Decitabine and Melphalan Fail to Reactivate p73 in p53 Deficient Myeloma Cells

(1) Background: TP53 deficiency remains a major adverse event in Multiple Myeloma (MM) despite therapeutic progresses. As it is not possible to target TP53 deficiency with pharmacological agents, we explored the possibility of activating another p53 family member, p73, which has not been well studied in myeloma. (2) Methods: Using human myeloma cell lines (HMCLs) with normal or abnormal TP53 status, we assessed TP73 methylation and expression. (3) Results: Using microarray data, we reported that TP73 is weakly expressed in 47 HMCLs and mostly in TP53 wild type (TP53wt) HMCLs (p = 0.0029). Q-RT-PCR assays showed that TP73 was expressed in 57% of TP53wt HMCLs (4 out of 7) and 11% of TP53 abnormal (TP53abn) HMCLs (2 out of 18) (p = 0.0463). We showed that TP73 is silenced by methylation in TP53abn HMCLs and that decitabine increased its expression, which, however, remained insufficient for significant protein expression. Alkylating drugs increased expression of TP73 only in TP53wt HMCLs but failed to synergize with decitabine in TP53abn HMCLs. (4) Conclusions: Decitabine and melphalan does not appear as a promising combination for inducing p73 and bypassing p53 deficiency in myeloma cells.


Introduction
Multiple Myeloma (MM) is characterized by an important biological heterogeneity related to recurrent chromosomal abnormalities, which occurs early in the disease development at the MGUS (Monoclonal Gammopathy of Undetermined Significance) stage [1]. The recurrent chromosomal abnormalities are IgH gene translocation with recurrent partners located on chromosomes 4, 6, 11, 16 and 20 or hyperdiploidy of odd chromosomes [1]. Different prognoses and overall survivals are associated with this first heterogeneity. However, and independently from this heterogeneity, the most adverse prognosis is related to chromosomal deletion of TP53 [2]. Mutations of TP53 were exclusively found in myeloma cells displaying a hemi-deletion of the short arm of chromosome 17 (del17p) [3]. The frequency of TP53 mutations, which is low at diagnosis (around 3%), increases with relapses and is high in plasma cell leukemia (PCL, >30%) and human myeloma cell lines (HMCLs, >70%), these latter mostly deriving from extramedullary MM and mainly from PCL [4][5][6][7][8]. Loss of function of mutant p53 is believed to be related to conformation deficiency: therefore, small molecules, such as RITA and Prima-1 Met , which were selected for their ability to induce cell death in p53 mutated cells were shown to bind to p53 protein [9]. However, in our hands, both molecules induced massive cell death but their efficacy was unrelated to TP53 status and p53 expression [10,11]. Because del (17)p and TP53 mutations, which are irreversible, are an adverse event whatever the treatment regimen, alternative treatments that bypass or circumvent p53 are needed. For instance, the p53 family member p73 could be of interest as p73 is also able to transactivate pro-apoptotic genes [12,13]. Moreover, TP73 is rarely mutated but frequently silenced by CpG methylation in hematological malignancies and in multiple myeloma (MM) [14][15][16][17]. TP73 expression might thus be reactivated by drugs preventing CpG methylation after DNA replication such as decitabine. To assess the interest in activating p73 in myeloma cells, we studied p73 expression and regulation, TP73 methylation and TP73 sequencing in a large panel of human myeloma cell lines HMCLs with a normal or abnormal TP53 status [7,18].

TP73 Is Preferentially Constitutively Expressed in TP53 wt HMCLs
We assessed expression of TP73 mRNA in 47 HMCLs using Affymetrix microarray data [7]. Although expression level was very low, HMCLs with normal TP53 status (TP53 wt ) appeared to significantly express higher level of TP73 mRNA than TP53 abn HMCLs (p = 0.0029, Mann-Whitney test, Figure 1). To confirm this observation, we further selected 25 HMCLs displaying either a normal (n = 7) or an abnormal TP53 status (n = 18, 3 TP53 deleted (TP53 del ) and 15 TP53 mutated (TP53 mut ) HMCLs) (Table 1) [7,11,18]. Status of TP53 and TP73 was determined by sequencing of RT-PCR products and/or whole exon sequencing. Q-RT-PCR assays showed that TP73 was expressed in four out of seven TP53 wt HMCLs (AMO1, MDN, MM1S, NAN11), in one out of three TP53 del HMCLs (JJN3) and in one out of fifteen TP53 mut HMCLs (XG11), p = 0.0463, Mann-Whitney test, ( Figure 1B). p73 expression was confirmed at the protein level using Western blotting for MDN, MM1S, NAN11, JJN3 and XG11 but not for AMO1 ( Figure 1C). Whole exon sequencing showed that only KMM1 and XG11 displayed a missense mutation (A211S and I626V, respectively). However, XG11, but not KM11, expressed TP73 and p73. The strong p73 expression in XG11 might be related to the presence of the mutation within the transactivation inhibitory domain, which was reported to be involved in p73 degradation [19].

