916 phosphorylation) in multiple CML cell lines(e.g., K562, KYO-1, LAMA84). PKD2 expression was also induced by IM in primary human CML stem cells. PKD2 promotes cell survival by activating NF-kB pathway, and IM treatment increased p65/RelA phosphorylation at S536 and its nuclear translocation, together with over 4 folds increase in expression of anti-apoptotic p65 target genes (e.g., CIAP2, Bfl-1/A1). Pharmacological inhibition using pan-PKD inhibitor, CRT0066101, completely abolishes IM induced p65 phosphorylation and increased the killing of CML cells by IM. This study for the first time reveals the critical role of PKC pathway in BCR-ABL independent IM resistance in CML. The validation of MEK/ERK and PKD/NF-kB pathway as critical downstream effectors provided specific targets for small molecule therapy in TKI-resistant CML and other malignancies caused by deregulated PKC pathways. Disclosures Cerny: Cellerant Therapeutics: Honoraria, Research Funding.

Tetrandrine citrate eliminates imatinib-resistant chronic myeloid leukemia cells in vitro and in vivo by inhibiting Bcr-Abl/β-catenin axis

To evaluate the effects of tetrandrine citrate, a novel tetrandrine salt with high water solubility, on the growth of imatinib (IM)-resistant chronic myeloid leukemia (CML) in vitro and in vivo, and reveal action molecular mechanisms. Cell viability in vitro was measured using methyl thiazolyl tetrazolium (MTT) assay. CML cell growth in vivo was assessed using a xenograft model in nude mice. Bcr-Abl and β-catenin protein levels were determined using Western blotting. Bcr-Abl messenger RNA (mRNA) was measured by reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry (FCM) was used to determine cell cycle status. Tetrandrine citrate inhibited the growth of IM-resistant K562 cells, primary leukemia cells, and primitive CD34(+) leukemia cells, and their inhibition concentration that inhibited 50% of target cells (IC(50)) ranged from 1.20 to 2.97 μg/ml. In contrast, tetrandrine citrate did not affect normal blood cells under the same conditions, and IC(50) values were about 10.12-13.11 μg/ml. Oral administration of tetrandrine citrate caused complete regression of IM-resistant K562 xenografts in nude mice without overt toxicity. Western blot results revealed that treatment of IM-resistant K562 cells with tetrandrine citrate resulted in a significant decrease of both p210(Bcr-Abl) and β-catenin proteins, but IM did not affect the Bcr-Abl protein levels. Proteasome inhibitor, MG132, did not prevent tetrandrine-mediated decrease of the p210(Bcr-Abl) protein. RT-PCR results showed that tetrandrine treatment caused a decrease of Bcr-Abl mRNA. FCM analysis indicated that tetrandrine induced gap 1 (G(1)) arrest in CML cells. Tetrandrine citrate is a novel orally active tetrandrine salt with potent anti-tumor activity against IM-resistant K562 cells and CML cells. Tetrandrine citrate-induced growth inhibition of leukemia cells may be involved in the depletion of p210(Bcr-Abl) mRNA and β-catenin protein.

Sonic hedgehog signaling regulates Bcr-Abl expression in human chronic myeloid leukemia cells

