EGFR Signaling Research Articles

Dual Target of EGFR and mTOR Suppresses Triple-Negative Breast Cancer Cell Growth by Regulating the Phosphorylation of mTOR Downstream Proteins.

To detect the activation of the EGFR and mTOR signaling pathways in the triple negative breast cancer cell line MDA-MB-468 and investigate the inhibitory effect of gefitinib, an epidermal growth factor receptor inhibitor, and everolimus, a target protein inhibitor of rapamycin, on triple negative breast cancer cells. Triple negative human breast cancer MDA-MB-468 cells were cultured and blank control group, single EGFR inhibitor gefitinib group, single mTOR inhibitor everolimus group, and two drug combination group were set up respectively to detect the effects of single and combined drugs on cell proliferation activity, cell cycle and apoptosis, and the expression of EGFR and mTOR signal pathway proteins in cell lines after single and combined drug intervention was detected again by Western blot. The level of EGFR and p-mTOR protein in triple negative breast cancer was higher than in non triple negative breast cancer (P<0.05). The level of mTOR, S6K1, p-EGFR, p-S6K1 was significantly increased when treated with EGF (0ng/mL, 10ng/mL, 100ng/mL) for 1h, compared to without EGF stimulation (P<0.05). The level of p-EGFR, p-mTOR, p-S6K1 protein increased significantly when the cells were exposed to EGF for 2h, respectively (P<0.05). EGFR inhibitor gefitinib alone and the mTOR inhibitor everolimus alone could significantly inhibit the proliferation of human triple negative breast cancer MDA-MB-468 cells in a dose-dependent manner (P<0.05). The level of p-4EBP1 protein in EGFR and mTOR signal pathway was significantly increased after the intervention of gefitinib alone, everolimus alone, and the combination of two drugs (P<0.05). EGFR and mTOR signaling pathways can be activated in triple negative breast cancer; Both the EGFR inhibitor gefitinib alone and the mTOR inhibitor everolimus alone can significantly inhibit the proliferation of human triple negative breast cancer MDA-MB-468 cells. The combination of the EGFR inhibitor gefitinib and the mTOR inhibitor everolimus may achieve anti-tumor effect similar to that of single drug by reducing the drug dose.

Abstract 1919: PTPRF negatively regulates EGFR signaling to inhibit cell migration in colon cancer

Abstract 1919: PTPRF negatively regulates EGFR signaling to inhibit cell migration in colon cancer

Plakophilin 3 facilitates G1/S phase transition and enhances proliferation by capturing RB protein in the cytoplasm and promoting EGFR signaling

Plakophilin 3 (PKP3) is a component of desmosomes and is frequently overexpressed in cancer. Using keratinocytes either lacking or overexpressing PKP3, we identify a signaling axis from ERK to the retinoblastoma (RB) protein and the E2F1 transcription factor that is controlled by PKP3. RB and E2F1 are key components controlling G1/S transition in the cell cycle. We show that PKP3 stimulates the activity of ERK and its target RSK1. This inhibits expression of the transcription factor RUNX3, a positive regulator of the CDK inhibitor CDKN1A/p21, which is also downregulated by PKP3. Elevated CDKN1A prevents RB phosphorylation and E2F1 target gene expression, leading to delayed S phase entry and reduced proliferation in PKP3-depleted cells. Elevated PKP3 expression not only increases ERK activity but also captures phosphorylated RB (phospho-RB) in the cytoplasm to promote E2F1 activity and cell-cycle progression. These data identify a mechanism by which PKP3 promotes proliferation and acts as an oncogene.

Role of identified proteins in the proteome profiles of CDK4/6 inhibitor-resistant breast cancer cell lines.

