A Roadmap of CAR-T-Cell Therapy in Glioblastoma: Challenges and Future Perspectives.

Glioblastoma (GBM) is the most common primary malignant brain tumor, with a median overall survival of less than 2 years and a nearly 100% mortality rate under standard therapy that consists of surgery followed by combined radiochemotherapy. Therefore, new therapeutic strategies are urgently needed. The success of chimeric antigen receptor (CAR) T cells in hematological cancers has prompted preclinical and clinical investigations into CAR-T-cell treatment for GBM. However, recent trials have not demonstrated any major success. Here, we delineate existing challenges impeding the effectiveness of CAR-T-cell therapy for GBM, encompassing the cold (immunosuppressive) microenvironment, tumor heterogeneity, T-cell exhaustion, local and systemic immunosuppression, and the immune privilege inherent to the central nervous system (CNS) parenchyma. Additionally, we deliberate on the progress made in developing next-generation CAR-T cells and novel innovative approaches, such as low-intensity pulsed focused ultrasound, aimed at surmounting current roadblocks in GBM CAR-T-cell therapy.

IRF8-driven reprogramming of the immune microenvironment enhances anti-tumor adaptive immunity and reduces immunosuppression in murine glioblastoma.

Background: Glioblastoma (GBM) has a highly immunosuppressive tumor immune microenvironment (TIME), largely mediated by myeloid-derived suppressor cells (MDSCs). Here, we utilized a retroviral replicating vector (RRV) to deliver Interferon Regulatory Factor 8 (IRF8), a master regulator of type 1 conventional dendritic cell (cDC1) development, in a syngeneic murine GBM model. We hypothesized that RRV-mediated delivery of IRF8 could "reprogram" intratumoral MDSCs into antigen-presenting cells (APCs) and thereby restore T-cell responses. Methods: Effects of RRV-IRF8 on survival and tumor growth kinetics were examined in the SB28 murine GBM model. Immunophenotype was analyzed by flow cytometry and gene expression assays. We assayed functional immunosuppression and antigen presentation by ex vivo T-cell-myeloid co-culture. Results: Mice with RRV-IRF8 pre-transduced intracerebral tumors had significantly longer survival and slower tumor growth compared to controls. RRV-IRF8 treated tumors exhibited significant enrichment of cDC1s and CD8+ T-cells. Additionally, myeloid cells derived from RRV-IRF8 tumors showed decreased expression of the immunosuppressive markers Arg1 and IDO1 and demonstrated reduced suppression of naïve T-cell proliferation in ex vivo co-culture, compared to controls. Furthermore, DCs from RRV-IRF8 tumors showed increased antigen presentation compared to those from control tumors. In vivo treatment with azidothymidine (AZT), a viral replication inhibitor, showed that IRF8 transduction in both tumor and non-tumor cells is necessary for survival benefit, associated with a reprogrammed, cDC1- and CD8 T-cell-enriched TIME. Conclusions: Our results indicate that reprogramming of glioma-infiltrating myeloid cells by in vivo expression of IRF8 may reduce immunosuppression and enhance antigen presentation, achieving improved tumor control.

Inhibition of D-2HG leads to upregulation of a proinflammatory gene signature in a novel HLA-A2/HLA-DR1 transgenic mouse model of IDH1R132H-expressing glioma.

BACKGROUND: Long-term prognosis of WHO grade II, isocitrate dehydrogenase (IDH)-mutated low-grade glioma (LGG) is poor due to high risks of recurrence and malignant transformation into high-grade glioma. Immunotherapy strategies are attractive given the relatively intact immune system of patients with LGG and the slow tumor growth rate. However, accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) in IDH-mutated gliomas leads to suppression of inflammatory pathways in the tumor microenvironment, thereby contributing to the 'cold' tumor phenotype. Inhibiting D-2HG production presents an opportunity to generate a robust antitumor response following tumor antigen vaccination and immune checkpoint blockade. METHODS: An IDH1R132H glioma model was created in syngeneic HLA-A2/HLA-DR1-transgenic mice, allowing us to evaluate the vaccination with the human leukocyte antigens (HLA)-DR1-restricted, IDH1R132H mutation-derived neoepitope. The effects of an orally available inhibitor of mutant IDH1 and IDH2, AG-881, were evaluated as monotherapy and in combination with the IDH1R132H peptide vaccination or anti-PD-1 immune checkpoint blockade. RESULTS: The HLA-A2/HLA-DR1-syngeneic IDH1R132H cell line expressed the IDH1 mutant protein and formed D-2HG producing orthotopic gliomas in vivo. Treatment of tumor-bearing mice with AG-881 resulted in a reduction of D-2HG levels in IDH1R132H glioma cells (10 fold) and tumor-associated myeloid cells, which demonstrated high levels of intracellular D-2HG in the IDH1R132H gliomas. AG-881 monotherapy suppressed the progression of IDH1R132H gliomas in a CD4+ and CD8+ cell-dependent manner, enhanced proinflammatory IFNγ-related gene expression, and increased the number of CD4+ tumor-infiltrating T-cells. Prophylactic vaccination with the HLA-DR1-restricted IDH1R132H peptide or tumor-associated HLA-A2-restricted peptides did not enhance survival of tumor-bearing animals; however, vaccination with both HLA-A2-IDH1R132H and DR1-IDH1R132H peptides in combination with the IDH inhibitor significantly prolonged survival. Finally, tumor-bearing mice treated with both AG-881 and a PD-1 blocking antibody demonstrated improved survival when compared with either treatment alone. CONCLUSION: The development of effective IDH1R132H-targeting vaccine may be enhanced by integration with HLA class I-restricted cytotoxic T cell epitopes and AG-881. Our HLA-A2/HLA-DR1-syngeneic IDH1R132H glioma model should allow us to evaluate key translational questions related to the development of novel strategies for patients with IDH-mutant glioma.

