PI3K/Akt/mTOR Signaling Pathway in Blood Malignancies-New Therapeutic Possibilities.

Blood malignancies remain a therapeutic challenge despite the development of numerous treatment strategies. The phosphatidylinositol-3 kinase (PI3K)/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway plays a central role in regulating many cellular functions, including cell cycle, proliferation, quiescence, and longevity. Therefore, dysregulation of this pathway is a characteristic feature of carcinogenesis. Increased activation of PI3K/Akt/mTOR signaling enhances proliferation, growth, and resistance to chemo- and immunotherapy in cancer cells. Overactivation of the pathway has been found in various types of cancer, including acute and chronic leukemia. Inhibitors of the PI3K/Akt/mTOR pathway have been used in leukemia treatment since 2014, and some of them have improved treatment outcomes in clinical trials. Recently, new inhibitors of PI3K/Akt/mTOR signaling have been developed and tested both in preclinical and clinical models. In this review, we outline the role of the PI3K/Akt/mTOR signaling pathway in blood malignancies' cells and gather information on the inhibitors of this pathway that might provide a novel therapeutic opportunity against leukemia.

Induction of Transcriptional Inhibitor HES1 and the Related Repression of Tumor-Suppressor TXNIP Are Important Components of Cell-Transformation Program Imposed by Oncogenic Kinase NPM-ALK.

This study reports that hairy and enhancer of split homolog-1 (HES1), known to repress gene transcription in progenitor cells of several cell lineages, was strongly expressed in cells and tissues of T-cell lymphoma expressing the oncogenic chimeric tyrosine kinase nucleophosmin (NPM)-anaplastic lymphoma kinase [ALK; ALK+ T-cell lymphoma (TCL)]. The structural analysis of the Orange domain of HES1 indicated that HES1 formed a highly stable homodimer. Of note, repression of HES1 expression led to inhibition of ALK+ TCL cell growth in vivo. The expression of the HES1 gene was induced by NPM-ALK through activation of STAT3, which bound to the gene's promoter and induced the gene's transcription. NPM-ALK also directly phosphorylated HES1 protein. In turn, HES1 up-regulated and down-regulated in ALK+ TCL cells, the expression of numerous genes, protein products of which are involved in key cell functions, such as cell proliferation and viability. Among the genes inhibited by HES1 was thioredoxin-interacting protein (TXNIP), encoding a protein implicated in promotion of cell death in various types of cells. Accordingly, ALK+ TCL cells and tissues lacked expression of TXNIP, and its transcription was co-inhibited by HES1 and STAT3 in an NPM-ALK-dependent manner. Finally, the induced expression of TXNIP induced massive apoptotic cell death of ALK+ TCL cells. The results reveal a novel NPM-ALK-controlled pro-oncogenic regulatory network and document an important role of HES and TXNIP in the NPM-ALK-driven oncogenesis, with the former protein displaying oncogenic and the latter tumor suppressor properties.

Gene expression and mutation-guided synthetic lethality eradicates proliferating and quiescent leukemia cells.

Quiescent and proliferating leukemia cells accumulate highly lethal DNA double-strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase-mediated (DNA-PK-mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA- and DNA-PK-deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK-deficient quiescent leukemia cells and BRCA/DNA-PK-deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients.

Personalized synthetic lethality induced by targeting RAD52 in leukemias identified by gene mutation and expression profile.

