PDGFR Pathways Research Articles

MEK Inhibitors Lead to PDGFR Pathway Upregulation and Sensitize Tumors to RAF Dimer Inhibitors in NF1-Deficient Malignant Peripheral Nerve Sheath Tumor.

Malignant peripheral nerve sheath tumor (MPNST) is a highly aggressive subtype of soft-tissue sarcoma with a high propensity to metastasize and extremely limited treatment options. Loss of the RAS-GAP NF1 leads to sustained RAF/MEK/ERK signaling in MPNST. However, single-agent MEK inhibitors (MEKi) have failed to elicit a sustained inhibition of the MAPK signaling pathway in MPNST. We used pharmacological, biochemical, and genetic perturbations of the receptor tyrosine kinase and MAPK signaling pathway regulators to investigate the mechanisms of MEKi resistance and evaluated combination therapeutic strategies in various preclinical MPNST models in vitro and in vivo. Here, we report that MEKi treatment resistance in MPNST involves two adaptive pathways: direct transcriptional upregulation of the receptor tyrosine kinase PDGFRβ and MEKi-induced increase in RAF dimer formation and activation of downstream signaling. Although the pharmacologic combination of a MEKi with a PDGFRβ-specific inhibitor was more effective than treatment with the MEKi alone, the combination of the MEKi and RAF dimer inhibitors led to a robust inhibition of MAPK pathway signaling. This combination treatment was effective in vitro and in vivo, as demonstrated by the significant increase in drug synergism and its high effectiveness in decreasing MPNST viability. Our findings suggest that the combination of MEKis and PDGFR and/or RAF dimer inhibitors can overcome MEKi resistance and may serve as a novel targeted therapeutic strategy for patients with NF1-deficient MPNST, which in turn could impact future clinical investigations for this patient population.

Ajuga taiwanensis Extract Promotes Wound-healing via Activation of PDGFR/MAPK Pathway.

Chronic and prolonged wounds are a serious public problem that may severely affect the quality of life and result in psychological pressure. Fibroblasts play a crucial role in the wound process and in skin pathology. Herbal drugs have long been used for wound care worldwide. Ajuga taiwanensis (Lamiaceae) is a folk medicine for antipyretics, anti-inflammation, and reducing swelling in Taiwan. This study aimed to investigate the effect of A.taiwanensis in wound healing and the underlying mechanisms. Under human dermal fibroblast (HDF) wound-healing activity-guided fractionation, we found that a sub-fraction (AT-M) of A. taiwanensis extract (AT) and the major ingredients significantly promoted wound healing and decreased IL-1β and - 6 expressions on HDFs. Furthermore, the fraction of AT-M enhanced wound healing on C57BL/6 mouse skins, increased PDGFR expressions, and activated the PDGFR/MAPK pathway. Taken together, A.taiwanensis extracts promote wound healing by the PDGFR pathway and lead to enhanced cell spreading and motility, thereby having a possible beneficial effect on wound healing.

Abstract A061: Grb7 is dispensible for normal development and ERBB2-driven mammary tumorigenesis, but maternal Grb7 deletion causes lactational insufficiency

Abstract Growth factor receptor-bound 7 (Grb7) is an adaptor protein implicated in signal transduction downstream of multiple receptor tyrosine kinases, including ERBB, FGFR and PDGFR pathways. Experimental studies have implicated Grb7 in regulating cell proliferation, survival, migration, and invasion through its large repertoire of protein-protein interactions. Here, we describe the generation and characterization of a Grb7 knockout (KO) mouse. These mice are viable and fertile. A lacZ knock-in reporter was used to visualize Grb7 promoter activity patterns in adult tissues, indicating widespread Grb7 expression in glandular epithelium, the central nervous system and other tissues. The sole defect observed in these animals was a failure of Grb7 KO females to successfully raise pups to weaning age, a phenotype that was independent of both paternal and pup genotypes. These data suggest a regulatory role for Grb7 in mammary lactational physiology. Grb7 is genomically adjacent to ERBB2 and commonly co-amplified with ERBB2 in human breast cancer. This adaptor protein physically interacts with ERBB2 and has been implicated in oncogenic signal propagation to intracellular pathways. We intercrossed Grb7 KO and MMTV-Neu mice to determine whether Grb7 is required for ERBB2-driven tumorigenesis. Surprisingly, there was no difference in time to tumor detection between cohorts, indicating that Grb7 is not obligately required for ERBB2-driven tumorigenesis. In conclusion, despite a considerable body of literature suggesting key roles for Grb7 in signal transduction pathways involved in both normal and malignant processes, our analysis of Grb7 KO mice demonstrates that Grb7 is largely dispensable for both development and ERBB2-driven tumorigenesis. These data raise the possibility that other Grb family members, Grb10 and Grb14, might compensate for Grb7 loss. Accordingly, a comprehensive assessment of all Grb family members may be needed to fully clarify their physiological roles. Citation Format: Kristopher A. Lofgren, Peter Feiszt, Paraic A. Kenny. Grb7 is dispensible for normal development and ERBB2-driven mammary tumorigenesis, but maternal Grb7 deletion causes lactational insufficiency [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A061.

