Graduate Student Indiana University School of Medicine, United States
Introduction: Multiple myeloma(MM) cells are critically dependent on the bone marrow microenvironment(BME) for survival. We have previously shown that a key pro-survival interaction is MM engagement with the dendritic cells(DCs) in the BME, leading to the production of the highly immunosuppressive enzyme indoleamine 2,3-dioxygenase 1(IDO1) by DCs. IDO1 catabolizes tryptophan(TRP)kynurenine(KYN), and this depletion of TRP in the ME suppresses T cell activation. KYN is a ligand for transcription factor aryl hydrocarbon receptor(AHR) that induces essential pro-survival programs in MM. Below we have made the novel observation that MM cells can catabolize TRP⇒KYN independently through upregulation of tryptophan 2,3 dioxygenase(TDO). High TDO expression was correlated with poor clinical outcomes in MM patients; direct TDO inhibition caused MM cell death. These findings suggest that understanding how MM cells metabolize TRP could identify novel treatment targets that may also abrogate the immunosuppressive ME.
Methods: Patient RNA expression data were taken from the CoMMpass database. We measured MM cell lines U266, 8226, MM1S and KMS11 for expression of TRP⇒KYN enzymes with western blot and qPCR. TDO was knocked down with shRNA or inhibited with TDO-specific inhibitor 680C91. In some experiments MM cell lines were co-cultured with monocyte-derived DC. Viability was measured through flow cytometry. AHR activation was measured through qPCR of its downstream target CYP1a1. KYN production was measured by ELISA.
Results: MM cells produce KYN in monoculture, indicating they produce TRP⇒KYN enzymes directly. MM cells do not express IDO1, but three of our four cell lines (8226, MM1S and KMS11) expressed the tryptophan metabolizing enzyme TDO. CoMMpass patients with the highest quartile of TDO expression have significantly lower rates of progression-free and overall survival. In vitro pharmacological inhibition or shRNA knockdown of TDO led to reduced cell viability and growth in all TDO+ cell lines but not the TDO- cell line U266. TDO inhibition led to reduced production of KYN and reduced expression of AHR downstream target CYP1a1. Co-culture with dendritic cells was only able to partially protect MM cell viability and restore KYN production from TDO inhibition.
Conclusions: TRP metabolism has a central role in MM cell survival and we have made the novel observation that MM cells can metabolize TRP independently through the expression of TDO. TDO expression is linked to patient survival. We predict that the depletion of TRP could repress anti-MM T cell activity and production of KYN could directly support MM survival through AHR activation. TDO upregulation may be one mechanism by which MM cells become independent of the BME, which clinically heralds highly treatment-refractory disease. Understanding the pro-MM survival roles of the TRP metabolizing enzymes could lead to novel therapies that directly target the MM cell and reverse the immunosuppressive ME, leading to better responses to immunotherapy.