Abemaciclib (Ab) and palbociclib (Pb) are CDK4/6 inhibitors used to cure advanced breast cancer (BC). However, acquired resistance is a major challenge. The molecular mechanisms and signature proteins of therapy resistance for Ab and Pb drugs need to be explored. Here we developed resistant cells for Ab and Pb drugs in MCF-7 cell lines and explored the mechanisms and signature proteins of therapy resistance in BC. Proteome profiling was performed using the label-free proteome-orbitrap-fusion-MS-MS technique. Gene ontology (GO)-terms, KEGG pathways and network analysis were performed for the proteome data. Drug-resistant cells showed increased drug tolerance, enhanced colony formation potential and an increased gap-healing tendency for the respective drug. Up-regulation of survival genes (BCL-2 and MCL-1) and down-regulation of apoptosis inducers were observed. Drug-resistance markers (MDR-1 and ABCG2 (BCRP)) along with ESR-1, CDK4, CDK6, and cyclin-D1 genes were up-regulated in resistant cells. A total of 237 and 239 proteins were found to be differentially expressed in the Ab and Pb-resistant cells, respectively. Down-regulated proteins induce apoptosis signalling and nucleotide metabolisms and restrict EGFR signalling; however, up-regulated proteins induce Erk, wnt-β-catenin, VEGFR-PI3K-AKT, glucose transportation, and hypoxia signalling pathways and regulate hydrogen peroxide signalling pathways. The panel of identified proteins associated with these pathways might have characteristics of molecular signature and new drug targets for overcoming drug resistance in breast cancer.

Autocrine EGF and TGF-α promote primary and acquired resistance to ALK/c-Met kinase inhibitors in non-small-cell lung cancer.

Drug resistance severely limits the clinical therapeutic value of molecularly targeted drugs. Growth factors gain a tremendous amount of focus due to the ability to promote drug resistance in non-small-cell lung cancer (NSCLC). However, whether tumor cells themselves can mediate drug resistance by secreting growth factors needs further clarification. Here, we first screened growth factors to identify autocrine epidermal growth factor (EGF) and transforming growth factor alpha (TGF-α) that caused primary resistance to the ALK inhibitor TAE684 in H3122 cells and the c-MET-specific inhibitor SGX-523 in EBC-1 cells. Next, we discovered increased autocrine production of EGF and TGF-α in established acquired resistant H3122/TR and EBC-1/SR cells. Importantly, overexpression of EGF and TGF-α in two NSCLC cell lines produced resistance to TAE684 and SGX-523. Clinically, NSCLC patients with high expression of EGF and TGF-α developed primary resistance to crizotinib. Mechanistically, autocrine EGF and TGF-α activated EGFR signaling pathways to survive targeted c-Met and ALK inhibition. Furthermore, combined treatment with gefitinib circumvented EGF- and TGF-α-mediated primary and acquired resistance to TAE684/SGX-523. Taken together, these results suggested increased autocrine EGF and TGF-α conferred primary and acquired resistance to ALK/c-Met kinase inhibitors in NSCLC.

Immunoregulatory framework and the role of miRNA in the pathogenesis of NSCLC - A systematic review.

With a 5-year survival rate of only 15%, non-small cell lung cancer (NSCLC), the most common kind of lung carcinoma and the cause of millions of deaths annually, has drawn attention. Numerous variables, such as disrupted signaling caused by somatic mutations in the EGFR-mediated RAS/RAF/MAPK, PI3K/AKT, JAK/STAT signaling cascade, supports tumour survival in one way or another. Here, the tumour microenvironment significantly contributes to the development of cancer by thwarting the immune response. MicroRNAs (miRNAs) are critical regulators of gene expression that can function as oncogenes or oncosuppressors. They have a major influence on the occurrence and prognosis of NSCLC. Though, a myriad number of therapies are available and many are being clinically tested, still the drug resistance, its adverse effect and toxicity leading towards fatality cannot be ruled out. In this review, we tried to ascertain the missing links in between perturbed EGFR signaling, miRNAs favouring tumorigenesis and the autophagy mechanism. While connecting all the aforementioned points multiple associations were set, which can be targeted in order to combat NSCLC. Here, we tried illuminating designing synthetically engineered circuits with the toggle switches that might lay a prototype for better therapeutic paradigm.

Regulation of the alveolar regenerative niche by amphiregulin-producing regulatory T cells.

