Potent Activity of the Bromodomain Inhibitor OTX015 in Multiple

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Potent Activity of the Bromodomain Inhibitor OTX015 in Multiple Myeloma Jixiang Shi, Sha Song, Huiying Han, Hongxia Xu, Moli Huang, Chen’ao Qian, Xiaojuan Zhang, Lu Ouyang, Yating Hong, Wenzhuo Zhuang, and Bingzong Li Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00554 • Publication Date (Web): 26 Jul 2018 Downloaded from http://pubs.acs.org on July 28, 2018

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Molecular Pharmaceutics

Potent Activity of the Bromodomain Inhibitor OTX015 in Multiple Myeloma Jixiang Shi1,2, +, Sha Song3, +, Huiying Han3, Hongxia Xu3, Moli Huang4, Chen’ao Qian4, Xiaojuan Zhang3, Lu Ouyang3, Yating Hong1, Wenzhuo Zhuang3, *, Bingzong Li1,* 1

Department of Haematology, the Second Affiliated Hospital of Soochow University, Suzhou, China; 2 Department of Haematology, the Central Hospital of Zibo, Zibo, China; 3 Department of Cell Biology, School of Biology & Basic Medical Sciences, Soochow University, Suzhou, China; 4 Department of Bioinformatics, School of Biology & Basic Medical Sciences, Soochow University, Suzhou, China; +

: Contribute equally

*: To whom correspondence should be addressed: Wenzhuo Zhuang Department of Cell biology School of Biology & Basic Medical Sciences, Soochow University Ren Ai Road 199 Suzhou 215123, China Phone: 86−512−65880103 Fax: 86-512-65880103 E-mail: [email protected] Bingzong Li Department of Haematology, The Second Affiliated Hospital of Soochow University San Xiang Road 1055 Suzhou 215006, China. Telephone: 86−512−67784069, E-mail: [email protected]

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Molecular Pharmaceutics 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 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Several studies demonstrate that the bromodomain inhibitor OTX015 has an anti-tumor activity in cancers. However, translation of these data to molecules suitable for clinical development has not yet to be accomplished in multiple myeloma (MM). Here, we identified genes and biologic processes which substantiated the anti-myeloma activity of OTX015 with global transcriptomics. OTX015 exerted a strong anti-proliferative effect and induced cell cycle arrest in vitro. Gene expression profiling uncovered that OTX015 targeted NF-κB, EGFR, cell cycle regulation and cancer proliferation signaling pathway. Gene expression signatures displayed various level of sensitivity to OTX015 were also identified. The data also showed that oral administration of OTX015 displayed significant anti-tumor activity in mice model of disseminated human myeloma. In addition, our study provided the first evidence and rationale that OTX015 could promote osteoblast differentiation of mesenchymal stem cells (MSCs) and inhibited osteoclast formation and resorption in vivo experiments. Herein our results expanded the understanding of the mechanism for BET inhibitors OTX015 in MM. Our study provided an empressive basis for the clinical application of the novel anti-myeloma agent OTX015 and uncovered signaling pathways may play key roles in myeloma cell proliferation.

KEYWORDS: Multiple myeloma, Bromodomain inhibitor, Mesenchymal stem cells, OTX015, Osteogenesis

INTRODUCTION


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Multiple myeloma (MM) is a plasma cell malignancy, characterized by hypocalcemia, renal insufficiency, anemia or bone lesions1. Over the past decade, with the development of novel agents such as the immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs), great progress has been made in the therapeutics of MM2. These agents have greatly improved the survival of MM patients, however, nearly all MM patients become refractory to PIs and IMIDs and MM still remains incurable in modern era3. New therapeutic strategies for refractory or relapsing myeloma patients are extremely needed. Bromodomain and extraterminal domain (BET) proteins comprise the universal expressed BRD2, BRD3, BRD4 and the testisrestricted BRDT, and mainly recognize acetylated lysine of histone 44. Bromodomain recognizing and binding to acetylated lysine of histone recruits the elongation complex and binds at the extra-terminal domain, where gene promoters would localize to trigger downstream transcription.5,6. BET proteins regulate gene expression and participate in cancer pathogenesis. Several studies have provided the rationale for targeting BET proteins as a strategy for the development of novel anti-cancer drugs7-10. BET inhibitors are in early phase clinical trials for acute myeloid leukemia, acute lymphoblastic leukemia, lymphoma and multiple myeloma11,12. OTX015, a new oral BET bromodomain inhibitor occupies the acetyl-binding pockets of BRD2, BRD3, BRD4, causing in their release from the active chromatin and the repression of downstream signaling to RNA polymerases13. OTX015 has entered clinical development in phase I studies for patients with hematologic malignancies11,14. It was



