Myristate Binding Pocket Research Articles

Select P-Loop Mutants in BCR::ABL1 Confer Moderate to High Degrees of Resistance to Asciminib

Introduction: NCCN CML Guidelines describe BCR::ABL1 mutants that are contraindicated for select ATP-competitive BCR:ABL1 TKIs due to their association with in vitro and clinical resistance. The allosteric ABL1 kinase inhibitor asciminib was recently approved in the US at a "low” dose of 40 mg twice daily (or 80 mg once daily) in patients with resistance or intolerance to two prior TKIs, and a "high” dose of 200 mg twice daily in CML patients with BCR::ABL1/T315I. Prior work demonstrated ~10x reduced sensitivity of BCR::ABL1/T315I to asciminib compared with native BCR::ABL1 in cellular proliferation studies. Based on its unique mode of binding to a myristate binding pocket, it has been hypothesized that asciminib may complement all approved ATP-competitive TKIs due to presumed non-overlapping mechanisms of resistance with respect to kinase domain mutation. Although several ATP-competitive kinase domain mutants (Q252H, V299L, E355G, F359C/I/V) confer a degree of in vitro resistance similar to T315I (Wylie et al, Nature 2017; Eide et al, Cancer Cell 2019), at the present time, treatment with low dose asciminib is indicated in cases that harbor these mutations. There are no formal guidelines for dose escalation in the event of poor response. To date, documentation of clinical activity of asciminib in patients with pre-existing BCR::ABL1 kinase domain mutations has been limited, and several kinase domain mutants have yet to be assessed for sensitivity to asciminib in vitro. NCCN CML Guidelines list mutations at two positions for which asciminib is contraindicated. These amino acid positions map to the asciminib binding pocket (A337, P465). Mutations at these residues retain sensitivity to ATP-competitive TKIs. Results: We recently treated two chronic phase CML patients whose disease harbored the imatinib-resistant BCR::ABL1/M244V mutation with asciminib 80 mg once daily. Both patients demonstrated failure to respond. In both cases, despite dose escalation (to 120 mg twice daily and 200 mg twice daily), asciminib was discontinued due to rising blood counts or BCR::ABL1 transcript level. One patient was subsequently switched to ponatinib 15 mg daily and has responded well; the other patient has maintained disease control on dasatinib. The in vitro activity of asciminib against BCR::ABL1/M244V has not been previously reported. We therefore assessed the sensitivity of Ba/F3 cells transduced with BCR::ABL1/M244V. BCR::ABL1/M244V confers a surprisingly high degree of resistance to asciminib (IC50 increased > 10-fold relative to BCR::ABL1/T315I). BCR::ABL1/M244V confers a surprisingly high degree of resistance to asciminib (IC50 increased > 10-fold relative to BCR::ABL1/T315I), albeit less than that conferred by the previously described asciminib-specific mutant BCR::ABL1/A337V. Interestingly, M244 resides at the base of the P-loop, at considerable distance from the asciminib binding pocket. We therefore tested three other resistance-conferring mutants in proximity to M244V that have not been previously profiled (L248R/V, Y253F), and found that two of these mutants (L248V and Y253F) indeed exhibit moderate resistance relative to native BCR::ABL1, similar to that observed with BCR::ABL1/T315I. These findings suggest that select alterations in the kinase P-loop can lead to conformational changes in myristate binding pocket. Conclusions: BCR::ABL1/M244V confers a high degree of in vitro resistance and is associated with clinical resistance to even high doses of asciminib. Asciminib should therefore be considered contraindicated in patients with the BCR::ABL1/M244V mutation. Furthermore, several additional commonly encountered BCR::ABL1 kinase domain P-loop mutations (L248V, Y253F, F359C/I/V) that confer clinical resistance to ATP-competitive TKIs confer substantial cross-resistance to asciminib in vitro. While assessment of clinical response in patients with these mutations to asciminib is required, it appears unlikely that the currently approved dose of asciminib will be active in CML cases that harbor these mutants, and they may lead to acquired resistance. These mutations represent critical vulnerabilities for ongoing investigational efforts to combine asciminib with imatinib in an attempt to suppress the outgrowth of drug-resistant BCR::ABL1 mutants.

