BCR-ABL In Chronic Myeloid Leukemia Research Articles

Sensitivity of imatinib-resistant T315I BCR-ABL CML to a synergistic combination of ponatinib and forskolin treatment.

Tyrosine kinase inhibitors (TKIs) have dramatically improved the life expectancy of patients suffering from chronic myeloid leukemia (CML); however, patients will eventually develop resistance to TKI therapy or adverse side effects due to secondary off-target mechanisms associated with TKIs. CML patients exhibiting TKI resistance are at greater risk of developing an aggressive and drug-insensitive disease. Drug-resistant CML typically arises in response to spontaneous mutations within the drug binding sites of the targeted oncoproteins. To better understand the mechanism of drug resistance in TKI-resistant CML patients, the BCR-ABL transformed cell line KCL22 was grown with increasing concentrations of imatinib for a period of 6 weeks. Subsequently, a drug-resistant derivative of the parental KCL22 cell line harboring the T315I gatekeeper mutation was isolated and investigated for TKI drug sensitivity via multi-agent drug screens. A synergistic combination of ponatinib- and forskolin-reduced cell viability was identified in this clinically relevant imatinib-resistant CML cell line, which also proved efficacious in other CML cell lines. In summary, this study provides new insight into the biological underpinnings of BCR-ABL-driven CML and potential rationale for investigating novel treatment strategies for patients with T315I CML.

Drug repositioning in sarcomas and other rare tumors

Drug repositioning in sarcomas and other rare tumors

Potential role of Notch signalling in CD34+ chronic myeloid leukaemia cells: cross-talk between Notch and BCR-ABL.

Notch signalling is critical for haemopoietic stem cell (HSC) self-renewal and survival. The role of Notch signalling has been reported recently in chronic myeloid leukaemia (CML) – a stem cell disease characterized by BCR-ABL tyrosine kinase activation. Therefore, we studied the relationship between BCR-ABL and Notch signalling and assessed the expression patterns of Notch and its downstream target Hes1 in CD34+ stem and progenitor cells from chronic-phase CML patients and bone marrow (BM) from normal subjects (NBM). We found significant upregulation (p<0.05) of Notch1, Notch2 and Hes1 on the most primitive CD34+Thy+ subset of CML CD34+ cells suggesting that active Notch signalling in CML primitive progenitors. In addition, Notch1 was also expressed in distinct lymphoid and myeloid progenitors within the CD34+ population of primary CML cells. To further delineate the possible role and interactions of Notch with BCR-ABL in CD34+ primary cells from chronic-phase CML, we used P-crkl detection as a surrogate assay of BCR-ABL tyrosine kinase activity. Our data revealed that Imatinib (IM) induced BCR-ABL inhibition results in significant (p<0.05) upregulation of Notch activity, assessed by Hes1 expression. Similarly, inhibition of Notch leads to hyperactivation of BCR-ABL. This antagonistic relationship between Notch and BCR-ABL signalling was confirmed in K562 and ALL-SIL cell lines. In K562, we further validated this antagonistic relationship by inhibiting histone deacetylase (HDAC) - an effector pathway of Hes1, using valproic acid (VPA) - a HDAC inhibitor. Finally, we also confirmed the potential antagonism between Notch and BCR/ABL in In Vivo, using publically available GSE-database, by analysing gene expression profile of paired samples from chronic-phase CML patients pre- and post-Imatinib therapy. Thus, we have demonstrated an antagonistic relationship between Notch and BCR-ABL in CML. A combined inhibition of Notch and BCR-ABL may therefore provide superior clinical response over tyrosine-kinase inhibitor monotherapy by targeting both quiescent leukaemic stem cells and differentiated leukaemic cells and hence must be explored.