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reported based on the results from the phase I that the patients with myeloma showed clinical responses to orally available OTX01511. Researches are being actively pursued and may guide the future development of OTX015. In this study, we assessed the activity of OTX015 in myeloma. Vitro experiments were performed to identify the genetic features and to reveal the mechanism of OTX015 anti-tumor activity. In mice model established with bone marrow– disseminated human multiple myeloma, orally OTX015 has a marked action on controlling tumor burden and also provided an impressive benefit in associated bone disease. Our study provided the rationale for further clinical testing of the novel agent OTX015 in MM.

MATERIALS and METHODS

Cell Lines and Primary Samples 8226 and MM.1S myeloma cell lines were cultured as previously described

15

. Cell

lines were authenticated by short tandem repeat profiling (Genetic Testing Biotechnology Corporation, Suzhou). Samples were collected at the Second Affiliated Hospital of Soochow University after informed consent was obtained according to the institutional guidelines. The study was approved by the Ethics Committee of the Affiliated Hospital of Soochow University. Bone marrow-derived MSCs were isolated and cultured as described previously

16-20

. The detailed clinical characteristics of these patients are provided in


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Table S1.

Statistical analysis We used R (version 3.3.2) to conduct all the statistical analyses in this study. We presented the data as mean ± SD (standard deviation). We used Student’s t test to compare the difference of mean values between case and control groups and to calculate p-values. As usual, we considered p-value lower than 0.05 as statistically significant.

Other assays are detailed in supplemental materials and methods.

RESULTS

OTX015 reduced proliferation and induced cell cycle arrest in myeloma cells. We first assessed the anti-proliferative activity of OTX015 in myeloma cell lines MM.1S and 8226. OTX015 was active in both cell lines in a dose-dependent manner (IC50 of OTX015 on MM.1S: 40 nmol/L. IC50 of OTX015 on 8226: 1600 nmol/L) (Figure 1A) after their exposure to the agent for 72 hours. OTX015 induced cell cycle arrest with G1 accumulation and decreased S phase (Figure 1B, 1C). But, the percentage of cells in sub-G1 did not increase in MM.1S and 8228 after OTX015 treatment. Then we evaluated the induction of apoptosis after OTX015 treatment in MM.1S and 8226 by Annexin-V staining. The population of apoptotic cells was not significantly higher in MM.1S and 8226 after OTX015 exposure compared to the



vehicle control (Figure 1D). Treatment with OTX015 did not significant increase the fraction of cells in sub-G1 and did not induce an increase in the proportion of Annexin V-positive cells, indicating that these cells were not undergoing active apoptosis. Therefore, OTX015 reduced cell proliferation and induced cell cycle arrest, but not apoptosis in myeloma cells.

Gene expression signature of OTX015 in myeloma cells To acquire a universal view on the transcriptional changes after OTX015 treatment, gene expression profiling (GEP) was performed on MM.1S and 8226 treated with vehicle or OTX015 (40 nmol/L for MM.1S, 1600 nmol/L for 8226) for 12 hours. Since the two cell lines had a great difference in IC50 value, the gene ranking metrics and GSEA of the GEP data from each cell line were performed, respectively, in order to study the coordinated changes in biologic networks associated with each phenotype. The data indicated that after OTX015 exposure, only about eight genes were overlapping between MM.1S and 8226 cell lines among the top (upregulated) and bottom (downregulated) 100 genes in the gene ranking metrics (Figure 2A, 2B), reflecting the distinct oncogenic programs and genetic profiles of the two cells. Previous study showed that c-Myc gene expression is potently suppressed by BET domain inhibition in solid tumors and hematologic cancers21-24. Consistent with these data, GEP data showed that c-Myc was downregulated after OTX015 exposure in both myeloma MM.1S and 8226 cells. C-Myc downregulation was confirmed by


