EGFR as a potent CAR T target in triple negative breast cancer brain metastases.

PURPOSE: There is currently no curative treatment for patients diagnosed with triple-negative breast cancer brain metastases (TNBC-BM). CAR T cells hold potential for curative treatment given they retain the cytolytic activity of a T cell combined with the specificity of an antibody. In this proposal we evaluated the potential of EGFR re-directed CAR T cells as a therapeutic treatment against TNBC cells in vitro and in vivo. METHODS: We leveraged a TNBC-BM tissue microarray and a large panel of TNBC cell lines and identified elevated epidermal growth factor receptor (EGFR) expression. Next, we designed a second-generation anti-EGFR CAR T construct incorporating a clinically relevant mAb806 tumor specific single-chain variable fragment (scFv) and intracellular 4-1BB costimulatory domain and CD3ζ using a lentivirus system and evaluated in vitro and in vivo anti-tumor activity. RESULTS: We demonstrate EGFR is enriched in TNBC-BM patient tissue after neurosurgical resection, with six of 13 brain metastases demonstrating both membranous and cytoplasmic EGFR. Eleven of 13 TNBC cell lines have EGFR surface expression ≥ 85% by flow cytometry. EGFR806 CAR T treated mice effectively eradicated TNBC-BM and enhanced mouse survival (log rank p < 0.004). CONCLUSION: Our results demonstrates anti-tumor activity of EGFR806 CAR T cells against TNBC cells in vitro and in vivo. Given EGFR806 CAR T cells are currently undergoing clinical trials in primary brain tumor patients without obvious toxicity, our results are immediately actionable against the TNBC-BM patient population.

CAR T cells for brain tumors: Lessons learned and road ahead.

Malignant brain tumors, including glioblastoma, represent some of the most difficult to treat of solid tumors. Nevertheless, recent progress in immunotherapy, across a broad range of tumor types, provides hope that immunological approaches will have the potential to improve outcomes for patients with brain tumors. Chimeric antigen receptors (CAR) T cells, a promising immunotherapeutic modality, utilizes the tumor targeting specificity of any antibody or receptor ligand to redirect the cytolytic potency of T cells. The remarkable clinical response rates of CD19-targeted CAR T cells and early clinical experiences in glioblastoma demonstrating safety and evidence for disease modifying activity support the potential of further advancements ultimately providing clinical benefit for patients. The brain, however, is an immune specialized organ presenting unique and specific challenges to immune-based therapies. Remaining barriers to be overcome for achieving effective CAR T cell therapy in the central nervous system (CNS) include tumor antigenic heterogeneity, an immune-suppressive microenvironment, unique properties of the CNS that limit T cell entry, and risks of immune-based toxicities in this highly sensitive organ. This review will summarize preclinical and clinical data for CAR T cell immunotherapy in glioblastoma and other malignant brain tumors, including present obstacles to advancement.

Activation of Src induces mitochondrial localisation of de2-7EGFR (EGFRvIII) in glioma cells: implications for glucose metabolism.

A common mutation of the epidermal growth factor receptor in glioma is the de2-7EGFR (or EGFRvIII). Glioma cells expressing de2-7EGFR contain an intracellular pool of receptor with high levels of mannose glycosylation, which is consistent with delayed processing. We now show that this delay occurs in the Golgi complex. Low levels of de2-7EGFR were also seen within the mitochondria. Src activation dramatically increased the amount of mitochondrial de2-7EGFR, whereas its pharmacological inhibition caused a significant reduction. Because de2-7EGFR is phosphorylated by Src at Y845, we generated glioma cells expressing a Y845F-modified de2-7EGFR. The de2-7EGFR(845F) mutant failed to show mitochondrial localisation, even when co-expressed with constitutive active Src. Low levels of glucose enhanced mitochondrial localisation of de2-7EGFR, and glioma cells expressing the receptor showed increased survival and proliferation under these conditions. Consistent with this, de2-7EGFR reduced glucose dependency by stimulating mitochondrial oxidative metabolism. Thus, the mitochondrial localisation of de2-7EGFR contributes to its tumorigenicity and might help to explain its resistance to some EGFR-targeted therapeutics.

