Harboring KRAS Mutations Research Articles

Genomic Profiling of Biliary Tract Cancers: Comprehensive Assessment of Anatomic and Geographic Heterogeneity, Co-Alterations and Outcomes.

Biliary tract cancers (BTCs) represent distinct biological and genomic entities. Anatomic and geographic heterogeneity in genomic profiling of BTC subtypes, genomic co-alterations, and their impact on long-term outcomes are not well defined. Genomic data to characterize alterations among patients with BTCs were derived from the AACR GENIE registry (v15.1) and other genomic data sets. Patterns of mutational co-occurrence, frequency of co-alterations, and their impact on long-term outcomes among BTC patients were examined. Alterations in IDH1 and FGFR2 genes were mostly noted among intrahepatic cholangiocarcinoma (iCCA) samples, TP53, ERBB2/HER2, and SMAD4 mutations were more frequent among gallbladder cancer (GBC) samples while extrahepatic cholangiocarcinoma (eCCA) more commonly harbored KRAS mutations (all Q < 0.001). Alterations in IDH1 and FGFR2 genes were more frequent among iCCA samples from Western vs. Eastern populations, while KRAS, SMAD4, and ERBB2 mutations were more commonly observed among Eastern populations(all Q < 0.05). FGFR2 gene was frequently co-mutated with BAP1 (log2OR: 1.55, Q < 0.001), while IDH1 gene was commonly co-mutated with PBRM1 (log2OR: 1.09, Q < 0.001). Co-alteration rate among patients with IDH1-mutant iCCAs, FGFR2-rearranged iCCAs, KRAS-mutant eCCA, and HER2-mutant GBCs were 80.8%, 85.2%, 76.7%, and 100%, respectively. Among patients with iCCA and FGFR2 fusions/rearrangements, harboring co-alterations in the TP53 pathway or PI3K pathway correlated with worse overall survival (OS), while patients with IDH1-mutant iCCA had worse OS when harboring co-alterations in the cell cycle pathway. Marked genomic heterogeneity exists among patients with BTCs based on anatomic and geographic location. The overwhelming majority of BTC patients with clinically significant mutations had concurrent genomic co-alterations. The current study highlights the molecular complexity of BTCs with multiple alterations that commonly co-exist and could potentially be targeted to treat BTCs.

Current Strategies and Future Dimensions in the Development of KRAS Inhibitors for Targeted Anticancer Therapy.

KRAS is a proto-oncogene that is found to be mutated in 15% of all metastatic cancers with high prevalence in pancreatic, lung, and colorectal cancers. Additionally, patients harboring KRAS mutations respond poorly to standard cancer therapy. As a result, KRAS is seen as an attractive target for targeted anticancer therapy. Over the last decade, this protein has evolved from being termed "undruggable" to producing two clinically approved drugs along with several more in clinical development, and many under preclinical investigations. This review details the development of various KRAS-targeted molecules with emphasis on the different drug design strategies employed by examining the following areas: (1) Direct inhibition of KRAS mutants using small molecule binders, (2) Inhibiting the activated state of KRAS mutants using a binary complex of small molecule binders and cyclophilin A, and (3) Targeted degradation of KRAS mutants using the PROTAC approach. We assess the pharmacological attributes and possible clinical benefits of the different molecules and look to the next frontiers in the application of KRAS inhibitors as anticancer agents.

Development of KRAS Inhibitors and Their Role for Metastatic Colorectal Cancer.

Colorectal cancer (CRC) is a heterogeneous group of diseases comprising several molecular subtypes. Comprehensive DNA sequencing is now standard practice to identify these subtype. Until recently, KRAS mutation status in metastatic CRC was primarily used as a biomarker to predict resistance to EGFR inhibition. However, with up to 40% of CRC cases harboring KRAS mutations, therapeutic targeting of RAS has been an area of great need. The development of KRASG12C inhibitors has led to the FDA approval of drugs for treating non-small cell lung cancer. Recently, these and other newly developed inhibitors have been investigated as monotherapies and in combination for metastatic KRASG12C-mutant CRC. This review examines the development of these inhibitors and highlights data supporting the inclusion of sotorasib and adagrasib, in combination with either panitumumab or cetuximab, in the NCCN Guidelines for CRC for the treatment of refractory metastatic disease.

KRAS Mutations in Cholangiocarcinoma: Prevalence, Prognostic Value, and KRAS G12/G13 Detection in Cell-Free DNA.

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. 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 tissue-based sequencing from 78 matched samples. 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. 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.

