Growth Factor Receptor Signaling Research Articles

Abstract PO4-06-07: Efficacy and safety of futibatinib in patients with locally advanced/metastatic triple-negative breast cancer harboring FGFR2 gene amplification: final results from the phase 2, open-label FOENIX-MBC2 study

Abstract Background: Triple-negative breast cancer (TNBC) accounts for ~15% of breast cancers and represents an aggressive subtype with limited targeted therapeutic options. Fibroblast growth factor receptor (FGFR) signaling plays an important role in breast tumorigenesis and metastasis. The phase 2 FOENIX-MBC2 study (NCT04024436) was designed to evaluate responses to futibatinib, a highly selective and potent irreversible covalent inhibitor of FGFR1–4 (FDA-approved for intrahepatic cholangiocarcinoma), in patients with metastatic breast cancer. Here, we report final efficacy and safety data for futibatinib in the cohort of patients with advanced/metastatic TNBC harboring FGFR2 gene amplification. Methods: Eligible patients had locally advanced/metastatic TNBC with measurable disease per RECIST v1.1, an ECOG performance status of 0/1, and progressive disease after ≥1 prior chemotherapy-containing regimen for advanced disease. Patients were positive for FGFR2 gene amplification determined by analysis of tumor tissue or circulating tumor DNA. Patients received oral futibatinib 20 mg once daily until disease progression, unacceptable toxicity, or other discontinuation criteria were met. The primary endpoint was objective response rate (ORR) by RECIST v1.1 in patients with centrally confirmed FGFR2 amplification. Responses (complete response and partial response) required confirmation after at least 4 weeks. Secondary endpoints included the 6-month progression-free survival (PFS) rate, PFS, duration of response (DOR), and overall safety. Results: Overall, 21 female patients with TNBC were enrolled (median age: 53 years) in this cohort. Patients had received a median 5 lines of prior systemic therapy. Overall, 2 patients had a confirmed partial response (ORR: 9.5%; 95% confidence interval [CI]: 1.2, 30.4), with a median DOR of 6.1 months (range: 3.1−9.2 months). Five (23.8%) patients had stable disease. PFS at 6 months was observed in 4 patients (19.0%; 95% CI: 5.4, 41.9) and the median PFS was 1.9 months (95% CI: 1.6, 4.0). Twenty patients (95.2%) had ≥1 treatment-related adverse event (TRAE), including 7 (33.3%) with a Grade 3 TRAE (no Grade 4 or 5). Common TRAEs (any grade) included hyperphosphatemia (85.7% of patients), constipation, diarrhea, fatigue, and dry skin (each in 23.8% of patients). Anemia was the only Grade 3 TRAE observed in more than 1 patient (n=2, 9.5%). Overall, 28.6% of patients had a serious adverse event (SAE; any cause), and 1 patient had an SAE leading to death (ischemic stroke). None of the SAEs or deaths were considered treatment-related. Conclusions: In heavily pretreated patients with metastatic TNBC harboring FGFR2 gene amplification, futibatinib monotherapy demonstrated modest anticancer activity. Futibatinib was tolerable and had an acceptable safety profile, consistent with previous data. Further biomarker work to identify patients who might benefit most from futibatinib is ongoing. Clinical trial information: NCT04024436 Citation Format: Antonio Giordano, Nisha Unni, Senthil Damodaran, Hope Rugo, Timothy Crook, Thomas Bachelot, Federico Piacentini, Hector Soto Parra, Ian Krop, Masashi Shimura, Gareth Tomlinson, Maciej Gil, Nicholas Turner, Fabrice André. Efficacy and safety of futibatinib in patients with locally advanced/metastatic triple-negative breast cancer harboring FGFR2 gene amplification: final results from the phase 2, open-label FOENIX-MBC2 study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-07.

The involvement of epidermal growth factor receptor/protein kinase Bsignaling in the tumor intrinsic PD-L1-induced malignant potential of oral squamous cell carcinoma.

Various antigen-presenting cells and tumor cells-expressing PD-L1 inhibits antitumor immune responses in the tumor microenvironment. Recently, numerous studies have shown that tumor cell intrinsic PD-L1 also plays important roles in tumor growth and progression. On the other hand, oral squamous cell carcinoma (OSCC) cells overexpress epidermal growth factor receptor (EGFR) and EGFR signal pathway exacerbates tumor progression. Therefore, this study assessed whether tumor-intrinsic PD-L1 facilitates malignant potential of OSCC cells through regulation of EGFR signaling. Two OSCC cell lines, SAS and HSC-3, were transfected with PD-L1 and EGFR-specific small interfering RNA (siRNA). Influences of PD-L1 knockdown on malignant potentials of OSCC cells were examined by Cell Counting kit-8 assay, transwell assay, sphere formation assay, flow cytometry, and Western blot. Effects of PD-L1 and EGFR knockdown on each expression were examined by quantitative real-time PCR (qRT-PCR), Western blot, and flow cytometry. Transfection of an PD-L1-siRNA into OSCC cells decreased the abilities of proliferation, stemness, and mobility of these cells significantly. PD-L1 knockdown also decreased EGFR expression through the promotion of proteasome- and lysosome-mediated degradation and following activation of the EGFR/protekin kinase B (AKT)signal pathway. Meanwhile, EGFR knockdown did not influence PD-L1 expression in SAS and HSC-3 cells, but treatment with a recombinant human EGF induced its expression. Treatment with erlotinib and cetuximab suppressed rhEGF-induced PD-L1 expression and localization in the cellular membrane of both OSCC cells. OSCC cells-expressing PD-L1 induced by EGF stimulation may promote malignancy intrinsically via the activation of the EGFR/AKT signaling cascade.

