Philadelphia-positive Chronic Myelogenous Leukemia Research Articles

A 14q32.31 Genomic-Imprinted DLK1-DIO3 microrna promotes Leukemogenesis By Inducing Stem Cell Quiescence and Inhibiting NK Cell Anti-Cancer Immunity

Leukemia emergence, maintenance, relapse and/or progression are causally linked to the presence of drug-resistant leukemia-initiating cells and impaired natural killer (NK) cell anti-tumor immune-response. Bone marrow microenvironment (BMM)- and/or leukemia-derived signals induce aberrant non-coding RNA expression and inhibit protein phosphatase 2A (PP2A) tumor suppressor activity. PP2A loss-of-function is essential for NK cell activity and leukemic but not normal stem and progenitor cell proliferation and survival. The human MIR300 gene is an intergenic miRNA that belongs to the 14q32.31 DLK1-DIO3 genomic-imprinted tumor suppressor miRNA cluster B. MIR300 was found involved in loss-of heterozygosity, inhibited in several tumor types with high mitotic index and during epithelial-to-mesenchymal transition (EMT), and associated with a cancer stem cell phenotype. By using primary cells from Philadelphia-positive (Ph+) chronic myelogenous leukemia (CML) in chronic (CP) and blastic (BC) phase, and complex karyotype (CK) acute myeloid leukemia (AML) patients, as paradigmatic examples of stem cell-derived neoplasms characterized by constitutive expression of oncogenic kinases, PP2A loss-of-function, altered microRNA expression and impaired NK cell proliferation and cytotoxicity, we found that MIR300 is a cell context-independent tumor suppressor with anti-proliferative and PP2A-dependent pro-apoptotic activities which are sequentially activated in a MIR300 dose-dependent manner through inhibition of CCND2/CDK6 and SET (PP2A inhibitor), respectively. To prevent PP2A-induced apoptosis, MIR300 is inhibited by oncogenic signals in CD34+CML (CP and BC) and CK-AML progenitors. Conversely, tumor-naïve BMM-induced C/EBPbeta-mediated signals (hypoxia and MSC exosomes) markedly upregulate MIR300 expression in primary CML and AML CD34+CFSEmax leukemic stem (LSC) and CD56+CD3-NK cells to induce/maintain quiescence (increased CD34+leukemic blasts in G0) and impair immune-response (suppression of NK cell proliferation and cytotoxic activity toward CD34+ leukemic blasts and CFSEmaxCD34+ CML-BC quiescent LSCs), respectively. Inhibition of MIR300 expression/activity rescues NK cell proliferation and anti-tumor cytotoxicity and prevented MSC- and hypoxia-induced growth-suppression of CD34+leukemic blasts by inhibiting degradation of MIR300 targets (e.g. SET, CCND2). We found that CML and AML LSCs escape MIR300-induced PP2A-mediated apoptosis through the hypoxia- and tumor-dependent TGFb1-FoxM1-mediated upregulation of TUG1 lncRNA. TUG1 is an oncogenic lncRNA described as a MIR300sponge and found upregulated in solid tumors, in which it has strong diagnostic, prognostic and therapeutic relevance and is associated with cancer stem cell maintenance and EMT. In quiescent CML and AML LSCs, TUG1 uncouples and limits MIR300 tumor suppressor functions to cytostasis by maintaining unbound MIR300 at levels sufficient to inhibit CCND2 and CDK6 but not SET expression. Exposure to clinically-relevant CpG-modified oligonucleotides modulating MIR300levels and/or inhibiting TUG1 MIR300-sponging activity, restores NK cell proliferation and cytotoxic activity, and suppresses human leukemic but not normal hematopoiesis by eradicating nearly all (> 95% reduction) CFSEmaxCD34+ and CD45+CD34+CD38-CD90+ LSCs and CD34+leukemic CML (CP and BC) and CK-AML blasts in vitro (CFCs, LTC-IC, and CFSEmaxCD34+cell tracking) and/or in NRG-SGM3 PDX mouse models of acute and chronic myeloid leukemias. Altogether, this work highlights the therapeutic importance of altering MIR300 expression in anti-LSC and NK cell-based approaches for myeloid leukemias, and indicates that tumor-naïve BMM-induced MIR300 tumor suppressor anti-proliferative and PP2A-activating functions may support leukemogenesis by promoting the formation and initial expansion of the quiescent LSC pool through the induction of LSC dormancy and inhibition of quiescent LSC killing by cytokine-activated NK cells, respectively. (G.S. and R.T. equally contributed to this work) Disclosures Stagno: BMS: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Deininger:TRM: Consultancy; Sangoma: Consultancy; Incyte: Honoraria; Novartis: Honoraria; Sangamo: Consultancy; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Humana: Honoraria; Ascentage Pharma: Consultancy, Honoraria; Blueprint: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Research Funding; Fusion Pharma: Consultancy; Adelphi: Consultancy. Milojkovic:BMS: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau. Apperley:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Baer:Takeda: Research Funding; Incyte: Research Funding; Kite: Research Funding; Forma: Research Funding; AI Therapeutics: Research Funding; Abbvie: Research Funding; Astellas: Research Funding.

