To Study Resistant Mechanisms and Reversal In An Imatinib Resistant Ph+ Positive Acute Lymphoblastic Leukemia Cell Line

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2135-2135
Author(s):  
Hongyun Xing ◽  
Yuping Gong ◽  
Ting Liu
Keyword(s):  
Cell Signaling ◽  
Cell Line ◽  
Lymphoblastic Leukemia ◽  
Kinase Domain ◽  
Rt Pcr ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Cell Signaling Pathways

Abstract Abstract 2135 Objective To establish an imatinib resistant Bcr-Abl positive acute lymphoblastic leukemia (ALL) cell line in vitro and to study imatibin resistance in Ph+ ALL. The reversal of the imatinib resistance by rapamycin, the second generation tyrosine kinase inhibitor and proteasome inhibitor was studied. Methods Ph(+) ALL SUP-B15 cell line was cultured in gradually increasing concentrations of imatinib to generate the imatinib resistant cell line at 6 μM imatinib. The cytotoxic effect of imatinib and other drugs was analyzed by MTT assay. RT-PCR, flow cytometry, Western blot analyses of proteins, DNA sequence analysis of ABL kinase domain were used to clarify the possible mechanisms of the imatinib resistance in the SUP-B15/RI cell line. Results We established the imatinib resistant Ph+ ALL cell line. The fusion bcr-abl gene was 6.1 times as high as that of the parental sensitive cell, and the mdr1 gene also increased 1.7 times in SUP-B15/RI cell line by the RT-PCR detection. However, the expression of hoct1 Abcl–2 and topoIIα gene were no difference between two cell lines by the RT-PCR detection. A K362S point mutation in the Abl kinase domain of SUP-B15/RI was found. The detection of cell signaling pathway of PI3K/AKT/mTOR, RAS/RAF, NF-κBA JNK and STAT showed the expression of PTEN and 4EBP-1 was down-regulated, AKT, mTOR and P70S6K was up-regulated and the expression of other cell signaling pathways in SUP-B15/RI was similar to its parental sensitive cell line. Dasatinib, nilotinib, and bortezomib could inhibit proliferation of SUP-B15/RI cells at nM concentration. SUP-B15/RI cell line also showed partial resistance to dasatinib and nilotinib, but not bortezomib. The combination of imatinib with rapamycin had synergistic effect to the resistance cell line. Conclusion In vitro, we establish imatinib resistant Ph + ALL cell line. Overexpression of bcr-abl and mdr1 gene, K362S point mutation in ABL kinase domain and up-regulation of the cell signaling pathways of PI3K/AKT/mTOR, RAS/RAF in SUP-B15/RI cell line were involved in the resistance mechanisms. The SUP-B15/RI cell line was also resistant to the second generation tyrosine kinaeses dasatinib and nilotinib,not bortezomib in vitro. However, the combination of imatinib with rapamycin can partially overcome the resistance. Blockade of the ubiquitin-proteasome could be a promising pathway to overcome resistance to imatinib. Disclosures: No relevant conflicts of interest to declare.

Correlation of Clinical Response to Nilotinib with BCR-ABL Mutation Status in Advanced Phase Chronic Myelogenous Leukemia (CML-AP) Patients with Imatinib-Resistance or Intolerance.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1940-1940 ◽  
Author(s):  
Giuseppe Saglio ◽  
Dong-Wook Kim ◽  
Andreas Hochhaus ◽  
Simona Soverini ◽  
P. Erben ◽  
...  
Keyword(s):  
Disease Progression ◽  
Cytogenetic Response ◽  
Kinase Domain ◽  
Mutant Clone ◽  
Imatinib Resistance ◽  
Mutation Status ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Kinase Domain Mutations

