TI17, a novel compound, exerts anti‐MM activity by impairing Trip13 function of DSBs repair and enhancing DNA damage

Abstract Background Thyroid hormone receptor interacting protein 13 (Trip13) is an AAA‐ATPase that regulates the assembly or disassembly protein complexes and mediates Double‐strand breaks (DSBs) repair. Overexpression of Trip13 has been detected in many cancers and is associated with myeloma progression, disease relapse and poor prognosis inmultiple myeloma (MM). Methods We have identified a small molecular, TI17, through a parallel compound‐centric approach, which specifically targets Trip13. To identify whether TI17 targeted Trip13, pull‐down and nuclear magnetic resonance spectroscopy (NMR) assays were performed. Cell counting kit‐8, clone formation, apoptosis and cell cycle assays were applied to investigate the effects of TI17. We also utilized a mouse model to investigate the effects of TI17 in vivo. Results TI17 effectively inhibited the proliferation of MM cells, and induced the cycle arrest and apoptosis of MM cells. Furthermore, treatment with TI17 abrogates tumor growth and has no apparent side effects in mouse xenograft models. TI17 specifically impaired Trip13 function of DSBs repair and enhanced DNA damage responses in MM. Combining with melphalan or HDAC inhibitor panobinostat triggers synergistic anti‐MM effect. Conclusions Our study suggests that TI17 could be acted as a specific inhibitor of Trip13 and supports a preclinical proof of concept for therapeutic targeting of Trip13 in MM.


| INTRODUCTION
Multiple myeloma (MM) is a malignant monoclonal tumor characterized by its prominent clonal proliferation of plasma cells in the bone marrow, which accounts for about 1% of neoplastic diseases and 10% of hematologic malignancies. 13][4] In addition, most MM patients will occur disease recurrence after primary treatment. 5Therefore, scientists have been trying to develop effective and safe drugs for many years.
Double-strand breaks (DSBs), the most cytotoxic types of DNA damage, are difficult to repair, and can lead to genome rearrangements and cell death. 6,7And nonhomologous end joining (NHEJ) 8 is the major pathway for the efficient repair of DSBs, since it can occur throughout the cell cycle and is favored in G1 cells. 91][12] Trip13 is expressed in multiple somatic tissues. 13,14Meanwhile, overexpression of Trip13 may promote the development of cancer. 157][18] In prostate cancer, Trip13 promotes the early repair of DSBs, and its overexpression is associated with poor prognosis. 19In head and neck cancer, Trip13 could promote error-prone NHEJ, enhance the repair of DSBs, and induce treatment resistance. 20These findings suggest that Trip13 protein can serve as a kind of important monitoring, through the provision of DNA repair mechanisms to help assess the DNA integrity.
Trip13 is a chromosomal instability gene in MM, which is related to drug resistance and disease relapse, 21 and it has also been considered to be an important highly expression gene in a 70-gene model of high-risk myeloma by extensive gene expression profiling. 22In a previous study, we have found that Trip13 is significantly up-regulate at relapse MM patients after initial chemotherapy. 23ere, this study details our efforts in identifying a novel small molecular inhibitor, TI17, which was identified from a compound library and confirmed to bind to Trip13.Then we further evaluated the potential anti-MM activity of TI17 in MM cells by impairing the Trip13 function of DSBs repair in vitro and in vivo.
All MM cell lines and A549, PC-3, 5-8F cells were maintained in RPMI-1640 medium (Gibco, BRL, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, BRL, USA) and 1% penicillin-streptomycin (PS; Gibco, BRL, USA).Human HS-5, MDA-MB-231, LM3, and A498 cells were cultured in DMEM/high glucose medium (Gibco, BRL, USA) containing 10% FBS and 1% PS.All cells were maintained in a humidified atmosphere of 5% CO 2 at 37°C.Culture medium was changed every other day.Primary cells assay was performed as described previously. 24Informed consent was obtained from each patient and healthy donor.The protocol for collection and usage of clinical samples was approved by the institutional review board of Shanghai Tenth People's Hospital, Shanghai, China.Informed consent was obtained in accordance with the Declaration of Helsinki.

