Upregulation of PD-L1 on Myeloma Cells By Immunomodulatory Agents Potentiates the Effect of Durvalumab
Authors: Ishibashi Mariko , Yamamoto Junichi , Ito Takumi , Handa Hiroshi , Inokuchi Koiti , Takahashi Hidemi , Tamura Hideto 
Published: October 2019

Abstract
Introduction: Programmed death-ligand 1 (PD-L1) shows increased expression on plasma cells from multiple myeloma (MM) patients as compared with those from monoclonal gammopathy of undetermined significance patients and healthy volunteers. PD-L1 inhibits tumor-specific cytotoxic T lymphocyte-mediated responses via interaction with PD-1. Our previous study showed that PD-L1 on MM cells is associated with aggressive myeloma behaviors including cell proliferation and drug resistance, and furthermore the interaction of PD-L1 with PD-1 molecules induced drug resistance in MM cells by antiapoptotic responses through the Akt signaling pathway (Tamura et al. Leukemia 2013, Cancer Immunol Res 2016). We next investigated the regulation of PD-L1 expression by immunomodulatory drugs (IMDs) and antimyeloma effects of the anti-PD-L1 antibody durvalumab (DUR) in combination with IMDs.
Methods: 1) The expression of mRNA and protein in MM cell lines (MOSTI-1, U266, KMS18, OPM2, MM.1S) and plasma cells from MM patients was analyzed using real-time PCR/DNA microarray and flow cytometry/Western blotting, respectively. 2) PD-L1 induction by IMDs was evaluated based on promoter activity in the luciferase assay. 3) Cereblon (CBRN)-knockdown and -knockout MM cell lines were established using the lentiviral shRNA and CRISPR/Cas9 system, respectively. 4) MM.1S cells were inoculated subcutaneously into immunodeficient NOD/Shi-scid, IL-2Rγnull (NOG) mice and then MM1.S-engrafted mice were administered lenalidomide (LEN; 25 mg/kg per day) orally for 12 days. 5) CD3+ T cells were isolated from peripheral blood mononuclear cells of healthy volunteers, and then T cells activated with anti-CD3/CD28 antibodies were co-cultured with X-irradiated MM cells for 4 days. T-cell proliferation and activation were assessed using the CFSE cell division assay and expression levels of activation markers CD25 and CD45RO, respectively.
Results: 1) PD-L1 expression was upregulated in MM cell lines by LEN and pomalidomide (POM) in a concentration-dependent manner, but not by thalidomide. Its expression on plasma cells, but not on lymphocytes and monocytes, obtained from relapsed/refractory MM patients was enhanced by LEN and POM. This PD-L1 induction was not seen in CRBN-knockdown and -knockout MM cell lines. To clarify this mechanism, gene expression profiling analysis was performed in MM cell lines with and without POM treatment, demonstrating that not only PD-L1 but also a proliferation-inducing ligand (APRIL) was enhanced by POM. APRIL expression was not induced by POM in CRBN-knockdown and -knockout MM cell lines. PD-L1 upregulation on MM cells by POM was inhibited by either recombinant B cell maturation antigen (BCMA)-Ig as an APRIL inhibitor, the antibody against BCMA, one of the receptors for APRIL, or MEK/ERK inhibitor U0126. Furthermore, PD-L1 and APRIL expression in MM.1S cells engrafted in NOG mice were upregulated by LEN treatment. On the other hand, LEN/POM also increased PD-L1 promoter activity in BCMA- and APRIL-negative HeLa cells. 2) When T cells were co-cultured with LEN- or POM-treated U266 cells (PD-L1-upregulated cells), the proliferation of CD4+ and CD8+ T cells was inhibited in comparison with that in untreated cells. This T-cell suppression did not occur when DUR and LEN/POM were added during co-cultivation of LEN/POM-treated U266 cells with T cells. Moreover, the population of both CD25+CD45RO+ double-positive CD4+ and CD8+ T cells co-cultured with POM-treated U266 cells was increased by combined POM and DUR treatment compared with DUR alone.
Conclusion: Our study revealed that IMDs enhanced PD-L1 expression on MM cells as well as APRIL production through CRBN binding, and one mechanism may involve the APRIL-BCMA interaction. Another mechanism may be directly involved in PD-L1 downregulation by IKZF1 degradation through CRBN. Furthermore, T-cell inhibition induced by PD-L1-upregulated MM cells was effectively recovered after combination treatment with DUR and LEN or POM. These results suggest that DUR combined with IMDs may be a reasonable treatment for relapsed/refractory MM patients.

Disclosures
Ito: Celgene: Honoraria. Handa:JVC KENWOOD Corporation: Other: Leadership Position/Advisory Role; SYSMEX CORPORATION: Other: Leadership Position/Advisory Role; TAMAGAWA SEIKI Co.,Ltd.: Other: Leadership Position/Advisory Role; Celgene: Honoraria, Other: Supplied with reagents and drugs., Research Funding. Tamura:Takeda Pharmaceutical: Honoraria; Ono Pharmaceutical: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria, Research Funding.