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Understanding resistance mechanisms in cancer is of utmost importance for the discovery of novel “druggable” targets. Efficient genetic screening, now even more possible with CRISPR‐Cas9 gene‐editing technology, next‐generation sequencing and bioinformatics, is an important tool for deciphering novel cellular processes, such as resistance to treatment in cancer. Imatinib specifically eliminates chronic myeloid leukemia (CML) cells by targeting and blocking the kinase activity of BCR‐ABL1; however, resistance to treatment exists. In order to discover BCR‐ABL1 independent mechanisms of imatinib resistance, we utilized the genome‐scale CRISPR knock‐out library to screen for imatinib‐sensitizing genes in vitro on K562 cells. We revealed genes that seem essential for imatinib‐induced cell death, such as proapoptotic genes (BIM, BAX) or MAPK inhibitor SPRED2. Specifically, reestablishing apoptosis in BIM knock‐out (KO) cells with BH3 mimetics, or inhibiting MAPK signaling in SPRED2 KO cells with MEK inhibitors restores sensitivity to imatinib. In this work, we discovered previously identified pathways and novel pathways that modulate response to imatinib in CML cell lines, such as the implication of the Mediator complex, mRNA processing and protein ubiquitinylation. Targeting these specific genetic lesions with combinational therapy can overcome resistance phenotypes and paves the road for the use of precision oncology., We utilized a genome‐scale CRISPR library to discover genes and pathways that could potentially be the target of novel therapeutic strategies for overcoming resistance to imatinib in chronic myeloid leukemia in vitro.

Background: Mutations in CALR observed in myeloproliferative neoplasms (MPN) were recently shown to be pathogenic via their interaction with MPL and the subsequent activation of the Janus Kinase &ndash, Signal Transducer and Activator of Transcription (JAK-STAT) pathway. However, little is known on the impact of those variant CALR proteins on endoplasmic reticulum (ER) homeostasis. Methods: The impact of the expression of Wild Type (WT) or mutant CALR on ER homeostasis was assessed by quantifying the expression level of Unfolded Protein Response (UPR) target genes, splicing of X-box Binding Protein 1 (XBP1), and the expression level of endogenous lectins. Pharmacological and molecular (siRNA) screens were used to identify mechanisms involved in CALR mutant proteins degradation. Coimmunoprecipitations were performed to define more precisely actors involved in CALR proteins disposal. Results: We showed that the expression of CALR mutants alters neither ER homeostasis nor the sensitivity of hematopoietic cells towards ER stress-induced apoptosis. In contrast, the expression of CALR variants is generally low because of a combination of secretion and protein degradation mechanisms mostly mediated through the ER-Associated Degradation (ERAD)-proteasome pathway. Moreover, we identified a specific ERAD network involved in the degradation of CALR variants. Conclusions: We propose that this ERAD network could be considered as a potential therapeutic target for selectively inhibiting CALR mutant-dependent proliferation associated with MPN, and therefore attenuate the associated pathogenic outcomes.

International audience; Genomic instability drives cancer progression by promoting genetic abnormalities that allow for the multi-step clonal selection of cells with growth advantages. We previously reported that the IL-9-dependent TS1 cell line sequentially acquired activating substitutions in JAK1 and JAK3 upon successive selections for growth factor independent and JAK inhibitor-resistant cells, suggestive of a defect in mutation avoidance mechanisms. In the first part of this paper, we discovered that the gene encoding mutL homolog-1 (MLH1), a key component of the DNA mismatch repair system, is silenced by promoter methylation in TS1 cells. By means of stable ectopic expression and RNA interference methods, we showed that the high frequencies of growth factor-independent and inhibitor-resistant cells with activating JAK mutations can be attributed to the absence of MLH1 expression. In the second part of this paper, we confirm the clinical relevance of our findings by showing that chronic myeloid leukemia relapses upon ABL-targeted therapy correlated with a lower expression of MLH1 messenger RNA. Interestingly, the mutational profile observed in our TS1 model, characterized by a strong predominance of T:A>C:G transitions, was identical to the one described in the literature for primitive cells derived from chronic myeloid leukemia patients. Taken together, our observations demonstrate for the first time a causal relationship between MLH1-deficiency and incidence of oncogenic point mutations in tyrosine kinases driving cell transformation and acquired resistance to kinase-targeted cancer therapies.