| Cell viability assay
Cell viability assay was performed according to a previous study. 24Briefly, cells were seeded into 96-well plates and then treated with TI17.Cell viability was determined using the Cell Counting Kit (CCK)-8 assays.

| Clonogenic assay
MM cell lines were inoculated in 12-well plates at 1000 cells per well and cultured at 37°C for 2 weeks.Cell colonies with 0.1% crystal violet staining for 30 min.Colonies with at least 50 cells were counted.

| Cell cycle analysis
Cell viability assay was determined with reference to previous study. 24Briefly, cells were exposed with TI17.Then cells were harvested, with propidium iodide (PI) (BD Pharmingen, Franklin Lakes, NJ, USA) incubation, and then analyzed by flow cytometry.

| Apoptosis analysis
Apoptosis assay was performed according to a previous study. 24Briefly, cells were treated with or without TI17.Then, cells were collected and stained, and then analyzed by flow cytometry.

| Pull-down analysis
Pull-down assay was determined with reference to previous study. 25Briefly, the cell lysate was then incubated with 50 μmol/L TI17-biotin or biotin in the presence of neutral fat sugar resins, and analyzed by immunoblotting.

| Tumor xenograft models
Tumor xenograft models were performed as described previously. 24Briefly, 3 × 10 6 OCI-MY5 cells were injected subcutaneously into the upper flank region of the BALB/C nude mice.Then mice were randomly divided into three groups: the control group, TI17(50 mg/kg) and TI17 (100 mg/kg).

| TUNEL assay
TUNEL assay were performed as described previously. 24ell apoptosis was evaluated by use a light microscope by three pathologists who do not know the original specimens.

| Statistical analysis
Results were presented as mean ± standard deviation (SD).SPSS v20.0 software (IBM, Armonk, NY, USA) was used for Student's t-test and one-way analysis of variance (ANOVA).p < 0.05 was considered statistically significant.

| TI17 directly targeted Trip13
Based on our previous research, we knew that Trip13 involved in the development and prognosis of MM.To search for such effective compounds, we first screened a compound library, and chose only one named TI17 (Figure 1A,B) for the further research.We first elucidated the binding mode of TI17 to Trip13 by using the ligand observed T1ρ nuclear magnetic resonance (NMR) analysis.Dose-dependent effects observed in the T1ρ titration experiments of Trip13 to TI17 indicate that the binding between these two molecules is specific (Figure 1C).Additionally, T1ρ titration data suggest a mutually exclusive binding of TI17 and ADP to Trip13 (Figure 1D).Meanwhile, we detected an affinitypulldown target verification system, in which TI17 was coupled to a biotin via a multi-step synthesis.Comparing with the control matrix, mixing the TI17-affinity biotin with the cell lysate reduced the endogenous Trip13 (Figure 1E).We also found that TI17 inhibited the growth of MM cells in a dose-dependent manner, while overexpression of TRIP13 increased sensitivity to TI17 as detected by CCK8 kit assays (Figure 1F,G).In further, we examined whether TI17 inhibited the ATPase activity of TRIP13.These data showed that the ATPase activity of TRIP13 was inhibited by TI17 in a dose-dependent manner (Figure 1H).These experimental results indicate that TI17 directly target Trip13.

