Abstract
Targeting nuclear mechanics is emerging as a promising therapeutic strategy for sensitizing cancer cells to immunotherapy. Inhibition of the mechano-sensory kinase ATR leads to mechanical vulnerability of cancer cells, causing nuclear envelope softness and collapse and activation of the cGAS-STING-mediated innate immune response. Finding novel compounds that interfere with the non-canonical role of ATR in controlling nuclear mechanics presents an intriguing therapeutic opportunity. We carried out a multiparametric high-content screen to identify small molecules that affect nuclear envelope shape and to uncover novel players that could either ameliorate or further compromise the nuclear mechanical abnormalities of ATR-defective cells. The screen was performed in HeLa cells genetically depleted for ATR. Candidate hits were also tested in combination with the chemical inhibition of ATR by AZD6738, and their efficacy was further validated in the triple-negative breast cancer cell lines BT549 and HCC1937. We show that those compounds enhancing the abnormal nuclear shape of ATR-defective cells also synergize with AZD6738 to boost the expression of interferon-stimulated genes, highlighting the power of multiparametric screens to identify novel combined therapeutic interventions targeting nuclear mechanics for cancer immunotherapy.
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Introduction
The Nuclear Envelope (NE), surrounded by the double lipid bilayer of nuclear membrane and the underlying filamentous Lamina, is an elastic connector that protects the genome of the cell and contributes to its organization1. The NE is a mechanosensing hub that mediates the transduction of mechanical stimuli generated outside and inside the nucleus2,3. Changes in the morphological and visco-elastic properties of the NE are associated with various pathologies, including cancer, neurodegenerative diseases, laminopathies, cardiovascular diseases, and muscular dystrophies4.
Multiple molecular pathways are involved in the control of nuclear envelope shape and elasticity2, underlining the biological complexity required to preserve its integrity. We previously reported that Ataxia Telangiectasia and Rad3-related protein (ATR) acts as a mechanosensor at the nuclear envelope following mechanical stress and is essential to preserve nuclear integrity and mechanics5,6,7. ATR is an essential PI3-kinase that protects genome integrity from replication stress8. ATR is an essential gene, and homozygous ATR inactivation is embryonically lethal in mice9. ATR is rarely mutated in cancers. Cancer cells exploit the ATR pathway to overcome replication stress and promote cell survival10. Thus, inhibiting the ATR pathway is selectively toxic in tumors with high levels of oncogene induced replication stress11,12. Germ line hypomorphic mutations in the ATR gene cause Seckel syndrome, a genetic disorder characterized by mental retardation, dwarfism, and microcephaly13.
ATR was shown to localize to the nuclear envelope in response to mechanical stress6, and its inhibition leads to softer nuclei and subsequent nuclear collapse during compression and interstitial migration5. Tumor cells are constantly exposed to different degrees of compressive forces that are generated within the tumor microenvironment or during metastatic migration across vascular tissue barriers. Nuclear compression under those circumstances can cause breaks in the nuclear envelope, especially in mechanically fragile nuclei14,15,16. It has been shown that nuclear envelope ruptures, micronuclei (MN) formation, and nuclear collapse due to fragile or weakened nuclei release into the cytosol genomic DNA fragments that are sensed by the Cyclic GMP AMP Synthase (cGAS), thus triggering the immune cell mediated cancer cell surveillance through the activation of the Stimulator of Interferon Genes (STING) pathway17. The pharmacological stimulation of cGAS-STING-mediated inflammation in combination with immunotherapy represents a promising anti-tumor strategy. Pointing in this direction, clinical trials are ongoing where ATR inhibitor AZD6738 (Ceralasertib), that alters the elastic properties of the NE, is combined with durvalumab, an immune-checkpoint response inhibitor (ICI) in triple negative breast cancers in order to sensitize the cancer cells to immunotherapy (ATRiBRAVE NCT05582538 clinical trial).
In the current study, we screened a library of 1512 small molecules, including oncological and non-oncological approved drugs, as well as small molecules at different stages of clinical investigation, with the aim of identifying small molecules or novel drugs that can rescue or augment the NE deformation phenotype caused by ATR depletion. All the 1512 compounds of the library were screened, independently on the molecular targets, with the purpose to identify novel and unanticipated function as NE modulators among small molecules, including drugs already approved for clinical use. For the scope, we have developed an image-based pipeline to perform high throughput analysis of nuclear morphometric parameters, including nuclear size, shape, NE invaginations, and Lamina Fragmentation. 1512 small molecules were screened for their effect on nuclear morphology in ATR-defective cells. While the compounds that rescue the nuclear phenotype could have potential therapeutic applications in the treatment of Seckel syndrome, those molecules that enhance NE fragility could offer novel anti-cancer drugs in combination therapy with ATR inhibitors and immune-checkpoint inhibitors.
Results
A multiparametric screen for the identification of small molecules worsening and/or reverting nuclear envelope morphometric perturbations of ATR defective cells
We conducted a high-content screening to analyze drug-induced nuclear membrane perturbations on HeLa cells depleted of ATR by short hairpin RNA (shATR) (Supplementary Fig. S1). To characterize the nuclear morphology, we developed a custom image analysis pipeline to extract morphometric parameters. The Primary Screening was conducted on shATR HeLa depleted cells using 1512 small molecules, including 950 approved drugs, 421 small molecules at different stages of clinical testing, and 141 targeted compounds not yet tested in humans. The library was assembled from different collections of Selleck Chemicals, Sigma, and MicroSource. The screening was performed using 384 multi-well plates, and shATR HeLa cells were treated with the compounds for 24 h at three different doses (10–1–0.1 μM). We conducted the primary screening using shATR HeLa cells since genetic depletion of ATR in HeLa cells has been shown to cause NE invaginations and nuclear softening5. This approach allows us to focus on compounds that specifically interfere with ATR-mediated NE integrity. shCTRL and shATR HeLa cells treated with DMSO were used as references for basal and ATR perturbed NE shape in each assay plate. Following 24 h of compound treatment, cells were fixed, permeabilized, and immunostained for nuclear Lamin A/C and DAPI. The acquired images were analyzed with an in-house developed script that, in addition to nuclei count, estimated different nuclear morphometric parameters (see “Materials and methods” and Fig. 1). Following quality control (QC) of data on all assay plates, intra- and inter-plate variability of every single parameter was reduced by data normalization and robust z-score transformations (see “Materials and methods” and Supplementary Figs. S2 and S3). The z-scores of three main nuclear morphometric parameters (roundness, Lamina Fragmentation, and percent of nuclei with invaginations) were used for hit identification, while all treatments causing a drastic reduction of nuclei (z-score < − 3, i.e., corresponding to around 35% nuclei reduction with respect to DMSO treated cells) were excluded from the analysis.
Overview of the nuclear morphology image analysis used in the High Content Screening. Representative images of shCTRL and shATR HeLa cells growing in control conditions (DMSO treated) are shown on the left side. Blue: DAPI, Green: Lamin A/C. Nuclei count, roundness and the percent of nuclei with invaginations were estimated by analyzing the DAPI staining. The Lamina Fragmentation was estimated by segmenting the nuclear membrane on the Lamin A/C (FITC staining). The segmentation result on the DAPI channel is shown in the middle panels. NE invaginations are marked in red and the X signs point out nuclei with invaginations. The Lamina segmentation obtained from the FITC channel is shown in black on the right panels. Descriptor vectors of representative images expressed as (nuclei number, roundness, Lamina Fragmentation, percentage of nuclei with invaginations) are for shCTRL (36, 0.97, 1.06, 2.7%) and for shATR (24, 0.94, 1.32, 50%). Scale bar 20 μm.
