Identification of a Novel Mode of Kinase Inhibitor Resistance: An F604S Exchange in FIP1L1-PDGFRA Modulates FIP1L1-PDGFRA Protein Stability in a SHP-2 and SRC-Dependent Manner

Abstract Abstract 1739 FIP1L1-PDGFR alpha is a constitutively activated protein kinase which was reported in chronic eosinophilic leukemia (CEL) and in cases of hypereosinophilic syndrome and mastocytosis with eosinophilia. Imatinib is clinically active against FIP1L1-PDGFRA positive disease. However, clinical resistance to imatinib has been observed in FIP1L1-PDGFRA positive leukemia and was shown to occur due to a secondary mutation (T674I) in the PDGFR alpha kinase domain. Using a screening strategy to identify imatinib resistant mutations, we generated numerous imatinib resistant cell clones. Analysis of the PDGFRA kinase domain in these cell clones revealed a broad spectrum of resistance mutations including the clinically reported exchange T674I. Interestingly, one of the abundant mutations was a Phe to Ser exchange at position 604 (F604S), which occurred alone or in combination with other exchanges. Surprisingly, FIP1L1-PDGFRA/F604S did not increase the biochemical or cellular IC50 value to imatinib when compared to wild-type (WT FP). However, F604S and F604S+D842H transformed Ba/F3 and mouse bone marrow more efficiently compared to WT and D842H, respectively. Immunoprecipitation and immunoblotting indicated increased amounts of FIP1L1-PDGFRA protein in F604S versus WT cells. Pulse chase analysis revealed that FIP1L1-PDGFRA/F604S is strongly stabilized compared to WT. SRC coimmunoprecipitated with FIP1L1-PDGFRA in WT, but not F604S cells. Co-expression of SRC in 293T cells augmented degradation of WT, but not F604S FIP1L1-PDGFRA, indicating that SRC is a negative regulator of FIP1L1-PDGFRA protein stability. Importantly both, the SRC inhibitor PD166326 and SRC siRNA mimicked the F604S phenotype and resulted in stabilization of the WT protein. Importantly, phosphatase inhibitor treatment of FIP1L1-PDGFRA/F604S led to destabilization and SRC recruitment indicating that phosphatases might be responsible for the enhanced stability of FIP1L1-PDGFRA/F604S. In fact, coimmunuprecipitaion experiments identified the phosphatase SHP2 as a specific binding partner of F604S and mapping experiments revealed that the phosphatase domain of SHP-2 directly interacted with FIP1L1-PDGFRA/F604S but not with wt- FIP1L1-PDGFRA. Together, these results suggest that stabilization of FIP1L1-PDGFRA/F604S is due to dephosphorylation by SHP-2 leading to lesser activation of the SRC and Cbl mediated ubiquitination machinery. Finally a novel exchange (L629P) identified in imatinib resistance CEL patient also leads to the stabilization of FIP1L1-PDGFRA protein similar to F604S. This indicates that stabilization of FIP1L1-PDGFRA is a common mode of drug resistance in FIP1L1-PDGFRA positive HES or CEL. In summary, imatinib resistance screening identified a novel class of resistance mutations in FIP1L1-PDGFRA, that do not act by impeding drug binding to the target, but increased target protein stability and abundance by interfering with SRC- mediated degradation. Disclosures: No relevant conflicts of interest to declare.

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Identification of a Novel Mode of Kinase Inhibitor Resistance: An F604S Exchange in FIP1L1-PDGFRA Modulates FIP1L1-PDGFRA Protein Stability in a SRC-Dependent Manner

Blood ◽

10.1182/blood.v112.11.2792.2792 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2792-2792

Author(s):

Sivahari P. Gorantla ◽

Nikolas von Bubnoff ◽

Christian Peschel ◽

Justus Duyster

Keyword(s):