Grb7 knockout mice develop normally but litters born to knockout females fail to thrive.

Growth factor receptor-bound 7 (Grb7) is an adaptor protein involved in signal transduction downstream of multiple receptor tyrosine kinases, including ERBB, FGFR, and PDGFR pathways. Experimental studies have implicated Grb7 in regulating cell proliferation, survival, migration, and invasion through its large repertoire of protein-protein interactions. Here, we describe the generation and characterization of a Grb7 knockout mouse. These mice are viable and fertile. A lacZ knock-in reporter was used to visualize Grb7 promoter activity patterns in adult tissues, indicating widespread Grb7 expression in glandular epithelium, the central nervous system, and other tissues. The sole defect observed in these animals was a failure of Grb7 knockout females to successfully raise pups to weaning age, a phenotype that was independent of both paternal and pup genotypes. These data suggest a regulatory role for Grb7 in mammary lactational physiology.

FDA-approved Abl/EGFR/PDGFR kinase inhibitors show potent efficacy against pandemic and seasonal influenza A virus infections of human lung explants

Influenza viruses (IVs) cause substantial global morbidity and mortality. Given the limited range of licensed antiviral drugs and their reduced efficacy due to resistance mutations, repurposing FDA-approved kinase inhibitors as fast-tracked host-targeted antivirals is an attractive strategy. We identified six FDA-approved non-receptor tyrosine kinase-inhibitors (NRTKIs) as potent inhibitors of viral replication of pandemic and seasonal IVs invitro. We validated their efficacy in a biologically and clinically relevant exvivo model of human precision-cut lung slices. We identified steps of the virus infection cycle affected by these inhibitors and assessed their effect(s) on host responses. Their overlapping targets suggest crosstalk between Abl, EGFR, and PDGFR pathways during IAV infection. Our data and established safety profiles of these NRTKIs provide compelling evidence for further clinical investigations and repurposing as host-targeted influenza antivirals. Moreover, these NRTKIs have broad-spectrum antiviral potential given that their kinase/pathway targets are critical for the replication of many viruses.

Identification of oncogenes and tumor-suppressor genes with hepatocellular carcinoma: A comprehensive analysis based on TCGA and GEO datasets.

Aim: Existing targeted therapies for hepatocellular carcinoma (HCC) are resistant and have limitations. It is crucial to find new HCC-related target genes. Methods: RNA-sequencing data of HCC were gathered from The Cancer Genome Atlas and Gene Expression Omnibus datasets. Initially, differentially expressed genes between normal and tumor tissues were identified from four Gene Expression Omnibus datasets, GSE36376, GSE102079, GSE54236, and GSE45267. GO terms and KEGG pathway enrichment analyses were performed to explore the potential biological functions of differentially expressed genes. A PPI network was constructed by using the STRING database, and up-regulated and down-regulated hub genes were defined through 12 topological approaches. Subsequently, the correlation bounded by up-regulated genes and down-regulated genes in the diagnosis, prognosis, and clinicopathological features of HCC was analyzed. Beyond a shadow of doubt, the key oncogene PBK and tumor suppressor gene F9 were screened out, and the specific mechanism was investigated through GSEA enrichment analysis and immune correlation analysis. The role of PBK in HCC was further verified by western blot, CCK8, transwell, and tube formation experiments. Results: CDCA5, CDC20, PBK, PRC1, TOP2A, and NCAPG are good indicators of HCC diagnosis and prognosis. The low expressions of F9, AFM, and C8B indicate malignant progression and poor prognosis of HCC. PBK was found to be closely related to VEGF, VEGFR, and PDGFR pathways. Experiments showed that PBK promotes HCC cell proliferation, migration, invasion, and tube formation in HUVEC cells. F9 was negatively correlated with the degree of immune infiltration, and low expression of F9 suggested a poor response to immunotherapy. Conclusion: The role of HCC-related oncogenes and tumor-suppressor genes in diagnosis and prognosis was identified. In addition, we have found that PBK may promote tumor proliferation through angiogenesis and F9 may be a predictor of tumor immunotherapy response.

