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[Cell Cycle 7:3, 1-7; 1 February 2008]; 2008 Landes Bioscience

The calcineurin/NFAT signaling pathway is unique to verte-brates and clear genetic evidences show that it plays criticalroles in orchestrating the intricate cellular interactions thatcharacterize vertebrate development and morphogenesis. In thissetting, the transcriptional regulators of the NFAT family func-

tion as molecular integrators of specific calcium signals with othersignaling pathways, including MAPkinase, WNT or NOTCH.Deregulation of calcineurin/NFAT signaling and/or abnormalexpression of its components have recently been reported in solidtumors of epithelial origin, lymphoma and lymphoid leukemia.Our studies in mouse models of human T-ALL/lymphomashows that persistent activation of calcineurin/NFAT signalingis pro-oncogenic in vivo and can be efficiently targeted by well-characterized calcineurin inhibitors. We further discuss factsand hypotheses concerning the molecular events that may actupstream and downstream of calcineurin and/or NFAT activationin different type of cancer cells.

The Calcineurin/NFAT Signaling Pathway Calcineurin (PP2B) is a calcium-calmodulin-dependent serine/

threonine phosphatase implicated in a number of biological processes(reviewed in refs. 13). In vertebrates, calcineurin is a heterodimercomposed of a catalytic subunit A (CnA, 5962kDa) and a regula-tory subunit B (CnB, 19kDa). Three genes encoding the catalyticsubunits have been described in vertebrates.CnA and areexpressed ubiquitously whereasCnA expression is restricted to testisand brain.2,4,5 In addition to the phosphatase catalytic domain, CnAcontains a CnB-binding domain, a calmodulin-binding domainas well as a carboxy-terminal autoinhibitory domain (Fig. 1). Thecalcineurin regulatory subunit is encoded by two genes:CnB2 ,which is specifically expressed in testis andCnB1 , which exhibits anubiquitous expression pattern. In mice, deletion of theCnB1 genecompletely impairs calcineurin enzymatic activity in somatic tissuesand results in embryonic lethality at day 11 of development due tosevere defects in vascular patterning.6

Under physiological conditions, engagement of cell-surfators coupled to phospholipase C activation (e.g., the antigenin mature T and B cells) results in the generation of I(1,4,5)trisphosphate (InsP3) and diacylglycerol (DAG). Whactivates the RAS/PKC pathway, InsP3 mediates the release from internal stores, which in turn induces the opening ofstore-operated calcium channels (CRAC). This results in tof extracellular calcium and the calcium/calmodulin-deactivation of calcineurin (Fig. 1; reviewed in ref. 7) and thdephosphorylation of its substrates, including NFAT (Nucleof Activated T cell) proteins (Fig. 2). NFAT is a family of 5 NFATc1, c2, c3, c4 and NFAT5, the latter being the only member not regulated by calcineurin. Mouse genetic studdemonstrated a strong epistatic relationship between calcinNFATs activation and function in many developmental p(reviewed in refs. 6 and 8 and references therein).

Perspective

The calcineurin/NFAT signaling pathwayA novel therapeutic target in leukemia and solid tumors

Hind Medyouf and Jacques Ghysdael*CNRS UMR146 and Institut Curie; Centre Universitaire; Orsay, France

Key words: calcineurin, NFAT, leukemia, lymphoma, solid tumors

*Correspondence to: Jacques Ghysdael; CNRS UMR146 and Institut Curie;Centre Universitaire; Bat 110; Orsay 91405 France; Tel.: 33.1.69863152; Fax:33.1.69.07.45.25; Email: [email protected]

Submitted: 11/05/07; Revised: 11/21/07; Accepted: 11/23/07

Previously published online as aCell Cycle E-publication:http://www.landesbioscience.com/journals/cc/article/5357

Figure 1. Mechanism of calcineurin activation. Caleincurin is composed ocatalytic (CnA) and a regulatory (CnB) subunit. In unstimulated cells, calmulin is not associated with calcincurin and the CnA C-terminal autoinhtory domain (AID) interacts with the catalytics cleft and inhibits calcinephosphatase activity. Signal-evoked increase in intracellular calcuim resuin calcium-dependent binding of calmodulin to calcineurin, thus relieving inhibitory activity of the AID on the catalytics domain and resulting in dephosphorylation of calcineurin substrates, including NFAT.

