Preclinical validation and phase I trial of 4-hydroxysalicylanilide, targeting ribonucleotide reductase mediated dNTP synthesis in multiple myeloma

Abstract

Background: Aberrant DNA repair pathways contribute to malignant transformation or disease progression and the acquisition of drug resistance in multiple myeloma (MM); therefore, these pathways could be therapeutically exploited. Ribonucleotide reductase (RNR) is the rate-limiting enzyme for the biosynthesis of deoxyribonucleotides (dNTPs), which are essential for DNA replication and DNA damage repair. In this study, we explored the efficacy of the novel RNR inhibitor, 4-hydroxysalicylanilide (HDS), in myeloma cells and xenograft model. In addition, we assessed the clinical activity and safety of HDS in patients with MM.

Methods: We applied bioinformatic, genetic, and pharmacological approaches to demonstrate that HDS was an RNR inhibitor that directly bound to RNR subunit M2 (RRM2). The activity of HDS alone or in synergy with standard treatments was evaluated in vitro and in vivo. We also initiated a phase I clinical trial of single-agent HDS in MM patients (ClinicalTrials.gov: NCT03670173) to assess safety and efficacy.

Results: HDS inhibited the activity of RNR by directly targeting RRM2. HDS decreased the RNR-mediated dNTP synthesis and concomitantly inhibited DNA damage repair, resulting in the accumulation of endogenous unrepaired DNA double-strand breaks (DSBs), thus inhibiting MM cell proliferation and inducing apoptosis. Moreover, HDS overcame the protective effects of IL-6, IGF-1 and bone marrow stromal cells (BMSCs) on MM cells. HDS prolonged survival in a MM xenograft model and induced synergistic anti-myeloma activity in combination with melphalan and bortezomib. HDS also showed a favorable safety profile and demonstrated clinical activity against MM.

Conclusions: Our study provides a rationale for the clinical evaluation of HDS as an anti-myeloma agent, either alone or in combination with standard treatments for MM.

Trial registration: ClinicalTrials.gov, NCT03670173, Registered 12 September 2018.

Keywords: 4-Hydroxysalicylanilid; DNA damage repair; Deoxyribonucleotides; Multiple myeloma; Ribonucleotide reductase.

Fig. 1

Cytotoxic activity of HDS against MM cells.  A Indicated MM cell lines were treated with vehicle or HDS (12.5, 25, 50, 100, 200, 300, 400 µM) for 72 h. Then the cell viability was determined by CCK-8 assay. Cell viability data are presented as the means of 3 independent experiments in a Heatmap. B Cell clone colonies formed by the H929 and ARP-1 cells treated with HDS (0, 50 and 100 µM); the colonies in each well were quantified. C Primary CD138⁺ MM cells from patients (Pt#1–Pt#9) and normal PBMCs from healthy donors (D#1–D#3) were exposed to HDS with the indicated concentrations for 72 h and then apoptosis was analyzed. Pt represents patient. D represents healthy donor. D H929 cells were treated with indicated concentrations of HDS alone or in the presence of IL-6 or IGF-1 for 72 h. Cell viability was determined by CCK-8 assay. E H929 cells treated with different concentrations of HDS (0, 50 and 100 µM) were cultured with or without BMSCs for 72 h, and cell growth was assessed using CCK-8 assay. F H929 cells were exposed to 0, 50 and 100 µM HDS for indicated time (24, 48 and 72 h). Cell apoptosis was determined by Annexin V/PI staining. Representative results of triplicate experiments were shown. G Representative fluorescent images of typical apoptotic cells evaluated by TUNEL staining (red) after 100 µM HDS treatment for 24 h. DAPI was used as a nuclear stain (blue). H Cell cycle analysis was performed using flow cytometry. Percentages showed cell population in S-phase. I The viability of H929 and ARP-1 cells transfected with scramble or corresponding shRNA plasmids with HDS treatment (0, 12.5, 25, 50, 100 and 200 µM, 72 h) were analyzed by a CCK-8 assay. Western blots showed RRM1 and RRM2 expressions in H929 and ARP-1 cells after transfected with RRM1 or RRM2 shRNA. Notably, the cell viability of RRM1-shRNA transfected cells and scramble-transfected cells were not significantly different at the corresponding HDS concentration. J CCK-8 assay was performed on RRM1-OE and RRM2-OE cells or empty vector-transfected cells. Western blots showed RRM1 and RRM2 overexpression in H929 and ARP-1 cells after transfected with RRM1 OE or RRM2 OE vectors. Empty vector-transfected cells served as controls. Data are presented as the means ± SD of 3 independent experiments. *P < 0.05; **P < 0.01; #, not significant

