KRAS Protein Research Articles

AACR Cancer Progress Report 2021: Discovery Science Driving Clinical Breakthroughs.

AACR Cancer Progress Report 2021: Discovery Science Driving Clinical Breakthroughs.

Escaping KRAS: Gaining Autonomy and Resistance to KRAS Inhibition in KRAS Mutant Cancers.

Simple SummaryWhile KRAS is a driver oncogene, tumor cells can acquire mutant KRAS independency by activating pathways that functionally substitute for mutant KRAS. These KRAS-independent tumor cells exhibit a mesenchymal phenotype, readily primed for potential metastasis. The activation of YAP and/or RSK-mTOR pathways and mutations in LKB1, KEAP1, and/or NRF2 are associated with mutant KRAS autonomy. These alterations rewire survival signaling and metabolic processes originally governed by mutant KRAS. The presence of KRAS-independent cells is associated with the heterogeneity of KRAS mutant cancers, as well as variable responses to therapies. Notably, KRAS G12C-specific inhibitors appear to be effective only in tumors dependent on mutant KRAS for their survival. Therefore, determining KRAS dependency will be critical for selecting patients who should be treated with mutant-specific inhibitors. Furthermore, elucidating underlying mechanisms of KRAS autonomy is crucial towards developing optimal treatment strategies for KRAS-independent tumors.Activating mutations in KRAS are present in 25% of human cancers. When mutated, the KRAS protein becomes constitutively active, stimulating various effector pathways and leading to the deregulation of key cellular processes, including the suppression of apoptosis and enhancement of proliferation. Furthermore, mutant KRAS also promotes metabolic deregulation and alterations in the tumor microenvironment. However, some KRAS mutant cancer cells become independent of KRAS for their survival by activating diverse bypass networks that maintain essential survival signaling originally governed by mutant KRAS. The proposed inducers of KRAS independency are the activation of YAP1 and/or RSK-mTOR pathways and co-mutations in SKT11 (LKB1), KEAP1, and NFE2L2 (NRF2) genes. Metabolic reprogramming, such as increased glutaminolysis, is also associated with KRAS autonomy. The presence or absence of KRAS dependency is related to the heterogeneity of KRAS mutant cancers. Epithelial-to-mesenchymal transition (EMT) in tumor cells is also a characteristic phenotype of KRAS independency. Translationally, this loss of dependence is a cause of primary and acquired resistance to mutant KRAS-specific inhibitors. While KRAS-dependent tumors can be treated with mutant KRAS inhibitor monotherapy, for KRAS-independent tumors, we need an improved understanding of activated bypass signaling pathways towards leveraging vulnerabilities, and advancing therapeutic options for this patient subset.

A structural model of a Ras-Raf signalosome.

The protein K-Ras functions as a molecular switch in signaling pathways regulating cell growth. In the human mitogen-activated protein kinase (MAPK) pathway, which is implicated in many cancers, multiple K-Ras proteins are thought to assemble at the cell membrane with Ras effector proteins from the Raf family. Here we propose an atomistic structural model for such an assembly. Our starting point was an asymmetric guanosine triphosphate-mediated K-Ras dimer model, which we generated using unbiased molecular dynamics simulations and verified with mutagenesis experiments. Adding further K-Ras monomers in a head-to-tail fashion led to a compact helical assembly, a model we validated using electron microscopy and cell-based experiments. This assembly stabilizes K-Ras in its active state and presents composite interfaces to facilitate Raf binding. Guided by existing experimental data, we then positioned C-Raf, the downstream kinase MEK1 and accessory proteins (Galectin-3 and 14-3-3σ) on and around the helical assembly. The resulting Ras-Raf signalosome model offers an explanation for a large body of data on MAPK signaling.

Cobimetinib Plus Gemcitabine: An Active Combination in KRAS G12R-Mutated Pancreatic Ductal Adenocarcinoma Patients in Previously Treated and Failed Multiple Chemotherapies.

