Cytokine CCL9 Mediates Oncogenic KRAS-Induced Pancreatic Acinar-to-Ductal Metaplasia by Promoting Reactive Oxygen Species and Metalloproteinases.

Pancreatic ductal adenocarcinoma (PDAC) can originate from acinar-to-ductal metaplasia (ADM). Pancreatic acini harboring oncogenic Kras mutations are transdifferentiated to a duct-like phenotype that further progresses to become pancreatic intraepithelial neoplasia (PanIN) lesions, giving rise to PDAC. Although ADM formation is frequently observed in KrasG12D transgenic mouse models of PDAC, the exact mechanisms of how oncogenic KrasG12D regulates this process remain an enigma. Herein, we revealed a new downstream target of oncogenic Kras, cytokine CCL9, during ADM formation. Higher levels of CCL9 and its receptors, CCR1 and CCR3, were detected in ADM regions of the pancreas in p48cre:KrasG12D mice and human PDAC patients. Knockdown of CCL9 in KrasG12D-expressed pancreatic acini reduced KrasG12D-induced ADM in a 3D organoid culture system. Moreover, exogenously added recombinant CCL9 and overexpression of CCL9 in primary pancreatic acini induced pancreatic ADM. We also showed that, functioning as a downstream target of KrasG12D, CCL9 promoted pancreatic ADM through upregulation of the intracellular levels of reactive oxygen species (ROS) and metalloproteinases (MMPs), including MMP14, MMP3 and MMP2. Blockade of MMPs via its generic inhibitor GM6001 or knockdown of specific MMP such as MMP14 and MMP3 decreased CCL9-induced pancreatic ADM. In p48cre:KrasG12D transgenic mice, blockade of CCL9 through its specific neutralizing antibody attenuated pancreatic ADM structures and PanIN lesion formation. Furthermore, it also diminished infiltrating macrophages and expression of MMP14, MMP3 and MMP2 in the ADM areas. Altogether, our results provide novel mechanistic insight into how oncogenic Kras enhances pancreatic ADM through its new downstream target molecule, CCL9, to initiate PDAC.

Discovery of Novel Noncovalent KRAS G12D Inhibitors through Structure-Based Virtual Screening and Molecular Dynamics Simulations.

The development of effective inhibitors targeting the Kirsten rat sarcoma viral proto-oncogene (KRASG12D) mutation, a prevalent oncogenic driver in cancer, represents a significant unmet need in precision medicine. In this study, an integrated computational approach combining structure-based virtual screening and molecular dynamics simulation was employed to identify novel noncovalent inhibitors targeting the KRASG12D variant. Through virtual screening of over 1.7 million diverse compounds, potential lead compounds with high binding affinity and specificity were identified using molecular docking and scoring techniques. Subsequently, 200 ns molecular dynamics simulations provided critical insights into the dynamic behavior, stability, and conformational changes of the inhibitor-KRASG12D complexes, facilitating the selection of lead compounds with robust binding profiles. Additionally, in silico absorption, distribution, metabolism, excretion (ADME) profiling, and toxicity predictions were applied to prioritize the lead compounds for further experimental validation. The discovered noncovalent KRASG12D inhibitors exhibit promises as potential candidates for targeted therapy against KRASG12D-driven cancers. This comprehensive computational framework not only expedites the discovery of novel KRASG12D inhibitors but also provides valuable insights for the development of precision treatments tailored to this oncogenic mutation.

Inhibition mechanism of MRTX1133 on KRASG12D: a molecular dynamics simulation and Markov state model study.

The mutant KRAS was considered as an "undruggable" target for decades, especially KRASG12D. It is a great challenge to develop the inhibitors for KRASG12D which lacks the thiol group for covalently binding ligands. The discovery of MRTX1133 solved the dilemma. Interestingly, MRTX1133 can bind to both the inactive and active states of KRASG12D. The binding mechanism of MRTX1133 with KRASG12D, especially how MRTX1133 could bind the active state KRASG12D without triggering the active function of KRASG12D, has not been fully understood. Here, we used a combination of all-atom molecular dynamics simulations and Markov state model (MSM) to understand the inhibition mechanism of MRTX1133 and its analogs. The stationary probabilities derived from MSM show that MRTX1133 and its analogs can stabilize the inactive or active states of KRASG12D into different conformations. More remarkably, by scrutinizing the conformational differences, MRTX1133 and its analogs were hydrogen bonded to Gly60 to stabilize the switch II region and left switch I region in a dynamically inactive conformation, thus achieving an inhibitory effect. Our simulation and analysis provide detailed inhibition mechanism of KRASG12D induced by MRTX1133 and its analogs. This study will provide guidance for future design of novel small molecule inhibitors of KRASG12D.