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qPCR and western blot (Figure 2C). BET-bromodomain inhibition does not always downregulated c-Myc expression, and alternative c-Myc-independent mechanisms can also be involved in the observed anti-proliferative effect13,25. C-Myc-independent signaling pathways that were enriched in both or in one of the two cell lines were identified (Figure 2D, 2E, Table S3, S4). These included important biologic processes: NF-κB, cell cycle regulation, cancer proliferation and EGFR signaling, similar to those reported after exposure to another BET domain inhibitor, JQ1, in myeloma22,23,26. The GEP analysis also showed that OTX015 effects on myeloma were generally consistent with that on lymphoma24.

Gene expression profile was associated with sensitivity to OTX015. In order to investigate molecular signatures that could predict the sensitivity to OTX015 in myeloma, we analyzed the sensitivity with the gene expression profile in MM.1S with lower IC50 (40 nmol/L) and 8226 with higher IC50 (1600 nmol/L). GSEA analysis revealed the enrichment of PTPN4, CCND1 and mitotic signaling genes in OTX015 sensitive myeloma cell line (MM.1S) (Figure 3A, Table S4). Whereas, transcripts negatively associated with OTX015 sensitivity were enriched of genes involved in methylation, proteasome and immune responses signaling pathway (Figure 3B, Table S5). Heat maps showed the top differentially ranked transcripts between MM.1S and 8226 cells (Figure 3C). Here, we identified gene expression signatures associated with OTX015 sensitivity in myeloma cells. These data may help to identify molecularly defined subsets of



patients with myeloma that are most likely to benefit from OTX015.

OTX015 reduced multiple myeloma tumor burden. To evaluate the therapeutic potential of OTX015 in vivo in myeloma, bioluminescent MM models (8226-luc) recapitulated MM patients clinical features were established27. Tumor-bearing mice were treated with OTX015 administered by oral gavage (25 mg/kg, once a day) or vehicle control. The models displayed an acceptable tolerance receiving daily oral doses of OTX015 up to 25mg/kg with no evident decrease of body weight compared with vehicle control. OTX015 treatment dramatically decreased the tumor burden measured by serial, whole-body, noninvasive bioluminescence imaging (Figure 4A and 4B). Moreover, OTX015-treated animals had a significant prolongation in overall survival compared with vehicle-treated animals (Figure 4C). These data firstly represented that OTX015 could significantly delay myeloma progression in vivo.

OTX015 promoted MSCs osteogenesis in vitro and restrained myeloma-ralated bone lesions in vivo. One of the main features of MM is the occurrence of bone damage caused by severe imbalance of bone remodeling28,29. The osteogenesis of human mesenchymal stem cells is characteristically impaired in myeloma patients, resulting in osteoblast deficiency28,29. It is valuable to investigate whether OTX015 is effective on osteogenic process of MSCs. Primary MSCs were derived from myeloma patients


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with bone lesions. MSCs1 and MSCs2 corresponded to patient #1 and patient #2, respectively. MSCs1 and MSCs2 were treated with OTX015 or vehicle control in osteogenic differentiation medium for 14 days. As assessed by Von Kossa staining and alizarin red staining assays, OTX015 significantly increased bone nodule formation (Figure 5A). We next studied the bone anabolic and antiresorptive effects of OTX015 in mice models with bone marrow–disseminated human myeloma. MicroCT analyses of trabecular and cortical bone were performed. The trabecular structures of distal femurs were measured to reveal that whether OTX015 could prevent osteolytic destruction. The results turned out that vehicle-treated group suffer much more severe bone loss while OTX015-group had a more complete bone structure. (Figure 5B). Trabecular bone volume and connectivity were significantly increased in OTX015-treated group, while trabecular separation was much lower compared to vehicle-group. (Figure 5C-E). We also assessed some bone turnover markers which indicated bone resorption or formation. The CTX, a bone resorption marker, was dramatically decreased in OTX015–treated group (Figure 5F). The bone formation marker P1NP increased significantly in the OTX015–treated group relative to the vehicle group (Figure 5G). Taken together, OTX015 was potentially beneficial to myeloma-related bone lesions in vivo in addition to its anti-myeloma activity in MM.