Advances in computational and translational approaches for malignant glioma.

Gliomas are the most common primary brain tumors in adults and carry a dismal prognosis for patients. Current standard-of-care for gliomas is comprised of maximal safe surgical resection following by a combination of chemotherapy and radiation therapy depending on the grade and type of tumor. Despite decades of research efforts directed towards identifying effective therapies, curative treatments have been largely elusive in the majority of cases. The development and refinement of novel methodologies over recent years that integrate computational techniques with translational paradigms have begun to shed light on features of glioma, previously difficult to study. These methodologies have enabled a number of point-of-care approaches that can provide real-time, patient-specific and tumor-specific diagnostics that may guide the selection and development of therapies including decision-making surrounding surgical resection. Novel methodologies have also demonstrated utility in characterizing glioma-brain network dynamics and in turn early investigations into glioma plasticity and influence on surgical planning at a systems level. Similarly, application of such techniques in the laboratory setting have enhanced the ability to accurately model glioma disease processes and interrogate mechanisms of resistance to therapy. In this review, we highlight representative trends in the integration of computational methodologies including artificial intelligence and modeling with translational approaches in the study and treatment of malignant gliomas both at the point-of-care and outside the operative theater in silico as well as in the laboratory setting.

Rapid In Vitro Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging.

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.

The Effects of Time to Treatment Initiation for Patients With Non-small-cell Lung Cancer in the United States.

BACKGROUND: The purpose of this study was to determine the effects of time from diagnosis to treatment (TTI) on survival in patients with nonmetastatic non-small-cell lung cancer (NSCLC). MATERIALS AND METHODS: The National Cancer Database was queried for patients with stages 1 to 3 NSCLC between 2004 and 2013. Patients with missing survival status/time, unknown TTI, or receipt of palliative therapy were excluded. Multivariable Cox proportional hazards modeling, logistic regression, and recursive partitioning analysis were performed to determine associated variables and survival outcomes. RESULTS: Altogether, 1,393,232 patients met inclusion criteria. The median follow-up was 36 months. The median TTI increased between 2004 and 2013 from 35 to 39 days (P < .001). On multivariable Cox proportional hazards modeling, TTI groups 31 to 60 days, 61 to 90 days, and > 90 days were independently related to poorer overall survival (OS) compared with TTI 1 to 30 days (hazard ratio, 1.04, 1.10, and 1.14; 95% confidence interval [CI], 1.02-1.06, 1.07-1.12, and 1.11-1.17, respectively; P < .001 for all). Recursive partitioning analysis revealed that TTI of ≤ 45 days was the most optimal threshold for survival (P < .001); patients with TTI ≤ 45 days had a median OS of 70.2 months (95% CI, 69.3-71.1 months) versus 61.5 months (95% CI, 60.5-62.4) (P < .001). There were significant disparities by age, race, ethnicity, and income for delayed (> 45 days) TTI (P < .001 for all). Subgroup analysis revealed that stage 1 and 2 patients with TTI > 45 days had a higher risk of mortality compared with TTI ≤ 45 days (hazard ratio, 1.15 and 1.05; 95% CI, 1.12-1.17 and 1.01-1.09, respectively) (P < .001). CONCLUSIONS: Increased TTI is independently associated with poorer survival in non-metastatic NSCLC. TTI ≤ 45 days is a clinically targetable time frame associated with improved outcomes and ought to be considered for patients with lung cancer undergoing definitive therapy.

Phase I Study of Yttrium-90 Radiolabeled M5A Anti-Carcinoembryonic Antigen Humanized Antibody in Patients with Advanced Carcinoembryonic Antigen Producing Malignancies.

