KRAS Mutations Research Articles

Association between KRAS mutation and alcohol consumption in Brazilian patients with colorectal cancer

Colorectal cancer (CRC) is a leading cause of morbidity and mortality worldwide. Detection before metastasis and efficient treatment of disease significantly improve patient survival and quality of life. However, limitations in diagnosis and postoperative surveillance are associated with low CRC detection and survival rates. Thus, this project aimed to evaluate the molecular profile of patients diagnosed with CRC, as molecular biomarkers constitute a new frontier for diagnosis, treatment and prognosis. Methods and Results: 42 patients were included in the study, predominantly male (59.5%), with a median age of 63 years (SD: 10.0; min: 41; max: 83). The majority of primary tumors were located in the rectum (38.1%), in the sigmoid (33.3%) and in the ascending (21.4%) colon. We evaluated the genes KRAS, NRAS, BRAF, EGFR and TP53 using Sanger sequencing. Somatic and germline mutations were found in the KRAS, EGFR and TP53 genes, with the most common somatic alteration being rs121913529 in KRAS. This variant was also strongly associated with alcoholism (p = 0.002). Furthermore, patients with somatic mutations in TP53 had significantly higher mortality compared to those with wild-type alleles (OR: 11.2; 95% CI 1.25–2.45). Conclusions: Our findings support a relationship between alcohol consumption and the rs121913529 mutation, which is classified as pathogenic for colorectal cancer. Thus, further studies investigating the link between alcohol consumption, colorectal carcinogenesis and tumor progression ought to be conducted.

Driver mutations associated with signatures of platinum sensitivity in germ cell tumors

We sought to evaluate the genomic and transcriptomic landscapes in primary and metastatic germ cell tumors (GCTs; N = 138) to uncover factors that drive cisplatin resistance. Prevalence was calculated for platinum-resistant alterations (PRAs; KRAS, TP53, and KIT mutations, and MDM2 amplification) and high copy number amplifications (CNA ≥ 6 copies). Tumors were designated as chemo-naïve (PreC, N = 66) or post-chemotherapy (PostC, N = 17). A transcriptomic signature associated with platinum sensitivity (PSS, high suggests increased sensitivity) was applied. KIT mutations were observed in 14.5% of primary versus 1.8% of met and 0% of lymph. TP53 mutations were identified in 10% of primary GCTs versus 17% of met and 16.7% of lymph. MDM2 CNAs were similar between sites. PRA-positive PreC GCTs had significantly lower average PSS scores compared to PRA-negative tumors. Lower PSS scores in chemo-naïve tumors were associated with PRAs, suggesting a potential mechanism for platinum resistance.

Oncogenic KRAS mutations modulate BAX-mediated cell death

Kirsten rat sarcoma viral oncogene homolog (KRAS) belongs to the GTPase RAS superfamily, which regulates several cell-signaling pathways involved in the control of important cellular functions, including apoptosis. Oncogenic mutations in KRAS are considered the most common gain-of-function mutations, affecting 30–50 % of colorectal cancer (CRC) patients. While RAS proteins usually play an anti-apoptotic role, little is known about the involvement of KRAS mutations in apoptosis regulation. Here, we aimed to elucidate the role of mutated human KRAS in the regulation of BAX, a key pro-apoptotic member of the Bcl-2 family. For this purpose, we took advantage of the simpler yeast model Saccharomyces cerevisiae, using cells deficient in the main yeast RAS isoform (ras2Δ) co-expressing wild-type KRAS (KRASWT) or the most frequent KRAS mutations found in CRC - KRASG12D, KRASG12V or KRASG13D, along with human BAX. We show that, in comparison with KRASWT, KRAS mutants confer resistance to BAX-induced death and cytochrome c (cyt c) release. The modulation of BAX by KRAS isoforms seems to result from a direct interaction between these proteins, as they co-localize at the mitochondria and there is evidence they may physically interact. We further show that acetic acid significantly increased cell death in cells expressing BAX and co-expressing oncogenic KRAS mutants, but not KRASWT. This suggests a potential mechanism explaining the increased sensitivity of CRC cells harboring a KRAS-activated pathway to acetate. These findings contribute to a clearer understanding of how KRAS regulate BAX function, a relevant aspect in tumor progression.