Homologous recombination repair (HRR) protects cells from the lethal effect of spontaneous and therapy-induced DNA double-stand breaks. HRR usually depends on BRCA1/2-RAD51, and RAD52-RAD51 serves as back-up. To target HRR in tumor cells, a phenomenon called "synthetic lethality" was applied, which relies on the addiction of cancer cells to a single DNA repair pathway, whereas normal cells operate 2 or more mechanisms. Using mutagenesis and a peptide aptamer approach, we pinpointed phenylalanine 79 in RAD52 DNA binding domain I (RAD52-phenylalanine 79 [F79]) as a valid target to induce synthetic lethality in BRCA1- and/or BRCA2-deficient leukemias and carcinomas without affecting normal cells and tissues. Targeting RAD52-F79 disrupts the RAD52-DNA interaction, resulting in the accumulation of toxic DNA double-stand breaks in malignant cells, but not in normal counterparts. In addition, abrogation of RAD52-DNA interaction enhanced the antileukemia effect of already-approved drugs. BRCA-deficient status predisposing to RAD52-dependent synthetic lethality could be predicted by genetic abnormalities such as oncogenes BCR-ABL1 and PML-RAR, mutations in BRCA1 and/or BRCA2 genes, and gene expression profiles identifying leukemias displaying low levels of BRCA1 and/or BRCA2. We believe this work may initiate a personalized therapeutic approach in numerous patients with tumors displaying encoded and functional BRCA deficiency.

Targeting RAD51 phosphotyrosine-315 to prevent unfaithful recombination repair in BCR-ABL1 leukemia.

Chronic myeloid leukemia chronic phase (CML-CP) CD34(+) cells contain numerous DNA double-strand breaks whose unfaithful repair may contribute to chromosomal instability and disease progression to blast phase (CML-BP). These phenomena are often associated with the appearance of imatinib-resistant BCR-ABL1 kinase mutants (eg, T315I) and overexpression of BCR-ABL1. Here we show that BCR-ABL1 (nonmutated and T315I mutant) promoted RAD51 recombinase-mediated unfaithful homeologous recombination repair (HomeoRR) in a dosage-dependent manner. BCR-ABL1 SH3 domain interacts with RAD51 proline-rich regions, resulting in direct phosphorylation of RAD51 on Y315 (pY315). RAD51(pY315) facilitates dissociation from the complex with BCR-ABL1 kinase, migrates to the nucleus, and enhances formation of the nuclear foci indicative of recombination sites. HomeoRR and RAD51 nuclear foci were strongly reduced by RAD51(Y315F) phosphorylation-less mutant. In addition, peptide aptamer mimicking RAD51(pY315) fragment, but not that with Y315F phosphorylation-less substitution, diminished RAD51 foci formation and inhibited HomeoRR in leukemia cells. In conclusion, we postulate that BCR-ABL1 kinase-mediated RAD51(pY315) promotes unfaithful HomeoRR in leukemia cells, which may contribute to accumulation of secondary chromosomal aberrations responsible for CML relapse and progression.

BCR/ABL stimulates WRN to promote survival and genomic instability.

BCR/ABL-transformed chronic myeloid leukemia (CML) cells accumulate numerous DNA double-strand breaks (DSB) induced by reactive oxygen species (ROS) and genotoxic agents. To repair these lesions BCR/ABL stimulate unfaithful DSB repair pathways, homologous recombination repair (HRR), nonhomologous end-joining (NHEJ), and single-strand annealing (SSA). Here, we show that BCR/ABL enhances the expression and increase nuclear localization of WRN (mutated in Werner syndrome), which is required for processing DSB ends during the repair. Other fusion tyrosine kinases (FTK), such as TEL/ABL, TEL/JAK2, TEL/PDGFßR, and NPM/ALK also elevate WRN. BCR/ABL induces WRN mRNA and protein expression in part by c-MYC-mediated activation of transcription and Bcl-xL-dependent inhibition of caspase-dependent cleavage, respectively. WRN is in complex with BCR/ABL resulting in WRN tyrosine phosphorylation and stimulation of its helicase and exonuclease activities. Activated WRN protects BCR/ABL-positive cells from the lethal effect of oxidative and genotoxic stresses, which causes DSBs. In addition, WRN promotes unfaithful recombination-dependent repair mechanisms HRR and SSA, and enhances the loss of DNA bases during NHEJ in leukemia cells. In summary, we postulate that BCR/ABL-mediated stimulation of WRN modulates the efficiency and fidelity of major DSB repair mechanisms to protect leukemia cells from apoptosis and to facilitate genomic instability.

The chromatin remodeling factor SRCAP modulates expression of prostate specific antigen and cellular proliferation in prostate cancer cells.

