Targeting breast cancer stem cells by novel HDAC3-selective inhibitors

Accepted Manuscript Targeting breast cancer stem cells by novel HDAC3-selective inhibitors Hao-Yu Hsieh, Hsiao-Ching Chuang, Fang-Hsiu Shen, Kinjal Detroja, Ling-Wei Hsin, Ching-Shih Chen PII:

S0223-5234(17)30680-3

DOI:

10.1016/j.ejmech.2017.08.069

Reference:

EJMECH 9713

To appear in:

European Journal of Medicinal Chemistry

Received Date: 3 July 2017 Revised Date:

30 August 2017

Accepted Date: 30 August 2017

Please cite this article as: H.-Y. Hsieh, H.-C. Chuang, F.-H. Shen, K. Detroja, L.-W. Hsin, C.-S. Chen, Targeting breast cancer stem cells by novel HDAC3-selective inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.08.069. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Targeting Breast Cancer Stem Cells by Novel HDAC3-Selective Inhibitors

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Hao-Yu Hsieh,1,2,3† Hsiao-Ching Chuang,2† Fang-Hsiu Shen,3 Kinjal Detroja,3 Ling-

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Wei Hsin,1* and Ching-Shih Chen*2,3

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Taiwan; 2Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy,

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The Ohio State University, Columbus, OH, USA; 3Institute of Biological Chemistry,

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Academia Sinica, Taipei, Taiwan;

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(†Equal contributions; *Co-corresponding authors)

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School of Pharmacy, College of Medicine, National Taiwan University, Taipei,

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ACCEPTED MANUSCRIPT Abstract

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Although histone deacetylase (HDAC) inhibitors have been known to suppress the

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cancer stem cell (CSC) population in multiple types of cancer cells, it remains unclear

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which HDAC isoforms and corresponding mechanisms contribute to this anti-CSC

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activity. Pursuant to our previous finding that HDAC8 regulates CSCs in triple-

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negative breast cancer (TNBC) cells by targeting Notch1 stability, we investigated

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related pathways and found HDAC3 to be mechanistically linked to CSC homeostasis

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by increasing β-catenin expression through the Akt/GSK3β pathway. Accordingly,

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we used a pan-HDAC inhibitor, AR-42 (1), as a scaffold to develop HDAC3-selective

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inhibitors, obtaining the proof-of-concept with 18 and 28. These two derivatives

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exhibited high potency and isoform selectivity in HDAC3 inhibition. Equally

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important, they showed in vitro and/or in vivo efficacy in suppressing the CSC

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subpopulation of TNBC cells via the downregulation of β-catenin.

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Histone deacetylase 3 (HDAC3), cancer stem cell (CSC), triple-negative breast cancer

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(TNBC), β-catenin

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INTRODUCTION

The concept of cancer stem cells (CSCs) or tumor-initiating cells has provided a

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new paradigm for the functional heterogeneity of cancer cells [1, 2]. Because of their

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tumorigenic properties and capacity for self-renewal and differentiation, this small

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subpopulation of cancer cells drives initiation, progression, and metastasis in many

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types of tumors [3].

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mesenchymal transition acts as a critical regulator of the drug-resistant phenotype of

Moreover, recent evidence suggests that epithelial-to-

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ACCEPTED MANUSCRIPT CSCs [4, 5]. Consequently, CSCs are more resistant to chemotherapeutic agents than

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the bulk population of non-CSCs within a tumor, allowing the surviving CSCs to

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repopulate the tumor and leading to tumor relapse. From a therapeutic perspective,

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there is an urgent unmet need for CSC-targeting therapeutic agents to achieve optimal

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patient outcomes. To date, a series of therapeutic agents targeting key signaling

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pathways, especially those mediated by Notch1, Hedgehog, and Wnt, have proved to

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be efficacious in eradicating CSCs in preclinical settings [6-8]. In addition, there is

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accumulating evidence of the in vitro and/or in vivo efficacy of pan- and class I

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HDAC inhibitors, such as suppressing the CSC subpopulation in different cancer cell

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lines [9]. However, two issues remain unclear with respect to the anti-CSC activity of

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HDAC inhibitors. First, as there are 11 Zn2+-dependent isoforms in the HDAC

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family, it is unclear which of these isoforms contribute to the regulation of CSCs.

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Second, the mechanism by which pan- or class I HDAC inhibitors suppress the CSC

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subpopulation has not been clearly defined.

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Previously, we reported a non-epigenetic function of HDAC8 in activating CSC-

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like properties in triple-negative breast cancer (TNBC) cells by upregulating the

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expression of Notch1 [10]. We demonstrated that HDAC8 protected Notch1 from

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Fbwx7-facilitated protein degradation, thereby leading to the accumulation of Notch1

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and the Notch intracellular domain NICD.

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account for the ability of the class I HDAC inhibitor MS-275 to effectively block

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mammosphere formation in breast cancer cell lines [10]. Because MS-275 is deficient

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in HDAC8 inhibitory activity, this suggested the involvement of another HDAC

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isoform, namely, HDAC3. In the present study, we obtained evidence that HDAC3

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plays a pivotal role in regulating CSCs through a distinct mechanism, i.e., to increase

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β-catenin protein stability by activating the Akt/glycogen synthase kinase (GSK)3β

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Nevertheless, this finding could not

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signaling pathway. Based on this biological finding, we embarked on developing HDAC3-selective

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inhibitors as anti-CSC agents via structural modifications of AR-42 [formerly, (S)-

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HDAC42; 1]. We had a three-fold rationale for employing this hydroxamate-based

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pan-HDAC inhibitor developed in our laboratory [11, 12]. First, AR-42 shows high

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potency in ablating cancer stem cells in acute myelogenous leukemia [13] and triple-

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negative breast cancer (TNBC) [10].

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pharmacokinetic properties in animal models [14].

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bioavailability of AR-42 was estimated to be 26% and 100% in mice and rats,

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respectively. Third, AR-42 has recently completed a phase I trial in patients with

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multiple myeloma and T-cell lymphoma, which showed that oral AR-42 was well

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tolerated with no apparent dose-limiting toxicities [15].

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perspective, AR-42-derived HDAC3 inhibitors might retain drug-like properties with

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lesser toxicity. Here, we report the successful conversion of AR-42 into a series of

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HDAC3-selective inhibitors, represented by 18 and 28, by replacing the hydroxamate

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moiety with o-aminoanilides as the Zn2+-chelating motif, followed by altering the

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structure of the cap group. Compounds 18 and 28 exhibited high potency and isoform

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selectivity toward HDAC3, and equally important, were able to mimic the suppressive

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effect of HDAC3 depletion on CSCs in vitro and/or in vivo through the inhibition of

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the Akt/β-catenin signaling pathway.

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signaling, has been shown to play a critical role in the maintenance of the CSC

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population in many types of cancer [16, 17], including TNBC [18]. This regulation is

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mediated, in part, through the transcriptional regulation of target genes associated

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with tumor initiation and cell renewal, including c-Myc [19, 20] and BMI-1 [19], by

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interacting with a complex network of binding partners [16, 17].