The new allosteric inhibitor asciminib is susceptible to resistance mediated by ABCB1 and ABCG2 overexpression in vitro.

Asciminib (previously ABL001), which binds the myristate-binding pocket of the Bcr-Abl kinase domain, is in phase I clinical trials as monotherapy and in combination with imatinib, nilotinib and dasatinib for the treatment of patients with refractory CML or Ph+ ALL. Asciminib sensitivity was evaluated in asciminib naïve BCR-ABL1+ cell lines K562 (negligible ABCB1/ABCG2 expression), K562-Dox (ABCB1-overexpressing through doxorubicin exposure) and K562-ABCG2 (ABCG2 overexpression via transduction) with results demonstrating asciminib efflux by both ABCB1 and ABCG2 transporters. K562-Dox and K562-ABCG2 cells demonstrated increased LD50asciminib vs K562 control cells: 256 and 299 nM respectively vs 24 nM, p < 0.001. Sensitivity was completely restored with specific inhibitors cyclosporine (ABCB1) and Ko143 (ABCG2): K562-Dox LD50asciminib+cyclosporine = 13 nM, K562-ABCG2 LD50asciminib+Ko143 = 15 nM (p < 0.001). When asciminib resistance was modelled in vitro, ABCB1 and ABCG2 overexpression was integral in the development of asciminib resistance. In K562 asciminib-resistant cells, ABCG2 expression increased prior to BCR-ABL1 overexpression and remained high (up to 7.6-fold greater levels in resistant vs control cells, p < 0.001). K562-Dox asciminib-resistant cells had increased ABCB1 expression (2.1-fold vs control cells p = 0.0033). KU812 asciminib-resistant cells overexpressed ABCB1 (5.4-fold increase, p < 0.001) and ABCG2 (6-fold increase, p < 0.001) before emergence of a novel myristate-binding pocket mutation (F497L). In all three cell lines, asciminib resistance was reversible upon chemical inhibition of ABCB1, ABCG2 or both (p < 0.001). When K562 asciminib-resistant cells were treated with asciminib in combination with clinically achievable doses of either imatinib or nilotinib, reversal of the resistance phenotype was also observed (p < 0.01). Overexpression of efflux transporters will likely be an important pathway for asciminib resistance in the clinical setting. Given the lack of evidence for ABCG2-mediated transport of nilotinib or imatinib at clinically relevant concentrations, our data provide an additional rationale for using asciminib in combination with either TKI.

A Low Concentration of ABL001 Potentiates In Vitro TKI-Induced Bcr-Abl Kinase Inhibition in CML Cells

