SHANK3 depletion leads to ERK signalling overdose and cell death in KRAS-mutant cancers.

The KRAS oncogene drives many common and highly fatal malignancies. These include pancreatic, lung, and colorectal cancer, where various activating KRAS mutations have made the development of KRAS inhibitors difficult. Here we identify the scaffold protein SH3 and multiple ankyrin repeat domain 3 (SHANK3) as a RAS interactor that binds active KRAS, including mutant forms, competes with RAF and limits oncogenic KRAS downstream signalling, maintaining mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) activity at an optimal level. SHANK3 depletion breaches this threshold, triggering MAPK/ERK signalling hyperactivation and MAPK/ERK-dependent cell death in KRAS-mutant cancers. Targeting this vulnerability through RNA interference or nanobody-mediated disruption of the SHANK3-KRAS interaction constrains tumour growth in vivo in female mice. Thus, inhibition of SHANK3-KRAS interaction represents an alternative strategy for selective killing of KRAS-mutant cancer cells through excessive signalling.

Honokiol enhances the sensitivity of cetuximab in KRASG13D mutant colorectal cancer through destroying SNX3-retromer complex.

Rationale : the proto-oncogene KRAS is frequently mutated in colorectal cancer (CRC), leading to inherent resistance against monoclonal antibodies targeting the epidermal growth factor receptor (EGFR), such as cetuximab. Therefore, addressing the primary resistance and expanding the indications for target therapy have become critical challenges. Methods : the screening of a natural product library against KRAS mutant CRC cells was conducted, leading to the discovery of a small molecule compound that sensitive to the KRASG13D mutation site. The anti-tumor activity of this small molecule compound in combination with cetuximab was evaluated using the KRASG13D mutant CRC models both in vivo and in vitro. This evaluation includes an examination of its effects on cell proliferation, viability, apoptosis, cell cycle progression, and tumor growth. Furthermore, RNA sequencing, western blot analysis, immunofluorescence, real-time quantitative PCR, and pull-down assays were employed to explore the molecular mechanisms underlying the synergistic anti-tumor effect of this small molecule compound in combination with cetuximab. Results : our study screened 882 compounds in KRAS mutant CRC cells and identified honokiol, a small molecule compound that exhibits specific sensitivity to KRASG13D mutant CRC cells. Furthermore, we revealed that the synergistic augmentation of cetuximab's sensitivity in vivo and in vitro models of KRASG13D mutant CRC in combination with honokiol. Mechanistically, honokiol suppresses SNX3-retromer mediated trafficking, thereby impeding lysosomal proteolytic capacity and inhibiting autophagy and macropinocytosis fluxes. Moreover, honokiol inhibits the conversion of RAS GDP to RAS GTP, heightening the susceptibility of KRASG13D CRC mutant cells to cetuximab. Conclusions : honokiol enhances the sensitivity of cetuximab by destroying SNX3 retromer in KRASG13D mutant CRC preclinical model. These findings present a promising strategy for expanding the indications of target therapy in KRAS mutant colorectal cancer patients.

Targeting cancer with small-molecule pan-KRAS degraders.

Mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) protein are highly prevalent in cancer. However, small-molecule concepts that address oncogenic KRAS alleles remain elusive beyond replacing glycine at position 12 with cysteine (G12C), which is clinically drugged through covalent inhibitors. Guided by biophysical and structural studies of ternary complexes, we designed a heterobifunctional small molecule that potently degrades 13 out of 17 of the most prevalent oncogenic KRAS alleles. Compared with inhibition, KRAS degradation results in more profound and sustained pathway modulation across a broad range of KRAS mutant cell lines, killing cancer cells while sparing models without genetic KRAS aberrations. Pharmacological degradation of oncogenic KRAS was tolerated and led to tumor regression in vivo. Together, these findings unveil a new path toward addressing KRAS-driven cancers with small-molecule degraders.

Targeted protein degradation using chimeric human E2 ubiquitin-conjugating enzymes.

