•Matrine ameliorated tactile hypersensitivity and pain sensitivity in mice afflicted with CIPN.•Matrine enhanced the functionality of the sciatic nerve and spinal cord neurons in CIPN mice.•Matrine, ...a natural KRAS inhibitor, is derived from the medicinal plant Sophora alopecuroides l.
Chemotherapy-induced peripheral neuropathy (CIPN) represents a prevailing and severe clinical concern, characterized by limited availability of clinically effective treatment strategies. Current evidence endorses matrine's potential as a neuroprotective and analgesic agent for CIPN. Nevertheless, the precise targets and mechanisms of action of matrine remain insufficiently explored, impeding comprehensive pharmacological investigation and clinical application.
This study endeavors to elucidate the analgesic and neuroprotective effects of matrine in mice with vincristine-induced neuropathic pain. A focal point is the identification of matrine's specific target and the underlying molecular mechanisms governing its analgesic and neuroprotective actions.
To discern matrine's analgesic effects in CIPN mice, we conducted behavioral experiments encompassing the Von Frey filament test and Hargreaves Test. Furthermore, we conducted electrophysiological and histopathological assessments involving HE staining, Nissl staining, and Fluoro-Jade B staining to evaluate matrine's effects on neuroprotection within dorsal root ganglia and the spinal cord of CIPN mice. Sequentially, thermal shift assay, GTP hydrolysis assay, and nucleotide exchange assay were executed to validate matrine's inhibitory effects on KRAS. Molecular docking and site-directed mutagenesis experiments were implemented to identify the precise binding pocket of matrine on KRAS. Lastly, matrine's inhibitory effects on downstream signaling pathways of KRAS were confirmed through experiments conducted at animal model.
Matrine exhibited a notable increase in mechanical withdrawal threshold and thermal withdrawal latency in vincristine-treated mice. This compound substantially ameliorated the neurofunctional blockade associated with sensory and motor functions induced by vincristine. Moreover, matrine mitigated pathological damage within DRG and the L4-L5 spinal cord regions. The study's MST experiments indicated matrine's substantial elevation of KRAS's melting temperature. The GTP hydrolysis and nucleotide exchange assays revealed concentration-dependent inhibition of KRAS activity by matrine. Molecular docking provided insight into the binding mode of matrine with KRAS, while site-directed mutagenesis verified the specific binding site of matrine on KRAS. Lastly, matrine's inhibition of downstream Raf/Erk1/2 and PI3K/Akt/mTOR signaling pathways of KRAS was confirmed in VCR mice.
Compared to previous studies, our research has identified matrine as a natural inhibitor of the elusive protein KRAS, often considered "undruggable." Furthermore, this study has revealed that matrine exerts its therapeutic effects on chemotherapy-induced peripheral neuropathy (CIPN) by inhibiting KRAS activation, subsequently suppressing downstream signaling pathways such as Raf/Erk1/2 and PI3K/Akt/mTOR. This investigation signifies the discovery of a novel target for matrine, thus expanding the potential scope of its involvement in KRAS-related biological functions and diseases. These findings hold the promise of providing a crucial experimental foundation for forthcoming drug development initiatives centered around matrine, thereby advancing the field of pharmaceutical research.
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The clear importance of mutated KRAS as a therapeutic target has driven the investigation of multiple approaches to inhibit oncogenic KRAS signaling at different molecular levels. ...However, no KRAS-targeted therapy has reached the clinic to date, which underlies the intrinsic difficulty in developing effective, direct inhibitors of KRAS. Thus, this article provides an overview of the history and recent progress in the development of pharmacological strategies to target oncogenic KRAS with small molecule agents. Mechanistically, these KRAS-targeted agents can be classified into the following four categories. (1) Small-molecule RAS-binding ligands that prevent RAS activation by binding within or outside the nucleotide-binding motif. (2) Inhibitors of KRAS membrane anchorage. (3) Inhibitors that bind to RAS-binding domains of RAS-effector proteins. (4) Inhibitors of KRAS expression. The advantage and limitation of each type of these anti-KRAS agents are discussed.