Detection and analysis of tumour biomarkers to strengthen the diagnosis of acute and chronic leukaemias

Molecular markers in leukaemia are essential to diagnose, establish prognosis factors and determine the correct treatment of patients; therefore, it is imperative to include molecular biology studies, so that, combined with cytomorphology and immunophenotyping studies, they constitute the differential diagnosis of these neoplasias. It is extremely important to implement a panel of molecular markers that allows us to detect oncogenes derived from chromosomal translocations, genes derived from epigenetic alterations and drug-resistant genes.A panel of molecular markers that included 11 genes derived from chromosomal translocations BCR-ABL major and minor breakpoints, E2A-PBX1, MLL-AF4, TEL-AML1, PML-RARα, AML1-ETO was standardised; cancer testis antigens (CTA) derived from NY-ESO1 and MAGE-A3 epigenetic alterations and multi-drug-resistant genes ABCB1 and ABCG2. 30 patients diagnosed with leukaemia from Mexico's General Hospital (Hospital General de Mexico) were included. They suffered from acute lymphoblastic leukaemia (ALL) and acute myeloblastic leukaemia (AML); bone marrow mononuclear cells were used, from which RNA was extracted for the synthesis of cDNA and RT-PCR for each of the markers. In acute lymphoblastic leukaemia (ALL), BCR-ABL biomarkers expressed under 30% (3/10), E2A-PBX1 10% (1/10), ABC-B1 80% (8/10), and ABC-G2 60% (6/10). Patients with acute myeloblastic leukaemia (AML) expressed 30% PML-RARα (3/10), 40% ABC-B1 (4/10), and 10% ABC-G2 (1/10). Lastly, in patients with chronic myeloid leukaemia (CML), BCR-ABL was over 100% (10/10), ABC-B1 20% (2/10), and ABC-G2 50% (5/10). The presence of transcripts from chimeric genes minor BCR-ABL and E2A-PBX1 in ALL; PML-RARα in AML; and major BCR-ABL in CML, confirms the importance that the panel of molecular markers has in strengthening the diagnosis and prognosis of these conditions.

Anti-cancer fatty-acid derivative induces autophagic cell death through modulation of PKM isoform expression profile mediated by bcr-abl in chronic myeloid leukemia

The fusion gene bcr-abl develops chronic myeloid leukemia (CML), and stimulates PI3K/Akt/mTOR signaling, leading to impaired autophagy. PI3K/Akt/mTOR signaling also plays an important role in cell metabolism. The Warburg effect is a well-recognized hallmark of cancer energy metabolism, and is regulated by the mTOR/c-Myc/hnRNP/PKM signaling cascade. To develop a new strategy for the treatment of CML, we investigated the associations among bcr-abl, the cascade related to cancer energy metabolism, and autophagy induced by a fatty-acid derivative that we had previously reported as being an autophagy inducer. Here we report that a fatty-acid derivative, AIC-47, induced transcriptional repression of the bcr-abl gene and modulated the expression profile of PKM isoforms, resulting in autophagic cell death. We show that c-Myc functioned as a transcriptional activator of bcr-abl, and regulated the hnRNP/PKM cascade. AIC-47, acting through the PPARγ/β-catenin pathway, induced down-regulation of c-Myc, leading to the disruption of the bcr-abl/mTOR/hnRNP signaling pathway, and switching of the expression of PKM2 to PKM1. This switching caused autophagic cell death through an increase in the ROS level. Our findings suggest that AIC-47 induced autophagic cell death through the PPARγ/β-catenin/bcr-abl/mTOR/hnRNP/PKM cascade.

Emerging Role of Tyrosine Kinases as Drugable Targets in Cancer.

Tyrosine kinases (TKs) play a significant role in cancerogenesis and cancer cell function. Initial developments in this field go back to the early 80s, but the success story really started with the selective BCR-ABL inhibitor, imatinib. Owing to the cancer-driving role of BCR-ABL in chronic myeloid leukemia (CML), excellent response rates lead to fast FDA approval in both the first and second treatments of CML patients. Since then, numerous TKs were identified. TK inhibitors have been developed accordingly, and technology to test for ideal drug–target interactions has profoundly improved. By now, medical oncologists and hematologists struggle to have a pool of potential TK inhibitors, where the most efficient one could be picked out to treat a specific cancer patient, which might also help overcome the occurring resistance mechanisms against TK inhibitors. Whether disease eradication can be achieved via single or sequential TK inhibitor treatment(s) needs to be tested in the present and in the future.