Background: M5A is a humanized monoclonal antibody (mAb) directed against carcinoembryonic antigen (CEA) The purpose of this first in human phase I dose-escalation trial was to characterize the toxicities and determine the maximum tolerated dose (MTD) of yttrium-90 (90Y)-DOTA-M5A as a single agent and in combination with gemcitabine (gem). Methods: Patients with advanced metastatic CEA-producing malignancies who had progressed on standard therapies were first administered indium-111 (111In)-DOTA-M5A. If tumor targeting was observed, the patient then received the therapy dose of 90Y-DOTA-M5A. Serial scans, blood sampling, and 24 h urine collections were then performed to estimate radiation doses to organs and total body. Assays for human antihuman antibody (HAHA) responses were performed out to 6 months. Results: Of the 18 patients who received 111In-DOTA-M5A, 16 received 90Y-DOTA-M5A therapy; 1 patient at 14 mCi/m2 with gem (150 mg/m2 days 1and 3), 3 patients at 12 mCi/m2 with gem, 6 patients at 12 mCi/m2 without gem, and 6 at 10 mCi/m2 without gem. Prolonged cytopenias resulted in discontinuation of dose escalation with gemcitabine. A single agent MTD of 10 mCi/m2 was established based on dose-limiting hematopoietic toxicities. HAHA immune response was identified in 2 of 16 patients (12.5%). Stable disease at 3 months was seen in 10 patients and 2 patients demonstrated an 88% and 64% decrease in CEA back to normal levels. In 2 patients 111In-DOTA-M5A imaging revealed previously unknown brain metastases. Conclusion: This study demonstrates the potential utility of the 90Y-DOTA-M5A anti-CEA mAb as a therapeutic antibody. There is decreased immunogenicity compared with murine and chimeric mAbs, allowing for the potential of multiple administrations. Combined modality therapy approaches incorporating this agent should continue to be evaluated.

Disparities Predict for Higher Rates of Cut-through Hysterectomies in Locally Advanced Cervical Cancer.

OBJECTIVES: The treatment of choice for locally advanced cervical cancer is definitive chemoradiation (CRT). Hysterectomy is not indicated due to higher-rates of cut-through resections leaving gross disease behind, requiring additional therapy with increasing morbidity and no benefit in overall survival (OS). The objectives of this study were to determine factors associated with cut-through hysterectomies and evaluate OS outcomes. MATERIALS AND METHODS: The National Cancer Database (NCDB) was queried for patients 18 years and older with clinical Federation of Gynecology and Obstetrics stage IB2 to IVA. All patients underwent upfront hysterectomy and had known margin status. Cut-through hysterectomy was classified as presence of microscopic or macroscopic disease at the margin. RESULTS: A total of 11,638 patients were included; 993 (8.5%) had positive margins. In patients with positive margins, 560 (56.4%) received postoperative CRT and 148 (14.9%) underwent postoperative radiation. Five-year OS was worse for those with cut-through resections when compared with those with negative margins, 66.0% versus 86.7%, respectively (hazard ratios, 3.08; P<0.001). Under multiple logistic regression, African American race (odds ratio [OR], 1.45; P=0.001), older age (OR per year increase, 1.03; P<0.001), patients with government insurance (OR, 1.21; P=0.019), and those treated at community practices (OR, 1.31; P=0.001) were more likely to undergo cut-through hysterectomies. CONCLUSIONS: A review of national patterns of care over the past decade confirms women with positive margins after hysterectomy for cervical cancer have significantly worse OS. Disparities in surgical results for women with cervical cancer exist. In response, further causality evaluation and corrective action are warranted to address these inequalities.

Glycerol facilitator GlpF and the associated aquaporin family of channels.

The aqua (glycero) porins conduct water (and glycerol) across cell membranes. The structure of these channels reveals a tripathic channel that supports a hydrophobic surface and, opposite to this, a line of eight hydrogen-bond acceptors and four hydrogen-bond donors. The eight carbonyls act as acceptors for water (or glycerol OH) molecules. The central water molecule in the channel is oriented to polarize hydrogen atoms outward from the center. This arrangement suggests how the structure prevents the potentially lethal conduction of protons across the membrane. The structure also suggests the mechanism behind the selectivity of aquaglyceroporins for glycerol, the basis for enantioselectivity among alditols, and the basis for the prevention of any leakage of the electrochemical gradient.

Pilot study on "pericytic mimicry" and potential embryonic/stem cell properties of angiotropic melanoma cells interacting with the abluminal vascular surface.