Raziskava korozijskih uravnav v Sloveniji podaja sistematično klasifikacijo korozijskih uravnav na podlagi njihovih geoloških in morfoloških značilnosti. Klasifikacija jih združuje v štiri ...kategorije: kraški ravnik, suho kraško polje, robna uravnava kontaktnega krasa in robna uravnava fluviokrasa. Kraški ravniki so največji in nastanejo z denudacijo v stabilnih tektonskih in hidrogeoloških razmerah. Suha kraška polja so zaprte živoskalne kotanje le mestoma prekrite s sedimenti. Robne uravnave kontaktnega krasa so nastale na stiku kraških in nekraških kamnin. Robne uravnave fluviokrasa so nastale z večfaznim razvojem kraških polj zaradi tektonske dejavnosti. Ta raziskava prispeva k boljšemu razumevanju korozijskih uravnav in lahko pomaga pri njihovi identifikaciji in upravljanju v kraških območij.
Kirsten Rat Sarcoma (KRAS) is a master oncogene involved in cellular proliferation and survival and is the most commonly mutated oncogene in all cancers. Activating KRAS mutations are present in over ...90% of pancreatic ductal adenocarcinoma (PDAC) cases and are implicated in tumor initiation and progression. Although KRAS is a critical oncogene, and therefore an important therapeutic target, its therapeutic inhibition has been very challenging, and only recently specific mutant KRAS inhibitors have been discovered. In this review, we discuss the activation of KRAS signaling and the role of mutant KRAS in PDAC development. KRAS has long been considered undruggable, and many drug discovery efforts which focused on indirect targeting have been unsuccessful. We discuss the various efforts for therapeutic targeting of KRAS. Further, we explore the reasons behind these obstacles, novel successful approaches to target mutant KRAS including G12C mutation as well as the mechanisms of resistance.
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•GTF3C6 is upregulated in LUAD tissues, LSL-KrasG12D/+;LSL-p53−/− LUAD mouse models and LUAD organoid.•Upregulation of GTF3C6 is associated with an unfavorable clinical prognosis in ...LUAD.•GTF3C6 is driven by KRAS/PI3K axis in LUAD.•GTF3C6 promotes anchorage-independent proliferation, migration, and invasion of LUAD cells via FAK pathway.
Effective targeting drugs for KRAS mutation-mediated Lung Adenocarcinoma (LUAD) are currently are limited.
Investigating and intervening in the downstream key target genes of KRAS is crucial for clinically managing KRAS mutant-driven LUAD. GTF3C6, a newly identified member of the general transcription factor III (GTF3) family, plays a role in the transcription of RNA polymerase III (pol III)-dependent genes. However, its involvement in cancer remains unexplored.
This study examined the expression, roles, and potential molecular mechanisms of GTF3C6 in LUAD tissues, LSL-KrasG12D/+;LSL-p53−/− LUAD mouse models, and LUAD patients-derived organoid using Western blot, qRT-PCR, immunofluorescence, immunohistochemistry, and gene manipulation assays.
We present the first evidence that GTF3C6 is highly expressed in LUAD tissues, LSL-KrasG12D/+;LSL-p53−/− LUAD mouse models, and LUAD organoids, correlating with poor clinical prognosis. Furthermore, GTF3C6 was found to promote anchorage-independent proliferation, migration, and invasion of LUAD cells. Mechanistically, KRAS mutation drives GTF3C6 expression through the PI3K pathway, and GTF3C6 knockdown reverses the malignant phenotype of KRAS mutation-driven LUAD cells. Additionally, the FAK pathway emerged as a crucial downstream signaling pathway through which GTF3C6 mediates the malignant phenotype of LUAD. Finally, GTF3C6 knockdown suppresses LUAD organoid formation and inhibits tumor growth in vivo.
Our findings demonstrate that GTF3C6, driven by KRAS mutation, promotes LUAD development by regulating FAK phosphorylation, suggesting its potential as a biomarker and therapeutic target in KRAS mutant-driven LUAD.
Patient-derived organoids (PDO) are promising tumor avatars that could enable ex vivo drug tests to personalize patients’ treatment in the frame of functional precision oncology (FPM). Yet, clinical ...evidence remain scarce. This study aims to evaluate whether PDO can be implemented in clinical practice to benefit patients with advanced refractory pancreatic adenocarcinoma (PDAC).