Oncogenic KRAS Mutations Confer a Unique Mechanotransduction Response to Peristalsis in Colorectal Cancer Cells.

Colorectal cancer (CRC) tumors start as polyps on the inner lining of the colorectum, where they are exposed to the mechanics of peristalsis. Our previous work leveraged a custom-built peristalsis bioreactor to demonstrate that colonic peristalsis led to cancer stem cell enrichment in CRC cells. However, this malignant mechanotransductive response was confined to select CRC lines that harbored an oncogenic mutation in the KRAS gene. Here, we explored the involvement of activating KRAS mutations on peristalsis-associated mechanotransduction in CRC. Peristalsis enriched cancer stem cell marker LGR5 in KRAS mutant lines, in a Wnt-ligand-independent manner. Conversely, LGR5 enrichment in wild type KRAS lines exposed to peristalsis were minimal. LGR5 enrichment downstream of peristalsis translated to increased tumorigenicity in vivo. Differences in mechanotransduction was apparent via unbiased gene set enrichment analysis, where many unique pathways were enriched in wild type vs. mutant lines. Peristalsis also triggered β-catenin nuclear localization independent of Wnt-ligands, particularly in KRAS mutant lines. The involvement of KRAS was validated via gain and loss of function strategies. Peristalsis induced β-catenin activation and LGR5 enrichment depended on the activation of the MEK/ERK cascade. Taken together, our results demonstrated that oncogenic KRAS mutations conferred a unique peristalsis-associated mechanotransduction response to colorectal cancer cells, resulting in cancer stem cell enrichment and increased tumorigenicity. These mechanosensory connections can be leveraged in improving the sensitivity of emerging therapies that target oncogenic KRAS. Implications: Oncogenic KRAS empowers colorectal cancer cells to harness the mechanics of colonic peristalsis for malignant gain, independent of other cooperating signals. .

More T cell receptors to the RAScue in cancer?

Treatment with T cells genetically engineered to express tumor-reactive T cell receptors (TCRs), known as TCR-gene therapy (TCR-T), is a promising immunotherapeutic approach for patients with cancer. The identification of optimal TCRs to use and tumor antigens to target are key considerations for TCR-T. In this issue of the JCI, Bear and colleagues report on their use of in vitro assays to characterize four HLA-A*03:01- or HLA-A*11:01-restricted TCRs targeting the oncogenic KRAS G12V mutation. The TCRs were derived from healthy donors or patients with pancreatic cancer who had received a vaccine against mutant KRAS. The most promising TCRs warrant testing in patients with KRAS G12V-positive cancers.

KRASG12D-Mutated Metastatic Colorectal Cancer: Clinical, Molecular, Immunologic, and Prognostic Features of a New Emerging Targeted Alteration.

KRASG12D mutation (mut) occurs in about 10%-12% of metastatic colorectal cancer (mCRC). Recently, novel KRASG12D inhibitors have been developed and are currently under investigation in phase I/II clinical trials in solid tumors including mCRC. We aimed at performing a comprehensive characterization of clinical, molecular, immunologic, and prognostic features of KRASG12D-mutated mCRC to inform the design and the interpretation of future trials. We performed a pooled analysis of phase III TRIBE and TRIBE2 studies comparing 5-fluorouracil, leucovorin, oxaliplatin, and irinotecan (FOLFOXIRI)/bevacizumab (bev) to doublets (5-fluorouracil, leucovorin, and oxaliplatin or 5-fluorouracil, leucovorin, and irinotecan)/bev. One hundred and thirty-six (16%) of 854 patients with available KRASG12D mutational status were KRASG12D mutated. KRASG12D-mutated patients had more frequently right-sided primary tumor and were less likely to present liver-only metastases with respect to other RAS mutated and all-wild-type (wt) patients. Compared with the BRAFV600E-mutated group, KRASG12D-mutated patients had more frequently left-sided primary tumor, resected primary tumor at the time of diagnosis, and Eastern Cooperative Oncology Group performance status 0. KRASG12D-mutated patients had better prognosis than BRAFV600E-mutated and worse prognosis than all wt patients. No prognostic difference was evident between KRASG12D mut and other RAS mut patients overall or according to other specific KRAS or NRAS hotspot mutations. No interaction effect was observed between KRASG12D mut and the benefit provided by FOLFOXIRI/bev compared with doublets/bev. PIK3CA mut were reported more frequently among KRASG12D-mutated tumors compared with both other RAS mut and all wt. A detail estimation of KRASG12D mut mCRC patients' characteristics and expected outcomes may be useful when planning future studies in this subgroup. The high prevalence of PI3K/PTEN/Akt pathway activating alterations may affect the efficacy of targeted strategies.