Decitabine Decreased TP73 Methylation and Induced TP73 Expression
To assess the methylation level in TP73 promoter, we performed nested methyl-specific PCR (MS-PCR) targeting the CpG island upstream of the gene ( Figure 2). PCR-1 was run on bisulfite converted DNA with primers that did not involve CpG. The PCR-1 product was then used for three nested PCRs; two, respectively, specific of unconverted CpG (methylated PCR, M-PCR) and converted CpG (unmethylated PCR, U-PCR) sequence; and one quantifying the PCR-1 product (PCR-Q, Figure 2). PCRs were run in HMCLs treated or not with decitabine to assess the TP73 methylation status ( Figure 3A). In parallel, we measured the expression level of TP73 by Q-PCR ( Figure 3B). In JIM3, KMS12PE and XG5, TP73 was found methylated and decitabine induced a decrease in M-PCR and an increase in U-PCR, which correlated with an increase in TP73 expression. However, despite the increase in TP73 expression, we could not detect any protein. Because anti-p73 Ab was directed against the N terminal part of the protein (amino acids 1-62) and could thus not detect ΔNp73, we performed three RT-PCRs to assess the total TP73 expression (exons 7-10), the full-length TAp73 (exons 2-6) and the ΔNp73 (exons 3′-6) isoforms, respectively. The TAp73 isoform was increased in the three TP53 mut HMCLs, although in KMS12PE the ΔNp73 isoform was the main increased isoform ( Figure 3C). The data suggested that the lack of p73 detection by Western blotting was not related to the lack of TAp73 transactivation, but rather to a weak transactivation.

Decitabine Decreased TP73 Methylation and Induced TP73 Expression
To assess the methylation level in TP73 promoter, we performed nested methyl-specific PCR (MS-PCR) targeting the CpG island upstream of the gene ( Figure 2). PCR-1 was run on bisulfite converted DNA with primers that did not involve CpG. The PCR-1 product was then used for three nested PCRs; two, respectively, specific of unconverted CpG (methylated PCR, M-PCR) and converted CpG (unmethylated PCR, U-PCR) sequence; and one quantifying the PCR-1 product (PCR-Q, Figure 2). PCRs were run in HMCLs treated or not with decitabine to assess the TP73 methylation status ( Figure 3A). In parallel, we measured the expression level of TP73 by Q-PCR ( Figure 3B). In JIM3, KMS12PE and XG5, TP73 was found methylated and decitabine induced a decrease in M-PCR and an increase in U-PCR, which correlated with an increase in TP73 expression. However, despite the increase in TP73 expression, we could not detect any protein. Because anti-p73 Ab was directed against the N terminal part of the protein (amino acids 1-62) and could thus not detect ∆Np73, we performed three RT-PCRs to assess the total TP73 expression (exons 7-10), the full-length TAp73 (exons 2-6) and the ∆Np73 (exons 3 -6) isoforms, respectively. The TAp73 isoform was increased in the three TP53 mut HMCLs, although in KMS12PE the ∆Np73 isoform was the main increased isoform ( Figure 3C). The data suggested that the lack of p73 detection by Western blotting was not related to the lack of TAp73 transactivation, but rather to a weak transactivation.

Melphalan and Cisplatin Induced TP73 Expression in TP53 wt HMCLs but Not in Decitabine-Treated TP53 mut HMCLs
To further study TP73 regulation, we treated four HMCLs that constitutively expressed p73 with alkylating drugs, melphalan and cisplatin and determined by RT-PCR and Western blotting the regulation of TP73 and p73. Regulation of p53 expression was assessed as a control of drug response [20]. Alkylating drugs increased the expression of the p73 full-length isoform in the TP53 wt MDN, MM1S and NAN11 HMCLs but not in TP53 deleted (TP53 del ) JJN3 HMCL ( Figure 3D). MDN also expressed the ∆Np73 isoform that was slightly increased by the drugs. By Western blotting, we confirmed that melphalan and cisplatin similarly increased p73 expression in the three TP53 wt HMCLs but failed to increase p73 expression in TP53 del JJN3 ( Figure 3E). As expected, p53 expression was increased by both drugs in the TP53 wt MDN, MM1S and NAN11 HMCLs but not in TP53 del JJN3 cells [20].
We then assessed whether melphalan could increase expression of p73 in decitabine-treated TP53 mut HMCLs. We selected the three HMCLs that displayed a strong increase in TP73 expression upon decitabin treatment, i.e., JIM3, KMS12PE and XG5 ( Figure 3B). As shown in Figure 3F, alkylating drugs failed to increase any p73 expression that remained undetectable in the 3 TP53 mut HMCLs.

