KRAS Mutations Research Articles

Abstract PR012: KRAS mutation-specific effects on the tumor immune microenvironment drive tumor progression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with abysmal survival rates. The highly immunosuppressive microenvironment of PDAC poses a major barrier to effective therapy. KRAS mutations are near-ubiquitous in PDAC, and studies indicate clinical outcomes vary depending on the specific KRAS genotype. Given that tumor genotype can shape the immune cell composition of tumors, we examined the immune cell composition of pancreatic tumors with specific KRAS mutations, focusing on KRASG12D mutations (representing 40% of PDAC cases which have the worst prognosis) and KRASG12R mutations (which make up 20% of PDAC cases and have a better prognosis). Orthotopically implanted Kras G12R/+ ;Trp53 KO mouse pancreatic tumors have a distinct immune profile in comparison to Kras G12D/+ ;Trp53 KO tumors, characterized by an influx of intratumoral CD3+ T cells and mature dendritic cells. Selective depletion of CD4+ T cells induces a rapid progression in KRASG12R, but not KRASG12D mouse tumors. RNA sequencing of KRASG12R tumor cells revealed enrichment of Type I interferon (IFN) pathways accompanied by related chemokines, CXCL9 and CXCL10 in comparison to KRASG12D tumors. Analysis of publicly available and real-world human PDAC datasets and enrichment of dendritic cells and other immune related cell types in KRASG12R tumors. Collectively, these data link the KRASG12R mutation to a unique immunogenic role. Citation Format: Andrew Wenger, Tal Baron, Erika Hissong, Rohit Chandiwani, Lukas Dow, Despina Siolas, Katherine Bacchi, Huanhuan Sun, Erika Hissong, Adrian Vega, Whitney Sisso, and Kawther Abdilleh. KRAS mutation-specific effects on the tumor immune microenvironment drive tumor progression in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR012.

Abstract A022: Disrupting redox homeostasis initiates anti-tumor immune responses in pancreatic cancer

Abstract Although immune therapy has made impressive progress in cancer treatment, pancreatic ductal adenocarcinoma (PDAC) remains an exception, likely due to its immunosuppressive tumor microenvironment that prevents immune responses against the tumor. Our current work aims to reactivate the suppressive immune infiltrate using radiation therapy and oxidative stress to potentiate antitumor immune responses in PDAC. Our lab previously found that PDAC cells are dependent on the antioxidant peroxiredoxin-4 (PRDX4) expression. PRDX4 prevents excessive levels of reactive oxygen species in cells and while high PRDX4 expression is associated with a more aggressive disease, it is dispensable in normal tissue. As such, disrupting redox homeostasis by inhibiting PRDX4 represents a targetable vulnerability in cancer cells. To investigate the radio-sensitizing potential of PRDX4, we knocked-down (KD) PRDX4 in human pancreatic tumor cells as well as in mouse cancer cells obtained from the spontaneous KPC model (driven by the KRas and P53 mutations), in combination with radiation. Presence and quantification of DNA damage were performed by immunofluorescence, and impaired cell survival was analyzed by clonogenic assays. Changes in immune secretion were assessed by RT-qPCR and Elisa and in vitro macrophage phenotyping was performed upon exposure to tumor cell conditioned media. Our data so far show an accumulation of cytosolic DNA upon PRDX4 KD in PDAC cells, with the presence of micronuclei as well as less aggregated DNA. Interestingly, targeting PRDX4 before radiation amplifies the release of radiation-induced cytosolic DNA while significantly decreasing cell growth and survival, compared to radiation alone. Cytosolic DNA has been shown to trigger innate immune signaling in tumor cells. Consistently, PRDX4 loss induces the upregulation of many pro-inflammatory genes, including CCL5, CCL20, and CXCL10, in a STING-dependent manner. Combining PRDX4 KD with radiation leads to a higher gene upregulation of these immune stimulatory chemokines, as well as an increased protein secretion within tumor cell media. These findings support an amplified immunomodulatory effect of radiation when combined with PRDX4 loss in PDAC cells. To assess whether targeting PRDX4 can remodel the tumor microenvironment, we applied tumor conditioned media from cancer cells on isolated naive mouse CD45+/CD11c+/F4/80+ macrophages. Media from cells treated with PRDX4 KD + radiation strongly increased the migration ability of macrophages while modulating the expression of a subset of chemokine-encoding genes in immune cells. Using the spontaneous KPC model depleted for PRDX4, along with syngeneic orthotopic models, we are now aiming to confirm this radio-sensitizing and immune-modulatory potential of PRDX4 in vivo. The aggressiveness of PDAC requires research towards novel treatment strategies and our data strongly support evidence for targeting cancer metabolism, particularly antioxidant systems, in synergy with existing cancer therapies, like radiation and immune therapy. Citation Format: Lucie Malbeteau, Emily Poulton, Vishal Pandya, Marianne Koritzinsky. Disrupting redox homeostasis initiates anti-tumor immune responses in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A022.

