Abstract
Inhibition of the autophagic pathway has recently revealed promising results in increasing pro-death activity of multiple cancer therapeutics. Here, we discuss our findings regarding the autophagy-blocking and anti-neoplastic effects of a synthetic sphingosine analog, FTY720, in mantle cell lymphoma (MCL). We also emphasize how FTY720 enhances the pro-death activity of the fully humanized monoclonal antibody milatuzumab by inhibiting the autophagy-lysosome dependent degradation of its therapeutic target, CD74. Our results provide justification for further evaluation of FTY720 and milatuzumab as a combination therapy for this aggressive B-cell malignancy.
As our knowledge of the role of autophagy in disease expands, it has become clear that this catabolic process is highly relevant to cancer biology. The effect of autophagy on cancer spans from initiation of tumorigenesis, to support of cancer cell growth and proliferation in both normal and stress-inducing environments.
Autophagic flux in nondiseased cells depends on the cellular physiological demands with respect to stress and nutrient availability. Cells in optimal growth environments with adequate supplies of nutrients (such as amino acids and glucose) require low (housekeeping) levels of autophagy to ensure elimination of long-lived or damaged proteins and organelles (autophagic cargo), thereby maintaining cellular homeostasis. Stressful conditions (nutrient deprivation or hypoxia), however, require enhanced autophagy to provide sufficient nutrients and energy produced from degradation and recycling of the autophagic cargo.
Enhanced autophagy also plays a major role in suppressing the activity of anticancer agents. This may be due to the fact that high levels of autophagy provide therapeutically treated cancer cells with improved intracellular conditions that facilitate survival. In line with this, a number of experiments performed in vivo using preclinical mouse models have demonstrated suppressed tumor growth and enhanced death of cancer cells, when autophagy has been abrogated genetically or pharmacologically. Taking advantage of these observations, several clinical trials using drug combination strategies have been initiated for multiple cancer types. These trials have used the well-characterized antimalaria drugs chloroquine or hydrochloroquine as autophagy-inhibiting agents. However, given the numerous side effects and unfavorable pharmacokinetics of these drugs, there is a growing interest in developing and discovering new agents that block autophagy in cancer cells.
Our recent work identified the synthetic analog of sphingosine, FTY720, an immuno suppressive drug recently approved for the treatment of relapsed multiple sclerosis, as an autophagy-inhibiting agent in mantle cell lymphoma (MCL). In direct correlation with recent findings that enhanced autophagic activity is necessary for malignant cell growth, we found that FTY720 treatment promotes MCL cell death that is associated with accumulation of enlarged autolysosomes and increased intracellular levels of LC3-II and p62. These findings support the hypothesis that FTY720 blocks autophagic flux and confirmed that MCL cell death is significantly enhanced in this fashion. FTY720 has been previously studied in ovarian cancer cells and acute lymphoblastic leukemia, where enhanced cell death is observed after incubation with FTY720. However, in these cell types, autophagic activity was found to be upregulated, indicating that cell death is, instead, related to an increase in autophagic flux. We therefore hypothesized that FTY720 may exert a dual effect on autophagyin MCL, perhaps with an activation of upstream steps in the autophagic process, characterized by accumulation of LC3, followed by a blockade of a later step(s) in the pathway by inhibition of autophagic protein degradation.
Similar to sphingosine, FTY720 is phosphorylated in vivo by the sphingosine kinase 2 enzyme and converted to p-FTY720, allowing for enhanced binding to sphingosine-1-phosphate (S1P) receptors. As previously reported, p-FTY720 induces autophagy in prostate cancer cell lines through its interaction with S1P receptors. However, upon treatment of MCL cells with a nonphosphorylatable FTY720 derivative, OSU-2S, we found comparable induction of cell death and similar features of autophagy blockage. Thus, FTY720 phosphorylation and its interaction with S1P receptors do not seem to be a requirement for FTY720-mediated cell death and inhibition of autophagy in MCL cells and may provide an explanation of why our results with MCL may differ from that seen in prostate cancer cells. We further demonstrated that FTY720-induced cell death is mediated by lysosomal membrane permeabilization with subsequent translocation of lysosomal hydrolases to the cytosol. In addition, we found that FTY720-mediated disruption of the autophagy-lysosome pathway leads to increased levels of CD74, a therapeutic target in MCL that normally undergoes lysosomal degradation. Since we have recently shown that CD74 is expressed on the surface of MCL cells and that milatuzumab, a fully humanized anti-CD74 monoclonal antibody, has significant anti-MCL activity, we examined a combination approach with FTY720 and milatuzumab in MCL. We demonstrated that the in vitro survival of four MCL cell lines and six MCL primary tumors treated with a combination of FTY720 and immobilized milatuzumab results in a statistically significant decrease in cell viability compared with either agent alone. MCL is a heterogeneous disease with significant variability of CD74 expression. Thus, this combination strategy presents an attractive therapeutic modality for the treatment of MCL patients. Of note, the combination treatment induces significantly enhanced cell death regardless of MCL subtype (classic vs. blastic variant). Even more interestingly, because of the ability of FTY720 to increase CD74 expression, we were able to significantly decrease the dose of these two agents without affecting the synergistic effect on MCL cell viability, suggesting that lower dosages may be used in vivo resulting in a more favorable toxicity profile.
Finally, the combination of FTY720 and milatuzumab was evaluated in a preclinical mouse model of MCL where we found FTY720-milatuzumab combination-treated mice to survive significantly longer (36 d) when compared with mice treated with vehicle control (28 d), trastuzumab (27 d) or to those that received single-agent treatment with FTY720 (31 d) or milatuzumab (33.5 d).
In summary, our findings not only identify FTY720 as a new autophagy-blocking agent, but also demonstrate, for the first time, that this potent anti-MCL agent can modulate protein levels of a druggable target (CD74), by preventing its degradation by the autophagy-lysosomal pathway. It will be essential to further investigate other targets that become modulated with FTY720 to discover novel treatment strategies for treating this aggressive disease. Although clinical evaluations are needed, our work identifies FTY720 as a promising autophagy-blocking agent for lymphoma therapeutics, and suggests that it can be used successfully in combination with other targeted therapies.
Acknowledgments
Milatuzumab was kindly provided by Dr. D. Goldenberg, Garden State Cancer Center, Center for Molecular Medicine and Immunology, Morris Plains, NJ USA. FTY720 was kindly provided by Dr. Natarajan Muthusamy, Division of Hematology, Department of Medicine, College of Medicine, Ohio State University, Columbus, Ohio, USA. OSU-2S, a nonphosphorylatable FTY720 derivative recently developed at Ohio State University was kindly provided by Dr. C.-S. Chen, Division of Medicinal Chemistry, Ohio State University, Columbus, OH USA. This research was supported by grants from the Lymphoma Research Foundation’s Mantle Cell Lymphoma Research Initiative (R.A.B), the American Cancer Society (ODSR 2009-2 and IRG-67-003-47, M.P-I).