Despite substantial therapeutic advancements, multiple myeloma (MM) remains incurable, calling for novel targeted therapies. Disease progression depends on the ability of malignant plasma cells (PC) to subvert the local microenvironment and reshape host immunity to support tumor growth. MM cells are addicted to specific molecules, including p62 and IRF4, which respectively control protein homeostasis and PC identity. Stable lentiviral silencing of either factor leads to in vitro extinction of MM cells within ~2 weeks. However, a mechanistic and functional connection between p62 and IRF4, and between these factors and environmental signals has never been investigated.
In view of the critical role of the stress-responsive adapter p62 in MM cells, we hypothesized that environmental arginine deprivation (see ABS#103715) may promote MM cell fitness by sustaining p62 and IRF4 expression via the stress-sensor kinase GCN2, thereby alleviating proteostatic stress and increasing drug resistance, while favoring immune escape at the same time.
Our hypothesis was strengthened by in vitro studies on four human myeloma cell lines (H929, OPM2, MM.1S and U266) that revealed that selective arginine deprivation increased the expression not only of p62 and IRF4, but also of Blimp-1 and PD1 in a GCN2-dependent manner. A positive correlation among p62 and Blimp-1 gene expression levels was observed in a large dataset of patient-derived primary PCs, representative of different PC dyscrasias (9 healthy controls; 20 MGUS; 33 smoldering MM; 170 MM; 24 primary and 12 secondary PCL) from two publicly available datasets (GSE66293, GSE47552). Lower GCN2 gene expression levels were found in more advanced stages of secondary PCL, further hinting at a role for GCN2 in responding to microenvironmental signals.
Moreover, the genetic ablation of GCN2 reduced p62 and IRF4, and led to in vitro extinction of MM cell lines (but not the mantle cell lymphoma Jeko with comparable basal IRF4 levels) within 5 days of culture.
We then hypothesized that the poor clinical response to anti-PD1 therapy in MM may depend on arginase 1 (Arg-1)-dependent activation of the identified GCN2-IRF4 pathway.
We found variable PD1 expression in primary samples, higher levels being associated to more aggressive disease. In vitro 24 h treatment with rh-Arg-1 increased PD1, whereas concomitant treatment with the selective Arg-1 inhibitors nor-NOHA and BEC-HCl prevented PD1 induction. Similarly, stable lentiviral GCN2 silencing abolished PD1 expression. Triggering GCN2 via arginine deprivation was associated to increased glutamine anaplerosis via GLUD1 (see ABS#103715).
Overall, our findings disclose a putative novel micro-environmental circuit co-opted by MM evolution, whereby exposure to an arginine-poor immunosuppressive environment may sustain plasma cell fitness via GCN2.
Ciceri: GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding.
Asterisk with author names denotes non-ASH members.