Treatment with an EYA2 inhibitor prolongs survival of SIX1-expressing murine T-cell leukemias Free

Background:

The CALM-AF10 (PICALM-MLLT10) fusion, found in 5-10% of pediatric T-ALLs (T-cell acute lymphoblastic leukemias), is associated with a poor prognosis. CALM-AF10 leukemias, which are dependent on the nuclear export protein XPO1, share many traits with other poor prognosis leukemias expressing translocations or rearrangements of the Lysine Methyltransferase 2A (KMT2A) gene, including aberrant activation of HOXA genes. In addition to HOXA gene activation, we previously demonstrated that CALM-AF10 also activates the homeobox gene SIX1. Together with its tyrosine phosphatase cofactor Eyes Absent 2 (EYA2), SIX1 transcriptionally activates developmental genes involved in cell proliferation and embryogenesis. SIX1 overexpression has been observed in mesenchymal and epithelial malignancies (e.g. breast, ovary, esophagus, gliomas), and SIX1 is involved in accelerating the epithelial mesenchymal transition and metastasis. The present studies evaluate the importance of SIX1 and its interaction with EYA2 in leukemogenesis, using hematopoietic stem cells and established leukemia cell lines. In addition, we assess the role of small molecule inhibitors of EYA2 in leukemia cells in vitroand in vivo.

Methods/Results:

Using the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database, which exploits a multiomic approach to evaluate profiles of multiple cancers, we established that increased SIX1 expression in relapsed T-ALL patients is associated with a worse event free survival (EFS) and overall survival (OS), while increased EYA2 expression is associated with worse EFS. We used the Broad Institute Cancer Dependency Map to identify that Jurkat leukemia cells, developed from a 14-year-old patient with T-ALL, had increased expression of SIX1, but not EYA2. RT-qPCR and immunoblot validated increased SIX1 expression. Compared to unaltered Jurkat leukemia cell lines, full CRISPR-Cas9 knockout of SIX1 abrogatedand partial knockout of SIX1 attenuated proliferation of Jurkat cells.

To gain insight into the SIX1/EYA2 interaction, we assessed the immortalization potential of fetal liver hematopoietic stem cells (FL-HPs) transduced with a wild-type SIX1 and a mutant of SIX1 unable to bind EYA2 (SIX1^EYA) through serial replating in methylcellulose colony assays. Wild-type SIX1, but not SIX1^EYA, was able to immortalize FL-HPs, indicating that EYA proteins are required for SIX1-dependent immortalization.

Since SIX1 expression is increased in Jurkat cells, and EYA2 is required for SIX1-induced immortalization of FL-HPs, we hypothesized that inhibition of EYA2 would slow the proliferation of SIX1-expressing Jurkat cells. We used two small molecule inhibitors of EYA2 phosphatase, 9987 and LG1-34 (PID:38861151); LG1-137, an analog of LG1-34 that lacks the tyrosine phosphatase activity of LG1-34, was used as a negative control. CellTiterGlo assays measuring proliferation 72 hours after treatment established that the IC₅₀ for 9987 in Jurkat cells was 33.1µM, and that the IC₅₀ of LG1-34 and LG1-137 was 0.74µM and 58.2µM respectively. LG1-34, but not LG1-137, showed a dose response effect on Jurkat leukemia cell proliferation with 99%, 68%, 41%, 20%, and 11% viability at doses of 0.1µM, 0.5µM, 2.5µM, 5.0µM, and 10µM, respectively. Intriguingly, addition of the XPO1 inhibitor KPT-330 to LG1-34 potentially enhances the inhibitory ability of LG1-34.

In vivo pharmacokinetics studies demonstrated that IV administration of LG1-34 resulted in higher plasma (2.8µM) and brain (2.8µM) concentrations compared with PO administration (1.3µM plasma and 0.8µM brain). In addition, the half-life of LG1-34 following PO administration (1.63h) is more than twice that for IV administration (0.72h). Safety studies showed no changes in weight or perturbations of CBC parameters following 28 days of LG1-34 treatment. Daily treatment of NSG mice transplanted with Jurkatleukemia cells with 100 mg/kg of LG1-34 (n=5) prolonged survival by over 10 days (p= 0.0075).Discussion:

SIX1 overexpression is associated with worse prognosis in T-ALL as well as other leukemias. As SIX1 requires EYA2 for immortalization of FL-HPs, 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 both in vitro and in vivo supports a role for SIX1/EYA2 in these leukemias.