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The calcineurin/NFAT signaling pathway in cancer

NFATc1, c2 and c3 are the only members expressed in thelymphoid lineage. In resting lymphocytes, NFAT is located in thecytoplasm as a hyperphosphorylated, inactive form. Under theseconditions, NFAT phosphorylation is insured by the combinedaction of several maintenance kinases, including CK1 and DYRK2

that target specific serine residues in the NFAT conserved regulatorydomain. Signaling through calcium/calcineurin results in NFATproteins dephosphorylation, causing a conformational switch thatunmasks their nuclear localization sequence (NLS) and allows theirtranslocation to the nucleus, where they bind to specific DNAresponse elements to regulate transcription in synergy with a numberof other transcriptional regulators. Mice with conditional deletionof theCnB1 gene early in thymocytes development exhibit an 80%decrease in thymic cellularity, perturbed pre-TCR signaling and thecomplete absence of TCR-mediated positive selection of mature Tcells.9 The importance of calcineurin downstream of TCR signalingin peripheral T cells is underscored by the fact that (1) two effective

immunosuppressant used in human medFK506 and cyclosporine A (CsA) are inhof calcineurin; (2) patients with a rare fohereditary severe combined immunodefi(SCID) show a selective defect in calcinNFAT activation.10 Targeted deletion ofCnB1in B cells shows an essential role for calcin cell proliferation in response to in vitrIgM stimulation of the antigen receptor (B1In contrast, B cell responses in vivo are pindependent of calcineurin, likely due to thgration of BCR signaling with other cell receptor signals. This study also showed tantigen-mediated selection of the B1 cell sentirely dependent upon calcineurin.11

Calcineurin and NFAT Factors in CanceCalcineurin and/or its downstream N

targets have recently been implicated in ca1Tissue sections from invasive ductal breas

noma patients display high expression of Nand NFAT5 in tumor cells.13 Studies in brecarcinoma cell lines show that NFAT esion and transcriptional activation are indownstream of integrin signaling, are indent of calcineurin activity and promotmigration and matrigel invasion, suggesrole for NFATc2/NFAT5 in carcinoma invness in vivo.13 Buchholz and colleagues shown that about 70% of pancreatic carcshow high level expression of nuclear NFAcompared to healthy pancreatic tissue.14 Usinhuman-derived pancreatic carcinomas celthe authors demonstrated that the nuclear ization of transcriptionally active NFATccalcineurin-dependent process since it wasited by CsA. Treatment with CsA also inhin vitro cell cycle progression and anchoragpendent proliferation of the Panc1 cell line

In addition to their proposed implication intumors, the involvement of calcineurin and NFAT is also sin hematologic malignancies. Marafioti et al. surveyed a largNon Hodgkin B-cell and T-cell lymphomas for NFATc1 exand nuclear localization.15 Nuclear localization of NFATc1 was in 70% of the cases of Burkitt lymphoma (BL) and about 30

cases of diffuse large B cell lymphoma (DLBCL). Nucleation of NFATc1 or dephosphorylation of both NFATc1 and Nwas also observed in DLBCL patient material16,17 and in aggressT cell lymphoma.17 NFAT activation in cell lines derived DLBCL and T cell acute lymphoblastic leukemia (T-ALLneurin-dependent as it was suppressed in response to CsA otreatment.16,17 CsA treatment inhibited cell cycle progressiinduced apoptosis in these lines. The in vivo relevance observations was obtained in mouse models of human lymphoma induced either by constitutive activation of tSTAT or NOTCH1 signaling pathways. In both systems, suactivation of the calcineurin/NFAT signaling module was

