Disulfide tethering is a site-directed method of drug discovery used to identify hits for challenging targets. We applied tethering to target oncogenic KRAS, a small GTPase once considered undruggable due to its high nucleotide affinity and a perceived absence of binding sites. We prepared a library of 2160 disulfide-containing fragments. We screened over 1000 compounds against a panel of 83 engineered cysteine mutants of KRAS G12D in the active conformation and screened the full library for a subset of 30 mutants. For select mutants and hits, we performed 2-mercaptoethanol competition assays (βME-50) to prioritize ligands. Ligandability analysis comparing hit rates across mutant residues enabled the identification of druggable hot spots. Our studies confirmed known binding sites, including the Switch-II / α-helix 3 pocket. In addition, we identified previously undescribed cryptic pockets and validated select hits using computational chemistry and NMR spectroscopy. These pockets represent promising opportunities for future drug discovery campaigns.

C/EBPβ regulates oncogene-induced senescence (OIS) and the senescence-associated secretory phenotype (SASP) through activation by ERK1/2 and CK2. In tumor cells, C/EBPβ activity is suppressed by its 3'UTR via a mechanism termed 3'UTR regulation of protein activity (UPA), which spatially segregates CEBPB transcripts from kinase-rich perinuclear endosomes. Here, we identify kinase-proximal mRNA decay as the underlying mechanism. The mRNA decay factors UPF1 and STAU1/2 localize to perinuclear endosomes and promote degradation of CEBPB transcripts, thereby preventing C/EBPβ phosphorylation and activation. Disruption of this pathway restores C/EBPβ activity and induces senescence. In vivo, deletion of a G/U-rich regulatory element (GRE) in the 3'UTR impairs the progression of Kras-driven lung tumors and biases cells toward an AT2-like differentiation state with reduced EMT-associated transcriptional reprogramming. RAS-expressing GRE Δ/Δ fibroblasts show enhanced OIS that requires upregulation of the pro-senescent cytokine S100a9. These findings identify perinuclear mRNA decay as a mechanism suppressing C/EBPβ activity and senescence in cancer.

Increased expression of lnc-FANCI-2, a newly discovered long noncoding RNA, is associated with cervical lesion progression from cervical intraepithelial neoplasia stage 1 (CIN1, low grade), CIN2–3 (high grade), to cervical cancer. Viral oncoprotein E7 of high-risk human papillomaviruses (HR-HPVs) and host transcription factor YY1 are two major factors promoting lnc-FANCI-2 expression. Using CRISPR-Cas9 technology, we knocked out the expression of lnc-FANCI-2 in the HPV16-positive cervical cancer cell line, CaSki cells. The selected knockout (KO) single-cell clones displayed altered cell morphology and proliferation with changes of cellular soluble receptors, but normal HPV16 E6 and E7 expression. Relative to the parental cells, lnc-FANCI-2 KO cells exhibited significantly increased RAS signaling and epithelial-mesenchymal transition, but decreased response to IFN signaling, along with increased p-Akt and p-Erk1/2 (two RAS signaling effectors), IGFBP3, MCAM, VIM, and CCND2 (cyclin D2) and decreased expression of RAC3. lnc-FANCI-2 in CaSki interacts with cellular proteins H13, HNRH1, K1H1, MAP4K4, and RNPS1. MAP4K4 knockdown led to enhance the expression of p-Erk1/2 and p-Akt. High lnc-FANCI-2 and low MCAM levels in cervical cancer tissues were found to be associated with patients’ survival. A key function of lnc-FANCI-2 intrinsically regulates RAS signaling to impact cervical lesion progression and cervical cancer prognosis.

KRAS , the most frequently mutated oncogene in human cancer, produces two isoforms, KRAS4a and KRAS4b, through alternative splicing. These isoforms differ in exon 4, which encodes the final 15 residues of the G-domain and hypervariable regions (HVRs), vital for trafficking and membrane localization. While KRAS4b has been extensively studied, KRAS4a has been largely overlooked. Our multidisciplinary study compared the structural and functional characteristics of KRAS4a and KRAS4b, revealing distinct structural properties and thermal stability. Position 151 influences KRAS4a’s thermal stability, while position 153 affects binding to RAF1 CRD protein. Nuclear magnetic resonance analysis identified localized structural differences near sequence variations and provided a solution-state conformational ensemble. Notably, KRAS4a exhibits substantial transcript abundance in bile ducts, liver, and stomach, with transcript levels approaching KRAS4b in the colon and rectum. Functional disparities were observed in full-length KRAS variants, highlighting the impact of HVR variations on interaction with trafficking proteins and downstream effectors like RAF and PI3K within cells.

