Targeting BCR-ABL Research Articles

Expression of CAMKIIγ, HSP70 and HSP90 Genes Are Differentially Associated with the Mutational Status of Tirosine Kinase Domain in Chronic Myeloid Leukemia Resistant Patients

Abstract 4877Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder characterized by the reciprocal chromosomal translocation between chromosome 9 and 22, t(9;22)(q34;q11), known as Philadelphia chromosome. At molecular level, this cytogenetic aberration associates with a chimeric BCR-ABL fusion gene responsible for the pathogenesis of the disease. Targeting BCR-ABL with tyrosine kinase inhibitors (TKIs) has led to a rapid clinical response in most CML cases; however, harboring of point mutations within the kinase domain (KD) of BCR-ABL is the most common mechanism responsible for acquired resistance to therapy.The objective of this work was to evaluate the expression profiles of 4 different genes involved in proliferation, differentiation or cell survival, in order to study their association with acquired missense mutations within the KD of BCR-ABL.Due to its clear involvement in cell proliferation and apotosis the following genes were analyzed. CAMKIIγ (Ca2+ /calmodulin dependent protein kinase IIγ) is a critical regulator of multiple signalling networks regulating the proliferation in myeloid leukaemia. HSP70 (Heat Shock protein 70) is a chaperone protein that protects cells from apoptotic and necrotic stimuli. HSP90 (Heat Shock protein 90) is a chaperone that facilitates folding of client proteins such as BCR-ABL. Ki-67 is a nuclear antigen associated with cellular proliferation and ribosomal RNA transcription.Total RNA was extracted from 50 TKI-resistant CML-patient’s PBMC samples; by quantitative real time-PCR (QRT-PCR) (SybrGreen method, melting analysis and β2-microglobulin as an endogenous control reference gene) we measured the gene expression of CaMKIIγ, Ki67, HSP70, HSP90 and BCR-ABL. All patients were treated with TKIs (26 imatinib, 13 nilotinib, 11 dasatinib) and showed primary lack or loss of haematological and/or cytogenetic response according to the ELN (European Leukaemia Net) criteria.To characterize TKI-resistant mutations within the KD of the chimeric BCR-ABL gene, ABL exons 4, 5, 6 and 7 were subjected to automated DNA sequencing following a nested- BCR-ABL-specific PCR. Mutations were observed in 27/50 cases at 15 different residues: 1 L248V, 1 G250E, 1 Y253H, 5 E255K/V, 1 E279K, 1 V289F, 4 T315I, 2 F317L, 1 L348M, 3 M351T, 1 E355G, 1 N358S, 2 F359V/C, 2 L387M and 1 L389V. Remaining 23 CML cases did not show mutations (detection limit 10–20%).Our results showed a significant increase in the expression of CaMKIIγ and HSP70 and decrease of HSP90 in mutated patients (MT) with respect to cases without mutations (WT) (p<0.01). On the contrary, transcript levels of Ki67 and BCR-ABL did not show significant differences between MT and WT, likely due to the resistant status of both groups.Taking into account these results we design a score (TKI-MT) to estimate the likelihoods for a patient to harbor TKI-resistance mutations using the transcript normalised expression of CaMKIIγ, HSP70 and HSP90 as follows: [Log10(CaMKIIγ) + Log10(HSP70) – Log10(HSP90)]. TKI-MT scores from 27 and 23 patients from the MT and WT groups respectively were analysed by use of ROC curves in order to find an optimal cut-off value to classify new unstudied cases. We found that patients with TKI-MT scores over a cut-off value of −0.79 showed 4.8 times more probabilities to present TKI-resistance mutations than those below −0.79 (OR 4.8, CI95% 1.3–17.6, P<0.02).We concluded that the expression of CaMKIIγ, HSP70 and HSP90 allowed prediction of mutations in the ABL tyrosine kinase domain with 82% of specificity in CML patients treated with TKIs and associated with lack or loss of response. Disclosures:No relevant conflicts of interest to declare.

An In-house Method for Molecular Monitoring of BCR-ABL

Objective: At present, there are a limited number of facilities in Turkey that can provide reliable real-time quantitative(RQ)-PCR BCR-ABL results. The present study aimed to test a cost-effective, in-house method of BCR-ABL quantification,including verification of the method by RQ-PCR validation tests.Material and Methods: BCR-ABL and ABL target sequences were cloned into pJET1.2 vectors, from whichcalibrators were prepared and used as templates in RQ-PCR reactions to generate standard curves. Dilutions of K562cells (representing an in vitro simulation of BCR-ABL transcript reduction) were analyzed.Results: Standard curves were generated from calibrators. These curves were then used to calculate the BCR-ABL andABL copy numbers; in which linear BCR-ABL results were obtained. Repetitive experiments showed that our methodologywas able to detect 1 BCR-ABL positive cell from amnong 1x105 cells.Conclusion: The method described herein is suitable for implementation with any RQ-PCR instrument and/or kit forquantify BCR-ABL transcripts.Conflict of interest:None declared.

Graft-versus-Leukemia (GVL) against Mouse Blast-Crisis Chronic Myelogenous Leukemia (BC-CML) and Chronic-Phase Chronic Myelogenous Leukemia (CP-CML): Shared Mechanisms of T Cell Killing, but Programmed Death Ligands Render CP-CML and Not BC-CML GVL Resistant

