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INTRODUCTION: Personalized and tumor-informed circulating tumor DNA (ctDNA) testing is feasible and allows for molecular residual disease (MRD) identification in patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: In this retrospective analysis of commercial cases from multiple US institutions, personalized, tumor-informed, whole-exome sequenced, and germline-controlled ctDNA levels were quantified and analyzed in patients with PDAC. Plasma samples (n = 1329) from 298 clinically validated patients were collected at diagnosis, perioperatively (MRD-window; within 2-12 weeks after surgery, before therapy), and during surveillance (>12 weeks post-surgery if no ACT or starting 4 weeks post-ACT) from November 2019 to March 2023. RESULTS: Of the initially diagnosed patients with stages I-III PDAC who went for resection, the median follow-up time from surgery was 13 months (range 0.1-214). Positive ctDNA detection rates were 29% (29/100) and 29.6% (45/152) during the MRD and surveillance windows, respectively. Positive ctDNA detection was significantly associated with shorter DFS within the MRD window (median DFS of 6.37 months for ctDNA-positive vs 33.31 months for ctDNA-negative patients; HR: 5.45, P

Background: Bronchiolar adenoma (BA) is a rare benign tumor originating in the bronchial mucosal epithelium and occurring primarily in the periphery of the lung. The most prominent histopathological feature of BA is a double-layer bronchial epithelium containing continuous basal cell layers. However, due to the high mutation frequency of the driver gene, there is still debate as to whether BA has the potential for malignant transformation. In frozen sections, basal cells are difficult to identify under the microscope, which makes it difficult to distinguish from mucinous adenocarcinoma, especially when BA malignancies transform into invasive mucinous adenocarcinoma (IMA), which can only be distinguished by histomorphological criteria, greatly increasing the difficulty of diagnosis. Case summary: In this paper, we present a case study of a 59-year-old man whose chest computed tomography (CT) revealed a progressively enlarging, high-density nodule over a four-year period in the outer basal segment of the right lower lobe. Consequently, he underwent thoracoscopic wedge resection of the right lower lobe. The postoperative pathological diagnosis revealed BA with mucous gland structure formation combined with partial basal cell loss, raising the possibility of malignant transformation into IMA. Regular postoperative follow-up showed no recurrence or metastasis. Hybridization Capture-based next-generation sequencing (NGS) analysis detected driver gene mutations in Kirsten Rat Sarcoma viral oncogene homolog (KRAS) and Cyclin-Dependent Kinases (CDK) 6 in the case, thereby inferring the malignant transformation of BA into IMA. Conclusion: In this case, the detection of driver gene KRAS mutation and loss of continuity in the basal cell layer within the mucous glandular structures of the nodule suggests the malignant transformation of BA into IMA, inferring the malignant potential of BA. [ABSTRACT FROM AUTHOR]

Gapmer-type antisense oligonucleotides (ASOs) are an emerging class of therapeutic agents that directly inhibit pathogenic mRNA. In this study, three new 4′-C-substituted thymidine analogs were generated using a synthetic strategy recently established by our group, namely, 4′-C-(N-ethyl) aminoethyl (4′-EAE-T), 4′-C-(N-butyl) aminoethyl (4′-BAE-T), and 4′-C-(N-octyl) aminoethyl (4′-OAE-T). Their properties were evaluated and compared with those of previously reported analogs, including 4′-C-aminoethyl (4′-AE-T) and 4′-C-(N-methyl) aminoethyl (4′-MAE-T). The novel nucleoside analogs were subsequently incorporated into gapmer-type ASOs featuring phosphorothioate (PS) linkages and locked nucleic acids (LNAs) in the wing regions. The incorporation of 4′-EAE-T and 4′-BAE-T analogs resulted in RNA binding affinities similar to that of the previously reported 4′-MAE-T analog, whereas a marked decrease in RNA affinity was noted for 4′-OAE-T, however, this reduction was mitigated when combined with other chemical modifications. Furthermore, the structural modifications conferred enhanced nuclease resistance under bovine serum conditions, with 4′-EAE-T resulting in the highest stability, followed by 4′-BAE-T and 4′-OAE-T. Additionally, oligonucleotides modified with the developed analogs preserved their RNase H cleavage susceptibility, albeit inducing minor alterations in the cleavage pattern. Finally, the oligonucleotides were applied in a gene silencing experiment targeting the KRAS gene, conducted without the use of transfection agents, displaying gene silencing activities comparable to that of the control, with the exception of the 4′-OAE-modified nucleotide, which exhibited low activity. [ABSTRACT FROM AUTHOR]

Simple Summary: Borderline Ovarian Tumors (BOTs) are a unique group of ovarian growths that fall between benign and malignant categories. These tumours primarily affect younger women, often raising concerns about preserving fertility during treatment. Currently, doctors rely on surgical findings and tumour characteristics to predict outcomes and guide treatment decisions. However, this approach may not always provide a complete picture. Our review explores the potential of using molecular markers, such as BRAF, KRAS, CA125, and others, to better understand and manage BOTs. By identifying these biomarkers, we aim to develop a more personalized approach to treatment, potentially allowing for less aggressive interventions in low-risk cases while ensuring appropriate care for those at higher risk of recurrence or progression. This research could lead to improved decision-making and outcomes for women diagnosed with BOTs. Borderline Ovarian Tumours (BOTs) are a heterogenous group of ovarian neoplasms which have increased mitotic activity but lack stromal invasion. We performed a narrative review of the literature, aiming to identify prognostic molecular biomarkers that can potentially be used for treatment personalisation. We identified and discussed BRAF/KRAS, Cancer Antigen 125 (Ca 125), Calprotectin, p16ink4a, and Microsatellite instability (MSI) as the most studied biomarkers related to BOTs. Overall, BRAF and KRAS mutations are associated with earlier-stage and favourable prognosis; KRASmt may indicate extraovarian disease in serous BOT (sBOT). Ca125, the only currently clinically used biomarker, can be assessed pre-operatively and has an established role in post-operative surveillance, especially when it is raised pre-operatively or a high potential for malignant transformation is suspected post-operatively. p16ink4a expression trends could also indicate the malignant transformation of the tumour. Calprotectin has an inferior specificity to Ca125 and is not yet established as a biomarker, whilst there is very limited evidence available for MSI. As new evidence is coming along with artificial intelligence platforms, these biomarkers can be integrated and used towards the development of a precision model for treatment stratification and counselling in women diagnosed with BOTs. [ABSTRACT FROM AUTHOR]

