Periodic Reporting for period 4 - MitoGuide (Integration and adaptation of impaired mitochondrial fitness in orchestrating T cell dysfunction in the tumor microenvironment)
Reporting period: 2023-08-01 to 2024-01-31
Mitochondria govern cellular energy production via the tricarboxylic acid (TCA) cycle and electron transport chain. Through the cooperation of these metabolic processes, nutrients fuel cellular metabolic demands in response to growth factors, nutrient availability and nutrient-sensing signaling cascades. In addition, metabolic intermediates of the TCA cycle are used to produce building blocks for cell proliferation. Therefore, tight control that balances mitochondrial activity is critical to bridging nutrient availability and metabolic demands in response to stimuli for cell proliferation and activation. This tight control of mitochondrial functions also must communicate with nuclear events, including DNA replication, transcription and damage responses, through retrograde signals generated from mitochondria to ensure proper cellular responses. Intriguingly, emerging evidence indicates that epigenetic alterations might be the major regulatory mechanism to link mitochondrial activity with nuclear reprogramming. Although mitochondrial activities are critical for T cell differentiation, it remains unclear whether mitochondrial dynamics can be modulated to drive T cell dysfunction via alterations in their epigenome and generation of metabolic checkpoints.
We established the critical foundations for the proposed study. Based on these findings and tools, we are investigating several directions in this study. We expect the results of these experiments will uncover the detailed mechanisms by which mitochondrial fitness determines T cell differentiation program and further reveal how targeting kinase or metabolic enzymes will be able to sustain T cell anti-tumor immunity. Here are the summarised update we made.
1. Delineating how deregulated mitochondrial dynamics drives a p53-mediated epigenetic reprogramming for T cell exhaustion.
2. Investigating whether accumulation of depolarized mitochondria drive a heme-dependent transcriptional network for T cell exhaustion.
3. Decipher how impaired mitochondrial fitness in tumor-infiltrating T cells supports epigenetic reprogramming leading to T cell exhaustion.
4. Validating kinase and metabolic enzyme identified from our in vivo CRISPR screening that could sustain TIL mitochondrial fitness.
We established the critical foundations for the proposed study. Based on these findings and tools, we are investigating several directions in this study. We expect the results of these experiments will uncover the detailed mechanisms by which mitochondrial fitness determines T cell differentiation program and further reveal how targeting kinase or metabolic enzymes will be able to sustain T cell anti-tumor immunity. Here are the summarised update we made.
1. Delineating how deregulated mitochondrial dynamics drives a p53-mediated epigenetic reprogramming for T cell exhaustion.
2. Investigating whether accumulation of depolarized mitochondria drive a heme-dependent transcriptional network for T cell exhaustion.
3. Decipher how impaired mitochondrial fitness in tumor-infiltrating T cells supports epigenetic reprogramming leading to T cell exhaustion.
4. Validating kinase and metabolic enzyme identified from our in vivo CRISPR screening that could sustain TIL mitochondrial fitness.
This project has been conducted on different fronts, which are structured into three research thrusts.
First, Mitophagy-guided T cell anti-tumor immunity, we investigate the contribution of damage mitochondria removal machinery (mitophagy) on supporting T cell anti-tumor immunity. In this part, we confirm that a) mitophagy is required to support T cell anti-tumor immunity, maintain responsiveness to PD-1 blockade, drive formation of stem-like tutor-specific TILs; b) chronic T cell receptor stimulation and hypoxia in tumors abolish mitophagy activity in T cells; c) TILs accumulating depolarised mitochondria as a result of impaired mitophagy commit to terminally exhausted program. This exhausted features are characterised by functional, transcriptomic and epigenetic features, including changes in DNA methylation and chromatin accessibility.
Second, mitochondria-nucleus communication on guiding T cell anti-tumor responses, we uncover that the declined mitochondrial fitness in T cells leads to permanent impairment of proliferative capacity and effector molecule expression and sustained expression of inhibitory receptors. As a result of these actions, T cells accumulating depolarised mitochondria engage senescence/apoptosis and fail to sustain responsiveness to immune checkpoint blockade treatment. Moreover, we conduct an in vivo CRISPR screening to identify metabolic enzymes and kinase that can be targeted for sustaining mitochondrial fitness in CD8 TILs. We are currently in the stage to validate those targets we identify and elucidate how this targeting approach can be exploited in T cell transfer therapy. In the end of this direction, we also finish proteome analysis and are in the process to finish metabolic analysis. We expect the integrated cross-omics analysis will allow us to further explore how mitochondria instruct T cell functionality and differentiation.
