Next‐generation bromodomain inhibitors of the SWI/SNF complex enhance DNA damage and cell death in glioblastoma

Abstract Glioblastoma (GBM) is an aggressive brain cancer with a poor prognosis. While surgical resection is the primary treatment, adjuvant temozolomide (TMZ) chemotherapy and radiotherapy only provide slight improvement in disease course and outcome. Unfortunately, most treated patients experience recurrence of highly aggressive, therapy‐resistant tumours and eventually succumb to the disease. To increase chemosensitivity and overcome therapy resistance, we have modified the chemical structure of the PFI‐3 bromodomain inhibitor of the BRG1 and BRM catalytic subunits of the SWI/SNF chromatin remodelling complex. Our modifications resulted in compounds that sensitized GBM to the DNA alkylating agent TMZ and the radiomimetic bleomycin. We screened these chemical analogues using a cell death ELISA with GBM cell lines and a cellular thermal shift assay using epitope tagged BRG1 or BRM bromodomains expressed in GBM cells. An active analogue, IV‐129, was then identified and further modified, resulting in new generation of bromodomain inhibitors with distinct properties. IV‐255 and IV‐275 had higher bioactivity than IV‐129, with IV‐255 selectively binding to the bromodomain of BRG1 and not BRM, while IV‐275 bound well to both BRG1 and BRM bromodomains. In contrast, IV‐191 did not bind to either bromodomain or alter GBM chemosensitivity. Importantly, both IV‐255 and IV‐275 markedly increased the extent of DNA damage induced by TMZ and bleomycin as determined by nuclear γH2AX staining. Our results demonstrate that these next‐generation inhibitors selectively bind to the bromodomains of catalytic subunits of the SWI/SNF complex and sensitize GBM to the anticancer effects of TMZ and bleomycin. This approach holds promise for improving the treatment of GBM.


| INTRODUC TI ON
Gliomas are the most common primary brain cancers in adults. While Grade IV glioma (GBM, glioblastoma) is the most aggressive and deadliest brain tumour, Grade I glioma is the least malignant glioma. The primary treatment modality for GBM is surgical resection combined with adjuvant temozolomide (TMZ) chemotherapy and radiation therapy, which only provides slight improvement in disease course and outcome. 1 The median time for GBM recurrence after surgery is 7 months and overall prognosis is dismal with a 5-year survival of only 5%. 2 The mammalian ATP-dependent chromatin remodelling SWI/ SNF complex, is an evolutionarily conserved multi-subunit complex that regulates gene expression, differentiation, DNA repair and development. 3 The two catalytic subunits, BRM (Brahma) and BRG1 (Brahma-related gene 1) reposition and/or remodel nucleosomes, which opens or closes chromatin to regulate gene transcription. 4 In adult glioma, BRG1 expression increases with histological tumour grade with the highest levels found in GBM patients. In contrast, BRM expression is inversely related to tumour grade with the lowest expression found in GBM patients. BRG1 functions as a tumour suppressor in cancers of the lung, ovaries, skin and blood with silencing or loss-of-function mutations enriched. [5][6][7][8][9] In contrast, BRG1 has tumour promoting activity in several other cancers, including GBM. 7,10 Mutations of BRG1 are rarely found in multiple genomic databases of GBM patients. 11 Moreover, we demonstrated that high BRG1 expression selectively localizes in GBM patient tumour tissue. 12 BRG1 contains an evolutionarily conserved bromodomain proteinprotein interaction module that binds acetyl-lysine on protein and histone tails. 13,14 For example, BET bromodomain-containing proteins regulate the expression of key oncogenes, and specific and potent BET inhibitors are now in cancer clinical trials. [15][16][17] Thus, bromodomains are attractive targets in cancer, yet no inhibitors selective for BRG1 have been identified. PFI-3 was developed as a highly selective small molecule bromodomain inhibitor of the BRG1 and BRM subunits of the SWI/SNF complex, which has minimal 'off-target' effects in primary human cells and no evidence of toxicity on the NCI-60 panel of tumour cell lines. 18,19 We found that, although PFI-3 does not affect GBM cell proliferation, PFI-3 increased the sensitivity of various established GBM cell lines to TMZ as well as overcame TMZ-resistance. 12,20 In the present study, we have developed active secondgeneration bromodomain inhibitors, denoted therapy enhancing drugs (TEDs), by making modifications to the chemical structure of PFI-3. As described herein, we identified TEDs that markedly enhanced the sensitivity of GBM cells to DNA damaging agents. In addition, we identified one TED that was highly selective for binding to the BRG1 subunit of the SWI/SNF complex. Importantly, it significantly enhances sensitivity of GBM to DNA damaging agents in BRG1 expressing GBM cells but not in BRG1 knockout GBM cells.

