Pharmacologic LDH inhibition redirects intratumoral glucose uptake and improves antitumor immunity in solid tumor models.

Tumor reliance on glycolysis is a hallmark of cancer. Immunotherapy is more effective in controlling glycolysis-low tumors lacking lactate dehydrogenase (LDH) due to reduced tumor lactate efflux and enhanced glucose availability within the tumor microenvironment (TME). LDH inhibitors (LDHi) reduce glucose uptake and tumor growth in preclinical models, but their impact on tumor-infiltrating T cells is not fully elucidated. Tumor cells have higher basal LDH expression and glycolysis levels compared with infiltrating T cells, creating a therapeutic opportunity for tumor-specific targeting of glycolysis. We demonstrate that LDHi treatment (a) decreases tumor cell glucose uptake, expression of the glucose transporter GLUT1, and tumor cell proliferation while (b) increasing glucose uptake, GLUT1 expression, and proliferation of tumor-infiltrating T cells. Accordingly, increasing glucose availability in the microenvironment via LDH inhibition leads to improved tumor-killing T cell function and impaired Treg immunosuppressive activity in vitro. Moreover, combining LDH inhibition with immune checkpoint blockade therapy effectively controls murine melanoma and colon cancer progression by promoting effector T cell infiltration and activation while destabilizing Tregs. Our results establish LDH inhibition as an effective strategy for rebalancing glucose availability for T cells within the TME, which can enhance T cell function and antitumor immunity.

Deleting Trim33 in myeloid cells improves the efficiency of radiotherapy through an interferon beta dependent anti-tumor immune response.

Radiotherapy (RT) triggers an immune response that contributes to anti-tumor effects. Induction of interferon beta (IFN-ß) is a key event in this immunogenicity of RT. We have previously shown that TRIM33, a chromatin reader, restrains IFN-ß expression in Toll-like receptor-activated myeloid cells. Here, we explored whether deleting Trim33 in myeloid cells might improve the radio-induced immune response, and subsequent efficiency of RT. We first established that Trim33-/- bone marrow-derived macrophages showed increased expression of IFN-ß in response to direct irradiation, or to treatment with irradiated cancer cells, further supporting our hypothesis. We then tested the efficiency of a single dose RT in three subcutaneous and one orthotopic tumor models. In all situations, myeloid deletion of Trim33 led to a significantly improved response after RT, leading to a complete and durable response in most of the treated mice bearing orthotopic oral tumors. This effect required the IFN-I pathway, and the presence of CD8+ T lymphocytes, but not NK cells. In addition, cured mice were capable of rejecting a secondary tumor challenge, demonstrating an in situ vaccination effect. We conclude that deleting Trim33 in myeloid cells improves RT efficiency, through a mechanism involving the IFN-I pathway and the immune response. Our work suggests that myeloid Trim33 is a host factor affecting the tumor response to RT, thus representing a new potential therapeutic target for modifying RT responses.

A genetic variant controls interferon-ß gene expression in human myeloid cells by preventing C/EBP-ß binding on a conserved enhancer.

Interferon ß (IFN-ß) is a cytokine that induces a global antiviral proteome, and regulates the adaptive immune response to infections and tumors. Its effects strongly depend on its level and timing of expression. Therefore, the transcription of its coding gene IFNB1 is strictly controlled. We have previously shown that in mice, the TRIM33 protein restrains Ifnb1 transcription in activated myeloid cells through an upstream inhibitory sequence called ICE. Here, we show that the deregulation of Ifnb1 expression observed in murine Trim33-/- macrophages correlates with abnormal looping of both ICE and the Ifnb1 gene to a 100 kb downstream region overlapping the Ptplad2/Hacd4 gene. This region is a predicted myeloid super-enhancer in which we could characterize 3 myeloid-specific active enhancers, one of which (E5) increases the response of the Ifnb1 promoter to activation. In humans, the orthologous region contains several single nucleotide polymorphisms (SNPs) known to be associated with decreased expression of IFNB1 in activated monocytes, and loops to the IFNB1 gene. The strongest association is found for the rs12553564 SNP, located in the E5 orthologous region. The minor allele of rs12553564 disrupts a conserved C/EBP-ß binding motif, prevents binding of C/EBP-ß, and abolishes the activation-induced enhancer activity of E5. Altogether, these results establish a link between a genetic variant preventing binding of a transcription factor and a higher order phenotype, and suggest that the frequent minor allele (around 30% worldwide) might be associated with phenotypes regulated by IFN-ß expression in myeloid cells.

Growing Murine Bone Marrow-Derived Macrophages.

Bone marrow-derived macrophages (BMDM) are primary macrophages obtained by in vitro differentiation of bone marrow cells in the presence of macrophage colony-stimulating factor (M-CSF or CSF1). They are easy to obtain in high yields, can be stored by freezing, and can be obtained from genetically modified mice strains. They are therefore widely used as prototypical macrophages for in vitro studies. In this chapter, we present the method for obtaining BMDMs and freezing them.