Divergent clinical and immunologic outcomes based on STK11 co-mutation status in resectable KRAS-mutant lung cancers following neoadjuvant immune checkpoint blockade.

Co-mutations of the KRAS and STK11 genes in advanced non-small cell lung cancer (NSCLC) are associated with immune checkpoint blockade (ICB) resistance. While neoadjuvant chemoimmunotherapy is now a standard of care treatment for resectable NSCLC, the clinical and immunologic impact of KRAS andSTK11 co-mutations in this setting are unknown. We evaluated and compared recurrence-free survival of resectable KRAS-mutated NSCLC tumors, with or without co-occuring STK11 mutations, treated with neoadjuvant ICB. Single cell transcriptomics was performed on tumor-infiltrating T cells from 7 KRASmut/STK11wttumors and 6 KRASmut/STK11mut tumors. Relative to KRASmut/STK11wttumors, KRASmut/STK11mut exhibited significantly higher recurrence risk. Single-cell transcriptomics showed enhanced oxidative phosphorylation with evidence of decreased PGE-2 signaling and increased IL-2 signaling in CD8+ tumor-infiltrating lymphocytes (TIL) from KRASmut/STK11mut tumors, a finding that was mirrored in KRASwt tumors that relapsed. TIL from KRASmut/STK11mut tumors expressed high levels of molecules associated with tumor residence, including CD39 and ZNF683 (HOBIT). These divergent T cell transcriptional fates suggest T cell maintenance and residence may be detrimental to anti-tumor immunity in the context of neoadjuvant ICB for resectable NSCLC, regardless of KRAS mutation status. Our work provides a basis for future investigations into the mechanisms underpinning PGE-2 and IL-2 signaling as they relate to T cell immunity to cancer and to divergent clinical outcomes in KRASmut/STK11mut NSCLC treated with neoadjuvant ICB.

Multi-omics-driven discovery of invasive patterns and treatment strategies in CA19-9 positive intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor with a poor prognosis, predominantly CA19-9 positive. High CA19-9 levels correlate with increased aggressiveness and worse outcomes. This study employs multi-omics analysis to reveal molecular features and identify therapeutic targets of CA19-9 positive ICC, aiming to support individualized treatment. Data from seven clinical cohorts, two whole-exome sequencing cohorts, six RNA sequencing/microarray cohorts, one proteomic cohort, 20 single-cell RNA sequencing samples, and one spatial transcriptome sample were analyzed. Key findings were validated on tissue microarrays from 52 ICC samples. CA19-9 positive ICC exhibited poorer OS (median 24.1 v.s. 51.5months) and RFS (median 11.7 v.s. 28.2months) compared to negative group (all P < 0.05). Genomic analysis revealed a higher KRAS mutation frequency in the positive group and a greater prevalence of IDH1/2 mutations in the negative group (all P < 0.05). Transcriptomic analysis indicated upregulated glycolysis pathways in CA19-9 positive ICC. Single-cell analysis identified specific glycolysis-related cell subclusters associated with poor prognosis, including Epi_SLC2A1, CAF_VEGFA, and Mph_SPP1. Higher hypoxia in the CA19-9 positive group led to metabolic reprogramming and promoted these cells' formation. These cells formed interactive communities promoting epithelial-mesenchymal transition (EMT) and angiogenesis. Drug sensitivity analysis identified six potential therapeutic drugs. This study systematically elucidated the clinical, genomic, transcriptomic, and immune features of CA19-9 positive ICC. It reveals glycolysis-associated cellular communities and their cancer-promoting mechanisms, enhancing our understanding of ICC and laying the groundwork for individualized therapeutic strategies.

