Whole-Exome Sequencing Identifies Recurrent Mutations of BCOR in Acute Myeloid Leukemia with Normal Karyotype

Abstract 71

Acute myeloid leukemia (AML) with normal cytogenetics (CN-AML) represents about half of all adult AML. NPM1 and CEBPA mutations define WHO provisional entities accounting for ∼60% of CN-AML, but the remaining cases (∼40%) remain poorly characterized. To address this issue, we carried out whole-exome-sequencing (WES) of leukemic and normal cells from one patient with CN-AML that lacked mutations in NPM1, CEBPA, FLT3-ITD, and MLL-PTD. Using this approach, we identified a clonal somatic mutation of BCOR, a gene located on chromosome Xp11.4, that was present in the leukemic but not normal cells of the index AML case. The BCOR (BCL6 co-repressor) gene encodes for an ubiquitously expressed nuclear protein that is involved in repressing the activity of BCL6 and other transcriptional factors. BCOR is a key transcriptional regulator of early embryonic development, mesenchymal stem cell function and hemopoiesis. Germline mutations of BCOR are responsible for the oculo-facio-cardio-dental (OFCD) genetic syndrome that is inherited in an X-linked pattern and comprises microphtalmia, dysmorphic appearance, dental abnormalities (radiculomegaly), hammer-toe deformity and cardiac defects. WES findings in the index case were subsequently validated and further studied in a total cohort of 514 AML patients. We first performed deep-sequencing analyses of all exons of the BCOR gene in an initial set of 82 AML cases that were selected because they showed the same genetic characteristics of our index patient (i.e. normal karyotype without NPM1, CEBPA, FLT3-ITD and MLL-PTD mutations). Disruptive BCOR mutations (i.e., nonsense mutations, out-of-frame small indels, and consensus splice-site mutations) were detected in 14/82 (17.1%) of these cases. We next assessed the frequency of BCOR mutations in a series of unselected CN-AML patients (n=262) and found that they occurred in 4.2% of cases, mostly showing the typical features of BCOR-mutated cases (absence of NPM1, CEBPA, FLT3-ITD and MLL-PTD mutations). Almost mutual exclusion of BCOR and NPM1 mutations was further confirmed in a separate series of 71 NPM1-mutated only AML patients. No BCOR mutations were observed in the 89 AML cases with recurrent cytogenetic abnormalities investigated, including t(8;21)(q22;q22) (n= 29), inv(16)(p13q22) (n=40), t(15;17)(q22;q12) (n=10), and t(11q23)/MLL (n=10), and in the 10 patients with double CEBPA-mutated AML studied. BCOR mutations were: i) scattered across the whole length of the coding sequence with no hotspots identified; ii) somatic in origin and disruptive molecular events similar to germline BCOR mutations causing the OFCD genetic syndrome; iii) associated with markedly decreased BCOR mRNA levels, absence of full-length BCOR and absent or low expression of a truncated BCOR protein; iv) almost mutually exclusive with NPM1 (only 1.5% of the 197 NPM1-mutated AML investigated carried BCOR mutations); v) rarely associated with FLT3-ITD; and vi) frequently associated with DNMT3A and RUNX1 mutations, suggesting cooperation with the respective mutated pathways. Clinically, BCOR mutations correlated with poor outcome among the cohort of 160 CN-AML patients evaluated (28.0% versus 66.3% overall survival at 2 years, P=0.024). We also searched for BCOR mutations in the human AML cell lines OCI-AML2, OCI-AML3, KG1a, U937, HL-60, HL-60R, HB4, AML193, and MVP-11. Only HL-60 and HL-60R (a ATRA-resistant derivative of HL-60) carried a BCOR mutation that consisted of a hemizygous G to T transition at position 4616 in exon 10, leading to the Glu1442X nonsense mutation. Western blot analysis of HL-60 cells resulted in the absence of the full-length BCOR protein (predicted MW: 192 kDa) and presence of a low intensity 156 kDa band likely corresponding to a truncated BCOR protein. In conclusion, our results implicate for the first time BCOR in the pathogenesis of CN-AML and suggest it may act as tumor suppressor gene.