Simple Summary: Non-small cell lung cancer (NSCLC) patients harboring KRAS mutations are targeted using monoclonal antibody (mAb) or tyrosine kinase inhibitors (TKI) therapies. To impede the KRAS activity, it was proposed to inhibit SRC kinase, an intermediary signal transductor between DDR2 and KRAS, using Dasatinib (Bcr-Abl TKI). However, clinical trials using Dasatinib failed to fully inhibit SRC or sensitize therapies towards SRC inhibitors, and its mechanistic failure remains partially understood. This report identifies AXL as a bypass resistant gene and investigates its role with SRC and KRAS activity. We found that AXL overexpression drives downstream KRAS and Akt pathways, and co-inhibiting AXL and SRC using SGI-7079 and Dasatinib effectively impeded both those pathways. Synergistic inhibition of AXL and SRC led to significant tumor growth suppression in A549 mice xenografts. The results from this study will be beneficial in understanding drug-resistance mechanisms and to strategize effective drug therapies in patients harboring KRAS mutations. Background/Objectives: KRAS-mutated NSCLC has been targeted using monoclonal antibody (mAb) or tyrosine kinase inhibitor (TKI) therapies. However, in time, these mutations appear to develop resistance against the targeted antibodies and TKI treatments. One possible explanation is the activation of pro apoptotic pathways through the AXL–SRC–Akt axis. In this study, we identify AXL as the bypass resistant gene and investigate its role with KRAS and SRC activity. Methods: In this study, we use Dasatinib and SGI-7079 to co-inhibit SRC and AXL respectively. In vitro studies were conducted using four cell lines, and AXL suppression was achieved using siRNA and in CRISPR-Cas9 mediated knockout models. Subsequently, we studied gene-protein expression analysis using Western blot, apoptotic markers using a cytochrome release assay and cytotoxicity using an MTT assay. A549 xenografts were studied for in vivo validation of our proposed hypothesis. Results: The results suggest that dual inhibition of AXL and SRC significantly reversed this resistance, both in in vivo and in vitro studies. Conclusions: Co-inhibition of AXL and SRC synergistically reduced KRAS activity and induced apoptosis in NSCLC. [ABSTRACT FROM AUTHOR]

Simple Summary: KRAS mutations are key drivers of colorectal cancer progression and resistance to treatment, significantly limiting therapeutic options for affected patients. Found in 30–40% of cases, these mutations promote persistent activation of oncogenic pathways such as MAPK/ERK and PI3K/AKT, contributing to tumor growth, poor prognosis, and reduced responsiveness to anti-EGFR therapies. This study aims to elucidate the biological role, clinical significance, and therapeutic potential of targeting KRAS mutations. Recent breakthroughs include the development of targeted inhibitors, such as sotorasib and adagrasib for G12C mutations, and experimental therapies for G12D. However, therapeutic responses in colorectal cancer remain suboptimal compared to other malignancies, largely due to resistance mechanisms and tumor heterogeneity. Promising strategies, including combination therapies, vaccines, and nucleic acid-based treatments, offer hope for improved outcomes. These findings underscore the importance of advancing personalized approaches to enhance care for patients with KRAS-mutant colorectal cancer. Background/Objectives: Colorectal cancer (CRC) remains a leading cause of cancer mortality globally, with KRAS mutations occurring in 30–40% of cases, contributing to poor prognosis and resistance to anti-EGFR therapy. This review explores the biological significance, clinical implications, and therapeutic targeting of KRAS mutations in CRC. Methods: A comprehensive analysis of the existing literature and clinical trials was performed, highlighting the role of KRAS mutations in CRC pathogenesis, their impact on prognosis, and recent advancements in targeted therapies. Specific attention was given to emerging therapeutic strategies and resistance mechanisms. Results: KRAS mutations drive tumor progression through persistent activation of MAPK/ERK and PI3K/AKT signaling pathways. These mutations influence the tumor microenvironment, cancer stem cell formation, macropinocytosis, and cell competition. KRAS-mutant CRC exhibits poor responsiveness to anti-EGFR monoclonal antibodies and demonstrates primary and acquired resistance to KRAS inhibitors. Recent breakthroughs include the development of KRAS G12C inhibitors (sotorasib and adagrasib) and promising agents targeting G12D mutations. However, response rates in CRC remain suboptimal compared to other cancers, necessitating combination therapies and novel approaches, such as vaccines, nucleic acid-based therapeutics, and macropinocytosis inhibitors. Conclusions: KRAS mutations are central to CRC pathogenesis and present a significant therapeutic challenge. Advances in KRAS-targeted therapies offer hope for improved outcomes, but resistance mechanisms and organ-specific differences limit efficacy. Continued efforts in personalized treatment strategies and translational research are critical for overcoming these challenges and improving patient survival. [ABSTRACT FROM AUTHOR]

Cholesterol metabolism is abnormally active in tumour cells. Metabolic enzymes related to cholesterol metabolism are upregulated in tumours, but their nonmetabolic functions remain unclear. We found that MVK (mevalonate kinase) is upregulated in lung adenocarcinoma tissues vs. normal tissues and that its expression can be induced by constitutively activated Kras. By investigating the molecular mechanisms involved, we discovered that MVK interacts with TBK1, inhibiting TBK1 phosphorylation and thereby suppressing cGAS-Sting signalling. In addition, we found a negative correlation between MVK expression and CD8+ T-cell infiltration via a public database analysis. In summary, our study demonstrates the importance of the nonmetabolic function of MVK in modifying the immunological milieu and provides new targets for lung adenocarcinoma therapy. [ABSTRACT FROM AUTHOR]

Pancreatic intraepithelial neoplasia (PanIN) is the most common precursor lesion of pancreatic ductal adenocarcinoma (PDAC), which has poor prognosis with a short median overall survival of 6-12 months and a low 5-year survival rate of approximately 3%. It is crucial to remove PanIN lesions to prevent the development of invasive PDAC, as PDAC spreads rapidly outside the pancreas. This review aims to provide the latest knowledge on PanIN risk, pathology, cellular origin, genetic susceptibility, and diagnosis, while identifying research gaps that require further investigation in this understudied area of precancerous lesions. PanINs are classified into PanIN 1, PanIN 2, and PanIN 3, with PanIN 3 having the highest likelihood of developing into invasive PDAC. Differentiating between PanIN 2 and PanIN 3 is clinically significant. Genetic alterations found in PDAC are also present in PanIN and increase with the grade of PanIN. Imaging methods alone are insufficient for distinguishing PanIN, necessitating the use of genetic and molecular tests for identification. In addition, metabolomics technologies and miRNAs are playing an increasingly important role in the field of cancer diagnosis, offering more possibilities for efficient identification of PanIN. Although detecting and stratifying the risk of PanIN poses challenges, the combined utilization of imaging, genetics, and metabolomics holds promise for improving patient survival in this field. [ABSTRACT FROM AUTHOR]