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Second, AR-42 exhibits favorable

From a translational

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For example, the oral

β-Catenin, a downstream effector of Wnt

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In this paper, we first describe our biological finding on the role of HDAC3 in

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regulating the CSC subpopulation in TNBC cells, followed by the structural

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modification of AR-42 to develop HDAC3-selective inhibitors and the evaluation of

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their in vitro and/or in vivo efficacies in suppressing CSCs.

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Evidence that HDAC3 is involved in regulating breast CSCs via the Akt-GSK3β β-

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β-catenin signaling pathway. To shed light onto the functional role of HDAC3, we

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used two different shRNAs (#4993 and #6267) to deplete HDAC3 in two TNBC cell

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lines, MDA-MB-231 and SUM-159 (Figure 1A). We isolated two stable clones from

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each treated cell type to assess the consequent effects, vis-à-vis parental cells, on

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different cellular functions, including cell proliferation, colony formation, and

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mammosphere formation, a surrogate measure of CSC expansion [21].

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knockdown of HDAC3 via these two shRNAs gave rise to consistent results in both

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cell lines (MDA-MB-231, Figure 1B; SUM-159, Figure 1C). As shown, HDAC3

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silencing led to decreases in cell proliferation rate (left), clonogenic survival (center),

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and mammosphere formation (right) as compared to control.

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To understand the mechanism by which HDAC3 regulates CSCs, we analyzed

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various signaling pathways in both cell lines by Western blot analysis after stable

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HDAC3 knockdown. Among the different pathways examined, the ability of HDAC3

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depletion to reduce β-catenin expression via the inhibition of the Akt-GSK3β

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signaling axis was especially noteworthy. As shown, ablation of HDAC3 led to

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ACCEPTED MANUSCRIPT reduced phosphorylation of Akt and GSK3β, accompanied by parallel changes in the

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expression of β-catenin in both cell lines (Figure 1A). Moreover, the suppressive

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effect of HDAC3 depletion on β-catenin expression was corroborated by parallel

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decreases in β-catenin target gene products associated with the maintenance of breast

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CSCs, including c-Myc [19, 20] and BMI-1 [19]. This finding is consistent with

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recent reports that blocking β-catenin signaling is effective in inhibiting breast CSCs

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[18, 19, 22]. Conversely, enforced expression of HDAC3 in MDA-MB-231 cells

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resulted in the activation of Akt-GSK3β signaling, leading to the accumulation of β-

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catenin and the aforementioned CSC regulators (Figure 1D).

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As CSCs are characterized by their capacity to form tumors from low cell

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numbers, we assessed the effect of HDAC3 depletion on tumor-initiation using MDA-

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MB-231 cells. Female NOD/SCID mice were injected bilaterally in the mammary fat

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pads with 50,000 MDA-MB-231 cells stably expressing HDAC3 shRNA (HDAC3KD;

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Figure 1E) on one side and an equal number of control MDA-MB-231 cells on the

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opposite side (N = 9). As shown, HDAC3 depletion suppressed breast tumorigenicity

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in vivo.

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generated from the control cells, while only 40% of the mice had HDAC3KD tumors

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(P < 0.025; log-rank test). Moreover, the HDAC3KD tumors grew at a slower rate

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than the control tumors (P = 0.0015).

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For example, at six days post-injection, 90% of the mice had tumors

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Together, these data supported the function of HDAC3 in regulating breast CSCs,

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in part via the Akt-GSK3β-β-catenin signaling pathway. Involvement in this pathway

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provided a mechanistic basis to develop HDAC3-selective inhibitors as anti-CSC

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agents. Accordingly, we embarked on the development of HDAC3-selective

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inhibitors by using compound 1 as a starting point according to the strategy delineated

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in the next section.

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ACCEPTED MANUSCRIPT 1 Use of the pan-HDAC inhibitor 1 (AR-42) as a scaffold to develop class I- and

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HDAC-3-selective inhibitors. There is substantial evidence that the Zn2+-chelating

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motif is critical for determining the selectivity of HDAC inhibitors [23, 24]. For

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example, compound 1 and most other hydroxamate-based HDAC inhibitors, such as

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SAHA, TSA, and LBH589, are broad-spectrum inhibitors, while many class I HDAC

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inhibitors (MS-275, CI994, and mocetinostat) and HDAC3-selective inhibitors (BG-

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45 and RGFP966) contain N-(2-aminophenyl)-benzamide and o-aminoanilide as their

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Zn2+-chelating motifs, respectively (Figure 2A).

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We hypothesized that the isoform selectivity of the aforementioned class I

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and HDAC3-selective inhibitors might be attributable to the bulkiness of the

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respective Zn2+-chelating motifs relative to the hydroxamate moiety, which hinders

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their access to the catalytic Zn2+-binding domain of non-class I HDAC isoforms.

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Based on this reasoning, we replaced the –NHOH moiety of 1 with o-aminoaniline,

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yielding 2. In addition, we prepared 3, which is the (R)-enantiomeric isomer of 2, to

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examine potential stereochemical effects on the potency/selectivity in inhibiting

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different HDAC isoforms.

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subsequent new derivatives are depicted in Scheme 1.

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General procedures for the syntheses of 2, 3, and

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ACCEPTED MANUSCRIPT Compounds 2 and 3, each at 1 µM, were subjected to HDAC isoform-profiling

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analysis (HDAC1-8) by a commercial vendor (Reaction Biology Cooperation,

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Malvern, PA). The isoform profiling data revealed that, compared to compound 1,

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this substitution of the Zn2+-chelating motif resulted in decreased, yet more selective,

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inhibitory activities against the various HDAC isoforms examined (Table 1).

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For example, while 1 inhibited HDAC1-3, HDAC6, and HDAC8 with high potencies

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(data provided by Arno Therapeutics), 2 and 3 showed substantially lower activities

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relative to 1 in inhibiting HDAC6 and HDAC8. Of note, 3 displayed higher potency

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than 2 toward HDAC1-3, indicating the stereochemical preference of the (R)-α-

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isopropyl substituent in binding to the catalytic Zn2+-binding domain of these HDAC

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isoforms. In addition to the above in vitro deacetylase assays, we measured three

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cell-based biomarkers by Western blot, including histone H3 acetylation (both pan-

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and H3K9-acetylation), tubulin acetylation [25], and Notch1 expression [10], to

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monitor the inhibitory activities of individual derivatives against histone

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deacetylation, HDAC6, and HDAC8, in MDA-MB-231 cells. As shown, treatment of

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cells with 1, 2, or 3 at the indicated concentrations increased histone H3 pan- and

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H3K9 acetylation in a consistent manner, demonstrating the inhibition of histone

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deacetylase activity.

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HDAC8; at 0.25 µM it facilitated tubulin acetylation and decreased the expression of

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Notch1 (Figure 3).