| Synthesis of TEDs
Utilising PFI-3 as a lead compound, novel TEDs were designed and synthesized, which were expected to enhance the sensitivity of GBM cells to DNA alkylating agents. As illustrated in Figure 1, we aimed to optimize the bromodomain inhibitory activity by eliminating the double-bond and modifying the aromatic A-ring and B-ring of the structure. This approach enabled us to gain a better understanding of the structure activity relationship of these bromodomain inhibitors and helped us to design analogues with improved biological activity.

| Cell death, viability and invasion assays
For apoptosis assays, cells were plated into 48-well plates (1 × 10 4 cells/well), and after 3 days of drug treatment the levels of apoptosis in the attached cells were determined according to the instructions Structure of PFI-3 and new  analogues IV-129, IV-191, IV-255 and  IV-275. using the cell death ELISA PLUS assay (Roche), which measures cytoplasmic histone-associated DNA fragments. 21 In addition, to determine the effect of various drugs on cell viability and death, the Live/Dead cell viability/cytotoxicity assay (Molecular Probes) as previously described. 22 Images were captured on a Zeiss LSM700 laser scanning confocal microscope. Invasion assays using transwell inserts (BD Biosciences) were performed as previously described. 23

| Cellular thermal shift assay (CETSA)
The binding activity of PFI-3 and TEDs to the bromodomain of the BRG1 and BRM subunits of SWI/SNF was assessed by previously described CETSA. 24 In brief, MT330 GBM cells transduced with an epitope tagged BRG1 or BRM bromodomain were treated with PFI-3 (30 μM), the indicated TEDs (30 μM) or DMSO as a vehicle control for 3 h. After heating over a temperature range from 44.5 to 55.6°C for 5 min, the cells were lysed, placed on ice at 4°C and then immunoblotted for BRG1, BRM or actin.

| Gene expression analysis
Total RNA was extracted using RNeasy mini kits (Qiagen Inc.).   Slides were washed with PBS and then fixed with 4% paraformaldehyde/PBS containing 0.3% Triton-X100 for 30 min at 25°C. After blocking with 10% goat serum and 1% BSA/PBS for 1 h, the slides were incubated with the indicated primary γH2AX antibody overnight at 4°C. After incubating with goat anti-rabbit (or rabbit antimouse) Alexa Fluor 488 secondary antibody at 25°C for 90 min, DNA was counterstained with DAPI (Vectra Laboratories). Images were captured on a Zeiss LSM700 laser scanning confocal microscope.

| Statistical analyses
At least two independent experiments were performed in duplicate, and data are presented as means ± standard deviation (S.D.). Data were analysed by anova (Analysis of Variance) and post-hoc least significant difference analysis or Student's t-tests.

| Ethics Statement
Not applicable.

| The design and synthesis of PFI-3 analogues
As illustrated in Figure 1, we aimed to improve the activity of PFI-3 through major structural changes to optimize the bromodomain inhibitory activity by replacing acryloyl (3-oxo-prop-1-enyl) moiety with an ester or amide moiety between the A ring and the N atom of bicyclic ring system. We have changed the substituents on both the aromatic A and B-ring of PFI-3 to give the resulting analogues illustrated in Figure 1. Our approach enabled us to gain a better understanding of the structure activity relationship of these bromodomain inhibitors and help us to design better analogues for enhancing the action of TMZ in treating GBM. We confirmed the structure of the new analogues using nuclear magnetic resonance and mass spectrometry, and their purity was confirmed by high performance liquid chromatography.

