Combination anti-CXCR4 and anti-PD-1 immunotherapy provides survival benefit in glioblastoma through immune cell modulation of tumor microenvironment.

BACKGROUND: Emerging evidence suggests that myeloid cells play a critical role in glioblastoma (GBM) immunosuppression. Disappointing results of recent checkpoint inhibitor trials suggest that combination immunotherapy with alternative agents could be fruitful in overcoming immunosuppression. Overexpression of chemokine receptor CXCR4 is associated with poor prognosis in GBM. We investigate the treatment effects of combination immunotherapy with anti-PD-1 and anti-CXCR4 in a murine glioma model. METHODS: C57BL/6 mice were implanted with GL261-Luc+ glioma cells and randomized into 4 arms: (1) control (2) anti-PD-1 (3) anti-CXCR4, and (4) anti-PD-1 and anti-CXCR4 therapy. Overall survival and median survival were assessed. Cell populations were assessed by flow cytometry. RESULTS: Combination therapy conferred a significant survival benefit compared to control and monotherapy arms. Mice that received combination therapy demonstrated immune memory and decreased populations of immunosuppressive tumor-infiltrating leukocytes, such as monocytic myeloid-derived suppressor cells and microglia within the brain. Furthermore, combination therapy improved CD4+/CD8+ ratios in the brain as well as contributed to increased levels of pro-inflammatory cytokines. CONCLUSIONS: Anti-CXCR4 and anti-PD-1 combination immunotherapy modulates tumor-infiltrating populations of the glioma microenvironment. Targeting myeloid cells with anti-CXCR4 facilitates anti-PD-1 to promote an antitumor immune response and improved survival rates.

A Novel, Fully Human Anti-fucosyl-GM1 Antibody Demonstrates Potent In Vitro and In Vivo Antitumor Activity in Preclinical Models of Small Cell Lung Cancer.

Purpose: The ganglioside fucosyl-GM1 (FucGM1) is a tumor-associated antigen expressed in a large percentage of human small cell lung cancer (SCLC) tumors, but absent in most normal adult tissues, making it a promising target in immuno-oncology. This study was undertaken to evaluate the preclinical efficacy of BMS-986012, a novel, nonfucosylated, fully human IgG1 antibody that binds specifically to FucGM1.Experimental Design: The antitumor activity of BMS-986012 was evaluated in in vitro assays using SCLC cells and in mouse xenograft and syngeneic tumor models, with and without chemotherapeutic agents and checkpoint inhibitors.Results: BMS-986012 showed a high binding affinity for FcγRIIIa (CD16), which resulted in enhanced antibody-dependent cellular cytotoxicity (ADCC) against FucGM1-expressing tumor cell lines. BMS-986012-mediated tumor cell killing was also observed in complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) assays. In several mouse SCLC models, BMS-986012 demonstrated efficacy and was well tolerated. In the DMS79 xenograft model, tumor regression was achieved with BMS-986012 doses of 0.3 mg/kg and greater; antitumor activity was enhanced when BMS-986012 was combined with standard-of-care cisplatin or etoposide. In a syngeneic model, tumors derived from a genetically engineered model of SCLC were treated with BMS-986012 or anti-FucGM1 with a mouse IgG2a Fc and their responses evaluated; when BMS-986012 was combined with anti-PD-1 or anti-CD137 antibody, therapeutic responses significantly improved.Conclusions: Single-agent BMS-986012 demonstrated robust antitumor activity, with the addition of chemotherapeutic or immunomodulatory agents further inhibiting SCLC growth in the same models. These preclinical data supported evaluation of BMS-986012 in a phase I clinical trial of patients with relapsed, refractory SCLC. Clin Cancer Res; 24(20); 5178-89. ©2018 AACR.

Dynamic ligand modulation of EPO receptor pools, and dysregulation by polycythemia-associated EPOR alleles.

