De Novo UBE2A Mutations Are Recurrently Acquired during CML Progression and Interfere with Myeloid Differentiation Pathways

Abstract

Despite the advent of tyrosine kinase inhibitors, a proportion of chronic myeloid leukemia (CML) patients in chronic phase (CP) fail to respond to Imatinib or to second generation drugs and progress to blast crisis (BC). Limited advances in the understanding of the molecular mechanisms responsible for CML transformation from CP to the aggressive BC were achieved until now. We present here a massive parallel sequencing analysis of 10 BC samples and of the corresponding autologous CP controls. Whole exome sequencing (WES) was performed to identify somatic variants occurring in BC and absent in the corresponding CP sample. A total of 41 non-synonymous single nucleotide variants (SNVs)/small indels were identified upon BC progression. Several mutations identified in BC samples such as RUNX1, IKZF1, NRAS, ASXL1 and ABL1 were already associated with progression; however we also identified, for the first time, recurrent mutations affecting the ubiquitin-conjugating enzyme E2A gene (UBE2A, formerly RAD6A, Xq24) in two out of ten cases (20%). In both cases UBE2A variant was present in the major clone (Table 1).Additional analysis on a larger cohort revealed the presence of UBE2A variants in 16.7% of advanced phases. No evidence of UBE2A variants could be found in the CP samples at onset.To investigate the functional role of UBE2A mutations, we stably transfected a BA/F3-BCR-ABL1 cell line with the wild-type(WT) and with two UBE2A variants, I33M and D114V. In order to understand the effect of UBE2A mutations, we initially assessed the activity of exogenous UBE2A by measuring the ubiquitination level of histone H2A, a known UBE2A substrate (Sung P et al 1988;2(11):1476). We observed a reduced amount of mono-ubiquitinated H2A in mutated UBE2A cells compared to WT, which suggests that UBE2A mutations could impair the activity of the enzyme. This result has been further confirmed by an in-vitro ubiquitination assay on total cell lysates, thus providing evidence of a damaging effect of the UBE2A mutations. To study the effect of UBE2A loss of function, we set-up a stable UBE2A silencing model (siUBE2A) in the human BCR-ABL-positive K562 cell line. RNA-Seq analysis of siUBE2A clones vs controls showed a significant enrichment for ontologies related to myeloid differentiation. Interestingly, the expression of CSF3R was dramatically downregulated both at mRNA (12.5 down-regulation) and protein levels. In line with the known role of CSF3R as an inducer of neutrophilic differentiation, we show here that siUBE2A causes a delay in the differentiation of K562 cells after phorbol-myristate-acetate or hydroxyurea treatment, suggesting that CSF3R downmodulation is one of the key players responsible for this phenomenon.In conclusion, we identified recurrent, somatic UBE2A mutations occurring in a significant fraction of advanced CML cases. We propose that the acquisition of UBE2A mutations affects myeloid developmental pathways, causing a down-modulation of CSF3R expression and therefore 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. [Display omitted] DisclosuresYeung:Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Saglio:Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Roche: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Kim:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Il-Yang: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Branford:Otsuka: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Gambacorti-Passerini:Pfizer: Consultancy, Honoraria, Research Funding; BMS: Consultancy.