Small Molecule Inhibitors of EYA2 Slow Proliferation of SIX1-Expressing Pediatric Leukemias

Background: SIX1 is a homeobox gene that is involved in cell proliferation, embryogenesis, and apoptosis. Together with its cofactor Eyes Absent 2 (EYA2), a protein phosphatase, SIX1 transcriptionally activates developmental genes, and is expressed at low levels post-embryologically. Overexpression of SIX1 has been observed in mesenchymal and epithelial malignancies including breast cancer, ovarian cancer, gliomas and esophageal cancer, and SIX1 is involved in accelerating the epithelial mesenchymal transition (EMT) and metastasis. SIX1 has also been implicated in MLL/KMT2A leukemias, although its precise leukemogenic role has not been defined. We have previously demonstrated that SIX1 expression is increased in CALM-AF10 leukemia cells. The present studies evaluate the importance of SIX1 and its interaction with EYA2 in leukemogenesis, using hematopoietic stem cells (HSC) and established leukemia cell lines. In addition, we assess the role of small molecule inhibitors of EYA2 in leukemia cells in vitro, and establish pharmacokinetic parameters in mice in vivo.

Methods/Results: We used the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database, which exploits a multiomic approach to evaluate profiles of multiple cancers, to determine that increased SIX1 expression is associated with worse event free survival (EFS) and overall survival (OS) in relapsed T-cell Acute Lymphoblastic Leukemia (T-ALL), Ambiguous Lineage Acute Leukemias (ALAL), and Acute Myeloid Leukemias (AML). While increased EYA2 expression is associated with worse EFS in T-ALL patients, it correlates with improved OS in B-ALL, ALAL, and AML patients. To evaluate whether established leukemia cell lines display increased SIX1 or EYA2 expression, we queried the Broad Institute Cancer Dependency Map. We identified increased SIX1 expression in Jurkat (T-ALL), SHI-1 (AML), and OCIM2 (AML) leukemia lines, though none had increased expression of EYA2. SIX1 expression in these cells was validated by immunoblot, and shRNA knockdown of SIX1 reduced proliferation of all three cell lines.

To determine the impact of SIX1 and its interaction with EYA2 on HSC immortalization, we overexpressed SIX1 in HSCs. Wild-type SIX1 immortalizes HSCs, while SIX1 bearing a mutation in its EYA binding domain does not immortalize, indicating that EYA proteins are required for SIX1-dependent immortalization. Using co-immunoprecipitation in SIX1-overexpressing leukemia cell lines, we confirmed that SIX1 and EYA2 directly interact. To interfere with SIX1 activity, we used two small molecule inhibitors of EYA2 phosphatase (9987 and the recently described LG1-34 (PID: 38861151)); LG1-137, an analog of LG1-34 that has decreased binding affinity for the EYA2 phosphatase, was used as a negative control. Treatment of two murine CALM-AF10 leukemia lines as well as the human leukemia cell lines Jurkat, SHI-1, and OCI-M2 with 9987 impaired proliferation at doses ranging from 20-40 μM. LG1-34, a more potent version of 9987, also impaired proliferation of all three cell lines at doses ranging from 0.1-5 μM. Importantly, LG1-137 had no effect on these cells, with IC₅₀ concentrations 30-90 fold higher than LG1-34. Preliminary in vivo pharmacokinetics studies demonstrated that IV administration leads to higher concentration in plasma (2.8 μM) and brain (2.8 μM) compared with PO administration (1.3 μM for plasma and 0.8 μM for brain), while the half-life of LG1-34 following PO administration (1.63 h) is more than twice that for IV administration (0.72 h).

Discussion: Overexpression of SIX1 is associated with worse prognosis in T-ALL, ALAL and AML. Since SIX1 requires EYA2 for immortalization of HSCs, we assessed the efficacy of available small molecule inhibitors of EYA2 to interfere with proliferation of SIX1-expressing leukemia cell lines. The ability of 9987 and LG1-34 to impair proliferation of SIX1-expressing leukemias supports a role for SIX1/EYA2 in these leukemias. These studies provide a framework for evaluating the efficacy of EYA2 inhibitors in murine leukemias as well as PDX models of leukemia. Future studies will assess the effect of EYA2 overexpression on immortalization in vitro, and will evaluate the effectiveness of EYA2 inhibitors in PDX and murine leukemia models.

No relevant conflicts of interest to declare.