Jerome Kluza

Objective Long-term treatment with tyrosine kinase inhibitors (TKI) represents an effective cure for chronic myeloid leukemia (CML) patients and discontinuation of TKI therapy is now proposed to patient with deep molecular responses. However, evidence demonstrating that TKI are unable to fully eradicate dormant leukemic stem cells (LSC) indicate that new therapeutic strategies are needed to control LSC and to prevent relapse. In this study we investigated the metabolic pathways responsible for CML surviving to imatinib exposure and its potential therapeutic utility to improve the efficacy of TKI against stem-like CML cells. Methods Using complementary cell-based techniques, metabolism was characterized in a large panel of BCR-ABL+ cell lines as well as primary CD34+ stem-like cells from CML patients exposed to TKI and L-Asparaginases. Colony forming cell (CFC) assay and flow cytometry were used to identify CML progenitor and stem like-cells. Preclinical models of leukemia dormancy were used to test the effect of treatments. Results Although TKI suppressed glycolysis, compensatory glutamine-dependent mitochondrial oxidation supported ATP synthesis and CML cell survival. Glutamine metabolism was inhibited by L-asparaginases such as Kidrolase or Erwinase without inducing predominant CML cell death. However, clinically relevant concentrations of TKI render CML cells susceptible to Kidrolase. The combination of TKI with Lasparaginase reactivates the intinsic apoptotic pathway leading to efficient CML cell death. Conclusion Targeting glutamine metabolism with the FDA-approved drug, Kidrolase in combination with TKI that suppress glycolysis represents an effective and widely applicable therapeutic strategy for eradicating stem-like CML cells.

Background: Air pollution-derived particulate matter (PM) is considered as one of the major environmental risk to health worldwide. Despite intensive research effort, the mechanisms underlying PM2.5 toxicity remain unclear. Objectives: To better clarify whether the mitochondrial dysfunction is related to the cell homeostasis disruption by PM2.5 exposure. Methods: Oxidative, inflammatory, apoptotic and mitochondrial endpoints were studied in BEAS-2B, normal human bronchial epithelial (NHBE) and COPD-diseased human bronchial epithelial (DHBE) cells acutely or repeatedly exposed to PM2.5 (2 and 10 µg/cm²). Results: The mitochondrial morphology was altered in the cells repeatedly exposed to the highest dose of PM2.5. Dose- and exposure-dependent oxidative damages were seen in PM2.5-exposed cells, notably COPD-DHBE. NRF2 gene expression and binding activity, and the mRNA levels of its target genes, were related to the exposure number for the lowest dose, but not related for the highest dose. There were dose- and exposure-dependent increases of NF-kB binding activity and target cytokine secretion in PM2.5-exposed cells, and notably COPD-DHBE. Mitochondrial ROS production, membrane potential depolarization, oxidative phosphorylation, and ATP production were modified in these cells, notably COPD-DHBE, repeatedly exposed to the highest dose. Conclusions: The repeated exposure to BEAS-2B, NHBE and in particular sensible COPD-DHBE cells caused an oxidative boost able to partially inactivate the NRF2 signaling pathway and to critically impair mitochondrial redox homeostasis, thereby producing a persistent mitochondrial dysfunction and a lowering cell energy supply.

In vitro exposure to cigarette smoke extract (CSE) modulate the phenotype of monocyte-derived dendritic cells (MDDC) from healthy subjects activated by LPS. Expression of CD83 (a), HLA-DR (b), CD80 (c), CD86 (d), CD40 (e) and CD54 (f) was evaluated by flow cytometry in MDDC exposed to CSE and then activated or not by LPS for 24 h. Data are reported as mean fluorescence intensity (MFI) ± S.E.M. of 20 experiments. *P

T-cells intracellular staining strategy of gaiting. Cells were first gated using foward scatter (FSC) and side scatter (SSC) and next, T-cells were separated on CD8+ and CD4+ based on fluorescence of these two markers on CD45+ cells. IL-17 ans IFN-γ positive cells were calculated in comparison with baseline fluorescence of isotype controls. (PDF 422 kb)

In vitro exposure to rotenone (Rot) or antimycin A (AmA) inhibits the phenotype of monocyte-derived dendritic cells (MDDC) activated by S.pneumoniae (Sp). Expression of HLA-DR (a), CD80 (b), CD86 (c), CD40 (d) and CD54 (e) were evaluated by flow cytometry in MDDC exposed to rotenone or antimycin A and then activated or not by S.pneumoniae for 24 h. Data represent mean ± S.E.M. of 6 experitments. There were no statistical differences between groups. (PDF 50 kb)

