The PI3K Inhibitor ON 01910.Na Inhibits Critical Survival Pathways and Induces Apoptosis in CLL Cells through Induction of NOXA and BIM.

Abstract 412

Chronic Lymphocytic leukemia (CLL), the most common leukemia in Western countries, is a clinically heterogeneous disease characterized by the accumulation of mature B lymphocytes. Disease progression is driven by cell survival and proliferation signals provided by the tumor microenvironment through different receptors including the B-cell receptor, TNF, chemokine and cytokine receptors, and through other cell-cell interactions. ON 01910.Na (Onconova, Therapeutics) belongs to the family of styryl benzyl sulfones, a novel family of non-ATP competitive compounds that are currently under development as potential anticancer treatments. These compounds have shown activity in a variety of xenograft mouse models. Interestingly, it has recently been described that ON 01910.Na exerts potent antitumor activity against Mantle Cell Lymphoma (MCL) cells by inhibition of PI-3K/Akt/mTOR pathway and downregulation of Cyclin D1 translation. Here, we evaluated the cytotoxic effect of ON 01910.Na after 48 hours of in vitro exposure on PBMCs from CLL and normal donors by flow cytometry using AnnexinV-PE. We co-stained with CD3-APC and CD19-FITC to assess the effect on B and T cells separately. ON 01910.Na induced apoptosis of the leukemic cells in all CLL samples tested (n=24), without affecting T-cell viability. Notably, ON 01910.Na did not affect B and T cells from normal donors at concentrations that induced apoptosis of CLL cells. Thirteen CLL samples were highly sensitive towards ON 01910.Na (mean B-cell IC₅₀ 0.71 μM) a concentration readily achieved in phase I clinical trials, eight samples were sensitive (mean B-cell IC₅₀ 1.38 μM) and three showed moderate sensitivity (mean B-cell IC₅₀ 4.26 μM). In contrast, the IC₅₀ of T-cells was >8 μM in all samples. No significant differences were found in ON 01910.Na cytotoxicity against CLL expressing mutated (n=9, mean B-cell IC₅₀ 1.45 μM) or unmutated (n=15, mean B-cell IC₅₀ 1.34μM) IgVH sequences. ON 01910.Na was also effective against tumor cells having 17p (n=4, mean B-cell IC₅₀ 1.45 μM) or 11q deletions (n=3, mean B-cell IC₅₀ 0.96 μM). ON 01910.Na activated the mitochondrial apoptotic pathway, as shown by flow cytometry using conformation specific antibodies to detect activation of Bax and Bak, leading to mitochondrial depolarization (measured by mitotracker) and caspase-3 activation. ON 01910.Na also induced the upregulation of the proapoptotic BH3-only proteins Noxa and Bim and decreased expression of Mcl-1 at 4 hours, well before the onset of apoptosis. Inhibition of Noxa expression by retroviral transfection reduced the sensitivity to drug by 60%. ON 01910.Na induced reactive oxygen species (ROS) that may contribute to Noxa induction. Consistently ROS blockade using N-acetyl-cysteine reduced ON 01910.Na cytotoxic activity. To further delineate the biological processes underlying ON 01910.Na induced apoptosis, we performed Gene Expression Profiling (GEP) in CLL cells treated in vitro for 4 and 10 hours with ON 01910.Na. GEP revealed the induction of Noxa and ATF3, consistent with an oxidative stress response. Noteworthy, Gene Set Enrichment Analysis (GSEA) of microarray data showed an induction of an AP-1 gene signature, (False Discovery Rate (FDR) = 0.10), validated by the nuclear accumulation of c-jun at 4 hours and a FOXO gene signature (FDR=0.13) that correlated with the upregulation of the proapoptotic BH3-only protein Bim. Strikingly, ON 01910.Na repressed B-cell receptor (FDR=0.023), NF-kB (FDR=0.084) and PI3K gene signatures. Consistent with inhibition of the BCR/PI3K/AKT axis, ON 01910.Na inhibited AKT phosphorylation by 65% after in vitro BCR activation of CLL cells. Our results identify ON 01910.Na as a promising agent in the treatment of CLL with an interesting dual mechanism of action: activation of apoptotic stress signals leading to Noxa and BIM up-regulation, combined with inhibition of the BCR/PI3K/AKT pathway that can block microenvironment-induced survival and proliferation signals. These data support the development of ON 01910.Na in CLL and a clinical trial has been initiated at our institution.