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V. Magistroni et al.
ulation of surface protein expression; P<0.001) and CD44 (38% decrease at mRNA level; P<0.01) after PMA treat- ment (Online Supplementary Figure S6).
Neutrophilic differentiation was similarly tested in the 32Dcl3 BCR/ABL1 cell lines over-expressing UBE2A WT or I33M. Treatment of the UBE2A I33M cell line with GCSF + IL-3 showed a delay in neutrophilic differentia- tion, as assessed by CD11b expression levels when com- pared with both UBE2A WT or control (Figure 4A). Cells treated with IL-3 alone were used as an internal control. FACS showed no difference in CD11b surface expression in UBE2A I33M cell line compared to controls after three days of treatment but showed a 37% decrease at day 6 which was also confirmed by confocal microscopy analy- sis (Figure 4B).
Discussion
In line with previous results,5,7,9 our analysis performed on matched CP/BC CML samples showed considerable somatic heterogeneity in BC phase. In all the samples, we detected a very low number of acquired SNV, correspon- ding to an average of 4.1 non-synonymous mutations per patient, a frequency far below the average reported for other hematopoietic neoplasms, such as acute myeloid leukemia (AML: 7.8) and chronic lymphocytic leukemia (CLL: 11.9).25 This can in part be explained by the charac- teristics of our analysis, where somatic variants occurring in BC were filtered against those in CP, therefore filtering- out all the driver and passenger variants pre-existing the evolution to BC. All BC samples showed the prevalence of transition events and, in particular, of C:G>T:A substi- tutions, accounting for 66.7% of all the SNV (Online Supplementary Figure S1). Approximately 85% of the C:G>T:A transitions were part of a CpG dinucleotide. Cytosines in CpG sites are known to be affected by a high mutation rate, caused by a spontaneous deamina- tion of methylated cytosines.26 This mutation pattern is also in accordance with a BCR-ABL1 dependent mutation signature, characterized by inhibition of the mismatch repair system (MMR) and by accumulation of reactive oxygen species (ROS), as previously reported.27 Mutations in RUNX1 and IKZF1, both involved in hematopoietic differentiation, have already been detected in the advanced stages of CML7 and are confirmed here as specific markers for BC progression. Along with this, the XPO1 gene (exportin-1) mutated here in a single patient with the E571K substitution, is also frequently mutated in clonal hematologic disorders, with the E571K mutation widely represented in chronic lymphocytic leukemia.28 SNV analysis showed the presence of a recur- rent mutation affecting the UBE2A gene (Xq24) (pt#3 and pt#8). UBE2A is an E2-ubiquitin conjugating enzyme that has never been found mutated in CML. Interestingly, the two patients harboring UBE2A mutations lacked any rec- ognizable copy number alteration (Table 2). WES and tar- geted resequencing of a broader cohort showed that somatic UBE2A mutations are found in a significant frac- tion (16.7%) of advanced CML phases, thus confirming the initial exome analysis and suggesting a driver role for UBE2A loss of function during disease progression.
The Saccaromyces Cerevisiae UBE2A homolog Rad6 par- ticipates in DNA repair, sporulation and cell cycle regula-
tion;29 in mammals a role for UBE2A in the regulation of transcription and chromatin reorganization through post- translational histone modifications has recently been hypothesized.30 Germline mutations of the UBE2A gene in humans have been associated with the X-linked Nascimento-type intellectual disability syndrome.31-33 In order to understand the effect of UBE2A mutations in a BCR-ABL1-positive model, we tested the activity of exogenous UBE2A both in the WT or mutated forms (D114V and I33M) in BA/F3 BCR-ABL1-positive cell lines. We observed a reduced amount of mono-ubiquitinated histone H2A, a known UBE2A substrate, after over- expression of mutated UBE2A compared to the WT (Figure 1C), which indicates that the UBE2A mutations analyzed in this study decrease the activity of the enzyme. This result has been further confirmed by in vitro assays for ubiquitination and enzymatic activity on total cell lysates (Figure 1D and E), thus providing evidence of a damaging effect of the two mutations on UBE2A func- tion. Accordingly, one of the four variants identified in our cohort is a N-terminal frameshift mutation, thus sup- porting this hypothesis. This evidence is further strength- ened by the distribution of UBE2A mutations throughout the entire protein, a pattern that is more common for genes undergoing inactivation. Mutations in the UBE2A paralog UBE2B were not detected in this study, which suggests a specific role for UBE2A in chronic myeloid leukemia. Stable silencing of UBE2A in the BCR-ABL- positive K562 cell line or overexpression of the I33M mutated form in a BCR-ABL1-positive 32Dcl3cl3 myeloid cell line showed profound downmodulation of CSF3R, a critical regulator of myeloid lineage differentiation and development.34,35 CSF3R, also known as granulocyte colony-stimulating factor receptor (GCSFR), is a member of the hematopoietin receptor superfamily35 and plays a key role in promoting neutrophilic differentiation but may also support the development of different types of hematopoietic progenitors.34 This suggests a potential role for CSF3R modulation in the suppression of myeloid differentiation in BC. Although the precise mechanism by which UBE2A controls CSF3R expression is still unknown, our data suggest that UBE2A-mediated CSF3R regulation occurs at transcriptional level. Alteration of CSF3R transcription could occur either by a direct activity of UBE2A on CSF3R promoter through epigenetic mech- anisms36 or indirectly by UBE2A-mediated ubiquitination of specific transcription factors. Further studies will be needed to clarify this process and to establish the rele- vance of CSF3R deregulation in the impairment of CML cell differentiation. In line with these findings, we showed that impairment of UBE2A function induces a delay in the differentiation of K562 and 32Dcl3- BCR/ABL1 cells after PMA, hydroxyurea or GCSF treat- ment, suggesting an important role for UBE2A as a mod- ulator of myeloid differentiation.
In conclusion, in this work we identified recurrent, somatic UBE2A mutations occurring in a significant pro- portion of advanced CML cases. We propose that the acquisition of somatic UBE2A mutations affects myeloid developmental pathways, promoting a differentiation blockade. Further studies will be required to thoroughly dissect the molecular mechanisms responsible for these effects and to define possible therapeutic strategies for UBE2A-mutated BC-CML cases.
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