BCR-ABL Tyrosine Kinase Activity Research Articles

The Bcr–Abl kinase regulates the actin cytoskeleton via a GADS/Slp-76/Nck1 adaptor protein pathway

Bcr–Abl is the transforming principle underlying chronic myelogenous leukaemia (CML). Here, we use a functional interaction proteomics approach to map pathways by which Bcr–Abl regulates defined cellular processes. The results show that Bcr–Abl regulates the actin cytoskeleton and non-apoptotic membrane blebbing via a GADS/Slp-76/Nck1 adaptor protein pathway. The binding of GADS to Bcr–Abl requires Bcr–Abl tyrosine kinase activity and is sensitive to the Bcr–Abl inhibitor imatinib, while the GADS/Slp-76 and Slp-76/Nck interactions are tyrosine phosphorylation independent. All three adaptor proteins co-localize with cortical actin in membrane blebs. Downregulation of each adaptor protein disrupts the actin cytoskeleton and membrane blebbing in a similar fashion and similar to imatinib. These findings highlight the importance of protein interaction dependent adaptor protein pathways in oncogenic kinase signaling.

Chronic myeloid leukemia: a review

Background: Chronic myeloid leukemia remains a rare disease in children accounting for 2-3% of all childhood leukemias.The field has witnessed great advances in the understanding of it's molecular biology and therapeutics. Methods: Various therapeutic advances in the field have been reviewed. Special focus is on emerging treatments: especially oral tyrosine kinase inhibitors which target the activity of the bcr-abl tyrosine kinase. Results: Initially therapy revolved around early allogenic stem cell transplantation. The introduction of the oral tyrosine kinase inhibitor imatinib and other such molecules has however changed the management paradigm. The high response rates and excellent tolerance to initial therapy has tilted the balance away from allogenic stem cell transplantation. Conclusions: Better understanding of the molecular basis of disease has resulted in the availability of a plethora of agents whose potential has yet to be fully explored. Treatment paradigms in children are in a state of flux : especially relating to the role and timing of the stem cell transplant and its integration with imatinib and other therapies.

In vitro Anti-leukaemia Activity of Pyrrolo[1,2-b][1,2,5]benzothiadiazepines (PBTDs)

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized by unregulated growth of myeloid leukemia cells in the bone marrow and accumulation of these cells in the blood. CML represents approximately 15-20% of all adult leukemia and the disease development is clearly linked to the constitutively active tyrosine kinase of the chimeric protein BCR-ABL. It is encoded by the Bcr-Abl fusion gene sequence as the result of chromosome 9/22 translocation (Philadelphia chromosome) or other aberrant cytogenetic events. The development of targeted agents that specifically inhibit the tyrosine kinase (TK) activity of BCR-ABL has revolutionized the treatment of CML. Imatinib is now the first-line treatment for chronic phase CML, and several newer tyrosine kinase inhibitors (TKIs) such as dasatinib and nilotinib have been added to the pharmacologic compendium. Despite the proven efficacy of TKIs to induce hematological and cytogenetic remission, the large majority of patients still have molecularly detectable disease. Therefore, new options are needed to improve therapeutic success in the treatment of leukemia. Pyrrolo[1,2-b][1,2,5]benzothiadiazepine 5,5-dioxides (PBTDs) induced apoptosis in human BCR-ABL expressing leukemia cells. The apoptotic activity was also observed in primary leukemic blasts, obtained from CML patients at onset or from patients in blast crisis and who were imatinib- dasatinib- and nilotinib resistant. These results suggests that these compounds are promising agent for the treatment of leukemia. Due to the fact that the phenomenon of resistance to TKIs remains a major issue in the treatment of patients with CML, the identification of new drugs may be of clinical relevance. This review summarises patents and papers dealing with the present understanding of mechanism of action and the most relevant data concerning TKs inhibition.

Tyrosine Kinase Proteins profiling of Nilotinib Resistant Chronic Myelogenous Leukemia Cells Unravels a Tyrosine Kinase-Mediated Bypass.