Coupled delivery of imatinib mesylate and doxorubicin with nanoscaled polymeric vectors for a sustained downregulation of BCR-ABL in chronic myeloid leukemia.

In this work, we have investigated the potential benefits of combining biodegradable pH sensitive core-shell PCL NPs loaded with IM and enzyme sensitive polyelectrolyte complexes (PECs) loaded with doxorubicin (DOX). Our in vitro studies confirmed the excellent antileukemic activity of dual drug loaded nanoparticles on CML cells. As compared with a drug alone, co-treatment with IM and DOX loaded in nanoparticles allowed a sustained downregulation of BCR-ABL and significant CML stem cell death. This furthermore showed that couple formulation of nanoparticles enhanced the drugs' kinetics and efficacy, combined with the pH sensibility of core-shell PCL NPs loaded with IM and enzyme sensitive polyelectrolyte complexes (PECs) loaded with DOX. Our study demonstrates that dual drug loaded nanoparticles work in a synergistic manner, lowering the dose and confirming that both drugs reach the target cell specifically, maximizing the cytotoxicity while minimizing the chances of cell resistance to any one drug.

Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines.

This protocol describes a highly reproducible antibody-based method that provides protein level and phosphorylation status information from nanogram quantities of protein cell lysate. Nanocapillary isoelectric focusing (cIEF) combines with UV-activated linking chemistry to detect changes in phosphorylation status. As an example application, we describe how to detect changes in response to tyrosine kinase inhibitors (TKIs) in the phosphorylation status of the adaptor protein CrkL, a major substrate of the oncogenic tyrosine kinase BCR-ABL in chronic myeloid leukemia (CML), using highly enriched CML stem cells and mature cell populations in vitro. This protocol provides a 2.5 pg/nl limit of protein detection (<0.2% of a stem cell sample containing <10(4) cells). Additional assays are described for phosphorylated tyrosine 207 (pTyr207)-CrkL and the protein tyrosine phosphatase PTPRC/CD45; these assays were developed using this protocol and applied to CML patient samples. This method is of high throughput, and it can act as a screen for in vitro cancer stem cell response to drugs and novel agents.

LASP1 is a novel BCR-ABL substrate and a phosphorylation-dependent binding partner of CRKL in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is characterized by a genomic translocation generating a permanently active BCR-ABL oncogene with a complex pattern of atypically tyrosine-phosphorylated proteins that drive the malignant phenotype of CML. Recently, the LIM and SH3 domain protein 1 (LASP1) was identified as a component of a six gene signature that is strongly predictive for disease progression and relapse in CML patients. However, the underlying mechanisms why LASP1 expression correlates with dismal outcome remained unresolved. Here, we identified LASP1 as a novel and overexpressed direct substrate of BCR-ABL in CML. We demonstrate that LASP1 is specifically phosphorylated by BCR-ABL at tyrosine-171 in CML patients, which is abolished by tyrosine kinase inhibitor therapy. Further studies revealed that LASP1 phosphorylation results in an association with CRKL - another specific BCR-ABL substrate and bona fide biomarker for BCR-ABL activity. pLASP1-Y171 binds to non-phosphorylated CRKL at its SH2 domain. Accordingly, the BCR-ABL-mediated pathophysiological hyper-phosphorylation of LASP1 in CML disrupts normal regulation of CRKL and LASP1, which likely has implications on downstream BCR-ABL signaling. Collectively, our results suggest that LASP1 phosphorylation might serve as an additional candidate biomarker for assessment of BCR-ABL activity and provide a first step toward a molecular understanding of LASP1 function in CML.

