Multi-species genome-wide CRISPR screens identify conserved suppressors of cold-induced cell death

Reviewer #1 (Public review):

Summary:

Through a series of CRISPR-Cas9 screens, the GPX4 antioxidant pathway was identified as a critical suppressor of cold-induced cell death in hibernator-derived cells. Hamster BHK-21 cells exposed to repeated cold and rewarming cycles revealed five genes (Gpx4, Eefsec, Pstk, Secisbp2, and Sepsecs) as critical components of the GPX4 pathway, which protects against cold-induced ferroptosis. A second screen with continuous cold exposure confirmed the essential role of GPX4 in prolonged cold tolerance. GPX4 knockout lines exhibited complete cell death within four days of cold exposure, and pharmacological inhibition of GPX4 further increased cell death, underscoring the necessity of GPX4's catalytic activity in cold conditions.

An additional CRISPR screen in human cold-sensitive K562 cells identified 176 genes for cold survival. The GPX4 pathway was found to confer significant resistance to cold in hibernators and human cells, with GPX4 loss significantly increasing cold-induced cell death.

Comparing hamster and human GPX4, overexpression of GPX4 in human K562 cells, whether hamster or human GPX4, dramatically improved cold tolerance, while catalytically dead mutants showed no such effect. These findings suggest that GPX4 abundance is a key limiting factor for cold tolerance in human cells, and primary cell types show strong sensitivity to GPX4 loss, highlighting that differences in cold tolerance across species may be due to varying GPX4-mediated protection.

Strengths:

(1) Innovative Approach: The study employs a series of unbiased genome-wide CRISPR-Cas9 screens in both hibernator- and non-hibernator-derived cells to investigate the mechanisms controlling cellular cold tolerance. Notably, this is the first genome-scale CRISPR-Cas9 screen conducted in cells derived from a hibernator, the Syrian hamster.

(2) Identification of the GPX4 Pathway: Identifying glutathione peroxidase 4 (GPX4) as a critical suppressor of cold-induced cell death significantly contributes to the field. Recently, GPX4 was also reported as a potent regulator of cold tolerance through overexpression screening (Sone et al.) in hamsters, which further supports this finding.

(3) Improved Cold Viability Assessment: The study identifies an important technical artifact in using trypan blue to assess cell viability following cold exposure. It reveals that cells stained immediately after cold exposure retain the dye, inaccurately indicating cell death. By introducing a brief rewarming period before viability assessment, the authors significantly improve the accuracy of detecting cold-induced cell death. This refinement in methodology ensures more reliable results and sets a new standard for future research on cold stress in cells.

Weaknesses:

(1) Mechanisms Regulating GPX4 Levels: While the study highlights GPX4 levels as a major determinant of cellular cold tolerance, it does not discuss how these levels are regulated or why they differ between hibernators and non-hibernators. This omission leaves an important aspect of GPX4's role in cold tolerance unexplored.

(2) Generalizability Across Species: Although the study demonstrates the role of GPX4 in several mammalian species, it does not investigate whether this mechanism extends to other vertebrates (e.g., fish and amphibians) that also face cold challenges. This limitation could restrict the broader evolutionary claims made by the study.

(3) Variability in Cold Sensitivity Across Human Cell Lines: The study observes significant variability in cold tolerance among different human cell lines but does not explain these differences clearly. This leaves a key aspect of human cell cold sensitivity insufficiently addressed.

Reviewer #2 (Public review):

Summary:

Lam et al., present a very intriguing whole genome CRISPR screen in Syrian Hamster cells as well as K562 cells to identify key genes involved in hypothermia-rewarming tolerance. Survival screens were performed by exposing cells to 4C in a cooled CO2 incubator followed by a rewarming period of 30 minutes prior to survival analysis. In this paradigm, Syrian hamster-derived cell lines exhibit more robust survival than human cell lines (BHK-21 and HaK vs HT1080, HeLa, RPE1, and K562). A genome-wide Syrian hamster CRISPR library was created targeting all annotated genes with 10 guides/gene. LV transduction of the library was performed in BHK-21 cells and the survival screen procedures involved 3 cycles of 4C cold exposure x4 days followed by 2 days of re-warming.