Abstract B011: RNA-based therapeutics to target the p53 pathway in cancer

Abstract Mutations in the TP53 gene and activation of the PI3K/AKT pathway are the two most frequent genetic alterations across multiple human tumors. We discovered a crucial mechanism of crosstalk between these two events consisting of two oncogenic lncRNAs, TROLL-2 and TROLL-3, whose expression is induced by mutant p53. We demonstrated that these lncRNAs drive tumor and metastasis formation in several orthotopic models, including breast cancer, lung adenocarcinoma, and melanoma. The investigation of these mouse models and a pan-cancer analysis of human cancers showed that these lncRNAs are markers of tumor progression and lead to the phosphorylation and subsequent activation of AKT by promoting the cytoplasmic localization of the scaffold protein WDR26. Notably, we have now proven that TROLL-2 and TROLL-3 are also involved in the response to cancer therapies. Indeed, the downregulation of these lncRNAs via siRNAs bypasses the AKT-driven resistance to chemotherapeutics (e.g. Adriamycin, docetaxel, and paclitaxel) in triple-negative breast cancer cells, to inhibitors of mutant KRAS (e.g. sotorasib) and EGFR (e.g. erlotinib) in lung adenocarcinoma cells, and to inhibitors of PARP (e.g. niraparib) in ovarian cancer cells. To target these lncRNAs in a clinically relevant manner, we designed LNA GapmeRs, also known as antisense oligonucleotides (ASOs), to downregulate these two lncRNAs directly. The efficacy of the ASOs was confirmed in ex vivo specimens of tumors and metastases of lung adenocarcinoma and ovarian cancer patients. These clinical specimens were also analyzed via single-cell RNA sequencing and phospho-proteomics to unveil the common vs. unique downstream effects of targeting each lncRNA. When administered together, the ASOs targeting TROLL-2 and TROLL-3 effectively decreased the levels of these lncRNAs, which in turn caused WDR26 sequestration in the nucleus, thus preventing the activation of the AKT pathway and the proliferation of these cancer specimens. Furthermore, we confirmed that the antitumor activities of these ASOs were also maintained in vivo, as proven by the decreased tumor proliferation observed in breast cancer xenograft models. Finally, to improve the delivery of these ASOs in vivo, the usage of non-viral systems, including lipid-based and metallic-based nanoparticles, is being tested in both ex vivo and in vivo preclinical models. Overall, our results unveiled TROLL-2 and TROLL-3 as suitable targets to counteract tumor growth and promote the efficacy of chemotherapeutics and targeted therapies in ex vivo and in vivo cancer models. Citation Format: Marco Napoli, Ashley K Lu, Avani A Deshpande, Jaden R Baldwin, Christina L Carr, Michael R Dunne, Omar K Farha, Michelle H Teplensky, Elsa R Flores. RNA-based therapeutics to target the p53 pathway in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B011.

Abstract A022: Novel technology affords real-time PCR the sensitivity required for minimal residual disease monitoring

Abstract Monitoring minimal residual disease (MRD) provides invaluable information for disease progression and therapeutic decision making. However, there are many challenges to reliably detect key mutations or biomarkers, such as the accessibility, turnaround time, and sensitivity of current methods. High sensitivity is crucial due to the limited DNA available in certain sample types such as liquid biopsy. Next generation sequencing (NGS) is capable of highly sensitive detection but is dependent on high sample input and read depth, which is not always feasible due to the variable nature of circulating tumor DNA (ctDNA) in blood or plasma. In addition, NGS is not an economical nor rapid option for recurrent longitudinal monitoring. Digital PCR (dPCR) offers the sensitivity required for MRD monitoring but has yet to be widely adopted for this application. While real-time and qPCR are commonly used in oncology research, RT-PCR does not have the sensitivity required for early detection of key MRD mutations. Here, we present novel patent-pending technology that enhances the sensitivity of RT-PCR, allowing this common platform to be used to reliably detect low abundant mutations. We performed comparisons between GT Molecular’s patent-pending technology paired with RT-PCR and commercially available research use only dPCR and NGS assays to assess the sensitivity this new technology provides for RT-PCR. Cell-free DNA (cfDNA) was extracted from real patient or contrived plasma samples using the QIAamp Circulating Nucleic Acid Kit and analyzed using RT-PCR and dPCR assays targeting KRAS or ESR1 mutations. Samples with KRAS or ESR1 mutations were tested at variant allele frequencies (VAFs) varying between 1%-0.01% to evaluate the ability of GT Molecular’s new technology to detect low abundant mutations using RT-PCR. KRAS and ESR1 mutations were reliably detected with RT-PCR at VAFs as low as 0.01%, demonstrating sensitivity comparable to dPCR. Samples demonstrating positivity for KRAS or ESR1 mutations via dPCR were also positive via RT-PCR. With this patent-pending technology, RT-PCR is now capable of achieving the sensitivity required for MRD monitoring, providing critical information from liquid biopsy samples at a fraction of the cost. Citation Format: Mary Stischer, Nick Allen, Stephanie Barbari, Sarah Kane. Novel technology affords real-time PCR the sensitivity required for minimal residual disease monitoring [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A022.

