Biochemical characterization of the interaction between KRAS and Argonaute 2

Oncogenic mutations in KRAS result in a constitutively active, GTP-bound form that in turn activates many proliferative pathways. However, because of its compact and simple architecture, directly targeting KRAS with small molecule drugs has been challenging. Another approach is to identify targetable proteins that interact with KRAS. Argonaute 2 (AGO2) was recently identified as a protein that facilitates RAS-driven oncogenesis. Whereas previous studies described the in vivo effect of AGO2 on cancer progression in cells harboring mutated KRAS, here we sought to examine their direct interaction using purified proteins. We show that full length AGO2 co-immunoprecipitates with KRAS using purified components, however, a complex between FL AGO2 and KRAS could not be isolated. We also generated a smaller N-terminal fragment of AGO2 (NtAGO2) which is believed to represent the primary binding site of KRAS. A complex with NtAGO2 could be detected via ion-mobility mass spectrometry and size exclusion chromatography. However, the data suggest that the interaction of KRAS with purified AGO2 (NtAGO2 or FL AGO2) is weak and likely requires additional cellular components or proteo-forms of AGO2 that are not readily available in our purified assay systems. Future studies are needed to determine what conformation or modifications of AGO2 are necessary to enrich KRAS association and regulate its activities.     

Synergistic Effects of Concurrent Blockade of PI3K and MEK Pathways in Pancreatic Cancer Preclinical Models

Patients with pancreatic cancer have dismal prognoses, and novel therapies are urgently needed. Mutations of the KRAS oncogene occur frequently in pancreatic cancer and represent an attractive target. Direct targeting of the predominant KRAS pathways have been challenging and research into therapeutic strategies have been now refocused on pathways downstream of KRAS, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK [MEK]). We hypothesized that concurrent inhibition of the PI3K and MEK pathways would result in synergistic antitumor activity, as it would circumvent the compensatory feedback loop between the two pathways. We investigated the combined effect of the PI3K inhibitor, GDC0941, and the MEK inhibitor, AZD6244, on cell viability, apoptosis and cell signaling in a panel of pancreatic cancer cell lines. An in vivo analysis was conducted on pancreatic cancer xenografts. While BxPC-3 (KRAS wild type) and MIA PaCa-2 (KRAS mutated) cell lines were sensitive to GDC0941 and AZD6244 as single agents, synergistic inhibition of tumor cell growth and induction of apoptosis were observed in both cell lines when the two drugs were combined. Interestingly, phosphorylation of the cap-dependent translational components, 4E-binding protein (p-4E-BP1) and S6 was found to be closely associated with sensitivity to GDC0941 and AZD6244. In BxPC-3 cell xenografts, survival differences were observed between the control and the AZD6244, GDC0941, and combination groups. Our study provides the rationale for concurrent targeting of the PI3K and MEK pathways, regardless of KRAS status, and suggests that phosphorylation of 4E-BP1and S6 can serve as a predictive biomarker for response to treatment.     

RNA aptamers targeting the carboxyl terminus of KRAS oncoprotein generated by an improved SELEX with isothermal RNA amplification

Mutations in the KRAS gene occur frequently in various human tumors and are known to lead to malignant transformation. We isolated RNA aptamers targeting activated mutant KRAS proteins using an improved SELEX method by isothermal RNA amplification. RNA aptamers were selected against mutant KRAS (G12V) proteins, as well as a biotinylated 15-amino-acid peptide from the carboxyl terminal of KRAS that contains a farnesylation site. All the selected RNA aptamers bound to the basic carboxy-terminal region of KRAS protein and the highest K(D) value was 2.3 microM. By an in vitro scintillation proximity assay, we demonstrated that KRAS aptamers inhibited farnesylation moderately. From these aptamers, we determined a consensus sequence (U)CCAAGCAC(AC) that, when concatamerized, exhibited higher binding affinity to the carboxy-terminal region of KRAS protein. Further improvement of binding affinity between aptamers and KRAS protein might provide a new therapeutic approach for activated mutant KRAS proteins.     