| TI17 inhibits proliferation of MM cells and overcomes the protective effect of the BM environment on MM cells in vitro
In this study, we further evaluated the expression of Trip13 by western blot in human MM cells, our findings shown that the protein level of Trip13 was elevated in MM cells (Figure S1).And our data demonstrated that the viability of these MM cells was obviously inhibited by TI17 treatment (Figure 2A).IC 50 of TI17 at concentrations of 1.25, 2.5, 5, 10, and 20 μM was detected in each these MM cells for inhibition of cell viability using CalcuSyn software, Version 2.1 (Figure 2A).Interestingly, we also found TI17 induced a time-and dose-dependent decline in the viability of ARP1 and OCI-MY5 cells (Figure 2B).
Importantly, as in Figure S2A, we observed the level of Trip13 was increased in other tumor cells, which is consistent with previous studies in these cancers. 13,19,20,26,27eanwhile, results presented that the activity of these cancer cells was slightly inhibited after TI17 intervention, compared to MM cells (Figure S2B,C).These data showed that TI17 also has anti-proliferation effect on other cancer cells, although this effect was less sensitive than on MM cell lines.
We found that the expression of Ki-67 was significantly decreased after TI17 intervention (Figure 2C,E).Furthermore, EDU incorporation assay demonstrated that TI17 significantly decreased DNA synthesis in ARP-1 and OCI-MY5 cells for 24 h (Figure 2D,E).In addition, the results of colonogenic survival assays further confirmed the anti-proliferation activity of TI17 towards at least two MM cells (ARP-1 and OCI-MY5) (Figure 2F).Taken together, these results collectively indicated that TI17 significantly inhibits the proliferation of MM cells.ARP1 and OCI-MY5 cells were cultured in the presence or absence of BMSC, IL-6 and IGF-1 at various concentrations of TI17 for 48 h.Results showed that TI17 treatment significantly inhibits the cell viability of ARP1 and OCI-MY5 cells when BMSC were present (Figure 2G), as well as IL-6 and IGF-1 (Figure 2H), while TI17 treatment has no significant effect on the viability of BMSCs.These results indicated that TI17 can not only directly impact on MM cells, but also overcome the protective effect of the BM microenvironment on MM cells.

| TI17 induced G 0 /G 1 arrest and apoptosis in MM cells in vitro
We next examined the effects of TI17 on the cell cycle in MM cells in vitro by flow cytometry analysis.As shown in Figure 3A,B, treatment of TI17 induced obvious cell accumulation of G 0 /G 1 DNA content in ARP1 and OCI-MY5 cells.Meanwhile, the expression levels of CDK4, CDK6, and cyclinD1 were markedly downregulated after TI17 treatment (Figure 3C,D), further conforming that TI17 could significantly induce cell cycle arrest.Furthermore, As presented in Figure 4A-D, administration of TI17 also caused significant increase in the rate of apoptosis in a concentration-and time-dependent manner in ARP-1 and OCI-MY5 cells, as well as in H929R and H929S (supplementary Figure S3A,B).Meanwhile, western blot analysis indicated that the expression levels of cleavage caspase-3, cleavage caspase-8, cleavage caspase9, as well as BAX were upregulated, while BCL-XL and BCL2 were down-regulated in ARP-1 and OCI-MY5 cells after TI17 intervention (Figure 4E).Meanwhile, we found that pan-caspase inhibitor Z-VAD-FMK significantly eliminated TI17-induced apoptosis of ARP-1 and OCI-MY5 cells (Figure 5A,B).These results indicated that TI17 triggers both exogenous and endogenous apoptotic pathways, and MM cells apoptosis induced by TI17 is mediated via caspases.
In order to determine whether TI17 has anti-MM activity on patient MM cells, we detected the effect of TI17 on patients CD138 + MM cells by flow cytometry analysis, and our findings showed that TI17 induced a significant dose-dependent increase apoptosis in primary CD138 + MM cells, while have no cytotoxic effect on CD138 − MM cells (Figure 5C,D), as well as normal PBMCs (Figure 5E,F), indicating TI17 is a favorable therapeutic index in MM.

| TI17 impaired DSBs repair and enhanced DNA damage responses in MM cells in vitro
Then we further performed whether Trip13 could interact with Ku70 and Ku80 proteins in MM cell lines by co-immunoprecipitated assay.As shown in Figure 6A, Trip13 could co-immunoprecipitate with Ku70 and Ku80 proteins but not control IgG in ARP-1 cells.Second, we observed that TI17, which can target Trip13, could inhibit the repair of NHEJ by decreasing the expression of Ku70 and Ku80 via using western blot analysis in ARP-1 cells (Figure 6B).
H2AX plays an important role in the DNA damage response and is phosphorylated at Ser-139 (γ-H2AX) in response to DSBs. 28Therefore, to assess the effect of TI17 on DSB-repair in MM cell lines, we investigated the expression of γ-H2AX by western blot and immunofluorescence analysis in ARP-1 and OCI-MY5 cells (Figure 6B).As shown in Figure 6C, TI17 markedly increased the expression of γ-H2AX in ARP-1 and OCI-MY5 cells.
Moreover, ataxia telangiectasia mutated (ATM), one of the phosphoinositide (PI) 3-like kinases, 29 which is triggered by DSBs can phosphorylate various downstream substrates such as H2AX and cell cycle checkpoint kinases Chk2. 30Therefore, we next detected the expression of phosphorylated (p)-ATM, as well as its downstream effector p-Chk2.Results showed that TI17 increased the levels of p-ATM and p-Chk2, while decreased the level of cdc25A (Figure 6B).

