The structure-function relationship of oncogenic LMTK3

Abstract

Elucidating signaling driven by lemur tyrosine kinase 3 (LMTK3) could help drug development. Here, we solve the crystal structure of LMTK3 kinase domain to 2.1Å resolution, determine its consensus motif and phosphoproteome, unveiling in vitro and in vivo LMTK3 substrates. Via high-throughput homogeneous time-resolved fluorescence screen coupled with biochemical, cellular, and biophysical assays, we identify a potent LMTK3 small-molecule inhibitor (C28). Functional and mechanistic studies reveal LMTK3 is a heat shock protein 90 (HSP90) client protein, requiring HSP90 for folding and stability, while C28 promotes proteasome-mediated degradation of LMTK3. Pharmacologic inhibition of LMTK3 decreases proliferation of cancer cell lines in the NCI-60 panel, with a concomitant increase in apoptosis in breast cancer cells, recapitulating effects of LMTK3 gene silencing. Furthermore, LMTK3 inhibition reduces growth of xenograft and transgenic breast cancer mouse models without displaying systemic toxicity at effective doses. Our data reinforce LMTK3 as a druggable target for cancer therapy.

Fig. 1. Crystal structure of LMTK3.

(A) Structure of the kinase domain of LMTK3 showing the main features [Protein Data Bank (PDB) 6SEQ]. Blue, the glycine-rich loop; red, activation loop; salmon, the P+1 loop. The activation loop and the P+1 loop constitute the activation segment. Yellow, the catalytic loop; magenta, the kinase insert region. (B) Superimposition of the kinase domains of LMTK3 (green) and JAK1 (cyan). The activation loop of LMTK3 is shown in red and adopts an inactive conformation. The activation loop of JAK1 is shown in blue and adopts the active state. The two JAK1 phosphotyrosine residues (Tyr¹⁰³⁴ and Tyr¹⁰³⁵) are shown as yellow sticks. (C) Superimposition of LMTK3 and JAK1 active site (PDB 5KHW) residues involved in ADP binding. Key residues that bind ADP in JAK1 are positioned slightly differently to those of LMTK3 because of the different catalytic states of the kinases. However, residues in JAK1 that interact with ADP are conserved within LMTK3, although two noticeable differences are seen (LMTK3 Arg²⁴⁹ and Cys²⁴² replaces Glu⁹⁶⁶ and Leu⁹⁵⁹), which are unlikely to affect ATP binding. (D) Superimposition of LMTK3 and EGFR active site (PDB 5CNO) residues involved in ATP binding. A comparison with the ATP-bound form of EGFR also shows strong conservation among residues of EGFR that bind ATP and amino acid residues at equivalent positions within LMTK3 and residues that are not conserved (Ala⁶⁹⁸, Met⁷⁶⁹, and Cys⁷⁷³ for Trp¹⁷², Cys²⁴², and Asp²⁴⁶ in LMTK3), form main-chain interactions with bound ATP. [Green, amino acid residues of LMTK3 with black residue numbers; cyan, amino acid residues of JAK1 (top) or EGFR (bottom) with blue residue numbers; red spheres, water molecules; blue dashes, hydrogen bonds; green sphere, magnesium ion; green dashes, bonds coordinating the magnesium ion.]

Fig. 2. Defining the LMTK3 consensus phosphorylation motif and identifying HSP27 as an LMTK3 substrate.

(A) A spatially 198 components arrayed PSPL was subjected to in vitro phosphorylation with active LMTK3cat. A representative image of the average log₂ values of two independent experiments is shown. (B) Scaled-sequence PhosphoLogo representation of the LMTK3 consensus phosphorylation motif. The size of the letter is proportional to the signal for the corresponding amino acid at the indicated position. (C) In vitro kinase assays using wild-type (WT) LMTK3cat as source of enzyme activity and peptide variants with individual amino acid substitutions at different positions. Data shown are the average of two separate experiments (±SEM). (D) Top: In vitro kinase assay using recombinant HSP27 as a substrate and WT LMTK3cat or kinase-dead (KD) LMTK3 mutant (LMTK3cat-KD) as source of enzyme activity. Bottom: Time course in vitro kinase assay using recombinant HSP27 and LMTK3cat. (E) Schematic representation of SILAC proteomic experiment. Western blotting analysis of LMTK3 and FLAG-LMTK3 protein levels showing the transient overexpression of full-length pCMV6-LMTK3 (FLAG-tag) plasmid. m/z, mass/charge ratio. (F) Volcano scatter plot showing the log₂ “normalized ratios” (H/L) against log₁₀ “intensity” (H+L) for each characterized phosphorylated protein (phosphopeptide) following overexpression of WT-LMTK3 in MCF7 cells. Proteins are displayed in circles based on P values from significant B test. Red, P < 0.001; yellow, 0.001 > P < 0.01; green, 0.01 > P < 0.05; blue, P > 0.05.

