A Virtual Screening Platform Identifies Chloroethylagelastatin A as a Potential Ribosomal Inhibitor.

Chloroethylagelastatin A (CEAA) is an analogue of agelastatin A (AA), a natural alkaloid derived from a marine sponge. It is under development for therapeutic use against brain tumors as it has excellent central nervous system (CNS) penetration and pre-clinical therapeutic activity against brain tumors. Recently, AA was shown to inhibit protein synthesis by binding to the ribosomal A-site. In this study, we developed a novel virtual screening platform to perform a comprehensive screening of various AA analogues showing that AA analogues with proven therapeutic activity including CEAA have significant ribosomal binding capacity whereas therapeutically inactive analogues show poor ribosomal binding and revealing structural fingerprint features essential for drug-ribosome interactions. In particular, CEAA was found to have greater ribosomal binding capacity than AA. Biological tests showed that CEAA binds the ribosome and contributes to protein synthesis inhibition. Our findings suggest that CEAA may possess ribosomal inhibitor activity and that our virtual screening platform may be a useful tool in discovery and development of novel ribosomal inhibitors.

Pomalidomide Alters Pancreatic Macrophage Populations to Generate an Immune-Responsive Environment at Precancerous and Cancerous Lesions.

During development of pancreatic cancer, alternatively activated macrophages contribute to fibrogenesis, pancreatic intraepithelial neoplasia (PanIN) lesion growth, and generation of an immunosuppressive environment. Here, we show that the immunomodulatory agent pomalidomide depletes pancreatic lesion areas of alternatively activated macrophage populations. Pomalidomide treatment resulted in downregulation of interferon regulatory factor 4, a transcription factor for M2 macrophage polarization. Pomalidomide-induced absence of alternatively activated macrophages led to a decrease in fibrosis at PanIN lesions and in syngeneic tumors; this was due to generation of an inflammatory, immune-responsive environment with increased expression of IL1α and presence of activated (IFNγ-positive) CD4+ and CD8+ T-cell populations. Our results indicate that pomalidomide could be used to decrease fibrogenesis in pancreatic cancer and may be ideal as a combination treatment with chemotherapeutic drugs or other immunotherapies. SIGNIFICANCE: These findings reveal new insights into how macrophage populations within the pancreatic cancer microenvironment can be modulated, providing the means to turn the microenvironment from immunosuppressive to immune-responsive.

Combined alkylation and histone deacetylase inhibition with EDO-S101 has significant therapeutic activity against brain tumors in preclinical models.

There is a clear unmet need for novel therapeutic agents for management of primary and secondary brain tumors. Novel therapeutic agents with excellent central nervous system (CNS) penetration and therapeutic activity are urgently needed. EDO-S101 is a novel alkylating and histone deacetylase inhibiting agent created by covalent fusion of bendamustine and vorinostat. We used murine models to perform CNS pharmacokinetic analysis and preclinical therapeutic evaluation of EDO-S101 for CNS lymphoma, metastatic triple-negative breast cancer of the brain, and glioblastoma multiforme. EDO-S101 has excellent CNS penetration of 13.8% and 16.5% by intravenous infusion and bolus administration respectively. It shows promising therapeutic activity against CNS lymphoma, metastatic triple-negative breast cancer of the brain, and glioblastoma multiforme with significant prolongation of survival compared to no-treatment controls. Therapeutic activity was higher with IV infusion compared to IV bolus. It should be evaluated further for therapeutic use in brain tumors.

T-Cell/Histiocyte-Rich Large B-Cell Lymphoma Presenting as a Primary Central Nervous System Lymphoma.

Primary central nervous system (PCNSL) lymphoma is an aggressive extranodal non-Hodgkin lymphoma, and most cases are classified as diffuse large B-cell lymphoma (DLBCL) by histology. T-cell/histiocyte-rich large B-cell lymphoma (TCRLBCL) represents a distinct subtype of diffuse large B-cell lymphoma and is characterized by the presence of scattered large neoplastic B-cells in a background of abundant T-cells and histiocytes. This is in contrast to the dense perivascular cuffing of neoplastic B-cells in classic DLBCL. T-cell/histiocyte-rich large B-cell lymphoma should be considered in PCNSL cases in which neoplastic B-cells are sparse and scattered. Immunohistochemistry will help identify the B-cells and surrounding infiltrate rich in Tlymphocytes and histiocytes. Future studies exploring the biology of TCRLBCL and the crosstalk between the neoplastic cells and the surrounding inflammatory infiltrate may provide exciting prospects for future therapies for TCRLBCL.

