Dimethyl itaconate selectively targets inflammatory and metabolic pathways in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) co-evolves with its own microenvironment where inflammatory stimuli including toll-like receptors (TLR) signaling can protect CLL cells from spontaneous and drug-induced apoptosis by upregulating IκBζ, an atypical co-transcription factor. To dissect IκBζ-centered signaling pathways, we performed a gene expression profile of primary leukemic cells expressing either high or low levels of IκBζ after stimulation, highlighting that IκBζ is not only an inflammatory gene but it may control metabolic rewiring of malignant cells thus pointing to a novel potential opportunity for therapy. We exploited the capacity of the dimethyl itaconate (DI), an anti-inflammatory electrophilic synthetic derivative of the metabolite Itaconate, to target IκBζ. CLL cells, murine leukemic splenocytes, and leukocytes from healthy donors were treated in vitro with DI that abolished metabolic activation and reduced cell viability of leukemic cells only, even in the presence of robust TLR prestimulation. RNA sequencing highlighted that in addition to the expected electrophilic stress signature observed after DI treatment, novel pathways emerged including the downregulation of distinct MHC class II complex genes. In conclusion, DI not only abrogated the proinflammatory effects of TLR stimulation but also targeted a specific metabolic vulnerability in CLL cells.

Disrupting pro-survival and inflammatory pathways with dimethyl fumarate sensitizes chronic lymphocytic leukemia to cell death.

Microenvironmental signals strongly influence chronic lymphocytic leukemia (CLL) cells through the activation of distinct membrane receptors, such as B-cell receptors, and inflammatory receptors, such as Toll-like receptors (TLRs). Inflammatory pathways downstream of these receptors lead to NF-κB activation, thus protecting leukemic cells from apoptosis. Dimethyl fumarate (DMF) is an anti-inflammatory and immunoregulatory drug used to treat patients with multiple sclerosis and psoriasis in which it blocks aberrant NF-κB pathways and impacts the NRF2 antioxidant circuit. Our in vitro analysis demonstrated that increasing concentrations of DMF reduce ATP levels and lead to the apoptosis of CLL cells, including cell lines, splenocytes from Eµ-TCL1-transgenic mice, and primary leukemic cells isolated from the peripheral blood of patients. DMF showed a synergistic effect in association with BTK inhibitors in CLL cells. DMF reduced glutathione levels and activated the NRF2 pathway; gene expression analysis suggested that DMF downregulated pathways related to NFKB and inflammation. In primary leukemic cells, DMF disrupted the TLR signaling pathways induced by CpG by reducing the mRNA expression of NFKBIZ, IL6, IL10 and TNFα. Our data suggest that DMF targets a vulnerability of CLL cells linked to their inflammatory pathways, without impacting healthy donor peripheral blood mononuclear cells.

Toll-like receptor 9 signaling in chronic lymphocytic leukemia cell lines.

Chronic lymphocytic leukemia (CLL) is a prototypic neoplasia in which malignant cells strongly depend on microenvironmental stimulations in the lymphoid tissues where they accumulate; leukemic cells are exposed to interaction with bystander and accessory cells, as well as inflammatory soluble mediators. Cell lines are frequently used to model the pathobiology of this disease; however, they do not always recapitulate leukemic cell growth and response to stimulation, and no data are available on Toll-like receptors (TLR) signaling in CLL cell lines. To address this gap, we analyzed HG3, MEC2, and PCL12 cell lines, before and after CpG stimulation, by RNA-sequencing followed by bioinformatic analyses and validation experiments. We identified NFKBIZ mRNA and the corresponding IkBz protein as robust markers of TLR9 activation in both MEC2 and PCL12, but not in HG3 cells. Next, we compared our current results with previous results obtained with primary CLL patient samples and were able to conclude that MEC2 is most similar to the patients' cells in terms of global responsiveness to TLR stimulation; in particular, MEC2 better resembles the samples of patients, as it is characterized by high expression levels of IkBz, but with a lower number of genes regulated.