A novel role for SIX1 and EYA2 and a novel EYA2 inhibitor in myeloid leukemogenesis Free

Background:

Patients with Acute Myeloid Leukemias (AML) harboring MLLT10 (AF10) fusion oncogenes have a 5-year survival rates of <10%. The PICALM-MLLT10 (CALM-AF10) fusion is found in 1-2% of AMLs and is associated with a poor prognosis. These leukemiasharbor increased HOXA gene expression, similar to leukemias with Lysine Methyltransferase 2A (KMT2A) gene rearrangements. We have recently shown that PICALM-MLLT10 activates the homeobox gene SIX1 in addition to HOXA genes. SIX1, together with its cofactor Eyes Absent 2 (EYA2), a protein tyrosine phosphatase, is involved in cell proliferation and embryogenesis and transcriptionally activates developmental genes. Overexpression of SIX1 has been observed in mesenchymal and epithelial malignancies including breast, ovarian, and esophageal cancers, in addition to being involved in accelerating the epithelial mesenchymal transition (EMT) and metastasis. The present studies evaluate the importance of SIX1 and its interaction with EYA2 in leukemogenesis, as well as the use of small molecule inhibitor in both in vitro and in vivo studies.

Methods/Results:

We first determined that increased SIX1 expression is associated with worse event free survival (EFS) and overall survival, while EYA2 correlates solely with EFS using the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database, which exploits a multiomic approach to evaluate profiles of multiple cancers. We next queried the Broad Institute Cancer Dependency Map and identified increased SIX1 expression in SHI-1 and OCI-M2 AML cell lines; neither displayed increased EYA2 expression. SIX1 expression in these cells was validated by immunoblot, and shRNA knockdown of SIX1 reduced proliferation in both cell lines. Overexpression of wild-type SIX1 in hematopoietic stem cells (HSCs) resulted in immortalization (tertiary colony formation in methylcellulose), but overexpression of a SIX1 mutant that is unable to bind EYA2 did not result in immortalization, suggesting that EYA proteins are required for SIX1 activity.

Based on these observations, we evaluated the effects of EYA2 inhibition by a small molecule inhibitor of the EYA2 protein tyrosine phosphatase (LG1-34) along with an inactive analog (LG1-137) as a negative control (PID:38861151). CellTiterGlo assays measuring proliferation 72 hours after treatment established that the IC₅₀ of LG1-34 for SHI-1 and OCI-M2 was 0.952 µM and 58.2 µM respectively, both showing over 10-fold increase in IC₅₀ for the inactive compound LG1-137. LG1-34, but not LG1-137, showed a dose response effect on SHI-1 cell proliferation with 91%, 68%, 46%, 3%, and 2% viability at doses of 0.1 µM, 0.5 µM, 2.5 µM, 5 µM, and 10 µM, respectively. A similar, but not as robust, dose response effect was seen in OCI-M2 cells, with cell proliferation 99%, 68%, 41%, 20%, and 11% viability at doses of 0.1 µM, 0.5 µM, 2.5 µM, 5 µM, and 10 µM, respectively, corresponding with the IC₅₀values obtained.

IV administration of LG1-34 yielded a half-life of LG1-34 following PO administration (1.63 h) is more than twice that for IV administration (0.72 h), while higher plasma (2.8 µM) and brain (2.8 µM) concentrations were obtained with IV compared with PO administration (1.3 µM plasma and 0.8 µM brain). Daily treatment with LG1-34 for 28 d showed no showed no changes in weight or perturbations of CBC parameters. Daily treatment of NSG mice transplanted with SHI-1leukemia cells with 100 mg/kg of LG1-34 (n=5) prolonged survival by over 10 days (p= 0.0254).

Discussion:

AMLs harboring a MLLT10 translocation are associated with a poor prognosis. We have shown that a PICALM-MLLT10 fusion expresses the homeobox gene SIX1, which is also associated with worse outcomes. We have demonstrated that SIX1 requires EYA2 for immortalization of HSCs, and that treatment with a small molecule inhibitor of EYA2 reduces proliferation of AML cells in vitro and prolongs survival of leukemia-bearing mice in vivo. The results suggest an important role for SIX1/EYA2 in myeloid leukemogenesis, and the potential for EYA2 inhibition as a novel therapeutic modality.