Many chemotherapeutic drugs produce double-strand breaks (DSB) on cancer cell DNA, thereby inducing cell death. However, the DNA damage response (DDR) enables cancer cells to overcome DNA damage and escape cell death, often leading to therapeutic resistance and unsuccessful outcomes. It is therefore important to develop inhibitors that target DDR proteins to render cancer cells hypersensitive to DNA damage. Here, we investigated the applicability of PFI-3, a recently developed bromodomain inhibitor specifically targeting the SWI/SNF chromatin remodeler that functions to promote DSB repair, in cancer treatment. We verified that PFI-3 effectively blocks chromatin binding of its target bromodomains and dissociates the corresponding SWI/SNF proteins from chromatin. We then found that, while having little toxicity as a single agent, PFI-3 synergistically sensitizes several human cancer cell lines to DNA damage induced by chemotherapeutic drugs such as doxorubicin. This PFI-3 activity occurs only for the cancer cells that require SWI/SNF for DNA repair. Our mechanism studies show that PFI-3 exerts the DNA damage–sensitizing effect by directly blocking SWI/SNF's chromatin binding, which leads to defects in DSB repair and aberrations in damage checkpoints, eventually resulting in increase of cell death primarily via necrosis and senescence. This work therefore demonstrates the activity of PFI-3 to sensitize cancer cells to DNA damage and its mechanism of action via SWI/SNF targeting, providing an experimental rationale for developing PFI-3 as a sensitizing agent in cancer chemotherapy.
This study, revealing the activity of PFI-3 to sensitize cancer cells to chemotherapeutic drugs, provides an experimental rationale for developing this bromodomain inhibitor as a sensitizing agent in cancer chemotherapy.
Chemotherapy and radiotherapy are the most widely used treatment modalities for cancer. Many chemotherapeutic drugs, such as doxorubicin, and ionizing radiation work by generating double-strand breaks (DSB) on cancer cell DNA and thereby inducing cell death. However, the DNA damage response (DDR), a complex network of cellular pathways that sense, signal, and repair DNA lesions, enables tumor cells to overcome DNA damage and thereby escape cell death, which often leads to therapeutic resistance and unsuccessful outcome in cancer treatment. Targeting DDR proteins can be a useful strategy to sensitize tumor cells to DNA damage and thereby increase the efficacy of cancer treatment. Therefore, it is important to discover DDR proteins whose inactivation renders cancer cells hypersensitive to DNA damage and develop inhibitors that specifically target these proteins (1–4).
SWI/SNF chromatin-remodeling complex functions in multiple cellular processes, including DNA repair (5). The mammalian SWI/SNF complexes (denoted SWI/SNF hereafter and also known as BAF) contain either BRG1 or BRM as a catalytic ATPase, which confers the complex with remodeling activity, and at least 10 noncatalytic subunits, including BAF180 (PBRM1), which play regulatory role. SWI/SNF exists in many different forms in the subunit compositions and is classified into two major groups, BAF and PBAF. While BAF contains either BRG1 or BRM as an ATPase, PBAF always contain BRG1 as an ATPase and BAF180 as an associating factor (6, 7). Both BAF and PBAF have direct role in DSB repair. Depletion or inactivation of SWI/SNF subunits, including BRG1 and BRM, reduces DSB repair and sensitizes cells to DSB-inducing agents, and SWI/SNF rapidly localizes at DSB-surrounding chromatin (8–16). We have previously shown that BRG1 lacking bromodomain, an approximately 110 amino acid protein domain that recognizes and binds acetyl-lysine motifs, is unable to stimulate DSB repair and BRG1 bromodomain exerts dominant negative activity against BRG1 to inhibit DSB repair, indicating that SWI/SNF binding to chromatin via bromodomain is important for DSB repair (17, 18). The follow-up work showed that blocking BRG1 chromatin binding by overexpression of BRG1 bromodomain inhibits DSB repair and sensitizes cancer cells to DSB-generating agents, such as doxorubicin and ionizing radiation. This proof-of-concept study verified that BRG1 bromodomain is a target to enhance the efficacy of cancer chemotherapy and radiotherapy (19).