Fig. 3. Identification of C28 as a potent inhibitor against LMTK3.

(A) Top: Experimental pipeline to identify LMTK3 inhibitors. Middle: HTRF data showing coverage of different inhibitors per active cluster. Green stars are the 868 compounds chosen for further confirmation, showing >50% mean inhibition (blue crosses are nonselected compounds). Selections were biased toward higher potency, sensible calculated physicochemical properties, and structural coverage within each cluster. Bottom: HTRF data showing the range of IC₅₀ values and purity of the top 160 compounds. (B) The IC₅₀ value for C28 against LMTK3cat was determined by in vitro kinase assays. (C) EC₅₀ values for C28 in FDCP1 and FDCP1-LMTK3 cell lines. Error bars represent the means ± SD from three independent experiments. (D) Table summarizing the IC₅₀ and EC₅₀ values of the top 38 compounds. (E) Chemical structure of C28. (F) Characteristic thermal denaturation curves of LMTK3 (black) and LMTK3/C28 complex (red) as monitored by DSF and (G) CD spectroscopy, indicating the increased protein thermodynamic stability upon ligand binding. T_m values from DSF were determined from the maximum in the first derivative of the fluorescence with respect to the temperature, or the midpoint in the transition region by fitting a Boltzmann sigmoidal to the CD data. Experiments were performed in triplicate. DMSO, dimethyl sulfoxide; OD, optical density; RFU, relative fluorescence units. (H) Kinetic analysis of HSP27 phosphorylation by LMTK3 in the absence or presence of C28. Kinetic parameters were determined from nonlinear regression fit of the initial reaction rates as a function of HSP27 concentration to the Michaelis-Menten equation using Prism 8. (I) Kinetic analysis as a function of ATP concentration for 0.6 μM HSP27 substrate, in the absence or presence of C28. Kinetic parameters were determined from nonlinear regression fit of the initial reaction rates as a function of ATP concentration to the Michaelis-Menten equation using Prism 8.

Fig. 4. Selectivity of C28 toward LMTK3.

(A) Selectivity profile of C28 (1 μM) against 140 kinases using radioactive filter binding assay. The data are displayed as percent activity remaining of assay duplicates with an SD. Only kinases with >50% decrease in their activity are shown. The relative IC₅₀ values are also presented. (B) Treespot interaction map depicting the kinome phylogenetic grouping, with kinases interacting with C28 (5 μM) represented as red circles. The larger the diameter of the circle, the higher the C28 binding affinity to the respective kinase. Kinases whose binding was inhibited by C28 to less than 10% of the control (DMSO) are shown. Lower numbers indicate most probable hits to bind to C28. Overlapping kinases identified in assays (radioactive filter binding and site-directed competition binding) are highlighted in yellow. (C) Western blots of LMTK3 and ERα in BC cell lines treated with increasing concentrations of C28 at different time points. Mean densitometry values of three independent experiments were calculated using ImageJ. GADPH, glyceraldehyde 3-phosphate dehydrogenase. (D) Effects of C28 on LMTK3 protein half-life in MDA-MB-231 cells. Cells were treated with CHX (cycloheximide) (100 μg/ml) and 10 μΜ C28 (or DMSO) for different time points. The relative LMTK3 protein levels (−/+ C28) were calculated and plotted against the time of treatment with CHX. (E) Western blots of total and phospho-HSP27 (normalized versus total) in MCF7, T47D, and MDA-MB-231 cell lines treated with 10 μΜ C28 for different time points. Mean densitometry values of three independent experiments are shown. (F) Western blots of total and phospho-HSP27 (normalized versus total) in BC cell lines treated with either control or LMTK3 small interfering RNAs (siRNAs) for 72 hours. Mean densitometry values of three independent experiments are shown. (G) Western blots of different kinases in BC cell lines treated with C28 (10 μΜ) at different time points.

Fig. 5. C28 promotes proteasome-mediated degradation of LMTK3, an HSP90 client protein.