A case series of patients with de novo Philadelphia-positive chronic myelogenous leukemia at tertiary care center SKIMS

The well-established molecular pathogenesis of chronic myelogenous leukemia (CML) and its consequences for laboratory testing and clinical management illustrate a classic paradigm for the importance of molecular diagnostics in targeted drug therapy. The success of the tyrosine kinase inhibitor (TKI), Imatinib, as the currently recommended first-line treatment of early chronic phase CMLhas both fueled the need for timely and reproducible molecular testing of the BCR-ABL fusion transcript in diagnosis and monitoring. As ongoing research continues to refine guidelines for monitoring residual disease in patients undergoing TKI therapy, an understanding of molecular technologies and their interpretation is critical. In this regard, we at Cancer Diagnostic and Research Centre, Immunology and Molecular Medicine established a gold standard RT-PCR and RQ-PCR technique to diagnose and monitor residual disease in CMLpatients. JMS 2016; 19(2):82-84

The prevalence of Trisomy 8 as the commonest additional chromosomal abnormality in Philadelphia positive chronic myelogenous leukemia

The prevalence of Trisomy 8 as the commonest additional chromosomal abnormality in Philadelphia positive chronic myelogenous leukemia

Nilotinib reverses ABCB1/P-glycoprotein-mediated multidrug resistance but increases cardiotoxicity of doxorubicin in a MDR xenograft model

The BCR-Abl tyrosine kinase inhibitor (TKI), nilotinib, was developed to surmount resistance or intolerance to imatinib in patients with Philadelphia-positive chronic myelogenous leukemia. Recent studies have shown that nilotinib induces potent sensitization to anticancer agents by blocking the functions of ABCB1/P-glycoprotein (P-gp) in multidrug resistance (MDR). However, changes in P-gp expression or function affect the cardiac disposition and prolong the presence of both doxorubicin (DOX) and doxorubicinol (DOXol) in cardiac tissue, thus, enhancing the risk of cardiotoxicity. In this study, we used a MDR xenograft model to evaluate the antitumor activity, tissue distribution and cardiotoxicity of DOX when co-administered with nilotinib. This information will provide more insight into the pharmacological role of nilotinib in MDR reversal and the risk of DOX cardiotoxicity. Our results showed that nilotinib significantly enhanced DOX cytotoxicity and increased intracellular rhodamine 123 accumulation in MG63/DOX cells in vitro and strongly enhanced DOX inhibition of growth of P-gp-overexpressing MG63/DOX cell xenografts in nude mice. Additionally, nilotinib significantly increased DOX and DOXol accumulation in serum, heart, liver and tumor tissues. Importantly, nilotinib induced a disproportionate increase in DOXol in cardiac tissue. In the co-administration group, CBR1 and AKR1A1 protein levels were significantly increased in cardiac tissue, with more severe necrosis and vacuole formation. These results indicate that nilotinib reverses P-gp- mediated MDR by blocking the efflux function and potentiates DOX-induced cardiotoxicity. These findings represent a guide for the design of future clinical trials and studies of pharmacokinetic interactions and may be useful in guiding the use of nilotinib in combination therapy of cancer in clinical practice.

Update on the management of Philadelphia chromosome positive chronic myelogenous leukemia: role of nilotinib.

Chronic myelogenous leukemia (CML) is a pluripotent stem cell disease characterized by the presence of the Philadelphia chromosome and the bcr-abl gene. The discovery of tyrosine kinase inhibitors (TKIs) revolutionized therapy for CML, such that durable response, increased overall survival, and increased progression-free survival of patients in chronic phase CML is now possible. Due to resistance and intolerance to imatinib, there was need for development of second- and third-generation TKIs for the treatment of CML. This review examines the role of nilotinib, an oral second-generation TKI, in the treatment of Philadelphia positive CML. The pharmacology, efficacy, and safety of nilotinib are critically evaluated. Patient-related issues, including tolerance, drug interactions, and quality of life issues are also examined.

Description and Management of Accelerated Phase and Blast Crisis in 21 CML Pediatric Patients

