Hirai N et al. (2020), Monomerization of ALK Fusion Proteins as a Ther...

ECB-ART-49867

Front Oncol 2020 Apr 02;10:419. doi: 10.3389/fonc.2020.00419.

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Monomerization of ALK Fusion Proteins as a Therapeutic Strategy in ALK-Rearranged Non-small Cell Lung Cancers.

Hirai N , Sasaki T , Okumura S , Minami Y , Chiba S , Ohsaki Y .

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Objective: Oncogenic echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) (EML4-ALK) fusion proteins found in non-small cell lung cancers (NSCLC) are constitutively phosphorylated and activated by dimerization via the coiled-coil domain (cc) within EML4. Here, we investigated whether disruption of ALK fusion protein oligomerization via competitive cc mimetic compounds could be a therapeutic strategy for EML4-ALK NSCLC. Methods: A Ba/F3 cell model was created that expressed an ALK intracellular domain in which the dimer/monomer state is ligand-regulated. This novel cell model was used to investigate the effect of disrupting ALK fusion protein oligomerization on tumor cell growth in vitro and in vivo using nude mice. Subsequently, the antiproliferative effects of endogenous cc domain co-expression and mimetic cc peptides were assayed in EML4-ALK cancer cell lines. Results: Cells induced to express monomeric ALK in vitro did not survive. When transplanted into mice, induction of monomers abrogated tumor formation. Using a fluorescent protein system to quantify protein-protein interactions of EML4-ALK and EML4cc, we demonstrated that co-expression of EML4cc suppressed EML4-ALK assembly concomitant with decreasing the rate of tumor growth in vitro and in vivo. In EML4-ALK cancer cell lines, administration of synthetic EML4cc peptide elicited a decrease of phosphorylation of ALK leading to reduction in tumor cell growth. Conclusions: Our findings support the monomerization of ALK fusion proteins using EML4cc peptides for competitive inhibition of dimerization as a promising therapeutic strategy for EML4-ALK NSCLC. Further studies are warranted to explore the use of specific cc peptide as a therapeutic option for other lung cancers harboring driver fusion genes containing a cc or oligomerization domain within the fusion partner.

