Genome-wide studies in multiple myeloma identify XPO1/CRM1 as a critical target validated using the selective nuclear export inhibitor KPT-276

Abstract

RNA interference screening identified XPO1 (exportin 1) among the 55 most vulnerable targets in multiple myeloma (MM). XPO1 encodes CRM1, a nuclear export protein. XPO1 expression increases with MM disease progression. Patients with MM have a higher expression of XPO1 compared with normal plasma cells (P<0.04) and to patients with monoclonal gammopathy of undetermined significance/smoldering MM (P<0.0001). The highest XPO1 level was found in human MM cell lines (HMCLs). A selective inhibitor of nuclear export compound KPT-276 specifically and irreversibly inhibits the nuclear export function of XPO1. The viability of 12 HMCLs treated with KTP-276 was significantly reduced. KPT-276 also actively induced apoptosis in primary MM patient samples. In gene expression analyses, two genes of probable relevance were dysregulated by KPT-276: cell division cycle 25 homolog A (CDC25A) and bromodomain-containing protein 4 (BRD4), both of which are associated with c-MYC pathway. Western blotting and reverse transcription-PCR confirm that c-MYC, CDC25A and BRD4 are all downregulated after treatment with KPT-276. KPT-276 reduced monoclonal spikes in the Vk*MYC transgenic MM mouse model, and inhibited tumor growth in a xenograft MM mouse model. A phase I clinical trial of an analog of KPT-276 is ongoing in hematological malignancies including MM.

Trial registration: ClinicalTrials.gov NCT01607892.

Figure 1

XPO1 expression in myeloma data sets. XPO1 expression increases with disease progression, as shown in gene expression data sets from MM patients. XPO1 expression level is highest in myeloma as compared with normal plasma cells (PCs), monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) (a). XPO1 expression levels are also shown in relation to common mutations in MM. XPO1 expression is highest in 11q13 and 6p21 TC groups (b). XPO1 expression level is significantly higher in HMCL than in primary patient samples, as shown by gene (c) and protein level (d).

Figure 2

KPT-276 has anti-myeloma activity in vitro. Twelve HMCL were treated with vehicle or KPT-276 in nanomolar concentrations, and after 72 h cell viability was reduced in most cell lines by at least 50% (a). Bone marrow cells obtained from myeloma patients were also treated with KPT-276 for 48 h, and drug-treated cells stained positive for AnnexinV, as compared with vehicle control. CD138+ plasma cells stained positive for AnnexinV in both sorted CD138+ (b) and unsorted whole bone marrow (c). FITC, fluorescein isothiocyanate.

Figure 3

KPT-276 downregulates cell cycle genes. Cells were treated with KPT-276, then collected at time points spanning 24 h. For immunoblotting, lysates were run on SDS-polyacrylamide gel electrophoresis and probed with antibodies for CDC25A, c-Myc and BRD4. Western blots show CDC25A and c-MYC protein expression was reduced starting at 12 h post-treatment, and BRD4 expression was reduced starting at 6 h post-treatment (a). To analyze BRD4 expression in response to KPT-276, RNA was extracted for reverse transcription-PCR analysis. BRD4 expression levels decreased after 6 h of drug exposure in two sensitive cell lines (b), (P values: *0.274, **0.247, ***0.044, ⁺0.192, ⁺⁺0.088, ⁺⁺⁺0.186).

Figure 4

KPT-276 is synergistic in combination with JQ1. Cells were treated with KPT-276 in combination with JQ1, both at nanomolar concentrations, for 72 h, then analyzed by MTT assay. HMCL viability was reduced at a greater rate in response to KPT-276 and JQ1 in combination, compared with treatment with either drug alone (a–c).

Figure 5

KPT-276 induces cell cycle arrest in MM1.S cells. After treating MM1.S cells for 24 h with varying doses of KPT-276, cell cycle arrest was observed in all drug-treated samples. The population of cells arrested in G1 increased from 31% in vehicle-treated cells (a) to 56% in cells treated with the highest dose of KPT-276 (d). The population of cells in S-phase decreased in all drug-treated cells (7–8.5%) (b–d), as compared with cells treated with DMSO vehicle (21%) (a).

Figure 6

KPT-276 reduces immunoglobulin levels and tumor volume in mouse models of myeloma. KPT-276 decreased tumor volume in an MM1.S xenograft model. As soon as 12 days after treatment initiation, tumor volume decreased substantially as compared with tumors in vehicle-treated mice. Tumor growth was inhibited for up to 30 days (a). Three transgenic Vk*Myc mice were treated with KPT-276 for 3 weeks, and the M-spike, a disease marker, was reduced in all three mice (b). KPT-276 activity in Vk*MYC mice is comparable to melphalan and carfilzomib (c).