Targeting FGFR1 by β,β-dimethylacrylalkannin suppresses the proliferation of colorectal cancer in cellular and xenograft models

BackgroundColorectal cancer (CRC) continues to be a major global health challenge, ranking as a top cause of cancer-related mortality. Alarmingly, the five-year survival rate for CRC patients hovers around a mere 10–30 %. The disruption of fibroblast growth factor receptor (FGFRs) signaling pathways is significantly implicated in the onset and advancement of CRC, presenting a promising target for therapeutic intervention in CRC management. Further investigation is essential to comprehensively elucidate FGFR1′s function in CRC and to create potent therapies that specifically target FGFR1. PurposeThis study aims to demonstrate the oncogenic role of FGFR1 in colorectal cancer and to explore the potential of β,β-dimethylacrylalkannin (β,β-DMAA) as a therapeutic option to inhibit FGFR1. MethodsIn this research, we employed a comprehensive suite of techniques including tissue array, kinase profiling, computational docking, knockdown assay to predict and explore the inhibitor of FGFR1. Furthermore, we utilized kinase assay, pull-down, cell proliferation tests, and Patient derived xenograft (PDX) mouse models to further investigate a novel FGFR1 inhibitor and its impact on the growth of CRC. ResultsIn our research, we discovered that FGFR1 protein is markedly upregulated in colorectal cancer tissues, suggesting a significant role in regulating cellular proliferation, particularly in patients with colorectal cancer. Furthermore, we conducted a computational docking, kinase profiling analysis, simulation and identified that β,β-DMAA could directly bind with FGFR1 within ATP binding pocket domain. Cell-based assays confirmed that β,β-DMAA effectively inhibited the proliferation of colon cancer cells and also triggered cell cycle arrest, apoptosis, and altered FGFR1-mediated signaling pathways. Moreover, β,β-DMAA effectively attenuated the development of PDX tumors in mice that were FGFR1-positive, with no notable toxicity observed. In summary, our study highlights the pivotal role of FGFR1 in colorectal cancer, suggesting that inhibiting FGFR1 activity could be a promising strategy for therapeutic intervention. We present strong evidence that targeting FGFR1 with β,β-DMAA is a viable approach for the management of colorectal cancer. Given its low toxicity and high efficacy, β,β-DMAA, as an FGFR1 inhibitor, warrants further investigation in clinical settings for the treatment of FGFR1-positive tumors.

The receptor tyrosine kinase IGF1R and its associated GPCRs are co-regulated by the noncoding RNA NEAT1 in Alzheimer’s disease

The study is based on the complexity of Insulin like growth factor receptor (IGF1R) signaling and its regulation by noncoding RNAs (ncRNAs). IGF1R signaling is an important cascade in Alzheimer’s disease (AD); however, its regulation and roles are poorly understood. Due to the presence of β-arrestin and GPCR Receptor Kinase binding sites, this protein has been termed a ‘functional hybrid’, as it can take part in both kinase and GPCR signaling pathways, further adding to its complexity. The objective of this study is to understand the underlying ncRNA regulation controlling IGF1R and GPCRs in AD to find commonalities in the network. We found through data mining that 45 GPCRs were reportedly deregulated in AD and built clusters based on GO/KEGG pathways to show shared functionality with IGF1R. Eight miRs were further discovered that could coregulate IGF1R and GPCRs. We validated their expression in an AD cell model and probed for common lncRNAs downstream that could regulate these miRs. Seven such candidates were identified and further validated. A combined network comprising IGF1R with nine GPCRs, eight miRs, and seven lncRNAs was created to visualize the interconnectivity within pathways. Betweenness centrality analysis showed a cluster of NEAT1, hsa-miR-15a-5p, hsa-miR-16-5p, and IGF1R to be crucial form a competitive endogenous RNA-based (ceRNA) tetrad that could relay information within the network, which was further validated by cell-based studies. NEAT1 emerged as a master regulator that could alter the levels of IGF1R and associated GPCRs. This combined bioinformatics and experimental study for the first time explored the regulation of IGF1R through ncRNAs from the perspective of neurodegeneration.

“Qi Nan” agarwood restores podocyte autophagy in diabetic kidney disease by targeting EGFR signaling pathway

BackgroundDiabetic kidney disease (DKD) is a microvascular complication of diabetes mellitus, contributing to end-stage renal disease with limited treatment options. The development of DKD is attributed to podocyte injury resulting from abnormal podocyte autophagy. Consequently, the restoration of podocyte autophagy is deemed a practicable approach in the treatment of DKD.MethodsDiabetic mice were induced by streptozotocin and high-fat diet feeding. Following 8 weeks of “QN” agarwood treatment, metrics such as albuminuria, serum creatinine (Scr), and blood urea nitrogen (BUN) were evaluated. Renal histological lesions were evaluated by H&E, PAS, Masson, and Sirius red staining. Evaluation of the effects of “QN” agarwood on renal inflammation and fibrosis in DKD mice through WB, q-PCR, and IHC staining analysis. Cytoscape 3.7.1 was used to construct a PPI network. With the DAVID server, the gene ontology (GO) functional annotation and the Kyoto encyclopedia of genes and genomes (KEGG) signaling pathways of the target enrichment were performed. Molecular docking and binding affinity calculations were conducted using AutoDock, while PyMOL software was employed for visualizing the docking results of active compounds and protein targets.ResultsThe results of this study show that “QN” agarwood reduced albuminuria, Scr, and BUN in DKD mice, and improved the renal pathological process. Additionally, “QN” agarwood was observed to downregulate the mRNA and protein expression levels of pro-inflammatory and pro-fibrotic factors in the kidneys of DKD mice. Network pharmacology predicts that “QN” agarwood modulates the epidermal growth factor receptor (EGFR) signaling pathway. “QN” agarwood can increase the expression of LC3B and Nphs1 in DKD mice while reducing the expression of EGFR.ConclusionThe present study demonstrated that “QN” agarwood ameliorated renal injury in DKD by targeting EGFR and restoring podocyte autophagy.Graphical

