Kirsten Rat Sarcoma Research Articles

Characteristics and outcomes of large (≥5 cm) colonic adenocarcinomas and comparing outcomes of minimally invasive and open surgery for stage I to III disease

BackgroundColon cancer prognosis is primarily dependent on the stage at diagnosis, but tumor size and location may also impact prognosis. This study aimed to assess the characteristics and outcomes of patients with ≥5 cm colonic adenocarcinomas and compare outcomes of open and minimally invasive surgery for stage I to III large colonic adenocarcinomas. MethodsThe National Cancer Database (2010–2019) was searched for patients with colonic adenocarcinomas ≥5 cm. Outcomes of patients who underwent minimally invasive surgery or open surgery were compared after propensity-score matching. The primary outcome was 5-year overall survival and, secondarily, hospital stay, surgical margins, and short-term mortality. ResultsA total of 126,959 patients were included (22.1% of all diagnosed adenocarcinomas). 56% of tumors were right-sided, 32.6% were left-sided, and 11.4% were in the transverse colon. Stage IV disease was recorded in 34.6% of patients. Lymphovascular invasion, perineural invasion, and Kirsten rat sarcoma viral oncogene homolog mutations were recorded in 35.7%, 14.9%, and 41.6% of patients. The rate of positive surgical margins was 9.8%. Median hospital stay was 6 (interquartile range: 4–8) days. 30- and 90-day mortality rates were 4.1% and 7.5%, respectively. After matching, 15,228 patients in the open surgery group were matched to a similar number in the minimally invasive surgery group. The minimally invasive surgery group was associated with significantly lower rates of 30- and 90-day mortality, positive surgical margins, shorter hospital stay, and longer median overall survival (110.6 vs 86.6 months, P < .001) than did open surgery. ConclusionLarge colonic adenocarcinomas are mostly right-sided or transverse and present at a more advanced stage with adverse pathologic features. Minimally invasive surgery was associated with better overall survival and short-term benefits when compared with open surgery.

Decoding the Versatile Landscape of Autophagic Protein VMP1 in Cancer: A Comprehensive Review across Tissue Types and Regulatory Mechanisms.

Autophagy, a catabolic process orchestrating the degradation of proteins and organelles within lysosomes, is pivotal for maintaining cellular homeostasis. However, its dual role in cancer involves preventing malignant transformation while fostering progression and therapy resistance. Vacuole Membrane Protein 1 (VMP1) is an essential autophagic protein whose expression, per se, triggers autophagy, being present in the whole autophagic flux. In pancreatic cancer, VMP1-whose expression is linked to the Kirsten Rat Sarcoma Virus (KRAS) oncogene-significantly contributes to disease promotion, progression, and chemotherapy resistance. This investigation extends to breast cancer, colon cancer, hepatocellular carcinoma, and more, highlighting VMP1's nuanced nature, contingent on specific tissue contexts. The examination of VMP1's interactions with micro-ribonucleic acids (miRNAs), including miR-21, miR-210, and miR-124, enhances our understanding of its regulatory network in cancer. Additionally, this article discusses VMP1 gene fusions, especially with ribosomal protein S6 kinase B1 (RPS6KB1), shedding light on potential implications for tumor malignancy. By deciphering the molecular mechanisms linking VMP1 to cancer progression, this exploration paves the way for innovative therapeutic strategies to disrupt these pathways and potentially improve treatment outcomes.

Red blood cells function as reservoirs of tumor DNA.

Novel screening techniques for early detection of lung cancer are urgently needed. Profiling circulating tumor cell-free DNA (ctDNA) has emerged as a promising tool for biopsy-free tumor genotyping. However, both the scarcity and short half-life of ctDNA substantially limit the sensitivity and clinical utility of ctDNA detection methodologies. Our discovery that red blood cells (RBCs) sequester mitochondrial DNA opens a new avenue for detecting circulating nucleic acids, as RBCs represent an unrecognized reservoir of circulating nucleic acid. Here, we show that RBCs acquire tumor DNA following coculture with lung cancer cell lines harboring Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR) mutations. RBC-bound tumor DNA is detectable in patients with early-stage non-small cell lung cancer (NSCLC) but not in healthy controls by qPCR. Our results collectively uncover a previously unrecognized yet easily accessible reservoir of tumor DNA, offering a promising foundation for future RBC-based tumor diagnostics.NEW & NOTEWORTHY We present a novel method for lung cancer detection by revealing RBCs as a reservoir for tumor DNA, overcoming the limitations of current circulating tumor ctDNA methodologies. By demonstrating that RBCs can capture tumor DNA, including critical mutations found in lung cancer, we provide a promising, biopsy-free avenue for early cancer diagnostics. This discovery opens up exciting possibilities for developing RBC-based diagnostic tools, significantly enhancing the sensitivity and clinical utility of noninvasive cancer detection.

