Abstract 1649: Pim kinase inhibitor, SGI-1776, induces apoptosis in acute myeloid leukemia primary cells

Abstract

Abstract Small molecule SGI-1776 was found to be potent and selective inhibitor of Pim family serine-threonine kinases [IC50: Pim-1 (7 nM), Pim-2 (363 nM) and Pim-3 (69 nM)] without effects on other kinases except Flt-3 and haspin. In preclinical studies in chronic lymphocytic leukemia, SGI-1776 treatment resulted in apoptosis induction that was associated with a decrease in RNA synthesis, Mcl-1 protein level, and c-Myc phosphorylation (Chen, LS et al, Blood 2009, 114: 4150). Increased levels of Pim-2 are reported in acute myeloid leukemia (AML) as well as Flt-3 activating mutation, providing a rationale to test SGI-1776 in AML. We evaluated SGI-1776 using FLT3-ITD AML cell lines (MOLM-13, MV4-11), xenograft models, and in primary cells from AML patients, and our results indicate that SGI-1776 is effective in AML. In MOLM-13 and MV4-11 cell lines, the IC50 for viability was < 10 nM after treatment with SGI-1776 for 72 h, which was associated apoptosis induction and reduction in phosphoBad (Ser112) and Bad protein. In xenograft models of both cell lines, treatment with 135mg/kg SGI-1776 resulted in a consistent decline in tumor volume after 14 days. To further these investigations in primary cells, we obtained cells from AML patients including FLT-3 positive and mutated FLT3-ITD patients, and evaluated the potential for SGI-1776 to induce cell death. In vitro incubation of AML cells (n = 4) with 1, 3, and 10 μM SGI-1776 for 24 h resulted in an average increase in apoptosis of 5%,12% and 19% respectively, measured using Annexin-staining compared with untreated cells. Apoptosis induction was verified via PARP cleavage immunoblot, and apoptosis was observed in cells from FLT3 negative or mutated FLT3-ITD patients. In the samples evaluated, there was disparity in cytogenetic factors, Flt-3 status and prior chemotherapeutic treatment history. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. There was no observed change in phosphorylation of Pim targets histone H3 (Ser10) or p21 (Thr145), however protein levels of both total and phospho-Bad(Ser112) decreased following SGI-1776 treatment. In contrast, there was no change in anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1, and levels of pro-apoptotic proteins, Bak and Bax, were also unaffected. However, there was a decrease in phospho-c-Myc(Ser 62), which is a Pim target and associated with stabilization of c-Myc protein. Consequently, we evaluated the potential inhibition of c-Myc driven transcription by SGI-1776 by measuring total RNA synthesis. Following treatment with 1, 3 or 10 μM SGI-1776, there was a decrease in total RNA synthesis to 75%, 42% and 25% of control respectively, measured using a radioactive uridine incorporation assay (n = 4). Taken together, SGI-1776 was demonstrated to be an effective agent against AML in cell lines, xenograft models, and primary cells, and may be an effective new agent in the treatment of AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1649.