Search

FMS-like tyrosine kinase 3 (FLT3) inhibitors have shown activity in the treatment of acute myelogenous leukemia (AML). Secondary mutations in target kinases can cause clinical resistance to therapeutic kinase inhibition. We have previously shown that sensitivity toward tyrosine kinase inhibitors varies between different activating FLT3 mutations. We therefore intended to determine whether different FLT3 inhibitors would produce distinct profiles of secondary, FLT3 resistance mutations. Using a cell-based screening approach, we generated FLT3–internal tandem duplication (ITD)–expressing cell lines resistant to the FLT3 inhibitors SU5614, PKC412, and sorafenib. Interestingly, the profile of resistance mutations emerging with SU5614 was limited to exchanges in the second part of the kinase domain (TK2) with exchanges of D835 predominating. In contrast, PKC412 exclusively produced mutations within tyrosine kinase domain 1 (TK1) at position N676. A mutation at N676 recently has been reported in a case of PKC412-resistant AML. TK1 mutations exhibited a differential response to SU5614, sorafenib, and sunitinib but strongly impaired response to PKC412. TK2 exchanges identified with SU5614 were sensitive to PKC412, sunitinib, or sorafenib, with the exception of Y842D, which caused a strong resistance to sorafenib. Of note, sorafenib also produced a highly distinct profile of resistance mutations with no overlap to SU5614 or PKC412, including F691L in TK1 and exchanges at position Y842 of TK2. Thus, different FLT3 kinase inhibitors generate distinct, nonoverlapping resistance profiles. This is in contrast to Bcr-Abl kinase inhibitors such as imatinib, nilotinib, and dasatinib, which display overlapping resistance profiles. Therefore, combinations of FLT3 inhibitors may be useful to prevent FLT3 resistance mutations in the setting of FLT3-ITD–positive AML. [Cancer Res 2009;69(7):3032–41]

Many protein kinase functions, including autophosphorylation in trans, require dimerization, possibly by activation segment exchange. Such dimers have been reported for a few autophosphorylating protein kinases, but not for mitogen-activated protein kinases (MAPKs). Activation of MAPKs proceeds not only via the well-characterized action of dual T/Y specificity MAPK kinases, phosphorylating both residues of the MAPK TxY activation loop motif, but also via a noncanonical activation pathway triggered by phosphorylation at Tyr323 and homodimerization. Here, we report the 2. 7-A-resolution structure of p38α MAPK from Salmo salar in a novel domain-swapped homodimeric form. The tyrosines of the conserved sequence YxAPE anchor the swapped activation segments in a configuration suitable for autophosphorylation in trans and provide a model for the noncanonical pathway. In the dimer, a structural unit containing Tyr323 is formed at a dimerization contact region that stabilizes the HRD catalytic loop in a unique inactive geometry. This feature is consistent with the requirement of Tyr323 phosphorylation for the initiation of the noncanonical pathway. Despite the interacting surface of more than 2600 A2, the dimer is not obligate, as gel filtration shows the dimerization to occur only at relatively high concentrations. The transition from monomer to dimer involves a relatively simple hinged displacement of helix EF and adjacent residues. Thus, dimer formation is likely to be transient, compatible with functional requirements for autophosphorylation, allowing further modulation, for example, by scaffolding mechanisms.