Purpose: The KRAS proto-oncogene is involved in the RAS/MAPK pathway. KRAS is present in the wild type or mutated forms. The oncogene KRAS is frequently mutated in various cancers. At the time that amino acid glycine is mutated, KRAS protein acquires oncogenic properties that result in the tumor cell growth, proliferation, and cancer progression. There has been limited understanding of the different mutations at codon 12. The consequences of such mutations is not fully understood. Various G12X mutations in pancreatic cancer patients have been examined, with the most common mutations being G12D (40%), G12V (30%), and G12R (15–20%). Now we are understanding that G12X mutations in the KRAS are not all equal.Methods: In a single-arm exploratory study, we accrued 13 KRAS-G12X-mutated pancreatic patients (KRAS G12D, G12V, and G12R). They were divided into two groups: group 1 consisted of seven patients with G12D and G12V and group 2 included six patients with the KRAS G12R mutation. All patients were treated with the combination of gemcitabine at 1250 mg/m2 intravenous weekly for 3 weeks and oral cobimetinib 20 mg b.i.d. for 3 weeks. This was followed by a week of rest before the initiation of the next cycle.Results: In the first cohort, seven patients were on treatment, all of whom progressed and died within the 2 months of the study. In the second cohort, one of six patients achieved partial response, and five achieved stable disease. Median progression-free survival was 6 months (9% confidence interval 3.0–9.3 months) and overall survival has been reached at 8 months. Common adverse reactions included rash, fatigue, nausea, and vomiting (grades 2 and 3). Cancer antigen CA19-9 decreased by >50% in all group 2 patients.Conclusion: Our pancreatic cancer patients were heavily pretreated (all had received FOLFIRINOX and gemcitabine/nab-paclitaxel) before the entry into our trial. Upon entry into our trial, all patients were treated with the combination of gemcitabine and oral cobimetinib. Therefore, this constituted the second exposure of the patients to gemcitabine. This study illustrates a new discovery, which can potentially target 15–20% of pancreatic cancer patients and allow for a significant improvement in their prognosis. We will be conducting randomized phase II trials to substantiate our findings.

Conformational Fluctuations in GTP-Bound K-Ras: A Metadynamics Perspective with Harmonic Linear Discriminant Analysis.

Biomacromolecules often undergo significant conformational rearrangements during function. In proteins, these motions typically consist in nontrivial, concerted rearrangement of multiple flexible regions. Mechanistic, thermodynamics, and kinetic predictions can be obtained via molecular dynamics simulations, provided that the simulation time is at least comparable to the relevant time scale of the process of interest. Because of the substantial computational cost, however, plain MD simulations often have difficulty in obtaining sufficient statistics for converged estimates, requiring the use of more-advanced techniques. Central in many enhanced sampling methods is the definition of a small set of relevant degrees of freedom (collective variables) that are able to describe the transitions between different metastable states of the system. The harmonic linear discriminant analysis (HLDA) has been shown to be useful for constructing low-dimensional collective variables in various complex systems. Here, we apply HLDA to study the free-energy landscape of a monomeric protein around its native state. More precisely, we study the K-Ras protein bound to GTP, focusing on two flexible loops and on the region associated with oncogenic mutations. We perform microsecond-long biased simulations on the wild type and on G12C, G12D, G12 V mutants, describe the resulting free-energy landscapes, and compare our predictions with previous experimental and computational studies. The fast interconversion between open and closed macroscopic states and their similar thermodynamic stabilities are observed. The mutation-induced effects include the alternations of the relative stabilities of different conformational states and the introduction of many microscopic metastable states. Together, our results demonstrate the applicability of the HLDA-based protocol for the conformational sampling of multiple flexible regions in folded proteins.

KRAS G12C fragment screening renders new binding pockets

ABSTRACT KRAS genes belong to the most frequently mutated family of oncogenes in cancer. The G12C mutation, found in a third of lung, half of colorectal and pancreatic cancer cases, is believed to be responsible for a substantial number of cancer deaths. For 30 years, KRAS has been the subject of extensive drug-targeting efforts aimed at targeting KRAS protein itself, but also its post-translational modifications, membrane localization, protein–protein interactions and downstream signalling pathways. So far, most KRAS targeting strategies have failed, and there are no KRAS-specific drugs available. However, clinical candidates targeting the KRAS G12C protein have recently been developed. MRTX849 and recently approved Sotorasib are covalent binders targeting the mutated cysteine 12, occupying Switch II pocket. Herein, we describe two fragment screening drug discovery campaigns that led to the identification of binding pockets on the KRAS G12C surface that have not previously been described. One screen focused on non-covalent binders to KRAS G12C, the other on covalent binders.

Current Landscape of Non-Small Cell Lung Cancer: Epidemiology, Histological Classification, Targeted Therapies, and Immunotherapy.

