Inhibition of HIF1α-Dependent Upregulation of Phospho-l-Plastin Resensitizes Multiple Myeloma Cells to Frontline Therapy.

The introduction of novel frontline agents in multiple myeloma (MM), like immunomodulatory drugs and proteasome inhibitors, has improved the overall survival of patients. Yet, MM is still not curable, and drug resistance (DR) remains the main challenge. To improve the understanding of DR in MM, we established a resistant cell line (MOLP8/R). The exploration of DR mechanisms yielded an overexpression of HIF1α, due to impaired proteasome activity of MOLP8/R. We show that MOLP8/R, like other tumor cells, overexpressing HIF1α, have an increased resistance to the immune system. By exploring the main target genes regulated by HIF1α, we could not show an overexpression of these targets in MOLP8/R. We, however, show that MOLP8/R cells display a very high overexpression of LCP1 gene (l-Plastin) controlled by HIF1α, and that this overexpression also exists in MM patient samples. The l-Plastin activity is controlled by its phosphorylation in Ser5. We further show that the inhibition of l-Plastin phosphorylation restores the sensitivity of MOLP8/R to immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs). Our results reveal a new target gene of DR, controlled by HIF1α.

BAT3 modulates p300-dependent acetylation of p53 and autophagy-related protein 7 (ATG7) during autophagy.

Autophagy is regulated by posttranslational modifications, including acetylation. Here we show that HLA-B-associated transcript 3 (BAT3) is essential for basal and starvation-induced autophagy in embryonic day 18.5 BAT3(-/-) mouse embryos and in mouse embryonic fibroblasts (MEFs) through the modulation of p300-dependent acetylation of p53 and ATG7. Specifically, BAT3 increases p53 acetylation and proautophagic p53 target gene expression, while limiting p300-dependent acetylation of ATG7, a mechanism known to inhibit autophagy. In the absence of BAT3 or when BAT3 is located exclusively in the cytosol, autophagy is abrogated, ATG7 is hyperacetylated, p53 acetylation is abolished, and p300 accumulates in the cytosol, indicating that BAT3 regulates the nuclear localization of p300. In addition, the interaction between BAT3 and p300 is stronger in the cytosol than in the nucleus and, during starvation, the level of p300 decreases in the cytosol but increases in the nucleus only in the presence of BAT3. We conclude that BAT3 tightly controls autophagy by modulating p300 intracellular localization, affecting the accessibility of p300 to its substrates, p53 and ATG7.

A high rate of telomeric sister chromatid exchange occurs in chronic lymphocytic leukaemia B-cells.

Cancer cells protect their telomere ends from erosion through reactivation of telomerase or by using the Alternative Lengthening of Telomere (ALT) mechanism that depends on homologous recombination. Chronic lymphocytic leukaemia (CLL) B cells are characterized by almost no telomerase activity, shelterin deregulation and telomere fusions. To characterize telomeric maintenance mechanisms in B-CLL patients, we measured their telomere length, telomerase expression and the main hallmarks of the ALT activity i.e. C-circle concentration, an extra-chromosomal telomere repeat (ECTR), and the level of telomeric sister chromatid exchange (T-SCE) rate. Patients showed relative homogenous telomere length although almost no TERT transcript and nearly no C-circle were evidenced. Nevertheless, compared with normal B cells, B-CLL cells showed an increase in T-SCE rate that was correlated with a strong down-regulation of the topoisomerase III alpha (TOP3A) expression, involved in the dissolution of Holliday Junctions (HJ), together with an increased expression of SLX1A, SLX4, MUS81 and GEN1, involved in the resolution of HJ. Altogether, our results suggest that the telomere maintenance mechanism of B-CLL cells do not preferentially use telomerase or ALT. Rather, the rupture of the dissolvasome/resolvasome balance may increase telomere shuffling that could homogenize telomere length, slowing telomere erosion in this disease.

MicroRNA as biomarkers and regulators in B-cell chronic lymphocytic leukemia.

