PDGFRα Expression Research Articles

Integrating transcriptomics and metabolomics to reveal the protective effect and mechanism of Bushen Kangshuai Granules on the elderly people.

Background: Aging is characterized by a decline in the adaptability and resistance of the body. In this study, Bushen Kangshuai Granules (BKG), as a kind of Chinese herbal formula, was developed and shown to alleviate aging-related symptoms. Methods: Self-controlled study combined with RNA-seq and metabonomics were used to expound the efficacy and safety of BKG and revealed the regulation mechanism of BKG treating aging. In vitro experiments were used to confirm the analytical results. The aging cell model of AC16 cells were treated with D-galactose. The RT-qPCR was used to detect the impact of BKG on telomere length. The DCFH-DA staining was used for detecting intracellular ROS. The targeted signaling pathway was selected and verified using Western blot. Results: After 8weeks of treatment, BKG significantly reduced SOD level (p = 0.046), TCM aging symptoms (p < 0.001) and TNF-α level (p = 0.044) in the elderly participants. High-throughput sequencing showed that BKG reversed the expression of 70 and 79 age-related genes and metabolites, respectively. Further enrichment analysis indicated that BKG downregulated the PI3K-AKT signaling pathway, extracellular matrix (ECM)-receptor interaction, and Rap1 signaling pathway, while up-regulating sphingolipid metabolism. The results of in vitro experiments show that, after D-gal treatment, the viability and telomere length of AC16 cells significantly decreased (p < 0.05), while the expression of ROS increased (p < 0.05), BKG significantly increased the telomere length of AC16 cells and reduced the level of ROS expression (p < 0.05). In addition, BKG decreased the expression of THBS1, PDGFRA, and EPS8L1(p < 0.05), consistent with the RNA-seq results. Our results also showed that BKG affects PI3K-AKT signaling pathway. Conclusion: BKG can significantly improve aging-related symptoms and increase SOD levels, which may be associated with the reversal of the expression of various aging-related genes. The PI3K-AKT signaling pathway and sphingolipid metabolism may be potential mechanisms underlying BKG anti-aging effects.

The Distant Molecular Effects on the Brain by Cancer Treatment.

Cancer survivors experience cancer-related cognitive impairment (CRCI) secondary to treatment. Chemotherapy and radiation therapy independently contribute to cognitive dysfunction; however, the underlying mechanisms leading to dysfunction remain unclear. We characterized brain gene expression changes in a mouse model of CRCI to identify the mechanistic underpinnings. Eleven-to-twelve-week-old SKH1 mice were treated with doxorubicin (DOX), hindlimb radiation (RT), concurrent hindlimb radiation and doxorubicin (DOX-RT), or no treatment (control). Sixteen days following treatment, gene expression was measured from murine brains using the NanoString nCounter® glial profiling panel. Gene expression was normalized and compared between groups. No two groups shared the same expression pattern, and only Gnb1 and Srpr were upregulated in multiple treatment groups. Brains from DOX-treated mice had upregulated Atf2, Atp5b, Gnb1, Rad23b, and Srpr and downregulated Sirt5 expression compared to control brains. Brains from RT-treated mice demonstrated increased Abcg2 and Fgf2 and decreased C1qa and C1qb expression compared to control brains. Brains from DOX-RT-treated mice had upregulated Adar, E2f3, Erlec1, Gnb1, Srpr, Vim, and Pdgfra expression and downregulated Rock2 and Inpp5f expression compared to control brains. The gene expression changes demonstrated here highlight roles for neuronal transmission and oxidative stress in the pathogenesis of doxorubicin-related CRCI and inflammation in RT-related CRCI.

Nuclear Glycoprotein A Repetitions Predominant (GARP) Is a Common Trait of Glioblastoma Stem-like Cells and Correlates with Poor Survival in Glioblastoma Patients.

Glioblastoma (GB) is notoriously resistant to therapy. GB genesis and progression are driven by glioblastoma stem-like cells (GSCs). One goal for improving treatment efficacy and patient outcomes is targeting GSCs. Currently, there are no universal markers for GSCs. Glycoprotein A repetitions predominant (GARP), an anti-inflammatory protein expressed by activated regulatory T cells, was identified as a possible marker for GSCs. This study evaluated GARP for the detection of human GSCs utilizing a multidimensional experimental design that replicated several features of GB: (1) intratumoral heterogeneity, (2) cellular hierarchy (GSCs with varied degrees of self-renewal and differentiation), and (3) longitudinal GSC evolution during GB recurrence (GSCs from patient-matched newly diagnosed and recurrent GB). Our results indicate that GARP is expressed by GSCs across various cellular states and disease stages. GSCs with an increased GARP expression had reduced self-renewal but no alterations in proliferative capacity or differentiation commitment. Rather, GARP correlated inversely with the expression of GFAP and PDGFR-α, markers of astrocyte or oligodendrocyte differentiation. GARP had an abnormal nuclear localization (GARPNU+) in GSCs and was negatively associated with patient survival. The uniformity of GARP/GARPNU+ expression across different types of GSCs suggests a potential use of GARP as a marker to identify GSCs.