| TEDs enhance the apoptosis-inducing activity of TMZ in GBM cells
The DNA damaging agent TMZ is frontline therapy for patients with GBM, and we previously found that the BRG1/BRM bromodomain inhibitor PFI-3 can enhance the sensitivity of GBM cells to the anticancer activity of TMZ in vitro. 25 Additional chemical modifications of the PFI-3 structure resulted in our lead molecule IV-129 ( Figure 1) that had greater activity than PFI-3 to enhance the sensitivity of MT330 and LN229 GBM cells to the apoptosis-inducing activity of 200 μM TMZ (Figure 2A), which was denoted as a TED. Further refinements of the IV-129 structure resulted in the generation of TEDs IV-255 and IV-275 that had even greater bioactivity in cell death assays in both GBM cell lines with TMZ ( Figure 2A). It was of particular interest that TED IV-191 had no effect on the induction of apoptosis by TMZ in both GBM cell lines.

| TEDs enhance the sensitivity of TMZ-resistant GBM cells to the apoptosis-inducing activity of TMZ
To further evaluate the ability of TEDs to enhance TMZ activity, we used two GBM cell lines (T98G and U87) that are highly resistant to the cell death-inducing activity of TMZ. While IV-129 was found to be much more potent than PFI-3 in sensitising U87 and T98G GBM cells in vitro to the cell-death inducing effects of TMZ ( Figure 2B),

| TEDs also sensitize GBM cells to bleomycin-induced cell death
Bleomycin is a chemotherapeutic drug that is used to treat various cancers. In addition, bleomycin induces DNA damage in a manner similar to that of ionising radiation 26

| The binding of TEDs to the bromodomain of BRG1 and BRM
The binding of the TEDs to the BRG1 and BRM bromodomains was assessed by the cellular thermal shift assay (CETSA), which measures thermostability of these bromodomains when complexed to the TEDs. 24,25 In brief, MT330 cells that express a BRG1 or BRM bromodomain construct were treated with TEDs for 2 h.
After heating for 5 min over a temperature range from 44.5 to 55.6°C, cells were lysed and immunoblotted for BRG1 or BRM.
As shown in Figure

| The identification of IV-255 as a BRG1-selective TED
Thermal shift assays show that IV-255 binds to the BRG1 bromodomain but does not bind the BRM bromodomain ( Figure 3A), suggesting that IV-255 has specificity for the BRG1 bromodomain. GBM cell lines with either BRG1 or BRM knocked out were generated by CRISPR/Cas9 gene editing, and the knockout was validated by immunoblotting. 25  As expected TMZ alone had no effect on γH2AX staining in U87 and T98G TMZ resistant cells (Figure 4). In contrast, in the presence of PFI-3 and TEDs γH2AX staining was increased in TMZ-treated cells.
To quantify the effect on γH2AX staining, we then determined the ratio of γH2AX staining relative to the DAPI nuclear counterstaining.
As shown in the graphs in Figure 4

| TEDs enhance the effect of TMZ on reducing GBM cell viability
Since we found that TEDs increase DNA damage induced by TMZ, which should lead to increase in cell death, we next simultaneously

| TEDS inhibit the invasiveness of GBM cells
The GBM cells are intrinsically high invasive, which plays an important role in GBM pathogenicity. Thus, based on our previous studies that the BRG-1 subunit plays a pro-oncogenic role in GBM, 25

| The effect of TEDs on IFN-regulated gene expression
In previous studies, we found that PFI-3 had a significant inhibitory effect on IFN-stimulated gene (ISG) expression. 28 Based on our finding that these newly developed TEDs were more potent inhibitors of the BRG1/BRM bromodomain than PFI-3, we next

