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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.

S1. Overexpression of ULK1 in melanoma cells has minor effects on transcription of IFNγ-induced immunostimulatory genes.S2. Effects of gene targeted inhibition of ULK1 in transcription of IFNγ-induced immunostimulatory genes in melanoma cells.S3. Effects of drug-targeted inhibition of ULK1 in transcription of IFNγ-induced immunostimulatory genes in melanoma cells.S4. Effects of siRNA-mediated knockdown of ULK1 on IFNγ-inducedSTAT1 phosphorylation.S5. Identification of putative cytoplasmic and nuclear ULK1 binding partners in melanoma cells upon IFNγ stimulation by mass spectrometry analysis.S6. Pharmacological inhibition of ULK1 enhances the anti-tumor effects of anti-PD-1 therapy in YUMMER1.7 mouse melanoma in vivo model.S7. Tumor infiltrating CD8+ T cells and TAMCs in vehicle-isotype-treated mice.S8. Tumor infiltrating CD8+ T cells and TAMCs in ULK inhibitor-treated mice.S9. Tumor infiltrating CD8+ T cells and TAMCs in anti-PD-1-treated mice.S10. Tumor infiltrating CD8+ T cells and TAMCs in ULKi plus anti-PD-1-treated mice.S11. Tumor infiltrating CD4+ T cells and Tregs in vehicle-isotype-treated mice.S12. Tumor infiltrating CD4+ T cells and Tregs in ULK inhibitor-treated mice.S13. Tumor infiltrating CD4+ T cells and Tregs in anti-PD-1treated mice.S14. Tumor infiltrating CD4+ T cells and Tregs in ULKi plus anti-PD-1-treated mice.S15. Tumor infiltrating CD8+ T cells and TAMCs in control, ULKi and/or anti-PD-1-treated mice.S16. Tumor infiltrating CD4+ T cells and Tregs in control, ULKi and/or anti-PD-1-treated mice.S17. Gating strategy for flow cytometric immunophenotyping of mouse melanoma tumors.

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.

List of the putative ULK1 interactor proteins identified by mass spectrometry analysis in A375 cells

Methods, Figures, legends and Tables

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.

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.

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) (BiTE(LLON)) engages peripheral and tumor-infiltrating lymphocytes harvested from patients' tumors and, in so doing, exerts anti-GBM activity ex vivo. The interaction of BiTE(LLON) 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 BiTE(LLON) (NSC(LLON)). When injected intracranially in mice with a brain tumor, NSC(LLON) show tropism for tumor, secrete BiTE(LLON), and remain viable for over 7 d. When injected directly into the tumor, NSC(LLON) provide a significant survival benefit to mice bearing various IL13Rα2(+) GBMs. Our results support further investigation and development of this therapeutic for clinical translation.

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.

Glioblastoma (GBM) is the most lethal primary brain tumor in adults. There is no treatment that provides durable relief for the vast majority of GBM patients. In this study, we’ve tested a bispecific antibody comprised of single-chain variable regions (scFvs) against T cell CD3ε and GBM cell interleukin 13 receptor alpha 2 (IL13Rα2). We demonstrate that this BiTE (BiTELLON) engages peripheral and tumor-infiltrating lymphocytes harvested from patient’s tumors, and in so doing exerts anti-GBM activityex vivo. The interaction of BiTELLONwith T cells and engagement of IL13Rα2-expressing GBM cells stimulates T cell proliferation as well as production of pro-inflammatory cytokines INFγ and TNFα. We have modified neural stem cells (NSCs) to produce and secrete the BiTE (NSCsLLON). When injected intracranially in mice with brain tumor, NSCsLLONshow tropism for tumor, secrete BiTELLON, and remain viable for several days. When injected directly into tumor, NSCLLONprovide significant survival benefit to mice bearing IL13Rα2+ GBM. Our results support further investigation and development of this therapeutic for clinical translation.

Diffuse intrinsic pontine glioma (DIPG) is among the deadliest of pediatric brain tumors. Radiation therapy is the standard of care treatment for DIPG, but offers only transient relief of symptoms for DIPG patients without providing significant survival benefit. Oncolytic virotherapy (OV) is an anticancer treatment that has been investigated for treating various types of brain tumors. Here, we have explored the use of mesenchymal stem cells (MSC) for OV delivery and evaluated treatment efficacy using preclinical models of DIPG. Our results show that DIPG cells and tumors exhibit robust expression of cell surface proteins that are important for OV entry, and that MSCs loaded with OV disseminate within and release OV throughout the tumor in mice bearing DIPG brainstem xenografts. When combining administration of OV-loaded MSCs with radiotherapy, mice bearing brainstem DIPG xenografts experience a significant survival benefit, relative to that conferred by either therapy alone (p

Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor affecting young children. Immunotherapies hold promise however the lack of immunocompetent models recreating a faithful tumor microenvironment (TME) remains a challenge for development of targeted immunotherapeutics. We propose to generate an immunocompetent DIPG mouse model through induced overexpression of interleukin 13 receptor alpha 2 (IL13Rα2), a tumor-associated antigen overexpressed by glioma cells. A model with an intact TME permits comprehensive preclinical assessment of IL13Rα2-targeted immunotherapeutics. Our novel model uses the retroviral avian leucosis and sarcoma virus (RCAS) for in vivo gene delivery leading to IL13Rα2 expression in proliferating progenitor cells. Transfected cells expressing IL13Rα2 and PDGFB, a ligand for platelet derived growth factor receptor, alongside induced p53 loss via the Cre-Lox system are injected in the fourth ventricle in postnatal pups. We validated the expression of PDGFB and IL13Rα2 transgenes in vitro and in vivo and will characterize the TME through evaluation of the peripheral and tumor immunologic compartments using immunohistochemistry and flow cytometry. We confirmed expression of transgenes via flow cytometry and western blotting. Comparison of survival dynamics in mice inoculated with PDGFB alone with PDGFB+IL13Rα2 demonstrated that co-expression of IL13Rα2 did not significantly affect mice survival compared to the PDGFB model. At time of application, we initiated experiments to characterize the TME. Preliminary data demonstrate establishment of tumors within and adjacent to the brainstem and expression of target transgenes. Preclinical findings in a model recapitulating the TME may provide better insight into outcomes upon translation to clinical application.

