DataSheet_1_ALK F1174S mutation impairs ALK kinase activity in EML4-ALK variant 1 and sensitizes EML4-ALK variant 3 to crizotinib.docx

ObjectiveTo assess the influence of F1174S mutation on kinase activity and drug sensitivity of the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) fusion (EML4-ALK) variants 1 and 3.MethodsWe constructed mammalian expression plasmids of both wildtype and F1174 mutant EML4-ALK variants 1 and 3, and then characterized them with cell models by performing immunoblotting, neurite outgrowth assay, focus formation assay as well as protein stability assay. Drug sensitivity to ALK tyrosine kinase inhibitors was also compared between wildtype and F1174 mutant EML4-ALK fusions. In addition, we characterized the effect of different F1174 kinase domain mutations in the context of EML4-ALK fusions.ResultsIn contrast to the oncogenic ALK-F1174S mutation that has been reported to be activating in the context of full-length ALK in neuroblastoma, EML4-ALK (F1174S) variant 1 exhibits impaired kinase activity leading to loss of oncogenicity. Furthermore, unlike the previously reported F1174C/L/V mutations, mutation of F1174 to S sensitizes EML4-ALK variants 3a and 3b to crizotinib.ConclusionThese findings highlight the complexity of drug selection when treating patients harboring resistance mutations and suggest that the F1174S mutation in EML4-ALK variant 1 is likely not a potent oncogenic driver. Additional oncogenic driver or other resistance mechanisms should be considered in the case of EML4-ALK variant 1 with F1174S mutation.</p

Generation and verification of the ALK kinase knock-out mouse strain.

<p>(A) Schematic overview of the ALK kinase knock-out mice. (i) Schematic representation of ALK protein structure. The region of the protein corresponding to the targeted exons 20–23 is indicated (black bar). (ii) Schematic overview of the CRE-ALK construct (conditional knock-out). FRT (blue) and loxP (red) sites are indicated. Restriction sites for Southern are marked with the restriction enzyme SpeI, black arrows indicate the position of the Southern probe used for genotyping. Green bars denoted with the numbers 15–22 respectively are exons. (iii) Schematic overview of ALK kinase KO (non-conditional knock-out). Flp-mediated recombination leads to the deletion of exons 20–23. The remaining FRT site still present after Flp mediated recombination. (B) Verification of the ALK kinase KO line by (i) southern blot, (ii) immunobloting with anti-ALK mAb135 which recognizes the extracellular domain of ALK and by (iii) PCR.</p

ALK knockout mice display reduced GnRH expression.

<p>(A) Confocal analysis of GnRH expression in P40 ALK kinase KO. GnRH (green), DAPI (blue). (B) Expression of hypothalamic GnRH at P40 in ALK kinase knockout males. Reconstituted OPT images of representative hypothalami are shown in anterior, lateral and ventral views. GnRH positive regions are presented as (iso-surface rendered) black dots on the iso-surfaces of dissected hypothalami. (C) Quantitation of GnRH positive regions from P40 hypothalami. Values represent the average volumes of GnRH stained regions in hypothalami, 3–4 mice per group. *P<0.05 indicates significant difference.</p

Effect of loss of ALK activity on puberty onset and testosterone levels.

<p> (A) The age at preputial separation was assessed in littermate control (n = 11) and ALK mutant (n = 12) male mice. (B) Body weight development in male mice (n = 10 each genotype). (C) ALK kinase KO male mice display significantly lower levels of testosterone at P40 as compared with controls. No significant differences between ALK kinase KO and wild type were observed at P60. Box plots indicate the median (black line), 25th and 75th percentiles (borders of boxes), 10th and 90th percentiles (whiskers) and the outliers (dots) from 20–25 mice per group. *P<0.05 indicates significant difference. (D) Testicular weight (n = 5 per postnatal day and genotype), as well as representative testicular size in wild type mice compared with ALK KO mice is shown. (E) Number of cauda epididymidis-retrieved spermatozoa per epididymis (n = 4 per genotype at P40, n = 6 at P60 for WT animals and n = 4 at P60 for ALK mutant mice). (F) Viability percentage of cauda epididymidis-retrieved spermatozoa (n = 4 per genotype and per age).</p

Crizotinib treatment in mice leads to reduction of serum testosterone levels.

<p>Adult male mice were treated with crizotinib daily (at 20 mg/kg). Blood samples were collected prior to crizotinib treatment, one day after the last treatment and five weeks after completion of treatment and analyzed for testosterone. Box plots indicate the median (black line), 25th and 75th percentiles (borders of boxes), 10th and 90th percentiles (whiskers) and the outliers (dots) from 13 mice per group. *P<0.05 indicates significant difference.</p

Targeted Disruption of ALK Reveals a Potential Role in Hypogonadotropic Hypogonadism

<div><p>Mice lacking ALK activity have previously been reported to exhibit subtle behavioral phenotypes. In this study of ALK of loss of function mice we present data supporting a role for ALK in hypogonadotropic hypogonadism in male mice. We observed lower level of serum testosterone at P40 in ALK knock-out males, accompanied by mild disorganization of seminiferous tubules exhibiting decreased numbers of GATA4 expressing cells. These observations highlight a role for ALK in testis function and are further supported by experiments in which chemical inhibition of ALK activity with the ALK TKI crizotinib was employed. Oral administration of crizotinib resulted in a decrease of serum testosterone levels in adult wild type male mice, which reverted to normal levels after cessation of treatment. Analysis of GnRH expression in neurons of the hypothalamus revealed a significant decrease in the number of GnRH positive neurons in ALK knock-out mice at P40 when compared with control littermates. Thus, ALK appears to be involved in hypogonadotropic hypogonadism by regulating the timing of pubertal onset and testis function at the upper levels of the hypothalamic-pituitary gonadal axis.</p></div

Abnormalities in seminiferous tubules from ALK kinase KO testes.