(A) Western blots of LMTK3, HSP70, and CDK4 in BC cell lines following treatment with increasing concentrations of NVP-AUY922 for 72 hours. (B) Western blots of LMTK3, HSP70, and CDK4 in BC cell lines following treatment with increasing concentrations of geldanamycin for 72 hours. (C) In vitro kinase assays using recombinant HSP27 as a substrate and LMTK3cat as source of enzyme activity, examining the effects of C28 treatment or CDC37 addition. (D) Western blots of cell lysates from BC cell lines incubated with 10 μM of MG132 for 4 hours before treatment with C28 (10 μM) for the indicated time points. LMTK3 protein levels in the detergent-soluble and detergent-insoluble fractions are presented. (E) Isothermal titration calorimetry examining the ability of C28 to bind HSP90α. Injection of 0.2 mM C28 into 20 μM human HSP90α showed no detectible heat of interaction. (F) Western blots of LMTK3, HSP90, HSP70, and CCD37 in BC cell lines following treatment with increasing concentrations of C28 for 48 hours. (G) Normalized dose-response curves comparing thermophoresis of NT647-LMTK3cat on titration of increasing concentrations of CDC37 in the presence (red, K_D = 2.21 ± 1.17 μM) or absence (blue, K_D = 1.29 ± 3.9 nM) of 20 μM C28. Error bars represent SE, n = 5. K_D values were calculated from fitted data. (H) Left: LMTK3 was immunoprecipitated (IP) from MCF7 cells stably overexpressing LMTK3, in the presence (48 hours) or absence of C28 and the complexes were immunoblotted for LMTK3, CDC37, and HSP90. Right: CDC37 was immunoprecipitated from MCF7 cells stably overexpressing LMTK3, in the presence (48 hours) or absence of C28, and the complexes were immunoblotted for CDC37, LMTK3, and HSP90. Western blots for the respective proteins in whole cell lysates (input) were also performed.

Fig. 6. C28 impairs the viability of various human cancer cell lines.

(A) Left: Viability of nontransformed and BC cell lines treated with increasing concentrations of C28 for 72 hours. The IC₅₀ values are means from three independent experiments. Right: Western blots of LMTK3 in nontransformed and BC cell lines. (B) One-dose screening of C28 (10 μM; 24 hours) on the NCI-60 panel of tumor cell lines. The percent growth of C28-treated cells is shown. Negative values represent lethality. NSCLC, non-small cell lung cancer. (C) Western blots of BCL-XL, BCL2 and cleaved poly(ADP-ribose) polymerase (PARP) in BC cell lines following siRNA silencing of LMTK3 for 72 hours. GADPH was used as a loading control. (D) MCF7, T47D, MDA-MB-231, and MCF12A cell lines were treated with increasing concentrations of C28 for 72 hours, and the percentages of apoptotic and dead cells were analyzed by annexin V and 7-AAD staining. Results are expressed as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Each experiment was conducted at least three times. (E) Western blots of LMTK3, BCL2, and BCL-XL and cleaved PARP in MCF7, T47D, MDA-MB-231, and MCF12A cell lines treated with increasing concentrations of C28 for 72 hours. GADPH was used as a loading control. (F) Western blots of BCL2 and cleaved PARP levels in T47D and MDA-MB-231 cell lines treated with 10 μM C28 for 72 hours followed by LMTK3 overexpression for 48 hours. Tubulin was used as a loading control. Mean densitometry values of three independent experiments are shown. (G) T47D and MDA-MB-231 cell lines were treated with 10 μM C28 for 72 hours, followed by LMTK3 overexpression for 48 hours, and the percentages of apoptotic and dead cells were analyzed by annexin V and 7-AAD staining. Results are expressed as means ± SEM. Each experiment was conducted at least two times.

Fig. 7. C28 impedes tumor growth of BC mouse models.

(A) PK parameters of C28 following a single intravenous (IV), intraperitoneal (IP), or oral (PO) dose administration in male BALB/c mice (n = 4 per time point). *, based on 1 mg/kg intravenous group. **, based on 5 mg/kg intravenous group. Intravenous dose of 5 mg/kg was lethal for some mice, and therefore, the dose was decreased to 1 mg/kg. (B) Tumor growth in vehicle- and C28-treated groups (n = 6 each) of MMTV-Neu transgenic mice. Unpaired t test was performed using Prism 8. Results are expressed as means ± SEM; *P < 0.05. (C) Representative images of hematoxylin and eosin (H&E) staining of tumor sections from MMTV-Neu animals treated with vehicle or C28 (10 mg/kg). Original magnification, ×10. Scale bar, 100 μm. (D) Representative IHC images of Ki-67 expression in tumor sections from MMTV-Neu animals treated with vehicle or C28 (10 mg/kg). Original magnification, ×40. Scale bar, 50 μm. The percentage of Ki-67–positive cells versus the total number of cells is shown. Data represent the average of four vehicle and five C28-treated samples. Results are expressed as means ± SEM; ***P < 0.001. (E) Representative IHC of LMTK3 expression in tumor sections from MMTV-Neu animals treated with vehicle or C28 (10 mg/kg). Original magnification, ×20. Scale bar, 100 μm. (F) Tumor growth in vehicle- and C28-treated groups (n = 14 each) of MDA-MB-231 mice xenografts. (G) Box-and-whisker plots comparing vehicle (n = 6)–treated and C28-treated (10 mg/kg, n = 6 and 30 mg/kg, n = 5) groups of MDA-MB-231–luciferase mice xenografts groups at day 21. Unpaired t test was performed using Prism 8. Results are expressed as means ± SEM; *P < 0.05. (H) Schematic model depicting the proposed mechanism of action of C28 inhibitor.