A small molecule Abl kinase inhibitor induces differentiation of Abelson virus–transformed pre-B cell lines

10.1038/ni870 ◽  
2002 ◽  
Vol 4 (1) ◽  
pp. 31-37 ◽  
Author(s):  
Stefan A. Muljo ◽  
Mark S. Schlissel
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
Abl Kinase ◽  
Abelson Virus

scholarly journals Repurposing dasatinib for diffuse large B cell lymphoma

2019 ◽  
Vol 116 (34) ◽  
pp. 16981-16986 ◽  
Author(s):  
Claudio Scuoppo ◽  
Jiguang Wang ◽  
Mirjana Persaud ◽  
Sandeep K. Mittan ◽  
Katia Basso ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Multikinase Inhibitor ◽  
Large B Cell Lymphoma ◽  
Large B Cell

To repurpose compounds for diffuse large B cell lymphoma (DLBCL), we screened a library of drugs and other targeted compounds approved by the US Food and Drug Administration on 9 cell lines and validated the results on a panel of 32 genetically characterized DLBCL cell lines. Dasatinib, a multikinase inhibitor, was effective against 50% of DLBCL cell lines, as well as against in vivo xenografts. Dasatinib was more broadly active than the Bruton kinase inhibitor ibrutinib and overcame ibrutinib resistance. Tumors exhibiting dasatinib resistance were commonly characterized by activation of the PI3K pathway and loss of PTEN expression as a specific biomarker. PI3K suppression by mTORC2 inhibition synergized with dasatinib and abolished resistance in vitro and in vivo. These results provide a proof of concept for the repurposing approach in DLBCL, and point to dasatinib as an attractive strategy for further clinical development in lymphomas.

The Dual SRC/ABL Kinase Inhibitor BMS-354825 Potently Inhibits the Growth of Myeloid Leukemic Cells Characterized by FLT3-ITD Expression, GM-CSF Dependency, or G-CSF Responsiveness Via an ABL-Independent Mechanism.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4480-4480
Author(s):  
Jukka Kanerva ◽  
Ogonna Nwawka ◽  
Kevin Hwang ◽  
Francis Y. Lee ◽  
Seth J. Corey
Keyword(s):  
Growth Inhibition ◽  
Cell Lines ◽  
Western Blotting ◽  
Mtt Assay ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Src Kinase ◽  
Myeloid Cell ◽  
Gm Csf ◽  
Abl Kinase