We identified two classes of compounds defined as ‘worsening’ or ‘reverting’ corresponding to the small molecules increasing or reducing the nuclear morphometric perturbations caused by ATR down-regulation in HeLa cells, respectively.
Compounds were considered potential hits based on the following criteria: (1) they significantly perturbed the NE by altering at least two of the three morphometric descriptors, (2) they affected the Lamina Fragmentation, (3) they significantly increase/decrease the percent of nuclei with invaginations. For the ‘worsening’ compounds, a median threshold z-score of ± 3 was applied to identify potential candidates, while a median threshold z-score of ± 2 was considered to identify potential reverting hit compounds (Fig. 2). Following these criteria, during the primary screening, 114 ‘worsening’ compounds and 35 ‘reverting’ compounds were identified (Supplementary Table 1 and Supplementary HCS_ScreeningData). To validate the results obtained during the primary screening, the selected compounds were retested at 3 doses (10–1–0.1 μM), in triplicate, on shATR HeLa cells. Applying the same criteria as the primary screening, a hit confirmation rate of 50% overall was obtained (46% for ‘worsening’ compounds and 66% for ‘reverting’ compounds; Supplementary HCS_ScreeningData). The potential hits were tested simultaneously on shCTRL HeLa cells to verify whether their effect on nuclear membrane perturbation was specific to ATR defective cells. The compounds showing a significant preferential effect on shATR Hela cells rather than on their respective control cells, on any of the three main morphometric parameters, were identified (see “Materials and methods” and Supplementary Fig. S4).
Results of the primary high content screening. Distribution of the z-scores for (a) the percentage of nuclei with invaginations, (b) Lamina Fragmentation and (c) roundness parameters. 18 Assay Plates are represented in the figure. Each compound was tested at three doses (10–1–0.1 μM). Each single dose is distributed in plate A, B and C respectively. Z-scores are calculated for each sample (light gray dots) after normalization on shATR HeLa treated with DMSO (blue dots). shCTRL HeLa cells treated with DMSO are depicted in red. Selected hits (Active-UP/DOWN) are highlighted with green stars according to cut-off value.
The comparative analysis of the effects induced by the small molecules on shCTRL and shATR HeLa cells, as well as additional visual inspection of the images, and multiple biological replicates, helped to identify 12 candidates, including small molecules increasing and/or reducing perturbation of the nuclear morphometric parameters (Table 1).
Representative images of the effects determined by ‘worsening’ and ‘reverting’ compounds on both shATR and shCTRL HeLa cells are reported in Supplementary Fig. S5. Due to the relevance of micronuclei formation in tumor progression and their relation to NE abnormalities and DNA damage, we added the quantification of micronuclei formation to the analysis of the selected small molecules (Supplementary Figs. S6 and S7). Sample images of nuclei with micronuclei and their segmentation in the image analysis are reported in Supplementary Fig. S8.
To further validate the preferential activity of the ‘worsening’ compounds on ATR-defective cells, the 8 ‘worsening’ small molecules were also tested in shATM HeLa cells. ATM, Ataxia-telangiectasia Mutated, is the second major player in the DNA damage response pathway, primarily involved in coordinating repair of DNA double-strand breaks18.
ATM belongs to the family of phosphoinositide 3-kinase (PI3K)-related kinases and shares structural similarities with ATR19,20. Previous work from our laboratory has shown that ATM and ATR play distinct roles in mechanotransduction5,21. While ATR deficiency leads to nuclear softness and increased NE invaginations, the absence of ATM induces hyper-stiffness in tumor cells by altering chromatin and cytoskeletal functions. Notably, ATM-defective tumor cells do not exhibit alterations in nuclear shape. The results of this analysis show that shATM HeLa cells are even less sensitive than shControl HeLa cells to the drugs that preferentially perturb the nuclear morphology in shATR cells (Table 1 and Supplementary HCS ScreeningData). Only the ROCK kinase inhibitor Y-39983 HCl and Abemaciclib induced NE shape alterations in shATM HeLa cells to a similar extent as in shATR HeLa cells; however, these molecules were causing a preferential worsening effect on shControl HeLa.
We conclude that three out of the eight ‘worsening’ compounds preferentially affect the NE morphology of ATR-defective cells (Table 1), suggesting that these molecules specifically interfere with molecular pathways critical for maintaining nuclear envelope stability in the absence of ATR activity.
Overview of compounds affecting ATR-mediated NE integrity and their molecular targets involved in DNA repair, inflammation, membrane metabolism, cytoskeleton, and protein translation
ATR-NE defects ‘Worsening’ compounds
Among the selected ‘worsening’ compounds, we found: small molecules that interfere with DNA repair, such as Olaparib, the first approved Parp inhibitor, and the potent and selective Chk1 inhibitor (PF-477736); four approved drugs, such as Mometasone Furoate, Abemaciclib, Dasatinib, and Ruxolitinib, as well as the Rock inhibitor (Y-39983) and the LXR agonist (GW3965).
Among the identified hits, Olaparib was one of the eight PARP inhibitors tested during the primary screening. Both Olaparib and AZD2461, were identified as hits based on our selection criteria. We then focused on Olaparib over AZD2461, primarily because Olaparib is a clinically approved drug for the treatment of BRCA-mutated ovarian cancer and other cancers22,23,24. Mometasone is a corticosteroid with anti-inflammatory activity. Besides Mometasone Furoate, additional drugs targeting the glucocorticoid receptor, such as Ciclesonide and Medrysone, were also identified as hit, being able to induce nuclear membrane perturbation, but they were not further characterized as showing an effect only on Lamina Fragmentation. Moreover, Mometasone showed a specificity towards HeLa cells in which ATR was depleted. Notably, Mometasone was recently identified as a novel Farnesoid X Receptor (FXR) ligand25.
Abemaciclib is an approved CDK4/6 inhibitor, showing also activity on kinases involved in transcription regulation such as DYRK1A and HIP26. Among CDK4/6 inhibitors27, three different drugs were tested during the screening, and both Abemaciclib and Palbociclib were identified as ‘worsening’ compounds, while Ribociclib did not show any effect on nuclear morphometric parameters. We then focused on Abemaciclib since it showed a stronger worsening effect on the percentage of invaginated nuclei.
Dasatinib28, a multityrosine kinase inhibitor, was identified as a hit during the primary screening. Beside Dasatinib, other approved multityrosine kinase inhibitors like Nilotinib and Bosutinib29,30 were identified as hits. However, unlike Dasatinib, these compounds were not further characterized, as their effects were observed only at the highest dose of 10 μM.
Ruxolitinib is a JAK inhibitor reported to bind to lipid membranes, causing an increased disorder of the lipid chains31,32. Ruxolitinb is the only Jak inhibitor identified as a hit, among other JAKi tested, suggesting that its role as a nuclear envelope remodeler could be related to eventual off-targets of the drug. Furthermore, the Rock inhibitor (Y-39983) was identified among the worsening compounds, which is expected considering the documented role of Rho-ROCK inhibition in nuclear envelope invaginations33, as well as the existence of deep cytoskeleton-NE interconnections, such as the perinuclear actin cap, that regulate nuclear shape34,35. Finally, the role that LXR plays in regulating glycosphingolipid synthesis could justify the identification of an LXR agonist (GW3965) among the worsening compounds.
