Impairing NAD+ synthesis is a powerful tool to increase the efficacy of the BTK inhibitor ibrutinib in Waldenström's macroglobulinemia. Free

Waldenström's Macroglobulinemia (WM) is a rare indolent B-cell lymphoma characterized by the infiltration of bone marrow with lymphoplasmacytic cells and the presence of monoclonal IgM in the serum. BTK inhibition is a cornerstone in the treatment of WM and Ibrutinib as first in class BTK inhibitor (BTKi) and second generation BTKis are widely used in the clinical management of this lymphoma subtype. However, many WM patients show resistance over time towards BTKi or are less responsive when carrying CXCR4 mutations or being non-mutated for MYD88. Thus, there is the need to identify novel targets which collaborate with BTKis in controlling WM growth. Metabolic pathway inhibition as a potential therapy has garnered less attention in WM so far. One such pathway is the NAD⁺ synthesis and recycling pathway, which is vital for tumor growth as NAD⁺ serves as the cellular energy currency. A significant fraction of NAD⁺ is recycled via the salvage pathway from Nicotinamide Mononucleotide (NAM) by the enzyme Nicotinamide phosphoribosyl transferase (NAMPT). Beside the salvage pathway, NAD⁺ homeostasis is maintained by the de-novo pathway via Tryptophan (Trp) and through dietary niacin via the Preiss-Handler pathway. Thus, cells can circumvent NAMPT shortage by using the two forementioned pathways to sustain NAD⁺ supply. Studies have shown that NAMPT can prevent cell death by sustaining mitochondrial NAD⁺ levels and its inhibition has been shown to potentiate BTK inhibition, thereby opening a potential therapeutic avenue. The study follows the hypothesis that simultaneous targeting of NAMPT and de-novo NAD⁺ synthesis by Trp starvation greatly improves therapeutic activity of the BTK inhibitor Ibrutinib in WM.

To test this the WM cell line BCWM1 was treated with the NAMPT inhibitor OT82 for 120h in a first step: 100% cellular death was achieved with 19.5nM OT82 in regular RPMI medium with a 4.9-fold increase in efficacy in Trp RPMI deficient medium. Moreover, the treatment with 4nM OT82 in regular media reduced the NAD⁺/NADH ratio from 1.681 ± 0.083 (mean ± SEM) to 0.315 ± 0.084. Of note, after Trp starvation that ratio decreased 63-fold (0.005±0.002), indicating enhanced efficacy of the inhibitor under Trp deficient conditions. The addition of 1µM Ibrutinib to OT82 had no noticeable effect on the NAD⁺/NADH ratio in tryptophan deficient media, but resulted in higher apoptosis levels than compared to Ibrutinib alone (67.84 ± 4.37% and 58.66 ± 1.01%, respectively). In addition, a reduction in the mitochondrial membrane potential was observed for Ibrutinib/OT82 (4895.7 ± 475.77 RFU) vs Ibrutinib alone in Trp deficient media (5610.66 ± 177.29 RFU).

In addition, we could demonstrate in the mitochondrial ROS assay that the Ibrutinib/OT82 combination in concert with Trp starvation induced 95.52 ± 0.15% MitoSox positive cells compared to 69.85 ± 0.16 % MitoSox positive cells with Ibrutinib alone in Trp deficient media after 48h. The addition of Trp to these conditions reduced the proportion of MitoSox positive cells to 60.78 ± 0.022% for the Ibrutinib/OT82 combination and to 49.89 ± 0.16 % cells with Ibrutinib alone, indicating the relevance of Trp deprivation for the cytotoxic effect of the Ibrutinib combination.

With regard to mitochondrial respiration treatment of BCWM.1 cells with 4nM OT82 and 1µM Ibrutinib in Trp deficient media for 48h induced a basal respiration rate of 5.61 ± 1.39 pmol/min/100ng DNA and maximal respiration rate of 5.89 ± 2.28 pmol/min/100ng DNA in the XF Mito Stress assay. With this it was substantially lower compared to the OT82/Ibrutinib (26.15 ± 1.22 pmol/min/100ng DNA and maximal respiration rate to 34.97 ± 2.22 pmol/min/100ng DNA) and to Ibrutinib alone (24.54 ± 1.22 pmol/min/100ng DNA and maximal respiration rate to 32.07 ± 2.06 pmol/min/100ng DNA) in regular medium.

Of note, IgM secretion measured by ELISA dropped 24-fold when BCWM1 cells were incubated with Ibrutinib/OT82 in Trp deficient medium (120h treatment) compared to non-treated cells in regular medium (76.63 ± 5.83ng/ml vs 1860 ± 28.54 ng/ml IgM, respectively).

In conclusion, these data demonstrate that impairing intracellular NAD+ synthesis by concurrent NAMPT inhibition and Trp depletion is a powerful tool to increase the therapeutic activity of the BTKi Ibrutinib. They furthermore underline that Tryptophan can serve as an auxiliary source of NAD⁺ for WM cells when intracellular NAMPT levels are limiting.