The human genome encodes 61 bromodomains found in 42 proteins and many of these proteins are chromatin regulators, such as chromatin remodeler and histone-modifying enzyme, which function in a wide array of biological processes, including DNA repair (20). Bromodomain are classified into eight families based on similarity of their sequence and structure. Despite of their sequence diversity, all bromodomains share a conserved fold comprising left-handed four helix bundles linked by loops with variable lengths that form a central deep and narrow hydrophobic cavity, which enables this protein domain to recognize the acetyl-lysine motifs in a sequence-dependent manner. This feature of the bromodomain fold has made it possible to develop specific small-molecule inhibitors against many different bromodomains (21–24). The first examples of such inhibitors were JQ1 and I-BET, which target the bromodomain and extraterminal domain (BET) family proteins and exhibited anticancer and antiinflammatory activities (25, 26). Subsequently, a number of small-molecule inhibitors targeting various other bromodomains were developed (27–29). One of those inhibitors is PFI-3. In vitro experiments showed that PFI-3 specifically binds to each bromodomain of BRG1 and BRM and selectively to the fifth bromodomain among the six bromodomains of BAF180, all belonging to the family VIII of bromodomains (30, 31).
In this study, we investigated whether PFI-3 inhibits SWI/SNF and sensitizes cancer cells to DNA damage to test its applicability in cancer treatment. We found that, while exhibiting little cytotoxic activity by itself, PFI-3 synergistically sensitizes several human cancer cells to DNA damage induced by the chemotherapeutic drugs, such as doxorubicin. Our data suggest that PFI-3 exerts this activity by direct blockade of SWI/SNF's chromatin binding, which leads to defective DNA repair and aberrant damage checkpoint, ultimately resulting in increase of cell death primarily via necrosis and senescence.
Materials and Methods
Cells and plasmids
A549, HT29, H460, H1299, and U2OS cells were purchased from ATCC and cultured according to the vendor's instructions. The cell lines were authenticated with DNA fingerprinting using short tandem repeat markers every 50 passages and were tested for the absence of Mycoplasma contamination using the e-Myco VALiD Mycoplasma PCR Detection Kit (Intron Biotechnology). The U2OS stable cells were generated by transfection with the vector-expressing GFP-conjugated dimeric form of BRG1 bromodomain (19) followed by selection with G418 at 800 μg/mL. The vectors expressing GFP-conjugated monomeric BRG1 bromodomain or BRM bromodomain were described previously (32).
Inhibitors and drugs
PFI-3 and BI-9564 was obtained from the Structural Genomics Consortium (https://www.thesgc.org) and Tocris (5072). Doxorubicin and etoposide were purchased from Sigma-Aldrich, and the BRG1/BRM-specific ATPase inhibitor from Chem Scene. The chemicals were dissolved in DMSO, kept in aliquots at −20°C, and thawed immediately before use for experiments.
Cell viability assay
Clonogenic assay was performed as described previously (17). After stained with 0.5% crystal violet, colonies were dissolved in 10% acetic acid before measuring absorbance at 590 nm with a spectraMax i3X plate reader (I3X-SC-ACAD, Molecular Devices). MTS assay was performed using the CellTilter 96 AQueous One Solution Cell Proliferation Assay kit (Promega), with absorbance at 490 nm measured by the above-mentioned plate reader.
Chromatin fractionation assay
Biochemical fractionation was performed as described previously (33). Briefly, cells were incubated in the fractionation buffer [50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 0.5% NP-40, 0.5 mmol/L phenylmethylsulfonylfluoride, 5 μg/mL aprotinin, 5 μg/mL leupeptin, 5 μg/mL pepstatin A, and 10 mmol/L NaF] on ice for 1 hour, and then centrifuged at 16,000 g for 20 minutes to separate supernatant (chromatin-unbound fraction) from pellet (chromatin-bound fraction).
The significance of differences between measurements was evaluated by Student t test in Microsoft Excel. A P value < 0.05 was deemed to indicate statistical significance. Combination index (CI) was calculated according to the Chou-Talalay algorithm with the CompuSyn software and drug interaction was described as reported previously (34).
