# Effect of Bromodomain and Extraterminal Inhibitors with Different Bromodomain Selectivity on Mesenchymal Stem Cells

Riho Tanimoto, Yung-Sing Wong, Mitsuhiro Ebara, Koichiro Uto

► To cite this version:

Riho Tanimoto, Yung-Sing Wong, Mitsuhiro Ebara, Koichiro Uto. Effect of Bromodomain and Extraterminal Inhibitors with Different Bromodomain Selectivity on Mesenchymal Stem Cells. Chemistry Letters, 2024, 10.1093/chemle/upae238. hal-04877640

HAL Id: hal-04877640

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Submitted on 9 Jan 2025

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# Effect of Bromodomain and Extraterminal Inhibitors with Different Bromodomain Selectivity on Mesenchymal Stem Cells

Riho Tanimoto,<sup>1,2</sup> Yung-Sing Wong,<sup>3</sup> Mitsuhiro Ebara,<sup>1,2,4</sup> and Koichiro Uto*<sup>1</sup>

## Abstract

This study examines the influence of bromodomain and extraterminal (BET) inhibitors with different selectivity for bromodomains on human mesenchymal stem cells. Our findings demonstrate that extraterminal inhibitors with higher selectivity for the second bromodomain (BD2) exhibit lower cytotoxicity in human mesenchymal stem cells, suggesting that the inhibition of osteogenic differentiation in human mesenchymal stem cells (hMSCs) is primarily due to the suppression of the first bromodomain of bromodomain-containing protein 4, rather than the inhibition of its second bromodomain.

**Keywords:** BET inhibitors, BD2 selectivity, human mesenchymal stem cells

## Graphical abstract

Bromodomain and extraterminal (BET) inhibitors
with different BD selectivity

<figure><img src="image_2.png" /></figure>

(+)-JQ1

Pan-BET inhibitor

<figure><img src="image_3.png" /></figure>

I-BET726

BD2 selective BET inhibitor

<figure><img src="image_4.png" /></figure>

RVX-208

BD2 inhibitor

<figure><img src="image_5.png" /></figure>

<footnote n="1"><sup>1</sup>Research Center for Macromolecules and Biomaterials, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044</footnote>

<footnote n="2"><sup>2</sup>Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571</footnote>

<footnote n="3"><sup>3</sup>Univ. Grenoble Alpes, CNRS, DPM, 470, rue de la Chimie, 38000 Grenoble, France</footnote>

<footnote n="4"><sup>4</sup>Graduate School of Advanced Engineering, Tokyo University of Science, 6-3-1 Niijuku, Katsushika, Tokyo 125-8585</footnote>

<footnote>*Corresponding author: National Institute for Materials Science, 1-1 Namiki, Tsukuba-shi, Ibaraki 305-0044. Email: UTO.Koichiro@nims.go.jp</footnote>
<figure><img src="image_6.png" /><figcaption>**Fig. 1.** Experimental design to evaluate the effects of BET inhibitors with varying selectivity (JQ1, I-BET726, and RVX-208) on human mesenchymal stem cells (hMSCs).</figcaption></figure>

