Emodin Inhibits Resistance to Imatinib by Downregulation of Bcr-Abl and STAT5 and Allosteric Inhibition in Chronic Myeloid Leukemia Cells.

Imatinib-resistance is a significant concern for Bcr-Abl-positive chronic myelogenous leukemia (CML) treatment. Emodin, the predominant compound of traditional medicine rhubarb, was reported to inhibit the multidrug resistance by downregulating P-glycoprotein of K562/ADM cells with overexpression of P-glycoprotein in our previous studies. In the present study, we found that emodin can be a potential inhibitor for the imatinib-resistance in K562/G01 cells which are the imatinib-resistant subcellular line of human chronic myelogenous leukemia cells with overexpression of breakpoint cluster region-abelson (Bcr-Abl) oncoprotein. Emodin greatly enhanced cell sensitivity to imatinib, suppressed resistant cell proliferation and increased potentiated apoptosis induced by imatinib in K562/G01 cells. After treatment of emodin and imatinib together, the levels of p-Bcr-Abl and Bcr-Abl were significantly downregulated. Moreover, Bcr-Abl important downstream target, STAT5 and its phosphorylation were affected. Furthermore, the expression of Bcr-Abl and signal transducers and activators of transcription 5 (STAT5) related molecules, including c-MYC, MCL-1, poly(ADP-ribose)polymerase (PARP), Bcl-2 and caspase-3, were changed. Emodin also decreased Src expression and its phosphorylation. More importantly, emodin simultaneously targeted both the ATP-binding and allosteric sites on Bcr-Abl by molecular docking, with higher affinity with the myristoyl-binding site for enhanced Bcr-Abl kinase inhibition. Overall, these data indicated emodin might be an effective therapeutic agent for inhibiting resistance to imatinib in CML treatment.

Andrographolide and its potent derivative exhibit anticancer effects against imatinib-resistant chronic myeloid leukemia cells by downregulating the Bcr-Abl oncoprotein.

Chronic myelogenous leukemia (CML) is clinically treated with imatinib, which inhibits the kinase activity of the Bcr-Abl oncoprotein. However, imatinib resistance remains a common clinical issue. Andrographolide, the major compound of the medicinal plant Andrographis paniculata, was reported to exhibit anticancer activity. In this study, we explored the therapeutic potential of andrographolide and its derivative, NCTU-322, against both imatinib-sensitive and imatinib-resistant human CML cell lines. Both andrographolide and NCTU-322 downregulated the Bcr-Abl oncoprotein in imatinib-resistant CML cells through an Hsp90-dependent mechanism similar to that observed in imatinib-sensitive CML cells. In addition, NCTU-322 had stronger effects than andrographolide on downregulation of Bcr-Abl oncoprotein, induction of Hsp90 cleavage and cytotoxicity of CML cells. Notably, andrographolide and NCTU-322 could induce differentiation, mitotic arrest and apoptosis of both imatinib-sensitive and imatinib-resistant CML cells. Finally, the anticancer activity of NCTU-322 against imatinib-resistant CML cells was demonstrated in vivo. In summary, our data demonstrated that andrographolide and NCTU-322 inhibit Bcr-abl function via a mechanism different from that of imatinib, and they induced multiple anticancer effects in both imatinib-sensitive and resistant CML cells. Our findings demonstrate that andrographolide and NCTU-322 are potential therapeutic agents again CML.

C-Abl is not activated in DNA damage-induced and Tap63-mediated oocyte apoptosis in human ovary.

