Stem cell-like reprogramming is required for leukemia-initiating activity in B-ALL.

B cell acute lymphoblastic leukemia (B-ALL) is a multistep disease characterized by the hierarchical acquisition of genetic alterations. However, the question of how a primary oncogene reprograms stem cell-like properties in committed B cells and leads to a preneoplastic population remains unclear. Here, we used the PAX5::ELN oncogenic model to demonstrate a causal link between the differentiation blockade, the self-renewal, and the emergence of preleukemic stem cells (pre-LSCs). We show that PAX5::ELN disrupts the differentiation of preleukemic cells by enforcing the IL7r/JAK-STAT pathway. This disruption is associated with the induction of rare and quiescent pre-LSCs that sustain the leukemia-initiating activity, as assessed using the H2B-GFP model. Integration of transcriptomic and chromatin accessibility data reveals that those quiescent pre-LSCs lose B cell identity and reactivate an immature molecular program, reminiscent of human B-ALL chemo-resistant cells. Finally, our transcriptional regulatory network reveals the transcription factor EGR1 as a strong candidate to control quiescence/resistance of PAX5::ELN pre-LSCs as well as of blasts from human B-ALL.

Inhibition of ubiquitin-specific protease 7 sensitizes acute myeloid leukemia to chemotherapy.

Resistance of acute myeloid leukemia (AML) to therapeutic agents is frequent. Consequently, the mechanisms leading to this resistance must be understood and addressed. In this paper, we demonstrate that inhibition of deubiquitinylase USP7 significantly reduces cell proliferation in vitro and in vivo, blocks DNA replication progression and increases cell death in AML. Transcriptomic dataset analyses reveal that a USP7 gene signature is highly enriched in cells from AML patients at relapse, as well as in residual blasts from patient-derived xenograft (PDX) models treated with clinically relevant doses of cytarabine, which indicates a relationship between USP7 expression and resistance to therapy. Accordingly, single-cell analysis of AML patient samples at relapse versus at diagnosis showed that a gene signature of the pre-existing subpopulation responsible for relapse is enriched in transcriptomes of patients with a high USP7 level. Furthermore, we found that USP7 interacts and modulates CHK1 protein levels and functions in AML. Finally, we demonstrated that USP7 inhibition acts in synergy with cytarabine to kill AML cell lines and primary cells of patients with high USP7 levels. Altogether, these data demonstrate that USP7 is both a marker of resistance to chemotherapy and a potential therapeutic target in overcoming resistance to treatment.

Regulation of CHK1 by the Ubiquitin-Proteasome System.

The checkpoint kinase 1 (CHK1) is a master regulator of genome integrity in vertebrate cells. Despite its important cell cycle functions, its regulation is still incompletely understood. Cassidy et al. provide novel insights on the regulation of the CHK1 abundance by the HECT E3 ligase HUWE1 during unperturbed cell cycle as well as in response to replicative stress. These results may help us to apprehend the underlying mechanism of tumorigenesis.

A PIM-CHK1 signaling pathway regulates PLK1 phosphorylation and function during mitosis.

Although the kinase CHK1 is a key player in the DNA damage response (DDR), several studies have recently provided evidence of DDR-independent roles of CHK1, in particular following phosphorylation of its S280 residue. Here, we demonstrate that CHK1 S280 phosphorylation is cell cycle-dependent and peaks during mitosis. We found that this phosphorylation was catalyzed by the kinase PIM2, whose protein expression was also increased during mitosis. Importantly, we identified polo-like kinase 1 (PLK1) as a direct target of CHK1 during mitosis. Genetic or pharmacological inhibition of CHK1 reduced the activating phosphorylation of PLK1 on T210, and recombinant CHK1 was able to phosphorylate T210 of PLK1 in vitro Accordingly, S280-phosphorylated CHK1 and PLK1 exhibited similar specific mitotic localizations, and PLK1 was co-immunoprecipitated with S280-phosphorylated CHK1 from mitotic cell extracts. Moreover, CHK1-mediated phosphorylation of PLK1 was dependent on S280 phosphorylation by PIM2. Inhibition of PIM proteins reduced cell proliferation and mitotic entry, which was rescued by expressing a T210D phosphomimetic mutant of PLK1. Altogether, these data identify a new PIM-CHK1-PLK1 phosphorylation cascade that regulates different mitotic steps independently of the CHK1 DDR function.This article has an associated First Person interview with the first author of the paper.

