p53 haploinsufficiency and functional abnormalities in multiple

ORIGINAL ARTICLE

p53 haploinsufficiency and functional abnormalities in multiplemyelomaPJ Teoh1,2, TH Chung2, S Sebastian3, SN Choo4, J Yan2, SB Ng4,5, R Fonseca3 and WJ Chng1,2,6

Hemizygous deletion of 17p13, which harbors the TP53 gene, has been identified in 410% of newly diagnosed multiple myeloma(MM) patients and is associated with poor prognosis. To date, there is no conclusive evidence that TP53 is the critical gene.Furthermore, the functional effect of TP53 haploinsufficiency is not well characterized. By utilizing human myeloma cell lines,we showed that TP53 hemizygous loss was associated with decreased basal expression level with a partially or severely inactivatedp53 response upon genotoxic and non-genotoxic stress. The pathway deficiency was manifested as defective p53 transcriptionalactivities, together with significant resistance to apoptosis. In some cases with p53 WT/� and no p53 protein expression, theremaining allele was silenced by promoter hypermethylation. We also developed a p53 target gene signature to summarize thecomplexity of the p53 pathway abnormalities in MM and showed that it is strongly associated with genomic complexity and patientsurvival. In conclusion, this study identified TP53 as the critical gene located in 17p13, and revealed its haploinsufficiency propertiesin MM. Furthermore, we have elucidated that multiple mechanisms can deregulate the p53 functions and that this has importantprognostic impact in MM.

Leukemia (2014) 28, 2066–2074; doi:10.1038/leu.2014.102

INTRODUCTIONMultiple myeloma (MM) is the second most commonly diagnosedhematologic malignancy, accounting for almost 15% of bloodcancer and 1% of all types of human cancer.1,2 Despite theemergence of promising therapies and the advances in themolecular understanding of the disease, MM remains incurable.1–3

Mapped to the position of chromosome 17p13, TP53 encodes for atumor suppressor protein, p53, which acts as a critical regulatorof cellular proliferation, cell-cycle checkpoint, DNA repair andapoptosis.3–6 p53 inactivation has been associated withoncogenesis, cancer progression and drug resistance.

In contrast to other human cancers, p53 abnormalities are rarein MM; however, the incidence increases in the advanced stages ofthe disease, suggesting an important role in disease progres-sion.7,8 Mutations of TP53 have been reported only in a smallsubset of patients at the time of diagnosis (B3%).7–9 On the otherhand, chromosome 17p13 deletion, that contains the TP53 gene isobserved in about 10% of new cases, and has been associatedwith poor prognosis.7–11 In almost all cases, 17p13 deletions aremonoallelic and the remaining allele of p53 is rarely mutated.7–9

To date, it is unknown whether the remaining allele is stillfunctional or has been inactivated by other alternativemechanisms, such as MDM2 overexpression or epigeneticsilencing. The biological effects of loss of heterozygosity on thep53 pathway have also not been well studied in MM. Given thatmany other genes are also residing at 17p13, there is until now noconclusive evidence that TP53 is the critical gene in this region. Inview of the above, we hypothesized that if TP53 is a critical geneon 17p13 for myeloma development and progression, it must be ahaploinsufficient tumor suppressor. Therefore, we postulated that

p53 downstream function will be affected by reduction in p53gene dosage arising from one copy loss of its gene.

In this study, we sought to understand the complexity of thep53 pathway by using a variety of human myeloma cell lines(HMCLs) that includes mutated, deleted and wild-type versions ofTP53. We found that the basal p53 dosage is critical to maintainthe integrity of the p53 pathway and the responsiveness of thecells towards drug treatment, thus providing strong evidence forp53 haploinsufficiency in MM. We identified other mechanisms bywhich p53 may be silenced and also showed that besides 17p13-deletion, increased expression of MDM2 is also associated withpoor outcome. We also identified a gene expression signaturesummarizing the p53 pathway abnormalities that is associatedwith poorer clinical outcome.