There is a controversy in literature as to whether c-Abl is crucial for the induction of TAp63-mediated apoptosis and whether that inhibition of c-Abl with imatinib, which was designed to inhibit the oncogenic kinase BCR-ABL and c-kit, protects oocytes from chemotherapy-induced apoptosis in mice. No human data are available on this issue. We therefore aimed to explore whether genomic damage induced by chemotherapy drug cisplatin activates c-Abl along with TAp63 and the inhibition of c-Abl with imatinib prevents cisplatin-induced oocyte death and follicle loss in human ovary. Exposure to cisplatin induced DNA damage, activated TAp63 and SAPK/JNK pathway, and triggered apoptosis in the oocytes and granulosa cells. However, TAp63 activation after cisplatin was not associated with any increase in the expression of c-Abl. Imatinib did not prevent cisplatin-induced apoptosis of the granulosa cells or oocytes. Moreover, treatment with this drug resulted in the formation of bizarre shaped follicles lacking oocytes and increased follicular atresia by inducing apoptosis of granulosa cells and oocytes. Similar toxic effects were observed when ovarian tissue samples were incubated with a c-kit antagonist drug anti-CD117, but not with another c-Abl tyrosine kinase inhibitor GNF-2, which lacks an inhibitory action on c-kit. Intraperitoneal administration of imatinib to the xenografted animals produced similar histomorphological abnormalities in the follicles in human ovarian grafts and did not prevent cisplatin-induced follicle loss when co-administered with cisplatin. Our findings provide, for the first time, a molecular evidence for ovarian toxicity of this drug in human. Furthermore, this study together with two previous case reports of a severely compromised ovarian response to gonadotropin stimulation and premature ovarian failure in patients, while receiving imatinib, further heighten the concerns about its potential gonadotoxicity on human ovary and urge caution in its use in young female patients.

Efficacy and safety of autologous peripheral blood stem cell transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia: A study protocol for a multicenter exploratory prospective study (Auto-Ph17 study).

INTRODUCTION: The prognosis of Philadelphia chromosome positive acute lymphoblastic leukemia (Ph + ALL) has been dramatically improved since the introduction of tyrosine kinase inhibitors (TKIs). Although allogeneic hematopoietic cell transplantation (allo-HCT) is a major treatment option, the role of autologous peripheral blood stem cell transplantation (auto-PBSCT) has been reconsidered, especially in patients who achieved early molecular remission. METHODS AND ANALYSIS: This is a multicenter exploratory study for Ph + ALL patients aged between 55 and 70 years who achieved complete molecular remission within 3 cycles of chemotherapy. The target sample size is 5, and the registration period is 2 years. The primary endpoint is Day100- mortality after transplantation, and the secondary endpoints are survival, relapse rate, nonrelapse mortality, and adverse events.This study is divided into 3 phases: peripheral blood stem cell harvest, transplantation, and maintenance. Chemomobilization is performed using a combination of cyclophosphamide (CPM), doxorubicin, vincristine (VCR), and prednisolone (PSL). As a preparative regimen, the LEED regimen is used, which consists of melphalan, CPM, etoposide, and dexamethasone. Twelve cycles of maintenance therapy using a combination of VCR, PSL, and dasatinib are performed.In association with relapse, the minimal residual disease (MRD) of BCR-ABL chimeric gene and T-cell subsets are analyzed both before and after auto-PBSCT. ETHICS AND DISSEMINATION: The protocol was approved by the institutional review board of Nagoya University Hospital and all the participating hospitals. Written informed consent was obtained from all patients before registration, in accordance with the Declaration of Helsinki. Results of the study will be disseminated via publications in peer-reviewed journals. TRIAL REGISTRATION: Trial registration number UMIN000026445.

Dysregulation of catalase activity in newborn myocytes during hypoxia is mediated by c-Abl tyrosine kinase.