Therapeutic targeting of RAS: New hope for drugging the “undruggable”

RAS is the most frequently mutated oncogene in cancer and a critical driver of oncogenesis. Therapeutic targeting of RAS has been a goal of cancer research for more than 30 years due to its essential role in tumor formation and maintenance. Yet the quest to inhibit this challenging foe has been elusive. Although once considered “undruggable”, the struggle to directly inhibit RAS has seen recent success with the development of pharmacological agents that specifically target the KRAS(G12C) mutant protein, which include the first direct RAS inhibitor to gain entry to clinical trials. However, the limited applicability of these inhibitors to G12C-mutant tumors demands further efforts to identify more broadly efficacious RAS inhibitors. Understanding allosteric influences on RAS may open new avenues to inhibit RAS. Here, we provide a brief overview of RAS biology and biochemistry, discuss the allosteric regulation of RAS, and summarize the various approaches to develop RAS inhibitors.     

利用CRISPR/Cas9技术靶向KRAS或TP53突变抑制SW620细胞的增殖

摘要 目的：运用CRISPR/Cas9基因编辑工具研究靶向SW620细胞系中KRAS或TP53突变对细胞增殖活性的影响。方法：针对SW620细胞中KRAS和TP53的突变位点设计sgRNA,并利用TIDE法检测sgRNA的切割效率。通过细胞增殖实验检测靶向KRAS或TP53突变后SW620细胞增殖活性的改变,并应用Annexin V-FITC/PI双染法检测细胞凋亡水平的变化。结果：分别构建了靶向SW620细胞系中KRAS和TP53突变的sgRNA质粒,并通过TIDE分析验证了sgRNA的内源切割效率;细胞增殖实验及细胞凋亡检测显示,靶向突变的KRAS或TP53基因后,SW620细胞增殖活性明显减弱,凋亡水平明显升高（P<0.05）。结论：本研究基于CRISPR/Cas9技术实现了对SW620细胞系中突变的KRAS和TP53的基因编辑,发现靶向KRAS或TP53突变能够明显抑制SW620细胞的增殖活性并促进细胞凋亡,为结直肠癌相关靶点治疗提供了体外实验依据。     

Strategies for targeting energy metabolism in Kirsten rat sarcoma viral oncogene homolog -mutant colorectal cancer

Alterations in cellular energy metabolism play critical roles in colorectal cancer (CRC). These alterations, which correlate to KRAS mutations, have been identified as energy metabolism signatures. This review summarizes the relationship between colorectal tumors associated with mutated KRAS and energy metabolism, especially for the deregulated energy metabolism that affects tumor cell proliferation, invasion, and migration. Furthermore, this review will concentrate on the role of metabolic genes, factors and signaling pathways, which are coupled with the primary energy source connected with the KRAS mutation that induces metabolic alterations. Strategies for targeting energy metabolism in mutated KRAS CRC are also introduced. In conclusion, deregulated energy metabolism has a close relationship with KRAS mutations in colorectal tumors. Therefore, selective inhibitors, agents against metabolic targets or KRAS signaling, may be clinically useful for colorectal tumor treatment through a patient-personalized approach.     

KRAS G12V mutation upregulates PD-L1 expression via TGF-β/EMT signaling pathway in human non-small-cell lung cancer

Although clinical data suggest remarkable promise for targeting programmed cell death protein-1 (PD-1) and ligand (PD-L1) signaling in non-small-cell lung cancer (NSCLC), it is still largely undetermined which subtype of patients will be responsive to checkpoint blockade. In the present study, we explored whether PD-L1 was regulated by mutant Kirsten rat sarcoma viral oncogene homolog (KRAS), which is frequently mutated in NSCLC and results in poor prognosis and low survival rates. We verified that PD-L1 levels were dramatically increased in KRAS mutant cell lines, particularly in NCI-H441 cells with KRAS G12V mutation. Overexpression of KRAS G12V remarkably elevated PD-L1 messenger RNA and protein levels, while suppression of KRAS G12V led to decreased PD-L1 levels in NCI-H441 cells. Consistently, higher levels of PD-L1 were observed in KRAS-mutated tissues as well as tumor tissues-derived CD4⁺ and CD8⁺ T cells using a tumor xenograft in B-NDG mice. Mechanically, both in vitro and in vivo assays found that KRAS G12V upregulated PD-L1 via regulating the progression of epithelial-to-mesenchymal transition (EMT). Moreover, pembrolizumab activated the antitumor activity and decreased tumor growth with KRAS G12V mutated NSCLC. This study demonstrates that KRAS G12V mutation could induce PD-L1 expression and promote immune escape via transforming growth factor-β/EMT signaling pathway in KRAS-mutant NSCLC, providing a potential therapeutic approach for NSCLC harboring KRAS mutations.     