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Compound 1 was highly potent in inhibiting HDAC6 and

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ACCEPTED MANUSCRIPT 1 Consistent with the low in vitro HDAC6 and HDAC8 inhibitory activities, 2 and

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3 effected no apparent changes in the levels of tubulin acetylation and Notch1

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expression, with the exception of 3 at 2.5 µM against Notch1. Throughout the course

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of this structural modification, we used these two biomarkers to measure HDAC6-

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and HDAC8-inhibitory activities of individual derivatives.

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Previously, it has been reported that fluorine-containing benzamides as the Zn2+-

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chelating motif could enhance HDAC3 selectivity [26, 27]. Consistent with this

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finding, the isoform selectivity of 2 and 3 could be further improved by adding a

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fluorine atom at the para-position of the aminoaniline moiety, resulting in 4 and 5,

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respectively. As shown, 5 was deficient in HDAC6- and HDAC8-inhibitory activities

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(<10% inhibition at 1 µM; Table 1), further confirmed by the lack of appreciable

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changes in tubulin acetylation and Notch1 expression, respectively (Figure 3). In

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particular, 5 exhibited a slight preference for inhibiting HDAC3 over HDAC1 and

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HDAC2 [Table 1; IC50, 0.75, 1.2 and 2 µM, respectively; determined by Reaction

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Biology Cooperation (Figure 2B)]. However, substituting the fluorine atom of 5 with

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CF3 completely abolished the HDAC inhibitory activity of the resulting compound 6

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(Table 1), consistent with the lack of increase in histone H3 acetylation in drug-

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treated cells (Figure 3). This loss of HDAC inhibitory activity might be associated

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with the bulkiness of the CF3 group, which restricted ligand access to the active site

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pocket. From a structural perspective, 5 provided a proof-of-concept that HDAC3-

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selective inhibitors could be developed from a pan inhibitor by replacing hydroxamate

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with N-(2-amino-4-fluorophenyl)carboxamide as the Zn2+-chelating motif.

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therefore conducted further structural modifications of 5 to augment its HDAC3

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We

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inhibitory potency and isoform selectivity by altering the structure of the cap group

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and/or linker. It is noteworthy that substitution of the α-substituent of the cap group of 5, i.e.,

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(R)-isopropyl, with either (S)- or (R)-ethyl (7 and 9, respectively) or methyl (8 and 10,

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respectively) resulted in multifold increases in the potency in HDAC3 inhibition

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(Figure 2B; IC50, 5, 1 µM; 7, 0.17 µM; 8, 0.23 µM; 9, 0.11 µM; 10, 0.21 µM).

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Equally important, this increase in HDAC3 inhibitory activity was also accompanied

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by improved isoform selectivity. There were multifold increases in the selectivity

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ratio, defined as the ratio of IC50 of HDAC1 to that of HDAC3 (IC50HDAC1/IC50HDAC3),

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of individual compounds (5, 1.2; 7, 14.7; 8, 13; 9, 10.9; 10, 8.6).

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To assess the effect of altering the linker on the potency and/or selectivity, we

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replaced the 4-aminobenzoyl linker of the above three enantiomeric pairs of

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compounds (4 and 5, 7 and 9, and 8 and 10) with a heterocyclic linker, 5-

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aminopicolinoyl, yielding the corresponding optically active derivatives 11-16. This

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modification led to increases in HDAC3 inhibitory potencies in 11, 13, 14, and 15.

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Compound 14, in particular, had a nearly tenfold increase in activity relative to 5

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(IC50, 0.08 µM versus 1 µM). 14 also showed improved isoform selectivity with a

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selectivity ratio of 6.5. In contrast, 12 and 16 had a modest loss of HDAC3-inhibitory

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activity, but an increased HDAC1-inhibitory activity, as compared to 7 and 10.

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Interestingly, replacing the 5-aminopicolinoyl linker of 14 with an isomeric 6-

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aminonicotinoyl moiety to produce compound 17 resulted in a substantial loss of

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activities against not only HDAC3 (IC50, 1.7 µM), but also HDAC1 (> 10 µM) and

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HDAC2 (8.4 µM) (Figure 2B). Together, these data suggest subtle interplay between

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the linker and α-substituent in interacting with the binding pockets of different HDAC

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isoforms. Because there were no apparent changes in tubulin acetylation and Notch 1

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expression, it was evident that compounds 11 and 13-16 lacked inhibitory activities

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toward HDAC6 and HDAC8 (Figure 3). Pursuant to the above findings, we further used achiral p-substituted 2-

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phenylacetyl groups (R = H, F, and CF3) as the cap group in conjunction with either

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4-aminobenzoyl (to generate derivatives 18-20) or 5-aminopicolinoyl (to generate

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derivatives 21-23).

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inhibitory potency (IC50, 0.18 µM) as well as isoform selectivity of HDAC3 over

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HDAC1 (2.3 µM) and HDAC2 (3.2 µM). The modifications to generate 19-23 did

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not improve potency or isoform selectivity relative to 18, and in the case of 23, even

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abolished the HDAC-inhibitory activity (IC50 > 10 µM for all three isoforms). This

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intricate structure-activity relationship (SAR) was also illustrated by a switch in

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isoform selectivity from HDAC3 to HDAC1 when the F atom was removed from the

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Zn2+-chelating motif of 20 to generate 24 (Table 1). The structural basis of this

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selectivity switch is as yet unclear. Moreover, replacement of the phenylacetyl group

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of 18 and 21 with a 3-phenylpropionyl moiety decreased and abrogated the HDAC3-

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inhibitory potency in the resulting compounds 25 (IC50, 0.8 µM) and 26 (no

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appreciable activity at 10 µM), respectively. We proceeded to synthesize a series of

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derivatives with conformationally restricted cap groups, 27 - 31, among which 28 was

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notable for its high HDAC3-inhibitory potency (IC50, 0.35 µM) and isoform

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selectivity (Table 1; Figure 2B).

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inhibitory potency and isoform selectivity, which reiterates the integral role of the F

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atom on the Zn2+-chelating motif in interacting with the catalytic pockets of HDAC3

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versus other HDAC isoforms.

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Of these six derivatives, 18 exhibited the highest HDAC3-

Relative to 28, 27 exhibited lower HDAC3-

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ACCEPTED MANUSCRIPT In vitro and/or in vivo efficacy of 18 and 28 in suppressing breast CSCs. Based on

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the above SAR, we evaluated 18 and 28 for their in vitro and/or in vivo efficacies in

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inhibiting breast CSCs. Compounds 18 and 28 exhibited only a modest suppressive

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effect on cell viability (IC50 ≥ 10 µM) by MTT assays (Figure 4A, left), which was

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also confirmed by the growth inhibition assay using crystal violet staining (right; IC50

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≥ 10 µM). In contrast, both compounds were highly effective in blocking colony

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formation and mammosphere formation in suspension medium (IC50, 0.5 – 1 µM)

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(Figure 4B).

