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C. Ciardullo et al.
that can enhance expression of these pro-apoptotic BH3- only proteins might represent a clinically relevant thera- peutic option for CLL.
The variable clinical course of CLL is driven, at least in part, by molecular heterogeneity which is underscored by the variety of genetic lesions observed, from classical markers of CLL to new genetic lesions uncovered by whole-genome and whole-exome sequencing.15-19 Among the genetic lesions identified, TP53 deletions and/or muta- tions are restricted to ~10% of CLL cases at diagnosis and are associated with decreased survival and clinical resist- ance to chemotherapeutic treatment.15,16 Since the preva- lence of TP53 defects at diagnosis is low, the majority of CLL patients retain a functional p53, and in these patients the possibility of activating p53 should be explored as a therapeutic strategy.
Given the central role of p53 in preventing aberrant cell proliferation and maintaining genomic integrity, there is increasing interest in developing pharmacological strate- gies aimed at manipulating p53 in a non-genotoxic man- ner, maximizing the selectivity and efficiency of cancer cell eradication.20,21 The levels and activity of functional p53 are mainly regulated through direct interaction with the human homolog of the murine double-minute 2 (MDM2) protein.22,23 MDM2 is an E3 ubiquitin ligase which controls the half-life of p53 via ubiquitin-depen- dent proteasomal degradation.22 In response to cellular stress, the p53-MDM2 interaction is disrupted and p53 undergoes post-translational modifications on multiple sites to promote transcription of target genes that trigger cell-cycle arrest, apoptosis and/or cell senescence.20-23 Since the discovery of the first selective small molecule MDM2 inhibitor, Nutlin-3a, newer compounds have been devel- oped with increased potency and improved bioavailabili- ty.24,25 These non-genotoxic compounds bind to MDM2 in the p53-binding pocket with high selectivity and can release p53, leading to effective stabilization of the protein and activation of the p53 pathway.24,25 Initial preclinical and clinical studies have demonstrated promising efficacy of this class of drugs in a number of p53 wildtype adult and pediatric cancers, as single agents or in combination with other targeted therapies.26-34 However, the contribu- tion of transcription-dependent pathways to the p53- mediated response in CLL has not been systematically explored, and, importantly, the effect of p53 reactivation and the p53 gene expression signature in normal cells implicated in the dose-limiting hematologic toxicity is yet to be elucidated.
In this study, we compared the effects of a second-gen- eration and clinically relevant MDM2 inhibitor, RG7388, in patient-derived primary CLL cells and normal blood and bone marrow cells, including CD34+ hematopoietic progenitors, and report the contrasting transcriptional induction profile of p53-target genes and consequent pref- erential pro-apoptotic responses of CLL cells to RG7388 exposure, compared with those of normal hematopoietic cells.
Methods
Patients and cell isolation
Peripheral blood samples (n=55) from CLL patients (Online Supplementary Table S1) were collected into EDTA-coated tubes. Informed consent was obtained in accordance with the
Declaration of Helsinki, and with approval from the National Health Service Research Ethics Committee. CLL patients’ samples were collected and stored under the auspices of the Newcastle Academic Health Partners Biobank (http://www.ncl.ac.uk/biobanks/collections/nbrtb/). CLL was diagnosed according to the International Working Group on CLL-164 National Cancer Institute’s 2008 criteria.35
Normal peripheral blood mononuclear cells (PBMC), bone mar- row mononuclear cells (BMMC) and CD34+ hematopoietic stem cells (CD34+ cells) were isolated from six, five and three healthy donors, respectively. Details on the isolation and culture of leukemic and normal cells are provided in the Online Supplementary Methods.
Reagents
The small-molecule MDM2 inhibitor RG7388 was custom syn- thesized as part of the Newcastle University/Astex Pharmaceuticals Alliance and CRUK Drug Discovery Program at the Northern Institute for Cancer Research, Newcastle University. RG7388 was dissolved in dimethylsulfoxide to make a 10 mM stock solution and stored in small aliquots at −20°C.
Nutlin-3a was purchased from Cambridge Bioscience (Cambridge, UK), ibrutinib from Axxora (Enzo Life Sciences, Exeter, UK), and venetoclax (ABT199) from Selleckchem, Absource Diagnostics (Munich, Germany).
Functional assessment of the p53 pathway
The functional status of p53 in CLL samples was determined by observing the modulation of p53 and transcriptional target gene protein products, MDM2 and p21, following short-term exposure to MDM2 inhibitors.36 The TP53 mutational status of CLL samples was assessed by next-generation sequencing (using Roche 454 GS FLX and Illumina MiSeq platforms) in 54/55 samples. The pres- ence of a 17p deletion was assessed by fluorescence in situ hybridization and/or multiplex ligation-dependent probe amplifi- cation analysis in 54/55 samples. In one case (CLL 0255), we were unable to perform DNA analysis; the functional status of p53 for this case was, therefore, evaluated in vitro using short-term expo- sure of the CLL cells to MDM2 inhibitors, and this sample was identified as p53-non-functional.
Ex vivo cytotoxicity assay
Cells (5x106/mL) in 100 μL of medium per well of a 96-well
plate were exposed to a range of concentrations of RG7388 for 48 h. Cytotoxicity was assessed using an XTT Cell Proliferation Kit II (SigmaAldrich, UK), as detailed in the Online Supplementary Methods.
Western blot analysis
Cells (5x106/mL) were seeded in 1 mL per well of a 24-well plate and exposed to a range of concentrations of RG7388. Cells were harvested and lysed at 6 h and 24 h. Protein concentration was measured using a PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, UK). The protocol is described in detail in the Online Supplementary Methods.
Real-time reverse transcriptase polymerase chain reaction gene expression analysis
Cells (5x106/mL) were seeded in 2 mL per well of a 12-well plate and exposed to a range of concentrations of RG7388 for 6 h and 24 h. Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Manchester, UK). The concentration and purity of the RNA were measured using a NanoDrop ND-1000 spectrophotometer. RNA was reverse-transcribed with a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, UK). Relative quantifi-
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haematologica | 2019; 104(12)