Targeting Overexpressed IDO on Stromal Cells As a Potential Therapeutic Strategy in Multiple Myeloma

【Background】

Interactions of multiple myeloma (MM) cells with cellular and non-cellular components in the bone marrow milieu, such as cytokines, metabolites and exosomes, are important in the pathogenesis of MM. Stromal cells are an important component of the MM tumor microenvironment (TME) and promote the development of drug resistance. Indoleamine 2,3-dioxygenase 1 (IDO), an enzyme that metabolizes tryptophan to kynurenine, has been reported to cause T cell exhaustion and increase regulatory T cells, thus acting as an immunosuppressive factor in the TME of some cancers. However, the status of IDO has not been fully investigated regarding the immune milieu of MM. We previously demonstrated that both primary MM samples and MM cell lines exhibit little or no IDO expression, but that its expression on stromal cell lines was upregulated by co-culture with MM cells (A. Asano et al. Hematol Oncol 2023; 41: 424-433). Here, we analyzed the mechanism of IDO upregulation in stromal cells co-cultured with MM cells, and investigated whether the upregulation of IDO expression is prevented by IDO inhibitors.

【Materials and Methods】

Stromal cells were incubated with MM cell lines or primary MM cells derived from patients, and the expression of IDO and related immune checkpoint markers was evaluated. Mechanisms of IDO upregulation were investigated using two strategies, RNA sequencing (RNA-Seq) of stromal cells co-incubated with MM cell lines and cytokine assays of MM cell culture supernatant. In addition, cardiac glycosides (CGs), which are reported to be inhibitors of IDO expression (MA Shandell et al. J Biol Chem 2022; 298, 101707) were tested for their effects on the expression of IDO on stromal cells co-incubated with MM cells. Similarly, using cytokine-neutralizing antibodies, the roles of specific cytokines for the upregulation IDO expression were examined.

【Results】

Of the several MM cell lines tested, XG-7 and IM-9 were found to upregulate IDO expression in the stromal cell lines UBE6T-7 and HS-5. IDO upregulation was also observed when primary stromal cells from patients with MM were tested. Expression of PD-L1, reported to be an activation marker associated with IDO expression, was also upregulated. Cytokine detection assays showed that MM cell lines that upregulated IDO on stromal cells tended to produce more IL-12 than cell lines that did not. RNA-Seq analysis of UBE6T-7 co-incubated with XG-7 and IM-9 showed significant upregulation of the transcription factors STAT1, STAT2 and IRF1 and pathways enriched in viral defense responses, innate immune responses, lipoprotein metabolism and cellular responses to IFN-γ. We also found IL-12 receptor β subunit 1, which is associated with inflammatory responses, was upregulated in UBE6T-7 co-incubated with both XG-7 and IM-9. These results suggest that specific cytokines and their receptors are involved in elevating IDO levels in stromal cells. Next, to evaluate the effect of CGs, we added ouabain or digoxin to cultures of stromal cells previously co-incubated with MM cell lines. We found that these CGs prevented the upregulation of IDO expression on stromal cells. Based on the results of RNA-seq analysis and cytokine detection assays, we hypothesize that IL-12 and related signaling pathways are associated with the elevation of IDO on stromal cells. Adding Ustekinumab, an IL-12 neutralizing antibody, tended to suppress the upregulation of IDO expression on stromal cells co-incubated with MM cells. According to a previous report, IL-32γ is highly expressed in MM patients and induces IDO production in macrophages via activation of JAK-STAT pathways. Accordingly, we found that recombinant human IL-32γ upregulated IDO expression on stromal cells to some extent.

【Conclusion】

MM cells may enhance the immunosuppressive function of stromal cells by upregulating IDO expression. Two cytokines, IL-12 and IL-32γ, and related pathways may be associated with IDO upregulation on stromal cells via activated JAK-STAT pathways. This immunosuppressive milieu may contribute to the poor efficacy, refractoriness, and acquired resistance to immunotherapies, including monoclonal antibodies, bispecific antibodies, and chimeric antigen receptor T-cell therapy. IDO inhibitors, CGs, and targeting the IL-12 and IL-32γ pathways may improve the TME in MM and positively influence immunotherapy outcomes.