Preclinical Validation of Alpha-Enolase (ENO1) As a Novel Immunometabolic Target in Multiple Myeloma
Authors: Ray Arghya , Song Yan , Chauhan Dharminder , Anderson Kenneth C. 
Published: February 2020

Introduction. Bone marrow plasmacytoid dendritic cells (pDCs) in patients with multiple myeloma (MM) both promote tumor growth, survival, and drug resistance, as well as induce decreased T/NK effector cell function and immune suppression. Delineation of the mechanism(s) mediating pDCs-MM-T-NK cells interactions may therefore identify novel therapeutic targets to enhance anti-MM immunity. Using gene expression profiling, we show that pDC-MM interactions trigger significant upregulation of the immunosuppressive metabolic enzyme alpha-Enolase (ENO1) in both pDCs and MM cells. ENO-1 functions as a glycolytic enzyme and plasminogen receptor which is overexpressed on the surface of tumor cells and MM pDCs. Here, we utilized our coculture models of patient autologous pDC-T-NK-MM cells to examine whether targeting ENO1, either alone or in combination can enhance anti-MM immunity in the BM milieu.
Methods Gene expression profiles of MM cells cultured in the presence vs absence of pDCs were compared, and a heat map was generated (&gt;1.5-fold change was considered significant, CI &gt; 95%). MM cells were co-cultured with pDCs for 24h, followed by multicolor flow analyses to determine the pDC-induced change in ENO1 expression. Cytotoxic T lymphocyte (CTL) and NK cell activity assays: MM patient BM CD8+ T or NK-cells were cocultured with autologous pDCs (pDC1:T/NK10 ratio) in the presence or absence of ENO1 inhibitor/ENO1i ENOblock (0.2 µM) or anti-ENO1 Ab for 5 days; MM pre-stained cells were added for 24h (10T/NK:1MM), followed by quantification of viable MM cells by FACS. Anti-PD-L1 Ab (5 ug/ml) or HDAC 6 selective inhibitor ACY-241 (0.2 uM) were utilized for combination studies with ENO1i. CD107a expression was quantified in a degranulation assay.
Results GEP analysis showed that pDCs induce upregulation of ENO1 transcript in MM (1.8-fold vs MM alone; n = 3; CI &gt; 95%). Protein expression analysis showed that ENO1 is expressed in both pDC and MM cells; and importantly, that pDC-MM coculture further increases ENO1 expression in MM cells (5-6-fold; n=3; p = 0.003). The ENO+ MM cell population is also increased after pDC-MM cell coculture (3-4-fold vs MM; mean ± SD; n = 3; p = 0.005). Blockade of ENO1 with ENO inhibitor (ENOi) activates pDCs, as evidenced by increase in pDCs maturation/activation markers (CD80/CD83/CD86). Importantly, ENOi restores the ability of pDCs to trigger T cell activation and proliferation (n = 3; p = 0.018). Above all, ENOi increases pDC-induced MM-specific CD8+ CTL activity (p = 0.006), as well as NK cell-mediated cytolytic activity against autologous tumor cells (p = 0.005). Moreover, pDC-mediated MM-specific CD8+ CTL activity was effective even against allogeneic HLA-A2+ U266 MM cells (p = 0.008). Consistent with CTL and NK cell activation, ENO1i increases expression of surface CD107a on CD8+ T and NK cells (p = 0.006 and p= 0.0112, respectively; n = 7). The combination of ENO1i and anti-PD-L1 Ab induces more robust allogeneic and autologous MM-specific CD8+ CTL activity than ENO1i alone (% MM lysis: ENO1i: 34%; ENO1i + anti-PD-L1 Ab: 54%; n = 7; p = 0.0013). Finally, the combination of ENOi and ACY-241 also enhances anti-MM immune responses (% MM lysis: ENO1i: 35%; ENO1i + ACY-241: 54%; n = 8; p = 0.0044).
Conclusions Our preclinical data provide the basis for novel immune-based therapeutic approaches targeting immunosuppressive metabolic alpha-enolase enzyme ENO1 to restore anti-MM immunity and improve patient outcome.

Disclosures
Chauhan: Stemline Therapeutics: Consultancy; C4 Therapeutics.: Equity Ownership. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.