Abstract BMS-354825 is a dual SRC and ABL inhibitor, which has been shown effective in imatinib-resistant BCR-ABL+ cells, and it is in phase I trials for patients with imatinib-resistant leukemia. We conducted a study to evaluate growth inhibition and inhibition of Src v. Abl protein tyrosine kinases in human myeloid cell lines: MV4-11 expressing an internal tandem duplication of Flt3 (Flt3-ITD), the murine pro-B cell line Ba/F3 that expresses the Flt3-ITD, the GM-CSF dependent Mo7e, and the G-CSF-responsive BaF3-GR (Ba/F3 cells expressing the human G-CSF receptor). We compared BMS-354825 with PP1, a SRC kinase inhibitor with in vitro IC50 at sub-micromolar concentrations. We sought to correlate growth inhibition with SRC or ABL inhibition. In these myeloid cell lines, LYN is the predominant SRC kinase. Methods: Growth inhibition was assessed by Trypan blue exclusion and MTT assay using drug concentrations 0.1 uM – 10 uM. Drugs were added daily to the cell suspension during the 3-day experiment. After a 60 min incubation at concentrations 0.1 nM – 1 uM, SRC or ABL kinase inhibition was analyzed by blotting with a polyclonal phospho-SRC (Tyr416) antibody or a polyclonal phospho-ABL (Tyr245) antibody. Results: In MV4-11 cells BMS-354825 and PP1 caused similar growth inhibition IC50 at 5 uM. By western blotting, inhibition of phospho-Src 416 occurred at 1 nM concentrations of both compounds. Protein expression of ABL was not detected in MV4-11 cells. In Ba/F3-ITD cells, the IC50 for BMS-354825 was 1–10 uM (grown in IL-3) and 0.01 uM (without IL-3). The IC50 for PP1 was 1–10 uM (grown in IL-3) and 0.1 uM (without IL-3). Inhibition of phospho-SRC occurred at 10 nM. In Mo7e cells, grown in the presence of GM-CSF, the IC50 was 5 uM for BMS-354825 v. 10 uM for PP1 by MTT assay. By western blotting, inhibition of phospho-SRC 416 occurred at 1 nM for both BMS-354825 and PP1. To determine specific contribution of LYN to Mo7e growth, we treated Mo7e cells with LYN siRNA. With 70% knock-down of LYN, there was 50% growth inhibition. ABL was present in Mo7e cells, but no phosphoAbl was demonstrated (K562 cells served as positive control). In BaF3-GR cells grown in G-CSF, the IC50 was 5 uM for BMS-354825 vs. 10 uM for PP1 by MTT assay. In western blotting, inhibition of phospho-Src 416 was detected at 10 nM BMS-354825. ABL was present in Ba/F3GR cells, but no phospho-ABL was demonstrated (K562 cells served as positive control). Conclusions: BMS-354825 is more potent than PP1 in causing growth inhibition and SRC kinase inhibition in Mo7e and Ba/F3GR cells that serve as models for acute myeloid leukemia. It is unlikely that ABL is the drug target, because MV4-11 cells do not express ABL and phospho-ABL was not found in Mo7e or Ba/F3 cells. These results suggest that inhibition of SRC tyrosine kinases contributes predominantly to growth inhibition caused by the dual SRC/ABL kinase inhibitor BMS-354825 in myeloid cell lines expressing Flt3-ITD and cytokine-driven proliferation and survival via the IL-3/GM-CSF Receptor or G-CSF Receptor.

CHIR258, a Novel Multi-Targeted Tyrosine Kinase Inhibitor, for the Treatment of t(4;14) Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 641-641 ◽  
Author(s):  
Suzanne Trudel ◽  
Zhi Hua Li ◽  
Ellen Wei ◽  
Marion Wiesmann ◽  
Katherine Rendahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Myeloma Cells

Abstract The t(4;14) translocation that occurs uniquely in a subset (15%) of multiple myeloma (MM) patients results in the ectopic expression of the receptor tyrosine kinase, Fibroblast Growth Factor Receptor3 (FGFR3). Wild-type FGFR3 induces proliferative signals in myeloma cells and appears to be weakly transforming in a hematopoeitic mouse model. The subsequent acquisition of FGFR3 activating mutations in some MM is associated with disease progression and is strongly transforming in several experimental models. The clinical impact of t(4;14) translocations has been demonstrated in several retrospective studies each reporting a marked reduction in overall survival. We have previously shown that inhibition of activated FGFR3 causes morphologic differentiation followed by apoptosis of FGFR3 expressing MM cell lines, validating activated FGFR3 as a therapeutic target in t(4;14) MM and encouraging the clinical development of FGFR3 inhibitors for the treatment of these poor-prognosis patients. CHIR258 is a small molecule kinase inhibitor that targets Class III–V RTKs and inhibits FGFR3 with an IC50 of 5 nM in an in vitro kinase assay. Potent anti-tumor and anti-angiogenic activity has been demonstrated in vitro and in vivo. We employed the IL-6 dependent cell line, B9 that has been engineered to express wild-type FGFR3 or active mutants of FGFR3 (Y373C, K650E, G384D and 807C), to screen CHIR258 for activity against FGFR3. CHIR258 differentially inhibited FGF-mediated growth of B9 expressing wild-type and mutant receptors found in MM, with an IC50 of 25 nM and 80 nM respectively as determined by MTT proliferation assay. Growth of these cells could be rescued by IL-6 demonstrating selectivity of CHIR258 for FGFR3. We then confirmed the activity of CHIR258 against FGFR3 expressing myeloma cells. CHIR258 inhibited the viability of FGFR3 expressing KMS11 (Y373C), KMS18 (G384D) and OPM-2 (K650E) cell lines with an IC50 of 100 nM, 250 nM and 80 nM, respectively. Importantly, inhibition with CHIR258 was still observed in the presence of IL-6, a potent growth factors for MM cells. U266 cells, which lack FGFR3 expression, displayed minimal growth inhibition demonstrating that at effective concentrations, CHIR258 exhibits minimal nonspecific cytotoxicity on MM cells. Further characterization of this finding demonstrated that inhibition of cell growth corresponded to G0/G1 cell cycle arrest and dose-dependent inhibition of downstream ERK phosphorylation. In responsive cell lines, CHIR258 induced apoptosis via caspase 3. In vitro combination analysis of CHIR258 and dexamethasone applied simultaneously to KMS11 cells indicated a synergistic interaction. In vivo studies demonstrated that CHIR258 induced tumor regression and inhibited growth of FGFR3 tumors in a plasmacytoma xenograft mouse model. Finally, CHIR258 produced cytotoxic responses in 4/5 primary myeloma samples derived from patients harboring a t(4;14) translocation. These data indicate that the small molecule inhibitor, CHIR258 potently inhibits FGFR3 and has activity against human MM cells setting the stage for a Phase I clinical trial of this compound in t(4;14) myeloma.