Decorin suppresses stemness and migration potential of malignant peripheral nerve sheath tumor through inhibiting epidermal growth factor receptor signaling

Cancer stem cells (CSCs) play pivotal roles in the growth, invasion, metastasis, chemo-resistance in malignant peripheral nerve sheath tumor (MPNST). The current characterization of CSCs in MPNST is not complete. Decorin is a critical regulator of microenvironment, but its expression and function in CSCs of MPNST has not been studied. In the current study, Decorin levels and its relationship with lung and liver metastasis were determined in clinical specimens. Decorin expression in CD133-positive or CD44-positive CSCs was analyzed by RT-qPCR on cytospun MPNST cells after flow cytometry-based cell sorting. Decorin-positive cells were separated from Decorin-negative cells in transfected MPNST cell lines using a designed plasmid expressing red fluorescent protein (RFP) under a Decorin promoter. Tumor sphere formation, tumor growth, cell invasion, cell migration, and the resistance to chemotherapy-induced apoptosis were determined on Decorin-positive versus Decorin-negative MPNST cells. In vivo tumor growth was analyzed in mice receiving subcutaneous transplantation of Decorin-positive versus Decorin-negative MPNSTs. We found that Decorin levels were significantly downregulated in MPNST specimens, compared to non-tumorous adjacent tissue. Significantly lower Decorin levels were detected in MPNSTs with lung or liver metastasis compared to those without. Poorer patient survival was detected in Decorin-low MPNST, compared to Decorin-high subjects. More Decorin-negative cells were detected in CD133-positive MPNST cells than CD133-negative MPNST cells, and in CD44-positive MPNST cells than in CD44-negative MPNST cells. Compared to Decorin-positive MPNST cells, Decorin-negative MPNST cells generated significantly more tumor spheres in culture, were more invasive and migratory, and were more resistant to chemotherapy-induced apoptosis, likely due to the inhibition of epidermal growth factor receptor signaling by Decorin. Decorin-negative MPNST cells grew significantly larger tumor in vivo. Thus, depletion of Decorin may occur in CSCs in MPNSTs, serving possibly as a new therapeutic target.

DNA Damage Responses in Tumors Are Not Proliferative Stimuli, but Rather They Are DNA Repair Actions Requiring Supportive Medical Care.

In tumors, somatic mutagenesis presumably drives the DNA damage response (DDR) via altered regulatory pathways, increasing genomic instability and proliferative activity. These considerations led to the standard therapeutic strategy against cancer: the disruption of mutation-activated DNA repair pathways of tumors. Justifying that cancer cells are not enemies to be killed, but rather that they are ill human cells which have the remnants of physiologic regulatory pathways. 1. Genomic instability and cancer development may be originated from a flaw in estrogen signaling rather than excessive estrogen signaling; 2. Healthy cells with genomic instability exhibit somatic mutations, helping DNA restitution; 3. Somatic mutations in tumor cells aim for the restoration of DNA damage, rather than further genomic derangement; 4. In tumors, estrogen signaling drives the pathways of DNA stabilization, leading to apoptotic death; 5. In peritumoral cellular infiltration, the genomic damage of the tumor induces inflammatory cytokine secretion and increased estrogen synthesis. In the inflammatory cells, an increased growth factor receptor (GFR) signaling confers the unliganded activation of estrogen receptors (ERs); 6. In breast cancer cells responsive to genotoxic therapy, constitutive mutations help the upregulation of estrogen signaling and consequential apoptosis. In breast tumors non-responsive to genotoxic therapy, the possibilities for ER activation via either liganded or unliganded pathways are exhausted, leading to farther genomic instability and unrestrained proliferation. Understanding the real character and behavior of human tumors at the molecular level suggests that we should learn the genome repairing methods of tumors and follow them by supportive therapy, rather than provoking additional genomic damages.

Insulin Signaling Differentially Regulates the Trafficking of Insulin and Amyloid Beta Peptides at the Blood-Brain Barrier.