KRAS Prenylation Is Required for Bivalent Binding with Calmodulin in a Nucleotide-Independent Manner

Deregulation of KRAS4b signaling pathway has been implicated in 30% of all cancers. Membrane localization of KRAS4b is an essential step for the initiation of the downstream signaling cascades that guide various cellular mechanisms. KRAS4b plasma membrane (PM) binding is mediated by the insertion of a prenylated moiety that is attached to the terminal carboxy-methylated cysteine, in addition to electrostatic interactions of its positively charged hypervariable region with anionic lipids. Calmodulin (CaM) has been suggested to selectively bind KRAS4b to act as a negative regulator of the RAS/mitogen-activated protein kinase (MAPK) signaling pathway by displacing KRAS4b from the membrane. However, the mechanism by which CaM can recognize and displace KRAS4b from the membrane is not well understood. In this study, we employed biophysical and structural techniques to characterize this mechanism in detail. We show that KRAS4b prenylation is required for binding to CaM and that the hydrophobic pockets of CaM can accommodate the prenylated region of KRAS4b, which might represent a novel CaM-binding motif. Remarkably, prenylated KRAS4b forms a 2:1 stoichiometric complex with CaM in a nucleotide-independent manner. The interaction between prenylated KRAS4b and CaM is enthalpically driven, and electrostatic interactions also contribute to the formation of the complex. The prenylated KRAS4b terminal KSKTKC-farnesylation and carboxy-methylation is sufficient for binding and defines the minimal CaM-binding motif. This is the same region implicated in membrane and phosphodiesterase6-δ binding. Finally, we provide a structure-based docking model by which CaM binds to prenylated KRAS4b. Our data provide new insights into the KRAS4b-CaM interaction and suggest a possible mechanism whereby CaM can regulate KRAS4b membrane localization.     

A High-Throughput Assay for Small Molecule Destabilizers of the KRAS Oncoprotein

Mutations in the Ras family of small GTPases, particularly KRAS, occur at high frequencies in cancer and represent a major unmet therapeutic need due to the lack of effective targeted therapies. Past efforts directed at inhibiting the activity of the Ras oncoprotein have proved difficult. We propose an alternative approach to target Ras by eliminating Ras protein from cells with pharmacological means. In this study, we developed a cell-based, high-content screening platform to identify small molecules that could promote the degradation of the KRAS oncoprotein. We generated an EGFP-KRAS^G12V fluorescence reporter system and implemented it for automated screening in 1536-well plates using high-throughput cellular imaging. We screened a library of clinically relevant compounds at wide dose range and identified Ponatinib and AMG-47a as two candidate compounds that selectively reduced the levels of EGFP-KRAS^G12V protein but did not affect EGFP protein in cells. This proof-of-principle study demonstrates that it is feasible to use a high-throughput screen to identify compounds that promote the degradation of the Ras oncoprotein as a new approach to target Ras.     

Synthesis and evaluation of apoptosis induction of thienopyrimidine compounds on KRAS and BRAF mutated colorectal cancer cell lines

Monoclonal antibodies (MoAb) and tyrosine kinase inhibitors (TKI) targeting the EGFR (Epidermal Growth Factor Receptor) pathways are currently used in colorectal cancer treatment. Despite the improvement of median overall survival, resistance is observed notably due to KRAS and BRAF gene mutations. We synthesized four series of thienopyrimidines whose scaffold is structurally close to TKI used in clinical practice. We evaluated apoptosis induced by these compounds using flow cytometry on KRAS and BRAF mutated cell lines. Our results confirm that the mutated cell lines (HCT116 and HT29) are more resistant to apoptosis than the non-mutated cell line (Hela). Interestingly, among the 13 compounds tested, three of them (5b, 6b and 6d) and gefitinib exhibited a noteworthy pro-apoptotic effect, especially on mutated cell lines with an IC₅₀ value between 70 and 110 μM. These three compounds seem particularly attractive for the development of novel treatments for colorectal cancer patients harboring EGFR pathway mutations.     

Prospective analysis of KRAS wild-type patients with metastatic colorectal cancer using cetuximab plus FOLFIRI or FOLFOX4 treatment regimens

Cetuximab, a monoclonal antibody targeting epidermal growth factor receptor, has proven to be efficient in the treatment of metastatic colorectal cancer. We made a prospective study of the efficacy and toxicities of cetuximab-combination first-line (FOLFOX4) versus second/third-line (FOLFIRI) chemotherapy in 98 KRAS wild-type patients who had metastatic colorectal cancer. Wild-type KRAS had been identified by direct sequencing. Associations between clinical response/progression-free survival/overall survival/toxicities and cetuximab-combination chemotherapy timing were evaluated. The overall response rate was significantly higher for first-line treatment than for second/third-line treatment (relative risk = 1.707, 95% confidence interval = 1.121-2.598). Both progression-free survival and overall survival indicated significantly longer survival of first-line treatment than second/third-line treatment patients. This study is a validation of a molecular analysis of KRAS wild-type status for the prediction of response to cetuximab-combination chemotherapy for metastatic colorectal cancer patients; its predictive role was less prominent in the second/third-line than in the first-line treatment patients.     

