Ferroptosis is executed through caspase-5 cleavage of gasdermin E in ovarian cancer cells

Reviewer #1 (Public review):

Summary:

This report seeks to understand the mechanisms whereby the ferroptosis inducers ML162 and erastin cause cell death in several tumor cell lines. They present evidence that caspase-5 is activated and required for ferroptosis, but other caspases, including caspase-1 and -4, are not important. Surprisingly, caspase-5 cleaved and activated GSDME, instead of the expected gasdermin target GSDMD

Strengths:

The magnitude of effect for triggering ferroptosis by ML162 and erastin is strong, and the strength of inhibition by YVAD is also very strong, making these effects convincing. The lack of effect of DEVD, which inhibits apoptotic caspases, is also convincing. Also, the lack of effect of necrostatin is convincing. These negative results strengthen the positive results seen with YVAD.

Inhibition by disulfiram is convincing.

Caspase-5 knockout single-cell clones and the ability to complement these with caspase-5, but not catalytically inactive caspase-5 in Figure 5, is strong data.

Weaknesses:

(1) Prior publications have asserted that ferroptosis is caspase-independent. Can the authors repeat some of these experiments directly to reveal whether there was an error in the published work that resulted in missing the phenotype for a caspase in ferroptosis? In my experience, caspase inhibitors sometimes only delay cell death because they are not 100% effective, especially over hours of time. Can the authors repeat the prior experiments to reveal whether this caveat affected previously published data? At the least, the authors should use the z-VAD-fmk and Boc-D-FMK inhibitors to determine whether they give the same effects as YVAD to rule out a very unlikely possibility that these "pan-caspase" inhibitors do not inhibit caspase-5.

a) The original report describing ferroptosis by Dixon and Stockwell, doi: 10.1016/j.cell.2012.03.042, shows that erastin treatment-induced ferroptosis is not affected by z-VAD-fmk in 3 cell lines.
b) A later report from Dr. Stockwell states in data not shown that a different pan-caspase inhibitor (Boc-D-Fmk) does not block erastin-driven ferroptosis. Doi 10.1016/S1535-6108(03)00050-3
c) An earlier 2008 report from Dr. Stockwell shows that z-VAD-fmk and Boc-D-fmk do not rescue cells treated with RSL-3 or RSL-5 treated cell lines derived from BJ cells. doi 10.1016/j.chembiol.2008.02.010
d) A recent paper shows a delay of ferroptosis after RSL3 treatment by pan-caspase inhibitor Q-VD-OPh. Doi 10.1038/s41418-025-01514-7. The delay was about 8 hours in time, so cells were still dying.
e) Gpx4 knockout cells or erastin or RSL3 treatment are unaffected by z-VAD-FMK. Doi 10.1038/ncb3064
f) I encourage the authors to do more thorough searching of the literature to find more publications that have used caspase inhibitors.

(2) The authors should discuss how mouse cells can undergo ferroptosis while they do not encode caspase-5, and the evolutionary conservation of caspase-5 in general. If caspase-5 is not encoded by an animal (as is the case with mice), can their cells undergo ferroptosis?

(3) Disulfiram is not a specific inhibitor. It is a nonspecific inhibitor that modifies cysteine residues of many proteins. This should be described in more detail so the reader can appreciate the strengths and weaknesses of the inhibitor.

(4) I encourage the authors to assess IL-1β processing by Western blot and show that this is inhibited by YVAD. Because ELISA can detect release of the pro form after lytic cell death by other mechanisms.

(5) ASC knockdown in Supplementary Figure 3a for two cell lines is not sufficient to draw any conclusions in Figure 3a.

(6) Caspase-5 can be more specifically inhibited by LEVD inhibitors. Can the authors show that these work as well?

(7) I would like to see a positive control in Figure 5a to show what a strong caspase signal activity looks like.

(8) Since caspase-3 is known to cleave GSDME, the authors need to assess whether caspase-3 is also activated, and whether other caspase-3 target proteins are also cleaved. There are many to choose from. Caspase-3 western blots, including with the cleaved caspase-3-specific antibody, are critical. This is in addition to the blot shown in Supplementary Figure 10. Positive controls should be included. It is important to continue to add controls to rule out caspase-3, with more than just negative data with DEVD inhibitors and the western blot in Figure S10.

(9) The data in Figure 6c are not strong.

(10) One would expect that any mode of activation of caspase-5 should lead to its proteolytic activity upon its preferred substrates, so LPS should cause caspase-5 to cleave GSDME and not GSDMD. Additional data to strongly activate caspase-5 with LPS should be investigated to see if this leads to GSDME cleavage and pyroptosis via GSDME and not GSDMD.