The blood-brain barrier (BBB) is instrumental in clearing toxic metabolites from the brain, such as amyloid-β (Aβ) peptides, and in delivering essential nutrients to the brain, like insulin. In Alzheimer's disease (AD) brain, increased Aβ levels are paralleled by decreased insulin levels, which are accompanied by insulin signaling deficits at the BBB. Thus, we investigated the impact of insulin-like growth factor and insulin receptor (IGF1R and IR) signaling on Aβ and insulin trafficking at the BBB. Following intravenous infusion of an IGF1R/IR kinase inhibitor (AG1024) in wild-type mice, the BBB trafficking of 125I radiolabeled Aβ peptides and insulin was assessed by dynamic SPECT/CT imaging. The brain efflux of [125I]iodo-Aβ42 decreased upon AG1024 treatment. Additionally, the brain influx of [125I]iodoinsulin, [125I]iodo-Aβ42, [125I]iodo-Aβ40, and [125I]iodo-BSA (BBB integrity marker) was decreased, increased, unchanged, and unchanged, respectively, upon AG1024 treatment. Subsequent mechanistic studies were performed using an in vitro BBB cell model. The cell uptake of [125I]iodoinsulin, [125I]iodo-Aβ42, and [125I]iodo-Aβ40 was decreased, increased, and unchanged, respectively, upon AG1024 treatment. Further, AG1024 reduced the phosphorylation of insulin signaling kinases (Akt and Erk) and the membrane expression of Aβ and insulin trafficking receptors (LRP-1 and IR-β). These findings reveal that insulin signaling differentially regulates the BBB trafficking of Aβ peptides and insulin. Moreover, deficits in IGF1R and IR signaling, as observed in the brains of type II diabetes and AD patients, are expected to increase Aβ accumulation while decreasing insulin delivery to the brain, which has been linked to the progression of cognitive decline in AD.

Abstract LB262: Reprogramming the tumor microenvironment by targeting IGF2-IGF1R signaling, enhancing viro-immunotherapy

Abstract While an FDA-approved oncolytic herpes simplex-1 virus (oHSV) has shown therapeutic promise with superior safety, accumulating clinical data revealed that its therapeutic efficacy is observed in a small subset of patients. Here, we show that NFκB-mediated Insulin-like Growth Factor 2 (IGF2)/Insulin-like Growth Factor-1 Receptor (IGF1R) signaling pathway as a key modulator for oHSV therapy-mediated tumor resistance. RNA sequencing on the oHSV-infected primary glioblastoma (GBM) and breast cancer (BC) cells identified IGF2 as one of the top 10 upregulated secretomes. Transcriptomic analysis also revealed significant upregulation/enrichment of genes related to IGF2-IGF1R-MAPK pathway in oHSV-treated tumor cells. Infection with oHSV in GBM and BC cells up-regulated IGF2 transcription via direct binding of NFκB in the IGF2 promoter 3. Compromising the IGF2-IGF1R signaling pathway using IGF2 neutralizing antibody significantlDB7 BC tumors in vivo. To circumvent this and promptly translate our findings in clinical settings, we have developed a novel oHSV, oHSV-D11mt, by incorporating modified IGF2R domain 11 into the parental oHSV genome to function as IGF2 decoy receptor as a single-agent modality. oHSV-D11mt specifically bind to IGF2 not IGF1, blocks oHSV-induced IGF2-IGF1R signaling, increases tumor cell killing, decreases oHSV-induced neutrophils/PMN-MDSCs infiltration, immune suppressive/proangiogenic cytokine secretion, and increases CD8+ cytotoxic T lymphocytes (CTLs) activation, resulting in increased virus propagation and mice survival. These findings suggest that IGF2-IGF1R signaling is a novel target to overcome oHSV therapy resistance and oHSV-D11mt could be used for improved viro-immunotherapy. Citation Format: Min Hye Noh, Alexandra Miller, Grace Nguyen, Hiroshi Nakashima, E. Antonio Chiocca, Balveen Kaur, Zhongming Zhao, Tae Jin Lee, Ji Young Yoo. Reprogramming the tumor microenvironment by targeting IGF2-IGF1R signaling, enhancing viro-immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB262.

Abstract LB301: BRAFV600E drives WNT signalling in colorectal cancer via aberrant DNA methylation

Abstract Objective: The BRAF V600E mutation drives the generation of an aggressive subtype of colorectal cancer (CRC). Although activation of WNT signalling is a generic hallmark of CRC, mutations in APC are uncommon in BRAF V600E-mutant CRC. Instead, mutations in RNF43 are the suspected WNT signalling driving event in these tumours. Here, we aimed to uncover how the WNT pathway is activated in BRAF V600E-mutant colon cancer. Design: CRISPR-LbCpf1-corrected BRAF (V600E) and RNF43 (P441fs) organoids were analysed for their growth potential in the presence or absence of growth factors, for their ability to form tumours, and by immunoblotting, RNA sequencing, and DNA methylation assays. Results: BRAF E600V organoids regained dependency on epidermal growth factor (EGF) receptor signalling for ERK pathway activation and proliferation, and completely lost tumorigenic potential. Correction of BRAF V600E, but not RNF43 P441fs, caused suppression of WNT target genes, while genes reflecting epithelial differentiation and negative regulators of WNT signalling were upregulated in BRAF E600V organoids. DNA methylation analysis revealed promoter hypermethylation of WNT antagonist genes in BRAF V600E organoids. Gene body hypermethylation, associated with transcriptional upregulation, was observed in key WNT-driven intestinal stem cell marker genes LGR5 and EPHB2, and in the WNT-effector TCF4. Treatment of BRAF V600E organoids with the DNA demethylating agent 5-aza-2′-deoxycytidine resulted in upregulation of WNT antagonist genes and a marked decrease in WNT target gene expression. Conclusions: BRAFV600E, rather than mutant RNF43, is a prominent force behind WNT pathway activation, and is essential for maintaining tumorigenic capacity. BRAFV600E causes widespread dysregulation of DNA methylation leading to activation of WNT signalling. Citation Format: Layla El Bouazzaoui, Jeroen M. Bugter, Emre Küçükköse, André Verheem, Jasmin B. Post, Nicola Fenderico, Inne H. Borel Rinkes, Hugo J. Snippert, Madelon M. Maurice, Onno Kranenburg. BRAFV600E drives WNT signalling in colorectal cancer via aberrant DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB301.