Background/Objective: Studies have shown an association between colorectal cancer (CRC) sidedness and gene mutations that may affect CRC clinical behavior. This study examined the association between specific KRAS, NRAS, and BRAF hot-spot mutations and primary CRC sidedness. Methods: We performed a retrospective cohort analysis of 722 patients diagnosed with primary CRC and tested for KRAS, NRAS, and BRAF hot-spot mutations at the University of Texas Medical Branch (UTMB) from January 2016 through July 2023. Multivariable logistic regressions analyses were conducted. Results:KRAS, NRAS, and BRAF hot-spot mutations rates were 37.8%, 4.6%, and 6.1%, respectively. Right-sided primary CRC had the highest prevalence of mutated tumors (64%). KRAS and BRAF hot-spot mutations were significantly different according to tumor sidedness. KRAS p.Gly12Asp, p.Gly12Val, and p.Gly13Asp showed a significantly increased likelihood of right-sided primary CRC compared to KRAS wildtype, 128%, 134%, and 221% higher, respectively. Conversely, KRAS p.Gly12Val and p.Gly13Asp mutations were associated with decreased likelihood of rectal cancer (53% lower) and left-sided tumors (56% lower), respectively. BRAF p.Val600Glu mutation, as opposed to BRAF wildtype, was associated with a 278% higher likelihood of right-sided CRC. No significant associations were observed between NRAS mutations and primary CRC sidedness. Conclusions: In primary CRC, specific mutations in KRAS (p.Gly12Asp, p.Gly12Val, and p.Gly13Asp) and BRAF p.Val600Glu were associated with increased likelihood of right-sided tumors. KRAS p.Gly12Val and p.Gly13Asp mutations were associated with decreased likelihood of rectal cancer and left-sided tumors, respectively. These findings suggest that tumorigenesis and mutational processes differ based on tumor sidedness. Further studies are needed to substantiate these findings. [ABSTRACT FROM AUTHOR]

Simple Summary: This review presents an overview of recent findings on various epigenetic and post-transcriptional modifications of the epidermal growth factor receptor (EGFR), which activates the oncogenes RAS and MYC. Alterations in EGFR, RAS, and MYC play a crucial role in dysregulated processes that contribute to oncogenesis. Focused, targeted research aimed at understanding the distinct effects of these oncogenes and their carcinogenic modifications has facilitated the successful treatment of various malignancies using inhibitors of EGFR tyrosine kinase activity and other therapeutic agents. The epidermal growth factor receptor (EGFR) regulates gene expression through two primary mechanisms: as a growth factor in the nucleus, where it translocates upon binding its ligand, or via its intrinsic tyrosine kinase activity in the cytosol, where it modulates key signaling pathways such as RAS/MYC, PI3K, PLCγ, and STAT3. During tumorigenesis, these pathways become deregulated, leading to uncontrolled proliferation, enhanced migratory and metastatic capabilities, evasion of programmed cell death, and resistance to chemotherapy or radiotherapy. The RAS and MYC oncogenes are pivotal in tumorigenesis, driving processes such as resistance to apoptosis, replicative immortality, cellular invasion and metastasis, and metabolic reprogramming. These oncogenes are subject to regulation by a range of epigenetic and post-transcriptional modifications. This review focuses on the deregulation of EGFR, RAS, and MYC expression caused by (epi)genetic alterations and post-translational modifications. It also explores the therapeutic potential of targeting these regulatory proteins, emphasizing the importance of phenotyping neoplastic tissues to inform the treatment of cancer. [ABSTRACT FROM AUTHOR]

KRAS is one of the most mutated genes, driving alternations in metabolic pathways that include enhanced nutrient uptaking, increased glycolysis, elevated glutaminolysis, and heightened synthesis of fatty acids and nucleotides. However, the beyond mechanisms of KRAS-modulated cancer metabolisms remain incompletely understood. In this review, we aim to summarize current knowledge on KRAS-related metabolic alterations in cancer cells and explore the prevalence and significance of KRAS mutation in shaping the tumor microenvironment and influencing epigenetic modification via various molecular activities. Given that cancer cells rely on these metabolic changes to sustain cell growth and survival, targeting these processes may represent a promising therapeutic strategy for KRAS-driven cancers. [ABSTRACT FROM AUTHOR]

Background: Lung cancer is a globally pervasive and deadly disease, claiming more than 1 million lives annually. Therefore, the identification of mutations in crucial cancer-related genes is paramount for guiding optimal chemotherapy strategies. The distribution of EGFR, KRAS, ALK, and ROS1 mutations varies across diverse ethnic populations. Nonetheless, there is limited data available on the prevalence of these mutations and their correlation with PD-L1 expression among Iranian lung cancer patients. Aim: This study involved an analysis of EGFR, KRAS, ALK, and ROS1 gene mutations in lung cancer patients, followed by an assessment of the correlation between PD-L1 expression and clinicopathological variables. Methods: Mutational profiling was conducted by examining EGFR (exons 18–21) and KRAS (exon 2) through pyrosequencing. Detection of ALK and ROS1 rearrangements, alongside PD-L1 expression, was carried out using immunohistochemistry techniques. Results: EGFR mutations were identified in 23.4% of cases, exhibiting a notably higher occurrence in females (p = 0.001). KRAS mutations were present in 7.1% of cases, with no significant association found between KRAS mutations and sex (p = 0.229). ALK rearrangements were found in 4.9% of cases, while ROS1 rearrangements were present in 0.6% of patients. The overall prevalence of PD-L1 protein expression was 36.85%. Notably, PD-L1 expression was detected in 24.8% of cases with EGFR mutations, 20% of cases with KRAS mutations, 64.7% of cases with ALK rearrangements, and in 100% of cases with ROS1 rearrangements. Conclusion: Although no correlation was found between PD-L1 expression and EGFR, KRAS mutations, and ROS1 rearrangements, a noteworthy association was identified between ALK rearrangements and elevated PD-L1 expression. [ABSTRACT FROM AUTHOR]

