Epigenetic Activity of Peroxisome Proliferator-Activated Receptor Gamma Agonists Increases the Anticancer Effect of Histone Deacetylase Inhibitors on Multiple Myeloma Cells.

Epigenetic modifications play a major role in the development of multiple myeloma. We have previously reported that the PPARγ agonist pioglitazone (PIO) enhances, in-vitro, the cytotoxic effect of the Histone deacetylase inhibitor (HDACi), valproic acid (VPA), on multiple myeloma cells. Here, we described the development of a new multiple myeloma mouse model using MOLP8 cells, in order to evaluate the effect of VPA/PIO combination on the progression of myeloma cells, by analyzing the proliferation of bone marrow plasma cells. We showed that VPA/PIO delays the progression of the disease and the invasion of myeloma cells in the bone marrow. Mechanistically, we demonstrated that VPA/PIO increases the cleavage of caspase 3 and PARP, and induces the acetylation of Histone 3 (H3). Furthermore, we provided evidence that PPARγ agonist is able to enhance the action of other HDACi such as Vorinostat or Mocetinostat. Using PPARγ antagonist or siPPARγ, we strongly suggest that, as described during adipogenesis, PIO behaves as an epigenetic regulator by improving the activity of HDACi. This study highlights the therapeutic benefit of PIO/VPA combination, compared to VPA treatment as a single-arm therapy on multiple myeloma and further highlights that such combination may constitute a new promising treatment strategy which should be supported by clinical trials

Flow cytometry analysis.

<p>CD38+CD138+ MOLP8 cell invasion in BM, as well as peripheral blood, was observed and compared between the different animal groups. Histogram representation shows the average of tumor burden for each group.</p

Molecular mechanism explaining the increase of acetylation level by PPARγ agonist.

<p>Schematic representation of molecular mechanism, explaining the increase of acetylation after PPARγ agonist treatment.</p

Effect of treatment on weight evolution in different MM mice groups.

<p><b>a.</b> Effect observed of treatment on weight evolution in different MM mice groups and <b>b.</b> Kaplan-Meier curves sustaining the effect of treatments on the paralysis delay. Implantation to paralysis delay between MM Group (blue), VPA Group (green) and VPA/PIO co-treatment Group (red).</p

FLC quantification in the different groups of mice at day18.

<p>Average ± standard deviation (SD, n = 3)</p><p>^b non-significant (n/s, n = 1), the concentration of the λ light chain is in mg/mL.</p><p>FLC quantification in the different groups of mice at day18.</p

Analysis of cell death mechanism and H3 acetylation level.

<p><b>a.</b> Immunoblotting of caspase 3 and PARP cleavages, <b>b.</b> H3 acetylation at day 25 and 30 in untreated (-) or treated (+) animals with VPA or/and PIO as indicated. *<i>As negative control of day 30 was used the BM flush of the last survived mouse of the group</i>, <i>with fully developed MM disease that was euthanized at day 25</i>. <b>c.</b> Effect of the combination of PIO with other HDACi on the H3 acetylation. MOLP8 cell line, treated with each of the three different HDACi (MGCD 0.2μM, SAHA 0.25μM and VPA 1mM) alone or combined with PIO 30μM. <b>d.</b> Study of the level of H3 acetylated of MOLP8 cells after treatment with the combination HDAC inhibitors/PIO and with the combination HDAC inhibitors/ PIO /PPARγ antagonist. Partial inhibition of H3 acetylation can be observed, by the pharmacological PPARγ antagonist, BADGE (0.5μM), or by genetic approaches using siGFP (as positive control) and siPPARγ after 48 hours of treatment.</p

Experimental design.

<p>Experimental design of the treatment protocol of MM mouse model.</p

Development of MM mouse model.

<p><b>a.</b> correlation between MM development and loss of weight in MM mice. Weight of mice inoculated with MOLP8 cell line reported as a median of the 6 mice and correlated the disease development. <b>b.</b> Paralysis development of back limbs, <b>c.</b> x-ray analysis of skeletons, showed osteolytic lesions in MM Group compared to healthy mice. <b>d.</b> Flow cytometry analysis of bone marrow-derived MOLP8 tumor cells. Cells, cultured in-vitro, are used as a control. <b>e.</b> H&E staining performed on longitudinal tibia sections showing a different cellularity and bone structure in the MM Group compared to healthy mice (Healthy Group). Also, H&E staining performed on spleen, lungs and liver of Healthy Group, is showing infiltration of malignant plasma cells in the MM Group. Internal panels are showing higher magnification (50x) for the identification of malignant cells infiltration.</p

Determination of genes and microRNAs involved in the resistance to fludarabine in vivo in chronic lymphocytic leukemia

Background: Chronic lymphocytic leukemia (CLL) cells are often affected by genomic aberrations targeting key regulatory genes. Although fludarabine is the standard first line therapy to treat CLL, only few data are available about the resistance of B cells to this purine nucleoside analog in vivo. Here we sought to increase our understanding of fludarabine action and describe the mechanisms leading to resistance in vivo. We performed an analysis of genomic aberrations, gene expression profiles, and microRNAs expression in CLL blood B lymphocytes isolated during the course of patients' treatment with fludarabine. Results: In sensitive patients, the differentially expressed genes we identified were mainly involved in p53 signaling, DNA damage response, cell cycle and cell death. In resistant patients, uncommon genomic abnormalities were observed and the resistance toward fludarabine could be characterized based on the expression profiles of genes implicated in lymphocyte proliferation, DNA repair, and cell growth and survival. Of particular interest in some patients was the amplification of MYC (8q) observed both at the gene and transcript levels, together with alterations of myc-transcriptional targets, including genes and miRNAs involved in the regulation of cell cycle and proliferation. Differential expression of the sulfatase SULF2 and of miR-29a, -181a, and -221 was also observed between resistant and sensitive patients before treatment. These observations were further confirmed on a validation cohort of CLL patients treated with fludarabine in vitro. Conclusion: In the present study we identified genes and miRNAs that may predict clinical resistance of CLL to fludarabine, and describe an interesting oncogenic mechanism in CLL patients resistant to fludarabine by which the complete MYC-specific regulatory network was altered (DNA and RNA levels, and transcriptional targets). These results should prove useful for understanding and overcoming refractoriness to fludarabine and also for predicting the clinical outcome of CLL patients before or early during their treatment