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in chronic myeloid leukemia (CML) and in Bcr-Abl–positive acute lymphoblastic leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second-generation inhibitors of Bcr-Abl tyrosine kinase such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or imatinib-intolerant disease. In the current study, we generated nilotinib-resistant cell lines and investigated their mechanism of resistance. Overexpression of BCR-ABL and multidrug resistance gene (MDR-1) were found among the investigated mechanisms. We showed that nilotinib is a substrate of the multidrug resistance gene product, P-glycoprotein, using verapamil or PSC833 to block binding. Up-regulated expression of p53/56 Lyn kinase, both at the mRNA and protein level, was found in one of the resistant cell lines and Lyn silencing by small interfering RNA restored sensitivity to nilotinib. Moreover, failure of nilotinib treatment was accompanied by an increase of Lyn mRNA expression in patients with resistant CML. Two Src kinase inhibitors (PP1 and PP2) partially removed resistance but did not significantly inhibit Bcr-Abl tyrosine kinase activity. In contrast, dasatinib, a dual Bcr-Abl and Src kinase inhibitor, inhibited the phosphorylation of both BCR-ABL and Lyn, and induced apoptosis of the Bcr-Abl cell line overexpressing p53/56 Lyn. Such mechanisms of resistance are close to those observed in imatinib-resistant cell lines and emphasize the critical role of Lyn in nilotinib resistance. [Cancer Res 2008;68(23):9809–16]

International audience; The het-e gene of the filamentous fungus Podospora anserina is involved in vegetative incompatibility. Co-expression of antagonistic alleles of the unlinked loci het-e and het-c triggers a cell death reaction that prevents the formation of viable heterokaryons between strains that contain incompatible combinations of het-c and het-e alleles. The het-elA gene encodes a polypeptide that contains a putative GTP-binding site and WD40 repeats. The role of these two domains in the reactivity of the HET-E protein in incompatibility was analyzed. An in vitro assay confirmed that the first domain is functional and can bind GTP and not ATP, suggesting that GTP-binding is essential for triggering the incompatibility reaction. The relationship between the number of WD40 repeats and the reactivity of the protein in incompatibility was investigated by estimating this number in different wild-type and mutant het-e alleles. It was deduced that reactive alleles contain a minimal number of ten WD40 repeats. These results demonstrate that the reactivity of the HET-E protein depends on two functional elements, a GTP-binding domain and several WD40 repeats. These motifs are present in separate polypeptides in trimeric G proteins, suggesting that HET-E polypeptides are also involved in signal transduction. Disruption of the het-e locus does not impair the phenotype of strains but DNA hybridization analyses revealed that het-e may belong to a multigenic family.

International audience; Vegetative incompatibility is widespread in fungi but its molecular mechanism and biological function are still poorly understood. A way to study vegetative incompatibility is to investigate the function of genes whose mutations suppress this phenomenon. In Podospora anserina, these genes are known as mod genes. In addition to suppressing vegetative incompatibility, mod mutations cause some developmental defects. This suggests that the molecular mechanisms of vegetative incompatibility and development pathway are interconnected. The mod-E1 mutation was isolated as a suppressor of the developmental defects of the mod-D2 strain. We show here that mod-E1 also partially suppresses vegetative incompatibility, strengthening the link between development and vegetative incompatibility. mod-E1 is the first suppressor of vegetative incompatibility characterized at the molecular level. It encodes a member of the Hsp90 family, suggesting that development and vegetative incompatibility use common steps of a signal transduction pathway. The involvement of mod-E in the sexual cycle has also been further investigated.

