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V. Magistroni et al.
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Figure 1. Activity of UBE2A mutants. (A) Western blot analysis of total cell lysates from BA/F3_BCR-ABL cell lines stably transfected with pMIGR-UBE2A vectors encoding for wild-type (WT) or mutated (D114V or I33M) UBE2A. Empty vector has been used as negative control. (B) Real-time quantitative polymerase chain reac- tion (RT-qPCR) of total RNA extracted from BA/F3_BCR-ABL_pMIGR/UBE2A cell lines. The values are normalized on the EMPTY cells (***P<0.001). (C) Western blot of total cell lysates from BA/F3_BCR-ABL_pMIGR/UBE2A cell lines. The signal at 14KDa corresponds to histone H2A. The signal at ~23KDa corresponds to monoubiquitinated histone H2A (mUbH2A) according to Wu et al.37 (D) Western blot analysis of the in vitro ubiquitination reaction performed with in vitro translated UBE2A (WT and mutated forms) and GST-ubiquitin on total BA/F3_BCR-ABL lysate. (Right) The densitometric analysis of GST-ub signal (>170KDa) from three inde- pendent experiments obtained with ImageJ software.38 The fold change is obtained normalizing the signal on the WT sample (WT vs. D114V *P=0.022; WT vs. I33M **P=0.0069). (E) Histogram showing the enzymatic activity of in vitro expressed UBE2A using the AMP Glow assay (WT vs. D114V *P=0.0056; WT vs. I33M **P=0.0036).
progression to BC. Initially whole exome sequencing (WES) data from ten patients were analyzed. CP samples were used as baseline controls for each patient to identify somatic variants selectively occurring in BC (Table 1), thus allowing the recognition of molecular events occurring exclusively upon CML progression. By using this approach we identified mutations on genes already asso- ciated with BC, such as RUNX1, IKZF1, NRAS, ASXL1 and ABL1.7,13 A total of 41 non-synonymous single nucleotide variants (SNV) and small indels were identified, with a mean of 4.1 mutations/patient acquired upon BC progression. Of these events, 31 were transitions, seven transversions and three indels, with the C>T substitution being the most frequent (63.4%) (Online Supplementary Figure S1). In one patient (patient #7) no acquired exonic SNV could be detected during CML progression.
Analysis of SNV data showed the presence of two recur- rently mutated genes in this cohort: ABL1, with mutations F486S, E255V and T315I occurring on the BCR-ABL1 fusion gene and leading to TKI resistance (30%, 95%CI: 0.574, 0.026), and UBE2A (Xq24), an E2-ubiquitin conju-
gating enzyme required for post-replicative DNA damage repair15 (20%, 95%CI: 0.447, 0.000), which has never been previously reported as mutated in CML patients. UBE2A mutations occurred on two non-contiguous residues: D114V and I33M (Tables 1 and 2). Patient #3 (male) showed an UBE2A variant frequency of 93%, as expected given that the gene is localized on the X chromo- some. Patient #8 (female) carried a heterozygous UBE2A mutation (mutation ratio: 39%). The high mutation ratio observed in both patients suggests that UBE2A is present in the dominant BC clone (Table 1).
UBE2A mutations are recurrent and acquired in late chronic myeloid leukemia
The evidence of recurrent, somatic UBE2A mutations has never been reported in BC cases; however, they had been previously found in other clonal disorders both of solid and hematopoietic origin, confirming their potential role in tumor progression.19 To further characterize the pat- tern and the frequency of UBE2A mutations in a larger cohort of patients, we sequenced 31 additional CML CP
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haematologica | 2019; 104(9)