Abstract 6623: Ala59 mutants of KRAS cooperate with Nf1 loss to enhance colon tumorigenesis

Abstract Understanding the role of KRAS allele heterogeneity in tumor development, and its influence on therapeutic efficacy, is critical for the improvement of cancer treatment. The Kirsten rat sarcoma (KRAS) gene is the most frequently mutated oncogene in colorectal cancer (CRC) and patients with CRC tumors positive for KRAS mutations have poorer outcomes and limited treatment options. Extensive work over the past four decades has shown that common oncogenic alleles of KRAS are associated with hyperactivation of the MAPK signaling pathway (i.e. RAF/MEK/ERK), and it is for this reason that patients with KRAS mutant tumors are excluded from targeted therapies against EGFR. Recently, we characterized two alleles of KRAS, KRASA59T and KRASA59E, that paradoxically promote KRAS activation but are impaired in their ability to promote BRAF dimerization (Johnson et al. Mol Cell. 2022. PMID: 35202574). Consistent with these biochemical functions, KRASA59T and KRASA59E are enriched in CRC tumors that already have genetic alterations in genes that regulate MAPK signaling (e.g. EGFR, BRAF, NRAS, NF1). Further, unlike other described oncogenic alleles of KRAS, clinical data suggest that these KRASA59T and KRASA59E may not confer intrinsic resistance to anti-EGFR therapies at all. While KRASA59T and KRASA59E may not promote BRAF dimers, here we show that KRASA59T and KRASA59E does promote dimerization between ARAF and CRAF, and using an unpublished conditional mouse mode of K-Ras (i.e. LSL-K-RasA59E) we tested (1) whether Ala59 alleles are oncogenic or (2) require cooperation with additional MAPK pathway activating mutations, and (3) whether Ala59 alleles can promote intrinsic resistance to EGFR inhibition. Here, we show that endogenous K-RasA59E can disturb colon homeostasis and decrease mouse survival in an autochthonous colon tumor model, demonstrating that A59E alone is an oncogenic allele of KRAS in the colon. Breeding of LSL-K-RasA59E mice with mice harboring a conditional knockout of Nf1 (i.e. Nf1fl/fl), a GTPase activating protein (GAP) that promotes activation of wildtype RAS, show that combined expression of K-RasA59E and wildtype RAS activation cooperatively perturb colon homeostasis and decrease mouse survival. Thus, while KRASA59E may not require a second genetic alteration to activate the MAPK signaling pathway, it can cooperate with these alterations to increase the severity of disease. Finally, using mouse colon derived organoids, we show that endogenous K-RasA59E neither promotes EGF independence of organoid growth nor confers on these organoids a resistance to EGFR inhibition. Thus, our data support a unique role for RAF dimer function in in KRAS mutant tumors of the colon in both development and sensitivity to drugs, as well as argue for the inclusion of KRASA59T and KRASA59E bearing CRC tumors in anti-EGFR therapies. Citation Format: Christian William Johnson, Elizabeth M. Terrell, Josh Cook, Bing Shui, Eve O'Donoghue, Shannon Hull, Moon Hee Yang, Shikha Sheth, Rona Yaeger, Scott Kopetz, Deborah K. Morrison, Kevin Haigis. Ala59 mutants of KRAS cooperate with Nf1 loss to enhance colon tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6623.

Abstract 5033: Sub-0.01% KRAS G12C dPCR variant detection by mutation specific blocker (MSB) cassettes

Abstract Purpose: Kirsten rat sarcoma viral oncogene homologue (KRAS) is one of the most well-known oncogenes and is found commonly mutated in numerous cancer subtypes. Despite its prevalence as a driver of disease state, until recently KRAS-targeting therapeutics have proven difficult to develop due to lack of specificity (Huang 2021). Now, several treatments have been approved for KRAS mutated cancers that are driven specifically by the G12C variant, and additional compounds are under investigation. Enrollment on these therapies is conditional on the G12C mutation presence, and thus accurate, sensitive detection of G12C is critical. Herein, we describe the development of KRAS G12C-targeting MSB dPCR assays capable of detecting G12C variants down to less than 0.01% variant allele frequency in whole blood, FFPE, and cfDNA samples. Methods: Multiple KRAS G12C MSB cassettes were developed, composed of four components: a mutation-specific forward primer with an engineered penultimate nucleotide mismatch, a non-extendable blocking oligo for the wild-type (WT) KRAS G12 sequence, a TaqMan probe, and a standard reverse primer. Initial cassette performance was assessed using synthetic KRAS G12C and KRAS G12 WT sequences inserted into an IDT plasmid backbone and tested directly or spiked into whole blood or extracted genomic DNA obtained from healthy donors prior to testing. Data obtained with the MSB cassettes across multiple matrices was compared to a BioRad off-the-shelf KRAS G12C droplet digital PCR (ddPCR) assay for accuracy. Cassette designs achieving the desired specificity and sensitivity were subsequently run on cell-free DNA samples possessing KRAS G12C, KRAS G12D, and WT sequences to evaluate performance in patient material. Results: The enhanced WT suppression design of the assay allowed the lower limit of detection and limit of blank to remain at 6 copies/reaction (cps/rxn) and 3 cps/rxn, respectively, across 30 ng/rxn to 350 ng/rxn gDNA background. At the theoretical maximum input of 200,000 cps/rxn, the assay can achieve 0.003% frequency detection in a single well, with robust detection down to 5 ng/rxn input, making it ideal for samples with limited availability. The assay demonstrated high accuracy, with a correlation coefficient ≥ 0.95 to known values and the BioRad reference assay, and high precision, with less than 10% coefficient of variation among allele frequencies of the same sample. Conclusion: With the emergence of targeted therapies for KRAS G12C driven cancers, detection of this mutation has become crucial in the diagnosis. While multiple methods are available for variant identification, MSB cassettes represent a powerful approach to achieve a sensitivity of molecular detection not possible with other techniques. This enhanced sensitivity directly impacts patient access to potentially life-saving therapies, ensuring all possible options are available to diagnose the patients sensitively and early. Citation Format: Ashley Beams, Kevin Lee, Samantha Addison, Nathan Riccitelli. Sub-0.01% KRAS G12C dPCR variant detection by mutation specific blocker (MSB) cassettes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5033.