Pomalidomide shows significant therapeutic activity against CNS lymphoma with a major impact on the tumor microenvironment in murine models.

Primary CNS lymphoma carries a poor prognosis. Novel therapeutic agents are urgently needed. Pomalidomide (POM) is a novel immunomodulatory drug with anti-lymphoma activity. CNS pharmacokinetic analysis was performed in rats to assess the CNS penetration of POM. Preclinical evaluation of POM was performed in two murine models to assess its therapeutic activity against CNS lymphoma. The impact of POM on the CNS lymphoma immune microenvironment was evaluated by immunohistochemistry and immunofluorescence. In vitro cell culture experiments were carried out to further investigate the impact of POM on the biology of macrophages. POM crosses the blood brain barrier with CNS penetration of ~ 39%. Preclinical evaluations showed that it had significant therapeutic activity against CNS lymphoma with significant reduction in tumor growth rate and prolongation of survival, that it had a major impact on the tumor microenvironment with an increase in macrophages and natural killer cells, and that it decreased M2-polarized tumor-associated macrophages and increased M1-polarized macrophages when macrophages were evaluated based on polarization status. In vitro studies using various macrophage models showed that POM converted the polarization status of IL4-stimulated macrophages from M2 to M1, that M2 to M1 conversion by POM in the polarization status of lymphoma-associated macrophages is dependent on the presence of NK cells, that POM induced M2 to M1 conversion in the polarization of macrophages by inactivating STAT6 signaling and activating STAT1 signaling, and that POM functionally increased the phagocytic activity of macrophages. Based on our findings, POM is a promising therapeutic agent for CNS lymphoma with excellent CNS penetration, significant preclinical therapeutic activity, and a major impact on the tumor microenvironment. It can induce significant biological changes in tumor-associated macrophages, which likely play a major role in its therapeutic activity against CNS lymphoma. POM should be further evaluated in clinical trials.

LRRTM3 interacts with APP and BACE1 and has variants associating with late-onset Alzheimer's disease (LOAD).

Leucine rich repeat transmembrane protein 3 (LRRTM3) is member of a synaptic protein family. LRRTM3 is a nested gene within α-T catenin (CTNNA3) and resides at the linkage peak for late-onset Alzheimer's disease (LOAD) risk and plasma amyloid ß (Aß) levels. In-vitro knock-down of LRRTM3 was previously shown to decrease secreted Aß, although the mechanism of this is unclear. In SH-SY5Y cells overexpressing APP and transiently transfected with LRRTM3 alone or with BACE1, we showed that LRRTM3 co-localizes with both APP and BACE1 in early endosomes, where BACE1 processing of APP occurs. Additionally, LRRTM3 co-localizes with APP in primary neuronal cultures from Tg2576 mice transduced with LRRTM3-expressing adeno-associated virus. Moreover, LRRTM3 co-immunoprecipitates with both endogenous APP and overexpressed BACE1, in HEK293T cells transfected with LRRTM3. SH-SY5Y cells with knock-down of LRRTM3 had lower BACE1 and higher CTNNA3 mRNA levels, but no change in APP. Brain mRNA levels of LRRTM3 showed significant correlations with BACE1, CTNNA3 and APP in ∼400 humans, but not in LRRTM3 knock-out mice. Finally, we assessed 69 single nucleotide polymorphisms (SNPs) within and flanking LRRTM3 in 1,567 LOADs and 2,082 controls and identified 8 SNPs within a linkage disequilibrium block encompassing 5'UTR-Intron 1 of LRRTM3 that formed multilocus genotypes (MLG) with suggestive global association with LOAD risk (p = 0.06), and significant individual MLGs. These 8 SNPs were genotyped in an independent series (1,258 LOADs and 718 controls) and had significant global and individual MLG associations in the combined dataset (p = 0.02-0.05). Collectively, these results suggest that protein interactions between LRRTM3, APP and BACE1, as well as complex associations between mRNA levels of LRRTM3, CTNNA3, APP and BACE1 in humans might influence APP metabolism and ultimately risk of AD.

Apoptotic non-beta cells suppress beta cell antigen-reactive T cells and induce beta cell antigen-specific regulatory T cells.