Abstract The 2nd-generation bcr-abl inhibitor nilotinib is more potent than imatinib (IC50 <30 nM) against unmutated bcr-abl and active against 32/33 imatinib-resistant BCR-ABL mutants in vitro. We investigated the in vivo activity of nilotinib stratified by the baseline BCR-ABL mutation status in 127 imatinib-resistant or -intolerant CML-AP patients (pts) enrolled in an open-label phase II trial of nilotinib. Eighty-five pts (85/127, 67%) were screened prior to nilotinib therapy for BCR-ABL kinase domain mutations by direct sequencing. Of the 85 pts, 75 (88%) were resistant to imatinib and 10 (12%) were intolerant using standard published criteria. Twenty-two different baseline mutations involving 19 amino acids were identified in 50 (59%) pts analyzed. Other 35 (41%) pts did not have a baseline mutation. The most frequent mutation types identified included M351T (8 pts), G250E (7 pts), Y253H (6 pts), M244V (5 pts), F359V (5 pts) and T315I (5 pts). Twenty-two percent of pts with baseline mutations (11/50) showed more than one mutation (9 with two, 1 with three, and 1 with four mutations). All baseline mutations occurred in imatinib-resistant pts but none in intolerant pts. After 12 months of therapy, confirmed (confirmed in two consecutive analyses 4 week apart) hematologic response (HR) was achieved in 48% (21/50), major cytogenetic response (MCR) in 20% (10/50), and complete cytogenetic response (CCR) in 16% (8/50) of imatinib-resistant pts with baseline mutation versus 44% (12/25), 40% (10/25), and 20% (2/25) of imatinib-resistant pts without baseline mutation, respectively. Responses appeared to be affected by the in vitro sensitivity of the mutant clone against nilotinib. Pts with less sensitive mutation (cellular IC50 of >200nM: Y253H, E255K, E255V, F359C) representing 13% (11/85) of all patients assessed for baseline mutation, showed 13% (1/11) HR and 13% (1/11) MCyR compared to 74% (17/28) and 18% (5/28) respectively in the mutant group with IC50 of ≤200 nM. The nilotinib resistant T315I mutation occurred in 5 pts. Only one of these 5 pts who had T315I and G250E dual mutation achieved HR conceivably reflecting the sensitivity of G250E or non-mutant clone to nilotinib. At the time of data analyses, 50% of pts with baseline mutation were free of disease progression versus 62% of pts without baseline mutation. Rate of progression was 64% (7/11) in the group with less sensitive mutations and 60% (3/5) in pts. with T315I. However, the mutants most frequently associated with progression were F359V and M244V both having 4/5 pts (80%) progressed. In summary, BCR-ABL kinase domain mutations were identified at baseline in 59% of all pts in this cohort and in 67% of pts with imatinib resistance. Responses were observed across a broad spectrum of mutant genotypes. The rate of responses and disease progression may be affected by the baseline mutation types, although a larger data set with longer follow up is needed to further establish the correlation.

BCR-ABL truncation due to premature translation termination as a mechanism of resistance to kinase inhibitors

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 7028-7028
Author(s):  
C. Yeh ◽  
W. Ma ◽  
H. Kantarjian ◽  
Z. J. Zhang ◽  
J. Cortes ◽  
...  
Keyword(s):  
Cell Line ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Kinase Domain ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Imatinib Resistance ◽  
Abl Kinase

7028 Background: The major mechanism underlying imatinib resistance in patients with chronic myeloid leukemia (CML) is clonal expansion of leukemic cells with point mutations in the BCR-ABL tyrosine kinase. We describe three novel ABL premature termination mutations leading to BCR-ABL truncation in leukemia patients with multidrug (imatinib/nilotinib/dasatinib) resistance. Methods: Peripheral blood or bone marrow samples from drug-resistant CML patients were collected. Total nucleic acids were purified and subjected to two rounds of PCR analysis, with the first PCR designed to eliminate amplification of the wild-type, non-translocated ABL gene. Bi-directional sequencing was then performed. HL60 cells (a Ph-negative myeloid leukemia cell line) and peripheral blood of healthy subjects were used as negative controls; a human CML cell line (K562) was used as a positive control. Results: We identified an exon 7 deletion in three CML patients, a 4-nt insertion (908insCAGG) near the exon 5/6 junction in one CML case, and an exon 6 point mutation (997C>T) in one patient with acute lymphoblastic leukemia (ALL). These mutations all create premature stop codons and cause termination at residues 381, 315, and 333, respectively, leading to truncated proteins with only the first quarter of the kinase domain (P-loop) or lacking the C-terminus of ABL including the A-loop. Conclusions: These novel mutations, and the previously documented 35-nt insertion in exon 8, may constitute a new class of mutations that 1) cause truncation of the BCR-ABL kinase; (2) abolish the regulatory element in the ABL kinase domain and the downstream C-terminal region; and (3) confer significant drug resistance. [Table: see text]

