Histone deacetylase inhibitor panobinostat induces calcineurin degradation in multiple myeloma
Yoichi Imai, Eri Ohta, Shu Takeda, Satoko Sunamura, Mariko Ishibashi, Hideto Tamura, Yan-hua Wang, Atsuko Deguchi, Junji Tanaka, Yoshiro Maru, Toshiko Motoji
Yoichi Imai, Eri Ohta, Shu Takeda, Satoko Sunamura, Mariko Ishibashi, Hideto Tamura, Yan-hua Wang, Atsuko Deguchi, Junji Tanaka, Yoshiro Maru, Toshiko Motoji
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Research Article Hematology Oncology

Histone deacetylase inhibitor panobinostat induces calcineurin degradation in multiple myeloma

Abstract

Multiple myeloma (MM) is a relapsed and refractory disease, one that highlights the need for developing new molecular therapies for overcoming of drug resistance. Addition of panobinostat, a histone deacetylase (HDAC) inhibitor, to bortezomib and dexamethasone improved progression-free survival (PFS) in relapsed and refractory MM patients. Here, we demonstrate how calcineurin, when inhibited by immunosuppressive drugs like FK506, is involved in myeloma cell growth and targeted by panobinostat. mRNA expression of PPP3CA, a catalytic subunit of calcineurin, was high in advanced patients. Panobinostat degraded PPP3CA, a degradation that should have been induced by inhibition of the chaperone function of heat shock protein 90 (HSP90). Cotreatment with HDAC inhibitors and FK506 led to an enhanced antimyeloma effect with a greater PPP3CA reduction compared with HDAC inhibitors alone both in vitro and in vivo. In addition, this combination treatment efficiently blocked osteoclast formation, which results in osteolytic lesions. The poor response and short PFS duration observed in the bortezomib-containing therapies of patients with high PPP3CA suggested its relevance to bortezomib resistance. Moreover, bortezomib and HDAC inhibitors synergistically suppressed MM cell viability through PPP3CA inhibition. Our findings underscore the usefulness of calcineurin-targeted therapy in MM patients, including patients who are resistant to bortezomib.

Authors

Yoichi Imai, Eri Ohta, Shu Takeda, Satoko Sunamura, Mariko Ishibashi, Hideto Tamura, Yan-hua Wang, Atsuko Deguchi, Junji Tanaka, Yoshiro Maru, Toshiko Motoji

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Figure 2

PPP3CA protein is degraded by HDAC (histone deacetylase) inhibitors in MM cells.

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PPP3CA protein is degraded by HDAC (histone deacetylase) inhibitors in M...
The degree of protein expression change estimated by quantitative analyses of bands is displayed as indicated. (A) U266 and KMS-11 were treated with 20 nM panobinostat for 48 h followed by Western blot analysis. Actin served as a loading control. Nine (U266) and 3 (KMS-11) biologically independent experiments were performed. To determine the expression of PPP3CA mRNA in treated cells for 24 h, we performed relative quantification real-time PCR (n = 6). Four (U266) and 2 (KMS-11) biologically independent experiments were performed. (B) PPP3CA expression in U266 treated with DMSO (vehicle control) or 600 nM 17-AAG for 24 h. Three biologically independent experiments were performed. (C) IP assays using IgG2b or anti-PPP3CA antibody in U266. TCL, total cell lysate. Five biologically independent experiments were performed. (D) Protein expression in U266 treated with 20 nM panobinostat, 5 nM lactacystin, or both panobinostat and lactacystin for 48 h. Two biologically independent experiments were performed. (E) Protein expression in U266 treated with 20 nM panobinostat, 2 nM romidepsin, or 2 µM ACY-1215 for 48 h. Two biologically independent experiments were performed. (F) IP assays using anti-PPP3CA antibody in U266 treated with 2 µM ACY-1215 for 16 h. Two biologically independent experiments were performed. (G) Protein expression in U266 treated with 2 µM ACY-1215, 5 nM lactacystin, or both ACY-1215 and lactacystin for 48 h. Two biologically independent experiments were performed. (H) HSP70 protein expression in U266 treated with 600 nM 17-AAG for 24 hours. Two technically independent experiments were performed. (I) HSP70 expression in U266 treated with 20 nM panobinostat, 10 µM FK506, or both panobinostat and FK506 as indicated for 36 h. Cotreatment with 0.75 µM ACY-1215 and 10 µM FK506 for 48 h was also performed. Two biologically independent experiments were performed.