Establishing the Role of EPS15, DVL2 and Cortactin in CALM-AF10 Leukemogenesis
Authors: Kazi Rafi , Aumann Waitman Kurt , Bagchi Pritha , Tope Donald , Wechsler Daniel S. 
Published: November 2021

Abstract
Background:
Leukemia is the most common type of childhood cancer. Although the prognosis for many pediatric leukemias has improved, leukemias associated with the t(10;11) CALM-AF10 translocation remain difficult to treat. CALM-AF10 leukemias account for ~5-10% of childhood T-cell acute lymphoblastic leukemia (T-ALL)as well as a subset of acute myeloid leukemia (AML). CALM-AF10 leukemias exhibit increased expression of proleukemic HOXA genes, but relatively little is known about the cellular mechanisms that drive CALM-AF10 leukemogenesis. Our laboratory has demonstrated that the CALM protein contains a nuclear export signal (NES) that is critical for CALM-AF10-dependent leukemogenesis. The NES interacts with the CRM1/XPO1 nuclear export receptor, which shuttles proteins from the nucleus to the cytoplasm through the nuclear pore complex. We have shown that transcriptional activation of HOXA genes by CALM-AF10 is dependent on its interaction with CRM1. Importantly, CRM1 does not contain a recognized DNA binding domain, and it is not currently understood how the CALM-AF10/CRM1 complex interacts with regulatory regions of HOXA genes. To identify proteins that mediate the interaction between the CALM-AF10/CRM1 complex and DNA, we took advantage of a proximity-based labeling approach using BioID2, a second-generation biotin ligase. When fused to a protein of interest and in the presence of biotin, BioID2 biotinylates proteins in close proximity to the ligase. These biotinylated proteins can then be identified by mass spectrometry (MS).
Methods:
We prepared an expression plasmid in which BioID2 was cloned in-frame with CALM-AF10. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with BioID2-CALM-AF10 and grown in the presence or absence of biotin. MS was performed to identify candidate interacting proteins. We validated direct interactions of candidate proteins with CALM-AF10 using co-immunoprecipitation experiments in HEK293 cells transfected with a CALM-AF10 plasmid. We confirmed that candidate proteins are present in murine CALM-AF10 leukemia cells via Western blotting. In order to efficiently knockout (KO) candidate proteins, we have generated a human U937 cell line (which harbors a t(10;11) CALM-AF10 translocation) with a stable incorporated Cas9. To assess whether KO of EPS15, DVL2 or CTTN affects HOXA5 expression, we performed RT-qPCR in U937-Cas9 cells lines with confirmed KO.
Results:
We carried out three independent transfections/MS experiments, which identified 71, 95 and 61 proteins, respectively. Of the proteins identified, 12 candidates were common to all three experiments . Importantly, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), a protein known to interact with AF10, and Nuclear pore complex protein 214 (NUP214), a protein that interacts with CRM1 and that is involved in leukemogenic translocations. We chose EPS15, DVL2 and CTTN for further study, as each of these proteins plays a role in leukemogenesis. We performed initial validation of direct interactions via co-immunoprecipitation and found that all three proteins co-precipitate with CALM-AF10. Western blotting showed that all three proteins are expressed in a murine CALM-AF10 leukemia cell line. We effectively knocked out EPS15 protein expression in U937 cells, and showed that HOXA5 expression is reduced in the setting of EPS15 knockout.
Conclusion:
We used biotin ligase-dependent proximity-based labeling to identify candidate proteins that potentially interact with the CALM-AF10 fusion protein. Our identification of DOT1L validates the approach, since DOT1L is known to interact with CALM-AF10. We have started to investigate three candidate proteins - EPS15, DVL2 and CTTN - all of which are involved in leukemogenic transformation. We have shown that EPS15, DVL2 and CTTN are expressed in murine CALM-AF10 leukemia cells and directly interact with the CALM-AF10 fusion protein. Knockout of EPS15 in U937 cells results in decreased HOXA5 expression, suggesting the importance of EPS15 in CALM-AF10 leukemogenesis. Evaluation of the roles of these proteins in leukemogenesis may lead to identification of novel pathways involved in CALM-AF10 leukemogenesis.

Disclosures
No relevant conflicts of interest to declare.