Following respiratory viral infection, regeneration of the epithelial barrier is required to preserve lung function and prevent secondary infections. Lung regulatory T (Treg) cells are critical for maintaining blood oxygenation following influenza virus infection through production of the EGFR ligand amphiregulin (Areg); however, how Treg cells engage with progenitors within the alveolar niche is unknown. Here, we describe local interactions between Treg cells and an Areg-responsive population of Col14a1+EGFR+ lung mesenchymal cells that mediate type II alveolar epithelial (AT2) cell-mediated regeneration following influenza virus infection. We propose a mechanism whereby Treg cells are deployed to sites of damage and provide pro-survival cues that support mesenchymal programming of the alveolar niche. In the absence of fibroblast EGFR signaling, we observe impaired AT2 proliferation and disrupted lung remodeling following viral clearance, uncovering a crucial immune/mesenchymal/epithelial network that guides alveolar regeneration.

A Novel Mechanism Underlying the Inhibitory Effects of Trastuzumab on the Growth of HER2-Positive Breast Cancer Cells.

To improve the efficacy of trastuzumab, it is essential to understand its mechanism of action. One of the significant issues that makes it difficult to determine the precise mechanism of trastuzumab action is the formation of various HER receptor dimers in HER2-positive breast cancer cells. So far, studies have focused on the role of HER2-HER3 heterodimers, and little is known regarding EGFR-HER2 heterodimers. Here, we study the role of trastuzumab on the cell signaling and cell proliferation mediated by EGFR-HER2 heterodimers in BT474 and SRBR3 cells. EGF stimulates the formation of both EGFR homodimer and EGFR-HER2 heterodimer. Trastuzumab only binds to HER2, not EGFR. Therefore, any effects of trastuzumab on EGF-induced activation of EGFR, HER2, and downstream signaling proteins, as well as cell proliferation, are through its effects on EGFR-HER2 heterodimers. We show that trastuzumab inhibits EGF-induced cell proliferation and cell cycle progression in BT474 and SKBR3 cells. Interestingly trastuzumab strongly inhibits EGF-induced Akt phosphorylation and slightly inhibits EGF-induced Erk activation, in both BT474 and SKBR3 cells. These data suggest the presence of a novel mechanism that allows trastuzumab to inhibit EGR-induced Akt activation and cell proliferation, without blocking EGF-induced EGFR-HER2 heterodimerization and activation. We show that trastuzumab inhibits EGF-induced lipid raft localization of the EGFR-HER2 heterodimer. Disruption of the lipid raft with MβCD blocks HER2-mediated AKT activation in a similar way to trastuzumab. MβCD and trastuzumab synergically inhibit AKT activation. We conclude that trastuzumab inhibits EGF-induced lipid raft localization of EGFR-HER2 heterodimer, which leads to the inhibition of Akt phosphorylation and cell proliferation, without blocking the formation and phosphorylation of the EGFR-HER2 heterodimer.

Egfr signaling promotes juvenile hormone biosynthesis in the German cockroach

BackgroundIn insects, an interplay between the activities of distinct hormones, such as juvenile hormone (JH) and 20-hydroxyecdysone (20E), regulates the progression through numerous life history hallmarks. As a crucial endocrine factor, JH is mainly synthesized in the corpora allata (CA) to regulate multiple physiological and developmental processes, including molting, metamorphosis, and reproduction. During the last century, significant progress has been achieved in elucidating the JH signal transduction pathway, while less progress has been made in dissecting the regulatory mechanism of JH biosynthesis. Previous work has shown that receptor tyrosine kinase (RTK) signaling regulates hormone biosynthesis in both insects and mammals. Here, we performed a systematic RNA interference (RNAi) screening to identify RTKs involved in regulating JH biosynthesis in the CA of adult Blattella germanica females.ResultsWe found that the epidermal growth factor receptor (Egfr) is required for promoting JH biosynthesis in the CA of adult females. The Egf ligands Vein and Spitz activate Egfr, followed by Ras/Raf/ERK signaling, and finally activation of the downstream transcription factor Pointed (Pnt). Importantly, Pnt induces the transcriptional expression of two key enzyme-encoding genes in the JH biosynthesis pathway: juvenile hormone acid methyltransferase (JHAMT) and methyl farnesoate epoxidase (CYP15A1). Dual-luciferase reporter assay shows that Pnt is able to activate a promoter region of Jhamt. In addition, electrophoretic mobility shift assay confirms that Pnt directly binds to the − 941~ − 886 nt region of the Jhamt promoter.ConclusionsThis study reveals the detailed molecular mechanism of Egfr signaling in promoting JH biosynthesis in the German cockroach, shedding light on the intricate regulation of JH biosynthesis during insect development.