1 Bromodomain and extraterminal (BET) proteins,
2 which recognize acetylated histones, act as
3 transcriptional regulators controlling the expression of
4 genes involved in key cellular processes such as
5 proliferation, cell cycle progression, and apoptosis.<sup>1-3</sup>
6 Therefore, they have become prominent targets in
7 epigenetic research. There are four BET proteins in
8 humans: bromodomain-containing proteins 2, 3, 4
9 (BRD2, BRD3, BRD4), and bromodomain testis-
10 associated protein (BRDT). Each contains two
11 bromodomains, the first bromodomain (BD1) and the
12 second bromodomain (BD2). The pioneering BET
13 inhibitors JQ1<sup>4</sup> and I-BET<sup>5</sup> have demonstrated inhibitory
14 effects on cell growth and apoptosis in cancer cells and
15 anti-inflammatory activity, respectively, paving the way
16 for a new class of drugs. JQ1, a pan-BET inhibitor, does
17 not differentiate between BRDs. It exhibits equal
18 affinity for BD1 and BD2 pockets, with its cytotoxicity
19 commonly attributed to this lack of selectivity. BD2 can
20 also interact with proteins other than histones,
21 particularly transcription factors. Very recently, its
22 involvement in the rapid induction of gene expression
23 following an external stimulus such as inflammation
24 has been demonstrated.<sup>6, 7</sup> Conversely, BD1 primarily
25 regulates tissue-specific expression in the steady state,
26 and its inhibition is more relevant to cell growth arrest
27 and cancer therapy. These differences in their roles
28 may contribute to the lower cytotoxicity observed with
29 selective BD2 inhibitors. This BD selectivity arises from
30 differences in chemical scaffolds. For example, I-
31 BET726 (also called GSK1324726A) inhibits
32 bromodomains through a THQ structure that mimics
33 acetylated lysine residues, resulting in ~10-fold
34 preference for BRD4(BD2) over BRD4(BD1).<sup>8</sup> Similarly,
35 RVX-208 (apabetalone) is a BD2-selective inhibitor that
36 binds to BD2 and causes a conformational change with
37 aromatic stacking with a large entropy change<sup>3</sup>, which
38 has approximately or more than 10-fold high selectivity
39 for BRD4(BD2) over BRD4(BD1)<sup>9, 10</sup>. As mentioned
40 earlier, the chemical structure of the basic skeleton of
41 BET inhibitors and the dynamic properties of BRDs
42 enable the design of inhibitors with diverse effects and
43 selectivity.

44 The effects of BET inhibitors may vary depending on
45 the cell type, which warrants further investigation. For
46 instance, JQ1, a well-known BET inhibitor that induces
47 apoptosis in cancer cells, causes cell cycle arrest at the
48 G1 phase in mesenchymal stem cells (MSCs).<sup>11</sup> Given
49 the intricate relationship between epigenetic regulation
50 and cell function and fate, the impact of BET inhibitors
51 on adult stem cells, such as MSCs, is an important area
52 of investigation. Maintaining MSCs in an
53 undifferentiated state, which tends to differentiate
54 preferentially toward the osteogenic lineage<sup>12</sup> when
55 cultured on conventional substrates such as tissue
56 culture polystyrene (TCPS), is often desirable for
57 therapeutic applications.<sup>13</sup> The use of BET inhibitors
58 may prevent the MSCs' osteogenic differentiation
59 bias.<sup>14</sup> Additionally, BET-BD2 selective inhibitors, which
60 exhibit low cellular toxicity in MSCs, could address this
61 problem. Previous studies have shown that the pan-

62 BET inhibitor JQ1 induced cellular toxicity in MSCs<sup>15</sup>.
63 Therefore, in this study, we compared the effects of
64 JQ1 with two known BET-BD2 inhibitors, RVX-208 and
65 I-BET726, on human MSC (hMSC) functions. To date,
66 no studies have systematically investigated this
67 comparison. Understanding the effects and cytotoxicity
68 of BET inhibitors on MSC functions, in particularly their
69 cytotoxicity and on differentiation, is essential for
70 evaluating the safety profile of these drugs<sup>11</sup> and, by
71 extension, could lead to a better understanding of the
72 mechanisms involved in MSC differentiation. This
73 study addresses this gap by providing new insights into
74 the structure-function relationships of BET inhibitors in
75 the context of MSC biology.
76