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	Figure 1. Effect of conditional monomerization of DmrB-ALK_wt. (A) Illustration of the inducible dimerization of wild-type ALK intracellular domain. ALK intracellular domain encoded by exons 20–29 was ligated to DmrB (DmrB-ALK_wt). Dimer/monomer state of the fusion protein was regulated by B/B homodimerizer (B/B) which acts as a ligand for DmrB. (B) Proliferation of Ba/F3 cells expressing DmrB-ALK_wt (Ba/F3 DmrB-ALK_wt) after B/B withdrawal or B/B continuation. Survival and proliferation of Ba/F3 DmrB-ALK_wt were dependent on B/B continuation. Error bars: SD. (C) Tumor volume curve of Ba/F3 DmrB-ALK_wt xenografts in nude mice. Nude mice were treated with B/B for 5 w (group 1), 3.5 w (group 2, solid line) and then withdrawn for 1.5 w (group 2, dashed line) or mock solution for 5 w. Tumor formation and growth was dependent on continuous B/B treatment. Error bars: SD. (D) Western blotting of the DmrB-ALK_wt protein with an anti-HA antibody. The DmrB-ALK_wt protein was immunoprecipitated with an anti-HA antibody and electrophoretically separated on a native polyacrylamide gel under non-denaturing conditions. Under the B/B treatment, monomeric DmrB-ALK_wt (predicted molecular weight of 75.5 kDa) was not observed (left lane). After withdrawal of B/B, monomeric DmrB-ALK_wt proteins were detected (right lane). (E) Western blotting of Ba/F3 DmrB-ALK_wt with B/B treatment (B/B+) or B/B withdrawal (B/B–). Phosphorylation of ALK, ERK1/2, AKT, and Stat3 proteins were significantly attenuated in the B/B withdrawal condition. Total and phosphorylated proteins were probed on the same blot sequentially.
	Figure 2. Effect of conditional DmrB-ALK_mF1174L monomerization. (A) Illustration of inducible dimerization of the ALK mutant F1174L intracellular domain. ALK intracellular domain with mutation from phenylalanine to leucine at amino acids 1174 of ALK (F1174L; highlighted in violet) was ligated to DmrB (DmrB-ALK_mF1174L). The dimer/monomer state of the fusion protein was regulated by B/B homodimerizer (B/B) which acts as a ligand for DmrB. (B) Proliferation of Ba/F3 cells expressing DmrB-ALK_mF1174L (Ba/F3 DmrB-ALK_mF1174L) after B/B withdrawal or B/B continuation. Ba/F3 DmrB-ALK_mF1174L could survive and proliferate after the withdrawal of B/B. Error bars: SD. (C) Tumor volume curve of Ba/F3 DmrB-ALK_mF1174L xenografts in nude mice. Nude mice were treated with B/B or mock solution for 3.5 w. Tumor xenografts were formed in both groups. Error bars: SD. (D) Kaplan Meier curve of Ba/F3 DmrB-ALK_mF1174L mice xenografts. Median overall survival of mice, 4 w for B/B-treated group and 5 w for mock-treated group, and was not significantly different between the groups. (E) Western blot of Ba/F3 DmrB-ALK_mF1174L with B/B treatment (B/B+) or B/B withdrawal (B/B–). Phosphorylation of ALK, ERK1/2, AKT, and Stat3 proteins was maintained in the B/B withdrawal condition. Total and phosphorylated proteins were probed on the same blot sequentially.
	Figure 3. Growth inhibition of endogenous EML4-coiled-coil domain in Ba/F3 cells expressing EML4-ALK. (A) Schematic of EML4-ALK and EML4-coiled-coil (cc) domain interaction analysis using FluoppiTM technology. hAG-EML4-ALK, hAG (homo tetramer Azami-GFP)-tagged EML4-ALK; Ash-EML4-ALK, Ash (homo-oligomerized protein assembly helper)-tagged EML4-ALK; Ash-EML4-cc, Ash-tagged EML4-cc domain. hAG-tagged EML4-ALK (EA) and Ash-tagged EA, or hAG-tagged EA and Ash-tagged cc were co-transfected into Ba/F3 cells (Ba/F3 EA/EA and Ba/F3 EA/cc cells, respectively). Protein-protein interaction between EA-EA or EA-cc can be quantified through fluorescent intensity. In the Ba/F3 EA/EA, tagged EA proteins assemble to make oligomers, and binding of the Ash- and Azami-GFP tags makes large fluorescent foci (Azami-GFP hyper). In the Ba/F3 EA/cc, Azami-GFP tagged EA proteins alone emit fluorescence (homo tetramer Azami-GFP), but only a few large fluorescent foci are formed due to Ash-hAG tag binding (Azami-GFP hyper). (B) Flow cytometric analyses of Ba/F3 EA/EA and Ba/F3 EA/cc. In Ba/F3 EA/EA (left), all cells expressed homo tetramer Azami-GFP (green arrow) and 2.57% of cells expressed Azami-GFP hyper (red arrow). In Ba/F3 EA/cc (right), all cells expressed homo tetramer Azami-GFP (green arrow), but only 0.17% of cells expressed Azami-GFPhyper (red arrow), indicating hyper fluorescent foci formation derived from Azami-GFP-EA and Ash-cc interactions was a rare fraction. X axis; GFP fluorescent intensity. Y axis; PI. (C) Cell growth assay of Ba/F3 EA/EA and Ba/F3 EA/cc. Ba/F3 EA/EA cells that contained oligomerized EA protein served as control. Cell growth of Ba/F3 EA/cc was significantly slower compared with Ba/F3 EA/EA. Error bars: SD. (D) Tumor growth assay in mice models. The animals of both Ba/F3 EA/EA and Ba/F3 EA/cc groups developed tumors. The averaged tumor growth delay of group Ba/F3 EA/cc was higher than that of group EA/EA.
	Figure 4. EML4-cc peptides inhibit growth of H3122 and Ba/F3 cells expressing EML4-ALK. (A) Peptide sequence and 3D structure of EML4cc constructs. The EML4cc peptide consists of the original EML4-coiled-coil domain spanning amino acids T17 to K42 and were chemically synthetized. The 3D structure of EML4cc was generated by PyMOL2.0.6. (B) H3122 and A549 cells were treated with PBS, EML4cc peptide, or EML4cc conjugated with TAMRA (red fluorescence). After 6 h of treatment with PBS or 1 μg/ml of EML4cc peptides, the cells were observed under a fluorescence microscope. Red fluorescence of TAMRA was detected in the cytoplasm of both H3122 and A549 cells. (C) Cell growth of H3122 and A549 cell lines. EML4cc peptides inhibited the growth of H3122 cells, but not that of A549 cells. Error bars: SD. (D) Western blotting of H3122 extracts treated with cc peptides. The phosphorylation of ALK was decreased at 3 h after peptide treatment; the same trend was visible at 6 h. The phosphorylation of ERK and AKT was decreased at 6 h after cc peptide treatment. (E) The efficacy of the combination of Alectinib and cc peptides. After 24 h from concurrent administration of Alectinib and cc peptide treated with Xfect, the viability of H3122 cells treated with Alectinib and cc peptide was significantly decreased in comparison with that of cells treated with Alectinib alone. Error bars: SD.

References [+] :

Amano, Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3. 2015, Pubmed