Background: Resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) can either originate from mutations in the BCR-ABL1 gene, which are mostly well characterized, or emerge from unknown alternative mutations elsewhere in the genome. Small hairpin (sh)RNA screens have been used to discover such genes but are becoming limited due to sup-optimal protein depletion and non-reliable off-target effects. More efficient screening techniques in human cells are now available as a result of the increasing understanding of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) /Cas9 system. Aims: Our goal is to uncover imatinib (IM)-sensitizing genes that cause IM resistance when knocked-out. Characterizing these genes may help understand the mechanisms of IM uptake, metabolism, degradation and/or activity in CML cells. Additionally, we also expect to unveil alternative, BCR-ABL1 independent, oncogenic pathways in CML cells. Methods: In order to find other genes involved in IM resistance, we performed a genome scale CRISPR knock-out (GeCKO) screen, which contains 121,413 sgRNAs that target 20,914 protein coding genes and miRNAs. We transduced one sgRNA per cell and challenged the K562-GeCKO cell pool to IM selection. We compared the abundance of sgRNAs between pre/post-IM treatment by next generation sequencing (NGS). Results: After IM selection, the sgRNAs from surviving cells were identified by NGS and unveiled potential IM-sensitizing genes. The most enriched sgRNAs (FDR < 0.01) targeted genes involved in transcriptional (KLF1, MED24) and translational (EIF2AK1, UBE2M) regulation, apoptosis (BAX, BCL2L11) and cell cycle regulation (BAP1, SPRED2). Subsequent screens on LAMA84 cells are currently underway in order to validate our findings. Additionally, the establishment of individual gene knock-out cell lines are in progress in order to fully understand the role of each gene in IM resistance. Summary/Conclusion: Using a CRISPR knock-out screen, we produced a list of 19 genes (FDR < 0.05) that may play a role in IM resistance. Encouragingly, a subset of these genes (BAX, BAP1, BCL2L11 and SPRED2) have already been correlated to CML progression and/or TKI resistance in the past. We aim to bolster our findings by establishing individual gene KO cell lines and study resistance in LAMA84 cells. The utilization of CRISPR libraries may not only help understand TKI resistance in CML, but also help identify numerous novel genes involved in drug resistances for a myriad of different diseases. Disclosures Mahon: ARIAD: Honoraria; PFIZER: Honoraria; BMS: Consultancy, Honoraria; NOVARTIS PHARMA: Consultancy, Honoraria, Research Funding.

International audience; The het-c locus of the filamentous fungus Podospora anserina controls heterokaryon formation through genetic interaction with alleles of the unlinked loci het-e and het-d. We have isolated four wild-type and two mutant alleles of the het-c locus. A comparison of the predicted proteins encoded by the different wild-type alleles revealed an unusual high level of amino-acid replacements compared to silent polymorphisms but only one amino-acid difference is sufficient to modify the specificity of het-c alleles. Chimeric genes constructed in vitro may exhibit a new specificity different from that of any known wild-type allele.

Despite the excellent efficacy of imatinib in chronic myeloid leukemia (CML), the response in patients is heterogeneous, which may in part be caused by pharmacogenetic variability. Imatinib has been reported to be a substrate of the P-glycoprotein pump. In the current study, we focused on the ABCB1 (MDR1) genotype. We analyzed the 3 most relevant single nucleotide polymorphisms of MDR1 in 90 CML patients treated with imatinib. Among the patients homozygous for allele 1236T, 85% achieved a major molecular response versus 47.7% for the other genotypes (P = .003). For the 2677G>T/A polymorphism, the presence of G allele was associated with worse response (77.8%, TT/TA; vs 47.1%, GG/GA/GT; P = .018). Patients with 1236TT genotype had higher imatinib concentrations. One of the haplotypes (1236C-2677G-3435C) was statistically linked to less frequent major molecular response (70% vs 44.6%; P = .021). Hence, we demonstrated the usefulness of these single nucleotide polymorphisms in the identification of CML who may or may not respond optimally to imatinib.