RAS research has entered the world of translational and clinical science. Progress has been based on our appreciation of the role of RAS mutations in different types of cancer and the effects of these mutations on the biochemical, structural, and biophysical properties of the RAS proteins themselves, particularly KRAS, on which most attention has been focused. This knowledge base, while still growing, has enabled creative chemical approaches to targeting KRAS directly. Our understanding of RAS signaling pathways in normal and cancer cells plays an important role for developing RAS inhibitors but also continues to reveal new approaches to targeting RAS through disruption of signaling complexes and downstream pathways.

In cancer research, RAS biology has been focused on only a handful of tumor types. While RAS genes have long been suspected as common contributors to a wide spectrum of cancer types, robust evidence is required to firmly establish their critical oncogenic significance. We present a data mining study using DepMap genome-wide CRISPR screening data, which provide substantial evidence to support the prominent pervasive oncogenic role and tissue-specific permissiveness of RAS gene mutations. Differential analysis of CRISPR effect scores identifies K- or N-RAS genes as the most differential gene in contrasts of (K-, N-, combined) RAS mutant versus wild-type cell lines across multiple tissue types. The distinguished tissue-specific pattern of KRAS vs. NRAS as top differential genes in subsets of tissue types and evidence from genome data supported the idea of KRAS- and NRAS-engaged tissue types. To our knowledge, this is the first report of prominent pervasive oncogenic role of RAS mutations revealed by gene dependency data that is beyond the current understanding of the oncogenic role of RAS genes and their well-known involved tissue types. Our findings strongly support RAS mutations as primary oncogenic drivers beyond traditionally recognized cancer types and offer insights into their tissue-specific permissiveness.

Supplementary Methods, Figure Legends 1-5 from miRNA-7 Attenuation in Schwannoma Tumors Stimulates Growth by Upregulating Three Oncogenic Signaling Pathways

Oncogenic forms of KRAS proteins are known to be drivers of pancreatic, colorectal, and lung cancers. The goal of this study is to identify chemical leads that inhibit oncogenic KRAS signaling. We first developed an isogenic panel of mouse embryonic fibroblast (MEF) cell lines that carry wild-type RAS, oncogenic KRAS, and oncogenic BRAF. We validated these cell lines by screening against a tool compound library of 1402 annotated inhibitors in an adenosine triphosphate (ATP)-based cell viability assay. Subsequently, this MEF panel was used to conduct a high-throughput phenotypic screen in a cell viability assay with a proprietary compound library. All 126 compounds that exhibited a selective activity against mutant KRAS were selected and prioritized based on their activities in secondary assays. Finally, five chemical clusters were chosen. They had specific activity against SW620 and LS513 over Colo320 colorectal cancer cell lines. In addition, they had no effects on BRAFV600E, MEK1, extracellular signal-regulated kinase 2 (ERK2), phosphoinositide 3-kinase alpha (PI3Kα), AKT1, or mammalian target of rapamycin (mTOR) as tested in in vitro enzymatic activity assays. Biophysical assays demonstrated that these compounds did not bind directly to KRAS. We further identified the mechanism of action and showed that three of them have CDK9 inhibitory activity. In conclusion, we have developed and validated an isogenic MEF panel that was used successfully to identify RAS oncogenic or wild-type allele-specific vulnerabilities. Furthermore, we identified sensitivity of oncogenic KRAS-expressing cells to CDK9 inhibitors, which warrants future studies of treating KRAS-driven cancers with CDK9 inhibitors.

The incidence and mortality rates of prostate cancer are significantly higher in African-American men when compared to European-American men. We tested the hypothesis that differences in tumor biology contribute to this survival health disparity. Using microarray technology, we obtained gene expression profiles of primary prostate tumors resected from 33 African-American and 36 European-American patients. These tumors were matched on clinical parameters. We also evaluated 18 non-tumor prostate tissues from 7 African-American and 11 European-American patients. The resulting datasets were analyzed for expression differences on the gene and pathway level comparing African-American with European-American patients. Our analysis revealed 162 transcripts to be differentially expressed between African-American and European-American prostate cancer patients at a false discovery rate of 5% or less. Using a disease association analysis, we identified a common relationship of these transcripts with autoimmunity and inflammation. These findings were corroborated on the pathway level with numerous differentially expressed genes clustering in immune response, defense response, antigen presentation, B-cell/T-cell function, cytokine signaling, and inflammatory response pathways. Most commonly, the genes involved in these pathways were more highly expressed in tumors of African-American patients when compared with those of European-American patients. Amongst the immune-specific genes over-expressed by African-American patients was indoleamine 2,3-dioxygenease, HLA-E, and HLA-G. All three of these genes are well-known contributors of immunologic tolerance in tumors. Furthermore, a distinctive interferon signature was identified in the African-American prostate tumors, suggesting the possibility of viral involvement in disease etiology in this African-American population. In conclusion, the gene expression profiles of prostate tumors indicate prominent differences in tumor immunobiology between African-American and European-American men. The profiles portray the existence of a distinct tumor microenvironment in these two patient groups.