Reactive oxygen species- and DNA damage response-dependent NK cell activating ligand upregulation occurs at transcriptional levels and requires the transcriptional factor E2F1.

Increasing evidence indicates that cancer cell stress induced by chemotherapeutic agents promote antitumor immune responses and contribute to their full clinical efficacy. In this article, we identify the signaling events underlying chemotherapy-induced NKG2D and DNAM-1 ligand expression on multiple myeloma (MM) cells. Our findings indicate that sublethal doses of doxorubicin and melphalan initiate a DNA damage response (DDR) controlling ligand upregulation on MM cell lines and patient-derived malignant plasma cells in Chk1/2-dependent and p53-independent manner. Drug-induced MICA and PVR gene expression are transcriptionally regulated and involve DDR-dependent E2F1 transcription factor activity. We also describe the involvement of changes in the redox state in the control of DDR-dependent upregulation of ligand surface expression and gene transcriptional activity by using the antioxidant agent N-acetyl-L-cysteine. Finally, in accordance with much evidence indicating that DDR and oxidative stress are major determinants of cellular senescence, we found that redox-dependent DDR activation upon chemotherapeutic treatment is critical for MM cell entry in premature senescence and is required for the preferential ligand upregulation on senescent cells, which are preferentially killed by NK cells and trigger potent IFN-γ production. We propose immunogenic senescence as a mechanism that promotes the clearance of drug-treated tumor cells by innate effector lymphocytes, including NK cells.

Inhibition of glycogen synthase kinase-3 increases NKG2D ligand MICA expression and sensitivity to NK cell-mediated cytotoxicity in multiple myeloma cells: role of STAT3.

Engagement of NKG2D and DNAX accessory molecule-1 (DNAM-1) receptors on lymphocytes plays an important role for anticancer response and represents an interesting therapeutic target for pharmacological modulation. In this study, we investigated the effect of inhibitors targeting the glycogen synthase kinase-3 (GSK3) on the expression of NKG2D and DNAM-1 ligands in multiple myeloma (MM) cells. GSK3 is a pleiotropic serine-threonine kinase point of convergence of numerous cell-signaling pathways, able to regulate the proliferation and survival of cancer cells, including MM. We found that inhibition of GSK3 upregulates both MICA protein surface and mRNA expression in MM cells, with little or no effects on the basal expression of the MICB and DNAM-1 ligand poliovirus receptor/CD155. Moreover, exposure to GSK3 inhibitors renders myeloma cells more efficient to activate NK cell degranulation and to enhance the ability of myeloma cells to trigger NK cell-mediated cytotoxicity. We could exclude that increased expression of ß-catenin or activation of the heat shock factor-1 (transcription factors inhibited by active GSK3) is involved in the upregulation of MICA expression, by using RNA interference or viral transduction of constitutive active forms. On the contrary, inhibition of GSK3 correlated with a downregulation of STAT3 activation, a negative regulator of MICA transcription. Both Tyr(705) phosphorylation and binding of STAT3 on MICA promoter are reduced by GSK3 inhibitors; in addition, overexpression of a constitutively active form of STAT3 significantly inhibits MICA upregulation. Thus, we provide evidence that regulation of the NKG2D-ligand MICA expression may represent an additional immune-mediated mechanism supporting the antimyeloma activity of GSK3 inhibitors.

Heat shock protein-90 inhibitors increase MHC class I-related chain A and B ligand expression on multiple myeloma cells and their ability to trigger NK cell degranulation.

Modulation of the host immune system represents a promising therapeutic approach against cancer, including multiple myeloma. Recent findings indicate that the NK group 2D (NKG2D)- and DNAX accessory molecule-1 (DNAM-1)-activating receptors play a prominent role in tumor recognition and elimination by cytotoxic lymphocytes, suggesting that the levels of NKG2D and DNAM-1 ligand expression on tumor cells may be a critical factor to improve the immune response against cancer. In this study, we tested the effect of 17-allylaminogeldanamycin and radicicol, drugs targeting the heat shock protein-90 (HSP-90) chaperone protein and displaying antimyeloma activity, on the expression of NKG2D and DNAM-1 ligands in human myeloma cell lines. We demonstrate that HSP-90 inhibitors are able to up-regulate both MHC class I chain-related (MIC) A and MICB protein surface and mRNA expression in human myeloma cell lines, without any significant effect on the basal expression of the DNAM-1 ligand poliovirus receptor CD155, or induction of nectin-2 and UL16-binding proteins. Activation of the transcription factor heat shock factor-1 by HSP-90 inhibitors is essential for the up-regulation of MICA/MICB expression and knockdown of heat shock factor-1 using small hairpin RNA interference blocks this effect. Moreover, in vitro and in vivo binding of heat shock factor-1 to MICA and MICB promoters indicates that it may enhance NKG2D ligand expression at the transcriptional level. Finally, exposure to HSP-90 inhibitors renders myeloma cells more efficient to activate NK cell degranulation and a blocking Ab specific for NKG2D significantly reduces this effect. Thus, these results provide evidence that targeting NKG2D ligands expression may be an additional mechanism supporting the antimyeloma activity of HSP-90 inhibitors and suggest their possible immunotherapeutic value.