Graft-versus-leukemia (GVL) against chronic-phase chronic myelogenous leukemia (CP-CML) is potent, but it is less efficacious against acute leukemias and blast-crisis chronic myelogenous leukemia (BC-CML). The mechanisms underlying GVL resistance are unknown. Previously, we found that alloreactive T cell targeting of GVL-sensitive bcr-abl-induced mouse CP-CML (mCP-CML) required TCR-MHC interactions and that multiple and redundant killing mechanisms were in play. To better understand why BC-CML is resistant to GVL, we performed a comprehensive analysis of GVL against mouse BC-CML (mBC-CML) induced by the retroviral transfer of the bcr-abl and NUP98/HOXA9 fusion cDNAs. Like human BC-CML, mBC-CML was GVL resistant, and this was not due to accelerated kinetics or a greater leukemia burden. To study T cell recognition and killing mechanisms, we generated a panel of gene-deficient leukemias by transducing bone marrow from gene-deficient mice. T cell target recognition absolutely required that mBC-CML cells express MHC molecules. GVL against both mCP-CML and mBC-CML required leukemia expression of ICAM-1. We hypothesized that mBC-CML would be resistant to some of the killing mechanisms sufficient to eliminate mCP-CML, but we found instead that the same mechanisms were effective against both types of leukemia, because GVL was similar against wild-type or mBC-CML genetically lacking Fas, TRAIL-R, Fas/TRAIL-R, or TNFR1/R2 or when donor T cells were perforin(-/-). However, mCP-CML, but not mBC-CML, relied on expression of programmed death-1 ligands 1 and 2 (PD-L1/L2) to resist T cell killing, because only GVL against mCP-CML was augmented when leukemias lacked PD-L1/L2. Thus, mBC-CML cells have cell-intrinsic mechanisms, distinct from mCP-CML cells, which protect them from T cell killing.

The ABL Switch Control Inhibitor DCC-2036 Is Active against the Chronic Myeloid Leukemia Mutant BCR-ABLT315I and Exhibits a Narrow Resistance Profile

Acquired point mutations within the BCR-ABL kinase domain represent a common mechanism of resistance to ABL inhibitor therapy in patients with chronic myeloid leukemia (CML). The BCR-ABL(T315I) mutant is highly resistant to imatinib, nilotinib, and dasatinib, and is frequently detected in relapsed patients. This critical gap in resistance coverage drove development of DCC-2036, an ABL inhibitor that binds the switch control pocket involved in conformational regulation of the kinase domain. We evaluated the efficacy of DCC-2036 against BCR-ABL(T315I) and other mutants in cellular and biochemical assays and conducted cell-based mutagenesis screens. DCC-2036 inhibited autophosphorylation of ABL and ABL(T315I) enzymes, and this activity was consistent with selective efficacy against Ba/F3 cells expressing BCR-ABL (IC(50): 19 nmol/L), BCR-ABL(T315I) (IC(50): 63 nmol/L), and most kinase domain mutants. Ex vivo exposure of CML cells from patients harboring BCR-ABL or BCR-ABL(T315I) to DCC-2036 revealed marked inhibition of colony formation and reduced phosphorylation of the direct BCR-ABL target CrkL. Cell-based mutagenesis screens identified a resistance profile for DCC-2036 centered around select P-loop mutations (G250E, Q252H, Y253H, E255K/V), although a concentration of 750 nmol/L DCC-2036 suppressed the emergence of all resistant clones. A decreased concentration of DCC-2036 (160 nmol/L) in dual combination with either nilotinib or dasatinib achieved the same zero outgrowth result. Further screens for resistance due to BCR-ABL compound mutations (two mutations in the same clone) identified BCR-ABL(E255V / T315I) as the most resistant mutant. Taken together, these findings support continued evaluation of DCC-2036 as an important new agent for treatment-refractory CML.

Predictability of the response to tyrosine kinase inhibitors via in vitro analysis of Bcr-Abl phosphorylation

It would be of great value to predict the efficacy of tyrosine kinase inhibitors (TKIs) in the treatment of individual CML patients. We propose an immunoblot system for detecting the phosphorylation of Crkl, a major target of Bcr-Abl, in blood samples after in vitro incubation with TKIs. When the remaining phosphorylated Crkl after treatment with imatinib was evaluated as the “residual index (RI)”, high values were found in accordance with imatinib resistance. Moreover, RI reflected the outcome of imatinib- as well as second generation TKIs with a high sensitivity and specificity. Therefore, this system should be useful in the selection of TKIs.

Growth of chronic myeloid leukemia cells is inhibited by infection with Ad-SH2-HA adenovirus that disrupts Grb2-Bcr-Abl complexes

The persistence of Bcr-Abl-positive cells in patients on imatinib therapy indicates that inhibition of the Bcr-Abl kinase activity alone might not be sufficient to eradicate the leukemia cells. Many downstream effectors of Bcr-Abl have been described, including activation of both the Grb2-SoS-Ras-MAPK and Grb2-Gab2-PI3K-Akt pathways. The Bcr-Abl-Grb2 interaction, which is mediated by the direct interaction of the Grb2 SH2 domain with the phospho-Bcr-Abl Y177, is required for activation of these signaling pathways. Therefore, disrupting their interaction represents a potential therapeutic strategy for inhibiting the oncogenic downstream signals of Bcr-Abl. Adenovirus Ad-SH2-HA expressing the Grb2 SH2 domain was constructed and applied in this study. As expected, Ad-SH2-HA efficiently infected CML cells and functioned by binding to the phospho-Bcr-Abl Y177 site, competitively disrupting the Grb2 SH2-phospho-Bcr-Abl Y177 complex. They induced potent anti-proliferation and apoptosis-inducing effects in CML cell lines. Moreover, the Ras, MAPK and Akt activities were significantly reduced in the Ad-SH2-HA treated cells. These were not observed with the point-mutated control adenovirus Ad-Sm-HA with abolished phospho-Bcr-Abl Y177 binding sites. These data indicate that, in addition to the direct targeting of Bcr-Abl, selective inhibition of its downstream signaling pathways may be a therapeutic option for CML, and the Ad-SH2-HA-mediated killing strategy could be explored as a promising anti-leukemia agent in CML.

Molecular Mechanism Regulating Foxo In Leukemia Initiating Cells of Chronic Myeloid Leukemia.