Additional materials and methods
In situ cell extraction, comet assay, Annexin-V staining, immunoblotting, transfection, RNA sequencing (RNA-seq) analysis, and the sequences of siRNAs and the sources of antibodies were provided in Supplementary Data.
PFI-3 inhibits chromatin binding of its target bromodomains and the corresponding SWI/SNF proteins within cells
To verify the activity of PFI-3 (Fig. 1A) within the cells, we first examined its ability to inhibit chromatin binding of BRG1 bromodomain by in situ cell extraction using U2OS cells stably expressing a dimeric form of BRG1 bromodomain linked to GFP and Myc, which has higher chromatin affinity than a monomeric form (19). After treatment of the cells with PFI-3 along with trichostatin A (TSA, to enhance assay window) for 2 hours, we detergent-extracted unbound bromodomains and determined the GFP intensity on the cells by fluorescence microscopy (Fig. 1B). PFI-3 treatment at 30 and 50 μmol/L reduced the GFP intensity in a dose-dependent manner, whereas the same concentrations of BI-9564, a specific inhibitor against bromodomains of the BRD7 and BRD9 subunits of SWI/SNF (35), showed no effect (Fig. 1C), indicating that PFI-3 inhibited the chromatin binding of BRG1 bromodomain specifically. Then, we confirmed these results by chromatin fractionation. PFI-3 at 30 μmol/L, but not BI-9564, significantly inhibited the chromatin binding of BRG1 bromodomain regardless of TSA treatment (Fig. 1D; Supplementary Fig. S1A and S1B). PFI-3 inhibited the chromatin binding of BRG1 bromodomain as well as BRM bromodomain in a dose-dependent manner (Fig. 1E and F). We also confirmed that PFI-3 inhibited chromatin binding of the fifth bromodomain but not the other five bromodomains of BAF180 (Fig. 1G).
Next, we determined whether PFI-3 dissociates endogenous SWI/SNF proteins from chromatin. Treatment of U2OS cells with PFI-3 at 50 μmol/L for 2 hours dissociated BRG1, BRM, and BAF180 from chromatin regardless of TSA treatment whereas BI-9564 showed no effect (Fig. 1H; Supplementary Fig. S1C). This PFI-3 activity was dose dependent (Fig. 1I). PFI-3 appears to be highly stable within the cells because its activity was still effective after treatment for 24 hours (Supplementary Fig. S1D). We confirmed these results by in situ detergent extraction combined with immunofluorescence microscopy. When the cells were treated with increasing PFI-3 up to 100 μmol/L, the fluorescent signals specific for BRG1 (Fig. 1J) or BRM (Fig. 1K) proportionally decreased with the signals barely detected at 50 μmol/L and higher concentrations, indicating that PFI-3 dissociated BRG1 and BRM from chromatin effectively and in a dose-dependent manner. As a negative control, BI-9564 did not dissociate BRG1 from chromatin even at 50 μmol/L and higher concentrations (Fig. 1J). These results collectively demonstrated that PFI-3 inhibits chromatin binding of its target bromodomains and the corresponding endogenous SWI/SNF proteins at pharmacologically significant concentrations.
