Expression of Alternative CALM-AF10 Fusions Causes Different Hematological Malignancies in Mice.
Authors: Stoddart Angela , Fernald Anthony A. , Tennant Thelma R. , Davis Elizabeth M. , Anastasi John , Le Beau Michelle M. 
Published: April 2020

Abstract
The clathrin assembly lymphoid myeloid leukemia gene, CALM, was first described as a translocation partner for AF10 in morphologically distinct subsets of acute leukemia with a recurring 10;11 translocation. As the name implies, CALM is involved in the regulation of clathrin-mediated endocytosis. We hypothesize that expression of the CALM-AF10 protein disrupts endocytosis of growth receptors, leading to constitutive activation and, thereby, contributes to the development of leukemia. The CALM-AF10 fusion transcripts are characterized by two breakpoints in CALM at positions 1926 and 2091. Using the 293T human renal epithelial cell line as a model, we demonstrated that CALM1926-AF10 localized diffusely to the cytoplasm, whereas CALM2091-AF10 had a more prominent punctate appearance. This difference in distribution was likely attributable to protein domains in CALM rather than AF10, which is a transcription factor that localizes to the nucleus. The difference in localization extended to their effects on endocytosis. Whereas ∼4–8% of cells expressing truncated CALM2091, CALM1926, or AF10 displayed impaired transferrin uptake, this value was ∼60% in cells expressing CALM2091-AF10 and only ∼13% in cells expressing CALM1926-AF10. Next, we used a retroviral transduction-transplantation murine model to test the impact of CALM-AF10 expression on the development of leukemia. Mice transplanted with either CALM1926-AF10 or CALM2091-AF10 transduced fetal liver cells developed disease within a short period of time (∼8 to 21 weeks post-transplantation). Interestingly, expression of CALM1926-AF10 led to the development of a myeloproliferative disease (MPD)-like leukemia, with a predominance of mature neutrophils, whereas expression of CALM2091-AF10 led to the development of an acute myeloid leukemia (AML) with &gt;30% immature myeloid blasts, with one exception. One mouse transplanted with progenitor cells expressing CALM2091-AF10 developed a MPD accompanied by a granulocytic sarcoma; however, examination of RNA from the spleen of this diseased mouse revealed that it had undergone a splicing event consistent with the size expected for the 1926 breakpoint. Both diseases were transplantable to secondary recipients confirming the leukemic nature of the CALM-AF10 expressing cells. Previous studies show that patients with the t(10;11) (p13;q14-21) develop either AML or T cell-acute lymphoblastic leukemia (T-ALL). Most of the patients with T-ALL express the CALM1926-AF10 fusion. Although there are differences in presentation between humans and mice, the hematological disease may be a reflection of the conditions of the mouse model. To identify possible secondary cooperating mutations, spectral karyotyping analysis of the mouse AMLs and MPDs was performed. However, to date we have not identified any significant abnormalities. We hypothesize that defective endocytosis synergizes with transcriptional deregulation, leading to an aggressive form of leukemia.