Figure 2. Schematic view of the calcineurin/NFAT signaling pathway. Engagement by theirligand of cell surface receptors coupled to the activation of phospholipase C or phospholipaseC results in the hydrolysis of phophatidylinositol 4,5 bisphosphate (PIP2) into diacylglycerol(DAG) and inositol 1,4,5 triphosphate (InsP3). InsP3 binds to a receptor in the endoplasmicreticulum (ER) to release calcium ions stored in the ER. Store depletion results in the opening ofcalcium release-activated channels at the plasma membrane and in the import of extracellularcalcium. The resulting increase in intracellular calcium activates calmodulin and a series ofcalmodulin-dependent enzymes, including the protein phosphatase calcineurin (Cn). Caclium- andcalmodulin-activated calcineurin dephosphorylates the 1214 serine residue in NFAT regulatory

domain that are constitutively phosphorylated in quiescent cells. Phosphorylated NFAT is cyto-solic and its calcium-mediated dephosphorylation by calcineurin leads to a concerted change inconformation, leading to its nuclear translocation. Nuclear NFAT cooperates with a number ofother transcriptional regulators to integrate calcium signaling events with other signaling inputs atthe transcriptional level. Cycolsporine A (CsA) and FK506 (Tacrolimus) are unrelated compoundsthat inhibit calcineurin activity towards its protein substrates following their interactions with spe-cific immunophilins, namely cyclophilin A and FKBP12.

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in leukemic cells and treatment of diseased mice with either CsA orFK506 resulted in leukemia regression linked to inhibition of tumorcell proliferation, induction of apoptosis and stoechiometric NFATrephosphorylation.17 Moreover, transduction of leukemic cells with acalcium-independent, constitutively activated mutant of calcineurinwas found to enhance their aggressiveness and to enhance leukemiaprogression in vivo, consistent with an intrinsic requirement forcalcineurin in leukemic cells. It is possible that treatment of leukemicmice with CsA or FK506 also inhibited a function(s) in cells of thetumor microenvironment (e.g., bone marrow stromal cells; bloodvessel cells) that could assist leukemic cell proliferation or survival.Taken together, these data provide compelling evidence that calci-neurin activation contributes to the pathogenesis of T-ALL and otheraggressive lymphoid malignancies.

NFAT Proteins as Potential Mediators of Calcineurin Activityin Cancer

Members of the NFAT family are prominent targets of calcineurinwhich are involved in the regulation of a large number of genes crit-ical for proliferation, growth, migration, differentiation and survival

of cells in many lineages (reviewed in ref. 3). Since deregulation ofthese phenotypic traits is commonly observed in cancer cells, anoncogenic potential for the NFAT proteins has long been suspectedbut only recently reported.

Calcium/calcineurin/NFAT signaling was discovered as an essen-tial pathway acting downstream of the TCR to induce expressionof cytokine genes and drive proliferation of initially quiescent Tcells.18,19 Furthermore, the importance of this signaling pathway tocell cycle progression, in particular at the G1-to-S transition is wellestablished (reviewed in refs. 20 and 21). In line with this, proteinsof the NFAT family have been shown to directly regulate positivelyor negatively the expression of genes implicated in cell cycle control,including CDK4,22 cyclin A223 and p21 (WAF/Cip1).24 MoreoverNFAT loss-of-function or gain-of-function leads to deregulation ofD-type and E-type cyclins expression in several cell types, includinglymphocytes.11,25,26 The calcineurin/NFAT signaling module isalso involved in cell survival as exemplified by the increased apop-tosis observed in developing DP thymocytes of NFATc3-deficientmice,27,28 but the molecular mechanisms involved are still debated.Importantly, although acting downstream from well characterizedpro-survival factors such as neurotrophins in neuronal cells, thecalcineurin/NFAT pathway is not involved in survival of these cells,indicating clear differences depending upon the cellular context.29 During normal development, the calcineurin/NFAT signalingmodule plays a critical role in vasculogenesis and angiogenesis and inthe regulation of the VEGF pathway 30-32 and its abnormal deregula-tion in cancer cells could be involved in tumor neo-angiogenesis.