Identifying mechanisms involved in osteogenesis induction by OTX015 To investigate the underlying mechanisms of osteogenesis induction by OTX015 in



MSCs, gene expression profilings of MSCs treated with another BET inhibitor JQ1 were analyzed using GEO database (GSE70577)30. As shown in Figure 6A, mRNA levels of BMP6 were upregulated in JQ1-treated MSCs compared to untreated cells. Bone homeostasis is regulated by various signaling pathways. Notable signaling pathways including bone morphogenetic protein (BMP)31,32, Wnt33,34, and Notch35 are involved in MSCs osteoblast differentiation. BMPs were purified from bovine bone firstly and verified to induce bone formation31,32. Among the family members, BMP2, BMP6, and BMP7 can induce bone formation. BMP6 increases vitamin D or PTH -induced osteocalcin expression in MSCs36. Administered BMP6 increases bone formation and suppresses bone resorption, which restores bone in aged ovariectomized rats37. Then the alteration of BMP6 level after OTX015 exposure in MSCs was assessed using qPCR and western blot assay. As expected, BMP6 expression was upregulated in OTX015-treated MSCs to vehicle control cells (Figure 6B). Collectively, these data suggested that the osteogenesisc effect of BET inhibitor OTX015 in MSCs may be attributed to induction of BMP6.

DISCUSSION Over the last decade, some BET inhibitors have entered clinical evaluation and clinical activity has been observed in hematologic malignancies11,13. The BET inhibitor OTX015 is currently undergoing some clinical trials and it is urgently necessary to characterize the biological activity of OTX015 in myeloma. Here, we showed that OTX015 resulted in cell growth inhibition and cell-cycle arrest in


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myeloma cell lines in which it decreased the expression of c-Myc. Our study demonstrated that OTX015 was effective in the inhibition myeloma progression and in prevention of myeloma-related bone lesions, resulting in some survival benefits in a systemic myeloma xenograft model. OTX015 showed anti-proliferative activity in myeloma cell lines with median IC50 of 40nmol/L in MM.1S cells or 1600 nmol/L in 8226 cells. In gene expression profiling data analysis, to capture the overlapping but also distinct oncogenic programs, we analyzed our GEP data separately for each cell line to reveal the distinct myeloma-initiating genetic events. Similar to that reported for other BET inhibitors which are effective in anti-myeloma22,23,38, we found that OTX015 caused c-Myc downregulation in both myeloma cell lines. Otherwise, OTX015 targeted gene expression profiling showed that OTX015 affected NF-κB, EGFR, cell cycle regulation and cancer proliferation signaling pathway, which appeared similar to which reported in GEP studies involving OTX015 in lymphoma24. We also identified gene expression signatures associated with the different sensitivity to OTX015. These data may be helpful in identifying subgroups of myeloma patients that will potentially benefit from OTX015. Only a slight induction of apoptosis was observed after OTX015 exposure, which did not reach statistical significance. Previous study reported that JQ1 exerts the anti-myeloma activity through cell cycle inhibition, but only a modest induction of apoptosis23. We found that, similar to JQ1, OTX015 induced cell cycle arrest and thus effected on the inhibition of the myeloma proliferation. Overall, these phenotypes of



suppressed proliferation and cell cycle arrest were specially anticipated OTX015 effects in myeloma cells. We also examined the effect of OTX015 on myeloma growth in vivo experiments. OTX015 was previously reported to exert anti-lymphoma activity in vivo24 and our data showed that it had considerable efficacy against myeloma in a systemic xenograft model generated by intravenously injecting 8226-luc cells into NPG mice. This strong anti-myeloma activity accompanied by a significant prolongation in overall survival observed in OTX015-treated groups of mice. In addition, OTX015 was proved to be effective in treating myeloma-related bone lesions sustained by promoting bone-forming and anti-catabolic activities. Our study provided the first evidence and rationale for the clinical evaluation of BET inhibitor in the treatment of myeloma bone disease and other bone pathologies. Overall, this study provided mechanistic insight and extended the evidence that BET inhibitors OTX015 possessed strong anti-myeloma activity in vitro and in vivo. In common with the data coming from the current phase I study, our data provided the basis for further clinical research of OTX015.