Abl Kinase Domain Mutations Leading to Relapse of Ph+ Acute Lymphoblastic Leukemia (ALL) Occur Commonly and Can Be Detected At Initial Diagnosis: Molecular Results From CALGB 10001

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2541-2541
Author(s):  
Gregory Koval ◽  
Meir Wetzler ◽  
Dorothy Watson ◽  
Kouros Owzar ◽  
Clara D. Bloomfield ◽  
...  
Keyword(s):  
Combination Chemotherapy ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Kinase Domain ◽  
Resistant Clone ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Kinase Domain Mutations ◽  
Kinetics Of

Abstract Abstract 2541 CALGB 10001 tested the feasibility of performing allogeneic or autologous stem cell transplantation (SCT) following combination chemotherapy and tyrosine kinase inhibition with imatinib in patients <60 years old with newly diagnosed Ph+ ALL. Patients received imatinib with each course of chemotherapy prior to SCT. A minimum of 12 months of maintenance therapy with imatinib was recommended following SCT or until minimal residual disease was no longer detected using quantitative real-time PCR (q-PCR). One goal of this study was to determine the incidence of ABL kinase domain mutations in patients who relapsed during or following treatment on CALGB 10001, and to evaluate the kinetics of the resistant clone. 58 patients (median age, 44 yrs) enrolled on CALGB 10001 between 11/2003 and 2/2010. After 3–4 courses of imatinib plus chemotherapy, 15 patients received TBI/etoposide and an alloSCT from a matched sibling donor; 9 (60%) remain in CR (median, 665+ days; range, 152+ to 1208+ days), 2 subsequently relapsed (days +236 and +354), and 4 had transplant-related mortality (TRM). 17 patients received TBI/etoposide/cyclophosphamide and autologous SCT; 10 (59%) remain in CR (median, 742+ days; range, 196+ to 1656+), 9 relapsed (range, 257–522 days), and none had TRM (updated from Wetzler et al, ASH 2007; abstract #2869). Thus, a total of 19 patients have relapsed (8 prior to SCT; 2 after alloSCT; 9 after autoSCT,) at a median of 5.9 months following SCT. All patients were monitored for BCR/ABL1 by q-PCR at sequential time points during and following treatment. Using direct sequencing methods (sensitivity 15–25%), an ABL kinase domain mutation was found in 12 of the 19 (63%) at relapse. The mutations included: 4 with Y253H, 3 T315I, 3 E255K, and 1 E255V. One patient had both a T315I and E255V mutation. All of these mutations are associated with imatinib resistance. To evaluate the kinetics of the resistant clone, we used a quantitative mutation-specific PCR assay (Gruber et al, Leukemia 19:2159, 2005). The sensitivity of the assay was shown to be 1% for T315I and E255V and 0.1% for Y253H and E255K. Using the q-PCR method, we found the resistant clone was detectable at diagnosis in 6 of the 12 patients with kinase domain mutations. In 4 patients, the resistant clone was not detectable until the time of relapse; however, for 3 of these cases the relapse occurred very early, within 2 months of starting treatment. In the remaining 2 cases, the resistant clone was first detected during treatment at 1 and 9 months prior to relapse (Figure 1). In most cases, the kinetics of the resistant clone mirrored the overall BCR/ABL1 population (Figure 2). Nevertheless, the resistant clone proportionally increased in size over time, and at relapse, the resistant clone became the dominant BCR-ABL1 clone. In summary, our series demonstrates that ABL kinase domain mutations occur commonly at relapse in Ph+ ALL despite aggressive therapy with imatinib plus combination chemotherapy followed by autoSCT and in some cases, alloSCT. The relapses appear to be due to the emergence of an imatinib-resistant subclone that is frequently present but subclinical at the time of diagnosis. Improved TKI-based therapies with activity against imatinib-resistant subclones instituted early during treatment of Ph+ ALL are needed to prevent emergence of clonal resistance and eventual relapse. Use of sensitive q-PCR assays to screen for these commonly pre-existing mutations at diagnosis, perhaps using a multiplex method, would provide useful clinical information to allow for optimal therapeutic management of Ph+ ALL.Figure 1.Figure 1. Figure 2.Figure 2. Disclosures: No relevant conflicts of interest to declare.

