PS1362 TRYPTOPHAN DEPRIVATION DUE TO INCREASED IDO‐1 TRIGGERS A METABOLIC ADAPTIVE RESPONSE AND DOWN‐REGULATES CD38 IN MULTIPLE MYELOMA
Authors: Romano A. , Puglisi F. , Parrinello N.L. , Giallongo C. , La Cava P. , Todoerti K. , Tibullo D. , Camiolo G. , Del Fabro V. , Cambria D. , Conticello C. , Palumbo G.A. , Cenci S. , Neri A. , Di Raimondo F. 
Published: June 2019

Background:

There is an increasing interest about the role of amino acid degrading enzymes in cancer immunotherapy. Indoleamine 2,3 dioxygenase (IDO) is the rate‐limiting enzyme in the kynurenine pathway, catabolizing tryptophan (trp) to kynurenine (kyn), sustaining immune evasion and inhibiting effector T cells.

Aims:

Investigating the effects of trp deprivation in multiple myeloma (MM).

Methods:

By combining different assays (ELISA, RT‐PCR, immuofluorescence, flow cytometry and western blot) we evaluated the metabolic adaptive response to trp shortage in four human MM cell lines (HMCLs, MM1.s, H929, U266, OPM2), chosen for their cytogenetic alterations and previously characterized for differential expression of CD38 and c‐myc and primary BM samples of MGUS and MM. We validated in‐vitro results looking at the expression levels of key metabolic genes in a large series of highly purified BM PC samples from healthy donors (4N), 129 MM, 24 primary plasma cell leukemia (pPCL), 12 secondary PCL (sPCL) cases from a proprietary dataset (GSE66293). Then, we investigated the effects of IDO inhibition to delay MM engraftment in vivo.

Results:

In a series of 20 healthy, 25 MGUS, 30 newly diagnosed MM patient, 20 relapsed/refractory subjects IDO was progressively increased. As consequence, trp was reduced and Kyn increased. Trp deprivation induced cell cycle arrest, while progressive trp shortage (range 1000–10 nM) did not affect proliferation in vitro, as evaluated up to 72 hours. Long‐term trp deprivation altered the cellular dependence on mitochondrial ATP generation via oxidative phosphorylation and increased glutamine anaplerosis, while surface expression of CD38 and intra‐cellular amount of c‐myc was reduced. Metabolic re‐shape was associated to increased isocitrate dehydrogenase (IDH‐1), enolase‐1 (ENO‐1), phosphoglycerate kinase 1 (PGK‐1), and dihydrolipoamide dehydrogenase (DLD), suggesting that branched chain amino acids with tolerogenic meaning like trp are used in MM to sustain NADH availability and energy production.

On the other hand, long‐term trp deprivation induced expression of CXCR4 and IRF4 in a time dependent manner, associated to increased p‐STAT3 without affecting other transcription factors relevant for MM cells like c‐myc or Helios. Counterintuitively, monoclonal componenent secretion was not affected, and both intra‐cellular and extra‐cellular lambda chain amount in U266 cells did not change after 72 hours of trp starvation.

We confirmed these findings in short‐term culture of CD138+ primary plasma cells with autologous feeder cells in presence of trp‐deprived media: after 18 hours, IRF4 and CXCR4 expression was increased and CD38 reduced.

In the GSE66293 MM, progression was associated to increased expression of ENO‐1, PGK‐1, and DLD, but were independent from CD38 expression, suggesting that changes in transcription of genes involved in bioenergetics could be clinically relevant, but not able to reflect the complex networking between malignant PCs and microenvironment.

In zebrafishes IDO inhibitor epacadostat could not delay MM engraftment. Increased doses of epacadostat (range 500–1000 nM) were cytotoxic against both human T‐CD4+cells and T‐CD8+ cells and for this reason could not be further tested in pre‐clinical in‐vivo models.

Summary/Conclusion:

Trp shortage is associated to progression from MGUS through MM due to increased IDO, contributing to metabolic adaptation. However, other soluble factors are involved in co‐opting adaptive metabolic phenotype, since IDO inhibition is not sufficient to delay MM engraftment in vivo.