Purpose: CKS1B is significantly upregulated in multiple myeloma and associated with poor prognosis. The identification of novel therapies is essential for effective treatment of patients resistant to chemotherapy. The NEDD8 inhibitor MLN4924 selectively targets SCFSkp2 activation and offers a more specific approach to protein degradation inhibition than total proteasomal inhibition. The goal of this study was to evaluate whether MLN4924 is effective in high CKS1B conditions and identify mechanisms regulating drug potency.

Experimental Design: Bortezomib and MLN4924 sensitivity was assessed through proliferation, viability, clonogenic potential, and senescence induction in cells overexpressing CKS1B. The mechanism for MLN4924 sensitivity was elucidated by immunoblot analysis of SCFskp substrates and confirmed by shRNA knockdown. The clinical relevance of the NEDD8 pathway was examined in gene expression profiles (GEP) derived from healthy people, patients with monoclonal gammopathy of undetermined significance (MGUS), and multiple myeloma.

Results: Cells overexpressing CKS1B were resistant to bortezomib but sensitive to MLN4924. Treatment of CKS1B-overexpressing cells with MLN4924 decreased proliferation, clonogenicity, and induced senescence. MLN4924, but not bortezomib, induced stabilization of p21 and knockdown of p21 resulted in loss of MLN4924 sensitivity. Patients with MGUS and multiple myeloma exhibited increased expression of NEDD8 pathway genes relative to normal plasma cells. Multiple myeloma patients with high NEDD8 expression were linked to bortezomib resistance in clinical trials, and had inferior outcomes.

Conclusions: Our data demonstrate that cells with elevated CKS1B expression are resistant to bortezomib but sensitive to MLN4924 and offer a mechanism through the stabilization of p21. These findings provide rationale for targeting the NEDD8 pathway in multiple myeloma patients exhibiting elevated expression of CKS1B. Clin Cancer Res; 21(24); 5532–42. ©2015 AACR.

Translational Relevance

A subpopulation of patients with overexpression of CKS1B is resistant to available therapeutics, and correlated with poor prognosis, highlighting the immediate need for novel, effective therapeutics. Current therapeutic options, including the pan-proteasomal inhibitor bortezomib, are associated with toxicitiy arising from nonspecific proteasomal inhibition. MLN4924 inhibits the neddylation and activation of SCFSkp2 offering a more targeted method of inhibiting protein degradation than bortezomib. Here, we report that cells with elevated expression of CKS1B are resistant to bortezomib but remain sensitive to MLN4924 treatment, highlighting the efficacy of neddylation inhibition in high-risk multiple myeloma patients. We further define the mechanistic role for p21 in MLN4924 sensitivity in elevated CKS1B environments. Examination of gene expression profiles (GEP) from patients with multiple myeloma and monoclonal gammopathy of undetermined significance (MGUS) reveal elevation of NEDD8 machinery expression and further reinforce the importance of targeting SCFSkp2 activation. Our data support the further examination of MLN4924 in preclinical combinatorial studies with currently available treatments for multiple myeloma.

Multiple myeloma is a malignancy of plasma cells that accumulate in the bone marrow, secrete antibody, and interfere with the production of normal blood cells. Multiple myeloma is now the second most common hematologic malignancy in the United States, with a 5-year survival rate less than 50% (1). In the past decade, proteasome inhibition and immunomodulators have revolutionized multiple myeloma therapies. In particular, bortezomib, which targets the 26S proteasome subunit β5, has induced a high level of positive response rates (2, 3). However, toxicities associated with global proteasomal inhibition and resistance to bortezomib in multiple myeloma are major concerns, prompting the further development of novel therapies. These improved inhibitors are designed to more specifically target critical factors in protein turnover and are now part of ongoing clinical trials. One such molecule, MLN4924, is an inhibitor of NEDD8-activating enzyme (NAE), preventing the conjugation of the small ubiquitin-like protein NEDD8 (neural precursor cell expressed developmentally downregulated protein 8) to cullin-RING ubiquitin E3 ligases (CRL). This inhibitor has shown promise against multiple myeloma in vitro by inhibiting the degradation of skp1/cullin-1/F-box SKP2 (SCFSkp2) substrates, including p27 and p21 (4).

CDC28 kinase subunit 1 (CKS1B) is a necessary cofactor of the CRL complex, SCFSkp2, which regulates cellular entry into S-phase and possesses antiapoptotic activity through p27-dependent and -independent pathways, and is a critical factor in multiple myeloma drug resistance (5). CKS1B is an essential protein for normal cell division and growth (6), and is expressed at a high level in various cancer tissues, including hepatocellular carcinoma (7), colon (8), lung (9), oral squamous cell carcinoma (10), breast cancer (11), and others. In multiple myeloma, amplification of region 1q21, which includes the CKS1B gene, identifies a subpopulation with poor prognosis, an aggressive clinical course, and few therapeutic options (12, 13). CKS1B amplification is also associated with transformation from both the benign state of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma and further progression to plasma cell leukemia (14). Previously, we identified CKS1B as one of 70 high-risk genes inversely associated with survival in newly diagnosed multiple myeloma (15) and high nuclear expression of CKS1B is an adverse prognostic factor for relapsed/refractory multiple myeloma patients (16). These findings provide compelling evidence that CKS1B represents a strong candidate target gene for therapy.

Neddylated SCFSkp2 represents the activated complex essential for the ubiquitination of CRL substrates (17). In this study, we test the efficacy of MLN4924 in multiple myeloma cells overexpressing CKS1B. We explore how MLN4924 affects cell viability and senescence in CKS1B-overexpressing multiple myeloma cells. Our study identifies a novel function for MLN4921 in the upregulation of p21 expression, which is independent of the CKS1B-mediated SCFSkp2 complex formation and NEDD8-dependent SCFSkp2 activation, resulting in killing multiple myeloma cells.

Gene expression profiling

The data of gene expression profile (GEP) were collected from a publicly available website that include 351 newly diagnosed patients with multiple myeloma who participated in the Total Therapy 2 (TT2) clinical trial [Zhan (18); Shaughnessy (15)] and 264 relapsed multiple myeloma in the Assessment of Proteasome Inhibition for Extending Remission (APEX) phase III clinical trial [Mulligan (19)]. Permutation analyses were performed to correlate NAE1, UBA3, UBC12, and NEDD8 expression with patient survival in the APEX trial (n = 264). The significance of gene expression with drug response in the APEX trial was analyzed by a Student t test.