Dual regulation of Myc by Abl

The tyrosine kinase c-Abl (or Abl) and the prolyl-isomerase Pin1 cooperatively activate the transcription factor p73 by enhancing recruitment of the acetyltransferase p300. As the transcription factor c-Myc (or Myc) is a known target of Pin1 and p300, we hypothesized that it might be regulated in a similar manner. Consistent with this hypothesis, overexpression of Pin1 augmented the interaction of Myc with p300 and transcriptional activity. The action of Abl, however, was more complex than predicted. On one hand, Abl indirectly enhanced phosphorylation of Myc on Ser 62 and Thr 58, its association with Pin1 and p300 and its acetylation by p300. These effects of Abl were exerted through phosphorylation of substrate(s) other than Myc itself. On the other hand, Abl interacted with the C-terminal domain of Myc and phosphorylated up to five tyrosine residues in its N-terminus, the principal of which was Y74. Indirect immunofluorescence or immunohistochemical staining suggested that the Y74-phosphorylated form of Myc (Myc-pY74) localized to the cytoplasm and coexisted either with active Abl in a subset of mammary carcinomas or with Bcr-Abl in chronic myeloid leukemia. In all instances, Myc-pY74 constituted a minor fraction of the cellular Myc protein. Thus, our data unravel two potential effects of Abl on Myc: first, Abl signaling can indirectly augment acetylation of Myc by p300, and most likely also its transcriptional activity in the nucleus; second, Abl can directly phosphorylate Myc on tyrosine: the resulting form of Myc appears to be cytoplasmic, and its presence correlates with Abl activation in cancer.

Mechanisms of Resistance to Targeted Therapies in Acute Myeloid Leukemia and Chronic Myeloid Leukemia

Small molecule kinase inhibitors of BCR-ABL in chronic myeloid leukemia (CML) and of FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) in acute myeloid leukemia (AML) have been successful at achieving remissions in these diseases as monotherapy, but these leukemias do not initially respond in a subset of patients (primary resistance) and they progress in an additional group of patients after an initial response (secondary resistance). Resistance to these agents can be divided into mechanisms that allow reactivation kinase activity and those that bypass reliance on oncogenic signaling mediated by the target kinase. Elucidation of clinical resistance mechanisms to targeted therapies for patients can provide important insights into disease pathogenesis and signaling.

Overcoming Gleevec-resistance by blocking oncogene addiction in malignant hematologic cells

In some types of tumors, malignant cells are highly dependent on the constitutive activation of a certain protein encoded by oncogene, despite existence of additional carcinogenic genetic changes. This phenomenon is referred to oncogene addiction. Typical examples include cytoplasmic tyrosine kinase Bcr-Abl in chronic myeloid leukemia (CML), receptor tyrosine kinase KIT in systemic mastocytosis and gastrointestinal stromal tumors (GISTs), and PDGFRα in hypereosinophilic syndrome (HES). In 2001, the approval of Gleevec (STI571, imatinib mesylate, Norvartis) by FDA (Food and Drug Administration, USA) initiated a revolutionary targeted therapy against cancer with small molecule tyrosine kinase inhibitors. Gleevec blocks the signaling pathway of tyrosine kinase by competitively occupying the ATP-binding pocket of Bcr-Abl, KIT and PDGFRα, and therefore kills these oncogene-addicted tumor cells. Patients with CML and HES have gained much better prognosis with the treatment of Gleevec. However, in some patients (particularly in accelerating and blast crisis phases), relapse due to resistance to Gleevec is an emerging problem. Acquired point mutations within the target genes (Bcr-Abl, KIT and PDGFRα) are a major mechanism of resistance to Gleevec in some patients with hematologic malignance. The mutations are believed to block the binding of Gleevec to ATP binding pockets of these tyrosine kinases. In this case, novel tyrosine kinase inhibitor such as nilotinib and dasatinib (also called the second-generation of tyrosine kinase inhibitor) have been shown activity against Gleevec-resistant patients bearing some point mutations but the “gate-keeper” mutations (e.g., T315I Bcr-Abl, T674I PDGFRα). Therefore, development of more novel small molecule tyrosine kinase inhibitors is still needed. This talk covered the advance in the field of overcoming Gleevec resistance in terms of novel compounds and strategeies. Pan J et al reported that EXEL-0862 is effective against Gleevec-resistant D816V KIT and T674I PDGFRα (2007). Recently, in vitro and animal data supported that several novel tyrosine kinase inhibitors including (but not limited) AP24534 (ponatinib) and DCC-2036 have been demonstrated effective against T315I Bcr-Abl. However, the efficacy and safety of these pounds (EXEL-0862, AP24534 and DCC-2036) in patients remains to be defined. An alternative approach for overcoming Gleevec-resistance is to decrease the expression of “addicted” oncogenes, which are driving forces of the tumor cells, to kill the malignant cells. Our group discovered several compounds which are effective against Gleevec-resistant tumor cells regardless of resistance to imatinib. The compounds kill cells harboring gate-keeper mutants of tyrosine kinases by lowering the expression of the oncoproteins (Bcr-Abl, KIT and PDGFRα). Examples include triptolide, pristimerin and SNS-032 (transcription inhibitors), homoharringtonine (a translation inhibitor), and celastrol (a hsp90 inhibitor). In summary, Gleevec-resistance remains a challenge in leukemia. The findings from us and others suggest that several aforementioned compounds are promising agents to overcome Gleevec resistance, and warrant clinical trials.

