A recombinant chimeric protein specifically induces mutant KRAS degradation and potently inhibits pancreatic tumor growth.
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ABSTRACT:
SUBMITTER: Pan T
PROVIDER: S-EPMC5190097 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Tumour-associated KRAS mutations are the most prevalent in the three RAS-family isoforms and involve many different amino-acids. Therefore, molecules able to interfere with mutant KRAS protein are potentially important for wide-ranging tumour therapy. We describe the engineering of two RAS degraders based on protein macromolecules (macrodrugs) fused to specific E3 ligases. A KRAS-specific DARPin fused to the VHL E3 ligase is compared to a pan-RAS intracellular single domain antibody (iDAb) fused to the UBOX domain of the CHIP E3 ligase. We demonstrate that while the KRAS-specific DARPin degrader induces specific proteolysis of both mutant and wild type KRAS, it only inhibits proliferation of cancer cells expressing mutant KRAS in vitro and in vivo. Pan-RAS protein degradation, however, affects proliferation irrespective of the RAS mutation. These data show that specific KRAS degradation is an important therapeutic strategy to affect tumours expressing any of the range of KRAS mutations.
| S-EPMC7319959 | biostudies-literature
Project description:High frequency of KRAS and TP53 mutations is a unique genetic feature of pancreatic ductal adenocarcinoma (PDAC). TP53 mutation not only renders PDAC resistance to chemotherapies but also drives PDAC invasiveness. Therapies targeting activating mutant KRAS are not available and the outcomes of current PDAC treatment are extremely poor. Here, we report that MMRi62, initially identified as an MDM2-MDM4-targeting small molecule with p53-independent pro-apoptotic activity, shows anti-PDAC activity in vitro and in vivo. We show that MMRi62 inhibits proliferation, clonogenic, and spheroid growth of PDAC cells by induction of cell death. MMRi62-induced cell death in PDAC is characteristic of ferroptosis that is associated with increased autophagy, increased reactive oxygen species, and lysosomal degradation of NCOA4 and ferritin heavy chain (FTH1). In addition to induced degradation of FTH1, MMRi62 also induces proteasomal degradation of mutant p53. Interestingly, MMRi62-induced ferroptosis occurs in PDAC cell lines harboring either KRAS and TP53 double mutations or single TP53 mutation. In orthotopic xenograft PDAC mouse models, MMRi62 was capable of inhibiting tumor growth in mice associated with downregulation of NCOA4 and mutant p53 in vivo. Strikingly, MMRi62 completely abrogated metastasis of orthotopic tumors to distant organs, which is consistent with MMRi62's ability to inhibit cell migration and invasion in vitro. These findings identified MMRi62 as a novel ferroptosis inducer capable of suppressing PDAC growth and overcoming metastasis.
| S-EPMC10258866 | biostudies-literature
Project description:Stabilization of the MYC oncoprotein by KRAS signaling critically promotes the growth of pancreatic ductal adenocarcinoma (PDAC). Thus, understanding how MYC protein stability is regulated may lead to effective therapies. Here, we used a previously developed, flow cytometry-based assay that screened a library of >800 protein kinase inhibitors and identified compounds that promoted either the stability or degradation of MYC in a KRAS-mutant PDAC cell line. We validated compounds that stabilized or destabilized MYC and then focused on one compound, UNC10112785, that induced the substantial loss of MYC protein in both two-dimensional (2D) and 3D cell cultures. We determined that this compound is a potent CDK9 inhibitor with a previously uncharacterized scaffold, caused MYC loss through both transcriptional and posttranslational mechanisms, and suppresses PDAC anchorage-dependent and anchorage-independent growth. We discovered that CDK9 enhanced MYC protein stability through a previously unknown, KRAS-independent mechanism involving direct phosphorylation of MYC at Ser62 Our study thus not only identifies a potential therapeutic target for patients with KRAS-mutant PDAC but also presents the application of a screening strategy that can be more broadly adapted to identify regulators of protein stability.