MATERIALS AND METHODSDetermination of 17p13 minimally deleted regionArray-based comparative genomic hybridization (aCGH) data of MultipleMyeloma Research Consortium Reference Collection (MMRC)12 (GeneExpression Omnibus (GEO) [2] ID: GSE26849) were segmented usingcircular binary segmentation algorithm13 and each segment’s deletionstatus was determined based on the distribution of the segment’s averagelogratio values. Specifically, segments with average log ratio p� 0.2 and4� 0.4 were designated as hemizygous deletion while segments withaverage logratio p� 0.4 were designated as homozygous deletion. Theminimally deleted region was finally consolidated by aggregating allhemi- and homozygous deleted regions in chromosome 17 of the MMRCsamples. Genes overlapping with this region were collected based onRefSeq genes with NCBI36/hg17 genome annotation.14

1Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 2Cancer Science Institute of Singapore, National University of Singapore,Singapore; 3Department of Hematology-Oncology, Mayo Clinic, Scottsdale, AZ, USA; 4 Department of Pathology, National University Cancer Institute of Singapore, NationalUniversity Health System, Singapore; 5Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore and 6Department ofHaematology-Oncology, National University Cancer Institute of Singapore, National University Health System, Singapore. Correspondence: Dr WJ Chng, Cancer Science Instituteof Singapore, National University of Singapore, NUHS Tower Block #7, 1E Kent Ridge Road, Singapore 119228, Singapore.E-mail: [email protected] 7 January 2014; revised 20 February 2014; accepted 6 March 2014; accepted article preview online 14 March 2014; advance online publication, 11 April 2014

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Cell cultureThe culturing conditions and p53 status of the HMCLs are stated inSupplementary Information.

Cell viability and proliferation assayCell viability was assessed by MTS colorimetric assay. Cells were seeded in96-well plates in 100ml complete medium at a density of 20� 104 cells per100ml per well and incubated with the indicated drugs for 72 h. MTSreagent (Promega, Madison, WI, USA) was added and further incubated foran additional 2 h. The optical density of the cells was then read with amicroplate reader (Tecan, Maennedorf, Switzerland) at 490 nm. Eachexperiment was made in triplicates, and the mean value was calculated.The percentage of cell viability was normalized to the dimethylsulphoxide(DMSO) control.

Western blot analysisWhole-cell lysates were prepared from the cell pellet using RIPA lysisbuffer. Equal amounts of protein extracts were resolved by SDS-PAGE andwere transferred to PVDF membrane (Bio-Rad, Hercules, CA, USA). Theprimary and secondary antibodies used in probing the blots are listed inthe Supplementary Information. The blots were developed using thechemiluminescent detection system (ECL) (Santa Cruz, Dallas, TX, USA).

Quantitative Real Time PCR (qRT-PCR)Total RNA of treated cells was isolated using the Qiagen (Venlo, Limburg,Netherlands) RNeasy mini kit and 1ug RNA was used for cDNA synthesisusing iScript Reverse Transcription Supermix (Bio-rad). Real Time PCR wasperformed with the cDNA by SYBR green method. Primer list for the genestested is available in the Supplementary Information. The fold change ofthe mRNA expression was calculated using the ddCT method, normalizingagainst endogenous control GAPDH.

Apoptosis assayTreated cells were harvested and stained for flow cytometry analysis withAnnexin V-FITC and propidium iodide (BD Pharmingen, San Jose, CA, USA).Stained cells were analyzed with BD LSRII flow cytometer and the datawere analyzed with BD FACs DIVA software (BD Biosciences, San Jose, CA,USA).

Methylation specific PCRGenomic DNA from DMSO and 5-Azacytidine (5-Aza) treated cells wasisolated with Qiagen DNeasy Blood & Tissue Kit. Sodium bisulfateconversion of the genomic DNA was performed with Qiagen EpitectBisulfite Kit. 2 mg of the isolated DNA was used for bisulfite conversion. Themodified DNA was purified and PCR was carried out using 50 ng of DNAwith methylated and unmethylated primers specific for p53-promoter. Theprimer sequences (Supplementary Information) and the cycling conditionshave been reported previously.15 PCR products were electrophoresed on2% agarose gels.

TP53 shRNA lentiviral knockdownThe lentiviral constructs expressing non-targeting (shCtrl) and p53 shRNAs(Sigma-Aldrich, St Louis, MO, USA) were used for this study. Detailedexplanation for generation of stable p53-knockdown cell line is describedin Supplementary Information.

Ectopic expression of p53JJN3 was chosen for the re-expression of p53 studies because it does notexpress p53 at the mRNA and the protein level. pCMV-p53 vector wastransfected into JJN3 at the titre of 0.1, 0.25 and 0.5 ug usingElectroporation method (Neon Transfection System, Life Technologies,Carlsbad, CA, USA). Positive transfected cells were selected usingPuromycin for 48 h.

ImmunohistochemistryDouble staining of patient samples on a tissue microarray was conductedwith CD138 and p53 or MDM2-specific antibodies. The set of tissuemicroarray used has been previously described.16 The protocol and theexact conditions for CD138/p53 and CD138/MDM2 double staining isfound in the Supplementary Information.