The Src-Abl Kinase Inhibitor Dasatinib (BMS-354825) Shows Anti-Proliferative and Anti-Apoptotic Effects in Chronic Lymphocytic Leukemia (CLL) Cells In Vitro.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3101-3101
Author(s):  
Aditya Veldurthy ◽  
Michaela Patz ◽  
Christian P. Pallasch ◽  
Clemens M. Wendtner ◽  
Michael Hallek ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Parp Cleavage ◽  
Apoptosis Induction ◽  
Abl Kinase ◽  
Two Samples ◽  
Apoptotic Effects ◽  
Dasatinib Treatment

Abstract Introduction: Dasatinib is an ATP competitive, dual-specific Src-Abl kinase inhibitor used for the treatment of imatinib-resistant Bcr-Abl-positive leukemias. Originally it was developed from the aminothiazole scaffold as an inhibitor of Src family kinases (SFKs). Since the aberrant expression and activity of the SFK Lyn seems to enhance the survival of CLL cells, dasatinib is currently tested in clinical studies for CLL. In order to explore the anti-leukemic potential of dasatinib we tested its effects on freshly isolated CLL cells and on CLL cell lines. Methods and Results: In freshly isolated CLL cells, dasatinib showed a dose and time dependent reduction of global tyrosine phosphorylation which was paralleled by a decreased phosphorylation of the activating tyrosine residue of SFKs. The comparison of the inhibitory effect of several protein tyrosine kinase inhibitors on overall tyrosine phosphorylation in cellular lysates of freshly isolated CLL cells showed that 0.1 μM dasatinib had a more pronounced effect than 5 μM of nilotinib, or 10 μM of PP1 or imatinib. In the CLL cell lines, Mec1 and JVM-3, 0.1 μM of dasatinib appeared to interfere with survival signaling, since decreased levels of the activated phosphorylated forms of Akt, Erk1/2 and p38 were observed after exposure to dasatinib for 2 hours. In these cell lines, dasatinib treatment increased p53 protein levels and induced PARP cleavage and caspase activity. Pro-apoptotic effects of dasatinib were observed by an increase of annexin V-positive cells and a decrease of metabolic activity measured as XTT reduction, alone and in combination with fludarabine. Among six patient samples, two clones were particularly sensitive to dasatinib and fludarabine-resistant. While in these two samples the apoptosis induction by 5 μM dasatinib surpassed that by 5 μM fludarabine, the same dose of the two drugs led to similar apoptosis induction in the fludarabine-sensitive samples. Dasatinib treatment sensitized CLL cells for fludarabine-induced apoptosis. This enhancement of apoptosis induction was more pronounced in fludarabine-resistant patient samples and JVM-3 cells than in Mec1 and fludarabine-sensitive CLL cells. Conclusion: The Src-Abl kinase inhibitor dasatinib shows potent inhibitory effects on the survival of CLL cells in vitro.