Functional and Transcriptomic Characterization of Postnatal Maturation of ENS and SIP Syncytium in Mice Colon.

The interplay of the enteric nervous system (ENS) and SIP syncytium (smooth muscle cells-interstitial cells of Cajal-PDGFRα+ cells) plays an important role in the regulation of gastrointestinal (GI) motility. This study aimed to investigate the dynamic regulatory mechanisms of the ENS-SIP system on colon motility during postnatal development. Colonic samples of postnatal 1-week-old (PW1), 3-week-old (PW3), and 5-week-old (PW5) mice were characterized by RNA sequencing, qPCR, Western blotting, isometric force recordings (IFR), and colonic motor complex (CMC) force measurements. Our study showed that the transcriptional expression of Pdgfrα, c-Kit, P2ry1, Nos1, and Slc18a3, and the protein expression of nNOS, c-Kit, and ANO1 significantly increased with age from PW1 to PW5. In PW1 and PW3 mice, colonic migrating movement was not fully developed. In PW5 mice, rhythmic CMCs were recorded, similar to the CMC pattern described previously in adult mice. The inhibition of nNOS revealed excitatory and non-propulsive responses which are normally suppressed due to ongoing nitrergic inhibition. During postnatal development, molecular data demonstrated the establishment and expansion of ICC and PDGFRα+ cells, along with nitrergic and cholinergic nerves and purinergic receptors. Our findings are important for understanding the role of the SIP syncytium in generating and establishing CMCs in postnatal, developing murine colons.

Dipeptidyl Peptidase 4 Stimulation Induces Adipogenesis-Related Gene Expression of Adipose Stromal Cells.

Adipogenesis has emerged as a new therapeutic target for regulating metabolism and achieving anti-inflammatory and anti-atherosclerotic effects via the release of adiponectin. However, at present, the effects and mechanism of action of dipeptidyl peptidase 4 (DPP4) stimulation on adiponectin production and adipogenesis have not been clarified. Here, we investigated the effects of DPP4 stimulation with monocyte chemoattractant protein-1 (MCP-1) on platelet-derived growth factor receptor alpha (PDGFRα) expression in adipose tissue and blood adiponectin levels. Stromal vascular fractions (SVFs) purified from human subcutaneous adipose tissue and inguinal adipose tissue of obese and diabetic (Leprdb/db) mice were treated with 50 ng of MCP-1 and plasma from control (Lepr+/+) mice supplemented with 10 ng or 50 ng of MCP-1. Treatment of SVFs from human subcutaneous adipose tissues with 50 ng of MCP-1 significantly increased AdipoQ, DPP4, peroxisome proliferator-activated receptor gamma (PPARγ), fatty-acid-binding protein (FABP4), and SERBF1 mRNA expression. MCP-1-supplemented plasma increased adiponectin, CCAAT-Enhancer-binding protein alpha (C/EBPα), DPP4, IL-33, and PDGFRα mRNA expression and adiponectin and DPP4 protein expression, while decreasing the expression of IL-10 mRNA in SVFs compared with the levels in the plasma treatment group. MCP-1-supplemented plasma was shown to increase PPARγ, PPARγ2, adiponectin, DPP4, and FABP4 and decrease IL-10 mRNA expression in PDGFRα cells from adipose tissue. Meanwhile, MCP-1-supplemented plasma increased MCP-1, PDGFRα, TNFα, adiponectin, and IL-1β and decreased IL-10 and FOXP3 mRNA expression in DPP4 cells. Moreover, the injection of MCP-1-supplemented plasma into adipose tissue increased the proportion of DPP4+ cells among PDGFRα+ cells from adipose tissue and plasma adiponectin levels of Leprdb/db mice compared with the levels in the plasma injection group. Our results demonstrate that DPP4+ cells are important adipose progenitor cells. Stimulation of DPP4 with MCP-1 increases adipogenesis-related gene expression and the population of DPP4+ cells among PDGFRα+ cells in SVFs and blood adiponectin levels. DPP4 stimulation could be a novel therapy to increase local adipogenesis and systemic adiponectin levels.

Generation and characterization of PDGFRα-GFP knock-in mice for visualization of PDGFRα+ fibroblasts in vivo

The platelet-derived growth factor (PDGF) and its receptor, PDGFRα, are critical for tissue development and injury repair. To track PDGFRα-expressing cells in vivo, we generated a knock-in mouse line that expresses green fluorescent protein (GFP) under the control of the PDGFRα promoter. This genetic tool enabled us to detect PDGFRα expression in various organs during both neonatal and adult stages. Additionally, we confirmed the correlation between endogenous PDGFRα and transgenic PDGFRα expression using mouse injury models, showing the potential of this genetic reporter for studying PDGFRα-mediated signaling pathways and developing therapeutic strategies. Overall, the PDGFRα-GFP knock-in mouse line serves as a valuable tool for investigating the biology of PDGFRα and its role in normal development and disease.