Clinicopathological relevance of SMAD4 and RUNX3 in patients with resected pancreatic cancer

Abstract Objectives Treating pancreatic ductal adenocarcinoma (PDAC) is a major clinical challenge, owing to its poor prognosis. Understanding the underlying molecular interactions may be crucial in determining PDAC intractability. We elucidated the combinatory role of SMAD family member 4 (SMAD4) and Runt-related transcription factor 3 (RUNX3) in PDAC. Methods Formalin-fixed and paraffin-embedded tissues were obtained from 101 patients who underwent surgical resection of PDAC. SMAD4 and RUNX3 expression in the tissues was evaluated using immunohistochemistry. SMAD4 and KRAS mutations were evaluated using Sanger sequencing. SMAD4 copy number variations were evaluated using quantitative real-time PCR. These molecular characteristics were compared with clinicopathological features of patients. Results Retained SMAD4 expression in PDAC tissues was associated with higher tumor, node, metastasis (TNM) stage, and metastatic recurrence. Defective RUNX3 expression was associated with higher TNM stages. Further analysis to determine significance of retained SMAD4 indicated that all these cases had defective RUNX3 expression; therefore, the combined expression status of retained SMAD4 and defective RUNX3 expression in tissues was determined to be associated with higher TNM stage and metastatic recurrence than defective SMAD4 expression with defective or retained RUNX3 expression. Survival analysis showed that patients with tumors with retained SMAD4 and defective RUNX3 expression had relatively worse overall survival (OS) rates. KRAS mutations were not associated with SMAD4/RUNX3 expression or OS. Conclusions Despite inherent limitations of this retrospective study and small sample size, our findings highlight the potential combined role of SMAD4 and RUNX3 in node metastasis and metastatic recurrence in patients with resected pancreatic cancer.

HPB SO33 - Transcriptomic characterisation of the diabetogenic pancreatic ductal adenocarcinoma phenotype

Abstract Background New-onset diabetes mellitus (NOD) can manifest as a consequence of pancreatic ductal adenocarcinoma (PDAC), although not all PDAC patients develop NOD. For many patients, hyperglycaemia begins 3 years prior to PDAC diagnosis, with 30% of PDAC patients experiencing NOD by the time their cancer is diagnosed. The mechanisms underlying the onset of diabetes mellitus (DM) in PDAC and its consequences for disease development, detection and treatment are not well understood. The aim of this study was to characterise the transcriptomic differences between PDAC tumours that present with NOD compared to those that do not. Method Eight tumour samples from patients undergoing surgery for PDAC (4 without DM (HbA1c &amp;lt;35), 4 with NOD (HbA1c &amp;gt;65, within 3 years of cancer diagnosis) underwent single cell-RNA sequencing (scRNAseq) after taking intra-operative biopsies using Chromium 10X Genomics technology. Unique cell clusters were identified using CellRanger and cell types annotated using scATOMIC and singleR datasets. Manual annotation was undertaken when results were not agreeable after consulting the literature. Differential expression between groups was performed using DESeq2. Significant genes were identified as having a log2 fold-change &amp;gt;1.5 and p&amp;lt;0.05. Gene set enrichment was performed using Reactome. Analysis was conducted in RStudio. Results Single cell suspensions were created with a mean cell count of 1.2 million cells and a mean viability of 90%. PDAC cells and the full complement of tumour microenvironmental cells were recovered. Significant differential expression was seen in the PDAC cells themselves, CD4+ cells, macrophages, mast cells and fibroblasts. Cancer associated fibroblasts (CAFs) seemed not to differ. PDAC cells with NOD seemed to be enriched in MYC oncogenes whereas those without DM seemed to be enriched in KRAS mutations. There was a mixture of “basal” and “classical” transcriptional subtypes between the two groups. Conclusion Differences between diabetogenic PDAC and non-diabetogenic PDAC have been observed at the transcriptional level. Diabetogenic tumours seem to be promoted by MYC oncogenes and be more inflammatory than non-diabetogenic tumours. These findings require validation which will be done on independent samples using spatial transcriptomics to allow endocrine-exocrine crosstalk to be determined. Understanding the molecular pathways in diabetogenic pancreatic tumours could enhance future strategies to detect PDAC earlier and to target diabetes-associated vulnerabilities for therapeutic purposes.