Bosutinib, a Src/Abl Kinase Inhibitor Induces Apoptosis in CLL B Cells by Targeting Multiple Tyrosine Kinases and Overcomes Stroma Protection.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2368-2368
Author(s):  
Asish K Ghosh ◽  
Debabrata Mukhopadhyay ◽  
Neil E. Kay
Keyword(s):  
B Cells ◽  
B Cell ◽  
Tyrosine Kinases ◽  
Therapeutic Agent ◽  
Kinase Inhibitor ◽  
Src Kinase ◽  
Abl Kinase ◽  
Apoptotic Proteins ◽  
Constitutively Active

Abstract Abstract 2368 Poster Board II-345 Background: B-cell chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of CD5+ B lymphocytes in the peripheral blood, lymphoid organs and bone marrow. Despite aggressive therapy, CLL is still incurable partly because of intrinsic defect in apoptosis induction. A novel therapeutic agent, Bosutinib, was initially developed as an inhibitor of Src and Abl kinases and is currently in phase II clinical trials for the treatment of several human malignancies. Recently, Bosutinib has been shown to inhibit phosphorylation of a novel receptor tyrosine kinase, Axl which has been reported to be overexpressed in several types of human cancers including colon, prostatic, thyroid, breast, gastric, renal and lung. Previously, Dasatinib, another Src/Abl kinase inhibitor, showed cytotoxic effects on CLL B cells by decreasing levels of activated, phosphorylated forms of Akt, Erk1/2 and p38 and reducing expression of anti-apoptotic proteins Mcl-1 and Bcl-xL in CLL B cells. Here, we wished to examine receptor or non-receptor tyrosine kinases active in primary CLL B-cells and determine their status after exposure to Bosutinib as well as the latter drug's effect on CLL B-cell viability. Methods: We used freshly isolated CLL B cells after obtaining written consent from patients. Bosutinib was used at various doses (2.5, 5.0, 10.0 and 20.0 μM) to treat CLL B cells in vitro for 24/48 hrs. Induction of apoptosis was assessed by annexin/propidium staining. To examine the impact of stroma on Bosutinib induced CLL B-cell death, primary CLL-bone marrow stromal cells (BMSC) were cocultured with CLL B cells at a cell density ratio of 1:20 and treated with various doses of Bosutinib for 24 hrs. Expression status of various kinases and downstream targets were analyzed in CLL B cell lysates with or without Bosutinib-treatment by Western blot using specific antibodies. Results: Treatment of CLL B-cells with Bosutinib induces a massive apoptotic cell death in a dose- and time-dependent manner (IC50 for 24 h; ∼10 μM and IC50 for 48 h: 5-10 μM) which involves PARP cleavage as demonstrated by Western blot analysis. Moreover, Bosutinib-treatment reduced expression of several key anti-apoptotic proteins, Mcl-1, XIAP and Bcl-2 reported to be overexpressed in CLL B cells. Interestingly, we detected that the majority of CLL B-cells express constitutively active Axl. Importantly, Bosutinib treatment inhibited phosphorylation of Axl in CLL B cells resulting in inhibition of AKT-activation, one of its downstream signaling pathways. Previous studies have suggested a possible physical association between Axl and Src kinase. We observed that expression of constitutively active Axl was associated with the presence of highly phosphorylated Src kinase when compared with that in CLL B-cells with low or unphosphorylated Axl. These observations suggest that phosphorylation of Axl may be an upstream event for Src activation in CLL. We found inhibition of constitutively active Axl resulted in subsequent inhibition of Src kinase activation in CLL B-cells following Bosutinib-treatment. We also detected inhibition of ZAP70/Syk-phosphorylation in CLL B cells upon Bosutinib-treatment. Finally, we found Bosutinib was able to overcome stomal protection of CLL B cells at a dose of 10 μM in an in vitro coculture system suggesting its potential as a therapeutic agent against CLL. Conclusion: Together, these observations suggest that Bosutinib induces apoptosis in CLL B-cells, even in the presence of stromal cells, in association with the down regulation of multiple kinases including the novel receptor-tyrosine kinase, Axl, and reduces expression of the anti-apoptotic proteins critical to CLL B-cell survival. In total, these findings for the first time indicate that Bosutinib has the potential to be a very potent therapeutic agent for CLL patients. Disclosures: Kay: Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding.