Protein Stability ◽

Kinase Activity ◽

Kinase Domain ◽

Sh2 Domain ◽

Drug Binding ◽

Dependent Manner ◽

Resistance Mutations ◽

Cell Clones ◽

Imatinib Resistant ◽

Pdgfr Alpha

Abstract FIP1L1-PDGFR alpha is a constitutively activated protein kinase which was reported in chronic eosinophilic leukemia (CEL) and in cases of hypereosinophilic syndrome and mastocytosis with eosinophilia. Imatinib is clinically active against FIP1L1-PDGFRA positive disease. However, clinical resistance to imatinib has been observed in FIP1L1-PDGFRA positive leukemia and was shown to occur due to a secondary mutation (T674I) in the PDGFR alpha kinase domain. Using a screening strategy to identify imatinib resistant mutations, we generated numerous imatinib resistant cell clones. Analysis of the PDGFRA kinase domain in these cell clones revealed a broad spectrum of resistance mutations including the clinically reported exchange T674I. Interestingly, one of the abundant mutations was a Phe to Ser exchange at position 604 (F604S), which occurred alone or in combination with other exchanges. Surprisingly, FIP1L1-PDGFRA/F604S in contrast to D842H and F604+D842H did not increase the biochemical or cellular IC50 value to imatinib when compared to wild-type (wt). However, F604S and F604S+D842H transformed Ba/F3, NIH3T3 and mouse bone marrow more efficiently compared to wt and D842H, respectively. Also, F604S and F604S+D842H showed strong activation of Stat5, ERK and Akt compared to wt and D842H. Immunoprecipitation and immunoblotting indicated increased amounts of FIP1L1-PDGFRA protein in F604S versus wt cells. Moreover, SRC coimmunoprecipitated with FIP1L1-PDGFRA in wt, but not F604S cells. We hypothesized that F604S might interfere with FIP1L1-PDGFRA protein stability, and that SRC might be involved in this process. GST pull down experiments using SRC-SH2 domain showed lesser binding of FIP1L1-PDGFRA/F604S compared to wt. Similarly, using a GST-PDGFRA fragment, more SRC was precipitated with wt compared to F604S. Importantly both, the SRC inhibitor PD166326 and SRC siRNA mimicked the F604S phenotype and resulted in stabilization of the wt protein. Also, co-expression of SRC in 293T cells augmented degradation of wt, but not F604S FIP1L1-PDGFRA, indicating that SRC is a negative regulator of FIP1L1-PDGFRA protein stability. Similar results were obtained with an exchange in near proximity to F604. Kinase-defective SRC had no effect on FIP1L1-PDGFRA stability, indicating that kinase activity of SRC is necessary for its effect on FIP1L1-PDGFRA stability. Moreover, kinase defective FIP1L1-PDGFRA (G610R) was not degraded indicating that kinase activity of FIP1l1-PDGFRA is necessary for its own degradation. Taken together, imatinib resistance screening in FIP1L1-PDGFRA identified a novel class of resistance mutations, that do not act by impeding drug binding to the target, but rather increase target protein levels by interfering with its SRC mediated degradation.

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Mutation in Abl kinase with altered drug-binding kinetics indicates a novel mechanism of imatinib resistance

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2111451118 ◽

2021 ◽

Vol 118 (46) ◽

pp. e2111451118

Author(s):

Agatha Lyczek ◽

Benedict-Tilman Berger ◽

Aziz M. Rangwala ◽

YiTing Paung ◽

Jessica Tom ◽

...

Keyword(s):

Binding Site ◽

Kinase Domain ◽

Protein Kinase Inhibitors ◽

Drug Binding ◽

The Body ◽

Binding Kinetics ◽

Wild Type ◽

Resistance Mutations ◽

Imatinib Resistance ◽

Abl Kinase

Protein kinase inhibitors are potent anticancer therapeutics. For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for chronic myeloid leukemia by 80%, but 22 to 41% of patients acquire resistance to imatinib. About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain, where more than a hundred different mutations have been identified. Some mutations are located near the imatinib-binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug-binding site, and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies. Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease relevant or resistance causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than twofold compared to Abl wild type. Surprisingly, one-third of mutations in the Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified three clinical Abl mutations that bind imatinib with wild type–like affinity but dissociate from imatinib considerably faster. Given the relevance of residence time for drug efficacy, mutations that alter binding kinetics could cause resistance in the nonequilibrium environment of the body where drug export and clearance play critical roles.

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BCR-ABL Kinase Dynamics and Drug Resistance.

Blood ◽

10.1182/blood.v106.11.1996.1996 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1996-1996 ◽

Cited By ~ 1

Author(s):

Mohammad Azam ◽

Valentina Nardi ◽

William C. Shakespear ◽

Robert R. Latek ◽

Darren Veach ◽

...

Keyword(s):

