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.

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.

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.

BCR-ABL enhances differentiation of long-term repopulating hematopoietic stem cells.

In a previously developed inducible transgenic mouse model of chronic myeloid leukemia, we now demonstrate that the disease is transplantable using BCR-ABL(+) Lin(-)Sca-1(+)c-kit(+) (LSK) cells. Interestingly, the phenotype is more severe when unfractionated bone marrow cells are transplanted, yet neither progenitor cells (Lin(-)Sca-1(-)c-kit(+)), nor mature granulocytes (CD11b(+)Gr-1(+)), nor potential stem cell niche cells (CD45(-)Ter119(-)) are able to transmit the disease or alter the phenotype. The phenotype is largely independent of BCR-ABL priming before transplantation. However, prolonged BCR-ABL expression abrogates the potential of LSK cells to induce full-blown disease in secondary recipients and increases the fraction of multipotent progenitor cells at the expense of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow. BCR-ABL alters the expression of genes involved in proliferation, survival, and hematopoietic development, probably contributing to the reduced LT-HSC frequency within BCR-ABL(+) LSK cells. Reversion of BCR-ABL, or treatment with imatinib, eradicates mature cells, whereas leukemic stem cells persist, giving rise to relapsed chronic myeloid leukemia on reinduction of BCR-ABL, or imatinib withdrawal. Our results suggest that BCR-ABL induces differentiation of LT-HSCs and decreases their self-renewal capacity.

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.

MOZ-TIF2, but not BCR-ABL, confers properties of leukemic stem cells to committed murine hematopoietic progenitors.

To better understand the origin of leukemic stem cells, we tested the hypothesis that all leukemia oncogenes could transform committed myeloid progenitor cells lacking the capacity for self-renewal, as has recently been reported for MLL-ENL. Flow-sorted populations of common myeloid progenitors and granulocyte-monocyte progenitors were transduced with the oncogenes MOZ-TIF2 and BCR-ABL, respectively. MOZ-TIF2-transduced progenitors could be serially replated in methylcellulose cultures and continuously propagated in liquid culture, and resulted in an acute myeloid leukemia in vivo that could be serially transplanted. In contrast, BCR-ABL transduction conferred none of these properties to hematopoietic progenitors. These data demonstrate that some, but not all, leukemia oncogenes can confer properties of leukemic stem cells to hematopoietic progenitors destined to undergo apoptotic cell death.

Modeling the cancer patient with genetically engineered mice: prediction of toxicity from molecule-targeted therapies.

Current trends foretell the use of cancer treatments customized to each patient. Genetic and molecular profiling of tumors and an increasing number of molecule-targeted therapies contribute to making this a reality. However, as targets of anticancer therapies become specific proteins or pathways, unanticipated side effects may emerge. In addition, the chronic use of these treatments may contribute to the development of degenerative toxicity not predicted by short-term clinical trials. Here we review and propose how genetically engineered mouse models can serve as valuable tools to predict targeted therapy toxicity, as well as to identify allelic variants that predispose individuals to side effects.

Targeting tumor cells.

Several recent scientific and technical developments have made it possible to postulate the use of the 'magic bullet' concept; that is, the identification of specific antigens present on tumor cells that can be targeted either by therapeutic antibodies or by small molecules. The use of monoclonal antibodies in cancer, in particular, has moved beyond the proof-of-concept stage, and many such antibodies are presently being tested in the clinic. Several antibodies have been successfully developed and are now in use against various cancers, and we can expect many more to become available in the next few years. The use and development of these new therapeutics represent significant opportunities but also new challenges.

Novel role of pleckstrin homology domain of the Bcr-Abl protein: analysis of protein-protein and protein-lipid interactions.

The Bcr-Abl protein is a marker for malignant transformation in chronic myeloid leukemia and in acute lymphoblastic leukemia. There are three Bcr-Abl chimeras known so far, p190, p210 and p230. The only structural difference between the three Bcr-Abl proteins is the presence of DH and PH domains from the Bcr gene in p210 and p230. The Bcr-Abl DH domain is functioning as a guanine nucleotide exchange factor for Rho family of small GTPases. The PH domain confers binding to phosphoinositides but some PH domains have also been found to bind specific target proteins. Here we show that the PH domain from Bcr-Abl binds a number of proteins involved in vital cellular processes. These proteins include PLCvarepsilon, Zizimin1, tubulin and SMC1. The revelation of the role of the Bcr-Abl PH domain in leukemogenesis is likely to provide clues to the molecular mechanisms underlying the phenotypes of Bcr-Abl positive leukemia and could therefore provide tools for the identification of targets for the development of therapeutic treatments.

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.

Reduction of apoptosis in Rb-deficient embryos via Abl knockout.

The retinoblastoma protein RB regulates cell proliferation, differentiation and apoptosis. Homozygous knockout of Rb in mice causes embryonic lethality owing to placental defects that result in excessive apoptosis. RB binds to a number of cellular proteins including the nuclear Abl protein and inhibits its tyrosine kinase activity. Ex vivo experiments have shown that genotoxic or inflammatory stress can activate Abl kinase to stimulate apoptosis. Employing the Rb-null embryos as an in vivo model of apoptosis, we have shown that the genetic ablation of Abl can reduce apoptosis in the developing central nervous system and the embryonic liver. These results are consistent with the inhibitory interaction between RB and Abl, and provide in vivo evidence for the proapoptotic function of Abl.

