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Supplemental Figure S3. Suppression of Akt activation in resistant clones

Supplemental Methods and Figure Legends

Overcoming tolerance to tumor-associated antigens remains a hurdle for cancer vaccine-based immunotherapy. A strategy to enhance the anti-tumor immune response is the inclusion of adjuvants to cancer vaccine protocols. In this report, we generated and systematically screened over twenty gene-based molecular adjuvants composed of cytokines, chemokines, and T cell co-stimulators for the ability to increase anti-tumor antigen T cell immunity. We identified several robust adjuvants whose addition to vaccine formulations resulted in enhanced T cell responses targeting the cancer antigens STEAP1 and TERT. We further characterized direct T cell stimulation through CD80-Fc and indirect T cell targeting via the dendritic cell activator Flt3L-Fc. Mechanistically, intramuscular delivery of Flt3L-Fc into mice was associated with a significant increase in infiltration of dendritic cells at the site of administration and trafficking of activated dendritic cells to the draining lymph node. Gene expression analysis of the muscle tissue confirmed a significant up-regulation in genes associated with dendritic cell signaling. Addition of CD80-Fc to STEAP1 vaccine formulation mimicked the engagement provided by DCs and increased T cell responses to STEAP1 by 8-fold, significantly increasing the frequency of antigen-specific cells expressing IFNγ, TNFα, and CD107a for both CD8+ and CD4+ T cells. CD80-Fc enhanced T cell responses to multiple tumor-associated antigens including Survivin and HPV, indicating its potential as a universal adjuvant for cancer vaccines. Together, the results of our study highlight the adjuvanting effect of T cell engagement either directly, CD80-Fc, or indirectly, Flt3L-Fc, for cancer vaccines.

Proceedings of the National Academy of Sciences of the United States of America 116(20), 201821442 - (2019). doi:10.1073/pnas.1821442116, Epidermal growth factor receptor (EGFR) signaling is initiated by a large ligand-favored conformational change of the extracellular domain (ECD) from a closed, self-inhibited tethered monomer, to an open untethered state, which exposes a loop required for strong dimerization and activation. In glioblastomas (GBMs), structurally heterogeneous missense and deletion mutations concentrate at the ECD for unclear reasons. We explore the conformational impact of GBM missense mutations, combining elastic network models (ENMs) with multiple molecular dynamics (MD) trajectories. Our simulations reveal that the main missense class, located at the I-II interface away from the self-inhibitory tether, can unexpectedly favor spontaneous untethering to a compact intermediate state, here validated by small-angle X-ray scattering (SAXS). Significantly, such intermediate is characterized by the rotation of a large ECD fragment (N-TR1), deleted in the most common GBM mutation, EGFRvIII, and that makes accessible a cryptic epitope characteristic of cancer cells. This observation suggested potential structural equivalence of missense and deletion ECD changes in GBMs. Corroborating this hypothesis, our FACS, in vitro, and in vivo data demonstrate that entirely different ECD variants all converge to remove N-TR1 steric hindrance from the 806-epitope, which we show is allosterically coupled to an intermediate kinase and hallmarks increased oncogenicity. Finally, the detected extraintracellular coupling allows for synergistic cotargeting of the intermediate with mAb806 and inhibitors, which is proved herein., Published by National Acad. of Sciences, Washington, DC

With the evolution of technology, there is now a deeper understanding of glioblastoma as an inter- and intraheterogeneous disease comprising a multitude of genetically and epigenetically different cancer cells. Greater characterization of glioblastoma at the molecular level has improved its initial pathophysiological staging and classification. With this knowledge comes the hope that more efficacious therapies to combat this highly lethal disease are on the horizon. One possibility for intervention is represented by the targeting of epidermal growth factor receptor (EGFR), which is amplified and mutated in a large subset of patients. In this review, we provide a brief overview of EGFR and its mutated form, EGFR variant III, describing the downstream cellular pathways activated by each receptor, available animal models, therapeutic strategies to inhibit the receptor, and possible intervention routes to efficiently target this receptor and prevent the emergence of resistant mechanisms which to date have hampered a successful therapeutic outcome.

