Treatment of acquired drug resistance in multiple myeloma by combination therapy with XPO1 and topoisomerase II inhibitors

Abstract

Background: Acquired drug resistance is the greatest obstacle to the successful treatment of multiple myeloma (MM). Despite recent advanced treatment options such as liposomal formulations, proteasome inhibitors, immunomodulatory drugs, myeloma-targeted antibodies, and histone deacetylase inhibitors, MM is still considered an incurable disease.

Methods: We investigated whether the clinical exportin 1 (XPO1) inhibitor selinexor (KPT-330), when combined with pegylated liposomal doxorubicin (PLD) or doxorubicin hydrochloride, could overcome acquired drug resistance in multidrug-resistant human MM xenograft tumors, four different multidrug-resistant MM cell lines, or ex vivo MM biopsies from relapsed/refractory patients. Mechanistic studies were performed to assess co-localization of topoisomerase II alpha (TOP2A), DNA damage, and siRNA knockdown of drug targets.

Results: Selinexor was found to restore sensitivity of multidrug-resistant 8226B25, 8226Dox6, 8226Dox40, and U266PSR human MM cells to doxorubicin to levels found in parental myeloma cell lines. NOD/SCID-γ mice challenged with drug-resistant or parental U266 human MM and treated with selinexor/PLD had significantly decreased tumor growth and increased survival with minimal toxicity. Selinexor/doxorubicin treatment selectively induced apoptosis in CD138/light-chain-positive MM cells without affecting non-myeloma cells in ex vivo-treated bone marrow aspirates from newly diagnosed or relapsed/refractory MM patients. Selinexor inhibited XPO1-TOP2A protein complexes (proximity ligation assay), preventing nuclear export of TOP2A in both parental and multidrug-resistant MM cell lines. Selinexor/doxorubicin treatment significantly increased DNA damage (comet assay/γ-H2AX) in both parental and drug-resistant MM cells. TOP2A knockdown reversed both the anti-tumor effect and significantly reduced DNA damage induced by selinexor/doxorubicin treatment.

Conclusions: The combination of an XPO1 inhibitor and liposomal doxorubicin was highly effective against acquired drug resistance in in vitro MM models, in in vivo xenograft studies, and in ex vivo samples obtained from patients with relapsed/refractory myeloma. This drug combination synergistically induced TOP2A-mediated DNA damage and subsequent apoptosis. In addition, based on our preclinical data, we have initiated a phase I/II study with the XPO1 inhibitor selinexor and PLD (ClinicalTrials.gov NCT02186834). Initial results from both preclinical and clinical trials have shown significant promise for this drug combination for the treatment of MM.

Keywords: Acquired drug resistance; Liposomal doxorubicin; Mouse models; Multiple myeloma; Relapsed/refractory myeloma; XPO1 inhibition.

Figures

Fig. 1

XPO1 inhibition sensitizes drug-resistant human MM cell lines to doxorubicin (DOX). Human 8226B25 (a), U266PSR (b), 8226Dox6 (c), and 8226Dox40 (d) drug-resistant and parental MM cell lines were treated concurrently for 20 h with selinexor (300 nM) or KOS-2464 (10 nM) +/− doxorubicin (2 μM) and assayed for apoptosis by flow cytometry (activated caspase 3) (n = 3). XPO1 inhibitors sensitized drug-resistant cells to DOX compared with single-agent treatment

Fig. 2

Selinexor and KOS-2464 sensitize newly diagnosed and relapsed patient MM cells to doxorubicin. Bone marrow mononuclear cells were isolated and treated with selinexor or KOS-2464 +/− doxorubicin and fluorescently labeled with antibodies against activated caspase 3, CD138, and light chain (kappa or lambda). Newly diagnosed (n = 19) and relapsed (n = 22) CD-138/light-chain double-positive MM patient samples were all sensitized by selinexor and KOS-2464 to doxorubicin (P ≤ 1.37 × 10−8) versus single-agent treatment as shown by increased apoptosis (a, c). Non-myeloma CD138/light-chain double-negative patient cells were not sensitized to apoptosis by XPO1 inhibitors (b, d)

Fig. 3

In vivo studies. PLD/selinexor treatment reduced drug-resistant U266PSR (a) and parental U266 (b) tumor growth compared with single-agent treatment with PLD (P = 0.001) or selinexor (P = 0.009) in drug-resistant U266PSR (a) and PLD (P = 0.0003) or selinexor (P = 0.0079) in parental U266 (b). c, d: Selinexor/PLD significantly improved survival in U266PSR-challenged mice compared with single-agent selinexor (P = 0.0095) or PLD (P = 0.0018) (c) and in U266 compared with selinexor (P = 0.0002) or PLD (P = 0.0024) (d)

Fig. 4

Selinexor inhibits XPO1-TOP2A binding. Parental H929, U266, and 8226 and drug-resistant U266PSR, 8226Dox6, and 8226B25 plateau-density human MM cells (3 × 106/ml) were treated with selinexor (300 nM), cytospun, and assayed for intracellular co-localization of XPO1 and TOP2A by proximity ligation assay. A red fluorescent signal was generated only when XPO1 and TOP2A were in close proximity (<40 nm). a Selinexor blocked proximity co-localization of XPO1 and TOP2A. Inset, selinexor treatment did not affect XPO1 or TOP2A protein levels at 4 h as shown by Western blot. b Analysis of the number of XPO1-TOP2A foci showed that selinexor significantly decreased the number of foci in the nucleus and whole cells of both parental and drug-resistant MM cells

Fig. 5

Comet DNA fragmentation assay. High-density human 8226 and U226 and drug-resistant cell lines 8226Dox6, 8226B25, and U266PSR MM (2 × 106/mL) were treated with selinexor (100 nM) followed by doxorubicin (5 μM). DNA fragmentation (double-strand breaks) was measured by the neutral comet assay. Doxorubicin or selinexor, when used as single agents, increased DNA double-strand cleavage compared with untreated controls. However, selinexor + doxorubicin further increased DNA fragmentation over doxorubicin or selinexor alone

Fig. 6

Mechanism for selinexor and doxorubicin synergy. a Parental MM cell lines 8226 and U226 and drug-resistant 8226B25, 8226Dox6, and U266PSR cells were treated with doxorubicin (2 μM), selinexor (300 nM), or the combination for 20 h. Both parental and drug-resistant cell lines, when treated with the combination of selinexor and doxorubicin, had significantly increased DNA damage as shown by increased phospho-H2AX (Ser139) expression (n = 4). b SiRNA knockdown of TOP2A resulted in a substantial decrease in DNA damage (phospho-H2AX (Ser139)) and apoptosis, suggesting that selinexor and doxorubicin induced DNA damage, and subsequent apoptosis is TOP2A dependent. Inset, TOP2A knockdown (Western blot)