Background: Given the poor prognosis of metastatic pancreatic adenocarcinoma (mPDAC), closer disease monitoring through liquid biopsy, most frequently based on serial measurements of cell-free mutated KRAS (KRASmut cfDNA), has become a highly active research focus, aimed at improving patients' long-term outcomes. However, most of the available data show only a limited predictive and prognostic value of single-parameter-based methods. We hypothesized that a combined longitudinal analysis of KRASmut cfDNA and novel protein biomarkers could improve risk stratification and molecular monitoring of patients with mPDAC. Methods: We prospectively collected 160 plasma samples from 47 patients with mPDAC at our institution. Highly sensitive single-target ddPCR assays were employed to detect and quantify KRASmut cfDNA. Additionally, analysis of ten protein biomarkers was performed through Enzyme-linked Immunosorbent Assay (ELISA), and Carbohydrate-Antigen 19-9 (CA 19-9) dynamics were registered. Results: KRASmut cfDNA was detectable in 37/47 (78.7%) patients throughout the course of study, and CA 19-9 levels were elevated in 40 out of 47 (85.1%) patients. KRASmut cfDNA increase at the time of the first follow-up could predict inferior progression-free survival (PFS) (Hazard ratio (HR) = 3.40, p = 0.0003) and overall survival (OS) (HR = 4.91, p < 0.0001). In contrast to CA 19-9 kinetics, which were not predictive of outcome, integrated analysis of KRASmut cfDNA combined with six evaluated circulating protein biomarkers allowed basal risk stratification at the time of the first follow-up (HR = 10.2, p = 0.0014). Conclusions: A combined longitudinal analysis of KRASmut cfDNA with selected protein biomarkers offers significantly improved prognostic value for patients with mPDAC compared to single-parameter methods. This innovative approach is a step forward in the molecular monitoring of mPDAC and should be validated in further prospective studies. [ABSTRACT FROM AUTHOR]

Background The prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) is poor. Secondary brain metastasis (Br-M) occurs in less than 1% of patients. Clinical characteristics and molecular alterations have not been characterized in this rare patients' subset. Materials and methods The Foundry software platform was used to retrospectively query electronic health records for patients with Br-M secondary to PDAC from 2005 to 2023; clinical, molecular, and overall survival (OS) data were analyzed. Results Br-M was diagnosed in 44 patients with PDAC. Median follow-up was 78 months; median OS from initial PDAC diagnosis was 47 months. Median duration from PDAC diagnosis to Br-M detection was 24 months; median OS from Br-M diagnosis was 3 months. At Br-M diagnosis, 82% (n  = 36) of patients had elevated CA19-9. Lung was the most common preexisting metastatic location (71%) with Br-M, followed by liver (66%). Br-M were most frequently observed in the frontal lobe (34%, n  = 15), cerebellar region (23%, n  = 10), and leptomeninges (18%, n  = 8). KRAS mutations were detected in 94.1% (n  = 16) of patients who had molecular data available (n  = 17) with KRASG12V being the most frequent subtype 47% (n  = 8); KRASG12D in 29% (n  = 5); KRASG12R in 18% (n  = 3). Patients who underwent Br-M surgical resection (n  = 5) had median OS of 8.6 months, while median OS following stereotactic radiosurgery only (n  = 11) or whole-brain radiation only (n  = 20) was 3.3 and 2.8 months, respectively. Conclusion Br-M is a late PDAC complication, resulting in an extremely poor prognosis especially in leptomeningeal disease. KRAS was mutated in 94.1% of the patients and the KRASG12V subtype was prevalent. [ABSTRACT FROM AUTHOR]

Background/Aim: Cholangiocarcinoma (CCA) is an aggressive hepatobiliary malignancy characterized by genomic heterogeneity. KRAS mutations play a significant role in influencing patient prognosis and guiding therapeutic decision-making. This study aimed to determine the prevalence and prognostic significance of KRAS mutations in CCA, asses the detection of KRAS G12/G13 mutations in plasma cell-free DNA (cfDNA), and evaluate the prognostic value of KRAS G12/G13 mutant allele frequency (MAF) in cfDNA in relation to clinicopathological data and patient survival. Materials and Methods: A retrospective analysis of 937 CCA patients was performed using data from cBioPortal to examine KRAS mutation profiles and their association with survival. Plasma from 101 CCA patients was analyzed for KRAS G12/G13 mutations in the cfDNA using droplet digital PCR, and the results were compared with tissuebased sequencing from 78 matched samples. Results: KRAS driver mutations were found in 15.6% of patients, with common variants being G12D (37.0%), G12V (24.0%) and Q61H (8.2%). Patients harboring KRAS mutations exhibited decreased overall and recurrence-free survival. KRAS G12/G13 mutations were detected in 14.9% of cfDNA samples, showing moderate concordance with tissue sequencing, and achieving 80% sensitivity and 93% specificity. Elevated KRAS G12/G13 MAF in cfDNA, combined with high CA19-9 levels, correlated with poorer survival outcomes. Conclusion: The presence of KRAS mutations was associated with poor survival in CCA, underscoring the importance of KRAS mutations as prognostic markers. The detection of KRAS mutations in cfDNA demonstrated potential as a promising non-invasive alternative for mutation detection and, when combined with CA19-9 levels, may improve prognostic efficacy in CCA. [ABSTRACT FROM AUTHOR]

“Viral mimicry” refers to the induction of an innate immune response and interferon signaling by endogenous stimuli such as double‐stranded RNA (dsRNA). This response has been shown to have strong cancer therapeutic potential, including by enhancing the effectiveness of immune checkpoint inhibition (ICI) therapies, and may represent a tumor suppression mechanism that needs to be overcome for malignant transformation to proceed. In a recent study, Zhou and colleagues identify KRAS, a frequently mutated oncogene, as a negative regulator of dsRNA and viral mimicry in an ICI‐resistant colorectal cancer model. Oncogenic KRASG12D mutations downregulate the RNA‐binding protein DDX60 by activating the AKT signaling pathway, which inhibits STAT3, a critical transcription factor regulating DDX60 and other interferon‐stimulated genes. Overexpression of DDX60, which competitively binds to dsRNA to prevent RISC‐mediated degradation, or targeting of KRASG12D elevated dsRNA levels, resulting in viral mimicry activation and potentiation of ICI treatment. These results establish KRAS as a promising target to sensitize immune “cold” tumors to ICI therapy and demonstrate the potential role of oncogenic mutations in viral mimicry evasion during tumorigenesis.

The Kirsten rat sarcoma (KRAS) oncogene was considered “undruggable” until the development of sotorasib, a KRASG12C selective inhibitor that shows favorable effects against lung cancers. MRTX1133, a novel KRASG12D inhibitor, has shown promising results in basic research, although its effects against pancreatic cancer are limited when used alone. Therefore, there is an urgent need to identify effective drugs that can be used in combination with KRAS inhibitors. In this study, we found that administration of the KRAS inhibitors sotorasib or MRTX1133 upregulated STAT3 phosphorylation and reactivated ERK through a feedback reaction. The addition of the MEK inhibitor trametinib and the JAK2 inhibitor fedratinib successfully reversed this effect and resulted in significant growth inhibition in vitro and in vivo. Analyses of sotorasib‐ and MRTX1133‐resistant cells showed that trametinib plus fedratinib reversed the resistance to sotorasib or MRTX1133. These findings suggest that the JAK2‐mediated pathway and reactivation of the MAPK pathway may play key roles in resistance to KRAS inhibitors in pancreatic cancers. Accordingly, simultaneous inhibition of KRAS, MEK, and JAK2 could be an innovative therapeutic strategy against KRAS‐mutant pancreatic cancer.