In the adult heart, catalase (CAT) activity increases appropriately with increasing levels of hydrogen peroxide, conferring cardioprotection. This mechanism is absent in the newborn for unknown reasons. In the present study, we examined how the posttranslational modification of CAT contributes to its activation during hypoxia/ischemia and the role of c-Abl tyrosine kinase in this process. Hypoxia studies were carried out using primary cardiomyocytes from adult (>8 weeks) and newborn rats. Following hypoxia, the ratio of phosphorylated to total CAT and c-Abl in isolated newborn rat myocytes did not increase and were significantly lower (1.3- and 4.2-fold, respectively; P < .05) than their adult counterparts. Similarly, there was a significant association (P < .0005) between c-Abl and CAT in adult cells following hypoxia (30.9 ± 8.2 to 70.7 ± 13.1 au) that was absent in newborn myocytes. Although ubiquitination of CAT was higher in newborns compared to adults following hypoxia, inhibition of this did not improve CAT activity. When a c-Abl activator (5-(1,3-diaryl-1H-pyrazol-4-yl)hydantoin [DPH], 200 µmol/L) was administered prior to hypoxia, not only CAT activity was significantly increased (P < .05) but also phosphorylation levels were also significantly improved (P < .01) in these newborn myocytes. Additionally, ischemia-reperfusion (IR) studies were performed using newborn (4-5 days) rabbit hearts perfused in a Langendorff method. The DPH given as an intracardiac injection into the right ventricle of newborn rabbit resulted in a significant improvement (P < .002) in the recovery of developed pressure after IR, a key indicator of cardiac function (from 74.6% ± 6.6% to 118.7% ± 10.9%). In addition, CAT activity was increased 3.92-fold (P < .02) in the same DPH-treated hearts. Addition of DPH to adult rabbits in contrast had no significant effect (from 71.3% ± 10.7% to 59.4% ± 12.1%). Therefore, in the newborn, decreased phosphorylation of CAT by c-Abl potentially mediates IR-induced dysfunction, and activation of c-Abl may be a strategy to prevent ischemic injury associated with surgical procedures.

c-Abl antagonizes the YAP oncogenic function.

YES-associated protein (YAP) is a central transcription coactivator that functions as an oncogene in a number of experimental systems. However, under DNA damage, YAP activates pro-apoptotic genes in conjunction with p73. This program switching is mediated by c-Abl (Abelson murine leukemia viral oncogene) via phosphorylation of YAP at the Y357 residue (pY357). YAP as an oncogene coactivates the TEAD (transcriptional enhancer activator domain) family transcription factors. Here we asked whether c-Abl regulates the YAP-TEAD functional module. We found that DNA damage, through c-Abl activation, specifically depressed YAP-TEAD-induced transcription. Remarkably, c-Abl counteracts YAP-induced transformation by interfering with the YAP-TEAD transcriptional program. c-Abl induced TEAD1 phosphorylation, but the YAP-TEAD complex remained unaffected. In contrast, TEAD coactivation was compromised by phosphomimetic YAP Y357E mutation but not Y357F, as demonstrated at the level of reporter genes and endogenous TEAD target genes. Furthermore, YAP Y357E also severely compromised the role of YAP in cell transformation, migration, anchorage-independent growth, and epithelial-to-mesenchymal transition (EMT) in human mammary MCF10A cells. These results suggest that YAP pY357 lost TEAD transcription activation function. Our results demonstrate that YAP pY357 inactivates YAP oncogenic function and establish a role for YAP Y357 phosphorylation in cell-fate decision.

Exposure of neuroblastoma cell lines to imatinib results in the upregulation of the CDK inhibitor p27(KIP1) as a consequence of c-Abl inhibition.