Abstract 2175Poster Board II-152Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myeloid Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second generation inhibitors such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or –intolerant disease. We have shown that one of the mechanisms of resistance to nilotinib is an increasing expression of the p53/56 Lyn kinase, both at mRNA and protein level in cell lines. This result was confirmed in vivo in nilotinib-resistant CML patients (Mahon et al. Cancer Res., 2008, 68(23):9809-16.). To elucidate Lyn mediated-nilotinib resistance, a phosphoproteomic study was performed by Stable Isotope Labelling with Amino acid in Cell culture (SILAC) which highlights the potential role of downstream tyrosine kinases. Among different candidate proteinsThe Spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl were the most relevant in the nilotinib resistant cell line as compared to the sensitive counterpart. Syk hyperphosphorylation was confirmed in the nilotinib resistant cell line using western blot at least on tyrosine residues Y323 and Y525/526, two critical tyrosine residues respectively involved in Lyn-mediated Syk phosphorylation and autophosphorylation-associated Syk activation. Lyn interacts with Syk as detected in Syk immunoprecipitates in nilotinib resistant cells. Furthermore, Syk-Lyn interaction is inhibited by dasatinib suggesting the requirement of Lyn kinase activity and Syk phosphorylation. Targeting Syk expression in nilotinib resistant cells by siRNA or tyrosine kinase activity by pharmacological inhibitors leads respectively to a partial (35%) or to a full restoration of nilotinib sensitivity. Moreover, the identification of Axl by SILAC is correlated to a 9 fold increase of its level of expression in the resistant cell line and the inhibition of Axl tyrosine kinase activity decreases proliferation of both nilotinib sensitive and resistant CML cells. All together these results disclose a new pathway for tyrosine kinase inhibitors resistance in CML involving at least the two Lyn downstream tyrosine kinases Syk and Axl. Disclosures:Mahon:Amgen: Honoraria; Novartis Pharma: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria.

Intracerebellar Granulocytic Sarcoma Presenting as a First Manifestation of Relapse in Chronic Myelogenous Leukemia (CML) During Imatinib Mesylate Therapy: A Case Report and Literature Review.

Abstract 4289 IntroductionGranulocytic sarcoma (GS) is an unusual solid tumor of extramedullary localization consisting of immature granulocytic precursors. It is mostly encountered in the course of acute myelogenous leukemia (AML), myelodysplastic or myeloproliferative disorders, with an estimated incidence of 7.9% in CML (Terjanian et al). GS can occur before, concomitantly, or after the overt development of these hematologic disorders. Most common localizations are lymph nodes, bones, skin and soft tissues, but there is paucity of published data of isolated intracranial GS manifesting as relapse in CML. Case reportA 34-year-old male with Philadelphia chromosome-positive (Ph+) CML diagnosed in August 2005 in the chronic phase of the disease. After initiation of imatinib therapy at doses of 400 mg/d he achieved complete hematologic, cytogenetic and molecular responses. He remained in clinical remission for 3 years when he presented with bifrontal headache and dizziness. Physical exam revealed no abnormalities. Peripheral blood count showed WBC 9,200/mm3 with normal differential and no immature forms, platelet 180,000/mm3, and hemoglobin 14.4 g/dl. Magnetic resonance imaging of the brain revealed a 3.8 cm enhancing mass lesion within the left cerebellar hemisphere. He underwent posterior fossa exploration with resection of the solid tumor. Microscopic examination of the cerebellar mass demonstrated a diffuse cellular infiltrate of atypical mononuclear cells with high nuclear to cytoplasmic ratios and fine, immature chromatin, consistent with blasts. Flow cytometric analysis confirmed that the majority of the cells in the specimen were composed of blasts which coexpressed the CD33 and CD4 markers. Fluorescence in situ hybridization analysis on the cerebellar tissue detected the presence of BCR/ABL rearrangement in 60% of nuclei. Taken together, the morphologic, immunophenotypic, and molecular features are diagnostic of a myeloid sarcoma representing transformation of chronic myeloid leukemia. DiscussionA literature search was performed to identify all cases of intracranial GS in patients with diagnosis of CML reported in medical literature. Only 3 cases were identified in the literature to date. This case appears to be the first described in the imatinib mesylate era. Table 1 summarizes the clinical data of these previously recorded cases and the present case.Table 1Case no.ReferenceAge / sexGS locationCML Status (systemic)No. years since DxPrior therapiesOutcome1Simpson et al, 198951 FLeft parietal lobeRelapse2.5-Hydroxyurea, busulfan, α-interferonAlive 4 months after cranial RT with no evidence of blastic transformation2Mahendra et al, 199440 MRight frontal lobeRelapse8.5-Busulfan, hydroxyurea -Allogeneic SCTDied 3.8 months after his allograft3Grondin et al, 199631 MMultiple brain nodulesRelapse1.5-HydroxyureaAlive immediately after whole brain RT with near complete resolution of brain lesions4Present case report34 MLeft cerebellar hemisphereRemission3.3-Imatinib mesylateDied 7 months after dx of intracranial relapse. Subsequent to tumor resection and posterior fossa RT, patient achieved a non-durable response, ultimately developing cerebellar and leptomeningeal relapsesAbbreviations: CML: chronic myelogenous leukemia, Dx: diagnoses, GS: granulocytic sarcoma, RT radiotherapy, SCT: stem cell transplantPharmacokinetic analyses (Pfeifer et al, Leis at al, Wolff et al) have demonstrated that imatinib poorly penetrates the blood–brain barrier, with resultant CNS drug levels significantly lower than corresponding plasma levels. Measured CSF levels were below the concentration required for 50% inhibition of the BCR–ABL tyrosine kinase activity, suggesting that Ph+ leukemic patients treated with this selective tyrosine kinase inhibitor are at high risk of CNS relapse even in the setting of complete remission of systemic disease.This case illustrates an unusual site of recurrence of CML. To our knowledge, this is the first case of a granulocytic sarcoma presenting as a solitary parenchymal brain tumor in a patient receiving imatinib for CML. Noticeably, CNS relapse occurred despite lack of systemic evidence of disease. As described in human and animal model studies this may be the result of effective systemic antileukemic activity of imatinib therapy, but with limited activity in the CNS. Poor penetration of imatinib into the cerebrospinal fluid may allow the CNS to become a sanctuary reservoir for relapse of Ph+ CML. Disclosures:No relevant conflicts of interest to declare.