ATR-NE defects ‘Reverting’ compounds
Among the ‘Reverting’ compounds, we identified the two approved drugs, Everolimus and Bexarotene. Everolimus is an mTOR inhibitor approved for the treatment of different cancers36, while Bexarotene is a synthetic retinoid approved by the FDA for the treatment of cutaneous T cell lymphoma37.
These data align with the documented positive effect of all-trans retinoic acid (ATRA) on the recovery of nuclear shape dysfunctions in Hutchinson-Gilford Progeria Syndrome (HGPS) fibroblasts38 and the rescue of multiple cellular defects in laminopathy patients’ fibroblasts by Everolimus39,40. Other retinoid receptor agonists were tested during the primary screening: Fenritinide was not identified as hit, while Etretinate was not subsequently confirmed.
The ‘worsening’ compounds further compromise the mechanical properties of ATR defective cells
ATR deficiency causes nuclear envelope invaginations and nuclear softness, leading to compromised nuclear mechanical properties and cell death following mechanical stress5. Moreover, ATR downregulation induces cytoplasmic de-localization of YAP, a key mechano-sensing transcriptional activator5,41. Nuclear membrane deformations are frequently found in cancer and have functional implications for tumor cells, leading to micronuclei formation, altered cell mechanics, and nuclear envelope rupture in conditions of mechanical stress5,16,42. Based on this assumption, we investigated the functional consequences of the worsening compounds on the mechanical properties of ATR-deficient cells. We focused on the approved drugs that preferentially affected the nuclear morphology in shATR HeLa cells: Mometasone, Dasatinib, and Olaparib. Using Atomic Force Microscopy (AFM), we measured the cell elasticity of HeLa shATR cells treated for 24 h with or without the ‘worsening’ compounds at the indicated doses, which corresponded to the doses that influenced nuclear morphology without causing a significant reduction in the number of nuclei.
Interestingly, Olaparib, as well as Mometasone and Dasatinib decreased the stiffness of shATR HeLa cells, indicating that the increased NE defects have functional consequences for the mechanical properties of the cells (Fig. 3a).
Analysis of the cellular mechanical properties of HeLa shATR cells treated with ‘worsening’ compounds. (a) Elastic modulus measurements using AFM. Cellular stiffness was measured on shATR HeLa cells treated for 24 h with DMSO or the indicated compounds: Olaparib and Mometasone were tested at 10 μM, Dasatinib at 1 μM. At least 50 cells were detected in three independent experiments. (b) Quantification of nuclear to cytoplasmic YAP signal ratio. shATR HeLa cells were treated with the indicated compounds at two doses for 24 h, then fixed and stained for YAP. The ratio of nuclear and cytoplasmic intensity signals was calculated after background subtraction. Data is reported as mean ± SD of three independent experiments with at least 3 technical replicates for the treated samples and at least 12 technical replicates for the controls in each experiment. **p ≤ 0.01, ****p ≤ 0.0001 by ordinary one-way ANOVA test using Bonferroni’s correction for multiple comparisons. (c) Immunofluorescence images showing YAP cellular distribution under the indicated conditions. Scale bar 20 μm.
In addition, we monitored YAP1 (Yes-associated protein 1) subcellular localization by immunofluorescence as an alternative readout of cell mechanics in HeLa shATR cells treated with the above indicated compounds. YAP1 acts as a mechanosensor by responding to mechanical cues such as tissue stiffness and cell shape, thereby influencing cell proliferation, differentiation, and survival41. ATR depletion causes accumulation of YAP in the cytoplasm5, and interestingly, we found that all the tested drugs further increased the fraction of cytoplasmic YAP in shATR HeLa cells (Fig. 3b,c), in line with our AFM measurements. Altogether, these results suggest that the worsening effect of the compounds in the nuclear envelope morphology has functional consequences in terms of YAP localization and nuclear mechanics.
The worsening compounds compromise the nuclear envelope stability of tumor cells treated with the ATRi AZD6738
Moving forward, we explored whether the worsening compounds could induce similar nuclear deformations when combined with chemical inhibition of ATR kinase activity instead of gene downregulation.
Therefore, the eight ‘worsening’ compounds were tested on HeLa cells both as a single agent or in combination with the well-known and characterized ATR inhibitor AZD6738, currently subject of clinical trial (ATRiBRAVE NCT05582538)43.
At first, we determined the impact of AZD6738 on the nuclear morphology of HeLa cells. Interestingly, treatment with AZD6738 for 48 h increases the number of nuclear invaginations and micronuclei in HeLa cells (Fig. 4a,b). We then tested the selected compounds in combination with ATRi AZD6738. Hela cells were first treated with 2 μM AZD6738 for 24 h, followed by an additional treatment of 24 h with DMSO or the selected small molecules. The effect of the small molecules administered as a single agent was analyzed in parallel (Fig. 4a,b). At the end of the treatment, HeLa cells were fixed, stained for Lamin A/C and DAPI, and analyzed as previously reported. We found that all the tested molecules but Mometasone were able to impact on the nuclear envelope shape of the HeLa cells when given as a single agent (Fig. 4a,b). Noticeably, the combined treatment of AZD6738 with Olaparib and PF-477736 (a CHK1 inhibitor) resulted in a significantly synergistic effect (as evaluated by Highest Single-Agent model, HSA), compared to the single treatment, increasing both the percentage of invaginated nuclei and micronuclei formation. These results were further supported by an increased Lamina Fragmentation and a decreased roundness of the nuclei subjected to the combined treatments (Supplementary Fig. S9).
Effects of the ‘worsening’ compounds combined with AZD6738 on NE invaginations and MN in HeLa cells. HeLa cells were treated with AZD6738 2 μM. 24 h later, the selected compounds were added as single agents (pink bars) or in combination with AZD6738 (red bars) for further 24 h. All the compounds were used at 10uM with the exception of Dasatinib and PF-477736 that were tested at the dose of 1 μM. Doses were selected considering those that impact nuclear morphology without inducing a reduction in the number of nuclei by more than 35%. The effect of the compounds on NE invaginations (a) or micronuclei (b) is shown in the bar graphs. Data are reported as mean ± SD of three biological replicates with at least 3 technical replicates for the treated samples and at least 9 technical replicates for the DMSO conditions. Statistical analysis by ordinary one-way ANOVA test using Bonferroni’s correction for multiple comparisons was applied, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Moreover, the combination of AZD6738 with Ruxolitinib and Abemaciclib showed a significant increased effect with respect to the single agent treatment (as evaluated by Highest Single-Agent model, HSA) considering the number of micronuclei (Fig. 4b).
Conversely, it was not possible to appreciate an increased effect on any of the nuclear morphometric parameters by the combination of the other compounds.
Importantly, whether the effect on the nuclear envelope is caused by long-term ATR depletion or short-term ATR kinase inhibition, most of the compounds produced consistent results when combined with AZD6738 instead of being tested on HeLa ATR depleted cells. In addition, to rule out the possibility that the combined treatment could induce significant toxicity in normal human cells, the effect of the worsening compounds, both as single agents and in combination with ATRi, was characterized in the human primary fibroblast cell line (IMR90).