| DISCUSS ION
The SWI/SNF chromatin remodelling complex binds to the promoters and enhancers of specific genes to regulate cell proliferation, differentiation, metabolism and DNA repair. [31][32][33][34][35][36] Genomic alterations of the BRG1 and BRM subunits of SWI/SNF in several human malignancies is consistent with their function as tumour suppressors in these cancers. 3,11 However, in many cancers neither BRG1 nor BRM are mutated but instead they are overexpressed, thus suggesting that they may also play a pro-tumorigenic role. 12,20,37 We recently found that found that BRG1 expression was higher in GBM tumour tissue compared to non-tumour tissue, while BRM was expressed at lower levels. 25 Gene deletion studies and knockdown studies on BRG1 in GBM cells suggest that BRG1 a pro-tumorigenic role in GBM, and that BRG1 may be an attractive therapeutic target in GBM. BRM. 19,38 We found that, although PFI-3 alone does not alter cell proliferation, PFI-3 enhanced GBM cells sensitivity to the DNA alkylating TMZ. 12,20,24 Herein, we described the development of a new generation of structural analogues that further enhance GBM cell sensitivity to TMZ, and they are thus denoted TEDs. we also found that another structural analogue of PFI-3, did not bind to either bromodomain or enhance the sensitivity of GBM cells to TMZ. These data indicate that, although this compound is structurally similar to other potent TEDs, IV-191 is an inactive analogue. Moreover, we found that TEDs enhance the sensitivity of GBM cells to bleomycin that is often used as a radiomimetic, suggesting that TEDs may also be radiosensitizers as well as chemosensitizers.
We also examined the mechanism whereby TEDs enhance TMZ- The BRG-1/BRM inhibitor PFI-3 exerts its DNA-sensitising effects of doxorubicin in cancer cells by inhibiting DNA repair. 41 In contrast, the results described herein suggest that TEDs increase the amount of DNA breaks induced by TMZ and bleomycin. Moreover, consistent with our cell death ELISA studies, we show that TEDs markedly decrease the number of viable GBM cells and increase the number of dead GBM cells using live/dead cell assays.
Furthermore, consistent with BRG1 playing a pro-tumorigenic role in GBM, we found that TEDs decrease GBM cancer cell invasion. This effect has therapeutic importance since high invasiveness is a hallmark of GBM. We previously found that BRG1 selectively regulated the expression of several well-known ISGs in GBM cells, 12 which is not surprising as BRG1 is a catalytic subunit of the SWI/ SNF complex remodelling complex. SWI/SNF inhibition would be expected to inhibit the nucleosome remodelling and downstream chromatin changes required to activate many genes. The activation of a subset of ISGs, while less straightforward to understand mechanistically, may be due to the loss of expression of an ISG transcriptional repressor. 42 The role of the SWI/SNF complex in promoting ISG transcription has been the focus of several earlier studies. 43,44 Moreover, PFI-3 at higher concentrations than we used was found to decrease the expression of several genes. 28  Taken together, we found that TEDs selectively bind to the bromodomains of catalytic subunits of the SWI/SNF complex and sensitize GBM to the anticancer effects of TMZ in vitro. A limitation of the present study is that the anticancer effects of TEDs have not been tested in animal GBM models in vivo, which will be an important focus of future studies. project administration (lead); resources (lead); supervision (lead); validation (lead); writing -original draft (lead); writing -review and editing (lead). Lawrence Pfeffer: Conceptualization (lead); data curation (supporting); formal analysis (lead); funding acquisition (lead); investigation (supporting); methodology (supporting); resources (lead); supervision (lead); validation (supporting); visualization (lead); writing -original draft (lead); writing -review and editing (lead).

ACK N O WLE D G E M ENTS
The authors thank Dr. R.N. Laribee and Dr. R. Narayanan for their helpful comments and discussions. This work was supported in part by a CORNET grant from UTHSC, internal funds from UTHSC-COM, and NIH/NCI grant 1R01CA-281977-01.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study and the BRG1 KO and BRM KO GBM cells generated during and/or analysed during the current study are available from the corresponding author on reasonable request.