Predictive genomic and transcriptomic analysis on endoscopic ultrasound-guided fine needle aspiration materials from primary pancreatic adenocarcinoma: a prospective multicentre study

We apply endoscopic ultrasound-guided fine needle aspiration biopsy to cytopathologically diagnose and sample nucleic acids from primary tumours regardless of the disease stage. 397 patients with proven pancreatic adenocarcinoma were included and followed up in a multicentre prospective study. DNA and mRNA were extracted from materials of primary tumours obtained by endoscopic ultrasound-guided fine needle aspiration biopsy and analysed using targeted deep sequencing and RNAseq respectively. The variant allele frequency of the KRAS mutation was used to evaluate the tumour cellularity, ranging from 15 to 20% in all cells, regardless of the tumour stage. The molecular profile of metastatic primary tumours significantly differed from other types of tumours, more frequently having TP53 mutations (p=0.0002), less frequently having RNF43 mutations, and possessing more basal-like mRNA component (p=0.001). Molecular markers associated with improved overall survival were: mutations in homologous recombination deficiency genes in patients who received first-line platinum-based chemotherapy (p=0.025) and wild-type TP53 gene in patients with locally advanced tumours who received radio-chemotherapy (p=0.01). The GemPred transcriptomic profile was associated with a significantly better overall survival in patients with locally advanced or metastatic pancreatic cancer who received a gemcitabine-based first-line treatment (p=0.019). The combination of genomic and transcriptomic analyses of primary pancreatic tumours enables us to distinguish metastatic tumours from other tumour types. Our molecular strategy may assist in predicting overall survival outcomes for platinum or gemcitabine-based chemotherapies, as well as radio-chemotherapy. Institut National Du Cancer (BCB INCa_7294), CHU of Toulouse, Inserm and Ligue Nationale Contre le Cancer (CIT program).

Programmable DNA-based nanostructure sensors for mutation detection coupled with asymmetric amplification in precision genome profiling

We introduce a DNA-based nanostructure sensor for the rapid, precise identification of single-nucleotide polymorphisms (SNPs), a key factor in determining disease susceptibility and tailoring medical treatment. The proposed two-state sensor design facilitates multiplexed, sequence-specific detection of double-stranded genomic DNA (gDNA), overcoming traditional obstacles presented by base pairing interference. Utilizing detector strands with binding penalties, the sensor achieves high SNP specificity, discernible by distinct gel electrophoresis band positions. Coupled with asymmetric PCR (asy-PCR), our integrated system amplifies genetic profiling effectiveness, especially in clinical biopsies, enhancing gDNA detection sensitivity and specificity for concurrent multiple mutation identifications. This cost-effective method ($0.41 per test) streamlines mutation screening, enabling high-throughput analysis with limited equipment—requiring only a single staining agent and a unified electrophoresis apparatus. Validated clinically with 70 colorectal cancer samples, the system boasts a 98.57% sensitivity and 95.71% specificity for KRAS mutations, surpassing traditional PCR detection and uncovering low-frequency mutations untraceable by direct sequencing. Our findings suggest this asy-PCR enhanced DNA-based nanostructure sensor system has the potential to revolutionize genetic diagnostics, offering expansive applications in detecting viral variants and microbial strains, thus advancing personalized genetic testing.