Clinical characteristics and survival outcomes in patients with pulmonary sarcomatoid carcinoma: a multicenter retrospective study

Abstract Purpose The clinicopathologic features, mutational status, immunohistochemical markers, and prognosis of Pulmonary sarcomatoid carcinoma (PSC) remain uncertain. Methods This study included 81 PSC and 337 lung adenocarcinomas (LUAD). Progression-free survival (PFS), overall survival (OS), and other clinical data were examined. Results 46% PSC patients harbored KRAS mutation and 23% harbored EGFR mutation. Univariable analysis identified type and cTNM stage as significant predictor of PFS (type: HR 0.216; 95% CI 0.133–0.349; P &lt; 0.001, cTNM stage: HR 0.483; 95% CI 0.269–0.846; P = 0.014) and OS (type: HR 0.269; 95% CI 0.156–0.465; P &lt; 0.001, cTNM stage: HR 0.435; 95% CI 0.219–0.865; P = 0.018). Multivariable analysis confirmed sex, type and cTNM stage as independent predictors of PFS (sex: HR 2.026; 95%CI 1.027–3.996; P = 0.042; type: HR0.140; 95% CI 0.083–0.238; P &lt; 0.001, cTNM stage: HR0.305; 95% CI 0.165–0.564; P &lt; 0.001) and OS (type: HR0.231; 95% CI 0.132–0.404; P &lt; 0.001, cTNM stage: HR 0.394; 95% CI 0.194–0.797; P = 0.010). Significant differences in PFS (P &lt; 0.0001) and OS (P = 0.022) were observed between PSC and LUAD, and for PC compared with SCC (PFS: P = 0.00036, OS: P = 0.0053). Additionally, PSC patients treated with immunotherapy showed significantly better OS (P = 0.0019) compared with those treated without immunotherapy. Conclusions PSC exhibits high KRAS and EGFR mutation rates, and spindle cell carcinoma has a worse prognosis. Immunotherapy shows potential as a treatment for advanced PSC.

BIOACTIVE COMPOUNDS OF Moringa oleifera AS KRASG12C INHIBITORS IN COLORECTAL CANCER: IN SILICO STUDY

KRAS is a GTPase enzyme that regulates cell growth and division. Mutations in KRAS can lead to its permanent activation, resulting in uncontrolled cell growth and cancer progression. Approximately 30–44% of colorectal cancer cases harbor KRAS mutations, with 1–3% involving the KRASG12C variant. Historically considered "undruggable," recent advancements, such as Sotorasib, have demonstrated the potential to target KRASG12C effectively, making it a promising focus for drug discovery. Moringa oleifera, a plant rich in phytochemicals, is a potential source of bioactive compounds with therapeutic applications. In this study, 218 compounds derived from M. oleifera were screened using molecular docking, targeting KRASG12C. Quercetin (3) exhibited the lowest binding affinity (-9.37 kcal/mol) and showed interactions with key residues, including GLN100A, VAL104A, LYS17A, and TYR97A, suggesting a binding mechanism similar to that of Sotorasib as native ligand. The physicochemical analysis further revealed high gastrointestinal absorption, good lipophilicity, and favorable bioavailability scores for Quercetin (3), supporting its potential as a drug candidate. These findings highlight the potential of M. oleifera compounds, particularly quercetin (3), as inhibitors of KRASG12C in colorectal cancer.

First Insight into the Mutational Landscape of BRAF and KRAS Genes in Lung Cancer Patients from Morocco

Background: Lung cancer (LC) is a lethal malignancy with a late diagnosis and poor prognosis. During the last decade, the identification of oncogenic driver alterations has noticeably changed the therapeutic landscape and contributed to the emergence of the “oncogene addiction” concept and precision medicine in oncology. Among these alterations, the spotlight has turned to driver mutations in the KRAS and BRAF genes, which have garnered significant attention due to the emergence of targeted therapies and the potential for personalized treatment strategies. Objective: Hence, the present study aimed to evaluate the mutational landscape of the KRAS and BRAF genes in LC Moroccan patients along with their frequencies and their correlation with clinicopathological features. Methods: A total of 60 fresh biopsies were collected from patients with primary LC and were subjected to PCR-DNA sequencing of exon 2 of KRAS and exon 15 of BRAF genes in order to detect the most common mutations known by their implication in response to targeted therapies. Results: Sequencing analysis revealed that mutations in KRAS and BRAF genes represented respectively 8.3% and 6.7% of cases; one patient had two KRAS mutations (G12A and K5E), and none had simultaneous BRAF and KRAS mutations. The vast majority of patients harboring KRAS mutations were men, formal smokers with adenocarcinomas, and at advanced stage (stage III). BRAF mutations were mainly detected in men and nonsmokers with adenocarcinoma. Statistical analyses showed no significant correlation between KRAS and BRAF substitutions and clinico-pathological features (p&gt;0.05). Conclusion: The presence of these mutations will be used as a valuable molecular biomarker to select potential candidates eligible for effective personalized medicine using available agents targeting these mutations. Much effort is needed to identify other druggable mutations to generalize personalized LC therapy for better management of this devastating disease.