AML with t(4;12)(q12;p13): A Detailed Genomic and Transcriptomic Analysis Reveals Genomic Breakpoint Heterogeneity, Absence of Pdgfra Fusion Transcripts and Presence of Pdgfra Overexpression in a Subset of Cases

Background: Gene fusions are a frequent event in acute myeloid leukaemia (AML). It is estimated that about 40% of AML patients harbour a cytogenetic abnormality resulting in a gene fusion. However, AML with t(4;12)(q12;p13) is very rare, but it is often accompanied by an aggressive clinical course and a poor prognosis. As frequently indicated by fluorescence in situ hybridization analyses (FISH), patients are assumed to harbor an ETV6::PDGFRA fusion, which impacts the choice of therapy, as these patients can be treated with tyrosine kinase inhibitors. The genomic region on chromosome 4 involved in ETV6 rearrangements is densly packed with genes in close spatial proximity. Therefore, the resolution of chromosome banding analysis and also FISH is not sufficient to fully elucidate the fusion partners involved. Recent studies have pointed out that many of these patients do not harbor an ETV6::PDGFRA fusion but other rather unknown fusion partners have been elucidated. Aim: (1) Evaluation of the genomic breakpoints in patients with t(4;12)(q12;p13) ETV6-rearranged AML; (2) determine the alteration of PDGFRA expression in patients with t(4;12). Patients and Methods: 10 patients (F: 4, M: 6; median age: 63 [44-82] years) with a t(4;12)(q12;p13) indicated by FISH and chromosome analysis were included. Samples were analysed by whole genome and whole transcriptome sequencing (WGS/WTS). Sequencing was performed on NovaSeq instrument (Illumina). Fusions were called with Arriba, STAR-Fusion and Manta. For gene expression analysis WTS data were log2 transformed and normalized with the trimmed mean of M-values (TMM) method. Results: Based on chromosome analysis and FISH patients showed break events in 4q12 and 12p13. In all cases a FISH signal constellation typical for an ETV6 rearrangement was observed. The mutation spectrum for each patient of the most commonly mutated genes in AML was examined by use of WGS data. The median number of mutations was 3, with mutations in ASXL1 being the most common (6 patients) and none of the patients featuring mutations in the PDGFRA gene. WTS analysis revealed in-frame fusions in 6 of 10 patients involving genes located on chromosomes 4 and 12 (4 patients with ETV6::CHIC2, 1 patients with ETV6::SCFD2, 1 patient with ETV6::ADAMTS3). None of the 10 examined patients showed a gene fusion, or a fusion transcript involving PDGFRA. Based on WTS data, 7 of the 10 patients showed a PDGFRA overexpression. WGS breakpoint analysis showed that genetic breakpoints on chromosome 12 cluster in introns 1 (4 patients) and 2 (6 patients) of the ETV6 gene. In contrast chromosome breaks on chromosome 4 are heterogeneously distributed in the 4q12 region. Breakpoint comparison indicated that patients with chromosome breaks in a range from chr4:54,804,230-54,962,206 (7 patients) display PDGFRA overexpression. In contrast patients with chromosome breaks further downstream chr4:55,021,511-73,409,362 (3 patients) did not exhibit an increased PDGFRA expression. This suggests an ectopic expression of PDGFRA triggered by the translocation and independent of the existence of an in-frame fusion transcript. A chromosome break in the range of chr4:54,804,230-54,962,206 (seen in 7 patients) might result in the inactivation of a CTCF binding site (chr4:54,796,345-54,796,360) upstream of these chromosome breaks that is already known to induce PDGFRA overexpression when disrupted. Conclusions: Analysis of AML with t(4;12)(q12;p13) translocation by WGS and WTS provides detailed information that separates two subsets distinguished by distinct breakpoint cluster regions on 4q12 and PDGFRA gene expression. The clinical impact of increased PDGFRA gene expression on response to TKI and prognosis as well as the presence of additional fusion transcripts has to be evaluated in further studies.

Astrocyte lipocalin-2 modestly effects disease severity in a mouse model of multiple sclerosis while reducing mature oligodendrocyte protein and mRNA expression

Roles for lipocalin-2 (LCN2, also referred to as neutrophil gelatinase associated lipocalin, NGAL) in the progression of disease in multiple sclerosis and its animal models have been reported; however, the importance of astrocyte-derived LCN2, a major source of LCN2, have not been defined. We found that clinical scores in experimental autoimmune encephalomyelitis (EAE) were modestly delayed in mice with conditional knockout of LCN2 from astrocytes, associated with a small decrease in astrocyte GFAP expression. Immunostaining and qPCR of spinal cord samples showed decreased oligodendrocyte proteolipid protein and transcription factor Olig2 expression, but no changes in PDGFRα expression. These results suggest astrocyte LCN2 contributes to early events in EAE and reduces damage to mature oligodendrocytes at later times.