Background: Triple - negative breast tumours, characterized by the absence of estrogen, progesterone and HER 2 receptors expression, are difficult to treat . Indeed, there is no targeted therapy for these tumours. Moreover, their response rate to conventional therapies is low, they often develop resistances and the recurrence rate is high. Energy metabolism is often modified in cancer cells and this is an interesting target to develop new therapeutic strategies. In this context, we are developing original derivatives of troglitazone , a thiazolidinedione that exhibits anticancer activity and metabolic effects . The objective of the present study was to characterize the effects of EP 13, a desulfured troglitazone derivative, on the triple - negative breast cancer cell line MDA - MB - 231. Methods : The triple - negative breast cancer cell line MDA - MB 231 was exposed to EP 13 for different time periods. Cell number was assessed by crystal violet staining assay. Apoptosis and cellula...

Besides its influence on survival, growth, proliferation, invasion and metastasis, cancer cell metabolism also greatly influences the cellular responses to molecular-targeted therapies. To review the recent advances in elucidating the metabolic effects of BRAF and MEK inhibitors (clinical inhibitors of the MAPK/ERK pathway) in melanoma and discuss the underlying mechanisms involved in the way metabolism can influence melanoma cell death and resistance to BRAF and MEK inhibitors. We also underlined the therapeutic perspectives in terms of innovative drug combinations. BRAF and MEK inhibitors inhibit aerobic glycolysis and induce high levels of metabolic stress leading to effective cell death by apoptosis in BRAF-mutated cancer cells. An increase in mitochondrial metabolism is required to survive to MAPK/ERK pathway inhibitors and the sub-population of cells that survives to these inhibitors are characterized by mitochondrial OXPHOS phenotype. Consequently, mitochondrial inhibition co...

The synthesis and DNA binding characteristics of a polyamide-intercalator conjugate, designed to inhibit NF-Y binding to the ICB-2 site of the topoisomerase IIalpha promoter and up-regulate the expression of the enzyme in confluent cells, are reported. Thermal denaturation and CD titration studies demonstrated binding to the cognate sequence (5'-AAGCTA-3'). Formation of ligand-induced CD bands at approximately 330 nm provided indication that the molecule interacts selectively in the minor groove of DNA. Intercalation was evidenced by a fivefold increase in emission of the intercalator moiety upon binding to the ICB-2 hairpin oligonucleotide. An increase in viscosity of a solution of calf-thymus DNA on addition of the conjugate provided further evidence. The binding affinity of the conjugate was ascertained using SPR (5.6x10(6) M(-1)), which according to a gel shift assay was capable of inhibiting the binding of NF-Y at a concentration of 50 microM. DNaseI footprinting, using the topoIIalpha promoter sequence, highlighted the specificity of the conjugate for the cognate site (5'-AAGCTA-3'). Finally, through Western blot analysis, confluent murine NIH 3T3 cells treated with conjugate were found to have enhanced expression of topoIIalpha. These results suggest that the conjugate can enter the nucleus, bind to its target site, presumably as a stacked dimer, and up-regulate the expression of topoIIalpha by blocking the binding of NF-Y.

The synthesis and DNA-binding properties of a novel naphthalimide-polyamide hairpin (3) designed to target the inverted CCAAT box 2 (ICB2) site on the topoisomerase IIalpha (topoIIalpha) promoter are described. The polyamide component of 3 was derived from the minor-groove binder, 2, and tailored to bind to the 5'-TTGGT sequence found in and flanking ICB2. The propensity of mitonafide 4 to intercalate between G-C base pairs was exploited by the incorporation of a naphthalimide moiety at the N terminus of 2. Hybrid 3 targeted 5'-CGATTGGT and covered eight contiguous base pairs, which included the underlined ICB2 site. DNase I footprinting analysis with the topoIIalpha promoter sequence demonstrated that 3 bound selectively to the ICB2 and ICB3 sites. Thermal-denaturation studies confirmed these results, and the highest degree of stabilization was found for ICB2 and -3 in preference to ICB1 (4.1, 4.6, and 0.6 degrees C, respectively). CD studies confirmed minor-groove binding and suggested a 1:1 binding stoichiometry. Emission-titration experiments established intercalative binding. Surface plasmon resonance results showed strong binding to ICB2 (2.5x10(7) M(-1)) with no observable binding to ICB1. Furthermore, the binding constant of 3 to ICB2 was larger than that of the parent polyamide 2. The increased binding affinity was primarily due to a reduction in the dissociation-rate constant of the polyamide-DNA complex, which can be attributed to the N-terminal naphthalimide moiety. In addition, the binding site of 3 was larger than that of 2, which innately improved sequence selectivity. We conclude that the polyamide-naphthalimide 3 selectively binds to the ICB2 site by simultaneous intercalation and minor-groove binding, and warrants further investigation as a model compound for the regulation of topoIIalpha gene expression.