Ethanol exposure of human pancreatic normal ductal epithelial cells induces EMT phenotype and enhances pancreatic cancer development in KC (Pdx1-Cre and LSL-KrasG12D ) mice.

Alcohol is a risk factor for pancreatic cancer. However, the molecular mechanism by which chronic alcohol consumption influences pancreatic cancer development is not well understood. We have recently demonstrated that chronic ethanol exposure of pancreatic normal ductal epithelial cells (HPNE) induces cellular transformation by generating cancer stem cells (CSCs). Here, we examined whether chronic ethanol treatment induces epithelial-mesenchymal transition in HPNE cells and promotes pancreatic cancer development in KC (Pdx1-Cre, and LSL-KrasG12D ) mice. Our data demonstrate that chronic ethanol exposure of HPNE cells induces SATB2 gene and those cells became highly motile. Ethanol treatment of HPNE cells results in downregulation of E-Cadherin and upregulation of N-Cadherin, Snail, Slug, Zeb1, Nanog and BMI-1. Suppression of SATB2 expression in ethanol-transformed HPNE cells inhibits EMT phenotypes. KC mice fed with an ethanol-containing diet show enhanced pancreatic cancer growth and development than those fed with a control diet. Pancreas isolated from KC mice fed with an ethanol-containing diet show higher expression of stem cell markers (CD133, CD44, CD24), pluripotency-maintaining factors (cMyc, KLF4, SOX-2, and Oct-4), N-Cadherin, EMT-transcription factors (Snail, Slug, and Zeb1), and lower expression of E-cadherin than those isolated from mice fed with a control diet. Furthermore, pancreas isolated from KC mice fed with an ethanol-containing diet show higher expression of inflammatory cytokines (TNF-α, IL-6, and IL-8) and PTGS-2 (COX-2) gene than those isolated from mice fed with a control diet. These data suggest that chronic alcohol consumption may contribute to pancreatic cancer development by generating inflammatory signals and CSCs.

Loss of Heterozygosity for KrasG12D Promotes Malignant Phenotype of Pancreatic Ductal Adenocarcinoma by Activating HIF-2α-c-Myc-Regulated Glutamine Metabolism.

Loss of heterozygosity (LOH) for KRAS, in which a wild-type KRAS allele is progressively lost, promotes invasive and migratory abilities of pancreatic ductal adenocarcinoma (PDAC) cells and tissues. Moreover, the occurrence of KrasG12D-LOH activates nonclassical glutamine metabolism, which is related to the malignant behavior of PDAC cells. Herein, we aim to demonstrate the regulatory link between hypoxia-inducible factor-2α (HIF-2α) and glutamine metabolism that mediates malignant phenotypes in KrasG12D-LOH PDAC cells. HIF-2α-shRNA knockdown lentivirus transfection and metabolite analysis were performed in KrasG12D-LOH and KrasG12D cell lines, respectively. Cell proliferation, migration, and invasion were examined using Cell Counting Kit-8, colony formation, and Transwell assays. Cell cycle phase and apoptosis were determined using flow cytometry. Western blotting and real-time quantitative PCR were also performed. Additionally, a subcutaneous xenograft mouse model was established. LOH stimulated HIF-2α activity and transactivated c-Myc, which has a central regulatory effect on glutamine metabolism independent of hypoxia. Meanwhile, HIF-2α silencing repressed KrasG12D-LOH PDAC cell proliferation, invasion, and migration. HIF-2α knockdown inhibited glutamine uptake and GOT1 expression via a c-Myc-dependent pathway. Collectively, KrasG12D-LOH can activate HIF-2α to regulate c-Myc-mediated glutamine metabolism and promote malignant phenotypes. Moreover, targeting HIF-2α-c-Myc regulated nonclassical glutamine metabolism, providing a new therapeutic perspective for KrasG12D-LOH PDAC.