Cyclin-Dependent Kinase 7/9 Inhibitor SNS-032 Abrogates FIP1-like-1 Platelet-Derived Growth Factor Receptor α and Bcr-Abl Oncogene Addiction in Malignant Hematologic Cells

The "gate-keeper" mutations T674I platelet-derived growth factor receptor α (PDGFRα) in hypereosinophilic syndrome (HES) and T315I Bcr-Abl in chronic myeloid leukemia (CML) are resistant to imatinib and the second-generation small-molecule tyrosine kinase inhibitors (TKI). However, to combat acquired resistance to imatinib, an alternative approach is to decrease the expression of the addicted gene to efficiently kill resistant malignant hematologic cells. The purpose of this study was to evaluate the strategy of shutting down the transcription and expression of FIP1-like-1 (FIP1L1)-PDGFRα and Bcr-Abl with SNS-032, an inhibitor of cyclin-dependent kinase 7 (CDK7) and CDK9 in phase I clinical trials. The effects of SNS-032 on PDGFRα and Bcr-Abl signaling pathways, apoptosis, and cell cycling were analyzed in TKI-resistant cells of HES and CML. The in vivo antitumor activity of SNS-032 was assessed with xenografted BaF3-T674I FIP1L1-PDGFRα and KBM5-T315I Bcr-Abl cells in nude mouse models. SNS-032 inhibited the phosphorylation on Ser5 and Ser2 of RNA polymerase II. SNS-032 decreased both the mRNA and protein levels of FIP1L1-PDGFRα and Bcr-Abl and inhibited the proliferation of malignant cells expressing FIP1L1-PDGFRα or Bcr-Abl. It also decreased the phosphorylation of downstream molecules. It induced apoptosis by triggering both the mitochondrial pathway and the death receptor pathway. This CDK7/9 inhibitor potently inhibits FIP1L1-PDGFRα-positive HES cells and Bcr-Abl-positive CML cells regardless of their sensitivity to imatinib. SNS-032 may have potential in treating hematologic malignancy by abrogating oncogene addiction.

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

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

NADPH oxidases regulate cell growth and migration in myeloid cells transformed by oncogenic tyrosine kinases

Transformation by tyrosine kinase oncogenes in myeloid malignancies, including BCR-ABL in chronic myeloid leukemia, FLT3ITD in acute myeloid leukemia (AML) or JAK2V617F in myeloproliferative neoplasms (MPN), is associated with increased growth and cytoskeletal abnormalities. Using targeted approaches against components of the superoxide-producing NADPH-oxidases, including NOX2, NOX4 and the common p22phox subunit of NOX1-4, myeloid cells were found to display reduced cell growth and spontaneous migration. Consistent with a role of NOX as regulators of membrane proximal signaling events in non-phagocytic cells, NOX2 and NOX4 were not involved in the excess production of intracellular reactive oxygen species and did not significantly increase oxygen consumption. All NOX family members are controlled in part through levels of the rate-limiting substrate NADPH, which was found to be significantly elevated in tyrosine kinase oncogene transformed cells. Also, reduced phosphorylation of the actin filament crosslinking protein MARCKS in response to suppression of p22phox hints at a novel effector of NOX signaling. MARCKS was also found to be required for increased migration. Overall, these data suggest a model whereby NOX links metabolic NADPH production to cellular events that directly contribute to transformation.