Polyclonality overcomes fitness barriers in Apc-driven tumorigenesis.

Loss-of-function mutations in the tumour suppressor APC are an initial step in intestinal tumorigenesis1,2. APC-mutant intestinal stem cells outcompete their wild-type neighbours through the secretion of Wnt antagonists, which accelerates the fixation and subsequent rapid clonal expansion of mutants3-5. Reports of polyclonal intestinal tumours in human patients and mouse models appear at odds with this process6,7. Here we combine multicolour lineage tracing with chemical mutagenesis in mice to show that a large proportion of intestinal tumours have a multiancestral origin. Polyclonal tumours retain a structure comprising subclones with distinct Apc mutations and transcriptional states, driven predominantly by differences in KRAS and MYC signalling. These pathway-level changes are accompanied by profound differences in cancer stem cell phenotypes. Of note, these findings are confirmed by introducing an oncogenic Kras mutation that results in predominantly monoclonal tumour formation. Further, polyclonal tumours have accelerated growth dynamics, suggesting a link between polyclonality and tumour progression. Together, these findings demonstrate the role of interclonal interactions in promoting tumorigenesis through non-cell autonomous pathways that are dependent on the differential activation of oncogenic pathways between clones.

Adjuvant Modified FOLFIRINOX for Resected Pancreatic Adenocarcinoma (PDAC): Clinical Insights and Genomic Features from a Large Contemporary Cohort.

Adjuvant mFOLFIRINOX (mFFX) is standard of care for fit individuals with resected pancreatic ductal adenocarcinoma (PDAC). There are limited data on adjuvant mFFX outcomes outside clinical trials. Institutional databases queried to identify patients with resected PDAC who received ≥1 dose adjuvant mFFX. Primary endpoints: recurrence free survival (RFS), overall survival (OS). Secondary endpoints: clinical factors, genomic features associated with outcomes. RFS and OS were estimated with Kaplan-Meier. Cox proportional hazards regression model was used to associate clinico-genomic features correlated with survival outcomes. N = 147 identified between 01/2015-01/2023. Median age: 67 years; N = 57 (39%) >70 years. Unfavorable prognostic features: N = 52 (36%) N2 nodal status, N = 115 (78%) lymphovascular invasion), and N = 133 (90%) perineural invasion. Median time from surgery to start mFFX: 1.78 months (m) (IQR 1.45, 2.12). N = 124 (84%) completed 12 doses; N = 98 (67%) stopped oxaliplatin early for neuropathy (median 10 doses; range 4-12). Further dosing characteristics are summarized in Supplementary Table 3. With median follow up of 35.1 m, median RFS (mRFS) 26 m (95% CI 19, 39) and median OS (mOS) not reached. For >70 cohort, mRFS 23 m (95% CI 14, NR) and mOS 51 m (95% CI 37, NR). mFFX started <8 weeks from resection associated with improved RFS (HR 0.62; 95% CI 0.41, 0.96; p = .033) and OS (HR 0.53; 95% CI 0.3, 0.94; p = .030). KRAS mutation and whole genome doubling trended to shorter RFS and OS. Homologous recombination deficiency status did not confer improved survival outcomes. Adjuvant mFFX is effective and tolerable in resected PDAC in a non-trial setting, including for patients >70 years.

Updates in Immunotherapy for Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with limited effective therapeutic options. Due to a variety of cancer cell-intrinsic factors, including KRAS mutations, chemokine production, and other mechanisms that elicit a dysregulated host immune response, PDAC is often characterized by poor immune infiltration and an immune-privileged fibrotic stroma. As understanding of the tumor microenvironment (TME) evolves, novel therapies are being developed to target immunosuppressive mechanisms. Immune checkpoint inhibitors have limited efficacy when used alone or with radiation. Combinations of immune therapies, along with chemotherapy or chemoradiation, have demonstrated promise in preclinical and early clinical trials. Despite dismal response rates for immunotherapy for metastatic PDAC, response rates with neoadjuvant immunotherapy are somewhat encouraging, suggesting that incorporation of immunotherapy in the treatment of PDAC should be earlier in the disease course. Precision therapy for PDAC may be informed by advances in transcriptomic sequencing that can identify immunophenotypes, allowing for more appropriate treatment selection for each individual patient. Personalized and antigen-specific therapies are an increasing topic of interest, including adjuvant immunotherapy using personalized mRNA vaccines to prevent recurrence. Further development of personalized immune therapies will need to balance precision with generalizability and cost.

