A Rare Case of Quadruple Negative GIST with an Unusual Presentation in a Young Female

Gastrointestinal stromal tumors (GISTs) are an uncommon malignancy, with origin in the interstitial cells of Cajal located in the myenteric plexus. The incidence is 5000 new cases every year in the US. It is important to determine genetic alterations in GIST. Approximately 90% of GISTs have a gain of function mutation in either the c-KIT protooncogene (which encodes for the receptor tyrosine kinase KIT) accounting for 75%, or the platelet derived growth factor receptor alpha (PDGFRA) protooncogene which accounts for 15%. Only 5-10% constitute Wild Type (WT) GISTs with mutations observed in BRAF, NF1, & SDH. While Imatinib, a Tyrosine Kinase Inhibitor (TKI), is used as adjuvant therapy for most KIT-positive tumors, it cannot be used in TKI-resistant tumors that harbor alternative genetic mutations. We present a rare case of quadruple negative (negative for all aforementioned genes) GIST with a mutation identified as ETV6-NTRK3 fusion. This mutation was first described in a case of rectal quadruple negative WT GIST.

Adult medial habenula neurons require GDNF receptor GFRα1 for synaptic stability and function

The medial habenula (mHb) is an understudied small brain nucleus linking forebrain and midbrain structures controlling anxiety and fear behaviors. The mechanisms that maintain the structural and functional integrity of mHb neurons and their synapses remain unknown. Using spatiotemporally controlled Cre-mediated recombination in adult mice, we found that the glial cell–derived neurotrophic factor receptor alpha 1 (GFRα1) is required in adult mHb neurons for synaptic stability and function. mHb neurons express some of the highest levels of GFRα1 in the mouse brain, and acute ablation of GFRα1 results in loss of septohabenular and habenulointerpeduncular glutamatergic synapses, with the remaining synapses displaying reduced numbers of presynaptic vesicles. Chemo- and optogenetic studies in mice lacking GFRα1 revealed impaired circuit connectivity, reduced AMPA receptor postsynaptic currents, and abnormally low rectification index (R.I.) of AMPARs, suggesting reduced Ca2+ permeability. Further biochemical and proximity ligation assay (PLA) studies defined the presence of GluA1/GluA2 (Ca2+ impermeable) as well as GluA1/GluA4 (Ca2+ permeable) AMPAR complexes in mHb neurons, as well as clear differences in the levels and association of AMPAR subunits with mHb neurons lacking GFRα1. Finally, acute loss of GFRα1 in adult mHb neurons reduced anxiety-like behavior and potentiated context-based fear responses, phenocopying the effects of lesions to septal projections to the mHb. These results uncover an unexpected function for GFRα1 in the maintenance and function of adult glutamatergic synapses and reveal a potential new mechanism for regulating synaptic plasticity in the septohabenulointerpeduncular pathway and attuning of anxiety and fear behaviors.

Cryo-Electron Microscopy Structure and Interactions of the Human Cytomegalovirus gHgLgO Trimer with Platelet-Derived Growth Factor Receptor Alpha

HCMV is a herpesvirus that infects a large percentage of the adult population and causes significant levels of disease in immunocompromised individuals and birth defects in the developing fetus. The virus encodes a complex protein machinery that coordinates infection of different cell types in the body, including a trimer formed of gH, gL, and gO subunits.

BRD9 inhibition promotes PUMA-dependent apoptosis and augments the effect of imatinib in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are primarily characterized by activating mutations of tyrosine kinase or platelet-derived growth factor receptor alpha. Although the revolutionary therapeutic outcomes of imatinib are well known, the long-term benefits of imatinib are still unclear. The effects of BRD9, a recently identified subunit of noncanonical BAF complex (ncBAF) chromatin remodeling complexes, in GISTs are not clear. In the current study, we evaluated the functional role of BRD9 in GIST progression. Our findings demonstrated that the expression of BRD9 was upregulated in GIST tissues. The downregulation or inhibition of BRD9 could significantly reduce cellular proliferation, and facilitates apoptosis in GISTs. BRD9 inhibition could promote PUMA-dependent apoptosis in GISTs and enhance imatinib activity in vitro and in vivo. BRD9 inhibition synergizes with imatinib in GISTs by inducing PUMA upregulation. Mechanism study revealed that BRD9 inhibition promotes PUMA induction via the TUFT1/AKT/GSK-3β/p65 axis. Furthermore, imatinib also upregulates PUMA by targeting AKT/GSK-3β/p65 axis. In conclusion, our results indicated that BRD9 plays a key role in the progression of GISTs. Inhibition of BRD9 is a novel therapeutic strategy in GISTs treated alone or in combination with imatinib.