Generation of the p53 pathway gene signatureWe started off with a list of validated p53 targets that are not known to beaffected by mutations in MM;6 we first examined correlations for all pairsusing UAMS17 and HOVON18 gene expression profile data sets and thenfurther validation was done in an independent APEX data set.19 Detailedexplanation is described in the Supplementary Information.

Statistical analysisAll statistical analysis was done with an independent t-test. Survivalanalysis was done by using Kaplan–Meier function, with log rank test. IC50of nutlin-treated cells was analyzed using CompuSyn (Combosyn, Inc,Paramus, NJ, USA).

RESULTSTP53 was located in the minimally deleted region of 17p13In order to identify the minimally deleted region in 17p13chromosome, we analyzed the MMRC data set and found that10% of the 254 samples have 17p13 deletion (Figure 1a). Nohomozygous deletions were noted (Figure 1a). The minimal regiondeleted in these samples was mapped between 7 512 900 and 8189 409 bp (676 509 bp) (Figure 1b) and contained 32 genes,including TP53 (Supplementary Table 1).

Different array of p53 expression pattern associated with differentp53 statusNext, we investigated how p53 copy loss may affect the p53dosage. TP53 allelic status of the HMCLs was confirmed using

Figure 1. Analysis of the cases in MMRC data set. (a) aCGH analysis of the TP53 allelic status. (b) Gene mapping of 17p13(del) cases. Theminimal deleted region was encompassing 7.5–8.2Mbp. The vertical line represents TP53 gene.

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fluorescence in situ hybridization (Supplementary Figure 1) andaCGH (data not shown) prior to all downstream studies. In order togauge the p53 basal activity in the selected HMCLs, endogenousmRNA and protein expression levels were investigated. TheHMCLs displayed a different array of p53 mRNA and proteinexpression (Figures 2a and b), corresponding to their respectivep53 status. Normalizing to WT/WT (H929) cells, the mutant cells8226 and U266 were expectedly showing constitutively highexpression of p53,20,21 p53� /� KMS11 was deficient inexpression, whereas the WT/� HMCLs generally displayed areduction in p53 expression as compared with the WT/WT andmutant cells. Interestingly, despite sharing the same genotype, theWT/� cells have differential expression pattern of p53 amongthem. XG6 has the highest basal p53 mRNA and protein, albeit450% lower mRNA expression, and at least three folds lower proteinexpression when compared to H929 (WT/WT). Baseline p53expression in KMS18 was very low at the transcript level andundetectable at the protein level. On the other hand, JJN3 showedcomplete absence of p53 mRNA and protein. The results indicatedthat hemizygous deletion of TP53 is associated with the reductionof cellular p53 dosage and the reduction in its gene dosage canaffect its endogenous mRNA and protein expression, suggestingp53 haploinsufficiency.22 Despite the existence of one non-mutantp53 allele, KMS18 and JJN3 had very low or no mRNA and proteinexpression, indicating that in these cells other mechanisms maybe involved in silencing the remaining allele.

WT/� cells were more resistant to apoptosis as compared toWT/WT cellsNext we assessed the effects of p53 genotype and gene dosageon p53 functions. A DNA-damaging agent, etoposide, was used totreat the panel of HMCLs and their physiological response wasinterrogated by performing PARP-cleavage assay and annexin-Vlabeling. H929 (WT/WT) cells showed the highest amount ofcleaved PARP and annexin-V-positive cells (B20%) (Figures 3a and b).Both mutant cell lines (8226 and U266) expectedly showed anegligible amount of cell death, a phenotype attributable to theirimpaired p53 responses.5,20 Not surprisingly, the p53-deficientKMS11 also recorded a minimal apoptotic response. Thereafter,these mutant and p53� /� HMCLs were used as negativecontrols. Intriguingly, XG6 (WT/� ), which showed a detectable

amount of p53 protein at baseline, exhibited an apoptosisresponse, however, at a much lower degree (B11%) than inH929 (WT/WT). In contrast, WT/� cells with very low or completeabsence of basal p53 (KMS18 and JJN3) showed resistance toetoposide-induced apoptosis. These data collectively indicatethat p53 functional response to genotoxic stress is alreadycompromised when one allele is lost.