EGFR is the most common genetically altered oncogene in glioblastoma (GBM), but small-molecule EGFR tyrosine kinase inhibitors (TKI) have failed to yield durable clinical benefit. Here, we show that in two novel model systems of acquired resistance to EGFR TKIs, elevated expression of urokinase plasminogen activator (uPA) drives signaling through the MAPK pathway, which results in suppression of the proapoptotic BCL2-family member protein BIM (BCL2L11). In patient-derived GBM cells and genetic GBM models, uPA is shown to suppress BIM levels through ERK1/2 phosphorylation, which can be reversed by siRNA-mediated knockdown of uPA. TKI-resistant GBMs are resensitized to EGFR TKIs by pharmacologic inhibition of MEK or a BH3 mimetic drug to replace BIM function. A link between the uPA–uPAR–ERK1/2 pathway and BIM has not been previously demonstrated in GBM, and involvement of this signaling axis in resistance provides rationale for a new strategy to target EGFR TKI-resistant GBM. Cancer Res; 75(2); 394–404. ©2014 AACR.

Emerging evidence suggests that alpha-processing single transmembrane proteins, amyloid precursor protein (APP) and anti-aging protein Klotho, are likely to be involved in the progression of Alzheimer’s disease (AD). The natural phthalide Ligustilide (LIG) has been demonstrated to protect against aging- and amyloid-β (Aβ)-induced brain dysfunction in animal models. The present study is to investigate the effects of LIG on cognitive deficits and metabolism of both APP and Klotho and its underlying mechanism in AD double-transgenic (APP/PS1) mice and cultured human cells. Our results show that treatment with LIG significantly ameliorated memory impairment and Aβ levels and plaques burden. Specifically, LIG might act as a potent enhancer of α-secretase, disintegrin, and metalloprotease 10 (ADAM10), leading to upregulation of alpha-processing of both APP and Klotho and subsequent increases in the levels of both soluble APP fragment (sAPPα) and soluble Klotho (sKL) with inhibition of IGF-1/Akt/mTOR signaling in AD mice and cultured cells. Moreover, the specific ADAM10 inhibitor (G1254023X) effectively reversed LIG-induced alpha-processing of both APP and Klotho in vitro, while Klotho gene knockdown by small interfering RNA significantly blunted LIG-mediated inhibition of IGF-1/Akt/mTOR signaling in vitro. Taken together with the reported neuroprotective effects of both sAPPα and sKL as well as autophagy induction by Akt/mTOR pathway inhibition, our findings suggest that neuroprotection of LIG against AD is associated with induction alpha-processing of APP and Klotho and potential Aβ clearance. Whether LIG might induce Aβ autophagic clearance and the underlying mechanisms need to be further studied.

Glioblastoma is the most malignant tumor of the brain with a median overall survival of 12-15 months. One of the factors that make the treatment of this disease difficult is its inherent and typical heterogeneity promoted by different genetic drivers. Here we show that neural stem cells (NSCs) derived from human induced pluripotent stem cells (iPSCs), with different combinations of these genetic drivers introduced by CRISPR/Cas9-mediated editing display mutations-specific in vitro proliferation and drug resistance characteristics and give rise to distinct intracranial tumors when engrafted in immunocompromised mice. NSCs deficient in PTEN and NF1, a genotype associated with the mesenchymal phenotype, generate highly-invasive intracranial tumors with spindle-shaped cells. Sphere cultures of these tumor cells yield euploid cell lines. In contrast, NSCs deficient in TP53 and expressing a PDGFRA activating mutation (PDGFRAD8-9), a genotype associated with the proneural glioblastoma subtype, give rise to aggressive tumors with high cellularity and characteristics of glioblastomas, such as mitosis, necrosis, and vascular proliferation. Cells obtained from these tumors show aneuploidy and display extrachromosomal DNA (ecDNA), including double-minute-like structures, which commonly harbor amplified oncogenes as seen in many cancer types including glioblastomas. This model represents the first human pluripotent-cell-derived cancer model showing pathognomonic progression with ecDNA amplification. Additionally, RNA-seq analysis of both models confirms their mesenchymal and proneural glioblastoma gene expression signatures, respectively. Using these engineered human cell derived xenograft models (eHDX), we aim to advance our understanding of the effect of each genomic abnormality on the neoplastic process and to provide a platform for the development of subtype specific targeted therapies.