Design and development of dual targeting CAR protein for the development of CAR T-cell therapy against KRAS mutated pancreatic ductal adenocarcinoma using computational approaches

Mutant KRAS promotes the proliferation, metastasis, and aggressiveness of various cancers including pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), and colorectal adenocarcinoma (CRC) respectively. Mutant KRAS therapeutics are limited, while Sotorasib and Adagrasib were the only FDA-approved drugs for the treatment of KRASG12C mutated NSCLC. Chimeric antigen receptor (CAR) T-cell therapy has been emerged as an effective strategy against hematological malignancies and being extended towards solid cancers including PDAC. mesothelin (MSLN) and Carcinoembryonic Antigen (CEA) were reported to be highly overexpressed in KRAS-mutated PDAC. Meanwhile, in clinical trials, several CAR T-cell therapy studies are mainly focused towards these two cancer antigens in PDAC, however, the dual targeting of these two neoantigens is not reported. In the present study, we have designed and developed a novel dual-targeting CAR protein by employing various bioinformatics approaches such as functional analysis (antigenicity, allergenicity, antigen binding sites & signalling cascades), qualitative analysis (physicochemical, prediction, refinement & validation of 2D and 3D structures), molecular docking, and in silico cloning. Our results revealed that the designed CAR protein specifically binds with both MSLN & CEA with significant binding affinities, and was predicted to be stable & non-allergenic. Additionally, the protein–protein interaction network reveals the T-cell mediated antitumor responses of each domain in the designed CAR. Conclusively, we have designed and developed a dual targeting (MSLN & CEA) CAR protein towards KRAS-mutated PDAC using computational approaches. Alongside, we further recommend to engineer this designed CAR in T-cells and evaluating their therapeutic efficiency in in vitro and in vivo studies in the near future.

Comprehensive structure-function analysis reveals gain- and loss-of-function mechanisms impacting oncogenic KRAS activity.

To dissect variant-function relationships in the KRAS oncoprotein, we performed deep mutational scanning (DMS) screens for both wild-type and KRAS G12D mutant alleles. We defined the spectrum of oncogenic potential for nearly all possible KRAS variants, identifying several novel transforming alleles and elucidating a model to describe the frequency of KRAS mutations in human cancer as a function of transforming potential, mutational probability, and tissue-specific mutational signatures. Biochemical and structural analyses of variants identified in a KRAS G12D second-site suppressor DMS screen revealed that attenuation of oncogenic KRAS can be mediated by protein instability and conformational rigidity, resulting in reduced binding affinity to effector proteins, such as RAF and PI3-kinases, or reduced SOS-mediated nucleotide exchange activity. These studies define the landscape of single amino acid alterations that modulate the function of KRAS, providing a resource for the clinical interpretation of KRAS variants and elucidating mechanisms of oncogenic KRAS inactivation for therapeutic exploitation.

Intramuscular hemangioma capillary type with HRAS mutation: Expanding the molecular genetic spectrum with an emphasis on overlap with arteriovenous malformations and distinct from infantile hemangioma