Regarding nuclear morphometric parameters, IMR90 fibroblasts exhibited reduced sensitivity to AZD6738 compared to HeLa tumor cells, with the exception of the percentage of micronuclei (Supplementary Fig. S10a, Fig. S9a,b, Fig. 4a,b). We observed an increase in micronuclei induced by AZD6738, an increase in the percentage of nuclei with invagination caused by the ROCK inhibitor, and an increase in lamina fragmentation induced by Dasatinib, Ruxolitinib, Abemaciclib, GW3695, and the ROCK inhibitor. Interestingly, there were no significant changes with the combined treatments, unlike what was observed in HeLa cells. Moreover, the possible cytotoxic effects of the treatments were assessed using the ATP bioluminescent assay.
We observed only a slight reduction in cell viability (less than 30% compared to DMSO-treated cells) following treatment with the ATR inhibitor, which alignswith the essential role of ATR kinase activity. Importantly, the combined treatments did not lead to a reduction in cell viability greater than 35%, with the exception of the ROCK inhibitor (Supplementary Fig. S10b).
Validation of the selected hits as single agents and in combination with AZD6738 in different cancer cell lines
We performed additional experiments with the selected eight small molecules using different cancer cell lines in order to rule out a possible HeLa cell-type specific action of the compounds.
We chose Triple Negative Breast Cancer (TNBC) cell lines as an alternative cellular model considering that several ATR inhibitors, including AZD6738, are now undergoing phase II clinical trials in the treatment of this type of cancer43.
We screened two TNBC cell lines with a different BRCA1 functional status: HCC1937 (BRCA1 defective) and BT549 (BRCA1 functional).
At first, we checked the effect of the AZD6738 and the other eight small molecules selected so far on nuclear morphometric parameters as single agents (Fig. 5a,b). Notably, all the tested molecules showed an effect on the nuclear morphometric parameters of HCC1937 and BT549 cells. HCC1937 showed the highest sensitivity towards the compounds compared to BT549, considering all the analyzed parameters (Fig. 5a).
Effect of the 8 ‘worsening’ compounds on the nuclear morphometric parameters of human TNBC cell lines. Each molecule was tested at two doses for 24 h on HCC1937 cells (a) or BT549 cells (b). Doses were selected considering those that did not reduce the count of nuclei by more than 35% for the respective cell line. Data are reported as mean ± SD of two independent experiments with at least 3 technical replicates for the treated samples and at least 6 technical replicates for the DMSO conditions in each experiment. Statistical analysis by ordinary one-way ANOVA test using Bonferroni’s correction for multiple comparisons was applied, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Indeed, all the tested molecules led to an increase of the percent of nuclei with invaginations in HCC1937 cells. Conversely, BT549 cells showed a milder increase in the percent of nuclei with invaginations, which was limited to some compounds: AZD6738, Mometasone, Ruxolitinib, and, to a lesser extent, the Rock inhibitor Y39983 and the LXR agonist GW3965 (5b). The higher basal level of invaginated nuclei of BT549 cells compared to HCC1937 could justify the observed differences among the two cell lines. Conversely, the majority of the tested compounds induced significant effects on Lamina Fragmentation both in HCC1937 and BT549 cells. Moreover, most of the compounds, with the exception of Dasatinib and GW3965, increased micronuclei formation in HCC1937 cells, while in BT549 cells, this effect was restricted to PF-477736, Ruxolitinib, and Abemaciclib.
We then tested the combination of AZD6738 with the worsening compounds in HCC1937 cells since the very high basal level of invaginated nuclei in the BT549 cells could mask the results. The small molecules were tested both as single agents and in combination with an ATR inhibitor, as previously done for HeLa cells (Fig. 6). Of note, the dose of Dasatinib was reduced due to the very high sensitivity of HCC1937 cells to the drug in terms of cell viability. All the tested molecules but Mometasone, Y39983, and Abemaciclib, once combined with AZD6738, showed an increased effect on the number of invaginated nuclei compared to the single-agent treatment. The CHK1 inhibitor also increased the number of micronuclei in combination with the ATR inhibitor (Fig. 6). Moreover, all the tested molecules but Y39983 caused an increased reduction of nuclear roundness when combined with AZD6738, while no increased effect of the combined treatment was observed on the Lamina Fragmentation (Supplementary Fig. S11).
Effects of combined treatments with AZD6738 on NE invaginations and MN in the TNBC cell line HCC1937. Combined treatments are reported in red, while single treatments are in pink. Data are reported as mean ± SD of three independent experiments with at least 3 technical replicates for the treated samples and at least 9 technical replicates for the DMSO conditions in each experiment. All the compounds were tested at 10 μM, except PF-477736 and Dasatinib tested at 1 μM and 0.1 μM respectively. AZD6738 was tested at 3 μM. Doses were selected considering those that impact nuclear morphology without inducing a reduction in the nuclei count greater than 35%. The effect of the compounds on NE invaginations (a) or micronuclei (b) is shown in the bar graphs. Statistical analysis by ordinary one-way ANOVA test using Bonferroni’s correction for multiple comparisons was applied, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Compounds targeting nuclear mechanics in ATRi-treated cells further stimulate a cGAS-STING inflammatory response
NE deformations and remodeling lead to alterations in nuclear mechanics and promote nuclear membrane fragility, which can expose nuclear DNA to the cytosol, leading to perinuclear cGAS recruitment and activation of a STING-mediated interferon response3. cGAS-STING activation can be revealed by the increased expression of ISG genes44. Therefore, we investigated whether Mometasone, Dasatinib, Olaparib and PF-477736, which worsen NE defects in ATR-defective cells, also cause a boosting of ISG genes. HCC1937 cells were treated with Mometasone, Dasatinib, Olaparib and PF-477736 both as single agent and in combination with AZD6738, as previously reported. Following treatment with the indicated compounds, we monitored the gene expression levels of a selected panel of Interferon Stimulated Genes (ISG), by qPCR (Fig. 7). AZD6738 and Olaparib led to the induction of several of the tested ISG genes when administered as single agents (Fig. 7). Interestingly, with the exception of Mometasone, the combined treatment of the compounds with ATRi showed an increased effect on the induction of at least some of the ISG tested (Fig. 7). Notably, the induction of ISG genes was monitored following 24 h of combined treatment with AZD6738 and the worsening compounds, to replicate the screening conditions. Likely, extending the combined treatment duration could yield a higher additive effect on ISG induction. These results suggest that the nuclear mechanical perturbation caused by the combined treatment with the ‘worsening’ compounds and ATRi has functional implications for the activation of the cGAS-STING inflammatory pathway.
The ‘worsening’ compounds stimulate a cGAS-STING-mediated inflammatory ISG response in HCC1937 cells when combined with AZD6738. Heat-map graph showing the induction of a selected panel of ISG genes normalized to the DMSO condition in HCC1937 cells treated with ATRi AZD6738 3 μM for 48 h; AZD6738 was administered alone or in combination with the ‘worsening’ compounds (24 h treatment). Mometasone and Olaparib were tested at 10 μM, while PF-477736 and Dasatinib were tested at 1 μM and 0.1 μM respectively. Bar graphs show the ISG induction levels as an average of three independent biological replicates (left panel). Statistical analysis by ordinary one-way ANOVA test using Bonferroni’s correction for multiple was applied, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Discussion
Changes in nuclear structure play a significant role in cancer progression and malignancy45, with tumor cell aberrant nuclear morphology serving as a key diagnostic criterion for tissue pathologists46. Despite this, anti-cancer therapies targeting the nuclear mechanical properties of tumor cells are still missing. In this study, we tested the hypothesis that nuclear irregularities caused by ATR defects could be modulated using small molecule compounds. We performed the first high-throughput screen on HeLa cells genetically depleted for ATR that were treated with a library of 1512 small molecules, and we quantified nuclear irregularities using an image analysis pipeline developed in house. Subsequent confirmation studies conducted in shCTRL and shATR HeLa cells led to the discovery of 8 ‘worsening’ and 4 ‘reverting’ compounds able to modulate the NE shape of HeLa cells in a negative or positive manner. Interestingly, the 8 ‘worsening’ compounds exhibited a similar effect on the nuclear shape of HeLa, HCC1937, and BT549 cells when administered alone or in combination with AZD6738. This data suggests that our primary screening identified molecules whose function as NE modulators is comparatively conserved across various cancer cell lines. Furthermore, a subgroup of compounds exhibited a synergistic effect, as determined by Highest Single-Agent model, when combined with ATRi or ATR genetic depletion, suggesting that their capacity to disrupt the nuclear envelope is specifically enhanced by the lack of ATR kinase activity.
