Metformin inhibits RAN translation through PKR pathway and mitigates disease in C9orf72 ALS/FTD mice.

Repeat associated non-AUG (RAN) translation is found in a growing number of microsatellite expansion diseases, but the mechanisms remain unclear. We show that RAN translation is highly regulated by the double-stranded RNA-dependent protein kinase (PKR). In cells, structured CAG, CCUG, CAGG, and G4C2 expansion RNAs activate PKR, which leads to increased levels of multiple RAN proteins. Blocking PKR using PKR-K296R, the TAR RNA binding protein or PKR-KO cells, reduces RAN protein levels. p-PKR is elevated in C9orf72 ALS/FTD human and mouse brains, and inhibiting PKR in C9orf72 BAC transgenic mice using AAV-PKR-K296R or the Food and Drug Administration (FDA)-approved drug metformin, decreases RAN proteins, and improves behavior and pathology. In summary, targeting PKR, including by use of metformin, is a promising therapeutic approach for C9orf72 ALS/FTD and other expansion diseases.

mRNA-based precision targeting of neoantigens and tumor-associated antigens in malignant brain tumors.

BACKGROUND: Despite advancements in the successful use of immunotherapy in treating a variety of solid tumors, applications in treating brain tumors have lagged considerably. This is due, at least in part, to the lack of well-characterized antigens expressed within brain tumors that can mediate tumor rejection; the low mutational burden of these tumors that limits the abundance of targetable neoantigens; and the immunologically "cold" tumor microenvironment that hampers the generation of sustained and productive immunologic responses. The field of mRNA-based therapeutics has experienced a boon following the universal approval of COVID-19 mRNA vaccines. mRNA-based immunotherapeutics have also garnered widespread interest for their potential to revolutionize cancer treatment. In this study, we developed a novel and scalable approach for the production of personalized mRNA-based therapeutics that target multiple tumor rejection antigens in a single therapy for the treatment of refractory brain tumors. METHODS: Tumor-specific neoantigens and aberrantly overexpressed tumor-associated antigens were identified for glioblastoma and medulloblastoma tumors using our cancer immunogenomics pipeline called Open Reading Frame Antigen Network (O.R.A.N). Personalized tumor antigen-specific mRNA vaccine was developed for each individual tumor model using selective gene capture and enrichment strategy. The immunogenicity and efficacy of the personalized mRNA vaccines was evaluated in combination with anti-PD-1 immune checkpoint blockade therapy or adoptive cellular therapy with ex vivo expanded tumor antigen-specific lymphocytes in highly aggressive murine GBM models. RESULTS: Our results demonstrate the effectiveness of the antigen-specific mRNA vaccines in eliciting robust anti-tumor immune responses in GBM hosts. Our findings substantiate an increase in tumor-infiltrating lymphocytes characterized by enhanced effector function, both intratumorally and systemically, after antigen-specific mRNA-directed immunotherapy, resulting in a favorable shift in the tumor microenvironment from immunologically cold to hot. Capacity to generate personalized mRNA vaccines targeting human GBM antigens was also demonstrated. CONCLUSIONS: We have established a personalized and customizable mRNA-therapeutic approach that effectively targets a plurality of tumor antigens and demonstrated potent anti-tumor response in preclinical brain tumor models. This platform mRNA technology uniquely addresses the challenge of tumor heterogeneity and low antigen burden, two key deficiencies in targeting the classically immunotherapy-resistant CNS malignancies, and possibly other cold tumor types.

Adeno-associated virus delivered CXCL9 sensitizes glioblastoma to anti-PD-1 immune checkpoint blockade.

There are numerous mechanisms by which glioblastoma cells evade immunological detection, underscoring the need for strategic combinatorial treatments to achieve appreciable therapeutic effects. However, developing combination therapies is difficult due to dose-limiting toxicities, blood-brain-barrier, and suppressive tumor microenvironment. Glioblastoma is notoriously devoid of lymphocytes driven in part by a paucity of lymphocyte trafficking factors necessary to prompt their recruitment and activation. Herein, we develop a recombinant adeno-associated virus (AAV) gene therapy that enables focal and stable reconstitution of the tumor microenvironment with C-X-C motif ligand 9 (CXCL9), a powerful call-and-receive chemokine for lymphocytes. By manipulating local chemokine directional guidance, AAV-CXCL9 increases tumor infiltration by cytotoxic lymphocytes, sensitizing glioblastoma to anti-PD-1 immune checkpoint blockade in female preclinical tumor models. These effects are accompanied by immunologic signatures evocative of an inflamed tumor microenvironment. These findings support AAV gene therapy as an adjuvant for reconditioning glioblastoma immunogenicity given its safety profile, tropism, modularity, and off-the-shelf capability.