AimsIntramuscular hemangioma capillary type (IHCT) is a rare entity that refers to fast-flow vascular lesions. This study aims to elucidate the relationships between clinicopathological, radiological, and molecular characteristics in IHCT patients. Methods and resultsWe reviewed all IHCT cases which were treated surgically in our pathology database from 2014 to 2023. Ten cases were analyzed via next-generation sequencing (NGS) and Sanger sequencing. The cohort consisted of 10 patients (6 males, 4 females) with a median age of 18 years (range: 1–37). Disease lesions were located in the trunk (n = 4), upper extremity (n = 2), lower extremity (n = 2), shoulder (n = 1), and neck (n = 1). IHCT is most commonly a progressively increasing painless mass. Histopathologically, all lesions exhibited aggregates, lobules, and anastomosing cords of capillary-type vessels separating or infiltrating the skeletal muscles. Four cases exhibited irregularly dilated vessels with thick walls, such as arteriovenous malformations (AVM) in the lesion's periphery. MRI findings commonly demonstrated a well-delineated, homogeneous mass. Somatic mutations were detected in seven of the ten IHCT cases. Four cases harbored mutations in MAP2K1 (p.Q58_E62del, p.K57_G61del, p.K57 N), two cases harbored mutations in KRAS (p.Q61R and p.L56V, p.G13R), and one case harbored a mutation in HRAS (p.D69_Q70insRWYSAMRD). Mutant allele frequencies detected by sequencing ranged from 9.98% to 15.97%. ConclusionsThe hemodynamic and molecular genetic phenotypes of IHCT closely resemble those observed in AVMs. Newly identified KRAS missense mutations, including cases with coexisting mutation types, and HRAS insertion mutations offer valuable insights into the genetic basis of vascular anomalies. These findings may also present potential targets for the development of novel pharmacotherapeutic interventions.

Stromal reprogramming overcomes resistance to RAS-MAPK inhibition to improve pancreas cancer responses to cytotoxic and immune therapy.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that is often resistant to therapy. An immune suppressive tumor microenvironment (TME) and oncogenic mutations in KRAS have both been implicated as drivers of resistance to therapy. Mitogen-activated protein kinase (MAPK) inhibition has not yet shown clinical efficacy, likely because of rapid acquisition of tumor-intrinsic resistance. However, the unique PDAC TME may also be a driver of resistance. We found that long-term focal adhesion kinase (FAK) inhibitor treatment led to hyperactivation of the RAS/MAPK pathway in PDAC cells in mouse models and tissues from patients with PDAC. Concomitant inhibition of both FAK (with VS-4718) and rapidly accelerated fibrosarcoma and MAPK kinase (RAF-MEK) (with avutometinib) induced tumor growth inhibition and increased survival across multiple PDAC mouse models. In the TME, cancer-associated fibroblasts (CAFs) impaired the down-regulation of MYC by RAF-MEK inhibition in PDAC cells, resulting in resistance. By contrast, FAK inhibition reprogramed CAFs to suppress the production of FGF1, which can drive resistance to RAF-MEK inhibition. The addition of chemotherapy to combined FAK and RAF-MEK inhibition led to tumor regression, a decrease in liver metastasis, and improved survival in KRAS-driven PDAC mouse models. Combination of FAK and RAF-MEK inhibition alone improved antitumor immunity and priming of T cell responses in response to chemotherapy. These findings provided the rationale for an ongoing clinical trial evaluating the efficacy of avutometinib and defactinib in combination with gemcitabine and nab-paclitaxel in patients with PDAC and may suggest further paths for combined stromal and tumor-targeting therapies.

KRAS-mediated upregulation of CIP2A promotes suppression of PP2A-B56α to initiate pancreatic cancer development.

Oncogenic mutations in KRAS are present in ~95% of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) and are considered the initiating event of pancreatic intraepithelial neoplasia (PanIN) precursor lesions. While it is well established that KRAS mutations drive the activation of oncogenic kinase cascades during pancreatic oncogenesis, the effects of oncogenic KRAS signaling on regulation of phosphatases during this process is not fully appreciated. Protein Phosphatase 2A (PP2A) has been implicated in suppressing KRAS-driven cellular transformation and low PP2A activity is observed in PDAC cells compared to non-transformed cells, suggesting that suppression of PP2A activity is an important step in the overall development of PDAC. In the current study, we demonstrate that KRASG12D induces the expression of an endogenous inhibitor of PP2A activity, Cancerous Inhibitor of PP2A (CIP2A), and phosphorylation of the PP2A substrate, c-MYC. Consistent with these findings, KRASG12D sequestered the specific PP2A subunit responsible for c-MYC degradation, B56α, away from the active PP2A holoenzyme in a CIP2A-dependent manner. During PDAC initiation in vivo, knockout of B56α promoted KRASG12D tumorigenesis by accelerating acinar-to-ductal metaplasia (ADM) and the formation of PanIN lesions. The process of ADM was attenuated ex vivo in response to pharmacological re-activation of PP2A utilizing direct small molecule activators of PP2A (SMAPs). Together, our results suggest that suppression of PP2A-B56α through KRAS signaling can promote the MYC-driven initiation of pancreatic tumorigenesis.