Abstract C027: Efficacy advantages of a novel pan-RAS inhibitor, ADT-1004, over mutant- specific KRAS inhibitors for suppressing pancreatic tumor growth in murine models

Abstract Background/Objective: Prognosis remains grim for localized or metastasized pancreatic ductal adenocarcinoma (PDAC) with 5-year survival rates of 3-16%. More than 90% of PDAC cases harbor KRAS mutations. KRASG12C inhibitors recently developed have limited use for PDAC because only 3% of cases express this mutation. We developed a novel pan-RAS inhibitor, ADT-1004 and evaluated antitumor activity in tumor models established from murine or human PDAC cell lines or patient-derived xenografts (PDX). ADT-1004 is an orally bioavailable prodrug of ADT-007, a highly potent and selective pan-RAS inhibitor (doi.org/10.1101/2023.05.17.541233). Methods: Murine PDAC cells harboring the KRASG12D mutation (KPC-Luc or 2838C3-Luc) were orthotopically implanted into the pancreas of C57BL/6J mice. Four PDX PDAC tumors harboring KRAS mutations were implanted orthotopically in NSG mice. To assess potential to overcome RAS inhibitor resistance, parental KRASG12C MIA PaCa-2 PDAC cells and a resistant derivative of MIA PaCa-2 cells were implanted subcutaneously. To assess selectivity, mice were implanted with RASWT BxPC-3 cells SC. Mice were treated orally with ADT-1004 (40mg/kg) 5x/week for 4 weeks. Sotorasib and adagrasib KRASG12C inhibitors were administered at the same dose, route, and schedule as ADT-1004. Results: ADT-007 potently and selectively inhibited the growth of human and mouse PDAC cell lines in vitro. For example, the IC50 value of ADT-007 for KRASG12C MIA PaCa-2 PDAC cells was 2 nM compared to 2500 nM for RASWT BxPC-3 PDAC cells. Growth inhibition by ADT-007 was dependent on activated RAS and associated with reduced GTP-RAS levels and MAPK/AKT signaling. ADT-1004 was well tolerated in mice at doses up to 175 mg/kg bid orally with sustained plasma levels of ADT-007 exceeding growth IC50 values. ADT-1004 displayed robust antitumor activity in mouse tumor models using human- or mouse-derived PDAC cell lines with G12D, G12V, G12C, or G13Q KRAS mutations, causing selective inhibitory effects on ERK phosphorylation in tumors but not in normal tissues. ADT-1004 also inhibited growth of KRASG12C mutant MIA PaCa-2 tumors to a comparable extent as sotorasib and adagrasib. Sotorasib and adagrasib were ineffective against xenografted tumors of resistant cells derived from MIA PaCa-2 cells, while ADT-1004 strongly inhibited tumor growth. Target selectivity of ADT-1004 was confirmed in vivo, as ADT-1004 did not affect the growth of RASWT BxPC-3 tumors. Conclusions: ADT-1004 inhibited tumor growth of KRAS mutant PDAC tumors by blocking activated RAS to disrupt downstream MAPK/AKT signaling. Side-by-side comparisons reveal antitumor efficacy advantages of ADT-1004 over mutant-specific KRAS inhibitors. These results highlight the promise of ADT-1004 for treatment of PDAC and other RAS-driven cancers by addressing the complex genetic landscape of many cancers and limitations from resistance to mutant specific KRAS inhibitions. Citation Format: Dhana Sekhar Reddy Bandi, Ganji P Nagaraju, Jeremy B Foote, Adam B Keeton, Xi Chen, Kristy L Berry, Yulia Y Maxuitenko, Upender Manne, Donald J Buchsbaum, Gary A Piazza, Bassel F El-Rayes. Efficacy advantages of a novel pan-RAS inhibitor, ADT-1004, over mutant- specific KRAS inhibitors for suppressing pancreatic tumor growth in murine models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C027.

