RESEARCH OVERVIEW

Dr. Maria G. Castro’s Research

Description of research program: My research program relates to brain cancer biology, molecular pathways which mediate DNA repair, genomic stability, and the development of immune-therapeutic approaches; manipulating the brain tumor microenvironment using gene therapy strategies, small molecules and blocking antibodies; neuro-immunology, i.e., in vivo migration of immune cells into the central nervous system and the brain tumor micro-environment; the role of pattern recognition receptors’ signaling in cancer immune suppression and brain inflammation; mechanisms mediated by myeloid deriver suppressor cells (MDSCs) in brain cancer biology and response to therapeutics.

Immune suppression in the tumor microenvironment (TME).

My lab has made major contributions related to the immune suppressive TME in glioma. My group uncovered the role of plasmacytoid dendritic cells as APCs within the TME; uptake of tumor antigens, migration to the draining LN and triggering an effective adaptive anti-tumor immune response, which results in the regression of a large tumor mass and anti-tumor immunological memory. We discovered that the immune cells which are present within the TME and are responsible for tumor immunity are BM derived. We described the role of Toll-like receptor 2 (TLR2) signaling on BM derived, tumor infiltrating dendritic cells (DCs) in mediating tumor regression, long-term survival and anti-tumor immunological memory in response to combined conditional cytotoxic/immune-stimulatory gene therapy. Further, we demonstrated that TLR2 activation was elicited by an endogenous, cancer cells’ derived TLR ligand, i.e., HMGB1 (a transcription factor that binds to DNA in living cells) which is released from dying cancer cells in response to cancer ablative therapies. We propose the use of circulating levels of HMGB1 as a biomarker to monitor therapeutic efficacy. We are also investigating the molecular mechanisms mediating immune cells’ trafficking into the TME and mediating effective anti-tumor immunity. Elucidation of the mechanisms which mediate trafficking and homing of immune cells into the GBM microenvironment will uncover novel therapeutic strategies.

Genetically engineered mouse glioma models, epigenetic remodeling of the response to DNA damaging and immune mediated therapies.

Glioma genetic models are needed to uncover mechanisms that mediate tumor progression, the interplay with the tumor microenvironment (TME) and response to therapeutics. My team generated the first genetically engineered mutant IDH1 mouse glioma model. Primary neurospheres (NS) were isolated from the tumors, which exhibit cancer stem cell-like properties. This has enabled us to develop a transplantable mIDH1 glioma model amenable to testing novel therapies. NS are derived from fully immune-competent (C57BL/6) mice, thus allowing examination of the TME and the impact of tumor mutations on the immune response. We assessed the effect of mIDH1 on transcription (using mRNA-seq) and on global DNA and histone methylation. The mIDH1 glioma model enabled us to identify promoter/enhancer region-specific changes in histone methylation marks (using chromatin immunoprecipitation followed by deep sequencing, or ChIP-seq). We uncovered epigenetic patterning of histone 3 hypermethylation and cytosine modifications using next generation sequencing (NGS) technologies, allowed us to identify novel pathways and gene regulatory networks providing novel insights into disease biology and novel therapeutic targets.

Regulatable, non-immunogenic gene delivery platforms for cancer therapeutics.

A challenge to successful gene therapy implementation is the host immune response against the vectors, which can hamper long-term therapeutic transgene expression. To this end, we created helper-dependent high-capacity Ad vectors, completely devoid of viral coding regions. These vectors are invisible to the immune system of the host. Also, due to their large cloning capacity, they enable the cloning of large inserts, tissue specific promoters and regulatory regions. Harnessing these characteristics, we developed HC-Ad that allow long-term and inducible therapeutic transgene expression in the CNS. We developed a conditional cytotoxic, immune stimulatory approach for GBM which is under consideration by the FDA for a new Phase I trial. We utilized this regulatable gene expression platform to encode a bacterial toxin harboring a targeting sequence specific for GBM. This led to tumor regression and long-term survival, in murine models and PDXS, with no toxicities. This will enable the implementation of gene therapy modalities to treat human disease.

Phase I immunotherapy-mediated clinical trial for brain cancer.

In light of the immunosuppressive nature of GBM, we hypothesized that stimulating an immune response directly from within the TME could elicit effective anti-tumor immunity. Increasing the number of brain tumor infiltrating APCs [elicited by expressing fms-like tyrosine kinase ligand (Flt3L) in the TME] in combination with the cytotoxic effects of TK (+GCV) induces effective tumor antigen uptake, migration of DCs to draining LN, and presentation of tumor antigen to naïve T- cells culminating in a strong anti-tumor immune response. In response to our treatment, anti-GBM cytotoxic CD8 T cells eliminate a large brain tumor mass in rodents. Further, the induction of immunological memory elicits the elimination of a second GBM implanted in the contralateral hemisphere of long-term survivors. This led to a Phase I trial: “A non- randomized, open-label dose-finding trial of combined cytotoxic and immune‐stimulatory strategy for the treatment of primary GBM, utilizing Ad- hCMV-TK, and Ad-hCMV-Flt3L”, which has recently completed accrual at our institution (IND number BB14574, clinicaltrials.gov number NCT01811992).).