▪Background ABL001 (Novartis) is an allosteric inhibitor that binds to the myristate binding pocket of Abl to hold Bcr-Abl in the inactive conformation. A Phase I multicentre trial of ABL001 in chronic myeloid leukemia (CML) is ongoing (Protocol CABL001X2101), and includes testing ABL001 in combination with the ATP-competitive Tyrosine Kinase Inhibitors (TKIs) imatinib (IM), nilotinib (NIL) and dasatinib (DAS) to determine if simultaneous targeting of the myristate and ATP binding pockets provides greater pharmacological inhibition of Bcr-Abl and promotes greater reduction in tumor burden. In vitro sensitivity to TKIs can be measured by assessing the phosphorylation status of the Bcr-Abl substrate CrkL. Using this method, high IM in vitro sensitivity at diagnosis (IC50IM <0.6 µM; White et al, Blood 2005) is predictive of molecular response for patients treated with 600 mg IM.Aim To investigate if the in vitro combination of ABL001 (at a pharmacologically-relevant concentration) with ATP-competitive inhibitors is more effective atBcr-Abl kinase blockade than IM, NIL or DAS as single agents.Methods Blood mononuclear cells (MNC) were isolated with informed consent either prior to TKI therapy (n=6 de novo chronic phase CML patients) or at TKI-switch (n=4 resistant, intolerant or non-compliant patients). For in vitro sensitivity, MNC were treated for 2 h with a pre-determined range of concentrations of IM, NIL and DAS -/+ 1.2 µM ABL001 (based on human PK parameters from Novartis, average peak plasma concentration on 40 mg BID = 578 ng/mL or 1.24 µM) or a much lower dose of 1.2 nM; or with ABL001 alone. Phosphorylated-Crkl (p-Crkl) levels were determined by western blot and the IC50 was calculated by densitometry.Results Kinase inhibition was markedly enhanced by addition of 1.2 µM ABL001 as compared to TKIs alone with mean IC50s without and with ABL001 of 1.52 µM ± 0.46 µM vs. 0.019 µM ± 0.012 µM (p=0.014), 79.1 nM ± 8.8 nM vs. 0.26 nM ± 0.22 nM (p<0.0001) and 2.3 nM ± 0.3 nM vs. 0.014 nM ± 0.007 nM (p=0.002) for IM, NIL and DAS respectively (Fig 1A-C). Since NIL and DAS IC50 values were reduced to picomolar levels in the presence of 1.2 µM ABL001 we then tested a 1000-fold lower ABL001 concentration of 1.2 nM. Similar results were obtained: the observed reductions in IC50s with 1.2 nM ABL001 were 86% (n=8, p=0.013), 72% (n=9, p<0.0001) and 90% (n=3, p=0.0024) for IM, NIL and DAS respectively (Fig 1A-D). In contrast, ABL001 alone was unable to completely inhibit phosphorylation of Crkl in 2 h, thus IC50 calculation was not possible. Collectively, 8 patients of various disease status were tested with IM and all had high IC50IM >0.6 µM, however, with the addition of just 1.2 nM ABL001, the IC50IM was reduced in all cases to <0.6 µM (Fig 1D, p=0.0078), a level that predicts excellent outcomes with IM therapy. The ABL001-induced reductions in TKI IC50s were of similar magnitudes in previously untreated patients as well as those switching TKIs for resistance or intolerance.Conclusions These data suggest that (1) in vitro kinase inhibition achieved with ABL001 in combination with IM, NIL or DAS is greater than that achieved with TKIs alone, even at a concentration 1000-fold lower than achievable on the current ABL001 dosing regimen; and (2) patients with high IC50IM who would be predicted to respond poorly to 600 mg IM have dramatically increased kinase inhibition with the addition of just 1.2nM ABL001. These results suggest that simultaneous targeting of themyristate and ATP binding pockets ofBcr-Abl may be more effective than targeting either site alone and provides a strong rationale for combining IM and ABL001, particularly in patients with low intrinsic sensitivity to IM. [Display omitted] DisclosuresYeung:Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Research Funding. White:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hughes:Australasian Leukaemia and Lymphoma Group (ALLG): Other: Chair of the CML/MPN Disease Group; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Identification of TKI-Sensitive Point Mutations That Activate c-ABL Kinase Activity and Transformation Potential and Confer in Vitro Resistance to the Allosteric ABL Inhibitor GNF-5

