Identification of Metabolic Enzymes ENO1 and KMO As Novel Immunotherapeutic Targets in Myeloma

Introduction. Our previous studies showed an integral role of bone marrow (BM) plasmacytoid dendritic cells (pDCs) in MM pathogenesis. pDCs are present in increased numbers in MM BM, promote tumor growth and survival, protect tumor from therapy-induced cytotoxicity; and suppress immune responses (Chauhan et al, Cancer Cell 2009, 16:309-323) . Aberrant pDC function is evidenced from their interactions with MM tumor and T/NK effector cells in the BM milieu (Ray et al, Leukemia 2015, 29:1441-1444) . Direct interruption of pDC-MM and pDC-T cells interactions and their sequelae utilizing novel targeted agents is necessary to further enhance both anti-tumor immunity and cytotoxicity. Based on DNA microarray analysis, we identified several novel targets on MM cells that are upregulated after coculture with pDCs, and focused on metabolic pathway enzymes that are upregulated both in pDC and MM cells. We identified alpha-Enolase (ENO1) and Kynurenine 3-monooxygenase (KMO) as the two most promising therapeutic targets, based on their roles in normal and cancer metabolism. ENO1 is a glycolytic enzyme, and its surface localization is a unique feature of the tumor cells. Moreover, ENO1 promotes tumor metastasis. KMO is one of the two rate-limiting enzymes involved in eukaryotic tryptophan catabolic kynurenine pathway; and importantly, its overexpression has been reported to inhibit functional immune responses in cancer. Here, we utilized our pDC/MM or pDC/T cells co-culture model to show that targeting ENO1 or KMO generates MM-specific cytotoxic T lymphocyte activity.

Methods DNA MicroArray Analysis: MM.1S cells were cocultured with pDCs for 72h, separated using CD138 Microbeads, and harvested. Poly RNA was subjected to microarray analysis using HG-U133 plus 2.0 plus Affymetrix chip. Gene expression patterns for MM.1S cells cultured in the presence vs absence of pDCs were compared, and heat map was generated ( >1.5-fold change in transcript was considered significant, CI > 95%). pDC-induced overexpression of target proteins : MM cells were cocultured with pDCs for 24h, followed by multicolor flow analyses to determine the pDC-induced change in target protein expression. CD138+ tumor cells were gated out, and KMO-FITC or ENO1-AlexaFluor488-stained cells were analyzed. C TL activity assays : MM patient BM CD8+ T cells were cocultured with autologous pDCs at 1:10 (pDC:T) ratio in the presence or absence of KMO inhibitor Ro 61-8048 (10 nM) or ENO1 inhibitor ENOblock (0.1-0.2 µM) or anti-ENO1 Ab for 5 days; pre-stained MM cells were added for 24-48h (E/T ratio 10:1, T:MM), followed by quantification of viable MM cells by FACS. Statistical parameters were calculated using GraphPad Prism. Pharmacological inhibitors were obtained from Selleck Chemicals.

Results. 1) Normalized microarray expression profile showed pDC-induced upregulation of both ENO1 and KMO transcripts in MM cells (1.772 and 2.153-fold, respectively, vs MM.1S alone; n=3; CI > 95%). 2) Both pDC and MM cells showed high KMO and surface ENO1 expression. Coculture of MM cells with pDCs further increased ENO1 surface expression levels in MM cells (5-6-fold increase; n=3; p = 0.003), as well as the ENO1-positive MM cell population (3-4-fold versus MM alone; mean ± SD; n=3; p = 0.005). 3) Like ENO1, pDCs also increased both KMO expression on MM cells and KMO-positive MM cells (MFI: 2-3-fold in the presence vs absence of pDCs; p = 0.006). 4) Functional studies showed that treatment of pDC-T cell cocultures with ENO1 antagonists triggers a robust MM-specific CD8+ CTL activity against autologous CD138-positive MM cells (2 fold in treatment vs control; p = 0.005). 5) pDC-mediated MM-specific CD8+ CTL activity was also confirmed using allogeneic HLA-A2+ U266 MM cells (1.8-2-fold vs control; p = 0.008). Finally, 6) KMO inhibitor Ro 61-8048 activates patient BM-CD8+T cells in autologous pDCs/T cell cocultures, and triggers robust allogeneic and autologous MM-specific CD8+ CTL activity (~2.0-fold vs control; p = 0.009).

Conclusions Our study shows that pDC-MM interactions upregulate tumor promoting and immunosuppressive metabolic enzymes in MM. Blockade of ENO1 or KMO enzymes tiggers MM-specific CD8-positive CTL activity. These data therefore identify novel immune-based therapeutic strategies targeting ENO1 and KMO to enhance MM cytotoxicity and restore anti-MM immunity.