Available evidence obtained from the different models studiedso far indicates that constitutive activation of NFAT throughcalcineurin-dependent or independent pathways clearly contrib-utes to the expression of one or more of the phenotypic traits thatcharacterize in vitro transformed- and tumor cells. First, enforcedexpression in the 3T3L1 pre-adipocyte cell line of a constitutivelynuclear and transcriptionally active NFATc1 mutant (caNFATc1)obtained by substitution of the phospshorylation/dephosphoryla-tion sites in NFATc1 regulatory domain by alanine, was sufficient toimpair terminal differentiation into adipocytes and to induce cellular

transformation.26 The bypass of the G1 cell cycle checkpoint long-term proliferation induced by caNFATc1 under reduceconditions was associated with upregulation of c-MYC, cand cyclin D3 expression, but whether this reflects direct ttional deregulation of these genes by NFATc1 was not deteEnforced expression of caNFATc1 also protected cells fromnormally induced in 3T3-L1 cells in response to complewithdrawal, an effect that was associated with the produautocrine/paracrine survival factors by transformed cells.26 In celines derived from pancreatic adenocarcinoma, CsA treatmsiRNA-mediated knockdown of NFATc1 inhibited theireration and anchorage-independent growth.14 Cell lines that resisCsA treatment were derived from tumors harboring an ampof thec-MYC protooncogene whereas NFATc1-dependent rtion ofc-MYC was observed in responsive cells. An NFATc1site was identified in thec-MYC promoter, that overlaps wipreviously identified TGF-response element. Promoter studtransient transfection assays showed NFATc1-mediated aof a c-MYC promoter construct, suggesting the direct detion ofc-MYC expression by overexpressed NFATc1 in pan

cancer.14

Importantly, enforced expression of c-MYC wato rescue Panc1 cells proliferation from CsA-mediated cainhibition, indicating that c-MYC is indeed a downstreamof calcineurin/NFATc1 activation in these cells. In DLBCLcell lines, Ford and colleagues provided evidence that siRated downregulation of NFATc1 expression results in decreproliferation, impaired cell survival as well as reduced expCD154, a ligand of the TNF family that binds CD40. Since express CD40, assembly of an autocrine stimulatory meresulting in the assembly of a CD40 signalososme is procontrol cell survival and proliferation of DLBCL cell lines33 NFATbinding sites were identified in the CD154 promoter and sbe important for promoter activity in DLBCL cell lines, a

that relied upon NFATc1 acting in synergy with specific methe NFB family.16 In the breast carcinoma cell line model, enexpression of NFATc2 was found to be sufficient to enhainvasion in-matrigel assays whereas overexpression of eitheor NFAT5 induced cell migration.13 Further studies have shthat NFATc2 overexpression promotes the in vitro invasivtype of breast carcinoma cell lines through the induction such asCOX2 (cyclooxygenase 2) and autotaxin in breast carcells.34,35 NFATc2 binding sites are found in the promotboth the autotaxin andCOX2 genes, but whether NFATc2 dirderegulates transcription of these genes in breast carcinomenot analyzed.

In line with their activity as either positive or negative reggenes involved in cell survival and proliferation, NFAT proalso been described as potential tumor suppressor genes cellular contexts. Ectopic expression of NFATc2 has been repromote apoptosis of Burkitt lymphoma derived cell linesably through the induction of Nur77, a member of the nuclear receptor superfamily, a transcriptional regulator in apoptosis.36 In addition, NFATc3-deficient mice infected murine lymphomagenic retrovirus SL3-3 develop T-cell lyfaster and with higher frequencies as compared to wild-typNFATc2-deficient mice,37 suggesting that NFATc3 could acttumor suppressor gene in the T cell lineage.