Compounds

MLN4924 and bortezomib were provided by Millenium Pahrmaceuticals. Each was resuspended in dimethyl sulfoxide (DMSO) to a concentration of 10mmol/L and used at the concentrations indicated.

Cell lines and cell culture

Human multiple myeloma cell lines ARP-1, OCI-My5, and KMS28PE with or without CKS1B overexpression (expression levels previously detailed in ref. 5) were cultured in RPMI-1640 (Life Technologies). HEK-293T cells were cultured in DMEM (Life Technologies). All media were supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin. All cells were cultured in humidified incubators at 37°C with 5% CO2.

Establishment of stable multiple myeloma cell lines

Overexpressing CKS1B.

The construction of multiple myeloma cell lines stably overexpressing CKS1B has been described previously (5). Briefly, the cDNA sequence for human and mouse CKS1B is equivalent. CKS1B was amplified from the PCDH-mouse-CKS1B-EF1-RFP vector using the following primers: CKS1B Xba1 forward: GTGtctagaATGTCCCACAAACAAATCTACTATTCG, CKS1B BamH1 reverse: GTGggatccTCATTTCTTTGGCTTCTTGGGC. CKS1B cDNA was cloned into a modified pCDH vector (Systems Biology). Lentiviruses were obtained by cotransfection of the pCDH-CKS1B vector with packaging vectors into HEK-293T cells following the standard protocol of ProFection Mammalian Transfection System (Promega). Viral supernatant was harvested after 36 hours and concentrated by ultracentrifugation. ARP-1 and OCI-My5 multiple myeloma cells were transfected with lentivirus containing CKS1B cDNA to yield CKS1B-overexpressing multiple myeloma cells. The efficiency of lentiviral transfection was determined by measuring the expression of green fluorescent protein (GFP) using flow cytometry. Approximately 95% transduction efficiency of multiple myeloma cells was consistently achieved. CKS1B-OE multiple myeloma cells were screened by treatment with puromycin to select for CKS1B-overexpressing cells.

p21 knockout

The lentiviral CRISPR/Cas9 GeCKO system was used for specific p21 knockout in CKS1B-overexpressing multiple myeloma cells. Two pairs of p21 single guide RNAs (sgRNA) were annealed, phosphorylated, and ligated into the lentiCRISPR lentiviral sgRNA vector: p21 forward primer: 5′-CACCG CCGCGACTGTGATGCGCTAA-3′; reverse, 5′-AAACTTAGCGCATCACAGTCGCGGC-3′. The U6 forward primer: 5′-GACTATCATATGCTTACCGT-3′ was used for sequencing sgRNA. Lentiviruses containing the sgRNAs were obtained by cotransfection of the lentiCRISPR-p21sgRNA vector with pVSVg and psPAX2 vectors with packaging vectors into HEK-293T cells. Viral supernatant was harvested after 48 hours. ARP-1 and OCI-My5 multiple myeloma cells were transduced with lentivirus containing the annealed p21 sgRNAs. All primers were purchased from Integrated DNA Technologies.

RT-PCR

mRNA was extracted using the RNeasy Mini Kit (Bio-Rad) from wild-type, empty vector-transfected, CKS1B-overexpressing, and p21 knockout cultured cells treated with or without bortezomib, MLN4924, or vehicle for 24 hours. Reverse-transcription PCR was performed with 5× iScript reverse transcription supermix (Bio-Rad) according to the manufacturer's protocols. Quantitative real-time PCR (qRT-PCR) was used to detect p21 and GAPDH expression in. Primers used for qRT-PCR were as follows: p21 forward, 5′-TGT CCG TCA GAA CCC ATG C-3′, p21 reverse, 5′-AAA GTC GAA GTT CCA TCG CTC-3′, and GAPDH forward, 5′-GAC ACC CAC TCC TCC ACC T-3′, reverse, 5′-ATG AGG TCC ACC ACC CTG T-3′. GAPDH transcript levels were used to normalize the amount of p21 cDNA in each sample.

Cell proliferation assay

A total of 1.5 × 105 cells/mL were seeded into 6-well plates in 2-mL media in triplicate. Cells were untreated, treated with 5 nmol/L bortezomib, or 1 μmol/L MLN4924. Media were replenished with fresh RPMI-1640 with or without drugs every 2 days. Cell proliferation and viability were evaluated for 7 days at the indicated time point using a hemocytometer paired with Trypan blue exclusion. Cell viability is expressed as the ratio of live cells relative to total cells.

Soft-agar clonogenic formation assay

ARP-1 and OCI-My5 multiple myeloma cells (1 × 105) were seeded in 12-well plates with or without bortezomib or MLN4924 at varying doses in fresh RPMI-1640. Cells were resuspended in 0.33% agar in MyeloCult H5100 cultures/10% FBS and allowed to clonally expand for 3 weeks. Cells were fed twice each week by placing two drops of medium on the layer with or without drugs. Images of all plates were taken on a Nikon Eclipse Ti microscope, and colony numbers were counted using the ImageJ. The colony efficiency was calculated as (colony number in the image) × (well area)/(actual area the photo represent)/10,000 × 100%.

Senescence β-galactosidase cell staining

Senescence β-galactosidase staining was used to detect β-galactosidase activity, a known characteristic of senescent cells. OCI-My5 and ARP-1 cells with or without the stable overexpression of CKS1B or stable knockdown of p21 were grown in 6-well plates. Cells were mock treated or treated for 48 hours with bortezomib or MLN4924, were harvested, washed with PBS, and fixed. Cells were resuspended in β-Galactosidase Staining Solution (5 mmol/L K3Fe(CN)6, 5 mmol/L K4Fe(CN)6·3H2O, 2 mmol/L MgCl2, PBS up to 500 mL, pH 6.0) and seeded into 6-well plates. Plates were sealed with paraformaldehyde and incubated at 37°C. The β-galactosidase staining solution was removed and staining was assessed under a microscope. Stained cells were counted and expressed relative to total cells. Graphs represent the average and standard deviations from four separate plates. This method was also used to assess senescence in CKS1B-overexpressing multiple myeloma cells treated with empty vector or stable p21 knockdown with or without the treatment of MLN4924. Representative plates are depicted in Supplementary Fig. S3.

