Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics ...and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient) and state 2 (active, effector binding enabled). Here, we use 31P NMR to delineate the differences in state 1 and state 2 populations present in WT and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analog GppNHp (guanosine-5'-(β,γ)-imido triphosphate). Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RAS-binding domain and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small-molecule inhibitor, BBO-8956, which is effective against both GDP- and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs state 1–state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1–RAS-binding domain is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action for this novel and active-conformation inhibitor.
Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics ...and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient) and state 2 (active, effector binding enabled). Here, we use 31P NMR to delineate the differences in state 1 and state 2 populations present in WT and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analog GppNHp (guanosine-5'-(β,γ)-imido triphosphate). Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RAS-binding domain and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small-molecule inhibitor, BBO-8956, which is effective against both GDP- and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs state 1–state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1–RAS-binding domain is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action for this novel and active-conformation inhibitor.
The role of KRAS G12C is of particular interest given the promising clinical activity of KRAS G12C-specific inhibitors. This study comprehensively investigated the clinicopathological characteristics ...and prognostic value of KRAS G12C mutation in patients with surgically resected lung adenocarcinoma.
Data were collected on 3828 patients with completely resected primary lung adenocarcinomas who underwent KRAS mutation analysis between 2008 and 2020. The association between KRAS G12C and clinicopathologic characteristics, molecular profiles, recurrence patterns, and postoperative outcome were explored.
Two hundred seventy-five patients (7.2%) were confirmed to harbor a KRAS mutation, of whom 83 (30.2%) had the G12C subtype. KRAS G12C was more frequent in men, former/current smokers, radiologic solid nodules, invasive mucinous adenocarcinoma, and solid predominant tumors. KRAS G12C tumors had more lymphovascular invasion and higher programmed death-ligand 1 expression than KRAS wild-type tumors. TP53 (36.8%), STK11 (26.3%), and RET (18.4%) mutations were the 3 most frequent in the KRAS G12C group. Logistic regression analysis showed patients with KRAS G12C mutation were prone to experience early recurrence and locoregional recurrence. KRAS G12C mutation was found to be significantly associated with poor survival after propensity score matching. Stratified analysis showed that the KRAS G12C was an independent prognostic factor in stage I tumors and part-solid lesions, respectively.
The KRAS G12C mutation had a significant prognostic value in stage I lung adenocarcinomas as well as in part-solid tumors. Furthermore, it exhibited a potentially aggressive phenotype associated with early and locoregional recurrence. These findings might be relevant as better KRAS treatments are developed for clinical application.
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KRAS mutation is the most frequent molecular alteration found in advanced NSCLC; it is associated with a poor prognosis without available targeted therapy. Treatment options for NSCLC have been ...recently enriched by the development of immune checkpoint inhibitors (ICIs), and data about its efficacy in patients with KRAS-mutant NSCLC are discordant. This study assessed the routine efficacy of ICIs in advanced KRAS-mutant NSCLC.
In this retrospective study, clinical data were extracted from the medical records of patients with advanced NSCLC treated with ICIs and with available molecular analysis between April 2013 and June 2017. Analysis of programmed death ligand 1 (PD-L1) expression was performed if exploitable tumor material was available.
A total of 282 patients with ICI-treated (in the first line or more) advanced NSCLC (all histological subgroups) who were treated with ICIs (anti–programmed death 1, anti–PD-L1, or anti–cytotoxic T-lymphocyte associated protein 4 antibodies), including 162 (57.4%) with KRAS mutation, 27 (9.6%) with other mutations, and 93 (33%) with a wild-type phenotype, were identified. PD-L1 analysis was available for 128 patients (45.4%), of whom 45.3% and 19.5% had PD-L1 expression of 1% or more and 50%, respectively (49.5% and 21.2%, respectively, in the case of the 85 patients with KRAS-mutant NSCLC). No significant difference was seen in terms of objective response rates, progression-free survival, or overall survival between KRAS-mutant NSCLC and other NSCLC. No significant differences in overall survival or progression-free survival were observed between the major KRAS mutation subtypes (G12A, G12C, G12D, G12V, and G13C). In KRAS-mutant NSCLC, unlike in non–KRAS-mutant NSCLC, the efficacy of ICIs is consistently higher, even though not statistically significant, for patients with PD-L1 expression in 1% or more of tumor cells than for those with PD-L1 expression in less than 1% of tumor cells, and this finding is especially true when PD-L1 expression is high (PD-L1 expression ≥50%).
For patients with KRAS-mutant NSCLC (all mutational subtypes), the efficacy of ICI is similar to that for patients with other types of NSCLC. PD-L1 expression seems to be more relevant for predicting the efficacy of ICIs in KRAS-mutant NSCLC than it is in other types of NSCLC.
Extracranial arteriovenous malformation (AVM) is most commonly caused by a somatic mutation in MAP2K1. We report two patients with vascular anomalies that had an unclear clinical diagnosis most ...consistent with either an AVM or congenital hemangioma. Lesions were cutaneous, reddish‐purple with telangiectasias, present at birth, and had defined borders. Histopathology indicated AVM and both lesions contained somatic KRAS mutations. A rare AVM phenotype exists that shares clinical features with congenital hemangioma.