A novel terpenoid class for prevention and treatment of KRAS-driven cancers: Comprehensive analysis using in situ, in vitro, and in vivo model systems.

Inhibiting the disease progression in KRAS-driven cancers after diagnosis has been a difficult task for clinicians to manage due to the lack of effective intervention/preventive therapies. KRAS-driven cancers depend on sustained KRAS signaling. Although developing inhibitors of KRAS signaling has proven difficult in the past, the quest for identifying newer agents has not stopped. Based on studies showing terpenoids as modulators of KRAS-regulated downstream molecular pathways, we asked if this chemical family has an affinity of inhibiting KRAS protein activity. Using crystal structure as a bait in silico, we identified 20 terpenoids for their KRAS protein-binding affinity. We next carried out biological validation of in silico data by employing in situ, in vitro, patient-derived explant ex vivo, and KPC transgenic mouse models. In this report, we provide a comprehensive analysis of a lup-20(29)-en-3b-ol (lupeol) as a KRAS inhibitor. Using nucleotide exchange, isothermal titration calorimetry, differential scanning fluorimetry, and immunoprecipitation assays, we show that lupeol has the potential to reduce the guanosine diphosphate/guanosine triphosphate exchange of KRAS protein including mutant KRASG12V . Lupeol treatment inhibited the KRAS activation in KRAS-activated cell models (NIH-panel, colorectal, lung, and pancreatic intraepithelial neoplasia) and patient tumor explants ex vivo. Lupeol reduced the three-dimensional growth of KRAS-activated cells. The pharmacokinetic analysis showed the bioavailability of lupeol after consumption via oral and intraperitoneal routes in animals. Tested under prevention settings, the lupeol consumption inhibited the development of pancreatic intraepithelial neoplasia in LSL-KRASG12D/Pdx-cre mice (pancreatic ductal adenocarcinoma progression model). These data suggest that the selected members of the triterpene family (such as lupeol) could be exploited as clinical agents for preventing the disease progression in KRAS-driven cancers which however warrants further investigation.

KrasG12D-LOH promotes malignant biological behavior and energy metabolism of pancreatic ductal adenocarcinoma cells through the mTOR signaling pathway.

Oncogenic Kras with loss of heterozygosity (LOH) is frequently detected in various tumours. However, the exact function and mechanism by which KrasG12D-LOH operates remain unclear. Therefore, the current study investigated the effect of KrasG12D-LOH on the malignant phenotype of pancreatic ductal adenocarcinoma (PDAC) cells. Our investigation revealed that KrasG12D-LOH is associated with increased proliferation, invasion and reduced apoptosis in PDAC cells. The results also exhibited enhanced glycolytic phenotype of KrasG12D-LOH PDAC cells. Hyperactive mTOR plays a significant role in the initiation and maintenance of tumors. To investigate the correlation between KrasG12D-LOH and mTOR, the mTOR signaling pathway was detected by western blot analysis. We found that KrasG12D-LOH up-regulated Akt, AMPK, REDD1 and mTOR in PDAC cells. In summary, our results demonstrated that KrasG12D-LOH promotes oncogenic Kras-induced PDAC by regulating energy metabolism and mTOR signaling pathway. These data may provide novel therapeutic perspectives for PDAC.

Screening for tumor suppressors: Loss of ephrin receptor A2 cooperates with oncogenic KRas in promoting lung adenocarcinoma.

Lung adenocarcinoma, a major form of non-small cell lung cancer, is the leading cause of cancer deaths. The Cancer Genome Atlas analysis of lung adenocarcinoma has identified a large number of previously unknown copy number alterations and mutations, requiring experimental validation before use in therapeutics. Here, we describe an shRNA-mediated high-throughput approach to test a set of genes for their ability to function as tumor suppressors in the background of mutant KRas and WT Tp53. We identified several candidate genes from tumors originated from lentiviral delivery of shRNAs along with Cre recombinase into lungs of Loxp-stop-Loxp-KRas mice. Ephrin receptorA2 (EphA2) is among the top candidate genes and was reconfirmed by two distinct shRNAs. By generating knockdown, inducible knockdown and knockout cell lines for loss of EphA2, we showed that negating its expression activates a transcriptional program for cell proliferation. Loss of EPHA2 releases feedback inhibition of KRAS, resulting in activation of ERK1/2 MAP kinase signaling, leading to enhanced cell proliferation. Intriguingly, loss of EPHA2 induces activation of GLI1 transcription factor and hedgehog signaling that further contributes to cell proliferation. Small molecules targeting MEK1/2 and Smoothened hamper proliferation in EphA2-deficient cells. Additionally, in EphA2 WT cells, activation of EPHA2 by its ligand, EFNA1, affects KRAS-RAF interaction, leading to inhibition of the RAS-RAF-MEK-ERK pathway and cell proliferation. Together, our studies have identified that (i) EphA2 acts as a KRas cooperative tumor suppressor by in vivo screen and (ii) reactivation of the EphA2 signal may serve as a potential therapeutic for KRas-induced human lung cancers.