PFI-3 sensitizes several human cancer cells to DNA damage
Given that PFI-3 inhibits SWI/SNF, we investigated whether it sensitizes cancer cells to DNA damage. For this study, we chose A549 (lung cancer) and HT29 (colon cancer) as model cells representing not only different cancer types but also distinct SWI/SNF gene mutations; the former is BRG1 deficient and the latter BRG1 proficient while both express BRM and BAF180. First, we confirmed that PFI-3 dissociates SWI/SNF from chromatin in these cancer cells by chromatin fractionation (Supplementary Fig. S2A and S2B) and in situ detergent extraction (Supplementary Fig. S2C and S2D). We then found that, while having little cytotoxic activity by itself, PFI-3 sensitized both A549 and HT29 cells to doxorubicin and etoposide in certain combinations of drug concentrations in the assays to determine short-term viability (Supplementary Fig. S3A and S3B) and long-term clonogenic ability (Fig. 2A and B). Calculation of the CI indicated that the PFI-3 effects on the cancer cells' sensitivity to doxorubicin and etoposide were synergistic (Fig. 2A and B). A similar degree of doxorubicin sensitization was observed for both cells whether a same amount of PFI-3 was treated multiple times with wash (Supplementary Fig. S3C–S3E) or divided into small portions and given several times without wash (Supplementary Fig. S3F–S3H), suggesting that stability was not an issue for the PFI-3 activity. PFI-3 exhibited a synergy with ionizing radiation to kill A549 but this effect was observed only at a very high concentration (120 μmol/L), and this high concentration of PFI-3 had some cytotoxic activity on the cells by itself (Supplementary Fig. S3I). However, PFI-3 at 120 μmol/L showed no combined effect with ionizing radiation on HT29 cells, which possibly could be due to its own cytotoxic activity relatively stronger on this cell type compared with A549 (Supplementary Fig. S3J). Then, we analyzed two more lung cancer cell lines, H460 and H1299, with similar SWI/SNF gene expression as HT29 and A549, respectively (Fig. 2C). PFI-3 exhibited the similar activity on H460 as A549 and HT29 cells; a synergy with doxorubicin but no cytotoxicity by itself (Fig. 2D). Interestingly, however, PFI-3 alone showed significant cytotoxicity on H1299 cells but no combined effect with doxorubicin (Fig. 2E). This difference was attributed to the fact that SWI/SNF is dispensable for DNA repair in H1299 cells (see below). All these results show that PFI-3 sensitizes cancer cells to DNA damage and this activity of PFI-3 is not general but rather depends on both cancer cell type and DNA damaging agent.
A recent study reported that inhibiting ATPase activity is more appropriate than targeting bromodomain for inducing a synthetic lethality of SWI/SNF-mutant cancer cells (36). We therefore compared the two strategies for cancer cell cytotoxicity in combination with doxorubicin. The recently developed dual BRG1 and BRM ATPase inhibitor (ATPasei; ref. 37) had a strong cytotoxic activity by itself on A549 and HT29 cells with the IC50 values of 0.50 and 1.49 μmol/L, respectively (Supplementary Fig. S3K). When treated in combination with doxorubicin, the ATPasei exhibited only an additive effect of cytotoxicity on A549 cells in contrast to PFI-3 showing a synergy as observed before (Fig. 2F). The reason the ATPasei has no synergy with doxorubicin is likely because of its own strong cytotoxic activity. Therefore, while inhibiting the ATPase activity appears to be far more potent in treatment of SWI/SNF mutant cancers, targeting bromodomain could have an advantage of synergy effect in combination treatment with doxorubicin.
PFI-3 sensitizes cancer cells to DNA damage by inhibiting SWI/SNF-driven DNA repair
Next, we investigated whether PFI-3 sensitizes the cancer cells to DNA damage by inhibiting DNA repair. First, we asked whether SWI/SNF functions in DNA repair in the cancer cells. As assessed by neutral comet assay detecting DSBs specifically, siRNA knockdown of either BRM or BRM/BAF180 led to a large defect in DSB repair after doxorubicin treatment in A549 cells (Fig. 3A and B), showing that SWI/SNF is critical for DSB repair in this cell type. Then, we determined the effects of PFI-3 on DNA repair. Notably, while not inducing DSBs by itself, PFI-3 largely inhibited the repair of doxorubicin-induced DSBs that would have otherwise efficiently proceeded with recovery time (Fig. 3C). We confirmed these results by the observations that γ-H2AX, the physiologic DSB indicator (38), after induction by doxorubicin decreased with recovery time under normal conditions but this decrease was attenuated by PFI-3 as assessed by immunoblotting (Fig. 3D) and immunofluorescence microscopy detecting foci formation (Fig. 3E and F). Importantly, depletion of BRM or BRM/BAF180 canceled the doxorubicin-sensitizing activity of PFI-3 (Fig. 3G and H), suggesting that PFI-3 exerts this activity by targeting SWI/SNF.