PurposeBcr-Abl fusion protein activates tyrosine kinase, resulting in the proliferation of leukemia cells, especially chronic myeloid leukemia (CML) cells. Imatinib (IM) effectively targets Bcr-Abl tyrosine kinase, but development of resistance to IM occurs with varying frequency. MethodsElucidation of the common regulatory pathway upstream of Bcr-Abl in IM-sensitive and IM-resistant CML cells is important for developing novel therapeutics against CML. ResultsThis study demonstrated that IM preferentially inhibited the viability and Bcr-Abl expression in IM-sensitive K562 (K562) cells, but not in Bcr-Abl overexpressing IM-resistant K562 (K562R) cells. Both K562 and K562R cells expressed Shh preproprotein, cleaved Shh C-terminal and N-terminal peptides, as well as mRNA level of major Shh signaling molecules, including sonic hedgehog (Shh), patched (PTCH), smoothened (Smo) and Gli-1. Moreover, Gli-1 translocation into nucleus was evident in these two cell lines, suggesting that both K562 and K562R cells possess activated and major components of the Shh signaling pathway. Silencing of Gli-1 by interference RNA was accompanied by inhibition of Bcr-Abl protein expression. Pharmacological suppression of Bcr-Abl expression was restored by the Smo agonist purmorpharmine. Treatment of Shh peptide in both K562 and K562R cells not only increased Shh and Gli-1 expression, but also up-regulated Bcr-Abl expression. Resveratrol, a known Bcr-Abl inhibitor, reduced Gli-1 activation and inhibited the viability of CML cells. ConclusionsShh signaling may regulate Bcr-Abl expression in human chronic myeloid leukemia cells. Novel compounds inhibiting both Shh signaling and Bcr-Abl expression, such as resveratrol, may have potential to be effective agents against CML independent of IM resistance.

Destabilization of Bcr-Abl/Jak2 Network by a Jak2/Abl Kinase Inhibitor ON044580 Overcomes Drug Resistance in Blast Crisis Chronic Myelogenous Leukemia (CML)

Bcr-Abl is the predominant therapeutic target in chronic myeloid leukemia (CML), and tyrosine kinase inhibitors (TKIs) that inhibit Bcr-Abl have been successful in treating CML. With progression of CML disease especially in blast crisis stage, cells from CML patients become resistant to imatinib mesylate (IM) and other TKIs, resulting in relapse. Because Bcr-Abl is known to drive multiple signaling pathways, the study of the regulation of stability of Bcr-Abl in IM-resistant CML cells is a critical issue as a possible therapeutic strategy. Here, we report that a new dual-kinase chemical inhibitor, ON044580, induced apoptosis of Bcr-Abl+ IM-sensitive, IM-resistant cells, including the gatekeeper Bcr-Abl mutant, T315I, and also cells from blast crisis patients. In addition, IM-resistant K562-R cells, cells from blast crisis CML patients, and all IM-resistant cell lines tested had reduced ability to form colonies in soft agar in the presence of 0.5 µM ON044580. In in vitro kinase assays, ON044580 inhibited the recombinant Jak2 and Abl kinase activities when the respective Jak2 and Abl peptides were used as substrates. Incubation of the Bcr-Abl+ cells with ON044580 rapidly reduced the levels of the Bcr-Abl protein and also reduced the expression of HSP90 and its client protein levels. Lysates of Bcr-Abl+ cell lines were found to contain a large signaling network complex composed of Bcr-Abl, Jak2, HSP90, and its client proteins as detected by a gel filtration column chromatography, which was rapidly disrupted by ON044580. Therefore, targeting Jak2 and Bcr-Abl kinases is an effective way to destabilize Bcr-Abl and its network complex, which leads to the onset of apoptosis in IM-sensitive and IM-resistant Bcr-Abl+ cells. This inhibitory strategy has potential to manage all types of drug-resistant CML cells, especially at the terminal blast crisis stage of CML, where TKIs are not clinically useful.

Effects of carboxyamidotriazole on in vitro models of imatinib‐resistant chronic myeloid leukemia

Although imatinib mesylate (IM) has revolutionized the treatment of chronic myeloid leukemia (CML), some patients develop resistance with progression of leukemia. Alternative or additional targeting of signaling pathways deregulated in bcr-abl-driven CML cells may provide a feasible option for improving clinical response and overcoming resistance. In this study, we show that carboxyamidotriazole (CAI), an orally bioavailable calcium influx and signal transduction inhibitor, is equally effective in inhibiting the proliferation and bcr-abl dependent- and independent-signaling pathways in imatinib-resistant CML cells. CAI inhibits phosphorylation of cellular proteins including STAT5 and CrkL at concentrations that induce apoptosis in IM-resistant CML cells. The combination of imatinib and CAI also down-regulated bcr-abl protein levels. Since CAI is already available for clinical use, these results suggest that it may be an effective addition to the armamentarium of drugs for the treatment of CML.