Effective-compounds of Jinshui Huanxian formula ameliorates fibroblast activation in pulmonary fibrosis by inhibiting the activation of mTOR signaling

BackgroundJinshui Huanxian formula (JHF) ameliorates idiopathic pulmonary fibrosis patients. Active compounds, including icariin, isoliquiritigenin, nobiletin, peimine, and paeoniflorin, deriving from JHF were combined as effective-component compatibility ECC of JHF II (ECC-JHF II), which is an effective therapeutic strategy for pulmonary fibrosis (PF) induced by bleomycin (BLM) in rats. PurposeThis study aimed to explore the underlying mechanism of ECC-JHF II on pulmonary fibrosis. MethodsA model of PF in rats was established through intratracheal instillation of BLM. Pulmonary function, pathological changes, and collagen deposition were examined. The gene and protein expressions in fibroblast activation were detected by quantitative real-time PCR and western blotting respectively. ResultsECC-JHF II significantly improved BLM-induced PF in rats, manifested as decreased collagen deposition, reduced pathological damage and improved pulmonary function. Furthermore, ECC-JHF II inhibited fibroblast activation by reducing the expression of α-smooth muscle actin (α-SMA) and fibronectin. We analyzed the targets of ECC-JHF II and differentially expressed genes (DEGs) of fibroblast activation induced by transforming growth factor-β1 (TGF-β1) and found that ECC-JHF II might regulate fibroblast activation by EGFR, PI3K-Akt or mTOR signaling pathway. In vitro experiments, we also found that ECC-JHF II suppressed the mTOR pathway, such as downregulating the phosphorylation levels of p70S6K in fibroblast activation induced by TGF-β1. After activating mTOR signaling, the inhibition of ECC-JHF II on fibroblast activation was blocked. These results suggested that ECC-JHF II potently ameliorated pulmonary fibrosis in rats and effectively suppressed fibroblast activation by interfering with mTOR signaling. ConclusionWe combined transcriptomics with the network analysis to predict the mechanism underlying ECC-JHF II suppression of fibroblast activation. In summary, ECC-JHF II improved BLM-induced pulmonary fibrosis, which might be associated with the suppression of fibroblast activation by inhibiting the mTOR signaling.

DNA methylation and expression of the egfr gene are associated with worker size in monomorphic ants.

The reproductive division of labour is a hallmark of eusocial Hymenoptera. Females are either reproductive queens or non-reproductive workers. In ants, workers often display further task specialisation that is associated with variation in size and/or morphology. Because female polyphenism is typically under environmental control, it is thought epigenetic mechanisms (such as DNA methylation) play a central role since they mediate gene-by-environment interactions. Methylation of the growth-promoting gene epidermal growth factor receptor (egfr) was indeed shown to control worker size in a highly polymorphic ant. However, it remains unknown if egfr methylation could also regulate worker size in monomorphic species. By combining experimental pharmacology and molecular biology, we show that worker size is associated with egfr methylation in two monomorphic ants. Furthermore, we functionally demonstrate that EGFR signalling affects worker size. These results indicate that worker size regulation by egfr methylation has been mechanistically conserved in ants but remains unexploited in monomorphic species.

The second-generation tyrosine kinase inhibitor afatinib inhibits IL-1β secretion via blocking assembly of NLRP3 inflammasome independent of epidermal growth factor receptor signaling in macrophage

Chronic inflammation might lead to many malignancies, and inadequate resolution could play a crucial role in tumor invasion, progression and metastases. Afatinib is a second-generation tyrosine kinase inhibitor targeting epidermal growth factor receptor in non-small cell lung cancer. Few studies showed the correlation of afatinib and the innate immune system especially macrophage. Our study showed that afatinib could block the activation of NLRP3 inflammasome in a dose-dependent manner in macrophage, and that afatinib could prevent the assembly of NLRP3 inflammasome. Besides, afatinib could inhibit NLRP3 inflammasome activation independent of EGFR signaling. Moreover, afatinib was able to alleviate the LPS-induced sepsis in vivo. These investigations provide significant experimental evidence in afatinib as therapeutic drug for non-small cell lung cancer or other tumors and NLRP3-related diseases, and explore new target for afatinib in macrophage.

Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila.

A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling-pathway-regulated directed movement of cell clusters. OR is instructed by the EGFR and Notch pathways and Frizzled/planar cell polarity (Fz/PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification. Here, we show that Abl kinase negatively regulates OR through its activity in the R3/R4 pair. Abl is localized to apical junctional regions in R4, but not in R3, during OR, and this apical localization requires Notch signaling. We demonstrate that Abl and Notch interact genetically during OR, and Abl co-immunoprecipitates in complexes with Notch in eye discs. Perturbations of Abl interfere with adherens junctional organization of ommatidial preclusters, which mediate the OR process. Together, our data suggest that Abl kinase acts directly downstream of Notch in R4 to fine-tune OR via its effect on adherens junctions.

Interleukin-10 induces expression of CD39 on CD8+T cells to potentiate anti-PD1 efficacy in EGFR-mutated non-small cell lung cancer

BackgroundAnti-PD-1(L1) therapies are less efficacious in patients with EGFR-mutated non-small-cell lung cancer. However, the underlying mechanism is poorly understood.MethodsThe characteristics of T cells in EGFR-mutated and wild-type tumors were analyzed...

Periostin promotes EMT via inhibition of RIN1-mediated endocytosis of EGFR in gliomas

PurposeApproximately 1/3 of brain tumors are gliomas. Previous glioma-related studies have reported increased expression of periostin (POSTN) in these cancerous tissues, but the role and mechanism of POSTN in glioma development remain unclear.MethodsNanoscale liquid chromatography coupled with tandem mass spectrometry (nano LC–MS/MS) and RNA sequencing were used to identify differential protein and mRNA expression in clinical glioma samples. Quantitative real-time PCR (qRT–PCR) was used to measure the expression of POSTN in tissues and cells. The effects of POSTN on glioma cell migration and invasion were examined using wound healing, Transwell, and three-dimensional spheroid assays in vitro and a nude mouse xenograft model in vivo. The effects of POSTN on the stability, endocytosis, and degradation of EGFR were examined by immunoblotting and immunofluorescence staining. Truncation mutation analysis was performed to investigate direct interactions between POSTN and EGFR. Immunohistochemical staining was carried out to confirm the clinical significance of POSTN.ResultsOverexpression of POSTN induced epithelial-to-mesenchymal transition (EMT) in glioma cells in vivo and in vitro. Mechanistically, POSTN downregulation inhibited EGFR signaling by promoting EGFR endocytosis and degradation. In addition, POSTN was found to bind to EGFR and RIN1, inhibiting EGFR endocytosis and degradation and thus activating the PI3K-Akt signaling pathway.ConclusionThese findings indicate the mechanism by which the POSTN/EGFR/RIN1 axis inhibits EGFR endocytosis and degradation, resulting in glioma cell EMT through the PI3K-AKT signaling pathway. Targeting POSTN/EGFR/RIN1 interactions may guarantee beneficial outcomes of glioma treatment.

ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells.

Cancer stem cells (CSCs) may serve as the cellular seeds of tumor recurrence and metastasis, and they can be generated via epithelial-mesenchymal transitions (EMTs). Isolating pure populations of CSCs is difficult because EMT programs generate multiple alternative cell states, and phenotypic plasticity permits frequent interconversions between these states. Here, we used cell-surface expression of integrin β4 (ITGB4) to isolate highly enriched populations of human breast CSCs, and we identified the gene regulatory network operating in ITGB4+ CSCs. Specifically, we identified ΔNp63 and p73, the latter of which transactivates ΔNp63, as centrally important transcriptional regulators of quasi-mesenchymal CSCs that reside in an intermediate EMT state. We found that the transcriptional program controlled by ΔNp63 in CSCs is largely distinct from the one that it orchestrates in normal basal mammary stem cells and, instead, it more closely resembles a regenerative epithelial stem cell response to wounding. Moreover, quasi-mesenchymal CSCs repurpose this program to drive metastatic colonization via autocrine EGFR signaling.

