Exploring switch II pocket conformation of KRAS(G12D) with mutant-selective monobody inhibitors.

The G12D mutation is among the most common KRAS mutations associated with cancer, in particular, pancreatic cancer. Here, we have developed monobodies, small synthetic binding proteins, that are selective to KRAS(G12D) over KRAS(wild type) and other oncogenic KRAS mutations, as well as over the G12D mutation in HRAS and NRAS. Crystallographic studies revealed that, similar to other KRAS mutant-selective inhibitors, the initial monobody bound to the S-II pocket, the groove between switch II and α3 helix, and captured this pocket in the most widely open form reported to date. Unlike other G12D-selective polypeptides reported to date, the monobody used its backbone NH group to directly recognize the side chain of KRAS Asp12, a feature that closely resembles that of a small-molecule inhibitor, MTRX1133. The monobody also directly interacted with H95, a residue not conserved in RAS isoforms. These features rationalize the high selectivity toward the G12D mutant and the KRAS isoform. Structure-guided affinity maturation resulted in monobodies with low nM KD values. Deep mutational scanning of a monobody generated hundreds of functional and nonfunctional single-point mutants, which identified crucial residues for binding and those that contributed to the selectivity toward the GTP- and GDP-bound states. When expressed in cells as genetically encoded reagents, these monobodies engaged selectively with KRAS(G12D) and inhibited KRAS(G12D)-mediated signaling and tumorigenesis. These results further illustrate the plasticity of the S-II pocket, which may be exploited for the design of next-generation KRAS(G12D)-selective inhibitors.

Inhibition of RAS-driven signaling and tumorigenesis with a pan-RAS monobody targeting the Switch I/II pocket.

RAS mutants are major therapeutic targets in oncology with few efficacious direct inhibitors available. The identification of a shallow pocket near the Switch II region on RAS has led to the development of small-molecule drugs that target this site and inhibit KRAS(G12C) and KRAS(G12D). To discover other regions on RAS that may be targeted for inhibition, we have employed small synthetic binding proteins termed monobodies that have a strong propensity to bind to functional sites on a target protein. Here, we report a pan-RAS monobody, termed JAM20, that bound to all RAS isoforms with nanomolar affinity and demonstrated limited nucleotide-state specificity. Upon intracellular expression, JAM20 potently inhibited signaling mediated by all RAS isoforms and reduced oncogenic RAS-mediated tumorigenesis in vivo. NMR and mutation analysis determined that JAM20 bound to a pocket between Switch I and II, which is similarly targeted by low-affinity, small-molecule inhibitors, such as BI-2852, whose in vivo efficacy has not been demonstrated. Furthermore, JAM20 directly competed with both the RAF(RBD) and BI-2852. These results provide direct validation of targeting the Switch I/II pocket for inhibiting RAS-driven tumorigenesis. More generally, these results demonstrate the utility of tool biologics as probes for discovering and validating druggable sites on challenging targets.

Intersectin - many facets of a scaffold protein.

Intersectin (ITSN) is a multi-domain scaffold protein with a diverse array of functions including regulation of endocytosis, vesicle transport, and activation of various signal transduction pathways. There are two ITSN genes located on chromosomes 21 and 2 encoding for proteins ITSN1 and ITSN2, respectively. Each ITSN gene encodes two major isoforms, ITSN-Long (ITSN-L) and ITSN-Short (ITSN-S), due to alternative splicing. ITSN1 and 2, collectively referred to as ITSN, are implicated in many physiological and pathological processes, such as neuronal maintenance, actin cytoskeletal rearrangement, and tumor progression. ITSN is mis-regulated in many tumors, such as breast, lung, neuroblastomas, and gliomas. Altered expression of ITSN is also found in several neurodegenerative diseases, such as Down Syndrome and Alzheimer's disease. This review summarizes recent studies on ITSN and provides an overview of the function of this important family of scaffold proteins in various biological processes.

Mutations in the α4-α5 allosteric lobe of RAS do not significantly impair RAS signaling or self-association.

