Caspase 3 and caspase 7 promote cytoprotective autophagy and the DNA damage response during non-lethal stress conditions in human breast cancer cells

We used the autophagy-activating role in response to nutrient deprivation of the Drosophila effector caspase Dcp-1 [34–36] as the basis for investigating the potential functional conservation between two closely related human caspases, effector CASP3 and CASP7. First, we employed standard MAP1LC3B/LC3B-based autophagy assays as these have been used successfully to monitor autophagy in breast cancer cell lines, including in SKBR3 lines, and under similar stress conditions we employed [44–47]. To determine the optimum time point for detecting a consistent upregulation of autophagy without signs of cell death, SKBR3 cells were subjected to amino acid starvation (Fig 1A and 1B). We observed a significant increase in autophagic flux at 8- and 24-h starvation, as measured by the increased LC3BII accumulation in the presence of BafA1 (Fig 1B). Across all time points, low levels of cleaved-PARP (at ~ 89 kDa) were observed in both fed (non-stressed) control cells and starved (stressed) cells as reported previously as being normal even in non-apoptotic viable cells [7,48]. Furthermore, there were no signs of cell death, as indicated by the absence of a marked increase in cleaved-PARP1 in the starvation conditions (Fig 1A), and thus we selected 8-h starvation as the optimal time point for detecting non-lethal stress-related starvation-induced autophagic flux in SKBR3 cells in this study. Next, to determine whether amino acid starvation-induced autophagy was CASP3- and/or CASP7-dependent, single knockdown (KD) and double knockdown (DKD) experiments were performed with siRNAs. The control scrambled-siRNA-treated cells showed an increase in LC3BII levels in the presence of BafA1, indicating an increase in autophagic flux when cells were starved (Fig 1C). In single CASP3 or CASP7 siRNA-treated cells, the starvation-induced autophagic flux was comparable to that of the control. However, a significant suppression of autophagic flux was observed in CASP3 and CASP7 (CASP3 + 7) DKD cells (Fig 1C and 1D).

Fig 1. Dual loss of CASP3 and CASP7 suppresses starvation-induced autophagy.

(A) Representative western blots of indicated proteins from SKBR3 cells in fed (F) or in amino acid starvation (S) for various time periods, in the absence or presence of 50 nM Bafilomycin A1 (BafA1) in the final 2 h. (B) Quantification of LC3B-based autophagy flux in starved cells relative to the fed control, shown in (A). All with BafA1. (C) Representative western blots of indicated proteins from SKBR3 cells transfected with scramble, CASP3 and/or CASP7 siRNAs (48 h) and incubated in fed conditions or starved for 8 h with BafA1 (50 nM) for the final 2 h. (D) Quantification of LC3BII-based autophagy flux in starved cells relative to the starved scramble-siRNA control, shown in (C). (E) Representative western blots of indicated proteins from CASP3, CASP7 single (KO), or double knockout (DKO) SKBR3 cells in fed or starved (8 h) conditions, with BafA1 (50 nM) in the final 2 h. (F) Quantification of LC3BII-based autophagy flux in starved cells relative to the parental control, shown in (E). (G) Representative western blots of indicated proteins from SKBR3 parental, DKO or CASP3 + 7-WT re-expression in DKO cells in fed or starved (8 h) conditions, with BafA1 (50 nM) in the final 2 h. (H) Quantification of LC3BII-based autophagy flux in starved cells relative to starved parental cells, shown in (G). (I) Representative immunofluorescence images of SKBR3 parental, DKO or CASP3 + 7-WT re-expression in DKO cells treated with 0.25 µM DALGreen in fed or starved (8 h) conditions. BafA1 (50 nM for 8 h) in both fed and starved conditions serve as controls. Scale bars, 20 µm. (J) Quantification of DALGreen immunofluorescence in starved cells shown in (I). Graph shows number of punctae per cell. n = 2, each with 10 random images covering total of 500–700 cells. In graphs, data are shown as mean ±  SEM. n =  at least 3 independent experiments except in (J). * P P P P S1 Fig. The numerical data presented in this figure can be found in S1 Data.

https://doi.org/10.1371/journal.pbio.3003034.g001

We hypothesized that the lack of a significant effect on starvation-induced autophagy upregulation in single CASP3 or CASP7 KD experiments might be due to the presence of residual caspase levels (S1A and S1B Fig). To address this, we generated CASP3 and CASP7 single knockout (KO) and double knockout (DKO) SKBR3 cell lines (S1C Fig). In single caspase KO cells, we found negligible or undetectable effects on starvation-induced autophagy compared to the control parental cell lines (Fig 1E and 1F). This suggests that CASP3 and CASP7 have partially overlapping functions and/or single KO cell lines may have activated compensatory mechanisms to overcome the other caspase’s loss. Consistent with this notion and with what we observed in DKD cells, there was a significant impairment of autophagic flux in CASP3 + 7 DKO cell lines (Fig 1E and 1F). Similarly, following 24 hours starvation, there was no induction of autophagic flux in CASP3 + 7 DKO cells (S1D and S1E Fig). To confirm the specificity of our findings, we generated multiple isogenic caspase DKO SKBR3 cell lines. In all five CASP3 + 7 DKO lines we tested, we observed a reduction in LC3B-II levels (in the presence of BafA1) indicating that amino acid starvation-induced autophagy was compromised (S1F Fig). To exclude the possibility of cell line-specific effects, we repeated our experiments using another breast cancer cell line, MDA-MB-231, and similarly found that autophagic flux induction was significantly compromised only upon CASP3 + 7 DKD and DKO (S1G–S1L Fig). Lastly, when wild-type constructs of both CASP3 and CASP7 were stably reintroduced into DKO cells, the starvation-induced upregulation of autophagy was partially rescued (Fig 1G and 1H).

The processing of LC3B to form LC3BII is a hallmark of autophagy [49]. However, since LC3B is also involved in autophagy-independent processes [50], we orthogonally measured autophagy by employing the DALGreen autolysosome fluorescent marker [51]. In accordance with the LC3B-based autophagy assay results, the levels of DALGreen positive puncta indicate that amino-acid deprivation-induced autophagy is significantly compromised in CASP3 + 7 DKO cells, and the dual re-expression of CASP3 and CASP7 fully rescued autophagy (Fig 1I and 1J). The reduction in number of autolysosomes in CASP3 + 7 DKO cells was not due to a difference in cell size (S1M and S1N Fig). These observations together indicate that CASP3 and CASP7 play a partially redundant positive regulatory role in non-lethal amino acid deprivation-induced autophagy.