Can Antitumor Immunity Help to Explain “Oncogene Addiction”?

"Oncogene addiction" refers to the process of tumor cell death that can occur after inactivation of a single oncogene. In this issue of Cancer Cell , Rakhra et al. argue that complete tumor clearance after molecular targeted therapies requires a functioning immune system, pointing the way toward radically new combination therapies.

Minimal Residual Disease following Allogeneic Hematopoietic Stem Cell Transplantation

Minimal residual disease (MRD), both before and after transplantation, is a clinically important yet relatively poorly defined aspect of allogeneic hematopoietic stem cell transplantation (alloHSCT). The clinical relevance of MRD in the context of alloHSCT has been demonstrated by its association with the development of clinical relapse. However, with the possible exception of chronic myeloid leukemia (CML), the specific techniques, timing, frequency, and clinical utility, relative to improvement in patient outcomes, for monitoring MRD in the setting of alloHSCT has yet to be clearly defined. A concise overview of monitoring techniques for detecting MRD, as well as treatment strategies and biological and clinical research initiatives for MRD suggested by the National Cancer Institute First International Workshop on the Biology, Prevention, and Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation, is covered in this article.

BCR–ABL-mediated upregulation of PRAME is responsible for knocking down TRAIL in CML patients

Tumor necrosis factor-related apoptosis-inducing ligand-TNFSF10 (TRAIL), a member of the TNF-α family and a death receptor ligand, was shown to selectively kill tumor cells. Not surprisingly, TRAIL is downregulated in a variety of tumor cells, including BCR-ABL-positive leukemia. Although we know much about the molecular basis of TRAIL-mediated cell killing, the mechanism responsible for TRAIL inhibition in tumors remains elusive because (a) TRAIL can be regulated by retinoic acid (RA); (b) the tumor antigen preferentially expressed antigen of melanoma (PRAME) was shown to inhibit transcription of RA receptor target genes through the polycomb protein, enhancer of zeste homolog 2 (EZH2); and (c) we have found that TRAIL is inversely correlated with BCR-ABL in chronic myeloid leukemia (CML) patients. Thus, we decided to investigate the association of PRAME, EZH2 and TRAIL in BCR-ABL-positive leukemia. Here, we demonstrate that PRAME, but not EZH2, is upregulated in BCR-ABL cells and is associated with the progression of disease in CML patients. There is a positive correlation between PRAME and BCR-ABL and an inverse correlation between PRAME and TRAIL in these patients. Importantly, knocking down PRAME or EZH2 by RNA interference in a BCR-ABL-positive cell line restores TRAIL expression. Moreover, there is an enrichment of EZH2 binding on the promoter region of TRAIL in a CML cell line. This binding is lost after PRAME knockdown. Finally, knocking down PRAME or EZH2, and consequently induction of TRAIL expression, enhances Imatinib sensibility. Taken together, our data reveal a novel regulatory mechanism responsible for lowering TRAIL expression and provide the basis of alternative targets for combined therapeutic strategies for CML.

Dasatinib Once-Daily Dose Regimen Provides Robust Anti-Leukemic Activity While Avoiding Suppression of T Cell Activation.