Kinase Domain ◽

Drug Binding ◽

Hinge Region ◽

Activation Loop ◽

In Vitro Mutagenesis ◽

Resistance Mutations ◽

Imatinib Resistance ◽

Abl Kinase ◽

Clinical Resistance

Abstract The aberrant signaling behavior caused by the expression of BCR-ABL is necessary and sufficient to cause chronic myeloid leukemia (CML), an observation which paved the way for the development of imatinib (GleevecTM), a small molecule inhibitor of the BCR-ABL kinase. Enthusiasm for the remarkable efficacy of imatinib has been tempered by the development of clinical resistance. The most common mechanisms for resistance are the development of kinase domain mutations and/or overexpression of the BCR-ABL gene, with mutations in the kinase accounting for ~90 % of all cases. The resistance-conferring lesions are found in regions of the kinase that are critical to its autoregulation, such as P-loop, C-helix, gatekeeper area, activation loop and the SH2-C-lobe interface. Mechanistically, these mutations effect either a steric blockade or a change in the dynamic equilibrium that favors the active kinase conformation that precludes imatinib binding. We have analyzed two dual Src-Abl kinase inhibitors, AP23464 and PD166326, against 58 BCR-ABL kinase variants conferring imatinib resistance. PD166326 binds to the Abl kinase domain in the open although enzymatically inactive conformation, while AP23464 targets the active conformation. Both of these compounds have effectively suppressed the cell growth of imatinib resistance variants, except for a recurrent mutation in the gatekeeper residue (T315I). The P-loop variants are more sensitive to AP23464 than PD166326. Interestingly, the imatinib resistant variants from the C-helix, hinge region, activation loop and SH2-C-lobe region, are hypersensitive to both compounds, as compared to native BCR-ABL. The BCR-ABL variants in the C-helix, gatekeeper area, and the activation loop are more sensitive to AP23464 than PD166326, while variants from the hinge region and the SH2-C-lobe interface are hypersensitive to PD166326. Altogether, these results define a differential requirement for a specific ABL conformation for drug binding of AP23464 and PD166326. In order to better understand their structure activity relationships and the patterns of resistance, we carried out an in-vitro mutagenesis-screen using different concentration of the drug either alone or in combination with imatinib. AP23464 mediates 2–3 time less resistance than PD166326. A higher concentration of all three compounds suppresses all resistance mutations, save for the notable exceptions, T315I and F317L/VandC. Resistance conferring mutations selected at 10–20 fold higher IC50 values are different. AP23464 efficiently suppresses the mutations from the P-loop (except E255K) and two mutations from the activation loop, while PD166326 remains refractory to the mutations in the C-helix and SH2-C-lobe interface. In combination with imatinib, AP23464 and PD166326 suppressed the emergence of most resistance mutations, with the notable exception of T315I. These in-vitro studies demonstrate that the combination of two or three different conformation specific inhibitors is needed to suppress the emergence of resistance. We are characterizing variants of AP23464 that we predict will show activity against the most challenging imatinib resistance mutant T315I.

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Cumulative mechanism of several major imatinib-resistant mutations in Abl kinase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1919221117 ◽

2020 ◽

Vol 117 (32) ◽

pp. 19221-19227 ◽

Cited By ~ 1

Author(s):

Marc Hoemberger ◽

Warintra Pitsawong ◽

Dorothee Kern

Keyword(s):

Resistance Mechanisms ◽

Kinase Domain ◽

Drug Binding ◽

Prolonged Treatment ◽

Cancer Drug ◽

Resistance Mutations ◽

Imatinib Resistant ◽

Order Of Magnitude ◽

Patient Resistance ◽

Emergence Of Resistance

Despite the outstanding success of the cancer drug imatinib, one obstacle in prolonged treatment is the emergence of resistance mutations within the kinase domain of its target, Abl. We noticed that many patient-resistance mutations occur in the dynamic hot spots recently identified to be responsible for imatinib’s high selectivity toward Abl. In this study, we provide an experimental analysis of the mechanism underlying drug resistance for three major resistance mutations (G250E, Y253F, and F317L). Our data settle controversies, revealing unexpected resistance mechanisms. The mutations alter the energy landscape of Abl in complex ways: increased kinase activity, altered affinity, and cooperativity for the substrates, and, surprisingly, only a modestly decreased imatinib affinity. Only under cellular adenosine triphosphate (ATP) concentrations, these changes cumulate in an order of magnitude increase in imatinib’s half-maximal inhibitory concentration (IC50). These results highlight the importance of characterizing energy landscapes of targets and its changes by drug binding and by resistance mutations developed by patients.

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

Blood ◽

10.1182/blood.v104.11.552.552 ◽

2004 ◽

Vol 104 (11) ◽

pp. 552-552 ◽

Cited By ~ 1

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

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Mutation in Abl kinase with altered drug binding kinetics indicates a novel mechanism of imatinib resistance

10.1101/2021.06.28.449968 ◽

2021 ◽

Author(s):

Agatha Lyczek ◽

Benedict-Tilman Berger ◽

Aziz M Rangwala ◽

YiTing Paung ◽

Jessica Tom ◽

...