The down-regulation of nuclear lamins42 as well as alterations to chromatin structure47 have been linked to abnormal nuclear shapes in human cancer. Pointing in this direction, a small molecule screen using 145 compounds against chromatin regulators identified epigenetic modulators, mainly histone deacetylase inhibitors, that could revert the NE abnormalities caused by TP53 or MTA2 depletion48. Some of those drugs were also identified as hits during our screening, but their cytotoxicity on HeLa cells led us to exclude them during the hit identification process.
Moreover, two different chemical screens investigated compounds able to revert the nuclear abnormalities caused by Lamin A dysfunction in Hutchinson-Gilford Progeria Syndrome (HGPS) model systems identifying retinoids and monoaminopyrimidines as promising candidates49,50.
Nevertheless, recent genetic screens on NE shape across various cell types indicate the involvement of additional nuclear and non-nuclear pathways in regulating nuclear morphology51,52. Based on this, screening small molecule libraries to modulate the nuclear envelope shape can lead to the discovery of a broader spectrum of targets beyond those of Lamins and epigenetic regulators. Our high content screen identified 12 small molecules that could ameliorate or compromise NE abnormalities of HeLa tumor cells. Interestingly, the identified compounds targeted a variety of molecular pathways, ranging from DNA repair, cytoskeleton, lipid metabolism, cell cycle, and inflammation. Likely, additional molecules could be identified by testing the entire library of compounds across multiple cancer cell lines, since we expect the occurrence of cell-type specific effects.
Among the worsening compounds, Mometasone, Dasatinib and Olaparib showed a preferential effect on ATR-deficient HeLa cells. In addition, Dasatinib, Olaparib and PF-477736 showed a synergistic effect in cells treated with AZD6738, including HCC1937 cell line. These molecules have a potential clinical application as anti-tumor therapy, being particularly promising in combination with ATRi. Moreover, Mometasone, Dasatinib, and Olaparib were shown to soften the nuclear mechanical properties of shATR HeLa cells by AFM, while Dasatinib, Olaparib and PF-477736 boosted an ISG response in HCC1937 when administered with ATRi. Considering the relevance of nuclear mechanics in promoting cell survival during interstitial migration as well as preventing NE ruptures and cGAS-STING recruitment, we speculate that single or combined treatments based on Mometasone, or Dasatinib, or Olaparib, or PF-477736 with AZD6738 and ICI should be explored as potential anti-cancer immunotherapy.
Mometasone furoate is a nanomolar glucocorticosteroid with antipruritic, anti-inflammatory, and vasoconstrictive properties, currently indicated to treat asthma, rhinitis, and inflammatory dermatoses53. Glucocorticoids have been used in oncology, with Mometasone inhibiting the growth of certain cancers54,55. However, no clinical trials have been conducted using mometasone as an anti-tumor therapy. Notably, the relevance of the glucocorticoids into remodeling nuclear envelope structure and permeability56 well suits our observations that these compounds can exert significant NE alterations in cancer cells.
Dasatinib, a multityrosine kinase inhibitor, is approved for treating chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. Dasatinib’s primary molecular targets include BCR-ABL, the SRC family (SRC, LCK, YES, and FYN), cKIT, PDGFRb, and major immune system regulators like TEC and BTK57. Considering a potential immunomodulatory role for Dasatinib58, it is noteworthy that SRC, a primary target of Dasatinib, has been shown to inhibit antitumor immune responses by directly phosphorylating cGAS59. Moreover, Dasatinib was found to modulate antitumor immune responses through an increase of T helper 1 and CD8+ T cells and a decrease of Treg cells60. Dasatinib was identified as a hit in our screen for its negative modulation of NE stability and nuclear mechanics, suggesting it might enhance anti-tumor inflammatory responses by causing NE ruptures. Interestingly, Dasatinib was also shown to decrease the stiffness of isolated nuclei61, in line with our findings.
Olaparib was the first clinically approved PARP inhibitor for treating BRCA-mutated ovarian cancer and other cancers22,23,24. Of note, it was shown that Olaparib triggers a Sting-dependent antitumor immunity in BRCA deficient models, via stimulating the activation, migration, and function of immune cells62,63. Moreover, an increased number of cGAS-positive micronuclei has been observed in BRCA2-deficient cancer cells following the combined treatment with Olaparib and ATRi VE82164.
PF-477736, a Chk1 inhibitor65, has shown efficacy in enhancing the activity of various chemotherapeutic agents and demonstrated antitumor activity across a broad spectrum of p53-deficient human cancer cells and preclinical models, without increasing side effects65,65,66,68.
The identification of PARP1 and CHK1 inhibitors among the worsening compounds is expected, to some extent, considering the role of their targets in DNA damage repair and, particularly, their functional interaction with ATR. Chk1 is a direct target of ATR following replication stress69, and its inhibition is supposed to resemble at least some of the phenotypes caused by ATR deficiency. However, we observed an additive effect with the combination of AZD6738 and PF-477736, which maybe attributed to residual ATR kinase activity in ATRi-treated cells. Alternatively, this effect could stem from a non-canonical role of CHK1 in maintaining NE stability.
PARP1 is activated by DNA damage and promotes PARylation of histones and the recruitment of DNA repair-related proteins70.
While no data are available regarding the combination of PF-477736 with ATRi, several studies have explored the combination of PARP and ATR inhibitors as a therapeutic strategy in different types of cancer, including the ATRi AZD673871,71,72,73,74,76. Our results further strengthen the evidence of a synergistic interaction between ATRi and Olaparib, highlighting their additional activity driving nuclear mechanical defects in tumor cells, and indicate that PF-477736 could achieve a similar effect. Possibly the nuclear mechanical defects generated by these drugs underlie their ability to trigger cGAS-STING activation. Interestingly, both PARP1 and CHK1 inhibitors were shown to trigger Sting-dependent antitumor immunity as single agents62,63,77,77,79.
Eventually, our high-throughput imaging screen uncovered novel properties as NE remodelers, for the drugs Mometasone and Dasatinib. These two FDA-approved small molecules induce nuclear softening in HeLa cells. In addition, Dasatinib exacerbates the NE morphological abnormalities seen in HCC1937 treated with AZD6738. Dasatinib treatment could also induce an ISG response in HCC1937 treated with ATRi, contrary to Mometasone, possibly due to the inhibitory effect of Mometasone on shp2, a negative regulator of PARP1 activity that limits cGAS-STING activation following DNA damage55,80. Yet, Mometasone treatment increased the nuclear envelope abnormalities as single agent in TNBC cells, suggesting that this molecule has the potential to increase the mechanical vulnerability of cancer cell nuclei, independently on the activation of a cGAS-STING-mediated ISG response.