Molecular alterations influencing the prognostic outcome in small pancreatic cancer (≤ 2 cm)

Abstract Purpose Pancreatic cancer (PC) is the most lethal cancer. The prognosis of small PC (tumor smaller than 20 mm) is better than that of larger PC tumors, indicating the importance of detecting early-stage PC for patient outcome. The aim of this study was to elucidate the molecular features in small PC (≤ 20 mm). Methods This study included 79 PC tumors (≤ 20 mm in pathological examination) resected between 2004 and 2022. c-Myc, Caveolin-1, Smad4, and Thrombospondin-1 were examined by immunostaining. These molecular alterations were compared in PC patients with tumor size ≤10 mm (n = 11) (14%) and 10 mm &lt; tumor size ≤20 mm (n = 68) (86%). Mutation analyses of KRAS, PIK3CA, and BRAF were performed by pyrosequencing in 22 PCs. Results PC with 10 mm &lt; tumor size ≤20 mm showed significantly worse overall survival and disease-free survival than PC with tumor size &lt;10 mm (P = 0.024 and P = 0.028). Tumor c-Myc and stromal Caveolin-1 expressions were significantly increased in tumors larger than 10 mm (P = 0.02 and P = 0.04). c-Myc and Caveolin-1 expressions were associated with poor disease-free survival and overall survival. KRAS, PIK3CA, and BRAF mutation status did not differ between the two groups. Conclusions Tumor c-Myc and stromal Caveolin-1 overexpressions were detected in tumors larger than 10 mm. Their overexpressions were associated with worse prognosis even in small PC. These molecular alterations in small PC may be a clue for the detection of early-stage PC.

Evaluation of SNaPshot and Sanger sequencing for the detection of KRAS and NRAS mutations in a sample of Venezuelan patients with colorectal cancer

Evaluation of SNaPshot and Sanger sequencing for the detection of KRAS and NRAS mutations in a sample of Venezuelan patients with colorectal cancer

Elevated serum direct bilirubin is predictive of a poor prognosis for primary myelodysplastic syndrome.

The aim of this study was to assess the prognostic significance of serum direct bilirubin (DBIL) for patients newly diagnosed with myelodysplastic syndromes (MDS). The clinical, laboratory, and follow-up data of MDS patients were collected, and the associations of DBIL levels with overall survival (OS) and leukemia-free survival (LFS) were analyzed. In total, 262 MDS patients were assigned to the high DBIL level group or the normal DBIL level group in the retrospective study. High DBIL was associated with older age, reduced hemoglobin, higher levels of β2-microglobin, lactate dehydrogenase, and serum ferritin, along with the number of co-mutations (> 1) and a higher frequency of ASXL1, KIT, and KRAS mutations. Multivariate analyses found that high DBIL level was an independent adverse predictor for OS (p = 0.002, hazard ratio = 2.723, 95%CI = 1.442-5.143) but not for LFS (p = 0.057, hazard ratio = 1.678, 95%CI = 0.986-2.857). A novel nomogram based on DBIL, sex, age, β2-microglobulin, lactate dehydrogenase, the Revised International Prognostic Scoring System (IPSS-R) was constructed, which demonstrated superior accuracy compared with the IPSS-R (C-index, 0.790 vs. 0.731, respectively). An elevated DBIL level was identified as an independent adverse prognostic factor for MDS patients. An individualized prediction model was established and validated to improve prediction of OS and LFS.

The PI3K-AKT-mTOR axis persists as a therapeutic dependency in KRASG12D-driven non-small cell lung cancer.

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. We contrasted tumor development between KrasG12C and KrasG12D genetically engineered mouse models (GEMMs). To corroborate findings and determine mutant subtype-specific dependencies, isogenic models of KrasG12C and KrasG12D 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 KRASG12C or KRASG12D-mutant NSCLC. 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. 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.