Treatment of a mutant KRAS lung cancer cell line with polyisoprenylated cysteinyl amide inhibitors activates the MAPK pathway, inhibits cell migration and induces apoptosis.

KRAS mutations are the most common oncogenic mutations in lung adenocarcinoma in Black Americans. Polyisoprenylated Cysteinyl amide Inhibitors (PCAIs) constitute a group of potential cancer therapy agents that we designed to specifically disrupt and suppress hyperactive G-protein signaling, such as that caused by mutated RAS proteins. Here we determine the effects of PCAIs on the viability, G-protein levels, downstream mediators, and apoptosis-related proteins on the KRAS-mutated, Black American-derived lung adenocarcinoma cell line, NCI-H23. Of the 17 PCAIs tested, compounds NSL-YHJ-2-27 and NSL-YHJ-2-46 showed the most potency with EC50 values of 2.7 and 3.3 μM, respectively. Western blotting was used to determine the effect of the PCAIs on the phosphorylation levels of MAPK pathway enzymes. After 48 h exposure to 5 μM of the PCAIs, NSL-YHJ-2-46, the MAPK proteins BRAF, MEK1/2, ERK1/2, and p90RSK were activated through phosphorylation by 90, 190, 150 and 120%, respectively. However, CRAF/RAF1 phosphorylation decreased by 40%, suggesting significant changes in the KRAS/MAPK signaling patterns. Furthermore, 5 μM of NSL-YHJ-2-27 depleted the singly polyisoprenylated monomeric G-proteins RAC 1/2/3 and CDC42 by 77 and 76%, respectively. The depletion of these key cytoskeletal proteins may account for the observed inhibition of cell migration and invasion, and spheroid invasion observed on exposure to NSL-YHJ-2-27 and NSL-YHJ-2-46. Treatment with 5 μM of NSL-YHJ-2-27 suppressed full-length inactive caspase 3 and 7 levels by 72 and 91%, respectively. An analysis of cells treated with the fluorescently labeled active caspase 3/7 irreversible inhibitor, CaspaTagTM Caspase-3/7 in situ reagent revealed a 124% increase in active caspase at 3 μM over controls. These findings clearly show the direct effects of the PCAIs on the RAS signaling pathway. Given the profound increases observed in RPS6KA1/p90RSK phosphorylation, future work will involve a determination whether the proapoptotic isoforms of RPS6KA1/p90RSK are phosphorylated due to the PCAIs treatments. These results support the potential use of the PCAIs as targeted therapies against cancers with KRAS mutations.

Targeted Degradation of SOS1 Exhibits Potent Anticancer Activity and Overcomes Resistance in KRAS-Mutant Tumors and BCR-ABL-Positive Leukemia.