Nutlin-3, an MDM2 inhibitor, was developed as a non-genotoxicactivator of WT p53. Its efficacy-inducing apoptosis in MM with WTp53 has been previously reported.23,24 However, its efficacy in WT/�MM cells, a more clinically relevant study model, has remainedunexplored. MM1s (WT/WT) was used as a positive control, as ithas been shown to respond to nutlin.23 As depicted in Figure 3c,the viability of MM1s (WT/WT), H929 (WT/WT) and XG6 (WT/� )showed a sharp decline with nutlin treatment, displaying o20%cell viability at 10 mM drug concentration, consistent with reportson nutlin’s efficacy on p53 WT cells. Nevertheless, it is important tonote that the IC50 of XG6 (WT/� ) (3.12 mM) was much higher thanthat of WT/WT (0.49 mM in H929 and 1.52 mM in MM1s) (Figure 3c).Both WT/� cells, KMS18 and JJN3, which have minimalendogenous p53, were refractory to the treatment, similar to thenegative controls. In line with the MTS results, nutlin treatmentcaused a marked increase in apoptosis in both H929 (B70%) andXG6 (B40%) (Figure 3d). In contrast, the cell lines with low or zerobasal p53 expression (KMS18 and JJN3) were resistant to apoptosisinduction, as were the negative controls U266, 8226 and KMS11(Figure 3d). Importantly, again, the increase in annexin-V-positivecells in XG6 (WT/� ) was significantly lower than in H929 (WT/WT)after treatment. Cell cycle analysis also showed that the sub-G1cell population was markedly increased only in H929 and XG6,but to a much lower extent in XG6 (Supplementary Figure 2).All these data are consistent in proving impaired growthinhibition and defective apoptosis in response to genotoxic andnon-genotoxic stress based on p53 gene dosage and proteinexpression level.

WT/� cells have non-functional p53 signaling pathwayNext, we investigated if the partial (XG6) or absolute resistance(KMS18 and JJN3) towards genotoxic or non-genotoxic stress-induced apoptosis was due to the attenuation of p53 signaling.Upon nutlin treatment, a differential expression profile of down-stream proteins was observed according to p53 status. Ofrelevance, WT/WT (H929 and MM1s) showed normal functionalp53 responses as evidenced by the marked upregulation of totalp53 and its targets MDM2, p21, PUMA and NOXA (Figure 4a).Negative control mutant cells (8226 and U266) with defectivetranscriptional activities, showed a complete abolishment ofdownstream p53 signaling, as did p53-deficient-KMS11. Interest-ingly, among the WT/� cells, XG6, which has a relatively higherbasal p53 level, showed a functional the p53 pathway, whereas,abrogated p53 signaling was observed in cells with low or zerobasal p53 (KMS18 and JJN3). When we treated the same set ofHMCLs with etoposide, we observed a similar protein expressionprofile (Supplementary Figure 3). These results highlighted theimportance of p53 basal expression and its functional activity inretaining the integrity of the p53 pathway and triggering drug-induced apoptosis.

Quantitative real-time PCR was also performed to check thetranscriptional activity of p53. Consistent with western blotanalysis, p53 transcriptional role was operational only in XG6and H929 and not in other cell lines with non-functional p53(Figure 4b). Importantly, the fold increase of the transcripts in XG6(WT/� ) was found to be generally lower than that in H929(WT/WT). Therefore, the presence of hemizygous loss does indeedimpair the transcriptional activity of p53, a condition typical forhaploinsufficiency, and its dosage is crucial in determiningfunctionality of the pathway.

Figure 2. HMCLs with different p53 status displayed an array of basalp53 mRNA (a) and protein (b) expressions. HMCLs were incubatedunder non-stressed conditions. p53 genotype was indicated at thex-axis (a). WT/� cells generally have lower p53 expression ascompared with WT/WT and mutant cells.


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Figure 3. WT/� cells were more resistant to genotoxic and non-genotoxic drug treatment as compared with WT/WT cells. (a) DMSO andetoposide (5mM) treated cells were harvested for PARP analysis after 24 h treatment. 1: DMSO, 2: Etoposide 5mM. (b) Cells were treated withDMSO (control) and 5mM etoposide for 24 h and were subjected to flow cytometry for the detection of annexin-V-positive cells. Percentage-specific apoptosis was calculated using the equation: % specific apoptosis¼ (Test- control)*100/100-control. (c) Cell viability was analyzed byMTS assay after the cells were nutlin treated for 72 h. Cell viability was calculated by normalizing against DMSO-treated cells. Dotted linesrepresent the IC50 value (Computed from CompuSyn software) of the indicated cell line. (d) Apoptosis (Annexin-V positive) was measured byflow cytometry after the cells were treated with nutlin for 48 h. Each bar represents mean±s.d., *Po0.05, **Po0.0001. Mutant cells U266 and8226 and p53� /� KMS11 served as negative controls.

