Aberrant activation of EGFR in human cancers promotes tumorigenesis through stimulation of AKT signaling. Here, we determined that the discoidina neuropilin-like membrane protein DCBLD2 is upregulated in clinical specimens of glioblastomas and head and neck cancers (HNCs) and is required for EGFR-stimulated tumorigenesis. In multiple cancer cell lines, EGFR activated phosphorylation of tyrosine 750 (Y750) of DCBLD2, which is located within a recently identified binding motif for TNF receptor-associated factor 6 (TRAF6). Consequently, phosphorylation of DCBLD2 Y750 recruited TRAF6, leading to increased TRAF6 E3 ubiquitin ligase activity and subsequent activation of AKT, thereby enhancing EGFR-driven tumorigenesis. Moreover, evaluation of patient samples of gliomas and HNCs revealed an association among EGFR activation, DCBLD2 phosphorylation, and poor prognoses. Together, our findings uncover a pathway in which DCBLD2 functions as a signal relay for oncogenic EGFR signaling to promote tumorigenesis and suggest DCBLD2 and TRAF6 as potential therapeutic targets for human cancers that are associated with EGFR activation.

PDF file, 29K, Vaccine Administration Schedule and Characteristics of Adverse Events.

Supplementary Figure 1: NT113 Chemical Structure; Supplementary Figure 2: A) Relative expression of endogenous wild-type EGFR in parental U87 cells (U87p) vs. U87 cells transduced with EGFRvIII retrovirus (U87vIII). B) Relative expression of EGFRwt, EGFRvIII, ERBB2 and ERBB4 in subcutaneous tumors of GBM12, GBM39, and GBM6.

PDF file, 23K, Adjuvant Therapies Prior to and After Vaccination.

Supplementary Figure 3: Comparison of U87 and U87vIII viability response to increasing concentrations of NT113

PDF file, 48K, Overall Survival Characteristics of Treated Patients.

PDF file, 9905K, Representative phenotypic analysis of circulating lymphocytes in a clinical responder (Patient #009).

PDF file - 22K, Figure S1. Glioma cells induce B7-H1 expression in peripheral monocytes from healthy donors. Monocytes from healthy donors were cultured alone in normal media or in co-culture with U251 glioma cells in contact or through a 0.2μm pore filter. The percentage of B7-H1 expressing cells was determined by flow cytometry. Compared to normal monocytes, cells cultured with glioma cells through a filter (non-contact) have significantly more B7-H1 positive monocytes (* p < 0.005) and cells co-cultured in contact demonstrate a further significant increase in B7-H1 expression (vs. normal, ** p<0.001; vs. non-contact, p<0.01). Columns represent mean percent expression plus-minus SEM from 4 independent samples. Each sample was tested in triplicate and averaged as a single data point.

Proportional Hazards Model of Overall Survival

Supplementary Table 1: Selected IC50 results from NT113 screen of 130 tyrosine kinases; Supplementary Table 2: Inhibitor Tissue Concentrations.

Supplementary Figure 4: Plasma PK results associated with oral administration of 5 mg/kg NT113 in rats.

PDF file, 17162K, Model of HSPPC-96 mechanism of action.

PDF file, 8781K, Representative phenotypic analysis of circulating lymphocytes in a clinical non-responder (Patient #003).

Methods pertaining to experiments presented as supplementary figures.

PDF file, 50K, Descriptive Characteristics of the Study Population.

PDF file - 42K, Figure S2. Glioma-derived soluble factors can further induce B7-H1 expression in non-autologous GBM patient peripheral monocytes, but not in autologous patient monocytes. Quantification of mean fluorescence intensity for B7-H1 staining measured by flow cytometry is shown for monocytes stimulated with IL-10 (10 ng/ml) or glioma-conditioned media (SF1U-CM). In monocytes from healthy control patients, B7-H1 intensity was significantly increased by IL-10, and further increased by conditioned media (* p<0.05). Similar results were observed with stimulation of non-autologous monocytes from a GBM patient (* p<0.05, **p<0.01). Monocytes from an autologous GBM patient, SF1U, had high initial B7-H1 expression and could not be further increased with stimulation. Columns represent mean fluorescence intensity plus-minus SEM for each sample repeated in triplicate.