Then, we analyzed the other three cell lines. siRNA knockdown of BRG1/BRM or BRG1/BRM/BAF180 showed that SWI/SNF is important for the repair of doxorubicin-induced DSBs in HT29 (Supplementary Fig. S4A and S4B) and H460 cells (Supplementary Fig. S4C and S4D). PFI-3 inhibited DSB repair after doxorubicin treatment in both HT29 (Fig. 3I) and H460 cells (Supplementary Fig. S4E). Interestingly, the kinetics of DNA repair in these two cell lines was different from A549 in that unrepaired DSBs decreased shortly after doxorubicin treatment and then continued to increase until the end of the time course analyzed (24 hours), which may be due to the differences in BRG1 expression and/or other genetic factors. Notably, depletion of BRM or BRM/BAF180 did not affect DSB repair after doxorubicin treatment in H1299 cells (Supplementary Fig. S4F and S4G), indicating that SWI/SNF is dispensable for DSB repair in this cell line. Consistently, PFI-3 exhibited no effect on DSB repair after doxorubicin treatment (Supplementary Fig. S4H). These results explain why PFI-3 had no DNA damage–sensitizing activity on H1299 cells. Thus, the reduced viability of H1299 by PFI-3 alone is likely attributed to the SWI/SNF functions that are essential for cell survival but unrelated to DNA repair. Taking all these findings together, we concluded that PFI-3 sensitize cancer cells to DNA damage by inhibiting SWI/SNF-driven DNA repair.
PFI-3 blocks DNA damage–induced SWI/SNF binding to chromatin
Next, we investigated the mechanism by which PFI-3 inhibits the SWI/SNF-driven DNA repair. First, we examined how SWI/SNF responds to DNA damage by determining its chromatin binding various times after doxorubicin treatment. Chromatin fractionation showed that BRM/BAF180 and BRG1/BRM rapidly bound to chromatin after doxorubicin treatment in A549 and HT29 cells, respectively, and this binding increased continuously until the end of the time course analyzed (24 hours; Fig. 4A and B). Therefore, although the repair kinetics are different between A549 and HT29 cells, a continuous chromatin binding of SWI/SNF seems to be important for the proper responses to doxorubicin damage in both cells. Then, we determined whether PFI-3 inhibits the DNA damage–induced SWI/SNF's chromatin binding by chromatin fractionation. Notably, the chromatin binding of BRM/BAF180 in A549 (Fig. 4C; Supplementary Fig. S5A) and BRG1/BRM in HT29 cells (Fig. 4D; Supplementary Fig. S5B) increased after doxorubicin treatment, which was abolished by PFI-3. We confirmed these findings by in situ detergent extraction, which showed that PFI-3 abolished the increase of doxorubicin-induced chromatin binding of BRM in A549 (Fig. 4E) and BRG1/BRM in HT29 cells (Fig. 4F). These results suggest that PFI-3 inhibits the repair of doxorubicin-induced DNA damage by blocking SWI/SNF chromatin binding.
PFI-3 affects DNA damage checkpoint after doxorubicin treatment
Given the DNA repair inhibition by PFI-3, we determined its impacts on the cell-cycle checkpoint by recovering A549 and HT29 cells from doxorubicin damage with or without PFI-3 for various times up to 48 hours (Fig. 5A). While both cells exhibited G2–M arrest after doxorubicin treatment, PFI-3 treatment led to some delay in the establishment of G2–M arrest in A549 cells and a strengthened and persistent G2–M arrest in HT29 cells (Fig. 5B; Supplementary Fig. S6A). Analysis of several checkpoint proteins, such as Chk1/2, Cdk1, and p53, showed that, while doxorubicin treatment even in the presence of PFI-3 activated the damage checkpoint, PFI-3 had significant impacts on the checkpoint activity in both cells but in somewhat different ways. In A549 cells, the activity of Chk1, but not Chk2, was significantly attenuated by PFI-3, as assessed by their activating phosphorylation at Ser-317/345 (39). The inhibitory phosphorylation of Tyr-15 on Cdk1, the master kinase that regulates the transition from G2 to mitosis (40), was largely defective in the presence of PFI-3. In contrast, the p53 activation was significantly enhanced by PFI-3, as determined by Ser-20 phosphorylation (41). In HT29 cells, PFI-3 rather increased the Chk1 activity after doxorubicin induction and exhibited little effect on the activity of the other checkpoint proteins (Fig. 5C; Supplementary Fig. S6B). PFI-3 itself had no effect on the cell-cycle profile or the damage checkpoint (Fig. 5B and C). Therefore, it appears that PFI-3 induces some aberrations in the doxorubicin-induced damage checkpoint in both A549 and HT29 cells, possibly via defective DNA repair, although its specific impacts on the damage checkpoint seem somewhat different between the two cell types.