Figure 4. WT/� cells have impaired the p53 signaling pathway. (a) HMCLs were treated for 16 h and cell lysate were prepared for western blotanalysis. WT/WT (H929 and MM1s) and XG6 (WT/� ) showed functionally active p53 pathway, with upregulation of p53 with downstreamtargets, p21, MDM2, PUMA and NOXA. Other WT/� cells showed complete abolishment of the pathway, with no increased expression ofdownstream genes (1: Nutlin 0 mM, 2: Nutlin 2.5 mM, 3: Nutlin 5 mM, 4: Nutlin 10 mM). (b) HMCLs were treated for 8 h followed by total RNAextraction and cDNA synthesis. The samples were subjected to real-time PCR analysis for p53 downstream targets MDM2, p21, PUMA andNOXA. p53 mutant and � /� cells acting as negative control.


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Knockdown and overexpression studies confirmed thehaploinsufficiency of TP53 in MMGiven that baseline p53 dosage was an important determinant ofthe integrity of the p53 pathway, we further investigated thehaploinsufficiency of TP53 by knocking down p53 in H929(WT/WT). Among the three independent shRNAs targeting p53,#21 was able to suppress the p53 expression level by up to B90%as compared to non-targeting shRNA (Figure 5a). Thereafter,H929-shp53 #21 stable cell line was used for subsequent studies.The importance of p53 gene dosage was demonstrated bysignificantly higher cell-viability in H929-shp53 compared withH929-shCtrl at increasing concentrations of nutlin (Figure 5b).Furthermore, p53 transcriptional activity in H929-shp53 cells wasseverely attenuated as manifested by an almost zero expression ofboth MDM2 and p21 (Figure 5c). Knockdown of p53 alsoobliterated the cells’ ability to undergo apoptosis with the virtualabsence of PARP cleavage upon nutlin treatment (Figure 5d).These results were replicated when p53 expression was dimin-ished in MM1s (WT/WT) with specific p53 siRNA (SupplementaryFig 4). This provides further evidence that p53 signaling andresponse is dependent on p53 gene dosage.

To further demonstrate this, the converse experiment wasperformed. Ectopic expression of p53 was done in JJN3 (WT/� , nop53 mRNA and protein). Transfection of 0.25 mg of pCMV-p53WTwas enough to upregulate p53 to approximately 40-folds of mRNAand four to five folds of protein (Figures 5e and f). Overexpressionof p53 strikingly contributed to the reactivation of the p53pathway, with increased expression of p21, MDM2 and PUMA(Figures 5f and g). These experiments further confirm thefunctional significance of TP53 haploinsufficiency in MM.

TP53 mRNA level affects patient survivalPrevious findings in the UAMS data set indicated that low level ofTP53 mRNA is associated with poorer disease outcome inpatients.25 To further verify this association in clinical samples,we analyzed the HOVON data set.18,26 We noticed that, as we usedprogressively lower cutoff levels of TP53 expression for risk groupseparation, there was a progressive widening of survival gapbetween two simulated risk groups due to the expanding overlapbetween low-TP53 sample group and true worse outcome group.As a result, the survival difference became significant enough atlow cutoffs (Supplementary Figure 5). This result validated theprevious claims from the UAMS dataset and provided furtherevidence from patient samples that TP53 expression level is ofclinical relevance. This is consistent with our functional experi-ments in human myeloma cell lines (HMCLs) showing haploinsuf-ficiency of p53 in myeloma.

Promoter DNA hypermethylation is one of the possible p53-silencing mechanisms in MMDespite still bearing one WT p53 allele, the p53 protein expressionof KMS18 and JJN3 was absent, indicating that other mechanismsmay be involved in silencing the remaining allele. One possiblemechanism by which the expression of the remaining allele issuppressed is by aberrant promoter methylation.15,27,28 To assessthis, we first treated the cells with 5-Azacytidine (5-Aza), a potentglobal DNA demethylator, to investigate if p53 expression couldbe rescued. 2.5 mM of 5-Aza treatment for 24 h rendered anincrease in p53 transcript in both cell lines (Figure 6a), togetherwith a significant decrease in the cell viability (Figure 6b).

Figure 5. TP53 is haploinsufficient in myeloma. (a) Up to 90% of knockdown was achieved by p53 shRNA clone #21. Cells from this clone wereused thereafter as an isogenic partner for H929-shCtrl. (b) Cell viability of H929-shCtrl and H929-shp53 was analyzed by MTS assay after theywere treated with nutlin for 72 h; *Po0.01, **Po0.0001. (c) H929-shCtrl and H929-shp53 cells were treated with nutlin for 16 h and wereharvested for western blot analysis (1: Nutlin 0 mM, 2: Nutlin 2.5 mM). (d) PARP cleavage analysis was performed after H929-shCtrl and H929-shp53 cells were treated with nutlin for 24 h. (e and f ) Confirmation of overexpression of p53 by qPCR (e) and western blot (f ). (g) Ectopicexpression of p53 into JJN3 restored the p53 transcriptional activities. Re-expression of p53 saw an increase in mRNA expression of itsdownstream targets, p21, MDM2 and PUMA.


