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Crystal Structure of EML1 Reveals the Basis for Hsp90 Dependence of Oncogenic EML4-ALK by Disruption of an Atypical Β-Propeller Domain

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Crystal Structure of EML1 Reveals the Basis for Hsp90 Dependence of Oncogenic EML4-ALK by Disruption of an Atypical Β-Propeller Domain Crystal structure of EML1 reveals the basis for Hsp90 dependence of oncogenic EML4-ALK by disruption of an atypical β-propeller domain Mark W. Richardsa, Edward W. P. Lawb,La’Verne P. Rennallsc, Sara Busaccab, Laura O’Regana, Andrew M. Frya, Dean A. Fennellb, and Richard Baylissa,1 aDepartment of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom; bDepartment of Cancer Studies and Molecular Medicine, University of Leicester, Leicester LE1 9HN, United Kingdom; and cSection of Structural Biology, Institute of Cancer Research, London SW3 6JB, United Kingdom Edited by Charles David Stout, The Scripps Research Institute, La Jolla, CA, and accepted by the Editorial Board February 24, 2014 (received for review December 9, 2013) Proteins of the echinoderm microtubule-associated protein (EMAP)- variant 1 and regression in some EML4-ALK–positive tumor like (EML) family contribute to formation of the mitotic spindle and models (7, 9, 10). Furthermore, clinical efficacy of an Hsp90 in- interphase microtubule network. They contain a unique hydropho- hibitor in EML4-ALK NSCLC has been confirmed (11), and bic EML protein (HELP) motif and a variable number of WD40 clinical trials are ongoing. However, because neither ALK nor repeats. Recurrent gene rearrangements in nonsmall cell lung cancer EML4 are native Hsp90 clients, it was proposed that Hsp90 sen- fuse EML4 to anaplastic lymphoma kinase (ALK), causing expression sitivity of EML4-ALK fusions was due to their protein-folding of several fusion oncoprotein variants. We have determined a 2.6-Å properties, which might expose hydrophobic residues that lead to crystal structure of the representative ∼70-kDa core of EML1, re- Hsp90 recruitment (12). EML4-ALK variants incorporating vari- β vealing an intimately associated pair of -propellers, which we term ous portions of EML4 differ in sensitivity to Hsp90 inhibitors, a TAPE (tandem atypical propeller in EMLs) domain. One propeller is suggesting that they have different protein-folding properties (12). highly atypical, having a discontinuous subdomain unrelated to In contrast to the abundance of studies on EML4-ALK fusions, a WD40 motif in place of one of its blades. This unexpected feature the basic functions of EML proteins are relatively understudied. BIOCHEMISTRY shows how a propeller structure can be assembled from subdo- The archetypal EML protein was identified as the most abundant mains with distinct folds. The HELP motif is not an independent MAP in dividing echinoderm eggs and embryos, where it pro- domain but forms part of the hydrophobic core that joins the two motes microtubule dynamics (13, 14). Microtubule interactions of β-propellers. The TAPE domain binds α/β-tubulin via its conserved, EML1-4 have been characterized in six EML proteins present in concave surface, including part of the atypical blade. Mapping the – characteristic breakpoints of each EML4-ALK variant onto our struc- humans (15 18). More members of the family have been identified ture indicates that the EML4 TAPE domain is truncated in many in Drosophila and Caenorhabditis elegans, which each have a single variants in a manner likely to make the fusion protein structurally homolog (19). The EML proteins contain a variable number of ∼ unstable. We found that the heat shock protein 90 (Hsp90) inhibitor WD40 repeats, and in EML1-4 these fall within an 70-kDa core ∼ ganetespib induced degradation of these variants whereas others region that begins with a conserved 60-amino-acid sequence that lacking a partial TAPE domain were resistant in both overexpression has been termed the hydrophobic EML protein (HELP) motif models and patient-derived cell lines. The Hsp90-sensitive EML4-ALK variants are exceptions to the rule that oncogenic fusion proteins Significance involve breakpoints in disordered regions of both partners. Echinoderm microtubule-associated protein (EMAP)-like (EML) structural biology | stratified medicine proteins normally function in the cytoskeleton. In some lung cancers, genetic abnormalities generate the oncogenic fusion earrangements in the short arm of chromosome 2 leading to protein EML4-anaplastic lymphoma kinase (ALK) on which the Rgenetic fusions between EML4 [echinoderm microtubule- cancer cells depend for survival. We have determined the mo- associated protein (EMAP)-like 4] and the gene encoding ana- lecular structure of a conserved, tubulin-binding region of plastic lymphoma kinase (ALK) occur in ∼5% of cases of non- EML1 that reveals an unexpected protein fold. This region is small cell lung cancer (NSCLC) (1). Multiple variants of the disrupted in ∼70% of EML4-ALK fusions found in patients, EML4-ALK fusion have been identified in NSCLC resulting causing them to be sensitive to drugs that target Hsp90, a cel- from translocations at different points within the