Compensatory Signaling From ROR1 and the Pre-B Cell Receptor Promote Survival of t(1;19) Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2466-2466
Author(s):  
Vincent T Bicocca ◽  
Bill H Chang ◽  
Markus Muschen ◽  
Brian J. Druker ◽  
Jeffrey W Tyner
Keyword(s):  
Cell Viability ◽  
B Cell ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Immunoblot Analysis ◽  
Cell Receptor ◽  
Cell Surface Receptor

Abstract Abstract 2466 BACKGROUND: Aberrant tyrosine kinase activity is commonly implicated in the pathogenesis of leukemia and other cancers. Identification of these leukemogenic tyrosine kinases has proven invaluable for diagnostic and prognostic stratification of patients as well as for the development of novel strategies for therapeutic intervention. We previously demonstrated that siRNA screening of mononuclear cells from leukemia patients can determine sensitivity to individual tyrosine kinases. With the goal of uncovering novel viability-dependent tyrosine kinases in leukemia patients, we have employed an RNAi-assisted protein target identification (RAPID) assay to screen cytogenetic subtypes of acute lymphoblastic leukemia (ALL). ALL is the most common pediatric cancer, accounting for one-quarter of all childhood malignancies. Childhood ALL has a primarily B cell precursor phenotype and is characterized by chromosomal abnormalities, primarily translocations and duplications. One of the most common recurring translocations associated with pediatric ALL, t(1;19)(q23;p13.3), generates the E2A-PBX1 fusion product. Here we show unique viability-dependent expression of a receptor tyrosine kinase, ROR1, in the t(1;19) ALL background. In addition, we identify a kinase inhibitor, dasatinib, with significant activity against t(1;19) ALL cells due to its capacity to inhibit tyrosine kinases necessary for transduction of pre-B cell receptor (preBCR) signaling. Finally, we show that ROR1 and the preBCR activate mutually compensatory signaling pathways, suggesting that optimal therapeutic regimens would include agents targeting both pathways. METHODS: To identify targets required for viability of leukemic cells, we screened cell lines as well as primary cells from ALL patients with siRNAs and determined cell viability using an MTS assay. ROR1 expression levels were determined by RT-PCR, immunoblot analysis and flow cytometry. Kinase inhibitor screening was performed on both cells lines and primary ALL cells by treating samples with a library of small-molecule inhibitors and cell viability was assessed by MTS. Signaling pathways disrupted by inhibitor treatment or ROR1 knockdown were interrogated by phospho-protein arrays and confirmed by immunoblot analysis. RESULTS: The RAPID assay identified a unique sensitivity to the cell surface receptor ROR1 in a subject identified with t(1;19) pediatric ALL. Similar sensitivity was not observed in ALL patients of alternative cytogenetic subtypes. Examination of additional ALL patient samples revealed conserved overexpression of the ROR1 transcript in t(1;19)-positive specimens with absence of ROR1 expression in t(1;19)-negative samples. Cell lines and early passage xenograft cells confirmed overexpression and functional dependence of t(1;19)-positive cells on ROR1. A subsequent kinase inhibitor screen of t(1;19) ALL cell lines and patient samples revealed universal sensitivity to the FDA-approved drug dasatinib. Further examination revealed the dasatinib targets, BTK and LYN, which signal downstream of the preBCR as the viability dependent targets of dasatinib in t(1;19) ALL. Inhibition of the preBCR results in transient loss of AKT activity and, surprisingly, upregulation of ROR1. Analysis of signaling pathways after silencing of ROR1 or dasatinib treatment revealed compensatory signaling pathways emanating from ROR1 and the preBCR that both serve to regulate AKT activity. Consequently, combination of ROR1 knockdown and dasatinib treatment resulted in additive impairment of cell viability compared with either treatment alone. CONCLUSION: The cell surface receptor ROR1 is consistently overexpressed in t(1;19) ALL. RNAi mediated downregulation of ROR1 impairs the viability of these cells. Similarly, t(1;19) cells are sensitive to the kinase inhibitor dasatinib due to activity against the preBCR. Combined targeting of ROR1 and the preBCR with dasatinib yields an additive viability effect due to compensatory signaling pathways aimed at regulating AKT. These results demonstrate a novel mechanism of AKT regulation in t(1;19) ALL as well as a therapeutic strategy for treatment of t(1;19) ALL. Disclosures: Druker: MolecularMD: Equity Ownership, OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Committee and t; Ariad Pharmaceuticals: Consultancy; OHSU patent #843: Mutated ABL Kinase Domains: Patents & Royalties; Bristol-Myers Squibb: OHSU has clinical trial contracts with Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Novartis: OHSU has clinical trial contracts with Novartis to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Cylene: Consultancy.