77 We administered JQ1, I-BET726, and RVX-208,
78 which differ in BD2 selectivity, to hMSCs and
79 compared their ability to inhibit osteogenic
culture medium containing 0 M BET inhibitor dissolved
80 in 0.1% DMSO. Measurements were performed after
81 metabolic activity was measured using an MTT assay
82 after incubation with each BET inhibitor (Fig. 2). The
83 vertical axis represents values normalized to the optical
84 density of control, where cells incubated with cell
85 1, 3, and 7 days of culture. JQ1 substantially reduced
86 hMSC proliferation, consistent with previous reports.<sup>11</sup>
87 Fig. 2(a) demonstrates that JQ1 at concentrations of 10
88 nM or higher induces a significant reduction in cell
89 number after 1 day of incubation compared to 0 M,
90 consistent with previous reports.<sup>11 Fig. 2 (b)</sup> shows that
91 JQ1 at concentrations of 1 µM or higher induces a
92 significant reduction in cell number after 3 days of
93 incubation compared to 0 M, which is almost
94 consistent with previous reports (their study reported
95 this effect at 0.5 µM or higher).<sup>11 Fig. S2</sup> shows the cell
96 numbers relative to that on day 0, prior to BET inhibitor
97 administration. According to Fig. S2 (a), JQ1 at
98
<figure><img src="image_7.png" /><figcaption>**Fig. 2.** Metabolic activity of hMSCs measured by the MTT assay after exposure to 10 pM–100 µM of JQ1, I-BET726, and RVX-208 for (a) 1 day, (b) 3 days, and (c) 7 days. \* p < 0.05 for JQ1 vs. 0 µM, \*\* p < 0.05 for I-BET726 vs. 0 µM, \*\*\* p < 0.05 for RVX-208 vs. 0 µM. N.S. indicates no significant difference compared to 0 µM. The units are converted as 1 pM = 10⁻⁶ µM, 1 nM = 10⁻³ µM, and 1 mM = 10⁻³ µM.</figcaption></figure>

concentrations of 10 µM or lower did not induce a significant reduction in cell number on days 1 and 3 compared to day 0. Therefore, the reduction in cell number induced by JQ1 at 10 nM–10 µM in Fig. 2 (a) and 1 µM–10 µM in Fig. 2 (b) may potentially be attributed to cell cycle arrest.¹¹ After 7 days of incubation (Fig. 2 (c)), both JQ1 and I-BET726 induced a significant reduction in cell number at concentrations of 0.1 µM or higher, which is one order of magnitude lower than the concentrations required after 3 days of incubation. This suggests that the longer incubation period may have contributed to this effect. Furthermore, although RVX-208 did not induce a significant reduction in cell number within the tested concentration range on Day 1, a significant reduction was observed at concentrations of 1 µM, while on Day 3 and Day 7, significant reductions were observed at concentrations of 0.1 µM or higher, respectively. The IC50s of JQ1, I-BET726, and RVX-208 after 7 days of incubation calculated from Fig. 2 (c) were approximately 2.4 µM, 3.6 µM, and 11.6 µM, respectively. A. Shen and Y. Hu et al. demonstrated that BRD4 inhibitors with high BD2 selectivity exhibit lower rates of apoptosis induction compared to pan-BRD4 inhibitors, even at higher concentrations.¹⁶ Consistent with their findings, Fig. S2 shows that I-BET726 and RVX-208, which exhibit greater BD2 selectivity compared to JQ1, did not significantly reduce cell numbers relative to day 0 even at a concentration of 100 µM, where JQ1 induced a substantial reduction in cell numbers. This is likely because BD2-selective inhibitors have minimal effects on pre-existing gene expression programs, as they exert limited impact on displacing chromatin-bound BET proteins.¹⁷ BD2-selective BET inhibitors are known to regulate gene expression primarily in response to stimuli that induce rapid gene induction,⁶ such as those involved in inflammatory responses, thereby enabling effective anti-inflammatory effects in inflammatory cells.¹⁸

We compared the ability of JQ1, I-BET726, and RVX-208 to inhibit osteogenic differentiation in hMSCs.

