Importance of Mitochondrial Metabolism As a Selective Therapeutic Approach in Chronic Lymphocytic Leukemia Patients

Objective: Chronic lymphocytic leukemia (CLL) is the most common blood cancer among adults in Western countries. Impairments in mitochondrial physiology play a role in CLL. Selectively targeting mitochondrial bioenergetics in leukemia cells may be an effective therapeutic approach for CLL. However, mitochondrial metabolism in CLL remains relatively unexplored. Nicotinamidephospho-robosyltransferase (NAMPT) is a key enzyme in the nitoinamide adenine dinucleotide (NAD) salvage pathway. FK866, a chemical inhibitor of NAMPT depletes cellular NAD and ATP levels and triggers apoptosis, suggesting NAMPT contributes to the prolonged survival of CLL cells. Idelalisib, an inhibitor of phosphatidylinositol-3-kinase-delta (PI3Kδ) is an approved therapy for B-cell malignancies including CLL. Herein, bioenergetics profile in 20 CLL patients have been defined and the effects of these drugs on this profile, cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and protein levels of mitochondrial complexes and pPI3Kδ were analyzed. We hypothesize that both inhibitors will alter CLL mitochondrial bioenergetics profiles and function, cell viability, ROS levels and enable lower doses and profound effects for combination therapies rather than single agent therapy.

Methods: A high resolution Oroboros Oxygraph 2K (Oroboros Instruments, Innsbruck, Austria), a Clarke-type oxygen electrode is used to measure mitochondrial respiration rates CLL cells at 37⁰C. Freshly isolated CLL cells (5-10 mil./ml) were added to the chamber in 2 ml of RPMI. After the measurement of basal respiration rates (BR), the following chemicals were added: oligomycin (2μM), FCCP: carbonyl cyanide p-trifluoromethoxy phenyhydrazone (2.5-12.5 μM), and antimycin A (2 μM). Oxygen consumption rate is expressed in pmol oxygen/s/mil. cells. Protein levels in CLL cell lysates were determined by Western blotting. Cell viability, MMP, and ROS were assessed by Novocyte flow cytometer.

Results: Bioenergetics profile (BR: 14.45 ± 3.40; FCCP-induced MR: 57.48 ± 14.94; reserve respiratory capacity, RRC: 43.03 ± 12.80; respiratory control ratio, RCR: 9.36 ± 3.99; proton leak: 6.77 ± 2.45 and residual respiration: 6.03 ± 1.49; Mean ± SD) of 20 CLL patients was individually illustrated and summarized. Our data show that CLL cells have a high mitochondrial RRC and require a higher concentration of uncoupler, namely FCCP to achieve MR than commonly used. BR was not significantly decreased by 24 hours of treatment with FK866 at 1 nM (FK1) nor idelalisib at 6.25 μM (ID6.25) or the combination. After 48 hours, basal respiration of ID6.25 or ID6.25 + FK1 - treated cells was significantly decreased compared to DMSO vehicle control. However, after 24 or 48 hours MR, RRC, and RCR were significantly decreased by each individual drug or its combination compared to DMSO vehicle control. After 24 hours, combination therapy did not significantly decrease BR, RRC, and RCR compared to the treatment of FK1 (except MR), but MR, RRC, and RCR were decreased significantly compared to ID6.25 (except BR). The combination treatment of these two drugs significantly decreased all bioenergetics parameters compared to each individual drug after 48 hours. Preliminary data with another NAMPT-inhibitor, GMX-1778 at 1nM also showed similar effect to FK866 and in combination with ID6.25. MMP was not affected by these drug treatments, cell viability corresponded to the effect of mitochondrial bioenergetics profile, however, ROS levels were increased. Protein expression of certain subunits of mitochondrial complexes I, III and V and pPI3Kδ were decreased after the combined treatment.

Conclusion:Bioenergetics profile can be directly evaluated by high resolution Oroboros oxygraph in CLL cells without any excess manipulation. CLL cells have immense mitochondrial RRC and highly sensitive to an uncoupler. Both FK866 and idelalisib affected bioenergetics profile in CLL cells at doses as low as 1 nM or 6.25 µM, respectively. The combined effect of these drugs is more profound than each agent alone on mitochondrial bioenergetics profile, cell viability, ROS and protein levels of mitochondrial complexes and pPI3Kδ. These novel data derived from the combined treatment with lower doses targeting mitochondrial metabolism provide rational therapeutic combination that may lead to reduce toxicity and increased drug efficacy in CLL.