Cancer treatment decisions are increasingly guided by which specific genes are mutated within each patient’s tumor. For example, agents inhibiting the epidermal growth factor receptor (EGFR) benefit many colorectal cancer (CRC) patients, with the general exception of those whose tumor includes a KRAS mutation. However, among the various KRAS mutations, that which encodes the G13D mutant protein (KRASG13D) behaves differently; for unknown reasons, KRASG13D CRC patients benefit from the EGFR-blocking antibody cetuximab. Controversy surrounds this observation, because it contradicts the well-established mechanisms of EGFR signaling with regard to RAS mutations. Here, we identified a systems-level, mechanistic explanation for why KRASG13D cancers respond to EGFR inhibition. A computational model of RAS signaling revealed that the biophysical differences between the three most common KRAS mutants were sufficient to generate different sensitivities to EGFR inhibition. Integrated computation with experimentation then revealed a nonintuitive, mutant-specific dependency of wild-type RAS activation by EGFR that is determined by the interaction strength between KRAS and the tumor suppressor neurofibromin (NF1). KRAS mutants that strongly interacted with and competitively inhibited NF1 drove wild-type RAS activation in an EGFR-independent manner, whereas KRASG13D weakly interacted with and could not competitively inhibit NF1 and, thus, KRASG13D cells remained dependent on EGFR for wild-type RAS activity. Overall, our work demonstrates how systems approaches enable mechanism-based inference in genomic medicine and can help identify patients for selective therapeutic strategies.

PurposeTo investigate the effect of various riboflavin/ultraviolet light (UVA) crosslinking (CXL) protocols on corneal enzymatic resistance.MethodsA total of 66 enucleated porcine eyes, with the corneal epithelium removed, were divided into 6 groups. Group 1 remained untreated. Groups 2 to 6 received riboflavin/dextran for 30 minutes. Group 3 underwent standard CXL (SCXL) with 3 mW/cm2 UVA for 30 minutes (total energy dose 5.4 J/cm2). Groups 4 and 5 underwent high intensity CXL (HCXL) using 30 mW/cm2 UVA for 3 minutes (5.4 J/cm2) and 30 mW/cm2 for 4 minutes (7.2 J/cm2), respectively. Group 6 was exposed to 8 minutes of 30 mW/cm2 UVA in a 10-second on/10-second off pulsed-radiation mode (p-HCXL; 7.2 J/cm2). A central 8-mm disk from each cornea was submerged in pepsin digest solution at 23°C and measured daily. After 13 days, the dry weight was recorded from 5 samples in each group.ResultsThe CXL-treated corneas took longer to digest than nonirradiated corneas (P < 0.0001). Differences in digestion time also were observed between CXL groups, such that, HCXL (5.4 J/cm2) < SCXL (5.4 J/cm2) < HCXL (7.2 J/cm2) < p-HCXL (7.2 J/cm2; P < 0.0001). The dry weight of the SCXL (5.4 J/cm2) group was higher than the HCXL (5.4 and 7.2 J/cm2; P < 0.001) and p-HCXL 7.2 J/cm2 (P <0.05) groups. No difference was detected between the HCXL and p-HCXL 7.2 J/cm2 groups.ConclusionsThe intensity and distribution of the crosslinks formed within the cornea vary with different UVA protocols. The precise location and amount of crosslinking needed to prevent disease progression is unknown.

Data presented here suggest that CXL is carbonyl dependent and involves the formation of AGE cross-links. Six possible cross-linking mechanisms are discussed.

PURPOSE. Extracellular matrix metalloproteinases (MMPs) are thought to play a crucial role in corneal degradation associated with the pathological progression of keratoconus. Currently, corneal cross-linking by riboflavin and ultraviolet A (RFUVA) has received significant attention for treatment of keratoconus. However, the extent to which MMPs digest cross-linked collagen and small leucine-rich proteoglycans (SLRPs) remains unknown. In this study, the resistance of RFUVA-cross-linked collagens and SLRPs to MMPs has been investigated. METHODS.To investigate the ability of MMPs to digest crosslinked collagen and SLRPs, a model reaction system using purified collagen type I, type IV, and nonglycosylated, commercially available recombinant SLRPs, keratocan, lumican, mimecan, decorin, and biglycan in solution in vitro has been compared using reactions inside an intact bovine cornea, ex vivo.RESULTS. Our data demonstrate that corneal cross-linked collagen type I and type IV are resistant to cleavage by MMP-1, MMP-2, MMP-9, and MMP-13, whereas non-cross-linked collagen I, IV, and natively glycosylated SLRPs are susceptible to degradation by MMPs. In addition, both cross-linked SLRPs themselves and cross-linked polymers of SLRPs and collagen appear able to resist degradation. These results suggest that the interactions between SLRPs and collagen caused by RFUVA protect both SLRPs and collagen fibrils from cleavage by MMPs. CONCLUSIONS.A novel approach for understanding the biochemical mechanism whereby RFUVA cross-linking stops keratoconus progression has been achieved. (Invest Ophthalmol Vis Sci.