| In vivo anti-MM activity of TI17
In order to determine the anti-MM efficacy of TI17 in vivo, we subsequently examined the tumor inhibition activity in a MM xenograft murine model.We gave TI17 or vehicles daily to mice intravenously.As seen in Figure 7A,B, administration of TI17 significantly reduced the growth of tumor in a concentration-dependent manner (50 mg/kg, p = 0.048; 100 mg/kg, p = 0.0039), compared with vehicle-treated animals.Importantly, there were no significant difference in body weight among the groups (Figure 7C).Furthermore, HE staining indicated that TI17 induced a significant increase in cell shrinkage and fragmentation of harvested tumors (Figure 7D).Meanwhile, results showed that no significant histological changes in heart, liver, lung, and kidney in all mice (Figure S4), indicating that TI17 had minimal side effects.As shown in Figure 7E,F, TI17 significantly decreased the tumor proliferation by decreasing Ki-67 expression and induced tumor apoptosis by increasing the number of cleaved-caspase 3-and TUNEL-positive cells.Meanwhile, our results showed that TI17 could significantly enhanced DNA damage by increasing the expression of γ-H2AX, which was consistent with our results in vitro.These results showed that TI17 has potent activity in inhibiting tumor growth in animals.

| TI17 effects on MM cells in combination with melphalan or HDAC inhibitor
ARP-1 and OCI-MY5 cells were treated in combination with TI17 and MEL or HDAC inhibitor panobinostat.We observed that TI17 increased cytotoxicity of these agents in MM cells (Figure 8A-D).Meanwhile, we calculated the CI values by using CalcuSyn software.TI17 was synergistic with MEL (CI <1), or panobinostat (CI against ARP-1, as well as OCI-MY5 cells.Importantly, we further found TI17 with MEL increased cell apoptosis in ARP-1 cells by flow cytometry (Figure 8E,F), indicating that the synergistic cytotoxicity induced by combination with TI17 and MEL may due to apoptosis cell death.

