Metabolic Reprogramming of Bone Marrow Stromal Cells in Chronic Lymphocytic Leukemia

In chronic lymphocytic leukemia (CLL), tumor cell survival depends on a multitude of supportive signals delivered by the immune and stromal surrounding cells of the microenvironment, follicular reticular cells in the lymph nodes and the bone marrow. Through cell-to-cell direct contact or soluble factors production, the microenvironment promotes chemotaxis, homing, survival and proliferation of CLL cells within the lymph nodes and the bone marrow by activating pathways in the tumor cells. On the other hand, CLL cells are able to educate the surrounding immune and stromal cells to set up a protective environment leading to tumor evasion and production of soluble factors by the laters, which highlights the importance of the crosstalk between the tumor cells and the microenvironment.

To gain insight into how CLL cells modify stromal cells, we analyzed the gene expression profile and the proteome of a bone marrow mesenchymal stromal cell line, HS5, as a surrogate of the microenvironment in co-culture with the CLL cell line MEC1. HS5 and MEC1 cells were co-cultured but separated by a transwell to avoid direct contact in order to analyze specifically the effect on the stromal cells of soluble factors produced by CLL cells. After 24 hours of co-culture, both RNA and proteins were extracted from HS5 cells for transcriptome and proteome analyses. Transcriptome analysis using RNA-sequencing identified a total of 99 significantly upregulated and 111 significantly downregulated genes in HS5 cells co-cultured with MEC1 cells compared to HS5 cells alone (>2 fold, adjusted p<0.05). Genes encoding cytokines (IL6, IL1B, IL8, CXCL8), growth factors (CSF2, CSF3), NF-kB signaling members (NFKB2, NFKBIZ, TNFAIP3), adhesion molecules (ICAM1) and glycolysis and oxygen homeostasis regulators (ENO2, CYR61, SOD2, NAMPT) were significantly upregulated. Gene set enrichment analysis (GSEA) of these differentially expressed genes revealed a significant enrichment of the IL6-dependent JAK/STAT3 pathway (Normalized enrichment score NES=2.2), the inflammatory response (NES=2.5), the glycolysis (NES=1.9) and the hypoxia (NES=3.5) (all with False Discovery Rate <0.001). Analysis of the proteomic data revealed less pronounced changes of the differentially expressed proteins but metabolism members appeared among the top upregulated proteins. These proteins included the reductases AKR1B1 and PLOD1, and two mitochondrial proteins involved in the respiratory chain: the acetyl CoA-dehydrogenase MCAD and the subunit of the NADH dehydrogenase NDUFA10. In line with these results, clustering and pathway analysis of the proteomic data showed a protein signature related to the oxidation-reduction process. Altogether, these results suggest that CLL cells produce soluble factors that specifically modify the gene expression profile of stromal cells and induce metabolic reprogramming related to a mitochondrial phenotype.

To confirm and strengthen these findings, we assessed the cellular oxygen consumption rate (OCR) and the extra-cellular acidification rate (ECAR) using the Seahorse Assay to quantify the mitochondrial respiration and the glycolysis respectively, in the HS5 cells cultured with conditioned media (CM) issued from primary CLL cells (n=3). Analysis of the OCR showed a significant increase of the maximal respiration (p=0.01) and of the spare respiratory capacity (p=0.03) in HS5 cells with CLL CM as compared to HS5 cells alone, indicating an enhanced mitochondrial respiration. Analysis of the ECAR also revealed a significant increase of the glycolysis capacity (p=0.03) in the HS5 cells upon exposure to CLL CM. In agreement with increased glycolysis, glucose uptake and ATP production were enhanced in HS5 cells cultured with the CM issued from CLL cells.

Altogether, these results demonstrate that CLL cells increase the production of energy in surrounding stromal cells by enhancement of the glycolysis and by promoting mitochondrial-based oxidative phosphorylation. This bioenergetic shift induced by CLL cells could enhance stromal cells fitness, which therefore will benefit to tumoral cell survival. We are currently investigating how this crosstalk interferes with the effect of BCR pathway inhibitors or BH3-mimetics. Targeting this axis may represent a therapeutic strategy of interest.

Thieblemont:Bayer: Honoraria; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses; Kite: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses; Gilead Sciences: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses; Cellectis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses, Research Funding; Hospira: Research Funding; Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses, Research Funding.