Jain, Kanika

Abstract Ewing sarcoma is an aggressive pediatric cancer that has remained refractory to current therapeutics. Immunotherapy has been unsuccessful in Ewing sarcoma, largely due to poor understanding of how its immune tumor microenvironment is regulated. We recently demonstrated that ubiquitin-specific protease 6 (USP6) can remodel the Ewing sarcoma immune landscape to engender an antitumorigenic tumor microenvironment. USP6 expression in Ewing sarcoma cells enhances surface expression of immunostimulatory ligands and receptors and induces production of multiple chemokines, driving recruitment and activation of tumor-suppressive immune lineages, including NK cells. We sought to harness this multifaceted immunostimulatory function into a novel therapeutic by delivering in vitro transcribed USP6 mRNA via ionizable lipid nanoparticles (LNP). Treatment of Ewing sarcoma cells with USP6 mRNA in vitro is capable of inducing the aforementioned antitumorigenic and immunostimulatory responses. In addition, USP6 mRNA–treated Ewing sarcoma cells elicit cytolytic activation of primary human CD8+ and CD4+ T lymphocytes and NK cells in vitro. Intratumoral delivery of USP6 mRNA LNPs suppresses growth of Ewing sarcoma xenografts, coincident with increased immune infiltration and activation. We further demonstrate that USP6 mRNA is capable of igniting an immunostimulatory program in other cancer types (including acute myeloid leukemia, melanoma, prostate cancer, head and neck cancer, and osteosarcoma) in vitro and suppressing acute myeloid leukemia xenograft growth in vivo. Treatment with USP6 mRNA LNPs was well-tolerated, with no observed gross toxicity. Together, these preclinical studies provide proof-of-concept for the immunogenic and antitumorigenic efficacy of USP6 mRNA LNPs and support its promise as a novel immunotherapeutic in diverse cancer types.

Ewing sarcoma is a rare and deadly pediatric bone cancer for which survival rates and treatment options have stagnated for decades. Ewing sarcoma has not benefited from immunotherapy due to poor understanding of how its immune landscape is regulated. We recently reported that ubiquitin-specific protease 6 (USP6) functions as a tumor suppressor in Ewing sarcoma, and identified it as the first cell-intrinsic factor to modulate the Ewing sarcoma immune tumor microenvironment (TME). USP6 induces intratumoral infiltration and activation of multiple innate immune lineages in xenografted nude mice. Here we report that natural killer (NK) cells are essential for its tumor-inhibitory functions, as NK cell depletion reverses USP6-mediated suppression of Ewing sarcoma xenograft growth. USP6 expression in Ewing sarcoma cells directly stimulates NK cell activation and degranulation in vitro, and functions by increasing surface levels of multiple NK cell-activating ligands. USP6 also induces surface upregulation of the receptor for the apoptosis-inducing ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), providing an additional route for enhanced sensitivity to NK cell killing. Furthermore, USP6-expressing Ewing sarcoma and NK cells participate in a paracrine immunostimulatory feedforward loop, wherein IFNγ secreted by activated NK cells feeds back on USP6/Ewing sarcoma cells to induce synergistic expression of chemokines CXCL9 and CXCL10. Remarkably, expression of USP6 in subcutaneous Ewing sarcoma xenografts induces systemic activation and maturation of NK cells, and induces an abscopal response in which growth of distal tumors is inhibited, coincident with increased infiltration and activation of NK cells. This work reveals how USP6 reprograms the Ewing sarcoma TME to enhance antitumor immunity, and may be exploited for future therapeutic benefit. Significance: This study provides novel insights into the immunomodulatory functions of USP6, the only cancer cell–intrinsic factor demonstrated to regulate the immune TME in Ewing sarcoma. We demonstrate that USP6-mediated suppression of Ewing sarcoma tumorigenesis is dependent on NK cells. USP6 directly activates NK cell cytolytic function, inducing both intratumoral and systemic activation of NK cells in an Ewing sarcoma xenograft model.

Abstract Ewing sarcoma is the second most common pediatric bone cancer, with a 5-year survival rate for metastatic disease of only 20%. Recent work indicates that survival is strongly correlated with high levels of tumor-infiltrating lymphocytes (TIL), whose abundance is associated with IFN-inducible chemokines CXCL10 and CCL5. However, the tumor-intrinsic factors that drive chemokine production and TIL recruitment have not been fully elucidated. We previously showed that ubiquitin-specific protease 6 (USP6) directly deubiquitinates and stabilizes Jak1, thereby inducing an IFN signature in Ewing sarcoma cells. Here, we show that this gene set comprises chemokines associated with immunostimulatory, antitumorigenic functions, including CXCL10 and CCL5. USP6 synergistically enhanced chemokine production in response to exogenous IFN by inducing surface upregulation of IFNAR1 and IFNGR1. USP6-expressing Ewing sarcoma cells stimulated migration of primary human monocytes and T lymphocytes and triggered activation of natural killer (NK) cells in vitro. USP6 inhibited Ewing sarcoma xenograft growth in nude but not NSG mice and was accompanied by increased intratumoral chemokine production and infiltration and activation of NK cells, dendritic cells, and macrophages, consistent with a requirement for innate immune cells in mediating the antitumorigenic effects of USP6. High USP6 expression in patients with Ewing sarcoma was associated with chemokine production, immune infiltration, and improved survival. This work reveals a previously unrecognized tumor-suppressive function for USP6, which engenders an immunostimulatory microenvironment through pleiotropic effects on multiple immune lineages. This further raises the possibility that USP6 activity may be harnessed to create a “hot” tumor microenvironment in immunotherapy. Significance: This study reveals a novel tumor-suppressive function for USP6 by inducing an immunostimulatory microenvironment, suggesting that USP6 activity may be exploited to enhance immunotherapy regimens.