Survivin silencing sensitizes imatinib-resistant CML cells to the cytotoxic effect of hydroxyurea

13119 Background: Imatinib Mesylate (IM), a semi-specific inhibitor of the BCR-ABL tyrosine kinase, is currently the treatment of choice for Chronic Myeloid Leukemia (CML). However, about one third of CML patients treated with IM develop resistance to the drug because of reactivation of BCR-ABL kinase activity. This phenomenon is usually ascribed to the amplification of the BCR-ABL gene or to the selection of leukemic clones harboring point mutations that abrogate IM binding. To identify novel anti-apoptotic signaling pathways employed by BCR-ABL and devise strategies capable of killing IM-resistant CML cells, we investigated the interplay between BCR-ABL and the Inhibitor of Apoptosis Protein Survivin. Methods: Murine hematopoietic cells (32D) transduced with p210 BCR-ABL and human cell lines either positive (K562, KCL22, KYO1 and LAMA84) or negative (HL60) for the BCR-ABL oncoprotein, were analyzed for Survivin expression by western blot before and after IM treatment. Three different pathways (MAPK, PI3K and JAK2/STA3) potentially involved in BCR-ABL-mediated induction of Survivin were studied using inhibitors specific for each signaling cascade. The effect of Survivin on the proliferation and viability of IM-sensitive and IM-resistant CML cells was investigated after silencing Survivin expression with small interfering RNAs. Results: BCR-ABL tyrosine kinase activity induced an over-expression of Survivin in both human and murine hematopoietic cell lines. This over-expression was both at the transcriptional and the translational level and required the JAK2/STAT3 pathway. Survivin silencing by siRNA increased IM cytotoxicity in IM-sensitive cells but failed to restore IM efficacy in IM-resistant cells. However, Survivin silencing sensitized CML cells to the cytotoxic effect of hydroxyurea and enhanced the efficacy of this compound on three different murine cell lines are insensitive to IM because of point mutations in the BCR-ABL kinase domain (Ba/F3p210Y253F, Ba/F3p210D276G and Ba/F3p210T315I). Conclusions: Reduction of Survivin expression improves the efficacy of IM and increases the sensitivity of IM-resistant CML cells to hydroxyurea. Survivin may represent an attractive therapeutic target for both IM-sensitive and IM-resistant CML patients. No significant financial relationships to disclose.

Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction

AbstractDasatinib (BMS-354825), a novel dual SRC/BCR-ABL kinase inhibitor, exhibits greater potency than imatinib mesylate (IM) and inhibits the majority of kinase mutations in IM-resistant chronic myeloid leukemia (CML). We have previously demonstrated that IM reversibly blocks proliferation but does not induce apoptosis of primitive CML cells. Here, we have attempted to overcome this resistance with dasatinib. Primitive IM-resistant CML cells showed only single-copy BCR-ABL but expressed significantly higher BCR-ABL transcript levels and BCR-ABL protein compared with more mature CML cells (P = .031). In addition, CrKL phosphorylation was higher in the primitive CD34+CD38– than in the total CD34+ population (P = .002). In total CD34+ CML cells, IM inhibited phosphorylation of CrKL at 16 but not 72 hours, consistent with enrichment of an IM-resistant primitive population. CD34+CD38– CML cells proved resistant to IM-induced inhibition of CrKL phosphorylation and apoptosis, whereas dasatinib led to significant inhibition of CrKL phosphorylation. Kinase domain mutations were not detectable in either IM or dasatinib-resistant primitive CML cells. These data confirm that dasatinib is more effective than IM within the CML stem cell compartment; however, the most primitive quiescent CML cells appear to be inherently resistant to both drugs.