We report investigational studies of a Pim-1 kinase inhibitor, SGI-1776, as a therapeutic agent for the treatment of chronic lymphocytic leukemia (CLL). Pim family proteins are serine/threonine kinase inhibitors of apoptosis, and elevated expression of Pim kinases have been detected in hematological malignancies and in certain solid tumors. Three Pim kinases have been identified to date, Pim-1, -2 and -3. Pim-1 has been shown to synergize with c-Myc in tumorigenesis, and increased expression of the human Pim-2 gene is observed in CLL and non-Hodgkin lymphomas. Specifically, Pim-1 and Pim-2 have been shown to be required for efficient pre–B-cell transformation by v-Abl oncogene. Small molecule SGI-1776 was evaluated using Ambit Biosciences’ KINOMEscan and was found to have IC50s in the nanomolar range for Pim-1, Pim-2, and Pim-3 using the Millipore IC50 Profiler. SGI-1776 was screened against a panel of kinases utilizing radiolabeled biochemical assays, and was found to be highly selective for Pim kinases without any effects on CDKs, Chk1, IKK, JNK, Abl, Raf, MAP kinases and protein kinase A and B. Specifically, the IC50 were measured as follows: Pim-1 (7 nM), Pim-2 (363 nM) and Pim-3 (69 nM.) The other two enzymes affected at nM concentration of SGI-1776 were Flt-2 and haspin. Using primary samples obtained from patients with CLL, we evaluated the potential for SGI-1776 to induce cell death. In vitro incubation of primary CLL cells (n=7), with 1, 3, and 10 μM SGI-1776 for 24 h resulted in an average increase in apoptosis of 10%, 22% and 38% respectively, compared with untreated cells. SGI-1776-induced apoptosis was observed in heterogeneous patient populations, and there was disparity in the expression levels of traditional CLL prognostic markers including ZAP-70, b2-microglobulin, IgVH mutation status, Rai stage and number of prior treatments. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. Phosphorylation of traditional Pim-1 kinase targets, phospho-Bad (Ser112) and histone H3 (Ser10) were not affected by SGI-1776 treatment in CLL, unlike in replicating cell types, suggesting an alternative mechanism in CLL. We then evaluated the potential inhibition of c-Myc driven transcription by measuring total RNA synthesis. Following treatment with 3 or 10 μM SGI-1776, there was a decrease in total RNA synthesis to approximately 50% of control, measured using a radioactive uridine incorporation assay (n = 3). There was also a reduction in Mcl-1 and c-Myc protein level, both of which have short transcript half-lives. The reduction in Mcl-1 protein was not due to apoptosis or cleavage by caspases, since Mcl-1 reduction occurred in the presence of caspase inhibitor ZVAD, nor was there an increase in cleaved Mcl-1. In contrast, there was no change in anti-apoptotic proteins Bcl-2, XIAP, survivin, stabilization of p53 or p21. Taken together, SGI-1776 consistently induced apoptosis in CLL cells. Although its mechanism of action is not fully elucidated, inhibition of RNA synthesis and reduction of Mcl-1 and c-Myc protein levels are associated with SGI-1776-induced apoptosis.

Disclosures: Redkar:SuperGen In.c: Employment, Equity Ownership. Bearss:SuperGen Inc.: Employment, Equity Ownership.

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