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. 2013 Sep 12;4(9):e798.
doi: 10.1038/cddis.2013.306.

Preclinical screening of histone deacetylase inhibitors combined with ABT-737, rhTRAIL/MD5-1 or 5-azacytidine using syngeneic Vk*MYC multiple myeloma

G M Matthews  1 , M Lefebure, M A Doyle, J Shortt, J Ellul, M Chesi, K M Banks, E Vidacs, D Faulkner, P Atadja, P L Bergsagel, R W Johnstone
Affiliations

Preclinical screening of histone deacetylase inhibitors combined with ABT-737, rhTRAIL/MD5-1 or 5-azacytidine using syngeneic Vk*MYC multiple myeloma

G M Matthews et al. Cell Death Dis. .

Abstract

Multiple myeloma (MM) is an incurable malignancy with an unmet need for innovative treatment options. Histone deacetylase inhibitors (HDACi) are a new class of anticancer agent that have demonstrated activity in hematological malignancies. Here, we investigated the efficacy and safety of HDACi (vorinostat, panobinostat, romidepsin) and novel combination therapies using in vitro human MM cell lines and in vivo preclinical screening utilizing syngeneic transplanted Vk*MYC MM. HDACi were combined with ABT-737, which targets the intrinsic apoptosis pathway, recombinant human tumour necrosis factor-related apoptosis-inducing ligand (rhTRAIL/MD5-1), that activates the extrinsic apoptosis pathway or the DNA methyl transferase inhibitor 5-azacytidine. We demonstrate that in vitro cell line-based studies provide some insight into drug activity and combination therapies that synergistically kill MM cells; however, they do not always predict in vivo preclinical efficacy or toxicity. Importantly, utilizing transplanted Vk*MYC MM, we report that panobinostat and 5-azacytidine synergize to prolong the survival of tumor-bearing mice. In contrast, combined HDACi/rhTRAIL-based strategies, while efficacious, demonstrated on-target dose-limiting toxicities that precluded prolonged treatment. Taken together, our studies provide evidence that the transplanted Vk*MYC model of MM is a useful screening tool for anti-MM drugs and should aid in the prioritization of novel drug testing in the clinic.