| DISCUSSION
Trip13 has a great amplification in high-risk MM patients. 22Moreover, our previous studies have shown that knockdown Trip13 could against the growth of MM both in vivo and in vitro, 23 suggesting Trip13 may represent an anti-MM target and inhibition of Trip13 could be a promising therapeutic strategy for MM.In the present study, we have identified a novel small molecular inhibitor, TI17, which can impair DSBs repair by directly binding to Trip13, displays high potent anti-MM activity in both in vitro and in vivo study.
We first found that TI17 was identified to target Trip13 via using the ligand observed T1ρ nuclear magnetic resonance (NMR) analysis.Furthermore, we observed TI17 could specifically bound endogenous Trip13 derived from MM cells and the proliferative inhibitions of TI17 was increased in Trip13-overexpressed MM cells.Additionally, inhibition of ATPase activity after TI17 treatment further suggested that TI17 directly targets Trip13.
Through the study, we have demonstrated that TI17 treatment inhibits the growth of a large amount of MM cells in vitro.And we also observed that TI17 could trigger MM cell lines growth inhibition when MM cells were cultured with BMSC and cytokines IL-6 and IGF-1.These findings further indicate that as a small molecular inhibitor target trip13, TI17 has obvious anti-MM activity in vitro.
The drugs of DNA damage and mitotic spindle damage always remain the main stream in the therapy of multiple cancer. 31Interestingly, our date indicated that TI17 induced accumulation of MM cells at the G0/G1 phase, suggesting that TI17 triggers DNA damage responses via inducing MM cells cycle arrest.Apoptosis could be triggered by endogenously or exogenously via death signal pathways. 32We observed that TI17 induced apoptosis of MM cells and primary CD138 + MM cells, while no obvious apoptosis on normal cells.Cleavage of caspase-3, caspase-8, and caspase-9 was further confirming its high potent apoptosis.
Trip13, belongs to the AAA family of chaperone proteins that involves in the assembly or disassembly of protein complexes. 33Ku is a DNA repair protein that is essential for binding to broken DNA and recruiting other proteins to promote the repair of DSBs. 34NHEJ is the primary pathway for DSBs repair in mammalian cells and is activated when DSBs ends were recognized by the Ku (Ku70 and Ku80) heterodimer. 35Consistently, we also observed the endogenous binding of Trip13 to Ku70 and Ku80 in MM cells by western blot of bead-bound proteins, further strongly indicate that Trip13 plays an important role in cell survival by enhance NHEJ repair.Additionally, we found the expression of Ku70 and Ku80 in MM cells were significantly decrease after treatment with TI17, indicating that TI17 could decrease the DNA end-binding protein Ku70/Ku80 and show an inability to repair DNA damage.As histone H2AX is phosphorylated at the Ser-139 (γ-H2AX) in response to DSBs, and DNA damage is prevalent in MM, evidenced by the presence of γ-H2AX. 36,37Furthermore, the absence of γ-H2AX indicates efficient DSB-repair, while higher γ-H2AX performs impaired DSB-repair. 38Our results indicated that treatment with TI17 increase the phosphorylation of H2AX and the presence of γ-H2AX foci in MM cells.ATM plays a key role in regulating cellular response to DSBs, and its activation can lead to phosphorylation of many downstream targets that mediate DNA damage response pathways. 30,39mportantly, we found that TI17 could phosphorylation of ATM by DSBs trigger, and phosphorylation of its downstream Chk2, followed by cdc25A decrease.Taken together, TI17 not only impaired the repair of DSBs, but also induced DNA damage responses in MM (Figure 9).Nonetheless, the detail molecular mechanism of TI17 directly targets to Trip13 effect on MM still needs to our further research.
In order to further observe the anti-MM efficacy of TI17, we next developed tumor xenograft models in vivo.Our in vivo study indicated that TI17 treatment obviously inhibited the tumor growth, while without significant general toxicity.Meanwhile, Immunohistochemical staining of harvest tumors confirmed that TI17 inhibited the proliferation of MM cells and promoted their apoptosis.More importantly, the increase of γ-H2AX expression in tumor further support TI17-induced DNA damage responses.Therefore, TI17 is a well-tolerated agent with potential therapeutic effect in MM.
As MM patients often occurs drug recurrence and poor prognosis, combinational therapy is needed after the initial treatment to increase the therapeutic effect and improve patient outcomes in MM. 40,41 CCK8 kit and flow cytometry analysis showed that TI17 in combination with MEL or panobinostat induced synergistic cytotoxicity in MM cells.Therefore, these data suggest that TI17 may have a promising application in clinical treatment.
Our studies showed that, TI17, a novel small molecular, effectively exert antitumor activity on MM by impairing Trip13 function of DSBs repair and enhancing DNA damage responses via inhibition of MM cells proliferation, accumulation of MM cells at G0/G1 phase, induction of MM cells apoptosis, and retardation of tumor growth in vivo.Collectively, our present study provides a new and effective therapeutic strategy for treating MM, and suggests the promise of therapeutics targeting Trip13 to improve MM treatment.

F
I G U R E 2 TI17 inhibited proliferation of MM cells and overcame the protective effect of the BM environment on MM cells in vitro.(A) MM cell lines were treated with DMSO or with TI17 for 48 h and cell viability was measured by CCK8 kit.(B) ARP-1 (left panel) and OCI-MY5 (right panel) cells were treated with DMSO or with TI17 for 24, 48, and 72 h, and analyzed for cell viability by CCK8 kit.Immunofluorescence staining of Ki-67 (C) and EDU (D) in cells after DMSO or TI17 treatment for 24 h.Red indicate Ki-67 or EDU positive cells.250× magnification.(E) Analysis of Ki-67 and EDU staining.(F) Cells were treated with DMSO or with TI17 for 14 days, and colony numbers were quantified with software ImageJ.50× magnification.(G) Cells were cultured with or without BMSC in the presence or absence of TI17 for 48 h and analyzed for cell viability by CCK8 kit.(H) ARP-1 (Left panel) and OCI-MY5 (Right panel) cells were cultured with or without IL-6 and IGF-1 in the presence or absence of TI17 for 48 h and analyzed for cell viability by CCK8 kit.All results are expressed as mean ± SD of three independent experiments and *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