Alteration in tyrosine phosphorylation of cardiac proteome and EGFR pathway contribute to hypertrophic cardiomyopathy

Alterations of serine/threonine phosphorylation of the cardiac proteome are a hallmark of heart failure. However, the contribution of tyrosine phosphorylation (pTyr) to the pathogenesis of cardiac hypertrophy remains unclear. We use global mapping to discover and quantify site-specific pTyr in two cardiac hypertrophic mouse models, i.e., cardiac overexpression of ErbB2 (TgErbB2) and α myosin heavy chain R403Q (R403Q-αMyHC Tg), compared to control hearts. From this, there are significant phosphoproteomic alterations in TgErbB2 mice in right ventricular cardiomyopathy, hypertrophic cardiomyopathy (HCM), and dilated cardiomyopathy (DCM) pathways. On the other hand, R403Q-αMyHC Tg mice indicated that the EGFR1 pathway is central for cardiac hypertrophy, along with angiopoietin, ErbB, growth hormone, and chemokine signaling pathways activation. Surprisingly, most myofilament proteins have downregulation of pTyr rather than upregulation. Kinase-substrate enrichment analysis (KSEA) shows a marked downregulation of MAPK pathway activity downstream of k-Ras in TgErbB2 mice and activation of EGFR, focal adhesion, PDGFR, and actin cytoskeleton pathways. In vivo ErbB2 inhibition by AG-825 decreases cardiomyocyte disarray. Serine/threonine and tyrosine phosphoproteome confirm the above-described pathways and the effectiveness of AG-825 Treatment. Thus, altered pTyr may play a regulatory role in cardiac hypertrophic models.

Imatinib facilitates gemcitabine sensitivity by targeting epigenetically activated PDGFC signaling in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with poor prognosis. Gemcitabine-based chemotherapy has become one of the main modalities of its management. However, gemcitabine resistance frequently occurs, leading to failure of PDAC therapy. Platelet-derived growth factors (PDGFs) and their receptors play important roles in cancer progression and chemoresistance. We aimed to investigate the biological function and therapeutic significance of platelet-derived growth factor C (PDGFC) in drug-resistant PDAC. Our study showed that PDGFC was abnormally highly expressed in gemcitabine-resistant PDAC. Silencing PDGFC expression can enhance the therapeutic effect of gemcitabine on PDAC. Mechanistically, the transcription of PDGFC is mediated by H3K27 acetylation, and PDGFC promotes gemcitabine resistance by activating the PDGFR-PI3K-AKT signaling pathway. The PDGFR inhibitor imatinib inhibits the PDGFR pathway. Imatinib and gemcitabine have a synergistic effect on the treatment of PDAC, and imatinib can significantly enhance the anti-tumor effect of gemcitabine in a drug-resistant PDAC patient-derived xenograft model. In conclusion, PDGFC is a potential predictor of gemcitabine-resistant PDAC. Imatinib inhibits PDGFR activation to promote gemcitabine sensitivity in PDAC. Combined modality regimen of imatinib and gemcitabine is likely to translate into clinical trial for the treatment of PDGFC-associated gemcitabine-resistant patients.

Frequency-dependent signaling in cardiac myocytes.

