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C ritical Assessment of Genome Interpretation

The Nucleophosmin-Anaplastic Lymphoma Kinase fusion protein (NPM-ALK): predict the effect of mutations in the kinase domain on kinase activity and Hsp90 binding affinity

Challenge: NPM-ALK

Dataset description: public

Variant data: registered users only, limited by CAGI Data Use Agreement

Last updated: 4 April 2016

This challenge closed at 9:00 PM PST (Pacific Standard Time) on 9 November 2015.

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Summary

NPM-ALK is a fusion protein in which the ALK tyrosine kinase is constitutively activated, contributing to cancer. NPM-ALK constructs with mutations in the kinase domain have been assayed in extracts of transfected cells. The challenge is to predict the kinase activity and the Hsp90 binding affinity of the mutant proteins relative to the reference NPM-ALK fusion protein.

Background

ALK is a receptor tyrosine kinase originally described in t(2;5)(p23;q35)-positive anaplastic large cell lymphoma (ALCL) as part of the NPM-ALK fusion gene. Although the physiological function and regulation of full-length ALK remains poorly characterized, aberrant expression of constitutively activated NPM-ALK has been clearly established as the leading cause of ALK-positive ALCL. In this tumor, the presence of an NPM oligomerization domain promotes ligand-independent NPM-ALK kinase dimerization and constitutive self-phosphorylation. Aberrantly activated NPM-ALK causes cell transformation through activation of several biological pathways related to cell proliferation, cell-cycle control and apoptosis.

Chromosomal aberrations involving ALK have been identified in several other tumor types including non-small cell lung carcinoma (NSCLC) and inflammatory myofibroblastic tumor (IMT). In all these cases dimerization via fusion partners leads to ligand-independent activation of ALK, resulting in constitutive kinase activity and continuous signalling. Cancer cells in which genomic alterations of ALK lead to expression of truncated variants are strongly dependent upon ALK signalling, and its inhibition leads to a marked decrease in tumor growth and survival.

Small-molecule inhibitors of ALK are among the most promising drugs in several high-risk cancers, since ALK activation by mutation, amplification, or gene rearrangement is highly oncogenic. The ALK kinase inhibitor Crizotinib, for instance, has been approved for the treatment of ALK-rearranged malignancies including adult NSCLC and paediatric ALCL. However, recent studies suggest that inhibitor efficacy may be hampered by several resistance mechanisms including point mutations in ALK [1-3]. In this context, the inhibition of the molecular chaperone Hsp90 represents an alternative approach to overcoming resistance to kinase inhibitors, since NPM-ALK, like many other kinases, is strictly dependent on molecular chaperones for its maturation and activity [4]. Conformational stability of ALK is known to be maintained by Hsp90, but the principles of this interaction, the specific domains or motifs recognized, and the impact of mutations on chaperone activity remain obscure.

Experiment

The Bonvini lab has examined the kinase activity and Hsp90 binding affinity of a series of NPM-ALK constructs harboring single amino acid mutations, multiple amino acid mutations, or deletions in the ALK catalytic domain to define the manner by which nascent NPM-ALK kinase is recognized by Hsp90, and how Hsp90 helps to facilitate NPM-ALK folding, activity, and/or stability. Structural motifs and specific residues in or immediately adjacent to the NPM-ALK catalytic domain were analyzed [5-6] to identify the determinants of Hsp90 interaction based on the tendency of NPM-ALK to fold. Understanding these dynamics may be of particular interest to predicting NPM-ALK sensitivity to Hsp90 inhibitors, as well as to addressing the role of Hsp90 in maintaining the active conformation of NPM-ALK in cancer cells.

Hek-293T cells were transiently transfected with wild-type (WT) or mutant NPM-ALK constructs. ALK kinase activity was assessed by Western blotting using site-specific antibodies against phosphorylated ALK (Tyr1604) and STAT3 (Tyr705). Kinase activity was assessed by comparing phosphorylation status of NPM-ALK and STAT3: 0 = no activity (no phosphorylation of ALK, no increase of STAT3 basal phosphorylation) 0.5 = less activity than WT (lower levels of phosphorylated ALK and STAT3 ) 1 = same activity as WT (comparable levels of phosphorylated ALK and STAT3.) 2 = more activity than WT (increased levels of phosphorylated ALK and STAT3.)

To assess the effects of mutations on NPM-ALK binding to Hsp90, fresh protein lysates obtained from transfected cells were co-immunoprecipitated (IP) using antibodies specific for ALK or Hsp90. Immunocomplexes were resolved by SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed with anti-Hsp90 or anti-ALK antibodies, respectively. NPM-ALK was detected in Hsp90 IP samples, and a comparable level of Hsp90 bound to NPM-ALK was detected in the reciprocal co-IP experiment. Binding was measured as the interaction intensity (band density) of each mutant relative to WT NPM-ALK. 0 = mutant protein does not bind to Hsp90 0.5 = mutant protein binds to Hsp90 less than WT NPM-ALK 1 = mutant protein binds to Hsp90 as much as WT NPM-ALK 2 = mutant protein binds to Hsp90 more than WT NPM-ALK.

