Abstract 3192: Marrow stromal cell enhanced survival of CLL cell is mediated through activation of cellular transcriptional machinery
Authors: Kumudha Balakrishnan, Jan Burger, William Wierda, Varsha Gandhi
Journal: Cancer Research (2010)

Abstract Chronic lymphocytic leukemia (CLL) has been considered a non-proliferating disease and clonal expansion of CD5+ B cells is due to intrinsic resistance to apoptosis. However recent studies elucidate that CLL is a dynamic disease, and cell survival and maintenance are due to extrinsic microenvironment that influence apoptotic-resistance. Lines of evidences show that CLL primary cells when cultured in vitro undergo spontaneous apoptosis, but when co-cultured with bone marrow stromal cells (MSC), there was significant inhibition of apoptosis. Using human MSC (NKTert) as a representative bone marrow stromal microenvironment we examine the possible mechanisms involved in the stabilization of CLL cells. In vitro incubation of primary samples resulted in rapid cell loss by 48 hr (0 hr - 1*107; 48 hr- median 9*106; range 7.7*106 - 9.3*106; n=7). Co-culturing CLL cells with MSC enhanced the cell survival measured by DiOC6 (median viability 70% without and 90% with NKTert; 48 hr; p=0.001) but did not increase the cell number (median 9.5*106 range 8.0*106 - 9.8*106; n=7; p=0.5840), suggesting that MSC does not induce proliferation of CLL cells in vitro. MSC induced a rapid induction of global RNA synthesis measured by uridine incorporation method (median 248% of control; range 109-1107%; n=6). Compared to RNA synthesis there was minor change in thymidine (median 79%; range 29-160%) and leucine incorporation (median 84%; range 25-304%). The elevation in RNA synthesis was associated with an increase in phosphorylation of RNA Pol II at ser2 position without a change in total enzyme level, thus contributing to the activation of cellular transcriptional machinery. The profound increase in the regulatory complex of transcription promoted increase in transcripts (measured by real time RT-PCR) of essential signaling molecules such as MAP Kinase (median 158% of control; range 82-177%) and AKT (median 157% of control; range 71-281%; n=7) and anti-apoptotic molecules such as Mcl-1 (median 262% of control; range 110-347%; n=7) and Bcl-2 (median 156% of control; range 90-173%; n=7). Consistence with the increase in transcripts, there was an induction of corresponding proteins such as Mcl-1 (median 134% of control; range 109-554%; n=8) (but not Bcl-2 and Bcl-xL) and activation of p-Erk at Thr202/Tyr204 (median 236% of control; range 89-648; n=8) and p-Akt at Ser-473 position (median −156% of control; range 90-552%; n=8). Furthermore, our results demonstrated that induction of Mcl-1 was in relation with alteration in GSK-3β activity. MSC induced phosphorylation of GSK-3β at ser9 position (median 137% of control; range 85-415%; n=8) thus stabilized the Mcl-1 protein levels. Taken together, our results demonstrate that transcription activation followed by interplay between signaling molecules of survival pathway and the downstream anti-apoptotic proteins may enhance the survival of CLL cells in presence of MSCs. 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 3192.