Comparative Analysis of Two BCR-ABL Small Molecule Inhibitors Reveals Overlapping but Distinct Mechanisms of Resistance.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 552-552 ◽  
Author(s):  
Michael R. Burgess ◽  
Neil P. Shah ◽  
Brian J. Skaggs ◽  
Francis Y. Lee ◽  
Charles L. Sawyers
Keyword(s):  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Point Mutations ◽  
Kinase Domain ◽  
Drug Binding ◽  
Saturation Mutagenesis ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Imatinib Resistant

Abstract A novel dual SRC/ABL kinase inhibitor, BMS-354825, is showing promise for the treatment of imatinib-resistant chronic myeloid leukemia not only in vitro (Shah NP, et al., Science 305:399), but also in a phase I clinical trial (ASH abstract: Sawyers CL, et al.) Resistance to imatinib is increasingly found in patients due to point mutations in the BCR-ABL kinase domain that do not impair kinase activity but prevent drug binding. BMS-354825 is more potent than imatinib and retains activity against 14 of 15 imatinib-resistant BCR-ABL mutants in vitro. The compound’s ability to inhibit imatinib-resistant forms of BCR-ABL is presumed to be due to its relaxed binding requirements, whereas imatinib requires the adoption of a closed conformation of the kinase to bind. We addressed the hypothesis that the relaxed binding requirements of BMS-354825 would limit the range of BCR-ABL mutations that confer drug resistance. To address this question, we employed a saturation mutagenesis experiment as described by others (Azam M, et al., Cell 112:831) and found that the spectrum of BMS-354825-resistant mutants was reduced compared to that of imatinib. In a series of such screens, mutations at only four amino acids have been isolated, two of which account for the vast majority of resistant clones. In contrast, Azam et al. isolated over 20 mutations in a screen for imatinib resistance, a finding which has been generally reproduced in our lab. All four BMS-354825-resistant mutations map to known BMS-354825 contact residues as shown by co-crystallographic studies (ASH abstract: Tokarski JS et al., Bristol-Myers Squibb). Mutations at L248, T315, and F317 show BMS-354825 resistance and have been previously reported to confer imatinib resistance. Mutation at V299 represents a novel mode of resistance. Interestingly, some point mutations conferring BMS-354825 resistance were at positions known to be mutated in cases of imatinib resistance, but the mutated residues differed. Furthermore, the identity of the mutated residue was crucial in conferring sensitivity or resistance to an individual drug as shown by comparison of cellular IC50’s (see table). For example, F317L was shown previously to confer imatinib resistance. F317V, on the other hand, demonstrates relative BMS-354825-resistance but is still exquisitely sensitive to imatinib. In a screen for mutants simultaneously resistant to both drugs, we consistently recover 30–50 fold fewer mutant clones compared to single drug treatment. All such clones isolated to date encode for T315I. Kinase domain point mutation is becoming an increasingly encountered clinical problem in diseases treated with small molecule inhibitors. Our findings suggest that combination therapy with imatinib and BMS-354825 may be of clinical utility in CML, particularly by delaying the development of resistance. IC50 for growth (nM) Baf3 Clone imatinib BMS-354825 p210 wt < 1,000 < 5 T315I > 10,000 > 500 T315A 1,000 100 F317L 2,000 10 F317V < 1,000 60 V299L 1,000 20 L248R > 10,000 20

BCR-ABL P-Loop Deletion Detected in Imatinib-Resistant CML Patients Corresponds to Splice Variant Associated with L248V Point Mutation, Retains BCR-ABL Kinase Activity, and Responds to Dasatinib Treatment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2168-2168
Author(s):  
Nikolas von Bubnoff ◽  
Philipp Erben ◽  
Martin Müller ◽  
Tanja Lahaye ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Splice Variant ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Point Mutations ◽  
Kinase Domain ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Exon 4