Targeting ATR/CHK1 pathway in acute myeloid leukemia to overcome chemoresistance.

Resistance of acute myeloid leukemia to current therapies leads to frequent relapses. Identification of molecular mechanisms involved in chemoresistance constitutes a key challenge to define new therapeutic concepts. Here, we show that the ATR/CHK1 pathway, essential in maintaining genomic stability, is involved in resistance and proliferation characteristics of leukemic cells.

CHK1 as a therapeutic target to bypass chemoresistance in AML.

The nucleoside analog cytarabine, an inhibitor of DNA replication fork progression that results in DNA damage, is currently used in the treatment of acute myeloid leukemia (AML). We explored the prognostic value of the expression of 72 genes involved in various aspects of DNA replication in a set of 198 AML patients treated by cytarabine-based chemotherapy. We unveiled that high expression of the DNA replication checkpoint gene CHEK1 is a prognostic marker associated with shorter overall, event-free, and relapse-free survivals and determined that the expression of CHEK1 can predict more frequent and earlier postremission relapse. CHEK1 encodes checkpoint kinase 1 (CHK1), which is activated by the kinase ATR when DNA replication is impaired by DNA damage. High abundance of CHK1 in AML patient cells correlated with higher clonogenic ability and more efficient DNA replication fork progression upon cytarabine treatment. Exposing the patient cells with the high abundance of CHK1 to SCH900776, an inhibitor of the kinase activity of CHK1, reduced clonogenic ability and progression of DNA replication in the presence of cytarabine. These results indicated that some AML cells rely on an efficient CHK1-mediated replication stress response for viability and that therapeutic strategies that inhibit CHK1 could extend current cytarabine-based treatments and overcome drug resistance. Furthermore, monitoring CHEK1 expression could be used both as a predictor of outcome and as a marker to select AML patients for CHK1 inhibitor treatments.

CDC25A governs proliferation and differentiation of FLT3-ITD acute myeloid leukemia.

We investigated cell cycle regulation in acute myeloid leukemia cells expressing the FLT3-ITD mutated tyrosine kinase receptor, an underexplored field in this disease. Upon FLT3 inhibition, CDC25A mRNA and protein were rapidly down-regulated, while levels of other cell cycle proteins remained unchanged. This regulation was dependent on STAT5, arguing for FLT3-ITD-dependent transcriptional regulation of CDC25A. CDC25 inhibitors triggered proliferation arrest and cell death of FLT3-ITD as well as FLT3-ITD/TKD AC-220 resistant cells, but not of FLT3-wt cells. Consistently, RNA interference-mediated knock-down of CDC25A reduced the proliferation of FLT3-ITD cell lines. Finally, the clonogenic capacity of primary FLT3-ITD AML cells was reduced by the CDC25 inhibitor IRC-083864, while FLT3-wt AML and normal CD34+ myeloid cells were unaffected. In good agreement, in a cohort of 100 samples from AML patients with intermediate-risk cytogenetics, high levels of CDC25A mRNA were predictive of higher clonogenic potential in FLT3-ITD+ samples, not in FLT3-wt ones.Importantly, pharmacological inhibition as well as RNA interference-mediated knock-down of CDC25A also induced monocytic differentiation of FLT3-ITD positive cells, as judged by cell surface markers expression, morphological modifications, and C/EBPα phosphorylation. CDC25 inhibition also re-induced monocytic differentiation in primary AML blasts carrying the FLT3-ITD mutation, but not in blasts expressing wild type FLT3. Altogether, these data identify CDC25A as an early cell cycle transducer of FLT3-ITD oncogenic signaling, and as a promising target to inhibit proliferation and re-induce differentiation of FLT3-ITD AML cells.

Targeting CHK1 inhibits cell proliferation in FLT3-ITD positive acute myeloid leukemia.