When compared to controls maintained at 37C, 9 genes were required for BHK-21 survival of cold cycling conditions and 5 of these 9 are known components of the GPX4 antioxidant pathway. GPX4 KO BHK-21 cells had reduced cell growth at 37C and profoundly worse cold tolerance which could be reduced by GPX4 expression. GPX4 inhibitors also reduced survival in cold. CRISPR KO screens and GPX4 KO in K562 cells revealed comparable results (though intriguingly glutathione biosynthesis genes were more critical to K562 cells than BHK-21 cells). Human or Syrian hamster GPX4 overexpression improved cold tolerance.

Strengths:

This is a very nicely written paper that clearly communicates in figures and text complicated experimental manipulations and in vitro genetic screening and cell survival data. The focus on GPX4 is interesting and relatively novel. The converging pharmacologic, loss-of-function, and gain-of-function experiments are also a strength.

Weaknesses:

A recently published article (Reference 43, Sone et al.) also independently explored the role of GPX4 in Syrian hamster cold tolerance through gain-of-function screening. Further exploration of the GPX4 species-specific mechanisms would be of great interest, but this is considered a minor weakness given the already very comprehensive and compelling data presented.

Reviewer #3 (Public review):

Summary:

This work aims to address a fundamental biological question: how do mammalian cells achieve/lose tolerance to cold exposure? The authors first tried to establish an experimental system for cell cold exposure and evaluation of cell death and then performed genome-scale CRISPR-Cas9 screening on immortalized cell lines from Syrian Hamster (BHK-21) and human (K562) for key genes that are associated with cell survival during prolonged cold exposure. From these screenings, they focused on glutathione peroxidase 4 (GPX4). Using genetic modifications or pharmacological interventions, and multiple cell models including primary cells from various mammalian species, they showed that GPX4 proteins are likely to retain their activities at 4 {degree sign}C, functioning to prevent cold-induced cell ferroptosis.

Strengths:

(1) This paper is neatly written and hence easy to follow.

(2) Experiments are well designed.

(3) The data showing the overall good cell survival after a prolonged cold exposure or repeated cold-warm cycles are helpful to show the advantages of the experimental instruments and methods the authors used, and hence the validity of their results.

(4) The CRISPR-Cas9 screening is a great attempt.

(5) Multiple cell types from hibernating mammals (cold tolerant) and cold-intolerant species are used to test their findings.

(6) Although some may argue that other labs have published works with different approaches that have pointed out the importance of GPX4 and ferroptosis in hamster cell survival from anoxia-reoxygenation or cold exposure models, hence hurting the novelty of this work, this reviewer thinks that it is highly valuable to have independent research groups and different methods/systems to validate an important concept.

Weaknesses:

(1) Only cell death was robustly surveyed; though cell proliferation was evaluated too in some experiments, other cellular functions, such as mitochondrial ATP production vs. glycolysis, and the extent of lipid peroxidation, could have been measured to reflect cellular physiology.

Validations on complex tissues or in vivo systems would have further strengthened the work and its impact.

CRISPR-Cas9 screening may have technical limitations as knock-out of some essential genes/pathways may lead to cell lethality during screening, and hence the relevance of these genes/pathways to cell cold tolerance may not be noted. From the data presented in this study, this reviewer thinks that the GPX4 pathway is likely a conserved mechanism for long-term cold survival, but not for cold sensitivity or acute cell death from cold exposure. In line with my such speculation, their CRISPR-Cas9 screening revealed genes in the GPX4 pathway from a relatively cold-sensitive human cell line, but the endogenous GPX4 pathway is seemingly operational in this cold-sensitive cell line. Also, these cells are viable after GPX4 knock-out. Dead cells from the acute cold exposure phase may detached, or their genomic DNAs have been severely damaged by the time of sample collection, hence not giving any meaningful sequencing reads. Crippling other factors/pathways such as FOXO1 (PMID: 38570500) or 5-aminolevulinic acid (ALA) metabolism (PMID: 35401816) have been shown to severely aggravate cold-induced cell death, including TUNEL-revealed DNA damage, within a much shorter time scale, whilst loss-function knockouts of FOXO1 or ALA Synthase 1 (ALAS1) are usually cell lethal. Thus, they and other possible essential genes may not be screenable from the current experimental protocol. These important points need to be taken into consideration by the authors.