Abstract Clonal selection of cells harboring point mutations of the BCR-ABL kinase domain are considered a major cause of resistance to imatinib. More than 40 different point mutations have been described that cause a variable degree of imatinib resistance, and display a differential response to alternative kinase inhibitors, like dasatinib or nilotinib. Here, we describe three cases (2 m, 1 f) with imatinib resistant chronic myelogenous leukemia (CML) associated with a specific deletion of 81 bp of ABL exon 4. Patients were diagnosed with chronic phase (CP) CML at the age of 52, 54, and 68 years. After initial interferon alpha based therapies for 32, 60, and 71 mo, imatinib therapy was initiated at dosages between 400–800 mg per day. After 18, 24, and 29 mo patients lost hematologic response in CP CML (n=2) or progressed to lymphoid blast crisis (BC, n=1). Molecular analysis of the ABL kinase domain revealed a deletion of a 81 bp fragment associated with a loss of amino acids 248–274 in all cases. In one patient, an additional M351T mutation was found. In the two cases with CP CML, dasatinib was commenced for imatinib resistance, resulting in a partial hematologic and minor cytogenetic response (60 and 70% Ph+ metaphases, respectively) after 14 mo of therapy. The patient with lymphoid BC was treated with vincristine and prednisone and died 24 mo after appearance of imatinib resistance. In two cases, sequencing of genomic DNA revealed an underlying CTG/GTG mutation associated with a L248V amino acid switch. The point mutation activated a cryptic splice site within ABL exon 4 leading to an in-frame splice variant characterized by the loss of a 81 bp 3′ portion of exon 4. We sought to evaluate the BCR-ABL kinase activity of the splice variant and the response to tyrosine kinase inhibitors in vitro. The 81 bp deletion of p210 BCR-ABL was cloned using cDNA from one of the patients. Using this construct, retrovirally transduced Ba/F3 cells were transformed upon growth factor withdrawal. These cells expressed BCR-ABL at the transcript and protein levels. Presence of the 81 bp deletion was confirmed by sequencing. Despite the presence of the corresponding 27 amino acid P-loop deletion (Δ248–274), Western blot indicated strong autophosphorylation of BCR-ABL, which decreased in the presence of imatinib to non-detectable levels at concentrations of 1.25μM and above. In the presence of imatinib/nilotinib/dasatinib, the growth of BCR-ABL expressing Ba/F3 cells was shifted from an IC50 of 125/30/0.5nM for wild-type BCR-ABL to 470/185/1.9nM for Δ248–274 cells. Thus, in vitro data demonstrate that deletion of almost the entire P-loop does not abrogate BCR-ABL kinase activity and results in only marginal resistance towards ABL kinase inhibitors. We conclude that deletions of BCR-ABL may be the result of alternative splicing generated by point mutations associated with resistance to imatinib. The Δ248–274 splice variant retains BCR-ABL kinase activity and sensitivity to imatinib, nilotinib, and dasatinib.

Loss of Response to Imatinib: Mechanisms and Management

Hematology ◽  
2005 ◽  
Vol 2005 (1) ◽  
pp. 183-187 ◽  
Author(s):  
Neil P. Shah
Keyword(s):  
Kinase Inhibitor ◽  
Kinase Domain ◽  
Imatinib Resistance ◽  
Early Clinical Trial ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Trial Experience ◽  
Highly Effective ◽  
Mutant Forms

AbstractThe treatment of chronic myeloid leukemia (CML) has been revolutionized by the small molecule BCR-ABL-selective kinase inhibitor imatinib. Although imatinib is highly effective initially and generally well-tolerated, relapse is increasingly encountered clinically. Until recently, for the majority of CML patients with disease no longer responsive to imatinib, as well as for patients with imatinib intolerance, few effective therapeutic options existed. Our understanding of the major mechanisms of imatinib resistance has led to the clinical development of two novel BCR-ABL inhibitors that harbor significant therapeutic promise in early clinical trial experience. These agents, dasatinib (BMS-354825) and AMN107, are more potent inhibitors of BCR-ABL than imatinib, and moreover, harbor activity against nearly all imatinib-resistant BCR-ABL kinase domain mutant forms tested in vitro. Notably, neither of these compounds is effective against the imatinib-resistant BCR-ABL/T315I mutation. The potential availability of highly effective medications for the treatment of imatinib-resistant and intolerant cases of CML is expected to further complicate the timing of other effective therapies, such as allogeneic stem cell transplantation. Additionally, periodic genotyping of the BCR-ABL kinase domain to screen for drug-resistant mutations may play an increasingly important role in the future management of CML cases.

scholarly journals In vitro Activity of Bcr-Abl Inhibitors AMN107 and BMS-354825 against Clinically Relevant Imatinib-Resistant Abl Kinase Domain Mutants

2005 ◽  
Vol 65 (11) ◽  
pp. 4500-4505 ◽  
Author(s):  
Thomas O'Hare ◽  
Denise K. Walters ◽  
Eric P. Stoffregen ◽  
Taiping Jia ◽  
Paul W. Manley ◽  
...  
Keyword(s):  
Kinase Domain ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Abl Kinase ◽  
Imatinib Resistant

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.

