Supplemental Figure 1: MAPK pathway mutations reduce inavolisib efficacy in PIK3CA hotspot mutant cell lines

Supplemental Figure 4: Combination treatment of inavolisib with cobimetinib is synergistic

Supplemental Dataset 1 contains the difference in GR AOC values in the chemicogenomic screen for CRC cell lines HCT 116 (Supplemental Figure 4C), HCT-15, and T84 as well as Breast Cancer cell line MFM-223.

Supplemental Table 1: References for reagents and material

Supplemental Figure 5: Combination treatment of inavolisib with cobimetinib in vivo

Resistance to MEK inhibition in YAPC cells is not due to reactivation of phospho-Extracellular-signal-regulated-kinases (ERK) levels.

Supplemental Table 2: Cell line properties and inavolisib viability data

Supplemental Table 3: Cell line properties and levels of PI3K and MAPK signaling proteins

RNAi screening identifies G2/M kinases and kinetochore components as modulators of TNFα-induced NF-κB reporter activity and cell viability. A, Schematic demonstrating identified canonical and noncanonical regulators of NF-κB from the RNAi screening. Identified hits are in bold and underlined. B, NF-κB reporter activity in UMSCC1κB after transfection with select siRNAs from the kinome 1, kinome 2, and DG RNAi screens. Canonical and noncanonical hits are shown. NF-κB activity is shown as a percentage of a negative control siRNA (set at 100%). C, Cell viability in UMSCC1κB after transfection with select siRNAs from the kinome 1, kinome 2, and DG RNAi screens. Canonical and noncanonical hits are shown. Cell viability is shown as a percentage of a negative control siRNA (set at 100%). D, Validation of NF-κB reporter activity in UMSCC1κB comparing primary and secondary screen with three independent siRNAs. Select siRNAs from the RNAi screen are shown compared with the DG results (Genome in figure). Canonical and noncanonical hits are shown. NF-κB activity is shown as a percentage of a negative control (set at 100%). Each RNAi screen contained three individual siRNA targeting each gene. Validation siRNA experiments used separate siRNAs not used in the RNAi screens. (A, Created with BioRender.com.)

Despite recent advances in the inhibition of EGFR (epidermal growth factor receptor), there remains a clinical need for new EGFR Exon20 insertion (Ex20Ins) inhibitors that spare EGFR WT. Herein, we report the discovery and optimization of two chemical series leading to ether 23 and biaryl 36 as potent, selective, and brain-penetrant inhibitors of Ex20Ins mutants. Building on our earlier discovery of alkyne 5 which allowed access to CNS property space for an Ex20Ins inhibitor, we utilized structure-based design to move to lower lipophilicity and lower CLint compounds while maintaining a WT selectivity margin. During optimization, aldehyde oxidase (AO) metabolism was identified as a human clearance risk, and through SAR exploration, lower AO metabolism was achieved. Potency and WT margin were optimized across a range of Ex20Ins mutants including the potential acquired resistance T790M mutant and efficacy demonstrated in an LXF2478 Ex20Ins ASV model with margin to EGFR WT in vivo.

Supplemental Figure 2: Effect of inavolisib and RAS knock-down on AKT and MAPK pathway activity in RAS WT cell lines

Supplemental Figure 3: GP2d and HCT 116 models expressing exogenous mutant KRAS have different sensitivity to inavolisib

PIK3CA and KRAS are among the most frequently mutated oncogenes and often co-mutated in colorectal cancers. Understanding the impact of KRAS codon–specific mutations on cross-talks between the PI3K and MAPK pathways and response to targeted therapies, such as the p110α-specific inhibitor inavolisib (GDC-0077), is critical for advancing precision oncology. Focusing on colorectal PIK3CA + KRAS co-mutated models, we found that KRASG12D-mutated cells were more sensitive to inavolisib than models with KRASG13D, or other MAPK pathway mutations, even though the PI3K and MAPK pathways were active in both genotypes. In most co-mutated models, regardless of the type of KRAS alteration, the combination of inavolisib with MAPK pathway inhibitors showed synergy in vitro and in vivo. Our work highlights how specific codon substitutions in KRAS differentially toggle pathway activity and alter sensitivity to inavolisib, which could inform whether patients would benefit more from single-agent inavolisib or combination with MAPK pathway inhibitors.

Anti-sense oligonucleotides (ASOs) are modified synthetic single-stranded molecules with enhanced stability, activity, and bioavailability. They associate with RNA through sequence complementarity and can reduce or alter mRNA expression upon binding of splice site positions. To target RNA in the nucleus or cytoplasm, ASOs must cross membranes, a poorly understood process. We performed an unbiased CRISPR/Cas9 knockout screen with a genetic splice reporter to identify genes that can increase or decrease ASO activity, resulting in the most comprehensive catalog of ASO-activity modifier genes. Here we reveal distinct targets, including AP1M1 and TBC1D23, linking ASO activity to transport of cargo between the Golgi and endosomes. AP1M1 absence strongly increases ASO activity by delaying endosome-to-lysosome transport in vitro and in vivo. Prolonged ASO residence time in the endosomal system may increase the likelihood of ASO escape. This insight into AP1M1 role in ASO trafficking suggests a way for enhancing the therapeutic efficacy of ASOs by manipulating the endolysosomal pathways.