The SOS1 Inhibitor MRTX0902 Blocks KRAS Activation and Demonstrates Antitumor Activity in Cancers Dependent on KRAS Nucleotide Loading.

KRAS is the most frequently mutated oncogene in human cancer and facilitates uncontrolled growth through hyperactivation of the receptor tyrosine kinase (RTK)/mitogen-activated protein kinase (MAPK) pathway. The Son of Sevenless homolog 1 (SOS1) protein functions as a guanine nucleotide exchange factor (GEF) for the RAS subfamily of small GTPases and represents a druggable target in the pathway. Using a structure-based drug discovery approach, MRTX0902 was identified as a selective and potent SOS1 inhibitor that disrupts the KRAS:SOS1 protein-protein interaction to prevent SOS1-mediated nucleotide exchange on KRAS and translates into an anti-proliferative effect in cancer cell lines with genetic alterations of the KRAS-MAPK pathway. MRTX0902 augmented the antitumor activity of the KRAS G12C inhibitor adagrasib when dosed in combination in eight out of 12 KRAS G12C-mutant human non-small cell lung cancer and colorectal cancer xenograft models. Pharmacogenomic profiling in preclinical models identified cell cycle genes and the SOS2 homolog as genetic co-dependencies and implicated tumor suppressor genes (NF1 and PTEN) in resistance following combination treatment. Lastly, combined vertical inhibition of RTK/MAPK pathway signaling by MRTX0902 with inhibitors of EGFR or RAF/MEK led to greater downregulation of pathway signaling and improved antitumor responses in KRAS-MAPK pathway-mutant models. These studies demonstrate the potential clinical application of dual inhibition of SOS1 and KRAS G12C and additional SOS1 combination strategies that will aide in the understanding of SOS1 and RTK/MAPK biology in targeted cancer therapy.

Design and Discovery of MRTX0902, a Potent, Selective, Brain-Penetrant, and Orally Bioavailable Inhibitor of the SOS1:KRAS Protein-Protein Interaction.

SOS1 is one of the major guanine nucleotide exchange factors that regulates the ability of KRAS to cycle through its "on" and "off" states. Disrupting the SOS1:KRASG12C protein-protein interaction (PPI) can increase the proportion of GDP-loaded KRASG12C, providing a strong mechanistic rationale for combining inhibitors of the SOS1:KRAS complex with inhibitors like MRTX849 that target GDP-loaded KRASG12C. In this report, we detail the design and discovery of MRTX0902─a potent, selective, brain-penetrant, and orally bioavailable SOS1 binder that disrupts the SOS1:KRASG12C PPI. Oral administration of MRTX0902 in combination with MRTX849 results in a significant increase in antitumor activity relative to that of either single agent, including tumor regressions in a subset of animals in the MIA PaCa-2 tumor mouse xenograft model.

CCAAT/enhancer-binding protein delta (C/EBPdelta) maintains amelogenin expression in the absence of C/EBPalpha in vivo.

C/EBPalpha is implicated to regulate mouse amelogenin gene expression during tooth enamel formation in vitro. Because enamel formation occurs during postnatal development and C/EBPalpha-deficient mice die at birth, we used the Cre/loxP recombination system to characterize amelogenin expression in C/EBPalpha conditional knock-out mice. Mice carrying the Cre transgene under the control of the human keratin-14 promoter show robust Cre expression in the ameloblast cell lineage. Mating between mice bearing the floxed C/EBPalpha allele with keratin-14-Cre mice generate C/EBPalpha conditional knock-out mice. Real-time PCR analysis shows that removal of one C/EBPalpha allele from the molar enamel epithelial organ of 3-day postnatal mice results in dramatic decrease in endogenous C/EBPalpha mRNA levels and coordinately altered amelogenin mRNA abundance. Conditional deletion of both C/EBPalpha alleles further diminishes C/EBPalpha mRNA levels; however, rather than ablating amelogenin expression, we observe wild-type amelogenin mRNA abundance levels. We examined C/EBPbeta and nuclear factor YA expression, two transcription factors that had previously been shown to modestly participate in amelogenin expression, in vitro but found no significant changes in either of their mRNA abundance levels comparing conditional knock-out mice with wild-type counterparts. Although the abundance of C/EBPdelta is also unchanged in C/EBPalpha conditional knock-out mice, in vitro we find that C/EBPdelta activates the mouse amelogenin promoter and synergistically cooperates with nuclear factor Y, suggesting that C/EBPdelta can functionally substitute for C/EBPalpha to produce an enamel matrix competent to direct biomineralization.

Physical dissection of the CCAAT/enhancer-binding protein alpha in regulating the mouse amelogenin gene.

The amelogenin gene is tightly regulated at the temporal and spatial level in accord with the developmental requirement for tooth formation. Previous studies have shown that CCAAT/enhancer-binding protein alpha (C/EBPalpha) is a transactivator of the mouse X-chromosomal amelogenin gene. C/EBPalpha contains four highly conserved regions (CR) named CR1, CR2, CR3, and CR4. Transient transfection assays showed that CR2 in isolation had an exceptional capacity to enhance transcription from the 2.3 kb mouse amelogenin promoter. The remaining conserved regions of C/EBPalpha, either in isolation or in selected combinations, were less effective in amelogenin transactivation than the full length C/EBPalpha. Msx2 has previously been shown to antagonize C/EBPalpha through protein-protein interactions with C/EBPalpha, and the carboxyl-terminus of Msx2 is required for protein-protein interactions. Co-immunoprecipitation analyses identified that the carboxyl-terminal domain (residues 218-359) of C/EBPalpha is required for the C/EBPalpha-Msx2 protein-protein interactions.

NF-Y and CCAAT/enhancer-binding protein alpha synergistically activate the mouse amelogenin gene.

Amelogenin is the major protein component of the forming enamel matrix. In situ hybridization revealed a periodicity for amelogenin mRNA hybridization signals ranging from low to high transcript abundance on serial sections of developing mouse teeth. This in vivo observation led us to examine the amelogenin promoter for the activity of transcription factor(s) that account for this expression aspect of the regulation for the amelogenin gene. We have previously shown that CCAAT/enhancer-binding protein alpha (C/EBPalpha) is a potent transactivator of the mouse X-chromosomal amelogenin gene acting at the C/EBPalpha cis-element located in the -70/+52 minimal promoter. The minimal promoter contains a reversed CCAAT box (-58/-54) that is four base pairs downstream from the C/EBPalpha binding site. Similar to the C/EBPalpha binding site, the integrity of the reversed CCAAT box is also required for maintaining the activity of the basal promoter. We therefore focused on transcription factors that interact with the reversed CCAAT box. Using electrophoretic mobility shift assays we demonstrated that NF-Y was directly bound to this reversed CCAAT site. Co-transfection of C/EBPalpha and NF-Y synergistically increased the promoter activity. In contrast, increased expression of NF-Y alone had only marginal effects on the promoter. A dominant-negative DNA binding-deficient NF-Y mutant (NF-YAm29) dramatically decreased the promoter activity both in the absence or presence of exogenous expression of C/EBPalpha. We identified protein-protein interactions between C/EBPalpha and NF-Y by a co-immunoprecipitation analysis. These results suggest that C/EBPalpha and NF-Y synergistically activate the mouse amelogenin gene and can contribute to its physiological regulation during amelogenesis.