CHK1 Ser/Thr kinase, a well characterized regulator of DNA damage response, is also involved in normal cell cycle progression. In this study, we investigate how CHK1 participates to proliferation of acute myeloid leukemia cells expressing the mutated FLT3-ITD tyrosine kinase receptor. Pharmacological inhibition of CHK1 as well as its shRNA mediated down regulation reduced the proliferation rate of FLT-ITD expressing leukemic cell lines in a cytostatic manner. Flow cytometry analysis revealed no accumulation in a specific phase of the cell cycle upon CHK1 inhibition. Accordingly, lentiviral-mediated CHK1 overexpression increased the proliferation rate of FLT3-ITD expressing cells, as judged by cell viability and [3H] thymidine incorporation experiments. By contrast, expression of a ser280 mutant did not, suggesting that phosphorylation of this residue is an important determinant of CHK1 proliferative function. Clonogenic growth of primary leukemic cells from patients in semi-solid medium was reduced upon CHK1 inhibition, confirming the data obtained with leukemic established cell lines. Surprisingly, 3 out of 4 CHK1 inhibitory compounds tested in this study were also potent inhibitors of the FLT3-ITD tyrosine kinase receptor. Altogether, these data identify CHK1 as a regulator of FLT3-ITD-positive leukemic cells proliferation, and they open interesting perspectives in terms of new therapeutic strategies for these pathologies.

Evaluation of checkpoint kinase targeting therapy in acute myeloid leukemia with complex karyotype.

There has been considerable interest in targeting cell cycle checkpoints particularly in emerging and alternative anticancer strategies. Here, we show that checkpoint abrogation by AZD7762, a potent and selective CHK1/2 kinase inhibitor enhances genotoxic treatment efficacy in immature KG1a leukemic cell line and in AML patient samples, particularly those with a complex karyotype, which display major genomic instability and chemoresistance. Furthermore, these data suggest that constitutive DNA-damage level might be useful markers to select AML patients susceptible to receive checkpoint inhibitor in combination with conventional chemotherapy. Moreover, this study demonstrates for the first time that AZD7762 inhibitor targets the CD34(+)CD38(-)CD123(+) primitive leukemic progenitors, which are responsible for the majority of AML patients relapse. Finally, CHK1 inhibition does not seem to affect clonogenic potential of normal hematopoietic progenitors.

Constitutive activation of the DNA damage signaling pathway in acute myeloid leukemia with complex karyotype: potential importance for checkpoint targeting therapy.

Genomic instability in solid tumors participates in the oncogenetic process and is associated with the activation of the DNA damage response pathway. Here, we report the activation of the constitutive DNA damage and checkpoint pathway associated with complex karyotypes in samples from patients with acute myeloid leukemia (AML). We show that antagonizing CHK1 kinase with a small inhibitory compound or by RNA interference strongly reduces the clonogenic properties of high-DNA damage level AML samples, particularly those with complex karyotypes. Moreover, we observe a beneficial effect of CHK1 inhibition in high-DNA damage level AML samples treated with 1-beta-d-arabinofuranosylcytosine. In contrast, CHK1 inhibition has no effect on the clonogenic properties of normal hematopoietic progenitors. All together, our results indicate that CHK1 inhibition may represent an attractive therapeutic opportunity in AML with complex karyotype.

Evaluation of Polo-like Kinase 1 inhibition on the G2/M checkpoint in Acute Myelocytic Leukaemia.

Polo-Kinase 1 (PLK1) is a key cell cycle regulator that is necessary for checkpoint recovery after DNA damage-induced G2 arrest. We have examined the effects of PLK inhibition in Acute Myelocytic Leukaemia (AML) cells, whose resistance to genotoxic agents is thought to be associated with checkpoint reinforcement. We report that in U937 AML cells, PLK1 participates in checkpoint recovery, and that inhibition of PLK by the GW843682X compound results in mitotic accumulation and apoptosis. We also found that when challenged with VP-16, inhibition of PLK1 prevented U937 cells from checkpoint exit. Finally, we found that treatment with GW843682X slightly reduced genotoxic-induced inhibition of colony formation efficiency of primary leukaemia cells (CFU-L) from AML patients.

Inhibition of proteasome activity impairs centrosome-dependent microtubule nucleation and organization.