Introduction:Pathologically activated tyrosine kinases represent attractive therapeutic targets, with deregulated ABL kinase representing one of the best-validated examples.While physiologic activation of c-ABL is tightly controlled by intramolecular interactions, ABL fusion proteins are constitutively activated by oligomerization driven by the fusion partner, as well as by loss of kinase autoinhibition due to removal of an autoinhibitory myristate site at the N-terminus of c-ABL (Nagar et al, Cell, 2003). Recent routine genomic tumor sequencing of various human malignancies has identified point mutations of unknown significance in numerous kinases, including c-ABL, and it is expected that the increasingly common practice of tumor genome sequencing will provide new opportunities for tailored targeted therapy. To maximize therapeutic benefit and avoid unnecessary cost and toxicity, it will be critical to distinguish "driver" from "passenger" mutations. We therefore sought to: (i) test the transforming potential of select clinically detected c-ABL mutants, (ii) prospectively identify novel additional activating c-ABL point mutations, and (iii) determine if active mutant c-ABL isoforms retain sensitivity to ABL TKIs.Results:Two of four clinically identified c-ABL mutations tested caused a dramatic increase in ABL kinase activity and transformed Ba/F3 cells to growth factor independence. A mutagenesis screen of c-ABL identified six additional transforming point mutations in the SH3 and kinase domains, including one that has been reported clinically in breast invasive carcinoma (cBioPortal.org). Relative to BCR-ABL, all c-ABL mutants showed heightened sensitivity to imatinib. Allosteric site inhibitors targeting the myristate-binding pocket in the BCR-ABL kinase domain (KD) have been shown to inhibit BCR-ABL kinase activity, purportedly by mimicking binding of the c-ABL myristate group and establishing c-ABL-like autoinhibitory constraints (Hantschel, Haematologica, 2012). While only two c-ABL mutations from the screen were near the allosteric site, all mutants exhibited decreased sensitivity to the allosteric inhibitor GNF-5. Recent studies have shown that derivative compounds of GNF-5 targeting this site in BCR-ABL are subvertable by on-target resistance mutations. We tested whether three BCR-ABL mutations described to confer in vitro, and in one case clinical, resistance to ABL001 (currently undergoing clinical trial evaluation) could impact c-ABL kinase activity presumably by perturbing this autoinhibitory pocket. In the context of c-ABL, all three ABL001-resistant mutations activated ABL kinase, transformed Ba/F3 cells, and conferred substantial resistance to allosteric inhibition relative to native BCR-ABL. These findings raise the possibility that c-ABL mutations may play a role in resistance to emerging allosteric ABL inhibitors. Importantly, previous work suggested that the genomic breakpoint on chromosome 9 in one third of CML cases is located 5' to ABL exon 1b, which would be predicted to result in simultaneous KD mutation expression in BCR-ABL and c-ABL (Uphoff et al, Leuk Res, 1999). To definitively demonstrate that co-expression of KD mutations in c-ABL and BCR-ABL can occur in primary patient samples, we analyzed RNA from five CML patients harboring a dominant BCR-ABL T315I mutation, and identified a heterozygous c-ABL 1b T315I mutation in one case.Conclusions:We have identified eight novel activating point mutations in c-ABL (three found in clinical isolates) that are potential therapeutic targets of ABL TKIs. Activating c-ABL mutants, including T315I, confer substantial resistance to the allosteric ABL inhibitor GNF-5 regardless of proximity to the inhibitor-binding site, implying resistance mechanisms beyond mere steric hindrance. Thus, mutations may induce distal conformational changes by disrupting a kinase conformation required for allosteric compound inhibition. In a subset of CML cases, c-ABL point mutants are co-expressed from the same allele as BCR-ABL, suggesting that the location of the chromosome 9 breakpoint may negatively impact clinical responsiveness to allosteric ABL inhibitors. Sequence analysis of c-ABL in patients with resistance to allosteric ABL inhibitors is required to validate c-ABL point mutants as a clinically important mechanism of resistance to this emerging class of targeted therapeutics. DisclosuresOff Label Use: the use of ABL TKIs for malignancies associated with point mutations in c-ABL. Shah:Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding; Plexxikon Inc.: Research Funding.

Discovery of allosteric BCR-ABL inhibitors from phenotypic screen to clinical candidate.

