Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers.

RAS-driven cancers comprise up to 30% of human cancers. RMC-6236 is a RAS(ON) multi-selective noncovalent inhibitor of the active, GTP-bound state of both mutant and wild-type variants of canonical RAS isoforms with broad therapeutic potential for the aforementioned unmet medical need. RMC-6236 exhibited potent anticancer activity across RAS-addicted cell lines, particularly those harboring mutations at codon 12 of KRAS. Notably, oral administration of RMC-6236 was tolerated in vivo and drove profound tumor regressions across multiple tumor types in a mouse clinical trial with KRASG12X xenograft models. Translational PK/efficacy and PK/PD modeling predicted that daily doses of 100 mg and 300 mg would achieve tumor control and objective responses, respectively, in patients with RAS-driven tumors. Consistent with this, we describe here objective responses in two patients (at 300 mg daily) with advanced KRASG12X lung and pancreatic adenocarcinoma, respectively, demonstrating the initial activity of RMC-6236 in an ongoing phase I/Ib clinical trial (NCT05379985). SIGNIFICANCE: The discovery of RMC-6236 enables the first-ever therapeutic evaluation of targeted and concurrent inhibition of canonical mutant and wild-type RAS-GTP in RAS-driven cancers. We demonstrate that broad-spectrum RAS-GTP inhibition is tolerable at exposures that induce profound tumor regressions in preclinical models of, and in patients with, such tumors. This article is featured in Selected Articles from This Issue, p. 897.

Discovery of RMC-5552, a Selective Bi-Steric Inhibitor of mTORC1, for the Treatment of mTORC1-Activated Tumors.

Hyperactivation of mTOR kinase by mutations in the PI3K/mTOR pathway or by crosstalk with other mutant cancer drivers, such as RAS, is a feature of many tumors. Multiple allosteric inhibitors of mTORC1 and orthosteric dual inhibitors of mTORC1 and mTORC2 have been developed as anticancer drugs, but their clinical utility has been limited. To address these limitations, we have developed a novel class of "bi-steric inhibitors" that interact with both the orthosteric and the allosteric binding sites in order to deepen the inhibition of mTORC1 while also preserving selectivity for mTORC1 over mTORC2. In this report, we describe the discovery and preclinical profile of the development candidate RMC-5552 and the in vivo preclinical tool compound RMC-6272. We also present evidence that selective inhibition of mTORC1 in combination with covalent inhibition of KRASG12C shows increased antitumor activity in a preclinical model of KRASG12C mutant NSCLC that exhibits resistance to KRASG12C inhibitor monotherapy.

Stress Resistance Screen in a Human Primary Cell Line Identifies Small Molecules That Affect Aging Pathways and Extend Caenorhabditis elegans' Lifespan.

Increased resistance to environmental stress at the cellular level is correlated with the longevity of long-lived mutants and wild-animal species. Moreover, in experimental organisms, screens for increased stress resistance have yielded mutants that are long-lived. To find entry points for small molecules that might extend healthy longevity in humans, we screened ∼100,000 small molecules in a human primary-fibroblast cell line and identified a set that increased oxidative-stress resistance. Some of the hits fell into structurally related chemical groups, suggesting that they may act on common targets. Two small molecules increased C. elegans' stress resistance, and at least 9 extended their lifespan by ∼10-50%. We further evaluated a chalcone that produced relatively large effects on lifespan and were able to implicate the activity of two, stress-response regulators, NRF2/skn-1 and SESN/sesn-1, in its mechanism of action. Our findings suggest that screening for increased stress resistance in human cells can enrich for compounds with promising pro-longevity effects. Further characterization of these compounds may reveal new ways to extend healthy human lifespan.

Machine learning enables detection of early-stage colorectal cancer by whole-genome sequencing of plasma cell-free DNA.

BACKGROUND: Blood-based methods using cell-free DNA (cfDNA) are under development as an alternative to existing screening tests. However, early-stage detection of cancer using tumor-derived cfDNA has proven challenging because of the small proportion of cfDNA derived from tumor tissue in early-stage disease. A machine learning approach to discover signatures in cfDNA, potentially reflective of both tumor and non-tumor contributions, may represent a promising direction for the early detection of cancer. METHODS: Whole-genome sequencing was performed on cfDNA extracted from plasma samples (N = 546 colorectal cancer and 271 non-cancer controls). Reads aligning to protein-coding gene bodies were extracted, and read counts were normalized. cfDNA tumor fraction was estimated using IchorCNA. Machine learning models were trained using k-fold cross-validation and confounder-based cross-validations to assess generalization performance. RESULTS: In a colorectal cancer cohort heavily weighted towards early-stage cancer (80% stage I/II), we achieved a mean AUC of 0.92 (95% CI 0.91-0.93) with a mean sensitivity of 85% (95% CI 83-86%) at 85% specificity. Sensitivity generally increased with tumor stage and increasing tumor fraction. Stratification by age, sequencing batch, and institution demonstrated the impact of these confounders and provided a more accurate assessment of generalization performance. CONCLUSIONS: A machine learning approach using cfDNA achieved high sensitivity and specificity in a large, predominantly early-stage, colorectal cancer cohort. The possibility of systematic technical and institution-specific biases warrants similar confounder analyses in other studies. Prospective validation of this machine learning method and evaluation of a multi-analyte approach are underway.