Simple SummaryThe abundance and the dynamic of the studies on NSCLC require frequent summaries of the current achievements in the field. In our review, we aimed to update the status of knowledge about NSCLC, combining its epidemiology, classification novelties, tumor molecular basis, and two of the most promising approaches in cancer treatment: targeted therapy and immunotherapy.Non-small cell lung cancer (NSCLC) is a subtype of the most frequently diagnosed cancer in the world. Its epidemiology depends not only on tobacco exposition but also air quality. While the global trends in NSCLC incidence have started to decline, we can observe region-dependent differences related to the education and the economic level of the patients. Due to an increasing understanding of NSCLC biology, new diagnostic and therapeutic strategies have been developed, such as the reorganization of histopathological classification or tumor genotyping. Precision medicine is focused on the recognition of a genetic mutation in lung cancer cells called “driver mutation” to provide a variety of specific inhibitors of improperly functioning proteins. A rapidly growing group of approved drugs for targeted therapy in NSCLC currently allows the following mutated proteins to be treated: EGFR family (ERBB-1, ERBB-2), ALK, ROS1, MET, RET, NTRK, and RAF. Nevertheless, one of the most frequent NSCLC molecular sub-types remains without successful treatment: the K-Ras protein. In this review, we discuss the current NSCLC landscape treatment focusing on targeted therapy and immunotherapy, including first- and second-line monotherapies, immune checkpoint inhibitors with chemotherapy treatment, and approved predictive biomarkers.

Synthesis, Molecular Docking and Biological Evaluation of Novel 3-substituted Pyrido[2,3-b]pyrazine Derivatives

A novel series of 3-substituted pyrido[2,3-b]pyrazine derivatives were synthesized using Suzuki and Buchwald-Hartwig coupling reactions. All the synthesized derivatives were screened for in-vitro anti-inflammatory activities as inhibitors of pro-cytokines TNF-alpha and IL-6. Among them, compounds 7a, 7b, 7d, 9a and 9c showed LPS-induced TNF-α secretion of 79 ± 1.2, 98.5 ± 1.6, 95.5 ± 2.1, 92.4 ± 1.9, and 88.9 ± 1.2 pg mL-1 respectively at a concentration of 20 μM. Similarly, compounds 7a, 7b and 7d, 9a and 9c showed LPS-induced IL-6 secretion of 59.5 ± 2.1, 77 ± 2.1, 89 ± 2.4, 65.4 ± 2.1, and 74 ± 1.9 pg mL-1 respectively at a concentration of 20 μM. In addition, the compounds were tested for cytotoxic activity against A549, MCF-7, K562 & Hela cancer cell lines and all the compounds showed good biological profiles on cancer cell lines. In silico studies revealed that compounds 9a and 9b could bind strongly with the active site of the TNF-α protein structure and formed three hydrogen bonds with Gln61, Tyr151 amino acids with binding energies of -5.9 and -5.7 kcal/mol. Compound 9b formed three strong hydrogen bonds with amino acids of the TGF-β protein with a binding energy of -6.0 kcal/mol. Compound 7e bound strongly with the KRAS protein structure, making seven strong hydrogen bonds with amino acids Gly13, Val29, Asn116, Asp119, Ser145 and Ala146, each with a binding energy of -8.0 kcal/mol. Compound 7b bound strongly with the KRAS protein structure, making four strong hydrogen bonds with amino acids Asn116, Lys117, Asp119, Ser145, each with a binding energy of -8.2 kcal/mol.

SiRNA-Loaded Hydroxyapatite Nanoparticles for KRAS Gene Silencing in Anti-Pancreatic Cancer Therapy.

Pancreatic carcinoma (PC) is greatly induced by the KRAS gene mutation, but effective targeted delivery for gene therapy has not existed. Small interfering ribonucleic acid (siRNA) serves as an advanced therapeutic modality and holds great promise for cancer treatment. However, the development of a non-toxic and high-efficiency carrier system to accurately deliver siRNA into cells for siRNA-targeted gene silencing is still a prodigious challenge. Herein, polyethylenimine (PEI)-modified hydroxyapatite (HAp) nanoparticles (HAp-PEI) were fabricated. The siRNA of the KRAS gene (siKras) was loaded onto the surface of HAp-PEI via electrostatic interaction between siRNA and PEI to design the functionalized HAp-PEI nanoparticle (HAp-PEI/siKras). The HAp-PEI/siKras was internalized into the human PC cells PANC-1 to achieve the maximum transfection efficiency for active tumor targeting. HAp-PEI/siKras effectively knocked down the expression of the KRAS gene and downregulated the expression of the Kras protein in vitro. Furthermore, the treatment with HAp-PEI/siKras resulted in greater anti-PC cells’ (PANC-1, BXPC-3, and CFPAC-1) efficacy in vitro. Additionally, the HAp-PEI exhibited no obvious in vitro cytotoxicity in normal pancreatic HPDE6-C7 cells. These findings provided a promising alternative for the therapeutic route of siRNA-targeted gene engineering for anti-pancreatic cancer therapy.

