X- chromosome inac t iva t ion
11 Mode ls for nuc lear lncRNA func t ion
FIGURE 11: Models for nuclear lncRNAfunction: Long non-coding RNAs (lncRNAs) regulating transcription in cis (part A) and in trans (part B). Aa) cis-acting lncRNA examples are; X-inactive specific transcript (Xist), Kcnq1 overlapping transcript 1 (Kcnq1ot1) and Airn (antisense Igf2r (insulin-like growth factor 2 receptor) RNA. These lncRNAsinducetheformation of repressive chromatin throughtherecruitment of DNA methyltransferase 3(DNMT3), which inducesDNA methylation; Polycomb repressive complex 2 (PRC2), which produces histone H3 lysine 27 trimethylation (H3K27me3); and histone lysine N-methyltransferase EHMT2, which isresponsible for producing H3K9me2 and H3K9me3 (Lee and Bartolomei, 2013). Ab) HOXA distal transcript antisense RNA (HOTTIP) functions through the recruitment of the mixed lineageleukemia 1(MLL1) complex, which drivestheformation ofthe activa t-ingH3K4me3 mark (Wanget al., 2011). Ba —HOXA transcriptantisense RNA (HO- TAIR) is atrans-acting regulator of theHOXD genes (Rinn et al., 2007). It ischaracter- izedby amodular scaffold structure thatallows therecruitment of two distinctrepres- sive complexes, PRC2 and theH3K4 demethylating complex KDM1A-coREST-REST (lysine-specific histone demethylase 1A-REST corepressor 1-RE1-silencing transcrip- tionfactor) on thesame genomic region (Tsai et al., 2010). Bb —The pluripotency RNAs lncRNA-ES1 and lncRNA-ES2 associate with both PRC2 and thetranscription factor sex-determining region Y-box 2(SOX2), which suggests that theselncRNAs control embryonic stem cell pluripotency by silencing SOX2-bound developmental genes (Ng et al., 2012) ;thisfunction isalternative toOCT4 -and SOX2-dependent ac- tivationof pluripotency genes. Bc — The lncRNAJpx (Jpxtranscript, Xist activator) that binds to the transcriptional repressor CTCF inhibits its binding to the Xist pro- moter, thusactivatingXist transcription(Sun et al., 2013). From (Fatica and Bozoni, Nat Rev Genet, 2014)
Xist expression itselfis controlled by otherlncRNAsin both apositive and aneg- ative manner. Tsix is one of thebest-characterized Xist regulators, which is a natural antisense non-codingtranscript. Tsix counteracts Xist expression byinducingrepressive epigenetic modifications at theXist promoter (Lee and Bartolomei, 2013). Xist act iva-tionalso requiresthelncRNAJpx (Tianet al., 2010), whichinducesXist transcription throughthe sequestration oftranscriptionalrepressor CTCF (Figure 11 Bc)(Fatica and Bozzoni, 2014).
Genomicimprinting
Genomicimprintingis briefly defined as parental-specific geneexpression. Imprinted genesare generallyassociated inclustersandareepigenetically marked in different and sex-dependent ways duringmale andfemale gametogenesis and embryonicdevel- opment. Similarly, genomicimprinting is regulated by cis-acting imprintingcontrol regions (ICRs)thatinfluenceallelic expression acrosslong distances.
Imprinted genes encode various species of ncRNAs, including lncRNAs(in general longer than 100 kb). In many cases, these lncRNAs bind to imprinted regions andare directly involved in silencing. Two best-characterized examples are thelncRNAs Kcnq1 overlapping transcript 1(Kcnq1ot1)and Airn (antisense Igf2r (insulin-like growth factor 2 receptor)RNA); theselncRNAs are paternallyexpressed, and function by repressing flanking protein-coding genes incis (Lee and Bartolomei, 2013). During embryonic development, Kcnq1ot1 establishes and maintains repressive DNA methylation on surrounding genes, whereas, in the placenta, it functions by recruiting the repressive histone modifiers PRC2 and euchromatic histone-lysine N-methyltransferase 2(EHMT2)(also known as G9a) on genesthat arelocated further away fromtheim- printed region (Mancini-Dinardoet al., 2006).
The other example isAirn, which silencesthe paternalIgf2r allele incis, whereas the maternal Igf2r allele remains expressed. Inembryo, Airnsilences paternal Igf2r througha mechanismthat does not require astable RNA product but that is based
on continuous Airntranscription, whichinterferes with the recruitment ofRNA po ly-merase II (Latos et al., 2012),but in placenta, mature Airn recruitsEHMT2 toinduce theformation ofrepressive chromatin (Sleutelset al., 2002)(Figure 11 Aa).
Regulation of HOX genes
In mammals, there are 39HOX genesthat are grouped in four clusters (HOXA,HOXB, HOXC and HOXD), whichallow precise spatiotemporalexpression ofevolutionary con-served family oftranscriptionfactors. In additionto protein-coding genes, these c lus-ters producelncRNAsthatshow similar spatiotemporalwindows ofexpression totheir flanking protein-coding genesandit has been shown that theyareimplicated inthe regulation ofHOX genes(Rinn et al., 2007).
HOTTIP isthecis-acting antisensetranscript of HOXA locus. HOTTIP regulates HOXA expression by interacting with the activating H3K4 methyl transferase, mixed lineageleukemia 1(MLL1)complex, and bytheformation of chromatinloopsthat con-nect distally expressed HOTTIP transcriptswith various HOXA gene promoters (Wang et al., 2011)(Figure 11 Ab).
