CALM Insufficiency Impairs Leukemia Cell Proliferation by Limiting Iron Uptake

The CALM-AF10 fusion protein that arises from the t(10;11) chromosomal translocation is found in both acute myeloid leukemia and 8–10% of T-cell acute lymphoblastic leukemia and is associated with a poor clinical outcome. The interaction of AF10 with the DOT1L histone methyltransferase has been shown to be necessary for CALM-AF10 mediated transformation. However, the role of the Clathrin Assembly Lymphoid Myeloid leukemia (CALM) protein in leukemogenesis remains uncertain. CALM plays a role in clathrin-mediated endocytosis. This process mediates the entry of various growth factor receptors and nutrients into cells and is essential for the internalization of iron-bound transferrin.

We hypothesize that loss of CALM function, as a result of CALM haploinsufficiency and/or a dominant negative CALM-AF10 effect, may contribute to transformation.

To study the effects of CALM deficiency, we used genetically modified fit1 mice that harbor a Calm gene mutation that results in a severely truncated and nonfunctional Calm protein. Fetal liver (FL) cells from Calm^−/−, Calm^+/− or Calm^WT/WT E14 embryos were retrovirally transduced with MLL-ENL or CALM-AF10 and studied in vitro or transplanted into syngeneic mice. Interleukin-3 dependent cell lines were established from Calm^−/− or Calm^WT control FL cells immortalized by CALM-AF10 or MLL-ENL.

CALM-AF10 leukemias induced in Calm^−/− or Calm^+/− precursors were considerably delayed compared with those induced in Calm^WT controls. Clathrin-mediated endocytosis of fluorescently labeled transferrin receptor (TfR) was impaired in Calm^−/− cells. This was accompanied by a 2–4 fold increase in surface expression of TfR by flow cytometry and an increase in total TfR protein as measured by Western blot in Calm^−/− cells. Calm -deficient cells also displayed lower levels of the iron storage protein ferritin, suggestive of an iron deficient state. Each of these features was reversed by re-expression of CALM via retroviral transduction of Calm^−/− cells with a CALM expression vector. Paralleling the prolonged latency of Calm^−/− leukemias, we found that Calm -deficient cells proliferated at a considerably slower rate than CALM -rescued cells. This cell proliferation defect could be rescued by supplementing the cells with iron (ferric ammonium citrate, 50 μM), indicating that reduced iron availability limits the expansion of Calm -deficient cells. Intriguingly, Calm^−/− cells were significantly more sensitive to the growth inhibitory effect of iron chelation (deferoxamine, 5 μM) than CALM -rescued cells.

These observations suggest that loss of CALM function impairs iron import and consequently limits the rate of cell proliferation. This raises the possibility that CALM haploinsufficiency present in CALM-AF10 leukemias might render cells particularly responsive to iron chelation therapy.

No relevant conflicts of interest to declare.