Tumor Diagnosis by Genetic Markers Protein P-53, p16, C-MYC, N-MYC, Protein K-Ras, and Gene Her-2 Neu is this possible? Tumor Diagnosis by Genetic Markers C-MYC, N-MYC, Protein P-53, p16

Currently, cancer is among the leading causes of death in the world. It is the correct diagnosis that gives the patient a better chance of healing. The detection of genetic markers, macromolecules that are produced directly by the tumor or by the host, has the potential to be one of the diagnostic tests. This paper is a review article, with the objective of addressing tumor markers c-MYC, n-MYC, protein p-53, protein Kras, and the gene HER-2 neu regarding the diagnosis and prognosis of patients with cancer, studies demonstrated that c-MYC is expressed in several malignant tumors because it affects proliferation and cellular metabolism, P-53 in breast and lung tumors is overexpressed, Regarding p16 it has been suggested that cytoplasmic also overexpression as kind of cancer predictor. K-ras is used to determine survival time and free time for neoplasm. It is an effective marker for monoclonal antibody therapy. The marker HER-2 neu and N-MYC are indicative of poor prognosis, conclusions: application of these markers is being increasingly used for expressing specificity and sensitivity in different areas of tumor characterization, in addition to being related to the therapeutic conduct to be followed in tumor treatment. Keywords: genetic marker; p16; Tumor marker; cancer diagnosis;p53

Molecular-genetic mechanisms of the signal cascade RAS-RAF-MEK-ERK associated with the development of the tumor process and the purpose of targeted drugs for colorectal cancer

Colorectal cancer occupies one of the leading positions in the world in the structure of cancer incidence. The vital processes of cancer cells largely depend on the production of growth factors and their receptors. One of these is epidermal growth factor (EGFR), which is a tyrosine kinase receptor for cell membranes. Normally, binding of EGFR ligands and transforming growth factor alpha (TGFα) induces receptor activation, which triggers ERK and PI3K signaling pathways that control cell proliferation, migration, invasion, and many other processes. It was found that in 80% of cases, colorectal cancer occurs as a result of EGFR overexpression, which leads to increased growth and division of tumor cells due to hyperactivation of the RAS-RAF-MEKERK signaling cascade. The RAS-RAF-MEK-ERK cascade is a pathway that regulates cell proliferation, cell cycle, and cell migration. In the development of human cancer, mutations of the RAS/RAF family are most often the cause of dysregulation of signal transduction through this pathway. According to current data, about a third of all malignant neoplasms are associated with mutations in the genes of the RAS family, which include HRAS, KRAS, NRAS, RRAS, and other homologous proteins. Proteins of the RAS family are involved in the activation of tyrosine kinase signaling pathways, which leads to gene mutations. This process determines proliferative activity, ability to differentiate, metastasis, avoidance of apoptosis, induction of angiogenesis. Permanent RAS activation leads to malignant cell degeneration. Thus, the expression and mutation of the EGFR gene are associated with various variants of tumor progression and poor prognosis in malignant neoplasms of various localizations. Over the past decades, significant advances have been made in the treatment of metastatic colorectal cancer. However, the expansion of the spectrum of effective anticancer drugs also creates a number of difficulties in choosing the optimal drug therapy regimens in patients with metastases of colorectal cancer.

LncRNA IGFL2-AS1 Promotes the Proliferation, Migration, and Invasion of Colon Cancer Cells and is Associated with Patient Prognosis.

