Bortezomib Enhances the Antitumor Activity of Adoptively Infused Natural Killer Cells In Vivo: A Novel Approach To Override KIR-Mediated Inhibition of NK Cell Cytotoxicity.
Authors: Lundqvist Andreas , Rao Sheila , Smith Aleah , Berg Maria , Su Su , Yokoyama Hisayuki , Srivastava Shivani , Childs Richard 
Published: April 2020

Natural killer (NK) cell killer immunoglobulin-like receptor (KIR) interactions with self MHC class I molecules can regulate NK cell function; such interactions typically inactivate NK cells potentially providing a dominant mechanism through which malignant cells evade host NK cell-mediated immunity. Recently we found that the proteasome inhibitor bortezomib up-regulated surface expression of tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) on a variety of different human malignant cells rendering them susceptible to NK cell-mediated apoptosis in vitro; this effect appears to override KIR ligand-mediated NK cell inactivation, overcoming tumor resistance to both allogeneic KIR ligand-matched and autologous NK cell cytotoxicity. We also found that murine tumors were sensitized by bortezomib to the cytotoxic effects of syngeneic NK cells; the killing of RENCA and LLC1 tumors in vitro by syngeneic BALB/c and C57BL/6 NK cells respectively was enhanced when tumors were exposed to 10nM of bortezomib for 18h. Here, we show that the combined treatment of bortezomib followed by syngeneic NK cell infusions significantly delays tumor growth in tumor bearing animals. While treatment with bortezomib or interleukin-2 activated syngeneic NK cells alone had little effect on tumor growth, the combined treatment significantly delayed growth of RENCA tumors in BALB/c mice and LLC1 in C57BL/6 mice (p&lt;0.01;figure). In contrast to human tumor cell lines where an increase in expression of TRAIL-R2 was observed following bortezomib exposure, no change in expression of death receptors was observed in either murine tumor line. Flow cytometry analysis showed caspase-8 activity was significantly enhanced in bortezomib-treated murine tumor cells upon co-culture with NK cells compared to untreated tumor cells. Concanamycin A treatment significantly reduced NK cell-mediated apoptosis (but not neutralizing antibodies to Fas ligand or TRAIL) demonstrating that the sensitizing effect was mediated through perforin. Moreover, bortezomib-treated tumor cells were resistant to killing by perforin-deficient NK cells in vitro and the reduction in tumor growth observed in tumor bearing animals treated with bortezomib and wild-type NK cells was not observed in animals treated with bortezomib and perforin-deficient NK cells. These findings demonstrate that bortezomib-induced tumor sensitization to NK cell perforin and/or TRAIL could be used as a novel strategy to potentiate anti-tumor effects of adoptively infused NK cells in patients with cancer.

Figure. Left - BALB/c mice were injected with RENCA tumor cells (100.000 cells i.v) and treated with bortezomib (5ug/mouse i.v) on days 5, 12 and 19 followed by injection of sygeneric NK cells (2×106 i.v) on days 6,13 and 20. All animals received IL-2 (100.000 U i.p on days 6–9,13–16 and 20–23). Animals were euthanized on day 25 and evaluated for pulmonary metastasies. Right - C57BL/6 mice were injected with LLC1 tumor cells (500.000 s.c) and treated with bortezomib (15ug/mouse i.p) on day 14 followed by a single injection of syngeneic NK cells (1×106 i.v) on day 15. All mice were treated with IL-2 (100.000U i.p on days 15–18). Data depicts tumor sizes on day 28 post tumor injection. Figure. Left - BALB/c mice were injected with RENCA tumor cells (100.000 cells i.v) and treated with bortezomib (5ug/mouse i.v) on days 5, 12 and 19 followed by injection of sygeneric NK cells (2×106 i.v) on days 6,13 and 20. All animals received IL-2 (100.000 U i.p on days 6–9,13–16 and 20–23). Animals were euthanized on day 25 and evaluated for pulmonary metastasies. Right - C57BL/6 mice were injected with LLC1 tumor cells (500.000 s.c) and treated with bortezomib (15ug/mouse i.p) on day 14 followed by a single injection of syngeneic NK cells (1×106 i.v) on day 15. All mice were treated with IL-2 (100.000U i.p on days 15–18). Data depicts tumor sizes on day 28 post tumor injection.