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Platinum pyrithione induces apoptosis in chronic myeloid leukemia cells resistant to imatinib via DUB inhibition-dependent caspase activation and Bcr-Abl downregulation.

ABSTRACT: Chronic myelogenous leukemia (CML) is characterized by the chimeric tyrosine kinase Bcr-Abl. T315I Bcr-Abl is the most notorious point mutation to elicit acquired resistance to imatinib (IM), leading to poor prognosis. Therefore, it is urgent to search for additional approaches and targeting strategies to overcome IM resistance. We recently reported that platinum pyrithione (PtPT) potently inhibits the ubiquitin-proteasome system (UPS) via targeting the 26 S proteasome-associated deubiquitinases (DUBs), without effecting on the 20 S proteasome. Here we further report that (i) PtPT induces apoptosis in Bcr-Abl wild-type and Bcr-Abl-T315I mutation cells including the primary mononuclear cells from CML patients clinically resistant to IM, as well as inhibits the growth of IM-resistant Bcr-Abl-T315I xenografts in vivo; (ii) PtPT downregulates Bcr-Abl level through restraining Bcr-Abl transcription, and decreasing Bcr-Abl protein mediated by DUBs inhibition-induced caspase activation; (iii) UPS inhibition is required for PtPT-induced caspase activation and cell apoptosis. These findings support that PtPT overcomes IM resistance through both Bcr-Abl-dependent and -independent mechanisms. We conclude that PtPT can be a lead compound for further drug development to overcome imatinib resistance in CML patients.

SUBMITTER: Lan X

PROVIDER: S-EPMC5550844 | biostudies-other | 2017 Jul

REPOSITORIES: biostudies-other

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Gambogic acid induces apoptosis in imatinib-resistant chronic myeloid leukemia cells via inducing proteasome inhibition and caspase-dependent Bcr-Abl downregulation.

Project description:Chronic myelogenous leukemia (CML) is characterized by the constitutive activation of Bcr-Abl tyrosine kinase. Bcr-Abl-T315I is the predominant mutation that causes resistance to imatinib, cytotoxic drugs, and the second-generation tyrosine kinase inhibitors. The emergence of imatinib resistance in patients with CML leads to searching for novel approaches to the treatment of CML. Gambogic acid, a small molecule derived from Chinese herb gamboges, has been approved for phase II clinical trial for cancer therapy by the Chinese Food and Drug Administration (FDA). In this study, we investigated the effect of gambogic acid on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl.CML cell lines (KBM5, KBM5-T315I, and K562), primary cells from patients with CML with clinical resistance to imatinib, and normal monocytes from healthy volunteers were treated with gambogic acid, imatinib, or their combination, followed by measuring the effects on cell growth, apoptosis, and signal pathways. The in vivo antitumor activity of gambogic acid and its combination with imatinib was also assessed with nude xenografts.Gambogic acid induced apoptosis and cell proliferation inhibition in CML cells and inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Our data suggest that GA-induced proteasome inhibition is required for caspase activation in both imatinib-resistant and -sensitive CML cells, and caspase activation is required for gambogic acid-induced Bcr-Abl downregulation and apoptotic cell death.These findings suggest an alternative strategy to overcome imatinib resistance by enhancing Bcr-Abl downregulation with the medicinal compound gambogic acid, which may have great clinical significance in imatinib-resistant cancer therapy.

| S-EPMC3938960 | biostudies-literature

MicroRNA-1301-Mediated RanGAP1 Downregulation Induces BCR-ABL Nuclear Entrapment to Enhance Imatinib Efficacy in Chronic Myeloid Leukemia Cells.