It is well-documented that autophagy is upregulated as a compensatory mechanism to maintain protein homeostasis, when proteasome function is compromised [52,53]. Next, we investigated whether this compensatory autophagy was also dependent on CASP3 and CASP7. A proteasome inhibitor (PI) MG132, at 0.5 μM, was chosen as the non-lethal stress condition for these experiments. (Figs 2A–2C and S2A–S2C). The reduction of proteasome activity was confirmed directly by proteasome activity assays (Figs 2C and S2C) and indirectly through the accumulation of ubiquitinated proteins (Figs 2A and S2A). In line with what we observed in amino acid starvation conditions, the PI-induced autophagic flux was significantly inhibited in CASP3 + 7 DKD or DKO cells (Fig 2D–2G). Although the effect was modest, single CASP7 KO also resulted in a significant reduction in PI-induced autophagy (Fig 2F and 2G). Similar results were observed in the MDA-MB-231 cell line (S2D and S2E Fig), and with the clinically approved proteasome inhibitor Bortezomib [54,55] (S2F and S2G Fig). PI-induced LC3BII upregulation was partially rescued by dual re-expression of CASP3 + 7, supporting the requirement for these effector caspases in PI-induced autophagy (Fig 2H and 2I). Collectively, these results indicate that CASP3 and CASP7 have positive regulatory roles in autophagy induction in response to starvation or proteasome inhibition-induced stress.

Fig 2. Dual loss of CASP3 and CASP7 suppresses proteasome inhibition-induced compensatory autophagy and sensitizes cells to proteasome inhibitors.

(A) Representative western blots of indicated proteins from SKBR3 cells treated with MG132 at increasing dosage for 24 h, with BafA1 (50 nM) in the final 2 h. (B) Quantification of LC3B-based autophagy flux in MG132-treated cells relative to untreated (0 μM MG132; DMSO vehicle only; NT) SKBR3 shown in (A). The levels of LC3BII were normalized to loading control and shown relative to the untreated control (NT). (C) Graph showing proteasome activity in response to increasing concentrations of MG132 as depicted by chymotrypsin, caspase and trypsin-like activities. (D) Representative western blots of indicated proteins from SKBR3 cells transfected with scramble, CASP3 and/or CASP7 siRNAs (48 h) and treated with vehicle DMSO only or MG132 (0.5 μM) for 24 h, with BafA1 (50 nM) in the final 2 h. (E) Quantification of LC3B-based autophagy flux in MG132-treated cells relative to the MG132-treated scramble-siRNA control, shown in (D). (F) Representative western blots of indicated proteins from CASP3, CASP7 single knockout, or DKO SKBR3 cells treated with vehicle DMSO or MG132 (0.5 μM) for 24 h, with BafA1 (50 nM) in the final 2 h. (G) Quantification of LC3B-based autophagy flux in MG132-treated cells relative to the MG132-treated parental control show in (F). (H) Representative western blots of indicated proteins from SKBR3 parental, DKO or CASP3 + 7-WT reintroduced into DKO cells treated with vehicle DMSO or MG132 (0.5 or 1.0 μM) for 24 h with BafA1 (50 nM) in the final 2 h. (I) Quantification of LC3BII-based autophagy flux in MG132-treated cells relative to DMSO-treated parental control, shown in (H). (J) Representative images of crystal violet assay plates of SKBR3 parental and CASP3 + 7 DKO cells treated with indicated concentrations of MG132 for 24 h and continued to grow in drug free media for another 3 days. (K) Quantification of cell viability shown in (J). Percentage of stained (viable) cells at each concentration was normalized to respective untreated cells. In graphs, data are shown as mean ±  SEM. n =  3 independent experiments. * P P P P S2 Fig. The numerical data presented in this figure can be found in S1 Data.

https://doi.org/10.1371/journal.pbio.3003034.g002

Since autophagy has been reported to promote cell death in some contexts [56–58], we investigated whether the CASP3- and CASP7-dependent stress-induced autophagy contributes to cell death or survival. Crystal violet cell viability assays were carried out following PI treatment (MG132 or Bortezomib) or starvation of parental and CASP3 + 7 DKO or DKD of SKBR3 or MDA-MB-231 cells (Figs 2J, 2K and S2H–S2O). We observed a modest, but significant reduction in cell viability in response to PI treatment or starvation in CASP3 + 7 DKD or DKO cells compared to control cells. Contrary to traditional caspase roles, these results suggest that CASP3 and 7 promote cell survival in non-lethal PI or starvation stress conditions.

To elucidate the molecular mechanisms by which CASP3 and CASP7 positively regulate cytoprotective autophagy, we analyzed their expression and processing pattern following amino acid starvation or PI treatments. CASP3 and CASP7 processing during apoptosis each result in large ~ p20 and small ~ p10/p12 cleaved-caspase fragments [37,59,60]. Intriguingly, our western blot analyses revealed a stable non-canonical CASP7 cleavage fragment(s) at ~ 30 kDa (p30) and/or 29 kDa (p29), and a CASP3 cleavage fragment at ~ 27 kDa (p27) in non-lethal starvation (Fig 3A and 3B) or PI conditions (Fig 3C and 3D). In fed or untreated (non-stressed) cells, these non-canonical fragments were either absent or less apparent. The CASP3 fragment at ~ 27 kDa was inconsistent and/or not readily detectable (e.g., Fig 3C) across all experiments, so we focused on the CASP7-p29/p30 fragments.

Fig 3. CASP7 is cleaved non-canonically generating stable p29/p30 fragments under non-lethal stress conditions.

(A–D) Representative western blots showing changes in CASP7 and CASP3 in SKBR3 or MDA-MB-231 cells in amino acid starvation (A, B) or treated with MG132 (C, D). Non-canonical CASP7 bands (p29/p30) at 30 kDa are indicated by blue arrows. A non-canonical CASP3 band (p27) at 27 kDa is also detected in some conditions. (E) Representative western blots comparing CASP7, CASP3, and PARP1 processing patterns in SKBR3 cells following 4-h incubation in fed conditions (F), non-lethal stress (starvation) or lethal stress (apoptosis induction by staurosporine; STS, 1 or 2 µM). (F) Representative western blots comparing CASP7 and PARP1 processing in SKBR3 parental cells stably transfected with vector control or BCL2 expression construct, following 4-h incubation in fed conditions (F), non-lethal stress (starvation) or lethal stress (apoptosis induction by staurosporine; STS, 1 or 2 µM). (G) Representative western blots showing the effect of rapamycin on the formation of CASP7-p29/p30 bands in SKBR3 cells. Cells in fed, starved (4 h), and/or treated with 10 nM rapamycin (Rap; 24 h). Blot was immunolabeled with mTOR activity reporters P70S60K and p70S6K-PO4. (H) Representative western blot showing CASP7 immunolabeling in tissues from a female CD-1 mouse. Ponceau S labeling serves as the loading control. n =  3 mice. (I) Representative western blot showing CASP7 immunolabeling in human breast cancer PDX specimens from indicated subtypes. Starved SKBR3 serves as the positive control for p29/p30 fragments. (J) Representative western blot showing CASP7 immunolabeling in parental MDA-MB-231 cells and derivative epirubicin-resistant (R8a, R8b, R3-1, R3-2) and 5Fu-resistant (R6-1, R6-2) cells in fed conditions. In each, at least n = 2 independent experiments. See also S3 Fig.