BCR-ABL Tyrosine Kinase Activity Modulates the Phosphorylation, Localization and Function of Interferon Regulatory Factor 5 (irf-5) in Chronic Myeloid Leukemia (CML) Cells.

Abstract 2165Poster Board II-142 Introduction:Interferon Regulatory Factors (IRFs) comprise ten helix-turn-helix transcription factors that are pivotal modulators of the immune response against viral infections and cancer. IRF-5, a direct target of the p53 tumor suppressor, is known to bind an extensive number of gene promoters regulating cell growth, differentiation and apoptosis. Increased or reduced levels of IRF-5 have been reported in several solid and hematologic malignancies but there is currently no evidence concerning IRF-5 and Chronic Myeloid Leukemia (CML). We therefore wanted to investigate if alterations in IRF-5 expression and function were involved in the pathogenesis of CML. Patients and Methods:Expression studies and colony-forming assays after lentiviral transduction were carried out on primary cells isolated from 10 CML patients (pts) at diagnosis and 6 healthy donors (controls). Experiments on immortalized cell lines were performed using K562, KYO-1 (CML lines) and HL60 (negative control). Results:RT-PCR and immunoblot assays showed that IRF-5 is expressed in primary hematopoietic cells derived from both CML pts and healthy donors as well as in immortalized CML cell lines. We also found that IRF-5 immunoprecipitates with the BCR-ABL oncoprotein of CML and that BCR-ABL induces its tyrosine phosphorylation. Exposure to the semi-specific BCR-ABL inhibitor Imatinib mesylate (IM) reduced the interaction between IRF-5 and BCR-ABL and decreased IRF-5 tyrosine phosphorylation. Cell fractionation experiments employing the nuclear export inhibitor Leptomycin B showed that, in CML cells, IRF-5 is almost exclusively localized in the cell cytoplasm because of excessive nuclear export. Interestingly, both IM and Interferon (IFN) treatment radically modified IRF-5 intracellular distribution restoring its nuclear localization. A computational sequence analysis identified tyrosine 104 as part of a consensus for the BCR-ABL kinase. Hence, we generated an IRF-5 Y104F mutant that would no longer be a substrate for BCR-ABL catalytic activity. Indeed, compared to native IRF-5, the Y104F mutant displayed low levels of tyrosine phosphorylation that remained unchanged after IM treatment. Furthermore, only a fraction of BCR-ABL immunoprecipitated with the Y104F construct that, in cell fractionation experiments, localized predominantly in the nuclear compartment. Finally, both CML cell lines and primary CD34-positive cells lentivirally transduced with IRF-5 Y104F showed a remarkable reduction in cell proliferation in methylcellulose colony-forming assays. This result was not observed after transduction with the gfp control. Conclusions:In CML cells, BCR-ABL associates with IRF-5 and causes its phosphorylation on tyrosine 104. This event favors IRF-5 nuclear export thereby abrogating its function as a transcriptional regulator. Inactivation of BCR-ABL catalytic activity by IM treatment or exposure to IFN restores IRF-5 nuclear localization. Furthermore, expression of an IRF-5 Y104F mutant that can't be phosphorylated by BCR-ABL reduces the proliferative activity of both primary and immortalized CML cells. Taken together these data suggest that IRF-5 is involved in the pathogenesis of CML and that IM or IFN-based treatments can restore IRF-5 localization and function antagonizing the proliferation of the leukemic clones. Disclosures:No relevant conflicts of interest to declare.

Role of Stromal Microenvironment in Non-Pharmacological Resistance of CML to Imatinib through Lyn/CXCR4 Interactions.