Project description:Chronic myeloid leukemia (CML) is a myeloproliferative disease. Imatinib (IM), the first line treatment for CML, is excessively expensive and induces various side effects in CML patients. Therefore, it is essential to investigate a new strategy for improving CML therapy. Our immunoblot data revealed that RanGTPase activating protein 1 (RanGAP1) protein levels increased by approximately 30-fold in K562 cells compared with those in normal cells. RanGAP1 is one of the important components of RanGTPase system, which regulates the export of nuclear protein. However, whether RanGAP1 level variation influences BCR-ABL nuclear export is still unknown. In this report, using shRNA to downregulate RanGAP1 expression level augmented K562 cell apoptosis by approximately 40% after treatment with 250 nM IM. Immunofluorescence assay also indicated that three-fold of nuclear BCR-ABL was detected. These data suggest that BCR-ABL nuclear entrapment induced by RanGAP1 downregulation can be used to improve IM efficacy. Moreover, our qRT-PCR data indicated a trend of inverse correlation between the RanGAP1 and microRNA (miR)-1301 levels in CML patients. MiR-1301, targeting the RanGAP1 3' untranslated region, decreased by approximately 100-fold in K562 cells compared with that in normal cells. RanGAP1 downregulation by miR-1301 transfection impairs BCR-ABL nuclear export to increase approximately 60% of cell death after treatment of 250 nM IM. This result was almost the same as treatment with 1000 nM IM alone. Furthermore, immunofluorescence assay demonstrated that Tyr-99 of nuclear P73 was phosphorylated accompanied with nuclear entrapment of BCR-ABL after transfection with RanGAP1 shRNA or miR-1301 in IM-treated K562 cells. Altogether, we demonstrated that RanGAP1 downregulation can mediate BCR-ABL nuclear entrapment to activate P73-dependent apoptosis pathway which is a novel strategy for improving current IM treatment for CML.

| S-EPMC4881950 | biostudies-literature

Cantharidin Overcomes Imatinib Resistance by Depleting BCR-ABL in Chronic Myeloid Leukemia.

Project description:Cantharidin (CTD) is an active compound isolated from the traditional Chinese medicine blister beetle and displayed anticancer properties against various types of cancer cells. However, little is known about its effect on human chronic myeloid leukemia (CML) cells, including imatinib-resistant CML cells. The objective of this study was to investigate whether CTD could overcome imatinib resistance in imatinib-resistant CML cells and to explore the possible underlying mechanisms associated with the effect. Our results showed that CTD strongly inhibited the growth of both imatinib-sensitive and imatinib-resistant CML cells. CTD induced cell cycle arrest at mitotic phase and triggered DNA damage in CML cells. The ATM/ATR inhibitor CGK733 abrogated CTD-induced mitotic arrest but promoted the cytotoxic effects of CTD. In addition, we demonstrated that CTD downregulated the expression of the BCR-ABL protein and suppressed its downstream signal transduction. Real-time quantitative PCR revealed that CTD inhibited BCR-ABL at transcriptional level. Knockdown of BCR-ABL increased the cell-killing effects of CTD in K562 cells. These findings indicated that CTD overcomes imatinib resistance through depletion of BCR-ABL. Taken together, CTD is an important new candidate agent for CML therapy.

| S-EPMC5223104 | biostudies-literature

BCR-ABL SH3-SH2 domain mutations in chronic myeloid leukemia patients on imatinib.

Project description:Point mutations in the kinase domain of BCR-ABL are the most common mechanism of drug resistance in chronic myeloid leukemia (CML) patients treated with ABL kinase inhibitors, including imatinib. It has also been shown in vitro that mutations outside the kinase domain in the neighboring linker, SH2, SH3, and Cap domains can confer imatinib resistance. In the context of ABL, these domains have an autoinhibitory effect on kinase activity, and mutations in this region can activate the enzyme. To determine the frequency and relevance to resistance of regulatory domain mutations in CML patients on imatinib, we screened for such mutations in a cohort of consecutive CML patients with various levels of response. Regulatory domain mutations were detected in 7 of 98 patients, whereas kinase domain mutations were detected in 29. One mutation (T212R) conferred in vitro tyrosine kinase inhibitor resistance and was associated with relapse, whereas most other mutations did not affect drug sensitivity. Mechanistic studies showed that T212R increased the activity of ABL and BCR-ABL and that T212R-induced resistance may be partially the result of stabilization of an active kinase conformation. Regulatory domain mutations are uncommon but may explain resistance in some patients without mutations in the kinase domain.

| S-EPMC2995357 | biostudies-literature

Differentiation of imatinib -resistant chronic myeloid leukemia cells with BCR-ABL-T315I mutation induced by Jiyuan Oridonin A.

