ARAF (A-Raf Proto-Oncogene)

Pharmacologic profiling further demonstrates allele-selective inhibition, with mutant-specific FRET and BRET responses observed upon treatment with targeted KRAS inhibitors. This biosensor platform enables live-cell, real-time, and quantitative monitoring of RAF-KRAS interactions, facilitating the analysis of oncogenic signaling dynamics and the evaluation of mutation-specific responses to targeted therapies under physiologically relevant conditions.

RAS or MEK inhibition co-targeting is effective against this resistance mechanism. This study provides preclinical rationale for clinical testing of exarafenib in BRAF Class II/III cancers and unveils RAS-mediated ARAF-KSR1 complex formation as a resistance mechanism and rational co-therapy strategies.

RAF1 is a key, non-redundant vulnerability in KRAS-driven lung adenocarcinoma. Co-targeting ARAF, EGFR, or DDR1 provides no additional therapeutic benefit in established disease. The absence of adverse effects from ARAF co-deletion suggests that RAF1 degraders with partial cross-activity towards ARAF are likely to be safe. These findings provide a strong preclinical rationale for developing RAF1-targeted degradation as a monotherapy for these malignancies.

The unique mechanism of action and improved tolerability positions IK-595 as an ideal combination partner. IK-595 is an MEK-RAF molecular glue that prolongs pathway inhibition while providing a broader therapeutic window as monotherapy and in combination.

Our cryo-EM structure shows how this acrylamide-containing analog of MEK inhibitor trametinib covalently targets Cys514, a residue unique to ARAF. Our studies highlight the importance of the activating RAF isoform as a determinant of MEK inhibitor sensitivity and provide proof-of-concept for development of MEK inhibitors that more effectively block ARAF-driven MEK signaling via covalent targeting of Cys514.

The exarafenib plus binimetinib combination demonstrated superior efficacy in diverse preclinical models. This study establishes ARAF-KSR1 complex formation as a novel resistance mechanism to pan-RAF inhibition and provides mechanistic rationale for combination strategies with potential to address the unmet clinical need for BRAF Class II and III-mutated NSCLC.

This study investigated A-RAF, B-RAF, C-RAF, and RalGDS gene expression in fibroblasts from SSc patients and healthy controls, focusing on their response to TGF-β treatment. While baseline expression levels were similar between groups, TGF-β significantly upregulated these genes in SSc fibroblasts more than in controls. This suggests that TGF-β signaling may contribute to the dysregulation of RAF and RalGDS pathways in SSc, highlighting their potential as biomarkers and therapeutic targets for the disease. Key Points • No significant difference in gene expression levels between SSc and healthy fibroblasts. • SSc and healthy fibroblasts exhibit upregulated RAF and RalGDS expression at the mRNA level in response to TGF-β treatment. • Positive correlation between the expression levels of A-RAF and B-RAF, B-RAF and C-RAF, and finally between C-RAF and RalGDS genes.

Type I inhibitor SB590885 is roughly equipotent across RAF isoforms and, as expected, type I.5 inhibitors are typically most potent against BRAFV600E. Crystal structures of CRAF in complex with type I.5 inhibitor PLX4720 reveal an asymmetric CRAF dimer with one CRAF subunit bound in the inactive state and the second bound in an αC-helix-in, active conformation with an altered inhibitor pose. Our findings have important implications for understanding the pharmacology of current RAF inhibitors and will inform development of new agents with distinct isoform selectivity.

Finally, we show that inhibitors blocking the BRAF RBD:KRAS interaction were able to suppress the in vitro activation of BRAF, underscoring the critical role of RAS binding in initiating the disassembly of the BRAF autoinhibited state. Thus, this assay provides valuable insights into the steps required for BRAF activation and can serve as an effective screening tool for identifying compounds that may inhibit this process and have therapeutic potential.

Our findings demonstrate that RAS binding not only recruits BRAF to the plasma membrane but initiates the disassembly of the autoinhibited monomer, which in the context of the membrane, facilitates BRAF dimerization and activation. This assay advances our understanding of BRAF regulation and provides a novel platform for drug discovery efforts targeting BRAF.

Type I inhibitor SB590885 is roughly equipotent across RAF isoforms and, as expected, type I.5 inhibitors are typically most potent against BRAFV600E. Crystal structures of CRAF in complex with type I.5 inhibitor PLX4720 reveal an asymmetric CRAF dimer with one CRAF subunit bound in the inactive state and the second bound in an aC-helix-in, active conformation with an altered inhibitor pose. Our findings have important implications for understanding the pharmacology of current RAF inhibitors and will inform development of new agents with distinct isoform selectivity.