Mutations in one of the three RAS genes (HRAS, KRAS, and NRAS) are present in nearly 20% of all human cancers. These mutations shift RAS to the GTP-loaded active state due to impairment in the intrinsic GTPase activity and disruption of GAP-mediated GTP hydrolysis, resulting in constitutive activation of effectors such as RAF. Because activation of RAF involves dimerization, RAS dimerization has been proposed as an important step in RAS-mediated activation of effectors. The α4-α5 allosteric lobe of RAS has been proposed as a RAS dimerization interface. Indeed, the NS1 monobody, which binds the α4-α5 region within the RAS G domain, inhibits RAS-dependent signaling and transformation as well as RAS nanoclustering at the plasma membrane. Although these results are consistent with a model in which the G domain dimerizes through the α4-α5 region, the isolated G domain of RAS lacks intrinsic dimerization capacity. Furthermore, prior studies analyzing α4-α5 point mutations have reported mixed effects on RAS function. Here, we evaluated the activity of a panel of single amino acid substitutions in the α4-α5 region implicated in RAS dimerization. We found that these proposed "dimerization-disrupting" mutations do not significantly impair self-association, signaling, or transformation of oncogenic RAS. These results are consistent with a model in which activated RAS protomers cluster in close proximity to promote the dimerization of their associated effector proteins (e.g., RAF) without physically associating into dimers mediated by specific molecular interactions. Our findings suggest the need for a nonconventional approach to developing therapeutics targeting the α4-α5 region.

Direct inhibition of RAS: Quest for the Holy Grail?

RAS GTPases (H-, K-, and N-RAS) are the most frequently mutated oncoprotein family in human cancer. However, the relatively smooth surface architecture of RAS and its picomolar affinity for nucleotide have given rise to the assumption that RAS is an "undruggable" target. Recent advancements in drug screening, molecular modeling, and a greater understanding of RAS function have led to a resurgence in efforts to pharmacologically target this challenging foe. This review focuses on the state of the art of RAS inhibition, the approaches taken to achieve this goal, and the challenges of translating these discoveries into viable therapeutics.

Caveolin-1 stabilizes ATP7A, a copper transporter for extracellular SOD, in vascular tissue to maintain endothelial function.

Caveolin-1 (Cav-1) is a scaffolding protein and a major component of caveolae/lipid rafts. Previous reports have shown that endothelial dysfunction in Cav-1-deficient (Cav-1-/-) mice is mediated by elevated oxidative stress through endothelial nitric oxide synthase (eNOS) uncoupling and increased NADPH oxidase. Oxidant stress is the net balance of oxidant generation and scavenging, and the role of Cav-1 as a regulator of antioxidant enzymes in vascular tissue is poorly understood. Extracellular SOD (SOD3) is a copper (Cu)-containing enzyme that is secreted from vascular smooth muscle cells/fibroblasts and subsequently binds to the endothelial cells surface, where it scavenges extracellular [Formula: see text] and preserves endothelial function. SOD3 activity is dependent on Cu, supplied by the Cu transporter ATP7A, but whether Cav-1 regulates the ATP7A-SOD3 axis and its role in oxidative stress-mediated vascular dysfunction has not been studied. Here we show that the activity of SOD3, but not SOD1, was significantly decreased in Cav-1-/- vessels, which was rescued by re-expression of Cav-1 or Cu supplementation. Loss of Cav-1 reduced ATP7A protein, but not mRNA, and this was mediated by ubiquitination of ATP7A and proteasomal degradation. ATP7A bound to Cav-1 and was colocalized with SOD3 in caveolae/lipid rafts or perinucleus in vascular tissues or cells. Impaired endothelium-dependent vasorelaxation in Cav-1-/- mice was rescued by gene transfer of SOD3 or by ATP7A-overexpressing transgenic mice. These data reveal an unexpected role of Cav-1 in stabilizing ATP7A protein expression by preventing its ubiquitination and proteasomal degradation, thereby increasing SOD3 activity, which in turn protects against vascular oxidative stress-mediated endothelial dysfunction.

Inhibition of RAS: proven and potential vulnerabilities.