A Novel BCR-ABL Mutation Predicts Resistance to Tyrosine Kinase Inhibitor (TKI) Therapy by a Unique Mechanism in Patients (pts) with Philadelphia-Positive (Ph+) Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4773-4773
Author(s):  
Alfonso Quintas-Cardama ◽  
Jorge Cortes ◽  
Hagop M. Kantarjian ◽  
Moshe Talpaz ◽  
Ji Wu ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Abl Kinase ◽  
High Level ◽  
A Minor ◽  
Unique Mechanism

Abstract Albeit most pts with chronic myeloid leukemia (CML) treated with imatinib (IM) have a favorable outcome, some will acquire resistance, mainly due to the development of Abl kinase domain mutations, which confer varying levels of TKI resistance. We describe a novel V304D mutation in pts with Ph+ leukemia who failed TKI therapy. Expression of V304D mutation in BCR-ABL failed to induce cytokine-independence in Ba/F3 cells. Studies in Cos-7 cells demonstrated that this mutant did not induce autophosphorylation and was deficient in kinase activity. We detected V304D mutation in 13 (18%) of 70 IM-resistant pts screened (12 CML, 1 Ph+ acute lymphoblastic leukemia [ALL]), and it was present in a median of 37% (range, 20% to 80%) resistant clones. Median age was 60 years (range, 30 to 81) and median time from diagnosis to IM therapy was 39 months (range, 1 to 91). Eleven (92%) of 12 pts with CML were in chronic phase (CP) at IM start and 1 was in blast phase (BP). Pts received IM for a median of 35 months (range, 2 to 66). Nine pts with CML had failed interferon and 2 (1 CML, 1 Ph+ALL) allogeneic stem cell transplantation prior to IM. Ten (83%) of 12 pts started IM at 400 mg/d but all eventually received ≥600 mg/d. Six pts with CML achieved a complete hematologic response (CHR), 1 BP returned to chronic phase (RCP), and 6 (5 CML, 1 Ph+ALL) had primary hematologic resistance (HR). No cytogenetic (CG) responses were observed and 7 pts with CML CP progressed (4 to AP and 3 to BP) after IM discontinuation. Four pts with CML (1 CP, 2 AP, 1 BP) received nilotinib after IM failure for a median of 2 months (range, 1 to 3.5). Two pts (1 CP, 1 AP) showed primary HR, 1 AP progressed to BP, and 1 BP (on 600 mg twice daily) had a transient (6 weeks) CHR before showing secondary HR. Twelve pts (11 CML, 1 Ph+ALL) received dasatinib: 7 at 70 mg twice daily, 1 at 90 mg daily, 1 at 140 mg daily, 1 at 180 mg daily, 1 at 90 mg twice daily, and 1 at 120 mg twice daily. Dasatinib was administered for a median of 8 months (range, 1 to 23). Two pts achieved CHR and a minor CG response in 1 analysis (75% and 65% Ph+ cells, respectively), 1 RCP, 1 no evidence of leukemia, and 8 (67%) primary HR. One of 4 pts who started dasatinib in CP progressed to AP. Responders to dasatinib had V304D mutation in 20%, 20%, and 25% of clones, respectively. Four pts exhibited concomitant Abl kinase mutations developed prior to dasatinib therapy: 3 with F317L and 1 with G250E. One pt had a 6 base pair in-frame insertion in the TK domain. T315I mutation evolved in 1 pt after dasatinib discontinuation. Eight pts discontinued dasatinib due to disease progression (7 died), 2 were lost to follow-up, and 2 remain on CHR after 17+ and 23+ months on dasatinib. In vitro studies of cells from one pt in CP with V304D mutation (50% of clones) failed to detect CrkL phosphorylation despite detectable expression of the Bcr-Abl protein. In summary, the V304D mutation in the Abl kinase domain results in kinase inactivation and is associated with high-level resistance to TKI therapy, transformation to AP/BP in CML and a particularly poor prognosis. Loss of kinase activity by mutation represents a very unique mechanism of kinase inhibitor resistance and predicts acquisition of other transforming events that support CML cell survival.