Background: Recent experimental data support the view that signaling activity at the membrane depends on its geometric parameters such as surface area and curvature. However, a mathematical, biophysical concept linking shape to receptor signaling is missing. The membranes of cardiomyocytes are constantly reshaped due to cycles of contraction and relaxation. According to constant-volume behavior of cardiomyocyte contraction, the length shortening is compensated by Z-disc myofilament lattice expansion and dynamic deformation of membrane between two adjacent Z-discs. Both morphological changes are strongly dependent on the frequency of contraction. Here, we developed the hypothesis that dynamic geometry of cardiomyocytes could be important for their plasticity and signaling. This effect may depend on the frequency of the beating heart and may represent a novel concept to explain how changes in frequency affect cardiac signaling. Methods: This hypothesis is almost impossible to answer with experiments, as the in-vitro cardiomyocytes are almost two-dimensional and flattened rather than being in their real in-vivo shape. Therefore, we designed a COMSOL multiphysics program to mathematically model the dynamic geometry of a human cardiomyocyte and explore whether the beating frequency can modulate membrane signal transduction. Src kinase is an important component of cardiac mechanotransduction. We first presented that Src mainly localizes at costameres. Then, the frequency-dependent signaling effect was studied mathematically by numerical simulation of Src-mediated PDGFR signaling pathway. The reaction-convection-diffusion partial differential equation was formulated to simulate PDGFR pathway in a contracting sarcomeric disc for a range of frequencies from 1 to 4 Hz. Results: Simulations exhibits higher concentration of phospho-Src when a cardiomyocyte beats with higher rates. The calculated phospho-Src concentration at 4, 2, and 1 Hz beat rates, comparing to 0 Hz, was 21.5%, 9.4%, and 4.7% higher, respectively. Conclusion: Here we provide mathematical evidence for a novel concept in biology. Cell shape directly translates into signaling, an effect of importance particularly for the myocardium, where cells continuously reshape their membranes. The concept of locality of surface-to-volume ratios is demonstrated to lead to changes in membrane-mediated signaling and may help to explain the remarkable plasticity of the myocardium in response to biomechanical stress.

SCAMP3 Regulates EGFR and Promotes Proliferation and Migration of Triple-Negative Breast Cancer Cells through the Modulation of AKT, ERK, and STAT3 Signaling Pathways.

Simple SummaryTriple-negative breast cancer (TNBC) cases constitute ~15% of breast cancer tumors. This subtype is characterized by the lack of hormone receptors, estrogen and progesterone, and HER2, and it is a more aggressive, metastatic, and recurrent subtype than others. The need to develop novel anti-TNBC drugs encourages scientists to direct studies to identify potential therapeutic molecular targets. Our study aimed to define the role of the secretory carrier membrane protein, SCAMP3, in TNBC, and its association with the epidermal growth factor receptor (EGFR), which is present in ~90% of tumors. We confirmed that SCAMP3 promotes cell proliferation and motility and is directly associated with EGFR redistribution and degradation. It also regulates the gene and protein expression of molecules from the EGFR and PDGFR pathways. Finally, SCAMP3 depletion delays the establishment of TNBC tumors. This study provides evidence for SCAMP3′s TNBC tumor-promoting role and its potential as a target therapy for this disease.Triple-negative breast cancer (TNBC) is the most aggressive, metastatic, and lethal breast cancer subtype. To improve the survival of TNBC patients, it is essential to explore new signaling pathways for the further development of effective drugs. This study aims to investigate the role of the secretory carrier membrane protein 3 (SCAMP3) in TNBC and its association with the epidermal growth factor receptor (EGFR). Through an internalization assay, we demonstrated that SCAMP3 colocalizes and redistributes EGFR from the cytoplasm to the perinucleus. Furthermore, SCAMP3 knockout decreased proliferation, colony and tumorsphere formation, cell migration, and invasion of TNBC cells. Immunoblots and degradation assays showed that SCAMP3 regulates EGFR through its degradation. In addition, SCAMP3 modulates AKT, ERK, and STAT3 signaling pathways. TNBC xenograft models showed that SCAMP3 depletion delayed tumor cell proliferation at the beginning of tumor development and modulated the expression of genes from the PDGF pathway. Additionally, analysis of TCGA data revealed elevated SCAMP3 expression in breast cancer tumors. Finally, patients with TNBC with high expression of SCAMP3 showed decreased RFS and DMFS. Our findings indicate that SCAMP3 could contribute to TNBC development through the regulation of multiple pathways and has the potential to be a target for breast cancer therapy.

Natural Sourced Inhibitors of EGFR, PDGFR, FGFR and VEGFRMediated Signaling Pathways as Potential Anticancer Agents.