The development of imatinib, an ATP-competitive inhibitor of the BCR-ABL oncoprotein, has revolutionized the treatment of chronic myelogenous leukemia (CML). Unfortunately, the leukemia eventually becomes resistant imatinib as a result of emergence of cells expressing drug insensitive BCR-ABL mutant proteins. This has motivated the development of several next-generation ATP-competitive drugs. This chapter describes the discovery and development of a complementary strategy involving inhibiting BCR-ABL by targeting an allosteric binding site. Compounds that bind to the myristate-binding pocket of BCR-ABL are able to induce formation of an "inactive" state and are able to overcome resistance mutations located in the ATP-binding pocket including the recalcitrant T315I "gatekeeper" mutation. Myristate-pocket inhibitors are also able to function synergistically with ATP-competitive inhibitors in cellular and murine models of CML and this dual inhibitory strategy is currently being investigated in the clinic.

Influence of N-Myristylation and Ligand Binding on the Flexibility of the Catalytic Subunit of Protein Kinase A

The catalytic (C) subunit of protein kinase A is regulated in part by cotranslational N-myristylation and ligand binding. Using a combination of time-resolved fluorescence anisotropy and molecular dynamics (MD) simulations, we characterized the effect of N-myristylation and ligand binding on C-subunit dynamics. Five single-site cysteine-substitution mutants of the C-subunit were engineered with and without N-terminal myristylation and labeled with fluorescein maleimide, and time-resolved fluorescence anisotropy decays were measured to assess the flexibility of the labeled regions in the presence and absence of ligands. A parallel set of in silico experiments were performed to complement the experimental findings. These experiments showed that myristylation produces both local and global effects on C-subunit dynamics. The local effects include stabilization of the N-terminus and myristate pocket, and the global effects include small increases in mobility along the C-tail at residue C343. Additionally, ligand binding was associated with an increase in mobility of the myristate binding pocket for both the myristylated and nonmyristylated enzyme on the basis of both the experimental and MD results. Also, MD simulations suggest that the myristylated protein exhibits increased dynamics when bound to ligands compared to the nonmyristylated protein.

Abstract 2221: Enhanced SH3:linker interaction allosterically sensitizes Abl kinases to small molecule inhibitors.

Abstract Bcr-Abl is the oncogenic protein-tyrosine kinase responsible for the pathogenesis of chronic myelogenous leukemia (CML). Clinical management of CML has been revolutionized by imatinib, a selective ATP-competitive inhibitor of Bcr-Abl kinase activity. Imatinib selectivity is due in part to its ability to recognize and stabilize the inactive conformation of the kinase domain. This conformation is stabilized by intramolecular binding of the SH3 domain to the SH2-kinase linker, which in turn packs against the back of the kinase domain. Imatinib resistance can be caused by point mutations that either affect drug binding directly (e.g., through mutation of the gatekeeper position, Thr315) or that allosterically disturb the downregulated kinase domain conformation (e.g., through mutation or phosphorylation of the SH3 domain). These observations suggest that strengthening the regulatory SH3:linker interaction present in Bcr-Abl may stabilize a downregulated kinase domain conformation, thus sensitizing the kinase domain to inhibitor action. To explore this possibility, we created a series of modified c-Abl and Bcr-Abl proteins with enhanced SH3-linker interactions. By systematically increasing the proline content of the linker, we identified high-affinity linkers that stabilized intramolecular SH3 binding without disturbing the overall regulation of the kinase core. Enhanced SH3:linker interaction completely reversed c-Abl core activation by a C-lobe myristate binding pocket mutation, and substantially reduced activation by mutations in the ATP binding site (T315I) as well as the SH2:C-lobe interface (Y158D). Remarkably, enhanced SH3:linker interaction dramatically sensitized Bcr-Abl not only to imatinib but also to the myristic acid binding pocket inhibitor, GNF-2. These effects were observed in the context of both wild-type and imatinib-resistant forms of Bcr-Abl. Hydrogen exchange mass spectrometry of recombinant Abl core proteins with high-affinity linkers revealed dynamic coupling between the SH3:linker interface, the GNF-2 binding site in the C-lobe myristate binding pocket, and the active site. Taken together, these studies provide strong evidence that regulatory SH3:linker interaction is retained in the context of Bcr-Abl, and supports the idea that small molecules enhancing natural regulatory interaction at the SH3:linker interface may have utility as chemical sensitizers of Bcr-Abl drug action in the kinase domain. Citation Format: Shoghag B. Panjarian, Roxana Iacob, Chen Shugui, Thomas Wales, John R. Engen, Thomas E. Smithgall. Enhanced SH3:linker interaction allosterically sensitizes Abl kinases to small molecule inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2221. doi:10.1158/1538-7445.AM2013-2221