Interestingly, these compounds target different molecular pathways and have never been tested in combination with ATRi as anti-tumor therapy in preclinical in vitro models. While further in vivo studies are needed to evaluate the toxicity of the combined treatment of AZD6738 with the ‘worsening’ compounds, our observations using IMR90 fibroblasts suggest that non-transformed human cells might be less sensitive to the nuclear envelope perturbations induced by these treatments.
Our results strongly suggest that the anti-tumor effects of Mometasone and Dasatinib administered with or without AZD6738 should be further studied, alone or in combination with ICI.
Despite we focused our attention on the ‘worsening compounds’, driven by their potential application as cancer therapy, our primary screen identified 4 compounds that were able to rescue the nuclear envelope phenotype of ATR defective cells. Everolimus, an mTOR specific inhibitor, significantly improved all the morphometric parameters of cells lacking ATR. The mTOR inhibitors Rapamycin and Everolimus were found to rescue the nuclear shape defects of HGPS cells and other laminopathies38,39,40,81. The proposed mechanism for these improvements is that mTOR inhibition stimulates the autophagy of the mutant progerin protein or other forms of abnormal lamin A proteins, thus promoting the removal of toxic insoluble aggregates deriving from abnormal lamin proteins. More recently, Everolimus showed additional benefits in HGPS tissue engineered blood vessels when used in combination with Lonafarnib, a farnesyltransferase inhibitor (FTI) that inhibits post-translational farnesylation of progerin, reducing its intercalation into the nuclear envelope82.
Our screening data suggest that Seckel syndrome patients might benefit, at least partially, from treatment with Everolimus, highlighting phenotypic similarities with laminopathy patients, possibly due to shared alterations in molecular pathways.
Additional research using in vitro and in vivo models of Seckel syndrome will be required to determine whether the ‘reverting’ compounds, particularly Everolimus, have the potential to ameliorate at least some of the clinical traits of individuals affected by Seckel syndrome.
This study provides proof of principle that high-throughput screening for small molecules capable of perturbing nuclear envelope morphology can identify drugs that induce nuclear collapse, activate the cGAS-STING pathway, and ultimately lead to cell death in tumor cells, particularly those whose nuclear mechanics have been weakened by ATR inhibitor treatment.
Nuclear mechanics impact several aspects of tumorigenesis, such as interstitial migration, cell cycle progression, chromosome segregation, and inflammation83,83,84,85,87. Exploring the effects of these compounds on cellular metastasis and the link between nuclear shape defects and migration is particularly intriguing, especially given previous chemical screening studies that have indicated a strong correlation between nuclear size and tumor cell migration ability88.
Although drug-associated nuclear phenotypes could often be indirect and we did not infer mechanistic causes of drug-induced nuclear shape perturbations, our findings suggest that the readout of nuclear deformation is a relatively good predictor of nuclear softness and vulnerability to NE ruptures. This also provides support to the hypothesis that targeting the nuclear envelope stability of cancers has the potential to boost an anti-tumor immune response by activating the cGAS-STING inflammatory pathway.
Materials and methods
Reagents and antibodies
We used the SIGMA mission lentiviral plasmids shCTRL pLKO.1-puro (SHC001) and shATR (TRCN0000219647) to generate HeLa cells with stable ATR knockdown.
ATRi AZD6738 Ceralasertib (S7693 Selleckchem) was used to inhibit ATR kinase activity.
Primary antibodies used for IF or western blot analysis: Anti-lamin A/C (636) sc-7292 (Santa Cruz), anti-YAP antibody 63.7 sc-101199 (Santa Cruz), anti-Vinculin Sigma-Aldrich (V9131), anti-ATR E1S3S 13934 (Cell Signaling).
Secondary antibodies for IF: Donkey anti-mouse Alexa Fluor 488 ThermoFisher (A-21202).
Secondary antibodies for Western-blot: Goat anti-Mouse IgG (H + L)-HRP Conjugate Bio-Rad (1706516), Goat anti-Rabbit IgG (H + L)-HRP Conjugate Bio-Rad (1706515).
DAPI staining was from Sigma-Aldrich (D9542).
CellTiter-Glo luminescent cell viability assay was used (G9243, Promega) with IMR90 cells.
Cell culture
HeLa cell line was obtained from ATCC and cultured in Minimum Essential Medium (MEM, from Euroclone) supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin/streptomycin (Pen/Strep), 1 mM NaPyruvate and 0.1mM NEAA. HCC1937 cell line was obtained from DSMZ and cultured in RPMI 1640 (EuroClone) supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin/streptomycin (Pen/Strep). 1% glutamine, 10%FBS SA-DMEM high glucose Lonza 12–614 supplemented with 100 U/ml penicillin/streptomycin (Pen/Strep) was used for HEK293T cells. IMR90 cell line was obtained from CORIELL and cultured in MEM supplemented with 15% FBS not heat inactivated, 0.1 mM Non-Essential Amino Acids, 2 mM glutamine and 100 U/ml Pen/Strep. All cell lines were maintained at 37˚C with 5% CO2 and were tested for mycoplasma contamination.
Lentiviral infection
Lentiviral particles were generated by transfection of HEK293T cells with shCTRL (pLKO.1-puro) or shATR (TRCN0000219647)5 plasmids purchased from Sigma and co-transfected with viral packaging plasmids. HeLa cells were then infected twice (24 h each) and 48 h later puromycin (2 μg/ml) was added as a selection marker. Infected cells were maintained under Puromycin selection until the day of cell seeding on 384-PhenoPlates (Perkin Elmer, 6057300) for drug screening or 24 mm round bottom slide glasses for AFM analysis.
Protein extraction and western blot analysis
Cell lysates were prepared in lysis buffer (Tris–HCl pH 8.0 50 mM, MgCl 2 1mM, NaCl 200 mM, CaCl 2 1 mM, Glycerol 10%, NP-40 1%, protease inhibitors (complete inhibitors, Roche), phosphatase inhibitors (phosphatase inhibitor cocktail 2, Sigma or PhosSTOP Merc Life Science). Laemmli buffer was added to the lysates (Tris–HCl pH 6.8 50 mM, SDS 2%, Glycerol 10%, bmercaptoethanol 0.1%, Bromophenol blue 0.0005%) and proteins were denatured at 95˙C for 10 min. Samples were separated by SDS-page electrophoresis using 4–20% CriterionTGX Biorad polyacrylamide precast gels and then transferred on a nitro-cellulose membrane for incubation with primary and secondary antibodies. Finally, proteins were visualized with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) or SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific).
Images were acquired using ChemiDoc (Bio-Rad, Molecular Imager ChemiDoc XRS+) and processed with Imagelab volume tool.