Erythropoietin (EPO) and its cell surface receptor (EPOR) are essential for erythropoiesis; can modulate non-erythroid target tissues; and have been reported to affect the progression of certain cancers. Basic studies of EPOR expression and trafficking, however, have been hindered by low-level EPOR occurrence, and the limited specificity of anti-EPOR antibodies. Consequently, these aspects of EPOR biology are not well defined, nor are actions of polycythemia- associated mutated EPOR alleles. Using novel rabbit monoclonal antibodies to intracellular, PY- activated and extracellular EPOR domains, the following properties of the endogenous hEPOR in erythroid progenitors first are unambiguously defined. 1) High- Mr EPOR forms become obviously expressed only when EPO is limited. 2) EPOR-68K plus -70K species sequentially accumulate, and EPOR-70K comprises an apparent cell surface EPOR population. 3) Brefeldin A, N-glycanase and associated analyses point to EPOR-68K as a core-glycosylated intracellular EPOR pool (of modest size). 4) In contrast to recent reports, EPOR inward trafficking is shown (in UT7epo cells, and primary proerythroblasts) to be sharply ligand-dependent. Beyond this, when C-terminal truncated hEPOR-T mutant alleles as harbored by polycythemia patients are co-expressed with the wild-type EPOR in EPO-dependent erythroid progenitors, several specific events become altered. First, EPOR-T alleles are persistently activated upon EPO- challenge, yet are also subject to apparent turn-over (to low-Mr EPOR products). Furthermore, during exponential cell growth EPOR-T species become both over-represented, and hyper-activated. Interestingly, EPOR-T expression also results in an EPO dose-dependent loss of endogenous wild-type EPOR's (and, therefore, a squelching of EPOR C-terminal- mediated negative feedback effects). New knowledge concerning regulated EPOR expression and trafficking therefore is provided, together with new insight into mechanisms via which mutated EPOR-T polycythemia alleles dysregulate the erythron. Notably, specific new tools also are characterized for studies of EPOR expression, activation, action and metabolism.

Allosteric inhibition of lysyl oxidase-like-2 impedes the development of a pathologic microenvironment.

We have identified a new role for the matrix enzyme lysyl oxidase-like-2 (LOXL2) in the creation and maintenance of the pathologic microenvironment of cancer and fibrotic disease. Our analysis of biopsies from human tumors and fibrotic lung and liver tissues revealed an increase in LOXL2 in disease-associated stroma and limited expression in healthy tissues. Targeting LOXL2 with an inhibitory monoclonal antibody (AB0023) was efficacious in both primary and metastatic xenograft models of cancer, as well as in liver and lung fibrosis models. Inhibition of LOXL2 resulted in a marked reduction in activated fibroblasts, desmoplasia and endothelial cells, decreased production of growth factors and cytokines and decreased transforming growth factor-beta (TGF-beta) pathway signaling. AB0023 outperformed the small-molecule lysyl oxidase inhibitor beta-aminoproprionitrile. The efficacy and safety of LOXL2-specific AB0023 represents a new therapeutic approach with broad applicability in oncologic and fibrotic diseases.

Immunogold electron microscopic demonstration of distinct submembranous localization of the activated gammaPKC depending on the stimulation.

We examined the precise intracellular translocation of gamma subtype of protein kinase C (gammaPKC) after various extracellular stimuli using confocal laser-scanning fluorescent microscopy (CLSM) and immunogold electron microscopy. By CLSM, treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a slow and irreversible accumulation of green fluorescent protein (GFP)-tagged gammaPKC (gammaPKC-GFP) on the plasma membrane. In contrast, treatment with Ca(2+) ionophore and activation of purinergic or NMDA receptors induced a rapid and transient membrane translocation of gammaPKC-GFP. Although each stimulus resulted in PKC localization at the plasma membrane, electron microscopy revealed that gammaPKC showed a subtle but significantly different localization depending on stimulation. Whereas TPA and UTP induced a sustained localization of gammaPKC-GFP on the plasma membrane, Ca(2+) ionophore and NMDA rapidly translocated gammaPKC-GFP to the plasma membrane and then restricted gammaPKC-GFP in submembranous area (<500 nm from the plasma membrane). These results suggest that Ca(2+) influx alone induced the association of gammaPKC with the plasma membrane for only a moment and then located this enzyme at a proper distance in a touch-and-go manner, whereas diacylglycerol or TPA tightly anchored this enzyme on the plasma membrane. The distinct subcellular targeting of gammaPKC in response to various stimuli suggests a novel mechanism for PKC activation.