PanIN-associated pericyte, glial, and islet remodeling in mice revealed by 3D pancreatic duct lesion histology.

Pericytes and glial cells are accessory cells of neurovascular networks, which have been reported to participate in scar formation after tissue injury. However, it remains unclear whether similar reactive cellular responses occur in pancreatic intraepithelial neoplasia (PanIN). In this study we developed three-dimensional (3D) duct lesion histology to investigate PanIN and the associated pericyte, glial, and islet remodeling. Transparent mouse pancreata with a Kras(G12D) mutation were used to develop 3D duct lesion histology. Deep-tissue, tile-scanning microscopy was performed to generate panoramic views of the diseased pancreas for global examination of early stage and advanced duct lesion formation. Fluorescence signals of ductal and neurovascular networks were simultaneously detected to reveal associated remodeling. Significantly, in Kras(G12D)-mutant mice, when the low-grade PanINs emerge, duct lesions appear as epithelial buds with perilesional pericyte and glial activation. When PanINs occur in large scale (induced by cerulein injections to the mutant mice), the 3D image data identifies 1) aggregation of PanINs in clusters in space; 2) overexpression of the pericyte marker NG2 in the PanIN microenvironment; and 3) epithelial in-growth to islets, forming the PanIN-islet complexes. Particularly, the PanIN-islet complexes associate with proliferating epithelial and stromal cells and receive substantial neurovascular supplies, making them landmarks in the atrophic lobe. Overall, perilesional pericyte and glial activation and formation of the PanIN-islet complex underline cellular heterogeneity in the duct lesion microenvironment. The results also illustrate the advantage of using 3D histology to reveal previously unknown details of neurovascular and endocrine links to the disease.

A genetically engineered mouse model developing rapid progressive pancreatic ductal adenocarcinoma.

The premalignant lesions of pancreatic cancer, pancreatic intraepithelial neoplasia (PanIN), have a high frequency of mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS), and genetic alterations in the retinoblastoma (Rb)-E2 factor (E2F) and transformed 3T3 cell double minute 2 (MDM2)-p53 pathways accelerate development of pancreatic ductal adenocarcinoma. The viral oncoprotein SV40 large T antigen (TAg) can inhibit the effects of the Rb family of molecules and of p53 on these pathways, and targeted expression of TAg in mouse pancreas is associated with the development of endocrine or acinar cell tumours. In this study, to determine whether the viral oncoprotein promotes pancreatic duct carcinogenesis initiated by oncogenic KRAS, we generated mice expressing temperature-sensitive SV40 large T antigen (tsTAg) on pancreatic epithelial cells in the presence or absence of Kras(G12D) . Mice with pancreas-specific tsTAg expression developed acinar cell dysplasia by 22 weeks without PanIN formation, while mice expressing both tsTAg and Kras(G12D) developed highly aggressive adenocarcinoma with a ductal cell phenotype within a short period, and died within 3 weeks. The tumours resembled human pancreatic ductal adenocarcinoma (PDAC) at the histological level, and oncogenic Kras and tsTAg synergistically activated E2f and Sre transcription in established PDAC cell lines. These results suggest that tsTAg synergistically promotes Kras(G12D) -associated PDAC formation, and our study identifies a new mouse model of PDAC that may allow a better understanding of the mechanism of carcinogenesis in pancreatic carcinoma, which shows a catastrophic clinical course.

Inhibition of GTPase KRASG12D: a review of patent literature.