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To further confirm the direct effects of 5-Aza on the demethylationof p53 promoter, methylation-specific PCR was performed.Methylation-specific PCR results showed a notable decrease ofthe methylated DNA and a distinct increase in the unmethylatedDNA in both KMS18 and JJN3 (Figure 6c). The current datacollectively suggest that the remaining allele of WT p53 in KMS18and JJN3 was epigenetically suppressed by promoter hyper-methylation. At the protein level, 5-Aza was also able to induce anelevation of p53 expression in KMS18 (Figure 6d). However, thiswas not seen at the protein level for JJN3 (data not shown),suggesting that other factors may further regulate proteinexpression of p53.

We analyzed a publicly available methylation array dataset(GSE21304)29 to assess the frequency of methylation of the p53promoter in MM patients. Of 161 patients in the data set, only 5(3%) had at least one probe in the p53 promoter regionmethylated (Supplementary Figure 6). This suggests that methylationof p53 is relatively uncommon in myeloma patients.

Clinical impact of p53 and MDM2 on patient survivalOur in vitro studies suggest that abnormalities in p53 pathwaysmay not be limited to 17p13 deletion or p53 mutations. Changesin the protein levels of p53 and key regulatory proteins such asMDM2 are not well studied in MM. Therefore, we performedimmunohistochemistry using antibodies against p53 and MDM2on a tissue microarray of MM samples (n¼ 90). The representativeimmunohistochemistry results for both p53 and MDM2 stainingare shown in Supplementary Figure 7. We observed an increase ofp53 expression with disease stage (Figure 7a) (P¼ 0.0012),indicating the importance of p53 abnormalities in diseaseprogression, an observation consistent with previousreports.7,9,11,30,31 However, there was no association foundbetween p53 protein expressions and survival.

MDM2 is an important negative regulator of p53 and itsoverexpression/amplification has been reported in various tumortypes.32–35 In line with this, we then analyzed the association

Figure 6. Promoter DNA hypermethylation is one of the possible p53 silencing mechanisms in MM (a) JJN3 and KMS18 were treated with 2.5mM5-Aza for 24h and the cells were harvested for RNA extraction and was followed by qPCR analysis with two p53 primers (1 and 2) spanningdifferent p53 regions. (b) Cell viability of KMS18 and JJN3 after 2.5mM of 5-Aza treatment for 24h. (c) Methylation-specific PCR was performed afterthe cells were treated by 5-Aza for 24h. Methylated and unmethylated p53 promoter-specific primers were used for PCR amplification. (d) KMS18was treated with 5-Aza for 48h and the cells were harvested for nuclear/ cytoplasmic extraction and were subjected to western blot analysis.

Figure 7. Clinical impact of p53 and MDM2 on patient survival.(a) p53 expression increases with disease stage. Clinical sampleswere analyzed by using immunohistochemistry with double stainingto specifically identify p53 expression in CD138þ myeloma cells.(b) Kaplan Meier analysis of newly diagnosed cases identified theassociation of high MDM2 expression with poor survival (Po0.0001).


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between MDM2 expressions and survival in newly diagnosedcases of MM. This analysis has essentially shown that MDM2expression (positive staining in X10% of plasma cells) isassociated with significantly worse prognosis (Figure 7b).

MM p53 target gene signature is associated with TP53 status andsurvivalOur results suggest that abnormalities in the p53 pathway are offunctional relevance in MM. As multiple abnormalities mayperturb the p53 pathway, and the defective p53 may manifestits effect through an attenuated p53 transcriptional function, wepostulated that an analysis of p53 transcriptional targets that arenot themselves known to be affected by gene mutations inmyeloma would allow us to identify a signature for abnormal p53pathway. Based on the list of validated p53 targets6 and byutilizing the publicly available MM data sets, HOVON and UAMS,we developed a signature of p53 targets, called MM p53 targetgene signature, consisting of PUMA, Gadd45 and THBS1. Thesethree genes were chosen as they are validated p53 target genesthat had consistent correlation with each other across thedata sets.

Analyzing the MM p53 target gene signature, we found thatHMCLs with p53 abnormalities (WT/� , mutant and � /� ) havesignificantly reduced signature indices as compared to WT/WT celllines (Po0.05) (Figure 8a), indicating that anomalies in TP53 geneitself would affect its transcriptional functions.