| S-EPMC6728149 | biostudies-literature
Project description:The oncogenic Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation was reported to be the signature genetic event in most cases of pancreatic ductal adenocarcinoma (PDAC). Hepassocin (HPS/FGL1) is involved in regulating lipid metabolism and the progression of several cancer types; however, the underlying mechanism of HPS/FGL1 in the KRAS mutant PDAC cells undergoing eicosapentaenoic acid (EPA) treatment remains unclear. We measured HPS/FGL1 protein expressions in a human pancreatic ductal epithelial (HPNE) normal pancreas cell line, a KRAS-wild-type PDAC cell line (BxPC-3), and KRAS-mutant PDAC cell lines (PANC-1, MIA PaCa-2, and SUIT-2) by Western blot methods. HEK293T cells were transiently transfected with corresponding KRAS-expressing plasmids to examine the level of HPS expression with KRAS activation. We knocked-down HPS/FGL1 using lentiviral vectors in SUIT-2 cells and measured the cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenicity assays. Furthermore, a lipidomic analysis was performed to profile changes in lipid metabolism after HPS/FGL1 knockdown. We found that the HPS/FGL1 level was significantly upregulated in KRAS-mutated PDAC cells and was involved in KRAS/phosphorylated (p)-signal transduction and activator of transcription 3 (STAT3) signaling, and the knockdown of HPS/FGL1 in SUIT-2 cells decreased cell proliferation through increasing G2/M cell cycle arrest and cyclin B1 expression. In addition, the knockdown of HPS/FGL1 in SUIT-2 cells significantly increased omega-3 polyunsaturated fatty acids (PUFAs) and EPA production but not docosahexaenoic acid (DHA). Moreover, EPA treatment in SUIT-2 cells reduced the expression of de novo lipogenic protein, acetyl coenzyme A carboxylase (ACC)-1, and decreased p-STAT3 and HPS/FGL1 expressions, resulting in the suppression of cell viability. Results of this study indicate that HPS is highly expressed by KRAS-mutated PDAC cells, and HPS/FGL1 plays a crucial role in altering lipid metabolism and increasing cell growth in pancreatic cancer. EPA supplements could potentially inhibit or reduce ACC-1-involved lipogenesis and HPS/FGL1-mediated cell survival in KRAS-mutated pancreatic cancer cells.
| S-EPMC8001293 | biostudies-literature
Project description:Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal cancers with a dismal prognosis for the patient. This is due to limited diagnostic options for the early detection of the disease as well as its rather aggressive nature. Despite major advances in oncologic research in general, the treatment options in the clinic for PDAC have only undergone minor changes in the last decades. One major treatment advance would be the successful targeting of the oncogenic driver KRASmut. In the past, the indirect targeting of KRAS has been exploited, e. g., via upstream inhibition of receptor tyrosine kinases or via downstream MEK or PI3K inhibition. However, the experience gained from clinical trials and from the clinic itself in the treatment of KRASmut cancer entities has dampened the initial euphoria. Lately, with the development of KRASG12C-specific inhibitors, not only the direct but also the indirect targeting of KRASmut has gained momentum again. Though preclinical studies and preliminary early clinical studies of monotherapies have shown promising results, they have been overshadowed by the swift development of resistances resulting in inconsistent responses in patient cohorts. Currently, several different combination therapies for KRASmut cancer are being explored. If they hold the promise they have made in preclinical studies, they might also be suitable treatment options for patients suffering from PDAC.
