SUMMARY We describe the landscape of genomic alterations in cutaneous melanomas through DNA, RNA, and protein-based analysis of 333 primary and/or metastatic melanomas from 331 patients. We establish a framework for genomic classification into one of four subtypes based on the pattern of the most prevalent significantly mutated genes: mutant BRAF, mutant RAS, mutant NF1, and Triple-WT (wild-type). Integrative analysis reveals enrichment of KIT mutations and focal amplifications and complex structural rearrangements as a feature of the Triple-WT subtype. We found no significant outcome correlation with genomic classification, but samples assigned a transcriptomic subclass enriched for immune gene expression associated with lymphocyte infiltrate on pathology review and high LCK protein expression, a T cell marker, were associated with improved patient survival. This clinicopathological and multidimensional analysis suggests that the prognosis of melanoma patients with regional metastases is influenced by tumor stroma immunobiology, offering insights to further personalize therapeutic decision-making.

Recently, a rare activating mutation of AKT1 (E17K) has been reported in breast, ovarian, and colorectal cancers. However, analogous activating mutations in AKT2 or AKT3 have not been identified in any cancer lineage. To determine the prevalence of AKT E17K mutations in melanoma, the most aggressive form of skin cancer, we analysed 137 human melanoma specimens and 65 human melanoma cell lines for the previously described activating mutation of AKT1, and for analogous mutations in AKT2 and AKT3. We identified a single AKT1 E17K mutation. Remarkably, a previously unidentified AKT3 E17K mutation was detected in two melanomas (from one patient) as well as two cell lines. The AKT3 E17K mutation results in activation of AKT when expressed in human melanoma cells. This represents the first report of AKT mutations in melanoma, and the initial identification of an AKT3 mutation in any human cancer lineage. We have also identified the first known human cell lines with naturally occurring AKT E17K mutations.

A moderately stable protein with typical folding kinetics unfolds and refolds many times during its cellular lifetime. In monomeric lambda repressor this process is extremely rapid, with an average folded state lifetime of only 30 milliseconds. A thermostable variant of this protein (G46A/G48A) unfolds with the wild-type rate, but it folds in approximately 20 microseconds making it the fastest-folding protein yet observed. The effects of alanine to glycine substitutions on the folding and unfolding rate constants of the G46A/G48A variant, measured by dynamic NMR spectroscopy, indicate that the transition state is an ensemble comprised of a disperse range of conformations. This structural diversity in the transition state is consistent with the idea that folding chains are directed towards the native state by a smooth funnel-like conformational energy landscape. The kinetic data for the folding of monomeric lambda repressor can be understood by merging the new energy landscape view of folding with traditional models. This hybrid model incorporates the conformational diversity of denatured and transition state ensembles, a transition state activation energy, and the importance of intrinsic helical stabilities.

Twelve amino acid substitutions of varying size and hydrophobicity were constructed at Val 143 in human carbonic anhydrase II (including Gly, Ser, Cys, Asn, Asp, Leu, Ile, His, Phe and Tyr) to examine the catalytic roles of the hydrophobic pocket in the active site of this enzyme. The CO2 hydrase and p-nitrophenyl acetate (PNPA) esterase activities, the pKa of the zinc-water ligand, the inhibition constant for cyanate (KOCN), and the binding constants for sulfonamide inhibitors were measured for various mutants and correlated with the size and hydrophobicity of the substituted amino acid. The kcat/KM for PNPA hydrolysis and KOCN are linearly dependent on the hydrophobicity of the amino acid at position 143. All of the activities of CAII are decreased by more than a factor of 10(3) when large amino acids (Phe and Tyr) are substituted for Val 143, but the CO2 hydrase activity is the most sensitive to the size and structure of the substituted amino acid. Addition of a single methyl group (V143I) decreases the activity 8-fold, while substitution of valine by tyrosine essentially destroys the enzyme function (kcat/KM for CO2 hydration is decreased by more than 10(5)-fold). KOCN does not increase until Phe is substituted for Val 143, suggesting that the cyanate and CO2 binding sites are not identical. The functional data in conjunction with X-ray crystallographic studies of four of the mutants [Alexander et al., 1991 (following paper in this issue)] allow interpretation of the mutants at a molecular level and mapping of the region of the active site important for CO2 association. The hydrophobic pocket, including residues Val 121 and Val 143, is important for CO2 and PNPA association; if the pocket is blocked, substrates cannot approach the zinc-hydroxide with the correct orientation to react. The interaction between Val 143 and CO2 is relatively weak (less than or equal to 0.5 kcal/mol) and nonspecific; the association site does not tightly hold CO2 in one fixed orientation for reaction with the zinc-hydroxide. This mechanism of catalysis may reflect a decreased requirement for specific orientation by CO2 since it is a symmetrical molecule.