Design,synthesis and biological evaluation of new Myo-inositol derivatives as potential RAS inhibitors

Ras is a small family of GTPases that control numerous cellular functions like cell proliferation, growth, survival, gene expression, and is closely engaged in cancer pathogenesis. The ras-targeted methodology entails a holy grail in oncology. Nevertheless, there are no specific molecules reported targeting the same, although it is a known oncogene for more than three decades. In this study, we have designed and synthesized new phosphate derivatives of Myo-inositol to inhibit the oncogenic KRAS pathway in breast cancer cells, which has been validated by cellular and theoretical studies. The synthesized compound 1b (C2-O-phosphate derivative of Myo-inositol 1,3,5-orthobenzoate) inhibited the downstream signaling pathway of oncogenic KRAS, RAF/MEK/ERK. Furthermore, we also found that this compound induced necrosis/apoptosis and causes cell cycle arrest. This class of molecules may work as a potential inhibitor of breast cancer caused by a mutation in KRAS and its downstream proteins. Though the efficacy of the molecules is in the micromolar scale, they have not been explored previously for RAS inhibition. Impressive preliminary results are presented in this article which could be further explored for its detailed biological studies to get better candidates as RAS inhibitors.     

D154Q Mutation does not Alter KRAS Dimerization

KRAS is one of the most frequently mutated oncogenes in human cancers. Despite nearly 40 years of research, KRAS remains largely undruggable, in part due to an incomplete understanding of its biology. Recently, KRAS dimerization was discovered to play an important role in its signalling function. The KRAS D154Q mutant was described as a dimer-deficient variant that can be used to study the effect of dimerization in KRAS oncogenicity. However, we show here that KRAS D154Q homo- and heterodimerized with KRAS WT using three separate protein–protein interaction assays, and that oncogenic KRAS dimerization was not negatively impacted by the presence of a secondary D154Q mutation. In conclusion, we advise caution in using this variant to study the purpose of dimerization in KRAS oncogenic behaviour.     

EGFR和KRAS在上皮性卵巢癌组织中的表达及临床意义

目的:探讨表皮生长因子受体(EGFR)和鼠Kirsten肉瘤病毒致癌基因(KRAS)蛋白在上皮性卵巢癌组织中的表达水平及临床意义。方法:利用免疫组化SP法对上皮性卵巢癌组织(病例组,n=57)、卵巢良性肿瘤组织(良性组,n=50)以及正常卵巢组织(对照组,n=50)中的EGFR、KRAS蛋白水平进行检测,并分析其在上皮性卵巢癌发生发展中的作用。结果:病例组的EGFR、KRAS蛋白阳性率高于良性组和对照组(P0.05),良性组和对照组的EGFR、KRAS蛋白阳性率差异无统计学意义(P0.05)。浆液性腺癌组EGFR、KRAS蛋白的阳性表达率高于非浆液性腺癌组,Ⅲ~Ⅳ期的上皮性卵巢癌组织中EGFR、KRAS蛋白阳性表达率高于Ⅰ~Ⅱ期,中、低分化组中EGFR、KRAS蛋白阳性表达率高于高分化组,差异均具有统计学意义(P0.05)。上皮性卵巢癌组织中EGFR与KRAS的蛋白的阳性表达率呈正相关关系(r=0.469,P0.05)。结论:EGFR及KRAS蛋白在上皮性卵巢癌组织中的表达水平明显升高,可能参与了上皮性卵巢癌的发生发展过程,且两者之间呈正相关关系,联合检测可作为早期诊断上皮性卵巢癌的重要指标。     