Proteins can be targeted for degradation by engineering biomolecules that direct them to the eukaryotic ubiquitination machinery. For instance, the fusion of an E3 ubiquitin ligase to a suitable target binding domain creates a 'biological Proteolysis-Targeting Chimera' (bioPROTAC). Here we employ an analogous approach where the target protein is recruited directly to a human E2 ubiquitin-conjugating enzyme via an attached target binding domain. Through rational design and screening we develop E2 bioPROTACs that induce the degradation of the human intracellular proteins SHP2 and KRAS. Using global proteomics, we characterise the target-specific and wider effects of E2 vs. VHL-based fusions. Taking SHP2 as a model target, we also employ a route to bioPROTAC discovery based on protein display libraries, yielding a degrader with comparatively weak affinity capable of suppressing SHP2-mediated signalling.

Scalable production of siRNA-encapsulated extracellular vesicles for the inhibition of KRAS-mutant cancer using acoustic shock waves.

Extracellular vesicles (EVs) have emerged as a potential delivery vehicle for nucleic-acid-based therapeutics, but challenges related to their large-scale production and cargo-loading efficiency have limited their therapeutic potential. To address these issues, we developed a novel "shock wave extracellular vesicles engineering technology" (SWEET) as a non-genetic, scalable manufacturing strategy that uses shock waves (SWs) to encapsulate siRNAs in EVs. Here, we describe the use of the SWEET platform to load large quantities of KRASG12C-targeting siRNA into small bovine-milk-derived EVs (sBMEVs), with high efficiency. The siRNA-loaded sBMEVs effectively silenced oncogenic KRASG12C expression in cancer cells; they inhibited tumour growth when administered intravenously in a non-small cell lung cancer xenograft mouse model. Our study demonstrates the potential for the SWEET platform to serve as a novel method that allows large-scale production of cargo-loaded EVs for use in a wide range of therapeutic applications.

Inactivation of HIPK2 attenuates KRASG12D activity and prevents pancreatic tumorigenesis.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) features KRAS mutations in approximately 90% of human cases and excessive stromal response, termed desmoplastic reaction. Oncogenic KRAS drives pancreatic carcinogenesis by acting on both epithelial cells and tumor microenvironment (TME). We have previously shown that Homeodomain-Interacting Protein Kinase 2 (HIPK2) cooperates with KRAS in sustaining ERK1/2 phosphorylation in human colorectal cancers. Here, we investigated whether HIPK2 contributes to oncogenic KRAS-driven tumorigenesis in vivo, in the onset of pancreatic cancer. METHODS: We employed an extensively characterized model of KRASG12D-dependent preinvasive PDAC, the Pdx1-Cre;LSL-KRasG12D/+ (KC) mice. In these mice, HIPK2 was inhibited by genetic knockout in the pancreatic epithelial cells (KCH-/-) or by pharmacologic inactivation with the small molecule 5-IodoTubercidin (5-ITu). The development of preneoplastic acinar-to-ductal metaplasia (ADM), intraepithelial neoplasia (PanIN), and their associated desmoplastic reaction were analyzed. RESULTS: In Hipk2-KO mice (KCH-/-), ERK phosphorylation was lowered, the appearance of ADM was slowed down, and both the number and pathologic grade of PanIN were reduced compared to Hipk2-WT KC mice. The pancreatic lesion phenotype in KCH-/- mice was characterized by abundant collagen fibers and reduced number of αSMA+ and pSTAT3+ desmoplastic cells. These features were reminiscent of the recently described human "deserted" sub-TME, poor in cells, rich in matrix, and associated with tumor differentiation. In contrast, the desmoplastic reaction of KC mice resembled the "reactive" sub-TME, rich in stromal cells and associated with tumor progression. These observations were confirmed by the pharmacologic inhibition of HIPK2 in KC mice. CONCLUSION: This study demonstrates that HIPK2 inhibition weakens oncogenic KRAS activity and pancreatic tumorigenesis providing a rationale for testing HIPK2 inhibitors to mitigate the incidence of PDAC development in high-risk individuals.

Crosstalk between FTH1 and PYCR1 dysregulates proline metabolism and mediates cell growth in KRAS-mutant pancreatic cancer cells.