Barriers and Opportunities for the MCH Workforce to Support Hurricane Preparedness, Response, and Recovery in Florida.

OBJECTIVES: In recent years, catastrophic hurricanes have devastated numerous areas, prompting a need to build resilience particularly in at-risk populations that rely on health care and social services. The Maternal and Child Health (MCH) workforce covers a wide breadth of services to pregnant women, families, and children with special health care needs. Research has noted the need to strengthen this workforce with training and skills to help their patients and clients prepare, respond, and recover from disasters. METHODS: Focus groups and interviews with 35 Florida parents and professionals impacted by Hurricanes Irma, Maria, and Michael were conducted to evaluate the stressors placed on systems of care serving mothers and infants in Florida. Journey mapping was used to explore opportunities for improving MCH training and services. RESULTS: Results highlight the importance of increased communication and collaboration between families and providers, coordination among health care and social services providers, effective public messaging, tailored preparedness materials and processes, and the need for post-disaster mental health services and employment resources. CONCLUSION: Ultimately, hurricane preparation and mitigation are key for improving community resilience and these efforts should be tailored to MCH populations as well as delivered by the providers who know their needs best.

Novel EGFRvIII-CAR transgenic mice for rigorous preclinical studies in syngeneic mice.

BACKGROUND: Rigorous preclinical studies of chimeric antigen receptor (CAR) immunotherapy will require large quantities of consistent and high-quality CAR-transduced T (CART) cells that can be used in syngeneic mouse glioblastoma (GBM) models. To this end, we developed a novel transgenic (Tg) mouse strain with a fully murinized CAR targeting epidermal growth factor receptor variant III (EGFRvIII). METHODS: We first established the murinized version of EGFRvIII-CAR and validated its function using a retroviral vector (RV) in C57BL/6J mice bearing syngeneic SB28 GBM expressing EGFRvIII. Next, we created C57BL/6J-background Tg mice carrying the anti-EGFRvIII-CAR downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. We bred these mice with CD4-Cre Tg mice to allow CAR expression on T cells and evaluated the function of the CART cells both in vitro and in vivo. To inhibit immunosuppressive myeloid cells within SB28 GBM, we also evaluated a combination approach of CART and an anti-EP4 compound (ONO-AE3-208). RESULTS: Both RV- and Tg-CART cells demonstrated specific cytotoxic activities against SB28-EGFRvIII cells. A single intravenous infusion of EGFRvIII-CART cells prolonged the survival of glioma-bearing mice when preceded by a lymphodepletion regimen with recurrent tumors displaying profound EGFRvIII loss. The addition of ONO-AE3-208 resulted in long-term survival in a fraction of CART-treated mice and those survivors demonstrated delayed growth of subcutaneously re-challenged both EGFRvIII+ and parental EGFRvIII- SB28. CONCLUSION: Our new syngeneic CAR Tg mouse model can serve as a useful tool to address clinically relevant questions and develop future immunotherapeutic strategies.

Introduction to immunotherapy for brain tumor patients: challenges and future perspectives.

Malignant gliomas, including glioblastoma (GBM) as the most aggressive type of adult CNS tumors, are notoriously resistant to current standard of care treatments, including surgery, systemic chemotherapy, and radiation therapy (RT). This lack of effective treatment options highlights the urgent need for novel therapies, including immunotherapies. The overarching goal of immunotherapy is to stimulate and activate the patient's immune system in a targeted manner to kill tumor cells. The success of immunotherapeutic interventions in other cancer types has led to interest in and evaluation of various experimental immunotherapies in patients with malignant gliomas. However, these primary malignant brain tumors present a challenge because they exist in a vital and sensitive organ with a unique immune environment. The challenges and current status of experimental immunotherapeutic approaches, including vaccines, immune-checkpoint blockade, chimeric antigen receptor T-cell therapy, and oncolytic viruses will be discussed, as well as the potential for combinatorial therapies.

Transcriptional control of subtype switching ensures adaptation and growth of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is a heterogeneous disease comprised of a basal-like subtype with mesenchymal gene signatures, undifferentiated histopathology and worse prognosis compared to the classical subtype. Despite their prognostic and therapeutic value, the key drivers that establish and control subtype identity remain unknown. Here, we demonstrate that PDA subtypes are not permanently encoded, and identify the GLI2 transcription factor as a master regulator of subtype inter-conversion. GLI2 is elevated in basal-like PDA lines and patient specimens, and forced GLI2 activation is sufficient to convert classical PDA cells to basal-like. Mechanistically, GLI2 upregulates expression of the pro-tumorigenic secreted protein, Osteopontin (OPN), which is especially critical for metastatic growth in vivo and adaptation to oncogenic KRAS ablation. Accordingly, elevated GLI2 and OPN levels predict shortened overall survival of PDA patients. Thus, the GLI2-OPN circuit is a driver of PDA cell plasticity that establishes and maintains an aggressive variant of this disease.