Imatinib mesylate is a tyrosine kinase inhibitor with selectivity for abelson tyrosine-protein kinase 1 (c-Abl), breakpoint cluster region (Bcr)-Abl fusion protein (Bcr-Abl), mast/stem cell growth factor receptor Kit (c-Kit), and platelet-derived growth factor receptor (PDGFR). Previous studies demonstrated that imatinib in the low micromolar range exerted antiproliferative effects on neuroblastoma cell lines. However, although neuroblastoma cells express c-Kit and PDGFR, the imatinib concentrations required to achieve significant growth inhibitory effects (≥ 10 µM) are substantially higher than those required for inhibition of ligand-induced phosphorylation of wild type c-Kit and PDGFR (≤ 1 µM), suggesting that additional mechanisms are responsible for the antitumor activity of imatinib on these cells. In this study, we show that treatment of neuroblastoma cell lines with 1-15 µM imatinib resulted in a dose dependent inhibition of 5-bromo-2'-deoxyuridine (BrdU) incorporation into newly synthesized DNA. The antiproliferative effect of imatinib was dependent on the upregulation of the cyclin-dependent kinase (CDK) inhibitor p27(KIP1) in the nuclear compartment as a result of increased p27(KIP1) protein stability. We demonstrate that the mechanism of p27(KIP1) stabilization relied on inhibition of p27(KIP1) phosphorylation on tyrosine residues by c-Abl. We provide evidence that in neuroblastoma cell lines a significant fraction of cellular c-Abl is phosphorylated on Tyr-245, consistent with an open and active conformation. Notably, exposure to imatinib did not affect Tyr-245 phosphorylation. Given the low affinity of active c-Abl for imatinib, these data provide a molecular explanation for the relatively high imatinib concentrations required to inhibit neuroblastoma cell proliferation.

C-Abl tyrosine kinase mediates neurotoxic prion peptide-induced neuronal apoptosis via regulating mitochondrial homeostasis.

Prion diseases are neurodegenerative disorders characterized by the accumulation of a disease-associated prion protein and apoptotic neuronal death. Previous studies indicated that the ubiquitous expression of c-Abl tyrosine kinase transduces a variety of extrinsic and intrinsic cellular signals. In this study, we demonstrated that a synthetic neurotoxic prion fragment (PrP106-126) activated c-Abl tyrosine kinase, which in turn triggered the upregulation of MST1 and BIM, suggesting the activation of the c-Abl-BIM signaling pathway. The peptide fragment was found to result in cell death via mitochondrial dysfunction in neuron cultures. Knockdown of c-Abl using small interfering RNA protected neuronal cells from PrP106-126-induced mitochondrial dysfunction, production of reactive oxygen species, and apoptotic events inducing translocation of Bax to the mitochondria, cytochrome c release into the cytosol, and activation of caspase-9 and caspase-3. Blocking the c-Abl tyrosine kinase also prevented neuronal cells from PrP106-126-induced apoptotic morphological changes. This is the first study reporting that c-Abl tyrosine kinase as a novel upstream activator of MST1 and BIM plays an important role in prion-induced neuron apoptosis via mitochondrial dysfunction. Our findings suggest that c-Abl tyrosine kinase is a potential therapeutic target for prion disease.

Andrographolide downregulates the v-Src and Bcr-Abl oncoproteins and induces Hsp90 cleavage in the ROS-dependent suppression of cancer malignancy.

Andrographolide is a diterpenoid compound isolated from Andrographis paniculata that exhibits anticancer activity. We previously reported that andrographolide suppressed v-Src-mediated cellular transformation by promoting the degradation of Src. In the present study, we demonstrated the involvement of Hsp90 in the andrographolide-mediated inhibition of Src oncogenic activity. Using a proteomics approach, a cleavage fragment of Hsp90α was identified in andrographolide-treated cells. The concentration- and time-dependent induction of Hsp90 cleavage that accompanied the reduction in Src was validated in RK3E cells transformed with either v-Src or a human truncated c-Src variant and treated with andrographolide. In cancer cells, the induction of Hsp90 cleavage by andrographolide and its structural derivatives correlated well with decreased Src levels, the suppression of transformation, and the induction of apoptosis. Moreover, the andrographolide-induced Hsp90 cleavage, Src degradation, inhibition of transformation, and induction of apoptosis were abolished by a ROS inhibitor, N-acetyl-cysteine. Notably, Hsp90 cleavage, decreased levels of Bcr-Abl (another known Hsp90 client protein), and the induction of apoptosis were also observed in human K562 leukemia cells treated with andrographolide or its active derivatives. Together, we demonstrated a novel mechanism by which andrographolide suppressed cancer malignancy that involved inhibiting Hsp90 function and reducing the levels of Hsp90 client proteins. Our results broaden the molecular basis of andrographolide-mediated anticancer activity.

eIF4B phosphorylation by pim kinases plays a critical role in cellular transformation by Abl oncogenes.