AbstractAbstract 3391Chronic myeloid leukemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL, a constitutively active tyrosine kinase. Although the development of imatinib, a small molecule inhibitor of ABL, represents a breakthrough in the treatment of CML, major part of patients treated in chronic phase CML are not off therapy due to resistance or intolerance. Recent studies have suggested that imatinib is a potent inhibitor against differentiated leukemia cells, but does not deplete leukemia-initiating cells (LICs) responsible for recurrence of CML. To date, therapeutics that can eradicate CML LICs, however, have remained under investigation. To overcome these clinical problems, here we studied the molecular mechanisms regulating maintenance of imatinib-resistant CML LICs by forkhead transcription factor Foxo3a.We first generated a mouse CML model by using retroviral induction of BCR-ABL-ires-GFP gene into mouse immature hematopoietic cells, and the cells were subsequently transplanted into irradiated recipient mice. These experiments showed that CML LICs were highly enriched in c-Kit+Lin−Sca-1+ (KLS+) population in BCR-ABL+ CML cells. Serial transplantation experiments for CML LICs originated from Foxo3a-deficient mice and littermate wild-type mice indicated that Foxo3a-deficiency reduced lethality of recipient mice at third transplantation. Although recipients that transplanted with wild-type LICs developed CML and acute lymphocytic leukemia (ALL) at third transplantation, we did not observe development of ALL or CML in recipients of Foxo3a deficient LICs after 45 days post-third transplantation, suggesting that the Foxo3a deficient LICs lose their potential to generate malignancies. In addition, a combination of Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo, indicating that Foxo3a plays an essential role for the maintenance of imatinib-resistant CML LICs (Naka et al., Nature 463, 676–680, 2010).Interestingly, when we examined sub-cellular localization of Foxo3a transcription factor in the CML LICs, we found two CML LIC populations; one population was the cells with nuclear localization of Foxo3a (Foxo3a transcription factor is active) and the other population was the cells with cytoplasmic localization of Foxo3a (Foxo3a is inactive). To understand the molecular mechanisms regulating Foxo3a in CML LICs, we next evaluated the activity of upstream BCR-ABL, PI3K, PDK1, and Akt signaling pathway by fluorescence immunohistochemistry. BCR-ABL activity that was determined by phosphorylation levels of CrkL, a down-stream target of BCR-ABL, was detected in almost all of the CML LICs. However, unexpectedly, phosphorylation levels of Akt in the CML LICs with nuclear localization of Foxo3a appeared to be lower than that in the CML LICs with cytoplasmic localization of Foxo3a, despite it is widely believed that BCR-ABL induces activation of Akt signal. Consistent with Akt phosphorylation status, we detected low levels phosphorylation of PDK1 and PI3K p85, upstream regulators for Akt, in the CML LICs with nuclear localization of Foxo3a. Interestingly, expression levels of the cell proliferation antigen Ki67 were lower in the CML LICs with nuclear Foxo3a than that in the CML LICs with cytoplasmic Foxo3a. These results suggest that Foxo3a responsible for maintenance of imatinib-resistant CML LICs may be regulated by molecular mechanisms that are involved in dormancy in CML LICs. Disclosures:No relevant conflicts of interest to declare.

Combined Targeting of BCR-ABL and JAK2 with ABL and JAK2 Inhibitors Is Effective Against CML Patients' Leukemic Stem/Progenitor Cells.

AbstractAbstract 3404Imatinib mesylate (IM) is a tyrosine kinase inhibitor (TKI) that induces clinical responses in most chronic myeloid leukemia (CML) patients. Nevertheless, early relapses and later emergence of IM-resistant disease pose serious concerns for many. The inadequacies of IM therapy are due, at least in part, to the unique properties of CML stem/progenitor cells that make them generally less responsive to IM and, indeed, other TKIs, and also confer on them a genetic instability that leads to a high rate of formation of BCR-ABL mutants. Improved treatment approaches to prevent the development of resistant subclones by targeting other key molecular elements active in CML stem/progenitor cells are thus clearly needed. One candidate is a complex that forms in CML stem/progenitor cells between the oncoproteins encoded by AHI-1 (Abelson helper integration site 1), BCR-ABL and the JAK2 kinase. This complex contributes to the transforming activity of BCR-ABL both in vitro and in vivo and also plays a role in the IM response/resistance of primary CML stem/progenitor cells. We now describe the results of experiments designed to test the ability of ABL and JAK2 inhibitors to block the activity of this protein complex in CML cells. K562 cells engineered to stably overexpress AHI-1 showed a significantly reduced sensitivity to both IM (at 1 and 5 μM) and TG101209, a JAK2 inhibitor, (at 0.5 and 1 μM), as determined by assays for cell viability, apopotosis, and colony-forming activity. K562 cells engineered to suppression AHI-1 showed an opposite effect, with a heightened sensitivity to IM at concentrations as low as 1 μM. In addition, IM together with TG101209 was more effective at killing AHI-1-overexpressing K562 cells, IM-resistant K562 cells and IM-resistant T315I-mutant cells than either treatment alone. Western blot and co-IP experiments demonstrated a significant reduction of p-BCR-ABL, p-JAK2 and p-STAT5 in cells treated with IM plus TG101209 compared to cells treated with IM or TG101209 alone. Importantly, treatment with 5 μM IM, 150 nM dasatinib (DA) or 5 μM nilotinib (NL) in combination with 100 nM TG101209 caused a significantly greater reduction in the viability of primary CD34+CD38− and CD34+CD38+ CML cells when these responses were compared to any of the TKIs or TG101209 alone (~2-4 fold, n=3). Apoptotic cells at 72 hours were also significantly increased for all drug combinations compared to single agent treatments (40%-52% for the combinations vs 15%-18% for the single agents). CFSE tracking analysis of cell division in these cells further demonstrated additive anti-proliferative activity from the TKI plus TG101219 combinations, although some rare undivided cells were not eliminated. Nevertheless, exposure of CD34+ CML cells from IM-nonresponders (n=4) to TG101209 plus IM or DA did cause a greater inhibition (81% and 85%) of patients' colony-forming cells as compared to the same cells treated with the combination of IM plus DA only, or IM or DA only (60%, 41% and 50% inhibition, p<0.05). Long-term culture-initiating cell assays were undertaken to compare the effect of these combination treatments versus the effects of TKIs or TG101209 alone on very primitive CML cells. The results again showed a more significant reduction of these cells treated with the combination (n=3). Intracellular staining revealed a greater reduction in the levels of p-CrKL and p-STAT5 in CD34+ CML cells treated for 24 hours with the combination of TKIs plus TG101219 as compared to single TKI-treated cells (~44% vs 65% for p-CrKL and 36% vs 57% for p-STAT5, n=3). Strikingly, the combination treatment produced an even greater inhibition of both p-CrKL and p-STAT5 after 72 hours while p-CrKL was almost fully reactivated with TKIs alone (~29% vs 89% for p-CrKL and 23% vs 50% for p-STAT5). These results point to the possibility of achieving improved therapeutic outcomes in CML patients by simultaneously targeting both BCR-ABL and JAK2 activities in the critical TKI-insensitive CML stem/progenitor reservoir. Disclosures:No relevant conflicts of interest to declare.