Immunoblots

Protein expression levels were determined by Western blot analysis. Briefly, cells were lysed using a Mammalian Cell Extraction Kit with protease inhibitor cocktail (Biovision). Protein concentrations were determined on a NanoDrop 1000 (Thermo) and 10ug of total protein lysate was separated on a Mini-PROTEAN TGX precast acrylamide gel (BIO-RAD). Gels were transferred to activated nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk in Tris buffered saline (TBS) containing 0.05% Tween-20 (TBS-T). Membranes were blotted with the indicated primary antibodies. All primary antibodies were diluted according to the manufacturer's directions. CKS1B, CUL1, p27 and all secondary antibodies were purchased from Santa Cruz Biotechnology, primary antibodies p21, P-RB(S608), P-RB(S780), P-RB(S807) and β-Actin were purchased from Cell Signaling Technology. β-Actin was used to normalize the amount of protein for each sample. Protein bands were visualized using HRP-conjugated secondary antibodies and SuperSignal West Pico (Pierce). Blots were subsequently stripped and reprobed for β-actin as loading control. Uncropped blots are presented in Supplementary Fig. S1.

Statistical analyses

One-way ANOVA (≥ three groups) and Student t tests were used to determine significance between experimental groups. Two-way ANOVA paired with the Tukey–Kramer method for multiple comparisons was used to compare multiple groups with multiple treatment levels. Significance was defined as P < 0.05.

Multiple myeloma cells overexpressing CKS1B are resistant to bortezomib but sensitive to MLN4924

CKS1B expression is increased in relapsed multiple myeloma and confers drug resistance, including resistance to bortezomib (5). In an attempt to overcome this drug resistance and toxicities associated with pan-proteasomal inhibitors, more specifically targeted inhibitors of protein degradation have emerged, including the NAE inhibitor MLN4924. Because of the established drug-resistance against bortezomib of multiple myeloma cells overexpressing CKS1B, we wanted to examine whether cells overexpressing CKS1B were resistant to treatment with MLN4924.

We compared the effects of bortezomib and MLN4924 on the proliferation and survival of ARP-1 and OCI-My5 multiple myeloma cell lines stably transfected with empty vector or CKS1B expression vector. We found that while cells expressing basal levels of CKS1B were susceptible to treatment with both bortezomib and MLN4924 (Fig. 1A, left), cells overexpressing CKS1B treated with bortezomib continued to proliferate in a manner comparable with untreated cells (Fig. 1A, right) while cells overexpressing CKS1B treated with MLN4924 failed to proliferate appreciably (Fig. 1A, right, green line). In examining cell viability, treatment with either bortezomib or MLN4924 in cells expressing basal levels of CKS1B resulted in diminished viability (Fig. 1B, left). In cells overexpressing CKS1B, treatment with bortezomib resulted in viability comparable with mock treatment (Fig. 1B, right) but treatment with MLN4924 yielded rapid collapse leading to total cell death within 6 days (Fig. 1B, right, green line). These results highlight that cells with high CKS1B are resistant to bortezomib treatment but remain sensitive to MLN4924.

Figure 1.

Multiple myeloma cells overexpressing CKS1B are resistant to bortezomib but sensitive to MLN4924. A, effects of bortezomib and MLN4924 on the in vitro proliferation of ARP-1 and OCI-My5 multiple myeloma cell lines with and without the overexpression of CKS1B. Cells were treated with either bortezomib (5 nmol/L) or MLN4924 (1 μmol/L) for 7 days. Proliferation was assessed by hemocytometer cell counts. B, effects of bortezomib and MLN4924 on cell viability of ARP-1 and OCI-My5 multiple myeloma cell lines with and without the overexpression of CKS1B. Cells were treated with either bortezomib (5 nmol/L) or MLN4924 (1 μmol/L) for 7 days. Cell viability is expressed as the ratio of viable cells to the total number of cells. C, effects of bortezomib and MLN4924 on the clonogenic capacity of ARP-1 and OCI-My5 multiple myeloma cell lines with and without the overexpression of CKS1B was assessed by counting colony formation in soft-agar. D, the effects of CKS1B expression on senescence in multiple myeloma cell lines was assessed by β-galactosidase staining. E, the effects of bortezomib and MLN4924 on senescence in ARP-1 and OCI-My5 cells overexpressing CKS1B was assessed by β-galactosidase staining. In all experiments, data are mean ± SD of three separate experiments.

Figure 1.

Multiple myeloma cells overexpressing CKS1B are resistant to bortezomib but sensitive to MLN4924. A, effects of bortezomib and MLN4924 on the in vitro proliferation of ARP-1 and OCI-My5 multiple myeloma cell lines with and without the overexpression of CKS1B. Cells were treated with either bortezomib (5 nmol/L) or MLN4924 (1 μmol/L) for 7 days. Proliferation was assessed by hemocytometer cell counts. B, effects of bortezomib and MLN4924 on cell viability of ARP-1 and OCI-My5 multiple myeloma cell lines with and without the overexpression of CKS1B. Cells were treated with either bortezomib (5 nmol/L) or MLN4924 (1 μmol/L) for 7 days. Cell viability is expressed as the ratio of viable cells to the total number of cells. C, effects of bortezomib and MLN4924 on the clonogenic capacity of ARP-1 and OCI-My5 multiple myeloma cell lines with and without the overexpression of CKS1B was assessed by counting colony formation in soft-agar. D, the effects of CKS1B expression on senescence in multiple myeloma cell lines was assessed by β-galactosidase staining. E, the effects of bortezomib and MLN4924 on senescence in ARP-1 and OCI-My5 cells overexpressing CKS1B was assessed by β-galactosidase staining. In all experiments, data are mean ± SD of three separate experiments.

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In order to assess the role of p53 on proliferation and cellular viability, we performed experiments using the p53-positive KMS28PE multiple myeloma cell line with or without the stable overexpression of CKS1B. We found that KMS28PE cells exhibited very similar proliferative patterns to both OCI-MY5 and ARP1 cells when treated with MLN4924 (Supplementary Fig. S2B). We also found that viability was affected very similarly to OCI-MY5 cells of the same CKS1B background (Supplementary Fig. S2C). Together, these findings suggest that p53 status does not contribute greatly to MLN4924 sensitivity.