Phosphoproteomic investigation of targets of protein phosphatases in EGFR signaling.

Receptor tyrosine kinases (RTKs) initiate cellular signaling pathways, which are regulated through a delicate balance of phosphorylation and dephosphorylation events. While many studies of RTKs have focused on downstream-activated kinases catalyzing the site-specific phosphorylation, few studies have focused on the phosphatases carrying out the dephosphorylation. In this study, we analyzed six protein phosphatase networks using chemical inhibitors in context of epidermal growth factor receptor (EGFR) signaling by mass spectrometry-based phosphoproteomics. Specifically, we focused on protein phosphatase 2C (PP2C), involved in attenuating p38-dependent signaling pathways in various cellular responses, and confirmed its effect in regulating p38 activity in EGFR signaling. Furthermore, utilizing a p38 inhibitor, we classified phosphosites whose phosphorylation status depends on PP2C inhibition into p38-dependent and p38-independent sites. This study provides a large-scale dataset of phosphatase-regulation of EGF-responsive phosphorylation sites, which serves as a useful resource to deepen our understanding of EGFR signaling.

A novel dihydroacridine derivative targets epidermal growth factor receptor-expressing cancer cells in vitro and in vivo.

Small molecules are considered a source of novel medicines targeting carcinogenic intracellular pathways including epidermal growth factor receptor (EGFR) signaling. The main goal of the study is to assess whether LHT-17-19 could be considered an effective target molecule against EGFR-expressing tumor cells in silico, in vitro, and in vivo. This was an in vivo, ex vivo, and in vivo experimental study. LHT-17-19 affinity to EGFR's kinase domain was assessed by the ligand's molecular docking. EGFR-expressing Hs746T human gastric cancer cell culture and patient-derived organoid (PDO) model of EGFR-positive breast cancer (BC) were used for in vitro assessment of the molecule anticancer property. IC50 and GI50 indexes were estimated using MTT- and MTS-based tests, respectively. Anticancer activity of LHT-17-19 against EGFR-expressing mutant lung carcinoma was studied on patient-derived xenograft (PDX) model established in 10 humanized BALB/c male mice. Continuous variables were presented as a mean ± standard deviation. Intergroup differences were assessed by two-way t-test. Kaplan-Meier's curves were used for survival analysis. High affinity of LHT-17-19 for the EGFR kinase domain with dG score -7.9 kcal/mol, EDoc-5.45 kcal/mol, and Ki 101.24 uM was due to intermolecular π-σ bonds formation and the ligand intramolecular transformation. LHT-17-19 induced anti-EGFR-expressing gastric cancer cells cytotoxicity with IC50 0.32 µM (95% confidence interval [CI] 0.11-0.54 µM). The derivative inhibited growth of EGFR-expressing BC PDO with GI50 16.25 µM (95% CI 4.44-28.04 µM). 2 mg/kg LHT-17-19 intravenously daily during 7 days inhibited PDX tumor growth and metastatic activity, prolonged animals' survival, and eliminated EGFR-mutant lung cancer cells from residual tumor's node. LHT-17-19 may be considered a molecular platform for further search of promising molecules, EGFR-expressing cancer cell inhibitors.

Sinomenine inhibits PDGF/PDGFR signaling pathway to reduce RA-FLS migration induced by NETs

This study aims to decipher the mechanism of sinomenine in inhibiting platelet-derived growth factor/platelet-derived growth factor receptor(PDGF/PDGFR) signaling pathway in rheumatoid arthritis-fibroblast-like synoviocyte(RA-FLS) migration induced by neutrophil extracellular traps(NETs). RA-FLS was isolated from the synovial tissue of 3 RA patients and cultured. NETs were extracted from the peripheral venous blood of 4 RA patients and 4 healthy control(HC). RA-FLS was classified into control group, HC-NETs group, RA-NETs group, RA-NETs+sinomenine group and RA-NETs+sinomenine+CP-673451 group. RNA-sequencing(RNA-seq) was conducted to identify the differentially expressed genes between HC-NETs and RA-NETs groups. Sangerbox was used to perform the Gene Ontology(GO) function and the Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment. Cytoscape was employed to build the protein-protein interaction(PPI) network. AutoDock Vina and PyMOL were used for molecular docking of sinomenine with PDGFβ and PDGFRβ. The cell proliferation and migration were determined by the cell counting kit-8(CCK-8) and cell scratch assay, respectively. Western blot was employed to determine the protein level of PDGFRβ. Real-time quantitative polymerase chain reaction(RT-qPCR) was carried out to determine the mRNA levels of matrix metalloproteinases(MMPs). The results revealed that neutrophils in RA patients were more likely to produce NETs. Compared with HC-NETs group, RA-NETs group showed up-regulated expression of PDGFβ and PDGFRβ. Compared with control group, RA-NETs group showed increased cell proliferation and migration and up-regulated protein level of PDGFRβ and mRNA levels of PDGFβ, PDGFRβ, MMP1, MMP3, and MMP9(P<0.05). Compared with RA-NETs group, RA-NETs+sinomenine group presented decreased cell proliferation and migration and down-regulated protein and mRNA level of PDGFRβ and mRNA levels of MMP1, MMP3, and MMP9(P<0.05). Compared with RA-NETs+sinomenine group, the proliferation ability of RA-NETs+sinomenine+CP-673451 group decreased(P<0.05). The findings prove that sinomenine reduces the RA-NETs-induced RA-FLS migration by inhibiting PDGF/PDGFR signaling pathway, thus mitigating RA.

The impact of nitric oxide on HER family post-translational modification and downstream signaling in cancer.