The interaction of tumor cells with the tumor vasculature is mainly studied for its role in tumor angiogenesis and intravascular metastasis of circulating tumor cells. In addition, a specific interaction of tumor cells with the abluminal surfaces of vessels, or angiotropism, may promote the migration of angiotropic tumor cells along the abluminal vascular surfaces in a pericytic location. This process has been termed extravascular migratory metastasis. The abluminal vascular surface may also provide a vascular niche inducing or sustaining stemness to angiotropic tumor cells. This pilot study investigated if angiotropic melanoma cells might represent a subset population with pericytic and embryonic or stem cell properties. Through microarray analysis, we showed that the interaction between melanoma cells and the abluminal surface of endothelial cells triggers significant differential expression of several genes. The most significantly differentially expressed genes have demonstrated properties linked to cancer cell migration (CCL2, ICAM1 and IL6), cancer progression (CCL2, ICAM1, SELE, TRAF1, IL6, SERPINB2 and CXCL6), epithelial to mesenchymal transition (CCL2 and IL6), embryonic/stem cell properties (CCL2, PDGFB, EVX1 and CFDP1) and pericytic recruitment (PDGFB). In addition, bioinformatics-based analysis of the differentially expressed genes has shown that the most significantly enriched functional groups included development, cell movement, cancer, and embryonic development. Finally, the investigation of pericyte/mesenchymal stem cells markers via immunostaining of human melanoma samples revealed expression of PDGFRB, NG2 and CD146 by angiotropic melanoma cells. Taken together, these preliminary data are supportive of the "pericytic mimicry" by angiotropic melanoma cells, and suggest that the interaction between melanoma cells and the abluminal vascular surface induce differential expression of genes linked to cancer migration and embryonic/stem cell properties.

De-repression of PDGFRß transcription promotes acquired resistance to EGFR tyrosine kinase inhibitors in glioblastoma patients.

UNLABELLED: Acquired resistance to tyrosine kinase inhibitors (TKI) represents a major challenge for personalized cancer therapy. Multiple genetic mechanisms of acquired TKI resistance have been identified in several types of human cancer. However, the possibility that cancer cells may also evade treatment by co-opting physiologically regulated receptors has not been addressed. Here, we show the first example of this alternate mechanism in brain tumors by showing that EGF receptor (EGFR)-mutant glioblastomas (GBMs) evade EGFR TKIs by transcriptionally de-repressing platelet-derived growth factor receptor ß (PDGFRß). Mechanistic studies show that EGFRvIII signaling actively suppresses PDGFRß transcription in an mTORC1- and extracellular signal-regulated kinase-dependent manner. Genetic or pharmacologic inhibition of oncogenic EGFR renders GBMs dependent on the consequently de-repressed PDGFRß signaling for growth and survival. Importantly, combined inhibition of EGFR and PDGFRß signaling potently suppresses tumor growth in vivo. These data identify a novel, nongenetic TKI resistance mechanism in brain tumors and provide compelling rationale for combination therapy. SIGNIFICANCE: These results provide the fi rst clinical and biologic evidence for receptor tyrosinekinase (RTK) "switching" as a mechanism of resistance to EGFR inhibitors in GBM and provide a molecular explanation of how tumors can become "addicted" to a non amplified, nonmutated, physiologically regulated RTK to evade targeted treatment.

mTOR complex 2 controls glycolytic metabolism in glioblastoma through FoxO acetylation and upregulation of c-Myc.

Aerobic glycolysis (the Warburg effect) is a core hallmark of cancer, but the molecular mechanisms underlying it remain unclear. Here, we identify an unexpected central role for mTORC2 in cancer metabolic reprogramming where it controls glycolytic metabolism by ultimately regulating the cellular level of c-Myc. We show that mTORC2 promotes inactivating phosphorylation of class IIa histone deacetylases, which leads to the acetylation of FoxO1 and FoxO3, and this in turn releases c-Myc from a suppressive miR-34c-dependent network. These central features of activated mTORC2 signaling, acetylated FoxO, and c-Myc levels are highly intercorrelated in clinical samples and with shorter survival of GBM patients. These results identify a specific, Akt-independent role for mTORC2 in regulating glycolytic metabolism in cancer.