Ferritin, comprising heavy (FTH1) and light (FTL) chains, is the main iron storage protein, and pancreatic cancer patients exhibit elevated serum ferritin levels. Specifically, higher ferritin levels are correlated with poorer pancreatic ductal adenocarcinoma (PDAC) prognosis; however, the underlying mechanism and metabolic programming of ferritin involved in KRAS-mutant PDAC progression remain unclear. Here, we observed a direct correlation between FTH1 expression and cell viability and clonogenicity in KRAS-mutant PDAC cell lines as well as with in vivo tumor growth through the control of proline metabolism. Our investigation highlights the intricate relationship between FTH1 and pyrroline-5-carboxylate reductase 1 (PYCR1), a crucial mitochondrial enzyme facilitating the glutamate-to-proline conversion, underscoring its impact on proline metabolic imbalance in KRAS-mutant PDAC. This regulation is further reversed by miR-5000-3p, whose dysregulation results in the disruption of proline metabolism, thereby accentuating the progression of KRAS-mutant PDAC. Additionally, our study demonstrated that deferasirox, an oral iron chelator, significantly diminishes cell viability and tumor growth in KRAS-mutant PDAC by targeting FTH1-mediated pathways and altering the PYCR1/PRODH expression ratio. These findings underscore the novel role of FTH1 in proline metabolism and its potential as a target for PDAC therapy development.

Combining RAS(ON) G12C-selective inhibitor with SHP2 inhibition sensitises lung tumours to immune checkpoint blockade.

Mutant selective drugs targeting the inactive, GDP-bound form of KRASG12C have been approved for use in lung cancer, but resistance develops rapidly. Here we use an inhibitor, (RMC-4998) that targets RASG12C in its active, GTP-bound form, to treat KRAS mutant lung cancer in various immune competent mouse models. RAS pathway reactivation after RMC-4998 treatment could be delayed using combined treatment with a SHP2 inhibitor, which not only impacts tumour cell RAS signalling but also remodels the tumour microenvironment to be less immunosuppressive. In an immune inflamed model, RAS and SHP2 inhibitors in combination drive durable responses by suppressing tumour relapse and inducing development of immune memory. In an immune excluded model, combined RAS and SHP2 inhibition sensitises tumours to immune checkpoint blockade, leading to efficient tumour immune rejection. These preclinical results demonstrate the potential of the combination of RAS(ON) G12C-selective inhibitors with SHP2 inhibitors to sensitize tumours to immune checkpoint blockade.

Molecular genetic analysis of colorectal carcinoma with an aggressive extraintestinal immunohistochemical phenotype.

Colorectal cancer (CRC) is a leading global cause of illness and death. There is a need for identification of better prognostic markers beyond traditional clinical variables like grade and stage. Previous research revealed that abnormal expression of cytokeratin 7 (CK7) and loss of the intestinal-specific Special AT-rich sequence-binding protein 2 (SATB2) are linked to poor CRC prognosis. This study aimed to explore these markers' prognostic significance alongside two extraintestinal mucins (MUC5AC, MUC6), claudin 18, and MUC4 in 285 CRC cases using immunohistochemistry on tissue microarrays (TMAs). CK7 expression and SATB2-loss were associated with MUC5AC, MUC6, and claudin 18 positivity. These findings suggest a distinct "non-intestinal" immunohistochemical profile in CRC, often right-sided, SATB2-low, with atypical expression of CK7 and non-colorectal mucins (MUC5AC, MUC6). Strong MUC4 expression negatively impacted cancer-specific survival (hazard ratio = 2.7, p = 0.044). Genetic analysis via next-generation sequencing (NGS) in CK7 + CRCs and those with high MUC4 expression revealed prevalent mutations in TP53, APC, BRAF, KRAS, PIK3CA, FBXW7, and SMAD4, consistent with known CRC mutation patterns. NGS also identified druggable variants in BRAF, PIK3CA, and KRAS. CK7 + tumors showed intriguingly common (31.6%) BRAF V600E mutations corelating with poor prognosis, compared to the frequency described in the literature and databases. Further research on larger cohorts with a non-colorectal immunophenotype and high MUC4 expression is needed.