https://doi.org/10.1371/journal.pbio.3003034.g003

To confirm and further explore the observed non-canonical processing, we analyzed CASP7 processing in different contexts. We compared the CASP7 processing profiles resulting from starvation stress to apoptosis-induced profiles (staurosporine-treated cells) (Fig 3E). A marked increase in cleaved-PARP and canonical cleaved-CASP3 (p20) were present only in staurosporine-treated cells, confirming that the amino acid starvation conditions employed were non-lethal. The CASP7-p29/30 fragment(s) were produced predominantly only in the non-lethal starvation conditions, whereas the canonical apoptosis-associated CASP7-p20 fragments were primarily observed in staurosporine-treated cells. Altogether, these results confirm that the CASP7 processing in response to non-lethal stress conditions is distinct from CASP7 processing during apoptosis.

Mitochondrial outer membrane permeabilization (MOMP) activates caspases in apoptotic and non-apoptotic conditions [61,62]. BCL2 overexpression inhibits MOMP and subsequent CASP3 and CASP 7 processing in apoptosis [63,64]. To investigate whether MOMP plays a role in CASP7 non-canonical processing in non-lethal stress, we overexpressed BCL2 in SKBR3 parental cells. As expected, BCL2 overexpression inhibited canonical CASP7 processing in the control staurosporine-treated cells. In contrast, BCL2 overexpression did not inhibit the formation of CASP7-p29/30 in the non-lethal starvation stress condition (Fig 3F). These results indicate that CASP7 non-canonical processing is independent of MOMP.

We next asked whether other known autophagy inducers would also generate non-canonical CASP7 cleavage. Rapamycin inhibits mTOR and induces autophagy [65,66]. In SKBR3 and MDA-MB-231 cell lines (S3A and S3B Fig), autophagy was induced upon treatment with rapamycin. However, rapamycin did not result in non-canonical CASP7 processing (Figs 3G and S3C). Further, CASP3 + 7 DKO had no effect on rapamycin-induced autophagy (S3A and S3B Fig). Collectively, these observations suggest that caspases act upstream or in parallel to mTOR in autophagy regulation.

Next, we investigated the prevalence and the context dependency of non-canonical CASP7 processing. Using fed (unstressed) mice, we confirmed the existence of in vivo non-canonical processing of CASP7 in several tissues, including in the brain, heart, spleen, and kidney (Fig 3H). Multiple breast cancer patient-derived xenograft (PDX) specimens also clearly showed the existence of CASP7-p29/30 fragments (Fig 3I). Further, a panel of anthracycline-resistant MDA-MB-231 cell lines were analyzed, with the majority showing a clear accumulation of CASP7-p29/30 fragments (Fig 3J). Altogether, these data confirm the existence of stable non-canonical CASP7 fragments in vitro and in vivo models and in both normal and cancer tissues.

Next, we investigated the identity of the CASP7-p29/30 fragments observed in non-lethal stress conditions. In apoptosis, CASP7 activation is initiated by removing the pro-domain [37], and no stable accumulation of CASP7 lacking the pro-domain has been reported. To determine whether the non-canonical CASP7-p29/30 fragment(s) could be the result of pro-domain removal, we stably transfected CASP3 + 7 DKO cells with a full length CASP7 wild-type construct (CASP7-WT), a pro-domain deletion construct (CASP7-ΔPro) (Fig 4A and 4B), or a pro-domain cleavage site-mutant construct (S4A Fig). In western blot analyses, the CASP7-p29/30 fragments formed in starvation did not align with the CASP7-ΔPro construct, which appeared at ~ 33.5 kDa (Figs 4A and S4A). Additionally, when starved, the CASP7-ΔPro-expressing cells also resulted in CASP7-p29/30 fragment(s) (Figs 4A and S4A). Collectively, these data show that the non-lethal cellular stress-associated CASP7 cleavage occurs at a non-canonical site(s) downstream of the pro-domain cleavage site.

Fig 4. CASP7-p29 and p30 are generated via processing at calpain cleavage sites.

(A) Representative western blot showing CASP7 immunolabeling in CASP3 + 7 DKO SKBR3 cells stably transfected with CASP7-WT or CASP7-delta-Pro (CASP7-Δpro) constructs in fed or starvation conditions. Arrowhead indicates CASP7-Δpro band and arrow indicates CASP7-p29/p30 bands. n = 2 independent experiments. (B) Schematic of subunits, canonical cleavage sites and non-canonical cleavage sites in CASP7 wild-type or in various CASP7 fragments used or observed in this study. Black scissors denote the canonical cleavage sites and the order of processing in apoptosis. Purple scissors, bars, and black arrows denote the putative calpain cleavage sites (non-canonical). (C) The sequences on the left show the putative calpain cleavage sites in CASP7-WT and the amino acid replacements made in the CASP7 construct with putative calpain-cleavage sites mutated (CASP7-CCM). Representative western blot of CASP7 from CASP3 + 7 DKO SKBR3 cells transfected with CASP7-WT or CASP7-CCM constructs grown in fed (F) or starved (S) conditions. n = 2 independent experiments. (D, E) Representative western blots of indicated proteins from SKBR3 cells transfected with scramble, calpain 1 or calpain 2 siRNAs (48 h) and then continued to be cultured in fed (F) conditions or starved (S) for 8 h (D) or treated with MG132 for 24 h (E), with BafA1(50 nM) in the final 2 h. (F–I) Quantification of LC3B-based autophagy flux and CASP7 bands shown in (D) and (E) relative to scramble-siRNA control. In graphs, data are shown as mean ±  SEM. n =  3 independent experiments. *P P P P S4 Fig. In F–I, one-way ANOVA with Dunnett’s post-test. The numerical data presented in this figure can be found in S1 Data.

