Biological Effects of the Pim Kinase Inhibitor, SGI-1776, in Multiple Myeloma

Abstract

Pim kinases are constitutively active serine/threonine/tyrosine kinases that are overexpressed in hematological malignancies such as multiple myeloma. Pim kinase substrates are involved in transcription, protein translation, cell proliferation, and apoptosis. SGI-1776 is a potent Pim kinase inhibitor that has proven to be cytotoxic to leukemia and lymphoma cells. Based on this background, we hypothesized that SGI-1776 treatment would result in myeloma cytotoxicity. To test this, myeloma cell lines and primary CD138+ cells from myeloma patients were treated with SGI-1776 in a dose- and time-dependent manner and effect on cell death and proliferation, induction of autophagy, as wells as changes in cell cycle profile were measured. SGI-1776 treatment resulted in limited apoptosis in cell lines (mean 30%) and CD138+ cells (<10%) as assessed by Annexin-V/PI. Limited effect was observed in cell cycle profile or growth in cell lines. However, DNA synthesis was decreased by 70% at 3 μM (all time points) in U266 though this was not observed in MM.1S. In accordance, immunoblot analyses revealed no change in transcription (c-Myc and H3), or apoptotic (Bad) proteins that are substrates of Pim kinases. In contrast, autophagy, as assessed by acridine orange staining, was induced with SGI-1776 treatment in both cell lines (U266 25-70%; MM.1S 8-52%) and CD138+ cells (19-21%). Immunoblot analyses of autophagy LC3b marker and translation initiation proteins (phospho p70S6K and 4E-BP1) corroborated autophagy induction. These data indicate that SGI-1776 treatment in myeloma cell lines and CD138+ myeloma cells elicits its deleterious effects through inhibition of translation and induction of autophagy.

Flow sorting and exome sequencing reveal the oncogenome of primary Hodgkin and Reed-Sternberg cells

“…PIM kinases are overexpressed in chronic lymphocytic leukemia, mantle cell lymphoma, and multiple myeloma; and in vitro inhibition results in cellular toxicities. 41,42,45 Inhibitors of WEE1 enhance killing of Burkitt lymphoma cells.…”

Protein profiling identifies mTOR pathway modulation and cytostatic effects of Pim kinase inhibitor, AZD1208, in acute myeloid leukemia

“…The first Pim kinase inhibitor to enter clinical trial was SGI-1776 for refractory prostate cancer and relapsed/refractory non-Hodgkin's lymphoma (http://clinicaltrials.gov/ct2/show/NCT00848601). Induction of apoptosis by SGI-1776 was demonstrated by our group in cell lines and in primary cells from patients with chronic lymphocytic leukemia (CLL)[9], AML, mantle cell lymphoma and multiple myeloma[10–12]. SGI-1776 treatment in cell lines has also been shown to reduce the expression of cell surface drug resistance proteins[13].…”

Combination of Pim kinase inhibitors and Bcl-2 antagonists in chronic lymphocytic leukemia cells

“…The lysate protein content was measured using a DC protein assay kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Aliquots (30–50 μg) of total protein were loaded onto 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (GE Osmonics Labstore, Minnetonka, MN) as previously described [11]. The membranes were blocked at room temperature for 1 h in Odyssey blocking buffer (LI-COR Inc., Lincoln, NE) and then incubated overnight at 4°C with the following primary antibodies: Bcl-2 (Dako, Carpinteria, CA), Mcl-1, Bcl-X L (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-4E-BP1 (Thr 37/46), total 4E-BP1, phospho-p70S6K (Thr 389), or GAPDH (Cell Signaling Technology, Danvers, MA), and PARP (BD Pharmingen).…”

“…These include acute myelogenous leukemia [3], chronic lymphocytic leukemia (CLL) [4–6], lymphomas [7–9], and multiple myeloma [10, 11]. Additionally, the expression of Pim kinase proteins is much higher in CLL cells than in normal lymphocytes [6].…”