Loss of Heterozygosity and Mutations in the RAS-ERK Pathway Genes in Tumor Cells of Various Loci in Multiple Myeloma.

Multiple myeloma (MM) is a disease characterized by spatiotemporal heterogeneity of tumor clones. Different genetic aberrations can be observed simultaneously in tumor cells from different loci, and as the disease progresses, new subclones may appear. The role of liquid biopsy, which is based on the analysis of tumor DNA circulating in the blood plasma, continues to be explored in MM. Here, we present an analysis of the STR profiles and mutation status of the KRAS, NRAS, and BRAF genes, evaluated in plasma free circulating tumor DNA (ctDNA), CD138+ bone marrow cells, and plasmacytomas. The prospective single-center study included 97 patients, with a median age of 55 years. Of these, 94 had newly diagnosed symptomatic MM, and three had primary plasma cell leukemia. It should be noted that if mutations were detected only in ctDNA, "non-classical" codons were more often affected. A variety of adverse laboratory and clinical factors have been associated with the detection of rare KRAS or NRAS gene mutations in bone marrow or ctDNA, suggesting that these mutations may be factors of an unfavorable prognosis for MM. Liquid biopsy studies provide undeniable fundamental information about tumor heterogeneity and clonal evolution in MM. Moreover, we focus on using liquid biopsy to identify new high-risk factors for MM.

Loss of p53 and SMAD4 induces adenosquamous subtype pancreatic cancer in the absence of an oncogenic KRAS mutation.

Pancreatic cancer is associated with an oncogenic KRAS mutation in approximately 90% of cases. However, a non-negligible proportion of pancreatic cancer cases harbor wild-type KRAS (KRAS-WT). This study establishes genetically engineered mouse models that develop spontaneous pancreatic cancer in the context of KRAS-WT. The Trp53loxP/loxP;Smad4loxP/loxP;Pdx1-Cre (PPSSC) mouse model harbors KRAS-WT and loss of Trp53/Smad4. The Trp53loxP/loxP;Tgfbr2loxP/loxP;Pdx1-Cre (PPTTC) mouse model harbors KRAS-WT and loss of Trp53/Tgfbr2. We identify that either Trp53/Smad4 loss or Trp53/Tgfbr2 loss can induce spontaneous pancreatic tumor formation in the absence of an oncogenic KRAS mutation. The Trp53/Smad4 loss and Trp53/Tgfbr2 loss mouse models exhibit distinct pancreatic tumor histological features, as compared to oncogenic KRAS-driven mouse models. Furthermore, KRAS-WT pancreatic tumors with Trp53/Smad4 loss reveal unique histological features of pancreatic adenosquamous carcinoma (PASC). Single-cell RNA sequencing (scRNA-seq) analysis reveals the distinct tumor immune microenvironment landscape of KRAS-WT (PPSSC) pancreatic tumors as compared with that of oncogenic KRAS-driven pancreatic tumors.

Hope on the Horizon: A Revolution in KRAS Inhibition Is Creating a New Treatment Paradigm for Patients with Pancreatic Cancer.

KRAS is the most frequently altered oncogene in pancreatic ductal adenocarcinoma, in which the aberrantly activated RAS signaling pathway plays pleiotropic roles in tumor initiation and maintenance. Nearly four decades after the discovery of the RAS oncoprotein, a multitude of pharmacologic inhibitors are now available that directly target mutant KRAS. This In Focus commentary, published simultaneously with the 2024 AACR Special Conference on Pancreatic Cancer, summarizes the current state of this rapidly changing field, including preclinical data and emerging clinical trends with respect to therapeutic efficacy, mechanisms of resistance, and potential combinations to maximize clinical benefit from this promising class of therapies.

Molecular diagnosis for detecting KRAS mutation in peritoneal washing fluid of pancreatic ductal adenocarcinoma.