BACKGROUND: Malignant gliomas (MG) are the most common and difficult-to-treat adult brain tumors. The outstanding efficacy of chimeric antigen receptor (CAR)-modified T cells against hematological malignancies gives hope that they can be programmed to target and eradicate solid tumors like MG. We recently demonstrated that murine CAR.CD28.ζ T cells targeting the tumor-associated antigen interleukin-13 receptor alpha 2 (IL13Rα2) have antitumor activity in two syngeneic models of MG in comparison to control animals. However, the majority of tumors recurred. The goal of this project was now to explore if transgenic expression of IL15 improves their anti-glioma activity. METHODS/RESULTS: We generated a retroviral vector encoding murine IL13Rα2-CAR.CD28.ζ, a 2A peptide, and secretable IL15 (IL13Rα2-CAR.IL15), and a control vector encoding a non-functional CAR (IL13Rα2-CAR.Δ), a 2A peptide, and IL15 (IL13Rα2-CAR.Δ.IL15). Murine CAR T cells were generated by activating and transducing murine CD3(+) T cells with retroviral particles. The effector function of IL13Rα2-CAR, IL13Rα2-CAR.Δ, IL13Rα2-CAR.IL15, and CAR.Δ.IL15 were compared in vitro and in vivo. Only CAR T cells expressing functional CARs killed IL13Rα2+ GL261 (GL261-IL13Rα2) glioma cells. In addition, only IL13Rα2-CAR.IL15 and IL13Rα2-CAR.Δ.IL15 T cells secreted IL15 as judged by ELISA. In vivo, GL261-IL13Rα2 glioma-bearing mice treated with a single i.t. dose of IL13Rα2-CAR.IL15 T cells had a significant improved survival in comparison to mice treated with IL13Rα2-CAR T cells. This therapeutic benefit was antigen-specific since CAR.Δ.IL15 T-cells did not improve animals’ survival. CONCLUSIONS: Our data demonstrates that IL13Rα2-CAR.IL15 T cells have greater antitumor activity in vivo than IL13Rα2-CAR T cells in an immune competent glioma model. Thus, 2(nd) genetic modifications have the potential to improve current CAR T-cell therapy approaches for MG.

BACKGROUND The outstanding efficacy of bi-specific T-cell engagers (BiTE against hematological malignancies offers hope that they can similarly target solid tumors like GBM. In this study, we have designed a BiTE protein with specificity to the tumor-associated antigen, IL13Rα2, and investigated how BiTE protein engages a host’s T cell immune response to promote anti-glioma activity in pre-clinical models of GBM. MATERIAL AND METHODS BiTE molecule consisting of two single chain variable regions (scFv) of antibodies against either murine or human CD3ε and scFv47 against human IL13Rα2 connected through a flexible linker (BiTEIL13Rα2) were sub-cloned in a lentiviral expression cassette. The BiTE molecule (BiTEIL13Rα2off) modified to abrogate the interaction of scFv47 with IL13Rα2 served as a negative control. The BiTE proteins were isolated from the supernatants of HEK293T cells using His-Tag affinity chromatography and validated in SDS-PAGE, Western Blot, and ELISA assays. The BiTEIL13Rα2-induced T cells activation was measured in (i) cytotoxicity assay against IL13Rα2+ glioma cells, (ii) flow cytometry measuring for CD69 and CD25 T cells’ activation markers, and (iii) the production of cytokines, IFNγ and TNFα. For in vivo analysis, VmDk and C57Bl/6 mice bearing established intracranial glioma were treated systemically with BiTE proteins. The survival of the mice was recorded and analyzed using the log-rank test. RESULTS Here we show that BiTEIL13Rα2 specifically binds to IL13Rα2 but not to IL13Rα1, whereas BiTEIL13Rα2off has no binding activity to both IL13 receptors. The co-culture of naïve murine or donor’s human CD3+ T cells with IL13Rα2+ glioma cells in the presence of BiTEIL13Rα2 but not with BiTEIL13Rα2off (i) activates CD3+CD8+ T cells as judged by upregulation of CD69, CD25, and production of IFNγ and TNFα and (ii) results in concentration- and antigen-dependent cytotoxicity in glioma cells. Furthermore, a direct comparison of CD3+ T cells obtained from the peripheral blood and tumor tissue of GBM patients revealed that BiTEIL13Rα2 induces a potent cytotoxic activity of CD3+ T cells against IL13Rα2+ glioma cells. Finally, treatment of immunocompetent mice bearing IL13Rα2+ murine glioma with BiTEIL13Rα2 resulted in a higher frequency of intratumoral CD8+ T cells, and significant (p CONCLUSION Our data demonstrate that BiTEIL13Rα2 protein activates CD3+ T cells in an antigen-specific fashion. Furthermore, systemic treatment with BiTEIL13Rα2 protein confers a significant survival benefit in pre-clinical syngeneic glioma models, warranting investigations in other IL13Rα2-expressing cancers and translation to clinical settings.