The human epidermal growth factor receptor (HER) family consists of four members, activated by two families of ligands. They are known for mediating cell-cell interactions in organogenesis, and their deregulation has been associated with various cancers, including breast and esophageal cancers. In particular, aberrant epidermal growth factor receptor (EGFR) and HER2 signaling drive disease progression and result in poorer patient outcomes. Nitric oxide (NO) has been proposed as an alternative activator of the HER family and may play a role in this aberrant activation due to its ability to induce s-nitrosation and phosphorylation of the EGFR. This review discusses the potential impact of NO on HER family activation and downstream signaling, along with its role in the efficacy of therapeutics targeting the family.

Abstract 2905: Spatial analysis of neuropilin 2 expression in the microenvironment of melanoma

Abstract Background: Neuropilin 2 (NRP2) is a co-receptor that enhances signaling of growth factor and cytokine receptors and is expressed in tumor and microenvironment (TME) cells in melanoma and other tumors. Neuropilin 2 expression in tumor cells is associated with more aggressive tumors. Expression in tumor associated macrophages enhances efferocytosis and immunologically silent tumor cell death. Methods: Whole excisional lymph node biopsies were obtained from 10 treatment naïve patients with metastatic cutaneous melanoma who subsequently received anti-PD1 treatment. A single 5mm formalin-fixed paraffin embedded tissue section was used to assess a panel of 39 analytes by cyclic multiplex immunofluorescence (MxIF). Fields of view (~1mm2, n=288) were selected from pathologist-annotated regions of the tumor core, tumor-immune interface and adjacent lymphoid tissue. Pixel-based single cell segmentation and a supervised classifier approach was applied to resolve 12 distinct tumor, stromal and immune cell phenotypes and functional states (NRP2 positive or negative) in 1.5 million cells. Results: Tumor recurrence was observed in 6 patients (relapsed), whereas 4 patients were recurrence-free (non-relapsed) during follow up. Among the different cell types, NRP2 expression was more frequent in macrophages (44%), tumor cells (40%), dendritic cells (40%), followed by Tregs (31%) and T helper cells (29%), and less frequent in neutrophils (24%), cytotoxic T cells (23%) and B cells (15%). In colocalization permutation analysis, NRP2 expressing macrophages clustered away from tumor cells in non-relapsed patients while a random spatial distribution of NRP2 expressing macrophages with respect to tumor cells was observed in relapsed patients. In comparing the odds of tumor cell interactions with NRP2-expressing macrophages to those with NRP2 non-expressing macrophages, we found in the tumor:stromal interface (10% tumor cells) that cells from both non-relapsed and relapsed patients were more likely to interact with NRP2 non-expressing macrophages (OR = 0.73, 95% CI = [0.71, 0.75] and OR = 0.56, 95% CI = [0.55, 0.58] for non-relapsed and relapsed patients, respectively). However, in the tumor core (90% tumor cells), tumor cells from relapsed patients had significantly higher odds of interacting with NRP2 expressing macrophages, (OR = 1.15, 95% CI = [1.12, 1.18]), and this was not seen for non-relapsed patients (OR = 0.68, 95% CI = [0.66, 0.71]). In FOVs dominated by tumor cells (75% tumor), tumor cells from relapsed patients had 4.97 times the odds of expressing NRP2 compared to tumor cells from non-relapsed patients (95% CI = [2.23, 11.09], p = 0.0001). Conclusions: These spatial analytics suggest complex interactions among NRP2 expression in tumor and TME of melanoma determining risk for relapse with anti-PD1 treatment. Citation Format: Anastasios Dimou, Raymond M. Moore, Caitlin Ward, Alexey Leontovich, Ruifeng Guo, Chen Wang, Shankar Suman, Betty Dicke, Jill M. Schimke, Noah A. Stueven, Chathu A. Atherton, Wendy Nevala, Svetomir N. Markovic. Spatial analysis of neuropilin 2 expression in the microenvironment of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2905.

Abstract 6584: Aberrant TRIM7 expression potentiates RACO-1 mediated proliferation and dysregulated interferon responsiveness in the setting of anti-PD-1 acquired resistance in cancer

Abstract While checkpoint inhibitor treatment has extended the survival of many cancer patients, most initial responders will later develop disease progression through a variety of poorly understood acquired resistance mechanisms (CPI-AR). One such recently described mechanism involves the transcriptional hyperactivation of genes associated with interferon (IFN) signaling, JAK/STAT, and antigen processing/presentation pathways. Aberrant IFN regulation in the setting of AR suggests that tumor cell intrinsic mechanisms have conferred a growth advantage despite the presence of immune-mediated pressure unleashed by PD-1/L1 antibody blockade. Analysis of transcripts progressively upregulated during acquisition of AR in vivo and transcripts upregulated by IFN exposure of isolated AR cell lines in vitro identified the E3 ubiquitin ligase TRIM7 as a candidate mediator of the AR phenotype. A specific pocket on the PRY/SPRY domain of TRIM7 has been shown to bind viral peptides, resulting in ubiquitination and degradation as a mechanism to mitigate viral propagation. Here we describe the evaluation of small molecule inhibitors (SMI) designed to the TRIM7 PRY/SPRY viral-pocket domain, where binding specificity was confirmed using mass spec analysis. These SMI competitively inhibited TRIM7 mediated stabilization of RACO-1, resulting in decreased cancer cell proliferation which was associated with altered c-Jun/AP-1 transcription. TRIM7 SMI also disrupted STING and MAVS ubiquitination and restored IFN responsiveness and sensitivity to PD-1 inhibitor treatment. Finally, given the position of TRIM7 downstream of growth factor receptor and kras signaling, combination of TRIM7 inhibitors with kras, braf, and/or MEK inhibitors was shown to additively suppress proliferation of tumor cells with kras mutations. These results provide evidence that upregulation of TRIM7 mediates both a pro-proliferative and IFN resistant phenotype in the setting of CPI-AR, and that inhibition of TRIM7 with small molecule inhibitors may prevent or reverse AR to PD-1/L1 blockade. Citation Format: Thuy-Ai Tran Nguyen, Marc Morra, Kevin Li, Ulhas Bhatt, Joseph Mwangi, Yisel Rivera-Molina, Nathan Oien, Julio Medina, Taylor H. Schreiber, George Fromm. Aberrant TRIM7 expression potentiates RACO-1 mediated proliferation and dysregulated interferon responsiveness in the setting of anti-PD-1 acquired resistance in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6584.

