Predicting functional significance of cancer-associated p16INK4a mutations in CDKN2A

Abstract

Inherited mutations affecting the INK4a/ARF locus (CDKN2A) are associated with melanoma susceptibility in 40% of multiple case melanoma families. Over 60 different germline INK4a/ARF mutations have been detected in more than 190 families worldwide. The majority of these alterations are missense mutations affecting p16INK4a , and only 25% of these have been functionally assessed. There is therefore a need for an accurate and rapid assay to determine the functional significance of p16INK4a mutations. We reviewed the performance of several in vivo functional assays that measure critical aspects of p16INK4a function, including subcellular location, CDK binding and cell cycle inhibition. In this report the function of 28 p16INK4a variants, many associated with melanoma susceptibility were compared. We show that assessment of CDK4 binding and subcellular localization can accurately and rapidly determine the functional significance of melanomaassociated p16INK4a mutations. p16 INK4a -CDK6 binding affinity was unhelpful, as no disease-associated mutation showed reduced CDK6 affinity while maintaining the ability to bind CDK4. Likewise, in silico analyses did not contribute substantially, with only 12 of 25 melanomaassociated missense variants consistently predicted as deleterious. The ability to determine variant functional activity accurately would identify disease-associated mutations and facilitate effective genetic counselling of individuals at high risk of melanoma.

Multiple primary melanomas (MPMs) and criteria for genetic assessment: MultiMEL, a multicenter study of the Italian Melanoma Intergroup

“…44 The other mutations were described with evidence of pathogenicity. [45][46][47][48] The highest mutation rate in MPM cases was found in the northern regions of Italy, particularly in Liguria and Lombardy, followed by Veneto (35%, 24%, and 12%, respectively), whereas the percentage decreased in central regions, although remaining near 10% (Fig 2). When family history was taken into account, we observed that the prevalence of CDKN2A mutations in patients with MPM was as high as 44.4%.…”

The Cell-Cycle Regulator CDK4: An Emerging Therapeutic Target in Melanoma

“…INK4A lead to decreased binding to CDK4 but not CDK6 (35,36), underscoring a predominant role of CDK4 over CDK6 in the development of melanoma. Mutations in CDK4 (R24C) originally identified in melanoma-prone families (28) abolish the interaction with p16 INK4A and, thus, render CDK4 constitutively active (37), further highlighting the importance of CDK4 activation in melanoma.…”

Molecular biology of melanoma

“…The majority of mutations in CDKN2A affect p16 whereas only 2% of at-risk melanoma families have an ARF mutation. 18 Although much is known about this pathway, all of the cross-interactions are not understood. P16 functions to inhibit CDK-4 and keep the retinoblastoma protein in its suppressive state in which it functions to halt the cell cycle 18 (Fig 4).…”