Abstract 4111: Multi-peptide cancer vaccines targeting KRAS induce significant anti-tumor efficacy

Abstract Background and objective: Kirsten rat sarcoma viral oncogene homologue (KRAS) is frequently mutated in human cancers. KRAS mutants offer the potential for the development of cancer-specific immunotherapy, as epitopes harbouring mutations are strong neoantigens to which the immune system generates cancer-specific anti-tumor effects. However, previous clinical trials of KRAS-targeted vaccines failed to generate satisfactory anti-cancer efficacy. Evidence suggested that CD4 T cell activity plays a critical role in augmenting the cytotoxic effect of CD8 T cells. Here, we design long peptides based on the MHC II hotspots on the entire KRAS protein that activate both CD4 and CD8 T cells to kill KRAS-driven cancer cells. Long peptides also cover a wider range of HLA alleles to increase their utility in a broader spectrum of patients. Our objective is to create a multi-peptide vaccine simultaneously targeting the mutant and wild-type (WT) regions of human KRAS protein. Methods: By selecting MHC II hotspots via multiple epitope prediction algorithms, we designed and synthesized 8 long peptides with lengths between 25 to 30mers. One of them harboured a G12D mutation, while the others covered the WT regions of KRAS. In addition, the corresponding keyhole limpet hemocyanin (KLH) conjugated peptides were also generated. We first demonstrated their immunogenicity in mouse in vivo models. Different combinations of peptides adjuvanted with either CpG oligodeoxynucleotide + aluminum (CpG+alum) or complete/incomplete Freud’s adjuvant (CFA/IFA) were immunized into Balb/c mice before determining the immune response by mouse interferon γ (mIFNγ) ELISPOT assay. Subsequently, an in vivo prophylactic anti-tumor study was conducted by immunizing this multi-peptide vaccine in Balb/c mice and observing the growth inhibition of the CT26-derived tumors. Results: T cell responses were observed in two peptide mixes with or without G12D mutation, suggesting that both mutant-harbouring peptide and WT peptides were immunogenic. We showed that KLH-conjugated peptides and CpG+alum elicited stronger immune responses than the naked peptides and CFA/IFA respectively. Furthermore, peptide mapping showed that the G12D mutant-harbouring peptide and two other WT peptides may contribute to the overall T cell response in the Balb/c mouse. In the prophylactic anti-tumor study (n = 10), immunization of KRAS naked peptide and KLH-conjugated peptide vaccines significantly inhibited tumor growth by 51.2% (****p &amp;lt; 0.0001) and 44.7% (***p &amp;lt; 0.001) respectively, compared to control. Subsequent mIFNγ ELISPOT assay showed that the tumor inhibitory effect was attributed to the activation of antigen-specific T cell response of the vaccines. Conclusion: Our multi-peptide vaccines targeting both the mutant and WT regions of KRAS could generate KRAS-specific T cell responses and elicit significant anti-tumor effect in mouse models. Citation Format: Chi Han Samson Li, Melvin Toh. Multi-peptide cancer vaccines targeting KRAS induce significant anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4111.