https://doi.org/10.1371/journal.pbio.3003034.g004

To elucidate the precise identity of the non-canonical cleavage sites and fragments, we enriched for CASP7-p29/30 fragments by performing CASP7-immunoprecipitation (IP) under starvation conditions (S4B). Protein in the 29/30 kDa region was extracted and subjected to Edman sequencing, which revealed the location of a non-lethal stress-associated cleavage site 10 amino acids downstream of the CASP7 pro-domain cleavage site (Fig 4B and 4C). This non-canonical cleavage site and a second site nearby (Fig 4B and 4C), corresponding to p30 and p29, respectively, were shown previously to be cleaved in vitro by calpains, but were associated with further processing of CASP7 to yield p17 and p18 [39,40]. Using the DeepCalpain algorithm [67], we identified predicted calpain cleavage sites conserved in murine CASP7 (S4C Fig) and confirmed that they corresponded to the size of the CASP7-p30/p29 bands detected in murine samples (Fig 3H). To verify the cleavage sites, we created a CASP7-calpain cleavage mutant (CASP7-CCM) construct by substituting both calpain cleavage sites with alanine residues (see Materials and methods; Fig 4C). Upon starvation, CASP3 + 7 DKO SKBR3 cells stably transfected with CASP7-CCM failed to form abundant CASP7-p29/p30 compared to CASP7-WT cells (Fig 4C). Together, these results validate the non-canonical cleavage of CASP7 and suggest a potential involvement of calpains in regulating CASP7 to promote starvation or PI-induced autophagy and/or cell survival in non-lethal stress conditions.

To determine whether calpains have any direct involvement in CASP7 non-canonical processing, we performed single KD of the most abundant calpain family members calpain 1 and calpain 2 in SKBR3 cell lines and subjected these cells to non-lethal amino acid starvation or PI stress conditions (Fig 4D–4I). Single KD of calpain 1 or calpain 2 led to a significant increase in overall CASP7 levels compared to control (Fig 4D–4I), suggesting a role in CASP7 turnover. Next, the ratio of CASP7 p29/30 fragment(s) relative to its full length was quantified (Fig 4G and 4I). In both stress conditions, calpain 1 KD cells showed the highest level of autophagy induction (Fig 4F and 4H) and CASP7-p29/30 fragments ratios (Fig 4F and 4H). In contrast, in both stress conditions, calpain 2 KD showed a reduction of stress-induced autophagy (p S4D and S4E Fig), consistent with the possibility that CASP3 could also be processed by calpain 2 in this context. CASP3 was shown previously to be processed by calpains [68,69], further supporting the investigation of CASP3 processing in non-lethal stress conditions as an interesting avenue for future studies.

The well-known PARP1 binding site [43] in CASP7 is located between the two identified calpain cleavage sites (S4F Fig). PARP1 and associated PARylation activities were shown to play key roles in stress sensing, stress adaptation, and autophagy induction in multiple cell lines and in vivo mouse models [70–75]. Further, PARP1 is a CASP3 and CASP7 substrate, with the latter showing a higher affinity for PARP1 binding [76]. These factors prompted us to investigate PARP1 levels and PARP1 activity in the CASP3 + 7 DKO background. We predicted that cleaved-PARP1 levels would be reduced, and thus PARP1 activity would be increased in the CASP3 + 7 DKO cells. Unexpectedly, we detected a higher level of cleaved-PARP1 in CASP3 + 7 DKO cells compared to the SKBR3 parental cells (Fig 5A–5D). There was no evidence of cell death in the DKO cells, consistent with previous observations that cleaved-PARP1 (89 kDa) is not always associated with cell death [77,78]. Cleaved-PARP1 levels were further increased when cells were stressed with a non-lethal dosage of MG132 (Fig 5B and 5D). PARylation is a PARP catalytic activity-dependent process [79]. Next, PAR immunolabeling was performed as a readout for PARylation activity of PARP1 [80]. We observed a significant reduction in PAR labeling in CASP3 + 7 DKO cells compared to parental cells in both SKBR3 and MDA-MB-231 cell lines (Fig 5E–5H). These results indicate that in the absence of CASP3 and CASP7, PARP1 cleavage is enhanced and PARP1 activity (as measured by PAR level) is impaired.

Fig 5. PARylation and ATG gene expression are reduced in CASP3 + 7 DKO cells.

(A, B) Representative western blots of indicated proteins from parental and CASP3 + 7 DKO SKBR3 cells cultured in fed conditions (F) or starved (S) for 8 h (A) or treated with MG132 (0.5 μM) for 24 h (B). (C) Quantification of cleaved-PARP1 shown in (A). Values were normalized to parental fed conditions. (D) Quantification of cleaved-PARP1 shown in (B). Values were normalized to parental NT. NT = No Treatment (vehicle DMSO). (E, F) Representative western blots of PAR immunolabeling from parental and CASP3 + 7 DKO SKBR3 (E) or MDA-MB-231(F) cells treated with vehicle (DMSO) or MG132 (0.5 μM) for 24 h. (G, –H) Quantification of PAR levels shown in (E) and (F). Values were normalized to the parental NT condition. (I) RT-qPCR analyses of LC3B in SKBR3 parental, CASP3 + 7 DKO or CASP3 + 7-WT re-expression in CASP3 + 7 DKO SKBR3 cells. Cells were treated with vehicle (DMSO) or 0.5 μM of MG132 for 24 h. (J) Representative western blots of indicated proteins from SKBR3 parental and CASP3 + 7 DKO cells transfected with scramble or CASP2 siRNAs (72 h). (K) Quantification of cleaved-PARP1 levels (shown in J) relative to scramble-siRNA control. (L) Quantification of CASP2 levels relative to scramble-siRNA-treated parental control. (M) Quantification of PAR levels shown in (J) relative to scramble-siRNA-treated parental control. (N) Representative western blots of indicated proteins from SKBR3 parental and CASP3 + 7 DKO cells transfected with scramble, CASP2 or CASP8 siRNAs (42 h) and treated with vehicle DMSO or MG132 (0.5 μM) for 24 h, with BafA1(50 nM) in the final 2 h. (O) Quantification of LC3B-based autophagy flux in MG132-treated cells, shown in (N). In graphs, data are shown as mean ±  SEM. n =  3–6 independent experiments. *P P P P S5 Fig. The numerical data presented in this figure can be found in S1 Data.

https://doi.org/10.1371/journal.pbio.3003034.g005

In cellular stress conditions, PARP1 and PARylation were shown previously to positively regulate cytoprotective autophagy through multiple mechanisms [73–75,81], including the transcriptional upregulation of autophagy pathway components) [82]. Since CASP3 + 7 DKO lead to reduced PARylation, we predicted that CASP3 + 7 DKO would result in transcriptional downregulation of autophagy pathway components. Among the components evaluated by reverse transcription polymerase chain reaction reverse transcription polymerase chain reaction (RT-qPCR), we found that LC3B and ATG7 transcript levels were reduced in CASP3 + 7 DKO SKBR3 cells (S5A–S5E Fig). There was an increase in both transcripts in response to MG132-induced stress (Figs 5I and S5F), with DKO cells showing weaker upregulation compared to parental (Figs 5I and S5F). Dual re-expression of CASP3 + 7-WT constructs partially rescued transcript levels of these genes in DKO cells (Figs 5I and S5F). These results are consistent with the possibility that CASP3 and CASP7 regulate transcription of key autophagy genes, potentially through changes to PARP1 and PARylation.