Abstract 5799: Novel nanoparticle technology targets the delivery of polyIC to EGFR overexpressing cancers and induces a multimodal anti-tumor response

Abstract Aberrant activation of epidermal growth factor receptor (EGFR) signaling pathways is associated with tumor growth and progression. EGFR overexpression occurs in various types of solid cancers and correlates with poor outcome. Unfortunately, the clinical benefit from EGFR-targeted therapies is usually short-lived, due to the development of resistance. TargImmune has developed first-in-class Targeted Apoptotic Immunomodulators (TAIMs), which harness the body’s anti-viral responses to treat solid tumors. Our novel nanoparticle platform technology, Ta:RNA , selectively delivers synthetic dsRNA, polyinosinic:polycytidylic acid (polyIC) to tumor cells that overexpress a target receptor. PolyIC and other pattern recognition receptor (PRR) agonists have been used as adjuvants or for intratumoral administration but have limited efficacy as single agents. TargImmune’s platform allows systemic delivery of dsRNA, tailoring the nanoparticles to various solid cancers by simply changing the targeting moiety. Here we describe EGFR-Ta:RNA, which is targeted to tumors that overexpress EGFR. EGFR-Ta:RNA does not inhibit EGFR per se, and therefore activation of alternative or downstream oncogenes is not expected to cause resistance. The mechanism of action of EGFR-targeted Ta:RNA nanoparticles was extensively studied in vitro and in vivo, using confocal imaging, large-scale RNA sequencing studies, Western blot analysis and ELISA assays. In vitro, EGFR-Ta:RNA potency and selectivity were established by comparing cells with high receptor and low receptor expression. Imaging studies showed that the nanoparticles are internalized selectively into EGFR-overexpressing cancer cells by receptor-mediated endocytosis. RNAseq studies revealed: (i) a common transcriptional signature in EGFR-Ta:RNA-treated tumours and cancer cells; (ii) enrichment in pathways related to anti-viral response, inflammation, type I interferon signaling, and NFΚB signaling; (iii) enrichment in genes coding for cytokines. Western blot analysis and ELISA assays confirmed that the mechanism of action of EGFR-Ta:RNA is mediated via the induction of PRR downstream signaling, which results in cytokine secretion, apoptosis and immune cell activation. The efficacy of EGFR-Ta:RNA was demonstrated in multiple animal models, including both xenograft and immunocompetent models. The pharmacodynamic effect, exemplified by target engagement and activation of PRR signaling in tumors, was verified in vivo. In conclusion, TargImmune’s EGFR-Ta:RNA nanoparticles deliver polyIC in a tumor-targeted manner to evoke PRR signaling, inducing targeted tumor cell apoptosis as well as immune cell recruitment and activation against the heterogenous tumor. EGFR-Ta:RNA represents a novel, multimodal treatment approach for EGFR overexpressing cancers. Citation Format: Derrick Broka, Caroline De Feyter, Shoshana Klein, Meera Saxena, Joerg Schreiber, Alexei Shir, Michal Skowicki, Yaakov Benenson, Alexander Levitzki, Cornelia G. Palivan, Esteban Pombo-Villar, Babette Schade, Maya Zigler. Novel nanoparticle technology targets the delivery of polyIC to EGFR overexpressing cancers and induces a multimodal anti-tumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5799.

Abstract 2998: Regulation of epidermal growth factor receptor signaling by BRCA1-microRNA 146a network

Abstract Inherited defects in the breast cancer susceptibility gene, BRCA1 are associated with an increased risk for hereditary breast, ovarian, and other cancers. BRCA1-associated basal like breast cancers (BLBCs) are high-grade ductal carcinomas that do not express ER, PR or HER2, but frequently overexpress epidermal growth factor receptor (EGFR/ErbB1/HER1). The EGFR-driven signaling network plays a pivotal role in BLBC pathogenesis. EGFR overexpression is associated with tumor progression and metastasis, resistance to radiation and chemotherapy, and poor prognosis. Growth factor receptors are regulated by multiple miRNAs, each having many targets, coordinately controlling the complex pathways of downstream signaling. We have previously reported a post-transcriptional mechanism by which BRCA1 regulates EGFR expression through the induction of miR-146a and provided a rationale for the development of miR-146a based therapeutic strategies. In addition, we have shown that BRCA1 also exerts regulatory control over dozens of important miRNAs that appear to play a critical role in breast neoplasia. From TCGA analysis, we show that low expression of these miRNAs is associated with distinctively poor overall survival of TNBC patients. Using miR-146a loss or gain of function experiments in vitro, we show that BRCA1 and miR-146a regulate EGFR signaling, stemness, epithelial-to-mesenchymal transition (EMT), extracellular matrix (ECM) remodeling and chemoresistance. Using in vivo mouse models, we demonstrate that miR-146a overexpression delays tumor formation leading to better overall survival. BLBC unlike the other subtypes, lacks targeted therapy approaches making cytotoxic chemotherapy essentially the only option for treatment. Though EGFR inhibitors have been used to treat other cancers, clinical trials for breast cancer have so far been unsuccessful due to poor response rates. Since one miRNA can target multiple genes and regulate multiple signaling pathways, our study provides evidence to suggest that restoring miR-146a could suppress EGFR-RAS-MEK signaling and the other compensatory pathways, providing a targeted therapeutic option for BRCA1 associated BLBC. Citation Format: Easwari Kumaraswamy, Raeann M. Koren Shimak, Sumedha Gunewardena, Karen L. Wendt, Dani Alexander, Stacey L. Hembruff, Roy A. Jensen. Regulation of epidermal growth factor receptor signaling by BRCA1-microRNA 146a network [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2998.

