A saturation mutagenesis screen uncovers resistant and sensitizing secondary KRAS mutations to clinical KRASG12C inhibitors.

Mutant-specific inhibitors of KRASG12C, such as AMG510 (sotorasib) and MRTX849 (adagrasib), offer the unprecedented opportunity to inhibit KRAS, the most frequently mutated and heretofore undruggable oncoprotein. While clinical data are still limited, on-target mutations in KRASG12C at position 12 and other sites are emerging as major drivers of clinical relapse. We identified additional mutations in KRASG12C that impact inhibitor sensitivity through a saturation mutagenesis screen in the KRASG12C NCI-H358 non­small-cell lung cancer (NSCLC) cell line. We also identified individuals in population genetic databases harboring these resistance mutations in their germline and in tumors, including a subset that co-occur with KRASG12C, indicating that these mutations may preexist in patients treated with KRASG12C inhibitors. Notably, through structural modeling, we found that one such mutation (R68L) interferes with the critical protein­drug interface, conferring resistance to both inhibitors. Finally, we uncovered a mutant (S17E) that demonstrated a strong sensitizing phenotype to both inhibitors. Functional studies suggest that S17E sensitizes KRASG12C cells to KRASG12C inhibition by impacting signaling through PI3K/AKT/mTOR but not the MAPK signaling pathway. Our studies highlight the utility of unbiased mutation profiling to understand the functional consequences of all variants of a disease-causing genetic mutant and predict acquired resistant mutations in the targeted therapeutics.

CRISPR whole-genome screening identifies new necroptosis regulators and RIPK1 alternative splicing.

The necroptotic cell death pathway is a key component of human pathogen defense that can become aberrantly derepressed during tissue homeostasis to contribute to multiple types of tissue damage and disease. While formation of the necrosome kinase signaling complex containing RIPK1, RIPK3, and MLKL has been extensively characterized, additional mechanisms of its regulation and effector functions likely remain to be discovered. We screened 19,883 mouse protein-coding genes by CRISPR/Cas9-mediated gene knockout for resistance to cytokine-induced necroptosis and identified 112 regulators and mediators of necroptosis, including 59 new candidate pathway components with minimal or no effect on cell growth in the absence of necroptosis induction. Among these, we further characterized the function of PTBP1, an RNA binding protein whose activity is required to maintain RIPK1 protein abundance by regulating alternative splice-site selection.

Ubiquitin ligase RNF146 regulates tankyrase and Axin to promote Wnt signaling.

Canonical Wnt signaling is controlled intracellularly by the level of ß-catenin protein, which is dependent on Axin scaffolding of a complex that phosphorylates ß-catenin to target it for ubiquitylation and proteasomal degradation. This function of Axin is counteracted through relocalization of Axin protein to the Wnt receptor complex to allow for ligand-activated Wnt signaling. AXIN1 and AXIN2 protein levels are regulated by tankyrase-mediated poly(ADP-ribosyl)ation (PARsylation), which destabilizes Axin and promotes signaling. Mechanistically, how tankyrase limits Axin protein accumulation, and how tankyrase levels and activity are regulated for this function, are currently under investigation. By RNAi screening, we identified the RNF146 RING-type ubiquitin E3 ligase as a positive regulator of Wnt signaling that operates with tankyrase to maintain low steady-state levels of Axin proteins. RNF146 also destabilizes tankyrases TNKS1 and TNKS2 proteins and, in a reciprocal relationship, tankyrase activity reduces RNF146 protein levels. We show that RNF146, tankyrase, and Axin form a protein complex, and that RNF146 mediates ubiquitylation of all three proteins to target them for proteasomal degradation. RNF146 is a cytoplasmic protein that also prevents tankyrase protein aggregation at a centrosomal location. Tankyrase auto-PARsylation and PARsylation of Axin is known to lead to proteasome-mediated degradation of these proteins, and we demonstrate that, through ubiquitylation, RNF146 mediates this process to regulate Wnt signaling.

PAK4 mediates morphological changes through the regulation of GEF-H1.

Precise spatial and temporal regulation of Rho GTPases is required in controlling F-actin-based changes in cell morphology. The molecular mechanisms through which microtubules (MTs) modulate the activity of RhoGTPases and regulate the actin cytoskeleton are unclear. Here we show that p21-activated-kinase 4 (PAK4) mediates morphological changes through its association with the Rho-family guanine nucleotide exchange factor (GEF), GEF-H1. We show that this association is dependent upon a novel GEF-H1 interaction domain (GID) within PAK4. Further, we show that PAK4-mediated phosphorylation of Ser810 acts as a switch to block GEF-H1-dependent stress fiber formation while promoting the formation of lamellipodia in NIH-3T3 cells. We found that the endogenous PAK4-GEF-H1 complex associates with MTs and that PAK4 phosphorylation of MT-bound GEF-H1 releases it into the cytoplasm of NIH-3T3 cells, which coincides with the dissolution of stress fibers. Our observations propose a novel role for PAK4 in GEF-H1-dependent crosstalk between MTs and the actin cytoskeleton.

MRK, a mixed lineage kinase-related molecule that plays a role in gamma-radiation-induced cell cycle arrest.

Mitogen-activated protein (MAP) kinase pathways are three-kinase modules that mediate diverse cellular processes and have been highly conserved among eukaryotes. By using a functional complementation screen in yeast, we have identified a human MAP kinase kinase kinase (MAPKKK) that shares homology with members of the mixed lineage kinase (MLK) family and therefore was called MRK (MLK-related kinase). We report the structure of the MRK gene, from which are generated two splice forms of MRK, MRK-alpha and MRK-beta, encoding for proteins of 800 and 456 amino acids, respectively. By using a combination of solid phase protein kinase assays, transient transfections in cells, and analysis of endogenous proteins in stably transfected Madin-Darby canine kidney cells, we found that MRK-beta preferentially activates ERK6/p38gamma via MKK3/MKK6 and JNK through MKK4/MKK7. We also show that expression of wild type MRK increases the cell population in the G(2)/M phase of the cell cycle, whereas dominant negative MRK attenuates the G(2) arrest caused by gamma-radiation. In addition, exposure of cells to gamma-radiation induces MRK activity. These data suggest that MRK may mediate gamma-radiation signaling leading to cell cycle arrest and that MRK activity is necessary for the cell cycle checkpoint regulation in cells.

Requirement for PAK4 in the anchorage-independent growth of human cancer cell lines.

p21-activated protein kinase (PAK) serine/threonine kinases are important effectors of Rho family GTPases and have been implicated in the regulation of cell morphology and motility, as well as in cell transformation. To further investigate the possible involvement of PAK kinases in tumorigenesis, we analyzed the expression of several family members in tumor cell lines. Here we demonstrate that PAK4 is frequently overexpressed in human tumor cell lines of various tissue origins. We also have identified serine (Ser-474) as the likely autophosphorylation site in the kinase domain of PAK4 in vivo. Mutation of this serine to glutamic acid (S474E) results in constitutive activation of the kinase. Phosphospecific antibodies directed against serine 474 detect activated PAK4 on the Golgi membrane when PAK4 is co-expressed with activated Cdc42. Furthermore, expression of the active PAK4 (S474E) mutant has transforming potential, leading to anchorage-independent growth of NIH3T3 cells. A kinase-inactive PAK4 (K350A,K351A), on the other hand, efficiently blocks transformation by activated Ras and inhibits anchorage-independent growth of HCT116 colon cancer cells. Taken together, our data strongly implicate PAK4 in oncogenic transformation and suggest that PAK4 activity is required for Ras-driven, anchorage-independent growth.