The SNF2-related CBP activator protein (SRCAP) serves as a coactivator for several nuclear receptors including the androgen receptor (AR). SRCAP is an ATPase that is the core subunit of a large multiprotein complex and was shown to incorporate the histone variant H2A.Z into nucleosomes. In this report, we demonstrate that SRCAP is expressed in the epithelium of normal prostate and in prostate carcinoma cells, and is associated with AR in the nucleus. Using transient transfection assays we demonstrate that SRCAP activates hormone-dependent transcription of the androgen responsive, prostate specific antigen (PSA)-Luciferase reporter gene in human prostate cells. The in vivo occupancy of SRCAP at the endogenous PSA promoter is demonstrated using chromatin immunoprecipitation assays. ShRNA mediated knockdown of SRCAP resulted in decreased H2A.Z binding at the enhancer region of the PSA promoter and decreased expression of PSA in prostate cancer cells. Furthermore, inhibition of SRCAP expression significantly inhibited androgen dependent prostate cancer cell growth. These data identify SRCAP as a physiologically relevant mediator of PSA expression, and demonstrate that SRCAP plays a role in prostate cancer cell proliferation.

BCR/ABL and other kinases from chronic myeloproliferative disorders stimulate single-strand annealing, an unfaithful DNA double-strand break repair.

Myeloproliferative disorders (MPD) are stem cell-derived clonal diseases arising as a consequence of acquired aberrations in c-ABL, Janus-activated kinase 2 (JAK2), and platelet-derived growth factor receptor (PDGFR) that generate oncogenic fusion tyrosine kinases (FTK), including BCR/ABL, TEL/ABL, TEL/JAK2, and TEL/PDGFbetaR. Here, we show that FTKs stimulate the formation of reactive oxygen species and DNA double-strand breaks (DSB) both in hematopoietic cell lines and in CD34(+) leukemic stem/progenitor cells from patients with chronic myelogenous leukemia (CML). Single-strand annealing (SSA) represents a relatively rare but very unfaithful DSB repair mechanism causing chromosomal aberrations. Using a specific reporter cassette integrated into genomic DNA, we found that BCR/ABL and other FTKs stimulated SSA activity. Imatinib-mediated inhibition of BCR/ABL abrogated this effect, implicating a kinase-dependent mechanism. Y253F, E255K, T315I, and H396P mutants of BCR/ABL that confer imatinib resistance also stimulated SSA. Increased expression of either nonmutated or mutated BCR/ABL kinase, as is typical of blast phase cells and very primitive chronic phase CML cells, was associated with higher SSA activity. BCR/ABL-mediated stimulation of SSA was accompanied by enhanced nuclear colocalization of RAD52 and ERCC1, which play a key role in the repair. Taken together, these findings suggest a role of FTKs in causing disease progression in MPDs by inducing chromosomal instability through the production of DSBs and stimulation of SSA repair.

BCR/ABL inhibits mismatch repair to protect from apoptosis and induce point mutations.

BCR/ABL kinase-positive chronic myelogenous leukemia (CML) cells display genomic instability leading to point mutations in various genes including bcr/abl and p53, eventually causing resistance to imatinib and malignant progression of the disease. Mismatch repair (MMR) is responsible for detecting misincorporated nucleotides, resulting in excision repair before point mutations occur and/or induction of apoptosis to avoid propagation of cells carrying excessive DNA lesions. To assess MMR activity in CML, we used an in vivo assay using the plasmid substrate containing enhanced green fluorescent protein (EGFP) gene corrupted by T:G mismatch in the start codon; therefore, MMR restores EGFP expression. The efficacy of MMR was reduced approximately 2-fold in BCR/ABL-positive cell lines and CD34(+) CML cells compared with normal counterparts. MMR was also challenged by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which generates O(6)-methylguanine and O(4)-methylthymine recognized by MMR system. Impaired MMR activity in leukemia cells was associated with better survival, accumulation of p53 but not of p73, and lack of activation of caspase 3 after MNNG treatment. In contrast, parental cells displayed accumulation of p53, p73, and activation of caspase 3, resulting in cell death. Ouabain-resistance test detecting mutations in the Na(+)/K(+) ATPase was used to investigate the effect of BCR/ABL kinase-mediated inhibition of MMR on mutagenesis. BCR/ABL-positive cells surviving the treatment with MNNG displayed approximately 15-fold higher mutation frequency than parental counterparts and predominantly G:C-->A:T and A:T-->G:C mutator phenotype typical for MNNG-induced unrepaired lesions. In conclusion, these results suggest that BCR/ABL kinase abrogates MMR activity to inhibit apoptosis and induce mutator phenotype.

