Glutathione overproduction mediates lymphoma initiating cells survival and has a sex-dependent effect on lymphomagenesis.

Lymphoid tumor patients often exhibit resistance to standard therapies or experience relapse post-remission. Relapse is driven by Tumor Initiating Cells (TICs), a subset of tumor cells capable of regrowing the tumor and highly resistant to therapy. Growing cells in 3D gels is a method to discern tumorigenic cells because it strongly correlates with tumorigenicity. The finding that TICs, rather than differentiated tumor cells, grow in 3D gels offers a unique opportunity to unveil TIC-specific signaling pathways and therapeutic targets common to various cancer types. Here, we show that culturing lymphoid cells in 3D gels triggers reactive oxygen species (ROS) production, leading to non-tumor lymphoid cell death while enabling the survival and proliferation of a subset of lymphoma/leukemia cells, TICs or TIC-like cells. Treatment with the antioxidant N-acetylcysteine inhibits this lethality and promotes the growth of primary non-tumor lymphoid cells in 3D gels. A subset of lymphoma cells, characterized by an increased abundance of the antioxidant glutathione, escape ROS-induced lethality, a response not seen in non-tumor cells. Reducing glutathione production in lymphoma cells, either through pharmacological inhibition of glutamate cysteine ligase (GCL), the enzyme catalyzing the rate-limiting step in glutathione biosynthesis, or via knockdown of GCLC, the GCL catalytic subunit, sharply decreased cell growth in 3D gels and xenografts. Tumor cells from B-cell lymphoma/leukemia patients and λ-MYC mice, a B-cell lymphoma mouse model, overproduce glutathione. Importantly, pharmacological GCL inhibition hindered lymphoma growth in female λ-MYC mice, suggesting that this treatment holds promise as a therapeutic strategy for female lymphoma/leukemia patients.