Abstract 3941: Wild-type KRAS dosage in mutant KRAS lung cancer: Implications for tumorigenesis and therapeutic response

Abstract Lung adenocarcinomas (LUAD) are frequently driven by activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS). Previous studies have suggested that wild-type (wt)-RAS signaling may modulate cancer progression and drug resistance in these tumors. However, the interplay between oncogenic KRAS and wt-RAS-like GTPases in lung cancer biology and treatment remains debated. Here, we investigated the impact of increased wt-KRAS dosage in LUAD harboring mutant KRAS on lung cancer phenotypes and response to anti-KRAS therapies. Increased wt-KRAS dosage occurs in LUAD due to the loss of LZTR1, a proteostatic regulator of RAS-like GTPases. LZTR1 mutations, deletions, or downregulation co-occur with oncogenic KRAS mutations in more than 40% of LUAD patients. Using a global protein stability screen, we identified LZTR1 as the main regulator of the stability of wt-KRAS, but not of the oncogenic KRAS mutants. Proteomic analysis of LUAD cells confirmed that LZTR1 loss leads to elevated expression of wt-KRAS, but not of KRAS-G12D, indicating that LZTR1 loss affects wt-RAS rather than mutant KRAS signaling. LZTR1 loss in LUAD patients is associated with poor prognosis and hypoxic expression signatures. In a mouse model of Kras-mutated LUAD, Lztr1 haploinsufficiency promoted tumor initiation and progression. Moreover, Lztr1 loss results in hypoxic tumors with inflamed, tortuous, insufficiently perfused, and leaky vessels. Integrative proteomic and phosphoproteomic analysis revealed increased RAL/TBK1 pathway activity in LZTR1-depleted lung cancer cells and Lztr1-deleted tumors. This leads to enhanced secretion of proangiogenic and proinflammatory factors, such as VEGF and TNFα, by LZTR1-deficient cells and an increased number of neutrophils in Lztr1-deleted tumors. TNF-activated neutrophils present at vascular branching points can modulate vascular remodeling by influencing the formation and function of blood vessels. Pharmacological inhibition of RAL/TBK1 signaling with amlexanox, an anti-NF-κB/TBK1 drug, reduces TNFα secretion, decreases the number of neutrophils, and normalizes the tumor-associated vasculature. We demonstrated that increased wt-KRAS dosage, but not other small GTPases, mimicked the effects of LZTR1 loss on RAL/TBK1 signaling and VEGF secretion, suggesting that LZTR1 loss alters the heterogeneity of KRAS-mutated lung cancer by modulating wt-KRAS levels. Furthermore, MRTX1133 a selective inhibitor of KRAS-G12D, showed limited effect in the Kras-G12D, Lztr1-loss LUAD model, whereas the combination of MRTX1133 and amlexanox suppress tumor growth, likely due to the restoration of the tumor vasculature by RAL/TBK1 inhibition. Our study reveals novel insights into the role of wt-KRAS dosage in mutant KRAS lung cancer and identifies potential therapeutic strategies to target patients with dysregulated KRAS proteostasis due to LZTR1 loss. Citation Format: Tonci Ivanisevic, Greetje Vande Velde, Peihua Zhao, Wout Magits, Raj N. Sewduth, Anna A. Sablina. Wild-type KRAS dosage in mutant KRAS lung cancer: Implications for tumorigenesis and therapeutic response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3941.

Abstract 5356: Wild type Kirsten rat sarcoma (Kras) activation drives evasion of interferon-mediated immunity in hepatocellular carcinoma

Abstract Background: Immune checkpoint inhibitors (ICIs) exhibit heterogeneous efficacy in patients with advanced hepatocellular carcinoma (HCC). Currently, there are no effective biomarkers to select patients who could benefit the most from ICIs or to predict response outcomes. Increasing evidence indicates that oncogenic pathway activation is associated with the generation of an unfavorable tumor microenvironment, with consequent immunotherapy resistance. Therefore, an understanding of the immune evasive mechanism of HCC is urgently needed. Methods: Utilizing the antigen-expressing c-MycOE/Tp53KO HCC mouse model, a critical oncogenic signaling pathway in immune evasion was identified by data-independent acquisition mass spectrometry (DIA-MS) proteomics. Potential downstream pathways were investigated by scRNA-seq, immune profiling, western blot, and multiplexed immunofluorescence staining. We also examined the therapeutic potential of targeting wild-type Kras using the Kras inhibitor MRTX0902 and the MEK inhibitor trametinib to enhance the efficacy of immunotherapy in HCC. Results: We identified a significant upregulation of wild-type Kras in immune-escaped tumors, with concurrent activation of its ligand-driven EGFR and its downstream MEK/ERK signaling. Likewise, endogenous Kras overexpression in this model resulted in increased tumor burden with reduced survival time in mice, implicating the regulatory role of Kras signaling in immune evasion. Clinically, wild-type Kras showed elevated expression in HCC at both the mRNA and protein levels, and was associated with tumor recurrence and poorer patient survival. Through scRNA-seq analysis, we demonstrated that Kras hindered dendritic cell recruitment, leading to compromised Cd8+ T cell activity via the suppression of interferon-mediated Cxcl9. Wild-type Kras activation further impaired HCC recognition by T cells via the downregulation of MHC-I expression. Notably, the combination therapy of MRTX0902, trametinib, and anti-PD-1 effectively enhanced intratumoral Cd8+ T cell infiltration and demonstrated a favorable response in survival outcomes. Conclusion: This study showed that wild-type Kras, via the activation of its downstream MEK/ERK signaling, acts as a crucial immune evasive mechanism in HCC and contributes to resistance to anti-PD-1 therapy. It also provided new insights into the therapeutic potential of targeting wild-type Kras to enhance the efficacy of anti-PD-1 therapy, providing a foundation for further exploration and potential clinical applications in HCC. Citation Format: Mang Leng Lei, Terence Kin Wah Lee. Wild type Kirsten rat sarcoma (Kras) activation drives evasion of interferon-mediated immunity in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5356.