Although these BET inhibitors target BRD2, BRD3, and BRD4, BRD4 in particular is implicated in the suppression of osteogenic differentiation.¹⁴, ¹⁹⁻²⁵ We examined how differences in BRD4 selectivity among these BET inhibitors⁹, ¹⁰, ²⁶ affect their ability to inhibit osteogenic differentiation in hMSCs. ALP expression was evaluated in hMSCs incubated with each BET inhibitor to compare their ability to inhibit osteogenic differentiation, as ALP is a recognized early marker of this process. As shown in the control image (hMSCs cultured without BET inhibitor, using 0.1 % DMSO in cell culture medium), ALP expression was observed in part of the hMSCs after 8 days of culture on TCPS (1 day for cell adhesion and 7 days for culture control with BET inhibitor) (Fig. 3 (a)). Since hMSCs cultured on TCPS are reported to become irreversibly biased towards osteogenesis as the culture period increases,¹² this may result from prolonged mechanical stimulation of hMSCs from TCPS. To assess the ALP inhibitory potential, BET inhibitors were added to hMSCs and incubated for 7 days following 1 day of initial incubation for cell adhesion. As shown in Fig. 3(a), a reduction in ALP expression (shown in blue) was observed as the concentration of BET inhibitor increased. This result is consistent with previous reports showing that BRD4 inhibition by JQ1 inhibits MSC osteogenesis.¹⁴, ²⁴, ²⁵ These studies revealed that JQ1 concentrations higher than 10 nM²⁵ (60 nM in osteoblast differentiation medium¹⁴) significantly reduced MSC osteogenic differentiation as indicated by ALP expression and the later osteogenesis maker, Alizarin Red S. Similar to JQ1, since I-BET726 and RVX-208²³ are also considered to inhibit osteogenic differentiation through BRD4 inhibition, ALP expression was further quantified using a plate reader to compare inhibition levels between these BET inhibitors (Fig. 3 (b)).

As the results, JQ1 and I-BET726 significantly decreased ALP expression compared to the control at 0.1 µM, while RVX-208 significantly decreased it at 1 µM. Furthermore, JQ1, I-BET726, and RVX-208 nearly completely inhibited ALP expression at concentrations
<figure><img src="image_8.png" /><figcaption>**Fig. 3.** Inhibition of osteogenic differentiation in hMSCs cultured for 7 days with varying concentrations (10 nM–100 µM) of JQ1, I-BET726, or RVX-208, or without BET inhibitor (control). (a) Alkaline phosphatase (ALP) staining images showing osteogenic differentiation marker. The red-squared images highlight a significant reduction (~90%) in ALP expression compared to the control. (b) Quantitative analysis of ALP activity, measured by phenol concentration, which is the product of the ALP-catalyzed reaction. \* p < 0.05 for JQ1 vs. 0 µM, \*\* p < 0.05 for I-BET726 vs. 0 µM, \*\*\* p < 0.05 for RVX-208 vs. 0 µM. The units are converted as 1 nM = $10^{-3}$ µM and 1 mM = $10^{-3}$ µM.</figcaption></figure>

1 of 0.1, 1, and 10 µM, respectively. As shown in Fig. 2,
2 cell numbers at these concentrations were
3 approximately 60% of the control, and Fig. 3 (b)
4 indicates that the near-complete inhibition of ALP
5 expression at these concentrations was due to BRD
6 inhibition rather than cell death. Our results showed
7 that JQ1 inhibited osteogenic differentiation at lower
8 concentrations than I-BET726, which is consistent with
9 previous findings that JQ1 is a more effective BRD4
10 inhibitor.²⁶ This suggests that BET inhibitors with lower
11 IC50 values against BRD4 inhibit osteogenic
differentiation at lower concentrations. Furthermore, I-
12 BET726, being approximately 10 times more selective
13 for BD2 over BD1<sup>8</sup>, impacts steady-state gene
14 expression less than JQ1. This may also contribute to
15 I-BET726 requiring higher concentrations for ALP
16 inhibition.