<p> (A) Histological morphology of testes stained with standard H & E. Note the irregular arrangement of germ cells at the periphery (green arrow) of the seminiferous tubule and the increased presence of vacuoles (black arrow) in ALK kinase KO mice. Fewer GATA4-expressing cells within the seminiferous tubules are observed at P40 in ALK kinase KO mice as compared with wild type controls. This decrease is less obvious at P60 (insets). (B) Quantification of age-dependent loss of GATA4 immunoreactivity in the Sertoli cells within the seminiferous tubules of wild type and ALK kinase KO mice. N = 10 mice per age and per genotype.</p

Proteasome dependent degradation of receptor retained in intracellular compartment.

<p><b>A.</b> NIH3T3 cells stably expressing either the WT ALK or F1174L mutated ALK were non-treated or treated with Bafilomycin A1 (0.25 µM) or with Lactacystin (10 µM) for 16 hours. ALK immunoprecipitates from 1 mg of total cell lysate proteins were subjected to western blot analysis. ALK was immunoblotted with polyclonal REAB antibody. <b>B.</b> SH-SY5Y were non-treated or treated with Bafilomycin A1 (0.25 µM) or with Lactacystin (10 µM) for 16 hours. ALK immunoprecipitates from 1.5 mg of total cell lysate proteins were subjected to western blot analysis using polyclonal REAB antibody. The experiment was done in triplicates and quantified with LiCor Odyssey system. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033581#s2" target="_blank">Results</a> were expressed in percentage of control +/− s.e.m.</p

ALK down-regulation after agonist mAb 46 treatment.

<p><b>A. IMR-32 (WT) and B. SH-SY5Y (WT/F1174L)</b> were treated with mAb 46 for 15 min to 6 h. ALK immunoprecipitates from 1.5 mg of total cell lysate proteins were submitted to western blot. Total ALK were immunoblotted with polyclonal REAB and phosphorylated ALK were detected with monoclonal phosphotyrosine antibody 4G10 platinium. Tubulin acted as a loading control. <b>C.</b> SH-SY5Y cells were pre-treated or not with 50 nM NVP-TAE684 one hour before cell stimulation by agonist mAb 46 at 6 nM in time course manner (0 min to 6 h). ALK immunoprecipitates from 1.5 mg of total cell lysate proteins were submitted to western-blot analysis and immunoblotted as described. <i>Lower panel</i>: Experiments were done in triplicates and the 220 kD forms (the doublet was impossible to separate) and the 140 kD form of ALK were quantified with LiCor Odyssey software. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033581#s2" target="_blank">Results</a> are expressed in percentage of control +/− s.e.m. <b>D.</b> SH-SY5Y cells were pre-treated with either bafilomycin (0.25 µM) or lactacystin (10 µM) for 15′, then non treated or treated with agonist mAb 46 at 6 nM during 6 hours. ALK immunoprecipitates from 1.5 mg of total cell lysate proteins were submitted to Western-blot analysis as described. <i>Lower panel:</i> Experiments were done in triplicates total ALK (220 kD forms+140 kD form) was quantified with LiCor Odyssey software. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033581#s2" target="_blank">Results</a> are expressed in percentage of control +/− s.e.m.</p

Kinase activation dependent down-regulation was regulated by Cbl ubiquitin ligase and ALK ubiquitylation.

<p><b>A.</b> SH-SY5Y cells were treated or not with either agonist mAb 46 or antagonist mAb 30 at 6 nM during 15 or 60 min. Cbl immunoprecipitates from 2 mg total cell lysate proteins were submitted to western-blot analysis, and then immunoblotted with polyclonal anti Cbl and ALK recruitment with polyclonal REAB antibody. Phosphorylated ALK and Cbl were revealed with 4G10 antibody. <b>B.</b> SH-SY5Y cells were serum starved for 16 hours and non-treated or treated with either agonist mAb 46 or antagonist mAb 30 at 6 nM during 15 or 60 min. ALK immunoprecipitates from 2 mg total cell lysate proteins were submitted to western-blot analysis and then immunoblotted with polyclonal REAB. Cbl recruitment was revealed with polyclonal anti Cbl antibody. Phosphorylated ALK and Cbl were revealed with 4G10 antibody. <b>C.</b> SH-SY5Y were serum starved for 16 hours, and then non-treated or treated with either mAb 46 or mAb 30 at 6 nM for 15 minutes or 60 minutes. ALK immunoprecipitates were submitted to Western-blot analysis as described.</p