RAS is a membrane localized small GTPase frequently mutated in human cancer. As such, RAS has been a focal target for developing cancer therapeutics since its discovery nearly four decades ago. However, efforts to directly target RAS have been challenging due to the apparent lack of readily discernable deep pockets for binding small molecule inhibitors leading many to consider RAS as undruggable. An important milestone in direct RAS inhibition was achieved recently with the groundbreaking discovery of covalent inhibitors that target the mutant Cys residue in KRAS(G12C). Surprisingly, these G12C-reactive compounds only target mutant RAS in the GDP-bound state thereby locking it in the inactive conformation and blocking its ability to couple with downstream effector pathways. Building on this success, several groups have developed similar compounds that selectively target KRAS(G12C), with AMG510 and MRTX849 the first to advance to clinical trials. Both have shown early promising results. Though the success with these compounds has reignited the possibility of direct pharmacological inhibition of RAS, these covalent inhibitors are limited to treating KRAS(G12C) tumors which account for <15% of all RAS mutants in human tumors. Thus, there remains an unmet need to identify more broadly efficacious RAS inhibitors. Here, we will discuss the current state of RAS(G12C) inhibitors and the potential for inhibiting additional RAS mutants through targeting RAS dimerization which has emerged as an important step in the allosteric regulation of RAS function.

Inhibition of RAS function through targeting an allosteric regulatory site.

RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here we describe a functionally critical region, located outside the effector lobe of RAS, that can be targeted for inhibition. We developed NS1, a synthetic binding protein (monobody) that bound with high affinity to both GTP- and GDP-bound states of H-RAS and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H-RAS- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the α4-ß6-α5 region of RAS, which disrupted RAS dimerization and nanoclustering and led to blocking of CRAF-BRAF heterodimerization and activation. These results establish the importance of the α4-ß6-α5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.

Pharmacological targeting of RAS: Recent success with direct inhibitors.

RAS has long been viewed as undruggable due to its lack of deep pockets for binding of small molecule inhibitors. However, recent successes in the development of direct RAS inhibitors suggest that the goal of pharmacological inhibition of RAS in patients may soon be realized. This review will discuss the role of RAS in cancer, the approaches used to develop direct RAS inhibitors, and highlight recent successes in the development of novel RAS inhibitory compounds that target different aspects of RAS biochemistry. In particular, this review will discuss the different properties of RAS that have been targeted by various inhibitors including membrane localization, the different activation states of RAS, effector binding, and nucleotide exchange. In addition, this review will highlight the recent success with mutation-specific inhibitors that exploit the unique biochemistry of the RAS(G12C) mutant. Although this mutation in KRAS accounts for 11% of all KRAS mutations in cancer, it is the most prominent KRAS mutant in lung cancer suggesting that G12C-specific inhibitors may provide a new approach for treating the subset of lung cancer patients harboring this mutant allele. Finally, this review will discuss the involvement of dimerization in RAS function and highlight new approaches to inhibit RAS by specifically interfering with RAS:RAS interaction.

Akt2 (Protein Kinase B Beta) Stabilizes ATP7A, a Copper Transporter for Extracellular Superoxide Dismutase, in Vascular Smooth Muscle: Novel Mechanism to Limit Endothelial Dysfunction in Type 2 Diabetes Mellitus.

OBJECTIVE: Copper transporter ATP7A (copper-transporting/ATPase) is required for full activation of SOD3 (extracellular superoxide dismutase), which is secreted from vascular smooth muscle cells (VSMCs) and anchors to endothelial cell surface to preserve endothelial function by scavenging extracellular superoxide. We reported that ATP7A protein expression and SOD3 activity are decreased in insulin-deficient type 1 diabetes mellitus vessels, thereby, inducing superoxide-mediated endothelial dysfunction, which are rescued by insulin treatment. However, it is unknown regarding the mechanism by which insulin increases ATP7A expression in VSMCs and whether ATP7A downregulation is observed in T2DM (type2 diabetes mellitus) mice and human in which insulin-Akt (protein kinase B) pathway is selectively impaired. APPROACH AND RESULTS: Here we show that ATP7A protein is markedly downregulated in vessels isolated from T2DM patients, as well as those from high-fat diet-induced or db/db T2DM mice. Akt2 (protein kinase B beta) activated by insulin promotes ATP7A stabilization via preventing ubiquitination/degradation as well as translocation to plasma membrane in VSMCs, which contributes to activation of SOD3 that protects against T2DM-induced endothelial dysfunction. Downregulation of ATP7A in T2DM vessels is restored by constitutive active Akt or PTP1B-/- (protein-tyrosine phosphatase 1B-deficient) T2DM mice, which enhance insulin-Akt signaling. Immunoprecipitation, in vitro kinase assay, and mass spectrometry analysis reveal that insulin stimulates Akt2 binding to ATP7A to induce phosphorylation at Ser1424/1463/1466. Furthermore, SOD3 activity is reduced in Akt2-/- vessels or VSMCs, which is rescued by ATP7A overexpression. CONCLUSION: Akt2 plays a critical role in ATP7A protein stabilization and translocation to plasma membrane in VSMCs, which contributes to full activation of vascular SOD3 that protects against endothelial dysfunction in T2DM.