Abstract PO-093: Crenolanib improves PD-1 response and overall survival in immune checkpoint inhibitor resistant murine models of oral squamous cell carcinoma

Abstract Background: Present line indications for pembrolizumab are for patients with metastatic or unresectable recurrent (i.e. incurable) head and neck squamous cell carcinoma (HNSCC). However, only a minority of patients on immunotherapy will realize a durable survival benefit because &amp;gt;80% of patients with metastatic HNSCC do not respond to PD-1 blockade. Tumor microenvironment mesenchymal stem cells (MSCs) and cancer associated fibroblasts (CAFs) have been reported significantly contribute to chemotherapy and radiation resistance. Moreover, MSCs have been shown to contribute to an immunosuppressive tumor microenvironment by upregulating PD-L1 in breast cancer models. We have previously shown crenolanib improves MSC mediated cisplatin resistance in vitro through modulation of MSC-mediated activation of AKT signaling, therefore we hypothesize that targeting MSCs may be of therapeutic benefit alone and in combination with anti-PD1 immunotherapy. Methods: Oral cancer was induced in the buccal space of C56/BL6 mice with the murine oral cancer cell lines MOC1 or PD-1 resistant cell line, MOC2. When tumors reached approximately 5 × 5 mm, mice were treated with 15 mg/kg (low dose) or 30 mg/kg crenolanib (high dose) for five consecutive days over 3 weeks. Tissue were harvested for immunohistochemistry and immunofluorescence analysis and ex vivo cell cultures prepared for analysis by western immunoblotting and flow cytometry. Results: There was a significant reduction in tumor volume in mice bearing MOC1 and MOC2 tumors (p&amp;lt;0.03) treated with either low or high dose crenolanib compared to vehicle control. Overall survival was also significantly improved in mice bearing MOC1 tumors treated with high dose crenolanib compared to mice treated with vehicle control (p&amp;lt;0.04) and approached significance in MOC2 mice treated with low dose crenolanib. Tumor sections were imaged by immunofluorescence microscopy. There was a decrease in expression of PDGFR-α on MOC1 tumor cells and α-SMA on tumor microenvironment stromal cells in mice treated with crenolanib compared to vehicle control, suggesting crenolanib targets both cell types. There was a significant reduction in tumor volume (p&amp;lt;0.0001) and improved overall survival (p&amp;lt;0.0004) in mice bearing MOC2 tumors treated with combination crenolanib plus pembrolizumab compared to vehicle plus pembrolizumab. Conclusions: Preliminary in vivo data suggests crenolanib may be efficacious when used in combination with anti-PD1 immunotherapy by inhibiting the immunosuppressive effects of tumor microenvironment MSCs. Citation Format: Xiangfeng Shen, Katherine Gonzalez, Rico Castillo, Ashlyn Rickard, Yvonne Mowery, Tammara Watts. Crenolanib improves PD-1 response and overall survival in immune checkpoint inhibitor resistant murine models of oral squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-093.

Autologous Platelet Lysate Is an Alternative to Fetal Bovine Serum for Canine Adipose-Derived Mesenchymal Stem Cell Culture and Differentiation.

The use of fetal bovine serum (FBS) in regenerative medicine raises serious ethical and scientific concerns. We have cultured and differentiated the canine mesenchymal stem cells (cMSCs) in five different media combinations of autologous platelet lysate (A-PL) and FBS; consisting of 0% A-PL and 10% FBS (M-1), 2.5% A-PL and 7.5% FBS (M-2), 5% A-PL and 5% FBS (M-3), 7.5% A-PL and 2.5% FBS (M-4), and 10% A-PL and 0% FBS (M-5). The cMSCs were evaluated for their doubling time, differentiation efficiency, and expression of CD73, CD90, CD105, and PDGFRα. The mRNA expression of NT5E, THY1, ENG, PPARγ, FABP4, FAS, SP7, BGLAP, and SPP1 was also assessed. The results indicated non-significant differences in cellular proliferation/viability; positive expression of surface markers, and PDGFRα with substantial adipo/osteogenic differentiation. The expression of adipogenic (PPARγ, FABP4, FAS), and osteogenic (SP7, BGLAP, SPP1) genes were higher (p < 0.05) in the M5 group. In conclusion, A-PL in cMSCs culture did not negatively affect cellular proliferation and viability but also enhanced their genetic potential for multilineage differentiation. Our results indicate that A-PL can be used as an alternative for FBS to develop potent cMSCs under good manufacturing practice protocol for regenerative medicine.

Myeloid Cell-Specific Deletion of PDGFR-α Promotes Dysbiotic Intestinal Microbiota and thus Increased Colitis Susceptibility.

