Immunomodulatory EFFECTS of Lenalidomide and Pomalidomide ON INTERACTION of TUMOR and BONE MARROW Accessory CELLS IN MULTIPLE MYELOMA.
Authors: Gorgun Gullu , Calabrese Elisabetta , Hideshima Teru , Perrone Giulia , Bianchi Giada , Ikeda Hiroshi , Mani Mala , Hu Yiguo , Cristea Diana , Santo Loredana , Tai Yu-Tzu , Munshi Nikhil , Richardson Paul , Anderson Kenneth C. 
Published: October 2019

Abstract
Abstract 950
The bone marrow (BM) microenvironment consists of extracellular matrix and the cellular compartment including bone marrow stromal cells (BMSCs) and immune cells. Interaction between multiple myeloma (MM) cells and BM cells induces growth, survival, migration, and drug resistance in MM, via both cell-cell contact and cytokines. Even though MM cell interaction with BMSCs has been extensively studied, the role of immune cells in the MM BM milieu is not yet defined. The IMiDs® immunomodulatory agents lenalidomide (len) and pomalidomide (pom) target not only MM cells, but also MM cell-immune cell interactions and cytokine signaling. For example, we and others have shown that len stimulates T cell proliferation, secretion of IL2 and IFNγ, as well as promotes CTL and NK cell activity against MM cells. Here we examined the in vitro immunomodulatory effects of len or pom on cytokine signaling triggered by interaction of effector immune cells with MM cells and BMSCs. PBMCs or BMMNCs obtained from patients with rel/ref MM or healthy donors after informed consent. PBMCs were cultured either alone or with BMSC, in the absence or presence of len (1μM) or pom (1μM) for 1-48h. To determine whether len or pom regulate cytokine signaling in effector cells, we used flow cytometry to analyze their effects on suppressor of cytokine signaling proteins (SOCS, including SOCS1, SOCS2, SOCS3, CIS) expression in effector cells from both healthy donors and patients with MM. Len or pom diminished IL2 and IFNγ regulators SOCS1 and SOCS3 expression in effector cells from both BM and PB of MM patients. Additionally, coculture of MM cell lines, MM1S, U266, OPM1, RPMI, LR5 and DOX40, with healthy PBMCs induced SOCS1 and SOCS3 expression in effector cells; conversely, treatment with len or pom downregulated the SOCS1 and SOCS3 expression in effector cells. To assess effects of immunomodulatory agents on immune cell proliferation in their milieu, healthy or MM-PBMCs and MM-BMMNCs were prelabeled with CFSE and stimulated with PHA (5μg/ml) or anti-CD3 (1μg/ml) in the absence or presence of len or pom for 7 days. The proliferation of CD4T and CD8T, NKT, and NK cells was assessed by CFSE flow cytometric analysis. Len or pom induced CD4T cell (%Divided: Cont:55, len or pom >72), CD8 T cell (%Div: Cont:34, len or pom>60) and NKT cell (%Div: Cont:3.5, len or pom >8) proliferation, as well as stimulated IL2 (2-4 fold) and IFNγ (2 fold) production in effector cells from MM. It has been demonstrated that SOCS1 gene negatively regulates IL6 signaling and is silenced by methylation in MM cells. To understand the mechanism of cytokine inhibitory signaling in both effector cells and MM cells, we next analysed the interaction of effector cell with MM cells that were epigenetically modified to express SOCS1. SOCS1 methylation in MM cells was confirmed by SOCS1 gene methylation-specific polymerase chain reaction (SOCS1-MSP). Genomic DNA was isolated from MM cell lines (MM1S, RPMI8226, OPM1, INA6 and U266), sodium bisulfite-modified, and then subjected to MSP using MSP primers that specifically recognize unmethylated or methylated SOCS1 gene. SOCS1 gene was methylated and resulted in silenced SOCS1 protein expression in all MM cell lines. To delineate the role of SOCS in effector cell response against MM cells, MM cell specific cytotoxic T lymphocytes (CTL) were generated. T cells from healthy donors were stimulated with dendritic cells pulsed with apoptotic bodies of MM1S or U266 cells for 4 weeks, and cytotoxicity was measured by standard 51Cr-release assay. To reverse SOCS1 methylation, target MM cells were cultured with 5'-Azacytidine (Aza) or trichostatin A (TsA), alone or in combination with len or pom. CTLs were pretreated with len or pom for 24h and cocultured with DNA-modified or unmodified 51Cr-labeled target cells. Len induced more potent CTL response against MM cells that were treated with len and Aza combination (83% specific killing) than len alone (%50 specific killing). Len also showed more potent anti-MM activity, assessed by 3[H]thymidine proliferation assay, in the presence of Aza than alone (p<0.05). These data demonstrate that modulation of SOCS genes by blocking BMSC derived inhibitory cytokine signaling may enhance effector cell response and promote efficacy of len or pom in MM. Ongoing analysis of effects of len or pom on immune cells in the BM environment will both define their role in disease pathogenesis and suggest novel immune-based targeted therapies.

Disclosures:
Munshi: Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Research Funding; Novartis: Research Funding; Celgene: Research Funding.