Intersectin scaffold proteins and their role in cell signaling and endocytosis.

Intersectins (ITSNs) are a family of multi-domain proteins involved in regulation of diverse cellular pathways. These scaffold proteins are well known for regulating endocytosis but also play important roles in cell signaling pathways including kinase regulation and Ras activation. ITSNs participate in several human cancers, such as neuroblastomas and glioblastomas, while their downregulation is associated with lung injury. Alterations in ITSN expression have been found in neurodegenerative diseases such as Down Syndrome and Alzheimer's disease. Binding proteins for ITSNs include endocytic regulatory factors, cytoskeleton related proteins (i.e. actin or dynamin), signaling proteins as well as herpes virus proteins. This review will summarize recent studies on ITSNs, highlighting the importance of these scaffold proteins in the aforementioned processes.

Silencing Intersectin 1 Slows Orthotopic Neuroblastoma Growth in Mice.

Neuroblastoma accounts for 15% of all pediatric cancer deaths. Intersectin 1 (ITSN1), a scaffold protein involved in phosphoinositide 3-kinase (PI3K) signaling, regulates neuroblastoma cells independent of MYCN status. We hypothesize that by silencing ITSN1 in neuroblastoma cells, tumor growth will be decreased in an orthotopic mouse tumor model. SK-N-AS neuroblastoma cells transfected with empty vector (pSR), vectors expressing scrambled shRNA (pSCR), or shRNAs targeting ITSN1 (sh#1 and sh#2) were used to create orthotopic neuroblastoma tumors in mice. Volume was monitored weekly with ultrasound. End-point was tumor volume >1000 mm. Tumor cell lysates were analyzed with anti-ITSN1 antibody by Western blot. Orthotopic tumors were created in all cell lines. Twenty-five days post injection, pSR tumor size was 917.6±247.7 mm, pSCR was 1180±159.9 mm, sh#1 was 526.3±212.8 mm, and sh#2 was 589.2±74.91 mm. sh#1-tumors and sh#2-tumors were smaller than pSCR (P=0.02), no difference between sh#1 and sh#2. Survival was superior in sh#2-tumors (P=0.02), trended towards improved survival in sh#1-tumors (P=0.09), compared with pSCR-tumors, no difference in pSR tumors. Western blot showed decreased ITSN1 expression in sh#1 and sh#2 compared with pSR and pSCR. Silencing ITSN1 in neuroblastoma cells led to decreased tumor growth in an orthotopic mouse model. Orthotopic animal models can provide insight into the role of ITSN1 pathways in neuroblastoma tumorigenesis.

Protein Kinase A (PKA) Phosphorylation of Shp2 Protein Inhibits Its Phosphatase Activity and Modulates Ligand Specificity.

Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Src homology 2 domain-containing phosphatase (Shp2) is critical for cardiac function because mutations resulting in loss of Shp2 catalytic activity are associated with congenital cardiac defects and hypertrophy. We identified a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that Shp2 is a component of the protein kinase A anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits Shp2 phosphatase activity. We identified two key amino acids in Shp2 that are phosphorylated by PKA. Thr-73 contributes a helix cap to helix αB within the N-terminal SH2 domain of Shp2, whereas Ser-189 occupies an equivalent position within the C-terminal SH2 domain. Utilizing double mutant PKA phosphodeficient (T73A/S189A) and phosphomimetic (T73D/S189D) constructs, in vitro binding assays, and phosphatase activity assays, we demonstrate that phosphorylation of these residues disrupts Shp2 interaction with tyrosine-phosphorylated ligands and inhibits its protein-tyrosine phosphatase activity. Overall, our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic ß-adrenergic stimulation and PKA activation. Therefore, although induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote this compensatory response.

Probing RAS Function Using Monobody and NanoBiT Technologies.

