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 4

Calcineurin inhibition enhances apoptosis and blocks proliferation, and PPP3CA regulates NF-κB signaling in MM cells.

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Calcineurin inhibition enhances apoptosis and blocks proliferation, and ...
(A) Apoptotic protein expression of KMS-11 treated with 20 nM panobinostat, 10 µM FK506, or both panobinostat and FK506 for 36 h is displayed. Two biologically independent experiments were performed. (B) KMS-11 was treated with 20 nM panobinostat, 10 µM FK506, or both panobinostat and FK506 for 36 h. Induced apoptosis was evaluated by flow cytometry. Annexin V (+) and propidium iodide (PI) (–): early apoptotic cells. Annexin V (+) PI (+): late apoptotic cells. Two biologically independent experiments were performed. (C) BrdU assays in KMS-11 treated with 15 nM panobinostat, FK506, or both panobinostat and FK506 as indicated for 48 h (n = 6). Two biologically independent experiments were performed. (D) Cell growth and PPP3CA expression in U266 treated with 20 nM panobinostat, 10 µM cyclosporine A, or both panobinostat and cyclosporine A as indicated for 48 h (n = 5). Two biologically independent experiments were performed. (E) NFATc1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1) protein in U266 treated with 20 nM panobinostat, FK506, or both panobinostat and FK506 as indicated for 72 h. TATA-binding protein (TBP) served as a loading control. Four biologically independent experiments were performed. (F and G) Cytoplasmic (C) and nuclear (N) NF-κB p50 in U266 lentivirally transduced with control vector (sh-cont) or shRNA against PPP3CA (sh-PPP3CA) (F) and control vector (vector) or PPP3CA (FLAG-PPP3CA) (G). α-tubulin and TBP served as a loading control. N/C ratios of NF-κB p50 are also displayed. Two biologically independent experiments were performed. (H) C and N NF-κB p50 in U266 treated with DMSO or 10 µM FK506 for 48 h followed by Western blot analysis. Two technically independent experiments were performed.