Abstract Cholesterol metabolism is abnormally active in tumour cells. Metabolic enzymes related to cholesterol metabolism are upregulated in tumours, but their nonmetabolic functions remain unclear. We found that MVK (mevalonate kinase) is upregulated in lung adenocarcinoma tissues vs. normal tissues and that its expression can be induced by constitutively activated Kras. By investigating the molecular mechanisms involved, we discovered that MVK interacts with TBK1, inhibiting TBK1 phosphorylation and thereby suppressing cGAS-Sting signalling. In addition, we found a negative correlation between MVK expression and CD8+ T-cell infiltration via a public database analysis. In summary, our study demonstrates the importance of the nonmetabolic function of MVK in modifying the immunological milieu and provides new targets for lung adenocarcinoma therapy.

The present review demonstrates the major tumor suppressor genes, including TP53, CDKN2A and SMAD4, associated with pancreatic cancer. Each genes role, prevalence and impact on tumor development and progression are analyzed, focusing on the intricate molecular landscape of pancreatic cancer. In addition, this review underscores the prognostic significance of specific mutations, such as loss of TP53, and explores some potential targeted therapies tailored to these molecular signatures. The findings highlight the importance of genomic analyses for risk assessment, early detection and the design of personalized treatment approaches in pancreatic cancer. Overall, this review provides a comprehensive analysis of the molecular intricacies of pancreatic tumors, paving the way for more effective and tailored therapeutic interventions.

Cetuximab has been indicated as the mainstay of metastatic colorectal cancer (CRC) therapy, of which application was impeded by chemoresistance that was casually attributed to KRAS mutation. This study sought to determine whether YY1 mediated the resistance of CRC cells harbouring KRAS mutation (KRASmut) to cetuximab. The expression of YY1 between cetuximab response and resistance was investigated in cancerous tissues from CRC patients received cetuximab therapy comprising eight KRAS wild-type (KRASwt) and 12 KRASmut. The relationship between YY1 expression and cetuximab resistance was explored based on KRASmut and KRASwt CRC cell lines. To explore the role of YY1 in the cetuximab resistance of KRASmut CRC cells, the response to cetuximab was investigated in cetuximab-resistant cells (SW620-R) with YY1 silence and cetuximab sensitive cells (HCT116) with YY1 overexpression. EGFR/Akt/ERK signalling activation, as well as mRNA and active GTP-bound KRAS level were assessed after the treatment. In KRASmut CRC tissues, YY1 expression was correlated with the histological grade and the cetuximab resistance. Significantly markable differences in YY1 expression between cetuximab-resistant and the parental cell lines were found in KRASmut cells. Silencing YY1 re-sensitized SW620-R cells to cetuximab and led to an elevation of the active GTP-binding KRAS. Conversely, the capability against cetuximab and GTP-binding KRAS activation of HCT116 cells was enhanced by overexpressing YY1. The blockage of EGFR/Akt/ERK signalling by cetuximab was re-observed in SW620-R cells after silencing YY1 but impaired in HCT116 by overexpressing YY1. The YY1 mediates the resistance of KRASmut CRC cells to cetuximab. [ABSTRACT FROM AUTHOR]

Mutations in the KRAS gene in non-small cell lung cancer (NSCLC) are common drivers. Gene expression and mutation data of NSCLC were collected from the TCGA dataset. DEGs between KRAS mutations and wild type were identified, and enrichment analysis was performed. The differences in immune cell infiltration between the 2 groups were evaluated using ssGSEA, and TIDE scoring, immune checkpoint therapy sensitivity, and drug treatment sensitivity analysis were performed. The expression of PD-L1 and CTLA-4 in tumour tissues was detected by western blot. CD8+PD-1 and CD8+CTLA-4 cells were detected by flow cytometry. The frequencies of KRAS-G12C, KRAS-G12V, and KRAS-G12D mutations were the highest. A total of 1323 DEGs were predominantly enriched in the PI3K-Akt signalling pathway, cell adhesion molecules, and metabolism of xenobiotics by cytochrome P450. Additionally, most immune cell infiltration levels in KRAS mutations were lower than in KRAS wild type. Sensitivity to immune checkpoint inhibitors and drug treatments increased in KRAS mutations. Western blot revealed significantly higher expressions of PD-L1 and CTLA-4 in KRAS mutations compared to KRAS wild type. The infiltration of CD8+PD-1+ T cells and CD8+CTLA-4+ T cells was higher in KRAS mutations than in KRAS wild type. KRAS-G12C, KRAS-G12V, and KRAS-G12D mutations may enhance NSCLC drug resistance through immunosuppression.

The rise in the incidence of cancer globally has led to a heightened interest in targeted therapies as a form of anticancer treatment. Key oncogenic targets, including epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and kirsten rat sarcoma viral oncogene homologue (KRAS), have emerged as focal points in the development of targeted agents. Research has investigated the impact of gut microbiota on the efficacy of various anticancer therapies, such as immunotherapy, chemotherapy, and radiotherapy. However, a notable gap exists in the literature regarding the relationship between gut microbiota and targeted agents. This review emphasizes how specific gut microbiota and gut microbiota metabolites, including butyrate, propionate, and ursodeoxycholic acid, interact with oncogenic pathways to modulate anti-tumor effects. Conversely, deoxycholic acid, lipopolysaccharide, and trimethylamine n-oxide may exert pro-tumor effects. Furthermore, modulation of the gut microbiota influences glucose and lipid metabolism, thereby enhancing the response to anti-KRAS agents and addressing diarrhea induced by tyrosine kinase inhibitors. By elucidating the connection between gut microbiota and the EGFR/VEGF/KRAS pathways, this review provides valuable insights for advancing targeted cancer therapy and optimizing treatment outcomes in clinical settings.