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This discriminative effect suggests the ability of these two HDAC3 inhibitors to

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selectively target the CSC subpopulation in MDA-MB-231 cells. Consistent with

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effects of HDAC3 depletion, treating MDA-MB-231 cells in a monolayer culture with

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18 or 28 in the presence of 5% FBS led to concurrent downregulation of Akt

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phosphorylation and β-catenin expression, accompanied by increases in histone H3

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acetylation (Figure 4C). Moreover, RT-PCR analysis revealed no changes in mRNA

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expression, indicating that this downregulation of β-catenin was mediated at the

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protein level and suggesting that this decrease in β-catenin expression was attributable

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to protein degradation associated with drug-induced Akt deactivation.

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We next obtained two lines of evidence verifying the inhibitory effect of 28 on

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breast CSCs. First, the CD44+/CD24low breast cancer population is known to be

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enriched for breast cancer initiating stem cells [28]. Flow cytometric analysis

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demonstrated the concentration-dependent, suppressive effect of 28 on this

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subpopulation of MDA-MB-231 cells (Figure 4D). Second, we tested the in vivo

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ACCEPTED MANUSCRIPT efficacy of 28 in suppressing tumor initiation by MDA-MB-231 cells in NOD/SCID

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mice. After pretreating with 5 µM 28 or DMSO vehicle for 48 h, 50,000 MDA-MB-

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231 cells were injected into the mammary fat pads of female NOD/SCID mice (N =

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10 for the control and drug-treated groups, respectively). Mice were treated with 28

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at 100 mg/kg or vehicle by oral gavage, starting day 2 post-injection and continuing

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through day 12. Consistent with HDAC3 depletion effects, treatment with 28

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exhibited a suppressive effect on the tumorigenicity of MDA-MB-231 cells relative to

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vehicle control, though the P value of 0.053 bordered on being statistically

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significant. In addition, the drug-treated tumors grew at a slower rate than the control

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tumors (P = 0.0038) after 26 days of treatment.

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Discussion

Although the potential of using HDAC inhibitors to eradicate the CSC

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subpopulation has been demonstrated in multiple types of cancer [9], the underlying

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mechanism has not been fully elucidated. We previously demonstrated that HDAC8 is

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involved in regulating TNBC CSCs by maintaining the expression of Notch1 via

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protein stabilization [10]. Following this connection, we investigated the mechanistic

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link between HDAC3 and CSC homeostasis in TNBC cells. We obtained evidence

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that the suppressive effect of genetic depletion or pharmacological inhibition of

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HDAC3 on CSCs might be attributable to the inhibition of the Akt-GSK3β β-catenin

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signaling axis. It is well recognized that the activation status of Akt is controlled by a

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myriad of signaling effectors, including upstream kinases, phosphatases, and negative

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regulators [29]. Considering the importance of Akt in mediating cellular functions, the

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mechanism by which HDAC3 regulates Akt activation represents an intriguing

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ACCEPTED MANUSCRIPT therapeutic target. We speculate that the link between HDAC3 and Akt activation

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might not mediated through a general chromatin modeling mechanism based on the

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following rationale. From a mechanistic perspective, HDAC3 may mediate diverse

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biological functions in a cell context-specific manner via two distinct mechanisms

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[30]. First, HDAC3 forms complexes with the corepressors SMRT and N-CoR to

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facilitate the transcriptional repression of genes, especially those encoding nuclear

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receptors.

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transcription factors through deacetylation, including RelA, SRY, p53, and

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CBP/p300.

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Second, HDAC3 can also regulate transcriptional activity of various

In principle, HDAC3 may regulate the expression of an upstream

regulator of Akt phosphorylation via one of these two mechanisms, which is currently

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under investigation.

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According to the Human Protein Atlas, HDAC3 is overexpressed in multiple

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types of cancer, including that of breast, liver, lung, skin, and pancreas

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(www.proteinatlas.org/ENSG00000171720-HDAC3/cancer),

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involvement in tumorigenesis. The present study further demonstrated that HDAC3

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represents a potential anti-CSC target. Accordingly, we used the pan-HDAC inhibitor

17

1 previously developed in our laboratory as a starting point for synthesizing HDAC3-

18

selective inhibitors. From a drug development perspective, this strategy exploits the

19

advantages of retaining the drug-like properties of the parent molecule in the course

20

of structural modifications. The proof-of-concept of this strategy was in the efficacy

21

of the two HDAC3-selective inhibitors 18 and 28, both of which showed high

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HDAC3-inhibitory potency and isoform selectivity. Based on the previous reports

23

that use of F-containing benzamides as the Zn2+-chelating motif could enhance

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HDAC3 selectivity [26, 27], a key element of this “pan-to-HDAC3-selective

25

inhibitor” conversion was the replacement of the hydroxamate moiety with N-(2-

its

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suggesting

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ACCEPTED MANUSCRIPT amino-4-fluorophenyl)carboxamide, which resulted in the dissociation of HDAC6-

2

and HDAC8-inhibitory activities in resulting derivatives. Comparing the isoform

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selectivity between the compound pair of 27 versus 28 clearly indicates that the

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importance of the fluorine atom at the Zn2+-binding motif in improving ligand

5

recognition at the HDAC3 binding pocket. Moreover, optimal isoform selectivity

6

toward HDAC3 was achieved by varying the α-substituent on the 2-phenylacetyl

7

moiety, revealing the importance of this cap group in determining ligand interactions

8

with HDAC isoforms. In contrast, replacing the 4-aminobenzoyl linker of 5 and 9

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with a 5-aminopicolinoyl substructure was not beneficial. Although the resulting

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compounds 14 and 15 had improved HDAC3-inhibitory activity (IC50, 80 nM and 60

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nM, respectively), this change also gave rise to increased HDAC1- and HDAC2-

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inhibitory activities, therefore compromising the isoform selectivity.

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The suppressive effect of 18 and 28 on CSCs was confirmed by their inhibition of CD44+/CD24low

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clonogenic

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subpopulation of MDA-MB-231 cells (Figure 4B). Consistent with the HDAC3

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knockdown data, this anti-CSC effect correlated with the ability of 18 and 28 to

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inhibit Akt signaling, thereby downregulating the protein expression of β-catenin

18

(Figure 4C).

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suppressive effect of HDAC3 depletion on breast tumorigenicity in vivo in the MDA-

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MB-231 animal model.

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translational potential of HDAC3 inhibitors in combination with chemotherapeutic

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agents to target the CSC subpopulation in cancer treatment.

mammosphere

formation,

and/or

the

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Equally important, oral 28 at 100 mg/kg was able to mimic the

Together, these data provide a proof-of-concept for the

23 24 25

CONCLUSION Based on our finding that HDAC3 regulates TNBC CSCs by targeting the Akt-

15

ACCEPTED MANUSCRIPT GSK3β-β-catenin pathway, we used the pan HDAC inhibitor 1 as a scaffold to

2

develop HDAC3-selective inhibitors by replacing hydroxamate with the N-(2-amino-

3

4-fluorophenyl)carboxamide as the Zn2+-chelating motif, obtaining the proof-of-

4

concept with 18 and 28. These two derivatives exhibited high potency and isoform

5

selectivity in HDAC3 inhibition, and equally important, showed in vitro efficacy in

6

suppressing the CSC subpopulation of TNBC cells via the downregulation of β-

7

catenin. Moreover, oral 28 mimicked the suppressive effect of HDAC3 knockdown

8

on in vivo tumorigenesis in nude mice. These findings have simulated further

9

evaluation of 28 in enhancing the in vivo efficacy of different chemotherapeutic

10

agents and the use of 28 as a lead compound to develop more potent and selective

11

HDAC3 inhibitors. Based on the aforementioned SAR analysis, we will focus on

12

modifying the cap group of 28 by replacing the α,α,-dimethyl-phenylacetyl moiety

13

with rigid, non-aromatic functional groups to improve the HDAC3-inhibitory

14

potency, which is currently underway.