Chromium (VI)-induced ALDH1A1/EGF axis promotes lung cancer progression.

Cr(VI) is broadly applied in industry. Cr(VI) exposure places a big burden on public health, thereby increasing the risk of lung squamous cell carcinoma (LUSC). The mechanisms underlying Cr(VI)-induced LUSC remain largely elusive. Here, we report that the cancer stem cell (CSC)/tumour-initiating cell (TIC)-like subgroup within Cr(VI)-transformed bronchial epithelial cells (CrT) promotes lung cancer tumourigenesis. Mechanistically, Cr(VI) exposure specifically increases the expression levels of aldehyde dehydrogenase 1A1 (ALDH1A1), a CSC marker, through KLF4-mediated transcription. ALDH1A1 maintains self-renewal of CrT/TICs and facilitates the expression and secretion of EGF from CrT/TICs, which subsequently promotes the activation of EGFR signalling in differentiated cancer cells and tumour growth of LUSC. In addition, the ALDH1A1 inhibitor A37 and gemcitabine synergistically suppress LUSC progression. Importantly, high ALDH1A1 expression levels are positively correlated with advanced clinical stages and predict poor survival in LUSC patients. These findings elucidate how ALDH1A1 modulates EGF secretion from TICs to facilitate LUSC tumourigenesis, highlighting new therapeutic strategies for malignant lung cancers.

Abstract B022: The NEDD8 pathway as a therapeutic target in HER2-amplified colorectal cancer

Abstract Colorectal cancer (CRC) is a heterogeneous disease with a wide spectrum of clinical outcomes, from indolent resectable disease to aggressive-metastatic cases. Primary and acquired resistance limits the efficacy of available treatments, and the identification of effective drug combinations is needed to further improve patients' outcomes. We previously found that the NEDD8-activating enzyme inhibitor pevonedistat induces tumor stabilization in preclinical models of poorly differentiated, clinically aggressive CRC resistant to available therapies. To identify drugs that can be effectively combined with pevonedistat, we performed a drop-out loss-of-function synthetic lethality screening with a shRNA library covering 200 drug-target genes in four different CRC cell lines. Multiple screening hits were found to be involved in the EGFR signaling pathway, suggesting that, rather than inhibition of a specific gene, interference with the EGFR pathway at various levels could be effectively leveraged for combination therapies based on pevonedistat. Exploiting both BRAF-mutant and RAS/RAF wild-type CRC models, we validated the therapeutic relevance of our findings by showing that combined blockade of NEDD8 and EGFR pathways led to increased growth arrest and apoptosis both in vitro and in vivo. Pathway modulation analysis showed that compensatory feedback loops induced by single treatments were blunted by the combinations. Our results suggested possible therapeutic opportunities in specific CRC clinical settings. We further focused on HER2-amplified CRC and found marked sensitivity to pevonedistat in a CRC cell line carrying HER2 amplification. In these cells, long-term in vitro treatment with a combination of lapatinib and trastuzumab induces the emergence of colonies of long-term persisters (i.e., cells surviving to several weeks of HER2/EGFR blockade). These persisters display extremely slow growth and senescence features, however they fully recover upon removal of the blockade, mimicking disease relapse in patients after treatment suspension. Interestingly, persister colonies maintained sensitivity to pevonedistat, displaying a marked decrease when pevonedistat was added after six weeks of HER2/EGFR blockade, independently of the continuation of the blockade. These results unveil the possibility of employing pevonedistat in HER2-amplified CRC patients subsequently to tumor stabilization or regression by HER-2/EGFR blockade. Citation Format: Federica Invrea, Alessandro Carugo, Consalvo Petti, Giulio Draetta, Enzo Medico. The NEDD8 pathway as a therapeutic target in HER2-amplified colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B022.