Abstract 4248In patients with chronic-phase chronic myeloid leukemia (CML), imatinib resistance is of increasing importance. We have recently reported that the constitutively activated Bcr-Abl tyrosine kinase in CML suppresses CXCL12/CXCR4-mediated migration of CML cells to the bone marrow (BM) stroma. This finding can explain the characteristic leukocytosis in CML. In turn, tyrosine kinase inhibitor imatinib inhibits Bcr-Abl, enhances migration of CML cells towards CXCL12-producing BM stromal cells which in turn promotes cell quiescence and development of the microenvironment-mediated, non-pharmacological drug resistance (Jin, Mol Cancer Ther 2008;7:48). In this study, we further investigated the molecular mechanisms of imatinib-induced CML cell migration and adherence to the bone marrow-derived stromal cells (MSC). Src-related kinase Lyn regulates survival and responsiveness of CML cells to inhibition of BCR-ABL kinase and is known to interact with CXCL12/CXCR4 signaling. Lyn frequently localizes in lipid raft fractions, which act as signal transduction platforms for a variety of intracellular processes. Therefore, we investigated the effects of imatinib on the localization of activated Lyn in the lipid raft structures of KBM-5 CML cells under co-culture conditions with CXCL12-secreting MSC or recombinant CXCL12. Confocal microscopy and discontinuous sucrose density gradient fractionation demonstrated that CXCR4 and phosphorylated CXCR4 localized in the higher-density detergent-soluble non-raft cell surface regions in KBM5 cells in the presence and absence of imatinib, with or without MSC, which suggests that CXCR4 does not directly associate with lipid rafts. In contrast, Lyn was present both in the low-density raft and in the high-density non-raft fractions, which contained CXCR4. We have further demonstrated co-localization of CXCR4 with Lyn, and their direct interaction was confirmed by co-immunoprecipitation. Notably, the active form of phosphorylated p-LynTyr396 clustered in lipid rafts, while inactive p-LynTyr507 in non-raft fractions. In suspension KBM-5 cultures imatinib depleted both, p-LynTyr396 and p-LynTyr507. In contrast, under MSC co-culture conditions imatinib repressed p-LynTyr507, but failed to deplete p-LynTyr396 in lipid rafts, and p-LynTyr396 further accumulated in non-raft fractions, likely associating with CXCR4. Knock-down of Lyn by siRNA, Src inhibitor treatment or lipid raft destruction by methyl-b cyclodextrin (MbCD) abrogated imatinib-induced KBM5 migration to MSCs and CXCL12, indicating the critical role of p-LynTyr396 in cell migration. Since the a4b1 integrin VLA-4 represents a cooperative molecular pathway guiding BM homing in addition to CXCL12/CXCR4, we next investigated the localization and expression of VLA-4 in KBM5 cells. Imatinib decreased VLA-4 protein expression both in lipid raft and non-raft fractions without affecting VLA-4 gene expression levels as determined by quantitative RT-PCR. Interestingly, VLA-4 reduction by imatinib or lipid raft destruction by MbCD did not affect the ability to adhere to fibronectin.In conclusion, these findings demonstrate that under conditions mimicking BM microenvironment imatinib restores CXCL12-dependent migration through interactions between CXCR4 and active p-Lyn Tyr396 in non-raft microdomains of CML cells and that p-Lyn Tyr396 localized in lipid rafts is contributing to the CML cell migration. We propose that while CXCR4 is segregated from lipid raft fractions, imatinib through Bcr-Abl kinase inhibition induces the compartmental changes of multivalent Lyn complex between lipid raft and non-raft fractions, restoring the interactions between Lyn and CXCR4 and stimulating cell migration.Our findings indicate that leukemic BM microenvironment may be involved in imatinib resistance in a subset of CML patients through activation of Lyn kinase, consistent with reported higher clinical activity of Bcr-Abl/Src inhibitor dasatinib in patients with imatinib-resistant CML. We propose that BM stroma cells produce abundant CXCL12 and attract migrating cells through adhesive interactions with the extracellular matrix, which may in turn facilitate lodging into BM niches of imatinib-exposed CML cells and promote non-pharmacological resistance to this agent. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

ABC transporter A3 facilitates lysosomal sequestration of imatinib and modulates susceptibility of chronic myeloid leukemia cell lines to this drug

Inhibition of BCR-ABL tyrosine kinase activity has evolved as a mainstay of therapy for patients with chronic myeloid leukemia. However, a fraction of leukemic cells persists under targeted therapy and can lead to disease progression on cessation of treatment. We analyzed bone marrow progenitor cells with the side population phenotype, and characterized the role of the intracellular ABC transporter A3 in imatinib detoxification. BCR-ABL-positive leukemic cells contribute to the side population cell compartment in untreated patients. Such leukemic side population cells, as well as CD34-positive progenitors from chronic myeloid leukemia samples, strongly express the intracellular ABCA3. Functionally, ABCA3 levels are critical for the susceptibility of chronic myeloid leukemia blast cell lines to specific BCR-ABL inhibition by imatinib. The transporter is localized in the limiting membrane of lysosomes and multivesicular bodies, and intracellular [(14)C]-labeled imatinib accumulates in such organelles. The lysosomal storage capacity increases with ABCA3 expression, thus regulating imatinib sequestration. The intracellular ABC transporter A3 is expressed in chronic myeloid leukemia progenitor cells and may contribute to intrinsic imatinib resistance by facilitating lysosomal sequestration in chronic myeloid leukemia cells.