Development of PD3 and PD3-B for PDEδ inhibition to modulate KRAS activity

Despite extensive efforts over 40 years, few effective KRAS inhibitors have been developed to date, mainly due to the undruggable features of KRAS proteins. In addition to the direct approach to KRAS via covalent inhibition, modulation of the prenyl-binding protein PDEδ that binds with farnesylated KRAS has emerged as an alternative strategy to abrogate KRAS activity. For the verification of new therapeutic strategies, chemical probes with the dual functions of visualisation and pharmacological inhibition against oncogenic proteins are enormously valuable to understand cellular events related to cancer. Here, we report indolizino[3,2-c]quinoline (IQ)-based fluorescent probes (PD3 and PD3-B) for PDEδ inhibition. By using the unique fluorescent characteristics of the IQ scaffold, a fluorescence polarisation (FP)-based binding assay identified PD3 as the most effective PDEδ probe among the tested PD analogues, with a low K_d value of 0.491 µM and long retention time in the binding site of PDEδ. In particular, a FP-based competition assay using deltarasin verified that PD3 occupies the farnesylation binding site of PDEδ, excluding the possibility that the FP signals resulted from non-specific hydrophobic interactions between the ligand and protein in the assay. We also designed and synthesised PD3-B (5), an affinity-based probe (ABP) from the PD3 structure, which enabled us to pull down PDEδ from bacterial lysates containing a large number of intrinsic bacterial proteins. Finally, KRAS relocalization was verified in PANC-1 cells by treatment with PD3, suggesting its potential as an effective probe to target PDEδ.     

huBC1-IL12, an immunocytokine which targets EDB-containing oncofetal fibronectin in tumors and tumor vasculature,shows potent anti-tumor activity in human tumor models

IL-12 is a cytokine which showed anti-tumor effects in clinical trials, but also produced serious toxicity. We describe a fusion protein, huBC1-IL12, designed to achieve an improved therapeutic index by specifically targeting IL-12 to tumor and tumor vasculature. huBC-1 is a humanized antibody that targets a cryptic sequence of the human ED-B-containing fibronectin isoform, B-FN, present in the subendothelial extracellular matrix of most aggressive tumors. B-FN is oncofetal and angiogenesis-associated, and is undetectable in most normal adult tissues. The original murine BC-1 antibody has been used successfully for immunoscintigraphy to image brain tumor mass in glioblastoma patients. In huBC1-IL12, each of the IgG heavy chains is genetically fused to the N-terminus of the IL-12 p35 subunit, which in turn is disulfide-bonded to the p40 subunit, resulting in a hexameric molecule of MW of ∼300 kDa. Since human IL-12 has no biological activity in mice, we produced huBC1-muIL12 as a surrogate molecule for animal tumor models. Despite the relatively poor PK profile of this molecule in mice and the apparent drawbacks of xenogeneic models in SCID mice, which lack T and B cells, one cycle of treatment with huBC1-muIL12 was efficacious in the PC3mm2, A431, and HT29 subcutaneous tumor models and PC3mm2 lung metastasis model. This molecule also was found to have surprisingly low toxicity in immunocompetent mice. A fusion protein that contains human IL-12 (huBC1-huIL12), which is a suitable molecule for investigation as a therapeutic, has also been produced. This protein has been shown to have a longer serum half-life than huBC1-muIL12 in mice, and retains both antigen binding and IL-12 activity in in vitro assays.     

Peptide Scaffold-Based Discovery of Nonpeptide Natural Medicines to Target PI3K p85 SH2 Domain

Targeting phosphoinositide 3-kinase (PI3K) has been recognized as an attractive strategy for anticancer therapy. The PI3K is a heterodimer composed of a catalytic subunit p110 and a regulatory subunit p85. Here, instead of targeting the catalytic p110 that has been considered previously, we purposed targeting the peptide-recognition domain SH2 of regulatory p85 with natural medicines obtained by using a peptide scaffold-based screening scheme. In the procedure, a core binding motif was extracted from the cocrystallized complex of a cognate phosphopeptide with the domain, which was considered as basic scaffold to perform high-through virtual screening against a structurally diverse, nonredundant library of natural products. A number of hit compounds with high binding potency to the domain and significant conformational similarity with the peptide scaffold were identified; in vitro affinity assay confirmed that five hits have moderate or high affinity for the domain with measured dissociation constants K_d range between 25 and 360 μM, which are comparable to or even better than that of the cognate phosphopeptide SDpYMNMTP and its core motif peptide pYMNM (K_d?=?15 and 32 μM, respectively). Structural analysis and nonbonded comparison of SH2 interactions with phosphopeptides and potent hit compounds revealed that only negatively charged phosphate and, sometime, sulfate can confer domain-binding capability to small-molecule compounds, but carboxylate cannot. A similar binding mode of compounds with phosphopeptide is important for the compounds to have high affinity and specificity.