Missense mutations in the RAS family of oncogenes (HRAS, KRAS, and NRAS) are present in approximately 20% of human cancers, making RAS a valuable therapeutic target (Prior et al., Cancer Res 80:2969-2974, 2020). Although decades of research efforts to develop therapeutic inhibitors of RAS were unsuccessful, there has been success in recent years with the entrance of FDA-approved KRASG12C-specific inhibitors to the clinic (Skoulidis et al., N Engl J Med 384:2371-2381, 2021; Jänne et al., N Engl J Med 387:120-131, 2022). Additionally, KRASG12D-specific inhibitors are presently undergoing clinical trials (Wang et al., J Med Chem 65:3123-3133, 2022). The advent of these allele specific inhibitors has disproved the previous notion that RAS is undruggable. Despite these advancements in RAS-targeted therapeutics, several RAS mutants that frequently arise in cancers remain without tractable drugs. Thus, it is critical to further understand the function and biology of RAS in cells and to develop tools to identify novel therapeutic vulnerabilities for development of anti-RAS therapeutics. To do this, we have exploited the use of monobody (Mb) technology to develop specific protein-based inhibitors of selected RAS isoforms and mutants (Spencer-Smith et al., Nat Chem Biol 13:62-68, 2017; Khan et al., Cell Rep 38:110322, 2022; Wallon et al., Proc Natl Acad Sci USA 119:e2204481119, 2022; Khan et al., Small GTPases 13:114-127, 2021; Khan et al., Oncogene 38:2984-2993, 2019). Herein, we describe our combined use of Mbs and NanoLuc Binary Technology (NanoBiT) to analyze RAS protein-protein interactions and to screen for RAS-binding small molecules in live-cell, high-throughput assays.

HSP70-mediated mitochondrial dynamics and autophagy represent a novel vulnerability in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC), the most prevalent type of pancreatic cancer, is one of the deadliest forms of cancer with limited therapy options. Overexpression of the heat shock protein 70 (HSP70) is a hallmark of cancer that is strongly associated with aggressive disease and worse clinical outcomes. However, the underlying mechanisms by which HSP70 allows tumor cells to thrive under conditions of continuous stress have not been fully described. Here, we report that PDAC has the highest expression of HSP70 relative to normal tissue across all cancers analyzed. Furthermore, HSP70 expression is associated with tumor grade and is further enhanced in metastatic PDAC. We show that genetic or therapeutic ablation of HSP70 alters mitochondrial subcellular localization, impairs mitochondrial dynamics, and promotes mitochondrial swelling to induce apoptosis. Mechanistically, we find that targeting HSP70 suppresses the PTEN-induced kinase 1 (PINK1) mediated phosphorylation of dynamin-related protein 1 (DRP1). Treatment with the HSP70 inhibitor AP-4-139B was efficacious as a single agent in primary and metastatic mouse models of PDAC. In addition, we demonstrate that HSP70 inhibition promotes the AMP-activated protein kinase (AMPK) mediated phosphorylation of Beclin-1, a key regulator of autophagic flux. Accordingly, we find that the autophagy inhibitor hydroxychloroquine (HCQ) enhances the ability of AP-4-139B to mediate anti-tumor activity in vivo. Collectively, our results suggest that HSP70 is a multi-functional driver of tumorigenesis that orchestrates mitochondrial dynamics and autophagy. Moreover, these findings support the rationale for concurrent inhibition of HSP70 and autophagy as a novel therapeutic approach for HSP70-driven PDAC.

Emerging roles for intersectin (ITSN) in regulating signaling and disease pathways.

Intersectins (ITSNs) represent a family of multi-domain adaptor proteins that regulate endocytosis and cell signaling. ITSN genes are highly conserved and present in all metazoan genomes examined thus far. Lower eukaryotes have only one ITSN gene, whereas higher eukaryotes have two ITSN genes. ITSN was first identified as an endocytic scaffold protein, and numerous studies reveal a conserved role for ITSN in endocytosis. Subsequently, ITSNs were found to regulate multiple signaling pathways including receptor tyrosine kinases (RTKs), GTPases, and phosphatidylinositol 3-kinase Class 2beta (PI3KC2ß). ITSN has also been implicated in diseases such as Down Syndrome (DS), Alzheimer Disease (AD), and other neurodegenerative disorders. This review summarizes the evolutionary conservation of ITSN, the latest research on the role of ITSN in endocytosis, the emerging roles of ITSN in regulating cell signaling pathways, and the involvement of ITSN in human diseases such as DS, AD, and cancer.

Secreted frizzled related-protein 2 is prognostic for human pancreatic cancer patient survival and is associated with fibrosis.

