Here, we generated T cells expressing a mutation-specific transgenic T cell receptor (TCR) to target different immunogenic mutations in cyclin-dependent kinase 4 (CDK4) that naturally occur in human melanoma.
These data strongly support the idea that deregulation of the CDK4/cyclin D pathway, via CDKN2 or CDK4 mutations, is of biological significance in the development of melanoma.
In the case of CDK4, only one specific mutation, resulting in the substitution of a cysteine for an arginine at codon 24 (R24C), has been found to be associated with melanoma.
Nearly 100% of melanomas have a defect in the p16(INK4A):cyclin D-CDK4/6:RB pathway, leading to abnormal cell cycle control and unregulated cellular proliferation.
No mobility shifts indicating sequence alterations were observed in 273 tumors and 49 cell lines from diverse kinds of tumors These results suggest that in contrast to melanoma, in many other types of human neoplasms the mutation of the CDK4 gene is very rare.
The cellular activities of four melanoma-associated p16(INK4a) mutations (Arg24Pro, Ala36Pro, Met53Ile, and Val126Asp) were compared by use of inducible expression in stably transfected melanoma cells, deficient in expression of the endogenous protein, and compared with their ability to bind CDK4.
A proportion of melanoma-prone individuals in both familial and non-familial contexts has been shown to carry inactivating mutations in either CDKN2A or, rarely, CDK4.
Therefore, to localize additional loci involved in melanoma susceptibility, we have performed a genomewide scan for linkage in 49 Australian pedigrees containing at least three CMM cases, in which CDKN2A and CDK4 involvement has been excluded.
Human BRAF-V600-mutant melanoma (A375) xenograft-bearing balb/c nude mice (n = 21) were imaged by <sup>18</sup>F-FDG-PET/CT and DW-MRI before (day 0) and after (day 7) a 1-week BRAF and CDK 4/6 inhibitor combination therapy (n = 12; dabrafenib, 20 mg/kg/d; ribociclib, 100 mg/kg/d) or placebo (n = 9).
In summary, our results show frequent involvement of the p16 gene in familial melanoma and confirm the role of the CDK4 gene as a melanoma-predisposing gene.
Polymerase chain reaction and Southern blot analysis confirmed the frequent deletion or rearrangement of the CDK4-inhibitor gene in melanomas, gliomas, lung cancers and leukaemias, and the absence of detectable gene transcripts.
The gene for cyclin-dependent kinase 4, CDK4, has been found in mutated form in the germline from individuals belonging to 2 melanoma kindreds in the United States.
Among patients included in the North Sardinia tumor registry, 316 patients first evaluated for familial recurrence of melanoma were submitted to mutation analysis in CDKN2A and CDK4 genes.
To examine the contribution of rare and common variation within known MM genes in sporadic US MM patients, coding regions of known MM susceptibility genes [cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase 4, melanocortin 1 receptor (MC1R) and tyrosinase (TYR)] were resequenced in 109-135 MM cases.
Surprisingly, at the DNA level alone, 96% (43/45) of melanoma cell lines examined were found to be deleted/mutated/methylated for CDKN2A (34/45), homozygously deleted for CDKN2A's neighbor and homolog CDKN2B (6/45), and/or mutated/amplified for CDK4 (5/45).
We identified germline mutations in highly CM-associated genes (CDKN2A and CDK4) and low/medium-penetrance variants (MC1R and MITF) in patients with multiple primary CMs or individuals with one or more CM and a positive family history for CM or pancreatic cancer among first- or second-degree relatives.
As the pivotal residues around the most predominant R24C activating CDK4 mutation are invariant between CDK2 and CDK4, we speculated that the pivotal arginine (position 22 in CDK2), or a nearby residue, may be mutated in some melanomas, resulting in the diminution of its binding and inhibition by p27KIP1 or p21CIP1.
Of these, 238 cases (34 familial, 14 non-familial multiple primary and 190 non-familial single primary melanomas) were consecutively enrolled for screening of the CDKN2A and CDK4 genes.
: The entire CDKN2A coding region and exon 2 of the CDK4 gene of an affected member of each of 52 families from southern Sweden with at least two cases of melanoma in first- or second-degree relatives were screened for mutations by use of polymerase chain reaction-single-strand conformation polymorphism analysis.Statistical tests were two-sided.
Rare high-penetrance factors are expressed in familial clustering of melanoma and include mutations in CDKN2A (encoding p16(INK4a) and p14(ARF)) and CDK4.