Nuclease footprinting techniques were initially developed to investigate protein-deoxyribonucleic acid (DNA) interactions but these tools of molecular biology have also become instrumental for probing sequence-selective binding of small molecules to DNA. Here, the method is described and technical details are given for performing deoxyribonuclease (DNase) I footprinting with DNA-binding drugs. An example is presented where DNase I is used (as well as DNase II and micrococcal nuclease) to probe the patterns of sequence-selective recognition of DNA by the anticancer antibiotic actinomycin D. DNase I is a convenient endonuclease for detecting and locating the position of actinomycin-binding sites within GC-rich sequences.

Tafluposide (F11782), an epipodophyllotoxin derivative currently undergoing phase I clinical trials, is structurally close to the established anti-cancer drug etoposide, but mechanistically distinct. It is a dual inhibitor of topoisomerases I and II which impairs the binding of the enzyme to DNA, but does not stabilize the cleavage complex. Nevertheless, both etoposide and tafluposide induce DNA strand breaks and are potent pro-apoptotic agents. In this study, we have compared the cellular response of HL-60 human promyelocytic leukemia cells treated with etoposide and tafluposide. We show that tafluposide induces delayed, but extensive, DNA strand breaks, whereas etoposide provokes rapid and massive DNA damage. The two drugs trigger similar types of alterations at the mitochondrial and cell cycle levels, and lead to the generation of comparable levels of reactive oxygen species, but with different kinetics. Our data suggest that modification of the mitochondrial mass plays an important role in apoptosis induced by DNA-damaging anti-cancer agents, at least in the epipodophyllotoxin series. We suggest that drug-induced mitochondrial alterations can be divided into three successive steps: (i) hyperpolarization, (ii) depolarization and (iii) increase of the mitochondrial mass.

The N-terminal formamido group on imidazole- and pyrrole-containing polyamides causes stacked polyamides to bind in the minor groove of DNA in the staggered motif, and it also increases the binding affinity compared to those of non-formamido compounds. To further investigate the role of the N-terminal acylamido in affecting sequence specificity and binding affinity, six polyamide analogues containing the core triheterocyclic structure IPI were designed and synthesized, and the acylamido moiety reported herein includes the following: formamido (f-IPI, 1), acetamido (Ac-IPI, 2), trifluoroacetamido (Tf-IPI, 3), N-methylureido (Mu-IPI, 4), N-methylpyrrole-2-carboxamido (PIPI, 5), and the (13)C-labeled formamido-IPI compound ((13)C-f-IPI, 6). In addition, two nonacylated IPI compounds were also synthesized and examined, namely, the amino-containing (NH(2)-IPI, 7) and non-formamido (nf-IPI, 8) compounds. The binding characteristics of compounds 1-8 were investigated using methods of molecular biology and biochemistry, which included biophysical techniques, such as DNA melts, circular dichroism, isothermal titration calorimety, and surface plasmon resonance and DNase I footprinting. With the exception of nf-IPI and NH(2)-IPI, all other compounds preferentially interacted with the cognate sequence,…

Imidazole and pyrrole-containing polyamides belong to an important class of compounds that can be designed to target specific DNA sequences, and they are potentially useful in applications of controlling gene expression. The extent of polyamide curvature is an important consideration when studying the ability of such compounds to bind in the minor groove of DNA. The current study investigates the importance of curvature using polyamides of the form f-Im-Phenyl-Im, in which the imidazole heterocycles are placed in ortho-, meta-, and para-configurations of the phenyl moiety. The synthesis and biophysical evaluation of each compound binding to its cognate DNA sequence (5'-ACGCGT-3') and a negative control sequence…

... 4. Lamellarins (Bailly, 2004), a group of pyrrole alkaloids of marine origin typified by the hexacyclic compound lamellarin D, were characterized as a highly potent topoisomerase I poison and a promising anticancer agent (Facompré et al., 2003; Marco et al., 2005; Kluza et al ...