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EXPERIMENTAL SECTION

All commercially available reagents were used without further purification unless

18

otherwise stated. Anhydrous THF was obtained by distilling commercial reagent over

19

sodium, and anhydrous DMF was commercially available. Silica gel for column

20

chromatography was purchased from Merck Silica gel 60 (0.040 – 0.063 mm).

21

Routine 1H and 13C nuclear magnetic resonance spectra were recorded on the Bruker

22

NMR AV 400 or Bruker AVII 500 NMR. Samples were dissolved in deuterated

23

chloroform (CDCl3) or dimethyl sulfoxide (DMSO-d6), and tetramethylsilane (TMS)

24

was used as a reference. Mass spectrometry analyses were performed with a Waters,

25

LCT orthogonal acceleration time-of-flight (oa-TOF) LC/MS system. All compounds

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ACCEPTED MANUSCRIPT 1

for bioassays were identified with 1H NMR,

13

2

confirmed to be higher than 95%. Purities of all tested compounds were determined

3

by a Shimadzu LCMS-2020 system (comprised of a LC-20AD pump, a SPD-M30A

4

detector, an SIL-20AC autosampler and a Shim-pack GIST C18 2µm, 100 X 2.1 mm

5

I.D. C18 column). The general procedures for the synthesis of compounds 2-31 are

6

depicted in Scheme 1, and detailed synthesis is described in the Supplementary

7

Information.

8

compounds were conducted by Reaction Biology Cooperation (Malvern, PA).

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C NMR and HRMS, and purities

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HDAC isoform profiling and IC50 determination of individual

9

Cell Lines, Cell Culture, Reagents, and Antibodies. The breast cancer cell

11

lines, MDA-MB-231 and SUM-159, were purchased from American Type Culture

12

Collection (Manassas, VA) and Asterand Biosciences (Detroit, MI), respectively.

13

MDA-MB-231 cells were maintained in DMEM medium containing 10% fetal bovine

14

serum, and SUM159 cells were cultured in Ham’s F-12 (Life Technologies)

15

supplemented with 5% fetal bovine serum, 5 µg/ml insulin, and 1 µg/ml

16

hydrocortisone. All cells were cultured at 37°C in a humidified incubator containing

17

5% CO2. AR-42 (1) was a kind gift from Arno Therapeutics (Flemington, NJ).

18

Antibodies for various proteins were purchased from following sources: HDAC3, β-

19

catenin, Histone H3, tubulin, acetyl-tubulin and β-actin (Santa Cruz, CA); p-Ser473-

20

Akt, AKT, p-Ser9-GSK3β, GSK3β, c-Myc, BMI-1, and Ac-H3K9 (Cell Signaling

21

Technology; Beverly, MA); pan-Ac-H3 (Millipore; Billerica, MA); APC-conjugated

22

CD44 and PE-conjugated CD24, (Thermo Fisher Scientific, Waltham, MA);

23

horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Jackson

24

ImmunoResearch Laboratories; West Grove, PA).

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ACCEPTED MANUSCRIPT 1

Generation of HDAC3-depleted stable clones or HDAC3-overexpressing

2

MDA-MB-231 cells.

3

pMD2.G (#12259, Addgene) and shRNA of HDAC3 (TRCN0000194993,

4

TRCN0000196267; RNAi Core of Academia Sinica, Taipei, Taiwan) were

5

cotransfected in 293T cells with Lipofectamine 2000 (Life Technologies) according

6

to the manufacturers’ instructions. Viral particles were used for infection of target

7

cells and stable clones were maintained with puromycin (Life Technologies; Grand

8

Island, NY). pLAS.Void plasmid served as negative control. The HDAC3 expression

9

plasmid (#13819, Addgene; Cambridge, MA) was transfected with Lipofectamine

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2000 in MDA-MB-231 cells according to the manufacturers’ instructions.

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For lentivirus generation, psPAX2 (#12260, Addgene),

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Cell Lysis and Immunoblotting. Drug-treated cells were collected and then

13

lysed in a buffer containing 1% sodium dodecyl sulfate (SDS), 10 mM EDTA, 50

14

mM Tris–HCl (pH 8.1), and 1% protease and phosphatase inhibitor mixture. Lysates

15

were sonicated and then centrifuged at 13,000 rpm for 10 min. Protein concentrations

16

in the supernatants were determined (Micro BCA Protein Assay Kit, Pierce

17

Biotechnology, Rockford, IL) and equal amounts of proteins were resolved via SDS-

18

PAGE and transferred to a nitrocellulose membrane (GE Healthcare Life Sciences,

19

Pittsburgh, PA). The membrane was washed twice with Tris-buffered saline

20

containing 0.1% Tween-20 (TBST), blocked with TBST containing 5% non-fat milk

21

for 30 min, and then incubated with primary antibody (1:1000 dilution) in TBST at

22

4°C overnight. After washing with TBST, the membrane was incubated with goat

23

anti-rabbit or anti-mouse IgG–HRP conjugates (1:5000 dilution) for 1 h at room

24

temperature. The immunoblots were visualized by enhanced chemiluminescence.

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ACCEPTED MANUSCRIPT RNA Extraction and RT-PCR. Total RNA was isolated with TRIzol (Thermo

2

Fisher Scientific) according to the manufacturer's protocol. From each sample, 2 µg

3

total RNA was reverse-transcribed into cDNA using the iScriptTM cDNA Synthesis

4

Kit (Bio-Rad; Hercules, CA) and the cDNA were separated by electrophoresis. PCR

5

products were resolved by electrophoresis in 2% agarose gels and visualized by

6

ethidium bromide staining. The sequences of primers used for RT-PCR were as

7

follows. β-catenin, forward primer: GCTGATTTGATGGAGTTGGA; reverse primer:

8

GCTACTTGTTCTTGAGTGAA;

9

GCTCGTCGTGGACAACGGCTC, reverse primer: CAAACATGATCTGGGTCAT-

primer:

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forward

CTTCTC.

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β-actin,

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Colony Formation Assays. MDA-MB-231 or SUM-159 cells were seeded in

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six-well plates at a density of 1,000 cells per well and were left to attach overnight.

14

Vehicle control (DMSO) or increasing concentrations of test agents were added to the

15

cells in the presence of 5% FBS for 48 h. The drug mixture was washed out, and the

16

plates were incubated with media for 7-14 days until colonies were visible. Each drug

17

concentration was assessed in triplicate. The colonies were fixed with 4%

18

formaldehyde (Sigma-Aldrich) and stained with crystal violet (5 mg/ml in 2%

19

ethanol, Sigma-Aldrich). Colonies containing more than 50 cells were counted. Cell

20

survival is expressed as a percentage and was determined from the numbers of

21

colonies present in the drug-treated groups relative to the vehicle-treated control

22

group.

