Dissecting the metabolic dependencies of cancer and T cells with in vivo CRISPR screens
(2022) MOBN03 20212Degree Projects in Molecular Biology
- Abstract
- Due to their aberrant growth behavior, cancer cells maintain and depend on aerobic glycolysis even in the presence of physiological oxygen levels to provide building blocks for cell proliferation. This and other dependencies in the metabolism of cancer cells have emerged as promising targets for therapy development. However, metabolic interventions also target highly proliferating cells of the immune system, like Cd8+ T cells, that also indulge in aerobic glycolysis. To systematically probe the impact of cancer-associated metabolic dependencies in Cd8+ T cells, we performed CRISPR-Cas9-based screens using a focused library of 732 functionally validated CRISPR-sgRNAs targeting 318 differential metabolic dependencies of cancer cells in... (More)
- Due to their aberrant growth behavior, cancer cells maintain and depend on aerobic glycolysis even in the presence of physiological oxygen levels to provide building blocks for cell proliferation. This and other dependencies in the metabolism of cancer cells have emerged as promising targets for therapy development. However, metabolic interventions also target highly proliferating cells of the immune system, like Cd8+ T cells, that also indulge in aerobic glycolysis. To systematically probe the impact of cancer-associated metabolic dependencies in Cd8+ T cells, we performed CRISPR-Cas9-based screens using a focused library of 732 functionally validated CRISPR-sgRNAs targeting 318 differential metabolic dependencies of cancer cells in starvations conditions in proliferating Cd8+ T cells in vitro and in vivo. Despite transplantation bottlenecks, this focused, pre-validated CRISPR-sgRNA library was well-represented in vivo, enabling the robust detection of both positive and negative selection events. Results of our comparative screens in vitro show no prominent dependencies on glycolysis or oxidative phosphorylation genes, suggesting that both routes of basic energy metabolism can be exploited by Cd8+ T cells in culture in a redundant manner. By contrast, Cd8+ T cell in vivo strongly depend on both, glycolysis, and oxidative phosphorylation, suggesting that strategies to interfere with these pathways will potentially impair anti-tumor T-cell responses. Through comparing T-cell dependency profiles to results of in vivo CRISPR screens in a mouse model of pancreatic adenocarcinoma, we identify and experimentally validate several metabolic regulators that are strongly required in cancer cells, while their loss has no or (in two cases) even beneficial effects on the proliferation of T-cells in vivo. Collectively, our results establish the technical feasibility of candidate-focused CRISPR-Cas9 screens in vivo and shed new light on selective metabolic liabilities of cancer cells and cancer-associated Cd8+ T cells in the tumor microenvironment. (Less)
- Popular Abstract
- Cancer cells are omnivores, immune cells are picky
Pancreatic ductal adenocarcinoma (PDAC) is a highly prevalent disease with an unmet need for effective therapies. PDAC, like other solid tumors, has a distinct tumor microenvironment with specific metabolic necessities. Tumor cells have adapted to the tumor microenvironment and compete for nutrients with immune cells. Especially CD8+ T cells, equipped to kill tumor cells, are affected in their killing function by depleted nutrients in the tumor microenvironment. The metabolic interaction of tumor and T cells, likely gives rise to selective addictions or dependencies, which potentially offer entry points for therapy.
Major metabolic pathways for nutrient breakdown and energy... (More) - Cancer cells are omnivores, immune cells are picky
Pancreatic ductal adenocarcinoma (PDAC) is a highly prevalent disease with an unmet need for effective therapies. PDAC, like other solid tumors, has a distinct tumor microenvironment with specific metabolic necessities. Tumor cells have adapted to the tumor microenvironment and compete for nutrients with immune cells. Especially CD8+ T cells, equipped to kill tumor cells, are affected in their killing function by depleted nutrients in the tumor microenvironment. The metabolic interaction of tumor and T cells, likely gives rise to selective addictions or dependencies, which potentially offer entry points for therapy.