Author response:

Reviewer #1 (Public review):

Summary:

Through a series of CRISPR-Cas9 screens, the GPX4 antioxidant pathway was identified as a critical suppressor of cold-induced cell death in hibernator-derived cells. Hamster BHK-21 cells exposed to repeated cold and rewarming cycles revealed five genes (Gpx4, Eefsec, Pstk, Secisbp2, and Sepsecs) as critical components of the GPX4 pathway, which protects against cold-induced ferroptosis. A second screen with continuous cold exposure confirmed the essential role of GPX4 in prolonged cold tolerance. GPX4 knockout lines exhibited complete cell death within four days of cold exposure, and pharmacological inhibition of GPX4 further increased cell death, underscoring the necessity of GPX4's catalytic activity in cold conditions.

An additional CRISPR screen in human cold-sensitive K562 cells identified 176 genes for cold survival. The GPX4 pathway was found to confer significant resistance to cold in hibernators and human cells, with GPX4 loss significantly increasing cold-induced cell death.

Comparing hamster and human GPX4, overexpression of GPX4 in human K562 cells, whether hamster or human GPX4, dramatically improved cold tolerance, while catalytically dead mutants showed no such effect. These findings suggest that GPX4 abundance is a key limiting factor for cold tolerance in human cells, and primary cell types show strong sensitivity to GPX4 loss, highlighting that differences in cold tolerance across species may be due to varying GPX4-mediated protection.

Strengths:

(1) Innovative Approach: The study employs a series of unbiased genome-wide CRISPR-Cas9 screens in both hibernator- and non-hibernator-derived cells to investigate the mechanisms controlling cellular cold tolerance. Notably, this is the first genome-scale CRISPR-Cas9 screen conducted in cells derived from a hibernator, the Syrian hamster.

(2) Identification of the GPX4 Pathway: Identifying glutathione peroxidase 4 (GPX4) as a critical suppressor of cold-induced cell death significantly contributes to the field. Recently, GPX4 was also reported as a potent regulator of cold tolerance through overexpression screening (Sone et al.) in hamsters, which further supports this finding.

(3) Improved Cold Viability Assessment: The study identifies an important technical artifact in using trypan blue to assess cell viability following cold exposure. It reveals that cells stained immediately after cold exposure retain the dye, inaccurately indicating cell death. By introducing a brief rewarming period before viability assessment, the authors significantly improve the accuracy of detecting cold-induced cell death. This refinement in methodology ensures more reliable results and sets a new standard for future research on cold stress in cells.

Weaknesses:

(1) Mechanisms Regulating GPX4 Levels: While the study highlights GPX4 levels as a major determinant of cellular cold tolerance, it does not discuss how these levels are regulated or why they differ between hibernators and non-hibernators. This omission leaves an important aspect of GPX4's role in cold tolerance unexplored.

(2) Generalizability Across Species: Although the study demonstrates the role of GPX4 in several mammalian species, it does not investigate whether this mechanism extends to other vertebrates (e.g., fish and amphibians) that also face cold challenges. This limitation could restrict the broader evolutionary claims made by the study.

(3) Variability in Cold Sensitivity Across Human Cell Lines: The study observes significant variability in cold tolerance among different human cell lines but does not explain these differences clearly. This leaves a key aspect of human cell cold sensitivity insufficiently addressed.

We thank the reviewer for the positive evaluation and thoughtful comments on the manuscript. We acknowledge that our study does not delve into the mechanisms regulating GPX4 levels, including differences between hibernators and non-hibernators, differences between cell types, or the possibility that GPX4 levels are dynamically regulated by environmental conditions. We consider these as interesting open questions that could be addressed in future studies.

While our study focused entirely on mammalian species, we agree that examining cold tolerance mechanisms across a broader range of vertebrates, including fish and amphibians, could enhance our evolutionary perspective. Interestingly, previous work has indicated that C.elegans adapt to cold temperatures through ferritin mediated Fe2+ detoxification. This suggests that cold induces Fe2+-mediated toxicity in C.elegans as well as mammalian cells, but that the mechanisms through which distantly related species counteract cold-mediated cell death may vary.

Finally, we agree that the variability in cold sensitivity across human cell lines could be further explored, and we will strongly consider conducting follow up experiments to examine the extent to which this variability is driven by levels of GPX4.

We are grateful for these insightful comments, as they highlight important avenues for future research. Addressing these questions will enable a more comprehensive understanding of GPX4's role in cold tolerance and its evolutionary significance across diverse organisms.