Early cancer detection and disease stratification or classification are critical to successful treatment. Accessible, reliable, and informative cancer biomarkers can be medically valuable and can provide some relevant insights into cancer biology. Recent studies have suggested improvements in detecting malignancies by the use of specific extracellular microRNAs (miRNAs) in plasma. In chronic lymphocytic leukemia (CLL), an incurable hematologic disorder, sensitive, early, and noninvasive diagnosis and better disease classification would be very useful for more effective therapies. We show here that circulating miRNAs can be sensitive biomarkers for CLL, because certain extracellular miRNAs are present in CLL patient plasma at levels significantly different from healthy controls and from patients affected by other hematologic malignancies. The levels of several of these circulating miRNAs also displayed significant differences between zeta-associated protein 70 (ZAP-70)(+) and ZAP-70(-) CLL. We also determined that the level of circulating miR-20a correlates reliably with diagnosis-to-treatment time. Network analysis of our data, suggests a regulatory network associated with BCL2 and ZAP-70 expression in CLL. This hypothesis suggests the possibility of using the levels of specific miRNAs in plasma to detect CLL and to determine the ZAP-70 status.

Cooperative effects of Janus and Aurora kinase inhibition by CEP701 in cells expressing Jak2V617F.

The Janus kinase 2 mutant V617F occurs with high frequency in myeloproliferative neoplasms. Further mutations affecting the Janus kinase family have been discovered mostly in leukaemias and in myeloproliferative neoplasms. Owing to their involvement in neoplasia, inflammatory diseases and in the immune response, Janus kinases are promising targets for kinase inhibitor therapy in these disease settings. Various quantitative assays including two newly developed screening assays were used to characterize the function of different small-molecule compounds in cells expressing Jak2V617F. A detailed comparative analysis of different Janus kinase inhibitors in our quantitative assays and the subsequent characterization of additional activities demonstrated for the first time that the most potent Jak2 inhibitor in our study, CEP701, also targets Aurora kinases. CEP701 shows a unique combination of both activities which is not found in other compounds also targeting Jak2. Furthermore, colony forming cell assays showed that Janus kinase 2 inhibitors preferentially suppressed the growth of erythroid colonies, whereas inhibitors of Aurora kinases preferentially blocked myeloid colony growth. CEP701 demonstrated a combined suppression of both colony types. Moreover, we show that combined application of a Janus and an Aurora kinase inhibitor recapitulated the effect observed for CEP701 but might allow for more flexibility in combining both activities in clinical settings, e.g. in the treatment of myeloproliferative neoplasms. The newly developed screening assays are high throughput compatible and allow an easy detection of new compounds with Janus kinase 2 inhibitory activity.

Oncolytic H-1 parvovirus binds to sialic acid on laminins for cell attachment and entry.

H-1 parvovirus (H-1PV) is a promising anticancer therapy. However, in-depth understanding of its life cycle, including the host cell factors needed for infectivity and oncolysis, is lacking. This understanding may guide the rational design of combination strategies, aid development of more effective viruses, and help identify biomarkers of susceptibility to H-1PV treatment. To identify the host cell factors involved, we carry out siRNA library screening using a druggable genome library. We identify one crucial modulator of H-1PV infection: laminin γ1 (LAMC1). Using loss- and gain-of-function studies, competition experiments, and ELISA, we validate LAMC1 and laminin family members as being essential to H-1PV cell attachment and entry. H-1PV binding to laminins is dependent on their sialic acid moieties and is inhibited by heparin. We show that laminins are differentially expressed in various tumour entities, including glioblastoma. We confirm the expression pattern of laminin γ1 in glioblastoma biopsies by immunohistochemistry. We also provide evidence of a direct correlation between LAMC1 expression levels and H-1PV oncolytic activity in 59 cancer cell lines and in 3D organotypic spheroid cultures with different sensitivities to H-1PV infection. These results support the idea that tumours with elevated levels of γ1 containing laminins are more susceptible to H-1PV-based therapies.