International audience; Understanding the molecular basis of resistance to imatinib, a tyrosine kinase inhibitor used as front-line therapy in chronic myeloid leukemia, remains a challenge for successful treatment. In an attempt to identify new mechanisms of resistance, we performed a comparative proteomic analysis of an imatinib-resistant cell line generated from the erythroblastic cell line K562 (K562-r) for which no known mechanism of resistance has been detected. Bidimensional gel electrophoresis was carried out to compare the protein expression pattern of imatinib-sensitive and of imatinib-resistant K562 cells. Among the 400 matched spots on five pairs of gels, only 14 spots had a significantly increased or decreased expression leading to the identification of 24 proteins identified as scaffold proteins, metabolic enzymes, DNA translation and maturation, and chaperon proteins. Among the chaperon family, only Hsp70 and Hsc70 are overexpressed in K562-r, results confirmed by Western blotting. We recently reported the participation of Hsp70 overexpression in imatinib resistance whereas a role for Hsc70 has yet to be determined. Hsc70 is not involved in imatinib resistance as the inhibition of its expression by siRNA does not restore sensitivity to imatinib. In contrast, the induced decreased expression of Hsc70 was accompanied by a greater overexpression of Hsp70. This proteomic study therefore suggests opposing roles of Hsp70 and Hsc70 in imatinib resistance.

The two allelic genes s and S are responsible for heterogenic incompatibility between wild type strains of the fungus Podospora anserina. The s gene has been cloned by SIB selection and expression in a strain containing a neutral allele of this locus. The S gene was isolated from a genomic library using the DNA of the s locus as a probe. The physical map of the DNA fragments carrying the two genes are highly dissimilar and restriction polymorphism exists at the s locus between s and S strains. Nevertheless, homology between the two alleles was revealed by cross-hybridization at the DNA and RNA levels.

International audience; Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative disorder characterized by the expression of the recombinant oncoprotein Bcr-Abl. In this investigation, we studied an imatinib-resistant cell line (K562-r) generated from the K562 cell line in which none of the previously described mechanisms of resistance had been detected. A threefold increase in the expression of the heat-shock protein 70 (Hsp70) was detected in these cells. This increase was not associated to heat-shock transcription factor-1 (HSF-1) overexpression or activation. RNA silencing of Hsp70 decreased dramatically its expression (90%), and was accompanied by a 34% reduction in cell viability. Overexpression of Hsp70 in the imatinib-sensitive K562 line induced resistance to imatinib as detected by a large reduction in cell death in the presence of 1 muM of imatinib. Hsp70 level was also increased in blast cells of CML patients resistant to imatinib, whereas the level remained low in responding patients. Taken together, the results demonstrate that overexpression of Hsp70 can lead to both in vitro and in vivo resistance to imatinib in CML cells. Moreover, the overexpression of Hsp70 detected in imatinib-resistant CML patients supports this mechanism and identifies potentially a marker and a therapeutic target of CML evolution.

International audience; A Podospora anserina BAC library of 4800 clones has been constructed in the vector pBHYG allowing direct selection in fungi. Screening of the BAC collection for centromeric sequences of chromosome V allowed the recovery of clones localized on either sides of the centromere, but no BAC clone was found to contain the centromere. Seven BAC clones containing 322,195 and 156,244bp from either sides of the centromeric region were sequenced and annotated. One 5S rRNA gene, 5 tRNA genes, and 163 putative coding sequences (CDS) were identified. Among these, only six CDS seem specific to P. anserina. The gene density in the centromeric region is approximately one gene every 2.8kb. Extrapolation of this gene density to the whole genome of P. anserina suggests that the genome contains about 11,000 genes. Synteny analyses between P. anserina and Neurospora crassa show that co-linearity extends at the most to a few genes, suggesting rapid genome rearrangements between these two species.