PATH-61. GENOMIC SIGNATURE IN NON-SMALL CELL LUNG CANCER PATIENTS METASTATIC TO BRAIN-A SINGLE INSTITUTION EXPERIENCE

Abstract BACKGROUND Lung cancer is leading cause of cancer-related death in US, with 10-30 % of NSCLC patients developing brain metastasis, which challenges curative treatment and life expectancy. Somatic mutations in KRAS and EGFR genes have been commonly implicated with brain metastasis. Other studies have shown higher amplification frequencies in MYC, YAP1, and MMP13 and deletions in CDKN2A/B. METHODS We conducted a retrospective study on observational cohort of NSCLC patients from January 2022 to July 2023, using IRB-approved data in patients with confirmed brain metastasis (CBS). Beyond demographic information, data on genomic signatures was obtained from maximum parallel/next-generation sequencing on tumor biopsy samples. Comparative analysis was conducted to predict commonalities in brain metastasis gene signatures. RESULTS The study involved 143 newly diagnosed lung cancer patients, with 29 having CBS. Genomic data was available in 24 patients. Most common abnormality was noted in TP53 gene in about 47% of patients, followed by LRP1B (12%). Mutations in KRAS, EGFR, NF1, MET, SMARCA4, KEAP1, and STK11 genes were seen in two patients each. Interestingly, 3 of 4 patients with LRP1B had brain metastasis. Actionable mutations/rearrangements were significantly lower compared to general prevalence of these mutations (KRAS gene 8% and EGFR gene 5%). CONCLUSIONS Lung cancer with brain metastasis has an abysmal prognosis due to poor CNS penetration of chemotherapeutic agents. Surgical resection and RT remain cornerstones of treatment, but certain TKI has shown some potential. Our study uncovered a notably higher prevalence of TP53 mutation among patients with brain metastasis. While patient cohort was small, those harboring an LRP1B mutation exhibited significantly higher incidence of brain metastasis. Contrary to prevalent epidemiological data, our patient population demonstrated a lower frequency of targetable mutations (specifically in EGFR and KRAS genes). We hypothesize that this observation is due to a markedly higher smoking prevalence in our region.

PATH-11. TECTAL GLIOMA: CLINICAL, PATHOLOGICAL, AND MOLECULAR FEATURES

Abstract Tectal glioma (TG) is a rare lower-grade glioma (LrGG) that occurs in the tectum, mainly affecting children. The clinical course is usually indolent, with common symptoms including headache, gait disturbance, and ataxia resulting from obstructive hydrocephalus. TG shares pathological similarities with pilocytic astrocytoma (PA), but recent genetic analyses have revealed distinct features, such as alterations in KRAS and BRAF. Here, we report the clinical, radiological, pathological, and molecular characteristics of TG cases in our hospital. We conducted a retrospective review of cases clinically diagnosed as TG and surgically treated at our institute between January 2005 and March 2023. Molecular analysis, including genome-wide DNA methylation profiling, was performed. Six cases were identified and the median age at diagnosis was 30.5 years (range: 6–45 years). The mean tumor diameter was 22.8 mm (range: 14.7–33.2 mm), with obstructive hydrocephalus observed in all cases. Contrast enhancement of the tumor was noted in four cases and cyst formation occurred in two cases. Four patients underwent biopsy or biopsy with endoscopic third ventriculostomy, and two patients underwent tumor resection. Postoperatively, three patients received radiotherapy and two were treated with chemotherapy. The median overall survival was 6.2 years (range: 1.4–7.3 years), and the median progression-free survival was 3.4 years (range: 1.4–7.3 years). Histological diagnoses included three cases of PA, one case of astrocytoma, and two cases of high-grade glioma. Among the three patients who underwent molecular evaluation, including genome-wide methylation analysis, two had KRAS mutation and one had H3-3A K27M mutation. Our results demonstrate the diverse histological and molecular characteristics of TG distinct from other LrGGs. Given the heterogeneous pathological background and the risk of pathological progression in TG, we emphasize the importance of comprehensive diagnosis, including molecular evaluation.

KRAS mutations promote the intratumoral colonization of enterotoxigenic bacteroides fragilis in colorectal cancer through the regulation of the miRNA3655/SURF6/IRF7/IFNβ axis