Epidermal growth factor receptor (EGFR) inhibition is crucial in treating RAS wild-type metastatic colorectal cancer, yet current testing methods may miss rare RAS variants affecting treatment efficacy. We analyzed 4122 colorectal cancer patients receiving anti-EGFR antibodies from the Center for Cancer Genomics and Advanced Therapeutics database, identifying 54 patients (1.3%) with rare RAS variants undetectable by standard testing. These patients showed significantly lower response rates to anti-EGFR therapy (28.3%) compared to RAS wild-type cases (44.6%, p = 0.003). Disease control rates were also lower in rare variant cases (60.9%) versus wild-type cases (80.0%). Most common rare variants included KRAS Q22K, A59E, and A11_G12insGA. Comprehensive genomic profiling revealed additional alterations in TP53 (90.7%), APC (87.0%), and non-V600E BRAF mutations (25.9%). Our findings suggest that rare RAS variants predict poor anti-EGFR therapy response, highlighting the potential benefit of comprehensive genomic profiling before treatment initiation. This study provides real-world evidence supporting the clinical relevance of rare RAS variants in treatment decision-making for colorectal cancer. Future studies should focus on developing cost-effective comprehensive testing strategies and evaluating alternative treatment approaches for patients with rare RAS variants.

Aim: Immune checkpoint inhibitors improved the survival of advanced non-small cell lung cancer. However, only 20% of patients respond to these treatments and the search for predictive biomarkers of response is still topical. The objective of this work is to analyze the anti-PD-1 monotherapy benefit based on genetic alterations diagnosed by next generation sequencing (NGS), in advanced non-small cell lung cancer. Methods: Patients with advanced non-small cell lung cancer treated with immunotherapy were retrospectively included in this monocentric study. Clinical data, immunohistochemical expression of PD-L1 and molecular data, with a 22-genes NGS panel, were collected. Results: 107 patients were included. The median age was 65 years [59; 73], 70 were men (65%), 96 had adenocarcinoma (90%), 33 were receiving a first line (31%). 54 patients had KRAS mutation (50%) and 56 had TP53 mutation (52%). The remaining mutations were present in fewer than 10 patients. There was no statistically significant differences in median of progression-free or overall survival based on KRAS-only, TP53-only or KRAS-TP53 mutations co-mutated compared to double wild-type patients (P = 0.46 and P = 0.72 respectively). Conclusions: The search for a predictive composite biomarker remains a major issue in the coming years.

Inducing the degradation of KRAS represents a novel strategy to combat cancers with KRAS mutation. In this study, we identify ubiquitin-specific protease 2 (USP2) as a novel deubiquitinating enzyme of KRAS in multiple myeloma (MM). Specifically, we demonstrate that gambogic acid (GA) forms a covalent bond with the cysteine 284 residue of USP2 through an allosteric pocket, inhibiting its deubiquitinating activity. Inactivation or knockdown of USP2 leads to the degradation of KRAS, resulting in the suppression of MM cell proliferation in vitro and in vivo. Conversely, overexpressing USP2 stabilizes KRAS and partially abrogates GA-induced apoptosis in MM cells. Furthermore, elevated USP2 levels may be associated with poorer prognoses in MM patients. These findings highlight the potential of the USP2/KRAS axis as a therapeutic target in MM, suggesting that strategically inducing KRAS degradation via USP2 inhibition could be a promising approach for treating cancers with KRAS mutations.

Background AML is a complex and heterogeneous disease. The KRAS gene is one of the important genes in the pathogenesis of acute myeloid leukemia (AML). Mutant RAS can promote oncogenesis via different mechanisms including oncogenic transcription, cell cycle progression, cellular survival, growth, metabolism, and cell migration. Therefore, it is important to identify the genomic landscape of AML. The aim of the study is to identify KRAS variants in AML and their association with clinic pathological criteria and possible effects on prognosis using NGS.Method Hotspot mutations in the KRAS gene were studied using Ion S5 next-generation sequencing system. Bone marrow samples of newly diagnosed AML patients were collected to identify hotspot mutations in the KRAS gene. DNA amplicons were subjected to sequencing and were analyzed using ion torrent software. Patients were classified according to the FAB classification system. Patients are also classified according to the cytogenetic groups and the ELN risk stratification system.Results KRAS mutations were detected in exon 2, 3, whereas no mutations in KRAS exon 1. Interestingly, Novel mutations were detected in KRAS in AML Egyptian patients. Also, there was no statistically significant association of RAS mutations with different clinical and prognostic parameters. However, KRAS mutant patients tended to have increased PB WBC counts, percentage of PB, and bone marrow blasts.Conclusion NGS is considered a useful tool to identify KRAS variants that could be useful for risk stratification and tailored therapy in AML patients

Aim: Since the importance of epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog gene (KRAS) mutation status in predicting treatment response in non-small cell lung cancer (NSCLC) patients is well known, we aimed to evaluate whether initial fluorine-18-fluorodeoxyglucose (18F-FDG) positron emission tomography-computed tomography (PET/CT) imaging could non-invasively predict EGFR or KRAS mutation states in this patient group. Methods: This retrospective observational study examined patients with NSCLC who underwent 18F-FDG PET/CT for staging from August 2021 to January 2024. Age, sex, smoking status, pathological data, EGFR and KRAS mutation status, and metabolic and volumetric PET parameters were recorded. Groups were based on gene mutation status as follows: EGFR-mutations (mt) vs. EGFR wild-type (EGFR-wt) and KRAS-mt vs. KRAS-wt. Results: Ninety-nine patients with a mean age of 62.96±9.66 (range: 37-87) were included. The EGFR-mt group had lower metabolic tumor volume (MTV) (p=0.015) and total lesion glycolysis (TLG) (p=0.017) values. MTV had an area under the receiver operating characteristic curve (AUC) of 0.667 [95% confidence interval (CI): 0.547-0.788, p=0.015], and with a ≤24.9 cut-off, yielded 60.87% sensitivity, 68.42% specificity, and 66.67% accuracy to detect EGFR-mt. For TLG, the AUC was 0.664 (95% CI: 0.540-0.788, p=0.017) and a ≤408.1 cut-off yielded 86.96% sensitivity, 43.42% specificity, 53.54% accuracy, and 91.67% NPV. KRAS-mt was detected in 34 (34.34%) patients, and there were no significant differences between the KRAS-mt and KRAS-wt groups in terms of PET parameters. Conclusion: Primary tumor parameters derived from initial 18F-FDG PET/CT can predict EGFR mutation status but not KRAS mutation status. The high negative predictive value of TLG can be used to rule out EGFR-mt status, possibly preventing unnecessary treatments in patients without favorable genetic properties, especially when genetic analyses are not possible.