Identification of fungal sphingolipid C9-methyltransferases by phylogenetic profiling.

Fungal glucosylceramides play an important role in plant-pathogen interactions enabling plants to recognize the fungal attack and initiate specific defense responses. A prime structural feature distinguishing fungal glucosylceramides from those of plants and animals is a methyl group at the C9-position of the sphingoid base, the biosynthesis of which has never been investigated. Using information on the presence or absence of C9-methylated glucosylceramides in different fungal species, we developed a bioinformatics strategy to identify the gene responsible for the biosynthesis of this C9-methyl group. This phylogenetic profiling allowed the selection of a single candidate out of 24-71 methyltransferase sequences present in each of the fungal species with C9-methylated glucosylceramides. A Pichia pastoris knock-out strain lacking the candidate sphingolipid C9-methyltransferase was generated, and indeed, this strain contained only non-methylated glucosylceramides. In a complementary approach, a Saccharomyces cerevisiae strain was engineered to produce glucosylceramides suitable as a substrate for C9-methylation. C9-methylated sphingolipids were detected in this strain expressing the candidate from P. pastoris, demonstrating its function as a sphingolipid C9-methyltransferase. The enzyme belongs to the superfamily of S-adenosylmethionine-(SAM)-dependent methyltransferases and shows highest sequence similarity to plant and bacterial cyclopropane fatty acid synthases. An in vitro assay showed that sphingolipid C9-methylation is membrane-bound and requires SAM and Delta4,8-desaturated ceramide as substrates.

The Pichia pastoris formaldehyde dehydrogenase gene (FLD1) as a marker for selection of multicopy expression strains of P. pastoris.

The methylotrophic yeast Pichia pastoris is a popular host for the production of a variety of recombinant proteins. We describe the use of a novel selectable marker, the P. pastoris formaldehyde dehydrogenase gene (FLD1) for DNA-mediated transformations of this yeast. The product of the FLD1 gene (Fld1p) is required for growth of P. pastoris on methanol as a carbon source and methylamine as a nitrogen source. In both these C(1) pathways, Fld1p oxidizes formaldehyde to formate, which is subsequently further oxidized by a second dehydrogenase to carbon dioxide. We show that the FLD1 gene can be used as a marker in transformations of a P. pastoris fld1 host by selection on plates containing methylamine. Furthermore, we demonstrate that populations of these transformants can be enriched for strains that receive multiple copies of an FLD1-based vector by their increased resistance to formaldehyde. We provide the FLD1 selection system in a set of P. pastoris expression vectors that are composed almost entirely of P. pastoris DNA (except for the recombinant gene) and are devoid of antibiotic resistance genes or other sequences of bacterial origin. The vectors are useful for the selection of strains containing multiple copies of an expression vector and may be ideal for certain large-scale recombinant protein production processes where strains containing non-P. pastoris DNA sequences, particularly bacterial antibiotic resistance genes and replication origins, are considered a potential biological hazard to be avoided.

Molecular characterization of the Hansenula polymorpha FLD1 gene encoding formaldehyde dehydrogenase.

Glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required forthe catabolism of methanol as a carbon source and certain primary amines, such as methylamine as nitrogen sources in methylotrophic yeasts. Here we describe the molecular characterization of the FLD1 gene from the yeast Hansenula polymorpha. Unlike the recently described Pichia pastoris homologue, the H. polymorpha gene does not contain an intron. The predicted FLD1 product (Fld1p) is a protein of 380 amino acids (ca. 41 kDa) with 82% identity to P. pastoris Fld1p, 76% identity to the FLD protein sequence from n-alkane-assimilating yeast Candida maltosa and 63-64% identity to dehydrogenase class III enzymes from humans and other higher eukaryotes. The expression of FLD1 is strictly regulated and can be controlled at two expression levels by manipulation of the growth conditions. The gene is strongly induced under methylotrophic growth conditions; moderate expression is obtained under conditions in which a primary amine, e.g. methylamine, is used as nitrogen source. These properties render the FLD1 promoter of high interest for heterologous gene expression. The availability of the H. polymorpha FLD1 promoter provides an attractive alternative for expression of foreign genes besides the commonly used alcohol oxidase promoter.