Scribble mis-localization induces adaptive resistance to KRAS G12C inhibitors through feedback activation of MAPK signaling mediated by YAP-induced MRAS.

Tumor cells evade targeted drugs by rewiring their genetic and epigenetic networks. Here, we identified that inhibition of MAPK signaling rapidly induces an epithelial-to-mesenchymal transition program by promoting re-localization of an apical-basal polarity protein, Scribble, in oncogene-addicted lung cancer models. Mis-localization of Scribble suppressed Hippo-YAP signaling, leading to YAP nuclear translocation. Furthermore, we discovered that a RAS superfamily protein MRAS is a direct target of YAP. Treatment with KRAS G12C inhibitors induced MRAS expression, which formed a complex with SHOC2, precipitating feedback activation of MAPK signaling. Abrogation of YAP activation or MRAS induction enhanced the efficacy of KRAS G12C inhibitor treatment in vivo. These results highlight a role for protein localization in the induction of a non-genetic mechanism of resistance to targeted therapies in lung cancer. Furthermore, we demonstrate that induced MRAS expression is a key mechanism of adaptive resistance following KRAS G12C inhibitor treatment.

Hot-Spot-Specific Probe (HSSP) for Rapid and Accurate Detection of KRAS Mutations in Colorectal Cancer.

Detection of oncogene mutations has significance for early diagnosis, customized treatment, treatment progression, and drug resistance monitoring. Here, we introduce a rapid, sensitive, and specific mutation detection assay based on the hot-spot-specific probe (HSSP), with improved clinical utility compared to conventional technologies. We designed HSSP to recognize KRAS mutations in the DNA of colorectal cancer tissues (HSSP-G12D (GGT→GAT) and HSSP-G13D (GGC→GAC)) by integration with real-time PCR. During the PCR analysis, HSSP attaches to the target mutation sequence for interference with the amplification. Then, we determine the mutation detection efficiency by calculating the difference in the cycle threshold (Ct) values between HSSP-G12D and HSSP-G13D. The limit of detection to detect KRAS mutations (G12D and G13D) was 5-10% of the mutant allele in wild-type populations. This is superior to the conventional methods (≥30% mutant allele). In addition, this technology takes a short time (less than 1.5 h), and the cost of one sample is as low as USD 2. We verified clinical utility using 69 tissue samples from colorectal cancer patients. The clinical sensitivity and specificity of the HSSP assay were higher (84% for G12D and 92% for G13D) compared to the direct sequencing assay (80%). Therefore, HSSP, in combination with real-time PCR, provides a rapid, highly sensitive, specific, and low-cost assay for detecting cancer-related mutations. Compared to the gold standard methods such as NGS, this technique shows the possibility of the field application of rapid mutation detection and may be useful in a variety of applications, such as customized treatment and cancer monitoring.

Ras inhibitor farnesylthiosalicylic acid conjugated with IR783 dye exhibits improved tumor-targeting and altered anti-breast cancer mechanisms in mice.

Ras has long been viewed as a promising target for cancer therapy. Farnesylthiosalicylic acid (FTS), as the only Ras inhibitor has ever entered phase II clinical trials, has yielded disappointing results due to its strong hydrophobicity, poor tumor-targeting capacity, and low therapeutic efficiency. Thus, enhancing hydrophilicity and tumor-targeting capacity of FTS for improving its therapeutic efficacy is of great significance. In this study we conjugated FTS with a cancer-targeting small molecule dye IR783 and characterized the anticancer properties of the conjugate FTS-IR783. We showed that IR783 conjugation greatly improved the hydrophilicity, tumor-targeting and therapeutic potential of FTS. After a single oral administration in Balb/c mice, the relative bioavailability of FTS-IR783 was increased by 90.7% compared with FTS. We demonstrated that organic anion transporting polypeptide (OATP) and endocytosis synergistically drove the uptake of the FTS-IR783 conjugate in breast cancer MDA-MB-231 cells, resulting in superior tumor-targeting ability of the conjugate both in vitro and in vivo. We further revealed that FTS-IR783 conjugate could bind with and directly activate AMPK rather than affecting Ras, and subsequently regulate the TSC2/mTOR signaling pathway, thus achieving 2-10-fold increased anti-cancer therapeutic efficacy against 6 human breast cancer cell lines compared to FTS both in vivo and in vitro. Overall, our data highlights a promising approach for the modification of the anti-tumor drug FTS using IR783 and makes it possible to return FTS back to the clinic with a better efficacy.

