Identification of Alpha-Enolase (ENO1) As a Novel Immunometabolic Target in Multiple Myeloma

Introduction. Our previous studies demonstrated an integral role of bone marrow (BM) plasmacytoid dendritic cells (pDCs) in MM pathogenesis. 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). Here we performed DNA microarray analysis to identify and functionally validate the aberrant moleculer mechanisms mediating pDC-MM interactions with a goal to restore anti-tumor immunity and increase cytotoxicity. Our data shows that pDC-MM interactions trigger significant upregulation of the metabolic enzyme alpha-Enolase (ENO1) in both pDCs and MM cells. ENO1 is known to play a role in metabolic pathways in normal and cancer cells. It functions as a glycolytic enzyme and plasminogen receptor on the surface of tumor cells and MM pDCs. The surface localization of overexpressed ENO1 is a unique feature of tumor cells. We utilized our pDC/MM or pDC/T cells coculture models to show that targeting ENO1 generates MM-specific CTL activity and enhances NK-cell mediated lytic activity. Additionally, we examined whether the combination of ENO1 antagonist with anti-PD-L1 Ab or HDCAi ACY-241 enhances anti-tumor immunity and cytotoxicity in MM.

MethodsMicroArray: MM cells were co-cultured with pDCs for 72h, and then 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, and heat map was generated ( >1.5-fold change 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 ENO1 expression. CTL or NK-lytic activity assays: MM patient BM CD8+ T or NK-cells were cocultured with autologous pDCs (pDC:T/NK; 1:10 ratio) in the presence or absence of ENO1 inhibitor/ENO1i ENOblock (0.1-0.2 µM; Selleck Chemicals) or anti-ENO1 Ab for 5 days; 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. Combination studies were performed using ENO1 antagonists and anti-PD-L1 Ab (5 ug/ml) or ACY-241 (0.1-0.2 uM). Degranulation assay was done to quantify cell surface CD107a. Osteoclast (OCL) function was measured using TRAP staining. Osteoblast (OBL) formation and subsequent bone mineralization were assessed using Alizarin Red staining.

Results. 1) Normalized microarray expression profile showed pDC-induced upregulation of ENO1 transcript in MM (1.772-fold vs MM alone; n=3; CI > 95%). 2) Both pDC and MM cells showed high surface ENO1 expression, and pDC-MM coculture further increased surface ENO1 expression in MM cells (5-6 fold; n=5; p = 0.003), as well as the ENO1⁺ MM cell population (3-4 fold vs MM; mean ± SD; n=5; p = 0.005). 3) ENO1 antagonists directly activate pDCs and upregulate functional markers CD80 (1.2-fold) and CD83 (1.3-fold). 4) Treatment of pDC-T cell co-cultures with ENO1i triggers a robust MM-specific CD8+ CTL activity against autologous CD138+ 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; p = 0.008). 6) In concert with above findings, ENO1 blockade also increased surface CD107a expression on NK and CD8+T cells (CD107a+ NK: 1.4-fold; p = 0.003; CD107a+ CD8+T: 1.3 fold; p= 0.001). 7) Importantly, combination of ENO1i and anti-PD-L1 Ab triggers a more robust allogeneic and autologous MM-specific CD8+ CTL activity than ENO1i alone (%MM lysis: ENO1i: 20%; ENO1i + anti-PD-L1 Ab: 54%; n=7; p = 0.006). Combination of ENOi and ACY-241 also enhances the anti-tumor immunity in MM vs ENOi alone. Finally, 8) ENO1i blocked monocyte-derived osteoclast formation, as well as restored MM patient BM-derived osteoblast formation.

Conclusions pDC-MM interactions upregulate immunosuppressive metabolic alpha-enolase enzyme ENO1. Importantly, blockade of ENO1 tiggers MM-specific CD8⁺ CTL and NK-cell-medaited cytoxicity against MM cells. Our findings therefore identify novel immune-based therapeutic strategies targeting ENO1 to enhance MM cytotoxicity and restore anti-MM immunity.