Considering the association between p53 dysfunction withgenomic instability and patients’ survival,21,36,37 we wanted toknow how the expression of this p53 pathway signature could actas a surrogate for the above phenotypes. In the HOVON and UAMSdata sets, we saw negative correlations between the p53 signatureand chromosomal instability, indicating that lower p53signature (defective p53 pathway) is associated with greater

chromosomal instability (HOVON (r¼ � 0.329, P¼ 1.502E� 8),UAMS (r¼ � 0.272, P¼ 6.125E� 11)) (Figure 8b). Consistently,these results were further validated in APEX data set (r¼ � 0.369,P¼ 1.832E� 7)) (Figure 8b). In addition, patients with high andlow expression of the MM p53 target gene signature hadsignificantly different survival in the HOVON (P¼ 0.00175), APEX(P¼ 0.00628) and UAMS (P¼ 0.0203) data sets (Figure 8c).

DISCUSSIONThe clinical relevance of 17p13 deletion in myeloma is already wellestablished. MM patients with 17p13(del) have very poor outcomeand its prevalence increases with more advanced stages such asplasma cell leukemia and extramedullary disease.4,38 TP53, whichencodes the p53 tumor suppressor, is situated within this deletedlocus. While it is logical to implicate p53 as the critical tumor-suppressor gene deleted in this region, there is surprisingly littlepublished data in myeloma that directly supports this notion.Analysis of the MMRC data set showed that TP53 was consistentlypresent in the minimally deleted region of 17p13. Our datacorroborated with the results published by Boyd et al.9 from theMedical Research Council (MRC) dataset analysis whereby TP53was also found to be contained within their 17p13 minimallydeleted region. Essentially, only two genes, TP53 and SAT2 withinthis minimal area, have significantly lower expression in17p13(del) cases.9 Furthermore, functional p53 has been shownto be important in arresting MM development and progression asevidenced by the very poor survival in patients with p53abnormalities.4,7,12,25 These data support p53 as the very likelycritical gene in 17p13.

It is crucial to note that homozygous deletion of p53 isextremely rare, with monoallelic deletion being predominantlyfound.4,9,38 In view of this, if p53 is the critical tumor suppressor on

Figure 8. p53 pathway signature and MM characteristics. (a) Comparison of p53 pathway signature index distribution in MM cell linesaccording to TP53 locus status; intact locus (WT/WT), hemizygous deletion (WT/� � ) and homozygous deletion, mutation, no expression(� � /� � ;Mut;No). (b) Correlation between p53 pathway signature index and chromosomal instability index in MM. (c) Comparison of MMpatient prognosis based on p53 signature index. HOVON and APEX (top 25% vs bottom 25%), UAMS (top 50% vs bottom 50%).


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17p13, it must act as a haploinsufficient tumor suppressor gene(TSG) in promoting myelogenesis. We therefore assessed p53function in a panel of HMCLs to see if p53 function was affectedby the presence of monoallelic deletion. Indeed, we demonstratedthat p53 activity was already compromised in p53WT/� cells.Even though XG6 was still responsive to the drugs, the responsewas almost 50% lower than WT/WT cells, suggesting that one copyloss was enough to suppress p53 expression at baseline and itsactivity in response to stress. The dosage dependence of p53function and its haploinsufficiency were further validated byperforming p53 knockdown in H929 (WT/WT) and p53 re-expression in JJN3 (no mRNA and protein). Our in vitro datagenerated using these isogenic cell lines were coherent with thefindings from mouse model studies, whereby p53þ /� mice weremore susceptible to an array of tumors as compared with itsp53þ /þ counterparts.39,40 Further evidence for p53haploinsufficiency was provided by an in vitro study using p53heterozygous thymocytes that revealed an intermediate partialresistance to apoptosis induced by ionizing radiation andetoposide.41 To the best of our knowledge, our finding is thefirst to report the evidence of p53 haploinsufficency in myeloma.

Importantly, we have also shown that the basal p53 expressionwas crucial in ascertaining the functionality of the p53 pathway inWT/� . In some WT/� cells, such as KMS18 and JJN3, basal p53protein expression was absent and the p53 response pathwayswere completely abrogated. Our data were further supported by aprevious study revealing a defective induction of p21 and MDM2 inHCT116 p53þ /� with low p53 mRNA and protein expression.22

Furthermore, MDM2 promoter induction by p53 was also found tobe far less efficient in p53þ /� mice.39,40 Coupled with clinicalstudies that have importantly shown that patients with lowest levelof p53 mRNA have the worst clinical outcomes,9,25,42 our datastrongly suggest that TP53 is indeed the essential gene in 17p13.