Tyrosine Kinase Inhibitor Imatinib Mesylate Induces Generalized Skin Hypopigmentation In Chinese Patients with Chronic Myelogenous Leukemia.

AbstractAbstract 4475Imatinib mesylate is a selective tyrosine kinase inhibitor targeting BCR-ABL fusion protein. In the therapy of either interferon refractory or newly diagnosed chronic myeloid leukemia (CML), imatinib significantly improved the prognosis of the disease, albeit with typically mild to moderate adverse effects. In recent years, increasing attention has been focused on the skin hypopigmentation developed after the initiation of imatinib therapy, especially in Asian countries such as India, with the incidence between 33% and 85%, though sparsely reported in western countries. The inhibition of c-kit by imatinib may be a possible explanation, but the exact mechanism for this hypopigmentation caused by imatinib and its potential risk in patients is poorly understood. Here we report our clinical observation of this side effect of imatinib therapy among Chinese CML patients in our institution, as well as a brief in vitro study aiming to investigate the mechanism of the hypopigmentory effect and evaluate its potential risk. Among 116 patients who took imatinib for CML therapy, 90 of them (77.6%) had reported to develop generalized hypopigmentation or whitening in the skin. This side effect usually developed within 3 months after initiation of imatinib, persistent during the therapy, and reversible upon imatinib discontinuation, according to our interviews and observation. More interestingly, in three of these CML patients who originally had vitiligo, the cosmetic appearance seemed to be improved during imatinib therapy due to the whitening effect on normal pigmented skin areas. To further confirm and understand this side effect, we examined the impact of imatinib on cultured normal human melanocytes derived from the foreskins of healthy Chinese adults. Melanocytes were treat with various concentrations of imatinib (1~16 μ M) for different periods of time (1~7 days). Cell proliferation and apoptosis were detected by cell counting kit-8 and flow cytometric assay, respectively. The melanogenic activity of the melanocytes was detected by melanin content assay and tyrosinase activity assay. Moreover, the role of stem cell factor (SCF) and its receptor c-kit in imatinib induced hypopigmentation were investigated. We found that imatinib not only concentration-dependently inhibited melanocyte proliferation in vitro (IC50=6.23 μ M), but also resulted in massive melanocyte apoptosis at concentrations higher than 8 μ M (P<0.01). Even at low concentrations (1-8 μ M), imatinib was able to cause decreases in total melanin content (P<0.05) and tyrosinase activity (P<0.05) which were responsible for skin pigmentation. In addition, imatinib (1-8 μ M) completely abrogated SCF (5 ng/ml) induced melanocyte proliferation (P<0.01) and melanogenesis (P<0.01), through the inhibition of the phosphorylation of c-kit receptor (Tyr 703 and Tyr 719) and the downstream signal transduction, which is pivotal for melanocyte proliferation and melanin synthesis after ultraviolet exposure. These results reveal that imatinib may cause destruction or dysfunction of melanocytes in patients through the inactivation of SCF/c-kit signaling, leading to inability of the skin to produce melanin and perform protection against ultraviolet radiation. Therefore, CML patients who take imatinib are necessarily to be warned for this side effect, and diminish the intensive exposure to sunlight. Although an ethnic or genetic basis for the prevalence difference can not be excluded yet, we tend to believe that the hypopigmentation effect of imatinib was probably underestimated in caucasian patients due to their light skin color. Since the melanocytes used in this study were all isolated from the skin samples of Chinese volunteers, further study may be done to determine whether the similar results can be achieved in primary melanocytes from other ethnic group. Disclosures:No relevant conflicts of interest to declare.

GDC-0449, the Small Molecule Inhibitor of Hedgehog-Gli Pathway for the Treatment of BCR-ABL Positive Leukemia Cells and In Combination with Dasatinib