Partially or Fully BCR-ABL Independent Mechanisms of in Vitro Resistance to Ponatinib

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2481-2481 ◽  
Author(s):  
Qian Yu ◽  
Anna M Eiring ◽  
Matthew S. Zabriskie ◽  
Jamshid Khorashad ◽  
David J Anderson ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Synthetic Lethality ◽  
Immunoblot Analysis ◽  
Kinase Domain ◽  
Resistant Cell ◽  
Abl Kinase ◽  
Kinase Domain Mutation

Abstract Abstract 2481 Ponatinib (AP24534) is a pan-BCR-ABL inhibitor developed for treatment-refractory chronic myeloid leukemia (CML) and has significant activity in patients who fail second-line dasatinib and/or nilotinib tyrosine kinase inhibitor (TKI) therapy. A pivotal phase II trial (clinicaltrials.gov NCT01207440) is underway. BCR-ABL kinase domain mutation-mediated ponatinib resistance has been investigated in vitro (Cancer Cell 16, 2009, 401). Here, we developed ponatinib-resistant, BCR-ABL+ cell lines lacking a kinase domain mutation and investigated mechanisms of resistance to ponatinib and other TKIs. Methods: Four BCR-ABL+ CML cell lines (K562, AR230, BV173, and 32D(BCR-ABL)) were maintained in liquid culture containing ponatinib (0.1 nM) for 10 days. The ponatinib concentration was increased in small increments for a minimum of 90 days, yielding corresponding ponatinib-resistant cell lines. BCR-ABL kinase domain sequencing of sensitive and resistant cells confirmed BCR-ABL to be unmutated. Real-time qPCR was used to compare the expression of BCR-ABL in ponatinib-sensitive and -resistant cell lines. Immunoblot analysis (total and tyrosine-phosphorylated BCR-ABL) was used to the compare levels of BCR-ABL protein and to determine whether resistance to ponatinib corresponded with reduced (partially BCR-ABL-independent) or complete inhibition of BCR-ABL tyrosine phosphorylation (fully BCR-ABL-independent). Cell proliferation assays were performed on resistant and sensitive cell lines in the presence of ponatinib, nilotinib, and dasatinib. A small-molecule inhibitor screen composed of >90 cell-permeable inhibitors that collectively target the majority of the tyrosine kinome as well as other kinases (Blood 116, 2010, abstract 2754) is currently being applied to the 32D(BCR-ABL)R cell line in the presence of 24 nM ponatinib to assess synthetic lethality, with results analyzed using a companion drug sensitivity algorithm. As a second strategy to generate resistant lines, N-ethyl-N-nitrosourea (ENU) mutagenesis was done to investigate BCR-ABL kinase domain-mediated resistance in myeloid K562, AR230, BV173, and 32D(BCR-ABL) cells. After ENU exposure, cells were washed and cultured in 96-well plates with escalating ponatinib. Results: The four BCR-ABL+ cell lines initially grew in the presence of 0.1 nM but not 0.5 nM ponatinib. Upon gradual exposure to escalating ponatinib, each of the cell lines exhibited a degree of adaptation to growth in the presence of the inhibitor (range: 10 to 240-fold). Real-time qPCR showed a modest two-fold increase in BCR-ABL expression level in K562R, AR230R and BV173R cell lines relative to the respective parental lines. Based on immunoblot analysis, cell lines segregated into two categories of ponatinib resistance: partially (K562R and AR230R) or fully BCR-ABL-independent (BV173R and 32D(BCR-ABL)R). Cell proliferation assays showed that ponatinib resistant cell lines also exhibited resistance to nilotinib and dasatinib. The 32D(BCR-ABL)R cell line exhibited a level of ponatinib resistance comparable to that of the Ba/F3 BCR-ABLE255V cell line, which carries the most ponatinib-resistant BCR-ABL mutation. BCR-ABL tyrosine phosphorylation was efficiently blocked by low concentrations of ponatinib (<5 nM) in the 32D(BCR-ABL)R cell line, yet these cells remained viable in the presence of up to 24 nM ponatinib. The effects of providing a second kinase inhibitor along with ponatinib (24 nM) in order to probe for synthetic lethality are under study. Possible involvement of a second, moderately ponatinib-sensitive target is suggested by the sharp ponatinib maximum at 24 nM; even 26 nM ponatinib is toxic to 32D(BCR-ABL)R cells. Thus far, ENU mutagenesis screens in human CML cell lines failed to yield resistant clones and only a few were recovered from the murine 32D(BCR-ABL)R cell line (3/1440 wells; the only BCR-ABL mutant recovered was BCR-ABLL387F). Conclusions: The ponatinib resistant, BCR-ABL+ cell lines described here exhibit either a partially or fully BCR-ABL independent mechanism of resistance. The molecular details of both processes will be reported, with an emphasis on the striking level of resistance (240-fold over starting conditions) exhibited by the 32D(BCR-ABL)R cell line. Our in vitro results indicate that BCR-ABL independent mechanisms may contribute to ponatinib resistance in myeloid CML cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A common phosphotyrosine signature for the Bcr-Abl kinase