SOS1 is an essential guanine nucleotide exchange factor for RAS that also plays a critical role in the activation of the small GTPase RAC mediated by BCR-ABL in leukemogenesis. Despite this, small molecule inhibitors targeting SOS1 have shown limited efficacy in clinical trials for KRAS mutant cancers, and their potential as a therapeutic approach for chronic myeloid leukemia (CML) remains largely unexplored. In this study, we developed a potent SOS1 PROTAC SIAIS562055, which was designed by connecting a CRBN ligand to an analogue of the SOS1 inhibitor BI-3406. SIAIS562055 exhibited sustained degradation of SOS1 and inhibition of downstream ERK pathways, resulting in superior anti-proliferative activity compared to small molecule inhibitors. SIAIS562055 also potentiated the activity of both KRAS inhibitors in KRAS-mutant cancers and ABL inhibitors in BCR-ABL+ CML. In KRAS-mutant xenografts, SIAIS562055 displayed promising antitumor potency as a monotherapy and enhanced ERK inhibition and tumor regression when combined with KRAS inhibitors, overcoming acquired resistance. In CML cells, SIAIS562055 promoted the active uptake of BCR-ABL inhibitors by upregulating the carnitine/organic cation transporter SLC22A4. SIAIS562055 and BCR-ABL inhibitors synergistically enhanced inhibition of ABL phosphorylation and downstream signaling, demonstrating robust antitumor activities in both mouse xenografts and primary CML patient samples. In summary, this study suggests that PROTAC-mediated SOS1 degradation represents an effective therapeutic strategy for treating not only KRAS-mutant cancers but also BCR-ABL-harboring leukemia.

Tectal glioma: clinical, radiological, and pathological features, and the importance of molecular analysis.

Tectal glioma (TG) is a rare lower grade glioma (LrGG) that occurs in the tectum, mainly affecting children. TG shares pathological similarities with pilocytic astrocytoma (PA), but recent genetic analyses have revealed distinct features, such as alterations in KRAS and BRAF. We conducted a retrospective review of cases clinically diagnosed as TG and treated at our institute between January 2005 and March 2023. Six cases were identified and the median age was 30.5years. Four patients underwent biopsy and two patients underwent tumor resection. Histological diagnoses included three cases of PA, one case of astrocytoma, and two cases of high-grade glioma. The integrated diagnosis, according to the fifth edition of the World Health Organization Classification of Tumours of the central nervous system, included two cases of PA and one case each of diffuse high-grade glioma; diffuse midline glioma H3 K27-altered; glioblastoma; and circumscribed astrocytic glioma. Among the three patients who underwent molecular evaluation, two had KRAS mutation and one had H3-3A K27M mutation. Our results demonstrate the diverse histological and molecular characteristics of TG distinct from other LrGGs. Given the heterogeneous pathological background and the risk of pathological progression in TG, we emphasize the importance of comprehensive diagnosis, including molecular evaluation.

Bringing Hope to Improve Treatment in Pancreatic Ductal Adenocarcinoma—A New Tool for Molecular Profiling of KRAS Mutations in Tumor and Plasma Samples

Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) incidence is rising, and prognosis remains poor due to late diagnosis and limited effective therapies. Currently, patients are treated based on TNM staging, without molecular tumor characterization. This study aimed to validate a technique that combines the amplification refractory mutation system (ARMS) with high-resolution melting analysis (HRMA) for detecting mutations in codon 12 of KRAS in tumor and plasma, and to assess its prognostic value. Methods: Prospective study including patients with newly diagnosed PDAC with tumor and plasma samples collected before treatment. Mutations in codon 12 of KRAS (G12D, G12V, G12C, and G12R) were detected using ARMS–HRMA and compared to Sanger sequencing (SS). Univariate and multivariate analyses were used to evaluate the prognostic significance of these mutations. Results: A total of 88 patients, 93% with ECOG-PS 0–1, 57% with resectable disease. ARMS–HRMA technique showed a higher sensitivity than SS, both in tumor and plasma (77% vs. 51%; 25 vs. 0%, respectively). The most frequent mutation was G12D (n = 32, 36%), followed by G12V (n = 22, 25%). On multivariate analysis, patients with G12D and/or G12C mutations, either in tumor or plasma, had lower PFS (HR 1.792, 95% CI 1.061–3.028, p = 0.029; HR 2.081, 95% CI 1.014–4.272, p = 0.046, respectively) and lower OS (HR 1.757, 95% CI 1.013–3.049, p = 0.045; HR 2.229, 95% CI 1.082–4.594, p = 0.030, respectively). Conclusions: ARMS–HRMA is a rapid and cost-effective method for detecting KRAS mutations in PDAC patients, offering the potential for stratifying prognosis and guiding treatment decisions. The presence of G12D and G12C mutations in both tumor and plasma is associated with a poorer prognosis.