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.

Peroxisome proliferator-activated receptor gamma agonists potentiate the cytotoxic effect of valproic acid in multiple myeloma cells.

The main challenge in using chemotherapy to treat multiple myeloma (MM) is drug resistance. In order to evaluate the anti-neoplastic properties of a new drug combination in MM, two clinically available drugs, valproic acid (VPA) a histone deacetylase (HDAC) inhibitor and pioglitazone, a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, were tested in vitro on MM cell lines and MM patient cells. The sensitivity towards VPA alone was observed on several MM cell lines tested and also on primary myeloma cells and peripheral blood mononuclear cells from healthy donors. Importantly, the addition of a PPARgamma agonist to the VPA treatment increased the cytotoxic effect of VPA in a synergistic/additive manner on the different MM cell lines and MM patient cells. This effect was observed at the physiological range of VPA used to treat epileptic patients. The mechanisms underlying this increase induced a cell cycle arrest and caspase-dependent apoptosis. The potentiation of the effect of VPA by pioglitazone was mediated by higher acetylation levels of histones H3 and H4 compared to levels induced by HDAC inhibitors alone. This association reveals a new promising chemotherapeutic combination to be tested in MM.

Valproic acid induces non-apoptotic cell death mechanisms in multiple myeloma cell lines.

Multiple myeloma (MM) is an incurable hematological disorder characterized by dysregulated proliferation of terminally differentiated plasma cells. Aberrant histone acetylation has been observed in the development of numerous malignancies. Histone deacetylase inhibitors such as valproic acid (VPA) are promising drugs for cancer therapy since they have been reported to have antiproliferative effects and to induce differentiation in carcinoma and leukemic cells. Considering the advantage of being already in clinical use for epilepsy treatment, valproic acid might be a promising therapeutic candidate drug in the management of multiple myeloma. In this study, we show that the short fatty acid VPA has a time and dose-dependent cytotoxic effect on the MM cell lines OPM2, RPMI and U266. The influence of VPA on cell cycle and apoptosis have been evaluated by flow cytometry. Our results show that the three cell lines are blocked in G0/G1 phase. The observed sensitivity to VPA can be partially explained by late apoptosis. Since caspase 3 is activated in all tested cell lines after VPA treatment, a caspase-dependent pathway seems to be involved but not activated by the classic apoptotic pathways. We have also studied another mechanism of cell death, the senescence-like phenotype, but did not find any evidence for its implication. Thus, treatment with VPA may imply other alternative cell death mechanisms.

From molecular characteristics to cellular events in apoptosis-resistant HL-60 cells.

The ability to induce apoptosis in tumor cells is critical to elicit a positive response to cytotoxic chemo-therapy. In this study, we investigated the effect of the topoisomerase I inhibitors camptothecin and SN-38, known to cause an unusual form of DNA damage, on apoptotic pathways using the leukemic cell line HL-60 and its vincristine-resistant variant HL-60 VCR. Both camptothecin and SN-38 induced high levels of apoptosis in sensitive cells when compared to the multidrug-resistant ones. Interestingly, a higher BCL-2/BAX ratio was observed in HL-60 VCR at the basal state and during treatments. Moreover, these cells which did not exhibit Bcr-abl translocation or bcrp efflux pump, overexpressed topoisomerase I protein. The data provide evidence that BCL-2 protein could protect HL-60 VCR from mitochondrial membrane depolarization and block ROS production in these cells. Finally, our results suggest that dysregulation of proteins associated with DNA replication and apoptotic process could contribute to the multidrug-resistance phenotype.

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.

BAG6/BAT3 modulates autophagy by affecting EP300/p300 intracellular localization.

