Abstract 1365: BHLHE40 orchestrates remodeling of the intratumoral immune cell populations in response to immune checkpoint therapy
Authors: Shavkunov Alexander S. , Salmon Avery J. , Miao Qi , Keshari Sunita , Williams Charmelle D. , Pineda Josué E. , Chen Ken , Edelson Brian T. , Gubin Matthew M. 
Published: June 2022

Abstract
The success of immune checkpoint therapy (ICT) in generating durable clinical responses is remarkable but not all cancer patients respond for reasons that are incompletely understood. In our previous studies of immune responses to ICT in the mouse T3 sarcoma model, we found strong upregulation of the BHLHE40 transcription factor in a number of immune cell populations. It was particularly prominent in the tumor antigen-specific CD8+ and CD4+ T cells, which are crucial for ICT-induced tumor rejection and elimination. We used global and cell-type specific Bhlhe40 knockout (KO) mice to investigate its role in tumor rejection. Both global Bhlhe40-/- and CD4-Cre+ Bhlhe40f/f (CD4+ and CD8+ T cell-specific Bhlhe40 deletion) mice bearing the 1956 methylcholanthrene-induced sarcoma line failed to respond to either anti-PD-1 or anti-CTLA-4 ICT, in stark contrast to wild type (WT) mice. At the same time, Bhlhe40 deletion in macrophages and granulocytes (LysM-Cre+ Bhlhe40f/f mice) did not have an apparent effect on ICT-mediated tumor rejection. To gain insight into the functional changes orchestrated by BHLHE40 in response to ICT, we used single-cell RNA sequencing (scRNAseq) to analyze the transcriptional profiles of the CD45+ tumor-infiltrating immune cells from 1956 tumor-bearing Bhlhe40+/+ and Bhlhe40-/- mice treated with control, anti-PD-1 or anti-CTLA-4 mAb. While no major changes in the relative fractions of effector T cell clusters were observed, ICT induced BHLHE40-dependent remodeling within individual CD4+ and CD8+ clusters. Most notably, Bhlhe40-/- mice were strongly deficient in ICT-induced Ifng expression by CD8+ T cells, with intracellular cytokine staining revealing low IFNγ production by both CD4+ and CD8+ T cells. GSEA and Ingenuity Pathway analysis indicated changes in energy metabolism, hypoxia adaptation, NF-κB signaling, IFNγ response, and actin cytoskeleton remodeling in a number of Bhlhe40-/- T cell clusters. This was accompanied by upregulation of the inhibitory receptor Tigit mRNA, altered expression of chemokine/chemokine receptor genes, as well as granzyme gene family members. In addition to changes in the transcriptional profile shared between anti-PD-1 and anti-CTLA-4 treatments, each checkpoint inhibitor induced its own specific alterations both in WT and KO mice. Analysis of macrophage populations also revealed a notable difference between Bhlhe40+/+ and Bhlhe40-/- mice. The shift of macrophages from the CX3CR1+ CD206+ to the iNOS+ phenotype, typically observed during effective ICT, was dramatically reduced in the Bhlhe40-/- mice, which was confirmed by flow cytometry. Our results provide the basis for understanding the role of BHLHE40 in the ICT-induced tumor rejection, the pathways and effector mechanisms under its control, and the potential ways for therapeutic targeting of this regulatory network to improve the efficacy of cancer immunotherapy.
Citation Format: Alexander S. Shavkunov, Avery J. Salmon, Qi Miao, Sunita Keshari, Charmelle D. Williams, Josué E. Pineda, Ken Chen, Brian T. Edelson, Matthew M. Gubin. BHLHE40 orchestrates remodeling of the intratumoral immune cell populations in response to immune checkpoint therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1365.