X- chromosome inac t iva t ion
12 S truc ture of the g lucocor t ico id recep tor
FIGURE 12: Structure of the glucocorticoid receptor; the coding regions are indi- cated by gray boxes encoded within nine exons; is. The N-terminaldomain contains themajor transactivationfunction domain, AF-1. The DBD consists of two -helices, which coordinate twozinc atoms. This domainisimportant for dimerization, nuclear translocation,and binding glucocorticoid-response elements and subsequent transac- tivation. The LBD undergoes conformational change when itbinds ligand, enabling interaction withcoregulator proteins. Additional functional domains are indicatedby thethick horizontallinesmarked beneaththe structure: the HSP-binding domain; the homodimerization domain; themajor transactivationdomains AF-1and AF-2; and the region thatfunctionally interactswith AP-1and NFB. Abbreviations: AF-1, hormone- independent transactivation function domain; AF-2, transactivation domain; AP-1, adapter protein complex 1; DBD, DNA-binding domain; HSP, heat-shock protein; LBD, ligand-binding domain; NFB, nuclear factor B; P, sites of phosphorylation on serine or threonineresidues; SUMO, sites of SUMOylation (post-translationalmodifi- cation mediated by small, ubiquitin-related modifier proteins); U, site of ubiquitination (post-translationalmodification mediated by ubiquitin). From (McMaster and Ray, Nat Clin Pract Endocrinol Metab, 2008)
Nucleocytoplasmictranslocation of GCR
Inthe absence ofligand, GCRcan befound mostlyinthecytoplasm as part of a he tero-oligomeric complex, which contains chaperone heat shock proteins (HSPs) 90, 70 and 50,immunophilins and other proteins. HSP90 regulates cytoplasmic retention of GCR by exposing theligand-binding site and maskingthetwo nuclearlocalization sequences (NLS), NL1 and NL2, which are locatedrespectively adjacentto the DBD andin the LBD ofthe receptor. Uponligand-inducedactivation, the receptorundergoes aconfor- mational changethatresults in dissociationfromthis multiproteincomplex andtrans lo-cationintothe nucleus(Pratt, 1993).
Within the nucleus, the receptor forms a homodimer to GREs in the promoter
regions of target genes, or itinteracts as a monomer with other transcription factors protein-1(AP-1), nuclear factor-κB (NF-κB), p53 and signaltransducers andactivators oftranscription (STATs) andregulates geneexpression (Nicolaideset al., 2010). Some of these factors resultin Glucocorticoid dependent-lympholyticactions andthat’s the reasonwhy they areincludedinmany therapeuticregimens forthetreatment ofvarious forms of leukemia and lymphoma. As mentionedearlier, significant number of acute lymphoblastic leukemia patientsrespond well to GC treatment duringinitial phases, although prolongedtreatments sometimes results in steroid-resistance (Hillmannet al., 2000).
3 .4 Emerg ing therapeut ic strateg ies
As discussedabove, thereis currently no consensus onthe optimaltreatmentfor BPDCN. Radiotherapy or local chemotherapy is inefficient and only patients receiving systemic polychemotherapy regimens arelikelytoachieve complete remission,even if responses are short and mostly followed by drugresistance andrelapsein most patients. Molecular profiling inBPDCN suggests that a number ofnovel targeted agents might be usefulin this disease.
3.4.1 Inhibitors of signaling pathways
3.4.1.1 IL3 receptor pathway
Granulocyte-macrophage colony-stimulating factor (GM-CSF),interleukin-3(IL-3), and IL-5 are members of a small family ofcytokines thatregulate the production andactiv- ities ofmany hematopoietic cells. Theiractivities are mediated through binding to the few but highaffinity membranereceptors.
IL-3 isexpressed mostly byactivated Tlymphocytes andmast cells andisimpor- tant fortheregulation ofmast cells function. One ofthe biologicactivities of IL-3is
exerted at thelevel of the progenitor compartment, wherethiscytokine stimulates the
of NF-κB. Intheabsence ofIκBs, NF-κB isfreeand can translocate tothe nucleus (Perkins, 2007).
As mentioned earlierin arecentstudy bySapienzaet al., geneexpression profiling (GEP)andfunctional tests show a preclinical rational for NFκB pathway inhibitors (Bortezomib)in BPDCN.
Bortezomib is awell-known proteasomeinhibitor currentlyemployed inthetrea t-ment of multiple myeloma and some non-Hodgkinlymphomas and block proteasome degradation andthus prevents the NF-kB activation (Sapienza et al., 2014). Bor te-zomib has been already demonstratedto be very effective andinduce rapidly BPDCN cell deathinex-vivo experiments (Hiraiet al., 2011).
3.4.2 Epidrugs
As explained inprevious sections, gene mutationsimpinging on epigenome modifiers arerecurrent in BPDCN(TET2 mutations, ASXL1 and MLL alterations) Epigenetic therapycan at leastin theory counter the ensuing epigenetic disturbancesin BPDCN andshould show some anti-leukemic activity. Gene derepressionconsequent toloss of PRC2 function could be countered by HAT (histone acetyltransferaseinhibitors) or even BET(bromo andextra terminal domain) proteininhibitors whileas mentionedear- lierDNMT inhibitors could counterDNA hypermethylation phenotypesassociated to TET2 mutation. More detailedfunctional studiesincluding epigenome profiling(ChIP- seq andDNA methylation profiling/ sequencing)will berequiredto determinethe best strategies and which patients are mostlikelyto benefitfrom epidrugtherapy.
3.4.2.1 Hypomethylating agent 5-Azacytidine
As mentioned earlier partial or complete chromosomallosses and mutations are com-mon in myeloid malignancies. But loss of gene function can also result from epigenetic transcriptional silencing throughmethylation ofpromoter-associated CpG
islands and/or post-translational deacetylation of histones (Haase et al., 2007). Aber- rant DNA methylation has been described in cancer cells, and it is hypothesized that azanucleoside therapy induces DNA hypomethylation andre-expression of aberrantly silenced genes particularlytumor suppressor genes in patientswith these disorders(F ig-ure 13).
FIGURE 13: Proposed mechanism of action of Azanucleosides. From (Quinta´s-