Abstract A014: Identifying and overcoming resistance to RAS targeted therapy in colorectal cancer

Abstract KRAS is the most frequently mutated oncogene in human cancer and a potent oncogenic driver. In colorectal cancer (CRC), pathogenic KRAS mutations are observed in up to 50% of cases, yet there is little understanding of how distinct mutations dictate tumor biology including initiation, progression and therapy response. The recent approval of targeted KRASG12C inhibitors brings promise to transform the therapeutic landscape for many patients who lack effective, personalized treatment. Clinically acquired resistance is already observed, highlighting the need to identify mechanisms of resistance and approaches to overcome them. The aim of this study is to understand how distinct Kras alterations drive tumorigenesis and identify reasons underlying the failure of KRAS inhibitors in CRC. To explore the clinical significance of distinct KRAS mutations in tumor development and therapy response, we used Cre- conditional (Lox- stop- Lox) Kras mutant mouse strains crossed to the colon specific promoter Fabp1-Cre and Apc(flox/flox) mice. In the absence of Apc, we demonstrated that distinct Kras mutations G12C and G13D drive unique phenotypes in the colon which demonstrates that subtle mutational changes can dramatically alter the physiological response in vivo, and make distinct KRAS alleles an attractive target for targeted therapeutics. In tumor bearing models of CRC, we observed that KrasG12D drives large numbers of smaller tumors, while KrasG13D drives smaller numbers of larger tumors. To investigate the differences observed in tumorigenesis, we are studying the potential role of Kras to prime the colon for APC loss and the consequences of Kras alterations on the stem cell dynamics in driving tumorigenesis. Further, we employed our unique LSL-Kras mutant mouse strains and colon derived organoids to assess the therapeutic response of G12C and pan Kras inhibitors alone or in combination with upstream target drugs including inhibitors targeting EGFR signaling (EGFR, SHP2, SOS1 and MEK) and the Wnt signaling pathway. Together, this work has identified that distinct mutations in KRAS creates colon cancer specific vulnerabilities which dictates tumor biology. The unique in vivo and ex vivo organoid models provide an ideal platform to identify the underlying cause of failure of G12C inhibitors and developing effective approaches to target RAS in CRC. Citation Format: Marie J. Parsons, Maria Zafra, Sukanya Goswami, Maria Teresa Calvo Fernandez, John E. Wilkinson, Lukas E. Dow. Identifying and overcoming resistance to RAS targeted therapy in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A014.

Immune micro-environment and drug analysis of peritoneal endometriosis based on epithelial-mesenchymal transition classification.

Epithelial-mesenchymal transition (EMT) is a complex event that drives polar epithelial cells transform from adherent cells to motile mesenchymal cells, in which are involved immune cells and stroma cells. EMT plays crucial roles in migration and invasion of endometriosis. The interaction of endometrial implants with the surrounding peritoneal micro-environment probably affects the development of peritoneal endometriosis. To date, very few studies have been carried out on peritoneal endometriosis sub-type classification and micro-environment analysis based on EMT. The purpose of this study is to investigate the potential application of EMT-based classification in precise diagnosis and treatment of peritoneal endometriosis. Based on EMT hallmark genes, 76 peritoneal endometriosis samples were classified into two clusters by consistent cluster classification. EMT scores, which calculated by Z score of 8 epithelial cell marker genes and 8 mesenchymal cell marker genes, were compared in two clusters. Then, immune scores and the abundances of corresponding immune cells, stroma scores and the abundances of corresponding stroma cells were analyzed by the "xCell" package. Futhermore, a diagnostic model was constructed based on 9 diagnostic markers which related to immune score and stroma score by Lasso-Logistic regression analysis. Finally, based on EMT classification, a total of 8 targeted drugs against two clusters were screened out by drug susceptibility analysis via "pRRophetic" package. Hallmark epithelial-mesenchymal transition was the mainly enriched pathway of differentially expressed genes between peritoneal endometriosis tissues and endometrium tissues. Compared with cluster 2, EMT score and the abundances of most infiltrating stroma cell were significantly higher, while the abundances of most infiltrating immune cells were dramatically less. The diagnostic model could accurately distinguish cluster 1 from cluster 2. Pathway analysis showed drug candidates targeting cluster 1 mainly act on the IGF-1 signaling pathway, and drug candidates targeting cluster 2 mainly block the EGFR signaling pathway. In peritoneal endometriosis, EMT was probably promoted by stroma cell infiltration and inhibited by immune cell infiltration. Besides, our study highlighted the potential uses of the EMT classification in the precise diagnosis and treatment of peritoneal endometriosis.