Alterations in translation occur in cancer cells, but the precise pathogenic processes and mechanistic underpinnings are not well understood. In this study, we report that interactions between Pim family kinases and the translation initiation factor eIF4B are critical for Abl oncogenicity. Pim kinases, Pim-1 and Pim-2, both directly phosphorylated eIF4B on Ser406 and Ser422. Phosphorylation of eIF4B on Ser422 was highly sensitive to pharmacologic or RNA interference-mediated inhibition of Pim kinases. Expression and phosphorylation of eIF4B relied upon Abl kinase activity in both v-Abl- and Bcr-Abl-expressing leukemic cells based on their blockade by the Abl kinase inhibitor imatinib. Ectopic expression of phosphomimetic mutants of eIF4B conferred resistance to apoptosis by the Pim kinase inhibitor SMI-4a in Abl-transformed cells. In contrast, silencing eIF4B sensitized Abl-transformed cells to imatinib-induced apoptosis and also inhibited their growth as engrafted tumors in nude mice. Extending these observations, we found that primary bone marrow cells derived from eIF4B-knockdown transgenic mice were less susceptible to Abl transformation, relative to cells from wild-type mice. Taken together, our results identify eIF4B as a critical substrate of Pim kinases in mediating the activity of Abl oncogenes, and they highlight eIF4B as a candidate therapeutic target for treatment of Abl-induced cancers.

miR-29b suppresses CML cell proliferation and induces apoptosis via regulation of BCR/ABL1 protein.

MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally and are critical for many cellular pathways. Recent evidence has shown that aberrant miRNA expression profiles and unique miRNA signaling pathways are present in many cancers. Here, we demonstrate that miR-29b is markedly lower expressed in CML patient samples. Bioinformatics analysis reveals a conserved target site for miR-29b in the 3'-untranslated region (UTR) of ABL1. miR-29b significantly suppresses the activity of a luciferase reporter containing ABL1-3'UTR and this activity is not observed in cells transfected with mutated ABL1-3'UTR. Enforced expression of miR-29b in K562 cells inhibits cell growth and colony formation ability thereby inducing apoptosis through cleavage of procaspase 3 and PARP. Furthermore, K562 cells transfected with a siRNA targeting ABL1 show similar growth and apoptosis phenotypes as cells overexpression of miR-29b. Collectively, our results suggest that miR-29b may function as a tumor suppressor by targeting ABL1 and BCR/ABL1.

Sphingomyelin synthase 1 activity is regulated by the BCR-ABL oncogene.

Sphingomyelin synthase (SMS) produces sphingomyelin while consuming ceramide (a negative regulator of cell proliferation) and forming diacylglycerol (DAG) (a mitogenic factor). Therefore, enhanced SMS activity could favor cell proliferation. To examine if dysregulated SMS contributes to leukemogenesis, we measured SMS activity in several leukemic cell lines and found that it is highly elevated in K562 chronic myelogenous leukemia (CML) cells. The increased SMS in K562 cells was caused by the presence of Bcr-abl, a hallmark of CML; stable expression of Bcr-abl elevated SMS activity in HL-60 cells while inhibition of the tyrosine kinase activity of Bcr-abl with Imatinib mesylate decreased SMS activity in K562 cells. The increased SMS activity was the result of up-regulation of the Sms1 isoform. Inhibition of SMS activity with D609 (a pharmacological SMS inhibitor) or down-regulation of SMS1 expression by siRNA selectively inhibited the proliferation of Bcr-abl-positive cells. The inhibition was associated with an increased production of ceramide and a decreased production of DAG, conditions that antagonize cell proliferation. A similar change in lipid profile was also observed upon pharmacological inhibition of Bcr-abl (K526 cells) and siRNA-mediated down-regulation of BCR-ABL (HL-60/Bcr-abl cells). These findings indicate that Sms1 is a downstream target of Bcr-abl, involved in sustaining cell proliferation of Bcr-abl-positive cells.