Abstract 4032: ATRA induces an interferon response to reprogram the immunosuppressive tumor microenvironment and overcomes resistance to immune checkpoint inhibition in murine models of LKB1-deficient non-small cell lung cancer

Abstract Lung cancer remains the deadliest form of cancer, claiming 1.8 million lives worldwide in 2020. While treatment options have improved with the advent of immune checkpoint inhibitors (ICI), many patients do not respond to immunotherapy or develop resistance following initial response. A significant subset of non-small cell lung cancer (NSCLC) patients harbors somatic co-mutations in Kirsten rat sarcoma virus (KRAS) and Liver kinase 1 [LKB1, also known as serine/threonine kinase 11 (STK11)] genes, whose tumors are characterized by an immunosuppressive tumor microenvironment (TME) that are resistant to ICI. Our studies revealed that all-trans retinoic acid (ATRA), a metabolite derived from vitamin A, sensitized a murine model of NSCLC with increased tumor mutational burden (Kras G12D P53-/-Lkb1-/-; KPL-3M) to PD-1 blockade. The ATRA and anti-PD-1 combination therapy improved local and systemic T cell activation and generated systemic tumor-specific immunity. To understand ATRA-mediated anti-tumor effects, we performed single cell RNA sequencing (scRNA-seq) of KPL-3M murine tumors with or without 6 daily ATRA treatments. scRNA-seq analysis revealed a reduction of neutrophils as well as an enrichment in T cell and natural killer (NK) cell populations in the TME. Furthermore, scRNA-seq analysis revealed elevated expression of interferon (IFN) downstream genes in multiple immune subpopulations in the TME. Preliminary ex vivo studies indicated that ATRA increases NOX2 levels, intracellular reactive oxygen species (ROS), and IFN signaling in the neutrophils in KPL-3M tumors. Our findings suggest that ATRA may reshape the TME by activating IFN signaling in multiple cell subtypes to sensitize resistant tumors to ICI immunotherapy. Citation Format: William P. Crosson, Rui Li, Ramin Salehi-Rad, Raymond J. Lim, Jensen Abascal, Bitta P. Kahangi, Edgar Perez Reyes, Michael Oh, Camelia Dumitras, Nico Edgar, Ryan Chew, Rashel Jocobo, Adam Tfayli, Zhe Jing, Kostyantyn Krysan, Linh Tran, Steven M. Dubinett, Bin Liu. ATRA induces an interferon response to reprogram the immunosuppressive tumor microenvironment and overcomes resistance to immune checkpoint inhibition in murine models of LKB1-deficient non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4032.

Abstract 1806: Induction of KRAS G13D dimers as a therapeutic strategy for colorectal cancer

Abstract Colorectal cancer (CRC) is the third leading cause of cancer deaths. Most CRCs arise from polyps in the colon/rectum that acquire gene mutations, including frequent activation of the oncogene Kirsten rat sarcoma virus (KRAS) observed in 40% of cases, particularly at codons 12, 13, and 61. KRAS oligomerization is critical for MAPK pathway signaling, which drives CRC pathogenesis. This study evaluated compounds that bind mutant KRAS G13D to induce non-physiological dimerization. Screening of a 4.1 billion compound DNA-encoded library led to the identification of pyridinylpiperazine/quinoline compounds. These compounds demonstrated the ability to induce RAS dimer formation, subsequently reducing MAPK signaling, exhibiting similarities to BI-2852, which is known to induce formation of non-physiologic RAS dimers. Structure-activity studies yielded indole, piperidine, and morpholine variants showing improved activity in thermal shift assays (DSF) and in cell-based assays evaluating RAS signaling (p-ERK inhibition). Citation Format: Deekshi Angira, anbo Li, Zhiwei Zhou, Seungheon Lee, Joseph Ready, Kenneth Westover. Induction of KRAS G13D dimers as a therapeutic strategy for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1806.