Peptide Derivatives of Platelet-Derived Growth Factor Receptor Alpha Inhibit Cell-Associated Spread of Human Cytomegalovirus

Cell-free human cytomegalovirus (HCMV) can be inhibited by a soluble form of the cellular HCMV-receptor PDGFRα, resembling neutralization by antibodies. The cell-associated growth of recent HCMV isolates, however, is resistant against antibodies. We investigated whether PDGFRα-derivatives can inhibit this transmission mode. A protein containing the extracellular PDGFRα-domain and 40-mer peptides derived therefrom were tested regarding the inhibition of the cell-associated HCMV strain Merlin-pAL1502, hits were validated with recent isolates, and the most effective peptide was modified to increase its potency. The modified peptide was further analyzed regarding its mode of action on the virion level. While full-length PDGFRα failed to inhibit HCMV isolates, three peptides significantly reduced virus growth. A 30-mer version of the lead peptide (GD30) proved even more effective against the cell-free virus, and this effect was HCMV-specific and depended on the viral glycoprotein O. In cell-associated spread, GD30 reduced both the number of transferred particles and their penetration. This effect was reversible after peptide removal, which allowed the synchronized analysis of particle transfer, showing that two virions per hour were transferred to neighboring cells and one virion was sufficient for infection. In conclusion, PDGFRα-derived peptides are novel inhibitors of the cell-associated spread of HCMV and facilitate the investigation of this transmission mode.

P13.18 Inhibition of epidermal growth factor receptor and platelet-derived growth factor receptor-alpha exerts synergistic efficacy in glioblastoma

BACKGROUND Despite our understanding of the genetic changes that precipitate gliomagenesis, targeted therapy has failed in glioblastoma (GBM) with median survival not significantly improved over the past two decades. Epidermal growth factor receptor (EGFR) alterations, including amplification and activating mutations, are among the most common genetic changes in GBM, occurring in more than half of cases. EGFR is located on Chr. 7, and Chr. 7 gain is one of the earliest events precipitating gliomagenesis. Various EGFR inhibitors, including tyrosine kinase inhibitors, monoclonal antibodies, vaccines, and CAR-T cells have failed in GBM due to intrinsic heterogeneity and receptor tyrosine kinase bypass pathways that mediate therapeutic resistance. New targeted therapeutic approaches to leverage synergistic combinations are desperately needed to improve GBM prognosis. Using the TCGA and other GBM databases, we have previously demonstrated that the presence of PDGFRAamplification in patients with EGFR-amplified GBM carries significantly worse survival. EGFR and PDGFRA co-expression occur in more than one-third of GBM patients. The PDGFRA ligand PDGFA is also located on Chr. 7, and its expression is significantly increased with Chr. 7 gain and EGFR copy number increase. Therefore, Chr. 7 gain inherently leads to co-activation of both EGFR and PDGFRA signaling pathways. MATERIALS AND METHODS We used models of patient-derived glioblastoma cells to test combined inhibition of epidermal growth factor receptor and platelet-derived growth factor receptor-alpha in vitro. RESULTS Using patient-derived GBM models with Chr. 7 gain, we found that combined inhibition of both EGFR and PDGFRA using a variety of FDA-approved EGFR-targeted agents (Erlotinib, Gefitinib, Dacomitinib, Neratinib, and Osimertinib) and Crenolanib, respectively, leads to synergistic cytotoxicity in vitro. We found that inhibition of either EGFR or PDGFRA alone led to receptor cross-activation, and EGF and PDGF-AA-induced receptor tyrosine kinase activation was blocked by Neratinib and Crenolanib. Immunoprecipitation experiments and proximity ligation assays demonstrated that combined inhibition prevents EGFR and PDGFRA heterodimerization and pathways of therapeutic resistance. This combined inhibition led to decreased activation of downstream signaling pathways, including phosphatidylinositol 3-kinase and mitogen-activated protein kinase. CONCLUSIONS We show that combined inhibition of EGFR and PDGFRA exerts synergistic cytotoxicity in GBM and prevents resistance pathways that emerge during single-agent targeted therapy against these receptor tyrosine kinases. These pathways are targetable with FDA-approved agents that could be used in patients with GBM with Chr. 7 gain.

FIP1L1-PDGFRA-Associated Hypereosinophilic Syndrome as a Treatable Cause of Watershed Infarction