Our study also highlights the complexity of p53 pathways inmyeloma. Besides deletion and mutations, we found thatmethylation of TP53 promoter was another mechanism ofinactivating p53. DNA methylation negatively regulates varioustumor suppressor genes, such as p16, SOCS-1 and mir-34B inMM.43–46 Nevertheless, methylation of TP53 has been lessfrequently reported.27,47 Previous studies have shown that p53promoter was densely methylated in certain HMCLs and treatmentwith zebularine, another DNA methyltransferase inhibitor, wasable to rescue the expression of p53, and subsequently inducedapoptosis.27,47 However, the p53 allelic and mutational status ofthe cell lines was not taken into account in their analyses. Ouranalysis of a large methylation array patient data set suggests thatmethylation of the p53 promoter is a relatively rare event inmyeloma patients. This also implies that patients with very lowTP53 expression are likely to have recruited other mechanisms forsilencing the remaining allele. In this regard, aberrant expressionand function of p53-regulating miRNAs that have been reported inmyeloma48 may be another mechanism leading to low TP53expression.

In addition, expression of p53-regulatory protein such as MDM2may also be critical, considering the frequent findings on its geneamplification and protein overexpression in various tumor types,including MM.32–35,49 We found that high MDM2 expression wasassociated with significantly poorer survival. This is in line with theprevious study reporting on the presence of MDM2 geneamplification in patient samples38 and the incidence of high MDM2expression in the advanced stage of MM in plasma cell leukemia.49

However, these studies did not report any significant correlationbetween MDM2 abnormalities and clinical impact. Thus, we believethat ours may be the first to show an unfavorable outcome in thenewly diagnosed patients with high MDM2 expression.

All the different abnormalities described above would perturbthe p53 downstream function. Since p53 executes its tumor-suppressive properties by transactivating downstream genes, a

good way to summarize its downstream function would bethrough the expression of its target genes. We found that theexpression of such a target gene signature (MM p53 target genesignature) was different between HMCLs with and without p53abnormalities (mutations, deletions and no expression). Impor-tantly, we also found a strong association between the p53 targetgene signatures and patients’ overall survival in three indepen-dent myeloma data sets. These data indicate that p53 genesignature could be used to reflect the functionality of TP53 andcan potentially be a surrogate marker for p53 inactivation andsubsequent patient prognosis. This notion is supported by aprevious study that has highlighted the importance of p53 targetgenes in myeloma progression, by reporting close association oftheir p53 signature with p53 expression and poor clinicaloutcome.25 Essentially, our signature consists of only threegenes, thus, performing analysis on a small number of markersin the clinic would seem more feasible and efficient. The currentdata suggest that p53 signatures could be used as an alternativemeans to represent p53 dysfunctions should data on p53abnormalities be not available, although this will need to befurther validated clinically.

Our study also provides some potential avenue for therapeuticintervention in MM with 17p13(del). When there is no proteinexpression, it would be relevant to check for methylation of thep53 locus and if present, 5-Aza may be used to de-repress p53expression. Notably, p53 function was found to be still active insome p53 hemizygous deletion cases, although at a compromisedefficiency. Hence, it would be a feasible strategy to target thesecases with a p53 pathway reactivating agent. Indeed, thisfeasibility was attested by XG6,whereby nutlin was still able torestore the p53 pathway, albeit at a lower potency than WT/WTcells. Hence, it is imperative to investigate potential combinationtherapies that would enhance the single agent efficacy. Forinstance, nutlin was reported to demonstrate great synergism withvarious genotoxic drugs, including bortezomib, with little toxicityto normal bone marrow cells.3,23,50

In conclusion, we provide compelling evidence that TP53 is acritical gene within the 17p13 minimally deleted region and is ahaploinsufficient tumor suppressor in myeloma. Our findingssuggest that not all cases of 17p13 deletion are similar and thatthe p53 baseline protein expression level is critical. In 17p13hemizygous deletion cases, mechanisms such as promoterhypermethylation may silence the remaining allele, leading toLOH. Furthermore, abnormalities of the more extended p53network, such as MDM2 and other downstream targets (MM p53target gene signature), may be of clinical relevance. This may paveway for the development of novel biomarkers for MM.

CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGEMENTSWJC is supported by NMRC Clinician Scientist Investigator award. This work is partlysupported by Singapore Cancer Syndicate Grant. This research is supported by theNational Research Foundation Singapore and the Singapore Ministry of Educationunder the Research Centers of Excellence initiative. Rafael Fonseca is a ClinicalInvestigator of the Damon Runyon Cancer Research Fund. This work is supported bygrants SPORE CA90297052, P01 CA62242, R01 CA83724, ECOG CA 21115 T, PredolinFoundation, Mayo Clinic Cancer Center and the Mayo Foundation.

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p53 haploinsufficiency and functional abnormalities in multiple