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.

Src kinase signaling in leukaemia.

Role of Src kinases in acute lymphoblastic leukaemia has been recently demonstrated in leukaemia mouse model. Retained activation of Src kinases by the BCR-ABL oncoprotein in leukaemic cells following inhibition of BCR-ABL kinase activity by imatinib indicates that Src activation by BCR-ABL is independent of BCR-ABL kinase activity and provides an explanation for reduced effectiveness of the BCR-ABL kinase activity inhibitors in Philadelphia chromosome-positive acute lymphoblastic leukaemia. Simultaneous inhibition of kinase activity of both BCR-ABL and Src kinases results in long-term survival of mice with acute lymphoblastic leukaemia. Leukaemic stem cells exist in acute lymphoblastic leukaemia, and complete eradication of this group of cells would provide a curative therapy for this disease.

Contribution of ABL kinase domain mutations to imatinib resistance in different subsets of Philadelphia-positive patients: by the GIMEMA Working Party on Chronic Myeloid Leukemia.

PURPOSE: ABL kinase domain mutations have been implicated in the resistance to the BCR-ABL inhibitor imatinib mesylate of Philadelphia-positive (Ph+) leukemia patients. EXPERIMENTAL DESIGN: Using denaturing high-performance liquid chromatography and sequencing, we screened for ABL kinase domain mutations in 370 Ph+ patients with evidence of hematologic or cytogenetic resistance to imatinib. RESULTS: Mutations were found in 127 of 297 (43%) evaluable patients. Mutations were found in 27% of chronic-phase patients (14% treated with imatinib frontline; 31% treated with imatinib post-IFN failure), 52% of accelerated-phase patients, 75% of myeloid blast crisis patients, and 83% of lymphoid blast crisis/Ph+ acute lymphoblastic leukemia (ALL) patients. Mutations were associated in 30% of patients with primary resistance (44% hematologic and 28% cytogenetic) and in 57% of patients with acquired resistance (23% patients who lost cytogenetic response; 55% patients who lost hematologic response; and 87% patients who progressed to accelerated phase/blast crisis). P-loop and T315I mutations were particularly frequent in advanced-phase chronic myeloid leukemia and Ph+ ALL patients, and often accompanied progression from chronic phase to accelerated phase/blast crisis. CONCLUSIONS: We conclude that (a) amino acid substitutions at seven residues (M244V, G250E, Y253F/H, E255K/V, T315I, M351T, and F359V) account for 85% of all resistance-associated mutations; (b) the search for mutations is important both in case of imatinib failure and in case of loss of response at the hematologic or cytogenetic level; (c) advanced-phase chronic myeloid leukemia and Ph+ ALL patients have a higher likelihood of developing imatinib-resistant mutations; and (d) the presence of either P-loop or T315I mutations in imatinib-treated patients should warn the clinician to reconsider the therapeutic strategy.

Rapid microtubule-dependent induction of neurite-like extensions in NIH 3T3 fibroblasts by inhibition of ROCK and Cbl.

A number of key cellular functions, such as morphological differentiation and cell motility, are closely associated with changes in cytoskeletal dynamics. Many of the principal signaling components involved in actin cytoskeletal dynamics have been identified, and these have been shown to be critically involved in cell motility. In contrast, signaling to microtubules remains relatively uncharacterized, and the importance of signaling pathways in modulation of microtubule dynamics has so far not been established clearly. We report here that the Rho-effector ROCK and the multiadaptor proto-oncoprotein Cbl can profoundly affect the microtubule cytoskeleton. Simultaneous inhibition of these two signaling molecules induces a dramatic rearrangement of the microtubule cytoskeleton into microtubule bundles. The formation of these microtubule bundles, which does not involve signaling by Rac, Cdc42, Crk, phosphatidylinositol 3-kinase, and Abl, is sufficient to induce distinct neurite-like extensions in NIH 3T3 fibroblasts, even in the absence of microfilaments. This novel microtubule-dependent function that promotes neurite-like extensions is not dependent on net changes in microtubule polymerization or stabilization, but rather involves selective elongation and reorganization of microtubules into long bundles.

ABL oncogenes and phosphoinositide 3-kinase: mechanism of activation and downstream effectors.

The BCR-ABL oncogene is responsible for most cases of chronic myelogenous leukemia and some acute lymphoblastic leukemias. The fusion protein encoded by BCR-ABL possesses an aberrantly regulated tyrosine kinase activity. Imatinib mesylate (Gleevec, STI-571) is an inhibitor of ABL tyrosine kinase activity that has been remarkably effective in slowing disease progression in patients with chronic phase chronic myelogenous leukemia, but the emergence of imatinib resistance underscores the need for additional therapies. Targeting signaling pathways activated by BCR-ABL is a promising approach for drug development. The study of signaling components downstream of BCR-ABL and the related murine oncogene v-Abl has revealed a complex web of signals that promote cell division and survival. Of these, activation of phosphoinositide 3-kinase (PI3K) has emerged as one of the essential signaling mechanisms in ABL leukemogenesis. This review describes molecular mechanisms by which PI3K is activated and the downstream PI3K effectors that propagate the signal to promote myeloid and lymphoid transformation. Of particular recent interest is the mammalian target of rapamycin, a PI3K-regulated kinase that regulates protein synthesis and contributes to leukemogenesis.

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.