The molecular mechanisms of mitotic cell cycle progression involve very tightly restricted types of machinery which are highly regulated by a fine balance between the positive and negative accelerators (or regulators). These regulators include several checkpoints that have proteins acting as enzymes and their activating partners. These checkpoints incessantly monitor the external as well as internal environments such as growth signals, favorable conditions for growth, cell size, DNA integrity of the cell and hence function to maintain the highly ordered cell cycle progression by sustaining cell homeostasis and promoting error-free DNA replication and cell cycle division. To progress through the mitotic cell cycle, the cell has to successfully drive past the cell cycle checkpoints. Due to the abnormal behavior of some cell cycle proteins, the cells tend to divide continuously overcoming the tight regulation of cell cycle checkpoints. Such anomalies may lead to unwanted cell division, and this deregulation of cell cycle events is considered as one of the main reasons behind tumor development, and thus, cancer progression. So the understanding of the molecular mechanisms in cancer progression might be insightful for designing several cancer treatment strategies. The deregulation in the checkpoints is caused due to the changes in the tyrosine residues of TPKs via PDGFR, EGFR, FGFR, and VEGFR-mediated signaling pathways. Therefore, the inhibitors of PDGFR, EGFR, FGFR, and VEGFR-mediated signaling pathways could be potential anticancer agents. The resistance and toxicity in the existing synthetic anticancer chemotherapeutics may decrease the life span of a patient. For long, natural products have played an essential alternative source of therapeutic agents due to having least or no side effect and toxicity. The present study is an attempt to promote natural anticancer drug development focusing on the updated structural information of PDGFR, EGFR, FGFR, and VEGFR inhibitors isolated from the plant sources. The data used in this review has been collected from internet resources, viz. GOOGLE Web, GOOGLE SCHOLAR, and PubMed Central. The citation of each report was first checked, after which the articles were selected as an authentic reference for the present study. Around 200 journal articles were initially selected, of which around 142 were finally chosen for presenting the study on the natural sourced inhibitors of EGFR, PDGFR, FGFR, and VEGFR-mediated signaling pathways which may help to enhance the potential cancer treatment.

Abstract 11594: Deep Learning of the Retina Enables Phenome- and Genome- Wide Analyses of the Microvasculature

Introduction: The retinal fundus is a window for non-invasive assessment of the microvasculature. However, the range of phenotypes and inherited genetic variants associated with the retinal microvasculature remain unknown. Hypothesis: Deep learning of retinal fundus images enables large-scale quantification of vascular indices and an unbiased assessment of phenotypes and genetic variants linked to the microvasculature. Methods: We utilized 97,895 retinal fundus images from 54,813 UK Biobank participants (ages 40-70y, 55% female). Using convolutional neural networks to segment vessels, we calculated vascular branching complexity using fractal dimension (FD), and vascular density, and associated these indices with 1,100 incident ICD-based conditions (median 10y follow-up) and 88 quantitative traits, adjusting for age, sex, smoking status, and ethnicity. Genome-wide association study (GWAS) among 38,932 unrelated individuals was performed. Results: Low retinal vascular FD and density were significantly associated with increased risk of incident hypertension, congestive heart failure, renal failure, type 2 diabetes, sleep apnea, anemia, and multiple ocular conditions. Low retinal vascular FD and density were also linked to quantitative traits including elevated blood pressure, anemia indices, abnormal pulmonary function tests, measures of metabolic disease (high HbA1c, % body fat, BMI), and ocular traits (decreased visual acuity and increased intraocular pressure). GWAS of vascular FD and density identified loci enriched among pathways linked to angiogenesis (VEGF, PDGFR, angiopoietin, and WNT signaling pathways), and inflammation (interleukin, cytokine signaling). Conclusions: Through phenotypic and genotypic analyses, our study showed that the retinal vasculature may serve as a biomarker for future cardiometabolic and ocular disease, and provided insights on genes and biological pathways influencing microvascular indices.

Phase I study using crenolanib to target PDGFR kinase in children and young adults with newly diagnosed DIPG or recurrent high-grade glioma, including DIPG.