F
I G U R E 3 TI17 induced G 0 /G 1 arrest in MM cells in vitro.(A, B) Cell-cycle analysis of TI17-treated cells.(C, D) Western blot analysis for CDK4, CDK6, cyclin D in cells after DMSO or TI17 treatment for 24 h.All results are expressed as mean ± SD of three independent experiments and **p ≤ 0.01, ***p ≤ 0.001.

F I G U R E 4
TI17 induced apoptosis in MM cells in vitro (A-D) Cells were treated with DMSO or with TI17 for 24, 48, 72 h, and measured the cell apoptosis by Annexin V/PI double staining using flow cytometry.(E) Western blot analysis for cleaved-caspase 3, cleaved-caspase 8, cleaved-caspase 9, Bcl-2, Bcl-xl, and BAX in cells after DMSO or TI17 treatment for 48 h.All results are expressed as mean ± SD of three independent experiments and *p ≤ 0.05, **p ≤ 0.01.

F I G U R E 5
TI17-triggered apoptosis mediated by caspases and TI17 induces patient MM cells apoptosis.(A, B) Cells were pretreated with pan-caspase (Z-VAD-FMK) for 2 h, then added DMSO or TI17 (10 μM) for another 48 h, and measured the cell apoptosis by Annexin V/ PI double staining using flow cytometry.(C, D) Primary patient MM cells (CD138 + , CD138 − ) were treated with DMSO or TI17 for 48 h, and detected the cell apoptosis by anti-CD138/Annexin-V/7-AAD staining using flow cytometry.(E, F) Normal PBMCs were treated with DMSO or with TI17 for 48 h, and measured the cell apoptosis by Annexin V/PI double staining using flow cytometry.All results are expressed as mean ± SD of three independent experiments and *p ≤ 0.05, **p ≤ 0.01.

F
I G U R E 6 TI17 enhanced DNA damage in MM cells in vitro.(A) Trip13 was immunoprecipitated in the presence or absence of ethidium bromide and immunoblotted with anti-Ku70, anti-Ku80 and anti-Trip13 antibodies in ARP-1 cells.(B) Western blot analysis for Ku70, Ku80, γ-H2AX, ATM, p-ATM, Chk2, p-Chk2, and cdc25A in cells after TI17 treatment for 48 h.(C) Immunofluorescence staining of γ-H2AX in cells after TI17 treatment for 24 h.Green indicate γ-H2AX positive cells.1260× magnification.

F I G U R E 7
In vivo anti-MM activity of TI17.(A) OCI-MY5 cells were injected subcutaneously into mice and mice were treated with vehicle, 50 mg/kg, and 100 mg/kg TI17 every day for 18 days via intraperitoneal injection after tumors formed within 2 weeks.Tumor size was measured every other day.Five mice/group.(B) Gross appearance of tumors.(C) Mice body weight was measured every other day.(D) HE staining of tumor sections for detected the tumor histological after TI17 treatment.200× magnification.(E, F) Immunohistochemical staining of Ki-67, cleaved-caspase 3, TUNEL, and γ-H2AX in vivo after TI17 treatment.400× magnification.All results are expressed as mean ± SD of three independent experiments and *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

F I G U R E 8
TI17 effects on MM cells in combination with MEL or HDAC inhibitor.ARP-1 (A, B) and OCI-MY5 (C, D) cells were combined treatment with TI17 and MEL or HDAC inhibitor panobinostat for 48 h, and cell viability was evaluated by using CCK8 kit, CI <1 indicates synergistic activity by using CalcuSyn software, Version 2.1.(E, F) ARP-1 cells were treated with DMSO or TI17 (10 μM) and MEL (5 μM) for 48 h, and measured the cell apoptosis by Annexin V/PI double staining using flow cytometry.All results are expressed as mean ± SD of three independent experiments and *p ≤ 0.05, **p ≤ 0.01.