Targeting sphingolipid metabolism in chronic lymphocytic leukemia

Elevated levels of circulating C16:0 glucosylceramides (GluCer) and increased mRNA expression of UDP-glucose ceramide glycosyltransferase (UGCG), the enzyme responsible for converting ceramides (Cer) to GluCer, represent unfavorable prognostic markers in chronic lymphocytic leukemia (CLL) patients. To evaluate the therapeutic potential of inhibiting GluCer synthesis, we genetically repressed the UGCG pathway using in vitro models of leukemic B cells, in addition to UGCG pharmacological inhibition with approved drugs such as eliglustat and ibiglustat, both individually and in combination with ibrutinib, assessed in cell models and primary CLL patient cells. Cell viability, apoptosis, and proliferation were evaluated in vitro, and survival and apoptosis were examined ex vivo. UGCG inhibition efficacy was confirmed by quantifying intracellular sphingolipid levels through targeted lipidomics using mass spectrometry. Other inhibitors of sphingolipid biosynthesis pathways were similarly assessed. Blocking UGCG significantly decreased cell viability and proliferation, highlighting the oncogenic role of UGCG in CLL. The efficient inhibition of UGCG was confirmed by a significant reduction in GluCer intracellular levels. The combination of UGCG inhibitors with ibrutinib demonstrated synergistic effect. Inhibitors that target alternative pathways within sphingolipid metabolism, like sphingosine kinases inhibitor SKI-II, also demonstrated promising therapeutic effects both alone and when used in combination with ibrutinib, reinforcing the oncogenic impact of sphingolipids in CLL cells. Targeting sphingolipid metabolism, especially the UGCG pathway, represents a promising therapeutic strategy and as a combination therapy for potential treatment of CLL patients, warranting further investigation. Supplementary Information The online version contains supplementary material available at 10.1007/s10238-024-01440-x.

For UGCG knockdown in JVM2 and HG3 cells, shRNAs sequences targeting human UGCG are displayed in Supplementary Table 4. Lentivirus production and transduction steps were conducted as described previously 1 .Selections of cells containing the shRNAs were performed by adding 0.5 µg/mL puromycin for HG3 cells and 1 µg/mL puromycin for JVM2 cells.Validation of the knockdown was performed by RT-qPCR and immunoblotting for UGCG expression and by measuring intracellular sphingolipid levels for UGCG enzyme activity.shRNA demonstrating sufficient repression of UGCG expression were selected for further functional studies.

Cell-based assays
To assess cell viability, JVM2 and HG3 cells were plated at a density of 5 x 10 4 cells/well and 1 x 10 4 cells/well, respectively, in in 96-well U-bottom tissue culture plates (BD Bioscience, Franklin Lakes, NJ, USA).Cells were treated with various concentrations of sphingolipid inhibitors alone or in combination with ibrutinib.After 72h incubation, 20 μl of CellTiter aqueous one solution cell proliferation reagent (MTS Promega, Madison, WI, USA) was added to each well.Absorbance at 490 nm was measured in the Infinite M1000 plate reader (Tecan, Männedorf, Switzerland) after a 4-h incubation with CellTiter in the cell culture incubator at 37°C.Assays were replicated at least three times, in triplicate.
Apoptosis assays were carried out using 2 x 10 6 cells as previously described 1 using Violet Annexin V/Dead cell Apoptosis Kit (Invitrogen, Eugene, OR, USA).Assays were replicated in biological triplicate.
To assess cell proliferation in cells with UGCG knockdown, cells counting was performed for 96h.
HG3 and JVM2 cells were plated at a density of 1 x 10 5 cells/mL in T25 flasks (Corning, Durham, NC, USA) in duplicate for each condition.Every 24h, 10 µL of cells were sampled and mixed with 50% of trypan blue (Wisent) and stained cells were counted using a TC-10 automated cell counter in triplicate (Bio-Rad, Hercules, CA, USA).Cell numbers resulted from the average of six values per condition.Cell proliferation assays were replicated at least in a biological duplicate.

Gene and protein expression analyses
To validate UGCG knockdown, UGCG expression was quantified by RT-qPCR and immunoblotting.RNA extraction, reverse transcription and qPCR were performed as previously described 1 .Relative expression levels were calculated using the 2-∆∆CT method and normalized with 36B4 as the reference gene.Primer sequences are provided in Supplementary Table 3.

Cancer cell lines dependency on UGCG
UGCG dependency data by CRISPR/Cas9 system were issued from the Public 23Q4 dataset released by the DepMap project from Broad Institute.The data was processed using Chronos algorithm3.Data are available online at https://depmap.org/portal/and UGCG dependency analyses across cancer cell lines were performed using the "Perturbation Effects" online tool.