New agents in the treatment of chronic myelogenous leukemia.

The discovery of molecularly targeted agents that selectively inhibit bcr-abl tyrosine kinase activity, such as imatinib, has revolutionized the treatment and natural history of chronic myelogenous leukemia (CML). Treatment of chronic-phase CML with imatinib showed complete cytogenetic response rates of more than 40% in patients after failure of interferon-alpha, and more than 80% in patients with newly diagnosed CML. Patients with CML can now expect excellent long-term survival, often without major side effects. In most patients, however, residual leukemic burden remains detectable using a sensitive reverse transcription-polymerase chain reaction method. In addition, many patients undergoing imatinib therapy will either not respond or lose their response over time because of resistance or intolerance. The introduction of second-generation tyrosine kinase inhibitors (TKIs) re-establishes response in approximately half of these patients. Several agents are being developed for treating patients who experience suboptimal response to second-generation TKIs and for those who develop resistance caused by the emergence of highly resistant BCR-ABL1 mutations. This article provides an overview of novel targeted agents available for CML.

Interaction of nilotinib, dasatinib and bosutinib with ABCB1 and ABCG2: implications for altered anti‐cancer effects and pharmacological properties

ABC multidrug transporters (MDR-ABC proteins) cause multiple drug resistance in cancer and may be involved in the decreased anti-cancer efficiency and modified pharmacological properties of novel specifically targeted agents. It has been documented that ABCB1 and ABCG2 interact with several first-generation, small-molecule, tyrosine kinase inhibitors (TKIs), including the Bcr-Abl fusion kinase inhibitor imatinib, used for the treatment of chronic myeloid leukaemia. Here, we have investigated the specific interaction of these transporters with nilotinib, dasatinib and bosutinib, three clinically used, second-generation inhibitors of the Bcr-Abl tyrosine kinase activity. MDR-ABC transporter function was screened in both membrane- and cell-based (K562 cells) systems. Cytotoxicity measurements in Bcr-Abl-positive model cells were coupled with direct determination of intracellular TKI concentrations by high-pressure liquid chromatography-mass spectrometry and analysis of the pattern of Bcr-Abl phosphorylation. Transporter function in membranes was assessed by ATPase activity. Nilotinib and dasatinib were high-affinity substrates of ABCG2, and this protein mediated an effective resistance in cancer cells against these compounds. Nilotinib and dasatinib also interacted with ABCB1, but this transporter provided resistance only against dasatinib. Neither ABCB1 nor ABCG2 induced resistance to bosutinib. At relatively higher concentrations, however, each TKI inhibited both transporters. A combination of in vitro assays may provide valuable preclinical information for the applicability of novel targeted anti-cancer TKIs, even in multidrug-resistant cancer. The pattern of MDR-ABC transporter-TKI interactions may also help to understand the general pharmacokinetics and toxicities of new TKIs.

Chronic myeloid leukemia patients with the e13a2 BCR-ABL fusion transcript have inferior responses to imatinib compared to patients with the e14a2 transcript

Chronic myeloid leukemia is characterized by a reciprocal translocation between chromosomes 9 and 22, creating the fusion gene BCR-ABL. The clinical significance of the type of BCR-ABL transcript in newly diagnosed patients in chronic phase treated with imatinib 400 mg from initial diagnosis remains unknown. We analyzed the clinical outcome of 78 newly diagnosed chronic phase patients, aged 16 or over, treated with imatinib 400 mg. Of these, 71 expressed either e13a2 or e14a2 transcripts. BCR-ABL transcripts were assayed by quantitative real-time polymerase chain reaction. After 12 months of treatment, 54% of the e14a2 patients had achieved a complete cytogenetic response, compared to 25% of the e13a2 patients (p=0.01). Kaplan-Meier analysis of the time to achieve complete cytogenetic response revealed that e14a2 patients had more rapid response rates, compared to e13a2 patients (p=0.006). e14a2 patients had a higher event-free survival rate in the first 12 months of treatment, although overall survival did not differ significantly between the patients with the two types of transcript. Human organic cation transporter protein 1 mRNA levels did not differ between the patients with the two types of transcript. The pre-treatment pCrKL/CrKL ratio (a surrogate marker of BCR-ABL tyrosine kinase activity) was higher in patients with e13a2 transcripts than in those with e14a2 (p=0.017). Patients expressing the e14a2 transcript type have a higher rate and more rapid complete cytogenetic responses than e13a2-expressing patients, which may be due to higher BCR-ABL tyrosine kinase activity. Knowledge of the transcript type may yield additional prognostic information, although this requires testing on larger datasets.