Centrosomes are dynamic organelles that consist of a pair of cylindrical centrioles, surrounded by pericentriolar material. The pericentriolar material contains factors that are involved in microtubule nucleation and organization, and its recruitment varies during the cell cycle. We report here that proteasome inhibition in HeLa cells induces the accumulation of several proteins at the pericentriolar material, including gamma-tubulin, GCP4, NEDD1, ninein, pericentrin, dynactin, and PCM-1. The effect of proteasome inhibition on centrosome proteins does not require intact microtubules and is reversed after removal of proteasome inhibitors. This accrual of centrosome proteins is paralleled by accumulation of ubiquitin in the same area and increased polyubiquitylation of nonsoluble gamma-tubulin. Cells that have accumulated centrosome proteins in response to proteasome inhibition are impaired in microtubule aster formation. Our data point toward a role of the proteasome in the turnover of centrosome proteins, to maintain proper centrosome function.

Cell adhesion regulates CDC25A expression and proliferation in acute myeloid leukemia.

The effects of cell adhesion on leukemia cell proliferation remain poorly documented and somehow controversial. In this work, we investigated the effect of adhesion to fibronectin on the proliferation of acute myeloid leukemia (AML) cell lines (U937 and KG1a) and CD34+ normal or leukemic primary cells. We observed an increased rate of proliferation of AML cells when adhered to fibronectin, concomitant with accelerated S-phase entry and accumulation of CDC25A. Conversely, normal CD34+ cell proliferation was decreased by adhesion to fibronectin with a concomitant drop in CDC25A expression. Importantly, we showed that both small interfering RNA (siRNA)-mediated CDC25A down-regulation and a recently developed CDC25 pharmacologic inhibitor impaired this adhesion-dependent proliferation, establishing a functional link between CDC25A accumulation and adhesion-dependent proliferation in leukemic cells. CDC25A accumulation was found only slightly dependent on transcriptional regulation and essentially due to modifications of the proteasomal degradation of the protein as shown using proteasome inhibitors and reverse transcription-PCR. Interestingly, CDC25A regulation was Chk1 dependent in these cells as suggested by siRNA-mediated down-regulation of this protein. Finally, we identified activation of the phosphatidylinositol 3-kinase/Akt pathway as an adhesion-dependent regulation mechanism of CDC25A protein expression. Altogether, our data show that in leukemic cells adhesion to fibronectin increases CDC25A expression through proteasome- and Chk1-dependent mechanisms, resulting in enhanced proliferation. They also suggest that these adhesion-dependent proliferation properties of hematopoietic cells may be modified during leukemogenesis.

JAMP, a Jun N-terminal kinase 1 (JNK1)-associated membrane protein, regulates duration of JNK activity.

We report the identification and characterization of JAMP (JNK1 [Jun N-terminal kinase 1]-associated membrane protein), a predicted seven-transmembrane protein that is localized primarily within the plasma membrane and associates with JNK1 through its C-terminal domain. JAMP association with JNK1 outcompetes JNK1 association with mitogen-activated protein kinase phosphatase 5, resulting in increased and prolonged JNK1 activity following stress. Elevated expression of JAMP following UV or tunicamycin treatment results in sustained JNK activity and a higher level of JNK-dependent apoptosis. Inhibition of JAMP expression by RNA interference reduces the degree and duration of JNK activation and concomitantly the level of stress-induced apoptosis. Through its regulation of JNK1 activity, JAMP emerges as a membrane-anchored regulator of the duration of JNK1 activity in response to diverse stress stimuli.

The small ubiquitin-like modifier (SUMO) is required for gonadal and uterine-vulval morphogenesis in Caenorhabditis elegans.

The small ubiquitin-like modifier (SUMO) modification alters the subcellular distribution and function of its substrates. Here we show the major role of SUMO during the development of the Caenorhabditis elegans reproductive system. smo-1 deletion mutants develop into sterile adults with abnormal somatic gonad, germ line, and vulva. SMO-1::GFP reporter is highly expressed in the somatic reproductive system. smo-1 animals lack a vulval-uterine connection as a result of impaired ventral uterine pi-cell differentiation and anchor cell fusion. Mutations in the LIN-11 LIM domain transcription factor lead to a uterine phenotype that resembles the smo-1 phenotype. LIN-11 is sumoylated, and its sumoylation is required for its activity during uterine morphogenesis. Expression of a SUMO-modified LIN-11 in the smo-1 background partially rescued pi-cell differentiation and retained LIN-11 in nuclear bodies. Thus, our results identify the reproductive system as the major SUMO target during postembryonic development and highlight LIN-11 as a physiological substrate whose sumoylation is associated with the formation of a functional vulval-uterine connection.