Tyrosine phosphorylation of a SNARE protein, syntaxin 17: implications for membrane trafficking in the early secretory pathway.

The T-cell protein tyrosine phosphatase is expressed as two splice variants - TC45, a nuclear protein, and TC48, which is localized predominantly in the ER (endoplasmic reticulum). Yeast two-hybrid screening revealed direct interaction of TC48 with Syntaxin17, a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein localized predominantly in the ER and to some extent in the ER-Golgi intermediate compartment. Syntaxin 17 did not interact with TC45. C-terminal 40 amino acids of TC48 were sufficient for interaction with syntaxin 17. Overexpressed syntaxin 17 was phosphorylated at tyrosine upon pervanadate treatment (a tyrosine phosphatase inhibitor/tyrosine kinase activator) of COS-1 cells. Mutational analysis identified Tyr156 in the cytoplasmic domain as the major site of phosphorylation. Endogenous syntaxin 17 was phosphorylated by pervanadate treatment in CHO and MIN6 cells but was not phosphorylated in a variety of other cell lines tested. c-Abl was identified as one of the kinases, which phosphorylates syntaxin 17 in MIN6 cells. Phosphorylation of endogenous and overexpressed syntaxin 17 was reduced in the presence of IGF receptor and EGF receptor kinase inhibitors. Serum depletion reduced pervanadate-induced phosphorylation of endogenous syntaxin 17. TC48 coexpression reduced phosphorylation of syntaxin 17 by pervanadate and purified TC48 directly dephosphorylated syntaxin 17. ß-COP dispersal by overexpressed syntaxin 17 was reduced after pervanadate-induced phosphorylation. A phospho-mimicking mutant (Y156E) of syntaxin 17 showed reduced interaction with COPI vesicles. These results suggest that tyrosine phosphorylation of syntaxin 17 is likely to have a role in regulating syntaxin 17 dependent membrane trafficking in the early secretory pathway.

Advancing the STATus of MPN pathogenesis.

In this issue of Blood, Yan et al and Walz et al exploit mouse genetics to investigate the contribution of signal transducer and activator of transcription 5 (STAT5) to the abnormal in vivo growth of hematopoietic cells expressing JAK2(V617F) or BCR-ABL. Eliminating STAT5 expression had dramatic effects in both contexts, and this new work and other recent studies support the therapeutic potential of targeting pathways regulated by this important signaling molecule in patients with myeloproliferative neoplasms (MPNs).

Essential role for Stat5a/b in myeloproliferative neoplasms induced by BCR-ABL1 and JAK2(V617F) in mice.

STAT5 proteins are constitutively activated in malignant cells from many patients with leukemia, including the myeloproliferative neoplasms (MPNs) chronic myeloid leukemia (CML) and polycythemia vera (PV), but whether STAT5 is essential for the pathogenesis of these diseases is not known. In the present study, we used mice with a conditional null mutation in the Stat5a/b gene locus to determine the requirement for STAT5 in MPNs induced by BCR-ABL1 and JAK2(V617F) in retroviral transplantation models of CML and PV. Loss of one Stat5a/b allele resulted in a decrease in BCR-ABL1-induced CML-like MPN and the appearance of B-cell acute lymphoblastic leukemia, whereas complete deletion of Stat5a/b prevented the development of leukemia in primary recipients. However, BCR-ABL1 was expressed and active in Stat5-null leukemic stem cells, and Stat5 deletion did not prevent progression to lymphoid blast crisis or abolish established B-cell acute lymphoblastic leukemia. JAK2(V617F) failed to induce polycythemia in recipients after deletion of Stat5a/b, although the loss of STAT5 did not prevent the development of myelofibrosis. These results demonstrate that STAT5a/b is essential for the induction of CML-like leukemia by BCR-ABL1 and of polycythemia by JAK2(V617F), and validate STAT5a/b and the genes they regulate as targets for therapy in these MPNs.