[Uracil-DNA glycosylases]. / Glikozylazy DNA uracylu ssaków.

Uracil is one of four nitrogen bases, most frequently found in normal RNA. Uracyl can be found also in DNA as a result of enzymatic or non-enzymatic deamination of cytosine as well as misincorporation of dUMP instead of dTMP during DNA replication. Uracil from DNA can be removed by DNA repair enzymes with apirymidine site as an intermediate. However, if uracil is not removed from DNA a pair C:G in parental DNA can be changed into a T:A pair in the daughter DNA molecule. Therefore, uracil in DNA may lead to a mutation. Uracil in DNA, similarly to thymine, forms energetically most favorable hydrogen bonds with adenine, therefore uracil does not change the coding properties of DNA. Uracil in DNA is recognized by uracil DNA glycosylase (UDGs), which initiates DNA base excision repair, leading to removing of uracil from DNA and replacing it by thymine or cytosine, when arose as a result of cytosine deamination. Eukaryotes have at least four nuclear UDGs: UNG2, SMUG1, TDG i MBD4, while UNG1 operates in the mitochondrium. UNG2 is involved in DNA repair associated with DNA replication and interacts with PCNA and RPA proteins. Uracil can also be an intermediate product in the process of antigen-dependent antibody diversification in B lymphocytes. Enzymatic deamination of viral DNA by host cells can be a defense mechanism against viral infection, including HIV-1. UNG2, MBD4 and TDG glycosylases may cooperate with mismatch repair proteins and TDG can be involved in nucleotide excision repair system.

[The role of oncogenic tyrosine kinases in the cellular response to anticancer therapy]. / Rola onkogennych kinaz tyrozynowych w odpowiedzi komórek na terapie przeciwnowotworowa

Oncogenic tyrosine kinases (OTKs) expressed in malignant tumors stimulate cell proliferation, inhibition of apoptosis, and drug resistance. There are at least three mechanisms of response to chemo- and radiotherapy in OTK-positive cells: overexpression of anti-apoptotic proteins (such as Bcl-xL and Bcl-2) and blocking of the activation of pro-apoptotic proteins (such as caspase 3), arrest in the G2/M phase of the cell cycle, and modulation of DNA repair mechanisms. Furthermore, OTKs elevate the level of reactive oxygen species (ROS)-dependent spontaneous DNA damage. The accumulation of mutations in genetic material increases the metastatic potential following further cancer development. Oxidation-damaged DNA bases are repaired primarily via the mechanisms of base excision repair (BER) and nucleotide excision repair (NER). However, during DNA replication, the areas of single-stranded DNA produced by BER and NER can be converted to double-strand breaks (DSBs), which are then repaired via non-homologous end-joining (NHEJ) and homologous recombination repair (HHR) mechanisms. The HHR pathway is activated in OTK-positive cells due to the elevated level of RAD51 protein expression. In addition, RecQ helicases, such as BLM, play a significant role in this process. Understanding the mechanisms activated by OTKs may help in the development of novel therapeutic strategies that use OTKs as a target.

Brain tumor formation in tuberous sclerosis depends on Erk activation.

