Early Target Genes of CALM/AF10 as Revealed by Gene Expression Profiling.

Abstract

The t(10;11)(p12;q14) is a recurring chromosomal translocation that is found in acute myeloid and acute lymphoblastic leukemia as well as in malignant lymphoma. This translocation results in the fusion of AF10, a putative zinc finger transcription factor containing an N-terminal LAP/PHD zinc finger motif, a nuclear localization signal, an AT-hook domain, and a leucine zipper and with the CALM gene (Clathrin assembly protein lymphoid myeloid leukemia gene) that encodes a clathrin assembly protein. In the monocytic cell line U937 both the CALM/AF10 and the AF10/CALM fusion mRNAs can be identified. The CALM/AF10 fusion mRNA codes for the CALM/AF10 fusion protein which contains almost the complete CALM protein fused in frame to about 90% of the AF10 protein without the N terminal two PHD zinc fingers. CALM/AF10 is highly leukemogenic with its expression in primary bone marrow cells leading to the development of an aggressive acute leukemia with a short latency of 10 weeks in a murine bone marrow transplant model. We set out to identify immediate target genes of CALM/AF10. The fusion gene was cloned into pRTS-1, an episomally replicating tetracycline/doxycycline inducible (tet-on) expression vector. The construct was stably transfected into the Burkitt's lymphoma B cell line DG75. The expression of CALM/AF10 after induction was confirmed by RT-PCR. Gene expression profiling experiments were conducted using the Affymetrix® Human Genome U133 Plus 2.0 Array analyzing non-induced and induced (24 and 72 hours after induction) samples. As controls, DG75 cells transfected with the pRTS-1 vector without the CALM/AF10 fusion gene were used. The results were analyzed using the R statistical package and dChip (DNA Chip Analyzer) software. The expression of 1237 genes was found to be changed at least 2 fold 24 hours after the induction of CALM/AF10. 594 (48%) genes were downregulated, while 643 (52%) were upregulated. Downregulated genes included genes involved in DNA repair (DDB2, TOP2A, BRCA1), cell cycle check point control (CCNE2, CHEK1, CDC2), and chromosome maintenance (MCM3, MCM7, MCM10). Upregulated genes included signal transduction molecules like RAB8B (a member of the RAS oncogene family) and STAT family members (STAT1 and STAT2), chromatin remodeling factors like BAZ2A (bromodomain adjacent to zinc finger domain, 2A), and MAML3 (master mind like 3), a positive regulator of Notch signaling. Pathway analysis using KegArray showed an enrichment of differentially regulated genes in processes like cell cycle regulation and DNA replication and repair. Interestingly, no significant upregulation of Hox genes was observed, as was previously reported in a study of CALM/AF10 positive patient samples (Dik et al., 2005). The changes in the expression levels of some selected genes were confirmed using a Taqman® Low Density Array (LDA). This analysis showed a statistically significant correlation (r = 0.78, p = 0.001) between the real time PCR results and the expression levels obtained from the Affymetrix arrays. Our results demonstrate that the expression of the leukemogenic CALM/AF10 fusion protein leads to a severe deregulation of critical cellular processes giving a first hint at the direct target genes of this fusion protein.