EGFR mutation-induced alternative splicing of Max contributes to growth of glycolytic tumors in brain cancer.

Alternative splicing contributes to diverse aspects of cancer pathogenesis including altered cellular metabolism, but the specificity of the process or its consequences are not well understood. We characterized genome-wide alternative splicing induced by the activating EGFRvIII mutation in glioblastoma (GBM). EGFRvIII upregulates the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 splicing factor, promoting glycolytic gene expression and conferring significantly shorter survival in patients. HnRNPA1 promotes splicing of a transcript encoding the Myc-interacting partner Max, generating Delta Max, an enhancer of Myc-dependent transformation. Delta Max, but not full-length Max, rescues Myc-dependent glycolytic gene expression upon induced EGFRvIII loss, and correlates with hnRNPA1 expression and downstream Myc-dependent gene transcription in patients. Finally, Delta Max is shown to promote glioma cell proliferation in vitro and augment EGFRvIII expressing GBM growth in vivo. These results demonstrate an important role for alternative splicing in GBM and identify Delta Max as a mediator of Myc-dependent tumor cell metabolism.

An essential requirement for the SCAP/SREBP signaling axis to protect cancer cells from lipotoxicity.

The sterol regulatory element-binding proteins (SREBP) are key transcriptional regulators of lipid metabolism and cellular growth. It has been proposed that SREBP signaling regulates cellular growth through its ability to drive lipid biosynthesis. Unexpectedly, we find that loss of SREBP activity inhibits cancer cell growth and viability by uncoupling fatty acid synthesis from desaturation. Integrated lipid profiling and metabolic flux analysis revealed that cancer cells with attenuated SREBP activity maintain long-chain saturated fatty acid synthesis, while losing fatty acid desaturation capacity. We traced this defect to the uncoupling of fatty acid synthase activity from stearoyl-CoA desaturase 1 (SCD1)-mediated desaturation. This deficiency in desaturation drives an imbalance between the saturated and monounsaturated fatty acid pools resulting in severe lipotoxicity. Importantly, replenishing the monounsaturated fatty acid pool restored growth to SREBP-inhibited cells. These studies highlight the importance of fatty acid desaturation in cancer growth and provide a novel mechanistic explanation for the role of SREBPs in cancer metabolism.

The mTOR kinase inhibitors, CC214-1 and CC214-2, preferentially block the growth of EGFRvIII-activated glioblastomas.

PURPOSE: mTOR pathway hyperactivation occurs in approximately 90% of glioblastomas, but the allosteric mTOR inhibitor rapamycin has failed in the clinic. Here, we examine the efficacy of the newly discovered ATP-competitive mTOR kinase inhibitors CC214-1 and CC214-2 in glioblastoma, identifying molecular determinants of response and mechanisms of resistance, and develop a pharmacologic strategy to overcome it. EXPERIMENTAL DESIGN: We conducted in vitro and in vivo studies in glioblastoma cell lines and an intracranial model to: determine the potential efficacy of the recently reported mTOR kinase inhibitors CC214-1 (in vitro use) and CC214-2 (in vivo use) at inhibiting rapamycin-resistant signaling and blocking glioblastoma growth and a novel single-cell technology-DNA Encoded Antibody Libraries-was used to identify mechanisms of resistance. RESULTS: Here, we show that CC214-1 and CC214-2 suppress rapamycin-resistant mTORC1 signaling, block mTORC2 signaling, and significantly inhibit the growth of glioblastomas in vitro and in vivo. EGFRvIII expression and PTEN loss enhance sensitivity to CC214 compounds, consistent with enhanced efficacy in strongly mTOR-activated tumors. Importantly, CC214 compounds potently induce autophagy, preventing tumor cell death. Genetic or pharmacologic inhibition of autophagy greatly sensitizes glioblastoma cells and orthotopic xenografts to CC214-1- and CC214-2-induced cell death. CONCLUSIONS: These results identify CC214-1 and CC214-2 as potentially efficacious mTOR kinase inhibitors in glioblastoma, and suggest a strategy for identifying patients most likely to benefit from mTOR inhibition. In addition, this study also shows a central role for autophagy in preventing mTOR-kinase inhibitor-mediated tumor cell death, and suggests a pharmacologic strategy for overcoming it.