Introduction: Philadelphia-positive chronic myelogenous leukemia (CML) is a rare disease in children and constitutes approximately 3-5% of all childhood leukemias. With tyrosine kinase inhibitors (TKI), the frequency of accelerated phase (AP) or blast crisis (BC) is remarkably reduced, estimated to 1% to 1.5% per year in adults compared with more than 20% per year in the pre-TKI era. But no data are available among children.Purpose: We described the characteristics, the treatment and the outcome of 21 children with CML, who evolved in accelerated phase and/or blast crisis under TKI.Results: From 2001 to april 2015, 415 European patients were enrolled in the CML pediatric database. Twenty-one patients (5.1%), in chronic phase (CP) treated by TKI, presented AP or BC.The median age of AP/BC cohort was 13.2 years (range: 4.5-16.9 years) with a sex ratio M/F at 2. At CML diagnosis, 15 patients (71%) had high risk Sokal Score with a median score of 1,4 (range: 0,16-2,4). All patients harbored t(9;22)(q34;q11) but one had a complex translocation t(1;9;22)(q12;q34;q11) and another one presented additional inv(3)(q21q26). Imatinib was the first line TKI for all patients. Before AP or BC, only five patients (24%) obtained a complete cytogenetic response (CCyR) and three achieved MMR. For incomplete molecular response or progression to accelerated phase, 8 patients (38%) were switched to dasatinib. Median duration of TKI before AP or BC was 11 months (range: 3 months-56.5 months). Six patients evolved to AP with a median interval of 8.7 months (range: 1 months-24 months), leading to blast crisis for 4 patients with a median time of 3.5 months (range: 0.3-5.4 months). Among the 2 patients remaining in AP, imatinib was increased for one and the other was switched for dasatinib, all before hematopoietic stem cells transplantation (HSCT). One patient died of post-transplant complication and the other one is still alive in complete molecular response without TKI. Nineteen patients presented BC, including 4 after AP. Thirteen patients (62%) presented ALL, five (24%) AML and one a bi phenotypic leukemia. Central nervous system (CNS) was involved for two patients with ALL, one isolated, one combined. At AP or BC, nine patients (43%) presented new additional cytogenetic abnormalities. Eighteen patients with BC were treated according to AML or ALL protocols, combined with second generation TKI for twelve patients. Only one patient underwent preparative regimen, without intensive chemotherapy before HSCT. Ten patients reached complete remission. Four patients died before HSCT, by progressive disease for 2 and by fatal infection for 2. Overall, 15 patients in BC were transplanted. Before HSCT, median molecular response was 0.2% (range: 0-29%) and only four patients had a complete molecular response. After transplant, seven patients received second generation TKI. Four patients died, including three related to transplant toxicity. Thirteen patients were alive, but one with ALL BC relapsed 26 months post-transplant and was waiting for second HSCT. With a median follow-up of 4.4 years, 4-year overall survival was 59% (66% for ALL BC versus 40% for AML BC).Conclusion: Incidence of AP/BC after imatinib for CP CML is at 5%, in the CML pediatric database. Despite second generation TKI, combined with HSCT, outcome remains poor. Post-transplant indication of TKI is heterogenic. Recommendations would be useful for practice. [Display omitted] DisclosuresSuttorp:Novartis, Bristol Meyer Squib: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Study on the Effects of Rapamycin Reverse AMN107 resistance on k562/AO2 Cells

Abstract 5017 AimRapamycin(RAPA) is the inhibitor of m-TOR. The activation of m-TOR results in changes in multiple cellular processes, eg, catabolism, anabolism, proliferation, growth and apoptosis. Rapamycin, which directly inhibits the Akt downstream molecule mammalian target of rapamycin (mTOR), effectively inhibits survival and proliferation in the development of BCR- ABL–induced chronic myelogenous leukemia(CML) from PTENfl/fl. Nilotinib (AMN107), a BCR-ABL tyrosine kinase inhibitor, was developed to surmount resistance or intolerance to imatinib in patients with Philadelphia positive chronic myelogenous leukemia. We aim to introduce the CML cell line (k562/A02) resistant to AMN107 and investigate the effect of RAPA reverse AMN107 resistance on k562/A02 cell line. MethodK562/A02 be added in the culture medium final concentration 1μmol/ L of AMN107 drugs a week before the withdrawal. For all experiments, cells were on the logarithmic phase.K562/A02 cells were treated with 400nmol/L rapamycin(400nmol/L RAPA group),100nmol/L rapamycin(100nmol/L RAPA group), 20μmol/L nilotinib(20μmol/L AMN107 group), 400nmol/L rapamycin +20μmol/L nilotinib (400nmol/L RAPA+20μmol/L AMN107 group), 100nmol/L rapamycin +20μmol/L nilotinib (100nmol/L RAPA+20μmol/L AMN107 group). Tetrazolium—based colorimetric assay (MTT) was used to detect the inhibiton effect of cell growth. The expression of Survivin, m-TOR, Bcl-2, and Caspase-3 were detected by western blot; Cell apoptosis was inspected by Annexin V/PI double staining method; The expression of Survivin, m-TOR, Bcl-2 and Caspase-3 in mRNA level was determined using reverse transcriptase—polymerase chain reaction (RT-PCR). BCR-ABL gene was detected by Real Time-PCR when K562/AO2 cells in different groups were treated by drugs for 48h. ResultK562/A02 cells were successfully introduced to resistant toAMN107. Combination of 400 and 100 nmol/L RAPA with AMN107 could significantly enhance the sensitivity of k562/A02 to AMN107. The reverse factor was 1.97 and 2.73 fold respectively. RT-PCR and Western blot method indicated that the expression of Survivin and Bcl-2 was increased by RAPA+AMN107 group, but the MDR1 and Caspase-3 was decreased. The expression of BCR-ABL fusion gene in100 nmol/L RAPA group was higher than the blank groups, the copy number is 5.6×10−2. But the copy number in 400nmol/L RAPA +20μmol/L AMN107 group is1.64×10−2 that is lowest. Annexin V/PI showed that the apoptosis rates were (8.21±0.05)%, (32.07±0.03)%, (28.88±0.11)%, (16.57±0.05)%, (38.49±0.04)%, and(42.31±0.07)% respectively in control group, AMN107 group, 400nmol/L RAPA group, 100nmol/L RAPA group, 100nmol/L RAPA +20μmol/L AMN107 group, and 400nmol/L RAPA +20μmol/L AMN107 group. ConclusionWe successfully introduced K562/A02 resistant toAMN107. Moreover, RAPA can enhance the sensitivity of AMN107 resistant 562/A02 cells. Combined of RAPA and AMN107 has significant synergistic growth inhibiting effect and apoptosis inducing effect on AMN107-resistant K562/A02 cells. The mechanism may be related at down-regulation of expression MDR1 and up-regulation of expression Survivin. Disclosures:No relevant conflicts of interest to declare.

