Targeting ULK1 Decreases IFNγ-Mediated Resistance to Immune Checkpoint Inhibitors.

Immune checkpoint inhibitors (ICI) have transformed the treatment of melanoma. However, the majority of patients have primary or acquired resistance to ICIs, limiting durable responses and patient survival. IFNγ signaling and the expression of IFNγ-stimulated genes correlate with either response or resistance to ICIs, in a context-dependent manner. While IFNγ-inducible immunostimulatory genes are required for response to ICIs, chronic IFNγ signaling induces the expression of immunosuppressive genes, promoting resistance to these therapies. Here, we show that high levels of Unc-51 like kinase 1 (ULK1) correlate with poor survival in patients with melanoma and overexpression of ULK1 in melanoma cells enhances IFNγ-induced expression of immunosuppressive genes, with minimal effects on the expression of immunostimulatory genes. In contrast, genetic or pharmacologic inhibition of ULK1 reduces expression of IFNγ-induced immunosuppressive genes. ULK1 binds IRF1 in the nuclear compartment of melanoma cells, controlling its binding to the programmed death-ligand 1 promoter region. In addition, pharmacologic inhibition of ULK1 in combination with anti-programmed cell death protein 1 therapy further reduces melanoma tumor growth in vivo. Our data suggest that targeting ULK1 represses IFNγ-dependent immunosuppression. These findings support the combination of ULK1 drug-targeted inhibition with ICIs for the treatment of patients with melanoma to improve response rates and patient outcomes. IMPLICATIONS: This study identifies ULK1, activated downstream of IFNγ signaling, as a druggable target to overcome resistance mechanisms to ICI therapy in metastatic melanoma.

Discovery of a signaling feedback circuit that defines interferon responses in myeloproliferative neoplasms.

Interferons (IFNs) are key initiators and effectors of the immune response against malignant cells and also directly inhibit tumor growth. IFNα is highly effective in the treatment of myeloproliferative neoplasms (MPNs), but the mechanisms of action are unclear and it remains unknown why some patients respond to IFNα and others do not. Here, we identify and characterize a pathway involving PKCδ-dependent phosphorylation of ULK1 on serine residues 341 and 495, required for subsequent activation of p38 MAPK. We show that this pathway is essential for IFN-suppressive effects on primary malignant erythroid precursors from MPN patients, and that increased levels of ULK1 and p38 MAPK correlate with clinical response to IFNα therapy in these patients. We also demonstrate that IFNα treatment induces cleavage/activation of the ULK1-interacting ROCK1/2 proteins in vitro and in vivo, triggering a negative feedback loop that suppresses IFN responses. Overexpression of ROCK1/2 is seen in MPN patients and their genetic or pharmacological inhibition enhances IFN-anti-neoplastic responses in malignant erythroid precursors from MPN patients. These findings suggest the clinical potential of pharmacological inhibition of ROCK1/2 in combination with IFN-therapy for the treatment of MPNs.

Regulation of IFNα-induced expression of the short ACE2 isoform by ULK1.

The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been shown to hijack angiotensin converting enzyme 2 (ACE2) for entry into mammalian cells. A short isoform of ACE2, termed deltaACE2 (dACE2), has recently been identified. In contrast to ACE2, the short dACE2 isoform lacks the ability to bind the spike protein of SARS-CoV-2. Several studies have proposed that expression of ACE2 and/or dACE2 is induced by interferons (IFNs). Here, we report that drug-targeted inhibition or silencing of Unc51-like kinase 1 (ULK1) results in repression of type I IFN-induced expression of the dACE2 isoform. Notably, dACE2 is expressed in various squamous tumors. In efforts to identify pharmacological agents that target this pathway, we found that fisetin, a natural flavonoid, is an ULK1 inhibitor that decreases type I IFN-induced dACE2 expression. Taken together, our results establish a requirement for ULK1 in the regulation of type I IFN-induced transcription of dACE2 and raise the possibility of clinical translational applications of fisetin as a novel ULK1 inhibitor.

Neural stem cells secreting bispecific T cell engager to induce selective antiglioma activity.

Glioblastoma (GBM) is the most lethal primary brain tumor in adults. No treatment provides durable relief for the vast majority of GBM patients. In this study, we've tested a bispecific antibody comprised of single-chain variable fragments (scFvs) against T cell CD3ε and GBM cell interleukin 13 receptor alpha 2 (IL13Rα2). We demonstrate that this bispecific T cell engager (BiTE) (BiTELLON) engages peripheral and tumor-infiltrating lymphocytes harvested from patients' tumors and, in so doing, exerts anti-GBM activity ex vivo. The interaction of BiTELLON with T cells and IL13Rα2-expressing GBM cells stimulates T cell proliferation and the production of proinflammatory cytokines interferon γ (IFNγ) and tumor necrosis factor α (TNFα). We have modified neural stem cells (NSCs) to produce and secrete the BiTELLON (NSCLLON). When injected intracranially in mice with a brain tumor, NSCLLON show tropism for tumor, secrete BiTELLON, and remain viable for over 7 d. When injected directly into the tumor, NSCLLON provide a significant survival benefit to mice bearing various IL13Rα2+ GBMs. Our results support further investigation and development of this therapeutic for clinical translation.

Mesenchymal Stem Cells Successfully Deliver Oncolytic Virotherapy to Diffuse Intrinsic Pontine Glioma.

PURPOSE: Diffuse intrinsic pontine glioma (DIPG) is among the deadliest of pediatric brain tumors. Radiotherapy is the standard-of-care treatment for DIPG, but offers only transient relief of symptoms for patients with DIPG without providing significant survival benefit. Oncolytic virotherapy is an anticancer treatment that has been investigated for treating various types of brain tumors. EXPERIMENTAL DESIGN: Here, we have explored the use of mesenchymal stem cells (MSC) for oncolytic virus (OV) delivery and evaluated treatment efficacy using preclinical models of DIPG. The survivin promoter drives the conditional replication of OV used in our studies. The efficiency of OV entry into the cells is mediated by fiber modification with seven lysine residues (CRAd.S.pK7). Patients' samples and cell lines were analyzed for the expression of viral entry proteins and survivin. The ability of MSCs to deliver OV to DIPG was studied in the context of a low dose of irradiation. RESULTS: Our results show that DIPG cells and tumors exhibit robust expression of cell surface proteins and survivin that enable efficient OV entry and replication in DIPG cells. MSCs loaded with OV disseminate within a tumor and release OV throughout the DIPG brainstem xenografts in mice. Administration of OV-loaded MSCs with radiotherapy to mice bearing brainstem DIPG xenografts results in more prolonged survival relative to that conferred by either therapy alone (P < 0.01). CONCLUSIONS: Our study supports OV, CRAd.S.pK7, encapsulated within MSCs as a therapeutic strategy that merits further investigation and potential translation for DIPG treatment.