INTRODUCTION: KRAS is a critical oncogenic protein intricately involved in tumor progression, and the difficulty in targeting KRAS has led it to be classified as an 'undruggable target.' Among the various KRAS mutations, KRASG12D is highly prevalent and represents a promising therapeutic target, yet there are currently no approved inhibitors for it. AREA COVERED: This review summarizes numerous patents and literature featuring inhibitors or degraders of KRASG12D through searching relevant information in PubMed, SciFinder and Web of Science databases from 2021 to February 2024, providing an overview of the research progress on inhibiting KRASG12D in terms of design strategies, chemical structures, biological activities, and clinical advancements. EXPERT OPINION: Since the approval of AMG510 (Sotorasib), there has been an increasing focus on the inhibition of KRASG12D, leading to numerous reports of related inhibitors and degraders. Among them, MRTX1133, as the first KRASG12D inhibitor to enter clinical trials, has demonstrated excellent tumor suppression in various KRASG12D-bearing human tumor xenograft models. It is important to note, however, that understanding the mechanisms of acquired resistance caused by KRAS inhibition and developing additional combination therapies is crucial. Moreover, seeking covalent inhibition of KRASG12D also holds significant potential.

Atorvastatin inhibits pancreatic carcinogenesis and increases survival in LSL-KrasG12D-LSL-Trp53R172H-Pdx1-Cre mice.

There are several studies supporting the role of HMG-CoA reductase inhibitors such as atorvastatin against carcinogenesis, in which inhibiting the generation of prenyl intermediates involved in protein prenylation plays the crucial role. Mutation of Kras gene is the most common genetic alteration in pancreatic cancer and the Ras protein requires prenylation for its membrane localization and activity. In the present study, the effectiveness of atorvastatin against pancreatic carcinogenesis and its effect on protein prenylation were determined using the LSL-KrasG12D-LSL-Trp53R172H-Pdx1-Cre mouse model (called Pankras/p53 mice). Five-week-old Pankras/p53 mice were fed either an AIN93M diet or a diet supplemented with 100 ppm atorvastatin. Kaplan-Meier survival analysis with Log-Rank test revealed a significant increase in survival in mice fed 100 ppm atorvastatin (171.9 ± 6.2 d) compared to the control mice (144.9 ± 8.4 d, P < 0.05). Histologic and immunohistochemical analysis showed that atorvastatin treatment resulted in a significant reduction in tumor volume and Ki-67-labeled cell proliferation. Mechanistic studies on primary pancreatic tumors and the cultured murine pancreatic carcinoma cells revealed that atorvastatin inhibited prenylation in several key proteins, including Kras protein and its activities, and similar effect was observed in pancreatic carcinoma cells treated with farnesyltransferase inhibitor R115777. Microarray assay on the global gene expression profile demonstrated that a total of 132 genes were significantly modulated by atorvastatin; and Waf1p21, cyp51A1, and soluble epoxide hydrolase were crucial atorvastatin-targeted genes which involve in inflammation and carcinogenesis. This study indicates that atorvastatin has the potential to serve as a chemopreventive agent against pancreatic carcinogenesis.

Identifying Adult Stomach Tissue Stem/Progenitor Cells Using the Iqgap3-2A-CreERT2 Mouse.

Identification of unique gene markers of normal and cancer stem cells is an effective strategy to study cells of origin and understand tumor behavior. Lineage tracing experiments using the Cre recombinase driven by a stem cell-specific promoter in the CreERT2 reporter mouse model enables identification of adult stem cells and delineation of stem cell activities in vivo. In our recent research on the mouse stomach, Iqgap3 was identified as a homeostatic stem cell marker located in the isthmus of the stomach epithelium. Lineage tracing with the Iqgap3-2A-CreERT2;Rosa26-LSL-tdTomato mouse model demonstrated stem cell activity in Iqgap3-expressing cells. Using the Iqgap3-2A-CreERT2 mouse model to target oncogenic KrasG12D expression to Iqgap3-expressing cells, we observed the rapid development of precancerous metaplasia in the stomach and proposed that aberrant Iqgap3-expressing cells may be critical determinants of early carcinogenesis. In this chapter, we detail a lineage tracing protocol to assess stem cell activity in the murine stomach. We also describe the procedure of inducing KrasG12D expression in Iqgap3-expressing homeostatic stem cells to explore their role as cells of origin and to trace the early cellular changes that precede neoplastic transformation.