ABSTRACT KRAS mutations are associated with poor prognosis in colorectal cancer (CRC). Although the association between the gut microbiota and CRC has been extensively documented, it is unclear whether KRAS mutations can regulate the gut microbiota. Metagenomics has identified changes in the diversity of the gut microbiota in CRC due to KRAS mutations. Specifically, KRAS mutations positively correlate with the abundance of the bacteroides. Understanding how to regulate the classic carcinogenic bacterium within the bacteroides, such as enterotoxigenic bacteroides fragilis (ETBF), to enhance treatment efficacy of tumors is a key focus of research. Mechanistically, we found that the reduction of miR3655 is indispensable for KRAS mutation-promoted proliferation of CRC and the abundance of ETBF. miR3655 targets SURF6 to inhibit its transcription. Further transcriptomic sequencing revealed that SURF6 promotes intratumoral colonization of ETBF in CRC by inhibiting the nuclear translocation and transcription levels of the IRF7, affecting the activation of the IFNβ promoter. Regulating miR3655 and SURF6 can promote IFNβ secretion in CRC, directly killing ETBF. These data indicate that KRAS mutations affect the intratumoral colonization of ETBF in CRC through the miR3655/SURF6/IRF7/IFNβ axis. This provides new potential strategies for treating CRC associated with KRAS mutations or high levels of ETBF.

Using Genomic Heterogeneity to Inform Therapeutic Decisions for Metastatic Colorectal Cancer: An Application of the Value of Heterogeneity Framework.

Mutations in KRAS and NRAS are predictive of poor response to cetuximab and panitumumab, two anti-epidermal growth factor receptor (EGFR) monoclonal antibodies used in metastatic colorectal cancer (mCRC). Our objective was to explore the value of using KRAS and NRAS mutation status to inform third-line anti-EGFR therapy for mCRC using the value of heterogeneity (VOH) framework. We used administrative data to identify mCRC patients who were potentially eligible for third-line therapy in 2006-2019 in British Columbia (BC), Canada. We compared three alternative stratification policies in place during the study period: the unstratified policy where anti-EGFR therapy was not offered (2006-2009), stratification by KRAS mutation (2009-2016), and stratification by KRAS+NRAS mutation (2016-2019). We used inverse-probability-of-treatment weighting to balance covariates across the three groups. Cost and survival time were calculated using a 3-year time horizon and adjusted for censoring, with bootstrapping to characterize uncertainty. Mean net monetary benefit (NMB) was calculated at a range of threshold values. The VOH of using KRAS and NRAS mutation status to inform treatment selection was calculated as the change in NMB with increasing stratification, under current (static VOH) or perfect (dynamic VOH) information. We included 2664 patients in the analysis. At a willingness-to-pay of CA$100,000/ life-year gained (LYG), stratification on KRAS mutation status provided a static VOH of CA$1565 per patient; further stratification on KRAS+NRAS provided additional static VOH of CA$594. The static VOH exceeded the marginal cost of genomic testing under both policies. Stratification of anti-EGFR therapy by KRAS and NRAS mutation status can provide additional value at a threshold of CA$100,000/LYG. There is diminishing marginal value and increasing marginal costs as the policy becomes more stratified. The VOH framework can illustrate the value of subgroup-specific decisions in a comprehensive way, to better inform targeted treatment policies.

KRAS inhibitors may prevent colorectal cancer metachronous metastasis by suppressing TGF‑β mediated epithelial‑mesenchymal transition.

In colorectal cancer (CRC), KRAS mutations enhance metachronous metastasis, a condition without prognostic biomarkers or preventive measures. The present study demonstrated that KRAS mutation may be a risk factor for CRC metachronous metastasis through meta‑analysis of public databases. A risk scoring model was constructed using machine learning for predicting metachronous metastasis in KRAS‑mutant CRC. Wound healing and Transwell assay indicated that KRAS inhibitors strongly suppress migration and invasion capabilities of high‑risk CRC cells and these findings were validated through ex vivo organoid and a mouse model of splenic‑liver metastasis. Mechanistically, RNA sequencing, reverse transcription‑quantitative PCR and western blot analyses revealed that KRAS inhibitors suppressed epithelial‑mesenchymal transition (EMT) and transforming growth factor β (TGF‑β) signaling. Notably, addition of TGF‑β1 protein partially reversed the inhibitory effects of KRAS inhibitors on CRC. These results suggested that KRAS inhibitors may prevent CRC metachronous metastasis by downregulating TGF‑β‑mediated EMT, suggesting they can be used prophylactically in high‑risk KRAS‑mutant CRC.

