Targeting Tryptophan Catabolic Kynurenine Pathway Enhances Anti-Tumor Immunity and Cytotoxicity in Multiple Myeloma

Introduction. Plasmacytoid dendritic cells (pDCs) play an integral role in MM pathogenesis (Chauhan et al, Cancer Cell 2009, 16:309-323). pDCs interactions with tumor cells and T/NK effector cells in the MM-BM milieu induce immune suppression and MM cell proliferation (Chauhan et al, Cancer Cell 2009, 16:309-323;Ray et al, Leukemia 2015, 29:1441-1444). A direct interruption of pDC-MM and pDC-T cells interactions with novel agents will enhance cytotoxicity and anti-tumor immunity. In this context, our DNA microarray analysis identified upregulation of tryptophan catabolic kynurenine (Kyn) pathway during pDC-MM interactions. To date, the role of Kyn pathway in MM is unclear, but two rate-limiting enzymes of Kyn pathway, kynurenine 3-monooxygenase (KMO) and indoleamine 2,3-dioxygenase 1 (IDO1), have been implicated in various cancers. Overexpression of these enzymes causes hyper-accumulation of terminal metabolites, thereby triggering immune suppression via disruption of bidirectional signaling axes between antigen-presenting cells and T cells. In our study, we found that KMO and IDO1 are increased in both pDCs and MM cells during pDC-MM interactions. To assess the functional significance of these findings, we used our pDC/MM or pDC/T cells co-culture models, and show that targeting Kyn pathway generates MM-specific CTL activity. Moreover, the combination of KMO/IDO1 inhibitors with anti-PD-L1 Ab enhances anti-tumor immunity and cytotoxicity in MM.

MethodsMicroArray: MM cells were cocultured with pDCs for 72h; separated using CD138 Microbeads; poly RNA was subjected to microarray analysis using HG-U133 plus 2.0 plus Affymetrix chip. Gene expression patterns for MM cells cultured in the presence vs absence of pDCs were compared ( >1.5-fold change was considered significant, CI > 95%). pDC-induced overexpression of target proteins: MM cells were cocultured with pDCs for 24h; analyzed by flow to detect KMO/IDO1 expression. CTL or NK activity assays: Minimally cytotoxic concentrations of inhibitors were used to assess immune functions. MM-BM CD8+ T or NK- cells were cultured with autologous pDCs (pDC:T/NK; 1:10 ratio) in the presence or absence of KMO (Ro 61-8048: 100 nM) or IDO1 (INCB 024360: 0.1 µM) inhibitors (Selleck Chemicals) for 5 days; drug washed, pre-stained MM cells were added for 24-48h (E/T ratio 10:1, T/NK:MM), followed by quantification of viable MM cells by FACS. Degranulation assay was quantified using cell surface CD107a. Osteoclast and osteoblast formations were assessed from TRAP and Alizarin Red staining, respectively.

Results. 1) Normalized microarray expression profile showed pDC-induced upregulation of KMO in MM cells (2.153 fold vs MM alone; n=3; CI > 95%). 2) Both pDCs and MM cells expressed KMO and IDO1; pDC-MM coculture further increased KMO expression in MM cells (2-3-fold vs MM; p = 0.006); KMO⁺ MM cell populations also increased after coculture (~1.2 fold; p <0.05). Importantly, IDO1 expression in MM cells is also enhanced after co-culture; validating the importance of Kyn pathway in the context of pDC-MM interactions (MFI: 1.5-2 fold; p=0.006); IDO1⁺ MM cell populations increased by ~1.5 fold after coculture (p <0.05). 3) KMOi directly activates pDCs and upregulates functional markers on pDCs (CD80: 1.19 fold; CD83: 1.4 fold; p <0.05). 4) KMOi activates patient BM-CD8⁺T cells in autologous pDCs/T cell coculture; triggers robust allogeneic and autologous MM-specific CD8⁺ CTL activity (~2.0 fold vs control; p = 0.009). 5) In agreement with above findings, KMOi also increased surface CD107a expression on NK and CD8+T cells (CD56+NK: 1.41 fold; p = 0.004; CD8+T: 1.6 fold; p= 0.001). 6) Importantly, the combination of KMOi or IDO1i and anti-PD-L1 Ab (5 µg/ml) triggers a more robust MM-specific CD8⁺ CTL activity than single agent (%MM viability: KMOi: 72%; KMOi + anti-PD-L1 Ab: 57%; IDOi: 82%; IDOi + anti-PD-L1 Ab: 70%; n=7; p = 0.01). Finally, 7) Both KMOi and IDO1i blocked monocyte-derived osteoclast formation, as well as restored MM patient BM-derived osteoblast formation.

Conclusions pDC-MM interactions upregulate immunosuppressive enzymes in Kyn pathway; and importantly, blockade of Kyn pathway enzymes induces MM-specific CD8⁺ CTL activity. Our preclinical data therefore provides provides the basis for novel immune-based therapeutic approaches targeting kynurenine (Kyn) pathway enzymes KMO and IDO1 to enhance MM cytotoxicity and restore anti-MM immunity.