To test whether CASP3 and CASP7 regulate autophagy through PARP1, we investigated whether overexpression of PARP1 could rescue autophagy in the CASP3 + 7 DKO background. Moreover, we compared rescue ability of wild-type PARP1 (PARP1-WT), catalytically inactive PARP1 (PARP1-CI), and PARP1 with the DEVD cleavage site mutated (PARP1-DEVA, PARP1-AEVA). Consistent with endogenous PARP1, the PARP1-WT transfected CASP3 + 7 DKO cells resulted in a significantly increased accumulation of cleaved-PARP1 compared to the PARP1-WT transfected parental cells (S5G and S5H Fig). As expected, levels of cleaved PARP1 were significantly reduced in the PARP1-DEVA and PARP1-AEVA expressing cells (S5G and S5H Fig). While the cleavage-resistant PARP1-DEVA and PARP1-AEVA cells also showed a trend of increased LC3B transcription relative to PARP1-WT (S5J Fig), the PARylation and autophagy flux levels showed no differences (S5G, S5I, S5K, and S5L Fig). The PARP1-CI transfected cells did show significantly reduced PARylation compared to the vector control, consistent with the reported dominant negative effect of catalytically inactive PARP1 (S5G and S5I Fig) [83], but induction of cell death and very low expression levels confounded interpretation of results. Overall, while the association between PARP1 cleavage and LC3B transcription remains consistent, it is not clear whether the reduced autophagy flux in the CASP3 + 7 DKO background is due to the enhanced PARP1 cleavage.

Next, to identify the protease(s) responsible for PARP1 cleavage in the absence of CASP3 and 7, we evaluated multiple candidate proteases using a siRNA approach. The knockdown of CASP6, calpain 1, calpain 2, cathepsin B (CTSB), and cathepsin D (CTSD) resulted in a further increase in PARP1 cleavage (S5M–S5O Fig). We next investigated caspase 2 (CASP2) and caspase 8 (CASP8), both shown to localize to the nucleus in some contexts [84,85]. We discovered that knockdown of CASP2, but not CASP8, consistently reduced PARP1 cleavage in CASP3 + 7 DKO cells (Figs 5J, 5K, and S5P). Interestingly, the relative expression level of CASP2 was significantly higher in CASP3 + 7 DKO cells compared to the parental cells (Fig 5J and 5L), further supporting a role for CASP2 in this context. Consistent with the lack of rescue by the DEVD cleavage site mutants, PARylation and autophagic flux were not rescued in the CASP2 knockdown CASP3 + 7 DKO cells (Fig 5M–5O). Collectively, our data suggest that CASP2 is at least partially responsible for the increased PARP1 cleavage in the CASP3 + 7 DKO cells. Further investigation of the contexts and role of CASP2 in PARP1 regulation is warranted.

In addition to transcriptional remodeling, PARP1 plays a central role in DNA repair and DNA damage response (DDR) pathways in response to cell stress [71,86]. Both starvation and proteasome inhibition were reported to induce DNA damage and activate autophagy as a part of stress-induced DDR [87,88]. Western blot analyses revealed that MG132 stress-induced phospho-H2AX (γ-H2AX) accumulation was significantly reduced in CASP3 + 7 DKO cells compared to parental cells without reduction in total H2AX levels (Fig 6A and 6B).

Fig 6. CASP7-p29/30 promote phosphorylation of DDR pathway protein H2AX.

(A) Representative western blots of indicated proteins from SKBR3 parental and CASP3 + 7 DKO cells treated with vehicle DMSO or MG132 (0.5 µM) for 24 h. Ponceau S staining was used as the loading control. (B) Quantification of γ-H2AX/total H2AX shown in (A). (C) Representative images from alkaline comet assay from SKBR3 parental and CASP3 + 7 DKO cells treated with vehicle DMSO or MG132 (0.5 µM) for 24 h. Cells treated with H₂O₂ (100 μM for 4 h) serve as the positive control. (D) Quantification of tail moments of comets shown in (C). Tukey’s box-and whisker plots are based on tail moments determined by CometScore from 200 cells in each of two independent assays. (E) Representative western blots of indicated proteins from SKBR3 parental, CASP3 + 7 DKO or single KO cells treated with vehicle DMSO or MG132 (0.5 µM) for 24 h. Ponceau S staining was used as the loading control. (F) Quantification of γ-H2AX/total H2AX shown in (E). (G) Representative western blots of indicated proteins from CASP3 + 7 DKO SKBR3 cells stably transfected with indicated CASP7 constructs treated with vehicle DMSO or MG132 (0.5 µM) for 24 h. Ponceau S staining was used as the loading control. (H) Quantification of γ-H2AX/total H2AX shown in (G). (I) Representative images from alkaline comet assay from CASP3 + 7 DKO SKBR3 cells stably transfected with indicated CASP7 constructs and treated with vehicle DMSO or MG132 (0.5 µM) for 24 h. Cells treated with H₂O₂ (100 μM for 4 h) serve as the positive control. (J) Quantification of tail moments of comets shown in (I). Tukey’s box-and-whisker plots are based on tail moments obtained from 200 cells in each of two independent assays. (K) Representative images of crystal violet assay plate. CASP7-CCM, CASP7-p30, and CASP7-p29 SKBR3 cells treated with indicated concentrations of proteasome inhibitor MG132 for 24 h and continued to grow in drug free media for another 3 days. (L) Quantification of cell viability data presented in (K). The percentage of stained (viable) cells at each concentration was normalized to respective untreated cells. (M) Representative western blots of indicated proteins from CASP3 + 7 DKO SKBR3 cells re-expressing vector, CASP7-WT (catalytically active) or CASP7-inactive (catalytically inactive) constructs, treated with vehicle DMSO or MG132 (0.5 µM) for 24 h. H3 serves as the loading control. (N) Quantification of γ-H2AX/total H2AX shown in (M). (O) Representative western blots of LC3B-based autophagy flux in CASP3 + 7 DKO SKBR3 cells re-expressing the indicated CASP7 constructs, treated with vehicle DMSO or MG132 (0.5 µM) for 24 h, with BafA1(50 nM) in the final 2 h. (P) Quantification of autophagy flux in MG132 treated cells relative to MG132 treated CCM expressing cells, shown in (O). In graphs, data are shown as mean ±  SEM. n =  3 independent experiments except for comet assays n = 2. *P P P P S6 Fig. The numerical data presented in this figure can be found in S1 Data.