We examined the ability of CKS1B-overexpressing cells to form colonies in soft agar in the presence of bortezomib or MLN4924. ARP-1 and OCI-My5 cell lines with or without CKS1B overexpression were seeded in the presence or absence of bortezomib or MLN4924. Treatment of either EV cell line with bortezomib or MLN4924 resulted in decreased colony formation (Fig. 1C). However, upon CKS1B overexpression, colony formation significantly increased upon botezomib challenge. Conversely, colony formation remained very low in CKS1B-overexpressing cells upon challenge with MLN4924. (Fig. 1C). These results indicate that cells with basal levels of CKS1B are sensitive to both bortezomib and MLN4924, whereas cells overexpressing CKS1B are resistant to bortezomib but remain sensitive to MLN4924.

Skp2 is a critical component of the E3 ligase complex SCFSkp2. Targeting Skp2 leads to accumulation of p21 and p27 and increased cellular senescence (20–22). CKS1B is an essential accessory protein for the function of SCFSkp2. With a known role for CKS1B in SCFSkp2 function and because of the important role for SCFSkp2 in senescence, we examined the effects of bortezomib and MLN4924 on senescence in CKS1B-overexpressing multiple myeloma cell lines. We examined the effect of CKS1B knockdown on senescence in ARP-1 and OCI-My5 multiple myeloma cell lines using shRNA to deplete CKS1B expression (Fig. 1D). In each cell line, knockdown of CKS1B resulted in greatly increased senescence relative to treatment with a scrambled control. These results suggest that the role for CKS1B in SCFSkp2 integrity relates to senescence and that a loss of CKS1B has comparable effects to the loss SCFSkp2 in the promotion of senescence.

Next, we examined the effects of bortezomib and MLN4924 on senescence in ARP-1 and OCI-My5 cell lines stably overexpressing CKS1B. In both cell lines, the overexpression of CKS1B slightly increased senescence in untreated cells relative to cells transfected with empty vector (Fig. 1E). Treatment of parental cell lines with either bortezomib or MLN4924 resulted in a marked increase in senescence. However, upon CKS1B overexpression, there was a significant decrease in senescence upon bortezomib treatment. This decrease was not exhibited upon MLN4924 treatment. These results indicate that MLN4924, but not bortezomib, is able to induce senescence in CKS1B-overexpressing cells.

Taken together, these data illustrate that multiple myeloma cells over expressing CKS1B are resistant to treatment with the proteasomal inhibitor bortezomib but sensitive to the NAE inhibitor MLN4924.

CKS1B regulates neddylation-related signaling

In order to examine the relationship between CKS1B and neddylation, we examined the neddylation of Cullin-1 in ARP-1 and OCI-My5 multiple myeloma cell lines upon the modulation of CKS1B expression. In both cell lines, the overexpression of CKS1B led to increased neddylation of Cullin-1 as evidenced using an antibody targeted against NEDD8 (Fig. 2A). Conversely, knockdown of CKS1B expression with shRNA led to a decrease in neddylated Cullin-1 in each cell line (Fig. 2B). Taken together, these data highlight the intimate relationship between CKS1B and the neddylation of Cullin-1. We next explored the effects of MLN4924 on the neddylation of Cullin-1 in ARP-1 and OCI-My5 cells overexpressing CKS1B. Consistent with the role of MLN4924 as an NAE inhibitor, treatment with MLN4924 in each cell line led to a substantial decrease in the neddylation of Cullin-1 (Fig. 2C).

Figure 2.

CKS1B regulates neddylation-related signaling. A, protein expression was measured in ARP-1 and OCI-My5 cells stably transfected with empty vector (EV) or CKS1B (OE). A 30 μg/lane of whole-cell lysates were resolved on an SDS-PAGE gel and analyzed by immunoblotting with the indicated antibody. B, immunoblot of protein lysates derived from ARP-1 and OCI-My5 cells stably transduced with either scrambled control or CKS1B-targeted shRNA. A 30-μg whole-cell lysate was resolved using SDS-PAGE and lysates were blotted with the indicated antibodies. C, ARP-1 or OCI-My5 cells with or without the overexpression of CKS1B were immunoblotted for Cullin-1 with or without MLN4924 (1 μmol/L) treatment (1 hour).

Figure 2.

CKS1B regulates neddylation-related signaling. A, protein expression was measured in ARP-1 and OCI-My5 cells stably transfected with empty vector (EV) or CKS1B (OE). A 30 μg/lane of whole-cell lysates were resolved on an SDS-PAGE gel and analyzed by immunoblotting with the indicated antibody. B, immunoblot of protein lysates derived from ARP-1 and OCI-My5 cells stably transduced with either scrambled control or CKS1B-targeted shRNA. A 30-μg whole-cell lysate was resolved using SDS-PAGE and lysates were blotted with the indicated antibodies. C, ARP-1 or OCI-My5 cells with or without the overexpression of CKS1B were immunoblotted for Cullin-1 with or without MLN4924 (1 μmol/L) treatment (1 hour).

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Paired with our earlier findings, these results demonstrate that drug-resistant cells with increased CKS1B also have increased Cullin-1 neddylation. Because increased neddylation of Cullin-1 leads to increased activation of SCFSkp2, a negative regulator of p21, p27, and senescence, we hypothesized that the drug resistance of CKS1B-overexpressing cells against bortezomib, but not the NAE inhibitor MLN4924, stems from an increase in the ubiquitin-mediated degradation of downstream targets of the CRL–SCF complex. Therefore, we sought to examine the effect of drug treatment on SCFSkp2-regulated proteins in cells overexpressing CKS1B.

MLN4924 overcomes CKS1B-induced drug resistance by inhibiting p21 ubiquitin-mediated degradation

In order to examine the effect of drug treatment on SCFSkp2-regulated proteins in cells overexpressing CKS1B, we performed immunoblot analysis of known downstream targets of CRL/SCF (Fig. 3A).

Figure 3.