During 2021-2022, 87 patients were prospectively enrolled in an IRB-approved protocol. Inclusion criteria were: histologically-confirmed PDAC, tumor site accessible. A panel of 25 approved antitumor therapies (chemogram) was tested and compared to patient responses to assess PDO predictive values and map the drug sensitivity landscape in PDAC.
Fifty-four PDOs were generated from 87 pretreated patients (take-on rate 62%). The main PDO mutations were KRAS (96%), TP53 (88%) and CDKN2A/B (22%), with 91% concordance rate with their tumor of origin. The mean turnaround-time to chemogram was 6.8 weeks. In 91% of cases, ≥1 hit was identified (gemcitabine (n=20/54), docetaxel (n=18/54) and vinorelbine (n=17/54) with a median of 3 hits/patient range:0-12).
Our cohort included 34 evaluable patients with full clinical follow-up. We report a chemogram sensitivity of 83.3% and specificity of 92.9%. The overall-response rate and progression-free survival were higher when patients received a “hit” treatment as compared to patients that received a “non-hit” drug (as part of routine management).
Finally, we leveraged our PDO collection as a platform for drug validation and combo identification. We tested the anti-KRASG12D (MRTX1133), alone or combined, and identified a specific synergy with anti-EGFR therapies in KRASG12D variants.
We report the largest prospective study aiming at implementing PDO-based FPM and identify very robust predictive values in this clinical setting. In a clinically relevant turnaround-time, we identify putative hits for 91% of patients, providing unexpected potential survival benefits in this very aggressive indication. While this remains to be confirmed in interventional precision oncology trials, PDO collection already provide powerful opportunities for drugs and combinatorial treatment development.
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•CRISP prospectively collected real-world data on advanced NSCLC and KRAS mutations.•Of 1039 patients, 39.5 % had KRAS mutations, 38.9 % of these were KRAS G12C-mutated.•KRAS G12C-mutated advanced ...NSCLC has poor outcome under current standard therapy.•Valuable historical control for upcoming clinical studies on KRAS-inhibitors.
After decades of unsuccessful efforts in inhibiting KRAS, promising clinical data targeting the mutation subtype G12C emerge. Since little is known about outcome with standard treatment of patients with G12C mutated non-small cell lung cancer (NSCLC), we analyzed a large, representative, real-world cohort from Germany.
A total of 1039 patients with advanced KRAS-mutant or -wildtype NSCLC without druggable alterations have been recruited in the prospective, observational registry CRISP from 12/2015 to 06/2019 by 98 centers in Germany. Details on treatment, best response, and outcome were analyzed for patients with KRAS wildtype, G12C, and non-G12C mutations.
Within the study population, 160 (15.4 %) patients presented with KRAS G12C, 251 (24.2 %) with non-G12C mutations, 628 (60.4 %) with KRAS wildtype. High PD-L1 expression (Tumor Proportion Score, TPS > 50 %) was documented for 28.0 %, 43.5 %, and 28.9 % (wildtype, G12C, non-G12C) of the tested patients; 68.8 %, 89.3 %, and 87.7 % of the patients received first-line treatment combined with an immune checkpoint-inhibitor in 2019. TPS > 50 % vs. TPS < 1 % was associated with a significantly decreased risk of mortality in a multivariate Cox model (HR 0.39, 95 % CI 0.26−0.60, p=<0.001). There were no differences in clinical outcome between KRAS wildtype, G12C or non-G12C mutations and KRAS mutational status was not prognostic in the model.
Here we describe the so far largest prospectively recruited cohort of patients with advanced NSCLC and KRAS mutations, with special focus on the G12C mutation. These data constitute an extremely valuable historical control for upcoming clinical studies that employ KRAS inhibitors.