AbstractAbstract 2136Imatinib has shown clinical efficacy against Philadelphia chromosome (Ph) positive leukemia cells and it is now the standard care for initial therapy. However, recent studies reported imatinib are not effective in quiescent primitive chronic myeloid leukemia (CML) stem cells. Moreover, many Ph-positive leukemia patients develop resistance or fail to respond to imatinib by mutation in the ABL kinase domain in clinically. These results indicated that alternative combination therapy such as BCR-ABL targeting tyrosine kinase inhibitors (TKIs) and nontoxic agents are required to cure the Ph positive leukemia patients. Hedgehog (Hh)- Glioma-associated oncogene homolog (Gli) signaling regulates self-renewal of stem cells and implicates in a large number of human cancers. One of the Hh inhibitor, GDC-0449 is a potent small molecule inhibitor of Hedgehog-Gli pathway. It has been reported GDC-0449 showed high target specificity and demonstrated antiproliferative activity against tumors and it is now in clinical trial. Therefore, combination therapy using a BCR-ABL tyrosine kinase inhibitors and a Hedgehog-Gli inhibitor, GDC-0449 may help prevent CML relapse and these approaches may be expected to improve the outcomes of Ph-positive leukemia patients. In this study, we investigated the GDC-0449 efficacy by using the BCR-ABL positive cell lines, OM9;22, K562 and primary samples when leukemic cells were protected by the feeder cell line, S9 cells. We examined a comprehensive drug combination experiment using GDC-0449 and dual Src/ABL tyrosine kinase inhibitor, dasatinib. Gli proteins (Gli1, Gli2 and Gli3) were existed in Ph-positive cell lines. We found the cell numbers of OM9;22 were significantly increased with the feeder cell line, S9 cells compared to without S9 cells. The treatment of dasatinib exhibits cell growth inhibition partially against OM9;22 cells in the presence of feeder cell line, S9 cells. Caspase-3 activity by 100 nM dasatinib treatment was also reduced in the presence of S9 cells. 72 h of combined treatment of Ph-positive leukemia cells with 10 μM of GDC-0449 and 100 nM of dasatinib in the presence of feeder cell line, caused significantly more cytotoxicity than each drug alone. We next investigated the efficacy and intracellular signaling of GDC-0449. The treatment of GDC-0449 exhibits cell growth inhibition and induced apoptosis against OM9;22 cells in a dose and time dependent manner. Expression of Gli1 and Gli2 proteins were reduced after GDC-0449 treatment. 10 μM of GDC-0449 also inhibited the growth of Ph-positive primary samples by colony assay. Another Hh inhibitor, SANT-2 also exhibits cell growth inhibition against OM9;22 cells in a dose dependent manner. Data from this study suggested that administration of the Hh inhibitor, GDC-0449 may be a powerful strategy against Ph-positive leukemia cells and enhance cytotoxic effects of dasatinib in the presence of feeder cell. Disclosures:Ohyashiki: Nippon Shinyaku Co., Ltd.: Research Funding.

Immunoprofiling of patients with chronic myeloid leukemia at diagnosis and during tyrosine kinase inhibitor therapy

Tyrosine kinase inhibitors (TKIs) are the current standard treatment in chronic myeloid leukemia (CML). In addition to the BCR-ABL target oncoprotein, they also inhibit off-target kinases (e.g. c-KIT, TEC, SRC), some of which have physiological functions in immune responses. In vitro studies have implied immunosuppressive effects of TKI treatment. As comprehensive in vivo data are missing, we aimed at analyzing the detailed immunoprofile of patients with CML at diagnosis and during therapy. We collected 88 peripheral blood (PB) and 73 bone marrow (BM) samples from 54 patients with CML at diagnosis, during imatinib and dasatinib therapies. Leukocytes and lymphocyte subclasses were analyzed with an extensive flow cytometry panel including markers for activation, differentiation and memory status. At diagnosis, a lower proportion of B cells and dendritic cells and an increased amount of NKT-like cells were detected in the BM. During imatinib therapy, all these changes normalized and the immunoprofile resembled healthy controls. However, dasatinib patients were clearly divided into two distinct groups: one similar to healthy controls and the other showing immunoactivation characterized by significant elevations of CD8+, NK- and NKT-like cells in PB. T cells of the latter group strongly expressed CD57+, HLA-DR and CD45RO and had low CD62L antigen levels characteristic of late memory cytotoxic lymphocytes. Our results indicate that while both TKIs show immunosuppressive effects in vitro, they have a significant and differential effect on the numbers and proportions of immune effector cells in vivo. In particular, in a distinct subgroup of dasatinib-treated patients, immune reactivity is markedly enhanced warranting careful follow-up.

Phase III trial of nilotinib in patients with advanced gastrointestinal stromal tumor (GIST): First results from ENEST g3.

10017^ Background: Nilotinib (N), a selective tyrosine kinase inhibitor (TKI) targeting KIT, PDGFRA, and BCR-ABL with activity against imatinib (IM)-sensitive and -resistant GIST cells, has shown efficacy in phase I and II trials. ENEST g3, a phase III, randomized, open-label, multicenter trial was performed to test N vs best supportive care (BSC) with physician choice to continue or stop IM or sunitinib (S) in patients (pts) with advanced/metastatic GIST who had failed IM and S. Methods: Pts were randomized 2:1 to N 400 mg bid or to the control arm (C = BSC, BSC+IM, or BSC+S, at physician discretion). Primary endpoint was progression-free survival (PFS) per RECIST assessed by blinded central radiology review (CRR). Results: 248 pts were included in the intent-to-treat (ITT) population: N=165, C=83 (BSC=6, BSC+IM=54, BSC+S=23). No significant difference in PFS between the N and C arms was observed based on CRR (p=0.5555; median PFS=109 vs. 111 days, respectively). There was major discordance in local inve...

Delivery of therapeutic shRNA and siRNA by Tat fusion peptide targeting bcr–abl fusion gene in Chronic Myeloid Leukemia cells

Gene silencing by RNA interference (RNAi) is a promising therapeutic approach for a wide variety of diseases for which the biological cause is known. The main challenge remains the ineffective RNAi delivery inside the cells. Non-viral gene delivery vectors have low immunogenicity compared to viral vectors, but are constrained by their reduced transfection efficiency. Silencing of the bcr–abl gene expression by RNAi confers therapeutic potential in Chronic Myeloid Leukemia (CML), but is limited by the cytotoxicity of the existing delivery methods. Here, we present evidence that the fusion between the cell penetrating peptide (CPP) HIV-Tat (49–57) and the membrane lytic peptide (LK15), Tat–LK15, mediates high transfection efficiency in delivering short hairpin RNA (shRNA) and small interfering RNA (siRNA) targeting the BCR–ABL oncoprotein in K562 CML cells. Our results show that shRNA complexes induce a more stable gene silencing of bcr-abl when compared to silencing mediated by siRNA complexes. In addition, silencing of the BCR-ABL oncoprotein by both shRNA and siRNA delivered by Tat-LK15 is more efficient and longer lasting than that achieved using Lipofectamine and more importantly without considerable cytotoxicity. In these terms Tat–LK15 can be an alternative to DNA/siRNA delivery in difficult-to-transfect leukemic cells.

