RUNX1 DNA-Binding Mutations and RUNX1-PRDM16 Cryptic Fusion in BCR/ABL+ Leukemias Are Frequently Associated with Secondary Trisomy 21 and May Contribute to Clonal Evolution and Imatinib Resistance

Background: Besides the extensively studied point mutations in the BCR/ABL TK domain as the clinically most important cause of imatinib (IM) resistance either in CML and Philadelphia-positive (Ph+) ALL, additional acquired genetic events contributing to resistance/disease progression are not fully understood. Many evidences suggest that the enhanced survival and differentiation arrest of the CML blast crisis (BC) cells depends on the cooperation of BCR/ABL with other genes deregulated during disease progression. A recent study has identified RUNX1/AML1 transcription factor gene as a modulator of the cellular response towards IM in vitro and in vivo in mice, suggesting its possible involvement in disease persistence in IM-resistant CML patients. Purpose: We investigated RUNX1 molecular abnormalities (point mutations or cryptic chromosomal RUNX1 translocation to a novel recently described gene partner PRDM16 at 1p36) in 18 Ph+ leukemias. The observation that RUNX1 mutated allele is frequently duplicated by acquired trisomy of the altered chromosome 21 in acute myeloblastic leukemias (AML) prompt us to focus our analysis on a selected cohort (1 CP-CML, 3 AP-CML, 8 myeloid BC, 1 lymphoid BC, 2 de novo Ph+ ALL-B, 1 de novo AML and 1 therapy-related- Ph+ ALL) presenting acquired trisomy 21 along disease course. Methods: From peripheral blood leucocytes, recurrent mutations were investigated by sequencing DNA PCR fragments corresponding to exon 3 to 8 of RUNX1. RUNX1-PRDM16 fusion was investigated by RT-PCR (i.e. RUNX1 exon 5-PRDM16 exon 2 junction) and by FISH using the TEL/AML1 dual color/dual fusion translocation probes (Vysis, Downer’s grove, IL, USA). Results: We report a high frequency (33%) of recurrent point mutations (4 in myeloid BC CML and 1 in CP-CML) within the DNA-binding region of RUNX1. We did not find any mutation in de novo BCR/ABL+ ALLs or lymphoid BC CMLs. Onset of RUNX1 mutations was detected at diagnosis or before the acquisition of trisomy 21. We also report a high frequency of RUNX1-PRDM16 fusion for 3 out of 7 investigated patients: 2 CMLs and, for the first time reported to our knowledge, 1 therapy-related BCR/ABL+ ALL. RUNX1-PRDM16 is probably transcribed at low levels since only few bone marrow metaphases with trisomy 21 were t(1;21)(p36;q22) positive. Two patients presented both RUNX1 mutations and RUNX1-PRDM16 fusion. All these events are associated with a poor survival: 14 out of 18 patients died with a median survival of 3 months after the diagnosis of the acquired clonal trisomy 21. Furthermore, none of the patients in our study raised durable optimal responses to IM. Conclusion: Our data support the concept of a cooperative effect of BCR/ABL with molecular RUNX1 abnormalities on the differentiation arrest phenotype observed during progression of CML and in BCR/ABL+ ALL. Our findings also support that it may exist a heterogeneous genetic status at diagnosis of CML, underlying the need of further investigations able to define more precisely the molecular disease characteristics at diagnosis for better therapeutic decision adjustments.