The incidence of inflammatory bowel diseases [IBD] is steadily increasing, and thus the identification of new targets to improve therapy is a major goal. Growth factors of the PDGF family and their receptors are expressed early in intestinal development and are found in mononuclear cells and macrophages in adult tissues. Macrophages play a distinct role in the pathogenesis of IBD since their function is crucial to maintaining tolerance. We aimed to study the role of myeloid expression of PDGFR-α in mediating intestinal homeostasis in mouse IBD and infectious models. Our results show that loss of myeloid PDGFR-α increases susceptibility to dextran saline sulphate-induced colitis. Accordingly, LysM-PDGFR-α-/- mice showed higher colitis scores, and reduced levels of anti-inflammatory macrophages compared to control mice. This effect was mediated via a pro-colitogenic microbiota, which developed in the absence of myeloid PDGFR-α and caused increased colitis susceptibility in gnotobiotic mice upon faecal microbiota transplantation compared to controls. Furthermore, LysM-PDGFR-α-/- mice had a leaky gut, accompanied by impaired phagocytosis, resulting in a severe barrier defect. Taken together, our results indicate a protective role for myeloid PDGFR-α in maintaining gut homeostasis by promoting a protective intestinal microbiota and providing an anti-inflammatory macrophage phenotype.

Alterations of oligodendrocyte progenitor cells (OPCs) with survival time in humans following high impact brain trauma

BackgroundAs there is a lack of comprehensive literature regarding the molecular environment of the human brain emphasizing on oligodendrocyte progenitor cells (OPCs) following high impact brain trauma. The protagonist of OPCs post severe traumatic brain injury (sTBI) provides a significant thrust towards estimating time elapsed since trauma as well as developing novel therapeutic approaches. The present study was carried out to study post trauma alterations pertaining to myelin sheath and oligodendrocyte response with survival time. Materials and methodsIn the present study, victims (both male and female) of sTBI (n = 64) were recruited and contrasted with age and gender matched controls (n = 12). Post mortem brain samples from corpus callosum and grey white matter interface were collected during autopsy examination. Extent of myelin degradation and response of OPC markers Olig-2 and PDGFR-α were evaluated using immunohistochemistry and qRT-PCR. STATA 14.0 statistical software was used for data analysis with P-value<0.05 considered statistically significant. ResultsTimewise qualitative correlation with extent of demyelination performed using LFB-PAS/IHC-MBP, IHC Olig-2 and mRNA expression revealed tendency towards remyelination in both corpus callosum and grey white matter interface. Number of Olig-2 positive cells was significantly higher in sTBI group as compared to control group (P-value: 0.0001). Moreover, mRNA expression studies of Olig-2 showed significant upregulation in sTBI patients. mRNA expression of Olig-2 and PDGFR-α in sTBI patients showed significant variation with respect to survival time (p value:0.0001). ConclusionDetailed assessment of post TBI changes implementing various immunohistochemical and molecular techniques shall potentially reveal intriguing and important inferences in medicolegal practices and neurotherapeutics.

HGG-19. CLINICAL RESPONSE TO THE PDGFRA/KIT INHIBITOR AVAPRITINIB IN PEDIATRIC AND YOUNG ADULT HIGH-GRADE GLIOMA PATIENTS WITH H3K27M OR PDGFRA GENOMIC ALTERATIONS

Abstract PDGFRA is commonly altered in pediatric and young adult high-grade gliomas (pHGGs) including histone 3 lysine 27-mutated diffuse midline gliomas (H3K27M DMG), a fatal disease with no current options for cure. We performed comprehensive genomic and transcriptomic analyses of n=259 pediatric high-grade glioma cases which revealed PDGFRA genomic alterations in ~15% of patients. H3K27M DMGs had significantly higher PDGFRA expression compared to H3 wild-type tumors regardless of genomic alteration. Tumors with PDGFRA gene amplification demonstrated significantly elevated PDGFRA expression in both H3K27M DMGs and H3 wild-type pHGGs relative to tumors with wild-type or point mutated PDGFRA. We tested a range of PDGFRA inhibitors against a panel of patient derived pediatric H3K27M DMG, pHGG, and adult HGG. Amongst the inhibitors tested, avapritinib, a potent small molecule inhibitor with relatively selective activity against both wild-type and mutant PDGFRA showed potent toxicity against a wide array of pediatric and adult cell lines. This molecule also demonstrated supra-micromolar blood brain barrier penetration in pre-clinical in vivo models, and demonstrated significant decrease in tumor growth and improved survival in orthotopic mouse xenograft models. Finally, building on this preclinical activity, we report the first clinical experience using avapritinib in eight pediatric and young adult patients with high-grade glioma (H3K27M DMG and/or PDGFRA altered). Avapritinib usage showed no significant acute toxicities within this patient cohort. Most importantly, our preliminary data reveal radiographic response evaluated by RAPNO criteria in 50% of patients, a striking outcome rarely seen in this patient population. In summary, we report that avapritinib, a selective, CNS penetrant small molecule inhibitor of PDGFRA has potent activity in preclinical models and produced promising clinical responses with good tolerability in pediatric and young adult patients with high-grade glioma, suggesting a promising role for avapritinib therapy in pediatric high grade glioma.