The Ubiquitin+Proteasome Protein Degradation Pathway as a Therapeutic Strategy in the Treatment of Solid Tumor Malignancies

A concept that currently steers the development of cancer therapies has been that agents directed against specific proteins that facilitate tumorigenesis or maintain a malignant phenotype will have greater efficacy, less toxicity and a more sustained response relative to traditional cytotoxic chemotherapeutic agents. The clinical success of the targeted agent Imatinib mesylate as an inhibitor of the tyrosine kinase associated with the breakpoint cluster region-Abelson oncogene locus (BCR-ABL) in the treatment of Philadelphia-positive chronic myelogenous leukemia (CML) has served as a paradigm. While intellectually gratifying, the selective targeting of a single driver event by a small molecule, e.g., kinase inhibitor, to dampen a tumor-promoting pathway in the treatment of solid tumors is limited by many factors. Focus can alternatively be placed on targeting fundamental cellular processes that regulate multiple events, e.g., protein degradation, through the Ubiquitin (Ub)+Proteasome System (UPS). The UPS plays a critical role in modulating numerous cellular proteins to regulate cellular processes such as signal transduction, growth, proliferation, differentiation and apoptosis. Clinical success with the proteasome inhibitor bortezomib revolutionized treatment of B-cell lineage malignancies such as Multiple Myeloma (MM). However, many patients harbor primary resistance and do not respond to bortezomib and those that do respond inevitably develop resistance (secondary resistance). The lack of clinical efficacy of proteasome inhibitors in the treatment of solid tumors may be linked mechanistically to the resistance detected during treatment of hematologic malignancies. Potential mechanisms of resistance and means to improve the response to proteasome inhibitors in solid tumors are discussed.

Abstract 1531: BCR-Abl tyrosine kinase inhibitor Nilotinib (Tasigna®) reverses ABCB1/Pgp- and ABCG2/BCRP-mediated multidrug resistance

Abstract Over-expression of ABC transporters are involved in the extrusion of therapeutic drugs, leading to MDR, which can eventually produce cancer chemotherapy failure. Previously, it was shown that specific TKIs could modulate several human multidrug resistance (MDR) ATP-binding cassette (ABC) proteins. Recently discovered second generation, BCR-Abl tyrosine kinase inhibitor (TKI), Nilotinib was developed to surmount resistance or intolerance to imatinib in patients with Philadelphia positive chronic myelogenous leukemia. Here, we report for the first time that nilotinib not only potentiates the cytotoxicity of widely used therapeutic substrates of ABCB1, such as colchicine, vincristine, and paclitaxel, but also that of ABCG2 substrates such as mitoxantrone and doxorubicin. Nilotinib also significantly enhances the intracellular accumulation of methotrexate (MTX) in cells transfected with ABCG2. Similarly, nilotinib significantly increases the accumulation of paclitaxel in cell lines overexpressing ABCB1. Furthermore, nilotinib produces a concentration-dependent inhibition of the ABCG2-mediated transport of MTX, as well as E217βG a physiological substrate of ABCG2. Uptake studies in membrane vesicles over-expressing ABCG2 have indicated that nilotinib inhibits ABCG2 similar to other established TKIs as well as fumitremorgin C. In addition, the kinetic analysis demonstrated that nilotinib is a potent competitive inhibitor of MTX transport by ABCG2 with a Ki value of 0.69+/-0.083 μM. To sum it up, our results indicate that nilotinib could reverse ABCB1- and ABCG2-mediated MDR by blocking the efflux function of these transporters. These findings may be useful in clinical practice for cancer combination therapy with nilotinib and may be vital to the design of present and future clinical trials with nilotinib, elucidating potential pharmacokinetic interactions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1531.

Prognostic factors associated with complete cytogenetic response in patients with chronic myelogenous leukemia on imatinib mesylate therapy

Imatinib mesylate, a selective Bcr-Abl tyrosine kinase inhibitor, has proved to be most effective therapy of Philadelphia chromosome-positive chronic myelogenous leukemia. Imatinib induces complete haematological and cytogenetic response in high percentage of patients. The aim of this study was to identify potential prognostic factors before beginning treatment with imatinib associated with complete cytogenetic response. We analyzed 20 patients with newly diagnosed Philadelphia positive chronic myelogenous leukemia treated at our institution from June 2006 until May 2009. These patients were treated with imatinib mesylate in oral dose of 400 to 800 mg daily. Complete blood counts were performed every month, while serum chemistry evaluations and bone marrow evaluations including morphology and cytogenetics were performed every 6 months. Of the 20 patients analyzed in this study, 19 (95%) achieved complete haematologic response within three months. In all patients cytogenetic analyses were done and all have achieved absolute cytogenetic response. The best cytogenetic response rate at any time during study treatment among 20 patients was: complete cytogenetic response in 15, partial cytogenetic response in three and minor cytogenetic response in two patients. Among 11 observed base-line patients' characteristics five were independent predictors of a high rate of complete cytogenetic response; the absence of blasts and basophils in peripheral blood, the presence of less than 5 percent of bone marrow blasts, white blood cell count less than 10 x 10(9)/L and the absence of splenomegaly (p < 0.01). Our results showed that some pre-treatment characteristics of patients might be the cause of differences in treatment outcome. On the basis of this analysis, we identified several pre-treatment patients' characteristics to be independent prognostic factors for achievement of complete cytogenetic response.

Human GST5.8, Expressed in a Cell Cycle Specific Manner, May Offer a Method to Alter 4-Hydroxynonenal Concentrations of Philadelphia-Positive Chronic Myelogenous Leukemia Cells.