Steady-state cell apoptosis plays an important role in maintenance of self-tolerance. Based on this notion, the use of apoptotic cells to restore self-tolerance to beta cell antigens is a rational approach to type 1 diabetes (T1D) prevention. Our previous study demonstrated that transfusion of apoptotic beta cells induced immune tolerance to beta cell antigens in NOD mice. However, concerned about the limited beta cell source for future clinical applications, we attempted in the present study to develop a more practical approach for T1D prevention using apoptotic non-beta cells. We found that UVB-irradiation-induced apoptotic NOD splenic stromal cells significantly suppressed beta cell antigen-specific T cell proliferation in vitro and in vivo. Furthermore, TCR-transgenic CD4(+) T cells primed by the antigens to which they were specific in the presence of UVB-irradiated stromal cells were rendered unresponsive to the antigen restimulation, a result that was partially attributed to the induced IL-10-producing regulatory T cells. Of more interest, transfusion of UVB-irradiated stromal cells appeared to induce beta cell antigen-responding IL-10-producing regulatory T cells in vivo. Most importantly, transfusion of UVB-irradiated stromal cells effectively prevented T1D in NOD mice, which is consistent with these findings. This study suggests that it is possible to use apoptotic non-beta cells such as peripheral blood mononuclear cells to induce beta cell antigen-specific tolerance, thereby preventing T1D in humans.

Infusion of UVB-treated splenic stromal cells induces suppression of beta cell antigen-specific T cell responses in NOD mice.

Our previous study has demonstrated that transfusion of UVB-irradiation-induced apoptotic beta cells effectively prevents type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. However, the limitation of beta cell source would preclude the clinical application of this approach. Therefore, in the present study, we have attempted to establish a more practical approach by utilizing apoptotic non-beta cells to prevent T1D. We find that apoptotic splenic stromal cells significantly suppress beta cell antigen-reactive T cell proliferation in vitro and in vivo. Moreover, beta cell antigen-specific T cells primed by beta cell antigens in the presence of apoptotic stromal cells have markedly reduced responsiveness to the re-stimulation of the same beta cell antigen. We also find that beta cell antigen-specific IL-10-producing CD4+ T cells are induced in the presence of apoptotic splenic stromal cells. As expected, transfusion of apoptotic stromal cells effectively protected NOD mice from developing T1D. Furthermore, the proliferation of adoptively transferred beta cell antigen-specific TCR-transgenic T cells in pancreatic draining lymph nodes is markedly suppressed in UVB-stroma-treated mice, indicating that UVB-stroma treatment induces immune tolerance to multiple beta cell antigens. This study provides an effective and convenient approach for managing T1D by utilizing apoptotic non-beta cells.

Transfusion of apoptotic beta-cells induces immune tolerance to beta-cell antigens and prevents type 1 diabetes in NOD mice.

In vivo induction of beta-cell apoptosis has been demonstrated to be effective in preventing type 1 diabetes in NOD mice. Based on the notion that steady-state cell apoptosis is associated with self-tolerance and the need for developing a more practical approach using apoptotic beta-cells to prevent type 1 diabetes, the current study was designed to investigate apoptotic beta-cells induced ex vivo in preventing type 1 diabetes. The NIT-1 cell line serves as a source of beta-cells. Apoptotic NIT-1 cells were prepared by ultraviolet B (UVB) irradiation. Three weekly transfusions of UVB-irradiated NIT-1 cells (1 x 10(5)/mouse) or PBS were used to determine whether transfusions of UVB-irradiated NIT-1 cells induce immune tolerance to beta-cell antigens in vivo and prevent type 1 diabetes. The suppression of anti-beta-cell antibodies, polarization of T-helper (Th) cells, and induction of regulatory T-cells by UVB-irradiated NIT-1 cell treatment were investigated. The transfusions of apoptotic NIT-1 cells suppress anti-beta-cell antibody development and induce Th2 responses and interleukin-10-producing regulatory type 1 cells. Importantly, this treatment significantly delays and prevents the onset of diabetes when 10-week-old NOD mice are treated. Adoptive transfer of splenocytes from UVB-irradiated NIT-1 cell-treated mice prevents diabetes caused by simultaneously injected diabetogenic splenocytes in NOD-Rag(-/-) mice. Moreover, the proliferation of adoptively transferred carboxyfluorescein diacetate succinimidyl ester-labeled beta-cell antigen-specific T-cell receptor-transgenic T-cells in UVB-irradiated NIT-1-cell treated mice is markedly suppressed. The transfusion of apoptotic beta-cells effectively protects against type 1 diabetes in NOD mice by inducing immune tolerance to beta-cell antigens. This approach has great potential for immune intervention for human type 1 diabetes.