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 within the heterogeneous tumor population and/or acquired mutations that reactivate 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 and colorectal 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 TICs, but only in KRASG12- or KRASG13-mutated LUAD and COAD cell lines that lacked PIK3CA co-mutations. Cell lines with KRASQ61 and/or PIK3CA mutations were insensitive to combination therapy with trametinib and BI-3406. In contrast, deletion of the RAF/MEK/ERK scaffold protein KSR1 prevented treatment-induced TIC upregulation and restored trametinib sensitivity across KRAS mutant cell lines in both PIK3CA-mutated and PIK3CA wildtype cancers. Our findings demonstrate that vertical targeting of RTK/RAS signaling is an effective strategy to target KRAS-mutated cancers, but the specific combination is dependent both on 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.

Genome-wide, loss-of-function screening can be used to identify novel vulnerabilities upon which specific tumor cells depend for survival. Functional Signature Ontology (FUSION) is a gene expression-based high-throughput screening (GE-HTS) method that allows researchers to identify functionally similar proteins, small molecules, and microRNA mimics, revealing novel therapeutic targets. FUSION uses cell-based high-throughput screening and computational analysis to match gene expression signatures produced by natural products to those produced by small interfering RNA (siRNA) and synthetic microRNA libraries to identify putative protein targets and mechanisms of action (MoA) for several previously undescribed natural products. We have used FUSION to screen for functional analogues to Kinase suppressor of Ras 1 (KSR1), a scaffold protein downstream of Ras in the Raf-MEK-ERK kinase cascade, and biologically validated several proteins with functional similarity to KSR1. FUSION incorporates bioinformatics analysis that may offer higher resolution of the endpoint readout than other screens which utilize Boolean outputs regarding a single pathway activation (i.e., synthetic lethal and cell proliferation). Challenges associated with FUSION and other high-content genome-wide screens include variation, batch effects, and controlling for potential off-target effects. In this review, we discuss the efficacy of FUSION to identify novel inhibitors and oncogene-induced changes that may be cancer cell-specific as well as several potential pitfalls within FUSION and best practices to avoid them.

Tumor propagating cells (TPCs) make up a small proportion of tumor cells responsible for self-renewal and long-term propagation of small-cell lung carcinoma (SCLC) tumors. Here, we show that Kinase Suppressor of Ras 2 (KSR2) is an important regulator of the self-renewing and clonogenic properties of SCLC cells. KSR2 is a molecular scaffold which promotes Raf/MEK/ERK signaling and AMPK signaling in SCLC. KSR2 is preferentially expressed in the ASCL1 subtype of SCLC tumors as well as the pulmonary neuroendocrine cells from which the SCLC tumors arise. The expression of KSR2 in SCLC and pulmonary neuroendocrine cells was previously unrecognized and serves as a novel model for understanding the role of KSR2-dependent signaling in normal and malignant tissues. Disruption of KSR2 in SCLC-A cell lines significantly reduces colony forming ability of TPCs in vitro and tumor initiating capacity in vivo. These data indicate that the expression of KSR2 is an essential driver of SCLC-A tumor propagating cell function, and therefore may play a role in SCLC tumor initiation. These findings shed light on a key distinct protein responsible for regulation in ASCL1 subtype SCLC tumors, and a potential subtype specific therapeutic target.

In human colorectal cancer (CRC) cells the Raf/MEK/ERK scaffold Kinase Suppressor of Ras 1 (KSR1)-dependent signaling is required for the epithelial-to-mesenchymal transition (EMT)-like phenotype. Here we show that KSR1 promotes the association of differentially spliced mRNA bearing recognition sites for the Serine/Arginine-Rich (SR) splicing factor SRSF9. CRISPR/Cas9 disruption of KSR1 destabilizes SRSF9 protein, which interacts preferentially with mRNA encoding Epithelial Stromal Interaction 1 (EPSTI1). EPSTI1 protein mediates Ras and KSR1-dependent induction of EMT. Analysis of EPSTI1 splice variants reveals that inclusion of exon 8 is critical to the ability of EPSTI1 to promote the E-to N-cadherin switch and CRC cell motile and invasive behavior. These data reveal a mechanism in CRC cells in which Ras-induced and KSR1-dependent signaling affects pre-mRNA splicing to control behaviors critical to cancer cell dissemination and metastasis.