BackgroundLncRNAs play an important role in tumor initiation and development. However, the underlying involvement of lncRNA expression in colorectal carcinoma remains to be clarified.MethodsAll analyses were performed in R software v4.0, SPSS v13.0, and GraphPad Prism 8. The “limma” package was used to identify differentially expressed lncRNAs between two groups with the threshold of |logFC| >1 and P <0.05. The “Survival” package was used to conduct survival analysis. HCT8 and SE480 cell lines were used to conduct further phenotype experiments, including transwell, wound-healing, CCK8 and colony formation assay. Gene set enrichment analysis was used to explore the biological pathway difference in high and low IGFL2-AS1 patients.ResultsThe lncRNA IGFL2-AS1 was highly expressed in colon adenocarcinoma (COAD) tissue and cell lines (HCT116, HCT8, HCT129, and SW480). The COAD patients with high IGFL2-AS1 were associated with a worse prognosis. Meanwhile, the knockdown of IGFL2-AS1 could significantly suppress the proliferation and invasion of COAD cells. Gene set enrichment analysis showed that the top five biological pathways involving IGFL2-AS1 were angiogenesis, epithelial–mesenchymal transition, KRAS signaling, myogenesis, and coagulation. Western blot results showed that the inhibition of IGFL2-AS1 could significantly reduce the N-cadherin, HIF1A and KRAS protein expression, yet increase the E-cadherin protein level. IGFL2-AS1 was also positively correlated with M0 macrophages, M2 macrophages, and neutrophils but negatively correlated with CD4+ memory T cells and CD8+ T cells.ConclusionIGFL1-AS1 could seriously worsen patient outcomes and facilitate COAD progression, thus serving as an independent tumor marker.

PBDEs affect inflammatory and oncosuppressive mechanisms via the EZH2 methyltransferase in airway epithelial cells

AimsWe aimed to investigate the effect of PBDEs (47, 99, 209) on cellular events involved in epigenetic modification, inflammation, and epithelial mesenchymal transition (EMT). Materials and methodsWe studied: 1) ERK1/2 phosphorylation; 2) Enhancer of Zester Homolog 2 (EZH2); 3) Histone H3 tri-methylated in lysine 27 (H3K27me3); 4) K-RAS; 5) silencing disabled homolog 2-interacting protein gene (DAB2IP), 6) let-7a; 7) Muc5AC/Muc5B, and 8) IL-8 in a 3D in vitro model of epithelium obtained with primary Normal Human Bronchial Epithelial cells (pNHBEs) or A549 cell line, chronically exposed to PBDEs (47, 99, 209). Key findingsPBDEs (10 nM, 100 nM and 1 μM) increased ERK1/2 phosphorylation, and EZH2, H3K27me3, and K-RAS protein expression, while decreased DAB2IP and Let-7a transcripts in pNHBEs ALI culture. Furthermore PBDEs (47, 99) (100 nM) increased Muc5AC and Muc5B mRNA, and PBDE 47 (100 nM) IL-8 mRNA via EZH2 in pNHBEs. Finally, PBDEs (100 nM) affected EZH2, H3K27me3, K-RAS protein expression, and DAB2IP, Let-7a transcripts and cell invasion in A549 cells. Gsk343 (methyltransferase EZH2 inhibitor) (1 mM) and U0126 (inhibitor of MEK1/2) (10 μM) were used to show the specific effect of PBDEs. SignificancePBDE inhalation might promote inflammation/cancer via EZH2 methyltransferase activity and H3K27me3, k-RAS and ERk1/2 involvement, generating adverse health outcomes of the human lung.

Regulation of KRAS protein expression by miR-544a and KRAS-LCS6 polymorphism in wild-type KRAS sporadic colon adenocarcinoma.

Colorectal carcinoma (CRC) results from the accumulation of genetic mutations and alterations in signaling pathways. KRAS is mutated in 40% of CRC cases and is involved in increased tumor cells proliferation and survival. Although KRAS mutations are a dominant event in CRC tumorigenesis, increased wild-type KRAS expression has a similar effect on accelerated tumor growth. In this study, we investigated the KRAS status in correlation with clinicopathological features in sporadic CRC and more importantly the role of let-7a-5p and miR-544a-3p in the regulation of wild-type KRAS protein expression in the tumor center (T1) and invasive tumor front (T2). Analysis showed that 39.1% of tumor samples had KRAS mutations. In wild-type KRAS tumors, 62.0% were positive for KRAS protein expression and there was a higher percentage of KRAS-positive tumor cells and a higher intensity of immunohistochemical reaction in T2 than in T1 samples. This could not be attributed to differences in KRAS mRNA levels, suggesting regulation via miR-544a-3p expression which was significantly decreased in T2 samples. Furthermore, we demonstrated that tumor samples carrying the KRAS-LCS6 variant allele had significantly higher protein expression of the wild-type KRAS. Our results suggest the role of the KRAS-LCS6 polymorphism and miR-544a-3p expression in the regulation of wild-type KRAS protein expression in sporadic CRC.