An LXR agonist promotes glioblastoma cell death through inhibition of an EGFR/AKT/SREBP-1/LDLR-dependent pathway.

Glioblastoma (GBM) is the most common malignant primary brain tumor of adults and one of the most lethal of all cancers. Epidermal growth factor receptor (EGFR) mutations (EGFRvIII) and phosphoinositide 3-kinase (PI3K) hyperactivation are common in GBM, promoting tumor growth and survival, including through sterol regulatory element-binding protein 1 (SREBP-1)-dependent lipogenesis. The role of cholesterol metabolism in GBM pathogenesis, its association with EGFR/PI3K signaling, and its potential therapeutic targetability are unknown. In our investigation, studies of GBM cell lines, xenograft models, and GBM clinical samples, including those from patients treated with the EGFR tyrosine kinase inhibitor lapatinib, uncovered an EGFRvIII-activated, PI3K/SREBP-1-dependent tumor survival pathway through the low-density lipoprotein receptor (LDLR). Targeting LDLR with the liver X receptor (LXR) agonist GW3965 caused inducible degrader of LDLR (IDOL)-mediated LDLR degradation and increased expression of the ABCA1 cholesterol efflux transporter, potently promoting tumor cell death in an in vivo GBM model. These results show that EGFRvIII can promote tumor survival through PI3K/SREBP-1-dependent upregulation of LDLR and suggest a role for LXR agonists in the treatment of GBM patients.

Oncogenic EGFR signaling activates an mTORC2-NF-κB pathway that promotes chemotherapy resistance.

UNLABELLED: Although it is known that mTOR complex 2 (mTORC2) functions upstream of Akt, the role of this protein kinase complex in cancer is not well understood. Through an integrated analysis of cell lines, in vivo models, and clinical samples, we demonstrate that mTORC2 is frequently activated in glioblastoma (GBM), the most common malignant primary brain tumor of adults. We show that the common activating epidermal growth factor receptor (EGFR) mutation (EGFRvIII) stimulates mTORC2 kinase activity, which is partially suppressed by PTEN. mTORC2 signaling promotes GBM growth and survival and activates NF-κB. Importantly, this mTORC2-NF-κB pathway renders GBM cells and tumors resistant to chemotherapy in a manner independent of Akt. These results highlight the critical role of mTORC2 in the pathogenesis of GBM, including through the activation of NF-κB downstream of mutant EGFR, leading to a previously unrecognized function in cancer chemotherapy resistance. These findings suggest that therapeutic strategies targeting mTORC2, alone or in combination with chemotherapy, will be effective in the treatment of cancer. SIGNIFICANCE: This study demonstrates that EGFRvIII-activated mTORC2 signaling promotes GBM proliferation, survival, and chemotherapy resistance through Akt-independent activation of NF-κB. These results highlight the role of mTORC2 as an integrator of two canonical signaling networks that are commonly altered in cancer, EGFR/phosphoinositide-3 kinase (PI3K) and NF-κB. These results also validate the importance of mTORC2 as a cancer target and provide new insights into its role in mediating chemotherapy resistance, suggesting new treatment strategies.

mTOR signaling in glioblastoma: lessons learned from bench to bedside.

Phosphatidyl-inositol-3 kinases (PI3Ks) constitute a family of intracellular lipid kinases that are frequently hyperactivated in glioblastoma. The PI3K complex links growth factor signaling with cellular proliferation, differentiation, metabolism, and survival. Mammalian target of rapamycin (mTOR) acts both as a downstream effector and upstream regulator of PI3K, thus highlighting its importance in glioblastoma. This review highlights laboratory and clinical evidence of mTOR's role in glioblastoma. Mechanisms of escape from mTOR inhibition are also discussed, as well as future clinical strategies of mTOR inhibition.