Reviewer #2 (Public review):

Summary:

Lam et al., present a very intriguing whole genome CRISPR screen in Syrian Hamster cells as well as K562 cells to identify key genes involved in hypothermia-rewarming tolerance. Survival screens were performed by exposing cells to 4C in a cooled CO2 incubator followed by a rewarming period of 30 minutes prior to survival analysis. In this paradigm, Syrian hamster-derived cell lines exhibit more robust survival than human cell lines (BHK-21 and HaK vs HT1080, HeLa, RPE1, and K562). A genome-wide Syrian hamster CRISPR library was created targeting all annotated genes with 10 guides/gene. LV transduction of the library was performed in BHK-21 cells and the survival screen procedures involved 3 cycles of 4C cold exposure x4 days followed by 2 days of re-warming.

When compared to controls maintained at 37C, 9 genes were required for BHK-21 survival of cold cycling conditions and 5 of these 9 are known components of the GPX4 antioxidant pathway. GPX4 KO BHK-21 cells had reduced cell growth at 37C and profoundly worse cold tolerance which could be reduced by GPX4 expression. GPX4 inhibitors also reduced survival in cold. CRISPR KO screens and GPX4 KO in K562 cells revealed comparable results (though intriguingly glutathione biosynthesis genes were more critical to K562 cells than BHK-21 cells). Human or Syrian hamster GPX4 overexpression improved cold tolerance.

Strengths:

This is a very nicely written paper that clearly communicates in figures and text complicated experimental manipulations and in vitro genetic screening and cell survival data. The focus on GPX4 is interesting and relatively novel. The converging pharmacologic, loss-of-function, and gain-of-function experiments are also a strength.

Weaknesses:

A recently published article (Reference 43, Sone et al.) also independently explored the role of GPX4 in Syrian hamster cold tolerance through gain-of-function screening. Further exploration of the GPX4 species-specific mechanisms would be of great interest, but this is considered a minor weakness given the already very comprehensive and compelling data presented.

We greatly appreciate the reviewer’s compliments and thoughtful comments on our manuscript. We agree with the reviewer that our approach (dual unbiased genome-scale screens in human and hamster cells) and the recent investigation by Sone et al (gain-of-function screening involving the insertion of hamster cDNA into human cells) mutually strengthen the importance of GPX4 in cold tolerance across cell types and species.

Reviewer #3 (Public review):

Summary:

This work aims to address a fundamental biological question: how do mammalian cells achieve/lose tolerance to cold exposure? The authors first tried to establish an experimental system for cell cold exposure and evaluation of cell death and then performed genome-scale CRISPR-Cas9 screening on immortalized cell lines from Syrian Hamster (BHK-21) and human (K562) for key genes that are associated with cell survival during prolonged cold exposure. From these screenings, they focused on glutathione peroxidase 4 (GPX4). Using genetic modifications or pharmacological interventions, and multiple cell models including primary cells from various mammalian species, they showed that GPX4 proteins are likely to retain their activities at 4 {degree sign}C, functioning to prevent cold-induced cell ferroptosis.

Strengths:

(1) This paper is neatly written and hence easy to follow.

(2) Experiments are well designed.

(3) The data showing the overall good cell survival after a prolonged cold exposure or repeated cold-warm cycles are helpful to show the advantages of the experimental instruments and methods the authors used, and hence the validity of their results.

(4) The CRISPR-Cas9 screening is a great attempt.

(5) Multiple cell types from hibernating mammals (cold tolerant) and cold-intolerant species are used to test their findings.

(6) Although some may argue that other labs have published works with different approaches that have pointed out the importance of GPX4 and ferroptosis in hamster cell survival from anoxia-reoxygenation or cold exposure models, hence hurting the novelty of this work, this reviewer thinks that it is highly valuable to have independent research groups and different methods/systems to validate an important concept.

Weaknesses:

(1) Only cell death was robustly surveyed; though cell proliferation was evaluated too in some experiments, other cellular functions, such as mitochondrial ATP production vs. glycolysis, and the extent of lipid peroxidation, could have been measured to reflect cellular physiology.

Validations on complex tissues or in vivo systems would have further strengthened the work and its impact.