Blood ◽  
2006 ◽  
Vol 107 (12) ◽  
pp. 4888-4897 ◽  
Author(s):  
Valerie L. Goss ◽  
Kimberly A. Lee ◽  
Albrecht Moritz ◽  
Julie Nardone ◽  
Erik J. Spek ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitor ◽  
Imatinib Treatment ◽  
Treatment Comparison ◽  
Abl Kinase ◽  
Abl Tyrosine Kinase ◽  
Abl Tyrosine Kinase Inhibitor ◽  
Disease Stages ◽  
Insight Into ◽  
Fusion Type

Abstract The Bcr-Abl fusion kinase drives oncogenesis in chronic myeloid leukemia (CML). CML patients are currently treated with the Abl tyrosine kinase inhibitor imatinib, which is effective in early stages of the disease. However, resistance to imatinib arises in later disease stages primarily because of a Bcr-Abl mutation. To gain deeper insight into Bcr-Abl signaling pathways, we generated phosphotyrosine profiles for 6 cell lines that represent 3 Bcr-Abl fusion types by using immunoaffinity purification of tyrosine phosphopeptides followed by tandem mass spectrometry. We identified 188 nonredundant tyrosine-phosphorylated sites, 77 of which are novel. By comparing the profiles, we found a number of phosphotyrosine sites common to the 6 cell lines regardless of cellular background and fusion type, several of which are decreased by imatinib treatment. Comparison of this Bcr-Abl signature with the profile of cells expressing an alternative imatinib-sensitive fusion kinase, FIP1L1-PDGFRα, revealed that these kinases signal through different pathways. This phosphoproteomic study of the Bcr-Abl fusion kinase highlights novel disease markers and potential drug-responsive biomarkers and adds novel insight into the oncogenic signals driven by the Bcr-Abl kinase.

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