EML4 gene (2). lular factor that stabilizes misfolded protein. Our findings will A similar genetic fusion has also been reported between EML1 potentially enable more effective, stratified therapy of EML4- and the gene encoding another tyrosine kinase, Abelson 1 (ABL1), ALK nonsmall cell lung cancer and suggest that the truncation in T-cell acute lymphoblastic leukemia (3). These fusions display of a globular domain at the translocation breakpoint may prove potent transforming activity due to constitutive activation of the generally predictive of Hsp90 inhibitor sensitivity in cancers tyrosine kinase but confer addiction to the oncogene, and inhi- driven by fusion oncogenes. bition of the tyrosine kinase induces apoptosis in transformed cells (4, 5). The ALK inhibitor crizotinib is a highly effective first-line Author contributions: M.W.R., E.W.P.L., L.O., A.M.F., D.A.F., and R.B. designed research; M.W.R., E.W.P.L., L.P.R., and S.B. performed research; M.W.R., E.W.P.L., S.B., D.A.F., and therapy for NSCLC patients with EML4-ALK fusions, but because R.B. analyzed data; and M.W.R., A.M.F., D.A.F., and R.B. wrote the paper. the acquirement of resistance is inevitable, often involving sec- The authors declare no conflict of interest. ondary mutations in the kinase portion of the fusion protein, ad- This article is a PNAS Direct Submission. C.D.S. is a guest editor invited by the Editorial ditional therapeutic strategies must be identified (6, 7). Board. Inhibitors of the molecular chaperone heat shock protein 90 Data deposition: The atomic coordinates and structure factors have been deposited in the (Hsp90) are being investigated as potential cancer therapeutics Protein Data Bank, www.pdb.org (PDB ID code 4ci8). because many oncoproteins are obligate Hsp90 clients and they 1To whom correspondence should be addressed. E-mail: [email protected]. are rapidly degraded by the proteasome when Hsp90 is inhibited This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (8). Hsp90 inhibitors induce the degradation of EML4-ALK 1073/pnas.1322892111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1322892111 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 (15). Many EML proteins also contain a predicted coiled-coil (CC) TAPE domain, with one highly atypical blade built from two in their N termini that is likely to mediate oligomerization. separate regions of the primary sequence (Fig. 1). Functional studies of EML proteins and their oncogenic fusions The overall topology of the TAPE domain resembles the tan- have had to be interpreted without knowledge of their molecular dem β-propeller of actin-interacting protein 1 (AIP1) (21, 22). The structures. For example, it has been assumed that the HELP motif two β-propellers are intimately associated with one another at an forms an independent domain that perhaps mediates specific angle of ∼50°. Residues 230–540 form a seven-bladed β-propeller interactions with microtubules (MTs) or the plasma membrane domain (green in Fig. 1). Each blade is a twisted, four-stranded, (15, 17, 18, 20). The extent to which the folding of the WD40 antiparallel β-sheet that radiates from the center of the domain and repeat region is disrupted in EML4-ALK fusions is also unclear is encoded by a separate WD40 repeat. As in AIP1, but unusually because the number, position, and arrangement of β-propellers in for β-propeller domains, the seventh blade of this N-terminal EML proteins are unknown. We determined the crystal structure β-propeller is self-contained rather than having its fourth strand of the ∼70-kDa core HELP/WD region of human EML1. The donated by a more N-terminal part of the sequence. Residues – β structure serves as an archetype for the whole family and has an 542 815 form the C-terminal -propeller of EML1, which is also unusual molecular architecture that we have termed the TAPE similar in overall size and topology to a classical seven-bladed β (tandem atypical propeller in EMLs) domain. The structure en- -propeller (blue). Its seventh blade (blade 14) is completed – abled us to dissect the molecular determinants of tubulin-binding by a fourth strand formed by residues (220 227) immediately β β and map the precise location of breakpoints in EML4-ALK patients N-terminal to the first -propeller. The two -propellers commu- onto the structure. We explain how the partial EML moieties that nicate through contacts between the face of blade 1 and the edges of blades 14 and 13. are present in each of the variants confer differences between β them with respect to Hsp90 inhibitor sensitivity. Blade 12 is not a canonical four-stranded -sheet, but is com- posed of two subdomains, which we have termed Blade12-N and Results Blade12-C, that correspond to two distant stretches of primary β Conserved Core Region of EML1 Forms a Tandem β-Propeller Structure. sequence. This generates a -propeller structure in which one of We expressed fragments of human EML1–4 corresponding to an the blades is replaced by a subdomain of sequence and structure D–F ∼70-kDa core region that is conserved across the EMAP/EML entirely unrelated to the other constituent repeats (Fig.
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