Targeting Bcr–Abl by combining allosteric with ATP-binding-site inhibitors

In an effort to find new pharmacological modalities to overcome resistance to ATP-binding-site inhibitors of Bcr-Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr-Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry, we show that GNF-2 binds to the myristate-binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analogue of GNF-2 with improved pharmacokinetic properties, when used in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I mutant human Bcr-Abl and displayed in vivo efficacy against this recalcitrant mutant in a murine bone-marrow transplantation model. These results show that therapeutically relevant inhibition of Bcr-Abl activity can be achieved with inhibitors that bind to the myristate-binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.

Contribution of BCR–ABL‐independent activation of ERK1/2 to acquired imatinib resistance in K562 chronic myeloid leukemia cells

BCR-ABL tyrosine kinase, generated from the reciprocal chromosomal translocation t(9;22), causes chronic myeloid leukemia (CML). BCR-ABL is inhibited by imatinib; however, several mechanisms of imatinib resistance have been proposed that account for loss of imatinib efficacy in patients with CML. Previously, we showed that overexpression of the efflux drug transporter P-glycoprotein partially contributed to imatinib resistance in imatinib-resistant K562 CML cells having no BCR-ABL mutations. To explain an additional mechanism of drug resistance, we established a subclone (K562/R) of the cells and examined the BCR-ABL signaling pathway in these and wild-type K562 (K562/W) cells. We found the K562/R cells were 15 times more resistant to imatinib than their wild-type counterparts. In both cell lines, BCR-ABL and its downstream signaling molecules, such as ERK1/2, ERK5, STAT5, and AKT, were phosphorylated in the absence of imatinib. In both cell lines, imatinib effectively reduced the phosphorylation of all the above, except ERK1/2, whose phosphorylation was, interestingly, only inhibited in the wild-type cells. We then observed that phospho-ERK1/2 levels decreased in the presence of siRNA targeting BCR-ABL, again, only in the K562/W cells. However, using an ERK1/2 inhibitor, U0126, we found that we could reduce phospho-ERK1/2 levels in K562/R cells and restore their sensitivity to imatinib. Taken together, we conclude that the BCR-ABL-independent activation of ERK1/2 contributes to imatinib resistance in K562/R cells, and that ERK1/2 could be a target for the treatment of CML patients whose imatinib resistance is due to this mechanism.

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.

Weekend Drug Holiday of Dasatinib in CML Patients Not Tolerating Standard Dosing Regimens. Reducing Toxicity with Maintained Disease Control.

Abstract 1119Poster Board I-141Dasatinib (DA) is a multitargeted tyrosine kinase inhibitor (TKI) approved for 2nd line treatment of chronic myelogenous leukemia (CML) patients after imatinib failure. DA-related toxicity mandates dose reduction in selected patients beyond the labelled reduced continuous dosing. In chronic phase (CP) patients, intermittent targeting of BCR-ABL by a once daily regimen reduces side effects with equal efficacy compared to the initially explored twice daily regimen. Thus, considering the short half-life of DA (3-5 hours) additional treatment interruptions to reduce the total weekly dose may not negatively affect treatment outcome while allowing continued treatment with an effective drug. In a retrospective analysis 33 CML patients (pts; 20 m, 13 f, median age 66 years, range 39-81) with intolerance (n=11) or resistance (n=22) to imatinib were investigated. Pts were selected based on the toxicity-guided administration of a dose reduced dasatinib regimen and were treated with an on/off regimen (3 to 5 days on, 4 to 2 days off) expecting a reduction of DA dependent off-target toxicity. Pts were followed by routine hematologic and cytogenetic assessment, and molecular monitoring (quantitative reverse transcriptase polymerase chain reaction, PCR) to safeguard clinical response to the altered drug schedule. Further, resistant pts were regularly screened for BCR-ABL mutations. Median time since CML diagnosis until start of DA treatment was 38 mo (range, 6-189). The median number of preceding treatment modalities was 3 (range, 1-5). The median follow up of interval treatment was 23 mo (range, 3-41). 30 patients were in CP, 2 in accelerated phase, and one in blast phase CML. 13/33 patients carried mutant BCR-ABL prior to onset of DA-treatment. Non-exclusive reasons for dose reduction were hematologic toxicity (17/33; 51%), and fluid retention (18/33, 55%), including 17 patients with pleural effusions. 27 patients (82%) suffered from grade III/IV (CTC) side effects. The median weekly dose of the DA weekend holiday schedule was 500mg (range, 320-500). During interval treatment, mean CTC grade for hematologic toxicity improved from grade 3.2 to 1.5 (p<0.001), and for fluid retention from grade 2.9 to 1.6 (p<0.001). All but 2 pts (89%) affected by fluid retention, and all but one patient suffering from hematologic toxicity (94%) achieved a lower CTC toxicity level by allowing drug holiday. In 6/33 pts, resistance mutations (T315I x 2, F317L x 3, L248V) were recovered. For response analysis, 2 pts were excluded due to early stem cell transplantation or loss of follow up. 13/31 (42%) did either show transient improved molecular response or remained on stable BCR-ABL load over time. 3/31 progressed to advanced phase CML. 18/31 (58%) pts showed the desired disease control according to established criteria despite reduced total weekly DA doses either demonstrated by achieving an improved response level (12/31), or keeping the response level achieved by continuous dosing (6/31). Fourteen of the 18 pts achieved or maintained major molecular response (MMR) with 5 pts repeatedly tested negative by PCR. The remainder four pts demonstrated response by achieving complete cytogenetic remission (CCyR, 2x) or reduced BCR-ABL load <1% according to the international scale. Of note, 10/12 pts with improved response have been treated for a minimum of 6 mo with continuous dosing DA regimens without having achieved the response level observed after allowing drug holiday. We conclude that weekend treatment interruption allows continuation of DA treatment for pts suffering from side effects. This retrospective analysis in pts resistant or intolerant to imatinib with up to 5 preceding treatment modalities suggests good and in many cases even improved efficacy of interval treatment compared to continuous dosing. These data mandate the initiation of clinical trials to investigate alternative intermittent targeting regimens. DisclosuresOff Label Use: Dasatinib, a multikinase tyrosine kinase inhibitor, has been used in a lower than labeled total weekly dose to reduce off target toxicity for CML patients.. Hochhaus:Bristol Myers Squibb: Research Funding.