Abstract B085: The HIF-2 transcription factor mediates resistance to ferroptosis in pancreatic adenocarcinoma

Abstract Ferroptosis is an iron-dependent form of cell death converging on lipid peroxidation, first identified by examining compounds with enhanced lethality to KRAS mutant cells. Two compounds widely used as ferroptosis inducers are erastin, which acts as an inhibitor of cystine import and thereby depletes cellular glutathione, and RSL-3, which inhibits glutathione peroxidase 4. Despite over 90% of pancreatic adenocarcinoma (PDAC) tumors harboring KRAS mutations, PDAC exhibits relative resistance to ferroptosis compared to other tumor types, and the mechanisms behind this resistance remain unclear. The pancreatic tumor environment is marked by severe hypoxia, due to poor vascularity and a dense fibrotic stroma, and so we hypothesized that hypoxia might mediate resistance to ferroptosis. Here we report that across a panel of human and murine PDAC cell lines, hypoxia induces resistance to both ferroptosis inducing agents, erastin and RSL-3. The effect of hypoxia was synergistic with exposure to pancreatic cancer tumor interstitial fluid, which induces a hypoxic-like gene expression profile even under normoxia and enhances hypoxic gene expression changes when combined with hypoxia. Our findings reveal that hypoxia-mediated resistance to ferroptosis is orchestrated by the hypoxia-inducible transcription factor HIF-2, as evidenced by its complete abolition in HIF-2 knockout cell lines, while the protective effect of hypoxia was maintained in HIF-1 knockout lines. RNA-seq analyses and chromatin immunoprecipitation studies (chIP) identified several HIF-2 target genes responsible for coordinating ferroptosis resistance mechanisms. Cysteine is a precursor for glutathione, and HIF-2 increased cystine import through upregulation of both components of the cystine-glutamate antiporter system Xc (SLC7A11 and SLC3A2) and increased cysteine biosynthesis via upregulation of transsulfuration pathway enzymes (CBS and CTH). In addition, HIF-2 upregulated Parkin, which we identify as a novel regulator of ferroptosis, thereby increasing mitophagy and decreasing reactive oxygen species. In vivo studies using human and murine PDAC xenograft models confirmed that HIF-2 knockout impaired tumor growth and increased susceptibility to the pro-ferroptotic agent sulfasalazine. Collectively, these data identify a mechanism by which PDAC evades ferroptosis and identifies a possible therapeutic strategy of combining HIF-2 inhibitors with pro-ferroptotic agents in pancreatic cancer. Citation Format: Maimon E Hubbi, Catherine Wang, Erin Hollander, Yasir Suhail, Kshitiz, Alexander Muir, Ben Z Stanger, Chi V Dang. The HIF-2 transcription factor mediates resistance to ferroptosis in pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B085.

The effects of KRAS Mutations on the Prognosis of Rectal Cancer Following Neoadjuvant Chemoradiotherapy and Surgery.

Rectal cancers with mutations in the KRAS gene have worse prognoses than wild-type malignancies. Variants at codon 12 of KRAS have particularly detrimental effects on prognosis. We aimed to analyze whether KRAS mutations act as adverse prognostic factors following neoadjuvant concurrent chemoradiotherapy (CRT) and surgery for rectal cancer treatment. We analyzed the effects of KRAS mutations on disease-free survival (DFS) and locoregional recurrence-free survival (LRFS) in 125 patients with cT2-4N0-2M0 rectal cancer who underwent surgery following CRT between June 2014 and March 2023 Inje University Busan Paik Hospital. The median follow-up period was 39.7 (range, 7.5-98.2) months. There were 25 patients (20.0%) who harbored KRAS mutations. Among them, 22 patients (17.6%) had codon 12 variants. Overall, 43 patients (34.4%) showed recurrence, of which 10 (8.0%) had locoregional recurrence and 35 (28.0%) had distant metastases (two occurred simultaneously). DFS was significantly reduced in the patients with KRAS mutations (p = 0.005). LRFS was also reduced in patients with KRAS mutations (p = 0.039). DFS and LRFS were also relatively low in the subgroup with KRAS mutations at codon 12 (n = 22) (p = 0.003 and p = 0.017, respectively). However, pathologic complete response rate following CRT was not affected by KRAS mutations (p = 0.197). Overall survival was also not associated with KRAS mutations (p = 0.486). KRAS mutation is related to decreased DFS and LRFS, when surgery is performed following neoadjuvant CRT to treat rectal cancer. These effects are particularly pronounced for KRAS mutations at codon 12.

Down-Regulation of AKT Proteins Slows the Growth of Mutant-KRAS Pancreatic Tumors.