BackgroundPlatelet-derived growth factor receptor (PDGFR) signaling has been directly implicated in pediatric high-grade gliomagenesis. This study evaluated the safety and tolerability of crenolanib, a potent, selective inhibitor of PDGFR-mediated phosphorylation, in pediatric patients with high-grade glioma (HGG).MethodsWe used a rolling-6 design to study the maximum tolerated dose (MTD) of once-daily crenolanib administered during and after focal radiation therapy in children with newly diagnosed diffuse intrinsic pontine glioma (DIPG) (stratum A) or with recurrent/progressive HGG (stratum B). Pharmacokinetics were studied during the first cycle at the first dose and at steady state (day 28). Alterations in PDGFRA were assessed by Sanger or exome sequencing and interphase fluorescence in situ hybridization or single nucleotide polymorphism arrays.ResultsFifty evaluable patients were enrolled in the 2 strata, and an MTD of 170 mg/m2 was established for both. Dose-limiting toxicities were primarily liver enzyme elevations and hematologic count suppression in both strata. Crenolanib AUC0–48h and CMAX did not differ significantly for crushed versus whole-tablet administration. Overall, PDGFRA alterations were observed in 25% and 30% of patients in stratum A and B, respectively. Neither crenolanib therapy duration nor survival outcomes differed significantly by PDGFRA status, and overall survival of stratum A was similar to that of historical controls.ConclusionsChildren tolerate crenolanib well at doses slightly higher than the established MTD in adults, with a toxicity spectrum generally similar to that in adults. Studies evaluating intratumoral PDGFR pathway inhibition in biomarker-enriched patients are needed to evaluate further the clinical utility of crenolanib in this population.

Elucidating resistance mechanism to PARP inhibitors for the development of novel therapeutic approaches in high-grade serous ovarian cancer.

6078 Background: PARP inhibitors (PARPi) have been established as a targeted therapeutic approach not only in patients with high-grade serous ovarian cancer (HGSOC) that have genetic loss of function of BRCA1/2-associated DNA repair. However, treatment efficacy varies and neither BRCA mutation, nor homolog recombination deficiency (HRD) status seem to be optimal predictors. Moreover, mechanisms of treatment resistance are poorly understood and novel approaches are urgently required. Methods: Here we created gene expression data of HGSOC patients (n = 52) before PARPi treatment to elucidate key signaling pathways of resistance to increase their efficacy in combinatorial therapeutic strategies. We performed a comprehensive bioinformatics analysis of the differentially expressed genes between the 25% extreme responders (n = 26; 13 each group), including gene set enrichment analysis (GSEA) and causal inference analysis with the CARNIVAL pipeline to elucidate the underlying molecular and regulatory mechanisms governing treatment efficacy and resistance. Results: In accordance with recent publications, we found higher levels of MYC activity in non-responders and deregulation of the Wnt/ß-catenin signaling pathway resulting in PARPi treatment resistance. The pathway enrichment analysis also revealed specific pathways especially PDGFR, FGFR, PI3K/mTOR and MAPK signaling pathway associated with resistant phenotype. Furthermore, we have identified key kinases, particularly JAK1/2 and SRC that might mediate resistance to PARP inhibition. In addition, differential gene expression analysis revealed folate receptor 1 (FOLR1) to be significantly higher expressed in non-responders (logFC = 2.66; p < 0.0026) with the potential as a serum-based biomarker not only for ovarian cancer, as it correlates closely with CA125, but also PARPi treatment efficacy. Conclusions: In conclusion, these findings define a network of pathways, that are crucial to mediate mechanism of PARPi resistance and identified key signaling kinases as therapeutic targets in ovarian cancer.

Chromatin accessibility analysis reveals that TFAP2A promotes angiogenesis in acquired resistance to anlotinib in lung cancer cells.

Anlotinib, a multitarget tyrosine kinase inhibitor, is effective as a third-line treatment against non-small cell lung cancer (NSCLC). However, acquired resistance occurs during its administration. To understand the molecular mechanisms of anlotinib resistance, we characterized chromatin accessibility in both the parental and anlotinib-resistant lung cancer cell line NCI-H1975 through ATAC-seq. Compared with the parental cells, we identified 2666 genomic regions with greater accessibility in anlotinib-resistant cells, in which angiogenesis-related processes and the motifs of 21 transcription factors were enriched. Among these transcription factors, TFAP2A was upregulated. TFAP2A knockdown robustly diminished tumor-induced angiogenesis and partially rescued the anti-angiogenic activity of anlotinib. Furthermore, transcriptome analysis indicated that 2280 genes were downregulated in anlotinib-resistant cells with TFAP2A knocked down, among which the PDGFR, TGF-β, and VEGFR signaling pathways were enriched. Meanwhile, we demonstrated that TFAP2A binds to accessible sites within BMP4 and HSPG2. Collectively, this study suggests that TFAP2A accelerates anlotinib resistance by promoting tumor-induced angiogenesis.