International audience; Among filamentous fungi capable of mycelial growth, het genes play crucial roles by regulating heterokaryon formation between different individuals. When fusion occurs between fungal mycelia that differ genetically at their het loci, the resulting heterokaryotic cells are quickly destroyed. It is unclear how het gene products of Podospora anserina trigger heterokaryon incompatibility. One unexplored possibility is that glycosphingolipids play a role because the het-c2 gene encodes a protein that displays 32% sequence identity and an additional 30% similarity to the mammalian glycolipid transfer protein. Here, P. anserina protoplasts containing wild-type het-c2 genes were shown to have greater glycosphingolipid transfer activity than protoplasts with disrupted het-c2 genes, a condition previously linked to altered cell compatibility following hyphal fusion. The observed glycolipid transfer activity could not be accounted for by nonspecific lipid transfer protein activity. Direct assessment showed that purified, recombinant HET-C2 accelerates the intermembrane transfer of glycolipid in vitro, but that the HET-C2 activity is mitigated much less by negatively charged membranes than the mammalian glycolipid transfer protein. The findings are discussed within the context of HET-C2 being a member of an emerging family of ancestral sphingolipid transfer proteins that play important roles in cell proliferation and accelerated death.

Vegetative incompatibility, which is very common in filamentous fungi, prevents a viable heterokaryotic cell from being formed by the fusion of filaments from two different wild-type strains. Such incompatibility is always the consequence of at least one genetic difference in specific genes (het genes). In Podospora anserina, alleles of the het-e and het-d loci control heterokaryon viability through genetic interactions with alleles of the unlinked het-c locus. The het-d2Y gene was isolated and shown to have strong similarity with the previously described het-e1A gene. Like the HET-E protein, the HET-D putative protein displayed a GTP-binding domain and seemed to require a minimal number of 11 WD40 repeats to be active in incompatibility. Apart from incompatibility specificity, no other function could be identified by disrupting the het-d gene. Sequence comparison of different het-e alleles suggested that het-e specificity is determined by the sequence of the WD40 repeat domain. In particular, the amino acids present on the upper face of the predicted β-propeller structure defined by this domain may confer the incompatible interaction specificity.

International audience; Vegetative incompatibility limits heterokaryon formation in fungi. It results from genetic differences at specific loci (het loci). Characterization of het genes and, more recently, of incompatibility reaction suppressors has, provided insight into the mechanisms involved. A link between development, vegetative incompatibility and signaling pathways has been established in Podospora anserina.

Cell death via vegetative incompatibility is widespread in fungi but molecular mechanism and biological function of the process are poorly understood. One way to investigate this phenomenon was to study genes named mod that modified incompatibility reaction. In this study, we cloned the mod-D gene that encodes a Gα protein. The mod-D mutant strains present developmental defects. Previously, we showed that the mod-E gene encodes an HSP90. The mod-E1 mutation suppresses both vegetative incompatibility and developmental defects due to the mod-D mutation. Moreover, we isolated the PaAC gene, which encodes an adenylate cyclase, as a partial suppressor of the mod-D1 mutation. Our previous results showed that the molecular mechanisms involved in vegetative incompatibility and developmental pathways are connected, suggesting that vegetative incompatibility may result from disorders in some developmental steps. Our new result corroborates the involvement of mod genes in signal transduction pathways. As expected, we showed that an increase in the cAMP level is able to suppress the defects in vegetative growth due to the mod-D1 mutation. However, cAMP increase has no influence on the suppressor effect of the mod-D1 mutation on vegetative incompatibility, suggesting that this suppressor effect is independent of the cAMP pathway.