MAPK-activated protein kinase 5 (MK5) was recently identified as a physiological substrate of the atypical MAPK ERK3. Complex formation between ERK3 and MK5 results in phosphorylation and activation of MK5, concomitant stabilization of ERK3, and the nuclear exclusion of both proteins. However, ablation of ERK3 in HeLa cells using small interfering RNA or in fibroblasts derived from ERK3 null mice reduces the activity of endogenous MK5 by only 50%, suggesting additional mechanisms of MK5 regulation. Here we identify the ERK3-related kinase ERK4 as a bona fide interaction partner of MK5. Binding of ERK4 to MK5 is accompanied by phosphorylation and activation of MK5. Furthermore, complex formation also results in the relocalization of MK5 from nucleus to cytoplasm. However unlike ERK3, ERK4 is a stable protein, and its half-life is not modified by the presence or absence of MK5. Finally, although knock-down of ERK4 protein in HeLa cells reduces endogenous MK5 activity by approximately 50%, a combination of small interfering RNAs targeting both ERK4 and ERK3 causes a further reduction in the MK5 activity by more than 80%. We conclude that MK5 activation is dependent on both ERK3 and ERK4 in these cells and that these atypical MAPKs are both physiological regulators of MK5 activity.

The following article, Alexeeva, M., Åberg, E., Engh, R.A. & Rothweiler, U. (2015). The structure of a dual-specificity tyrosine phosphorylation-regulated kinase 1A-PKC412 complex reveals disulfide-bridge formation with the anomalous catalytic loop HRD(HCD) cysteine. Acta Crystallographica Section D: Biological Crystallography, 71, 1207-1215, can be accessed at https://doi.org/10.1107/S1399004715005106. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a protein kinase associated with neuronal development and brain physiology. The DYRK kinases are very unusual with respect to the sequence of the catalytic loop, in which the otherwise highly conserved arginine of the HRD motif is replaced by a cysteine. This replacement, along with the proximity of a potential disulfide-bridge partner from the activation segment, implies a potential for redox control of DYRK family activities. Here, the crystal structure of DYRK1A bound to PKC412 is reported, showing the formation of the disulfide bridge and associated conformational changes of the activation loop. The DYRK kinases represent emerging drug targets for several neurological diseases as well as cancer. The observation of distinct activation states may impact strategies for drug targeting. In addition, the characterization of PKC412 binding offers new insights for DYRK inhibitor discovery.

The era of structure-based protein kinase inhibitor design began in the early 1990s with the determination of crystal structures of protein kinase A (PKA, or cyclic AMP-dependent kinase). Although many other protein kinases have since been extensively characterized, PKA remains a prototype for studies of protein kinase active conformations. It serves well as a model for the structural properties of AGC subfamily protein kinases, clarifying inhibitor selectivity profiles. Its reliable expression, constitutive activity, simple domain structure, and reproducible crystallizability have also made it a useful surrogate for the discovery of inhibitors of both established and emerging AGC kinase targets.

Myeloproliferation with prominent eosinophilia is associated with rearrangements of PDGFR-A or -B. The most common rearrangement is FIP1L1-PDGFRA (FP). The majority of patients with PDGFR-rearranged myeloproliferation respond to treatment with imatinib. In contrast to BCR-ABL-positive chronic myelogenous leukemia, only few cases of imatinib resistance and mutations of the FP kinase domain have been described so far. We hypothesized that the number of critical residues mediating imatinib resistance in FP in contrast to BCR-ABL might be limited. We performed an established systematic and comprehensive in vitro resistance screen to determine the pattern and frequency of possible TKI resistance mutations in FP. We identified 27 different FP kinase domain mutations including 25 novel variants, which attenuated response to imatinib, nilotinib or sorafenib. However, the majority of these exchanges did not confer complete inhibitor resistance. At clinically achievable drug concentrations, FP/T674I predominated with imatinib, whereas with nilotinib and sorafenib, FP/D842V and the compound mutation T674I+T874I became prevalent. Our results suggest that the PDGFR kinase domain contains a limited number of residues where exchanges critically interfere with binding of and inhibition by available PDGFR kinase inhibitors at achievable concentrations, which might explain the low frequency of imatinib resistance in this patient population. In addition, these findings would help to select the appropriate second-line drug in cases of imatinib-resistant disease and may be translated to other neoplasms driven by activated forms of PDGFR-A or -B.