Abstract 1723: Elucidation of novel therapeutic targets for acute myeloid leukemias with RUNX1-RUNX1T1 fusion

Abstract The RUNX1-RUNX1T1 fusion is a frequent chromosomal alteration in acute myeloid leukemias (AMLs). Although RUNX1-RUNX1T1 fusion protein has pivotal roles in the development of AMLs with the fusion, RUNX1-RUNX1T1 fusion protein is difficult to target since it lacks kinase activities. Here, we used a sophisticated bioinformatic tool to elucidate targetable signaling pathways in AMLs with RUNX1-RUNX1T1 fusion. After analysis of 93 AMLs from the TCGA database, the expressions of 293 genes were significantly correlated with the expression of the RUNX1-RUNX1T1 fusion gene. Based on the 293 genes, the COX, VEGFR, PDGFR, and FGFR pathways are predicted to be specifically activated in AMLs with RUNX1-RUNX1T1 fusion. Compared with the AML cells without the fusion, the specific activations of the AML cell lines with RUNX1-RUNX1T1 fusion were confirmed by the increased phosphorylation of VEGFRs, PDGFRs, and FGFRs. Moreover, the in vitro proliferation of AML cells with RUNX1-RUNX1T1 fusion decreased significantly more than that of AML cells without the fusion when the pathways were inhibited pharmacologically. The results indicate that novel targetable signaling pathways could be identified by the analysis of the gene expression features of AMLs with non-targetable genetic alterations. The elucidation of specific molecular targets for AMLs that have a specific genetic alteration would promote personalized treatment of AMLs and improve treatment outcomes for AML patients in clinic. Note: This abstract was not presented at the meeting. Citation Format: Jae Won Yun, Yoon Kyung Bae, So Yeong Cho, Hee-Jin Kim, Do-Hyun Nam, Sun-Hee Kim, Sejong Chun, Kyeung Min Joo, Hye Won Lee, Woong-Yang Park. Elucidation of novel therapeutic targets for acute myeloid leukemias with RUNX1-RUNX1T1 fusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1723.

Suppressive effects of sunitinib on a TLR activation-induced cytokine storm

The cytokine storm includes a clinically heterogeneous set of life-threatening conditions that are manifested by extremely elevated serum cytokine levels and related symptoms (e.g., septic shock) and is devilishly mediated by Toll-like receptor (TLR) agonists in most situations. A tyrosine kinase inhibitor (TKIs), sunitinib, was screened in our group previously and showed antagonistic activity for cytokine release in a TLR7 stimulation model. In this paper, we further studied its mechanisms on interesting phenomena. In vitro, nearly all of the eleven TKIs decreased the TNF-α levels induced by the TLR7 agonist, especially sunitinib. Furthermore, sunitinib displayed potent inhibition of the cytokine levels triggered by several types of TLR ligands, including TLR3, TLR4, TLR7/8 and TLR9, in mouse spleen lymphocytes, mouse BMDCs and human PBMCs. The in vivo results showed that sunitinib efficiently depressed the LPS-induced cytokine storm, i.e., rapid and intense production of TNF-α and IL-6. Sunitinib further increased the survival time and decreased damage to mice. As for the immunosuppressive mechanisms of sunitinib, at least the PDGFR-activated ERK and p38 pathways were critical, although we could not rule out the possibility of other pathways being involved. In conclusion, our study demonstrated the inhibitory actions of TKIs on the cytokine storm induced by TLR ligands, primarily through PDGFR pathways, which could be potentially used to reduce cytokine storms in septic shock.