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Figures

Figure 1
Figure 1
(a) Differential sensitivities of human MM cell lines to HDACi treatment. Single-agent dose–response curves constructed for each human MM cell line (JJN3, OPM-2, RPMI-8226 and U266) treated with vorinostat, panobinostat or romidepsin for 24 and 48 h. (b) On-target histone-H3 acetylation is demonstrated in a dose-dependent manner in human MM cell lines (JJN3, OPM-2, RPMI-8226 and U266) treated for 24 h with increasing doses of panobinostat (0, 1 5, 10 and 50 nM) and assessed by western blot
Figure 2
Figure 2
(a) Human MM cell lines demonstrate differential expression of Bcl-2 prosurvival proteins. JJN3, OPM-2, RPMI-8226 and U266 were assessed for the expression of antiapoptotic Bcl-2 proteins by western blot: Bcl-2, Bcl-XL, Bcl-W, Mcl-1 and A1. (b) Differential sensitivities of human MM cell lines to ABT-737. Single-agent dose–response curves were constructed in human MM cell lines (JJN3, OPM-2, RPMI-8226 and U266) treated with ABT-737 for 24 and 48 h. (c) Synergistic induction of apoptosis in human MM cell lines JJN3, OPM-2, RPMI-8226 and U266 following 48 h treatment with panobinostat in combination with ABT-737 after 48 h incubation. *P<0.05 versus single agents. Calcusyn was used to determine synergy when the two agents were combined: synergy is determined when CI<0.9, additively when CI is between 0.9 and 1.1 and antagonism when CI>1.1. CI values are shown on each graph
Figure 3
Figure 3
(a) Assessment of cell surface death receptor DR-4 and DR-5 on human MM cell lines JJN3, OPM-2, RPMI-8226 and U266, using flow cytometry against an isotype control antibody (n=3). Black histogram=isotype control; gray shaded histogram=DR4 or DR5 expression. (b) Differential sensitivities of human MM cell lines to rhTRAIL treatment. Single-agent dose–response curves were constructed in human MM cell lines (JJN3, OPM-2, RPMI-8226 and U266) treated with rhTRAIL for 24 and 48 h. (c) Synergistic induction of apoptosis in human MM cell lines OPM-2, RPMI-8226 and U266 following 48 h treatment with panobinostat and rhTRAIL (CI<0.9). The combination of panobinostat with rhTRAIL did not synergize in JJN3 cells (CI>1.1) and was only additive in OPM-2 cells (CI between 0.9 and 1.1). *P<0.05 versus single agents; analysis of c-FLIP (NF6) was undertaken in human MM cell lines (JJN3, OPM-2, RPMI-8226 and U266) following 8–16 h treatment with increasing doses of panobinostat below that shown to induce apoptosis (0, 1 and 5 nM). (d) Panobinostat significantly reduced c-FLIP mRNA expression levels in all cell types (8 h), whereas (e) protein expression was reduced in OPM-2, RPMI-8226 and U266 cells (16 h); and (f) assessment of cell surface DR-4/5 expression on human MM cell lines (JJN3, OPM-2, RPMI-8226 and U266) following treatment (16 h) with panobinostat (n=3). Panobinostat treatment significantly increased DR-5 expression on RPMI-8226 cells while appearing to reduce DR-4 expression on U266 cells (P<0.05). Black histogram=isotype control; dark gray shaded histogram=vehicle control; medium shade of gray histogram=1 nM panobinostat; light shade of gray histogram=5 nM panobinostat. At least n=3 biological replicates were carried out for each assessment
Figure 4
Figure 4
(a) Human MM cell lines display differential and dose-dependent sensitivities to 5-AZA. Single-agent dose–response curves were constructed in human MM cell lines (JJN3 and U266) treated with 5-AZA for 24 and 48 h. (b) Synergistic induction of apoptosis in JJN3 and U266 cells with panobinostat was combined with 5-AZA after 48 h (CI<0.9) *P<0.05 verses single agents: (c) JJN3 cells or (d) U266 cells were treated with panobinostat, 5-AZA or the combination of both agents at synergistic concentrations (described in Figure 4b) and assessed for changes in gene expression using next-generation RNA sequencing after 24 h. Gene set enrichment was assessed using CAMERA. Each Venn diagram depicts the number of MSigDB gene sets enriched within each treatment and within each cell line (two-sided P<0.05, n=3); (e) demonstrates the number of distinct or overlapping MSigDB gene sets enriched when JJN3 or U266 cells were treated with the combination of panobinostat with 5-AZA
Figure 5
Figure 5
Expression of Bcl-2 prosurvival proteins and surface DR-5 on bone marrow cells from C57BL/6 mice bearing Vk*MYC MM. (a) Prosurvival Bcl-2 family protein expression in the bone marrow of mice bearing Vk*MYC MM by western blot (n=3), and (b) assessment of surface DR-5 expression on B220−/CD138+ plasma cells from bone marrow of a mouse bearing Vk*MYC MM by FACS. Black histogram=isotype control; light gray histogram=DR-5 expression
Figure 6
Figure 6
In vivo treatment of C57BL/6 mice bearing Vk*MYC MM reveals lack of therapeutic activity when panobinostat is combined with ABT-737 above that of panobinostat treatment alone. (a) Single-agent therapy consisting of vehicle (D5W), high-dose panobinostat (25 mg/kg days 1–4, 15 mg/kg remainder, 5 days per week), ABT-737 (75 mg/kg, 5 days per week) or the combination of both agents in mice bearing Vk*MYC MM. Normalized M-spike data over the 18 days of treatment. (b) Survival of mice treated with vehicle (D5W, n=6), panobinostat (n=5), ABT-737 (n=5) or the combination of both agents (n=6). (c) Mice bearing Vk*MYC MM were then treated with lower doses of both agents as follows: vehicle (n=5); panobinostat (5 mg/kg, 5 days per week, n=5); ABT-737 (50 mg/kg, two times daily, n=5); or the combination of both agents (n=6), for 4 weeks. Results are depicted as normalized M-spike over the 26 days of treatment, and (d) survival of mice treated with the lower doses of both agents, alone and in combination. *P<0.05 versus vehicle
Figure 7
Figure 7
In vivo treatment consisting of panobinostat in combination with MD5-1 is not well tolerated and does not enhance survival of C57BL/6 mice bearing Vk*MYC MM over single-agent panobinostat treatment alone, whereas its combination with 5-AZA provides significant benefit. (a) Normalized M-spike of mice bearing Vk*MYC MM treated as follows: vehicle (D5W±control antibody UC81B9, n=8); panobinostat (10 mg/kg, n=6); MD5-1 (50 μg per mouse; days 1, 4, 8, 12; n=8); or the combination of both agents (n=8). *P<0.05 versus vehicle. (b) Survival of mice treated as per 7A, (c) normalized M-spike of mice bearing Vk*MYC MM treated as follows: vehicle (D5W, n=7); panobinostat (7.5 mg/kg, n=7); MD5-1 (50 μg per mouse; days 2, 5, 9, 12; n=6); or the combination of both agents (n=7); (d) survival of mice treated as per (c); (e) absence of on-target MD5-1-mediated toxicity by treatment of C57BL/6.DR5 KO mice bearing Vk*MYC tumor with panobinostat and MD5-1 combination therapy leads to significant increases in survival. Mice were treated as follows: vehicle (D5W±control antibody UC81B9, n=6); panobinostat (7.5 mg/kg, n=6); MD5-1 (50μg per mouse, days 2, 5, 9, 12; n=6); or the combination of both agents (n=7); (f) normalized M-spike of mice bearing Vk*MYC MM treated as follows: vehicle (D5W, n=6), panobinostat (10 mg/kg, n=6), 5-AZA (5 mg/kg, n=7) and the combination of both agents (n=7). (g) Survival of mice treated as per (f). *P<0.05 versus vehicle and #P<0.05 versus initial (pretreatment) SPEP
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