Reviewer #2 (Public review):

Summary:

In the submitted manuscript, Akter et al use a series of ferroptosis inhibitors in mesenchymal-like ovarian cancer cells and discover that the ferroptosis inducers induce cell death that is inhibited by pyroptosis inhibitors, namely YVAD-fmk and disulfiram, which inhibit pore formation by gasdermin D (GSDMD). Remarkably, the authors also saw the release of IL-1β in response to ferroptosis inducers. Unexpectedly, they did not observe the involvement of caspase-1 but rather observed that caspase-5 was activated in response to the ferroptosis inducers. Moreover, they found that caspase-5 directly cleaves GSDME in response to the ferroptosis inducers, establishing CASP5/GSDME as downstream executors of ferroptosis.

Strengths:

These findings are interesting because only CASP1 is known to induce IL-1β maturation, and their data suggest that CASP5 rather than CASP1, is responsible for IL-1β activation in the context of ferroptosis inducers. Notably, CASP3 is the only caspase reported to be able to cleave GSDME, so the identification of CASP5 as a driver of ferroptosis in this context is a significant finding. They genetically show that loss of CASP5 and GSDME knockdown inhibits cell death in response to the ferroptosis inducers ML162 and Erastin, which is evidence that they play a role in this context.

Weaknesses:

The major findings in this paper are interesting, but the data presented do not robustly support the claims made in this paper. For example, they claim that CASP5 is responsible for the activation of GSDME by cleaving it directly to induce cell death. They try to rule out the involvement of CASP1, ASC, and CASP4 using siRNA targeting these genes, but the knockdowns are incomplete, and the loading controls are inconsistent. They also claim they do not see GSDMD or CASP3 cleavage and activation but use negative data to make that claim. It is unclear if the antibodies used can detect cleaved GSDMD or CASP3 as they do not include a positive control to show that they can indeed detect these activation events if they were occurring. This needs to happen in the same experiment - they need to show in the same experiment with the same lysates that they can detect CASP5, GSDME and IL-1β activation but not CASP1, GSDMD, CASP4, or CASP3 activation. Of course, they should include agonists for positive controls of CASP1, CASP4 and GSDMD activation, which are lacking in the current manuscript.

Notably, the major evidence supporting a direct role for CASP5 cleavage of GSDME is one Coomassie gel using recombinant CASP5 and GSDME, but there were too many non-specific bands, and the full-length uncleaved protein could not be detected even in the untreated lanes. The authors need to show a gel where the protein can easily be identified and should also include a positive control protein like GSDMD to show the relative cleavage efficiency of GSDME compared to a known substrate. It would also be great to compare this to CASP3-mediated cleavage of GSDME. With recombinant proteins, calculating the catalytic efficiencies would be the best way to ascertain if this is biologically similar to other known substrates.

The way that ferroptosis is defined, it is caspase-independent, and pyroptosis is defined as gasdermin-mediated cell death. Given that these agents lead to activation of CASP5/GSDME, it would be more accurate to say that these ferroptosis inducers also induce CASP5/GSDME-dependent pyroptosis, as opposed to them being the executors of ferroptosis. This can be a distinct mechanism/pathway from the ferroptosis pathway, as multiple cell death pathways can be initiated in cells. Consistent with this, ferrostatin-1 also inhibited cell death, likely due to inhibition of the ferroptosis signaling cascade. It is unclear if this pathway is upstream of the caspases. How these ferroptosis triggers selectively activate CASP5 and not CASP4 to induce GSDME cleavage is a major unresolved question. Notably, it is also unclear if this biology is specific to the mesenchymal-like cells used in this study or if it expands to other cells.

Reviewer #3 (Public review):

Summary:

Akter et al. identify caspase 5 activation and Gasdermin E cleavage as a novel downstream executioner of ferroptotic cell lysis induced by erastin and ML162. These data are novel and very interesting to the wider cell death community.

Strengths:

Strengths of the study include the use and validation of findings in several mesenchymal ovarian cancer cell lines, the rigorous validation using small molecule approaches, siRNA-mediated silencing and CRISPR/Cas9-mediated knockouts with re-expression.

Weaknesses:

A weakness of the study is the fact that ferroptosis was not induced genetically (GPX4 ko) and, hence, off-targets of the mode of induction cannot be ruled out at this point (e.g. ML162 also targets TrxR1). Moreover, it would be vital to understand at which point in ferroptosis execution caspase 5 is activated in a time-resolved kinetic together with lipid ROS tracing to also obtain hints as to its possible activation.

Conclusion:

Despite the weaknesses described, this is a very interesting, timely, and well-executed study with the described limitations. The work provides important mechanistic insights into the interplay between ferroptosis and pyroptosis with possible consequences for inflammatory responses.