DIPG-28. IDENTIFICATION AND TARGETING OF METABOLIC VULNERABILITIES IN COMBINATION WITH PDGFRA-INHIBITION IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas (DMG) are driven by self-renewing, stem-like glioma cells stalled in an oligodendrocyte precursor cell (OPC)-like state that are characterized by high expression of PDGFRA. This lineage overexpression of PDGFRA, together with commonly found genomic amplifications and mutations in PDGFRA, has been shown to cooperate with H3-K27M mutations in tumor development. Therefore, targeting PDGFRA has long been proposed as a potential treatment strategy for DMG. Our previous work revealed that Avapritinib, a small molecule inhibitor of PDGFRA, is highly effective in DMG cells in vitro and in vivo. Following these preclinical findings, nine DMG patients were treated with Avapritinib for the first time. While 50% of all treated patients experienced a strong clinical response from Avapritinib, tumor progression and treatment escape were eventually observed in most patients. Here, we investigated which mechanisms DMG cells use to escape Avapritinib treatment and explored how these resistance mechanisms can be therapeutically exploited. Transcriptional analysis of a DMG cell line previously identified as Avapritinib-resistant revealed an upregulation of genes associated with fatty acid metabolism and oxidative phosphorylation (OXPHOS) following treatment with Avapritinib. Functional assays confirmed elevated OXPHOS in Avapritinib-resistant cells with significant (i) increase in mitochondrial energy transduction, (ii) increase in palmitate- (a product of fatty acid metabolism) driven oxygen consumption rate and (iii) greater incorporation of palmitate-derived carbons into the tricarboxylic acid cycle. These metabolic changes could be genetically reproduced in the same cell line after CRISPR-Cas9-mediated knockout of PDGFRA. To determine which therapies could target the dependency of Avapritinib-resistant cells on OXPHOS and fatty acid metabolism, we performed a metabolic drug screening and identified two metabolic drugs to have synergistic cytotoxic effects with Avapritinib. Together, we revealed distinct metabolic changes in Avapritinib-resistant DMG cells and showed that targeting these metabolic vulnerabilities might further increase the clinical benefit of Avapritinib.

Apposition of Fibroblasts With Metaplastic Gastric Cells Promotes Dysplastic Transition

Elements of field cancerization, including atrophic gastritis, metaplasia, and dysplasia, promote gastric cancer development in association with chronic inflammation. However, it remains unclear how stroma changes during carcinogenesis and how the stroma contributes to progression of gastric preneoplasia. Here we investigated heterogeneity of fibroblasts, one of the most important elements in the stroma, and their roles in neoplastic transformation of metaplasia. We used single-cell transcriptomics to evaluate the cellular heterogeneity of mucosal cells from patients with gastric cancer. Tissue sections from the same cohort and tissue microarrays were used to identify the geographical distribution of distinct fibroblast subsets. We further evaluated the role of fibroblasts from pathologic mucosa in dysplastic progression of metaplastic cells using patient-derived metaplastic gastroids and fibroblasts. We identified 4 subsets of fibroblasts within stromal cells defined by the differential expression of PDGFRA, FBLN2, ACTA2, or PDGFRB. Each subset was distributed distinctively throughout stomach tissues with different proportions at each pathologic stage. The PDGFRα+ subset expanded in metaplasia andcancer compared with normal, maintaining a close proximitywith the epithelial compartment. Co-culture of metaplasia- or cancer-derived fibroblasts with gastroids showing the characteristics of spasmolytic polypeptide-expressing metaplasia-induced disordered growth, loss of metaplastic markers, and increases in markers of dysplasia. Culture of metaplastic gastroids with conditioned media from metaplasia- or cancer-derived fibroblasts also promoted dysplastic transition. These findings indicate that fibroblast associations with metaplastic epithelial cells can facilitate direct transition of metaplastic spasmolytic polypeptide-expressing metaplasia cell lineages into dysplastic lineages.

Mechanism study of platelet derived growth factor receptor alpha on the bidirectional differentiation regulation of glioma-associated oncogene homolog 1-positive mesenchymal stem cells in mice