| S-EPMC7459579 | biostudies-literature
Project description:Pancreatic ductal adenocarcinoma (PDA) represents an unmet therapeutic challenge. PDA is addicted to the activity of the mutated KRAS oncogene which is considered so far an undruggable therapeutic target. We propose an approach to target KRAS effectively in patients using RNA interference. To meet this challenge, we have developed a local prolonged siRNA delivery system (Local Drug EluteR, LODER) shedding siRNA against the mutated KRAS (siG12D LODER). The siG12D LODER was assessed for its structural, release, and delivery properties in vitro and in vivo. The effect of the siG12D LODER on tumor growth was assessed in s.c. and orthotopic mouse models. KRAS silencing effect was further assessed on the KRAS downstream signaling pathway. The LODER-encapsulated siRNA was stable and active in vivo for 155 d. Treatment of PDA cells with siG12D LODER resulted in a significant decrease in KRAS levels, leading to inhibition of proliferation and epithelial-mesenchymal transition. In vivo, siG12D LODER impeded the growth of human pancreatic tumor cells and prolonged mouse survival. We report a reproducible and safe delivery platform based on a miniature biodegradable polymeric matrix, for the controlled and prolonged delivery of siRNA. This technology provides the following advantages: (i) siRNA is protected from degradation; (ii) the siRNA is slowly released locally within the tumor for prolonged periods; and (iii) the siG12D LODER elicits a therapeutic effect, thereby demonstrating that mutated KRAS is indeed a druggable target.
| S-EPMC3870687 | biostudies-literature
Project description:BACKGROUND:Pancreatic cancer is one of the most aggressive human malignancies. The development of a novel drug to treat pancreatic cancer is imperative, and it is thought that complementary and alternative medicine (CAM) could yield such a candidate. Agaricus blazei Murrill is a CAM that has been tested as an anticancer drug, but its efficacy against pancreatic cancer is poorly understood. To study the potential of A. blazei in the treatment of pancreatic cancer, we examined the effects of its hot water extract on the proliferation and global gene expression profile of human pancreatic cancer cells. METHODS:Three distinct human pancreatic cancer cell lines, MIAPaCa-2, PCI-35, and PK-8, and the immortalized human pancreatic duct-epithelial cell line, HPDE, were employed. The cells were incubated with the appropriate growth medium supplemented with the hot water extract of A. blazei at final concentrations of 0.005, 0.015%, or 0.045%, and cellular proliferation was assessed for five consecutive days using an MTT assay. Apoptosis was examined by using flow cytometry and the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Caspase-dependent apoptosis was assayed using immunoblotting. Global gene expression profiles were examined using a whole human genome 44?K microarray, and the microarray results were validated by using real-time reverse transcription PCR. RESULTS:The hot water extract of A. blazei significantly inhibited the proliferation of cultured pancreatic cancer cells through the induction of G0/G1 cell cycle arrest and caspase-dependent apoptosis; the effect was the smallest in HPDE cells. Furthermore, significant alterations in the global gene expression profiles of pancreatic cancer cells occurred following treatment with the hot water extract of A. blazei. Genes associated with kinetochore function, spindle formation, and centromere maintenance were particularly affected, as well as cyclins and cyclin-dependent kinases that are essential for cell cycle progression. In addition, proapoptotic genes were upregulated. CONCLUSIONS:The hot water extract of A. blazei may be useful for the treatment of pancreatic cancer and is a potential candidate for the isolation of novel, active compounds specific for mitotic spindle dysfunction.
| S-EPMC6280349 | biostudies-literature
Project description:Induction of compensatory mechanisms and ERK reactivation has limited the effectiveness of Raf and MEK inhibitors in RAS-mutant cancers. We determined that direct pharmacologic inhibition of ERK suppressed the growth of a subset of KRAS-mutant pancreatic cancer cell lines and that concurrent phosphatidylinositol 3-kinase (PI3K) inhibition caused synergistic cell death. Additional combinations that enhanced ERK inhibitor action were also identified. Unexpectedly, long-term treatment of sensitive cell lines caused senescence, mediated in part by MYC degradation and p16 reactivation. Enhanced basal PI3K-AKT-mTOR signaling was associated with de novo resistance to ERK inhibitor, as were other protein kinases identified by kinome-wide siRNA screening and a genetic gain-of-function screen. Our findings reveal distinct consequences of inhibiting this kinase cascade at the level of ERK.