ERK3 and ERK4 are atypical MAPKs in which the canonical TXY motif within the activation loop of the classical MAPKs is replaced by SEG. Both ERK3 and ERK4 bind, translocate, and activate the MAPK-activated protein kinase (MK) 5. The classical MAPKs ERK1/2 and p38 interact with downstream MKs (RSK1–3 and MK2–3, respectively) through conserved clusters of acidic amino acids, which constitute the common docking (CD) domain. In contrast to the classical MAPKs, the interaction between ERK3/4 and MK5 is strictly dependent on phosphorylation of the SEG motif of these kinases. Here we report that the conserved CD domain is dispensable for the interaction of ERK3 and ERK4 with MK5. Using peptide overlay assays, we have defined a novel MK5 interaction motif (FRIEDE) within both ERK4 and ERK3 that is essential for binding to the C-terminal region of MK5. This motif is located within the L16 extension lying C-terminal to the CD domain in ERK3 and ERK4 and a single isoleucine to lysine substitution in FRIEDE totally abrogates binding, activation, and translocation of MK5 by both ERK3 and ERK4. These findings are the first to demonstrate binding of a physiological substrate via this region of the L16 loop in a MAPK. Furthermore, the link between activation loop phosphorylation and accessibility of the FRIEDE interaction motif suggests a switch mechanism for these atypical MAPKs in which the phosphorylation status of the activation loop regulates the ability of both ERK3 and ERK4 to bind to a downstream effector.

FMS-like tyrosine kinase 3 (FLT3) inhibitors have shown activity in the treatment of acute myelogenous leukemia (AML). Secondary mutations in target kinases can cause clinical resistance to therapeutic kinase inhibition. We have previously shown that sensitivity toward tyrosine kinase inhibitors varies between different activating FLT3 mutations. We therefore intended to determine whether different FLT3 inhibitors would produce distinct profiles of secondary, FLT3 resistance mutations. Using a cell-based screening approach, we generated FLT3–internal tandem duplication (ITD)–expressing cell lines resistant to the FLT3 inhibitors SU5614, PKC412, and sorafenib. Interestingly, the profile of resistance mutations emerging with SU5614 was limited to exchanges in the second part of the kinase domain (TK2) with exchanges of D835 predominating. In contrast, PKC412 exclusively produced mutations within tyrosine kinase domain 1 (TK1) at position N676. A mutation at N676 recently has been reported in a case of PKC412-resistant AML. TK1 mutations exhibited a differential response to SU5614, sorafenib, and sunitinib but strongly impaired response to PKC412. TK2 exchanges identified with SU5614 were sensitive to PKC412, sunitinib, or sorafenib, with the exception of Y842D, which caused a strong resistance to sorafenib. Of note, sorafenib also produced a highly distinct profile of resistance mutations with no overlap to SU5614 or PKC412, including F691L in TK1 and exchanges at position Y842 of TK2. Thus, different FLT3 kinase inhibitors generate distinct, nonoverlapping resistance profiles. This is in contrast to Bcr-Abl kinase inhibitors such as imatinib, nilotinib, and dasatinib, which display overlapping resistance profiles. Therefore, combinations of FLT3 inhibitors may be useful to prevent FLT3 resistance mutations in the setting of FLT3-ITD–positive AML. [Cancer Res 2009;69(7):3032–41]