Abstract 2130: Association between miR-18a-3p, miR-143 and expression of the wild-type KRAS gene in patients with sporadic colorectal cancer

Abstract Introduction: Colorectal cancer (CRC) has the third-highest incidence and the fourth-highest mortality from cancer globally. In the genesis of CRC, mutations have been identified in the KRAS gene associated with prognosis and therapeutic management, however, the epigenetic mechanisms that modulate the expression of KRAS in CRC patients when there are no mutations in the gene are not well known. miR-18a-3p and miR-143 have been described to target KRAS and thus may regulate gene expression. Objective: To determine if there is a relationship between the expression of the wild-type KRAS gene (mRNA and protein) and the expression levels of miR-143 and miR-18a-3p in FFPE tissue samples from CRC patients. Methodology: RNA was extracted from FFPE tumor tissue samples from 51 cases of patients with CRC. Expression of wild-type KRAS mRNA, miR-143 and miR-18a-3p was evaluated by qRT-PCR and expression levels of KRAS protein by immunohistochemistry. Contingency tables, Pearson's Chi-square test and logistic regression analysis were used for data analysis. Results: Regarding the expression levels of wild-type KRAS protein, a higher number of cases with a cytoplasmic expression of the protein was identified (98%) compared to the expression in the cell membrane (31%). In 7,8% (4/51) of the cases, it was not possible to determine the mRNA expression of the KRAS gene, while in 21,3% (10/47) no expression was detected. In 78,7% of the cases in which KRAS mRNA expression was detected, low (40,4%; 19/47) and high (38,3%; 18/47) expression were categorized according to the results found. It was not evidenced by a correlation between protein expression levels and mRNA expression levels of the wild-type KRAS gene. When determining the expression levels of miR-18a-3p and miR-143, it was found that 64,7% (33/51) of cases had an absent expression of miR-18a-3p and 54,9% (28/51) of cases had an absent or very low expression for miR-143. By means of a logistic regression analysis adjusted by age, stage, sex and presence or absence of KRAS protein in the membrane, it was determined that the presence of miR-18a-3p is inversely related to KRAS mRNA levels significantly (p=0,048; OR=0,346) while miR-143 only shows a tendency to an inverse correlation with KRAS mRNA levels (p=0,119; OR=0,469). Conclusions: No correlation was found between mRNA and protein expression levels of the wild-type KRAS gene, suggesting that protein levels do not strictly depend on mRNA levels. A significant inverse association was found between miR-18a-3p and wild-type KRAS mRNA levels. Citation Format: Jehison-Alirio Herrera-Pulido, Orlando Ricaurte Guerrero, Jinneth Acosta Forero, Gustavo Hernández Suárez, Pablo Moreno-Acosta, María-Carolina Sanabria Salas, Humberto Arboleda Granados, Alfredo Romero Rojas, Martha Lucía Serrano López. Association between miR-18a-3p, miR-143 and expression of the wild-type KRAS gene in patients with sporadic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2130.

Abstract 2273: MAPK signaling pathway disruption explains the anticancer mechanism for polyisoprenylated cysteinyl amide inhibitors (PCAIs)

