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V. Magistroni et al.
sis.3,4 However, a fraction of these patients fail to respond to the treatment (primary resistance) or become resistant after an initial response.4-6 The persistence of BCR-ABL1 activity typically drives the progression to the advanced phase of the disease within 3-5 years. One of the open issues in CML concerns the dissection of the molecular mechanisms driving the transformation to BC, commonly considered as a heterogeneous disease at the molecular level.7-9 BC is mainly characterized by the rapid expansion of the differentiation-arrested BCR-ABL1-positive blast cells,10 therefore resembling an acute leukemia. In most cases of BC cases (approx. 70%), blasts maintain myeloid features, while in 20-30% blast lineage is lymphoid. BCR- ABL1 expression, which increases during CML progres- sion in conjunction with BCR transcription, seems to have a prominent role in this process, hyperactivating proliferative and anti-apoptotic signals and inducing genetic instability.5,11,12 Previous reports showed the exis- tence of a heterogeneous molecular signature among dis- tinct BC patients.5,7,8 However, these data were limited by the scarcity of matched CP/BC samples, due to the infre- quent progression to BC after the advent of TKI. Here we analyzed ten paired CP/BC samples through a whole- exome sequencing (WES) approach, identifying somatic variants specific for BC progression since these were not present in the autologous CP controls. Along with several mutations previously identified as BC driver events,5,7,13 we detected, for the first time, recurrent BC-specific mutations occurring on the UBE2A gene. These data sug- gest that the appearance of UBE2A variants in CML cells could contribute to BC progression through the impair- ment of myeloid differentiation.
Methods
Cell lines
The BA/F3-BCR-ABL1 and 32Dcl3-BCR/ABL1 cell lines were generated and maintained as described by Puttini et al.14 and Piazza et al.15 K562 and 293FT were purchased from DSMZ (Braunschweig, Germany) and Thermo-Fisher-Scientific (Waltham, MA, USA) respectively, and were maintained accord- ing to the manufacturers’ instructions.
Patients
Diagnosis and staging were performed according to the World Health Organization WHO-2008 classification.16 Peripheral blood (PB) or bone marrow (BM) of ten matched CML chronic phase/blast crisis samples, 31 CP-CML, 14 AP/BC-CML, 38 atyp- ical-CML (aCML), and 40 AML were collected at diagnosis and after obtaining written informed consent approved by the institu- tional ethics committee. The study was conducted in accordance with the Declaration of Helsinki. Samples were prepared as described by Piazza et al.17
Whole exome sequencing
Genomic DNA (gDNA) was extracted from purified cells with PureLink Genomic DNA kit (Thermo-Fisher-Scientific). 1 μg of gDNA from each sample was fragmented (500bp) with a Diagenode-Bioruptor sonicator system (Diagenode, Belgium) and processed according to the standard Illumina protocol. The Illumina TruSeq Exome Enrichment kit (Illumina Inc., San Diego, CA, USA) was used to enrich the genomic libraries for the exonic regions and samples were sequenced as described in the Online Supplementary Appendix.
Plasmids, transfections and lentiviral infections
BA/F3_BCR-ABL1-positive cells were transfected with pMIGR1_UBE2A vectors (Online Supplementary Appendix) as by Puttini et al.14 and were analyzed for GFP positivity with a FACSAria flow cytometer (BD Bioscience, San Jose, USA) and FACS-sorted when transfection efficiency was lower than 85%.
32Dcl3-BCR/ABL1 cells were electroporated using a Gene Pulser® II Electroporation System (BIORAD) with pMIGR1_UBE2A WT and I33M vectors as described by Puttini et al.14 To obtain stable UBE2A WT or I33M cell lines, GFP positive population was FACS-sorted with a MoFlo Astrios cell sorter equipped with Summit 6.3 software (both from Beckman Coulter, Miami, FL, USA).
For UBE2A silencing, K562 cells were infected with lentivirus obtained from MISSION-shRNA pLKO.1-based vectors (TRCN0000320625) (Sigma-Aldrich, Missouri, USA) and pack- aged using 293FT cell line. As a control, a pLKO.1MISSION non- target control vector (SHC002) (Sigma-Aldrich) was used. After infection K562 cells were maintained in 2 μg/mL puromycin for selection of silenced (K562_shUBE2A) and control cells (K562_shNC).
Quantitative real-time polymerase chain reaction
Total RNA was extracted using Trizol (Thermo-Fisher- Scientific) following the manufacturer’s instructions. 1 μg of total RNA was used to synthesize cDNA using reverse transcription reagents (Thermo-Fisher-Scientific) after pre-treatment with DNAseI (Thermo-Fisher-Scientific) to avoid contamination from genomic DNA. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using TaqMan® Brilliant II QPCR Master Mix (Agilent Technologies, CA, USA) on a Stratagene- MX3005P (Agilent-Technologies) under standard conditions. The housekeeping gene glucoronidase β gene (GUSB) was used as an internal reference.12 TaqMan® Gene Expression Assays (Thermo- Fisher-Scientific) were used (Online Supplementary Table S1).
In vitro translation and ubiquitination assay
In vitro translation of UBE2A proteins was performed with 1- Step Human Coupled IVT Kit-DNA (Thermo-Fischer-Scientific) following the manufacturer’s instructions. For ubiquitination assay we incubated 15 μg of UBE2A proteins with 1 μg of GST- Ubiquitin (Enzo-Life-Sciences, NY, USA), 0.2 ng of ubiquitin acti- vating enzyme (E1) (Enzo-Life-Sciences), 2 mM ATP, energy regeneration solution (BostonBiochem, MA, USA), 2 mM MgCl2, 2 mM KCl, 16 μg of BA/F3_BCR-ABL1 whole cell lysate in 50 mM TrisHCl (ph7.5). The reactions were incubated for 20 minutes at
37°C. The products were analyzed by western blotting.
For the enzymatic activity of WT and mutated UBE2A, 15 μg of UBE2A in vitro synthesized protein were used. The AMP-Glo Assay (Promega catalog v5011) was used in order to quantify the amount of AMP generated by the ubiquitin conjugation machin- ery, composed of 170 ng/μL ubiquitin protein, 15 ng/μL UBA1 and
50 μM ATP (SignalChem).
The production of AMP from ATP is directly proportional to the
enzymatic activity of the ubiquitination machinery and therefore it was used to measure the ubiquitination in the presence of WT and mutated UBE2A. The AMP signal was detected using the AMP detection solution (Promega) and a TECAN reading plate (Infinite F200Pro TECAN).
Neutrophilic differentiation
For induction of neutrophilic differentiation, 32Dcl3-BCR/ABL1 UBE2A WT and I33M cells were treated as previously described.18 32Dcl3-BCR/ABL1 cells expressing UBE2A WT or I33M were seeded at a density of 2x105 cells per milliliter and cultured in the
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