ErbB receptor tyrosine kinases EGFR (ErbB1), HER2 (ErbB2, neu), HER3 (ErbB3), and HER4 (ErbB4) are part of a complex network activating signaling pathways involved in cell growth and survival. Mutations causing errant ErbB activation are an oncodriver in many cancers including NSCLC. Inhibitors targeting ErbB mutations have transformed outcomes for patients; however, resistance to treatment develops rapidly. The various ErbB receptors have overlapping roles in oncogenesis and crosstalk between ErbB family members is associated with acquired resistance and metastases. For example, amplification of HER2 is a well-established mechanism of acquired resistance to EGFR-TKIs. The development of next-generation agents targeting multiple ErbB receptors has shown promise but has been limited by toxicity and poor brain penetration. Up to 80% of NSCLC patients will experience a brain lesion associated with their disease; treatment-resistant phenotypes metastasizing to the brain have become an important driver of morbidity and mortality and patients have limited therapeutic options. New agents are needed to address this important and growing unmet medical need. EO1001 is a first-in-class, oral, brain-penetrating, irreversible pan-ErbB inhibitor targeting ErbB1, ErbB2, and ErbB4 that is positioned for near-term entry into clinical development. In vitro testing: EO1001 exhibits excellent and balanced equipotent activity against all three important ErbB receptors including EGFR, HER2, and HER4 with low nM activity (0.4 to 7.4 nM), with high specificity vs. off-target receptors. In vivo studies: Following oral administration, EO1001 treatment resulted in a statistically significant improvement in outcomes compared to positive and negative controls in erbB-positive mouse orthotopopic models including N87 (Her2+), H1975 (EGFR/T790M), GBM12 (EGFR+), GBM39 (EGVRvIII+). EO1001 rapidly enters the CNS at high concentrations relative to plasma and inhibits signaling downstream of mutant ErbB receptors in tumor tissue. Treatment with EO1001 was generally well tolerated with no gastrointestinal side effects observed at efficacious doses in mouse xenograft models. Preclinical pharmacokinetic and toxicology studies have been completed. EO-1001 exhibits a half-life of 16-20 hours in rodent models. Toxicities typical of the ErbB inhibitor class, including gastrointestinal effects, weight loss, and decreased activity, were observed at higher dose groups in both rodent and non-rodent species. Extrapolation to human dosing suggests an attractive therapeutic window in comparison to other agents in the class. EO1001 has the potential to be a best-in-class CNS-penetrating pan-ErbB inhibitor amenable for use as a single agent and in combination regimens. First-in-man clinical testing with EO1001 is planned. Continued characterization of EO1001 activity against specific ErbB mutations will be undertaken in parallel.

AbstractPurpose: Upregulation of programmed death-ligand 1 (PD-L1) on circulating and tumor-infiltrating myeloid cells is a critical component of GBM-mediated immunosuppression that has been associated with diminished response to vaccine immunotherapy and poor survival. Although GBM-derived soluble factors have been implicated in myeloid PD-L1 expression, the identity of such factors has remained unknown. This study aimed to identify factors responsible for myeloid PD-L1 upregulation as potential targets for immune modulation. Experimental Design: Conditioned media from patient-derived GBM explant cell cultures was assessed for cytokine expression and utilized to stimulate naïve myeloid cells. Myeloid PD-L1 induction was quantified by flow cytometry. Candidate cytokines correlated with PD-L1 induction were evaluated in tumor sections and plasma for relationships with survival and myeloid PD-L1 expression. The role of identified cytokines on immunosuppression and survival was investigated in vivo utilizing immunocompetent C57BL/6 mice bearing syngeneic GL261 and CT-2A tumors. Results: GBM-derived IL6 was identified as a cytokine that is necessary and sufficient for myeloid PD-L1 induction in GBM through a STAT3-dependent mechanism. Inhibition of IL6 signaling in orthotopic murine glioma models was associated with reduced myeloid PD-L1 expression, diminished tumor growth, and increased survival. The therapeutic benefit of anti-IL6 therapy proved to be CD8+ T-cell dependent, and the antitumor activity was additive with that provided by programmed death-1 (PD-1)-targeted immunotherapy. Conclusions: Our findings suggest that disruption of IL6 signaling in GBM reduces local and systemic myeloid-driven immunosuppression and enhances immune-mediated antitumor responses against GBM.

BACKGROUND:RNAs within extracellular vesicles (EVs) have potential as diagnostic biomarkers for patients with cancer and are identified in a variety of biofluids. Glioblastomas (GBMs) release EVs containing RNA into cerebrospinal fluid (CSF). Here we describe a multi-institutional study of RNA extracted from CSF-derived EVs of GBM patients to detect the presence of tumor-associated amplifications and mutations in epidermal growth factor receptor (EGFR).METHODS:CSF and matching tumor tissue were obtained from patients undergoing resection of GBMs. We determined wild-type (wt)EGFR DNA copy number amplification, as well as wtEGFR and EGFR variant (v)III RNA expression in tumor samples. We also characterized wtEGFR and EGFRvIII RNA expression in CSF-derived EVs.RESULTS:EGFRvIII-positive tumors had significantly greater wtEGFR DNA amplification (P = 0.02) and RNA expression (P = 0.03), and EGFRvIII-positive CSF-derived EVs had significantly more wtEGFR RNA expression (P = 0.004). EGFRvIII was detected in CSF-derived EVs for 14 of the 23 EGFRvIII tissue-positive GBM patients. Conversely, only one of the 48 EGFRvIII tissue-negative patients had the EGFRvIII mutation detected in their CSF-derived EVs. These results yield a sensitivity of 61% and a specificity of 98% for the utility of CSF-derived EVs to detect an EGFRvIII-positive GBM.CONCLUSION:Our results demonstrate CSF-derived EVs contain RNA signatures reflective of the underlying molecular genetic status of GBMs in terms of wtEGFR expression and EGFRvIII status. The high specificity of the CSF-derived EV diagnostic test gives us an accurate determination of positive EGFRvIII tumor status and is essentially a less invasive "liquid biopsy" that might direct mutation-specific therapies for GBMs.