Background and Purpose: Ischemic stroke has been reported in various conditions associated with eosinophilia. FIP1L1-PDGFRA fusion ([Fip1-like 1-platelet-derived growth factor receptor alpha]; F/P ) leads to the proliferation of the eosinophilic lineage and thus to a clonal hypereosinophilic syndrome that is highly responsive to imatinib. Methods: We previously reported on a nationwide retrospective study of 151 patients with F/P -associated clonal hypereosinophilic syndrome. Patients from this cohort with a clinical history of ischemic stroke (as well as 2 additional cases) were further analyzed to better define their clinical picture and outcomes. Results: Sixteen male patients (median age, 51 [43–59] years) with low-to-intermediate cardiovascular risk were included. Median National Institutes of Health Stroke Scale was 4 (range, 1–6). Most cerebral imaging disclosed multiple bilateral infarctions of watershed distribution (69%). Despite frequent cardiac involvement (50%), cardiac thrombus was evidenced in a single patient and, according to the TOAST classification (Trial of ORG 10172 in Acute Stroke Treatment), 62.5% of strokes were presumably of undetermined etiology. Among the 15 patients treated with imatinib, and after a median follow-up of 4.5 years, stroke recurred in only 3 patients (consisting of either cardio embolic or hemorrhagic events, unrelated to the first episode). Conclusions: F/P + clonal hypereosinophilic syndrome is a diagnosis to consider in patients with unexplained ischemic stroke and hypereosinophilia (especially in the setting of multiple cortical borderzone distribution) and warrants prompt initiation of imatinib.

Gastrointestinal Stromal Tumors—A Mini Review

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract. They are potentially malignant, and have an unpredictable evolution. The origin of these tumors is in the interstitial cells of Cajal, which are cells that are interposed between the intramural neurons and the smooth muscle cells of the digestive tract. GISTs are characterized by mutations in the gene c-Kit, but also other mutations, such as those of the platelet-derived growth factor receptor alpha. The most common locations of these tumors are the stomach and small intestine, although they can occur at any level of the digestive tract and occasionally in the omentum, mesentery and peritoneum. Most cases of GISTs are sporadic, and about 5% of cases are part of family genetic syndromes. The correct diagnosis of GIST is determined by histopathological examination and immunohistochemistry. According to histopathology, there are three main types of GISTs: spindle cell type, epithelioid type and mixed type. The therapeutic management of GIST includes surgery, endoscopic treatment and chemotherapy. The prognosis of patients with GIST varies depending on a number of factors, such as risk category, GIST stage, treatment applied and recurrence after treatment.

Role of Detrusor PDGFRα+ Cells in Cyclophosphamide-Induced Detrusor Overactivity

Cyclophosphamide (CYP)-induced cystitis is a rodent model that shares many features common to the cystitis occurring in patients, including detrusor overactivity (DO). Platelet-derived growth factor receptor alpha positive (PDGFRα+) cells regulate muscle excitability in murine bladders during filling. PDGFRα+ cells express small conductance Ca2+-activated K+ channels (predominantly SK3) that provide stabilization of membrane potential during filling. We hypothesized that down-regulation of the regulatory functions of PDGFRα+ cells and/or loss of PDGFRα+ cells generates the DO in CYP-treated mice. After CYP treatment, transcripts of Pdgfrα and Kcnn3 were reduced, and PDGFRα and SK3 protein was also reduced in detrusor muscle extracts. The distribution of PDGFRα+ cells was also reduced. Inflammatory markers were increased in CYP-treated detrusor muscles. An SK channel agonist, CyPPA, increased outward current and hyperpolarization in PDGFRα+ cells. This response was significantly depressed in PDGFRα+ cells from CYP-treated bladders. Ex vivo cystometry showed increased transient contractions in CYP-treated bladders, and the sensitivity of these bladders to apamin was reduced, reflecting the reduction in the SK conductance expressed by PDGFRα+ cells. In summary, PDGFRα+ cells were reduced and the SK3 conductance was downregulated in CYP-treated bladders. These changes are consistent with the development of DO after CYP treatment.

Selection of Human Cytomegalovirus Mutants with Resistance against PDGFRα-Derived Entry Inhibitors

The human cytomegalovirus (HCMV) infects fibroblasts via an interaction of its envelope glycoprotein gO with the cellular platelet-derived growth factor receptor alpha (PDGFRα), and soluble derivatives of this receptor can inhibit viral entry. We aimed to select mutants with resistance against PDGFRα-Fc and the PDGFRα-derived peptides GT40 and IK40 to gain insight into the underlying mechanisms and determine the genetic barrier to resistance. An error-prone variant of strain AD169 was propagated in the presence of inhibitors, cell cultures were monitored weekly for signs of increased viral growth, and selected viruses were tested regarding their sensitivity to the inhibitor. Resistant virus was analyzed by DNA sequencing, candidate mutations were transferred into AD169 clone pHB5 by seamless mutagenesis, and reconstituted virus was again tested for loss of sensitivity by dose-response analyses. An S48Y mutation in gO was identified that conferred a three-fold loss of sensitivity against PDGFRα-Fc, a combination of mutations in gO, gH, gB and gN reduced sensitivity to GT40 by factor 4, and no loss of sensitivity occurred with IK40. The resistance-conferring mutations support the notion that PDGFRα-Fc and GT40 perturb the interaction of gO with its receptor, but the relatively weak effect indicates a high genetic barrier to resistance.