Abstract The Mitogen-activated protein kinase (MAPK) pathway is one of the key signaling pathways that regulates normal cell proliferation, differentiation, and survival. Abnormalities of the pathway play vital roles in cancer initiation and progression. Ras small GTPases that are key upstream mediators of the MAPK pathway are mutated in 20%-30% of human tumors. KRAS is the most frequently mutated isoform of the RAS proteins, accounting for up to 86% of RAS mutant cancer cases. Mutant KRAS is prevalent in lung, colon, and pancreatic cancers, which considered to be the most aggressive and account for the most cancer deaths in the US. To help mitigate this problem and fill the therapeutic need that has lingered for decades, polyisoprenylated cysteinyl amide inhibitors (PCAIs) were designed and synthesized to target the hyperactivity of aberrant monomeric G-Proteins. Recently, the PCAIs underwent a synthetic optimization to mitigate the excessive hydrophobicity of the earlier analogs. While the newer agents are more polar and have maintained the effectiveness of the earlier PCAIs against cancer cell lines driven by mutant KRAS proteins, the specific anticancer mechanisms are still unclear. Thus, we explored the effects of the newer PCAIs on the MAPK pathway using lung cancer A549 cells which harbor the KRAS mutation. When the cells were treated with NSL-YHJ-2-27 for 48 h, the KRAS protein levels dropped by 40% at 5μM compared to the controls. However, no significant difference was observed in the levels of total or phosphorylated B- and C-Raf proteins. Moreover, it was observed that the PCAIs immensely stimulated the MAPK pathway in a concentration-dependent manner following treatment with 1, 2, and 5μM of NSL-YHJ-2-27. The phosphorylation of MEK1/2, ERK1/2, and p90RSK significantly increased by 84%, 59%, and 160%, respectively, relative to controls following treatment for 48h at 5 µM of NSL-YHJ-2-27. Interestingly, a non-farnesylated analog, NSL-YHJ-2-62, did not elicit similar effects on the MAPK pathway. Although ERK stimulation has mostly been associated with cell survival and proliferation, various studies show that ERK activation can also mediate apoptosis depending on the intensity and duration of the stimulation. These data reveal that the Ras-MAPK signaling axis is involved in the anticancer effects of the PCAIs. The PCAIs may thus be considered as possible agents for the unmet therapeutic needs of patients with aberrantly hyperactive G-proteins. Citation Format: Nada Tawfeeq, Yonghao Jin, Nazarius Lamango. MAPK signaling pathway disruption explains the anticancer mechanism for polyisoprenylated cysteinyl amide inhibitors (PCAIs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2273.

Abstract CT210: Trial in Process: Phase 1 studies of BI 1701963, a SOS1::KRAS Inhibitor, in combination with MEK inhibitors, irreversible KRASG12C inhibitors or irinotecan.

Abstract The SOS1i::KRAS inhibitor BI 1701963 is the first direct RAS signaling modifier to enter phase I clinical trials both as a monotherapy as well as in combination. SOS1::KRAS inhibitors exhibit activity against a broad spectrum of KRAS alleles, including the major G12D/V/C and G13D oncoproteins, while sparing the interaction of KRAS with SOS2. Here, we present pre-clinical data showing enhanced pathway modulation and synergistic anti-tumor effects following vertical pathway inhibition of BI 1701963 in combination with mitogen-activated protein kinase inhibitors (MEKi; trametinib and BI 3011441) or KRAS G12C inhibitors (MRTX849 and BI 1823911). Furthermore, SOS1::KRAS inhibitor treatment sensitizes tumor cells to increased DNA damage in combination with irinotecan. Our results highlight the suitability of SOS1::KRAS inhibitors as a backbone in combination therapies targeting KRAS-dependent tumors. Pre-clinical combination data supported the start of multiple phase I trials investigating the safety, tolerability, recommended dose and preliminary efficacy of BI 1701963 alone and in combination with other anti-cancer agents. Combination trials of BI 1701963 with MEKi include cohorts of patients with KRAS mutant solid tumors, such as non-small cell lung cancer (NSCLC), colorectal cancer (CRC), cholangiocarcinoma and pancreatic adenocarcinoma. Trials exploring the combination of BI 1701963 with irreversible KRAS G12C inhibitors (MRTX849 and BI 1823911) will include cohorts of patients with KRAS G12C mutant NSCLC and CRC. In a further study, the combination of BI 1701963 with irinotecan is being evaluated in patients with KRAS mutant CRC. Primary endpoints include dose-limiting toxicities, treatment-emergent or -related adverse events. Secondary endpoints include pharmacokinetic and pharmacodynamic properties of combination regimens and preliminary efficacy. Citation Format: Marco H. Hofmann, Hengyu Lu, Ulrich Duenzinger, Daniel Gerlach, Francesca Trapani, Annette A. Machado, Joseph R. Daniele, Irene Waizenegger, Michael Gmachl, Dorothea Rudolph, Christopher P. Vellano, Marcelo Marotti, Vitomir Vucenovic, Timothy P. Heffernan, Joseph R. Marszalek, Mark P. Petronczki, Norbert Kraut. Trial in Process: Phase 1 studies of BI 1701963, a SOS1::KRAS Inhibitor, in combination with MEK inhibitors, irreversible KRASG12C inhibitors or irinotecan. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT210.