https://doi.org/10.1371/journal.pbio.3003034.g006

Traditionally, the level of γ-H2AX was measured to determine the extent of DNA damage [89]; however, γ-H2AX levels were also shown to represent the strength of DDR signaling activity [12,90,91]. To distinguish between these two possibilities, we used comet assays to directly measure DNA damage [92]. The level of DNA damage/fragmentation in parental versus CASP3 + 7 DKO SKBR3 cells was not significantly different when treated with the vehicle (DMSO) control (Fig 6C and 6D). In response to non-lethal MG132 stress conditions, there was an increase in DNA damage, but it was comparable in both parental and CASP3 + 7 DKO cells. In contrast, the positive control, H₂O₂, lethal stress conditions induced an increase in DNA damage only in the parental cell line (Fig 6C and 6D). This data indicates that the γ-H2AX reduction observed in CASP3 + 7 DKO cells in the MG132 non-lethal stress conditions is not due to a reduction in DNA damage, but possibly instead due to impaired DDR signaling.

Next, we investigated the γ-H2AX levels in CASP3 + 7 double and single KO cells (Fig 6E and 6F). The γ-H2AX levels were reduced in CASP3 + 7 DKO cells, but relatively increased in both CASP3 single KO and CASP7 single KO cells. Notably, the γ-H2AX level was highest in CASP3 KO cells, suggesting that CASP7 plays a prominent role, relative to CASP3, in the H2AX phosphorylation. To further investigate this possibility and given the proximity of the PARP1 binding site to the CASP7 calpain cleavage sites, we generated CASP3 + 7 DKO cell lines stably expressing CASP7-CCM, CASP7-p30, or CASP7-p29 (S6A Fig). We used CASP7-CCM as the full-length control since it does not undergo non-canonical cleavage. We compared γ-H2AX levels in the absence and presence of non-lethal MG132, and observed that the stress-induced γ-H2AX accumulation was increased in CASP7-p30 and CASP7-p29 expressing cells compared to CASP7-CCM expressing cells (Fig 6G and 6H). Among the three constructs, the greatest level of γ-H2AX was observed with CASP7-p29. Furthermore, comet assays revealed that the degree of DNA damage was not significantly different among the three cell lines in either vehicle (DMSO) control, non-lethal MG132-treated or lethal H₂O₂-treated conditions (Fig 6I and 6J). In fact, the degree of DNA damage appeared slightly reduced in CASP7-p29 expressing cells, where we detected the greatest level of γ-H2AX (Fig 6H and 6J). Crystal violet cell viability assays, following treatment with increasing concentrations of MG132 or Bortezomib (Figs 6K, 6L, S6B and S6C), showed no significant differences in cell viability despite the different γ-H2AX levels observed in the three cell lines. Overall, these results support that CASP7 non-canonical processing does not affect the degree of DNA damage, but rather plays a role in inducing different levels of γ-H2AX signaling (S6D Fig). Consistent with several previous studies showing that γ-H2AX is not an unambiguous marker for DNA double-strand breaks [12,90,91], our findings emphasize the need for careful interpretation of γ-H2AX levels.

To further explore CASP7 in this role, we employed catalytically active wild type (CASP7-WT) and catalytically inactive (CASP7-inactive, C > A) constructs stably expressed in CASP3 + 7 DKO cells. CASP7-WT construct expression, but not catalytically inactive construct, led to a significant increase in the γ-H2AX signaling in non-lethal MG132-treated cells (Fig 6M and 6N), revealing that CASP7 and its catalytic activity are required for the phosphorylation of H2AX.

Our data shows that inhibition of CASP3 and CASP7 leads to increased PARP1 cleavage and reduced PARP activity (PARylation), DDR signaling, and autophagy response. Since these effects phenocopy the genetic or pharmacological inactivation of PARP1 [70,71], we hypothesized that dual CASP3 + 7 inhibition would also phenocopy the synthetic lethal effects of PARP1 in cells that are defective in homologous recombination (HR) [93,94]. To test this, we generated CASP3 + 7 DKD in the BRCA1-deficient SUM149PT breast cancer cells and assessed their viability by the crystal violet assay. In striking contrast to the BRCA1/2-proficient SKBR3, MDA-MB231, and JIMT1 breast cancer cells, the dual inhibition of CASP3 + 7 was lethal in SUM149PT cells (Fig 7A–7C). In the western blot analyses, a distinct band of cleaved-PARP1 was visible in CASP3 + 7 DKD cells compared to the scrambled-siRNA control (Fig 7C). Western blot analyses also confirmed that autophagic flux was reduced in the CASP3 + 7 DKD SUM149PT cells compared to the scrambled-siRNA control or compared to the single KD of CASP3 or CASP7 (Fig 7D and 7E). To determine whether the lethal effects of CASP3 + 7 DKD was redundant or potentially additive with PARP1 inhibition, we treated the SUM149PT cells with the PARP1 inhibitor olaparib. The combination of CASP3 + 7 DKD and olaparib treatment resulted in a further reduction of SUM149PT cell viability compared to olaparib treatment alone (Fig 7F and 7G). These results suggest that combined inhibition of CASP3 and CASP7 induces synthetic lethality with loss of BRCA1.

Fig 7. Dual inhibition of CASP3 and 7 mimic effects of PARP1 inhibitors.