Enhanced SH3/Linker Interaction Overcomes Abl Kinase Activation by Gatekeeper and Myristic Acid Binding Pocket Mutations and Increases Sensitivity to Small Molecule Inhibitors*

Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control.

Enhanced SH3:Linker Interaction Suppresses Activating Mutations of the c-Abl Protein-Tyrosine Kinase.

AbstractAbstract 1208Bcr-Abl, the chimeric protein-tyrosine kinase expressed as a result of the Philadelphia chromosome translocation, plays a pivotal role in the initiation and maintenance of chronic myelogenous leukemia (CML). Imatinib (Gleevec) is an ATP-competitive Bcr-Abl inhibitor that selectively kills Bcr-Abl+ CML cells. Despite its clinical success, imatinib is less effective in the advanced stages of CML due to the emergence of drug resistance caused by point mutations in the Abl kinase domain. Second generation Bcr-Abl inhibitors such as dasatinib and nilotinib are active against most imatinib-resistant forms of Bcr-Abl, with the exception of the T315I “gatekeeper” mutant. The Abl gatekeeper residue (Thr315) is located between the ATP-binding site and an adjacent hydrophobic pocket, and forms a key hydrogen bond with imatinib. Additionally, the T315I mutation produces a strong activating effect on the downregulated c-Abl “core,” consisting of the myristoylated N-terminal Ncap, tandem SH3 and SH2 regulatory domains, the SH2-kinase linker, which forms a polyproline type II helix for internal SH3 docking, and the tyrosine kinase domain. Using hydrogen-exchange mass spectrometry, we recently found that the T315I mutation not only induced conformational changes in the Abl kinase domain as expected, but also at a distance in the RT-loop of the SH3 domain. Such changes may allosterically contribute to kinase domain activation by disturbing the negative regulatory influence of SH3:linker interaction. Recently, a new class of allosteric Bcr-Abl inhibitors has been reported that targets the myristate-binding pocket of Abl, which localizes to C-lobe of the kinase domain and away from the active site. Together with our finding that the T315I mutation perturbs SH3:linker interaction, these inhibitors support the existence of an extensive network of allosteric interactions that work together to regulate Abl kinase activity. In this project, we investigated whether enhanced SH3:linker interaction can allosterically reverse the activating effects of the T315I imatinib resistance mutation as well as mutations of the N-terminal myristoylation site and myristic acid binding pocket. We created modified versions of Abl [High Affinity Linker proteins (HALs)] by mutating multiple residues within the SH2-kinase linker to proline, thereby enhancing the SH3 domain binding affinity. Using mammalian cell-based expression assays and immunoblotting with phosphospecific antibodies, we identified five of eleven Abl-HAL proteins that did not exhibit changes in basal kinase activity. The Abl-HAL protein with the greatest enhancement of SH3:linker interaction was then combined with the T315I mutation, a myristoylation-defective mutant, and a myristic acid binding pocket mutation. Remarkably, this HAL substitution completely reversed the activating effect of the myristic acid binding pocket mutation, while substantially suppressing the activity of Abl T315I and the myristoylation-defective mutant. These results indicate that stabilization of SH3:linker interaction allosterically represses Abl activation by a wide variety of mechanisms, and suggests a new approach to allosteric control of Bcr-Abl kinase activity. Disclosures:No relevant conflicts of interest to declare.