Abstract Background For decades, KRAS has always been a huge challenge to the field of drug discovery for its significance in cancer progression as well as its difficulties in being targeted as an “undruggable” protein. KRAS regulates downstream signaling pathways through protein–protein interactions, whereas many interaction partners of KRAS remain unknown. Results We developed a workflow to computationally predict and experimentally validate the potential KRAS-interacting proteins based on the interaction mode of KRAS and its known binding partners. We extracted 17 KRAS-interacting motifs from all experimentally determined KRAS-containing protein complexes as queries to identify proteins containing fragments structurally similar to the queries in the human protein structure database using our in-house protein–protein interaction prediction method, PPI-Miner. Finally, out of the 78 predicted potential interacting proteins of KRAS, 10 were selected for experimental validation, including BRAF, a previously reported interacting protein, which served as the positive control in our validation experiments. Additionally, a known peptide that binds to KRAS, KRpep-2d, was also used as a positive control. The predicted interacting motifs of these 10 proteins were synthesized to perform biolayer interferometry assays, with 4 out of 10 exhibiting binding affinities to KRAS, and the strongest, GRB10, was selected for further validation. Additionally, the interaction between GRB10 (RA-PH domain) and KRAS was confirmed via immunofluorescence and co-immunoprecipitation. Conclusions These results demonstrate the effectiveness of our workflow in predicting potential interacting proteins for KRAS and deepen the understanding of KRAS-driven tumor mechanisms and the development of therapeutic strategies.

Abstract Introduction KRASG12C and KRASG12D inhibitors represent a major translational breakthrough for non-small cell lung cancer (NSCLC) and cancer in general by directly targeting its most mutated oncoprotein. However, resistance to these small molecules has highlighted the need for rational combination partners necessitating a critical understanding of signaling downstream of KRAS mutant isoforms. Methods We contrasted tumor development between Kras G12C and Kras G12D genetically engineered mouse models (GEMMs). To corroborate findings and determine mutant subtype-specific dependencies, isogenic models of Kras G12C and Kras G12D initiation and adaptation were profiled by RNA sequencing. We also employed cell line models of established KRAS mutant NSCLC and determined therapeutic vulnerabilities through pharmacological inhibition. We analysed differences in survival outcomes for patients affected by advanced KRAS G12C or KRAS G12D -mutant NSCLC. Results KRASG12D exhibited higher potency in vivo, manifesting as more rapid lung tumor formation and reduced survival of KRASG12D GEMMs compared to KRASG12C. This increased potency, recapitulated in an isogenic initiation model, was associated with enhanced PI3K-AKT-mTOR signaling. However, KRASG12C oncogenicity and downstream pathway activation were comparable with KRASG12D at later stages of tumorigenesis in vitro and in vivo, consistent with similar clinical outcomes in patients. Despite this, established KRASG12D NSCLC models depended more on the PI3K-AKT-mTOR pathway, while KRASG12C models on the MAPK pathway. Specifically, KRASG12D inhibition was enhanced by AKT inhibition in vitro and in vivo. Conclusions Our data highlight a unique combination treatment vulnerability and suggest that patient selection strategies for combination approaches using direct KRAS inhibitors should be i) contextualised to individual RAS mutants, and ii) tailored to their downstream signaling.

Abstract The scaffold protein PEAK1 acts downstream of integrin adhesion complexes and the epidermal growth factor receptor, orchestrating signaling events that control cell proliferation and cytoskeletal remodeling. In this study we investigated the role of PEAK1 in colorectal carcinoma (CRC) progression using various in vitro and in vivo models to replicate the stepwise pathogenesis of CRC. While we observed a cell-type specific role for PEAK1 in the proliferation and in human CRC cell lines in vitro, our in vivo experiments using different CRC mouse models driven by loss of Apc, with or without oncogenic Kras or Pten loss suggest that PEAK1 does not significantly contribute to tumor formation in vivo. However, the survival time of Peak1 −/− mice in the Apc fl/+ model appeared to be slightly increased. Furthermore, PEAK1 promotes EGF-induced Caco-2 cell proliferation and regulates spheroid polarization and lumenization. Given that the Caco-2 cells harbor mutations in the tumor suppressors APC and β-CATENIN, but not in other tumor suppressors or in proto-oncogenes, we conclude that the PEAK1’s impact on colon carcinogenesis is limited, potentially playing a role in the initial stage of the adenoma to carcinoma progression.

Abstract Background The clustered regulatory interspaced short palindromic repeats (CRISPR)-Cas13a system has strong potential for highly sensitive detection of exogenous sequences. The detection of KRAS G12 point mutations with low allele frequencies may prove powerful for the formal diagnosis of pancreatic ductal adenocarcinoma (PDAC). Results We implemented preamplification of KRAS alleles (wild-type and mutant) to reveal the presence of mutant KRAS with CRISPR-Cas13a. The discrimination of KRAS G12D from KRAS WT was poor for the generic KRAS preamplification templates and depended on the crRNA design, the secondary structure of the target templates, and the nature of the mismatches between the guide and the templates. To improve the specificity, we used an allele-specific PCR preamplification method called CASPER (Cas13a Allele-Specific PCR Enzyme Recognition). CASPER enabled specific and sensitive detection of KRAS G12D with low DNA input. CASPER detected KRAS mutations in DNA extracted from patients’ pancreatic ultrasound-guided fine-needle aspiration fluid. Conclusion CASPER is easy to implement and is a versatile and reliable method that is virtually adaptable to any point mutation.

Abstract Background Lung cancer, a leading global cause of cancer-related mortality, necessitates enhanced prognostic markers for improved treatment outcomes. We have previously shown a tumor suppressive role of cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1), which is targeted for degradation upon phosphorylation at S14 (pCASTOR1) in multiple types of cancer. This study focuses on the predictive value of pCASTOR1 in lung adenocarcinoma (LUAD) patients with KRAS mutations. Results Employing a newly developed pCASTOR1 specific antibody, we found that tumor cells exhibited significantly elevated pCASTOR1 scores compared to non-tumor cells (P

Liu Liang,1 Xuemin Guo,1 Wei Ye,1 Yuxiang Liu2 1Department of Laboratory Medicine, Meizhou People’s Hospital, Meizhou, People’s Republic of China; 2Department of Medical Oncology, Meizhou People’s Hospital, Meizhou, People’s Republic of ChinaCorrespondence: Liu Liang, Department of Laboratory Medicine, Meizhou People’s Hospital, Meizhou, People’s Republic of China, Email 19926117315@163.comBackground: The efficacy of targeted therapy for colorectal cancer (CRC) is affected by hub genes of epidermal growth factor receptor (EGFR) signaling pathways, such as KRAS. Immune cell infiltration may lead to gene mutation, but the relationship between KRAS status and peripheral immune-inflammatory indices has not been clarified in CRC.Methods: Clinical records of CRC patients were collected. The relationship between KRAS status and clinicopathological characteristics, peripheral immune-inflammatory indices (pan-immune inflammation value (PIV) (monocyte×neutrophil×platelet/lymphocyte), systemic immune inflammation index (SII) (platelet×neutrophil/lymphocyte), and system inflammation response index (SIRI) (monocyte×neutrophil/lymphocyte)) were analyzed.Results: 1033 CRC patients were collected, there were 514 (49.8%) patients with KRAS wild-type and 519 (50.2%) with KRAS mutation. Patients with KRAS mutation had higher proportions of female, III-IV stage, and lymph node metastasis and lower proportion of low grade of tumor budding (the presence of single tumor cells or small clusters of up to 5 cells in mesenchyma at the front of tumor invasion) than those with KRAS wild-type. The PIV, SII, and SIRI levels in KRAS mutation patients were significantly higher than those in KRAS wild-type patients. The proportion of aged ≥ 65 years old, dMMR, distant metastasis, and KRAS mutation were high in patients with high PIV, SII, and SIRI levels. Logistic regression analysis showed that non-low grade of tumor budding (odds ratio (OR): 1.970, 95% confidence interval (CI): 1.287– 3.016, p=0.002), and high SII level (≥ 807.81 vs < 807.81, OR: 1.915, 95% CI: 1.120– 3.272, p=0.018) were independently associated with KRAS mutation.Conclusion: Non-low grade of tumor budding, and high SII level were independently associated with KRAS mutation in CRC. It provides additional references for diagnosis and treatment options for patients with CRC.Keywords: colorectal cancer, KRAS, systemic immune inflammation index, tumor budding