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Available evidence thus indicates that, depending upon the tumortype considered, an array of deregulated target genes may be actingdownstream of activated calcineurin/NFAT to modulate the tumorphenotype. However, a number of questions remain to be answered.First, the in vivo relevance of these observations, mostly made in celllines, should be addressed in appropriate mouse models of the respec-tive cancers. Second, several NFAT factors are likely to be expressedin tumor cells and the question arises whether the different NFATplay a redundant or specific role in tumorigenesis. For example, clearevidence indicates that different NFATs can have either specific, orredundant or even antagonistic roles in diverse aspects of normal Tcell development (reviewed in ref. 8). This complexity also exists inother cell lineages but to what extent its perturbation contributes totumor development at different phases of tumorigenesis is at presentunknown and will likely be distinct in different cancer types.

Finally, although mouse genetic studies have demonstrated astrong epistatic relationship between calcineurin and NFATs activa-tion, it is important to note that these transcription factors are notthe only calcineurin downstream substrates. Indeed, calcineurin hasbeen shown to positively regulate a number of other targets such as:channels like InsP3R, the ryanodine or the NMDA receptors;38,39 enzymes like PKA,40 NO synthase;41 other transcription factors likeMEF2C,42 MEF2D43 and the co-activator TORC2;44 mechanismsinvolved in mRNA stabilization45 or miRNA expression.46 Severalof these downstream targets could potentially mediate calcineurineffects in tumor cells.

Upstream Signals Leading to Calcineurin/NFAT Activationin Cancer Cells

Little information is available concerning the nature of theupstream events that control calcineurin and NFAT activation intumor cells. Calcineurin-independent upregulation of NFATc2 andNFAT5 expression in mammary tumor cell lines is linked to expres-sion of4 integrin and the assembly of an 64 heterodimer, but themolecular events downstream of 64 have not been identified.13 Inthe TEL-JAK2 and ICN1-induced mouse models we have used,17 therapid emergence of leukemia is critically dependent upon pre-TCRsignaling and these clonal leukemias express a functional TCR.47,48 Since the pre-TCR and the TCR are well characterized receptorscoupled to calcium/calcineurin/NFAT activation,9,49 we consid-ered the possibility that sustained calcium/calcineurin signaling inleukemic cells could reflect acquired hypersensitivity of these cellsto pre-TCR/TCR-dependent signals. This is not the case since bothTEL-JAK2- and ICN1-induced leukemias generated in Rag2-defi-cient micewhich cannot rearrange the genes encoding TCR and and thus cannot express neither the pre-TCR nor a TCRpresentpersistent activation of the calcineurin/NFAT signaling module ina similar fashion as leukemias obtained in RAG2-proficient mice(ref. 17 and our unpublished data). In our models of TEL-JAK2and ICN1-induced T-ALL/lymphoma, continuous activation of thecalcineurin/NFAT module was interrupted when leukemic cells wereremoved from their in vivo environment (e.g., thymus, spleen, lymphnodes) and maintained in tissue culture for short periods of time.17 This in vitro inactivation is reversible as re-implantation of these cellsinto syngeneic hosts resulted in reactivation of the calcineurin/NFATpathway in the transplanted leukemias (our unpublished observa-tions). This suggests that in vivo activation of the calcineurin/NFAT

pathway in leukemic cells depends upon autocrine/paracrinor signals generated by other cells in the tumor micro-ment. This in vivo/in vitro dichotomy was found in other inincluding human lymphoma samples (our unpublished tions). This also suggests that activation of the calcineuripathway in lymphoid malignancies might be broader than panticipated.15-17

Our experiments also point to the fact that the TEL-JAICN1 initiating/maintenance oncoproteins are not sufficienvate the calcineurin/NFAT module as they remained activin vitro conditions.17 This does not exclude that they nevertcould assist other upstream events in calcineurin/NFAT acFor example, TEL-JAK2 is known to activate the endogenprotein kinase50 and persistent activation of AKT could linactivation of GSK3, an export NFAT kinase. Likewise,tinocytes, ICN1 has been shown to induce the Hes1-derepression of the gene encoding calcipressin1 (CSP1 ), an endogenoinhibitor of calcineurin.51 Whether this regulation occurs in Tcarrying activating mutations in NOTCH152 or in other maligncies or other cancers remains to be investigated.