Arsenic Trioxide (ATO) Interacts Synergistically with MEK Inhibitors to Induce Apoptosis in STI571-Resistant Bcr-Abl Mutants.

Abstract 2177Poster Board II-154Resistance toward Imatinib and other Bcr-Abl tyrosine kinase inhibitors remains an increasing clinical problem in the treatment of advanced stages of chronic myeloid leukemia. Thus novel therapeutic strategies are needed to address the emerging problem of Imatinib resistance. Previous preclinical studies reported that the MEK inhibitors PD184352 or PD0325901 (Pfizer), strikingly enhances ATO-mediated apoptosis in Acute Myelogenous Leukemia and in Multiple myeloma. The aim of this study was to investigate whether the combined treatment with PD184352 (PD) and ATO has cytotoxic effects on murine Ba/F3 cells expressing wild-type (wt) or various imatinib-resistant mutant forms of Bcr-Abl, including T315I. We first analyzed the pharmacologic interactions between PD and ATO using a fixed-ratio experimental design in Bcr-Abl Ba/F3p210wt, Ba/F3p210T315I and Ba/F3p210Y253F cell lines and found that the combined treatment with PD plus ATO resulted in the synergistic induction of apoptosis in all cell lines tested (Chou-Talalay method): the averaged Combination Index values calculated from the ED50 (50% effective dose), ED75 and ED90, in PD plus ATO treated cells were 0.72± 0.19, 0.61± 0.04 and 0.69± 0.09 in BCR-ABL Ba/F3p210wt, Ba/F3p210T315I, and Ba/F3p210Y253F respectively. Synergistic interaction between ATO and PD0325901, a derivative of PD184352, was demonstrated in all tested cell lines. In order to investigate the molecular effectors involved in PD/ATO-induced apoptosis we first evaluated its effects on Bcr-Abl protein expression and CrkL phosphorylation, a well-known downstream target of Bcr-Abl. Immunoblotting analyses demonstrated that treatment for 24 to 48 hours of Ba/F3p210wt and Ba/F3p210T315I cells with PD (1μM) or ATO (1μM) alone or in combination had no effects on levels of total Bcr-Abl or phospho—CrkL thereby indicating that the combination PD/ATO does not act via Bcr-Abl oncogenic signaling. Therefore, we studied whether p53 and the p53-related gene p73 are molecular targets of the combined treatment in Ba/F3p210wt and Ba/F3p210T315I cell lines. We found that monotreatment with neither PD nor ATO 1 μM (or their combination) was able to induce p53 accumulation, whereas the combination PD/ATO promoted the accumulation of the proapoptotic and antiproliferative TA-p73α protein and reduced the levels of the antiapoptotic and proproliferative dominant-negative ΔN-p73α in both cell lines. Consistent with these results, we found that PD greatly enhanced the ATO-induced Puma expression, mitochondrial depolarization, caspase-3 activation, and apoptosis in Ba/F3p210wt and Ba/F3p210T315I cells and functional knock-out of p73 gene expression by small interfering (si)RNAs significantly reduced (P< .05 Dunnett test) the PD/ATO induced mitochondrial depolarization. To determine whether the PD plus ATO efficacy observed in vitro for BaF3 cells expressing mutant forms of Bcr-Abl was recapitulated in vivo, we studied PD/ATO combination in a mouse model of Imatinib-resistant, Bcr-Abl—dependent disease. Severe combined immunodeficient mice were injected intravenously with Ba/F3 cells expressing Bcr-Abl-T315I isoform. Mice with Bcr-Abl-T315I—induced leukemia were treated with the MEK inhibitor PD0325901 (10 mg/kg; orally) plus ATO (3.75 mg/kg; intraperitoneally) or Imatinib (50 mg/kg, twice daily; intraperitoneally) or vehicle for three weeks. Untreated or Imatinib-treated mice harboring the T315I isoform developed aggressive disease, with massive liver and splenic infiltration, typically resulting in death in 32 days. However, mice harboring the T315I isoform showed significantly prolonged survival when treated with PD/ATO (43 days, P=.001, Kaplan-Meier method and compared using the log-rank test). Moreover, histopathological analysis of 20 days Imatinib-treated mice revealed infiltration of the liver and spleen. In contrast, histopathological analysis of organs from PD/ATO-treated mice demonstrated normal tissue architecture. Consistent with these results immumoblottig analysis of the lysates from livers and spleens revealed a marked expression of Bcr-Abl protein in mice treated with Imatinib or vehicle. Our preclinical in vitro and in vivo studies suggest that a strategy combining ATO with disruption of MEK pathway could represent an effective therapeutic strategy for the treatment of Imatinib-resistant Bcr-Abl-positive leukemias, including those harboring the T315I mutation. Disclosures:No relevant conflicts of interest to declare.