The oncogenic role of KRAS in colorectal cancer (CRC) progression is well-established. Despite this, identifying effective therapeutic targets for KRAS-mutated CRC remains a significant challenge. ...This study identifies pyruvate dehydrogenase phosphatase catalytic subunit 1 (PDP1) as a previously unrecognized yet crucial regulator in the progression of KRAS mutant CRC. A substantial upregulation of PDP1 expression is observed in KRAS mutant CRC cells and tissues compared to wild-type KRAS samples, which correlates with poorer prognosis. Functional experiments elucidate that PDP1 accelerates the malignance of KRAS mutant CRC cells, both in vitro and in vivo. Mechanistically, PDP1 acts as a scaffold, enhancing BRAF and MEK1 interaction and activating the MAPK signaling, thereby promoting CRC progression. Additionally, transcription factor KLF5 is identified as the key regulator for PDP1 upregulation in KRAS mutant CRC. Crucially, targeting PDP1 combined with MAPK inhibitors exhibits an obvious inhibitory effect on KRAS mutant CRC. Overall, PDP1 is underscored as a vital oncogenic driver and promising therapeutic target for KRAS mutant CRC.
•PDP1 is overexpressed in KRAS mutant CRC and correlates with a poor prognosis.•PDP1 promotes the progression of KRAS mutant CRC both in vitro and in vivo.•PDP1 strengthens the interaction between BRAF and MEK1 to activate MAPK signals.•KRAS mutation increases KLF5 expression, which transcriptionally regulates the expression of PDP1.•Inhibiting PDP1 enhances the therapeutic efficacy of MAPK inhibitors in KRAS mutant CRC.
With recent advances proving that effective inhibition of KRAS is possible, there have been significant efforts made to develop inhibitors of specific mutant alleles. Here we describe a detailed ...protocol that employs homogeneous time-resolved fluorescence (HTRF) to identify compounds acting on KRAS signaling in malignant cell lines. This method allows for high-throughput, cell-based screens of large compound libraries for the development of RAS-targeted therapeutics.
Abstract
Objective
Numerous scattered case studies continue to demonstrate a strong correlation between acquired KRAS mutations and epidermal growth factor receptor-tyrosine kinase inhibitor ...resistance in non-small cell lung cancer. However, the comprehensive understanding of the KRAS pathway following the failure of epidermal growth factor receptor-tyrosine kinase inhibitor therapy remains limited.
Methods
We conducted a retrospective evaluation of the next generation sequencing data from 323 patients with advanced non-small cell lung cancer and EGFR-activating mutations after experiencing progression with epidermal growth factor receptor-tyrosine kinase inhibitor therapy. Our analysis specifically focused on the acquired changes to the KRAS gene.
Results
Among the 323 patients with advanced non-small cell lung cancer and EGFR-activating mutations who experienced resistance to epidermal growth factor receptor-tyrosine kinase inhibitor therapy, 14 individuals (4.3%) developed resistance due to acquired KRAS alterations. Of these 14 patients, 10 cases (71.4%) were due to KRAS missense mutations, 1 case (7.2%) was due to KRAS gene fusion and 3 cases (21.4%) were due to KRAS amplification. Notably, we identified one newly demonstrated KRAS gene fusion (KRAS and LMNTD1), one KRAS G13D and one KRAS K117N. The emergence of acquired KRAS alterations was often accompanied by novel mutations and high tumor mutation burden, with TP53, CNKN2A, PIK3CA, MYC, STK11, CDK4, BRCA2 and ERBB2 being the most frequently observed concurrent mutations. The median progression-free survival and overall survival for the 14 patients were 5.2 and 7.3 months, respectively. Acquired KRAS missense variants were associated with significantly worse progression-free survival compared with other KRAS variant subtypes (P < 0.028).
Conclusions
This study provides significant evidence of the role of acquired KRAS variants in the development of resistance to epidermal growth factor receptor-tyrosine kinase inhibitor therapy. Our results contribute to the growing body of knowledge on the mutational profiles associated with resistance to epidermal growth factor receptor-tyrosine kinase inhibitor treatment. Furthermore, our study highlights the KRAS gene change as a significant mechanism of resistance to epidermal growth factor receptor-tyrosine kinase inhibitor therapy.
The study identified 14 cases showing acquired KRAS alterations associated with resistance to epidermal growth factor receptor-tyrosine kinase inhibitor. These findings contribute to our comprehension of the mutational profiles linked to epidermal growth factor receptor-tyrosine kinase inhibitor resistance.
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