Abstract 2198Poster Board II-175Dasatinib (SPRYCEL®), a small molecule tyrosine kinase inhibitor is 325-fold more potent against BCR-ABL than imatinib. Targeting BCR-ABL in chronic myeloid leukemia (CML), dasatinib offers the most favorable benefit-risk ratio with the dose regimen of 100-mg once daily (in comparison with 3 other treatment arms: 50 mg BID, 140 mg QD and 70 mg BID. Duration of cytogenetic response and progression-free survival were similar across all 4 arms, but there was significantly less frequent grade 3-4 neutropenia, thrombocytopenia, anemia and pleural effusion in the 100-mg QD arm compared to the other 3 arms combined (Shah et al, J Clin Oncol 26:3204, 2008). The undiminished efficacy of once daily dosing occurs despite the fact that orally administered dasatinib has a pronounced peak-to-trough plasma pharmacokinetics profile and a relatively short half-life (∼3-5 h), which allows for complete recovery of BCR-ABL kinase activity within 8-12 hrs after a daily dose. The clinical efficacy of once daily dasatinib supports the notion that continuous target (BCR-ABL) inhibition is not required for anti-leukemic activity although the truncated exposure may help to ameliorate other side-effects of “on” or “off” targets inhibition. In addition to BCR-ABL, dasatinib potently inhibits SRC-family kinases (SFKs) with an IC50 in the low single digit nM range; SFKs play key roles in T cell activation. Based on continuous exposure studies, it had been suggested that dasatinib may act as an immunomodulator in vivo. We investigated the effects of variation in exposure duration in vivo and in vitro on the anti-leukemic and T cell activation inhibition activities of dasatinib in preclinical models. Methods.Anti-leukemic activity was determined in vitro in K562 and KU812 CML cell lines, and in vivo as xenografts in K562. BCR-ABL kinase activity was monitored with a phospho-specific CRKL antibody. Human T cells were isolated from PBMC by rosetting with sheep red blood cells and stimulated with anti-CD3/CD28 antibodies for 48 h. Cytokines production were measured by ELISA. T cell proliferation was determined at 3 days by 3H-thymidine incorporation. Immunocompetency of dasatinib treated mice were determined using the mouse cardiac allograft model and the in vivo MLR T cell proliferation model. Results.Transient (1-6 h) dasatinib exposure of CML cells that caused >80% inhibition of phospho-CRKL is highly cytotoxic. Degree of cytotoxicity directly correlates with the magnitude of BCR-ABL kinase inhibition. In vivo single dose of 30 mg/kg dasatinib administered IV was highly cytotoxic to K562 xenografts as determined by in vivo-in vitro colony formation assay. Intermittent IV dosing regimens of dasatinib (Q4D or Q7D) were effective against K562 xenografts. Dosing regimen in mice (5 mg/kg, PO) that closely mimic the pharmacokinetics of 100 mg oral dose in human was equally efficacious as administering the same dose in 2 split doses (BID, 2.5 mg/kg, PO). In terms of effects on T cell activation, a linear relationship was observed between serum concentration and in vitro T cell proliferation IC50 values. Modeling the human PK profile, delayed dasatinib treatment of T cells after T cell stimulation in vitro led to a time dependent decrease in potency as measured by both IC50 and Emax values. Comparison of serum adjusted IC50 values from these studies to the human PK profile suggests that dasatinib at the approved 100-mg once-daily dose would permit T cell activation on a daily basis. A similar pattern was observed in preclinical in vivo models. Dasatinib was found to be completely protective in a mouse model of cardiac allograft rejection at a dose of 25 mg BID, whereas a dose of 15 mg BID was not protective. In the MLR model, dasatinib inhibits T cell proliferation at 50 mg/kg but at the clinically relevant dose of 5 mg/kg was completely devoid of T cell inhibitory effects. Taken together, these results suggest that dasatinib may be able to provide anti-leukemic activity while avoiding suppression of T cell activation at clinically relevant doses. Disclosures:Schieven:Bristol-Myers Squibb Co: Employment. Zhang:Bristol-Myers Squibb Co: Employment. Pitt:Bristol-Myers Squibb Co: Employment. McGlinchey:Bristol-Myers Squibb Co: Employment. Menard:Bristol-Myers Squibb Co: Employment. Smykla:Bristol-Myers Squibb Co: Employment. Susulic:Bristol-Myers Squibb Co: Employment. Wen:Bristol-Myers Squibb Co: Employment. Wiebesiek:Bristol-Myers Squibb Co: Employment. Townsend:Bristol-Myers Squibb Co: Employment. Lee:Bristol-Myers Squibb Co: Employment.