PDGFR-induced autocrine SDF-1 signaling in cancer cells promotes metastasis in advanced skin carcinoma

Advanced and undifferentiated skin squamous cell carcinomas (SCCs) exhibit aggressive growth and enhanced metastasis capability, which is associated in mice with an expansion of the cancer stem-like cell (CSC) population and with changes in the regulatory mechanisms that control the proliferation and invasion of these cells. Indeed, autocrine activation of PDGFRα induces CSC invasion and promotes distant metastasis in advanced SCCs. However, the mechanisms involved in this process were unclear. Here, we show that CSCs of mouse advanced SCCs (L-CSCs) express CXCR4 and CXCR7, both receptors of SDF-1. PDGFRα signaling induces SDF-1 expression and secretion, and the autocrine activation of this pathway in L-CSCs. Autocrine SDF-1/CXCR4 signaling induces L-CSC proliferation and survival, and mediates PDGFRα-induced invasion, promoting in vivo lung metastasis. Validation of these findings in patient samples of skin SCCs shows a strong correlation between the expression of SDF1, PDGFRA, and PDGFRB, which is upregulated, along CXCR4 in tumor cells of advanced SCCs. Furthermore, PDGFR regulates SDF-1 expression and inhibition of SDF-1/CXCR4 and PDGFR pathways blocks distant metastasis of human PD/S-SCCs. Our results indicate that functional crosstalk between PDGFR/SDF-1 signaling regulates tumor cell invasion and metastasis in human and mouse advanced SCCs, and suggest that CXCR4 and/or PDGFR inhibitors could be used to block metastasis of these aggressive tumors.

Molecular Characterization of Non-responders to Chemotherapy in Serous Ovarian Cancer.

Nearly one-third of patients with high-grade serous ovarian cancer (HGSC) do not respond to initial treatment with platinum-based therapy. Genomic and clinical characterization of these patients may lead to potential alternative therapies. Here, the objective is to classify non-responders into subsets using clinical and molecular features. Using patients from The Cancer Genome Atlas (TCGA) dataset with platinum-resistant or platinum-refractory HGSC, we performed a genome-wide unsupervised cluster analysis that integrated clinical data, gene copy number variations, gene somatic mutations, and DNA promoter methylation. Pathway enrichment analysis was performed for each cluster to identify the targetable processes. Following the unsupervised cluster analysis, three distinct clusters of non-responders emerged. Cluster 1 had overrepresentation of the stage IV disease and suboptimal debulking, under-expression of miRNAs and mRNAs, hypomethylated DNA, “loss of function” TP53 mutations, and the overexpression of genes in the PDGFR pathway. Cluster 2 had low miRNA expression, generalized hypermethylation, MUC17 mutations, and significant activation of the HIF-1 signaling pathway. Cluster 3 had more optimally cytoreduced stage III patients, overexpression of miRNAs, mixed methylation patterns, and “gain of function” TP53 mutations. However, the survival for all clusters was similar. Integration of genomic and clinical data from patients that do not respond to chemotherapy has identified different subgroups or clusters. Pathway analysis further identified the potential alternative therapeutic targets for each cluster.

Transcriptomic analysis of hepatocellular carcinoma reveals molecular features of disease progression and tumor immune biology

Hepatocellular carcinoma (HCC) develops in the context of chronic inflammatory liver disease and has an extremely poor prognosis. An immunosuppressive tumor microenvironment may contribute to therapeutic failure in metastatic HCC. Here, we identified unique molecular signatures pertaining to HCC disease progression and tumor immunity by analyzing genome-wide RNA-Seq data derived from HCC patient tumors and non-tumor cirrhotic tissues. Unsupervised clustering of gene expression data revealed a gradual suppression of local tumor immunity that coincided with disease progression, indicating an increasingly immunosuppressive tumor environment during HCC disease advancement. IHC examination of the spatial distribution of CD8+ T cells in tumors revealed distinct intra- and peri-tumoral subsets. Differential gene expression analysis revealed an 85-gene signature that was significantly upregulated in the peri-tumoral CD8+ T cell-excluded tumors. Notably, this signature was highly enriched with components of underlying extracellular matrix, fibrosis, and epithelial–mesenchymal transition (EMT). Further analysis condensed this signature to a core set of 23 genes that are associated with CD8+ T cell localization, and were prospectively validated in an independent cohort of HCC specimens. These findings suggest a potential association between elevated fibrosis, possibly modulated by TGF-β, PDGFR, SHH or Notch pathway, and the T cell-excluded immune phenotype. Indeed, targeting fibrosis using a TGF-β neutralizing antibody in the STAM™ model of murine HCC, we found that ameliorating the fibrotic environment could facilitate redistribution of CD8+ lymphocytes into tumors. Our results provide a strong rationale for utilizing immunotherapies in HCC earlier during treatment, potentially in combination with anti-fibrotic therapies.