Acute functional tolerance to ethanol mediated by protein kinase Cepsilon.

A low level of response to ethanol is associated with increased risk of alcoholism. A major determinant of the level of response is the capacity to develop acute functional tolerance (AFT) to ethanol during a single drinking session. Mice lacking protein kinase C epsilon (PKCepsilon) show increased signs of ethanol intoxication and reduced ethanol self-administration. Here, we report that AFT to the motor-impairing effects of ethanol is reduced in PKCepsilon (-/-) mice when compared with wild-type littermates. In wild-type mice, in vivo ethanol exposure produced AFT that was accompanied by increased phosphorylation of PKCepsilon and resistance of GABA(A) receptors to ethanol. In contrast, in PKCepsilon (-/-) mice, GABA(A) receptor sensitivity to ethanol was unaltered by acute in vivo ethanol exposure. Both PKCepsilon (-/-) and PKCepsilon (+/+) mice developed robust chronic tolerance to ethanol, but the presence of chronic tolerance did not change ethanol preference drinking. These findings suggest that ethanol activates a PKCepsilon signaling pathway that contributes to GABA(A) receptor resistance to ethanol and to AFT. AFT can be genetically dissociated from chronic tolerance, which is not regulated by PKCepsilon and does not alter PKCepsilon modulation of ethanol preference.

Immunocytochemical localization of a neuron-specific diacylglycerol kinase beta and gamma in the developing rat brain.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA) and is, therefore, a potential terminator of DG signaling. DG and PA are important intracellular second messengers. DG directly binds protein kinase C (PKC) then activates this multifunctional enzyme. Ca2+-dependent and brain-specific DGKs, alpha, beta, and gamma, are suggested to play pivotal roles in the central nervous system. To elucidate the DGK function in neuronal development, we studied the developmental changes of DGKalpha, beta, and gamma in the postnatal rat brain. By immunoblot analysis, DGKalpha and gamma subtypes were present at birth and then gradually increased, while DGKbeta was not present at birth or postnatal day 3, then increased rapidly from day 14 to reach maximum at day 28. Immunohistochemically, DGKbeta and gamma were distributed in different brain regions. In most brain regions, DGKgamma showed sustained expression throughout the postnatal developmental periods. Interestingly, a temporal expression of DGKgamma was observed in the medial geniculate nucleus during day 3 to 14, and a delay of DGKgamma expression was seen in Purkinje cells, which was coincident with dendritic growth of Purkinje cells. In the hippocampal pyramidal cell, both DGKbeta and gamma were abundant but subcellular localization was different. DGKgamma localized in the cytosol while DGKbeta localized along the membrane structure. These findings suggest that each DGK subtype has a spatio-temporally different function in the developmental neurons.

Phenotypic switch from paracrine to autocrine role of hepatocyte growth factor in an androgen-independent human prostatic carcinoma cell line, CWR22R.

Support mechanisms involved in growth of androgen-independent prostate cancer are primarily unknown. Hepatocyte growth factor (HGF)/Met has been suggested to be one of them based primarily on immunohistochemical studies. We conducted a series of experiments to assess the role of the HGF/Met system in an androgen-dependent human prostate carcinoma, CWR22 and its androgen-independent derivative, CWR22R. We found that action of HGF changed from paracrine to autocrine in progression to androgen-independent state. CWR22 tumors did not express HGF but expressed Met, whereas prostate stromal cells expressed HGF at a high level. Growth of CWR22 was stimulated either by addition of HGF to the culture or by the presence of prostate stromal cells. On the other hand, CWR22R cells expressed both HGF and Met. Knockdown of Met expression by RNA interference method suppressed the growth of CWR22R cells. Our data suggest that HGF is intimately involved in growth of human prostate cancer and that progression from the androgen-dependent to the androgen-independent state is associated with an adaptive switch in support mechanism from paracrine to autocrine. Our data offer one mechanism to account for androgen-independent human cancer growth.