F14512, an epipodophyllotoxin derivative equipped with a spermine moiety, is selectively taken up by the polyamine transport system over-active in tumor cells. F14512 was identified as a selective anticancer agent with a broad spectrum of antitumor activities and is currently undergoing phase I clinical trial in onco-hematology. However, the mechanism by which F14512 exerts its selective effects on neoplastic cells remains poorly understood. In this study, using mainly P388 leukemia cells, we showed that activation of the DNA damage response by F14512 did not induce immediate apoptosis but resulted in an early growth arrest. F14512-induced G2 arrest was accompanied by the appearance of a senescence-like phenotype (characterized by an increased β-galactosidase staining) with up-regulation of the cyclin-dependent kinase inhibitor p16, and cyclin D1. The early senescence-based cell cycle block was characterized by a marked increase of the level of the IAP protein survivin, but not cIAP2, in P388 cells as well as in three other leukemia and melanoma cell types. The Thr(34)-phosphorylated form of survivin was observed within 4 h after F14512 exposure. Inhibition of survivin by siRNA resulted in a switch from senescence-like growth arrest to apoptosis. Compared with the parental drug etoposide, F14512-induced DNA damage signaling pathway resulted in greater senescence like-growth arrest and delayed apoptosis. Collectively, our data show that senescence arrest and subsequent apoptosis are powerful mechanisms mediating the chemotherapeutic effects of F14512 and identify survivin as the molecular determinant responsible for a qualitative shift in cell fate from senescence to apoptosis upon treatment with F14512.

In recent years, our knowledge regarding the metabolism of melanoma has greatly advanced and consequently new therapeutic strategies are being developed. This review is focused on metabolic pathways contributing to melanoma proliferation, the influence of BRAF inhibitors on those metabolic pathways and finally a presentation of potential therapeutic strategies aimed at blocking metabolic signaling pathways and therefore melanoma development.

We report the synthesis of a series of novel diphenylcarbazoles designed to interact with DNA. The compounds bearing two or three dimethylaminoalkyloxy side chains were found to bind much more tightly to DNA, preferentially at AT-rich sites, than the corresponding hydroxy compounds. The DNA binding compounds exhibit potent cytotoxic activity toward P388 leukemia cells. The 3,6-diphenylcarbazole thus represent an interesting scaffold to develop antitumor agents interacting with nucleic acids.

Imidazole (Im) and Pyrrole (Py)-containing polyamides that can form stacked dimers can be programmed to target specific sequences in the minor groove of DNA and control gene expression. Even though various designs of polyamides have been thoroughly investigated for DNA sequence recognition, the use of H-pin polyamides (covalently cross-linked polyamides) has not received as much attention. Therefore, experiments were designed to systematically investigate the DNA recognition properties of two symmetrical H-pin polyamides composed of PyImPyIm (5) or f-ImPyIm (3e, f=formamido) tethered with an ethylene glycol linker. These compounds were created to recognize the cognate 5'-ACGCGT-3' through an overlapped and staggered binding motif, respectively. Results from DNaseI footprinting, thermal denaturation, circular dichroism, surface plasmon resonance and isothermal titration microcalorimetry studies demonstrated that both H-pin polyamides bound with higher affinity than their respective monomers. The binding affinity of formamido-containing H-pin 3e was more than a hundred times greater than that for the tetraamide H-pin 5, demonstrating the importance of having a formamido group and the staggered motif in enhancing affinity. However, compared to H-pin 3e, tetraamide H-pin 5 demonstrated superior binding preference for the cognate sequence over its non-cognates, ACCGGT and AAATTT. Data from SPR experiments yielded binding constants of 1.6x10(8)M(-1) and 2.0x10(10)M(-1) for PyImPyIm H-pin 5 and f-ImPyIm H-pin 3e, respectively. Both H-pins bound with significantly higher affinity (ca. 100-fold) than their corresponding unlinked PyImPyIm 4 and f-ImPyIm 2 counterparts. ITC analyses revealed modest enthalpies of reactions at 298 K (DeltaH of -3.3 and -1.0 kcal mol(-1) for 5 and 3e, respectively), indicating these were entropic-driven interactions. The heat capacities (DeltaC(p)) were determined to be -116 and -499 cal mol(-1)K(-1), respectively. These results are in general agreement with DeltaC(p) values determined from changes in the solvent accessible surface areas using complexes of the H-pins bound to…