High Content Screening Assay
For the high-throughput screening we used a library of 1512 compounds assembled from different collections of Selleck Chemicals, Sigma, MicroSource. The primary screening was conducted on HeLa shATR infected cells: 1500 cells were seeded into each well of 384-PhenoPlates (Perkin Elmer, 6057300) in 50 μl of complete medium. Cell seeding and all operations of liquid handling were conducted with a Hamilton Microlab STAR Liquid Handling robot (ML-STAR, Hamilton). Briefly, immediately after cell seeding, the 384-well plates were automatically moved into the workstation integrated incubator (Cytomat 2 C-Lin, ThermoScientific) for 24 h. For drug treatment, molecules were initially prepared from 10 mM stocks in 3-points 1:10 serial dilutions in DMSO, further diluted 1:8 in complete medium and finally added (5 μl) to the cells at the final doses of 10–1–0.1 μM. HeLa shATR cells treated with 0.3% DMSO were used as controls. After 24 h of treatment, cells were fixed in 4% paraformaldehyde (PFA; HiMedia TCL119) in PBS for 20 min at room temperature and washed once in PBS. The plates were then stained for LaminA/C or conserved at 4 °C and subsequently stained the day after. All the fixing and staining procedures were performed using the Wellwash Versa plate washer (ThermoFisher Scientific) and the Multidrop Combi reagent dispenser (ThermoFisher Scientific). Fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 15 min, washed once with PBS-0.1% Tween 20 (PBST) and blocked in PBST and 1% BSA for 45 min. Cells were then immuno-stained with primary antibody against Lamin A/C (1:500) in PBST with 1% BSA for 1 h at room temperature, washed three times with PBST in low plate agitation for 1 min and incubated for 1 h at room temperature with Alexa Fluor 488 Donkey anti-mouse (1:400) diluted in PBS 1% BSA. After two washes in PBS in low plate agitation for 1 min each, the cells were stained with DAPI (0.2 μg/ml in PBS) for 20 min at room temperature and washed once with PBS. Images from 17 fields for each well were acquired with a water objective 63×/1.15 NA on Operetta CLS high-content system (PerkinElmer) equipped with an Andor Zyla 5.5 camera. Z-stacks of 4 focal planes with 0.7 μm step size were acquired from each field via Harmony 4.9. The acquired images were transferred to Columbus platform (PerkinElmer, v2.9.1) and viability and nuclear morphology was quantified using custom image analysis pipeline.
For the Confirmation some selected compounds were repurchased from Selleck Chemicals: Mometasone furoate (S1987), Dasatinib (S1021), Olaparib (AZD2281, Ku-0059436) (S1060), PF-477736 (S2904), Ruxolitinib (S1378), Y-39983 Dihydrochloride (S7935), Abemaciclib (S5716), GW3965 HCl (S2630), Everolimus (S1120).
Screening image analysis—viability and nuclear morphometric descriptors estimation
All images were analyzed in Columbus v2.9.1 using a custom in-house developed Acapella script (Perkin Elmer). Prior segmentation, all 3D z-stacks were Maximum Projected to 2D images. Then, nuclei bodies were segmented from the DAPI channel, and their nuclear membrane was segmented from the Lamin A/C channel. The nuclei bodies were then used to estimate cells viability and a series of morphological descriptors (i.e. area, roundness, width to length ratio, invaginations count). At the same time, the nuclear membrane was used to estimate possible perturbations on the external shape of the nuclei (e.g. the Lamina Fragmentation). Single cells morphological descriptors were eventually combined on a mean per well basis and thus associated to every treatment of the individual cell type (shATR and shCTRL HeLa cells).
Apart from cell viability, we individuate three morphometric measurements that better captured the perturbations of the nuclear membrane:
-
a)
Roundness: the ratio between the perimeter of a circle of area of the object and the real perimeter of the object (perfect circular nuclei have roundness 1, otherwise is smaller than 1).
-
b)
Percent of nuclei with Invaginations: the fraction of nuclei that have at least one visible invagination with respect to the entire population of nuclei segmented in each well (between 0 and 100%). A visible invagination was defined as a relevant difference between two nuclear stencils: the nuclear body and its smoothed version (an approximation of the convex hull). The smoothed body was obtained by morphological closing the nuclear body, using as morphological operator a circle of an adaptive size, with diameter equal to the nuclear body radial mean radius. The morphological closing operation was performed on each individual stencil of the nucleus body in order to remove artefacts due to short distance to its neighbors or to the image border. Criteria like area and width to length of the candidate invagination allowed to distinguish and select relevant thorn shaped invaginations from smooth cavities (e.g. from beam shaped nuclei).
-
c)
Lamina Fragmentation: the ratio between the length of the segmented Lamina skeleton (nuclear membrane) and the real perimeter of the nucleus (greater or equal to 1).
In addition to the three morphometric measurements previously described, we used the percentage of nuclei with micronuclei as additional phenotypic descriptor for some of the late experiments. Micronuclei were defined as very small objects identified through the nuclei segmentation procedure on the DAPI channel. We then applied Voronoi tessellation around the identified nuclei to assess whether they contained micronuclei.
Screening statistical data analysis—statistical approaches for hit detection
All the viability and morphometric descriptors estimated for each well and thus associated to individual treatments on the different cell-lines went through a standard processing and analysis pipeline for Hit calling: intra- e inter-plate variability removal by applying plate-wise data normalization, data QC assessment, and screen-wise robust z-score estimation89. The normalization of each of the four parameters was performed plate-wise, using as reference the median value associated to reference/control wells (e.g. shATR-downregulated cells). The viability value (of each treatment) was normalized as ‘percentage fold change’ with respect to the reference wells. Roundness and Lamina Fragmentation values were normalized as ‘difference percent’ with respect to the reference wells (i.e. the value difference between the treated well and the reference was expressed as percentage of the reference value). The percent of nuclei with invaginations was normalized as difference with respect reference wells.
Each of the four dimensions of the description vector (a viability value, and the three morphometric descriptors: roundness, Lamina Fragmentation and percent of nuclei with invaginations) was processed individually up to robust z-score estimation. Prior hit calling, the viability was used to filter out treatments that reduced drastically the number of cells: (z-score < − 3). Ultimately, the hit calling strategy was tailored for the different screening phase. The Primary and the Confirmation Screenings used thresholds of + 3 or − 3 (or + 2 or − 2 for treatments with reverting effects—see “Results” section) on the different morphometric descriptors to select those treatments inducing perturbations on the nuclear membrane.
The Confirmation and the Validation Screenings were conducted by simultaneously treating both shATR-downregulated and shCTRL cells. Therefore, each individual treatment was associated a triple effect: two individual effects on each cell type, and one differential effect obtained by subtracting, after normalization, the effect on shCTRL cells from the effect on shATR-downregulated cells (See Supplementary Fig. S4). Then, each of the resulting nine morphometric descriptors (three times three morphometric descriptors: roundness, Lamina Fragmentation and percent of nuclei with invaginations) was further analyzed as described previously for the Primary Screening, filtering out those treatments having low viability values on any of the two cell types.
Immunofluorescence staining for YAP
All the fixing and staining procedures were performed using the Wellwash Versa plate washer and the Multidrop Combi reagent dispenser. Cells were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature, washed once in PBS and simultaneously blocked and permeabilized with 0.2% Triton X-100, 10% Donkey serum (Jackson ImmunoResearch, 017-000-121) in PBS for 1 h at room temperature. Cells were then immuno-stained with primary antibody against YAP (1:300) in PBS with 10% Donkey serum for 1 h at room temperature, washed three times with PBS in low plate agitation for 1 min and incubated for 1 h at room temperature with Alexa Fluor 488 Donkey anti-mouse (1:400) diluted in PBS 10% Donkey serum. After two washes in PBS in low plate agitation for 1 min each, the cells were stained with DAPI (0.2 μg/ml in PBS) for 20 min at room temperature and washed once with PBS. Images were acquired as described for Lamin A/C staining. We evaluated the ratio of YAP intensity in the nucleus with respect to YAP intensity in the cytoplasm, after performing the maximum projections of the images and correcting for background intensity. The YAP intensity of the nucleus was estimated by averaging the intensities of the Alexa 488 channel within the nucleus body segmented on the DAPI channel. Similarly, the YAP intensity of the cytoplasm was estimated by averaging the intensities of the Alexa 488 channel within a ring-shaped region around the nuclear body. Per object nuclear to cytoplasm YAP ratio were eventually combined from all the different fields on a mean per well basis.