PFI-3 increases doxorubicin-induced cell necrosis and senescence
Next, we investigated how PFI-3 decreases the cancer cell viability after doxorubicin treatment. Annexin-V staining showed that doxorubicin treatment induced cell death mostly via necrosis in both A549 and HT29 cells, which was increased by PFI-3 (Fig. 6A and B). Although its contribution to cell death was small, apoptosis after doxorubicin treatment was significantly increased by PFI-3 in HT29 cells but not A549 (Fig. 6A and B), which was confirmed by PARP cleavage (Fig. 6C). Although A549 and HT29 cells showed a similar degree of sensitivity to the combined treatment of doxorubicin and PFI-3 in clonogenic assay, the former exhibited much less necrosis and apoptosis compared with the latter. Notably, doxorubicin treatment increased senescence in A549 cells, which was largely enhanced by PFI-3 (Fig. 6D), suggesting that, in addition to necrosis, senescence also contributes significantly to the reduced viability of this cell after the combined treatment of doxorubicin and PFI-3. PFI-3 alone had no effect on the senescence (Fig. 6D). Therefore, we concluded that PFI-3 sensitizes A549 cells to doxorubicin by increasing both necrosis and senescence while it does so for HT29 primarily via necrosis and apoptosis to some extent.
Impacts of PFI-3 on gene expression in cancer cells
Finally, we investigated the impacts of PFI-3 on gene expression in A549 and HT29 cells. The cells after doxorubicin treatment were recovered with or without PFI-3 and analyzed for genome-wide transcriptome profiles by RNA-seq. Total 456 and 341 genes were upregulated or downregulated by ≥ 2 folds relative to control (DMSO) by treatment of PFI-3, doxorubicin, or both in A549 and HT29 cells, respectively (Supplementary Fig. S7A; Supplementary Tables S1 and S2). Unbiased hierarchical clustering of each of those gene sets revealed that the samples of doxorubicin and PFI-3/doxorubicin were grouped closely together whereas DMSO was clustered with PFI-3 (Supplementary Fig. S7A). This pattern became more obvious when clustering was performed for the genes that were categorized as associated with DDR, including DNA repair, cell-cycle control, cell death, and proliferation (151 genes in A549 and 42 in HT29; Fig. 7A and B; Supplementary Tables S3 and S4). This clustering also revealed that, while PFI-3 by itself exhibited a similar pattern as control, it further increased and decreased doxorubicin-upregulated and -downregulated gene expression, respectively (Fig. 7A and B), possibly the results of the amplification of DDR signaling reinforced by defective DNA repair.
Plotting Venn diagrams for each of the fc ≥ 2 genes in A549 and HT29 cells showed that, although many genes were regulated in common by PFI-3, doxorubicin, and PFI-3/doxorubicin, far more genes were regulated independently (Fig. 7C and D). In A549 cells, for example, only 25 of 159 genes (15.7%) were regulated in common by PFI-3 and doxorubicin, and the remaining 134 genes (84.3%) were regulated independently. Notably, although significant percentages of PFI-3-regulated (27 genes of 72, 37%) and doxorubicin-regulated genes (74 genes of 112, 66%) were overlapped with PFI-3/doxorubicin-regulated genes, the majority of the PFI-3/doxorubicin-regulated genes (248 genes of 334, 74%) were unique with no overlapping with the PFI-3- or doxorubicin-regulated genes (Fig. 7C and D). These results suggest that, while each having distinct cellular activities, PFI-3 and doxorubicin in combination impose different impacts from individual treatment, which may be reflected on their synergistic cytotoxicity on the cancer cells.
