Role of Tryptophan Metabolizing Enzymes in Multiple Myeloma Survival

Introduction:

Multiple myeloma (MM) is a hematological malignancy caused by abnormally proliferating plasma cells in the bone marrow. It is considered incurable, as patients almost always go through cycles of treatment, remission and treatment-resistant relapse before eventually perishing from the disease; very few remain myeloma-free. Recently new immunotherapies have been developed for MM, opening a promising new avenue for treatment, but even with immunotherapy MM patients continue to relapse and perish from MM. Our lab has previously found that MM interacts with the Dendritic Cells (DCs) in the Bone Marrow Microenvironment (BMME) and that this interaction leads to the production of the immunosuppressive enzyme Indoleamine 2,3-dioxygenase 1 (IDO1) by DCs. IDO1 catabolizes tryptophan (TRP) to kynurenine (KYN). The depletion of TRP in the BMME has been known to suppress T-effector activity, while the KYN activates the transcription factor Aryl Hydrocarbon Receptor (AHR) that induces a pro-survival signal in MM and Tregs. Understanding how MM is able to evade the immune system and support its survival in the BMME could lead to novel treatment targets in MM.

Methods:

Patient RNA expression data were taken from the CoMMpass database. MM cell lines U266, 8226, MM1S and KMS11 were measured for expression of TRP->KYN metabolizing enzymes through western blot and qPCR. Cells were treated with TDO inhibitor 680C91 or underwent a TDO knockdown using shRNA. Activation of AHR was measured through expression of Cytochrome P450 Family 1 Subfamily A Member 1 (CYP1A1), an aryl hydrocarbon hydrolase linked to cancer formation and a transcriptional downstream target. CYP1A1 expression was measured through qPCR and western blot. For some experiments MM cell lines were co-cultured with monocyte-derived DC. Cell viability was measured by flow cytometry and KYN production was measured by the Ehrlich KYN detection assay.

Results:

We have found that MM cells produce KYN in the absence of DC, indicating that they produce TRP metabolizing enzymes of their own. MM cells do not express IDO1, but they do express TRP metabolizing enzymes Tryptophan Dioxygenase 2 (TDO2) and Interleukin 4-induced gene-1 (IL4i1). TDO2 metabolizes TRP->KYN and IL4i1 metabolized TRP->kynurenic acid. TDO and IL4i1 expression has been noted in several cancer types, but their role in MM has not been previously described. Cell line U266 expressed IL4i1, while cell lines 8226, MM1S and KMS11 express both TDO2 and IL4i1. Patients in the highest quartile of TDO2 expression had significantly worse progression-free survival and overall survival outcomes. Pharmacological inhibition and shRNA knockdown of TDO led to decreased cell survival and proliferation in TDO+ cell lines. Inhibition of TDO led to decreased expression of both CYP1A1 and AHR, indicating that it prevented AHR activation, as well as decreased production of KYN.

Discussion

We have found that MM cells express tryptophan metabolizing enzymes TDO2 and IL4i1, indicating that they may be able to deplete TRP and produce KYN in the microenvironment independent from interaction with other cells in the BMME. The depletion of TRP and creation of KYN could repress T-effector activity and increase Treg activity, while supporting MM survival through the activation of AHR. As MM cells are able to create TRP metabolizing enzymes without BMME interaction, this indicates that they could create an immunosuppressive and pro-MM survival microenvironment even once they escape the BMME. Studying the production of TRP metabolizing enzymes in MM could lead to novel treatment targets that would reduce MM survival and make MM more sensitive to immune response.