Depletion of Pleckstrin Homology Domain Leucine-rich Repeat Protein Phosphatases 1 and 2 by Bcr-Abl Promotes Chronic Myelogenous Leukemia Cell Proliferation through Continuous Phosphorylation of Akt Isoforms

The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway commonly occurs in cancers and is a crucial event in tumorigenesis. Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The PI3K/Akt pathway is activated by Bcr-Abl chimera protein and mediates the leukemogenesis in CML. However, the mechanism by which Bcr-Abl activates the PI3K/Akt pathway is not completely understood. In the present study, we found that pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1 and PHLPP2) were depleted in CML cells. We investigated the interaction between PHLPPs and Bcr-Abl in CML cell lines and Bcr-Abl+ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of PHLPP1 and PHLPP2, which dephosphorylated Ser-473 on Akt1, -2, and -3, resulting in inhibited proliferation of CML cells. The reduction of PHLPP1 and PHLPP2 expression by short interfering RNA in CML cells weakened the Abl kinase inhibitor-mediated inhibition of proliferation. In colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; colony-forming unit-granulocyte, macrophage; and burst-forming unit-erythroid, treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced PHLPP1 and PHLPP2 expression and inhibited colony formation of Bcr-Abl+ progenitor cells, whereas depletion of PHLPP1 and PHLPP2 weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl+ progenitor cells. Thus, Bcr-Abl represses the expression of PHLPP1 and PHLPP2 and continuously activates Akt1, -2, and -3 via phosphorylation on Ser-473, resulting in the proliferation of CML cells.

CCN3: a key growth regulator in Chronic Myeloid Leukaemia

Chronic Myeloid Leukaemia (CML) is characterized by expression of the constitutively active Bcr-Abl tyrosine kinase. We have shown previously that the negative growth regulator, CCN3, is down-regulated as a result of Bcr-Abl kinase activity and that CCN3 has a reciprocal relationship of expression with BCR-ABL. We now show that CCN3 confers growth regulation in CML cells by causing growth inhibition and regaining sensitivity to the induction of apoptosis. The mode of CCN3 induced growth regulation was investigated in K562 CML cells using gene transfection and treatment with recombinant CCN3. Both strategies showed CCN3 regulated CML cell growth by reducing colony formation capacity, increasing apoptosis and reducing ERK phosphorylation. K562 cells stably transfected to express CCN3 showed enhanced apoptosis in response to treatment with the tyrosine kinase inhibitor, imatinib. Whilst CCN3 expression was low or undetectable in CML stem cells, primary CD34+ CML progenitors were responsive to treatment with recombinant CCN3. This study shows that CCN3 is an important growth regulator in haematopoiesis, abrogation of CCN3 expression enhances BCR-ABL dependent leukaemogenesis. CCN3 restores growth regulation, regains sensitivity to the induction of apoptosis and enhances imatinib cell kill in CML cells. CCN3 may provide an additional therapeutic strategy in the management of CML.

Phase II trial evaluating imatinib (I) in patients (pts) with anaplastic thyroid carcinoma (ATC)

6057 Background: There is no standard treatment for ATC. Response to doxorubicin ranges between 5–22%; median survival ranges between 3–6 months. Affymetrix gene chip showed PDGFR overexpression in ATC. In p53 mutated/deficient ATC cell lines, c-Abl is overexpressed, and selective inhibition of c-Abl resulted in cytostatic effect. (I) inhibits tyrosine kinase activity of Bcr-Abl and PDGF. We hypothesize that ATC that overexpress PDGFR or Abl will respond to (I). Methods: Pts ≥ 18 years old with histologically confirmed ATC, overexpressing PDGFR or c-Abl by immunohistochemistry who had measurable disease were eligible. Pts must have Zubrod performance status ≤ 1, and adequate hepatic and renal function. Prior chemotherapy, chemoradiation, radiation therapy, or surgery must have been completed at least 28 days prior to registration. (I) was administered at 400mg orally twice daily without drug holiday. Response to treatment was assessed after every 8 weeks. Pts with complete response (CR)/partial responses (PR)/stable disease (SD) were treated until disease progression. The study was terminated early due to poor accrual. Results: From February 2004 to May 2007, eleven pts from our institution were enrolled and were started on (I) (6 men; 5 women) with a median age of 63 years (ranges 53–80). At baseline, 4/11 pts (36%) had locoregional disease, 5/11 pts (45%) had distant metastases, and 2/11 pts (18%) had both. Nine pts had prior chemoradiation, and 7 pts had thyroidectomy. Out of 11 pts, 8 were evaluable for response; 3 were excluded for lack of follow up radiological evaluation. The overall responses at 8 weeks were CR 0/8; PR 2/8 (25%); SD 4/8 (50%); and PD 2/8 (25%). The median time to follow up was 26 months (ranges 23–30 months). The estimate of 6-month progression free survival was 27% (95% CI, 7–54%). The estimate of 6-month overall survival was 46% (95% CI, 17–71%). The most common G3 toxicity was lymphopenia in 45%; other G3 toxicities included edema (27%), anemia (18%), and hyponatremia (18%). There was no G4 or higher or treatment related death. Conclusions: (I) appears to have activity in advanced ATC and is well tolerated. Due to difficulty of accruing pts with a rare malignancy at a single institution, investigation of (I) in ATC may be warranted in a multi-institution setting. No significant financial relationships to disclose.