Objective: To investigate the role of platelet derived growth factor receptor alpha (PDGFRα) on bidirectional differentiation of glioma-associated oncogene homolog 1-positive mesenchymal stem cells (Gli1+-MSC). Methods: Breeding double reporter transgenic mice ROSAmT/mG/Gli1-CreERt2/PDGFRαfl (Experimental group) and ROSAmT/mG/Gli1-CreERt2 (Control group), 20 mice in each of the two groups at four weeks of age were selected, MSC were isolated from the mouse aortic epithelium. After tamoxifen inducement, the two groups of Gli1+-MSC were screened by green fluorescent protein (GFP) labeling and flow cytometry sorting. PDGFRα was conditionally knocked out in the experimental group, and the control group Gli1+-MSC expressed PDGFRα normally. The two groups of Gli1+-MSC were subjected to adipogenic induction and fibrogenic induction, the Western blotting was performed to detect PDGFRα, adipocyte markers [perilipin and CCAAT/enhancer binding protein alpha (C/EBPα)] and fibrogenic markers [alpha smooth muscle actin (α-SMA) and fibroblast-specific protein 1 (FSP-1)] and semi-quantitative analysis was performed. The degree of cellular adipose differentiation after bidirectional induction of Gli1+-MSC in both groups was observed by oil red O staining and analyzed semi-quantitatively. Results: After tamoxifen induction, Gli1+-MSC could be accurately isolated from flow cytometry by GFP labeling. Via adipogenic differentiation, the expression of PDGFRα in the experimental group (0.017±0.002) was significantly lower than that in the control group (0.184±0.012) (t=25.48，P=0.002). The protein expressions of perilipin (3.138±0.414) and C/EBPα (3.565±0.289) were significantly higher than those in the control group (2.312±0.218 and 2.179±0.103, respectively) (t=6.21，P=0.025；t=6.69，P=0.022). Thus, the knock-out of PDGFRα enhanced the adipogenic differentiation ability of Gli1+-MSC. After fibrogenesis induction, the protein expressions of PDGFRα, α-SMA and FSP-1 in the experimental group (0.030±0.001, 0.932±0.177 and 0.276±0.020, respectively) were significantly lower than those in the control group (0.439±0.006, 1.352±0.170 and 0.835±0.097, respectively) (t=149.40, P<0.001; t=66.38,P<0.001; t=11.41,P<0.08). This suggested that the knock-out of PDGFRα significantly inhibited Gli1+-MSC differentiation toward fibroblasts. After bidirectional induction, significantly less adipocyte formation was seen in the control group and more in the experimental group. Quantitative analysis showed that the amount of oil red O staining in the experimental group (0.461±0.042) was significantly higher than that in the control group (0.017±0.007) after bidirectional induction (t=23.20, P<0.01). Conclusions: PDGFRα plays an important role in the regulation of bidirectional differentiation of vascular adventitial Gli1+-MSC.

PDGFRα promotes organ resistance to immune-mediated damage

Abstract Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that exhibits great clinical heterogeneity. Development and clinical expression of autoimmune diseases is strongly influenced by genetic variants. The single nucleotide polymorphism (SNP) rs1364989, located close to PDGFRA, has been associated with the development of lupus nephritis through an unknown mechanism. Because PDGF, through its α and β receptors, regulates tissue response to inflammation, we hypothesized that rs1364989 could modulate the behavior of renal cells in the context of immune-mediated glomerulonephritis. To determine whether rs1364989 affects the expression of PDGFRα, we analyzed umbilical cord mesenchymal cells from carriers of risk and protective alleles. We observed that the presence of the risk allele was associated with reduced PDGFRα expression. Next, we analyzed whether inflammatory mediators could modulate PDGFRα expression in in vitro and in vivo settings. We documented that its levels increase during acute inflammation in a murine model of glomerulonephritis. In vitro, this effect could be reproduced by IL-17. Absence of PDGFRα increased kidney damage during immune complex-induced glomerulonephritis. An analogous effect was observed in joints of mice with collagen-induced arthritis. Our results demonstrate that the SLE-associated allele of rs1364989 reduces the expression of PDGFRα and that this receptor may activate a pathway that protects tissues in response to local inflammation. These findings highlight the importance of tissue resilience as a factor that can protect an organ from inflammatory damage in the setting of autoimmunity. This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT), Mexico

Abstract 3479: Modeling epigenetic lesions that cause gliomas

Abstract Epigenetic lesions that disrupt gene regulatory elements and expression are increasingly recognized as potential drivers of human cancers. However, we currently lack the in vitro and in vivo models required to functionally validate such lesions and their tumorigenic impact. Here we model aberrations that arise in Isocitrate Dehydrogenase (IDH) mutant lower-grade gliomas, which exhibit profound DNA hypermethylation as a direct consequence of mutant IDH. DNA hypermethylation may promote gliomagenesis by silencing tumor suppressor genes or, alternatively, by activating proto-oncogenes through disruption of CCCTC-binding factor (CTCF) insulators. CTCF insulator sites define the three-dimensional shape of the genome by dictating the boundaries of topologically associated domains (TADs). Enhancers and promoters can interact when located in the same TAD but are restricted from interacting across different TADs. In IDH mutant gliomas, CpG sites around CTCF binding sites are frequently methylated, effectively compromising CTCF binding and thus TAD organization, allowing for cross-TAD interactions and aberrant activation of genes. We discovered a CTCF insulator downstream of the PDGFRA proto-oncogene that is recurrently disrupted in IDH mutant gliomas. We demonstrate that disruption of the syntenic insulator in mouse oligodendrocyte progenitor cells (OPCs) allows an OPC-specific enhancer to contact and induce Pdgfra, thereby increasing proliferation. In contrast, insulator disruption did not affect Pdgfra expression in neural progenitor cells (NPCs), which lack the enhancer. We also model a second recurrent epigenetic lesion in IDH mutant gliomas, the methylation-dependent silencing of the CDKN2A tumor suppressor. We show that inactivation of Cdkn2a/p19ARF by de novo promoter methylation or mutation drives OPC proliferation and cooperates with Pdgfra insulator loss. Finally, we use lentiviruses to coordinately inactivate the Pdgfra insulator and Cdkn2a in mouse corpus callosum, resulting in low-grade gliomagenesis in vivo with histological features reminiscent of human IDH mutant gliomas. Our study recapitulates recurrent epigenetic lesions in mouse models and demonstrates that the combination of Pdgfra activation and Cdkn2a silencing can transform OPCs in vitro and drive gliomagenesis in vivo. Citation Format: Gilbert J. Rahme, Nauman M. Javed, Kaitlyn L. Puorro, Shouhui Xin, Volker Hovestadt, Sarah E. Johnstone, Bradley E. Bernstein. Modeling epigenetic lesions that cause gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3479.