| S-EPMC4816652 | biostudies-literature
Project description:IntroductionPancreatic ductal adenocarcinoma (PDAC) has the highest mortality rate among all solid tumors. Tumorigenesis is promoted by the oncogene KRAS, and KRAS mutations are prevalent in patients with PDAC. Therefore, a comprehensive understanding of the interactions between KRAS mutations and PDAC may expediate the development of therapeutic strategies for reversing the progression of malignant tumors. Our study aims at establishing and validating a prediction model of KRAS mutations in patients with PDAC based on survival analysis and mRNA expression.MethodsA total of 184 and 412 patients with PDAC from The Cancer Genome Atlas (TCGA) database and the International Cancer Genome Consortium (ICGC), respectively, were included in the study.ResultsAfter tumor mutation profile and copy number variation (CNV) analyses, we established and validated a prediction model of KRAS mutations, based on survival analysis and mRNA expression, that contained seven genes: CSTF2, FAF2, KIF20B, AKR1A1, APOM, KRT6C, and CD70. We confirmed that the model has a good predictive ability for the prognosis of overall survival (OS) in patients with KRAS-mutated PDAC. Then, we analyzed differential biological pathways, especially the ferroptosis pathway, through principal component analysis, pathway enrichment analysis, Gene Ontology (GO) enrichment analysis, and gene set enrichment analysis (GSEA), with which patients were classified into low- or high-risk groups. Pathway enrichment results revealed enrichment in the cytokine-cytokine receptor interaction, metabolism of xenobiotics by cytochrome P450, and viral protein interaction with cytokine and cytokine receptor pathways. Most of the enriched pathways are metabolic pathways predominantly enriched by downregulated genes, suggesting numerous downregulated metabolic pathways in the high-risk group. Subsequent tumor immune infiltration analysis indicated that neutrophil infiltration, resting CD4 memory T cells, and resting natural killer (NK) cells correlated with the risk score. After verifying that the seven gene expression levels in different KRAS-mutated pancreatic cancer cell lines were similar to that in the model, we screened potential drugs related to the risk score.DiscussionThis study established, analyzed, and validated a model for predicting the prognosis of PDAC based on risk stratification according to KRAS mutations, and identified differential pathways and highly effective drugs.
| S-EPMC10805828 | biostudies-literature
Project description:Background/aimThe entire mechanisms by which epigenetic modifiers contribute to the development of pancreatic cancer remain unknown. Although the histone methyltransferase G9a is a promising target in human cancers, its role in pancreatic carcinogenesis has been under-studied. The aim of the study was to examine the role of G9a in pancreatic carcinogenesis by a gene-targeting mouse model.Materials and methodsWe established pancreas-specific G9aflox/flox mice and crossed them with Ptf1aCre/; KrasG12D/+ (KC) mice, which spontaneously develop pancreatic cancer. The phenotypes of the resulting KC mice with G9a deletion were examined. We analyzed transcriptomic data by microarray and genome-wide chromatin accessibility by transposase-accessible chromatin using sequencing. We established pancreatic organoids from KC mice.ResultsG9a deficiency impaired the progression of pancreatic intraepithelial neoplasia (PanIN) and prolonged the survival of KC mice. The number of phosphorylated Erk-positive cells and Dclk1-positive cells, which are reported to be essential for the progression of PanIN, were decreased by G9a deletion. UNC0638, an inhibitor of G9a, suppressed the growth of organoids and increased global chromatin accessibility, especially around the regions including the protein phosphatase 2A genes.ConclusionThus, our study suggested the functional interaction of G9a, Dclk1 and Mapk pathway in the Kras-driven pancreatic carcinogenesis. The inhibition of G9a may suppress the initiation of oncogenic Kras-driven pancreatic carcinogenesis.
| S-EPMC7675651 | biostudies-literature