To determine whether and how well PFI-3-regulated genes overlap with SWI/SNF-regulated genes, we performed transcriptome profiling coupled with gene set enrichment analysis (GSEA) for the cancer cells treated with PFI-3 or knockdowned for SWI/SNF (knockdown for BRM or BRM/BAF180 in A549; BRG1/BRM or BRG1/BRM/BAF180 in HT29; Supplementary Fig. S7B; Supplementary Tables S5 and S6). For both of the upregulated and downregulated genes (fc ≥ 2), there was a highly significant correlation between PFI-3 treatment and SWI/SNF knockdown in A549 and HT29 cells (FDR q values = 0.00 for both) (Fig. 7E and F), suggesting that PFI-3 targets SWI/SNF specifically. Importantly, no DNA repair genes were found among the PFI-regulated genes, suggesting that PFI-3 inhibits DNA repair by directly targeting SWI/SNF rather than via gene expression.
Here, we investigated the applicability of PFI-3 as a sensitizer for cancer chemotherapy based on our previous proof-of-concept study validating SWI/SNF as an appropriate target (19). We verified that PFI-3 inhibits chromatin binding of its target bromodomains and displaces the corresponding endogenous SWI/SNF proteins from chromatin at pharmacologically significant concentrations. We then found that, while having little cytotoxicity by itself, PFI-3 synergistically sensitizes several human cancer cells to DNA damage induced by chemotherapeutic drugs, such as doxorubicin. Our data suggest that PFI-3 exerts this activity by blocking the damage-induced SWI/SNF binding to chromatin, which leads to defective DNA repair and aberrant damage checkpoint, eventually resulting in increase of cell death primarily via necrosis and senescence (Fig. 7G). This work, demonstrating the activity of PFI-3 to sensitize cancer cells to DNA damage by targeting SWI/SNF and its action mechanism, provides a strong experimental rationale for developing PFI-3 as a sensitizing agent for cancer chemotherapy.
Several lines of experimental evidence support our conclusion that PFI-3 sensitizes cancer cells to DNA damage by direct blockade of DNA repair via SWI/SNF targeting. First, PFI-3 not only dissociates SWI/SNF from chromatin under normal conditions but also inhibits it from binding to chromatin in response to DNA damage. Second, SWI/SNF depletion abolishes the activity of PFI-3 to sensitize cancer cells to DNA damage. Third, either SWI/SNF depletion or PFI-3 treatment inhibits DNA damage repair in cancer cells. In addition, DNA damage sensitization by PFI-3 occurs in the cancer cells that require SWI/SNF for DNA repair but not the cancer cells in which SWI/SNF is dispensable for DNA repair. Finally, a genome-wide transcriptome analysis shows that PFI-3 has no effect on the expression of DNA repair genes and that PFI-3-regulated genes well overlap with SWI/SNF-regulated genes.
Our results show that the phenotypic outcomes of PFI-3 in cancer cells are complex. PFI-3 sensitizes A549, HT29, and H460 cells to DNA damage with little of its own cytotoxic activity whereas it has cytotoxicity on H1299 cells by itself with no DNA damage sensitization. The former three cells, but not the latter, require SWI/SNF for DNA repair. Thus, how PFI-3 acts on cancer cells appears to depend on whether the cancer cells rely on SWI/SNF for their survival or DNA repair. In addition, PFI-3, albeit at high concentrations, sensitizes A549 cells, but not HT29, to ionizing radiation whereas it sensitizes both cells to the chemotherapeutic drugs, such as doxorubicin and etoposide, suggesting that the PFI-3 activity of sensitizing cancer cells to DNA damage depends on DNA damage source as well as cancer cell type. Furthermore, although we expected that BRG1-deficient cells would be more sensitive to PFI-3 than BRG1-proficient cells simply due to less PFI-3 targets, we observed no clear correlation between BRG1 deficiency and the cells' sensitivity to PFI-3. Thus, the sensitivity of cancer cells to PFI-3 in DNA damage does not depend on whether or not the cancer cells possess both BRG1- and BRM-based SWI/SNF complexes. Instead, other factors, such as chromatin affinity of involved bromodomains and/or role of SWI/SNF in DNA repair, are likely the determinants of the efficacy of PFI-3 in particular cancer cells. Although further study will be necessary to clarify the complexity of the phenotypic outcomes of PFI-3, it could be an advantage to develop PFI-3 as an anticancer agent for treatment of specific cancer using specific modality.