Serine/threonine kinase AKT isoforms play a well-established role in cell metabolism and growth. Most pancreatic adenocarcinomas (PDACs) harbor activation mutations of KRAS, which activates the PI3K/AKT signaling pathway. However, AKT inhibitors are not effective in the treatment of pancreatic cancer. To better understand the role of AKT signaling in mutant-KRAS pancreatic tumors, this study utilized proteolysis-targeting chimeras (PROTACs) and CRISPR-Cas9-genome editing to investigate AKT proteins. The PROTAC down-regulation of AKT proteins markedly slowed the growth of three pancreatic tumor cell lines harboring mutant KRAS. In contrast, the inhibition of AKT kinase activity alone had very little effect on the growth of these cell lines. The concurrent genetic deletion of all AKT isoforms (AKT1, AKT2, and AKT3) in the KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cell line also dramatically slowed its growth in vitro and when orthotopically implanted in syngeneic mice. Surprisingly, insulin-like growth factor-1 (IGF-1), but not epidermal growth factor (EGF), restored KPC cell growth in serum-deprived conditions, and the IGF-1 growth stimulation effect was AKT-dependent. The RNA-seq analysis of AKT1/2/3-deficient KPC cells suggested that reduced cholesterol synthesis may be responsible for the decreased response to IGF-1 stimulation. These results indicate that the presence of all three AKT isoforms supports pancreatic tumor cell growth, and the pharmacological degradation of AKT proteins may be more effective than AKT catalytic inhibitors for treating pancreatic cancer.

From morphology to methylome: epigenetic studies of Müllerian mesonephric-like adenocarcinoma reveal similarities to cervical mesonephric adenocarcinoma†.

Mesonephric adenocarcinomas (MAs) and mesonephric-like adenocarcinomas (MLAs) are rare, aggressive neoplasms that arise in the gynecologic tract and show overlapping morphologic, immunohistochemical, and molecular features. While MAs occur in the cervix and are thought to arise from mesonephric remnants, MLAs occur in the endometrium and ovary and are believed to originate from transdifferentiation of Müllerian lesions. Both MAs and MLAs show a variety of architectural patterns, exhibit frequent expression of GATA3 by immunohistochemistry, and harbor KRAS mutations. In a recent article published in The Journal of Pathology, Kommoss and colleagues used DNA methylation profiling to extend these similarities and showed that MLAs and MAs cluster together based on their epigenetic signatures and are epigenetically distinct from other Müllerian adenocarcinomas. They also showed that MLAs and MAs harbor a high number of global copy number alterations. This study provides evidence that MLAs more closely resemble MAs than Müllerian carcinomas on an epigenetic level. As a result, the authors argue that MLA should be renamed 'mesonephric-type adenocarcinoma.' Further research is needed to establish the relationship between these two entities, their etiology, and pathogenesis. © 2024 The Pathological Society of Great Britain and Ireland.

Abstract LB352: Unraveling therapeutic potentials of mRNA-based cancer vaccine concurrently targeting heterogeneous KRAS mutant cancer

Abstract The utilization of messenger RNA (mRNA)-based cancer vaccines represents a compelling therapeutic strategy, providing distinct advantages over alternative modalities such as peptide vaccines or small-molecule-based cancer therapies. This innovative approach enables the concurrent targeting of heterogeneous cancer populations characterized by multiple mutations while preserving the integrity of normal cells. The Kristen rat sarcoma viral oncogene (KRAS) mutation is prevalent in lung, pancreatic, and colon cancer, and patients harboring KRAS mutations face a particularly dismal prognosis. This study focuses on the optimization of an mRNA-based cancer vaccine designed to target various KRAS mutations, demonstrating its efficacy inducing potent anti-tumor immune responses in both ex vivo immune cell and cancer cell co-culture, as well as in vivo syngeneic mouse cancer models. For the ex vivo evaluation of the multi-targeting capabilities of the vaccine, human peripheral blood mononuclear cells (PBMCs) primed with our mRNA cancer vaccine were subsequently co-incubated with human normal and cancer cell lines harboring KRAS mutations to assess antigen-specific immune responses and the normal cell-sparing effect of the cancer vaccine. Four types of KRAS mutant cancer cells co-cultured with the vaccine-activated PBMCs showed substantially decreased cell viability. We found enrichment of cytotoxic and pro-inflammatory cytokines including ganzyme B, IFNγ, IL-6 and IL-10 in co-culture system with KRAS vaccine transfected monocyte and PBMCs. This suggests that multiple KRAS mutations can be targeted with a single type of mRNA vaccine candidate. In the LL/2 syngeneic model (murine Lewis lung carcinoma, KRAS G12C mutant), the in vivo efficacy of KRAS cancer vaccine candidates was evaluated, revealing a notable reduction in tumor growth in response to the vaccine treatment. Also, during the treatment period, there were no significant clinical symptoms in the mouse between groups indicating the vaccine doses were tolerable. These results indicate that the KRAS vaccine effectively inhibits the growth of an LL/2 tumor which has the immunosuppressive tumor microenvironment. The ability to address the challenges posed by diverse KRAS mutations underscores the promising potential of this multiple-KRAS mutation targeting mRNA-based cancer vaccine in the pursuit of enhanced therapeutic outcomes for patients afflicted with KRAS-mutant cancer. Citation Format: Chang Gyu Lim, Seung-Hyun Shin, Youngjin Han, Yong Ho Heo, Innah Kim, Ji Hee Lee, Miyoung Lee, Seongju Jeong, Yunjae Kim, Jooyun Byun, Daejin Kim, In Young Choi. Unraveling therapeutic potentials of mRNA-based cancer vaccine concurrently targeting heterogeneous KRAS mutant cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB352.

