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Background PTEN is a multi-functional tumor suppressor protein regulating cell growth, immune signaling, neuronal function, and genome stability. Experimental characterization can help guide the clinical interpretation of the thousands of germline or somatic PTEN variants observed in patients. Two large-scale mutational datasets, one for PTEN variant intracellular abundance encompassing 4112 missense variants and one for lipid phosphatase activity encompassing 7244 variants, were recently published. The combined information from these datasets can reveal variant-specific phenotypes that may underlie various clinical presentations, but this has not been comprehensively examined, particularly for somatic PTEN variants observed in cancers. Methods Here, we add to these efforts by measuring the intracellular abundance of 764 new PTEN variants and refining abundance measurements for 3351 previously studied variants. We use this expanded and refined PTEN abundance dataset to explore the mutational patterns governing PTEN intracellular abundance, and then incorporate the phosphatase activity data to subdivide PTEN variants into four functionally distinct groups. Results This analysis revealed a set of highly abundant but lipid phosphatase defective variants that could act in a dominant-negative fashion to suppress PTEN activity. Two of these variants were, indeed, capable of dysregulating Akt signaling in cells harboring a WT PTEN allele. Both variants were observed in multiple breast or uterine tumors, demonstrating the disease relevance of these high abundance, inactive variants. Conclusions We show that multidimensional, large-scale variant functional data, when paired with public cancer genomics datasets and follow-up assays, can improve understanding of uncharacterized cancer-associated variants, and provide better insights into how they contribute to oncogenesis.

The tetracycline antibiotics are widely used in biomedical research as mediators of inducible gene expression systems. Despite many known effects of tetracyclines on mammalian cells-including inhibition of the mitochondrial ribosome-there have been few reports on potential off-target effects at concentrations commonly used in inducible systems. Here, we report that in human cell lines, commonly used concentrations of doxycycline change gene expression patterns and concomitantly shift metabolism towards a more glycolytic phenotype, evidenced by increased lactate secretion and reduced oxygen consumption. We also show that these concentrations are sufficient to slow proliferation. These findings suggest that researchers using doxycycline in inducible expression systems should design appropriate controls to account for potential confounding effects of the drug on cellular metabolism.

RAS oncogenes (collectively NRAS , HRAS and especially KRAS ) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 61 1 . Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer 2 , 3 . Nevertheless, KRAS G12C mutations account for only around 15% of KRAS -mutated cancers 4 , 5 , and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations ( KRAS G12X ). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRAS G12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS -mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985). RMC-7977, a compound that exhibits potent inhibition of the active states of mutant and wild-type KRAS, NRAS and HRAS variants has a strong anti-tumour effect on RAS-addicted tumours and is well tolerated in preclinical models.

The majority of the human proteome is phosphorylated by the eukaryotic protein kinase (EPK) enzyme family. EPKs are subject to multiple layers of regulation that modulate both the magnitude and specificity of their activity and this regulation is crucial, as loss of control over activity often results in disease. Commonly, this regulation is governed by interactions between the catalytic kinase domain and other regions of the protein. A classic example of intramolecular EPK regulation was delineated in the tyrosine kinase Src, whose regulatory SH2 and SH3 domains interface with its catalytic domain to constrain activity. Although this mechanism of Src intramolecular regulation is broadly well established, our knowledge of how the individual catalytic domain residues contribute to regulation is less well understood. To investigate this, we use saturation mutagenesis to functionally interrogate thousands of single missense mutants of the catalytic domain of Src kinase. We identify 27 residues located throughout the catalytic domain that, when mutated, result in increased Src activity. As expected, several of these residues hyper-activate Src by disrupting known mechanisms of SH2 or SH3 domain regulation. However, we also identify a patch of residues on Src’s C-lobe which strongly affects its activity but whose mechanism cannot be explained by our current understanding of Src regulation. We investigate this patch and surprisingly find that mutations here affect activity in a manner dependent on Src’s membrane-binding SH4 domain. Moreover, these mutations cause Src to associate more with membranes both in vitro and in cells, implying these mutations alter SH4 domain conformation. Our results suggest a novel functional interaction between Src’s SH4 and catalytic domain that couples localization and activity and thus our study adds an additional layer to the canonical mechanisms of Src kinase regulation. In a follow-up project, we use this same system to systematically identify mutations that confer resistance to three different conformation-selective inhibitors of Src. We identify dozens of resistance mutations, including some that are specific to a given inhibitor. These “unique” resistance mutations are of particular interest, because these residues may contribute to the adoption of specific conformations of Src’s catalytic domain. In summary, this work uncovers a previously unidentified regulatory role for Src’s SH4 domain. Moreover, it lays the groundwork for future investigations into the nature of these newly identified resistance mutants for conformation-selective inhibitors.

Kinase inhibitors are effective cancer therapies but resistance often limits clinical efficacy. Despite the cataloguing of numerous resistance mutations, our understanding of kinase inhibitor resistance is still incomplete. Here, we comprehensively profiled the resistance of ∼3500 Src tyrosine kinase mutants to four different ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Src’s catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Src’s phosphotransferase activity were prone to the development of resistance. Unexpectedly, we found that a resistance-prone cluster of residues located on the top face of the N-terminal lobe of Src’s catalytic domain contributes to autoinhibition by reducing catalytic domain dynamics, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how drug resistance profiling can be used to define potential resistance pathways and uncover new mechanisms of kinase regulation.

Protein kinase inhibitors are effective cancer therapies, but acquired resistance often limits clinical efficacy. Despite the cataloguing of numerous resistance mutations with model studies and in the clinic, we still lack a comprehensive understanding of kinase inhibitor resistance. Here, we measured the resistance of thousands of Src tyrosine kinase mutants to a panel of ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Src catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Src phosphotransferase activity were prone to the development of resistance. Unexpectedly, a resistance-prone cluster of residues that are on the top face of the N-terminal lobe of the catalytic domain contributes to Src autoinhibition by reducing the dynamics of the catalytic domain, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how comprehensive profiling of drug resistance can be used to understand potential resistance pathways and uncover new mechanisms of kinase regulation. Competing Interest Statement The authors have declared no competing interest. Footnotes * https://github.com/eahler/2019_SrcInhibitor

Rising income inequality and wealth in America have attracted substantial public attention in recent years. As evidence has mounted that the gap between the affluent and the poor has been increasing, elected officials, scholars, journalists, and even figures from popular culture have discussed the causes and effects of this phenomenon. The focus of the debate has often been the economic trends rather than the views of a broad sampling of Americans. But public opinion can condition policy responses, and it is important to measure public opinion on economic inequality, its causes, and potential remedies. This IGS Research Brief seeks to understand public opinion in California about this issue. It describes and analyzes the results of an IGS Poll on income inequality, its perceived causes, and the views of Californians regarding possible responses.