International audience; In Podospora anserina a linear plasmid with telomeric ends behaves as an artificial acentric minichromosome. Transformation is at least 100 times more efficient than with integrative vectors. Genomic DNA was inserted in this plasmid in vitro and the mixture used to transform a leu1-1 strain. Many fungal clones containing the leu1 gene as a genomic insert in the linear plasmid were identified. The leu1 gene was rescued as a circular plasmid in Escherichia coli demonstrating that a direct cloning procedure can be applied for the fungus P. anserina. The conservation of telomeric sequences among filamentous fungi suggests that a telomere-based linear plasmid could provide a general cloning vector for filamentous fungi.

To identify cellular functions involved in vegetative incompatibility in filamentous fungi, we have initiated the cloning of Podospora anserina (Pa) mod genes. These genes interfere with the lethal reaction triggered by interaction between incompatible het genes. A gene ( Pa AC ) has been cloned by complementation of developmental defects caused by a mutation in the mod-D gene. This gene encodes a protein of 2145 amino acids (aa) that exhibits strong similarities with many adenylate cyclases (AC). About 65% aa identity has been found between the sequence of the polypeptide encoded by this Pa AC gene and the AC of Neurospora crassa . The organization of peptidic domains in the polypeptide encoded by Pa AC is closely related to that of Saccharomyces cerevisiae CYRI. Restriction-fragment-length polymorphism (RFLP) and genetic analysis have shown that Pa AC and mod-D are distinct genes

International audience; The het-e-1 gene of the fungus Podospora anserina is responsible for vegetative incompatibility through specific interactions with different alleles of the unlinked gene, het-c. Coexpression of two incompatible genes triggers a cell death reaction that prevents heterokaryon formation. The het-e1 allele has been cloned to get information on the function of the locus. It encodes a putative 1356-amino-acid polypeptide that displays two sequence motifs that have not yet been reported to be present on a single polypeptide. They are a GTP-binding domain and a repeated region that shares similarity with that of the beta-transducin. Contrary to other members of the beta-transducin family, sequence conservation between the repeated units is very strong and the number of repeats is different in wild-type het-e alleles.

International audience; Somatic or vegetative incompatibility is widespread in filamentous fungi. It prevents the coexistence of genetically different nuclei within a common cytoplasm. Cloning the het genes that control this process has been achieved in several species. This has provided essential information on the function of the genes in the biology of fungi and has also led to the formulation of models that may explain similar phenomena in other organisms.

International audience; Vegetative incompatibility is a lethal reaction that destroys the heterokaryotic cells formed by the fusion of hyphae of non-isogenic strains in many fungi. That incompatibility is genetically determined is well known but the function of the genes triggering this rapid cell death is not. The two allelic incompatibility genes, s and S, of the fungus Podospora anserina were characterized. Both encode 30 kDa polypeptides, which differ by 14 amino acids between the two genes. These two proteins are responsible for the incompatibility reaction that results when cells containing s and S genes fuse. Inactivation of the s or S gene by disruption suppresses incompatibility but does not affect the growth or the sexual cycle of the mutant strains. This suggests that these incompatibility genes have no essential function in the life cycle of the fungus.

International audience; The sequences of homologous DNA regions of two wild-type strains of the fungus Podospora anserina, revealed in one strain the presence of a 349bp insertion leading to a RFLP. This DNA sequence is repeated in the genome and some of its locations are different in various wild-type strains. This DNA element exhibits structural similarities with the yeast solo delta, sigma or tau elements.

Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative syndrome characterised by the expression of the recombinant oncoprotein Bcr-Abl. Imatinib inhibits Bcr-Abl tyrosine kinase activity leading to apoptosis of leukemic cells sparing normal hematopoiesis. Several mechanisms of resistance to imatinib have been identified both in vitro and in vivo: Bcr-Abl mutations, an over-expression of the Bcr-Abl kinase itself or other tyrosine kinase bypass. To identify unknown mechanism, we used an imatinib resistant cell line (K562-R) generated from the erythroblastic cell line K562 (K562-S) (Blood, 2000; 93: 1070–1079) for which all described mechanisms of resistance have been previously invalidated. Previous results from a proteomic study identified some chaperon proteins such as heat shock proteins with an increased expression level in K562-R. One of them, the heat shock protein 70, Hsp70, has a 3 fold increase expression level in K562-R cells, results which have been confirmed by western-blot analysis. To characterise the role of Hsp 70 in imatinib resistance, we inhibit Hsp 70 expression by RNA silencing (siRNA) in K562-R cells and over-express it in K562-S cells. Inhibition of Hsp70 protein expression by siRNA decrease Hsp70 expression rapidly over 90% at day 4 which is associated with a significant reduction of viability (66 ± 6%, n = 5, p < 0.03). Over expression of Hsp 70 in K562-S cells induced a significant increase of resistance to imatinib since the addition of imatinib only increases mortality by 27 ± 5 % in comparison to 52 ± 4 % for K562-S cells (n = 4, p < 0.001). Detection of HSF-1 phosphorylation, the major transcription factor involved in Hsp 70 expression, did not show significant differences between K562-S and K562-R cells although over a 3 fold increase is detected in the mRNA level of Hsp 70 in K562-R cells by quantitative PCR. Furthermore, the comparison of Hsp70 expression in mononuclear cells of 7 CML patients before imatinib treatment and at the relapse time shows that Hsp 70 is increased in imatinib resistant patients suggesting it could also play a role in resistance in vivo. Present study confirmed that over expression of Hsp 70 in the cell line K562-R is involved in the mechanism of imatinib resistance in vitro. Moreover, the correlation between the increase of Hsp 70 in CML patient cells and resistance suggests it could be an interesting marker and potentially a therapeutic target.

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myelogenous Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Imatinib is a selective inhibitor of Bcr-Abl tyrosine kinase and is now used in frontline therapy for CML. However clinical resistance is the main concern using this treatment, mediated by mutations within the kinase domain of Bcr-Abl, amplification of the BCR-ABL genomic locus or other as yet unknown mechanisms. AMN107 (Novartis Pharma AG, Basel, Switzerland) is a synthetic, second generation inhibitor of Bcr-Abl tyrosine kinase. In the current study, we tested AMN107 against different Bcr-Abl positive cell lines such as K562, LAMA84, AR230 or murine Ba/F3 cells transfected with BCR-ABL (BaF/BCR-ABL). In a 4 day proliferation assay (MTS) the dose of AMN107 that inhibited 90% of the cells (IC90) was 0.01 μM which was 100-fold lower than the IC90 of imatinib. In addition, proliferation of imatinib resistant cell lines which exhibited amplification of BCR-ABL was inhibited by 75% in the presence of 0.01 μM AMN107. However, Ba/F3 cells expressing the imatinib resistant BCR-ABL T315I mutant were only inhibited with 10μM of AMN107. Furthermore, K562-R, an imatinib-resistant cell line exhibiting a new mechanism of imatinib resistance (modification of chaperone proteins such as heat shock proteins) was also insensitive to AMN107; the IC90 for AMN107 at day 4 was 1μM versus 4μM for imatinib. Finally, we investigated potential resistance to AMN107 in Bcr-Abl positive cells. Resistant cell lines were generated after long-term (2 month) gradual dose-escalation exposure to the inhibitor. Up to now, we have obtained four cell lines, AR230-ra, K562-ra, LAMA 84-ra, and BaF/BCR-ABL-ra resistant to 8, 10, 10, and 100 nM of AMN107, respectively. Resistance was defined as the capacity to survive in the continuous presence of doses of AMN107 that kill in three days more than 90% of the parental cells in liquid culture. Preliminary investigations of AMN107 resistance using western blot and cytometry have shown that only BaF/BCR-ABL-ra overexpressed wildtype Bcr-Abl as we have already reported for imatinib resistance. Studies with these resistant cell lines investigating cross resistance with imatinib and looking for additional mechanisms of resistance are in progress.We conclude that in vitro, AMN107 is more powerful than imatinib in inhibiting the proliferation of BCR-ABL positive cell lines. In addition, we have demonstrated that it is possible to develop resistant cell lines to this new inhibitor of Bcr-Abl.