The Functional Activity of the OCT-1 Protein Is Predictive of Molecular Response and Survival in CP-CML Patients Treated with Imatinib: A 5 Year Update of the TIDEL Trial.

Abstract 507The major active influx protein for imatinib into target BCR-ABL positive cells is the organic cation transporter OCT-1. We have previously demonstrated that the functional activity of the OCT-1 protein (OCT-1 activity) is predictive of molecular response in TIDEL (trial of imatinib 600 mg/day with selective dose intensification in untreated CP-CML) The OCT-1 activity (OA) is measured in mononuclear cells from untreated CML patients by calculating the intracellular concentration of 14-C imatinib less the intracellular concentration in the presence of OCT-1 inhibition. To address the question of whether OA is predicting only the rate of response, we now investigate the impact of OA on response and progression at 5 years. There is a significant difference in the achievement of MMR (p=0.007) and CMR by 60 months (p=0.032) (Table 1). Six patients developed kinase domain mutations over the course of this study. 5/6 had low OA. Significantly, for the first time addressing Event Free Survival (events defined as loss of CHR, MCR or CCR, progression to AP or BC or change of therapy due to unsatisfactory efficacy), we demonstrate that more patients with high OA are event free at 5 years when compared to patients with low OA (Table 1).Table 160 month analysis of the % of patients achieving MMR, CMR and EFS rates.The % of patients achieving:- By 60 monthsMMRCMREFSMutationsOverall (n=56)71506412Low OA 0-7.2 ng/200,000 cells (n=29)55345221High OA >7.2 ng/200,000 cells (n=27)8967784p-value0.0070.0320.0380.047To determine whether the detrimental effect of low OA on survival was more significant in those patients with OA in the lowest quartile (Q1) we compared the response of Q1 patients to all other patients (Table 2). These data demonstrate importantly, that patients in Q1 have significantly poorer outcomes, than the remainder of the patient cohort.Table 2Overall, Event and Progression Free Survival in Q1 compared to the remainder of the cohort.Event Free SurvivalTransformation Free SurvivalOverall SurvivalQ1 (<4.0ng/200,000cells: n=14)437979Remainder (n=42)7110098p-value0.0230.0020.009In previous analyses we have shown that the effects of a low OA can be partially overcome by higher imatinib doses. Limiting the analyses to those patients receiving <600mg average daily dose over the first 12 months there was a significant difference in the achievement of MMR (low OA (n=11) 27%: high OA (n=12) 92% p=0.021) and EFS (36% vs 75% p=0.03). In patients receiving ≥600 mg there was no significant difference between the groups, reinforcing the importance of dose.In 45 patients we examined the expression of OCT-1 mRNA for prediction of MMR, CMR, EFS and mutation development. Dividing the patients into low and high OCT-1 expression about the median we found that the level of mRNA is not predictive of MMR (low–60% vs high 78 p=0.241) CMR (low–45% vs high 55 p=0.456) EFS (low–55% vs high 70 p=0.315) or mutation development (low–18% vs high 14% p=0.666). These data indicate that the level of OCT-1 mRNA is not sufficiently discriminating to predict response and progression.While our previous studies demonstrated that OA could predict the rate of decline in BCR-ABL over the first 12-24 months, this update demonstrates for the first time, that this assay can identify nearly all patients (>80%) who fail to achieve MMR in the long term. Most importantly OA is also strongly predictive of resistance and progression events. Functional assessment of OCT-1 Activity provides prognostic information that is more discriminating than assaying the level of OCT-1 mRNA. This long term study reinforces the notion that OA is an important predictive variable in CP-CML patients treated with IM. It provides further evidence that OA is a critical variable to consider in future trials of imatinib and a key factor to enable individualization of imatinib dose to optimize the long term outcome for CML patients. Disclosures:White:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding. Manley:Novartis: Employment. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

WP1130 Inhibits Signaling through BCR-ABL Ubiquitination and Cytoplasmic to Aggresome Trafficking to Induce Apoptosis of CML Cells.

Abstract 3303Poster Board III-191BCR-ABL remains the primary target for chronic myelogenous leukemia (CML) therapy but longstanding control and complete suppression of the disease may require additional strategies beyond kinase inhibition. We previously described a small molecule, WP1130, that mediated down-regulation of BCR-ABL, induced apoptosis of CML cells with limited toxicity against normal myeloid progenitors and decreased CML tumor growth in mice (Blood. 2007;109(8):3470-8). In this report we further investigated the BCR-ABL targeting activity and mechanism of action of WP1130 in CML cells. WP1130 rapidly (within 30 to 60 min) reduced cytoplasmic levels of BCR-ABL protein and suppressed Stat5 tyrosine phosphorylation without affecting Jak2, c-Abl, BCR, Erk, Akt, Lyn, Hck or gp130. Down-regulation of BCR-ABL by WP1130 was not affected by point mutations that alter imatinib or dasatinib binding or BCR-ABL kinase inhibitory activity. Examination of the cellular distribution of BCR-ABL in treated cells revealed that BCR-ABL was sequestered into compact, cytoskeletal-enriched aggresomal structures that were recovered in the detergent-insoluble fraction of the cell. WP1130-mediated translocation of BCR-ABL was associated with increased cellular poly-ubiquitination without 20S proteosome inhibition, disruption of Hsp90/BCR-ABL complexes or increased reactive oxygen species. WP1130 induced BCR-ABL trafficking into aggresomes correlated with its poly-ubiquitination (primarily K63) and lysates from WP1130 treated cells demonstrated reduced capacity to hydrolyze ubiquitinated substrate. Together, these results suggest that WP1130 induces poly-ubiquitination and trafficking of BCR-ABL into signaling incompetent aggresomes in CML cells through a unique mechanism that may involve inhibition of deubiquitinase activity and define a unique pathway to suppress BCR-ABL signaling and induce CML cell destruction. DisclosuresNo relevant conflicts of interest to declare.