P-097: Preclinical validation of Ecto-5’ Nucleotidase (NT5E/CD73) as a novel immunotherapeutic target in Multiple Myeloma

Abstract

<h3>Background</h3> The interaction of plasmacytoid dendritic cells (pDCs) with multiple myeloma (MM) cells, and T- or NK-cells in the bone marrow (BM) microenvironment triggers enhanced tumor growth, as well as inhibits innate and adaptive immune responses. In the present study, we analyzed the genetic changes in MM cells triggered by co-culture with pDCs using next generation sequencing (NGS), and identified that pDC-MM interactions induce metabolic enzyme Ecto-5' Nucleotidase (NT5E/CD73) in both pDCs and MM cells. CD73 has been implicated in cancer metabolism and immunosuppression via nucleotide metabolism pathway. <h3>Methods</h3> Purified MM patient pDCs (n=3) were co-cultured with MM cells (1pDC/5MM; n=3) for 48h, followed by separation of MM cells from pDCs by flow. Total RNA from MM cells was subjected to RNAseq analysis using Illumina NGS. Data were analyzed by VIPER workflow to generate differential expression. The log2FC (fold change) values in co-cultures vs MM alone, with a False Discovery Rate adjusted p value of <0.05, was considered significant. Cytotoxic T lymphocyte (CTL) assay: MM patient BM CD8+ T-cells were co-cultured with autologous pDCs (pDC:T/1:10) in the presence or absence of anti-CD73 Ab (0.5 µg/ml) for 3 days; pre-stained MM cells were added for 24h (10T:1MM), followed by FACS quantification of viable MM cells. <h3>Results</h3> A total of 9200 and 9250 genes were differentially expressed based on negative binomial distribution (DEseq2) and linear (Limma) RNA-seq models, respectively (p<0.05). MM cells cultured with (n=3) or without pDCs (n=3) clustered into two distinct groups, indicating contact-dependent transcriptional changes in MM cells after co-culture. pDC-MM interaction regulates several pathways including DNA replication/repair, cell cycle, and nucleotide metabolism. We showed that: 1) pDC-MM interactions induce transcription of adenosine-signaling pathway enzyme NT5E/CD73 in MM cells (1.34-fold increase after co-culture; p=0.0002); 2) Both pDCs and MM cells express CD73, and pDC-MM interactions further increase CD73 levels in MM cells (p=0.008); 3) pDC-MM interactions increase adenosine generation (5.5-fold), and importantly, CD73 blockade by anti-CD73 Ab decreases adenosine production (p=0.0167); 4) CD73 blockade activates MM pDCs, evidenced by the increased expression of CD40/CD83/CD86/HLA-DR on pDCs, and also restores pDCs ability to activate autologous T cells; 5) Targeting CD73 induces MM-specific CD8+ CTL activity against autologous MM cells; and 6) a combination of anti-CD73 Ab and TLR7 agonist triggers more potent MM-specific CD8+ CTL activity against autologous MM cells than either agent alone (%viable MM: anti-CD73 Ab:70%; TLR7 agonist:60%; combo:30%; p=0.009;n=7). <h3>Conclusions</h3> Our preclinical data therefore suggest that the therapeutic targeting of CD73, alone or in combination with TLR7 agonist, represents a promising novel strategy to restore host anti-MM immunity.