MLN4924 overcomes CKS1B-induced drug resistance by inhibiting p21 ubiquitin-mediated degradation. A, immunoblot analysis of protein expression following 48-hour treatment with bortezomib (5 nmol/L) or MLN4924 (1 μmol/L) in ARP-1 and OCI-My5 multiple myeloma cell lines stably transfected with empty vector (EV) or CKS1B (OE). Whole-cell lysates were resolved on an SDS-PAGE gel and analyzed by immunoblot using the indicated antibodies. B, p21 mRNA expression was determined in ARP-1 and OCI-My5 multiple myeloma cell lines stably transfected with empty vector (EV) or CKS1B (OE). qRT-PCR was performed to assess p21 mRNA expression following 48-hour treatment with bortezomib (5nmol/L) or MLN4924 (1 μmol/L) and results were normalized to the expression of GAPDH. Values are expressed relative to untreated cells transfected with empty vector. Data are mean ± SD of four separate experiments.

Figure 3.

MLN4924 overcomes CKS1B-induced drug resistance by inhibiting p21 ubiquitin-mediated degradation. A, immunoblot analysis of protein expression following 48-hour treatment with bortezomib (5 nmol/L) or MLN4924 (1 μmol/L) in ARP-1 and OCI-My5 multiple myeloma cell lines stably transfected with empty vector (EV) or CKS1B (OE). Whole-cell lysates were resolved on an SDS-PAGE gel and analyzed by immunoblot using the indicated antibodies. B, p21 mRNA expression was determined in ARP-1 and OCI-My5 multiple myeloma cell lines stably transfected with empty vector (EV) or CKS1B (OE). qRT-PCR was performed to assess p21 mRNA expression following 48-hour treatment with bortezomib (5nmol/L) or MLN4924 (1 μmol/L) and results were normalized to the expression of GAPDH. Values are expressed relative to untreated cells transfected with empty vector. Data are mean ± SD of four separate experiments.

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Consistent with Fig. 2, the use of a Cullin-1–specific antibody showed that the overexpression of CKS1B led to an increase in the neddylated form of Cullin-1 and treatment with MLN4924 completely blocked the neddylation of Cullin-1 (Fig. 3A, row 2). In both cell lines, inhibition of SCFSkp2 activation by blocking Cullin-1 neddylation with MLN4924, independent of CKS1B expression, led to increased expression of p27 and p21proteins relative to expression seen in cells without drug treatment (Fig. 3A, rows 7 and 8). Treatment of cells with basal level CKS1B expression with bortezomib resulted in increased levels of p27 and p21 as we expected. Although we expected the overexpression of CKS1B to lead to the downregulation of p27 and p21 in bortezomib-treated cells, we observed downregulation of p21 but continued overexpression of p27 (Fig. 3A, row 7 vs. row 8). Together, these results imply that bortezomib-induced increases in p27 are insensitive to CKS1B overexpression and suggest a mechanism of p27 regulation distinct from SCFSkp2 regulation of p21 and p27. We further examined the effects of drug treatment in cells with basal and elevated levels of CKS1B expression on the mRNA expression of the p21 gene CDKN1A. Consistent with the immunoblots, MLN4924 increased the low basal level expression of p21 dramatically in both parental and CKS1B-overexpressing cells, whereas treatment with bortezomib had negligible effects on the mRNA expression of p21 (Fig. 3B).

CKS1B-overexpressing cells are sensitive to MLN4924, but not bortezomib, and p21 is differentially regulated from p27 in CKS1B-overexpressing cells upon bortezomib. These results suggest a critical role for p21 in the regulation of CKS1B drug resistance. Accordingly, we investigated the role of p21 in drug sensitivity in multiple myeloma cell lines.

p21 is necessary for MLN4924 sensitivity in CKS1B-overexpressing cells

In order to investigate the role of p21 in drug sensitivity, we modulated p21 expression using cells stably expressing shRNA against p21. Because low basal expression of p21 is well established (20, 21), we confirmed the induction of p21 expression upon treatment of cells with MLN4924, an established DNA-damaging agent and activator of p21 (22, 23). CKS1B-overexpressing OCI-My5 multiple myeloma cells expressing shRNA against p21 substantially knocked down p21 protein expression upon MLN4924 treatment relative to cells transfected with empty vector (Fig. 4A).

Figure 4.

p21 is necessary for MLN4924 sensitivity in CKS1B-overexpressing cells. A, immunoblot analysis of p21 expression in OCI-My5-CKS1B OE multiple myeloma cells stably transfected with empty vector (EV) or p21 shRNA-expressing vector (p21KO) following 24-hour treatment with MLN4924. B, the effects of bortezomib and MLN4924 on cell viability were assessed in OCI-My5-CKS1B–overexpressing cells transfected with either empty vector (EV) or shRNA targeting p21 expression (p21KO). Cells were seeded in the presence of MLN4924 (0.5 μmol/L, 1 μmol/L) and viability was monitored for 6 days. Cell viability was assessed by hemocytometer cell counts using trypan blue exclusion staining and is expressed as the ratio of viable cells to the total number of cells. Data are mean ± SD of three separate experiments. C, the effect of MLN4924 (10 nmol/L, 100 nmol/L, 500 nmol/L) on the clonogenic capacity of OCI-My5-CKS1B–overexpressing multiple myeloma cells transfected with empty vector or shRNA targeting p21 (p21 KO) was assessed by counting colony formation in soft-agar. Data are mean ± SD of three separate experiments. D, the effects of MLN4924 (100 nmol/L) on senescence in OCI-My5-CKS1B–overexpressing cells with the expression of empty vector or shRNA targeting p21 (p21 KO) was assessed by β-galactosidase staining. Data are mean ± SD of three separate experiments.

Figure 4.

p21 is necessary for MLN4924 sensitivity in CKS1B-overexpressing cells. A, immunoblot analysis of p21 expression in OCI-My5-CKS1B OE multiple myeloma cells stably transfected with empty vector (EV) or p21 shRNA-expressing vector (p21KO) following 24-hour treatment with MLN4924. B, the effects of bortezomib and MLN4924 on cell viability were assessed in OCI-My5-CKS1B–overexpressing cells transfected with either empty vector (EV) or shRNA targeting p21 expression (p21KO). Cells were seeded in the presence of MLN4924 (0.5 μmol/L, 1 μmol/L) and viability was monitored for 6 days. Cell viability was assessed by hemocytometer cell counts using trypan blue exclusion staining and is expressed as the ratio of viable cells to the total number of cells. Data are mean ± SD of three separate experiments. C, the effect of MLN4924 (10 nmol/L, 100 nmol/L, 500 nmol/L) on the clonogenic capacity of OCI-My5-CKS1B–overexpressing multiple myeloma cells transfected with empty vector or shRNA targeting p21 (p21 KO) was assessed by counting colony formation in soft-agar. Data are mean ± SD of three separate experiments. D, the effects of MLN4924 (100 nmol/L) on senescence in OCI-My5-CKS1B–overexpressing cells with the expression of empty vector or shRNA targeting p21 (p21 KO) was assessed by β-galactosidase staining. Data are mean ± SD of three separate experiments.