NF-κB suppresses ROS levels in BCR-ABL(+) cells to prevent activation of JNK and cell death.

Elevated levels of reactive oxygen species (ROS) are found in most oncogenically transformed cells and are proposed to promote cellular transformation through mechanisms such as inhibition of phosphatases. BCR-ABL, the oncoprotein associated with the majority of chronic myeloid leukemias (CMLs), induces accumulation of intracellular ROS, causing enhanced signaling downstream of PI3K. Previously we have shown that the transcription factor nuclear factor-kappa B (NF-κB) is activated by BCR-ABL expression and is required for BCR-ABL-mediated cellular transformation. Inhibition of IκB kinase (IKKß) and NF-κB leads to cell death through an unknown mechanism. Here, we analyze the potential involvement of NF-κB in moderating BCR-ABL-induced ROS levels to protect from death. The data confirm that BCR-ABL promotes ROS and demonstrate that NF-κB prevents excessive levels. Inhibition of NF-κB leads to an increase in ROS levels and to cell death, which is at least partially controlled through ROS-induced c-Jun N-terminal kinase activity. The data demonstrate that one function for NF-κB in oncogenesis is the suppression of oncoprotein-induced ROS levels and that inhibition of NF-κB in some cancers, including CML, will increase ROS levels and promote cell death.

Glucosylceramide synthase inhibitor PDMP sensitizes chronic myeloid leukemia T315I mutant to Bcr-Abl inhibitor and cooperatively induces glycogen synthase kinase-3-regulated apoptosis.

Inactivation of glycogen synthase kinase (GSK)-3 has been implicated in cancer progression. Previously, we showed an abundance of inactive GSK-3 in the human chronic myeloid leukemia (CML) cell line. CML is a hematopoietic malignancy caused by an oncogenic Bcr-Abl tyrosine kinase. In Bcr-Abl signaling, the role of GSK-3 is not well defined. Here, we report that enforced expression of constitutively active GSK-3 reduced proliferation and increased Bcr-Abl inhibition-induced apoptosis by nearly 1-fold. Bcr-Abl inhibition activated GSK-3 and GSK-3-dependent apoptosis. Inactivation of GSK-3 by Bcr-Abl activity is, therefore, confirmed. To reactivate GSK-3, we used glucosylceramide synthase (GCS) inhibitor PDMP to accumulate endogenous ceramide, a tumor-suppressor sphingolipid and a potent GSK-3 activator. We found that either PDMP or silence of GCS increased Bcr-Abl inhibition-induced GSK-3 activation and apoptosis. Furthermore, PDMP sensitized the most clinical problematic drug-resistant CML T315I mutant to Bcr-Abl inhibitor GNF-2-, imatinib-, or nilotinib-induced apoptosis by >5-fold. Combining PDMP and GNF-2 eliminated transplanted-CML-T315I-mutants in vivo and dose dependently sensitized primary cells from CML T315I patients to GNF-2-induced proliferation inhibition and apoptosis. The synergistic efficacy was Bcr-Abl restricted and correlated to increased intracellular ceramide levels and acted through GSK-3-mediated apoptosis. This study suggests a feasible novel anti-CML strategy by accumulating endogenous ceramide to reactivate GSK-3 and abrogate drug resistance.

MicroRNA-451 in chronic myeloid leukemia: miR-451-BCR-ABL regulatory loop?

Chronic myeloid leukemia (CML) is caused by constituve activity of BCR-ABL tyrosine kinase. Despite of high efficiency of imatinib, selective BCR-ABL inhibitor, about 30% of patients develop resistance. Novel markers and targets for therapy are thus necessary. MicroRNAs are small intereference RNAs whose role in physiological and malignant hematopoiesis has been shown. This study is focused on miR-451 in CML. Following our observation of miR-451 downregulation in CML, we further show its relation to BCR-ABL activity. Our data together with current literature indicate a more complex relationship of miR-451 and BCR-ABL in CML.