Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy.

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 611. 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 cancer2,3. Nevertheless, KRASG12C mutations account for only around 15% of KRAS-mutated cancers4,5, 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 (KRASG12X). 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 KRASG12C 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).

Profiling of drug resistance in Src kinase at scale uncovers a regulatory network coupling autoinhibition and catalytic domain dynamics.

Kinase inhibitors are effective cancer therapies, but resistance often limits clinical efficacy. Despite the cataloging of numerous resistance mutations, our understanding of kinase inhibitor resistance is still incomplete. Here, we comprehensively profiled the resistance of ∼3,500 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.

Molecular determinants of Hsp90 dependence of Src kinase revealed by deep mutational scanning.

Hsp90 is a molecular chaperone involved in the refolding and activation of numerous protein substrates referred to as clients. While the molecular determinants of Hsp90 client specificity are poorly understood and limited to a handful of client proteins, strong clients are thought to be destabilized and conformationally extended. Here, we measured the phosphotransferase activity of 3929 variants of the tyrosine kinase Src in both the presence and absence of an Hsp90 inhibitor. We identified 84 previously unknown functionally dependent client variants. Unexpectedly, many destabilized or extended variants were not functionally dependent on Hsp90. Instead, functionally dependent client variants were clustered in the αF pocket and ß1-ß2 strand regions of Src, which have yet to be described in driving Hsp90 dependence. Hsp90 dependence was also strongly correlated with kinase activity. We found that a combination of activation, global extension, and general conformational flexibility, primarily induced by variants at the αF pocket and ß1-ß2 strands, was necessary to render Src functionally dependent on Hsp90. Moreover, the degree of activation and flexibility required to transform Src into a functionally dependent client varied with variant location, suggesting that a combination of regulatory domain disengagement and catalytic domain flexibility are required for chaperone dependence. Thus, by studying the chaperone dependence of a massive number of variants, we highlight factors driving Hsp90 client specificity and propose a model of chaperone-kinase interactions.

Integrating thousands of PTEN variant activity and abundance measurements reveals variant subgroups and new dominant negatives in cancers.

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.

A Combined Approach Reveals a Regulatory Mechanism Coupling Src's Kinase Activity, Localization, and Phosphotransferase-Independent Functions.

Multiple layers of regulation modulate the activity and localization of protein kinases. However, many details of kinase regulation remain incompletely understood. Here, we apply saturation mutagenesis and a chemical genetic method for allosterically modulating kinase global conformation to Src kinase, providing insight into known regulatory mechanisms and revealing a previously undiscovered interaction between Src's SH4 and catalytic domains. Abrogation of this interaction increased phosphotransferase activity, promoted membrane association, and provoked phosphotransferase-independent alterations in cell morphology. Thus, Src's SH4 domain serves as an intramolecular regulator coupling catalytic activity, global conformation, and localization, as well as mediating a phosphotransferase-independent function. Sequence conservation suggests that the SH4 domain regulatory interaction exists in other Src-family kinases. Our combined approach's ability to reveal a regulatory mechanism in one of the best-studied kinases suggests that it could be applied broadly to provide insight into kinase structure, regulation, and function.

Doxycycline alters metabolism and proliferation of human cell lines.

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.

The degree of Helicobacter pylori-triggered inflammation is manipulated by preinfection host microbiota.

Helicobacter pylori infects over 3 billion people worldwide and is the primary risk factor for gastric cancer. Most individuals infected with H. pylori develop only asymptomatic gastritis; however, some develop ulcers or gastric adenocarcinoma. We demonstrate that one previously unappreciated parameter influencing H. pylori disease outcome is variation in the preinfection host microbiota. Utilizing a mouse model, we altered the microbiota by antibiotic treatment and found that these alterations resulted in significantly lowered H. pylori-triggered inflammation. Specifically, antibiotic pretreatment reduced CD4(+) T-helper cells and Ifnγ transcript levels in gastric tissue after H. pylori infection. The bacterial communities in mice with a reduced response to H. pylori displayed many differences from those in untreated mice, including significantly more cluster IV and XIVa Clostridium spp., bacteria known to influence inflammation via regulatory T cell populations. Our findings suggest that microbiota composition, perhaps Clostridium spp., contributes to the variable disease outcome of H. pylori infection by altering the recruitment of CD4(+) T cells to the gastric compartment. Our results suggest that gastric microbiota could be used as a diagnostic tool to determine which patients are at risk for developing severe disease.

Metabolomics strategy reveals subpopulation of liposarcomas sensitive to gemcitabine treatment.

UNLABELLED: Unlike many cancers that exhibit glycolytic metabolism, high-grade liposarcomas often exhibit low 2[18F]fluoro-2-deoxy-D-glucose uptake by positron emission tomography (PET), despite rapid tumor growth. Here, we used liquid chromatography tandem mass spectrometry to identify carbon sources taken up by liposarcoma cell lines derived from xenograft tumors in patients. Interestingly, we found that liposarcoma cell lines consume nucleosides from culture media, suggesting nucleoside salvage pathway activity. The nucleoside salvage pathway is dependent on deoxycytidine kinase (dCK) and can be imaged in vivo by PET with 1-(2'-deoxy-2'-[18F]fluoroarabinofuranosyl) cytosine (FAC). We found that liposarcoma cell lines and xenograft tumors exhibit dCK activity and dCK-dependent FAC uptake in vitro and in vivo. In addition, liposarcoma cell lines and xenograft tumors are sensitive to treatment with the nucleoside analogue prodrug gemcitabine, and gemcitabine sensitivity is dependent on dCK expression. Elevated dCK activity is evident in 7 of 68 clinical liposarcoma samples analyzed. These data suggest that a subpopulation of liposarcoma patients have tumors with nucleoside salvage pathway activity that can be identified noninvasively using [18F]-FAC-PET and targeted using gemcitabine. SIGNIFICANCE: Patients with high-grade liposarcoma have poor prognoses and often fail to respond to chemotherapy. This report identifies elevated nucleoside salvage activity in a subset of liposarcomas that are identifiable using noninvasive PET imaging with FAC and that are sensitive to gemcitabine. Thus, we suggest a new treatment paradigm for liposarcoma patients that uses [18F]-FAC-PET in the clinic to delineate gemcitabine responders from nonresponders.