We recently reported that BAG6/BAT3 (BCL2-associated athanogene 6) is essential for basal and starvation-induced autophagy in E18.5 bag6(-/-) mouse embryos and in mouse embryonic fibroblasts (MEFs) through the modulation of the EP300/p300-dependent acetylation of TRP53 and autophagy-related (ATG) proteins. We observed that BAG6 increases TRP53 acetylation during starvation and pro-autophagic TRP53-target gene expression. BAG6 also decreases the EP300 dependent-acetylation of ATG5, ATG7, and LC3-I, posttranslational modifications that inhibit autophagy. In addition, in the absence of BAG6 or when using a mutant of BAG6 exclusively located in the cytoplasm, autophagy is inhibited, ATG7 is hyperacetylated, TRP53 acetylation is abrogated, and EP300 accumulates in the cytoplasm indicating that BAG6 is involved in the regulation of the nuclear localization of EP300. We also reported that the interaction between BAG6 and EP300 occurs in the cytoplasm rather than the nucleus. Moreover, during starvation, EP300 is transported to the nucleus in a BAG6-dependent manner. We concluded that BAG6 regulates autophagy by controlling the localization of EP300 and its accessibility to nuclear (TRP53) and cytoplasmic (ATGs) substrates.

Bronchial airway gene expression in smokers with lung or head and neck cancer.

Cigarette smoking is the major cause of cancers of the respiratory tract, including non-small cell lung cancer (NSCLC) and head and neck cancer (HNC). In order to better understand carcinogenesis of the lung and upper airways, we have compared the gene expression profiles of tumor-distant, histologically normal bronchial biopsy specimens obtained from current smokers with NSCLC or HNC (SC, considered as a single group), as well as nonsmokers (NS) and smokers without cancer (SNC). RNA from a total of 97 biopsies was used for gene expression profiling (Affymetrix HG-U133 Plus 2.0 array). Differentially expressed genes were used to compare NS, SNC, and SC, and functional analysis was carried out using Ingenuity Pathway Analysis (IPA). Smoking-related cancer of the respiratory tract was found to affect the expression of genes encoding xenobiotic biotransformation proteins, as well as proteins associated with crucial inflammation/immunity pathways and other processes that protect the airway from the chemicals in cigarette smoke or contribute to carcinogenesis. Finally, we used the prediction analysis for microarray (PAM) method to identify gene signatures of cigarette smoking and cancer, and uncovered a 15-gene signature that distinguished between SNC and SC with an accuracy of 83%. Thus, gene profiling of histologically normal bronchial biopsy specimens provided insight into cigarette-induced carcinogenesis of the respiratory tract and gene signatures of cancer in smokers.

The histone deacetylase inhibitor MGCD0103 induces apoptosis in B-cell chronic lymphocytic leukemia cells through a mitochondria-mediated caspase activation cascade.

Clinical trials have shown activity of the isotype-selective histone deacetylase (HDAC) inhibitor MGCD0103 in different hematologic malignancies. There are data to support the use of HDAC inhibitors in association with other cancer therapies. To propose a rational combination therapy, it is necessary to depict the molecular basis behind the cytotoxic effect of MGCD0103. In this study, we found that MGCD0103 was substantially more toxic in neoplastic B cells relative to normal cells, and we described the death pathways activated by MGCD0103 in B-cell chronic lymphocytic leukemia (CLL) cells from 32 patients. MGCD0103 decreased the expression of Mcl-1 and induced translocation of Bax to the mitochondria, mitochondrial depolarization, and release of cytochrome c in the cytosol. Caspase processing in the presence of the caspase inhibitor Q-VD-OPh and time course experiments showed that caspase-9 was the apical caspase. Thus, MGCD0103 induced the intrinsic pathway of apoptosis in CLL cells. Moreover, MGCD0103 treatment resulted in the activation of a caspase cascade downstream of caspase-9, caspase-dependent amplification of mitochondrial depolarization, activation of calpain, and Bax cleavage. We propose a model whereby the intrinsic pathway of apoptosis triggered by MGCD0103 in CLL is associated with a mitochondrial death amplification loop.