Mechanisms of Leukemogenesis by MLL-CALM: Characterization of a CALM-Derived Transcriptional Regulatory Domain.

Background:MLL translocations are common in infant leukemias, and >50 distinct translocation partners have been described. We recently identified the CALM gene as a novel MLL partner in an infant with aggressive AML. Interestingly, CALM was first discovered as a translocation partner for AF10, which had previously been identified as an MLL fusion partner in aggressive leukemias and lymphomas. The native CALM protein exhibits predominantly cytoplasmic localization, and participates in clathrin-dependent endocytosis and intracellular vesicle transport. We have previously shown that expression of MLL-CALM immortalizes murine hematopoietic progenitors, and that fusion of the carboxy terminus of CALM to MLL alters MLL transcriptional activity. We hypothesize that CALM possesses a specific transcriptional activation domain (TAD) which modulates MLL transcriptional activity of HOX genes, thereby contributing to leukemogenesis.

Objectives: 1) To determine whether native CALM localizes to the nucleus, 2) To delineate specific CALM domains which constitute the CALM TAD, and 3) To determine whether MLL-CALM activates transcription through the murine HOXA7 promoter.

Methods: Human fibroblast cells were treated with Leptomycin B (an antifungal antibiotic which specifically inhibits nuclear export) and stained with an anti-CALM antibody. We prepared a set of expression vectors in which various portions of CALM are fused to a GAL4 DNA-binding domain. These vectors were co-transfected with a GAL4-luciferase reporter plasmid into COS7 cells, and luciferase activity was measured 48 hours after transient transfection. Luciferase assays were also performed using MSCV-MLL-CALM or MSCV-CALM plasmids co-transfected with a HOXA7 promoter-luciferase reporter construct.

Results: After inhibition of nuclear export, native CALM localized to both the nucleus and cytoplasm. Significant luciferase activity was only observed with constructs containing distal CALM carboxy amino acids (aa 436–660). Mutation of an NR (Nuclear Receptor) Box motif (aa 510–514) did not affect CALM-dependent transcription. We found that two endocytosis-related NPF domains play opposite roles: deletion of NPF#1 (aa 437–439) dramatically reduced, while mutation of NPF#2 (aa 639–641) increased transcriptional activity. Expression constructs lacking GAL4 DNA binding domains had no effect on transcription, and GAL4 binding sites were required for luciferase activity in this system. Finally, MLL-CALM activated transcription of the murine HOXA7 promoter in comparison with native CALM or empty vector.

Conclusions: We have confirmed that native CALM is able to localize to the nucleus, and we have begun to identify specific critical residues in the CALM TAD. The presence of a CALM TAD in MLL-CALM suggests that altered transcriptional regulation of MLL-dependent HOX genes may play an important role in MLL-CALM dependent transformation. Our observations raise the possibility that other MLL partners with native cytoplasmic localization may possess unrecognized transcriptional activity, and provide new insight into both MLL-CALM and CALM-AF10 mediated leukemogenesis.