Mutant KRAS extracranial arteriovenous malformation
A series of novel thieno2,3-dpyrimidine analogs were designed and synthesized as KRAS G12D inhibitors via combinatorial virtual screening approach. Most compounds exhibited potent antiproliferative ...activity on KRAS G12D mutated cancer cell lines (Panc1, SW1990 and CT26) with IC50 values in the low micromolar range. Among them, compound KD-8 showed the highest antiproliferative activity with an average IC50 of 2.1 μM against three KRAS G12D-mutated cells (Panc1, SW1990 and CT26). KD-8 decreased the active form of KRAS (KRAS-GTP) in KRAS G12D mutated cancer cell lines (CT26 and SW1990) but not in KRAS G13D mutated cancer cells (HCT116). Moreover, KD-8 down-regulated the phosphorylated Raf and Erk in CT26 and SW1990 cancer cell lines but not in HeLa cells (KRAS WT). The binding affinity of KD-8 was further confirmed by the isothermal titration calorimetry (ITC) assay in which KD-8 exhibited a KD of 33 nM for binding to KRAS G12D protein. In addition, KD-8 (40 mg/kg or 60 mg/kg) exhibited significant antitumor efficacy in a CT26 tumor model with a tumor growth inhibition (TGI) of 42% or 53% without causing apparent toxicity. Taken together the above results suggest that KD-8 is a promising KRAS G12D inhibitor deserving further investigation.
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●Novel thieno2,3-dpyrimidine-based KRAS G12D Inhibitors were discovered as anticancer agents.●KD-8 could bind to KRAS G12D protein with high affinity (KD = 33 nM by ITC).●KD-8 decreased the active form of KRAS G12D (KRAS G12D-GTP) but not KRAS G13D.●KD-8 suppressed CT26 tumor growth with a TGI of 42%.
During G1-phase of the cell cycle, normal cells respond first to growth factors that indicate that it is appropriate to divide and then later in G1 to the presence of nutrients that indicate ...sufficient raw material to generate two daughter cells. Dividing cells rely on the “conditionally essential” amino acid glutamine (Q) as an anaplerotic carbon source for TCA cycle intermediates and as a nitrogen source for nucleotide biosynthesis. We previously reported that while non-transformed cells arrest in the latter portion of G1 upon Q deprivation, mutant KRas-driven cancer cells bypass the G1 checkpoint, and instead, arrest in S-phase. In this study, we report that the arrest of KRas-driven cancer cells in S-phase upon Q deprivation is due to the lack of deoxynucleotides needed for DNA synthesis. The lack of deoxynucleotides causes replicative stress leading to activation of the ataxia telangiectasia and Rad3-related protein (ATR)-mediated DNA damage pathway, which arrests cells in S-phase. The key metabolite generated from Q utilization was aspartate, which is generated from a transaminase reaction whereby Q-derived glutamate is converted to α-ketoglutarate with the concomitant conversion of oxaloacetate to aspartate. Aspartate is a critical metabolite for both purine and pyrimidine nucleotide biosynthesis. This study identifies the molecular basis for the S-phase arrest caused by Q deprivation in KRas-driven cancer cells that arrest in S-phase in response to Q deprivation. Given that arresting cells in S-phase sensitizes cells to apoptotic insult, this study suggests novel therapeutic approaches to KRas-driven cancers.
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•Series of coumarin-based KRAS-G12C inhibitors were found through Virtual Screening and Rational Structural Optimization.•K45 displayed potent activity and selectivity against KRAS ...mutant cells.•K45 inhibited active KRAS, p-ERK and p-AKT in KRAS-G12C mutant cells.•K45 induced apoptosis by increasing the expression BAD and BAX in H23 cells.
KRAS-G12C inhibitors has been made significant progress in the treatment of KRAS-G12C mutant cancers, but their clinical application is limited due to the adaptive resistance, motivating development of novel structural inhibitors. Herein, series of coumarin derivatives as KRAS-G12C inhibitors were found through virtual screening and rational structural optimization. Especially, K45 exhibited strong antiproliferative potency on NCI-H23 and NCI-H358 cancer cells harboring KRAS-G12C with the IC50 values of 0.77 μM and 1.50 μM, which was 15 and 11 times as potent as positive drug ARS1620, respectively. Furthermore, K45 reduced the phosphorylation of KRAS downstream effectors ERK and AKT by reducing the active form of KRAS (KRAS GTP) in NCI-H23 cells. In addition, K45 induced cell apoptosis by increasing the expression of anti-apoptotic protein BAD and BAX in NCI-H23 cells. Docking studies displayed that the 3-naphthylmethoxy moiety of K45 extended into the cryptic pocket formed by the residues Gln99 and Val9, which enhanced the interaction with the KRAS-G12C protein. These results indicated that K45 was a potent KRAS-G12C inhibitor worthy of further study.
The Role of Autophagy in Cancer Santana-Codina, Naiara; Mancias, Joseph D; Kimmelman, Alec C
Annual review of cancer biology,
03/2017, Letnik:
1, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Autophagy is a highly conserved and regulated process that targets proteins and damaged organelles for lysosomal degradation to maintain cell metabolism, genomic integrity, and cell survival. The ...role of autophagy in cancer is dynamic and depends, in part, on tumor type and stage. Although autophagy constrains tumor initiation in normal tissue, some tumors rely on autophagy for tumor promotion and maintenance. Studies in genetically engineered mouse models support the idea that autophagy can constrain tumor initiation by regulating DNA damage and oxidative stress. In established tumors, autophagy can also be required for tumor maintenance, allowing tumors to survive environmental stress and providing intermediates for cell metabolism. Autophagy can also be induced in response to chemotherapeutics, acting as a drug-resistance mechanism. Therefore, targeting autophagy is an attractive cancer therapeutic option currently undergoing validation in clinical trials.
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