A Novel β-catenin Inhibitor, AV65 Suppresses the Growth of CML Cell Lines Which Acquire Imatinib-Resistance Because of Abl Kinase Domain Mutations Including T315I and Hypoxia-Adaptation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1081-1081 ◽  
Author(s):  
Rina Nagao ◽  
Shinya Kimura ◽  
Eishi Ashihara ◽  
Miki Takeuchi ◽  
Ruriko Tanaka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Second Generation ◽  
Kinase Domain ◽  
Parental Cell ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
P Glycoprotein ◽  
Imatinib Resistant ◽  
Kinase Domain Mutations

Abstract Imatinib and the second-generation ABL tyrosine kinase inhibitors (TKIs) such as dasatinib and nilotinib have dramatically changed the management of CML. However, these agents are effective neither for CML cells harboring T315I mutation nor for CML stem cells. Genetic analysis of blasts from CML patients in blast crisis has identified numerous members of the Wnt/b-catenin pathway as being activated (Radich et al, PNAS 2006) and loss of b-catenin has been reported to impair the renewal of CML stem cells (Chao et al, Cancer Cell 2007). Thus, b-catenin signaling inhibition may be useful for CML treatment. We herein describe the effects of a novel b-catenin inhibitor, AV65 on various imatinib-sensitive and –resistant CML cell lines (Table 1). Eight imatinib-sensitive human CML cell lines and one normal cell line derived from hepatocyte were exposed to AV65 for 72 hours and its anti-proliferative effects were examined by MTT assay. AV65 induced apoptosis in these CML cell lines time- and does-dependent manners and inhibited the growth of all eight CML cell lines with IC50 at ranging from 9.8 to 33.1nM. While that for hepatocyte was 204.8nM. Interestingly, AV65 induced polyploidies in K562 but not in BV173. In addition, AV65 augmented the anti-proliferative effects of imatinib additively against K562 and BV173. These findings suggested that AV65 alone or the combination with imatinib was effective for imatinib-sensitive CML cells. To investigated the effects of AV65 against imatinib-resistant CML cells, four imatinib-resistant CML cell lines such as K562/IMR (bcr-abl amplification), MYL-R1 (LYN overexpression), KBM5/ STIR (harboring T315I) and K562/D1-9 (P-glycoprotein overexpression), and BaF3 cells expressing the wild type BCR-ABL or its ten different mutant BCR-ABL forms including T315I, were used. There was little difference on the induction of apoptosis and anti-proliferative effects of AV65 except K562/D1-9 between each parental cell line and its resistant clone and AV65 inhibited the growth of all examined BaF3 cells harboring various mutations including T315I with IC50 at ranging from 21.6 to 46.5nM. IC50 values of AV65 for K562 and K562/D1-9 were 11.0 and 60.1nM, respectively. These findings suggested that the effects of AV65 were independent either to BCR-ABL expression level, ABL mutations or LYN overexpression, but might be affected by P-glycoprotein. Next, the effects of AV65 against the hypoxia-adapted CML cell lines such as K562/ HA and KCL22/HA were also investigated. These cell lines were resistant to imatinib, dasatinib, INNO-406 (another second-generation ABL TKI; Kimura et al, Blood 2005) and alkylating agents via the up-regulation of glyoxalase-I, a detoxification enzyme for the cytotoxic byproducts of glycolysis. Intriguingly, AV65 inhibited the growth of hypoxiaadapted CML cell lines at almost same concentration compared with their parental cell lines. Although CML stem cell niche has not been definitely identified, it might located in hypoxia because the inoculated human leukemia cells to immunodeficient mice preferably localized on the surface of osteoblasts in the epiphysis (Ninomiya et al, Leukemia 2007) where could be hypoxic. Taken together, AV65 may be effective for CML stem cells in hypoxia. In conclusion, AV65 inhibited the growth of CML cell lines which acquire imatinib-resistance because of Abl kinase domain mutations including T315I and hypoxiaadaptation. Therefore, AV65 may become a promising agent for CML treatment including both imatinib and the second-generation Abl TKIs-resistant patients. Figure

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