Project description:Chronic myeloid leukemia (CML) results from BCR-ABL oncogene, which blocks CML cells differentiation and protects these cells from apoptosis. T315I mutated BCR-ABL is the main cause of the resistance mediated by imatinib and second generation BCR-ABL inhibitor. CML with the T315I mutation has been considered to have poor prognosis. Here, we determined the effect of Jiyuan oridonin A (JOA), an ent-kaurene diterpenoid compound, on the differentiation blockade in imatinib-sensitive, particularly, imatinib-resistant CML cells with BCR-ABL-T315I mutation by cell proliferation assay, apoptosis analysis, cell differentiation analysis, cell cycle analysis and colony formation assay. We also investigated the possible molecular mechanism by mRNA sequencing, qRT-PCR and Western blotting. We found that JOA at lower concentration significantly inhibited the proliferation of CML cells expressing mutant BCR-ABL (T315I mutation included) and wild-type BCR-ABL, which was due to that JOA induced the cell differentiation and the cell cycle arrest at G0/G1 phase. Interestingly, JOA possessed stronger anti-leukemia activity than its analogues such as OGP46 and Oridonin, which has been investigated extensively. Mechanistically, the cell differentiation mediated by JOA may be originated from the inhibition of BCR-ABL/c-MYC signaling in CML cells expressing wild-type BCR-ABL and BCR-ABL-T315I. JOA displayed the activity of inhibiting the BCR-ABL and promoted differentiation of not only imatinib -sensitive but also imatinib -resistant cells with BCR-ABL mutation, which could become a potent lead compound to overcome the imatinib -resistant induced by inhibitors of BCR-ABL tyrosine kinase in CML therapy.

| S-EPMC10197941 | biostudies-literature

Molecular screening and the clinical impacts of BCR-ABL KD mutations in patients with imatinib-resistant chronic myeloid leukemia.

Project description:The present study aimed to detect the frequency of kinase domain (KD) mutations in order to evaluate their clinical significance and functional importance in 45 patients with chronic myeloid leukemia (CML) who were resistant to imatinib therapy. Sanger sequencing was used (45 patients), along with allele-specific oligonucleotide polymerase chain reaction (ASO-PCR; 3 patients), for the screening of mutations. BCR/ABL KD was amplified by nested PCR and sequencing was performed. Secondly, ASO-PCR was performed to confirm the results of the sequence analysis for E255K mutations. Mutations were detected in 11/45 patients (24.44%) via Sanger sequencing. D241G (4.4%), C369C (4.4%), K285N (2.2%), A380T (2.2%) and A366V (2.2%) mutations were detected. E255K (8.8%) was detected by ASO-PCR and Sanger sequencing. Mutations are a primary reason for suboptimal responses, loss of response and resistance to imatinib. In particular, the E255K mutation, which is characterized by resistance to imatinib and nilotinib, was detected in four patients. Analyzing the mutations and monitoring patients with CML may improve their prognosis and survival rate. ASO-PCR assays will be beneficial for the routine monitoring of mutations.

| S-EPMC5777321 | biostudies-literature

A therapeutically targetable mechanism of BCR-ABL-independent imatinib resistance in chronic myeloid leukemia.

Project description:Resistance to the BCR-ABL inhibitor imatinib mesylate (IM) poses a major problem for the treatment of chronic myeloid leukemia (CML). IM resistance often results from a secondary mutation in BCR-ABL that interferes with drug binding. However, in many instances, there is no mutation in BCR-ABL, and the basis of such BCR-ABL-independent IM resistance remains to be elucidated. To gain insight into BCR-ABL-independent IM resistance mechanisms, we performed a large-scale RNA interference screen and identified IM-sensitizing genes (IMSGs) whose knockdown renders BCR-ABL(+) cells IM-resistant. In these IMSG knockdown cells, RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling is sustained after IM treatment because of up-regulation of PRKCH, which encodes the protein kinase C (PKC) family member PKC?, an activator of CRAF. PRKCH is also up-regulated in samples from CML patients with BCR-ABL-independent IM resistance. Combined treatment with IM and trametinib, a U.S. Food and Drug Administration-approved MEK inhibitor, synergistically kills BCR-ABL(+) IMSG knockdown cells and prolongs survival in mouse models of BCR-ABL-independent IM-resistant CML. Finally, we showed that CML stem cells contain high levels of PRKCH, and this contributes to their intrinsic IM resistance. Combined treatment with IM and trametinib synergistically kills CML stem cells with negligible effect on normal hematopoietic stem cells. Collectively, our results identify a therapeutically targetable mechanism of BCR-ABL-independent IM resistance in CML and CML stem cells.

| S-EPMC4162097 | biostudies-literature

Curcumin derivative C817 inhibits proliferation of imatinib-resistant chronic myeloid leukemia cells with wild-type or mutant Bcr-Abl in vitro.