ACKNOWLEDGMENTS

This work was supported in part by Natural Science Foundation of Jiangsu Province China (BK20161223, BK 20161218), National Natural Science Foundation of China (81670191, 81673448), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions and the Applied Basic


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Research Programs of Suzhou City (SYS201546), and

CONFLICT-OF-INTEREST DISCLOSURE The authors declare no competing financial interests.

Supporting information Gene expression profiling, GEO Dataset Analysis, gene set enrichment analysis (GSEA), cell proliferation assay, cell cycle analysis, apoptosis analysis, antibodies In vivo xenograft studies, Osteogenic differentiation, von Kossa staining and alizarin red, micro-computed tomography and markers of bone turnover in mouse serum analysis, characterization of myeloma patients, primers used in this study, lists of the GSEA significant gene sets affected by OTX015 in MM.1S and 8226, the enrichment of GSEA significant gene sets in OTX015 sensitive myeloma cell line (MM.1S), lists of the GSEA significant gene sets affected by OTX015 in 8226.

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FIGURE LEGENDS


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Figure 1. OTX015 reduced proliferation and induced cell cycle arrest in myeloma cells. (A) Dose-dependent inhibition of MM.1S and 8226 proliferation by OTX015 as assessed by CCK8 assay. MM.1S or 8226 were treated with 40 nmol/L OTX015 or 1600 nmol/L OTX015, respectively. Representative flow cytometry profiles showing cell cycle alterations induced by OTX015 in MM.1S and 8226 cells (B) (C). The apoptosis was measured by Annexin V /7-AAD apoptosis detection kit (D).

Figure 2. Gene expression profiling of OTX015 anti-myeloma activity. (A) Heat map showing the 50 up- and downregulated genes after treatment with OTX015 for 12 hours of MM.1S and 8226 cells. (B) Venn diagram showing that among the 100 upregulated genes in OTX015-treated cells, 4 are shared by the 2 cell lines (left), while among the 100 most downregulated genes, 4 are shared by the 2 cell lines (right). (C) MM.1S or 8226 were treated with vehicle or OTX015 for the indicated time. QPCR was performed to determine gene levels, with GAPDH used as an internal normalization control. C-Myc protein expression was determined by western blot. *, p < 0.05, **, p < 0.01 versus the vehicle control (D) Representative GSEA plots illustrating: The enrichment of genes involved in MM.1S or 8226 after treatment with OTX015. (E) Venn diagrams showing the number of gene sets significantly enriched among list of genes downregulated in OTX015-treated cells; (P < 0.05; FDR q < 0.05) either in both or in individual cell lines.

Figure 3. Gene expression profile was associated with sensitivity to OTX015



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(A) Representative GSEA plots illustrating: The enrichment of positive genes involved in MM.1S. (B) Representative GSEA plots illustrating: The enrichment of negative genes involved in MM.1S. (C) Heat maps showed the top differentially ranked transcripts in between MM.1S and 8226. Left panel: Top 50 up-regulated genes (red) in MM.1S; Right panel: Top 50 up-regulated genes (red) in 8226.

Figure 4. Oral administration of OTX015 decreased tumor burden in mice model of MM. (A) Representative whole-body bioluminescence images of NPG mice orthotopically xenografted after intravenous injection with 8226-luc+ cells and treated with OTX015 or vehicle control. (B) Tumor burden of NPG mice orthotopically xenografted after intravenous injection with 8226-luc+ cells. Upon detection of MM lesions diffusely engrafted in the skeleton, mice were randomly assigned to receive OTX015 or vehicle control. Data are presented as mean ± SD (n = 6/group). P values indicate statistical significance of the observed differences (Student’s t test with Bonferroni multiple comparison correction). **P

Potent Activity of the Bromodomain Inhibitor OTX015 in Multiple

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