Bosutinib is a Substrate of the ABC Efflux Transporter P-Glycoprotein.

Abstract 3763Poster Board III-699Bosutinib (Bos) (Wyeth Pharmaceuticals) is a dual Src-Abl tyrosine kinase inhibitor developed to inhibit the activity of BCR-ABL in chronic myeloid leukemia. P-glycoprotein (P-gp) is an ATP-binding cassette (ABC) transporter responsible of Imatinib (Im) efflux, and its increased expression can be related to Im resistance. The aim of the study was to define if P-gp is also responsible for the cellular efflux of Bos, and if P-gp altered expression can be related to Bos resistance. Four different K562 Ph+ cell lines were used. 1) K562S, Im sensitive; 2) K562DOX, cells resistant to Doxorubicin that overexpress P-gp (kind gift of JP Marie, Université Pierre et Marie Curie, Paris); 3) K562DOX siP-gp, K562DOX cells carrying a stable silencing of P-gp (kind gift of E Gunsilius, Innsbruck Medical University, Austria); 4) K562DOX H1, K562DOX cells carrying a control vector (kind gift of E Gunsilius). Real Time PCR confirmed that K562DOX and K562DOX H1 express higher levels of P-gp than K562DOX siP-gp (approximately 10 fold) and K562S (approximately 10000 fold). Western blot α-P-gp showed similar results. We then assessed Bos IC50 on the cell lines with a proliferation assay. K562DOX (IC50=175.3nM) and K562DOX H1 (IC50=102 nM) are resistant to Bos if compared to K562DOX siP-gp (IC50=9.1nM) and K562S (IC50=8.7nM). This data confirm that the P-gp overexpression is related to Bos resistance. Using C-14 radiolabeled Bosutinib (C-14 Bos) (Wyeth Pharmaceuticals) we set up an Intracellular Uptake and Retention (IUR) assay on K562S, K562DOX and K562DOX siP-gp cells. Cells were pre-treated for an hour with 0-60μM of Verapamil (Ver), a P-gp inhibitor, and then treated for two hours with 1μM C-14 Bos. In presence of Ver, K562DOX showed a significant increased amount of intracellular C-14 Bos ([] C-14 Bos Ver60μM/ [] C-14 Bos Ver0μM=5.8) that was not observed in K562S or K562DOX siP-gp. These data confirm that the intracellular concentration of Bos is strictly related to the expression levels and to the activity of P-gp. To test the biological effect of P-gp inhibition, we evaluated Bos IC50 by proliferation and apoptosis induction in K562DOX and K562S cells pre-treated with increasing concentrations of Ver. K562S did not show a significant decrease of Bos IC50 even at the highest concentration of Ver (22.5μM). In K562DOX the same concentration of Ver led to a 20 fold decrease of Bos IC50 (9nM), down to a level comparable to Bos IC50 in K562S cells (8.7nM). Thus, the inhibition of P-gp seems to restore the efficacy of Bos on proliferation inhibition and apoptosis induction in BCR-ABL+ cells. Finally, we evaluated the effect of P-gp inhibition on the phosphorylation levels of BCR-ABL with an α-phosphoTyrosine western blot. K562DOX and K562S cells were treated with Bos concentrations ranging from 0 to 80nM, and in absence or presence of Ver (5μM). Phosphorylation levels of BCR-ABL in K562S treated with Ver didn't show any difference when compared to untreated samples. In K562DOX, in absence of Ver, BCR-ABL was phosphorylated even in presence of high Bos concentration; the treatment with Ver restored the sensitivity to Bos, thus leading to a phosphorylation pattern similar to the one of K562S cells. This is compatible with increasing intracellular concentration of Bos after P-gp inhibition which results in increased molecular effect of Bos on BCR-ABL tyrosine phosphorylation. All together, these data confirm the relevance of expression levels and activity of P-gp for the intracellular concentration of Bos. The intracellular concentration of Bos is strictly related to its molecular activity on BCR-ABL and to its biological effects on Ph+ cells. Disclosures:Boschelli:Wyeth Pharmaceuticals: Employment.