Fig. 2

HDS interacted with RRM2 and inhibited RNR activity.  A T1ρ spectra acquired by using 200 µM HDS solely (colored in red) and 200 µM HDS in the presence of 5 µM RRM2 protein (colored in green) are presented. B STD spectrum acquired by using 200 µM HDS in the presence of 5 µM RRM2 protein (colored in red) is presented. C SPR biosensor was used to detect the binding of HDS to RRM2. Representative sensorgrams of the interaction of 0.78125 to 100.0 µM HDS with 200 µg/mL RRM2. D Cellular thermal shift assay to examine interactions of compounds (100 µM HDS, 0.5 µM gemcitabine, or 500 µM HU) with RRM1 and RRM2. Lower panel is the charts of percentages of non-denatured protein fraction. E MM cells were treated with HDS for 24 h. Then the intracellular RNR was extracted and measured by LC–MS/MS system. F MM cells were treated with HDS (0, 50, and 100 µM) for 24 h. Then the intracellular dNTPs were extracted and measured. Data are presented as the means ± SD of 3 independent experiments. **P < 0.01, ***P < 0.001

Fig. 3

The oncogenic roles of RRM1 and RRM2 in MM.  A Analysis of RRM1 and RRM2 expression in publicly available MM patient data sets from Mayo Clinic (data set GSE6477). Increased RRM1 or RRM2 expression is observed in plasma cells from patients with MGUS, SMM, MM and relapsed MM than from normal healthy donors. B The expression levels of RRM1 and RRM2 were significantly up-regulated in relapsed patients from TT2 and TT3 cohorts in comparison with patients at the baseline stage (based on data set GSE2658). C Kaplan-Meier analyses of OS about patients from TT2 (p < 0.001) and TT3 (p < 0.05) cohorts revealed inferior outcomes among the patients with high (quartiles 4) RRM1 or RRM2 expression compared with the remaining patients with low (quartiles 1–3) RRM1 or RRM2 expression (based on data set GSE2658). D Immunohistochemical analysis of RRM1 and RRM2 expression (positive cells are brown) in 3 representative BM specimens derived from normal and MM patients (ND#1, 5, 8 and MM#1, 7, 14). Original magnification ×20. E MM cell lines were cultured for 6 days and knockdown of RRM1 or RRM2 in MM cell lines induced significant growth inhibition. F MM cell lines were cultured for 6 days and overexpression of RRM1 or RRM2 in MM cell lines induced significant growth increase. G Differences in tumor size between different groups of nude mice on Day 22 after injection of H929 cells. H929 cells transduced with RRM1 or RRM2 shRNA and scramble control vectors were subcutaneously injected into mice (n = 5/group). Tumor volume was quantified and knockdown of RRM1 or RRM2 inhibited tumor growth. All data are expressed as means ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