Abstract 5719: Clinical response to the PDGFRα inhibitor avapritinib in high-grade glioma patients

Abstract PDGFRA has been shown to be commonly altered in high-grade gliomas (HGGs), including histone 3 lysine 27-mutated diffuse midline gliomas (H3K27M DMG), a disease with almost no long-term survivors. Here, we performed comprehensive genomic and transcriptomic analysis of 260 high-grade glioma cases, which revealed PDGFRA genomic alterations (mutations and/or amplifications) in 13% of patients. H3K27M DMGs had significantly higher PDGFRA expression compared to H3 wild-type tumors, and PDGFRA gene amplification resulted in even higher expression levels in H3K27M DMGs as well as H3 wild-type HGGs. We tested a panel of patient- derived pHGG/H3K27M DMG models against a range of PDGFRA inhibitors, including avapritinib, a potent small molecule inhibitor with relatively selective activity against both wild-type and mutant PDGFRA. Avapritinib showed supra-micromolar blood-brain barrier penetration in our pre-clinical models and demonstrated significant survival impact in an aggressive patient-derived H3K27M DMG mouse xenograft model. Finally, building on this preclinical activity, we report here the first clinical experience using avapritinib in eight pediatric and young adult patients with high-grade glioma (H3K27M DMG and/or PDGFRA altered). Avapritinib has thus far been well tolerated with no significant acute toxicities. Most importantly, our preliminary data reveal radiographic response evaluated by RAPNO criteria in 50% of patients, a striking outcome rarely seen in this patient population. In summary, we report that avapritinib is a selective, CNS-penetrant small molecule inhibitor of PDGFRA that shows potent activity in preclinical models and produces promising clinical responses with good tolerability in patients with high-grade glioma. This suggests a promising role for avapritinib therapy in this population with previously dismal outcomes. Citation Format: Lisa Mayr, Maria Trissal, Kallen Schwark, Jenna Labelle, Andrew Groves, Julia Furtner-Srajer, Jeffrey Supko, Liesa Weiler-Wichtl, Olivia Hack, Jacob Rozowsky, Joana G. Marques, Eshini Pandatharatna, Ulrike Leiss, Verena Rosenmayr, Frank Dubois, Noah F. Greenwald, Sibylle Madlener, Armin S. Guntner, Hana Pálová, Natalia Stepien, Daniela Lötsch-Gojo, Christian Dorfer, Karin Dieckmann, Andreas Peyrl, Amedeo A. Azizi, Alicia Baumgartner, Ondřej Slabý, Petra Pokorná, Pratiti Bandopadhayay, Rameen Beroukhim, Keith Ligon, Christof Kramm, Annika Bronsema, Simon Bailey, Ana Guerreiro Stücklin, Sabine Mueller, David T. Jones, Natalie Jäger, Jaroslav Štěrba, Leonhard Müllauer, Christine Haberler, Chandan Kumar-Sinha, Arul Chinnaiyan, Rajen Mody, Mary Skrypek, Nina Martinez, Daniel C. Bowers, Carl Koschmann, Johannes Gojo, Mariella Filbin. Clinical response to the PDGFRα inhibitor avapritinib in high-grade glioma patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5719.

Development of myelin in fetal and postnatal neocortex of the pig, the European wild boar Sus scrofa

Myelination of the neocortex of altricial species is mostly a postnatal event, and the appearance of myelin has been associated with the end of the critical period for ocular dominance plasticity in rodent visual cortex. Due to their precocality, ungulates may tell a different story. Here, we analyzed the development of PDGFRα positive oligodendrocyte precursor cells and expression of myelin proteins in the laminar compartments of fetal and postnatal porcine cortex from E45 onwards. Precursor cell density initially increased and then decreased but remained present at P90. MAG and MBP staining were detectable at E70 in subventricular zone and deep white matter, ascending into gyral white matter at E85, and into the gray matter and marginal zone at E100 (birth in pig at E114). Protein blots confirmed the declining expression of PDGFRα from E65 onwards, and the increase of MBP and MAG expression from E80 onwards. Somatosensory input elicited by spontaneous activity is considered important for the formation of the body representation. Indeed, PDGFRα, MBP and MAG expression started earlier in somatosensory than in visual cortex. Taken together, myelination proceeded in white and gray matter and marginal zone of pig cortex before birth with an areal-specific time course, and an almost mature pattern was present at P5 in visual cortex.