14‐3‐3 Ligand Prevents Nuclear Import of c‐ABL Protein in Chronic Myeloid Leukemia

Here we demonstrated that the 'loss of function' of not-rearranged c-ABL in chronic myeloid leukemia (CML) is promoted by its cytoplasmic compartmentalization bound to 14-3-3 sigma scaffolding protein. In particular, constitutive tyrosine kinase (TK) activity of p210 BCR-ABL blocks c-Jun N-terminal kinase (JNK) phosphorylation leading to 14-3-3 sigma phosphorylation at a critical residue (Ser(186)) for c-ABL binding in response to DNA damage. Moreover, it is associated with 14-3-3 sigma over-expression arising from epigenetic mechanisms (promoter hyper-acetylation). Accordingly, p210 BCR-ABL TK inhibition by the TK inhibitor Imatinib mesylate (IM) evokes multiple events, including JNK phosphorylation at Thr(183), p38 mitogen-activated protein kinase (MAPK) phosphorylation at Thr(180), c-ABL de-phosphorylation at Ser residues involved in 14-3-3 binding and reduction of 14-3-3 sigma expression, that let c-ABL release from 14-3-3 sigma and nuclear import, and address BCR-ABL-expressing cells towards apoptotic death. Informational spectrum method (ISM), a virtual spectroscopy method for analysis of protein interactions based on their structure, and mathematical filtering in cross spectrum (CS) analysis identified 14-3-3 sigma/c-ABL binding sites. Further investigation on CS profiles of c-ABL- and p210 BCR-ABL-containing complexes revealed the mechanism likely involved 14-3-3 precluded phosphorylation in CML cells.

BCR–ABL promotes neutrophil differentiation in the chronic phase of chronic myeloid leukemia by downregulating c-Jun expression

The mechanism that is responsible for mature neutrophil overproduction in the chronic phase (CP) of chronic myeloid leukemia (CML), a neoplastic disease of hematopoietic stem cells carrying a constitutively active tyrosine kinase BCR-ABL, remains obscure. In this study, microarray analysis revealed that c-Jun, a monopoiesis-promoting transcription factor, was downregulated in CML neutrophils. BCR-ABL directly inhibited c-Jun expression, as c-Jun downregulation in primary CML neutrophils and in the CML blast cell lines, KCL22 and K562, was reversed by the tyrosine kinase inhibitor imatinib. We established a myeloid differentiation model in KCL22 cells using zinc-inducible CCAAT/enhancer-binding protein (C/EBP)alpha (KCL22/alpha). Myeloid differentiation was observed in C/EBP-induced KCL22/alpha cells. Imatinib-induced c-Jun upregulation promoted the monocytic differentiation of KCL22/alpha cells. c-Jun knockdown in KCL22/alpha cells by a short interfering RNA redirected their differentiation from the monocytic to the neutrophilic lineage, even after imatinib treatment. A blockade of PI3K-Akt signaling with an Akt inhibitor upregulated c-Jun and induced the monocytic differentiation of KCL22, K562, and C/EBP-induced KCL22/alpha cells. Thus, BCR-ABL downregulates c-Jun expression by activating the PI3K-Akt pathway during CML-CP, thereby allowing C/EBPs to promote neutrophil differentiation.

Time-dependent effects of imatinib in human leukaemia cells: a kinetic NMR-profiling study

The goal of this study was to evaluate the time course of metabolic changes in leukaemia cells treated with the Bcr-Abl tyrosine kinase inhibitor imatinib. Human Bcr-Abl+ K562 cells were incubated with imatinib in a dose-escalating manner (starting at 0.1 μM with a weekly increase of 0.1 μM imatinib) for up to 5 weeks. Nuclear magnetic resonance spectroscopy and liquid-chromatography mass spectrometry were performed to assess a global metabolic profile, including glucose metabolism, energy state, lipid metabolism and drug uptake, after incubation with imatinib. Initially, imatinib treatment completely inhibited the activity of Bcr-Abl tyrosine kinase, followed by the inhibition of cell glycolytic activity and glucose uptake. This was accompanied by the increased mitochondrial activity and energy production. With escalating imatinib doses, the process of cell death rapidly progressed. Phosphocreatine and NAD+ concentrations began to decrease, and mitochondrial activity, as well as the glycolysis rate, was further reduced. Subsequently, the synthesis of lipids as necessary membrane precursors for apoptotic bodies was accelerated. The concentrations of the Kennedy pathway intermediates, phosphocholine and phosphatidylcholine, were reduced. After 4 weeks of exposure to imatinib, the secondary necrosis associated with decrease in the mitochondrial and glycolytic activity occurred and was followed by a shutdown of energy production and cell death. In conclusion, monitoring of metabolic changes in cells exposed to novel signal transduction modulators supplements molecular findings and provides further mechanistic insights into longitudinal changes of the mitochondrial and glycolytic pathways of oncogenesis.

