Perturbed Endocytosis by Leukemogenic CALM-Containing Fusion Proteins Is Associated with Prolonged Growth Factor Signaling and Enhanced Cellular Proliferation.

Background: Clathrin Assembly Lymphoid Myeloid leukemia (CALM) gene rearrangements occur in a subset of aggressive leukemias and lymphomas, in which CALM is fused to the MLL or AF10 genes. Previous studies have shown that expression of MLL-CALM or CALM-AF10 fusion proteins immortalizes murine hematopoietic progenitor cells, an observation that often correlates with leukemogenesis in vivo. While disruption of MLL or AF10 activity contributes to malignant transformation, perturbation of normal CALM function may also play a role. Indeed, the identification of significant hematologic abnormalities in fit1 mice that harbor calm mutations suggests a role for CALM in normal hematopoiesis. The native CALM protein is primarily cytoplasmic and functions in Clathrin-Dependent Endocytosis (CDE). We have demonstrated that expression of native CALM or a CALM C-terminal domain in COS7 cells impairs endocytosis of both Transferrin and Epidermal Growth Factor (EGF). Since cell surface growth factor (GF) signaling may be downregulated by receptor internalization via CDE, we hypothesize that disruption of CDE by CALM-containing fusion proteins interferes with attenuation of GF signaling in hematopoietic cells; this results in sustained proliferation that contributes to leukemogenesis.

Objectives: 1) To demonstrate that expression of CALM fusion proteins interferes with the endocytosis of hematopoietic GF; 2) To analyze the time course of phosphorylation of Janus kinases (JAK), downstream targets of GF signaling; 3) To determine whether cells expressing CALM-containing fusion proteins have a survival advantage.

Methods: COS7 cells transfected with CALM-containing constructs, and hematopoietic cell lines that natively harbor CALM-AF10 translocations (U937, P31/Fuji) were incubated with ¹²⁵I-EGF or ¹²⁵I-GM-CSF, respectively, and the amount of internalized radioactivity was measured. To measure the functional significance of impaired endocytosis, the pattern of JAK phosphorylation in these cells was analyzed by Western blot. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to measure cell proliferation rates.

Results: Expression of CALM fusion proteins in COS7 cells was associated with a 40–50% reduction in the rate of EGF endocytosis when compared to control cells. Hematopoietic cell lines that natively harbor CALM-AF10 translocations (U937, P31/Fuji) exhibited reduced rates of GM-CSF endocytosis in comparison with hematopoietic cells lacking such translocations (HL60, K562). EGF treatment of CALM fusion-expressing COS7 cells resulted in increased and prolonged JAK1 phosphorylation. JAK2 phosphorylation was similarly affected in GM-CSF-treated hematopoietic cell lines that natively express CALM-AF10 fusion proteins. Finally, expression of MLL-CALM or CALM-AF10 proteins in COS7 cells was associated with a reduced serum requirement in vitro, showing increased proliferation in low serum relative to controls.

Conclusion: We have demonstrated that CALM-dependent perturbation of endocytosis in hematopoietic cells is associated with increased GF signaling, as well as a proliferative advantage in low serum. These observations argue for a crucial and previously unappreciated role for CDE dysregulation in leukemogenic transformation.