HDS impaired DNA damage repair by inhibiting dNTP synthesis. A Gene expression profiling was performed after H929 cells were treated with 50 µM HDS for 48 h. Heat map showed significant pathways affected by HDS. B DNA synthesis in MM cells treated with HDS for 24 h was evaluated by EdU incorporation. EdU incorporation was observed using laser scanning confocal microscopy. The number of EdU-positive cells was quantified. C MM cells were incubated with HDS either without or with exogenous 50 µM dNTPs for 24 h. Percentage of EdU positive cells in flow cytometry was showed in the right panel. D NHEJ and HR in cells treated with HDS (0, 50 and 100 µM) for 24 h were quantified by the GFP and DsRed expression using flow cytometry. E MM cells were treated with 100 µM HDS for 24 h, and then the comet assay was performed. F Expression of cellular γ-H2AX in H929 and ARP-1 cells treated with or without 100 µM HDS for 24 h was detected by immunofluorescence. G Western blot analysis of DNA damage-related proteins in cell lysates of H929 and ARP-1 cells treated with indicated concentration of HDS for 24 h. H Immunofluorescence staining of cellular γ-H2AX in H929 and ARP-1cells treated with 100 µM HDS, either without or with 50 µM exogenous dNTPs for 24 h. I Western blot analysis of DNA damage-related proteins in MM cells incubated with HDS either without or with exogenous 50 µM dNTPs for 24 h. All data are expressed as means ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Synergistic effect of HDS with melphalan and bortezomib.  A H929 and (B) ARP-1 cells were co-treated with indicated concentration of HDS (12.5–400 µM) and various concentrations of Melphalan (2.5–80 µM) either alone or in combination for 48 h. Cell viability was assessed using a CCK-8 assay (left panel). CI values were calculated based on the median-effect principle. The right graph showed values from the left tables. CI < 1 indicated synergism of HDS and melphalan, as determined using CalcuSyn software. C, D The expression of relative apoptosis (C) and DNA damage proteins (D) of MM cells were monitored by western blot after treated with HDS (50 µM) in the presence (+) or absence (−) of melphalan (10 µM) alone or together. Representative results of triplicate experiments are shown. E, F H929 (E) and ARP-1 (F) cells were treated with bortezomib for 24 h, and then HDS was added for an additional 24 h. Cell viability was assessed using a CCK-8 assay (left panel). CI values were using CalcuSyn software (Right panel). G Primary CD138⁺ cells were isolated from four bortezomib-refractory patients. MM cells were treated with HDS (40 µM) and bortezomib (40 nM) alone or in combination for 48 h, followed by an assessment of cell apoptosis. Representative results of triplicate experiments were shown (left panel). Apoptotic cells were quantified on the right. Data are expressed as means ± SD of three independent experiments. ***P < 0.001

Fig. 6

HDS inhibited myeloma growth in vivo. A Nude mice bearing H929 tumors were daily given either HDS (50 mg/kg, 100 mg/kg) or vehicle through intravenous injection for 20 days. B Tumors on day 20. C The weight of mice. D Kaplan-Meier analyses of overall survival. (E) Tumor sections were stained with HE, Ki67, cleaved caspase-3 or TUNEL. The positive cells in tumor sections stained with Ki67, cleaved caspase-3 or TUNEL are the dark brown ones. Scale bars, 100 µM. F Nude mice bearing H929 tumors were intravenously injected with vehicle (daily), HDS (50 mg/kg, daily), bortezomib (0.5 mg/kg, every three days) or HDS (50 mg/kg, daily) plus bortezomib (0.5 mg/kg, every 3 days). Tumor volumes were showed. G Pictures of tumors in nude mice. H Using Kaplan-Meier and log-rank analysis, the median overall survival of animals treated with combination therapy was significantly prolonged. I Tumor sections were stained with TUNEL and γ-H2AX. Positive cells are the dark brown ones. Scale bar, 100 µM. Data are represented as means ± SD. *P < 0.05

Fig. 7

Clinical activity of HDS in MM patients. A The maximum change of M-protein from baseline level after HDS treatment. B Swim‐lane plot showed the treatment response and duration for 9 MM patients after HDS treatment. Arrows indicated patients who were still ongoing at the time of study closure. PD progressive disease, SD stable disease, MR minimal response. C The protein levels of RRM2 in CD138⁺ MM cells obtained from patients were evaluated, with GAPDH used as a loading control