Keyword(s):

Binding Site ◽

Kinase Domain ◽

Protein Kinase Inhibitors ◽

Drug Binding ◽

The Body ◽

Binding Kinetics ◽

Wild Type ◽

Resistance Mutations ◽

Imatinib Resistance ◽

Abl Kinase

Protein kinase inhibitors are potent anti-cancer therapeutics. For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for Chronic Myeloid Leukemia (CML) by 80%, but 22-41% of patients acquire resistance to imatinib. About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain, in which more than a hundred different mutations have been identified. Some mutations are located near the imatinib binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug binding site and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies (10). Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease-relevant or resistance-causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than two-fold compared to Abl wild type. Surprisingly, one-third of mutations in Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified a clinical Abl mutation that binds imatinib with wild type-like affinity but dissociates from imatinib three times faster. Given the relevance of residence time for drug efficacy, mutations that alter binding kinetics could cause resistance in the non-equilibrium environment of the body where drug export and clearance play critical roles.

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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 ◽

10.1182/blood.v110.11.1940.1940 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1940-1940 ◽

Cited By ~ 2

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.

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

Blood ◽

10.1182/blood.v116.21.2135.2135 ◽

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.

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Landscape of drug-resistance mutations in kinase regulatory hotspots

Briefings in Bioinformatics ◽

10.1093/bib/bbaa108 ◽

2020 ◽

Cited By ~ 3

Author(s):

Pora Kim ◽

Hanyang Li ◽

Junmei Wang ◽

Zhongming Zhao

Keyword(s):

Drug Resistance ◽

Large Scale ◽

Kinase Inhibitors ◽

Growth Factor Receptor ◽

Kinase Domain ◽

Systematic Investigation ◽

Drug Binding ◽

Data Sets ◽

Resistance Mutations ◽

Cancer Data

Abstract More than 48 kinase inhibitors (KIs) have been approved by Food and Drug Administration. However, drug-resistance (DR) eventually occurs, and secondary mutations have been found in the previously targeted primary-mutated cancer cells. Cancer and drug research communities recognize the importance of the kinase domain (KD) mutations for kinasopathies. So far, a systematic investigation of kinase mutations on DR hotspots has not been done yet. In this study, we systematically investigated four types of representative mutation hotspots (gatekeeper, G-loop, αC-helix and A-loop) associated with DR in 538 human protein kinases using large-scale cancer data sets (TCGA, ICGC, COSMIC and GDSC). Our results revealed 358 kinases harboring 3318 mutations that covered 702 drug resistance hotspot residues. Among them, 197 kinases had multiple genetic variants on each residue. We further computationally assessed and validated the epidermal growth factor receptor mutations on protein structure and drug-binding efficacy. This is the first study to provide a landscape view of DR-associated mutation hotspots in kinase’s secondary structures, and its knowledge will help the development of effective next-generation KIs for better precision medicine.

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Point Mutation and Alternative Splicing in the ABL Kinase Domain as a Mechanism for Imatinib Resistance.

Blood ◽

10.1182/blood.v108.11.4814.4814 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4814-4814

Author(s):

Rong Chen ◽

Steven Potts ◽

Wanlong Ma ◽

Hagop Kantarjian ◽

Francis Giles ◽

...

Keyword(s):

Alternative Splicing ◽

Tyrosine Kinase ◽

Point Mutations ◽

Sr Proteins ◽

Kinase Domain ◽

Tyrosine Kinase Domain ◽

Imatinib Resistance ◽

Exon Junction ◽

Abl Tyrosine Kinase ◽

Imatinib Resistant

Abstract Missense point mutations in the region encoding the ABL tyrosine kinase domain have been reported in approximately 35% of patients with imatinib-resistant chronic myeloid leukemia (CML). The reported mutations result in reactivation of the BCR-ABL tyrosine kinase. Screening patients with imatinib-resistant CML, we identified 42 different mutations in the ABL tyrosine kinase domain-encoding region, 2 of which were silent (no amino acid change): A864G and G909A. The A864G mutation was associated with a 54-nucleotide reduction in the length of the mRNA transcript, representing a loss of nucleotides 1089-1143; the G909A mutation was associated with a normal-length transcript. The nt1089-1143 transcript deletion represents a partial exon deletion in which the first half of exon 8 is skipped, suggesting that A864G leads to abnormal splicing. Splicing is regulated by 6- to 8-nucleotide exonic splicing enhancer (ESE) and exonic splicing silencer (ESS) motifs recognized by the SR proteins (a family of splicing factors). We therefore used ESEfinder to examine whether A864G or G909A alter ESE motifs, which could block the ability of SR proteins to recognize and bind. This search showed that A864G is at the 7th position of an AGCTGCAG ESE motif, a binding site for SR35, and is within 35 bp of the intron-exon junction. In total, ESEfinder predicted 18 putative SR35-binding ESEs within 50 bp of the intron-exon junction, covering 20% of the kinase domain. The AGCTGCAG motif is conserved in primates (chimpanzees and monkeys) but not in rodents, while A864G is found in mice Although similar links cannot be made with the G909A mutation, these data suggest that imatinib resistance may develop in some patients through alternative splicing and the expression of a truncated (or potentially elongated) protein.

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