The Kinase Inhibitor Sorafenib Can Disrupt the Survival Support Provided by the Microenvironment and Induce Apoptosis of Chronic Lymphocytic Leukemia Cells

Abstract 47

Chronic Lymphocytic Leukemia (CLL) is characterized by an accumulation of mature monoclonal B cells in the blood, secondary lymphoid tissue, and marrow. Despite their accumulation in vivo, CLL cells undergo spontaneous apoptosis in vitro unless rescued by extrinsic factors derived from the leukemia-cell microenvironment. Monocyte-derived Nurse-Like Cells (NLCs) and Marrow Stromal Cells (MSCs), representing the leukemic microenvironment, have been show to sustain CLL cell survival and more importantly to protect CLL cells from drug-induced apoptosis in vitro and possibly in vivo. Such protective niches are thought to prevent current therapies from achieving complete remission in patients. Investigating the mechanism(s) by which cells from the microenvironment promote CLL cell survival, particularly the signaling pathways triggered, will allow for the identification of new therapeutic targets aiming to disrupt these protective interactions.

NLCs and MSCs have been shown to produce the chemokine SDF-1 (CXCL12), which can enhance CLL cell survival. We recently found that ZAP-70+ aggressive CLL cells responded by an increased survival to this chemokine, compared to ZAP-70- indolent CLL cells, and that this response was accompanied by the activation of the ERK pathway. Attempting to abrogate this survival pathway, we found that sorafenib (BAY 43–9006, Nexavar) a multi-kinase inhibitor targeting among others Raf kinases and thereby the RAF/MEK/ERK pathway, strongly reduced CLL cell viability in a time and dose dependent manner. A regimen of one single dose of 10uM of sorafenib significantly reduced CLL cell viability to 18+/−10% cells after 48hrs compared to vehicle control (DMSO; 100%; n=5). The daily addition of 1uM sorafenib also significantly decreased CLL cell viability, leading to 31+/−21% and 11+/−5% viable cells after 6 and 7 days respectively, compared to DMSO (n=5). More importantly, our results show that sorafenib induces CLL cell death in the presence of NLCs and MSCs. A single dose of sorafenib (10uM) rapidly decreased the fraction of viable CLL cells overtime, passing from 40+/−16% after 1 day to 10+/−3% after 4 days (n=4) in the context of NLCs and to 25+/−3% after 2 days and 14+/−3% after 4 days in the presence of MSCs, when compared to vehicle control (>80%; n=4). In the presence of NLCs, the 1uM daily regimen also uncovered an increased sensitivity of ZAP-70+ CLL cells to this drug, reducing in 6 days their viability to 13+/−2% (n=4), which approximately half the fraction of viable cells remaining in the ZAP-70- group (40+/−16%; n=7).

We next studied sorafenib-mediated cytotoxicity by investigating its impact on the expression of pro-survival molecules. We found that Mcl-1, Bcl-2 and Bcl-x_L protein expression was reduced in CLL cells compared to vehicle control, when stimulated with CXCL12 (n=3). In the presence of NLCs and MSCs, only Mcl-1 expression was downregulated, which was also associated with a reduction of the active form of the transcription factor CREB, involved in Mcl-1 expression. Because Mcl-1 expression can be regulated by ERK and AKT pathways, we next investigated if they were abrogated by sorafenib. We indeed found that MEK, ERK, and AKT activation were reduced by this inhibitor compared to vehicle control (n=3). We therefore propose that the cytotoxic effect of sorafenib on CLL cells is due to its interference with at least these two major survival pathways.

Since sorafenib caused apoptosis of CLL cells in context of the microenvironment, we reasoned that it might also cause apoptosis of chemotherapy resistant CLL cells. To test this hypothesis, we studied cells from fludarabine-refractory patients. In the presence of NLCs, a single dose of 10uM sorafenib induced a significant reduction in CLL cell viability after 2 days: only 4+/−2% viable cells remained compared to 78+/−12% for the vehicle control (n=4). A comparable observation was made in the presence of MSCs: sorafenib potently induced apoptosis, leaving 12+/−3% live cells after 2 days, compared to vehicle control (71+/−16%; n=4). These results are very promising as they suggest that sorafenib could be an effective novel therapeutic for CLL, affecting the viability of the leukemic cells even in protective niches. Since sorafenib has been approved by the FDA in 2007 for the treatment of advanced hepatocellular carcinoma, a pilot study is currently being planned at UCSD to evaluate the potential of this drug in CLL in vivo.