Dr. Pedro R. Lowenstein’s Research

Description of research program: The current focus of my research program is to discover the cellular, molecular, and neuroanatomical basis underlying the growth patterns of malignant brain tumors, and the interactions between cancer cells with the tumor microenvironment, in both experimental models and in human patients suffering from malignant brain tumors. To do so, we are probing how brain glioma cells migrate throughout the brain and eventually kill the hosts’ neurons and glial cells. Understanding the precise molecular basis of glioma tumor cell growth and invasive behavior will uncover novel therapeutic targets aiming at inactivating the essential mechanisms used by tumors to grow and destroy normal brain tissue and, thus, kill the host. We are studying very early stages of tumor pattern formation using fluorescently labeled glioma cells in combination with advanced in vivo multiphoton imaging technologies. We are also developing endogenous models of brain tumors induced by gene products known to cause human gliomas (e.g., PDGF, CDKN2A deletion, EGFR amplification, etc.). The new information obtained will lead to the development of novel therapeutics for malignant glioma. Specifically, my team pioneered the development of the dual gene therapy combining immune-stimulatory expression of Flt3L and conditional cytotoxicity of HSV1-TK, both genes expressed from adenoviral vectors. The essential hypothesis is that Flt3L will attract dendritic cells to the brain tumor area, and aid in their differentiation. The cytotoxicity of HSV1-TK together with valacyclovir will kill dividing transduced tumor cells and thus release into the glioma microenvironment, tumor antigens, and innate immune stimuli such as HMGB1. This immunostimulatory gene therapy approach will elicit the development of a potent anti-glioma immune response. Experimentally, in preclinical glioma models, we have shown that dendritic cells are attracted to brain tumors, take up tumor antigens, become activated and migrate to lymph nodes, where a cytotoxic and memory T cell response is stimulated. For the current proposal I will be involved in all aspects of the work described in SA 1-3 and specifically oversee the translational experiments described in Aim 3. The long-term goal of my research program is to translate novel therapeutic strategies into phase I clinical trials. In support of our capacity to do so, our first clinical trial (IND# 14574) employing the combined Flt3L/HSV1-TK therapeutic strategy developed by ourselves has completed accrual at our Institution (https://clinicaltrials.gov/ct2/show/NCT01811992). Thus, the combination of the Flt3L/HSV1-TK therapeutic strategy with the administration of checkpoint inhibitors, as proposed in this application, is a rational and exciting scientific extension of our immune-stimulatory approach to the treatment of malignant brain tumors.

Glioma Immune Microenvironment – Cancer immunity is mainly focused on strategies to stimulate T- and B-cells to attack tumors. Although innate immune responses are known to be necessary to activate anti-tumor immune responses, attempts to use the innate immune system to attack tumors, i.e., gliomas, has been under explored. Our work showed for the first time the existence of powerful inhibitors of NK tumor killing expressed by glioma cells. Together with other work characterizing the interplay of innate and adaptive immune responses in anti-glioma immunity, this work will open the path to exploit the use of NK cells to reject gliomas.

Immunological Synapses – T-cells interact with target cells through a microanatomical structure called immunological synapse (IS). IS had been described using in vitro T or B cells in contact with target antigen presenting cells. However, evidence that immunological synapses were present in vivo during the course of normal immune responses was lacking. In 2006, we published the first bona fide evidence that IS existed in vivo in the context of an antiviral immune response in the brain. Subsequently we characterized details of the structure and function of immunological synapses. This work strengthened the support in favor of immunological synapses as the structure mediating intercellular communication during immune responses in whole animals.

Phase I Clinical Trial TK/Flt3L – Even if the immune system should be a powerful weapon to attack brain tumors most trials so far using vaccination to immunize against gliomas have only been marginally successful. Ideally, immunization should be induced from within the brain, where the glioma tumors are located. Until a few years ago, it was impossible to immunize against gliomas directly from the brain. Utilizing Flt3L we were able to recruit dendritic cells to the brain and initiate a systemic immune response against glioma tumors, which includes memory anti-glioma responses, and occurs in the absence of brain autoimmune responses. This approach is now being tested in a Phase I clinical trial (ClinicalTrials.gov Identifier: NCT01811992). This is a first-in-the-world trial of this endogenous immunotherapy approach which we have pioneered.

Neuroimmunology of Gene Therapy Vectors – The use of viral vectors as gene therapy vehicles to treat brain tumors opened up a new era in the use of genetic elements as therapeutics. Because of our interest in using adenoviral vectors to treat human patients we embarked on a detailed analysis of the innate and adaptive immune responses to adenoviral vectors injected into the brain. Our work described in detail the viral thresholds required to induce innate or adaptive immune responses, thus setting the standard for the use of viral vectors avoiding inflammation. This work set a baseline for viral immunological studies and standards in the field. In our own work, it has helped us advance our program towards the Phase I clinical trials we are now pioneering to treat malignant gliomas.