Targeting the tyrosine kinase activity of Bcr-Abl by imatinib mesylate is an attractive therapeutic strategy in chronic myelogenous leukemia (CML) and in Bcr-Abl positive acute lymphoblastic leukemia. However, resistance to imatinib monotherapy is currently a major issue preventing the successful treatment of CML patients. It may be mainly mediated by mutations within the kinase domain of Bcr-Abl and/or amplification of the BCR-ABL genomic locus. The K562-r imatinib-resistant cell line, derived in our laboratories from the sensitive parental K562-s has neither mechanism of resistance, nor overexpression of Src-kinases such as Lyn and Hck, as described for other cell lines. In the current study we used two-dimensional (2D) difference gel electrophoresis (DIGE) and MALDI-TOF-TOF mass spectrometry to compare the proteome of K562-r and K562-s. With the aid of the Image Master TM 2D platinium software, we detected 31 different proteins in K562-r and K562-s. These proteins were classified in 3 different groups. The first includes proteins involved in the synthesis and stability of RNA (hnRNP K, hnRNP H, CstF , transcription elongation factor A protein 1, PCBP2, TCP1), the second encompasses structural proteins (CAPG, fascin, tubulin, vimentin, laminA, C tubulin beta-1 chain, actin cytoplasmic 1, keratin type I type II), and the third was represented by different enzymes participating in general metabolic pathways (glyceraldehyde 3-phosphate dehydrogenase, malate dehydrogenase, mitochondrial precursor, glutamate dehydrogenase 2, pyruvate kinase, PURH protein). Furthermore, chaperone proteins such as heat-shock protein Hsp60, P60HOP or STI-1, Hsp105 and Hsp70 were differentially expressed in the sensitive and resistant cell lines. Since these proteins complex with Hsp90 and this complex has been reported to interact with the Bcr-Abl protein, we focused on these molecular chaperones. Hsp70 family proteins such as Hsc70 and Hsp74 were found to be more expressed 2.5-fold higher in K562-r than in K562-s, and/or exhibited post translational modifications (phosphorylation and acetylation) confirmed by Western blotting. Hsp70 was recently described as an inhibitor of apoptosis (Ray S et al., JBC 2004) and its overexpression in K562-r could thus contribute to its imatinib-resistant phenotype. Preliminary functional studies showed that whereas K562-s and K562-r were equally sensitive to the apoptotic effect of geldanamycin (an inhibitor of Hsp90), the combination of geldanamycin and a proteasome inhibitor (MG132) was more efficient in K562-r than in K562-s (viability of 16% and 40% respectively after 4 days in culture). Ongoing experiments utilizing siRNA against Hsp70 will help understand the link between the expression profile of Hsp proteins and the imatinib-resistant phenotype of this cell line. In conclusion, the use of a new experimental strategy, i.e. proteomic analysis by DIGE and mass spectrometry, allowed us to identify selected proteins whose patterns of expression and post-translational modification may underlie a new mechanism of resistance to imatinib in Bcr-Abl positive cells.

International audience; We have sequenced the ura5 gene of the filamentous fungus Podospora anserina. The deduced sequence for the orotidylic acid pyrophosphorylase (OMPppase) has been compared with the Escherichia coli enzyme which is the only known sequence for this enzyme. This comparison shows extensive blocks of homology. The expression of the ura5 gene has been studied in a ura5 mutant which has been transformed by a recombinant plasmid carrying the ura5 gene. We observed that strains carrying integrated multicopies of the transforming vector exhibit higher specific activity for OMPppase than wild type (wt). By recombination we have constructed a strain in which the level of this enzyme is 32 times higher than in the wt strain.