Glioblastoma is a devastating disease, and there is an urgent need to develop novel therapies, such as oncolytic HSV1 (OV) to effectively target tumor cells. OV therapy depends on tumor-specific replication leading to destruction of neoplastic tissues. Host responses that curtail virus replication limit its efficacy in vivo. We have previously shown that cysteine-rich 61 protein (CCN1) activates a type 1 IFN antiviral defense response in glioblastoma cells. Incorporating TCGA data, we found CCN1 expression to be a negative prognostic factor for glioblastoma patients. Based on this, we used neutralizing antibodies against CCN1 to investigate its effect on OV therapy. Use of an anti-CCN1 antibody in mice bearing glioblastomas treated with OV led to enhanced virus expression along with reduced immune cell infiltration. OV-induced CCN1 increases macrophage migration toward infected glioblastoma cells by directly binding macrophages and also by enhancing the proinflammatory activation of macrophages inducing MCP-1 expression in glioblastoma cells. Activation of macrophages by CCN1 also increases viral clearance. Neutralization of integrin αMβ2 reversed CCN1-induced macrophage activation and migration, and reduced MCP-1 expression by glioblastoma cells. Our findings reveal that CCN1 plays a novel role in pathogen clearance; increasing macrophage infiltration and activation resulting in increased virus clearance in tumors.

Epidermal Growth Factor Receptor (EGFR) missense-mutations in glioblastoma (GBM) typically occur within the extracellular domain (ECD) and, to-date, have not shown a clinical impact. These ECD mutations, while not as common as wild-type amplification or EGFRvIII, do represent a distinct patient population in need of investigation. This study investigates the oncogenic effect of the most common EGFR ECD missense-mutations (R108K, A289D/T/V, and G598V) in a retrospective cohort of 260 de novo GBM patients from the University of Pennsylvania. Extrapolation of each individual missense-mutation revealed a significant reduction in patient survival for the EGFR(A289D/T/V) mutants (p=0.028). This effect was confirmed with a follow-up cohort of 111 patients. The Cancer Imaging Phenomics Toolkit (CaPTk – www.cbica.upenn.edu/captk) was used to extract 2104 quantitative imaging phenomic (QIP) features, from multiparametric magnetic resonance imaging, across the various tumor sub-regions comprising the enhancing and non-enhancing tumor core, as well as the peritumoral edematous/invaded tissue. Multivariate machine learning techniques integrated these features, revealing radiographic signatures suggestive of increased invasion and proliferation for patients bearing the A289 mutation. The underlying mechanism of EGFR(A289V) in tumor growth was examined using engineered U87 glioma cells and patient-derived HK281 glioma spheres simulating expression of wild-type-EGFR or EGFR(A289V). Corroborating our findings in patients, mice bearing intracranial tumors with EGFR(A289V) mutations revealed significantly worse survival accompanied by highly invasive tumors. Cells expressing EGFR(A289V) yielded a highly active EGFR/ERK signaling pathway, resulting in increased expression and functionality of the matrix metalloproteinase MMP1, which can be attenuated by the use of ERK pathway inhibitors. Collectively, the findings of this study highlight a highly invasive and proliferative phenotype associated with the EGFR(A289V) missense-mutation. Moreover, given the tumor-specific and extracellular nature of the mutation, we postulate that it may be amenable to targeted therapy. Key data presented is from a manuscript with preliminary acceptance to Cancer Cell. *equal contribution