Insights into the Overcoming EGFRDel19/T790M/C797S Mutation: A Perspective on the 2-Aryl-4-aminothienopyrimidine Backbone.

The epithelial growth factor receptor (EGFR) signaling pathway has been proposed to benefit non-small cell lung cancer (NSCLC) treatment. In this manuscript, we investigated the modification of 2-aryl-4-aminoquinazoline, the classical backbone of the fourth-generation EGFR inhibitors, in addition to obtaining a series of novel 2-aryl-4-aminothienopyrimidine derivatives (A1~A45), we also gained further understanding of the modification of this framework. Derivatives were tested for cytotoxicity against cancer cell lines (cervical cancer cell line Hela, lung cancer cell lines A549, H1975, and PC-9, Ba/F3-EGFRDel19/T790M/C797S cells, and human normal hepatocytes LO2) as well as for the derivative's inhibitory activity against EGFRWT, EGFRL858R/T790M, and EGFRDel19/T790M/C797S kinase inhibitory activities. The results showed that most of the target compounds showed moderate to excellent activity against one or more cancer cell lines. Among them, the antitumor activity (IC50) of the most promising A9 against A549 and H1975 cell lines was 0.77±0.08 μM, 6.90±0.83 μM, respectively. At concentration of 10 μM, A9 can be employed as the fourth-generation of EGFR inhibitors with the ability to overcome the C797S drug resistance since it can suppress EGFRDel19/T790M/C797S cells and kinase by 98.90 % and 85.88 %, respectively. Moreover, the tumor-bearing nude mice experiment further shows that A9 can significantly inhibit the growth of tumor in vivo, with the tumor inhibition rate (TIR) of 55.92 %, which was equivalent to the positive group. After that, from the result of HE staining experiment and blood biochemical analysis experiment, A9 show low toxicity and good safety, which is worthy of further research and development.

Mechanism of ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix regulates HIF-1α signaling pathway mediated by renal hypoxia to ameliorate renal fibrosis in DKD rats

This study explored the mechanism of the ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix in ameliorating renal fibrosis in the rat model of diabetic kidney disease(DKD) based on the expression of hypoxia-inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF) and HIF-1α/platelet-derived growth factor(PDGF)/platelet-derived growth factor receptor(PDGFR) signaling pathways in the DKD rats. After 1 week of adaptive feeding, 50 male SPF-grade Wistar rats were randomized into a blank group(n=7) and a modeling group. After 24 h of fasting, the rats in the modeling group were subjected to intraperitoneal injection of streptozocin and fed with a high-sugar and high-fat diet to establish a DKD model. After modeling, the rats were randomly assigned into model(n=7), low-dose ultrafiltration extract(n=7), medium-dose ultrafiltration extract(n=7), irbesartan(n=8), and high-dose ultrafiltration extract(n=8) groups. After intervention by corresponding drugs for 12 weeks, the general conditions of the rats were observed. The body weights and blood glucose levels of the rats were measured weekly, and the 24 h urinary protein(24hUP) was measured at the 6th and 12th weeks of drug administration. After the last drug administration, the renal function indicators were determined. Masson staining was employed to observe the pathological changes of the renal tissue. The expression of prolyl hydroxylase domain 2(PHD2) and HIF-1α in the renal tissue was detected by immunohistochemistry(IHC). Real-time qPCR was employed to determine the mRNA levels of PHD2, VEGF, PDGF, and PDGFR in the renal tissue. Western blot was employed to determine the protein levels of HIF-1α, VEGF, PDGF, and PDGFR in the renal tissue. The results showed that compared with the model group, drug administration lowered the levels of glycosylated serum protein(GSP), aerum creatinine(Scr), and blood urea nitrogen(BUN) in a dose-dependent manner(P<0.05 or P<0.01) and mitigated the pathological changes in the renal tissue. Furthermore, drug administration up-regulated mRNA level of PHD2(P<0.05 or P<0.01), down-regulated the mRNA levels of VEGF, PDGF, and PDGFR(P<0.05 or P<0.01) and the protein levels of HIF-1α, VEGF, PDGF, and PDGFR(P<0.01) in the renal tissue, and increased the rate of PHD2-positive cells(P<0.01). In conclusion, the ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix effectively alleviated the renal fibrosis in DKD rats by inhibiting the expression of key proteins in the HIF-1α signaling pathway mediated by renal hypoxia and reducing extracellular matrix(ECM) deposition.