TMOD-04. T-TYPE CALCIUM CHANNELS DRIVE GLIOBLASTOMA INITIATION AND PROGRESSION

Glioblastoma is the most common primary malignant brain tumor with a dismal median survival of 15 months. Calcium signaling regulates a plethora of cellular processes making it an ideal target for therapeutic intervention. Our group identified T-type calcium channels (TTCCs) as upregulated in GBM cells, stem cells and human tumors. Analysis of the Cancer Genome Atlas (TCGA) demonstrates for the first time that >20% of GBM patients exhibit mutation, amplification or mRNA upregulation of TTCCs. TCGA and Chinese Glioma Genome Atlas (CGGA) data analyses demonstrate that patients with alterations in TTCC trend toward worse survival compared to normal TTCC expression. Our group utilized an FDA repurposed TTCC blocker Mibefradil to demonstrate inhibition of TTCCs decreases cancer associated signaling, transcription, malignancy parameters and in vivo tumor growth. Mibefradil treatment sensitized Gliomas stem like cells (GSC) xenografts to temozolomide (TMZ) chemotherapy and radiotherapy. To elucidate the roles of the individual TTCCs in vivo we created RCAS/Tva-shTTCC mice for silencing of Cav3.1 and Cav3.2. The silencing of the TTCCs individually decreased tumor growth as measured by MRI and H&E staining. Silencing of TTCCs extended survival of mice in vivo. Silencing of TTCC decreases proliferation (Ki67) and increases apoptosis (Caspase 3). Our findings suggest T-type calcium channels contribute to tumor initiation and progression.

Myelin-Independent Therapeutic Potential of Canine Glial-Restricted Progenitors Transplanted in Mouse Model of Dysmyelinating Disease

Background: Dysfunction of glia contributes to the deterioration of the central nervous system in a wide array of neurological disorders, thus global replacement of glia is very attractive. Human glial-restricted precursors (hGRPs) transplanted intraventricularly into neonatal mice extensively migrated and rescued the lifespan in half of the studied mice, whereas mouse GRPs (mGRPs) presented no therapeutic benefit. We studied in the same experimental setting canine GRPs (cGRP) to determine whether their therapeutic potential falls between hGRPs and mGRPs. Additional motivation for the selection of cGRPs was a potential for use in veterinary medicine. Methods: cGRPs were extracted from the brain of dog fetuses. The cells were transplanted into the anterior or posterior aspect of the lateral ventricle (LV) of neonatal, immunodeficient, dysmyelinated mice (Mbpshi, Rag2 KO; shiv/rag2). Outcome measures included early cell biodistribution, animal survival and myelination assessed with MRI, immunohistochemistry and electron microscopy. Results: Grafting of cGRP into posterior LV significantly extended animal survival, whereas no benefit was observed after anterior LV transplantation. In contrast, myelination of the corpus callosum was more prominent in anteriorly transplanted animals. Conclusions: The extended survival of animals after transplantation of cGRPs could be explained by the vicinity of the transplant near the brain stem.

BCL-2 Inhibitor ABT-737 Effectively Targets Leukemia-Initiating Cells with Differential Regulation of Relevant Genes Leading to Extended Survival in a NRAS/BCL-2 Mouse Model of High Risk-Myelodysplastic Syndrome

During transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment.

Nanoparticle-mediated delivery of IL-2 to T follicular helper cells protects BDF1 mice from lupus-like disease

We recently reported that poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with interleukin (IL)-2 and targeted to T cells inhibited the development of lupus-like disease in BDF1 mice by inducing functional T regulatory cells (Tregs). Here we show that the protection from disease and the extended survival of BDF1 mice provided by IL-2-loaded NPs targeted to T cells is not only due to an induction of Tregs but also contributed by an inhibition of T follicular helper (TFH) cells. These results identify a dual protective activity of IL-2 in the control of lupus autoimmunity, namely the inhibition of effector TFH cells, in addition to the previously known induction of Tregs. This newly recognized activity of IL-2 delivered by NPs can help better explain the beneficial effects of low-dose IL-2 immunotherapy in systemic lupus erythematosus (SLE), and might be considered as a new strategy to slow disease progression and improve outcomes in lupus patients.

Metallomic profile in non-cirrhotic hepatocellular carcinoma supports a phenomenon of metal metabolism adaptation in tumor cells

We have previously described a form of hepatocellular carcinoma (HCC) in non-cirrhotic liver (HCC-NC) developed by Peruvian patients. We analyzed the metallomic profile in hepatic tissues from two independent cohorts exhibiting HCC-NC. Clinical, histopathological data, and HCC and non-tumoral liver (NTL) samples of 38 Peruvian and 38 French HCC-NC patients, were studied. Twelve metals were quantified using ICP/MS: Mn, Fe, Cu, Co, Zn, As, Se, Rb, Mo, Cd, Pb, and Sn. Associations between metals and survival were assessed. Our data showed significant differences between cohorts. Mean ages were 40.6 ± 20, 67.5 ± 9 years old for Peruvians and French, respectively. Fifty percent of the Peruvian patients were positive for the HBsAg, versus 3% in French patients. Mn, Cu, Zn, As, Se, Rb, Mo, Cd, Sn metal concentrations were higher in NTL of Peruvians. Importantly, metal concentrations were lower in HCC areas compared to NTL tissues in both cohorts, except for Cu for which mean concentration was higher in HCC (p < 0.05). Se concentration in HCC was associated with extended survival only in Peruvians. Our data, obtained in Peruvian and French HCC-NC cohorts, highlights similarity in the metallomic profile of HCC compared to NTL during the hepatic tumorigenesis in these specific groups of patients.

Rate of Freeze Impacts the Survival and Immune Responses Post Cryoablation of Melanoma

The emergence of ablative therapies has revolutionized the treatment of inoperable solid tumors. Cryoablation stands out for its uniqueness of operation based on hypothermia, and for its ability to unleash the native tumor antigens, resulting in the generation of anti-tumor immune responses. It is not clearly understood how alterations in the rate of freeze impact the immune response outcomes. In this study, we tested fast freeze and slow freeze rates for their locoregional effectiveness and their ability to elicit immune responses in a B16F10 mouse model of melanoma. Tumor bearing mice treated with fast freeze protocol survived better than the ones treated with slow freeze protocol. Fast freeze resulted in a higher magnitude of CD4+ and CD8+ T-cell responses, and a significantly extended survival post re-challenge. Thus, fast freeze rate should be applied in any future studies employing cryoablation as an in vivo vaccination tool in conjunction with targeted immunotherapies.