Project description:To find new kinase inhibitors that overcome the imatinib resistance in treatment of chronic myeloid leukemia (CML), we synthesized C817, a novel derivative of curcumin, and tested its activities against wild-type (WT) and imatinib-resistant mutant Abl kinases, as well as in imatinib-sensitive and resistant CML cells in vitro.32D cells harboring WT or mutant Abl kinases (nucleotide binding P-loop mutants Q252H, Y253F, and imatinib contact residue mutant T315I), as well as K562/G01 cells (with whole Bcr-Abl gene amplication) were tested. Kinase activity was measured using Kinase-Glo Luminescent Kinase Assay Platform in recombinant WT and mutant (Q252H, Y253F, and T315I) Abl kinases. Cell proliferation and apoptosis were examined using MTT assay and flow cytometry, respectively. The phosphorylation levels of Bcr-Abl initiated signaling proteins were analyzed using Western blotting. Colony forming units (CFU) growth and long term culture-initiating cells (LTC-ICs) were used to test the effects of C817 on human leukemia progenitor/stem cells.C817 potently inhibited both WT and mutant (Q252H, Y253F, and T315I) Abl kinase activities in a non-ATP competitive manner with the values of IC₅₀ at low nanomole levels. In consistent with above results, C817 suppressed the growth of both imatinib-sensitive and resistant CML cells, including wild-type K562, K562/G01, 32D-T315I, 32D-Q252H, and 32D-Y253F cells with the values of IC₅₀ at low micromole levels. C817 (0.5 or 1 μmol/L) dose-dependently inhibited the phosphorylation of Bcr-Abl and downstream proteins STAT-5 and CrkL in imatinib-resistant K562/G01 cells. Furthermore, C817 significantly suppressed CFU growth and LTC-ICs, implicating that C817 could eradiate human leukemia progenitor/stem cells.C817 is a promising compound for treatment of CML patients with Bcr-Abl kinase domain mutations that confer imatinib resistance.

| S-EPMC4647898 | biostudies-other

Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity.

Project description:Imatinib therapy, which targets the oncogene product BCR-ABL, has transformed chronic myeloid leukemia (CML) from a life-threatening disease into a chronic condition. Most patients, however, harbor residual leukemia cells, and disease recurrence usually occurs when imatinib is discontinued. Although various mechanisms to explain leukemia cell persistence have been proposed, the critical question from a therapeutic standpoint--whether disease persistence is BCR-ABL dependent or independent--has not been answered. Here, we report that human CML stem cells do not depend on BCR-ABL activity for survival and are thus not eliminated by imatinib therapy. Imatinib inhibited BCR-ABL activity to the same degree in all stem (CD34+CD38-, CD133+) and progenitor (CD34+CD38+) cells and in quiescent and cycling progenitors from newly diagnosed CML patients. Although short-term in vitro imatinib treatment reduced the expansion of CML stem/progenitors, cytokine support permitted growth and survival in the absence of BCR-ABL activity that was comparable to that of normal stem/progenitor counterparts. Our findings suggest that primitive CML cells are not oncogene addicted and that therapies that biochemically target BCR-ABL will not eliminate CML stem cells.

| S-EPMC3007128 | biostudies-literature

Anti-rheumatic agent auranofin induced apoptosis in chronic myeloid leukemia cells resistant to imatinib through both Bcr/Abl-dependent and -independent mechanisms.

Project description:Resistance to Imatinib mesylate (IM) is an emerging problem for patients with chronic myelogenous leukemia (CML). T315I mutation in the Bcr-Abl is the predominant mechanism of the acquired resistance to IM and second generation tyrosine kinase inhibitors (TKI). Therefore it is urgent to search for new measures to overcome TKI-resistance. Auranofin (AF), clinically used to treat rheumatic arthritis, was recently approved by US Food and Drug Administration for Phase II clinical trial to treat cancer. In contrast to the reports that AF induces apoptosis by increasing intracellular reactive oxygen species (ROS) levels via inhibiting thioredoxin reductase, our recent study revealed that AF-induced apoptosis depends on inhibition of proteasomal deubiquitinases (UCHL5 and USP14). Here we report that (i) AF induces apoptosis in both Bcr-Abl wild-type cells and Bcr-Abl-T315I mutation cells and inhibits the growth of IM-resistant Bcr-Abl-T315I xenografts in vivo; (ii) AF inhibits Bcr-Abl through both downregulation of Bcr-Abl gene expression and Bcr-Abl cleavage mediated by proteasome inhibition-induced caspase activation; (iii) proteasome inhibition but not ROS is required for AF-induced caspase activation and apoptosis. These findings support that AF overcomes IM resistance through both Bcr/Abl-dependent and -independent mechanisms, providing great clinical significance for cancer treatment.

| S-EPMC4253423 | biostudies-literature

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