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In cancer research, RAS biology has been focused on only a handful of tumor types. While RAS genes have long been suspected as common contributors to a wide spectrum of cancer types, robust evidence is required to firmly establish their critical oncogenic significance. We present a data mining study using DepMap genome-wide CRISPR screening data, which provide substantial evidence to support the prominent pervasive oncogenic role and tissue-specific permissiveness of RAS gene mutations. Differential analysis of CRISPR effect scores identifies K- or N-RAS genes as the most differential gene in contrasts of (K-, N-, combined) RAS mutant versus wild-type cell lines across multiple tissue types. The distinguished tissue-specific pattern of KRAS vs. NRAS as top differential genes in subsets of tissue types and evidence from genome data supported the idea of KRAS- and NRAS-engaged tissue types. To our knowledge, this is the first report of prominent pervasive oncogenic role of RAS mutations revealed by gene dependency data that is beyond the current understanding of the oncogenic role of RAS genes and their well-known involved tissue types. Our findings strongly support RAS mutations as primary oncogenic drivers beyond traditionally recognized cancer types and offer insights into their tissue-specific permissiveness.

As part of our efforts to develop rhenium-oxo corroles as photosensitizers for oxygen sensing and photodynamic therapy, we investigated the potential β-perhalogenation of five ReO meso-tris(para-X-phenyl)corroles, Re[TpXPC](O) (X = CF3, H, F, CH3, and OCH3), with elemental chlorine and bromine. With Cl2, β-octachlorinated products Re[Cl8TpXPC](O) were rapidly obtained for X = CF3, H, and CH3, but X = OCH3 resulted in overchlorination on the meso-aryl groups. Full β-octabromination proved slower relative to Cu and Ir corroles, but the desired Re[Br8TpXPC](O) products were finally obtained for X = H and F after a week at room temperature. For X = CH3 and OCH3, these conditions led to undecabrominated products Re[Br11TpXPC](O). Compared to the β-unsubstituted starting materials, the β-octahalogenated products were found to exhibit sharp 1H NMR signals at room temperature, indicating that the aryl groups are locked in place by the β-halogens, and substantially redshifted Soret and Q bands. Single-crystal X-ray structures of Re[Cl8TpCF3PC](O), Re[Cl8TpCH3PC](O), and Re[Br8TpFPC](O) revealed mild saddling for one Cl8 structure and the Br8 structure. These structural variations, however, appear too insignificant to explain the slowness of the β-octabromination protocols, which seems best attributed to the deactivating influence of the high-valent Re center.

For nearly a decade, the difficulties associated with both the determination and reproducibility of Ras-dependency indexes (RDIs) have limited their application and further delineation of the biology underlying Ras dependency. In this report, we describe the application of a computational single sample gene set enrichment analysis (ssGSEA) method to derive RDIs with gene expression data. The computationally derived RDIs across the Cancer Cell Line Encyclopedia (CCLE) cell lines show excellent agreement with the experimentally derived values and high correlation with a previous in-house siRNA effector node (siREN) study and external studies. Using EMT signature-derived RDIs and data from cell lines representing the extremes in RAS dependency, we identified enriched pathways distinguishing these classes, including the Fas signaling pathway and a putative Ras-independent pathway first identified in NK cells. Importantly, extension of the method to patient samples from The Cancer Genome Atlas (TCGA) showed the same consensus differential expression patterns for these two pathways across multiple tissue types. Last, the computational RDIs displayed a significant association with TCGA cancer patients' survival outcomes. Together, these lines of evidence confirm that our computationally derived RDIs faithfully represent a measure of Ras dependency in both cancer cell lines and patient samples. The application of such computational RDIs can provide insights into Ras biology and potential clinical applications.

Six-coordinate iridium(III) triarylcorrole derivatives, Ir[TpXPC)]L2, where TpXPC = tris(para-X-phenyl)corrole (X = CF3, H, Me, and OCH3) and L = pyridine (py), trimethylamine (tma), isoquinoline (isoq), 4-dimethylaminopyridine (dmap), and 4-picolinic acid (4pa), have been examined, with a view to identifying axial ligands most conducive to near-infrared phosphorescence. Disappointingly, the phosphorescence quantum yield invariably turned out to be very low, about 0.02 - 0.04% at ambient temperature, with about a two-fold increase at 77 K. Phosphorescence decay times were found to be around ~5 µs at 295 K and ~10 µs at 77 K. Fortunately, two of the Ir[TpCF3PC)]L2 derivatives, which were tested for their ability to sensitize singlet oxygen formation, were found to do so efficiently with quantum yields Φ(1O2) = 0.71 and 0.38 for L = py and 4pa, respectively. Iridium corroles thus may hold promise as photosensitizers in photodynamic therapy (PDT). The possibility of varying the axial ligand and of attaching biotargeting groups at the axial positions makes iridium corroles particularly exciting as PDT drug candidates.

Although post-translational modification of the C-terminus of RAS has been studied extensively, little is known about N-terminal processing. Mass spectrometric characterization of KRAS expressed in mammalian cells showed cleavage of the initiator methionine (iMet) and N-acetylation of the nascent N-terminus. Interestingly, structural studies on GDP- and GMPPNP-bound KRAS lacking the iMet and N-acetylation resulted in Mg2+-free structures of KRAS with flexible N-termini. In the Mg2+-free KRAS-GDP structure, the flexible N-terminus causes conformational changes in the interswitch region resulting in a fully open conformation of switch I. In the Mg2+-free KRAS-GMPPNP structure, the flexible N-terminus causes conformational changes around residue A59 resulting in the loss of Mg2+ and switch I in the inactive state 1 conformation. Structural studies on N-acetylated KRAS-GDP lacking the iMet revealed the presence of Mg2+ and a conformation of switch regions also observed in the structure of GDP-bound unprocessed KRAS with the iMet. In the absence of the iMet, the N-acetyl group interacts with the central beta-sheet and stabilizes the N-terminus and the switch regions. These results suggest there is crosstalk between the N-terminus and the Mg2+ binding site, and that N-acetylation plays an important role by stabilizing the N-terminus of RAS upon excision of the iMet.

Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer's disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5-10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ.