Tissue and differentiation stage specific expression of CALM/AF10 is required for leukemogenesis

The translocation t(10,11)(p13;q14) resulting in the formation of the CALM/AF10 fusion gene is involved in various hematological malignancies including acute myeloid leukemia, T-cell acute lymphoblastic leukemia, and malignant lymphoma and is usually associated with poor prognosis. We established a knock-in mouse model allowing tissue-specific CALM/AF10 expression from the Rosa26 locus using a loxP-STOP-loxP cassette to study leukemic transformation by the CALM/AF10 fusion protein during hematopoiesis. vav-Cre induced pan-hematopoietic expression of the CALM/AF10 fusion gene led to acute leukemia with a median latency of 12 months. Leukemias were either myeloid or had myeloid feature and showed expression of the B cell marker B220. Gene expression profiling of leukemic bone marrow cells revealed the overexpression of Hoxa cluster genes and the Hox co-factor Meis1. The long latency to leukemia development suggested that additional, collaborative genetic lesions are required. We identified an average of 2 to 3 additional mutations per leukemia using whole-exome sequencing. When CALM/AF10 was expressed in the B lymphoid compartment using mb1-Cre or CD19-Cre inducer lines no leukemia development was observed. Our results indicate that CALM/AF10 needs to be expressed from the stem or early progenitor cell stage onward to permit the acquisition of additional mutations required for leukemic transformation. Total RNA was extracted from BM cells of leukemic (n=8) and pre-leukemic CA+/vav-Cre+ mice (n=3) and of wild type C57Bl/6 mice (n=3) and from the B cells of CA+/mb1-Cre+ mice (n=3), pre-leukemic CA+/vav-Cre+ mice (n=3) and of wild type C57Bl/6 mice (n=3) using TRIzol reagent (Life Technologies, Darmstadt, Germany). Leukemic bone marrow cells were sorted using Gr1, Mac1 and B220 antibodies, purchased from BD Biosciences and cells were sorted using BD FACSVantage SE system. cDNA was prepared using the Ambion WT expression kit (Life Technologies) and hybridized to Affymetrix Mouse Gene 1.0 ST Arrays (Affymetrix, Santa Clara, CA, USA) for whole transcriptome microarray analysis.