Abstract Mutant KRAS promotes the proliferation, metastasis, and aggressiveness of various cancers including pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), and colorectal adenocarcinoma (CRC) respectively. Mutant KRAS therapeutics are limited, while Sotorasib and Adagrasib were the only FDA-approved drugs for the treatment of KRASG12C mutated NSCLC. Chimeric antigen receptor (CAR) T-cell therapy has been emerged as an effective strategy against hematological malignancies and being extended towards solid cancers including PDAC. mesothelin (MSLN) and Carcinoembryonic Antigen (CEA) were reported to be highly overexpressed in KRAS-mutated PDAC. Meanwhile, in clinical trials, several CAR T-cell therapy studies are mainly focused towards these two cancer antigens in PDAC, however, the dual targeting of these two neoantigens is not reported. In the present study, we have designed and developed a novel dual-targeting CAR protein by employing various bioinformatics approaches such as functional analysis (antigenicity, allergenicity, antigen binding sites & signalling cascades), qualitative analysis (physicochemical, prediction, refinement & validation of 2D and 3D structures), molecular docking, and in silico cloning. Our results revealed that the designed CAR protein specifically binds with both MSLN & CEA with significant binding affinities, and was predicted to be stable & non-allergenic. Additionally, the protein–protein interaction network reveals the T-cell mediated antitumor responses of each domain in the designed CAR. Conclusively, we have designed and developed a dual targeting (MSLN & CEA) CAR protein towards KRAS-mutated PDAC using computational approaches. Alongside, we further recommend to engineer this designed CAR in T-cells and evaluating their therapeutic efficiency in in vitro and in vivo studies in the near future.

Background: KRAS is frequently mutated in the tumors of patients with metastatic colorectal cancer (mCRC) and thus represents a valid target for therapy. However, the strategies of targeting KRAS directly and targeting the downstream effector mitogen-activated protein kinase kinase (MEK) via monotherapies have shown limited efficacy. Thus, there is a strong need for novel, effective combination therapies to improve MEK-inhibitor efficacy in patients with KRAS -mutated mCRC. Objective: Our objective was to identify novel drug combinations that enhance MEK-inhibitor efficacy in patients with KRAS -mutated mCRC. Design: In this study, we performed unbiased high-throughput screening (HTS) to identify drugs that enhance the efficacy of MEK inhibitors in vitro, and we validated the drugs' efficacy in vivo. Methods: HTS was performed using three-dimensional CRC spheroids. Trametinib, the anchor drug, was probed with two "clinically ready" libraries of 252 drugs to identify effective drug combinations. The effects of the drug combinations on CRC cell proliferation and apoptosis were further validated using cell growth assays, flow cytometry, and biochemical assays. Proteomic and immunostaining studies were performed to determine the drugs' effects on molecular signaling and cell division. The effects of the drug combinations were examined in vivo using CRC patient-derived xenografts. Results: HTS identified paclitaxel as being synergistic with trametinib. In vitro validation showed that, compared with monotherapies, this drug combination demonstrated strong inhibition of cell growth, reduced colony formation, and enhanced apoptosis in multiple KRAS -mutated CRC cell lines. Mechanistically, combining trametinib with paclitaxel led to alterations in signaling mediators that block cell-cycle progression. Trametinib also enhanced paclitaxel-mediated microtubule stability resulting in significantly higher defects in mitosis. Finally, the combination of trametinib with paclitaxel exhibited significant inhibition of tumor growth in several KRAS -mutant patient-derived xenograft mouse models. Conclusion: Our data provide evidence supporting clinical trials of trametinib with paclitaxel as a novel therapeutic option for patients with KRAS -mutated, metastatic CRC. Plain language summary: Combining paclitaxel enhances the efficacy of trametinib in colorectal cancer cells with mutations in the oncogenic KRAS protein Metastatic colorectal cancer (mCRC) is the second leading cause of cancer death in the USA. About half of these patients have mutations in the oncogenic protein KRAS. In recent developments targeted therapies that block KRAS demonstrate modest benefit in a small percentage of patients with mCRC and when benefit is obtained, it is transient. Thus, there is an urgent need for developing novel effective therapeutic strategies that can significantly improve survival of most patients with mCRC. The MEK protein is activated by KRAS and is a key protein for cancer cell survival. As inhibitors for the MEK protein by themselves are not effective in improving outcomes in patients with mCRC, the research team performed a drug screen to identify drugs that can significantly enhance the efficacy of MEK-inhibitors in blocking the growth of cultured CRC cells and colorectal tumors grown in animal models. Through unbiased high throughput screening, this study identified the anti-cancer drug paclitaxel to strongly enhance the efficacy of the MEK-inhibitor trametinib. Compared to the drugs by themselves, when combined, these drugs led to significant increases in death of multiple CRC cell types that have mutations in the oncogenic KRAS gene. Also, the drug combination blocked colorectal tumors growth in mice significantly more than the drugs used as single agents. The study also made a novel observation that the MEK-inhibitor can enhance the cell killing ability of paclitaxel by likely increasing its bioavailability inside of CRC cells. Thus, blocking of oncogenic survival signaling by the MEK-inhibitor and increased cytotoxic effects of paclitaxel, work together in inducing higher cell death in CRC cells. These preclinical studies indicate that the combination of trametinib and paclitaxel may be a strong candidate regimen for further evaluation in clinical studies and has the potential to improve outcomes in patients with metastatic colorectal cancer with KRAS mutations. [ABSTRACT FROM AUTHOR]