Major metabolic pathways for nutrient breakdown and energy acquisition in cells are glycolysis (oxidation of glucose) and the TCA (tricarboxylic acid) cycle and mitochondrial respiration for further energy acquisition. Cancer cells upregulate glycolysis to generate more intermediate products, used in other pathways to synthesize cellular building blocks. Since T cells experience the same harsh tumor environment and share the need for biosynthetic intermediates, we want to determine which metabolic pathways and genes are necessary for survival of cancer and T cells to understand their vulnerabilities.
CRISPR loss-of-function screens have revolutionized the way we can systematically interrogate gene essentiality and dissect the molecular function of whole pathways. Previous efforts in the lab have identified several hundred candidate genes involved in metabolic adaption of cancer cells. We perform CRISPR loss-of-function screens in T cells to elucidate metabolic differences between cancer and T cells and determine necessities for T cell survival.
Targets for cancer therapy
We aim to identify genes that are required for cancer cells but not essential for T cell survival, which could be exploited for anticancer therapy. Furthermore, we want to identify genes that increase the fitness of T cells, while not boosting cancer cells
We found that cancers cells were neither dependent on TCA cycle nor glycolysis, while T cells depended on both pathways for survival. We further identified and validated several cancer dependencies as non-essential for T cell proliferation and survival in vivo (in the living organism). We further identified two candidate genes that boost the proliferation of T cells. Therefore, we provide a dataset of dependencies of T cells, as well as targets for future efforts to boost our immune T cells.
Master’s Degree Project in Molecular Biology 60 credits 2022
Department of Biology, Lund University
Advisor: Johannes Zuber/ Robert Kalis
Advisors Department: IMP (Research Institute of Molecular Pathology) (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9079242
- author
- Haider, Jakob
- supervisor
- organization
- course
- MOBN03 20212
- year
- 2022
- type
- H2 - Master's Degree (Two Years)
- subject
- language
- English
- id
- 9079242
- date added to LUP
- 2022-05-12 10:14:09
- date last changed
- 2022-05-12 10:14:09
@misc{9079242,
abstract = {{Due to their aberrant growth behavior, cancer cells maintain and depend on aerobic glycolysis even in the presence of physiological oxygen levels to provide building blocks for cell proliferation. This and other dependencies in the metabolism of cancer cells have emerged as promising targets for therapy development. However, metabolic interventions also target highly proliferating cells of the immune system, like Cd8+ T cells, that also indulge in aerobic glycolysis. To systematically probe the impact of cancer-associated metabolic dependencies in Cd8+ T cells, we performed CRISPR-Cas9-based screens using a focused library of 732 functionally validated CRISPR-sgRNAs targeting 318 differential metabolic dependencies of cancer cells in starvations conditions in proliferating Cd8+ T cells in vitro and in vivo. Despite transplantation bottlenecks, this focused, pre-validated CRISPR-sgRNA library was well-represented in vivo, enabling the robust detection of both positive and negative selection events. Results of our comparative screens in vitro show no prominent dependencies on glycolysis or oxidative phosphorylation genes, suggesting that both routes of basic energy metabolism can be exploited by Cd8+ T cells in culture in a redundant manner. By contrast, Cd8+ T cell in vivo strongly depend on both, glycolysis, and oxidative phosphorylation, suggesting that strategies to interfere with these pathways will potentially impair anti-tumor T-cell responses. Through comparing T-cell dependency profiles to results of in vivo CRISPR screens in a mouse model of pancreatic adenocarcinoma, we identify and experimentally validate several metabolic regulators that are strongly required in cancer cells, while their loss has no or (in two cases) even beneficial effects on the proliferation of T-cells in vivo. Collectively, our results establish the technical feasibility of candidate-focused CRISPR-Cas9 screens in vivo and shed new light on selective metabolic liabilities of cancer cells and cancer-associated Cd8+ T cells in the tumor microenvironment.}},
author = {{Haider, Jakob}},
language = {{eng}},
note = {{Student Paper}},
title = {{Dissecting the metabolic dependencies of cancer and T cells with in vivo CRISPR screens}},
year = {{2022}},
}