ERK (extracellular-signal-regulated kinase) 4 [MAPK (mitogen-activated protein kinase) 4] and ERK3 (MAPK6) are atypical MAPKs. One major difference between these proteins and the classical MAPKs is substitution of the conserved T-X-Y motif within the activation loop by a single phospho-acceptor site within an S-E-G motif. In the present study we report that Ser186 of the S-E-G motif in ERK4 is phosphorylated in vivo. Kinase-dead ERK4 is also phosphorylated on Ser186, indicating that an ERK4 kinase, rather than autophosphorylation, is responsible. Co-expression of MK5 [MAPK-activated protein kinase 5; also known as PRAK (p38-regulated/activated kinase)], a physiological target of ERK4, increases phosphorylation of Ser186. This is not dependent on MK5 activity, but does require interaction between ERK4 and MK5 suggesting that MK5 binding either prevents ERK4 dephosphorylation or facilitates ERK4 kinase activity. ERK4 mutants in which Ser186 is replaced with either an alanine residue or a phospho-mimetic residue (glutamate) are unable to activate MK5 and Ser186 is also required for cytoplasmic anchoring of MK5. Both defects seem to reflect an impaired ability of the ERK4 mutants to interact with MK5. We find that there are at least two endogenous pools of wild-type ERK4. One form exhibits reduced mobility when analysed using SDS/PAGE. This is due to MK5-dependent phosphorylation and only this retarded ERK4 species is both phosphorylated on Ser186 and co-immunoprecipitates with wild-type MK5. We conclude that binding between ERK4 and MK5 facilitates phosphorylation of Ser186 and stabilization of the ERK4–MK5 complex. This results in phosphorylation and activation of MK5, which in turn phosphorylates ERK4 on sites other than Ser186 resulting in the observed mobility shift.

Abstract 2912 Poster Board II-888 Constitutively activated variants of PDGFRA, PDGFRB can be found in a subset of patients with myeloid neoplasms associated with eosinophilia. The most common is FIP1L1-PDGFRA (FP). Patients with PDGFR-A and -B rearranged myeloproliferation respond to treatment with imatinib. However, single cases of clinical resistance due to a secondary FP/T674I mutation have been reported. In CML, more than 40 different exchanges have been described that confer imatinib resistance, and sequential treatment with imatinib and novel Abl kinase inhibitors has become reality. Nilotinib and sorafinib are potent alternative inhibitors of PDGFR-A and -B. We therefore hypothesized that available PDGFR kinase inhibitors might produce specific profiles of secondary FP kinase domain mutations mediating inhibitor resistance. To this aim, we selected clones of FP expressing Ba/F3 cells resistant to rising concentrations of imatinib, nilotinib, and sorafinib. In these, we identified 27 different PDGFRA kinase domain mutations. Imatinib, nilotinib and sorafenib produced distinct profiles of resistance mutations. During selection with imatinib, FP/T674I predominated with rising concentrations. FP/T674I corresponds to Bcr-Abl/T315I, which is frequently found in imatinib resistant CML. In contrast to imatinib, nilotinib and sorafenib produced a significantly lower frequency of resistant cell clones. Also, T674I disappeared at therapeutic nilotinib concentrations in favour of T674I+T874I and D842V. Sorafinib displayed a distinct profile of mutations including D842V, but not T674I. Following cloning and expression of all FP variants, dose-response analysis indicated that full cross-resistance to all three inhibitors was limited to D842V, whereas the gatekeeper T674I exchange retained sensitivity to sorafenib and nilotinib, and T674I+T874I was responsive to sorafenib only. In silico structure modelling indicated that differences in inhibitor response observed in distinct clusters of FP mutations identified in drug-resistant clones are based on differences of sorafenib versus imatinib and nilotinib in key drug - protein target interactions in PDGFR family kinases. Besides direct inhibitor binding effects, we propose that the identified exchanges shift an inactive-active conformation equilibrium and thereby affect binding of type II inhibitors like imatinib, nilotinib and sorafenib. Our results predict PDGFR variants that might come up in patients with myeloproliferation positive for PDGFR-A or -B fusions treated with imatinib, nilotinib or sorafenib. These findings will help in selection of an appropriate second line PDGFR kinase inhibitor when resistance to imatinib emerges, and will guide drug design. Moreover, our data can be translated to other neoplasms driven by activated forms of PDGFR-A or -B including GIST, CMML, and dermatofibrosarcoma protuberans. Disclosures: Off Label Use: Use of sorafenib and nilotinib in myeloproliferation with prominent eosinophilia.

2021-06-16 online workshop