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We examined cell viability in OCI-My5 cells overexpressing CKS1B with and without knockdown of p21 protein expression. The knockdown of p21 decreased cell sensitivity to MLN4924 in CKS1B-overexpressing cells and increased cell viability while prolonging survival relative to cells expressing p21 (Fig. 4B). Importantly, we have performed comparative proliferation assays between cells with and without knockdown of p21. We find no significant differences in proliferation, suggesting that changes in viability are not an artifact of altered proliferation rates (data not shown).

We further examined the clonogenic ability of p21 knockout cells to form colonies in soft agar in the presence of MLN4924 relative to cells expressing basal p21 levels. Consistent with a role for p21 in sensitivity to MLN4924, colony formation significantly increased in cells with p21 knockdown compared with cells expressing p21 upon MLN4924 treatment (Fig. 4C). This increase was not seen in untreated cells. These results indicate that cells with diminished levels of p21 are less sensitive to MLN4924 and have a higher clonogenic capacity in the presence of MLN4924.

We also explored the importance of p21 in the induction of senescence in cells overexpressing CKS1B. Although treatment of CKS1B-overexpressing cells with MLN4924 resulted in increased senescence relative to untreated cells, the amount of cells in senescence was dramatically reduced upon the knockdown of p21 (Fig. 4D).

These results demonstrate that MLN4924 sensitivity on cell proliferation and survival, clonogenic capacity, and senescence in CKS1B-overexpressing cells is dependent upon p21 and a loss of p21 shifts CKS1B-overexpressing cells toward insensitivity to MLN4924. Taken together, these findings reveal, for the first time, a crucial role for p21 in regulating cell sensitivity to MLN4924.

Neddylation machinery is dysregulated and associated with drug resistance and poor clinical outcomes in multiple myeloma.

Having demonstrated the importance of the NEDD8 pathway in regulating SCFSkp2 activity in multiple myeloma cell lines, we investigated the clinical importance of the NEDD8 pathway in multiple myeloma.

Although there is a broad distribution of gene expression in each group from TT2, we found that the mRNA expression of NAE1, UBA3, and UBC12 was, on average, significantly elevated in plasma cells from patients with MGUS and patients newly diagnosed with multiple myeloma (24) relative to normal plasma cells (NPC) from healthy donors (Fig. 5A).

Figure 5.

The neddylation pathway is dysregulated and associated with drug resistance in multiple myeloma. A, examination of the mRNA expression profiles of neddylation pathway genes. Signal levels from NAE1, UBA3, UBC12, and NEDD8 are shown on the y-axis. GEP signals were derived from participants in TT2 clinical trials of different clinical backgrounds. Patients were designated as healthy donors with NPCs, MGUS patients (MGUS), or multiple myeloma patients (MM) and are sorted on the x-axis. B, an examination of NEDD8 expression in patients categorized as unresponsive (No Response) or responsive (Response) to treatment with dexamethasone and/or bortezomib. Mean signal levels of patients and P values of differences in expression are given. C, GEPs from 5B were further segregated by treatment with bortezomib or dexamethasone. NEDD8 expression signal levels are plotted on the y-axis. The mean signal level and P value of differences are given between unresponsive and responsive patients.

Figure 5.

The neddylation pathway is dysregulated and associated with drug resistance in multiple myeloma. A, examination of the mRNA expression profiles of neddylation pathway genes. Signal levels from NAE1, UBA3, UBC12, and NEDD8 are shown on the y-axis. GEP signals were derived from participants in TT2 clinical trials of different clinical backgrounds. Patients were designated as healthy donors with NPCs, MGUS patients (MGUS), or multiple myeloma patients (MM) and are sorted on the x-axis. B, an examination of NEDD8 expression in patients categorized as unresponsive (No Response) or responsive (Response) to treatment with dexamethasone and/or bortezomib. Mean signal levels of patients and P values of differences in expression are given. C, GEPs from 5B were further segregated by treatment with bortezomib or dexamethasone. NEDD8 expression signal levels are plotted on the y-axis. The mean signal level and P value of differences are given between unresponsive and responsive patients.

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We also examined NEDD8 mRNA expression in patients from APEX trials that were responsive or unresponsive to standard multiple myeloma therapies (i.e., bortezomib and dexamethasone; refs. 19, 25). Although great variance was seen in populations, the average expression of NEDD8 was significantly elevated in patients exhibiting decreased response to clinical treatment (P = 0.015; Fig. 5B). This result is further segregated into response to either bortezomib or dexamethasone. As shown in Fig. 5C, average NEDD8 expression was significantly elevated in patients exhibiting decreased clinical response to bortezomib (P = 0.0005), but was not significantly altered in patients resistant to dexamethasone (P = 0.61). These results identify a subset of patients with increased NEDD8 expression who are, on average as a population, resistant to bortezomib but not to dexamethasone in the clinic.

The effect of NAE1, UBA3, UBC12, and NEDD8 expression on patient outcome was further analyzed using the Kaplan–Meier survival and log-rank tests. We did not find any significance in the TT2 trial that includes treatment with melphalan and thalidomide (data not shown; ref. 24). However, patients with elevated expression of proteins involved in the neddylation and activation SCFSkp2 had decreased overall survival relative to patients with low expression of neddylation pathway proteins. The elevated expression of UBA3 (P = 0.003), UBC12 (P = 0.001), or CKS1B (P = 0.015) each correlated with significantly poorer prognoses than patients with low expression of the same gene in the APEX trial, a phase III study that compared bortezomib with dexamethasone in post-relapse multiple myeloma patients (Fig. 6A). Populations were further classified on two variables of CKS1B and UBA3 expression (Fig. 6B, left) and CKS1B and UBC12 expression (Fig. 6B, right). In both scenarios, patients with (high/high) expression of either CKS1B-UBA3 (P = 0.029) or CKS1B-UBC12 (P = 0.006) demonstrated significantly decreased survival from patients with heterogenous (high/low) or (low/high) expression. In both cases, greatest survival was seen in (low/low) CKS1B-UBA3 or CKS1B-UBC12 populations, suggesting synergistic effects of CKS1B with either UBC12 or UBA3 in patient outcome.