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Mammosphere formation assays. MDA-MB-231 (1,000 cells/well) and

25

SUM159 (500 cells/well) cells were seeded onto ultra-low attachment 24-well flat

19

ACCEPTED MANUSCRIPT bottom plates (Corning) in serum-free culture medium (MammoCult™, STEMCELL

2

Technologies; Vancouver, Canada) supplemented with 1 µM hydrocortisone

3

(MammoCult™) and 2 µg/ml heparin (MammoCult™). Cells were then treated with

4

28 at the indicated concentrations in triplicate for 7 days. The number of

5

mammospheres was counted at 100X magnification.

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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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bromide

(MTT)

Assays. To determine the drug effects on cell viability, MDA-MB-231 cells were

9

seeded onto 96-well plates at a density of 5,000 cells per well in the presence of 10%

10

FBS. After overnight incubation, cells were exposed to test agents in the presence of

11

5% FBS for 24, 48, and 72 h. After treatment, cells were incubated with MTT

12

(Biomatik, Wilmington, DE) for an additional 1 h. The medium was then removed

13

from each well and replaced with DMSO to dissolve the reduced MTT dye for

14

subsequent colorimetric measurement of absorbance at 560 nm. Cell viabilities are

15

expressed as percentages of viable cells relative to the corresponding vehicle-treated

16

control group. For the cell proliferation analysis of HDAC3KD stable clones versus

17

parental MDA-MB-231 cells, a total of 500 cells per well were seeded in 96-well

18

plates and incubated for the indicated time at 37°C. The cell proliferation was

19

analyzed by detecting the absorbance of reduced MTT dye.

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Growth Inhibition Assay.

Inhibition of cell growth was determined by a

22

modified crystal violet assay [31]. MDA-MB-231 cells were plated in 96-well culture

23

plates (5000 cells/well). After overnight incubation, cells were treated with individual

24

test agents at the indicated concentrations in the presence of 5% FBS for 24, 48 and

25

72hrs. After treatment, cells were washed with PBS and stained with crystal violet

20

ACCEPTED MANUSCRIPT 1

solution (0.5% crystal violet in 20% methanol). After 20 min, the wells were washed,

2

dried and the stain was solubilized with 2% SDS.

3

measured by a plate reader. The percentage of growth inhibition was determined

4

using the equation (1-Nt/Nc) x 100, where Nt and Nc are the absorbencies of stain in

5

treated and vehicle control wells, respectively.

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Absorbance at 550nM was

Flow cytometry. DMSO- or 28-treated MDA-MB-231 cells were harvested,

8

washed with stain buffer (BD, Franklin Lakes, NJ), and incubated with specific FITC-

9

or phycoerythrin-conjugated monoclonal antibodies for CD44 and CD24 on ice in the

10

dark for 30 min. Cells were washed with stain buffer, and the pellets were collected

11

and suspended in PBS. The CD44+/CD24low subpopulation was analyzed using a

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FACSCalibur (BD Biosciences) flow cytometer.

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In Vivo Tumorigenesis Study.

Female NOD/SCID mice (NOD.CB17-

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Prkdcscid /NCrHsd; 5–6 weeks of age; Harlan, Indianapolis, IN) were group-housed

16

under constant photoperiod (12-h light/12-h dark) with ad libitum access to sterilized

17

food and water. All experimental procedures were done according to protocols

18

approved by The Ohio State University Institutional Animal Care and Use

19

Committee. To assess the effect of HDAC3 knockdown on tumor initiation in vivo,

20

stable HDAC3KD clone #6267 cells [50,000 cells/0.1 ml in 50% Matrigel (BD

21

Biosciences)] were implanted into the 4th inguinal mammary fat pads of NOD/SCID

22

mice. An equal number of MDA-MB-231 cells transfected with control lentiviral

23

constructs were injected into the contralateral mammary fat pads to serve as negative

24

control. Tumors were measured with calipers and the volumes were calculated using

25

V = (width2 x length) x 0.52. To assess the effect of 28 on tumor initiation in vivo,

26

MDA-MB-231 cells were pretreated with 5 µM 28 for 48 h, and control cells were

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ACCEPTED MANUSCRIPT treated with the same amount of DMSO. Treated cells were then collected by trypsin

2

(Gibco), the cell density was adjusted to 50,000 cells/0.1ml in 50% Matrigel, and

3

implanted into the left 4th inguinal mammary fat pads of NOD/SCID mice. The day

4

after cell implantation, mice were treated with 28 at 100 mg/kg body weight or

5

vehicle (0.5% methylcellulose/0.1% Tween 80 [v/v] in sterile water) once daily by

6

oral gavage.

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Statistical Analysis. In vitro experiments were performed at least three times

9

and data are presented as means ± SD. Group means were compared using one-way

10

ANOVA followed by Student’s t tests. For the in vivo experiments, differences in

11

tumor incidence and tumor volume were analyzed by log-rank test and Student’s t-

12

test, respectively. Differences were considered significant at P < 0.05.

13 ASSOCIATED CONTENT

15

Supporting Information

Synthetic procedures of 2-31 and purity of individual derivatives as determined by HPLC

18

II. NMR and HRMS spectra, and HPLC chromatograms

19

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III. Images of all Western blots in full from Figure 3

21

AUTHOR INFORMATION

22

Co-corresponding Authors

23

Professor Ching-Shih Chen, E-mail: [email protected] or

24

[email protected]; Professor Ling-Wei Hsin, E-mail: [email protected]

25

Author Contributions

22

ACCEPTED MANUSCRIPT The manuscript was written through contributions from all authors. All authors have

2

given approval of the final version of the manuscript. H.Y.H. and H.C.C. contributed

3

equally to this manuscript.

4

Notes

5

CSC is the inventor of AR-42, which was licensed to Arno Therapeutics by the Ohio

6

State University Foundation for clinical development. He has received payments

7

associated with the licensing of this agent according to the University guidelines. The

8

other authors declare no competing financial interest.

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9 ACKNOWLEDGMENTS

11

This work was supported by grant # MOST 105-2321-B-001-064 from the Team of

12

Excellent Research Program of the Ministry of Science and Technology (Taiwan), the

13

National Health Research Institutes (Taiwan) grant NHRI-EX105-10521BI, and

14

Lucius A. Wing Endowed Chair Fund from The Ohio State University Medical

15

Center. We thank Dr. Cindy Lee at the Institute of Biological Chemistry at Academia

16

Sinica for the scientific editing of this manuscript. We thank Dr. Xiaokui Mo for the

17

statistical analysis of the difference in tumor incidence.

