Abstract 516: Marrow stromal cell rendered stability to chronic lymphocytic leukemia cells is partly mediated by Pim kinase

Abstract

Abstract Chronic lymphocytic leukemia (CLL) was thought to arise due to dysregulated apoptosis rather than uncontrolled proliferation. Recent reports reveal that at least 1% of CLL cells undergo replication in marrow and the accumulation is not merely due to intrinsic defect in apoptosis but in part due to extrinsic signals delivered by microenvironment. Although several entities of marrow stromal cell (MSC) microenvironment have been elucidated, the critical elements involved in CLL survival remain obscure. Pim proteins are constitutively active Ser/Thr kinases and to date Pim-1, -2, -3 have been identified. Their activity on target proteins influences a number of cellular processes such as transcription, translation, proliferation and survival. Previously, we elucidated that Pim kinases are highly expressed in CLL cells compared to normal lymphocytes [Blood 114:4150, 2009]. Based on this background, we hypothesized that Pim and their substrates may essentially contribute to MSC-driven CLL cell survival. To test this, we co-cultured CLL lymphocytes with NKTert stromal cells (a human MSC line) that mimic marrow microenvironment. Evaluation of Pim in CLL cells by immunoblotting (n=10) revealed that Pim-1 and -2 expression increased with supporting MSC, with more pronounced increase in Pim-2. A similar trend was observed in total and phospho cMyc (Ser62, n=8) suggesting that Pim is a co-activator of MYC and MYC is a Pim-driven transcription factor, and their levels largely correlate. This is consistent with the observed increase in the global RNA synthesis (uridine incorporation) in CLL cells co-cultured with MSC. Histone H3, a transcriptional activator and a Pim target, is important for MYC driven transcription. In CLL cells co-cultured with NKTert cells, phospho Histone H3 (Ser10) diminished significantly starting as early as 1 hr (n=10). The mechanism underlying the decrease and changes at the early time points are under investigation. In conjunction with transcription, translation is also governed by Pim kinases through phosphorylation of translation suppressor protein 4E-BP1. In this regard, our preliminary data shows that in CLL cells 4E-BP1 phosphorylation status was not affected by MSC. This is in concordance with the steady-state levels of total protein synthesis (leucine incorporation) in presence of stromal support. CLL cells remained replicationally quiescent on MSC (Ki67 staining) and hence cell cycle substrates of Pim (p21, p27, cdc25a and c) were not analyzed. In terms of survival (annexin/PI binding), the viability of CLL cells on MSC increased by 10-25% at day 4, 5, 6 of incubation (n=4, 6, 5, respectively). Consistent with this data, in CLL cells Bad phosphorylation (Ser112), a Pim substrate, was elevated with stromal support. Taken together, we demonstrate that MSC-rendered stability to CLL cells by Pim kinases is in part mediated by increased transcription and disrupted apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 516. doi:10.1158/1538-7445.AM2011-516