Perturbed Endocytosis by CALM-Containing Fusion Proteins: A Leukemogenic Mechanism in AML.

Background: We recently identified a novel fusion protein, MLL-CALM, in an infant with fatal AML. Although disruption of normal MLL function clearly contributes to leukemogenesis, perturbed CALM function may also be important. The native CALM protein plays a key role in clathrin-dependent endocytosis (CDE) and intracellular vesicle transport. Interestingly, CALM was first described as a partner for the AF10 gene - itself an MLL partner - in aggressive leukemias and lymphomas. Recently, it was shown that mutations in calm are responsible for hematopoietic abnormalities in inbred fit1 mice. Taken together, these observations suggest an important role for CALM in both normal and malignant hematopoiesis. We hypothesize that disrupted CDE resulting from the presence of CALM-containing fusion proteins interferes with downregulation of growth factor signaling, thereby contributing to leukemogenesis.

Objective: 1) To determine whether expression of MLL-CALM or CALM-AF10 interferes with endocytosis of transferrin (TF), 2) To analyze the importance of distinct CALM domains which affect endocytosis, and 3) To determine whether MLL-CALM expression reduces the IL-3 growth factor dependence of FL5.12 cells.

Design/Methods: The relative ability of leukemia cell lines that harbor MLL and CALM translocations to endocytose AlexaFluor633-conjugated TF was assessed by flow cytometry. The degree of Texas Red (TR)-TF endocytosis in COS7 cells expressing GFP-tagged MLL-CALM and CALM-AF10 was quantitated using densitometry. CALM deletion constructs were prepared and endocytosis of Texas Red-TF was examined in COS-7 cells transfected with these plasmids. Finally, IL-3-dependent FL5.12 cells engineered to stably express MLL-CALM were grown in decreasing amounts of IL-3-containing WEHI conditioned medium and survival was determined.

Results: U937 and P31/Fujioka cells (that harbor the CALM-AF10 translocation) showed reduced endocytosis of AF633-TF, but cells that lack CALM translocations (THP-1, K562, MonoMac6, HL-60) showed robust AF633-TF uptake. COS7 cells expressing MLL-CALM or CALM-AF10 showed 40–50% less endocytosis of TR-TF compared with untransfected cells or cells transfected with empty vector. CALM aa 484–620 appear to be important in inhibiting endocytosis. IL-3-dependent FL5.12 cells transfected with an MLL-CALM expression vector grew better in low concentrations of IL-3-containing WEHI conditioned media in comparison with FL5.12 cells transfected with empty vector.

Conclusions: Expression of MLL-CALM or CALM-AF10 interferes with endocytosis, and MLL-CALM expression renders FL5.12 cells less dependent on exogenous IL-3. The ability of MLL-CALM and CALM-AF10 to interfere with CDE may result in sustained growth factor signaling and contribute to the development of aggressive hematopoietic malignancies.