(A) Representative images of crystal violet assays. SUM149PT, MDA-MB-231, SKBR3, or JIMT-1 cells transfected with scramble-siRNA or CASP3 and CASP7 siRNAs (10 nM) for 2 days and then cultured in siRNA-free media for another 3 days. (B) Quantification of cell viability from data represented in (A). Percentage of stained (viable) cells at each concentration was normalized to respective untreated cells. (C) Representative western blots showing CASP3 and CASP7 levels following siRNA treatment conditions used in crystal violet assays shown in (A). Immunolabelling of cleaved-PARP1 is also shown. n = 2 independent experiments. (D) Representative western blots of indicated proteins from SUM149PT cells transfected with scramble, CASP3 and/or CASP7 siRNAs (48 h) and treated with vehicle DMSO or MG132 (0.5 μM) for 24 h, with BafA1 (50 nM) in the final 2 h. (E) Quantification of LC3B-based autophagy flux in proteasome inhibitor (MG132) treated cells shown in (D). The levels of LC3BII in MG132 treated cells were normalized to loading control and shown relative to the MG132 treated scramble-siRNA control. (F, G) Representative images of crystal violet assay plates (F) and quantification of percentage of cell viability (G). Indicated siRNA transfected cells treated with indicated concentrations of Olaparib for 24 h and continued to be cultured in drug free media for another 3 days. Graph (G) show the percentage of stained (viable) cells at each concentration normalized to scramble-siRNA-treated and Olaparib untreated cells. (H) Dot plot showing DepMap cancer cell lines with bottom 10% of CASP3 + 7 protein expression (n = 16) and top 10% CASP3 + 7 protein expression (n = 10). Normalized CASP3/7 protein expression (scaled and mean centered) were extracted from Nusinow and colleagues using GRETTA. (I) Ranked CASP3 + 7 genetic interaction scores generated using GRETTA. The cell lines at the bottom 10% of CASP3 and CASP7 protein expression are compared against cells lines at the top 10%. Rank is based on lethal GI scores. The red points indicate true candidate CASP3 + 7 lethal genetic interactions (GIs). The top most lethal genetic interactors are labeled. In graphs, unless otherwise noted, data are shown as mean ±  SEM. n =  3 independent experiments. *P P P P U-test p-value S7 Fig. The numerical data presented in this figure can be found in S1 Data. The code related to Fig 7H and 7I is publicly available in a GitHub repository (https://github.com/MarraLab/Caspase_GRETTA_analysis) and archived on Zenodo (https://doi.org/10.5281/zenodo.14722298).

https://doi.org/10.1371/journal.pbio.3003034.g007

To further assess genetic interactions between BRCA1 and CASP3 and CASP7, we utilized Genetic inteRaction and EssenTiality neTwork mApper (GRETTA) [95], a tool that leverages the publicly available data from the Cancer Dependency Map (DepMap [96–98], to perform a pan-cancer in silico genetic interaction screen. Using GRETTA, we identified 16 DepMap cancer cell lines harboring low protein expression of both CASP3 and CASP7 and 10 cancer cell lines with high expression of both CASP3 and CASP7 across 13 cancer types (Figs 7H and S7A). Comparisons between these two groups resulted in the identification of 142 candidate CASP3 + 7 synthetic lethal interactors, where perturbation of these genes resulted in significantly higher lethal probabilities in the CASP3 + 7 low expressor lines compared to the CASP3 + 7 high expressor lines (S7B Fig). The candidate synthetic lethal interactors included HR repair pathway genes (S7B Fig), with BRCA1 as the top synthetic lethal hit (Fig 7I). Collectively, our results show that the dual inhibition of CASP3 and CASP7 mimics the cellular effects of PARP1 inhibitors, revealing a surprising new avenue for potential therapeutic exploitation. Counter-intuitive to traditional caspase roles, our findings demonstrate that combined inhibition of CASP3 and CASP7 induces synthetic lethality in BRCA1-defective cells.

To obtain cell lysate from PDX, breast tumor tissues originally derived from human were grown as xenografts in mice as detailed in Eirew and colleagues (2022) [115] harvested, frozen in DMEM/FCS with 6%–10% DMSO and provided by Dr.Sam Aparicio and Dr. Peter Eirew. Ethical approval for research using these human breast tumor tissues was obtained from the Research Ethics Board of BC Cancer and Simon Fraser University.

Proteins in cell/tissue lysates were quantitated using the Pierce BCA Protein Assay Kit (Invitrogen, 23225). For gel electrophoresis, 10–20 µg protein was prepared by adjusting the total volume using dH₂O and 4× SDS Loading buffer. Proteins were denatured by boiling at 95 °C for 10 min and then separated by gel electrophoresis (SDS PAGE) on a 4%–12% or 10% Bolt Bis–Tris Plus gel (Invitrogen NW04125BOX or NW00105BOX) with 1× NuPAGE MES SDS Running Buffer (Invitrogen, NP0002 at 180V for 40 min). Separated proteins were transferred to methanol-activated PVDF membranes (BioRad, 1620177) at 100 V for 90 min in 1× Nupage Transfer Buffer (Invitrogen NP0006) with 20% (v/v) methanol. Membranes were blocked in 2% (w/v) skim milk in 1× PBS-T (PBS, 0.1% Tween-20, pH7.4) for 1 h at room temperature (RT), and incubated with primary antibodies overnight at 4 °C at 1:500 or 1:1000 dilution (see S3 Table for antibodies used) in Odyssey (Li-COR , 927-60003) plus 1× PBS-T (0.1%) solution mixed at 1:4 ratio.

For siRNA transfections, SKBR3 and SUM149PT cells were plated at 2 × 10⁵ cells per well and MDA-MB-231 cells were plated at 1.5 × 10⁵ cells per well, in 6-well plates in a total of 2 ml standard media. Twenty-four hours after plating, cells were transfected with 10 nM of CASP siRNA, calpain siRNA, cathepsin siRNA, or corresponding Scramble control siRNA (Integrated DNA Technologies for CASP-siRNAs and cathepsin-siRNAs and Santa-Cruz for calpain-siRNAs) using 4 μl of Lipofectamine RNAiMAX (Invitrogen, 13778075) as per manufacturer’s recommendations. siRNAs are listed in S1 Table. 48 h after transfection, cells were subjected to well-fed/starved or untreated/treated conditions with and/or without 50 nM BafA1 treatment for the final 2 h and harvested for western blot analysis as described above. SUM149PT cell lines were transfected with 5 nM of siRNA in experiments used to detect the combined effect with Olaparib. In experiments with CASP2 or CASP8 siRNA, cells were harvested at 72 h post-transfection.

Single guide RNA sequences targeting the human CASP3 and CASP7 genes were designed using Crispor.org [116] and cloned into PX458 (Cas9-GFP vector with AMP resistance, Addgene, plasmid # 48138) or PX459 (Cas9-Puromycin vector with AMP resistance, Addgene, plasmid # 62988) backbone vector following the protocol provided by Addgene. For gRNA sequences, see S3 Table. Plasmids were then transformed into One Shot Stbl3 chemically competent Escherichia coli cells. The resulting CRISPR plasmids were isolated, sequenced and transfected into the SKBR3 or MDA-MB-231 cell line using Lipofectamine 3000 (Invitrogen, L3000-008). After 48 h , GFP-positive individual cells (cells with PX458 vector) were selected by FACS sorting into 96-well plates and cultured to generate monoclonal cell lines. For cells contain CRISPR plasmids in PX459 vector backbone, puromycin-resistant cells were selected by treating with puromycin (Sigma, P9620) at 10 µg/ml concentration and individual clones were obtained by performing serial dilution on the surviving clones. Isogenic knockout clones were validated by western blotting and sequencing.