Prediction challenge

Participants are asked to submit predictions of both the kinase activity and the Hsp90 binding affinity of each mutant protein relative to the reference NPM-ALK fusion protein. The submitted prediction should be a numeric value ranging from 0 (no activity) to 1 (reference level of activity) or >1 if the predicted activity is greater than the activity of the reference fusion protein (e.g. 0.7 means 70% of reference and 1.3 means 130% of reference activity). Each predicted activity must include a standard deviation indicating confidence. Where multiple mutations are listed for a mutant protein, the challenge is to predict the combined effect of those mutations. Participants may submit predictions on either kinase activity, Hsp90 binding affinity, or both. An optional brief comment on the basis of each prediction may be given. The predictions will be assessed against the values actually measured for each mutant in the two assays.

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Additional information

The NPM-ALK reference sequence in GenBank: U04946.1 The full-length ALK reference sequence in GenBank: NM_004304.4 The full-length ALK reference sequence in Ensembl: ENST00000389048. Note that most publications refer to coordinates in the full-length ALK sequence.

Prediction submission format

The prediction submission is a tab-delimited text file. Organizers provide a file template, which should be used for submission. In addition, a validation script is provided, and predictors should check the correctness of the format before submitting their predictions.

In the submitted file, each row includes the following columns:

mutant - Substituted or deleted residue(s) in the NPM-ALK fusion protein
kinase activity - Prediction of kinase activity relative to the reference NPM-ALK fusion protein (0, 0.5, 1, 2)
s.d.(activity) - Standard deviation of the prediction in column 2
comment_activity - Optional brief comment on the basis of the prediction in column 2
Hsp90_binding - Prediction of Hsp90 binding affinity relative to the reference NPM-ALK fusion protein (0 - 1, or >1)
s.d.(binding) - Standard deviation of the prediction in column 5
comment_binding - Optional brief comment on the basis of the prediction in column 5

In the template file, cells in columns 2-5 are marked with a "*". Submit your predictions by replacing the "*" with your value. No empty cells are allowed in the submission; if you cannot submit predictions for a mutant, leave the "*" in these cells. Please make sure you follow the submission guidelines strictly.

In addition, your submission should include a detailed description of the method used to make the predictions (similar in style to the Methods section in a scientific article). This information will be submitted as a separate file.

To submit predictions, you need to create or be part of a CAGI User group. Submit your predictions by accessing the link "All submission forms" from the front page of your group. For more details, please read the FAQ page.

References

Lu L, Ghose AK, Quail MR, Albom MS, Durkin JT, Holskin BP, Angeles TS, Meyer SL, Ruggeri BA, Cheng M. ALK mutants in the kinase domain exhibit altered kinase activity and differential sensitivity to small molecule ALK inhibitors. Biochemistry. 2009 Apr 28;48(16):3600-9. doi: 10.1021/bi8020923.
Ceccon M, Mologni L, Bisson W, Scapozza L, Gambacorti-Passerini C. Crizotinib-resistant NPM-ALK mutants confer differential sensitivity to unrelated Alk inhibitors. Mol Cancer Res. 2013 Feb;11(2):122-32. doi: 10.1158/1541-7786.MCR-12-0569. Epub 2012 Dec 13. PubMed PMID: 23239810.
Lovisa F, Cozza G, Cristiani A, Cuzzolin A, Albiero A, Mussolin L, Pillon M, Moro S, Basso G, Rosolen A, Bonvini P. ALK kinase domain mutations in primary anaplastic large cell lymphoma: consequences on NPM-ALK activity and sensitivity to tyrosine kinase inhibitors. PLoS One. 2015 Apr 13;10(4):e0121378. doi: 10.1371/journal.pone.0121378. eCollection 2015. PubMed PMID: 25874976; PubMed Central PMCID: PMC4395299.
Bonvini P, Gastaldi T, Falini B, Rosolen A. Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), a novel Hsp90-client tyrosine kinase: down-regulation of NPM-ALK expression and tyrosine phosphorylation in ALK(+) CD30(+) lymphoma cells by the Hsp90 antagonist 17-allylamino,17-demethoxygeldanamycin. Cancer Res. 2002 Mar 1;62(5):1559-66.
Bonvini P, Dalla Rosa H, Vignes N, Rosolen A. Ubiquitination and proteasomal degradation of nucleophosmin-anaplastic lymphoma kinase induced by 17-allylamino-demethoxygeldanamycin: role of the co-chaperone carboxyl heat shock protein 70-interacting protein. Cancer Res. 2004 May 1;64(9):3256-64. PubMed PMID: 15126367.
Tartari CJ, Gunby RH, Coluccia AM, Sottocornola R, Cimbro B, Scapozza L, Donella-Deana A, Pinna LA, Gambacorti-Passerini C. Characterization of some molecular mechanisms governing autoactivation of the catalytic domain of the anaplastic lymphoma kinase. J Biol Chem. 2008 Feb 15;283(7):3743-50. Epub 2007 Dec 10. PubMed PMID: 18070884.

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