The LIM domain protein UNC-95 is required for the assembly of muscle attachment structures and is regulated by the RING finger protein RNF-5 in C. elegans.

Here, we describe a new muscle LIM domain protein, UNC-95, and identify it as a novel target for the RING finger protein RNF-5 in the Caenorhabditis elegans body wall muscle. unc-95(su33) animals have disorganized muscle actin and myosin-containing filaments as a result of a failure to assemble normal muscle adhesion structures. UNC-95 is active downstream of PAT-3/beta-integrin in the assembly pathways of the muscle dense body and M-line attachments, and upstream of DEB-1/vinculin in the dense body assembly pathway. The translational UNC-95::GFP fusion construct is expressed in dense bodies, M-lines, and muscle-muscle cell boundaries as well as in muscle cell bodies. UNC-95 is partially colocalized with RNF-5 in muscle dense bodies and its expression and localization are regulated by RNF-5. rnf-5(RNAi) or a RING domain deleted mutant, rnf-5(tm794), exhibit structural defects of the muscle attachment sites. Together, our data demonstrate that UNC-95 constitutes an essential component of muscle adhesion sites that is regulated by RNF-5.

RNF5, a RING finger protein that regulates cell motility by targeting paxillin ubiquitination and altered localization.

RNF5 is a RING finger protein found to be important in the growth and development of Caenorhabditis elegans. The search for RNF5-associated proteins via a yeast two-hybrid screen identified a LIM-containing protein in C. elegans which shows homology with human paxillin. Here we demonstrate that the human homologue of RNF5 associates with the amino-terminal domain of paxillin, resulting in its ubiquitination. RNF5 requires intact RING and C-terminal domains to mediate paxillin ubiquitination. Whereas RNF5 mediates efficient ubiquitination of paxillin in vivo, protein extracts were required for in vitro ubiquitination, suggesting that additional modifications and/or an associated E3 ligase assist RNF5 targeting of paxillin ubiquitination. Mutant Ubc13 efficiently inhibits RNF5 ubiquitination, suggesting that RNF5 generates polychain ubiquitin of the K63 topology. Expression of RNF5 increases the cytoplasmic distribution of paxillin while decreasing its localization within focal adhesions, where it is primarily seen under normal growth. Concomitantly, RNF5 expression results in inhibition of cell motility. Via targeting of paxillin ubiquitination, which alters its localization, RNF5 emerges as a novel regulator of cell motility.

Effects of cadmium and zinc on solar-simulated light-irradiated cells: potential role of zinc-metallothionein in zinc-induced genoprotection.

Zinc is an essential oligoelement for cell growth and cell survival and has been demonstrated to protect cells from oxidative stress induced by UVA or from genotoxic stress due to UVB. In a recent work we demonstrated that the antioxidant role of zinc could be related to its ability to induce metallothioneins (MTs). In this study we identified the mechanism of zinc protection against solar-simulated light (SSL) injury. Cultured human keratinocytes (HaCaT) were used to examine MTs expression and localization in response to solar-simulated radiation. We found translocation to the nucleus, with overexpression of MTs in irradiated cells, a novel observation. The genoprotective effect of zinc was dependent on time and protein synthesis. DNA damage was significantly decreased after 48 h of ZnCl(2) (100 microM) treatment and is inhibited by actinomycin D. ZnCl(2) treatment (100 microM) led to an intense induction, redistribution, and accumulation of MT in the nucleus of irradiated cells. MT expression correlated with the time period of ZnCl(2) treatment. CdCl(2), a potent MT inducer, did not show any genoprotection, although the MTs were expressed in the nucleus. Overall our findings demonstrate that MTs could be a good candidate for explaining the genoprotection mediated by zinc on irradiated cells.