Our results also show that JQ1 and RVX-208
18 inhibited 50% of osteogenic differentiation within 0.01-
19 0.1 µM and within 1-10 µM, respectively (Fig. 3 (b)),
20 which aligns with reports that the IC50 of BRD4(BD1)
21 is approximately 0.1 µM for JQ1 and 1 µM for RVX-208.⁹
22
23 ¹⁰ The result that 1 µM RVX-208 had less ALP inhibition
24 than 0.1 µM JQ1 (Fig. 3 (b)) is consistent with previous
25 reports that 5 µM RVX-208 (apabetalone) resulted in
26 less ALP inhibition than 0.1 µM JQ1 in vascular smooth
27 muscle cells.²³ These results suggest that the IC50
28 values of JQ1 and RVX-208 against BRD4(BD1) are
29 reflected in the inhibition of osteogenic differentiation
30 rather than their IC50 values against BRD4(BD2),
31 because JQ1 and RVX-208 have quite different IC50

<figure><img src="image_9.png" /><figcaption>**Fig. 4.** Comparison of cytotoxicity and osteogenic differentiation inhibition levels against hMSCs: The size of the circles representing the three BET inhibitor reflects the ratio of IC50 for BRD4(BD1) to IC50 for BRD4(BD2). The color of the circles represents the BRD4 inhibition ability and the IC50 value for BRD4(BD1). BET inhibitors with higher BRD4 inhibition ability, particularly those with stronger inhibition against BRD4(BD1) (i.e., lower IC50 for BRD4(BD1)), show greater ALP inhibition ability. In contrast, inhibitors with higher BD2 selectivity (i.e., a higher IC50 for BRD4(BD1) / IC50 for BRD4(BD2)) exhibit lower cytotoxicity.</figcaption></figure>
1 against BRD4(BD1) rather than BRD4(BD2) (RVX-208 61
2 has more than 10-fold higher IC50 for BRD4(BD1) 62
3 compared to JQ1, while its IC50 for BRD4(BD2) is only 63
4 2-4 fold higher compared to JQ1) (Fig. S1).⁹,¹⁰ 64
5
6 In summary, we demonstrated BD2-selective BET 66 5
7 inhibitors, which have previously been reported to 67
8 exhibit low cytotoxicity and high anti-inflammatory 68
9 effects in macrophages, also exhibit low cytotoxicity in 69
10 MSCs (Fig. 4). Furthermore, we demonstrated that 70
11 BRD4(BD1) inhibition plays a central role in the 71 6
12 inhibitory effects of BET inhibitors on osteogenic 72
13 differentiation in MSCs. RVX-208 and JQ1 exhibit 73
14 different IC50 values for BRD4(BD1) compared to 74
15 BRD4(BD2), with RVX-208 requiring higher 75
16 concentrations to achieve ALP inhibition, as shown in 76
17 Fig. 4. This may be because BD1 inhibition primarily 77
18 modulates established gene expression programs, 78
19 whereas BD2 inhibition modulates post-stimulus gene 79
20 expression, driving rapid gene induction.⁶ In cells 80
21 undergoing steady-state gene expression, such as 81
22 those in osteogenic differentiation induced by long- 82 7
23 term culture on stiff substrates, BD1 inhibition primarily 83
24 affects the inhibition of osteogenic differentiation. 84
25 Therefore, BET inhibitors with high BD2 selectivity do 85
26 not directly exert high osteogenic differentiation 86
27 inhibitory effects on MSCs without external stimuli, 87
28 however, their low toxicity makes them useful for 88
29 medical applications focused on the recovery of 89
30 undifferentiated MSCs. This study shows that BET 90
31 inhibitors with a low IC50 for BRD4(BD1) and much 91
32 lower IC50 for BRD4(BD2) than BRD4(BD1) (BD2 92 8
33 selective BET inhibitor) may provide superior results in 93
34 MSCs by providing strong osteogenic differentiation 94
35 inhibition with low toxicity. 95

36

37 Supplementary data 95

38 Supplementary material is available at *Chemistry* 96

39 *Letters* 97

40 98

41 **Funding** 99

42 This work was supported by JSPS KAKENHI (21K19921, 100 9

43 JP20H05877), labex ARCANE and CBH-EUR-GS (ANR- 101

44 17-EURE-0003). 102 10

45 103

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