Discovery of potent and noncovalent KRASG12D inhibitors: Structure-based virtual screening and biological evaluation.

KRASG12D, the most common oncogenic KRAS mutation, is a promising target for the treatment of pancreatic cancer. Herein, we identified four potent and noncovalent KRASG12D inhibitors (hits 1-4) by using structure-based virtual screening and biological evaluation. The in vitro assays indicated that the four compounds had sub-nanomolar affinities for KRASG12D and showed a dose-dependent inhibitory effect on human pancreatic cancer cells. In particular, the hit compound 3 was the most promising candidate and significantly inhibited the tumor growth of pancreatic cancer in tumor-bearing mice. The hit compound 3 represented a promising starting point for structural optimization in hit-to-lead development. This study shows that hit compound 3 provides a basis for the development of the treatment of cancer driven by KRASG12D.

Oncogenic Kras-Mediated Cytokine CCL15 Regulates Pancreatic Cancer Cell Migration and Invasion through ROS.

Pancreatic ductal adenocarcinoma (PDAC) is well known for its high death rate due to prompt cancer metastasis caused by cancer cell migration and invasion within the early stages of its development. Here, we reveal a new function of cytokine CCL15, namely the upregulation of PDAC cell migration and invasion. We showed increased levels of CCL15 transcripts and protein expressions in human PDAC tissue samples, as well as in cultured cell lines. Furthermore, PDAC cells also expressed CCL15 receptors, including CCR1 and CCR3. Murine PDAC cell lines and tissues strengthened this finding. The manipulation of CCL15 in metastatic Panc-1 cells through CCL15 knockdown or CCL15 neutralization decreased Panc-1 cell motility and invasiveness. In addition, treating non-metastatic BxPC-3 cells with recombinant CCL15 accelerated the cell migration of BxPC-3. A reduction in the levels of reactive oxygen species (ROS) by either N-Acetyl-L-Cysteine treatment or p22phox knockdown led to a decrease in Panc-1 cell migration and a reversed effect on recombinant CCL15-promoted BxPC-3 cell movement. Importantly, the knockdown of oncogenic Kras in Panc-1 cells abolished CCL15 protein expression and impeded cell migration without affecting PDAC cell growth. Altogether, our work elucidates an additional molecular pathway of oncogenic Kras to promote PDAC metastasis through the upregulation of cell migration and invasion by the Kras downstream CCL15, a lesser-known cytokine within the cancer research field.

KRAS inhibitors: going noncovalent.

KRASG12D is the most frequent KRAS mutation in human cancer with particularly high frequencies in pancreatic and colorectal cancer. Informed by the structure of the KRASG12C inhibitor adagrasib, Hallin et al. have now, through multiple rounds of structure-based drug design, identified and validated a potent, selective, and noncovalent KRASG12D inhibitor, MRTX1133. This study demonstrated that MRTX1133 inhibited both the inactive and active state of KRASG12D and showed potent antitumor activity in several preclinical models of pancreatic and colorectal cancer, especially when combined with cetuximab, a monoclonal antibody against the EGFR, or BYL-719, a potent PI3Kα inhibitor.

Precise and efficient silencing of mutant KrasG12D by CRISPR-CasRx controls pancreatic cancer progression.

Rationale: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with few therapeutic targets and rare effective treatments. Over 90% of PDAC tumors bear a Kras mutation, and the single-site mutation G12D (KrasG12D) is most prevalent. Methods: Here, we applied the CRISPR-CasRx system to silence the mutant KrasG12D transcript in PDAC cells. We also used a capsid-optimized adenovirus-associated virus 8 vector (AAV8) to deliver the CRISPR-CasRx system into PDAC orthotopic tumors and patient-derived tumor xenografts (PDX). Results: Our data showed that guided by a KrasG12D-specific gRNA, CasRx is able to precisely and efficiently silence the mutant KrasG12D expression in PDAC cells. The knockdown of mutant KrasG12D by CasRx abolishes the aberrant activation of downstream signaling induced by mutant KrasG12D and subsequently suppresses the tumor growth and improves the sensitivity of gemcitabine in PDAC. Additionally, delivering CasRx-gRNA via AAV8 into the orthotopic KrasG12D PDAC tumors substantially improves the survival of mice without obvious toxicity. Furthermore, targeting KrasG12D through CasRx suppresses the growth of PDAC PDXs. In conclusion, our study provides a proof-of-concept that CRISPR-CasRx can be utilized to target and silence mutant KrasG12D transcripts and therefore inhibit PDAC malignancy.

