Analyzing the Role of αvβ8 Integrin in Glioma-Induced Angiogenesis

In this study we aimed to determine the functional roles for αvβ8 integrin in astrocytoma-induced angiogenesis. These studies originate from our analyses of αvβ8 integrin in developmental brain angiogenesis. αv and β8 knockout (KO) mice develop brain-specific vascular phenotypes that resemble vascular pathologies observed in the malignant astrocytoma, glioblastoma multiforme (GBM). Indeed, a murine xenograft model of astrocytoma suggested a role for the integrin in glioma-induced angiogenesis. Primary mouse astroglia were cultured from wild type (WT) and β8 KO neonates and were immortalized (HPV:E6/E7) and transformed (HRas:G12V). WT and β8 KO transformed astroglia were intracranially injected into athymic mice. WT tumors displayed pathological features of grade III astrocytomas, whereas β8 KO tumors resembled grade IV GBMs. KO tumors contained widespread edema and hemorrhage as well as pathological angiogenesis, as assessed by quantitation of microvascular density and blood vessel morphology. Additionally, exogenous expression of β8 integrin in β8 KO transformed astroglia resolved the pathologies observed in KO tumors giving further credence to the idea that loss of αvβ8 integrin expression correlates with tumorigenic potential of oncogene-transformed astroglia. To compliment our mouse model, several established human glioma cell lines were characterized for expression of αvβ8 integrin protein. Some of the cell lines displayed low expression of αvβ8 integrin, whereas others showed high levels, as compared to non-malignant human astrocytes. Intracranial implantation of high and low β8 integrin-expressing human glioma cell lines resulted in tumors exhibiting similar phenotypes to those observed in the mouse model; low expressers were marked by vascular pathologies indicative of β8 KO mouse tumors. Upon overexpression of β8 integrin in a low β8 integrin-expressing human glioma cell line, angiogenic pathologies were largely resolved. Moreover, intracranially injected αvHI- and αvLO-sorted GBM stem cells (GSCs) resulted in significantly different tumor sizes, where those GSCs endogenously expressing low levels of αv integrin formed two to three fold larger tumors. Furthermore, lentiviral knockdown of β8 integrin in transformed human astrocytes formed tumors that strikingly recapitulated the characteristics of the murine β8-/- tumors, exhibiting a significant increase in microvascular density leading to decreased overall survival. A paracrine mechanism was discovered involving endothelial cell homeostatic control governed by canonical TGFβ signaling initiated by αvβ8 integrin’s role in the latent cytokine’s activation. Diminished TGFβ signaling in tumor-associated endothelial cells promoted increased angiogenesis and decreased overall survival as a result of αvβ8 integrin’s loss on the tumor cell. Collectively, these data suggest an important functional role for αvβ8 integrin in glioma angiogenesis

Efficacy of BET bromodomain inhibition in Kras-mutant non-small cell lung cancer

PurposeAmplification of MYC is one of the most common genetic alterations in lung cancer, contributing to a myriad of phenotypes associated with growth, invasion and drug resistance. Murine genetics has established both the centrality of somatic alterations of Kras in lung cancer, as well as the dependency of mutant Kras tumors on MYC function. Unfortunately, drug-like small-molecule inhibitors of KRAS and MYC have yet to be realized. The recent discovery, in hematologic malignancies, that BET bromodomain inhibition impairs MYC expression and MYC transcriptional function established the rationale of targeting KRAS-driven NSCLC with BET inhibition.Experimental DesignWe performed functional assays to evaluate the effects of JQ1 in genetically defined NSCLC cells lines harboring KRAS and/or LKB1 mutations. Furthermore, we evaluated JQ1 in transgenic mouse lung cancer models expressing mutant kras or concurrent mutant kras and lkb1. Effects of bromodomain inhibition on transcriptional pathways were explored and validated by expression analysis.ResultsWhile JQ1 is broadly active in NSCLC cells, activity of JQ1 in mutant KRAS NSCLC is abrogated by concurrent alteration or genetic knock-down of LKB1. In sensitive NSCLC models, JQ1 treatment results in the coordinate downregulation of the MYC-dependent transcriptional program. We found that JQ1 treatment produces significant tumor regression in mutant kras mice. As predicted, tumors from mutant kras and lkb1 mice did not respond to JQ1.ConclusionBromodomain inhibition comprises a promising therapeutic strategy for KRAS mutant NSCLC with wild-type LKB1, via inhibition of MYC function. Clinical studies of BET bromodomain inhibitors in aggressive NSCLC will be actively pursued