Figure 6.

Dysregulation of the neddylation pathway is associated with poor prognosis in multiple myeloma. A, the Kaplan–Meier estimates of overall survival in patients with varied expression of neddylation pathway genes. Patients with elevated expression of UBA3 (left) showed poorer overall survival than those with low expression (29% vs. 81%; P = 0.003). Patients with elevated expression of UBC12 (middle) showed poorer overall survival than those with low expression (28% vs. 48%; P = 0.001). Patients with elevated expression of CKS1B (right) showed poorer overall survival than those with low expression (35% vs. 47%; P = 0.015). B, the Kaplan–Meier estimates of overall survival in patients with sorted CKS1B and neddylation enzyme GEPs. Left, patient survival sorted by high/low CKS1B expression and high/low UBA3 expression is depicted. Patients high in expression of CKS1B and UBA3 exhibited significantly (P = 0.029) poorer overall survival (26%) than those with either high CKS1B/low UBA3 expression (40%) or low CKS1B/highUBA3 expression (31%). Patients with low CKS1B/low UBA3 demonstrated the greatest survival rate (29%). Right, patient survival sorted by high/low CKS1B expression and high/low UBC12 expression is depicted. Patients high in expression of CKS1B and UBC12 (left) exhibited significantly (P = 0.006) poorer overall survival (20%) than those with either high CKS1B/low UBC12 expression (44%) or low CKS1B/high UBC12 expression (36%). Patients with low CKS1B/low UBA3 demonstrated the greatest survival rate (54%).

Figure 6.

Dysregulation of the neddylation pathway is associated with poor prognosis in multiple myeloma. A, the Kaplan–Meier estimates of overall survival in patients with varied expression of neddylation pathway genes. Patients with elevated expression of UBA3 (left) showed poorer overall survival than those with low expression (29% vs. 81%; P = 0.003). Patients with elevated expression of UBC12 (middle) showed poorer overall survival than those with low expression (28% vs. 48%; P = 0.001). Patients with elevated expression of CKS1B (right) showed poorer overall survival than those with low expression (35% vs. 47%; P = 0.015). B, the Kaplan–Meier estimates of overall survival in patients with sorted CKS1B and neddylation enzyme GEPs. Left, patient survival sorted by high/low CKS1B expression and high/low UBA3 expression is depicted. Patients high in expression of CKS1B and UBA3 exhibited significantly (P = 0.029) poorer overall survival (26%) than those with either high CKS1B/low UBA3 expression (40%) or low CKS1B/highUBA3 expression (31%). Patients with low CKS1B/low UBA3 demonstrated the greatest survival rate (29%). Right, patient survival sorted by high/low CKS1B expression and high/low UBC12 expression is depicted. Patients high in expression of CKS1B and UBC12 (left) exhibited significantly (P = 0.006) poorer overall survival (20%) than those with either high CKS1B/low UBC12 expression (44%) or low CKS1B/high UBC12 expression (36%). Patients with low CKS1B/low UBA3 demonstrated the greatest survival rate (54%).

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A marked increase in the frequency of high-risk designation in multiple myeloma patients from 13% at diagnosis to 76% at relapse provides strong evidence for disease evolution in multiple myeloma (15), suggesting the outgrowth of a drug-resistant subpopulation of multiple myeloma cells. Previously, we have demonstrated CKS1B expression results in increased multidrug resistance, while, clinically, CKS1B expression is increased in relapsed multiple myeloma patients (5), providing molecular evidence for CKS1B in a more drug-resistant multiple myeloma disease. The existence of a subset of patients who do not receive maximum benefit from currently available treatments in multiple myeloma highlight the critical need for improved therapeutic options.

The aim of this study was to evaluate if the NAE inhibitor MLN4924 is effective in treating multiple myeloma in cells with increased CKS1B expression. Here, we demonstrate that multiple myeloma cells overexpressing CKS1B are resistant to bortezomib but remain sensitive to MLN4924. Our results demonstrate that the treatment of cells with elevated CKS1B expression with MLN4924, but not bortezomib, results in decreased cellular proliferation and survival, reduced clonogenic expansion, and the induction of cellular senescence, strongly suggesting a role for NEDD8 regulation of CRL/SCF activation under conditions of CKS1B-induced drug resistance. SCFSkp2 is responsible for regulating the ubiquitin-mediated degradation of the cell-cycle checkpoint proteins p21 and p27. We found the overexpression of CKS1B led to increased neddylation of Cullin-1, whereas targeted knockdown of CKS1B reduced Cullin-1 neddylation. These findings demonstrate a novel role for CKS1B in the regulation of SCFSkp2 ubiquitin-mediating complex and suggest a mechanism for how cells with elevated CKS1B expression may evade growth regulation. Finally, extensive immunoblot analysis of SCFSkp2 degradation targets upon drug treatment in CKS1B-overexpressing cells identified the differential regulation of the CRL/SCF downstream target p21, showing the degradation of p21 upon bortezomib treatment but not MLN4924 treatment. The use of targeted shRNA against p21 resulted in a loss of sensitivity to MLN4924 in cellular proliferation, clonogenic expansion, and senescence induction. These results confirm a mechanism through p21 by which CKS1B-overexpressing cells exhibit drug resistance to bortezomib but sensitivity to MLN4924.

The clinical potential of MLN4924 inhibition of Cullin-1 activation through the neddylation pathway was highlighted by examining neddylation machinery expression patterns in multiple myeloma patients. Consistent with GEPs derived from patient populations, a great deal of variance was exhibited in each examined population. On average, we found that the expression of the E1 Nedd8-activating enzyme NAE1, the E1 Nedd8-activating enzyme UBA3, the E2 Nedd8-conjugating enzyme UBC12, to be significantly upregulated in multiple myeloma patients relative to patients with MGUS or NPCs derived from healthy donors. In the APEX trial, we also saw a significant increase in the expression of Nedd8 in patients unresponsive to the combinatorial treatment of bortezomib and dexamethasone after relapse relative to those with response and these patients had inferior outcomes.