Positive peritoneal washing cytology is an indicator of poor prognosis in patients with pancreatic ductal adenocarcinoma (PDAC); however, its sensitivity is relatively low. This study evaluated the performance of peptide nucleic acid (PNA)-directed PCR clamping as a molecular-based peritoneal washing cytology for sensitive detection of KRAS mutation in PDAC. Intraoperative peritoneal washing fluid (IPWF) obtained from patients with PDAC who underwent surgery was analyzed. PNA-directed PCR clamping was performed on DNA extracted from IPWF. Among 54 patients enrolled, threshold cycle (Ct) was significantly lower in patients with positive peritoneal washing cytology than in those with negative peritoneal washing cytology (P < 0.001) and in patients with peritoneal dissemination than in those without peritoneal dissemination (P < 0.01). The optimal Ct cut-off to predict KRAS mutations in IPWF was 36.42 based on a receiver operating characteristic curve. The sensitivity, specificity, and accuracy for molecular diagnosis were 100%, 80.0%, and 85.2%, respectively. Peritoneal dissemination recurrence was significantly more frequent in patients with a positive molecular diagnosis than in those with a negative diagnosis (38.9 vs. 8.0%, P = 0.013). The genomic approach might be clinically valuable for a more precise tumor cell detection in IPWF.

Combined KRAS and TP53 mutation in patients with colorectal cancer enhance chemoresistance to promote postoperative recurrence and metastasis.

The response of patients with colorectal cancer to chemotherapy is tightly correlated with their genomic variation. Among these, APC, TP53, KRAS, PIK3CA are the most frequently mutated genes in advanced colorectal cancer patients. However, the precise correlation between these mutations and the therapeutic effects of chemotherapy remains elusive. Here, we conducted genome sequencing to identify commonly mutated genes in colorectal cancer patients and comprehensively assessed their sensitivity to chemotherapy drugs by monitoring computer tomography (CT) scans and carcinoembryonic antigen (CEA) levels. Surprisingly, we discovered that the objective response rate to the standard first-line chemotherapy among patients harboring combined KRAS and TP53 mutations is dismal, and these patients are predisposed to recurrence and metastasis. Furthermore, advanced-stage patients with concurrent KRAS and TP53 mutations are susceptible to developing cancer-associated cachexia due to chemotherapy resistance or forced cessation of treatment. Our findings underscore the urgent need for the development of innovative and novel chemotherapeutic strategies to effectively manage colorectal cancer patients harboring combined KRAS and TP53 mutations.

Intersecting evidence: Bibliometric analysis and clinical trials illuminate immunotherapy in KRAS-mutation cancer: A review.

KRAS mutations play a critical role in the development and progression of several cancers, including non-small cell lung cancer and pancreatic cancer. Despite advancements in targeted therapies, the management of KRAS-mutant tumors remains challenging. This study leverages bibliometric analysis and a comprehensive review of clinical trials to identify emerging immunotherapies and potential treatments for KRAS-related cancers. Using the Web of Science Core Collection and Citespace, we analyzed publications from January 2008 to March 2023 alongside 52 clinical trials from ClinicalTrials.gov and WHO's registry, concentrating on immune checkpoint blockades (ICBs) and novel therapies. Our study highlights an increased focus on the tumor immune microenvironment and precision therapy. Clinical trials reveal the effectiveness of ICBs and the promising potential of T-cell receptor T-cell therapy and vaccines in treating KRAS-mutant cancers. ICBs, particularly in combination therapies, stand out in managing KRAS-mutant tumors. Identifying the tumor microenvironment and gene co-mutation profiles as key research areas, our findings advocate for multidisciplinary approaches to advance personalized cancer treatment. Future research should integrate genetic, immunological, and computational studies to unveil new therapeutic targets and refine treatment strategies for KRAS-mutant cancers.

Clinical characteristics of KRAS mutation subtypes in non-small cell lung cancer population in Xinjiang, China, and their impact on the prognosis of immunotherapy.