Cell lines derived from DLBCL and a subset of T-ALL show activation of the calcineurin/NFAT module,16,17 suggestithat their establishment selected clones carrying mutationto activation of this pathway. Such mutations have in fadescribed in lymphoma-derived cell lines. For example,mouse T cell lymphoma cell line was found to express a muof calcineurin in which negative regulation of phosphatasby the CnA autoinhibitory domain is impaired due to a mthat replaces an aspartic acid in position 477 by asparag53 Incontrast, constitutive activation of calcineurin in the SMlymphoma cell line was found to result from the exprestruncated version of the CnA catalytic subunit.54 Proteolytic actition of caspases appears to be an alternative mode of act

calcineurin. To date, two different proteases, namely caspascalpain, have been shown to induce calcineurin cleavage in enhances its phosphatase activity.55-57 In Jurkat T cells, activatiocaspase3 in response to PHA treatment leads to the cleavagto generate truncated polypeptides with enhanced catalytic a5The second protease, m-calpain, activates calcineurin throdistinct mechanisms: (i) the cleavage of CnA that removeinhibiory domain, (ii) the cleavage of CABIN1, an endinhibitor of calcineurin. However, no cleaved forms of Cdetected in our T-ALL mouse models in which we have shpersistent calcineurin activation contributes to the leukeprocess (our unpublished observations).

Mutations in other components of calcium/calmodulin scould be involved in the deregulation of calcineurin or othways in tumor cells. Under physiological conditions, caactivity is negatively regulated by a number of endogenouincluding the calcipressin family (CSP1, CSP2 and C5CABIN1 (CAIN),59 AKAP79 (KAP5),60,61 CHP (CalcineurHomologous Protein)62 and FKBP38.63 Therefore, dysregulaexpression of these negative regulators could contribute to calcineurin activation observed in tumor cells. It has been shT cells from mouse expressing a truncated version of CABis no longer capable of inhibiting calcineurin activity, ova number of cytokine genes (IL2, IL4, IL9, IL13, IFN) due t



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their hypersensitivity to TCR signals64 whereas downregulation ofDSCR1 favors the anchorage-dependent and anchorage-independentproliferation of a colorectal cancer-derived cell line.65 Persistentcalcineurin activation could also potentially result from deregulatedactivity of upstream components in calcium signaling. For example,the TRPV6 calcium channel is overexpressed in advanced stages ofprostate cancer and studies in the LNCaP prostate cancer cell lineshow that TRPV6-mediated calcium import is associated with NFATactivation and favors cell survival and proliferation.66

Calcineurin/NFAT Signaling Pathway as a Potential Targetfor Therapy

The growing body of evidence implicating the activation of thecalcineurin/NFAT signaling pathway in progression and/or mainte-nance of solid tumors, lymphoma and leukemia suggests that the useor development of inhibitors of the molecular events acting upstreamof calcineurin activation, of calcineurin itself or of critical effectors ofcalcineurin may be useful in the treatment of these pathologies. CsAand FK506 are structurally unrelated, well characterized immuno-suppressive agents that function as co-drugs after binding to specific

endogenous cytoplasmic cyclophilins, namely cyclophilinA (CypA)and FKB12, respectively. Both the CsA-CypA and FK506-FKBP12complexes physically interact with calcineurin to inhibit its ability todephosphorylate protein substrates. This property is at the heart ofthe potent inhibitory properties of CsA and FK506 in the responseof T cells to alloantigens and thus of the widespread clinical use ofCsA and FK506 to prevent the rejection of organ transplants andin the treatment of aggressive forms of rheumatoid arthritis andpsoriasis.7,69

