ARHGDIA, a mutant TP53‐associated Rho GDP dissociation inhibitor, is over‐expressed in gene expression profiles of TP53 disrupted chronic lymphocytic leukaemia cells

Chronic lymphocytic leukaemia (CLL) is a disease with a highly variable clinical course that can be predicted by the presence of specific cytogenetic abnormalities (Zenz et al, 2008). Among them, the recurrent 17p deletion, affecting TP53, is associated in up to two-thirds of cases with concomitant TP53 mutations, which in turn may also occur in the absence of 17p deletion (Zenz et al, 2008). These TP53 CLL are known to respond poorly to conventional immunochemotherapy, thus underscoring the need for alternative therapeutic strategies, either operating independently of the canonical TP53 pathway (Zenz et al, 2008) or interfering with key genes transcriptionally up-regulated due to gain-of-function activities of mutant TP53 (Brosh & Rotter, 2009). We read with great interest the paper by Lin et al (2013) defining a gene expression signature peculiar of TP53 CLL patients. As highlighted by the Authors, however, such signature appears dominated by the under-expression of TP53 and of other genes located on chromosome 17p, while none of the genes found to be over-expressed in the TP53 CLL category have been reported as up-regulated upon activation of molecular pathways involving mutant TP53 (Lin et al, 2013). With the similar aim of identifying the genes associated with mutant TP53 in CLL, we took advantage of a cohort of 63 CLL, all characterized for TP53 gene status. The main clinical and biological features of CLL patients and the methods employed are reported in Table SI and Appendix SI. CLL samples were always collected at presentation or before first time therapy. Patients provided informed consent in accordance with local Institutional Review Board requirements and declaration of Helsinki. By comparing the gene expression profile of CLL cells from 7 TP53 cases with that of 13 TP53 wild-type (TP53) cases, 72 differentially expressed genes were obtained (26 up-regulated, 46 down-regulated in TP53 cases; adjusted P < 0 05; Table SII), which correctly split TP53 CLL from TP53 CLL by hierarchical clustering (Fig 1A). In keeping with cytogenetic and previously published data (Fabris et al, 2008; Lin et al, 2013), 31 out of 46 down-regulated genes in the TP53 CLL category were located in the deleted 17p13 segment. Consistently, a gene set enrichment analysis (GSEA), focusing on the positional gene sets corresponding to ‘human chromosomes and cytogenetic bands’, selected the gene set related to the 17p13 segment as having the lowest nominal P-value due to a significant enrichment in down-regulated genes in the TP53 CLL category [P = 0 002, false discovery rate (FDR) q = 0 001, Fig 1B]. This gene set included several genes with proapoptotic activities, such as PSMB6, RPL26 and ZBTB4 (Nandi et al, 1997; Ofir-Rosenfeld et al, 2008; Weber et al, 2008), whose lower expression in TP53 CLL was validated by Quantitative Real Time polymerase chain reaction (QRT-PCR; Fig 1C). At variance with the report by Lin et al (2013) but in keeping with previous results (Fabris et al, 2008), we failed to identify the TP53 transcript as down-regulated in TP53 CLL. Such a discrepancy might be due to a different percentage of cells representing the TP53 wild-type subclone in the context of each TP53 CLL case in our or in the other patient cohorts (Fabris et al, 2008; Lin et al, 2013). Importantly, one of the few genes which was up-regulated in the TP53 CLL category, was ARHGDIA, a gene coding for a Rho GDP dissociation inhibitor protein known to have antiapoptotic activity, and mediating cellular resistance to chemotherapeutic agents (Zhang et al, 2005). The upregulation of ARHGDIA in TP53 compared to TP53 CLL cells was confirmed by QRT-PCR experiments (P = 0 02, Fig 1C). Of note, although ARHGDIA was not found up-regulated in the signature reported by Lin et al (2013), this gene was specifically up-regulated upon transfection of mutant TP53 in TP53 null cells as part of the gainof-function activities of mutant TP53 itself (Bossi et al, 2008; Brosh & Rotter, 2009). The constitutive levels of ARHGDIA transcripts were then investigated by QRT-PCR in 43 TP53 CLL, and results were compared to those obtained in normal B cells purified from peripheral blood samples of 15 healthy donors. Expression levels of ARHGDIA in CLL were overall higher compared to normal B cells (P < 0 0001, Fig 2A). ARHGDIA levels also correlated with the presence of the main cytogenetic lesions in CLL (data available in 52/63 cases; Table SI). As expected, ARHGDIA expression was higher in TP53 cases compared to TP53 CLL belonging to the other cytogenetic subgroups (Zenz et al, 2008), although with a great case-to-case variation (Fig. 2B). In keeping with previous reports (Lin et al, 2013), we describe here that the peculiar gene expression signature of TP53 CLL is mainly characterized by the Correspondence