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ABBREVIATIONS USED

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CSC, cancer stem cells; TNBC, triple-negative breast cancer; HDAC, histone

21

deacetylase; GSK3β, glycogen synthase kinase 3β; MTT, 3-(4,5-dimethylthiazol-2-

22

yl)-2,5-diphenyltetrazolium bromide; SAR, structure-activity relationship

23 24

REFRENCES

25 26

[1]

A. Pietras, Cancer stem cells in tumor heterogeneity, Adv Cancer Res, 112 (2011) 255-281.

23

ACCEPTED MANUSCRIPT

[6]

[7]

[8]

[9] [10]

[11]

[12]

EP

[13]

RI PT

[5]

SC

[4]

M AN U

[3]

A.K. Singh, R.K. Arya, S. Maheshwari, A. Singh, S. Meena, P. Pandey, O. Dormond, D. Datta, Tumor heterogeneity and cancer stem cell paradigm: updates in concept, controversies and clinical relevance, Int J Cancer, 136 (2015) 1991-2000. H. Clevers, The cancer stem cell: premises, promises and challenges, Nat Med, 17 (2011) 313-319. T. Shibue, R.A. Weinberg, EMT, CSCs, and drug resistance: the mechanistic link and clinical implications, Nat Rev Clin Oncol, (2017). F. Marcucci, G. Stassi, R. De Maria, Epithelial-mesenchymal transition: a new target in anticancer drug discovery, Nat Rev Drug Discov, 15 (2016) 311-325. K. Chen, Y.H. Huang, J.L. Chen, Understanding and targeting cancer stem cells: therapeutic implications and challenges, Acta Pharmacol Sin, 34 (2013) 732740. D.L. Dragu, L.G. Necula, C. Bleotu, C.C. Diaconu, M. Chivu-Economescu, Therapies targeting cancer stem cells: Current trends and future challenges, World J Stem Cells, 7 (2015) 1185-1201. N. Takebe, P.J. Harris, R.Q. Warren, S.P. Ivy, Targeting cancer stem cells by inhibiting Wnt, Notch, and Hedgehog pathways, Nat Rev Clin Oncol, 8 (2011) 97-106. M. Dvorakova, T. Vanek, Histone deacetylase inhibitors for the treatment of cancer stem cells, Med Chem Commun, 7 (2016) 2217-2231. M.W. Chao, P.C. Chu, H.C. Chuang, F.H. Shen, C.C. Chou, E.C. Hsu, L.E. Himmel, H.L. Huang, H.J. Tu, S.K. Kulp, C.M. Teng, C.S. Chen, Nonepigenetic function of HDAC8 in regulating breast cancer stem cells by maintaining Notch1 protein stability, Oncotarget, 7 (2016) 1796-1807. Q. Lu, Y.T. Yang, C.S. Chen, M. Davis, J.C. Byrd, M.R. Etherton, A. Umar, C.S. Chen, Zn2+-chelating motif-tethered short-chain fatty acids as a novel class of histone deacetylase inhibitors, J Med Chem, 47 (2004) 467-474. Q. Lu, D.S. Wang, C.S. Chen, Y.D. Hu, C.S. Chen, Structure-based optimization of phenylbutyrate-derived histone deacetylase inhibitors, J Med Chem, 48 (2005) 5530-5535. M.L. Guzman, N. Yang, K.K. Sharma, M. Balys, C.A. Corbett, C.T. Jordan, M.W. Becker, U. Steidl, O. Abdel-Wahab, R.L. Levine, G. Marcucci, G.J. Roboz, D.C. Hassane, Selective activity of the histone deacetylase inhibitor AR42 against leukemia stem cells: a novel potential strategy in acute myelogenous leukemia, Mol Cancer Ther, 13 (2014) 1979-1990. H. Cheng, Z. Xie, W.P. Jones, X.T. Wei, Z. Liu, D. Wang, S.K. Kulp, J. Wang, C.C. Coss, C.S. Chen, G. Marcucci, R. Garzon, J.M. Covey, M.A. Phelps, K.K. Chan, Preclinical Pharmacokinetics Study of R- and S-Enantiomers of the Histone Deacetylase Inhibitor, AR-42 (NSC 731438), in Rodents, AAPS J, 18 (2016) 737-745. D.W. Sborov, A. Canella, E.M. Hade, X. Mo, S. Khountham, J. Wang, W. Ni, M. Poi, C. Coss, Z. Liu, M.A. Phelps, A. Mortazavi, L. Andritsos, R.A. Baiocchi, B.A. Christian, D.M. Benson, J. Flynn, P. Porcu, J.C. Byrd, F. Pichiorri, C.C. Hofmeister, A phase 1 trial of the HDAC inhibitor AR-42 in patients with multiple myeloma and T- and B-cell lymphomas, Leuk Lymphoma, (2017) 1-9. W.H. Lien, E. Fuchs, Wnt some lose some: transcriptional governance of stem cells by Wnt/beta-catenin signaling, Genes Dev, 28 (2014) 1517-1532.

TE D

[2]

AC C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

[14]

[15]

[16]

24

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

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[17] T. Zhan, N. Rindtorff, M. Boutros, Wnt signaling in cancer, Oncogene, 36 (2017) 1461-1473. [18] S.G. Pohl, N. Brook, M. Agostino, F. Arfuso, A.P. Kumar, A. Dharmarajan, Wnt signaling in triple-negative breast cancer, Oncogenesis, 6 (2017) e310. [19] J. Xu, J.R. Prosperi, N. Choudhury, O.I. Olopade, K.H. Goss, beta-Catenin is required for the tumorigenic behavior of triple-negative breast cancer cells, PLoS One, 10 (2015) e0117097. [20] J. Xu, Y. Chen, D. Huo, A. Khramtsov, G. Khramtsova, C. Zhang, K.H. Goss, O.I. Olopade, beta-catenin regulates c-Myc and CDKN1A expression in breast cancer cells, Mol Carcinog, 55 (2016) 431-439. [21] G. Dontu, W.M. Abdallah, J.M. Foley, K.W. Jackson, M.F. Clarke, M.J. Kawamura, M.S. Wicha, In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells, Genes Dev, 17 (2003) 1253-1270. [22] G.B. Jang, J.Y. Kim, S.D. Cho, K.S. Park, J.Y. Jung, H.Y. Lee, I.S. Hong, J.S. Nam, Blockade of Wnt/beta-catenin signaling suppresses breast cancer metastasis by inhibiting CSC-like phenotype, Sci Rep, 5 (2015) 12465. [23] F.F. Wagner, U.M. Wesmall yi, M.C. Lewis, E.B. Holson, Small molecule inhibitors of zinc-dependent histone deacetylases, Neurotherapeutics, 10 (2013) 589-604. [24] F. Thaler, C. Mercurio, Towards selective inhibition of histone deacetylase isoforms: what has been achieved, where we are and what will be next, ChemMedChem, 9 (2014) 523-526. [25] C. Hubbert, A. Guardiola, R. Shao, Y. Kawaguchi, A. Ito, A. Nixon, M. Yoshida, X.F. Wang, T.P. Yao, HDAC6 is a microtubule-associated deacetylase, Nature, 417 (2002) 455-458. [26] H. Jia, J. Pallos, V. Jacques, A. Lau, B. Tang, A. Cooper, A. Syed, J. Purcell, Y. Chen, S. Sharma, G.R. Sangrey, S.B. Darnell, H. Plasterer, G. Sadri-Vakili, J.M. Gottesfeld, L.M. Thompson, J.R. Rusche, J.L. Marsh, E.A. Thomas, Histone deacetylase (HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate polyglutamine-elicited phenotypes in model systems of Huntington's disease, Neurobiol Dis, 46 (2012) 351-361. [27] X. Li, Y. Zhang, Y. Jiang, J. Wu, E.S. Inks, C.J. Chou, S. Gao, J. Hou, Q. Ding, J. Li, X. Wang, Y. Huang, W. Xu, Selective HDAC inhibitors with potent oral activity against leukemia and colorectal cancer: Design, structure-activity relationship and anti-tumor activity study, Eur J Med Chem, 134 (2017) 185206. [28] M. Al-Hajj, M.S. Wicha, A. Benito-Hernandez, S.J. Morrison, M.F. Clarke, Prospective identification of tumorigenic breast cancer cells, Proc Natl Acad Sci U S A, 100 (2003) 3983-3988. [29] Y. Liao, M.C. Hung, Physiological regulation of Akt activity and stability, Am J Transl Res, 2 (2010) 19-42. [30] P. Karagianni, J. Wong, HDAC3: taking the SMRT-N-CoRrect road to repression, Oncogene, 26 (2007) 5439-5449. [31] M. Feoktistova, P. Geserick, M. Leverkus, Crystal Violet Assay for Determining Viability of Cultured Cells, Cold Spring Harb Protoc, 2016 (2016) pdb prot087379.