Oral IRAK-4 Inhibitor CA-4948 Is Blood-Brain Barrier Penetrant and Has Single-Agent Activity against CNS Lymphoma and Melanoma Brain Metastases.

PURPOSE: An ongoing challenge in cancer is the management of primary and metastatic brain malignancies. This is partly due to restrictions of the blood-brain barrier and their unique microenvironment. These challenges are most evident in cancers such as lymphoma and melanoma, which are typically responsive to treatment in systemic locations but resistant when established in the brain. We propose interleukin-1 receptor-associated kinase-4 (IRAK-4) as a potential target across these diseases and describe the activity and mechanism of oral IRAK-4 inhibitor CA-4948. EXPERIMENTAL DESIGN: Human primary central nervous system lymphoma (PCNSL) and melanoma brain metastases (MBM) samples were analyzed for expression of IRAK-4 and downstream transcription pathways. We next determined the central nervous system (CNS) applicability of CA-4948 in naïve and tumor-bearing mice using models of PCNSL and MBM. The mechanistic effect on tumors and the tumor microenvironment was then analyzed. RESULTS: Human PCNSL and MBM have high expression of IRAK-4, IRAK-1, and nuclear factor kappa B (NF-κB). This increase in inflammation results in reflexive inhibitory signaling. Similar profiles are observed in immunocompetent murine models. Treatment of tumor-bearing animals with CA-4948 results in the downregulation of mitogen-activated protein kinase (MAPK) signaling in addition to decreased NF-κB. These intracellular changes are associated with a survival advantage. CONCLUSIONS: IRAK-4 is an attractive target in PCNSL and MBM. The inhibition of IRAK-4 with CA-4948 downregulates the expression of important transcription factors involved in tumor growth and proliferation. CA-4948 is currently being investigated in clinical trials for relapsed and refractory lymphoma and warrants further translation into PCNSL and MBM.

CXCL9 recombinant adeno-associated virus (AAV) virotherapy sensitizes glioblastoma (GBM) to anti-PD-1 immune checkpoint blockade.

The promise of immunotherapy to induce long-term durable responses in conventionally treatment resistant tumors like glioblastoma (GBM) has given hope for patients with a dismal prognosis. Yet, few patients have demonstrated a significant survival benefit despite multiple clinical trials designed to invigorate immune recognition and tumor eradication. Insights gathered over the last two decades have revealed numerous mechanisms by which glioma cells resist conventional therapy and evade immunological detection, underscoring the need for strategic combinatorial treatments as necessary to achieve appreciable therapeutic effects. However, new combination therapies are inherently difficult to develop as a result of dose-limiting toxicities, the constraints of the blood-brain barrier, and the suppressive nature of the GBM tumor microenvironment (TME). GBM is notoriously devoid of lymphocytes driven in part by a paucity of lymphocyte trafficking factors necessary to prompt their recruitment, infiltration, and activation. We have developed a novel recombinant adeno-associated virus (AAV) gene therapy strategy that enables focal and stable reconstitution of the GBM TME with C-X-C motif ligand 9 (CXCL9), a powerful call-and-receive chemokine for cytotoxic T lymphocytes (CTLs). By precisely manipulating local chemokine directional guidance, AAV-CXCL9 increases tumor infiltration by CD8-postive cytotoxic lymphocytes, sensitizing GBM to anti-PD-1 immune checkpoint blockade (ICB). These effects are accompanied by immunologic signatures evocative of an inflamed and responsive TME. These findings support targeted AAV gene therapy as a promising adjuvant strategy for reconditioning GBM immunogenicity given its excellent safety profile, TME-tropism, modularity, and off-the-shelf capability, where focal delivery bypasses the constrains of the blood-brain barrier, further mitigating risks observed with high-dose systemic therapy.