Cell treatments in combination with ATR inhibitor
1000 cells of HeLa shCTRL infected cells, 1500 cells of HCC1937 human cells and 3000 cells of IMR90 cells were seeded into each well of 384-PhenoPlates (Perkin Elmer, 6057300) in 50 μl of their specific complete medium. The day after, the cells were treated with the ATR inhibitor AZD6738 (Selleck Chemicals, S7693) at the dose of 2 μM for HeLa cells and 3 μM for HCC1937 and IMR90 (final DMSO 0.1%). 24 h later, the selected compounds were added as single agents or in combination at the indicated doses (final DMSO 0.3%), for further 24 h. Then cells were fixed and stained for Lamin A/C as indicated for the screening.
Calculation of synergy
We have used Highest Single-Agent (HSA) to estimate synergy. The HSA model predicts the combined effect EAB for two single compounds with the effects EA and EB is EAB = max(EA,EB).
Cell Viability Assay
For the IMR90 cell line, the CellTiter-Glo luminescent cell viability assay was performed according to the manufacturer’s instructions. Briefly, cells were treated with or without the ATR inhibitor AZD6738 and various concentrations of the selected inhibitors. After the treatment, an equal volume of CellTiter-Glo reagent was added. Luminescence was recorded on a Tecan Reader after 20 min of incubation at room temperature.
cDNA preparation and qPCR analysis for ISG gene induction
HCC1937 were seeded at 400,000 cells/well on a 6-well plate in 2ml of cell culture media. The following day cell media was changed with fresh media containing DMSO or ATRi AZD6738 (3 μM). 24 h later cell media was replaced with DMSO, ATRi AZD6738 (3 μM), the small molecules or the combination of AZD6738 (3 μM) and each small molecule (Mometasone (10 μM), Olaparib (10 μM), Dasatinib (0.1 μM), and PF-47736 (1 μM)).
At the end of the treatment, HCC1937 cells were washed twice with ice-cold PBS, RNA was prepared for each sample using the RNAeasy Mini kit (Qiagen, Cat# 74104) according to manufacturer instructions. cDNA was prepared for each sample starting from 1 μg of RNA using the High- Capacity cDNA Reverse Transcription Kit (ThermoFisher, Cat# 4368814) according to manufacturer instructions. cDNA samples were treated with 1 μL RNAse H (Promega, Cat# M4281) for 20 min at 37˙C and stored at − 80 °C. Gene expression analysis was performed by the qPCR-Service at Cogentech-Milano. 5 ng of cDNA was amplified in triplicate in a reaction volume of 10 μL containing the following reagents: 5 μL of TaqMan Fast Advanced Master Mix (ThermoFisher), 0.5 μL of TaqMan Gene expression assay 20× for selected ISG genes and GAPDH and actb as reference genes (ThermoFisher). Real-time PCR was carried out on the QS12k (ThermoFisher), using a pre-PCR step of 20 s at 95 °C, followed by 40 cycles of 1 s at 95 °C and 20 s at 60 °C. Samples were amplified with primers and probes for each target, and for all the targets one NTC sample was run. Raw data (Ct) were analyzed with Biogazelle qbase plus software and the fold change was expressed as Calibrated Normalized Relative Quantity.
AFM measurements
AFM indentation experiments on shATR cells treated with DMSO or small molecules were performed using Nanowizard 3 (JPK instruments, Germany) mounted on an Olympus inverted microscope. A modified silicon nitride cantilever (NovaScan, USA) with a spring constant of 0.03 N/m, functionalized with a 10 μm-diameter borosilicate microsphere, was used for the AFM indentation. 70,000 HeLa cells/well of a 6-well plate were seeded on 24 mm round glass coverslips without fibronectin coating. 24 h post cell seeding DMSO or drug treatments were added to the cells. 48 h post cell seeding the coverslips were mounted on the AFM stage and cells were maintained in the same cell culture medium at a controlled temperature of 37 °C throughout the measurements using a heater (JPK instruments, Germany). Indentation was carried out at the center of the cells, in correspondence of the nuclear region, using a loading rate of 1.5 μm/s. A ramp size of 3 μm and an indentation force of 2 nN were used. For each cell sample, about 50 cells were indented; for each cell, five force curves were recorded. All measurements were performed as previously described90.
The data points up to 0.5 μm from contact point were used to calculate the elastic (Young’s) modulus, by fitting the curves with the Hertz model:
where F is the indentation force, E is the Young’s modulus to be determined, ν is the Poisson’s ratio, R is the radius of the spherical bead, and δ is the indentation depth. The cell was assumed incompressible and a Poisson’s ratio of 0.5 was used.
An analysis of variance (ANOVA), with the Bonferroni post hoc test, was used to test statistical differences in the samples. AFM measurements were repeated three times.
Statistical analysis
Data analysis was performed using Prism9 (GraphPad) software, while screening data were analyzed with Matlab (MathWorks). Data are expressed as mean ± standard deviation (SD) for minimum n = 3 replicates, as indicated in figure legends. Comparisons of mean values were performed with ordinary one-way ANOVA test using Bonferroni’s correction for multiple comparisons, as specified in figure legends; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. In co-treatment experiments, the statistically significant differences were reported with respect to control cells treated with DMSO (CTRL) for the single treatments. Alternatively, for the combined treatments with AZD6738, the statistically significant differences have been only reported if present simultaneously in both comparisons: to AZD6738 and to its single treatment.
Data availability
Further information and requests for resources and reagents should be directed to and will be fulfilled by the corresponding authors.
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Acknowledgements
We thank Gururaj Rao Kidiyoor for useful suggestions, all the members of MF and CM laboratory for useful discussions. In addition, we thank the IFOM Cell Biology Unit and the Cogentech Quantitative PCR unit. We are grateful to Ravelli family for the generous donation supporting Experimental Therapeutics Program activity. Work in MF laboratory is supported by MIUR-PRIN-2015SJLMB9, AIRC-5X1000-22759, and AIRC-IG-21416.
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G.B., M.R.C and D.P. designed, performed the experiments and analyzed the data; M.R., G.F. and M.R.C. performed the screening campaign; A.A. developed pipelines for HCS and analyzed all the screenings; R.C. provided technical support ; M.P., G.M. and M.V. curated, selected and handled the chemical collection; F. A., G.B and Q.L performed AFM experiments; G.B., M.R.C., A.A., D.P., C.M., and M.F. wrote the manuscript. C.M. and M.F. conceived the study and supervised the project; M.F. funding acquisition. All authors reviewed and approved the manuscript.
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Cera, M.R., Bastianello, G., Purushothaman, D. et al. A multiparametric screen uncovers FDA-approved small molecules that potentiate the nuclear mechano-dysfunctions in ATR-defective cells. Sci Rep 14, 30786 (2024). https://doi.org/10.1038/s41598-024-80837-w
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DOI: https://doi.org/10.1038/s41598-024-80837-w