Abstract 4260 BackgroundChronic Myelogenous Leukemia studies in K562 cell line, obtained from the pleural effusion of a CML patient in blastic crisis, initially focused upon chromosomal translocations such as del(22) (q12) and t(15;17)(q21;q24). {{Lozzio,C.B. 1975}} In a 2001 Phase I Clinical Trial, STI-571, a tyrosine kinase inhibitor which treats the BCR/Abl fusion protein formed due to t(9;22)(q34;q11) found in the majority of CML patients effectively ceased proliferation of BCR-Abl expressing hematopoietic cells.{{Brian J. Druker, M.D., 2001}} However, recent studies demonstrate that Chronic Myelogenous Leukemia persists in patients exhibiting differing chromosomal abnormalities. {{Jamshid S Khorashad, MD, 2008}} Therefore, we examined methods for inducing differentiation in K562 cells to pursue the senescence of leukemic blasts beyond proliferation in heterogeneic CML populations. Using 20 uM 4-hydroxynonenal, a lipid peroxidation product, Cheng successfully produced differentiated K562 cells. {{Cheng,J.Z. 1999}} Our studies confirm this; however 20 um-HNE-treated K562 cells also aggregate. Here, we performed titration studies examining extremely low concentrations of 4-HNE and its correlation with K562 morphology to investigate if low-HNE-containing cells remained premature blasts. Barrera also demonstrated inhibition of cell proliferation, as well as the downregulation of c-myc expression in K562 cells, by HNE in a dose-dependent manner. {{Barrera,G. 2004}} However, he reported that these effects were transient and disappeared within 6–8 hours following HNE administration. Because levels of aldehydes, such as 4-HNE, formed by lipid peroxidation are regulated through conjugation to glutathione, we investigated glutathione-S-transferase activity in K562 cells, which has been shown to exhibit extremely high specificity for 4-hydroxynonenal and is known to reduce 4-HNE concentrations in tissue cells. {{Cheng,J.Z. 1999}} HypothesisHuman GST5.8 exhibits the highest specific activity of the glutathione-S-transferases toward lipid peroxidation product, 4-HNE as shown in tissue. {{Singhal,S.S. 1999}} So, we observed if higher concentrations of the enzymatic activity occurred at specific points in the K562 cell cycle, affecting HNE concentrations and contributing to cell cycle phases such as G0/G1 or S phase. MethodsWe examined human GST5.8 levels in K562 cells following synchronized release from arrest with sodium butyrate. In addition, we performed morphological analysis of HNE effects on K562 cells via Giemsa stained negative controls versus titrations of HNE-treated K562 cells (at 0.5-20 uM) for 2 hours. Also, Western Blots and FACS analyses identifying time points of highest human GST5.8 expression were produced. ResultsDensitometric analysis of hGST5.8 expression by time point revealed that human GST5.8 is present in K562 cells and that the enzymatic concentration peaks at 0 hr to 30 minutes following release from arrest or the G0/G1 time point. Our results confirmed that 20 um HNE causes differentiation, aggregation, and apoptosis in K562 cells. We also showed 0.5 uM concentration produces differentiation but very minimal aggregation. ConclusionsBecause human GST5.8 peaks during the G0/G1 time point, causing a reduction of the 4-hydroxynonenal concentration; it is reasonable to conclude that hGST5.8 may rise during G0/G1 in order to lower HNE concentrations as a dedifferentiation mechanism in normal cell cycle events. Here, we have demonstrated that human GST5.8 is expressed in a cell-cycle specific manner. Future studies involve transfection of hGST5.8 antisense into K562 to learn if consistent induction of differentiation is possible. Taken together, these studies indicate that hGST5.8 modulation may hold promise as a method to control 4-hydroxynonenal levels and thus differentiation in Ph(+) Chronic Myelogenous Leukemia cells. Disclosures:No relevant conflicts of interest to declare.

Imatinib Is Efficient but Has a Negative Impact On Growth in Children with Previously Untreated chronic Myelogenous Leukaemia (CML) in Early Chronic Phase (CP): Results of the French National Phase IV Trial.