Although our study examines MLN4924 in a drug-resistant, elevated CKS1B environment that recapitulates a portion of a known high-risk phenotype in clinical multiple myeloma patients, there have been previous studies in multiple myeloma systems that share consistent findings with us. In examining the in vitro treatment of multiple myeloma cells with MLN4924, McMillin and colleagues (26) have found MLN4924 to be effective in limiting viability a number of multiple myeloma cell lines, including in the bortezomib-sensitive ANBL6wt multiple myeloma cell line and the bortezomib-resistant ANBL6-5VR subline. More recently, Gu and colleagues (27) have demonstrated the utility of MLN4924 in the inhibition of both AKT and mTOR signaling in multiple myeloma cell lines and primary multiple myeloma cells. By working in p53 wild-type multiple myeloma cell lines (MM.1S and MM.1R) and multiple myeloma cell lines with mutant/null p53 status (LP1, OCI-MY5, RPMI8266, OPM2, and U266), their work also showed that sensitivity to MLN4924 is not related to p53 status (27), while our work was performed primarily in OCI-My5 multiple myeloma cells (p53-heterozygous wt/mutant; refs. 28, 29) and ARP-1 multiple myeloma cells (p53-null, homozygous deletions; ref. 30) cell lines, our efforts in the p53-positive KMS28PE multiple myeloma cell line resulted in nearly identical effects to cellular proliferation and viability upon MLN4924 treatment as seen in the OCI-MY5 cell line, consistent with the previously published findings.

MLN4924 induction of senescence occurs in part from the accumulation of factors leading to DNA damage (e.g., CDT1) and the accumulation of cell-cycle regulatory proteins (e.g., p21), the combination of which results in the robust activation of the DNA-damage response, leading to senescence (31). We demonstrate the treatment of bortezomib-resistant CKS1B-overexpressing cells with MLN4924 results in an increase in senescence in a p21-dependent manner. This important role of p21 in MLN4924-induced senescence is well supported by the findings of others (32). Although the consensus is that p21 is important for MLN4924-induced senescence, there still remain discrepancies between the findings of other groups. One group has found that the genetic inactivation of Skp2 was insufficient to induce senescence alone but critically dependent upon p21, p27, and ATF4 (33). Another group found that MLN4924-induced senescence is critically dependent upon p21 but independent of p53, p16, and pRb (34). A third group found that both p21 and p53 are important for initiating senescence but either factor is dispensable as SA-b-gal staining was diminished, but not absent, in p21−/− and p53−/− cells (32). Our results are consistent with most consistent with the second group (34). The striking differences between these studies may be accounted for in part by the different cell culture models used by the each group.

The overexpression of CKS1B in relapsed multiple myeloma patients contributes to bortezomib resistance (5) and relates to decreased survival, constituting a risk factor and highlighting the need for new therapies. We have shown here that genes in the neddylation pathway (NAE1, UBA3, UBC12, and NEDD8) are upregulated leading to activation of SCFSkp2 and correlated with inferior survival in multiple myeloma patients. These results are consistent with and expand upon the findings of McMillin and colleagues (26) who demonstrated decreased progression-free survival (PFS) in patients with elevated expression of NEDD8. By targeting NAE, the first step in the activation of CRL complexes, MLN4924 is a more selective inhibitor of protein degradation than the 20S proteasome inhibitor bortezomib. Targets of SCFSkp2 are enriched for proliferation and survival and inhibition by MLN4924 achieves a disproportionately antitumoral response without the cellular toxicity associated with the pan-proteasomal inhibition of bortezomib. Therefore, inhibitors of the neddylation pathway, and MLN4924 specifically, offer a promising alternative to bortezomib in the clinical treatment of multiple myeloma and other cancers.

It has been demonstrated that the treatment of multiple myeloma cells with MLN4924 results in a distinct GEP from the treatment of cells with bortezomib, and that treatment with MLN4924 does not elicit the compensatory upregulation of transcripts specific for ubiquitin/proteasome (26). MLN4924 and bortezomib both inhibit proteasomal degradation, yet through distinct pathways. This excitingly suggests the possibility of a combinatorial approach between these drugs though, to date, preliminary findings remain disparate. Gu and colleagues (27) have found synergy between bortezomib and MLN4924 in the induction of apoptosis. Conversely, in exhaustive titration experiments, McMillin and colleagues (26) found no synergy but also no antagonism between bortezomib and MLN4924 in affecting the viability of multiple myeloma cell lines.

In summary, our study has demonstrated that MLN4924 is effective at limiting cell growth and proliferation and inducing senescence in drug-resistant, high CKS1B-expressing cells. We demonstrate that p21 is critically important for cell sensitivity to MLN4924 and loss of p21 expression results in MLN4924 resistance. Our findings highlight the clinical potential of targeting the NEDD8 pathway and are supported by clinical data demonstrating multiple myeloma patients with elevated expression of NEDD8 machinery have inferior clinical outcomes relative to patients with lower expression. Although we have not found a survival advantage correlated with p21 expression in multiple myeloma patients (data not shown), clinical trials paired with MLN4924 have yet to be performed and analyzed. Our findings suggest that the expression of p21 may predict MLN4924 efficacy and screening patients for p21 expression may have great merit. Further examination of MLN4924 in preclinical and, possibly, in clinical combinatorial studies with currently available treatments (e.g., bortezomib, dexamethasone, doxorubicin) for multiple myeloma is warranted.

No potential conflicts of interest were disclosed.

Conception and design: Y. Zhou, G.S. Thomas, Z. Gu, G. Tricot, F. Zhan

Development of methodology: J. Huang, Y. Zhou, Z. Gu, Y. Yang, H. Xu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Huang, F. Zhan

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Huang, G.S. Thomas, Y. Yang, H. Xu, F. Zhan

Writing, review, and/or revision of the manuscript: J. Huang, G.S. Thomas, G. Tricot, F. Zhan

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): G.S. Thomas

Study supervision: G. Tricot, F. Zhan

This work was supported by: R01CA152105 (to F. Zhan) from the NCI; The Leukemia & Lymphoma Society Translational Research Program (to F. Zhan; 6094-12); institutional start-up funds from the Department of Internal Medicine, Carver College of Medicine, University of Iowa (to F. Zhan and G. Tricot); The University of Iowa Holden Comprehensive Cancer Center Support Grant P30 CA086862.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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