Xin Huang

Supplementary Figure S1 supports Figure 1 and contains cellular properties of NST-628 and a comparison with tramentib Supplementary Figure S2 supports Figure 2 and contains KSR1 data Supplementary Figure S3 supports Figure 2 and contains additional RAF-MEK crystal structures Supplementary Figure S4 supports Figure 2 and contains EGF stimulation experiment Supplementary Figure S5 supports Figure 2 and contains CRAF-MEK cryo-EM Supplementary Figure S6 supports Figure 3 and contains BRAF and MEK IPs, and CRAF IP/MS Supplementary Figure S7 supports Figure 4 and contains the full OMNI screen panel Supplementary Figure S8 supports Figure 4 and contains NST-628 RNAseq, signaling and proteomics studies Supplementary Figure S9 supports Figure 4 and contains NST-628 resistance studies Supplementary Figure S10 supports Figure 5 and contains HCT116 and IPC298 in vivo data Supplementary Figure S11 supports Figure 6 and contains NST-628 PK as well as SK-MEL-2 and MeWo in vivo data Supplementary Figure S12 Supports Figure 7 and contains KRAS G12C inhibitor combination studies

PDX model patient characteristics

Supplementary Table 9 from The Pan-RAF–MEK Nondegrading Molecular Glue NST-628 Is a Potent and Brain-Penetrant Inhibitor of the RAS–MAPK Pathway with Activity across Diverse RAS- and RAF-Driven Cancers

Data collection and refinement statistics for crystal structures; Cryo-EM data collection, processing, and model statistics

OMNI screen panel data with NST-628

Supplementary Table 7 from The Pan-RAF–MEK Nondegrading Molecular Glue NST-628 Is a Potent and Brain-Penetrant Inhibitor of the RAS–MAPK Pathway with Activity across Diverse RAS- and RAF-Driven Cancers

Supplementary methods with detailed protein production, mass spectrometry, in vivo, and in vitro methodology

Supplementary Table 1 from The Pan-RAF–MEK Nondegrading Molecular Glue NST-628 Is a Potent and Brain-Penetrant Inhibitor of the RAS–MAPK Pathway with Activity across Diverse RAS- and RAF-Driven Cancers

Alterations in the RAS–MAPK signaling cascade are common across multiple solid tumor types and are a driver for many cancers. NST-628 is a potent pan-RAF–MEK molecular glue that prevents the phosphorylation and activation of MEK by RAF, overcoming the limitations of traditional RAS–MAPK inhibitors and leading to deep durable inhibition of the pathway. Cellular, biochemical, and structural analyses of RAF–MEK complexes show that NST-628 engages all isoforms of RAF and prevents the formation of BRAF–CRAF heterodimers, a differentiated mechanism from all current RAF inhibitors. With a potent and durable inhibition of the RAF–MEK signaling complex as well as high intrinsic permeability into the brain, NST-628 demonstrates broad efficacy in cellular and patient-derived tumor models harboring diverse MAPK pathway alterations, including orthotopic intracranial models. Given its functional and pharmacokinetic mechanisms that are differentiated from previous therapies, NST-628 is positioned to make an impact clinically in areas of unmet patient need.Significance:This study introduces NST-628, a molecular glue having differentiated mechanism and drug-like properties. NST-628 treatment leads to broad efficacy with high tolerability and central nervous system activity across multiple RAS- and RAF-driven tumor models. NST-628 has the potential to provide transformative clinical benefits as both monotherapy and vertical combination anchor.