Inhibition of Fatty Acid Oxidation Leads to Apoptosis of Resting and Proliferating Chronic Lymphocytic Leukemia Cells in Vitro
Authors: Messmer Davorka , Lorrain Kymmy , Bravo Yalda , Stock Nicholas , Bundey Richard , Correa Lucia , Poon Michael M , Stebbins Karin , Cabrera Geraldine , Chen Austin , Jacintho Jason , Spaner David , Prasit Peppi , Lorrain Daniel 
Published: October 2019

Abstract
Abstract 3879
Tumor-specific metabolic changes could serve as new therapeutic targets. While much attention has been given to the metabolism of glucose in cancer cells and intervening therapies, relatively little attention has been given to fatty acids and fatty acid β-oxidation (FAO) in promoting cancer cell survival. Here, we reveal new findings implicating fatty acid metabolism in Chronic lymphocytic leukemia (CLL) and have developed a small molecule inhibitor of this pathway as a novel therapeutic approach to treat CLL. We have found that B cells isolated from CLL patients show increased levels of b-oxidation as compared to B cells isolated from PBMCs of healthy donor controls. NXT629, a small molecule inhibitor of the FAO pathway, dose-dependently reduced b-oxidation in CLL cells but did not reduce b-oxidation in B cells isolated from healthy volunteers. A negative control compound (NXT962) that has a similar chemical structure to NXT629, but does not significantly bind to the relevant target, did not inhibit FAO. NXT629 exerted dose-dependent cytotoxicity in CLL cells (CC50=10 mM, n=5 different patient samples) whereas NXT962 was inactive. Since the cellular microenvironment can protect CLL cells from spontaneous and drug-induced apoptosis, it is pivotal to evaluate novel drugs in this context. As expected, we found that macrophages protected CLL cells from spontaneous and fludarabine-induced apoptosis in vitro. However, NXT629 significantly reduced CLL cells viablity in the co-cultures. NXT629 (at 10 mM, n=2 different patient samples) induced apoptosis in 80% of cells after 7 days of co-culture in a setting where fludarabine was inactive.
Though most CLL cells in the periphery are in a resting state, CLL cells proliferate in lymphoid organs. To mimic these conditions, we examined the activity of FAO inhibition on CLL cells that were induced to proliferate in vitro. To induce proliferation, CLL cells were cultured in media containing human interleukin (IL)-4, IL-10, and allogeneic T blasts. Labeling CLL cells with carboxyfluorescein succinimidyl ester, allowed us to monitor several rounds of induced CLL-cell division via flow cytometry, and within 5 days the CLL cells had expanded 3-fold. We found that addition of NXT629 to such cultures resulted in a dose-dependent reduction in the number of leukemia cells induced to undergo cell-division (IC50=3.7 mM, n=6), whereas the negative control compound showed no activity. Furthermore, when used in combination with dexamethasone (0.3 mM), NXT629 (3 mM) showed a significant decrease in the number of viable cells (38% +/−3%) over either dexamethasone (65% +/−12%) or NXT629 (74%+/−15%) alone. The additivity of FAO inhibition and dexamethasone in vitro suggests that a combination warrants further testing and might yield improved clinical responses. Overall, these results suggest that fatty acids promote primary CLL cell survival and proliferation, and that targeting the FAO pathway could be a new therapeutic approach to treating CLL.

Disclosures:
Messmer: Inception Sciences: Employment, Equity Ownership. Lorrain:Inception Sciences: Employment, Equity Ownership. Bravo:Inception Sciences: Employment, Equity Ownership. Stock:Inception Sciences: Employment, Equity Ownership. Bundey:Inception Sciences: Employment, Equity Ownership. Correa:Inception Sciences: Employment, Equity Ownership. Poon:Inception Sciences: Employment, Equity Ownership. Stebbins:Inception Sciencees: Employment, Equity Ownership. Cabrera:Inception Sciences: Employment, Equity Ownership. Chen:Inception Sciences: Employment, Equity Ownership. Jacintho:Inception Sciences: Employment, Equity Ownership. Spaner:Inception Sciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Prasit:Inception Sciences: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Lorrain:Inception Sciences: Employment, Equity Ownership.