SapC-DOPS is a novel nanotherapeutic that has been shown to target and induce cell death in a variety of cancers, including glioblastoma (GBM). GBM is a primary brain tumor known to frequently demonstrate resistance to apoptosis-inducing therapeutics. Here we explore the mode of action for SapC-DOPS in GBM, a treatment being developed by Bexion Pharmaceuticals for clinical testing in patients. SapC-DOPS treatment was observed to induce lysosomal dysfunction of GBM cells characterized by decreased glycosylation of LAMP1 and altered proteolytic processing of cathepsin D independent of apoptosis and autophagic cell death. We observed that SapC-DOPS induced lysosomal membrane permeability (LMP) as shown by LysoTracker Red and Acridine Orange staining along with an increase of sphingosine, a known inducer of LMP. Additionally, SapC-DOPS displayed strong synergistic interactions with the apoptosis-inducing agent TMZ. Collectively our data suggest that SapC-DOPS induces lysosomal cell death in GBM cells, providing a new approach for treating tumors resistant to traditional apoptosis-inducing agents.

The epidermal growth factor receptor (EGFR) is both amplified and frequently mutated in glioblastoma. While downstream inhibition of EGFR signaling through small molecule tyrosine kinase inhibitors has shown little clinical efficacy in glioblastoma, monoclonal antibody targeting is proving to be a promising alternative. The monoclonal antibody 806 (mAb806) specifically binds the most common EGFR mutant, EGFRvIII, along with a subset of amplified wild type EGFR, but not EGFR expressed at normal levels. The selectivity of mAb806 is thought to be due to its targeting a tumor-specific epitope of EGFR which is only accessible as the receptor transitions between an inactive tethered conformation to an active ligand-bound dimer. Here we used molecular dynamics simulations coupled with in vitro assays in glioblastoma cells to characterize the transitional conformation of EGFR in mAb806 binding. Focusing on the mutations R108K and G63R, molecular dynamics simulation revealed a spontaneous untethering of EGFR to a compact conformation, which is characterized by a backwards rotation of domain I-II exposing both the dimerization arm and the mAb806-epitope. This configuration is structurally equivalent to EGFRvIII, suggesting that several glioblastoma mutations converge to remove domain I from the dimerization interface. Next, we evaluated the effect of these point mutations on the ability of mAb806 to bind glioblastoma cells in vitro. Using site-directed mutagenesis coupled with lentiviral-transduction, we expressed EGFR-R108K or EGFR-G63R in U87 glioblastoma cells. Investigating mAb806 binding by FACS, we show that mAb806 binds the mutant cells five times greater than control cells, validating our simulation predictions. Future studies will examine the effect of mAb806 in an in vivo tumor model. Significantly, as these point mutations have been found in several GBM patients, our results indicate the possibility of a broader patient spectrum for the use of ABT-806, a humanized version of mAb806 currently in phase I clinical trials.

In glioblastoma (GBM), heterogeneous expression of amplified and mutated epidermal growth factor receptor (EGFR) presents a substantial challenge for the effective use of EGFR-directed therapeutics. Here we demonstrate that heterogeneous expression of the wild-type receptor and its constitutively active mutant form, EGFRvIII, limits sensitivity to these therapies through an interclonal communication mechanism mediated by interleukin-6 (IL-6) cytokine secreted from EGFRvIII-positive tumor cells. IL-6 activates a NF-κB signaling axis in a paracrine and autocrine manner, leading to bromodomain protein 4 (BRD4)-dependent expression of the prosurvival protein survivin (BIRC5) and attenuation of sensitivity to EGFR tyrosine kinase inhibitors (TKIs). NF-κB and survivin are coordinately up-regulated in GBM patient tumors, and functional inhibition of either protein or BRD4 in in vitro and in vivo models restores sensitivity to EGFR TKIs. These results provide a rationale for improving anti-EGFR therapeutic efficacy through pharmacological uncoupling of a convergence point of NF-κB-mediated survival that is leveraged by an interclonal circuitry mechanism established by intratumoral mutational heterogeneity.