Tuberous sclerosis (TS) is an autosomal dominant disease associated with the formation of usually benign tumors or hamartomas. The disease is connected with upregulation of mammalian target of rapamycin, central regulator of protein translation, which is usually regarded to be activated by Akt kinase. Here, we show for the first time that in all four brain lesions and one angiomyolipoma from TS patients both extracellular signal-regulated kinase (Erk) and p90 ribosomal S6 kinase 1 activation as well as Erk-dependent phosphorylation of p70 ribosomal S6 kinase 1 are markedly elevated whereas Akt, participating in the classical pathway of mammalian target of rapamycin activation is not always activated. Erk activation is also present in TS-derived cell lines. Importantly, Erk inhibition leads to the decrease of proliferation potential of such lines. These results show that Erk is specifically implicated in the pathogenesis of hamartomas.

Enhanced phosphorylation of Nbs1, a member of DNA repair/checkpoint complex Mre11-RAD50-Nbs1, can be targeted to increase the efficacy of imatinib mesylate against BCR/ABL-positive leukemia cells.

Nbs1, a member of the Mre11-RAD50-Nbs1 complex, is phosphorylated by ATM, the product of the ataxia-telangiectasia mutated gene and a member of the phosphatidylinositol 3-kinase-related family of serine-threonine kinases, in response to DNA double-strand breaks (DSBs) to regulate DNA damage checkpoints. Here we show that BCR/ABL stimulated Nbs1 expression by induction of c-Myc-dependent transactivation and protection from caspase-dependent degradation. BCR/ABL-related fusion tyrosine kinases (FTKs) such as TEL/JAK2, TEL/PDGFbetaR, TEL/ABL, TEL/TRKC, BCR/FGFR1, and NPM/ALK as well as interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) also stimulated Nbs1 expression. Enhanced ATM kinase-dependent phosphorylation of Nbs1 on serine 343 (S343) in response to genotoxic treatment was detected in leukemia cells expressing BCR/ABL and other FTKs in comparison to normal counterparts stimulated with IL-3, GM-CSF, and SCF. Expression of Nbs1-S343A mutant disrupted the intra-S-phase checkpoint, decreased homologous recombinational repair (HRR) activity, down-regulated XIAP expression, and sensitized BCR/ABL-positive cells to cytotoxic drugs. Interestingly, inhibition of Nbs1 phosphorylation by S343A mutant enhanced the antileukemia effect of the combination of imatinib and genotoxic agent.

ATR-Chk1 axis protects BCR/ABL leukemia cells from the lethal effect of DNA double-strand breaks.

BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and mitomycin C (MMC, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assay. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. Elevated levels of drug-induced DSBs in leukemia cells were associated with higher activity of ATR kinase, and enhanced phosphorylation of histone H2AX on serine 139 (gamma-H2AX). gamma-H2AX eventually started to disappear in BCR/ABL cells, while continued to increase in parental cells. In addition, the expression and ATR-mediated phosphorylation of Chk1 kinase on serine 345 were often more abundant in BCR/ABL-positive leukemia cells than normal counterparts after genotoxic treatment. Inhibition of ATR kinase by caffeine but not Chk1 kinase by indolocarbazole inhibitor, SB218078 sensitized BCR/ABL leukemia cells to MMC in a short-term survival assay. Nevertheless, both caffeine and SB218078 enhanced the genotoxic effect of MMC in a long-term clonogenic assay. This effect was associated with the abrogation of transient accumulation of leukemia cells in S and G2/M cell cycle phases after drug treatment. In conclusion, ATR-Chk1 axis was strongly activated in BCR/ABL-positive cells and contributed to the resistance to DNA cross-linking agents causing numerous replication-dependent DSBs.

Imatinib mesylate (STI571) abrogates the resistance to doxorubicin in human K562 chronic myeloid leukemia cells by inhibition of BCR/ABL kinase-mediated DNA repair.