CRISPR-Cas9 screening may have technical limitations as knock-out of some essential genes/pathways may lead to cell lethality during screening, and hence the relevance of these genes/pathways to cell cold tolerance may not be noted. From the data presented in this study, this reviewer thinks that the GPX4 pathway is likely a conserved mechanism for long-term cold survival, but not for cold sensitivity or acute cell death from cold exposure. In line with my such speculation, their CRISPR-Cas9 screening revealed genes in the GPX4 pathway from a relatively cold-sensitive human cell line, but the endogenous GPX4 pathway is seemingly operational in this cold-sensitive cell line. Also, these cells are viable after GPX4 knock-out. Dead cells from the acute cold exposure phase may detached, or their genomic DNAs have been severely damaged by the time of sample collection, hence not giving any meaningful sequencing reads. Crippling other factors/pathways such as FOXO1 (PMID: 38570500) or 5-aminolevulinic acid (ALA) metabolism (PMID: 35401816) have been shown to severely aggravate cold-induced cell death, including TUNEL-revealed DNA damage, within a much shorter time scale, whilst loss-function knockouts of FOXO1 or ALA Synthase 1 (ALAS1) are usually cell lethal. Thus, they and other possible essential genes may not be screenable from the current experimental protocol. These important points need to be taken into consideration by the authors.

We thank the reviewer for highlighting the novelty of using genome-scale CRISPR-Cas9 screens and the validation of GPX4 function across cell types and mammalian species.

We acknowledge that our study primarily focused on measuring cell death using Trypan Blue dye exclusion. To validate the Trypan Blue assay, cell survival data was orthogonally measured using the LDH release assays (Fig. 1g). The proliferation potential of putatively live cells was assessed by counting the increase in live cells following 24 h at 37°C (Fig. 1b). Prompted by your question, we will add additional data to the final version of the manuscript in which we show that following 1 day at 4°C, K562 cells rapidly restarted their cell cycle and double in numbers every 21 hours (Author response image 1). This rate is indistinguishable from the replication rate of cells that were not previously exposed to 4°C, suggesting that the cells following cold exposure are both alive and functionally capable of replicating.

Author response image 1.

Population doubling time of K562 cells cultured at 37°C (pink) and cells that are rewarmed to 37°C following 1 day of 4°C exposure

We agree that assessing additional cellular functions, such as mitochondrial ATP production, glycolysis, lipid metabolism and peroxidation could provide a more comprehensive understanding of cellular physiology under cold stress and would be valuable future studies. Similarly, we appreciate the suggestion to validate our findings in complex tissues or in vivo models. We recognize that such validation could strengthen the implications of our study and enhance its translational potential; however, due to their complexity, we believe that these additional studies are beyond the scope of our current study.

We agree with the reviewer that CRISPR-Cas9 screens have limitations. For example our screen was designed to identify genes that are preferentially required for cellular fitness at 4°C versus 37°C. There are many genes that are required for cellular survival at 4°C as well as 37°C that are not discussed (Table S2, S5). Also, given that the screen is designed to disrupt a single gene per cell, genes that have redundant functions in cold-tolerance will likely be missed. Given the reviewer’s questions, we will expand the discussion of the paper to highlight limitations of the screen.

We apologize for any lack of clarity about the methods we employed during the screen and will expand the methods section to provide further details. For example, for the BHK-21 screen we eliminated dead cells by sequencing cells that reattached after rewarming to 37°C for either 30 minutes (15 day cold exposure screen) or 24 hours (4°C cycling screen). Indeed, at the point of cell collection for both BHK-21 and K562 screens, the fraction of live cells was greater than 92% and 95%, respectively. We respectfully disagree with the reviewer that our screens would miss genes that affect acute cold tolerance. Any cells that would have died either early or late during cold exposure would have not been sequenced, and thus the sgRNAs targeting a specific gene in those cells would appear depleted, regardless of whether these cells died early/acutely or later during cold exposure.

We thank the reviewer for pointing out two additionally highly relevant studies. Interestingly, the genes implicated in cold tolerance in these studies, FOXO1 and ALAS1, did not appear essential for survival at 37°C or 4°C in BHK-21 or K562 cells. There are several possibilities that could explain this finding: 1) our screen may not have successfully knocked out these genes, 2) other proteins may have compensated for their loss, or 3) these pathways may regulate cold tolerance in some but not all cell types. We apologize that in the current version of the manuscript we did not reflect on these recent studies. We will expand our discussion to include their findings.

Once again, we are grateful for the reviewer’s insights, which have highlighted key areas for further exploration as well as pointed to specific ways to improve our manuscript.