Pancreatic adenocarcinoma (PDAC) is one of the deadliest cancers, with five-year survival rates of 9%. We hypothesized that secreted frizzled-related protein 2 (SFRP2) may influence stromal growth in pancreatic cancer, since it increases fibrosis and collagen production in non-neoplastic pathologies. We assessed SFRP2 value as a biomarker and assessed its function in PDAC. SFRP2 gene expression in patients with PDAC was analyzed using TCGA data. Disease free survival (DFS) was analyzed using Kaplan Meier test. The effect of KRAS inhibition on SFRP2 expression in PDAC cells was assessed. The associations of stromal content with SFPR2 mRNA and protein with fibrosis were analyzed. The role of SFRP2 in mesenchymal transformation was assessed by western blot in fibroblasts. Of all cancers in TCGA, SFRP2 levels were highest in PDAC, and higher in PDAC than normal tissues (n= 234, p= 0.0003). High SFRP2 levels correlated with decreased DFS (p= 0.0097). KRAS inhibition reduced SFRP2 levels. Spearman correlation was 0.81 between stromal RNA and SFRP2 in human PDAC, and 0.75 between fibrosis and SFRP2 levels in PDAC tumors. SFRP2-treated fibroblasts displayed mesenchymal characteristics. SFRP2 is prognostic for PDAC survival, regulated by KRAS, and associated with PDAC fibrosis.

Targeting the "undruggable" RAS with biologics.

RAS proteins represent critical drivers of tumor development and thus are the focus of intense efforts to pharmacologically inhibit these proteins in human cancer. Although recent success has been attained in developing clinically efficacious inhibitors to KRASG12C, there remains a critical need for developing approaches to inhibit additional mutant RAS proteins. A number of anti-RAS biologics have been developed which reveal novel and potentially therapeutically targetable vulnerabilities in oncogenic RAS. This review will discuss the growing field of anti-RAS biologics and potential development of these reagents into new anti-RAS therapies.

Identification of the nucleotide-free state as a therapeutic vulnerability for inhibition of selected oncogenic RAS mutants.

RAS guanosine triphosphatases (GTPases) are mutated in nearly 20% of human tumors, making them an attractive therapeutic target. Following our discovery that nucleotide-free RAS (apo RAS) regulates cell signaling, we selectively target this state as an approach to inhibit RAS function. Here, we describe the R15 monobody that exclusively binds the apo state of all three RAS isoforms in vitro, regardless of the mutation status, and captures RAS in the apo state in cells. R15 inhibits the signaling and transforming activity of a subset of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., fast exchange mutants). Intracellular expression of R15 reduces the tumor-forming capacity of cancer cell lines driven by select RAS mutants and KRAS(G12D)-mutant patient-derived xenografts (PDXs). Thus, our approach establishes an opportunity to selectively inhibit a subset of RAS mutants by targeting the apo state with drug-like molecules.

Targeting the KRAS α4-α5 allosteric interface inhibits pancreatic cancer tumorigenesis.

RAS is the most frequently mutated oncogene in human cancer with nearly ~20% of cancer patients possessing mutations in one of three RAS genes (K, N or HRAS). However, KRAS is mutated in nearly 90% of pancreatic ductal carcinomas (PDAC). Although pharmacological inhibition of RAS has been challenging, KRAS(G12C)-specific inhibitors have recently entered the clinic. While KRAS(G12C) is frequently expressed in lung cancers, it is rare in PDAC. Thus, more broadly efficacious RAS inhibitors are needed for treating KRAS mutant-driven cancers such as PDAC. A RAS-specific tool biologic, NS1 Monobody, inhibits HRAS- and KRAS-mediated signalling and oncogenic transformation both in vitro and in vivo by targeting the α4-α5 allosteric site of RAS and blocking RAS self-association. Here, we evaluated the efficacy of targeting the α4-α5 interface of KRAS as an approach to inhibit PDAC development using an immunocompetent orthotopic mouse model. Chemically regulated NS1 expression inhibited ERK and AKT activation in KRAS(G12D) mutant KPC PDAC cells and reduced the formation and progression of pancreatic tumours. NS1-expressing tumours were characterized by increased infiltration of CD4 + T helper cells. These results suggest that targeting the #x3B1;4-#x3B1;5 allosteric site of KRAS may represent a viable therapeutic approach for inhibiting KRAS-mutant pancreatic tumours.