SG-Transunet: A segmentation-guided Transformer U-Net model for KRAS gene mutation status identification in colorectal cancer

Accurately identifying the Kirsten rat sarcoma virus (KRAS) gene mutation status in colorectal cancer (CRC) patients can assist doctors in deciding whether to use specific targeted drugs for treatment. Although deep learning methods are popular, they are often affected by redundant features from non-lesion areas. Moreover, existing methods commonly extract spatial features from imaging data, which neglect important frequency domain features and may degrade the performance of KRAS gene mutation status identification. To address this deficiency, we propose a segmentation-guided Transformer U-Net (SG-Transunet) model for KRAS gene mutation status identification in CRC. Integrating the strength of convolutional neural networks (CNNs) and Transformers, SG-Transunet offers a unique approach for both lesion segmentation and KRAS mutation status identification. Specifically, for precise lesion localization, we employ an encoder–decoder to obtain segmentation results and guide the KRAS gene mutation status identification task. Subsequently, a frequency domain supplement block is designed to capture frequency domain features, integrating it with high-level spatial features extracted in the encoding path to derive advanced spatial-frequency domain features. Furthermore, we introduce a pre-trained Xception block to mitigate the risk of overfitting associated with small-scale datasets. Following this, an aggregate attention module is devised to consolidate spatial-frequency domain features with global information extracted by the Transformer at shallow and deep levels, thereby enhancing feature discriminability. Finally, we propose a mutual-constrained loss function that simultaneously constrains the segmentation mask acquisition and gene status identification process. Experimental results demonstrate the superior performance of SG-Transunet over state-of-the-art methods in discriminating KRAS gene mutation status.

Sotorasib (Lumakras)

&#x0D; CADTH recommends that public drug plans should not reimburse Lumakras for the treatment of adult patients with Kirsten rat sarcoma viral oncogene homologue (KRAS) G12C-mutated locally advanced (not amenable to curative therapy) or metastatic non–small cell lung cancer (NSCLC) who have received at least 1 prior systemic therapy.&#x0D; The clinical evidence reviewed by CADTH was insufficient to conclude that treatment with Lumakras results in a clinically meaningful delay in disease progression compared with docetaxel. It was impossible to assess whether treatment with Lumakras would prolong survival relative to docetaxel.&#x0D; Patients identified the need for more effective treatments that delay disease progression, prolong survival, control disease symptoms, improve quality of life, reduce side effects, and offer an oral route of administration. While Lumakras offers an oral route of administration, it is not clear whether Lumakras meets the other needs identified by patients.&#x0D;

Targeting KRAS G12C Mutation in Colorectal Cancer, A Review: New Arrows in the Quiver.

Kirsten rat sarcoma virus oncogene homolog (KRAS) is the most frequently mutated oncogene in human cancer. In colorectal cancer (CRC), KRAS mutations are present in more than 50% of cases, and the KRAS glycine-to-cysteine mutation at codon 12 (KRAS G12C) occurs in up to 4% of patients. This mutation is associated with short responses to standard chemotherapy and worse overall survival compared to non-G12C mutations. In recent years, several KRAS G12C inhibitors have demonstrated clinical activity, although all patients eventually progressed. The identification of negative feedback through the EGFR receptor has led to the development of KRAS inhibitors plus an anti-EGFR combination, thus boosting antitumor activity. Currently, several KRAS G12C inhibitors are under development, and results from phase I and phase II clinical trials are promising. Moreover, the phase III CodeBreaK 300 trial demonstrates the superiority of sotorasib-panitumumab over trifluridine/tipiracil, establishing a new standard of care for patients with colorectal cancer harboring KRAS G12C mutations. Other combinations such as adagrasib-cetuximab, divarasib-cetuximab, or FOLFIRI-panitumumab-sotorasib have also shown a meaningful response rate and are currently under evaluation. Nonetheless, most of these patients will eventually relapse. In this setting, liquid biopsy emerges as a critical tool to characterize the mechanisms of resistance, consisting mainly of acquired genomic alterations in the MAPK and PI3K pathways and tyrosine kinase receptor alterations, but gene fusions, histological changes, or conformational changes in the kinase have also been described. In this paper, we review the development of KRAS G12C inhibitors in colorectal cancer as well as the main mechanisms of resistance.

Discovery of Novel Noncovalent KRAS G12D Inhibitors through Structure-Based Virtual Screening and Molecular Dynamics Simulations.

The development of effective inhibitors targeting the Kirsten rat sarcoma viral proto-oncogene (KRASG12D) mutation, a prevalent oncogenic driver in cancer, represents a significant unmet need in precision medicine. In this study, an integrated computational approach combining structure-based virtual screening and molecular dynamics simulation was employed to identify novel noncovalent inhibitors targeting the KRASG12D variant. Through virtual screening of over 1.7 million diverse compounds, potential lead compounds with high binding affinity and specificity were identified using molecular docking and scoring techniques. Subsequently, 200 ns molecular dynamics simulations provided critical insights into the dynamic behavior, stability, and conformational changes of the inhibitor-KRASG12D complexes, facilitating the selection of lead compounds with robust binding profiles. Additionally, in silico absorption, distribution, metabolism, excretion (ADME) profiling, and toxicity predictions were applied to prioritize the lead compounds for further experimental validation. The discovered noncovalent KRASG12D inhibitors exhibit promises as potential candidates for targeted therapy against KRASG12D-driven cancers. This comprehensive computational framework not only expedites the discovery of novel KRASG12D inhibitors but also provides valuable insights for the development of precision treatments tailored to this oncogenic mutation.