Protein-RNA interaction dynamics reveal key regulators of oncogenic KRAS-driven cancers

KRAS is one of the most frequently mutated oncogenes across various cancers. Oncogenic KRAS mutations rewire cellular signaling, leading to significant alterations in gene expression. RNA-binding proteins (RBPs) play a pivotal role in gene expression regulation by post-transcriptionally controlling various aspects of RNA metabolism. It has become clear that interactions between RBPs and RNA are frequently dysregulated in numerous cancers. However, how oncogenic KRAS mutations reshape the post-transcriptional regulatory network mediated by RBPs remains poorly understood. In this study, we systematically dissected oncogenic KRAS-driven alterations of RNA-RBP networks. We identified 35 cancer-associated RBPs with either increased or decreased RNA binding upon oncogenic KRAS activation, including PDCD11, which is essential for the viability of KRAS mutant cancers, and ELAVL2, which regulates cell migration in KRAS mutant lung cancers. Our study serves as a crucial resource for elucidating RBP regulatory networks in KRAS mutant cancers and may provide new avenues for therapeutic strategies targeting KRAS mutant malignancies.

Dependence of NPPS creates a targetable vulnerability in RAS-mutant cancers.

RAS is the most frequently mutated oncoprotein for cancer driving. Understanding of RAS biology and discovery of druggable lynchpins in RAS pathway is a prerequisite for targeted therapy of RAS-mutant cancers. The recent identification of KRASG12C inhibitor breaks the "undruggable" curse on RAS and has changed the therapy paradigm of KRAS-mutant cancers. However, KRAS mutations, let alone KRASG12C mutation, account for only part of RAS-mutated cancers. Targeted therapies for cancers harboring other RAS mutations remain the urgent need. In this study we explored the pivotal regulatory molecules that allow for broad inhibition of RAS mutants. By comparing the expression levels of nucleotide pyrophosphatase (NPPS) in a panel of cell lines and the functional consequence of increased NPPS expression in RAS-mutant cells, we demonstrated that cancer cells with various kinds of RAS mutations depended on NPPS for growth and survival, and that this dependence conferred a vulnerability of RAS-mutant cancer to treatment of NPPS inhibition. RAS-mutant cells, compared with RAS-wildtype cells, bored and required an upregulation of NPPS. Transcriptomics and metabolomics analyses revealed a NPPS-dependent hyperglycolysis in RAS-mutant cells. We demonstrated that NPPS promoted glucose-derived glycolytic intermediates in RAS-mutant cells by enhancing its interaction with hexokinase 1 (HK1), the enzyme catalyzing the first committed step of glycolysis. Pharmacological inhibition of NPPS-HK1 axis using NPPS inhibitor Enpp-1-IN-1 or HK1 inhibitor 2-deoxyglucose (2-DG), or genetic interfere with NPPS suppressed RAS-mutant cancers in vitro and in vivo. In conclusion, this study reveals an unrecognized mechanism and druggable lynchpin for modulation of pan-mutant-RAS pathway, proposing a new potential therapeutic approach for treating RAS-mutant cancers.

Endosomal Trafficking Bypassed by the RAB5B-CD109 Interplay Promotes Axonogenesis in KRAS-Mutant Pancreatic Cancer.

Perineural invasion (PNI) represents a unique biological feature associated with poor prognosis in pancreatic ductal adenocarcinoma (PDAC), especially in the presence of KRAS mutations. Extracellular vesicle (EV)-packaged circular RNAs (circRNAs) function as essential mediators of tumor microenvironment communication, triggering PDAC cell invasion and distant metastasis. However, the regulatory mechanisms of EV-packaged circRNAs in the PNI of KRAS-mutant PDAC have not yet been elucidated. Herein, a KRASG12D mutation-responsive EV-packaged circRNA, circPNIT, which positively correlated with PNI in PDAC patients is identified. Functionally, KRASG12D PDAC-derived EV-packaged circPNIT promoted axonogenesis and PNI both in vitro and in vivo. Mechanistically, the circPNIT-mediated Rab5B-CD109 interplay bypassed traditional endosomal trafficking to anchor Rab5B to the lipid rafts of multivesicular bodies and packaged circPNIT into CD109+ EVs. Subsequently, CD109+ EVs delivered circPNIT to neurons by binding to TRPV1 and facilitating DSCAML1 transcription-induced axonogenesis, which in turn enhanced the PNI by activating the GFRα1/RET pathway. Importantly, circPNIT-loaded CD109+ EVs are established to dramatically promote PNI in a KRASG12D/+ Trp53R172H/+ Pdx-1-Cre mouse model. Collectively, the findings highlight the mechanism underlying how EV-packaged circRNAs mediate the PNI of KRAS-mutant PDAC cells through the Rab5B endosomal bypass, identifying circPNIT as an effective target for the treatment of neuro-metastatic PDAC.