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

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 (>1.5-fold change was considered significant, CI > 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 > 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.