Abstract 863 Aims:In order to investigate the efficacy and the safety of imatinib in children and adolescents with untreated Philadelphia-positive CML, the CML working party of the Société Française des Cancers de l'enfant (SFCE) conducted an open label, multicentric phase IV study trial (Clinical Trials.gov.NCT00845221). Patients and methods.Patients less than 18 yrs of age with newly diagnosed CP CML were eligible. Imatinib was administered orally at a dose of 260 mg/m2 which is equivalent in terms of drug exposure to the total dose of 400 mg in adults. Forty four children (64% boys) with a median age of 11.5 yrs (range 10 months-17 yrs) have been enrolled from 15 French pediatric centres between July 2004 and December 2008. Side effects were reported prospectively using the NIH CTCv2.0 criteria. Results.Median follow up is currently 17 months (range:1-67). The complete hematologic response rate was 86% and 98% (ITT) at 3 months and 6 months, respectively. The rate of complete cytogenetic response (CCyR) was 62% (ITT) at 12 months. The rate of major molecular response (MMR) defined as a BCR-ABL/ABL ratio ≤ 0.1% according to the International Scale was 34% (ITT) at 12 months. The median daily dose of imatinib was close to the intended doses, 250 mg/m2 (range 176-405) with a median treatment duration of 16 months (2 w to 67 mo). Three pts (7%) had a dose reduction of more than 25% of the theoretic dose. Nine pts (20%) interrupted imatinib temporarily at least once for a median duration of dose interruption of 12 days (range 2-70). Ten pts (23%) discontinued imatinib. The reasons for discontinuation were the following: allogeneic HSCT in CCyR or MMR (2 pts) according to the investigator choice, adverse event (2 pts: muscle pain 1 pt, liver enzyme elevation 1 pt), disease progression or failure (6 pts: loss of CHR [L248V mutation] 1pt, loss of CCyR [L384M mutation] 1pt, no cytogenetic response 1 pt, no molecular response 1 pt, loss of MMR 2 pts). One of them (loss of hematologic response) transformed to blastic phase shortly after coming off study and died. Grade III or IV toxicity was recorded in 14 pts (32%). GradeIII-IV hematologic toxicity was recorded in 8 pts (18%) including neutropenia in 18% and thrombocytopenia in 5%. Nine pts (21%) developed grade III or IV non-hematologic toxicity: muscle pain (2.5%), arthralgia (5%), weight gain (13%), liver enzyme (GOT/GPT) increase (6.5%). Change of body height was observed during the first year of treatment with imatinib in the 22 pts with a sufficient follow-up: a significant decrease of height standard deviation scores (SDS) was observed with a median of the difference of -0.37 (range, -1.09 to +0.14)(p<0.0001) between the start of the treatment and 12 months later. Conclusion.Imatinib is efficient in children and adolescent with previously untreated CML in early chronic phase. However, we report for the first time, a negative impact of imatinib on the growth in a cohort of children and adolescents treated with imatinib. Disclosures:No relevant conflicts of interest to declare.

Nilotinib (AMN107, Tasigna ®) reverses multidrug resistance by inhibiting the activity of the ABCB1/Pgp and ABCG2/BCRP/MXR transporters

Nilotinib, a BCR-Abl tyrosine kinase inhibitor (TKI), was developed to surmount resistance or intolerance to imatinib in patients with Philadelphia positive chronic myelogenous leukemia. Recently, it was shown that several human multidrug resistance (MDR) ATP-binding cassette (ABC) proteins could be modulated by specific TKIs. MDR can produce cancer chemotherapy failure, typically due to overexpression of ABC transporters, which are involved in the extrusion of therapeutic drugs. Here, we report for the first time that nilotinib potentiates the cytotoxicity of widely used therapeutic substrates of ABCG2, such as mitoxantrone, doxorubicin, and ABCB1 substrates including colchicine, vincristine, and paclitaxel. Nilotinib also significantly enhances the accumulation of paclitaxel in cell lines overexpressing ABCB1. Similarly, nilotinib significantly increases the intracellular accumulation of mitoxantrone in cells transfected with ABCG2. Furthermore, nilotinib produces a concentration-dependent inhibition of the ABCG2-mediated transport of methotrexate (MTX), as well as E 217βG a physiological substrate of ABCG2. Uptake studies in membrane vesicles overexpressing ABCG2 have indicated that nilotinib inhibits ABCG2 similar to other established TKIs as well as fumitremorgin C. Nilotinib is a potent competitive inhibitor of MTX transport by ABCG2 with a K i value of 0.69 ± 0.083 μM as demonstrated by kinetic analysis of nilotinib. Overall, our results indicate that nilotinib could reverse ABCB1- and ABCG2-mediated MDR by blocking the efflux function of these transporters. These findings may be used to guide the design of present and future clinical trials with nilotinib, elucidating potential pharmacokinetic interactions. Also, these findings may be useful in clinical practice for cancer combination therapy with nilotinib.

Dasatinib for the treatment of Philadelphia chromosome-positive chronic myelogenous leukaemia after imatinib failure

Dasatinib, a potent, oral kinase inhibitor, is presently approved for Philadelphia-positive chronic myelogenous leukaemia (CML) following imatinib failure. In an in vitro study, dasatinib had 325-fold greater potency than imatinib for inhibiting unmutated BCR-ABL. Phase I and II data show that dasatinib 70 mg b.i.d. is effective after imatinib failure in various phases of CML. Comparative data of dasatinib versus high-dose imatinib in patients with resistance or intolerance to imatinib demonstrated that dasatinib was associated with improved response rates and progression-free survival. Side effects of dasatinib, including pleural effusions, are manageable with modification of dose or schedule. Phase III dose optimisation studies and future indications are also discussed.

Nilotinib Is Associated with Minimal Cross Intolerance to Imatinib in Patients with Imatinib-Intolerant Philadelphia-Positive (Ph+) Chronic Myelogenous Leukemia (CML) in Either Chronic Phase (CP) or Accelerated Phase (AP).

