Abstract
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.
Highlights
Several genetic alterations, including mutation or rearrangements, were recently identified as molecular targets of therapies for lung cancers with promising results [1,2,3]
All reported echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) variants, including even the shortest variant 5a/b, contain the EML4-cc domain; in the non-EML4 ALK fusion partners reported in non-small cell lung cancers (NSCLC) (e.g., KIF5B, TPM3, TPM4, or TPR-) the amino-terminal fragment of the fusion partner necessarily contains cc domains or oligomerization domains [13,14,15]
We created a construct in which the DmrB domain was fused directly to the N-terminus of the ALK intracellular domain and transfected Ba/F3 cells, whose growth are dependent on interleukin-3 (IL-3)
Summary
Several genetic alterations, including mutation or rearrangements, were recently identified as molecular targets of therapies for lung cancers with promising results [1,2,3]. ALK tyrosine kinase inhibitors (ALK-TKI) have anti-tumor activities in NSCLC with ALK rearrangements, but complete cancer control has not been achieved due to acquired resistance [1, 3]. Gain-of-function mutations (e.g., F1174L or R1275Q) in ALK were first identified in neuroblastoma and induce constitutive autophosphorylation of ALK [7,8,9]. Some of these activating mutations confer secondary resistance against ALK-TKI in ALK-rearranged cancers [3, 6]. All reported EML4-ALK variants, including even the shortest variant 5a/b, contain the EML4-cc domain; in the non-EML4 ALK fusion partners reported in NSCLC (e.g., KIF5B-, TPM3-, TPM4-, or TPR-) the amino-terminal fragment of the fusion partner necessarily contains cc domains or oligomerization domains [13,14,15]
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