Abstract 3147: Natural killer cells mediate tumor-inhibitory functions of USP6 in Ewing sarcoma
Authors: Jain Kanika , Henrich Ian C. , Quick Laura Nicole , Young Robert A. , Chou Margaret M. 
Published: June 2022

INTRODUCTION: Understanding how Natural Killer (NK) cell activity is regulated by cancer cells is critical for developing NK-cell based immunotherapies. Our studies show that the deubiquitinating enzyme ubiquitin-specific protease-6 (USP6) can induce an immune-stimulatory hot tumor microenvironment in Ewing sarcoma (ES), the 2nd most common pediatric bone cancer. High USP6 expression in ES is associated with significantly improved overall and progression-free survival. We found that USP6 expression in ES cells inhibits xenograft growth in nude mice, but not NSG mice, implicating a role for the innate immune system, particularly cytotoxic NK cells. Notably, USP6 expression enhanced intra-tumoral abundance, activation, and proliferative capacity of NK cells. In vitro cytotoxicity assays confirm that USP6 can directly augment the ability of ES cells to activate NK cells. Based on these observations, we hypothesize that NK cells are essential for USP6-mediated tumor suppression and seek to determine the mechanism by which their cytolytic function is activated by USP6.

METHOD: We used ES cell lines A673 and RD-ES expressing USP6 in a doxycycline (dox)-inducible manner, the immortalized human NK cell line NK-92, and primary human and mouse NK cells. Athymic nude or RAG2-/- mice were xenografted subcutaneously with ES cell lines and fed dox-infused water to induce USP6. Immune cell abundance and activation were monitored both in tumors and peripheral blood by flow cytometry. For in vivo depletion of NK cells, 300ug of anti-NK1.1 (PK136) ab was injected i.p. into RAG2-/- mice one day prior to ES xenografting, then once weekly thereafter.

RESULTS: USP6 induces in ES cells the surface expression of multiple NK activating ligands, as well as receptors for the apoptotic factor TRAIL (DR5) and Type I/II interferons (IFNAR1 and IFNGR1), both in vitro and in vivo. This leads to enhanced NK activation and renders ES cells hypersensitive to NK-mediated cytotoxicity. IFNy released by activated NK cells feeds back on the USP6-expressing ES cells, inducing synergistic expression of multiple IFNy-inducible genes, including chemokines CXCL9/10 (chemoattractants for NK cells) and ICAM-1 (essential for immunological synapse formation between NK cells and their targets). A potent feed forward loop for NK activation/ES cell killing is thus generated. Concordantly, in RAG2-/- mice, USP6 induces increased intratumoral infiltration and activation of NK cells, and also, strikingly, systemic activation of NK cells. Depletion of NK cells using anti-NK1.1(PK136) completely abrogates USP6-mediated inhibition of ES xenograft growth.

CONCLUSION: NK cells are essential for mediating the tumor inhibitory functions of USP6 in ES. We posit that USP6’s ability to activate NK cells both intratumorally and systemically underlies its association with improved ES patient prognosis, which we seek to exploit for therapeutic benefit in future studies.

Citation Format: Kanika Jain, Ian C. Henrich, Laura Nicole Quick, Robert A. Young, Margaret M. Chou. Natural killer cells mediate tumor-inhibitory functions of USP6 in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3147.