The UBE2F-CRL5ASB11-DIRAS2 axis is an oncogene and tumor suppressor cascade in pancreatic cancer cells

UBE2F, a neddylation E2, neddylates CUL5 to activate cullin-RING ligase-5, upon coupling with neddylation E3 RBX2/SAG. Whether and how UBE2F controls pancreatic tumorigenesis is previously unknown. Here, we showed that UBE2F is essential for the growth of human pancreatic cancer cells with KRAS mutation. In the mouse KrasG12D pancreatic ductal adenocarcinoma (PDAC) model, Ube2f deletion suppresses cerulein-induced pancreatitis, and progression of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia. Mechanistically, Ube2f deletion inactivates the Mapk-c-Myc signals via blocking ubiquitylation of Diras2, a substrate of CRL5Asb11 E3 ligase. Biologically, DIRAS2 suppresses growth and survival of human pancreatic cancer cells harboring mutant KRAS, and Diras2 deletion largely rescues the phenotypes induced by Ube2f deletion. Collectively, Ube2f or Diras2 plays a tumor-promoting or tumor-suppressive role in the mouse KrasG12D PDAC model, respectively. The UBE2F-CRL5ASB11 axis could serve as a valid target for pancreatic cancer, whereas the levels of UBE2F or DIRAS2 may serve as prognostic biomarkers for PDAC patients.

Prognostic value of KRAS G12V mutation in lung adenocarcinoma stratified by stages and radiological features

ObjectiveKRAS G12V is one of the most common KRAS mutation variants in lung adenocarcinoma (LUAD), and yet its prognostic value is still unrevealed. In this study, we investigated the clinicopathologic characteristics and prognostic value of the KRAS G12V mutation in LUAD. MethodsData of 3829 patients who underwent LUAD resection between 2008 and 2020 were collected. Mutations were classified as wild-type, G12V, or non-G12V. The clinicopathologic characteristics, postoperative outcomes, and recurrence pattern were analyzed among groups. ResultsIn total, 3554 patients were wild-type and 275 patients harbored a KRAS mutation: 60 patients with G12V (22.2%) and 215 patients with non-G12V (77.8%). The KRAS G12V mutation was more frequent in male patients, older patients (≥60 years), former/current smokers, those patients with radiologic solid nodules, and those with highly invasive histologic subtypes. Tumors carrying KRAS G12V mutation exhibited elevated programmed death-ligand 1 expression in comparison with wild-type tumors. KRAS G12V was more prevalent in older patients and had less lymphovascular invasion compared with other mutation types. FGF3, RET, and KDR co-mutations occurred more frequently in the KRAS G12V group. Multivariate analysis demonstrated that the KRAS G12V mutation was an independent prognostic factor in stage Ⅰ tumors, whereas the KRAS non-G12V mutation was not. KRAS G12V was associated with early recurrence and locoregional recurrence. ConclusionsThe KRAS G12V mutation was associated with aggressive clinical-pathologic phenotype and early recurrence. To note, this mutation exhibited a significantly worse prognosis in patients with part-solid and stage Ⅰ lung adenocarcinoma. Meanwhile, the prognostic significance of KRAS G12C and G12V variants was comparable.

Abstract 4048: Comparative effects of SOS1 inhibitor with IL-6 blockade and their combination on Kras mutant lung cancer

