Askew, J. W. | bioRxiv Science

bioRxiv2024

KSR1 regulates small-cell lung carcinoma tumor initiation and therapy resistance

Small-cell lung cancer (SCLC) has a dismal five-year survival rate of less than 7%, with limited advances in first line treatment over the past four decades. Tumor-initiating cells (TICs) contribute to resistance and relapse, a major impediment to SCLC treatment. Here, we identify Kinase Suppressor of Ras 1 (KSR1), a molecular scaffold for the Raf/MEK/ERK signaling cascade, as a critical regulator of SCLC TIC formation and tumor initiation in vivo. We further show that KSR1 mediates cisplatin resistance in SCLC. While 50-70% of control cells show resistance after 6-week exposure to cisplatin, CRISPR/Cas9-mediated KSR1 knockout prevents resistance in >90% of SCLC cells in ASCL1, NeuroD1, and POU2F3 subtypes. KSR1 KO significantly enhances the ability of cisplatin to decrease SCLC TICs via in vitro extreme limiting dilution analysis (ELDA), indicating that KSR1 disruption enhances the cisplatin toxicity of cells responsible for therapeutic resistance and tumor initiation. The ability of KSR1 disruption to prevent cisplatin resistant in H82 tumor xenograft formation supports this conclusion. Previous studies indicate that ERK activation inhibits SCLC tumor growth and development. We observe a minimal effect of pharmacological ERK inhibition on cisplatin resistance and no impact on TIC formation via in vitro ELDA. However, mutational analysis of the KSR1 DEF domain, which mediates interaction with ERK, suggests that ERK interaction with KSR1 is essential for KSR1-driven cisplatin resistance. These findings reveal KSR1 as a key regulatory protein in SCLC biology and a potential therapeutic target across multiple SCLC subtypes. Statement of ImplicationGenetic manipulation of the molecular scaffold KSR1 in small-cell lung cancer cells reveals its contribution to cisplatin resistance and tumor initiation.

cancer biology↗

bioRxiv2022

SOS1 and KSR1 modulate MEK inhibitor responsiveness to target resistant cell populations based on PI3K and KRAS mutation status

KRAS is the most commonly mutated oncogene. Targeted therapies have been developed against mediators of key downstream signaling pathways, predominantly components of the RAF/MEK/ERK kinase cascade. Unfortunately, single-agent efficacy of these agents is limited both by intrinsic and acquired resistance. Survival of drug-tolerant persister cells (DTPs) within the heterogeneous tumor population and/or acquired mutations that reactivate receptor tyrosine kinase (RTK)/RAS signaling can lead to outgrowth of tumor initiating cells (TICs) and drive therapeutic resistance. Here, we show that targeting the key RTK/RAS pathway signaling intermediates SOS1 or KSR1 both enhances the efficacy of, and prevents resistance to, the MEK inhibitor trametinib in KRAS-mutated lung (LUAD) and colorectal (COAD) adenocarcinoma cell lines depending on the specific mutational landscape. The SOS1 inhibitor BI-3406 enhanced the efficacy of trametinib and prevented trametinib resistance by targeting spheroid initiating cells (SICs) in KRASG12/G13-mutated LUAD and COAD cell lines that lacked PIK3CA co-mutations. Cell lines with KRASQ61 and/or PIK3CA mutations were insensitive to trametinib and BI-3406 combination therapy. In contrast, deletion of the RAF/MEK/ERK scaffold protein KSR1 prevented drug-induced SIC upregulation and restored trametinib sensitivity across all tested KRAS mutant cell lines in both PIK3CA- mutated and PIK3CA wildtype cancers. Our findings demonstrate that vertical inhibition of RTK/RAS signaling is an effective strategy to prevent therapeutic resistance in KRAS- mutated cancers, but therapeutic efficacy is dependent on both the specific KRAS mutant and underlying co-mutations. Thus, selection of optimal therapeutic combinations in KRAS-mutated cancers will require a detailed understanding of functional dependencies imposed by allele-specific KRAS mutations. Significance StatementWe provide an experimental framework for evaluating both adaptive and acquired resistance to RAS pathway-targeted therapies and demonstrate how targeting specific RAS pathway signaling intermediates SOS1 or KSR1 enhanced effectiveness of and prevented resistance to MEK inhibitors in KRAS-mutated cancer cells with genotypic precision. The contribution of either effector was dependent upon the mutational landscape: SOS1 inhibition synergized with trametinib in KRASG12/G13-mutated cells expressing WT PI3K but not in KRASQ61-mutated cells or if PIK3CA is mutated. KSR1 deletion inhibited MEK/ERK complex stability and was effective in cells that are unresponsive to SOS1 inhibition. These data demonstrate how a detailed understanding of functional dependencies imposed both by allele specific KRAS mutations and specific co-mutations facilitates the optimization of therapeutic combinations.

cancer biology↗