In the third direction, as a result of mitochondrial damage, cells will degrade mitochondrial protein to release heme. Intriguingly, heme has been shown to affect B cell differentiation via the regulation of Blimp-1 expression. Given that Blimp-1 is critical transcription factor for orchestrating T cell exhaustion, we aim to explore how heme-mediated program can affect T cell exhaustion in this context. Most importantly, we will develop interventions to abolish this process for ensuring T cell anti-tumor immunity. In the past 18 months, we established assay to measure regulatory heme (freely available in the cytoplasm and nuclei with binding ability to target proteins). Moreover, we confirmed that exhausted T cells do contain more regulatory heme compared to less exhausted T cells and progenitor exhausted T cells in engrafted tumor murine model, inducible melanoma murine model, chronic LCMV infection model and human in vitro T cell exhaustion condition. We further revealed the deregulation of heme can further disrupted circadian clock in T cells, which in turn leads to impaired metabolic adaptation. Morevoer, we are testing how we can improve T cell mediated anti-tumor immunity by targeting heme signaling axis in both T cell transfer and CAR T cell settings.
First, Mitophagy-guided T cell anti-tumor immunity, we investigate the contribution of damage mitochondria removal machinery (mitophagy) on supporting T cell anti-tumor immunity. In this part, we confirm that a) mitophagy is required to support T cell anti-tumor immunity, maintain responsiveness to PD-1 blockade, drive formation of stem-like tutor-specific TILs; b) chronic T cell receptor stimulation and hypoxia in tumors abolish mitophagy activity in T cells; c) TILs accumulating depolarised mitochondria as a result of impaired mitophagy commit to terminally exhausted program. This exhausted features are characterised by functional, transcriptomic and epigenetic features, including changes in DNA methylation and chromatin accessibility.
Second, mitochondria-nucleus communication on guiding T cell anti-tumor responses, we uncover that the declined mitochondrial fitness in T cells leads to permanent impairment of proliferative capacity and effector molecule expression and sustained expression of inhibitory receptors. As a result of these actions, T cells accumulating depolarised mitochondria engage senescence/apoptosis and fail to sustain responsiveness to immune checkpoint blockade treatment. Moreover, we conduct an in vivo CRISPR screening to identify metabolic enzymes and kinase that can be targeted for sustaining mitochondrial fitness in CD8 TILs. We are currently in the stage to validate those targets we identify and elucidate how this targeting approach can be exploited in T cell transfer therapy. In the end of this direction, we also finish proteome analysis and are in the process to finish metabolic analysis. We expect the integrated cross-omics analysis will allow us to further explore how mitochondria instruct T cell functionality and differentiation.
In the third direction, as a result of mitochondrial damage, cells will degrade mitochondrial protein to release heme. Intriguingly, heme has been shown to affect B cell differentiation via the regulation of Blimp-1 expression. Given that Blimp-1 is critical transcription factor for orchestrating T cell exhaustion, we aim to explore how heme-mediated program can affect T cell exhaustion in this context. Most importantly, we will develop interventions to abolish this process for ensuring T cell anti-tumor immunity. In the past 18 months, we established assay to measure regulatory heme (freely available in the cytoplasm and nuclei with binding ability to target proteins). Moreover, we confirmed that exhausted T cells do contain more regulatory heme compared to less exhausted T cells and progenitor exhausted T cells in engrafted tumor murine model, inducible melanoma murine model, chronic LCMV infection model and human in vitro T cell exhaustion condition. We further revealed the deregulation of heme can further disrupted circadian clock in T cells, which in turn leads to impaired metabolic adaptation. Morevoer, we are testing how we can improve T cell mediated anti-tumor immunity by targeting heme signaling axis in both T cell transfer and CAR T cell settings.
This project pivots around five aspects which are beyond the state of the art:
1) The understanding on the unexplored link between mitochondrial fitness and epigenetic reprogramming in T cell exhaustion.
2) We define how the tumor microenvironment could diminish mitochondrial fitness in tumor-infiltrating CD8 T cells.
3) Establishing new dimension of understanding about how metabolic interventions that target tumor metabolism and/or immunometabolism could be exploited for rejuvenating T cell anti-tumor responses.
4) We also unexpectedly reveal interleukin-10 can rejuvenate terminally exhausted T cells by modulating mitochondrial pyruvate uptake. This finding open a new horizon on T cell biology and serves as a springboard for us to generating new immunotherapy modalities.
5) The first CRISPR screening for supporting mitochondrial fitness in T cells could be exploited on refining CAR T cell therapy.
6) Validating kinase and metabolic enzyme identified from our in vivo CRISPR screening that could sustain TIL mitochondrial fitness.
1) The understanding on the unexplored link between mitochondrial fitness and epigenetic reprogramming in T cell exhaustion.
2) We define how the tumor microenvironment could diminish mitochondrial fitness in tumor-infiltrating CD8 T cells.
3) Establishing new dimension of understanding about how metabolic interventions that target tumor metabolism and/or immunometabolism could be exploited for rejuvenating T cell anti-tumor responses.
4) We also unexpectedly reveal interleukin-10 can rejuvenate terminally exhausted T cells by modulating mitochondrial pyruvate uptake. This finding open a new horizon on T cell biology and serves as a springboard for us to generating new immunotherapy modalities.
5) The first CRISPR screening for supporting mitochondrial fitness in T cells could be exploited on refining CAR T cell therapy.
6) Validating kinase and metabolic enzyme identified from our in vivo CRISPR screening that could sustain TIL mitochondrial fitness.