Abstract Despite recent advances in treatment, lung adenocarcinoma (LUAD), particularly harboring Kras mutations, is still the leading cause of cancer mortality. We and others have previously shown that Kras mutant LUAD development and progression is associated with intrinsic inflammatory characteristics driven by epithelial NF-κB activation, increased levels of IL-6 (which is transcriptionally regulated by NF-κB), and activation of the IL-6-responsive transcription factor STAT3. We further demonstrated that IL-6 blockade suppresses lung tumorigenesis by reducing tumor-associated immunosuppressive myeloid populations and induction of a T cell-mediated anti-tumor response. The recent discovery of a novel SOS1-targeting compound (BI-3406) that selectively inhibits the KRAS-SOS1 interaction and leads to anti-proliferative activity has opened up the possibility to more efficiently inhibit Kras mutant lung cancer. Accordingly, in this study we used a mouse model of KRAS mutant LUAD (CC-LR) to compare the therapeutic effects of BI-3406, IL-6 blockade, and MEK inhibitor (trametinib). We also tested whether inhibiting IL-6 driven pro-tumor immune response would provide a synergistic/additive response and increase the tumor inhibitory effect of either BI-3406 or Trametinib. Eight cohorts of 14-week-old female mice were treated with either anti-IL-6 antibody, trametinib, or BI-3406, or their combinations for 4 weeks, and their tumor burden was compared at the end of study. We observed a significant reduction in tumor multiplicity and tumor area in response to all monotherapies, with the highest effect in the trametinib treated cohort. Neither combination therapy led to decreased tumor burden over either of the monotherapy regimens in our model. However, we observed an increase in T helper 1 and CD8 T expressing IFNγ cell populations in the cohort treated with Trametinib and anti-IL-6 Ab combination. In addition, using qPCR analysis of RNA extracted from the whole lung we found a higher expression of IFNγ along with a reduced expression of IL-10 in the cohort treated with BI-3406 and anti-IL-6 Ab combination. This suggests that these combinations may have the potential to improve the anti-tumor immune responses. Hence, we conclude that further adjustment of the treatment regimen (doses, interval, duration) may allow to obtain improved anti-tumor efficacy with these combinations. Overall, this study provides a new research perspective for the improvement of currently available treatment modalities for patients with Kras mutant lung cancer that could be further explored preclinically. Citation Format: Katherine E. Larsen, Maria J. Arredondo Sancristobal, Melody Zarghooni, Avantika Krishna, Maria T. Grimaldo, Nastaran Karimi, Arnav Gaitonde, Michael J. Clowers, Seyed Javad Moghaddam. Comparative effects of SOS1 inhibitor with IL-6 blockade and their combination on Kras mutant 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 4048.

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 657: Discovery of a novel brain penetrant SHP2 allosteric inhibitor with anti-tumor effects

Abstract KRAS is the most frequently dysregulated oncogene with high prevalence in non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and pancreatic ductal adenocarcinoma (PDAC). Currently, FDA-approved sotorasib and adagrasib provide ground breaking through therapies for cancer patients harboring KRAS G12C mutation. However, there is still high unmet medical needs for inhibitors targeting broader KRAS-driven tumor, especially a substantial unmet medical need for agents that can penetrate BBB to control the brain metastases. SHP2, a key activator of KRAS located in the upstream of RAS pathway, facilitates the conversion of GDP-bound KRAS (off) state to GTP-bound KRAS (on) state serving as a key mechanism of KRAS activation. Therefore, SHP2 could be a promising therapeutic target to shut down the oncogenic RAS pathway as a broad combination partner with RAS/RAF/MEK/ERK pathway inhibitors. Here we report a highly potent and selective brain penetrant SHP2 inhibitor that is designed to target solid tumors metastazied to brain. KT-01766 showed excellent potency against SHP2 protein in various biochemical and cellular assays and showed the robust synergistic effect with RAS pathway inhibitors. KT-01766 as a mono therapy and in combination with KRAS G12C inhibitors demonstrated great anti-tumor efficacy with no apparent toxicity in mouse xenograft models harboring KRAS mutations. Furthermore, robust anti-tumor acitivty was confirmed in intracranial brain tumor model, supporting its potential to control brain-metastasized tumors. The present data suggest that KT-01766 is a potential candidate for combination therapy with agents targeting RAS pathway, as it is able to effectively suppress tumor growth both systemically and within the brain in KRAS mutated cancers. Citation Format: Miyeon Kim, Dongsu Kim, Yeejin Jeon, Kyeong Jin Yoon, Anna Jang, Mijung Lee, Dahye Jeon, Soyeon Jang, Jinhwan Kim, Eunji Kim, Jihoon Park, Victor Hong, Hayoun Jung, Sungpil Choi. Discovery of a novel brain penetrant SHP2 allosteric inhibitor with anti-tumor effects [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 657.

Abstract 2151: Additional impact of genetic ancestry over self-reported race/ethnicity to prevalence of KRAS mutations and allele-specific subtypes in non-small cell lung cancer