Nilotinib, a highly selective BCR-ABL tyrosine kinase inhibitor that is 30-fold more potent than imatinib, represents an important treatment option for pts with imatinib-resistant or -intolerant Ph+CML. Reported are results from a phase II, open-label study evaluating the safety and efficacy of nilotinib in imatinib-resistant or -intolerant Ph+CML. Imatinib resistance was defined as treatment with imatinib ≥600 mg/d with disease progression (≥50% increase in WBCs, blasts, basophils, or platelets) or no hematologic response after 4 wks. Imatinib intolerance was defined as no prior MCyR and discontinuation of imatinib due to Grade 3/4 AE or persistent (>1 mo) or recurrent Grade 2 AE (recurred >3x) despite optimal supportive care. Nilotinib-imatinib cross-intolerance was defined as treatment with nilotinib and occurrence (regardless of causality) of Grade 3/4 of the same AE that led to discontinuation of imatinib therapy. Planned starting dose was nilotinib 400 mg BID but could be escalated to 600 mg BID for lack of response. Of 320 pts with CML-CP, 94 (29.4%) were enrolled for imatinib-intolerance for either nonhematologic and/or hematologic AEs, of these, 71 (76%) had Grade 3/4 AEs at study entry. Of 127 pts with CML-AP, 24 (18.9%) were enrolled for nonhematologic and/or hematologic imatinib-intolerance; of these, 16 (67%) had Grade 3/4 AEs at study entry. Some pts had >1 AE satisfying criteria for intolerance. Only 2/71 (3%) pts with nonhematologic imatinib-intolerance experienced a recurrence of similar Grade 3/4 AEs during nilotinib therapy. Of 37 pts with hematologic intolerance to imatinib, 19/37 (51%) did not develop Grade 3/4 or similar events during nilotinib therapy. Median duration of nilotinib exposure was 350.5 days in CML-CP and 141.5 days in CML-AP with median dose intensities of 725.8 mg/d and 768.8 mg/d, respectively. Only 1 pt (CML-AP) required a dose escalation to 600 mg BID. Although nilotinib and imatinib have some molecular similarities, these results support previous findings of minimal occurrence of cross-intolerance. These results also suggest important differences in safety profiles between imatinib and nilotinib. Thrombocytopenia appears to be the only intolerant AE that may recur with nilotinib. These results support nilotinib's excellent tolerability and indicate that it can be used effectively in both CML-CP and -AP pts with imatinib-intolerance.CML-CP Pts with Intolerance (N=94)CML-AP Pts with Intolerance (N=24)Intolerance AEsImatinib IntoleranceGrade 3/4 AE during NilotinibImatinib IntoleranceGrade 3/4 AE during NilotinibNon-hematologic562150Rash/Skin26050Fluid Retention17050GI16110Liver Toxicity10130 ALT4010 AST4100Myalgia/arthralgia9020Hematologic291683Thrombocytopenia241351Neutropenia8432Anemia2110

Anagrelide and imatinib mesylate combination therapy in patients with chronic myeloproliferative disorders.

The tyrosine kinase inhibitor imatinib mesylate inhibits the function of the Bcr-Abl oncoprotein associated with Philadelphia-positive chronic myelogenous leukemia (CML). Anagrelide suppresses megakaryocyte proliferation and differentiation. The objectives of this study were to investigate the feasibility and safety of imatinib mesylate and anagrelide combination therapy in patients with Ph-positive CML or chronic myeloproliferative disorders (MPD) with persistent thrombocythemia. This study was a retrospective review of all available records of patients with chronic MPD presenting to the M.D. Anderson Cancer Center between October 1998 and May 2002, treated with imatinib mesylate combined with anagrelide. Of 22 patients identified, 18 had Ph-positive CML (chronic phase, 16 patients; accelerated phase, 2 patients), 1 had agnogenic myeloid metaplasia (AMM), 2 had essential thrombocythemia (ET) and 1 had MPD transformed into refractory anemia with excess blasts (RAEB). The median age was 57 years (range 26-82 years). The median dose of imatinib mesylate administered was 400 mg (range 300-800 mg) and the median dose of anagrelide was 1.5 mg (range 0.5-4.0 mg). Imatinib mesylate and anagrelide combination therapy was feasible and tolerable. Of the 18 patients with Ph-positive CML, 15 in chronic phase and 1 in accelerated phase achieved a complete hematologic response (CHR), and 9 of the 18 achieved cytogenetic response (complete in 8 patients). No responses were noted in patients with AMM, ET or MPD transformed into RAEB. The combination of imatinib mesylate and anagrelide was safe and was associated with an 89% CHR rate in patients with CML in chronic phase and persistent thrombocythemia.

Imatinib in combination with cytarabine for the treatment of Philadelphia-positive chronic myelogenous leukemia chronic-phase patients: Rationale and design of phase I/II trials

Imatinib (Gleevec) (formerly STI571) has been shown to selectively inhibit the tyrosine kinase domain of the oncogenic BCR-ABL fusion protein of chronic myelogenous leukemia (CML) cells. In recent phase I and II studies testing this new compound in patients who had failed to respond to interferon (IFN), hematological and cytogenetic responses were reported in most of those with chronic-phase CML. However, in some patients resistance has been associated with a single amino acid substitution in a threonine residue of the Abl kinase domain. In vitro studies examining the effects of imatinib plus cytarabine (Ara-C) using CML cell lines and colony-forming assays of CML patient samples have shown synergistic antiproliferative effects of this combination. Thus several groups decided to investigate this new combination with the hypothesis that cell resistance would be less frequent. The CML French Group performed a phase II trial to determine the safety and tolerability of a combination of imatinib and Ara-C for previously untreated patients with chronic-phase CML. Treatment was administered on 28-day cycles for 12 months. Patients were treated continuously with imatinib orally at a dose of 400 mg daily. Ara-C was given on days 14 to 28 of each cycle at an initial dose of 20 mg/m2/d via subcutaneous injection, hydroxyurea (HU) being stopped at least 7 days before imatinib. Recently, the Dutch group decided to explore a combination of high-dose Ara-C with imatinib in patients in chronic-phase CML. Preliminary results are encouraging. However, a long follow-up is required before concluding that these strategies will overcome cell resistance. Semin Hematol 40(suppl 2):92-97. © 2003 Elsevier Inc. All rights reserved.