Activation of the PD-1 Pathway Contributes to Immune Escape in EGFR-Driven Lung Tumors

The success in lung cancer therapy with Programmed Death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between Epidermal Growth Factor Receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, cytotoxic T lymphocyte antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased cytotoxic T cells and increased markers of T cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T cell function and lowering the levels of tumor-promoting cytokines. Expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non-small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape, and mechanistically link treatment response to PD-1 inhibition

Metabolic and Functional Genomic Studies Identify Deoxythymidylate Kinase as a Target in LKB1-Mutant Lung Cancer

The LKB1/STK11 tumor suppressor encodes a serine/threonine kinase which coordinates cell growth, polarity, motility, and metabolism. In non-small cell lung cancer, LKB1 is somatically inactivated in 25-30% of cases, often concurrently with activating KRAS mutation. Here, we employed an integrative approach to define novel therapeutic targets in KRAS-driven LKB1 mutant lung cancers. High-throughput RNAi screens in lung cancer cell lines from genetically engineered mouse models driven by activated KRAS with or without coincident Lkb1 deletion led to the identification of Dtymk, encoding deoxythymidylate kinase which catalyzes dTTP biosynthesis, as synthetically lethal with Lkb1 deficiency in mouse and human lung cancer lines. Global metabolite profiling demonstrated that Lkb1-null cells had striking decreases in multiple nucleotide metabolites as compared to the Lkb1-wt cells. Thus, LKB1 mutant lung cancers have deficits in nucleotide metabolism conferring hypersensitivity to DTYMK inhibition, suggesting that DTYMK is a potential therapeutic target in this aggressive subset of tumors

β8 integrin regulates neurogenesis and neurovascular homeostasis in the adult brain

Central nervous system (CNS) neurovascular units are multicellular complexes consisting of neural cells, blood vessels and a milieu of extracellular matrix (ECM) proteins. ECM-mediated adhesion and signaling events within neurovascular units probably contribute to proper CNS development and physiology; however, the molecular mechanisms that control these events remain largely undetermined. Previous studies from our group and others showed that ablation of the ECM receptor, αvβ8 integrin, in neural progenitor cells (NPCs) of the embryonic mouse brain results in severe developmental neurovascular pathologies and premature death. Here, we have investigated the functions for this integrin in the adult brain by studying mice harboring a homozygous-null β8 gene mutation generated on an outbred background that permits survival for several months. We show that adult β8–/– mice display widespread defects in neurovascular unit homeostasis, including increased numbers of intracerebral blood vessels with pronounced perivascular astrogliosis. Furthermore, in neurogenic regions of the adult brain, where NPCs cluster around blood vessels in neurovascular niches, β8 integrin is essential for normal control of NPC proliferation and survival. Analysis of NPCs cultured ex vivo reveals that the growth and survival defects correlate, in part, with diminished integrin-mediated activation of latent transforming growth factor β1 (TGFβ1), which is an ECM protein ligand for αvβ8 integrin. Collectively, these data identify essential functions for β8 integrin in regulating neurovascular unit physiology in the post-natal mouse brain

The impact of the MYB-NFIB fusion proto-oncogene in vivo

Recurrent fusion of the v-myb avian myelobastosis viral oncogene homolog (MYB) and nuclear factor I/B (NFIB) generates the MYB-NFIB transcription factor, which has been detected in a high percentage of individuals with adenoid cystic carcinoma (ACC). To understand the functional role of this fusion protein in carcinogenesis, we generated a conditional mutant transgenic mouse that expresses MYB-NFIB along with p53 mutation in tissues that give rise to ACC: mammary tissue, salivary glands, or systemically in the whole body. Expression of the oncogene in mammary tissue resulted in hyperplastic glands that developed into adenocarcinoma in 27.3% of animals. Systemic expression of the MYB-NFIB fusion caused more rapid development of this breast phenotype, but mice died due to abnormal proliferation in the glomerular compartment of the kidney, which led to development of glomerulonephritis. These findings suggest the MYB-NFIB fusion is oncogenic and treatments targeting this transcription factor may lead to therapeutic responses in ACC patients

Activation of the PD-1 Pathway Contributes to Immune Escape in EGFR-Driven Lung Tumors

The success in lung cancer therapy with Programmed Death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between Epidermal Growth Factor Receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, cytotoxic T lymphocyte antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased cytotoxic T cells and increased markers of T cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T cell function and lowering the levels of tumor-promoting cytokines. Expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non-small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape, and mechanistically link treatment response to PD-1 inhibition