Abstract 603: Deficiency of metabolic regulator PKM2 activates the pentose phosphate pathway to generate TCF1+ progenitor CD8+ T cells to improve checkpoint blockade
Authors: Markowitz Geoffrey J. , Ban Yi , Tavarez Diamile A. , Crowley Michael J. , Yoffe Liron , Martin Mitchell T. , Sandoval Tito A. , Gao Dingcheng , Cubillos-Ruiz Juan R. , McGraw Timothy E. , Altorki Nasser K. , Mittal Vivek 
Published: April 2023

Abstract
Background: TCF1high progenitor CD8+ T cells have been shown to mediate the efficacy of PD-1 checkpoint blockade. However, the mechanisms that govern generation of TCF1high cells are poorly understood.
Methods: We sequenced bulk RNA from tumor-infiltrating lymphocytes to identify differentially expressed genes based on tumor progression. We utilized in vitro co-cultures of tumor-specific T cells tumor cells to examine differentiation, effector function, and metabolism of T cells with different genetic and pharmacologic manipulations by flow cytometry, metabolic flux analyses, and metabolomic profiling. We performed in vivo adoptive transfers of control and manipulated tumor-specific T cells into tumor-bearing mice from both a non-small cell lung cancer and a melanoma model to examine effects of genetic manipulation on differentiation and effector function, as well as determine tumor burden and overall mouse survival both in the treatment-naïve and anti-PD-1 treated contexts.
Results: RNA Sequencing demonstrated a metabolically active response in tumor-infiltrating CD8+ T cells isolated from large and late-stage tumors. Using a genetic screen targeting glycolytic enzymes up-regulated in tumor-infiltrating CD8+ T cells, we demonstrate that PKM2 deficiency (PKM2KO) enriched for TCF1high progenitor cells. Antigen-specific PKM2KO CD8+ T cells from draining lymph nodes and tumors exhibited a central memory-like phenotype with reduced effector cytokine production, increased CD44/CD62L expression, and increased TCF1 and Eomes in non-small cell lung cancer and melanoma. Adoptive transfer of PKM2KO CD8+ T cells in combination with PD-1 blockade impaired tumor growth and improved survival. PKM2KO CD8+ T cells showed reduced glycolytic flux and accumulation of glycolytic intermediates with concomitant increases in pentose phosphate pathway (PPP) metabolites. Importantly, small molecule agonism of PPP was sufficient to skew activated CD8+ T cells towards the TCF1high population, which combined with PD-1 blockade to impair tumor growth and improve survival.
Conclusions: Here we show that targeting glycolytic flux by deletion of pyruvate kinase muscle 2 (PKM2) results in elevated pentose phosphate pathway activity, leading to generation of an altered differentiation state responsive to PD-1 blockade. Our study demonstrates a novel metabolic reprogramming that contributes to a memory-like T cell state amenable to checkpoint blockade.
Citation Format: Geoffrey J. Markowitz, Yi Ban, Diamile A. Tavarez, Michael J. Crowley, Liron Yoffe, Mitchell T. Martin, Tito A. Sandoval, Dingcheng Gao, Juan R. Cubillos-Ruiz, Timothy E. McGraw, Nasser K. Altorki, Vivek Mittal. Deficiency of metabolic regulator PKM2 activates the pentose phosphate pathway to generate TCF1+ progenitor CD8+ T cells to improve checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 603.