Abstract 2936: Ago2 induces Kras signaling and promotes tumor progression in mouse models of lung adenocarcinoma

Abstract Lung cancer is the deadliest malignancy in the United States. Non-small cell lung cancer (NSCLC) accounts for 85% of cases, and is frequently driven by activating mutations in the gene encoding the KRAS GTPase (e.g. KRASG12D). Our previous work demonstrated that Argonaute 2 (AGO2)—a component of the RNA induced silencing complex (RISC)—physically interacts with KRAS and stabilizes it at the protein level. In multiple cell lines, AGO2 knockdown reduces KRAS protein level and attenuates cell proliferation. We therefore hypothesized that AGO2 could promote KRASG12D-dependent NSCLC in vivo. To test the hypothesis, we evaluated the impact of Ago2 knockout in the KPC mouse model of NSCLC. In KPC mice, intratracheal delivery of adenoviral Cre drives lung-specific expression of a stop-floxed KRASG12D allele and biallelic ablation of p53. Simultaneous biallelic ablation of floxed Ago2 inhibited KPC lung nodule growth while reducing proliferative index and improving pathological grade. We next applied the KPHetC model, in which the clara cell-specific CCSP-driven Cre activates KRASG12D and ablates a single p53 allele. In these mice, Ago2 ablation also reduced tumor size and grade. In both models, Ago2 knockout inhibited ERK phosphorylation in tumor nodules, indicating impaired KRAS signaling. Furthermore, RNA-seq analysis of KPC nodules demonstrated broadly reduced expression of Kras-related genes. Taken together, our data demonstrate a novel pathogenic role for AGO2 in KRAS-dependent NSCLC. Given the prevalence of this malignancy and current difficulties in therapeutically targeting KRAS signaling, our work has future translational relevance. Citation Format: Jean C. Tien, Seema Chugh, Andrew E. Goodrum, Yunhui Cheng, Lisha Wang, Rahul Mannan, Xiaoming Wang, Vijaya L. Dommeti, Yuping Zhang, Alice Xu, Fengyu Su, Xuhong Cao, Sunita Shankar, Arul M. Chinnaiyan. Ago2 induces Kras signaling and promotes tumor progression in mouse models of lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2936.

Residue interaction networks of K-Ras protein with water molecules identifies the potential role of switch II and P-loop

The mutant K-Ras with aberrant signaling is the primary cause of several cancers. The proposed study investigated the influence of water molecules in K-Ras crystal structure, where they have a significant function by understanding their residue interaction networks (RINs). We analyzed the RINs of K-Ras with and without water molecules and determined their interaction properties. RINs were developed with the help of StructureViz2 and RINspector; further, the changes in K-Ras backbone flexibility were predicted with the DynaMine. We found that the residues K42, I142, and L159 are the hotspots from water, including the K-Ras-GTP complex with the highest residue centrality analysis (RCA) Z-score. The DynaMine prediction calculated the NMR S2 value for the frequently mutated positions G12, G13, and Q61 showing a minor shift in flexibility, which make up the P-Loop and switch II of the K-Ras protein. This flexibility shift can account for changes in conformational activity and the protein's GTPase activity, making it difficult to recognize by the effectors and exchange factors. Taken together, our study helps in understanding the functional importance of the water molecules in K-Ras protein and the impact of mutation that modulate the conformational state of the protein.

Single‐Molecule Bioelectronic Label‐Free Assay of both Protein and Genomic Markers of Pancreatic Mucinous Cysts’ in Whole Blood Serum

AbstractThe timely diagnosis of cystic pancreatic cancer precursors is of utmost importance to improve patients’ low survival rate. Fine‐needle aspiration cytology is endowed with low diagnostic sensitivity, while more effective is the assay of markers, such as a mutated KRAS, in the cyst fluids. Next‐generation sequencing, detecting down to a single copy of a genomic marker, enables early diagnosis but the diagnostic sensitivity of high‐grade cysts, likely to become malignant, is low. Assaying both mutated KRAS and MUC1 protein markers can improve diagnostic accuracy. Their detection in blood would also be minimally invasive. Here, the mucinous lesions markers, KRAS and MUC1, are both successfully assayed in blood serum at the physical limit with the label‐free “Single‐Molecule assay with a large Transistor—SiMoT.” This is a compelling proof of principle that the SiMoT platform holds high potential to enable a timely, minimally invasive, and accurate diagnosis of pancreatic cancer precursor cysts.