Discussion
In myeloma, regulation of p73 expression has not been deeply investigated yet. For instance, TP73 expression has been shown to be induced by PRIMA-1 Met in several HMCLs including JJN3 in which p73 was reported to be partly involved in PRIMA-1 Met -induced cell death [17,21]. However, we did not confirm these results in JJN3, suggesting that JJN3 might be misidentified between the different laboratories. In hematological malignancies, TP73 that does not display frequent mutations is known to be silenced by CpG methylation [14]. Indeed, whole exon sequencing showed that only two HMCLs (KMM1 and XG11) harbored a mutation in TP73 gene. We thus assessed TP73 methylation and regulation in a large number of HMCLs. Our results showed that TP73 is mainly silenced by methylation and that the methylation inhibitor decitabine reversed the methylation-mediated silencing. However, the TP73 expression remained too weak for allowing a detectable p73 expression by Western blotting. We showed that decitabine-induced expression of both TAp73 and ∆Np73 isoforms: ∆Np73, which is lacking the transactivation domain, is known to inhibit TAp73 binding to DNA [19]. In KMS12PE, the high decitabine-induced expression of ∆Np73 isoform might explain the lack of p73 detection by Western blotting because the anti-p73 Abs was directed against the N terminal part of the protein (aa 1-62). TP73 gene also harbors an intrinsic CpG island that governs ∆Np73 isoform transcription. Thus, TP73 gene appears particularly methylated in MM cells and demethylation might equally induce expression of TAp73 and ∆Np73 isoforms, the latter being an inhibitory isoform [19]. We found that p73 was constitutively expressed in six HMCLs: MDN, MM1S, NAN11 and XG10 expressed a wildtype p53 protein, JJN3 lacked p53 expression and XG11 expressed a mutant p53 protein. Of note, p73 was overexpressed in XG11 despite a moderate TP73 expression suggesting a stabilization/lack of degradation of the protein that might be related to the presence of the mutation in the transactivation inhibitory domain, which is involved in degradation [22]. In three out of four HMCLs that constitutively expressed p73 and displayed an unmethylated profile of CpG, alkylating drugs increased both TP73 and p73 expression: this regulation seemed to be restricted to TP53 wt HMCLs as it did not occur in the TP53 del JJN3 HMCL. This finding is consistent with the possible regulation of TP73 expression via p53/p21/E2F1, which can only occur in TP53 wt HMCLs [23]. Indeed, alkylating drugs were unable to increase p73 expression in the three TP53 mut HMCLs that displayed a TP73 expression after decitabine treatment. Moreover, MDM2, which stabilizes p73 instead of inducing its degradation [24], could take a part in the p73 increase induced by alkylating drugs, because MDM2 is a p53 target gene mostly expressed in TP53 wt HMCLs [18]. Nevertheless, the role of p53 in melphalan-induced p73 regulation, if any, requires further investigations. On the other hand, TP73 expression was shown to be regulated by HDAC: indeed, the HDAC inhibitor sodium butyrate increased the "free" E2F1 pool and therefore TP73 transactivation independently from p53 [25]. We did not investigate the HDAC-mediated regulation of p73 in myeloma cells but it would be of interest to study HDAC inhibitors in combination with decitabine in p53 deficient cells. In summary, our data show that decitabine and melphalan does not appear as a promising combination for bypassing p53 deficiency in myeloma cells.

Bisulfite Treatment, Methylation Specific PCRs and RT-PCR
DNAs were treated with bisulfite as indicated by the supplier. Methylation specific PCRs (M-PCR, U-PCR) and quantifying PCR (PCR-Q) were performed after a first amplification (PCR1).

Western Blotting
Expression of p53 and p73 was determined by Western blotting as previously described [11]. A minimum of 80 µg of proteins was loaded in each lane.