Dasatinib in chronic myeloid leukemia: a review

Deregulated BCR-ABL tyrosine kinase (TK) activity is the molecular marker for chronic myeloid leukemia (CML), which provides an identifiable target for developing therapeutic agents. Imatinib mesylate, a BCR-ABL TK inhibitor, is the frontline therapy for CML. Despite the stunning efficacy of this agent, a small number of patients develop a suboptimal response or resistance to imatinib. In newly diagnosed patients with chronic phase CML, the rate of resistance to imatinib at 4 years was up to 20%, increasing to 70% to 90% for patients in the accelerated/blastic phase. Resistance to imatinib led to the development of novel TK inhibitors such as dasatinib. Several clinical trials have reported more durable complete hematologic and cytogenetic responses with this agent in patients who are resistant or intolerant to imatinib. Dasatinib is well tolerated and has broad efficacy, resulting in durable responses in patients with any BCR-ABL mutation except for T3151 and mutations in codon 317 - most commonly F317L - including mutations that were highly resistant to imatinib, such as L248, Y253, E255, F359, and H396. Dasatinib is recommended for CML in chronic, blastic or accelerated phase that is resistant or intolerant to imatinib. Dasatinib was approved by the FDA at 100 mg once daily as the starting dose in patients with chronic phase CML and at 70 mg twice daily in patients with accelerated or blastic phase CML. Various clinical trial results provided evidence that resistance to one TK inhibitor can be reversed with the use of a different TK inhibitor (TKI). Other second-generation TKIs with activity in CML include nilotinib, bosutinib and INNO 406. New molecules, such as the inhibitor of Aurora family serine-threonine kinases, MK0457, which has antileukemic activity in CML associated with a T315I mutation, are being investigated. Allogeneic hematopoietic stem cell transplantation remains an option for selected patients.

Suppression of Vascular Endothelial Growth Factor (VEGF) Expression by Targeting the Bcr-Abl Oncogene and Protein Tyrosine Kinase Activity in Bcr-Abl-positive Leukaemia Cells

Studies have shown that vascular endothelial growth factor (VEGF), a major and potent inducer of angiogenesis, is directly triggered by the disease-related oncogene Bcr-Abl in Bcr-Abl-positive cells. In this study, inhibition of Bcr-Abl tyrosine kinase activity by imatinib significantly decreased VEGF expression in Bcr-Abl-positive K562 cells in vitro. Imatinib treatment in vivo of nude mice xenografted with K562 cells resulted in a significant reduction in tumour size and microvessel density compared with untreated tumours. In addition, interfering with Bcr-Abl oncogene expression with small interfering RNAs (siRNAs) not only induced a specific reduction of Bcr-Abl mRNA and protein expression, but also efficiently inhibited the expression of VEGF in K562 cells. Combined treatment with imatinib and Bcr-Abl-targeting siRNAs resulted in an enhanced effect on VEGF suppression in K562 cells. The combined application of Bcr-Abl-targeting siRNAs and imatinib may provide a potent novel therapeutic approach for chronic myeloid leukaemia.

The stem cell factor–c-KIT pathway must be inhibited to enable apoptosis induced by BCR–ABL inhibitors in chronic myelogenous leukemia cells

Imatinib is an effective first-line therapy for chronic myelogenous leukemia (CML) that acts by targeting the tyrosine kinase activity of BCR-ABL. To overcome resistance, second-generation inhibitors of BCR-ABL have been developed. Among these, nilotinib is more potent against BCR-ABL than imatinib, and is effective against many imatinib-resistant BCR-ABL mutants. In this study, an in vitro flow cytometry assay to analyze imatinib- and nilotinib-induced apoptosis in CML cells has been developed. Both the drugs induced significant apoptosis in CD34+ cells from 36 CML bone marrow samples (P<10(-4)), whereas CD34+ cells from BCR-ABL negative samples were unaffected. When the experiments were carried out in the presence of a cocktail of cytokines, nilotinib- but not imatinib-induced apoptosis was inhibited. This differential inhibition was confirmed on K562 cells. A blocking anti-CD117 antibody alleviated the antiapoptotic effect of cytokines against nilotinib. Moreover, using short hairpin RNA against BCR-ABL, we showed that K562 cells were not dependent on BCR-ABL signaling as long as the stem cell factor (SCF) receptor pathway was activated. We conclude that the c-KIT pathway may substitute for BCR-ABL tyrosine kinase to activate survival signals, and that c-KIT must be inhibited besides Bcr-Abl to allow apoptosis of CML cells.