Myristoylation and Membrane Binding Regulate c-Src Stability and Kinase Activity

Myristoylation is critical for membrane association of Src kinases, but a role for myristate in regulating other aspects of Src biology has not been explored. In the c-Abl tyrosine kinase, myristate binds within a hydrophobic pocket at the base of the kinase domain and latches the protein into an autoinhibitory conformation. A similar pocket has been predicted to exist in c-Src, raising the possibility that Src might also be regulated by myristoylation. Here we show that in contrast to the case for c-Abl, myristoylation exerts a positive effect on c-Src kinase activity. We also demonstrate that myristoylation and membrane binding regulate c-Src ubiquitination and degradation. Nonmyristoylated c-Src exhibited reduced kinase activity but had enhanced stability compared to myristoylated c-Src. We then mutated critical residues in the predicted myristate binding pocket of c-Src. Mutation of L360 and/or E486 had no effect on c-Src membrane binding or localization. However, constructs containing a T456A mutation were partially released from the membrane, suggesting that mutagenesis could induce c-Src to undergo an artificial myristoyl switch. All of the pocket mutants exhibited decreased kinase activity. We concluded that myristoylation and the pocket residues regulate c-Src, but in a manner very different from that for c-Abl.

Abstract B165: Abl kinase myristate-site inhibitor, mechanism and application

Abstract We recently reported a new class of allosteric inhibitors, exemplified by GNF-2, that selectively inhibit the proliferation of Bcr-Abl dependent cells. Here we demonstrate, using selection for resistant Bcr-Abl clones, site-directed mutagenesis, affinity chromatography, and steady-state kinetics, that GNF-2 inhibits Bcr-Abl kinase activity by binding to the Abl myristate binding pocket and stabilizes the auto-inhibited conformation. We demonstrate that the 2-hydroxyethyl amide analog of GNF-2, GNF-5, in combination with ATP-competitive inhibitors such as nilotinib and dasatinib can overcome the T315I “gatekeeper” mutant of Bcr-Abl which is resistant to all clinically approved Bcr-Abl inhibitors. These studies demonstrate that targeting of the Abl myristate binding site can provide an important pharmacological means to overcome mutations that cause resistance to ATP-competitive inhibitors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B165.

N-Myristoylated c-Abl Tyrosine Kinase Localizes to the Endoplasmic Reticulum upon Binding to an Allosteric Inhibitor

Allosteric kinase inhibitors hold promise for revealing unique features of kinases that may not be apparent using conventional ATP-competitive inhibitors. Here we explore the activity of a previously reported allosteric inhibitor of BCR-Abl kinase, GNF-2, against two cellular isoforms of Abl tyrosine kinase: one that carries a myristate in the N terminus and the other that is deficient in N-myristoylation. Our results show that GNF-2 inhibits the kinase activity of non-myristoylated c-Abl more potently than that of myristoylated c-Abl by binding to the myristate-binding pocket in the C-lobe of the kinase domain. Unexpectedly, indirect immunofluorescence reveals a translocation of myristoylated c-Abl to the endoplasmic reticulum in GNF-2-treated cells, whereas GNF-2 has no detectable effect on the localization of non-myristoylated c-Abl. These results indicate that GNF-2 competes with the NH(2)-terminal myristate for binding to the c-Abl kinase myristate-binding pocket and that the exposed myristoyl group accounts for the localization to the endoplasmic reticulum. We also demonstrate that GNF-2 can inhibit enzymatic and cellular kinase activity of Arg, a kinase highly homologous to c-Abl, which is also likely to be regulated through intramolecular binding of an NH(2)-terminal myristate lipid. These results suggest that non-ATP-competitive inhibitors, such as GNF-2, can serve as chemical tools that can discriminate between c-Abl isoform-specific behaviors.