The Inhibition of Wnt Restrain KRASG12V-Driven Metastasis in Non-Small-Cell Lung Cancer.

The KRAS mutations have been an obstacle to identify therapeutic targets in cancer treatment. In this work, we clarified the distinct metastasis pattern of non-small-cell lung carcinoma (NSCLC) induced by KRASG12V/KRASG12D mutations and inhibited the KRASG12V mediated metastasis by Wnt inhibitor. First, we found that KRASG12V induced more aggressive phenotype in vitro and in vivo experiments. The Gene Set Enrichment Analysis (GSEA) results of H838 KRASG12V cells showed a significant negative correlation with RhoA-related signaling. Following this clue, we observed KRASG12D induced higher activation of RhoA and suppressed activation of Wnt/ß-catenin in H838KRASG12D cells. The restored activation of Wnt/ß-catenin in H838KRASG12D cells could be detected when expression with a dominant-negative mutant of RhoA or treatment with RhoA inhibitor. Furthermore, the Wnt inhibitor abolished the KRASG12V-induced migration. We elucidated the importance of the axis of RhoA/Wnt in regulatory NSCLC metastasis driven by KRAS mutations. Our data indicate that KRASG12V driven NSCLC metastasis is Wnt-dependent and the mechanisms of NSCLC metastasis induced by KRASG12V/KRASG12D is distinct.

Development of a miniaturized 3D organoid culture platform for ultra-high-throughput screening.

The recent advent of robust methods to grow human tissues as 3D organoids allows us to recapitulate the 3D architecture of tumors in an in vitro setting and offers a new orthogonal approach for drug discovery. However, organoid culturing with extracellular matrix to support 3D architecture has been challenging for high-throughput screening (HTS)-based drug discovery due to technical difficulties. Using genetically engineered human colon organoids as a model system, here we report our effort to miniaturize such 3D organoid culture with extracellular matrix support in high-density plates to enable HTS. We first established organoid culturing in a 384-well plate format and validated its application in a cell viability HTS assay by screening a 2036-compound library. We further miniaturized the 3D organoid culturing in a 1536-well ultra-HTS format and demonstrated its robust performance for large-scale primary compound screening. Our miniaturized organoid culturing method may be adapted to other types of organoids. By leveraging the power of 3D organoid culture in a high-density plate format, we provide a physiologically relevant screening platform to model tumors to accelerate organoid-based research and drug discovery.

Early detection of pre-malignant lesions in a KRASG12D-driven mouse lung cancer model by monitoring circulating free DNA.

Lung cancer is the leading cause of cancer-related death. Two-thirds of cases are diagnosed at an advanced stage that is refractory to curative treatment. Therefore, strategies for the early detection of lung cancer are urgently sought. Total circulating free DNA (cfDNA) and tumour-derived circulating tumour DNA (ctDNA) are emerging as important biomarkers within a 'liquid biopsy' for monitoring human disease progression and response to therapy. Owing to the late clinical diagnosis of lung adenocarcinoma, the potential for cfDNA and ctDNA as early detection biomarkers remains unexplored. Here, using a Cre-regulated genetically engineered mouse model of lung adenocarcinoma development, driven by KrasG12D (the KrasLSL-G12D mouse), we serially tracked the release of cfDNA/ctDNA and compared this with tumour burden as determined by micro-computed tomography (CT). To monitor ctDNA, a droplet digital PCR assay was developed to permit discrimination of the KrasLox-G12D allele from the KrasLSL-G12D and KrasWT alleles. We show that micro-CT correlates with endpoint histology and is able to detect pre-malignant tumours with a combined volume larger than 7 mm3 Changes in cfDNA/ctDNA levels correlate with micro-CT measurements in longitudinal sampling and are able to monitor the emergence of lesions before the adenoma-adenocarcinoma transition. Potentially, this work has implications for the early detection of human lung adenocarcinoma using ctDNA/cfDNA profiling.A video abstract for this article is available at https://youtu.be/Ku8xJJyGs3UThis article has an associated First Person interview with the joint first authors of the paper.