PURPOSE: Non-small cell lung cancer (NSCLC) is a highly fatal malignancy. The Kirsten rat sarcoma viral oncogene (KRAS) gene profoundly impacts patient prognosis. This study aims to explore the correlation between KRAS mutation subtypes, clinical data, and the impact of these subtypes on immunotherapy. MATERIALS AND METHODS: Tumor samples from 269 NSCLC patients at the Affiliated Cancer Hospital of Xinjiang Medical University were analyzed. Patients received first- or second-line therapy without targeted therapy. Molecular and clinical data were used to analysis KRAS mutation subtypes and treatment outcomes. RESULTS: KRAS mutations predominantly included G12C, G12D, and G12V subtypes. TP53 had the highest mutation frequency among KRAS mutations, followed by MST1, STK11, and KMT2C. Gender differences were noted among KRAS mutation subtypes, with G12C and G12V mutations prevalent in males, while G12D mutations were less common among males. Smokers exhibited varied KRAS mutation subtypes, with G12C and G12V prevalent in smokers and G12D in nonsmokers. KRAS mutations were mainly in lung adenocarcinoma. TTF-1 and PD-L1 expression differed significantly among KRAS mutations. Patients with G12C and G12V mutations showed higher TMB levels and better immunotherapy outcomes compared to those without KRAS mutations. Conversely, patients with G12D mutations had poorer immunotherapy responses. CONCLUSIONS: KRAS mutation subtypes exhibit distinct clinical and molecular characteristics and varying responses to immunotherapy. G12C and G12V mutations correlate with better immunotherapy outcomes, while G12D mutations are associated with poorer responses.

Connecting the dots: LncRNAs in the KRAS pathway and cancer.

Long non-coding RNAs (lncRNAs) have been identified as important participants in several biological functions, particularly their complex interactions with the KRAS pathway, which provide insights into the significant roles lncRNAs play in cancer development. The KRAS pathway, a central signaling cascade crucial for cell proliferation, survival, and differentiation, stands out as a key therapeutic target due to its aberrant activation in many human cancers. Recent investigations have unveiled a myriad of lncRNAs, such as H19, ANRIL, and MEG3, intricately modulating the KRAS pathway, influencing both its activation and repression through various mechanisms, including epigenetic modifications, transcriptional regulation, and post-transcriptional control. These lncRNAs function as fine-tuners, delicately orchestrating the balance required for normal cellular function. Their dysregulation has been linked to the development and progression of multiple malignancies, including lung, pancreatic, and colorectal carcinomas, which frequently harbor KRAS mutations. This scrutiny delves into the functional diversity of specific lncRNAs within the KRAS pathway, elucidating their molecular mechanisms and downstream effects on cancer phenotypes. Additionally, it underscores the diagnostic and prognostic potential of these lncRNAs as indicators for cancer detection and assessment. The complex regulatory network that lncRNAs construct within the context of the KRAS pathway offers important insights for the creation of focused therapeutic approaches, opening new possibilities for precision medicine in oncology. However, challenges such as the dual roles of lncRNAs in different cancer types and the difficulty in therapeutically targeting these molecules highlight the ongoing debates and need for further research. As ongoing studies unveil the complexities of lncRNA-mediated KRAS pathway modulation, the potential for innovative cancer interventions becomes increasingly promising.

Cell-specific models reveal conformation-specific RAF inhibitor combinations that synergistically inhibit ERK signaling in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) presents significant challenges for targeted clinical interventions due to prevalent KRAS mutations, rendering PDAC resistant to RAF and MEK inhibitors (RAFi and MEKi). In addition, responses to targeted therapies vary between patients. Here, we explored the differential sensitivities of PDAC cell lines to RAFi and MEKi and developed an isogenic pair comprising the most sensitive and resistant PDAC cells. To simulate patient- or tumor-specific variations, we constructed cell-line-specific mechanistic models based on protein expression profiling and differential properties of KRAS mutants. These models predicted synergy between two RAFi with different conformation specificity (type I½ and type II RAFi) in inhibiting phospho-ERK (ppERK) and reducing PDAC cell viability. This synergy was experimentally validated across all four studied PDAC cell lines. Our findings underscore the need for combination approaches to inhibit the ERK pathway in PDAC.