T-ALL accounts for about 15% of pediatric and 25% of adult casesof ALL. Molecular characterization of T-ALL has identified a numberof genes that contribute to cell cycle and growth deregulation (e.g., lossof the CDKN2A locus), impaired differentiation (e.g., deregulatedexpression of specific Hox genes) and unlimited self-renewal capacityof leukemic cells. Irrespective of their stage of differentiation arrest,approximately 5060% of T-ALL harbor activating mutations in theNOTCH1 gene,52 implying a central role of deregulated NOTCHsignaling in several aspects of T-ALL biology. While 75% of childrenwith T-cell ALL are cured with combination chemotherapy, theremaining 25% suffer either from refractory or relapsed diseases. Thesefigures and the toxic side effects associated with available chemotherapyregimens clearly calls for the search of novel therapeutic options. Ourstudies in several mouse models of human T-cell leukemia, includingthe T-ALL/lymphoma induced by activated NOTCH1, show thatshort-term (714 days) treatment with either CsA or FK506 resultsin clear anti-leukemic effects and in prolonged survival of treatedversus non treated animals.15,17 These pre-clinical data suggests thatthe use of these compounds could provide a therapeutic benefit inremission induction or in the consolidation phase of T-ALL treatmentand possibly other malignancies that display high calcineurin/NFATactivation.13,14,16,32 However CsA and FK506 show severe toxic sideeffects (neurotoxicity, nephrotoxicity, gastrointestinal disturbances,hypertension) and their long term administration (10 years on longer)in transplanted patients is associated with the emergence of specificcancers due to suppression of tumor immunosurveillance mechanisms.These side effects may compromise the usefulness of CsA and FK506even in short term induction remission protocols. Nevertheless, case

reports have described long term remission of specific lyleukemias after CsA treatment67 and the additional inhibitory perties of CsA on ABC transporters have been proposed to basis of its therapeutic benefit in AML.68 Interestingly, derivativCsA and FK506 have recently been described, including L(an analog of FK50669) and ISATX247 (an analog of CsA 70,71), thashow comparable or even higher efficiency toward calcineution and reduced renal toxicity as compared to the respeccompound.69-71

The alledged limitations linked to direct inhibition of calactivity clearly requires the further dissection of the molecways involved in its activation in human malignancies. the molecular events leading to persistent calcineurin acticancers remain to be identified and, as discussed above arebe distinct in different cancer types. Prior studies have clearstrated that under physiological conditions, calcineurin actdependent upon the increase in intracellular calcium concthrough a capacitative calcium entry (CCE) via the CRAnels (reviewed in ref. 7 and references herein). Thereforebe expected that inhibition of CCE through specific inof the CRAC channels would have similar effects as inhcalcineurin with CsA and FK506 (reviewed in ref. 7). Twocompounds, namely BTP2 and capsaicin, which fulfill theshave recently been described72-74 and therefore could reprean alternative approach to inhibit calcineurin for the treatcancers where persistent calcineurin activation is implicattherapeutic options however are likely to suffer from the samtions as those linked to the direct inhibition of calcineurin.

It is therefore critical to identify and analyze the respecof the downstream effectors involved in the pro-oncogeniof calcineurin in different malignancies. As discussed abovdata support the implication of the NFAT family of tration factors in cancer and the relevant genes/pathways de

by constitutive NFAT activation may represent novel, ptherapeutic targets. However, some of the other previously calcineurin targets have also been implicated in cancer. Forthe MEF2D transcription factor is a member of the MEF2 fDNA binding proteins that activate transcription of genes in the control of muscle cell differentiation and in the resneuronal cells and T-lymphocytes to mitogenic and survivaIn murine retroviral insertional mutagenesis studies, MEbeen identified as a candidate oncogene involved in the pesis of leukemia.75,76 Thus, deregulation of MEF2 target geneoccurs in tumor cells harboring persistent calcineurin activaoffer alternative targets of therapeutic interest.

Acknowledgements

HM was supported by fellowships from the MinislEducation Nationale et de la Recherche and lAssociatioRecherche contre le Cancer (ARC). Our work was suppfunds from CNRS (Centre National de la Recherche ScienInstitut Curie, INCA (Institut National du Cancer), CancIle-de-France, ANR (Agence Nationale de la RecherchNationale contre le Cancer (quipe Labelise Ligue) and Afor International Cancer Research.



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