Dissection of Bcr-abl Structural Domains Relating to Kinase Auto-Inhibition Using a Forward Mutational Screen with the Non-ATP Competitive Inhibitor GNF-2.

The Bcr-abl kinase is the causative agent for chronic myeloid leukemia (CML) and has been established as the primary clinical target for treatment of the disease through extensive use of Imatinib. Imatinib is the defining member of a class of ATP-binding site competitive inhibitors that lock Bcr-abl in an inactive conformation. Mutational screens of Bcr-abl using Imatinib and its derivatives as probes have been highly informative in prediction of clinically relevant mutations of Bcr-abl as well as in revealing the structure/function relationship of the kinase in general. Using compounds with a distinct mechanism of action from the Imatinib class to interrogate Bcr-abl will contribute to both more complete understanding of kinase function as well as to potential combination therapies for more effective treatment of CML. GNF-2, a recently identified inhibitor of Bcr-abl, establishes a new class of non-ATP competitive Bcr-abl family kinase inhibitors that may be developed as therapeutic agents for CML. GNF-2 effectively impairs the in vivo kinase activity of Bcr-abl and the growth of Bcr-abl transformed cells. GNF-2 functions at least in part through association with the myristate binding pocket of Bcr-abl. In order to further elucidate the mechanism of GNF-2 action as well as clinically relevant GNF-2 resistant mutants of Bcr-abl, a mutational screen coupling Bcr-abl mutagenesis to selection of drug resistance was performed using GNF-2 as probe. A number of functionally distinct resistant Bcr-abl mutations were recovered. Over half of all GNF-2 resistant clones harbored Bcr-abl mutations affecting the myristate binding pocket or the abl-SH3 domain, suggesting two potential methods of mutational resistance.1.The myristate binding domain mutants support a direct resistance model whereby GNF-2 association with Bcr-abl is impaired by disruption of the myristate binding pocket.2.Given a previous report that GNF-2 cannot inhibit Bcr-abl kinase activity in vitro, a novel model emerges for indirect resistance to GNF-2 by SH3 mutants that lose affinity for an inhibitory associated protein.The indirect resistance model specifically suggests that GNF-2 association confers a structural state of wildtype Bcr-abl which facilitates association to a putative inhibitory binding partner, thereby affecting inhibition. Indeed, the strongest of several candidate inhibitory binding partners, the Abl-SH3 domain binding inhibitor Abi-2 was observed to co-immunoprecipitate with Bcr-abl in the presence of GNF-2. This association correlated with reduced Bcr-abl auto-phosphorylation levels. These observations provide preliminary support for an indirect mechanism of Bcr-abl inhibition by GNF-2. Additional experiments involving shRNA knockdown of Abi-2 are being completed to determine the requirement of this Bcr-abl binding partner for GNF-2 activity. Further characterization of the SH3 and myristate binding domain mutants in the context of Abi-2 and GNF-2 binding affinities may establish a previously undescribed indirect mechanism of Bcr-abl inhibition by an allosteric non-ATP inhibitor.

Nef protein of human immunodeficiency virus type 1 binds its own myristoylated N-terminus

HIV-1 Nef is a small protein (approx. 25 kDa) that is posttranslationally modified by myristoylation. To explain its complex activities, a 'Nef-cycle' is discussed, which postulates different molecular conformations of Nef. Using recombinant full-length non-myristoylated Nef and synthetic peptides, we demonstrate by fluorescence titration experiments that a peptide representing the myristoylated N-terminus of Nef is specifically bound by Nef. A non-myristoylated N-terminal fragment of Nef or a myristoylated control peptide does not bind to Nef. These results are the first direct experimental evidence of the existence of a myristate-binding pocket in Nef, a prerequisite of the postulated 'closed' Nef conformation.