Imatinib in combination with cytarabine for the treatment of Philadelphia-positive chronic myelogenous leukemia chronic-phase patients: Rationale and design of phase I/II trials

Imatinib (Gleevec) (formerly STI571) has been shown to selectively inhibit the tyrosine kinase domain of the oncogenic BCR-ABL fusion protein of chronic myelogenous leukemia (CML) cells. In recent phase I and II studies testing this new compound in patients who had failed to respond to interferon (IFN), hematological and cytogenetic responses were reported in most of those with chronic-phase CML. However, in some patients resistance has been associated with a single amino acid substitution in a threonine residue of the Abl kinase domain. In vitro studies examining the effects of imatinib plus cytarabine (Ara-C) using CML cell lines and colony-forming assays of CML patient samples have shown synergistic antiproliferative effects of this combination. Thus several groups decided to investigate this new combination with the hypothesis that cell resistance would be less frequent. The CML French Group performed a phase II trial to determine the safety and tolerability of a combination of imatinib and Ara-C for previously untreated patients with chronic-phase CML. Treatment was administered on 28-day cycles for 12 months. Patients were treated continuously with imatinib orally at a dose of 400 mg daily. Ara-C was given on days 14 to 28 of each cycle at an initial dose of 20 mg/m2/d via subcutaneous injection, hydroxyurea (HU) being stopped at least 7 days before imatinib. Recently, the Dutch group decided to explore a combination of high-dose Ara-C with imatinib in patients in chronic-phase CML. Preliminary results are encouraging. However, a long follow-up is required before concluding that these strategies will overcome cell resistance. Semin Hematol 40(suppl 2):92-97. © 2003 Elsevier Inc. All rights reserved.

Bcr-abl-positive cells secrete angiogenic factors including matrix metalloproteinases and stimulate angiogenesis in vivo in Matrigel implants.

To further elucidate the role of angiogenesis in the pathogenesis of chronic myelogenous leukemia (CML) we evaluated the effects of the bcr-abl translocation on the secretion of the angiogenic factors VEGF, FGF-2, HGF, IL-8 and matrix metalloproteinases (MMPs) as well as on the angiogenic potential in vivo of bcr-abl+ cells. First, we examined murine FL5.12 cells transfected with the bcr-abl constructs p185, p210 and p230 and found that the transfected cells secreted as much as four-fold more VEGF (p185 > p210 >p230) than wild-type (wt) cells, as well as MMP-9 and MMP-2. When Matrigel fragments containing these bcr-abl+ cells were implanted subcutaneously in SCID or Balb-C mice they became significantly more vascularized and hemoglobinized than implants containing normal or wt cells (p185 > p210 > p230). Similarly, we found that myeloblasts expanded from bone marrow (BM) CD34+ cells derived from Philadelphia-positive CML patients secreted up to 10 times more VEGF, FGF-2, HGF and IL-8 compared to myeloblasts derived from normal donors' BM CD34+ cells and that BM mononuclear cells (MNC) isolated from CML patients induced vascularization of Matrigel implants in mice. Moreover, we found that peripheral blood MNC expressed MMP-2 and membrane-type (MT)1-MMP in about 50% of CML patients studied, and MMP-9 in all of them. Furthermore, VEGF stimulated the secretion of MMP-9 in these primary CML cells. We conclude that stimulation of angiogenesis by angiogenic factors, including MMPs, could play an important role in the pathogenesis of CML, suggesting that therapies targeting the newly formed endothelium could be developed for CML.

In vitro effects of STI 571-containing drug combinations on the growth of Philadelphia-positive chronic myelogenous leukemia cells.

Chronic myelogenous leukemia (CML) is characterized by a molecular aberration, a fusion BCR-ABL gene encoding for aberrant tyrosine kinase activity, which is crucial in the pathogenesis of CML. In vitro, inhibition of BCR-ABL protein tyrosine kinase activity by a tyrosine kinase inhibitor, Imatinib mesylate (STI571; formerly CGP57148B), successfully suppressed proliferation/survival of the BCR-ABL positive clones. In clinical studies, hematologic and cytogenetic remissions have been achieved in most patients with chronic phase CML; in accelerated and blastic phases of CML, STI571 appeared less effective. In the current study, the authors tested combinations of STI571 and cytarabine and homoharringtonine (HHT), drugs with documented activity in CML. The single agents and their combinations were studied for in vitro effect on proliferation of BCR-ABL positive cell lines KBM5 and KBM7 by 3(4,5-dimethylthiazol-2yl)-2,5 diphenyl-tetrazolium bromide assay and on primary patient-derived BCR-ABL cells by clonogenic assays. The in vitro additive, synergistic, or antagonistic effects of cytarabine and HHT with STI571 were then investigated by computer-assisted analysis using the CalcuSyn software. STI571 consistently suppressed BCR-ABL positive cell proliferation with a dose-effect correlation. In the model system used, STI571/cytarabine and STI571/HHT combinations were more effective in inhibiting KBM5 and KBM7 cell growth than each drug as single agent. These results were also verified in primary CML-derived clonogenic cells in semisolid cultures. In this experimental system, our studies documented additive or synergistic effects with STI571 plus cytarabine or HHT, supporting the future use of STI571 combinations in clinical trials in patients with Philadelphia chromosome-positive leukemias.