Venetoclax in Combination with NAMPT Inhibitors Decreases Mitochondrial Bioenergetics through the Impaired AMPK/SIRT/PGC1α Signaling Pathway in CLL
Bibliographic record
Abstract
Abstract Objective: Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults. Altered mitochondrial metabolism has been shown to be involved in the pathogenesis of CLL. Nicotinamidephospho-ribosyltransferase (NAMPT) is a key enzyme in the nicotinamide adenine dinucleotide (NAD) salvage pathway. FK866 and GMX1778, chemical inhibitors of NAMPT, deplete cellular NAD and ATP levels and trigger apoptosis in CLL cells, suggesting NAMPT contributes to the prolonged survival of these cells. Venetoclax is an approved therapy for CLL and blocks the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein, leading to programmed cell death of CLL cells. The adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signaling axis senses the metabolic demands of cells and regulates mitochondrial function. Silent information regulator T1 (SIRT1) is a cytoplasmic enzyme that mediates NAD+-dependent deacetylation of target substrates. The importance of the AMPK/SIRT/PGC1α signaling pathway has to be explored yet in CLL. Venetoclax may trigger a switching off of AMPK and /or SIRT1 signaling leading to impaired PGC-1α expression/activity and diminished mitochondrial activity. The effects of these drugs on mitochondrial bioenergetics profile, cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and protein levels of Bcl-2, AMPK, SIRT1, and PGC1α were analyzed. We hypothesize that venetoclax, along with FK866 and GMX1778, will alter CLL mitochondrial bioenergetics profiles and function, cell viability, and ROS levels through the involvement of the AMPK/SIRT/PGC1α signaling pathway 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 of CLL cells at 370C. Freshly isolated CLL cells (10 mil./ml) were added to the chamber in 2 ml of RPMI. After the measurement of basal respiration rates, the following chemicals were added: oligomycin (2uM), FCCP: carbonyl cyanide p-trifluoromethoxy phenyhydrazone (2.5-12.5 uM), and antimycin A (2 uM). 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: Primary CLL cells treated with 1.25 nM of venetoclax for 24 or 48h showed significantly decreased basal respiration rates, maximal respiration rates, and spare respiratory capacity compared to DMSO vehicle control. These parameters were also significantly affected by 1nM of FK866 or 1 nM of GMX1778 except basal respiration rates. The combination treatment of venetoclax with FK866 or GMX1778 for 24 or 48h significantly decreased all these bioenergetics parameters compared to each individual drug. MMP was not affected by these drug treatments and cell viability corresponded to the effect of the mitochondrial bioenergetics profile. ROS levels after 24 hours in venetoclax treated CLL cells were significantly increased compared to DMSO, any single agent or in combination. However, after 48 hours of treatment, increased ROS levels were not statistically significant compared to DMSO. Protein levels of Bcl-2, P-AMPK, AMPK, SIRT1, and PGC1α were decreased by venetoclax alone or in combination with FK866 or GMX1778. Conclusion: Venetoclax, FK866 and GMX1778 affected bioenergetics profiles in CLL cells at doses as low as 1.25 nM, 1nM, and 1 nM, respectively. The combined effect of these drugs on the mitochondrial bioenergetics profiles and cell viability is more profound than each NAMPT inhibitor agent alone. Protein levels of Bcl-2, AMPK, SIRT1, and PGC1α in venetoclax treated samples were reduced compared to DMSO and each single agent NAMPT inhibitor. These novel data suggest the involvement of the AMPK/SIRT/PGC1α signaling pathway to target mitochondrial metabolism and provide rational therapeutic combinations that may lead to reduced toxicity and increased drug efficacy in CLL. Disclosures Banerji: Roche: Other: Unrestricted grant received in the past; Janssen: Other: Unrestricted grant received in the past; Gilead: Other: Unrestricted grant received in the past; Abbvie: Other: Unrestricted grant received in the past; Teva: Other: Unrestricted grant received in the past.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.004 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".