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ACCEPTED MANUSCRIPT 1 2 3

Scheme 1. General Synthetic Procedures for 2- 31.

4

Figure Legends

5

Figure 1. Evidence that HDAC3 plays a role in regulating the CSC subpopulation in

6

TNBC cells.

7

HDAC3 by two different shRNAs (#4994 and #6267) versus control shRNA

8

treatment (Ctl) on the expression and/or phosphorylation levels of HDAC3, Akt,

9

GSK3β, and β-catenin and its target gene products c-Myc and BMI-1 in MDA-MB-

10

231 and SUM-159 cells. (B & C) Functional analyses of the effect of the stable

11

depletion of HDAC3 by two different shRNAs versus control on cell proliferation

12

(left; N = 6), colony formation (center; N = 6), and mammosphere formation (right; N

13

= 6) in (B) MDA-MB-231 and (C) SUM-159 cells. (D) Western blot analysis of the

14

effect of the ectopic expression of HDAC3 versus control on the expression and/or

15

phosphorylation levels of HDAC3, Akt, GSK3β, and β-catenin and its target gene

16

products c-Myc and BMI-1 in MDA-MB-231.

17

knockdown (HDAC3KD) versus control shRNA on (left) tumor-initiating ability by

18

monitoring tumor incidence, and (right) xenograft tumor growth, measured at Day 27

19

after implantation, of MDA-MB-231 cells. Data are expressed as means ± S.D. (N =

20

9).

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(E) Effect of stable HDAC3

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(A) Western blot analysis of the effect of the stable depletion of

22

Figure 2. (A) Structures of 1 (AR-42) versus various class I HDAC inhibitors and

23

HDAC3-selective inhibitors. (B) Structure of 2-31 and the respective activities in

24

inhibiting the deacetylase activities of HDAC1, HDAC2, and HDAC3 (HDAC1/2/3),

25

which were expressed in two ways: a, % inhibition at 1 µM; b, IC50 values.

26

26

ACCEPTED MANUSCRIPT 1

Figure 3.

2

indicated concentrations on histone H3 pan and H3K9 acetylation, tubulin acetylation,

3

and Notch 1 expression in MDA-MB-231 cells after 48 h of treatment, in which 1

4

(0.25 µM) was used as a positive control in each blot. Images of some of these blots,

5

including those depicting 1 versus 3, 1 versus 10 and 16, 1 versus 15 and 13, and 1

6

versus 18, 24, 20, 27, and 28, were not contiguous because lanes of repeated samples

7

were cropped out from the blots.

8

Supplemental Information.

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Western blot analyses of the effects of representative derivatives at

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Images of these full blots are shown in

9

Figure 4. The in vitro and/or in vivo efficacy of 18 and 28 in suppressing the CSC

11

subpopulation of MDA-MB-231 cells, in part through the downregulation of β-

12

catenin expression. (A) Concentration- and time-dependent effects of 18 and 28 on

13

cell viability by MTT assays (left) and growth inhibition by crystal violet staining

14

(right). Value, means + S.D. (N = 6). (B) Concentration-dependent suppressive

15

effects of 18 and 28 on (upper) colony formation and (lower) mammosphere

16

formation. Value, means + S.D. (N = 6). (C) Upper, Western blot analysis of

17

concentration-dependent suppressive effects of 18 and 28 on Akt phosphorylation, β-

18

catenin expression, and histone H3 pan and H3K9 acetylation. Lower, RT-PCR

19

analysis of concentration-dependent effects of 28 on β-catenin mRNA expression. (D)

20

Flow cytometric analysis of concentration-dependent suppressive effects of 28 on the

21

CD44+/CD24low subpopulation. (E) Effect of daily 28 at 100 mg/kg via oral gavage

22

(N = 10) versus vehicle control (N = 10) on (left) tumor-initiating ability by

23

monitoring tumor incidence, and (right) xenograft tumor growth, measured at Day 27

24

after implantation of MDA-MB-231 cells. Data are expressed as means ± S.D.

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ACCEPTED MANUSCRIPT

Table 1. HDAC isoform inhibition profiles of 1 and representative derivatives, each at 1 µM. HDAC isoforms; % Inhibition at 1 µM*

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HDAC1 HDAC2 HDAC3 HDAC4 HDAC5 HDAC6 HDAC7 HDAC8 95%

72%

93%

21%

0%

100%

22%

87%

2

55%

31%

30%

0%

0%

20%

1%

7%

3

80%

57%

76%

9%

9%

16%

9%

38%

5

42%

26%

78%

0%

0%

0%

0%

6%

6

6%

4%

0%

3%

0%

0%

0%

0%

20

41%

32%

36%

24

78%

38%

33%

27

49%

34%

36%

28

8%

5%

87%

29

0%

9%

M AN U 0%

0%

0%

0%

5%

0%

0%

0%

0%

0%

6%

0%

4%

0%

0%

0%

0%

4%

0%

0%

3%

0%

0%

14%

TE D 82%

SC

1

0%

AC C

EP

* Each value represents the average of two determinations

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

1. HDAC inhibitors could suppress cancer stem cells in multiple types of cancer. 2. HDAC isoform, HDAC3, plays a key role in regulating triple-negative breast CSCs. 3. HDAC3 mechanistically links to CSC homeostasis by regulating β-catenin expression. 4. HDAC3 inhibitor, 28 suppresses triple-negative breast CSCs in vitro and in vivo.