Phase II study of the GI-4000 KRAS vaccine after curative therapy in patients with stage I-III lung adenocarcinoma harboring a KRAS G12C, G12D, or G12V mutation.

INTRODUCTION: Patients with early-stage lung cancer have a high risk of recurrence despite multimodality therapy. KRAS-mutant lung adenocarcinomas are the largest genetically defined subgroup, representing 25% of patients. GI-4000 is a heat-killed recombinant Saccharomyces cerevisiae yeast-derived vaccine expressing mutant KRAS proteins. The present phase II study assessed the feasibility, immunogenicity, and safety of the GI-4000 vaccine in patients with early-stage, KRAS-mutant lung cancer. MATERIALS AND METHODS: Patients with stage I-III KRAS-mutant lung cancer who completed curative therapy were enrolled. The patients received the genotype matched GI-4000 vaccine for ≤ 3 years or until intolerance, disease recurrence, or death. The KRAS antigen T-cell response was assessed using the interferon-gamma enzyme-linked immunospot assay in peripheral blood mononuclear cells. The study was powered to detect an immune response in ≥ 25% of patients. RESULTS: A total of 24 patients were enrolled over 28 months. No vaccine-related serious adverse events occurred. One patient withdrew consent because of pain at the injection site. The study met its primary endpoint, with 50% of patients developing an immune response to mutant KRAS. The median number of vaccinations received was 15 (range, 1-19). Ten patients experienced disease recurrence, and 6 died. Compared with the genotypically matched historical controls, the recurrence rates were equivalent but overall survival showed a favorable trend. CONCLUSION: GI-4000 was well tolerated and immunogenic when used as consolidation therapy in patients with stage I-III KRAS-mutant lung cancer. The patterns of recurrence and death observed in the present study can be used to design a randomized study of GI-4000 with overall survival as the primary endpoint.

RAS mutations in thyroid cancer.

In recent years, our understanding of the genetic alterations underlying thyroid oncogenesis has greatly expanded. The use of molecular markers, including RAS, in the management of thyroid carcinoma is also increasing. This review summarizes the current literature surrounding RAS and discusses its potential as a diagnostic and prognostic indicator in the management of thyroid cancer.

Co-expression of matrix metalloproteinase-7 (MMP-7) and phosphorylated insulin growth factor receptor I (pIGF-1R) correlates with poor prognosis in patients with wild-type KRAS treated with cetuximab or panitumumab: a GEMCAD study.

BACKGROUND: By transactivacion, phosphorylated insulin growth factor receptor I (IGF-1R) can activate epidermal growth factor receptor (EGFR). MMP-7, produced by colorectal cancer cells, also can activate IGF-1R by degrading IGFBP-3 and releasing IGF-I. METHODS: A cohort of patients (pts) with advanced colorectal cancer (CRC), under second- or third-line treatment with cetuximab or panitumumab, was tested using immunohistochemistry for expression of the activated form of IGF-1R (p-IGF-1R) and MMP-7. KRAS and BRAF mutation status was determined by sequencing and allelic discrimination analysis, respectively. Analyses were performed in primary CRC tumor samples or metastases, and the association of immunohistochemistry findings, mutational results, and treatment outcomes was investigated in both univariate and multivariate analyses. RESULTS: Expression of activated IGF-1R and MMP-7 was observed in 51 and 49% of pts, respectively. Co-expression of MMP-7 and pIGF-1R (double positivity, DP) was observed in 28 pts (25%). There was no association between KRAS or BRAF mutational status and DP (p=0.52). Pts with DP responded more poorly to first-line chemotherapy (p=0.005) and to anti-EGFR treatment (p=0.01) than non-DP pts. In wild type (WT) KRAS pts, those with DP have poorer PFS (2.7 months vs. 3.5m, p=0.036; HR 1.98, 95% CI 1.05-3.75) and OS (6.4 months vs. 8.6 m, p=0.010; HR 2.33, 95%CI 1.23-4.43) in the adjusted multivariate analysis. CONCLUSIONS: Our study suggests that concomitant expression of MMP-7 and activation of p-IGF-1R (DP) correlates with poor prognosis in WT KRAS pts treated with anti-EGFR.

Implications for KRAS status and EGFR-targeted therapies in metastatic CRC.