Imatinib mesylate (STI571), a specific inhibitor of BCR/ABL tyrosine kinase, exhibits potent antileukemic effects in the treatment of chronic myelogenous leukemia (CML). However, the precise mechanism by which inhibition of BCR/ABL activity results in pharmacological responses remains unknown. BCR/ABL-positive human K562 CML cells resistant to doxorubicin (K562DoxR) and their sensitive counterparts (K562DoxS) were used to determine the mechanism by which the STI571 inhibitor may overcome drug resistance. K562 wild type cells and CCRF-CEM lymphoblastic leukemia cells without BCR/ABL were used as controls. The STI571 specificity was examined by use of murine pro-B lymphoid Baf3 cells with or without BCR/ABL kinase expression. We examined kinetics of DNA repair after cell treatment with doxorubicin in the presence or absence of STI571 by the alkaline comet assay. The MTT assay was used to estimate resistance against doxorubicin and Western blot analysis with Crk-L antibody was performed to evaluate BCR/ABL kinase inhibition by STI571. We provide evidence that treatment of CML-derived BCR/ABL-expressing leukemia K562 cells with STI571 results in the inhibition of DNA repair and abrogation of the resistance of these cells to doxorubicin. We found that doxorubicin-resistant K562DoxR cells exhibited accelerated kinetics of DNA repair compared with doxorubicin-sensitive K562DoxS cells. Inhibition of BCR/ABL kinase in K562DoxR cells with 1 microM STI571 decreased the kinetics of DNA repair and abrogated drug resistance. The results suggest that STI571-mediated inhibition of BCR/ABL kinase activity can affect the effectiveness of the DNA-repair pathways, which in turn may enhance drug sensitivity of leukemia cells.

BCR/ABL modifies the kinetics and fidelity of DNA double-strand breaks repair in hematopoietic cells.

The oncogenic BCR/ABL tyrosine kinase facilitates the repair of DNA double-strand breaks (DSBs). We find that after gamma-irradiation BCR/ABL-positive leukemia cells accumulate more DSBs in comparison to normal cells. These lesions are efficiently repaired in a time-dependent fashion by BCR/ABL-stimulated non-homologous end-joining (NHEJ) followed by homologous recombination repair (HRR) mechanisms. However, mutations and large deletions were detected in HRR and NHEJ products, respectively, in BCR/ABL-positive leukemia cells. We propose that unfaithful repair of DSBs may contribute to genomic instability in the Philadelphia chromosome-positive leukemias.

Activation of mammalian target of rapamycin in transformed B lymphocytes is nutrient dependent but independent of Akt, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase, insulin growth factor-I, and serum.

The study examines the preponderance and mechanism of mammalian target of rapamycin (mTOR) activation in three distinct types of transformed B lymphocytes that differ in expression of the EBV genome. All three types [EBV-immortalized cells that express a broad spectrum of the virus-encoded genes (type III latency; EBV+/III), EBV-positive cells that express only a subset of the EBV-encoded genes (EBV+/I), and EBV-negative, germinal center-derived cells (EBV-)] universally displayed activation of the mTOR signaling pathway. However, only the EBV+/III transformed B cells displayed also activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway that is considered to be the key activator of mTOR and of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway that coactivates one of the immediate targets of mTOR, p70 S6K1. Activation of the PI3K/Akt and MEK/ERK, but not of the mTOR pathway, was inhibited by serum withdrawal and restored by insulin growth factor-I. In contrast, activation of mTOR, but not PI3K/Akt and MEK/ERK, was sensitive to nutrient depletion. Both direct Akt (Akt inhibitors I-III) and a PI3K inhibitor (wortmannin at 1 nmol/L) suppressed Akt phosphorylation without significantly affecting mTOR activation. Furthermore, rapamycin, a potent and specific mTOR inhibitor, suppressed profoundly proliferation of cells from all three types of transformed B cells. U0126, a MEK inhibitor, had a moderate antiproliferative effect only on the EBV+/III cells. These results indicate that mTOR kinase activation is mediated in the transformed B cells by the mechanism(s) independent of the PI3K/Akt signaling pathway. They also suggest that inhibition of mTOR signaling might be effective in therapy of the large spectrum of B-cell lymphomas.

BLM helicase is activated in BCR/ABL leukemia cells to modulate responses to cisplatin.