Does artificial intelligence need companionship to assist in drug discovery? The Kirsten rat sarcoma virus study

Abstract In this Opinion article, we confront the role of artificial intelligence (AI) in targeting and understanding resistance to targeted therapy using the most frequently mutated oncoprotein family in human cancer, rat sarcoma virus guanosine triphosphate hydrolases (RAS GTPases), here Kirsten RAS (KRAS), as an example. Aberrant regulation of the active GTP-bound state of KRAS is associated with tumourigenesis, aggressive disease, and poor prognosis. KRAS mutations (eg, G12C, G12D, G12V, G13D, inter al.) are drivers of numerous cancer types, including non-small cell lung, colorectal, and pancreatic cancers. These mutations have shown to play a significant role in cell behaviour and response to treatment. Since its discovery in the 1980s, it has been recognized that over-expression of KRAS and other RAS family members induces resistance to radiotherapy. Moreover, over the years preclinical and clinical studies showed that tumours with KRAS mutations exhibit different treatment sensitivities compared to tumours with wild-type KRAS.

Haploinsufficiency of Adenomatous Polyposis Coli Coupled with Kirsten Rat Sarcoma Viral Oncogene Homologue Activation and P53 Loss Provokes High-Grade Glioblastoma Formation in Mice.

Glioblastoma multiforme (GBM) is the most common and deadly type of brain tumor originating from glial cells. Despite decades of clinical trials and research, there has been limited success in improving survival rates. However, molecular pathology studies have provided a detailed understanding of the genetic alterations associated with the formation and progression of glioblastoma-such as Kirsten rat sarcoma viral oncogene homolog (KRAS) signaling activation (5%), P53 mutations (25%), and adenomatous polyposis coli (APC) alterations (2%)-laying the groundwork for further investigation into the biological and biochemical basis of this malignancy. These analyses have been crucial in revealing the sequential appearance of specific genetic lesions at distinct histopathological stages during the development of GBM. To further explore the pathogenesis and progression of glioblastoma, here, we developed the glial-fibrillary-acidic-protein (GFAP)-Cre-driven mouse model and demonstrated that activated KRAS and p53 deficiencies play distinct and cooperative roles in initiating glioma tumorigenesis. Additionally, the combination of APC haploinsufficiency with mutant Kras activation and p53 deletion resulted in the rapid progression of GBM, characterized by perivascular inflammation, large necrotic areas, and multinucleated giant cells. Consequently, our GBM models have proven to be invaluable resources for identifying early disease biomarkers in glioblastoma, as they closely mimic the human disease. The insights gained from these models may pave the way for potential advancements in the diagnosis and treatment of this challenging brain tumor.

Pulmonary Adenocarcinoma In Situ and Minimally Invasive Adenocarcinomas in European Patients Have Less KRAS and More EGFR Mutations Compared to Advanced Adenocarcinomas.

Pulmonary adenocarcinoma (ADC) is a very diverse disease, both genetically and histologically, which displays extensive intratumor heterogeneity with numerous acquired mutations. ADC is the most common type of lung cancer and is believed to arise from adenocarcinoma in situ (AIS) which then progresses to minimally invasive adenocarcinoma (MIA). In patients of European ethnicity, we analyzed genetic mutations in AIS (n = 10) and MIA (n = 18) and compared the number of genetic mutations with advanced ADC (n = 2419). Using next-generation sequencing, the number of different mutations detected in both AIS (87.5%) and MIA (94.5%) were higher (p < 0.001) than in advanced ADC (53.7%). In contrast to the high number of mutations in Kirsten rat sarcoma virus gene (KRAS) in advanced ADC (34.6%), there was only one case of AIS with KRAS G12C mutation (3.5%; p < 0.001) and no cases of MIA with KRAS mutation (p < 0.001). In contrast to the modest prevalence of epidermal growth factor receptor (EGFR) mutations in advanced ADC (15.0%), the fraction of EGFR mutant cases was higher in both in AIS (22.2%) and MIA (59.5%; p < 0.001). The EGFR exon 19 deletion mutation was more common in both MIA (50%; n = 6/12) and ADC (41%; n = 149/363), whereas p.L858R was more prevalent in AIS (75%; n = 3/4). In contrast to pulmonary advanced ADC, KRAS driver mutations are less common, whereas mutations in EGFR are more common, in detectable AIS and MIA.