EGFR regulates cancer-cell proliferation, apoptosis and tumor-induced neoangiogenesis, and has been validated as a relevant therapeutic target in several human cancers, including metastatic colorectal cancer (mCRC). The anti-EGFR monoclonal antibodies cetuximab and panitumumab are available for the treatment of patients with mCRC. Although EGFR is expressed in approximately 85% of patients with mCRC, the clinical efficacy of treatment with anti-EGFR antibodies is limited to a subset of patients. A series of potential biomarkers that could be useful in predicting response to EGFR inhibitors has been investigated. In patients with mCRC, activating mutations within KRAS can predict resistance to anti-EGFR monoclonal antibodies. Activating mutations in KRAS, which could result in EGFR-independent intracellular signal transduction activation, are found in approximately 35-40% of patients with mCRC. These mutations are almost exclusively detected in codons 12 and 13 of exon 2. KRAS mutations have been significantly associated with lack of response to cetuximab or panitumumab therapy in patients with mCRC, which suggests that EGFR-independent, constitutive activation of the RAS signaling pathway could impair response to anti-EGFR drugs. We summarize the experimental and clinical evidence supporting the use of KRAS testing for the optimal selection of patients with mCRC to be treated with anti-EGFR monoclonal antibodies.

Intradermal ras peptide vaccination with granulocyte-macrophage colony-stimulating factor as adjuvant: Clinical and immunological responses in patients with pancreatic adenocarcinoma.

K-RAS mutations are frequently found in adenocarcinomas of the pancreas, and induction of immunity against mutant ras can therefore be of possible clinical benefit in patients with pancreatic cancer. We present data from a clinical phase I/II trial involving patients with adenocarcinoma of the pancreas vaccinated by i.d. injection of synthetic mutant ras peptides in combination with granulocyte-macrophage colony-stimulating factor. Forty-eight patients (10 surgically resected and 38 with advanced disease) were treated on an outpatient basis. Peptide-specific immunity was induced in 25 of 43 (58%) evaluable patients, indicating that the protocol used is very potent and capable of eliciting immune responses even in patients with end-stage disease. Patients followed-up for longer periods showed evidence of induction of long-lived immunological memory against the ras mutations. CD4(+) T cells reactive with an Arg12 mutation also present in the tumor could be isolated from a tumor biopsy, demonstrating that activated, ras-specific T cells were able to selectively accumulate in the tumor. Vaccination was well tolerated in all patients. Patients with advanced cancer demonstrating an immune response to the peptide vaccine showed prolonged survival from the start of treatment compared to non-responders (median survival 148 days vs. 61 days, respectively; p = 0.0002). Although a limited number of patients were included in our study, the association between prolonged survival and an immune response against the vaccine suggests that a clinical benefit of ras peptide vaccination may be obtained for this group of patients.

Prenylation inhibitors in renal disease.

Members of the superfamily of Ras GTPase signalling proteins (monomeric G proteins) require post-translational carboxy-terminal prenylation to function. Prenylation is the covalent attachment of a hydrophobic prenyl group (either farnesyl or geranylgeranyl), which localises the GTPase to cell membranes. Ras proteins exert substantial control on cell proliferation and gene-transcription events, and prenylation inhibitors are now included in clinical trials for cancer. Many renal diseases are highly proliferative and are driven by a range of profibrotic cytokines. We hypothesise that inhibition of prenylation could be of substantial therapeutic benefit in such diseases, providing greater selectivity against abnormal cytokine-driven proliferation and fibrogenesis than current treatments available to nephrologists.

Mutated Ras peptides as vaccines in immunotherapy of cancer.

Mutations in codon 12 and 13 of K-RAS are frequently found in human cancer, including pancreatic- and colorectal adenocarcinomas. T cell responses specific for individual RAS mutations can be elicited in vitro by stimulation with synthetic peptides and in vivo following vaccination with antigen presenting cells pulsed ex vivo with synthetic peptides. The peptide-responding T cells are capable of responding to intact p21 ras, and can recognise and kill tumour cell lines and isolated tumour cells harbouring the corresponding RAS mutation. The responding cells can be of both CD4+ and CD8+ phenotype, and these T cell subsets recognise nested epitopes within the vaccine peptides. Mutant ras peptides are therefore possibly an important vaccine for specific immunotherapy in patients with pancreatic and colorectal carcinomas, and are currently being tested in vivo together with GM-CSF as an adjuvant in these cancer patients.