Bloom protein (BLM) is a 3'-5' helicase, mutated in Bloom syndrome, which plays an important role in response to DNA double-strand breaks and stalled replication forks. Here, we show that BCR/ABL tyrosine kinase, which also modulates DNA repair capacity, is associated with elevated expression of BLM. Downregulation of BLM by antisense cDNA or dominant-negative mutant inhibits homologous recombination repair (HRR) and increases sensitivity to cisplatin in BCR/ABL-positive cells. Bone marrow cells from mice heterozygous for BLM mutation, BLM(Cin/+), transfected with BCR/ABL display increased sensitivity to cisplatin compared to those obtained from the wild-type littermates. BCR/ABL promotes interactions of BLM with RAD51, while simultaneous overexpression of BLM and RAD51 in normal cells increases drug resistance. These data suggest that BLM collaborates with RAD51 to facilitate HRR and promotes the resistance of BCR/ABL-positive leukemia cells to DNA-damaging agents.

NPM/ALK downregulates p27Kip1 in a PI-3K-dependent manner.

OBJECTIVES: Anaplastic large-cell lymphomas (ALCL) are frequently associated with the chromosomal translocation t(2;5) (p23;q35) resulting in the NPM/ALK fusion gene that encodes a constitutively activated tyrosine kinase. We showed that NPM/ALK stimulated cell proliferation and that PI-3K/AKT pathway played an important role in this effect. p27Kip1 is a member of the CDK family inhibitory proteins regulating the entry into S phase. It was reported that p27Kip1 function is impaired in many tumors. In this study we investigated the role of PI-3K/AKT in NPM/ALK-dependent downregulation of p27Kip1 protein. MATERIALS AND METHODS: To investigate this phenomenon the pro-B cell line BaF3, BaF3 cell line stably expressing NPM/ALK, and ALCL SUP-M2 cell line were used. The p27Kip1 protein expression before and after LY294002, wortmannin, or epoxomicin treatment and phosphorylation status of AKT were measured in parental and NPM/ALK+ cells by Western analysis. Also, the localization of p27Kip1 protein was analyzed by fractionation and immunoblotting. RESULTS: p27Kip1 was found to be downregulated in NPM/ALK-transformed hematopoietic cells, but inhibition of proteasome-dependent degradation pathway by epoxomicin reversed this effect. In addition, treatment of NPM/ALK+ cells with LY294002, the PI-3K inhibitor, caused elevation of p27Kip1 protein expression and its nuclear localization. CONCLUSIONS: Taken together, we postulate that NPM/ALK-PI-3K pathway stimulates cell proliferation by regulation of the expression and nuclear localization of p27Kip1.

BCR/ABL recruits p53 tumor suppressor protein to induce drug resistance.

Tumors expressing the ABL oncoproteins (BCR/ABL, TEL/ABL, v-ABL) can avoid apoptosis triggered by DNA damaging agents. The tumor suppressor protein p53 is an important activator of apoptosis in normal cells; conversely its functional loss may cause drug resistance. The ABL oncoprotein-p53 paradigm represents the relationship between an oncogenic tyrosine kinase and a tumor suppressor gene. Here we show that BCR/ABL oncoproteins employ p53 to induce resistance to DNA damage in myeloid leukemia cells. Cells transformed by the ABL oncoproteins displayed accumulation of p53 upon DNA damage. In contrast, only a modest increase of p53 expression followed by activation of caspase-3 were detected in normal cells expressing endogenous c-ABL. Phosphatidylinositol-3 kinase-like protein kinases (ATR and also ATM) -dependent phosphorylation of p53-Ser15 residue was associated with the accumulation of p53, and stimulation of p21(Waf-1) and GADD45, resulting in G(2)/M delay in BCR/ABL cells after genotoxic treatment. Inhibition of p53 by siRNA or by the temperature-sensitive mutation reduced G(2)/M accumulation and drug resistance of BCR/ABL cells. In conclusion, accumulation of the p53 protein contributed to prolonged G(2)/M checkpoint activation and drug resistance in myeloid cells expressing the BCR/ABL oncoproteins.