The SIX1 homeobox Gene Is a Novel Therapeutic Target in CALM-AF10 Leukemogenesis

Background: The CALM-AF10 translocation is found 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we hypothesized that identification of non-HOXA CALM-AF10 effector genes could potentially yield novel therapeutic targets. To discover novel CALM-AF10-regulated genes, we took advantage of our prior observation that the nuclear export factor CRM1/XPO1 tethers CALM-AF10 to HOXA genes by interacting with a nuclear export signal within CALM. Using microarrays, we identified a set of genes that showed decreased expression in response to the CRM1 inhibitor, Leptomycin B (LMB), similar to Hoxa genes, in murine CALM-AF10 leukemia cells. Then using RNA-sequencing, we discovered a set of genes increased in murine hematopoietic stem cells transduced with CALM-AF10. There were 11 genes that were both decreased in response to LMB and increased in response to CALM-AF10, which included the Hoxa gene cluster, as well as Six1. Similar to HOXA genes, SIX1 is a homeobox gene that is associated with embryogenesis and is quiescent post-embryologically. Additionally, SIX1 and its cofactor EYA2 have been found to be overexpressed in numerous solid tumors, and inhibitor of the SIX1/EYA2 complex has recently been described. While there is evidence of a role for SIX1 in solid tumors, its role in leukemias has not been explored. Objective: To evaluate the role of SIX1 in CALM-AF10 leukemias. Design/Methods: RT-qPCR and Chromatin Immunoprecipitation (ChIP) were performed using bone marrow progenitors transduced with CALM-AF10 or an empty vector, with and without LMB. Methylcellulose colony assays assessed the ability of SIX1 to enhance self-renewal of hematopoietic progenitors. An inhibitor of the Six1/Eya2 interaction (compound 8430) was used to evaluate cell proliferation. Downstream targets of Six1 were evaluated using RT-qPCR in CALM-AF10 cells treated with Six1/Eya2 inhibitor (8430). Results: RT-qPCR confirmed overexpression of SIX1 in CALM-AF10 leukemia cells, and showed decreased SIX1 expression in the presence of LMB. Furthermore, ChIP revealed that CALM-AF10 binds to the SIX1 gene locus. Overexpression of SIX1 in fetal liver progenitors was sufficient to increase self-renewal potential. The 8430 Six1/Eya2 inhibitor slowed cell growth in CALM-AF10 cells compared to cells treated with DMSO alone. Fi