The effects of miR-217 inhibitor and mimic in the progression of Kirsten rat sarcoma viral oncogene homologue driven cancers

BackgroundKirsten rat sarcoma viral oncogene homologue (KRAS) is one of the most frequently mutated proto-oncogenes in approximately 90% of pancreatic ductal carcinoma (PDAC) and 45% of colorectal cancer (CC) cases. Studies in the past have identified microRNA-217 (miR-217) as a potential tumour-suppressing miRNA that is downregulated in various cancers. Using in silico prediction algorithms, several studies have identified miR-217 as a potential regulator of KRAS, and we investigated its role in PDAC and CC progression. MethodThe study was carried out in KRAS-driven cancer (KDC) cell lines PANC-1 (pancreatic cancer) and SW-480 (CC), which have mutant KRAS gene expression. The KDC cells are transfected with specific oligonucleotides for miR-217, anti-miR-217, and a negative control in serum-free media using lipofectamine. After fixing the IC50, using specific primers, gene expression studies were carried out by qPCR for KRAS downstream targets and genes associated with apoptosis and cell cycle. Anti-migration and anti-apoptotic effects were studied using the transwell migration assay and annexin-V/PI staining methods, and mitochondrial morphology was observed using a transmission electron microscope. ResultsThe present study demonstrates that overexpression of miR-217 in KDC cells mitigates proliferation and migration and promotes cell cycle arrest and apoptosis of KDC cells via the MAPK/ERK signalling pathway. Besides, decreased miR-217 expression rescues KDC cells from the effects mediated by KRAS downstream signalling. ConclusionThe outcome of the study indicates miR-217 suppresses tumour growth and promotes apoptosis in KDC and that these effects are associated with down regulation of MAPK/ERK signalling.

Radiological and clinical features of large consolidative-type pulmonary invasive mucinous adenocarcinoma.

This study aimed to investigate the radiological, pathological, and prognostic characteristics of large consolidative-type pulmonary invasive mucinous adenocarcinomas (IMA). We retrospectively reviewed 738 patients who confirmed IMA between January 2010 and August 2022, and two radiologists reviewed imaging data to determine subtypes. We included 41 patients with pathologically large consolidative-type IMA. We analyzed their radiological, pathological, and prognostic characteristics. The recurrence-free survival (RFS) and overall survival (OS) were determined using the Kaplan-Meier method. Most lesions were located in the lower lobe, with 46.3% patients showing multiple lesions. Halo, angiogram, vacuole, air bronchogram, and dead branch sign were observed in 97.6%, 73.2%, 63.4%, 61.0%, and 61.0% of the cases, respectively. Unevenly low enhancement was observed in 88.89% of patients. T3 and T4 pathological stages were observed in 50.0% and 30.6% of patients, respectively. Lymph node metastasis was observed in 16.7% patients, with no distant metastasis. Spread-through air spaces and intrapulmonary dissemination were observed in 27.8% and 19.4% patients, respectively. Moreover, Kirsten rat sarcoma viral oncogene mutations were found in 68.6% of cases, and no epidermal growth factor receptor mutations were seen. Among all mutation sites, G12V mutation is the most common, accounting for 40%. The average RFS and OS were 19.4 and 66.4months, respectively, with 3-year RFS and OS rates of 30.0% and 75.0%, respectively. Pleural invasion and lymph node metastasis were independent risk factors for diagnosis. Halo, vacuole, angiogram, and dead branch signs were frequently observed in consolidative-type IMA. Kirsten rat sarcoma viral oncogene mutations are common in consolidative-type IMA, especially site G12V, whereas epidermal growth factor receptor mutations were rare; therefore, gene immunotherapy was more difficult. Most patients were in stage T3-T4; however, lymph node metastasis was rare.

Effect of Strong CYP3A4 Inhibition, CYP3A4 Induction, and OATP1B1/3 Inhibition on the Pharmacokinetics of a Single Oral Dose of Sotorasib.

Sotorasib is a small molecule that irreversibly inhibits the Kirsten rat sarcoma viral oncogene homolog (KRAS) protein with a G12C amino acid substitution mutant protein. The impact of cytochrome P450 (CYP) 3A4 inhibition and induction on sotorasib pharmacokinetics (PKs) was evaluated in 2 separate studies in healthy volunteers (N = 14/study). The impact of CYP3A4 inhibition was interrogated utilizing repeat doses of 200mg of itraconazole, a strong CYP3A4 inhibitor, on 360mg of sotorasib PKs. The impact of CYP3A4 induction was interrogated utilizing multiple doses of 600mg of rifampin, a strong CYP3A4 inducer. Additionally, the impact of organic anion transporting polypeptide (OATP) 1B1/3 inhibition on 960mg of sotorasib PKs was interrogated after a single dose of 600mg of rifampin. CYP3A4 inhibition did not significantly impact sotorasib Cmax but did lead to a 26% increase in sotorasib AUCinf. CYP3A4 induction decreased sotorasib Cmax by 35% and AUCinf by 51%. OATP1B1/3 inhibition decreased sotorasib Cmax and AUCinf by 16% and 23%, respectively. These results support that sotorasib can be given together with strong CYP3A4 and OATP1B1/3 inhibitors but the co-administration of sotorasib and strong CYP3A4 inducers should be avoided.