Abstract Background: KRAS mutation is the most common oncogenic driver in patients with non-small cell lung cancer (NSCLC). However, the interplay between self-reported race and/or ethnicity (SIRE) and genetically inferred ancestry (GIA) on KRAS mutations and allele-specific subtypes is largely unknown. Methods: A total of 3918 NSCLC patients from the Chinese OrigiMed (OM) cohort (n=2039) and Boston Lung Cancer Survival (BLCS) cohort (n=1879) were included, all of whom had baseline covariates including age, sex, clinical stage, SIRE, histology and smoking status. KRAS mutation and subtypes were determined by targeted NGS panels in each cohort, and KRAS mutation outcomes, including KRAS mutation, KRAS subtypes, and KRAS transversion and transition were analyzed. GIA was inferred from paired germline data for patients in the BLCS cohort. Multivariable and multinomial logistic regressions were employed to assess the associations between SIRE, GIA and KRAS outcomes. Results: In the entire cohort, 804 (20.5%) patients harbored KRAS mutation, with a considerably higher prevalence of 31.5% observed in BLCS compared to 10.4% in OM cohort. Patients with self-identified Asian was associated with a significantly lower rate of KRAS mutation (OR = 0.44, 95% CI:0.22 - 0.81, P = 0.01), transversion substitutions (OR = 0.24, 95% CI:0.10 - 0.62, P = 0.003) and KRASG12C (OR = 0.17, 95% CI:0.04 - 0.72, P = 0.02) compared to SIRE-White patients after adjusting for confounders including smoking status, sex and histology. The association of SIRE and KRAS driver mutation was further modified by sex, with Asian women demonstrating lower risk compared to Asian men, while the opposite trend was observed in SIRE-White patients. Increased European ancestry was associated with a higher risk of KRAS mutation (OR per 10% increase = 1.05, 95% CI:1.00 - 1.09, P = 0.03), particularly with more transition substitutions (OR per 10% increase = 1.10, 95% CI:1.01 - 1.21, P = 0.03) and KRASG12D (OR per 10% increase = 1.18, 95% CI:1.04 - 1.34, P = 0.01). This association was even more pronounced in patients of increased Northern European ancestry. Furthermore, even among SIRE-White patients, a 10% increase in European ancestry was associated with an additional 6% increase in the likelihood of KRAS mutations (OR = 1.06, 95% CI: 1.01 - 1.12, P = 0.03), while a 10% increase in Admixed American ancestry was associated with a 6% decreased likelihood of KRAS mutations (OR = 0.94, 95% CI: 0.88 - 0.99, P = 0.04). Results were validated in an independent cohort of 1450 patients. Conclusion: Self-reported race/ethnicity and genetic ancestry are both associated with KRAS driver mutations and allele-specific subtypes of NSCLC. This work highlights the significance of integrating both into population-based cancer research, aiming to refine clinical decision-making processes and mitigate health disparities. Citation Format: Xinan Wang, Kangcheng Hou, Biagio Ricciuti, Joao V. Alessi, Xihao Li, Federica Pecci, Rounak Dey, Jia Luo, Mark M. Awad, Alexander Gusev, Xihong Lin, Bruce E. Johnson, David C. Christiani. Additional impact of genetic ancestry over self-reported race/ethnicity to prevalence of KRAS mutations and allele-specific subtypes in 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 2151.

Abstract 4325: CX-4945 (Silmitasertib) induces cell death through impairment of lysosomal utilization in KRAS mutant cholangiocarcinoma cell lines

Abstract Purpose: Macropinocytosis is a non-selective endocytosis that uptakes extracellular substances such as nutrients and macromolecules into the cells. In KRAS-driven cancer such as pancreatic ductal adenocarcinoma, the macropinocytosis and following lysosomal utilization are enhanced to overcome metabolic stress. In this study, we investigated the role of CK2 in micropinocytosis and the following metabolic process in the KRAS mutant CCA cell line. Materials and Methods: The BSA uptake indicating micropinocytosis was performed by flow cytometry using the HuCCT1, cholangiocarcinoma cell line harboring KRAS mutation. To validate macropinosome, the Rab7 and LAMP2 were labeled and analyzed via immunocytochemistry and Western blot. The CX-4945, CK2 inhibitor, was used to investigate the role of CK2 in micropinocytosis and following lysosomal metabolism. Results: The HuCCT1, a KRAS mutant CCA cell line, showed micropinocytosis. Although CX-4945, a CK2 inhibitor, induced morphological changes accompanied by accumulation of intracellular vacuoles and cell death rate, the level of micropinocytosis was not changed. The vacuoles accumulated in the cell were identified as late macropinosomes representing Rab7 before fusion with lysosomes. In addition, the CX-4945 suppressed LAMP2 expression following inhibition of the Akt-mTOR signaling pathway which interrupts mature macropinosome and lysosomal metabolic utilization. Conclusion: These results support the utilization of micropinocytosis as an energy source even in CCA cell line harboring KRAS mutation. The inhibition of CK2, the regulator of macropinosome maturation, by CX-4945, alters lysosome dependent metabolism and leads to cell death in KRAS mutant CCA cell lines. Citation Format: Chorong Kim, Da Sol Lee, Minwoo Han, Seonmin Lee, Kyu-pyo Kim, Changhoon Yoo. CX-4945 (Silmitasertib) induces cell death through impairment of lysosomal utilization in KRAS mutant cholangiocarcinoma cell lines [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 4325.