PFI-3 has been previously investigated for its cellular and biological activities (30, 31, 36). In the study reporting the development of PFI-3, the authors showed that PFI-3 displaces transfected GFP-BRM from chromatin in U2OS cells in a fluorescence recovery after photobleaching assay, and that PFI-3 mimics the effects of BRG1 depletion on stemness gene expression in embryonic stem cells, leading to deprivation of their stemness and deregulated lineage specification (30). Another study reported that, although PFI-3 inhibits chromatin binding of transfected BRM bromodomain, it does not displace endogenous BRM from chromatin in A549 cells in in situ cell extraction assay (36). Our results, showing that PFI-3 displaces the endogenous BRM from chromatin in in situ cell extraction and chromatin fractionation assays using U2OS and A549 cells, are in keeping with the former but not the latter study. Although not clearly understood, the discrepancy between the in situ extraction data of the latter study and ours could be attributed to different experimental conditions used. For example, we used much lower concentrations of detergent compared with that study, which might have increased the assay window. In addition, the latter study also showed that PFI-3 alone has no cytotoxic activity on any of the tested cancer cells, including A549, H1299, and H460 cells, which is contradictory to our results that, while having no effect on A549 and H460 cells, PFI-3 by itself has cytotoxicity on H1299 cells. One possible explanation for this discrepancy could be that the apparent BRM level in our H1299 cells is much lower than that in the H1299 cells used in that study, as judged by direct comparison of the BRM levels between H1299 and H460 cells (see Fig. 2C in this article and Fig. 2A in ref. 36). The low BRM level in our H1299 may render the cells sensitive to PFI-3 alone.
The success in evolving potent and highly specific inhibitors for the BET family of bromodomains and their entering into clinical trials has stimulated intensive research activities to develop inhibitors targeting other bromodomains (42). PFI-3 was one of the fruits of such research efforts. Although our work presented the possibility of potential application of PFI-3 as a sensitizer for cancer chemotherapy, many issues remain to be solved. Most importantly, the effective concentrations of PFI-3 on cancer cells are considerably high relative to the BET bromodomain inhibitors, such as JQ1 and PFI-1 (25, 43). It is thus worth to evolve structural analogs of PFI-3 as to have higher affinity to its target bromodomains and better cytotoxic efficacy on cancer cells, for instance, by using a molecular modeling approach. It is also of great value to pursue investigation to search in a systematic way for more cancer cell types that are sensitive to PFI-3 alone and/or sensitized to DNA-damaging chemotherapeutic drugs and ionizing radiation by PFI-3.
D. Lee reports a patent (KR102063398) issued to Ewha University, Industry Collaboration Foundation. Y.-S. Hwang reports a patent (KR102063397) issued to Ewha University, Industry Collaboration Foundation. J. Kwon reports patents KR102063397 and KR102063398 issued to Ewha University, Industry Collaboration Foundation. No disclosures were reported by the other authors.
D. Lee: Validation, investigation, methodology. D.-Y. Lee: Validation, investigation, methodology. Y.-S. Hwang: Validation, investigation, methodology. H.-R. Seo: Resources, supervision, investigation, methodology. S.-A. Lee: Resources, data curation, supervision, investigation. J. Kwon: Conceptualization, data curation, supervision, funding acquisition, investigation, writing–original draft.
This work was supported by grants 2015M2A2A7A01041767 (to J. Kwon), 2018R1A2B2007128 (to J. Kwon), and 2019R1A5A6099645 (to J. Kwon) from the National Research Foundation of Korea.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.