For CASP3 and BCL2, a clone from Sino Biological (HG10050-CH) or Addgene (N-FLAG-BCL2, 18003) was obtained, respectively, and used directly for transfection. A PARP1 clone (N-Myc-PARP1, HG11040-NM) was obtained from SinoBiological. CASP7 plasmids containing CASP7-WT, CASP7-inactive (CASP7-C186A), CASP7-ΔPro or non-cleavable at the prodomain were kindly gifted by Dr. Salvesen [117]. The site-directed mutagenesis method was employed to modify nucleotides in CASP7 or PARP1 as needed. CASP7-p30, CASP7-p29, and CASP7 calpain cleavage sites mutated (CASP7-CCM) constructs were generated by PCR using CASP7-WT as the template. The primers and restriction enzymes used are listed below. Since calpains are known to recognize the three-dimensional structure rather than the sequence of the targeted site of the substrate protein [67,118], and a single amino acid mutation does not prevent the recognition by calpains [40,119], we sequentially altered amino acid residues to generate CASP7-CCM. Multiple amino acid residues at the 1st and 2nd calpain cleavage sites were changed into ‘A’ until a significant reduction in non-canonical cleavage was obtained (Fig 4C). For the catalytically inactive PARP1 construct (PARP1-CI), three amino acid residues (H862, Y896, and E988) in the catalytic (CAT) domain of PARP1 were sequentially mutated [79]. DNA sequencing was performed to verify the sequence integrity of all the constructs.

Total RNA was isolated and purified using the RNeasy Plus Mini Kit (QIAGEN, 74104) as per manufacturer’s instructions. To remove any contaminating genomic DNA, RNA was treated with DNase 1 (Invitrogen, 18068-015) as per manufacturer’s instructions. The quantification of RNA was performed using a Nanodrop Spectrophotometer. The quantitative RT-qPCR was performed to quantitate selected ATG transcript expression levels using One-Step Plus SYBR Green reagent kit (Applied Biosystems, 4385617) in 15 µl reactions containing 100 ng of total RNA and 0.1 µM of each primer on the 7900 sequence detection system (Applied Biosystems). Manufacturer’s recommended cycling conditions were used. The ATG proteins and the sequences of forward and reverse primers designed using Primer–BLAST [120] are listed in S2 Table. The mRNA expression levels were determined by the Comparative Cycle Threshold method (2^{−[delta][delta]Ct}) normalizing to internal reference gene (βactin. The expression is presented as the fold change relative to the control. RT-qPCR analyses were done in two or three technical replicates and experiments were repeated at least thrice.

The level of DNA damage (single and double strand breaks) was evaluated by the comet assay (single cell-gel electrophoresis) as previously described [121]. Briefly, cells were washed in PBS (2-3×), trypsinised, harvested in cold PBS and the supernatant was removed after spinning for 5 min at 1,200 rpm at 4 °C. Each cell pellet was resuspended in cold PBS and gently mixed with 1% low melting agarose (Invitrogen, 16500-100) solution at a ratio of 1:10 (v/v). A drop from each cell-agarose mixture was immediately placed on a 1% agarose pre-coated glass microscopic slide, covered with a cover slip, and placed flat for 30 min at 4 °C. Next, the coverslips were removed gently, and the slide was immediately placed in 4 °C lysis solution (2.5 M NaCl, 100 mM disodium EDTA, 10 mM Tris base, and 200 mM NaOH) overnight at 4 °C. Next, slide was incubated in alkaline unwinding buffer (pH > 13) (200 mM NaOH, 1 mM EDTA) for 20 min at RT in the dark before being subjected to gel electrophoresis in alkaline buffer (200 mM NaOH, 1 mM EDTA) at ~ 20 V (300 mA) for 30 min at 4 °C. Slides were rinsed by immersing in dH₂O for 5 min (2×), DNA was fixed by immersing in 70% ethanol for 5 min, and dried at 37 °C for 10 min before stained with GelRed (Biotium, 41,003) (1/3,000 dilution) for 30 min at RT in darkness. The slide was rinsed in water, dried, and mounted with slow-fade mounting media. Images were taken using a fluorescent microscope at 10× objective and DNA damage was quantified as the comet tail moment (product of the tail length × the fraction of total DNA in the tail) using CometScore 2.0 software [122]. Approximately, 200 randomly selected nucleoids were scored per each treatment. Experiments were repeated twice.

In silico genetic interaction screening was performed using GRETTA (v0.99.2) [95] on the R statistical software (v4.2.2) [123]. We computed on the cancer DepMap public release version 22Q2 [124] data from FigShare https://figshare.com/articles/dataset/DepMap_22Q2_Public/19700056/2; accessed on 11 August 2022) according to Takemon and Marra (2023). GRETTA was used to identify cell lines with combined low or high CASP3 and CASP7 protein expression. We identified a group of pan-cancer cell lines below the 10th percentile of CASP3 and CASP7 protein expression (16 cell lines; low expressors) and those above the 90th percentile of both CASP3 and CASP7 protein expression (10 cell lines; high expressors) as the control comparator. The normalized (scaled and mean centered) gene and protein expression values for the selected cell lines were extracted from Nusinow and colleagues (2020) [98] using GRETTA for comparison between the low and high expressor groups.

Genetic interactors of combined CASP3 and CASP7 low expression were predicted using GRETTA, which performed pairwise Mann–Whitney U-tests between the high expressors and low expressors cancer cell line groups for all 17,386 genes to obtain p-values. P-values were adjusted for multiple testing using a permutation (10,000 randomized resampling). Candidate genetic interactors of combined low CASP3 and CASP7 expression were called using a threshold of adjusted Mann–Whitney U-test p-value  0.5 in at least one group as previously defined [95]. To highlight candidate lethal interactors, genetic interaction scores computed by GRETTA were used to generate lethal genetic interaction scores by collapsing genetic interaction scores below zero to zero. A higher interaction score indicates an increased likelihood that a gene knockout is lethal in the test group. Rank is based on lethal GI scores.

We used clusterProfiler (v4.6.0) [125] to annotate GO biological processes of genes that were candidate co-essential or genetic interactors (unadjusted p-value 95]. The number of distinct clusters was determined using gap statistics, which calculated the optimal number of clusters (up to 20 clusters) by iteratively bootstrapping 10,000 times.

In each graph, error bars represent standard error of at least n = 3 independent experiments. As indicated in the legends, statistical significance was calculated by Student’s t-tests (for comparisons between two samples) or analysis of variance (ANOVA) with appropriate post-tests for multiple comparisons (Tukey’s, Dunnett’s, or Sidak’s) using GraphPad Prism version 8.4.1 for Windows (GraphPad Software, San Diego, California, USA, www.graphpad.com). Significance (p-values) indicated are relative to the control unless otherwise indicated. P-values less than 0.05% were determined as significant.