HOTAIR (HOXC transcript antisenseRNA) was one ofthefirsttrans-acting lncR-NAs to beidentified anditacts as a repressor oftheHOXD cluster, whichislocated on a different chromosome. HOTAIR acts as a molecularscaffold with twoknown chro-matin modificationcomplexes; the 5’region ofthislncRNA bindstothe PRC2complex responsible for H3K27 methylation and the 3’region binds to LSD1, which mediates enzymatic demethylation of H3K4(Tsaiet al., 2010)(Figure 11 Ba).
Long noncoding RNA in cancerdevelopment
As for protein coding genes, ncRNAs contribute to the regulatory networks that pa r-ticipateto cancerdevelopment. Theirimplicationin cancerdevelopment has been de-tected by various techniquesincluding RNA-sequencing (RNA-seq), next generation sequencing, and methylation analysis(Trapnell et al., 2010). These approacheshave
ledtotheidentification ofseveral lncRNAs whoseexpression andthe epigeneticstate oftheir coding genes were associated with cancercells ortissues. Proteins, controlling chromatin organization including polycombrepressor complexes, PRC1 andPRC2, and members ofthetrithorax family constitutekey playersinthe molecular pathogenesis of cancer. As mentioned before,many evidences suggest that a major role oflncRNAsis to guidethesite-specific chromatin-modifyingcomplexes totarget genes andcontribute tothe epigenetic modification ofthese genes (Mattick and Gagen, 2001). Recent s tud-ieshave linkedthemiss-expression ofwell-characterized lncRNAstodiverse cancers, such as HOTAIR in breast cancer, AINRIL and SChLAP1 in prostate cancer (Gupta et al., 2010,Kotake et al., 2011, Prensneret al., 2013).
HOTAIR is one ofthefirst lncRNA discovered to be involved intumor igene-sis. Itsexpression is upregulated in primarytumors and upto 2000-foldinmetastatic breast cancer(Gupta et al., 2010).Overexpression ofHOTAIR in epithelial cancercells induces genome-wide re-targeting of PRC2toan occupancy pattern more resembling embryonic fibroblasts, resulting to altered histoneH3 lysine 27methylation, gene ex-pression, andincreased cancer invasiveness and metastasisin a PRC2 dependent man-ner(Gupta et al., 2010).
Prostate cancer is anotherexample in whichlncRNAs has beenimplicatedin d is-ease development and progression. As an example, SChLAP, alncRNA identified by RNA-seq in prostatecancer, shows highlevelexpression inaggressive formsis pred ic-tive of poor outcomes, andmetastasis. SChLAP1 appears to mediate this function by antagonizing the genome-wide localization and regulatory functions of the SWI/SNF chromatin-modifying complex (Prensner et al., 2013). SWI/SNF complexes interact with transcription factors, co-activators, and corepressors and are capable of mobilizing nucleosomes at target promoters and enhancers to modulate gene expression (Tolstorukov et al., 2013). Loss of SWI/SNF complex functionality promotes cancer progression, and it has been shown that multiple SWI/SNF components are somatically inactivatedin cancer(Reismanet al., 2009).
AINRIL (antisense non-coding RNA inthe INK4 locus) isa 3.8 kb-long non-coding RNA expressed inthe opposite directionfromINK4A-ARF-INK4Bgene cluster
and isimportantfor expression ofthe protein-coding genes in cis. ANRIL activates two polycomb repressorcomplexes, PRC1 and PRC2 by direct bindingto chromobox 7(CBX 7) and SUZ12, members of PRC1 and PRC2, respectively (Kotakeet al., 2011, Yap et al., 2010). Thisresultsin silencingtheINK4b-ARF-INK4alocus encodingtumor suppressors p15INK4b, p14ARF, and p16INK4a, whichare usually alteredinan esti- mated 30-40% of human tumors. These genes are implicatedincell cycle inhibition, senescence and stress-induced apoptosis.
Selective binding of lncRNAs,such as HOTAIRE, ANRIL and SChLAP1 with chromatin modifying complexes andexecute histone modifications at specificlocithus strongly supportsthe ideathatlncRNAsmay function asideal epigeneticregulators in cells (Kotake et al., 2011). LncRNAs are also implicated in othercellularfunctions such as DNA repairin responseto genotoxic stress (Prensneret al., 2014),but to our knowledge themain function oflncRNAs currentlyistheregulation of geneexpression, thereforeitis obvious thattheir maintainedexpression remains criticalin ordertokeep cellular homeostasis andprevent the neoplastictransformation.
2.2.3 Gene expression profiling of BPDCN - insights to molecular pathogenesis
Besides chromosomal abnormality and mutational analysis, gene expression profiling (GEP) provides important insights, which cannot beexplored bycytogenetic andmu- tational studies. For example Dijkmanet al performedGEP analysis on 5 BPDCN and 3 cutaneous-AML samples andhave identified high-level expression of Notch signa l-ing genes (HES6,RUNX2). Moreover, despitethe mono-allelic deletion ofFLT3 gene, increasedexpression ofFLT3 oncogene was also observed in BPDCN, coincidentwith expression of both myeloidandlymphoid specific gene expression (Dijkman et al., 2007).
Inasecond study, a global GEP analysis on 6 BPDCN cases, incomparison with normal pDCs, myeloid and lymphoid precursorcells has demonstrated predom-inant expression of myeloid lineage genes and resting pDCs. Thiswork also discov- ered evidence ofan aberrantactivation oftheNF-κB pathway in BPDCN, whichwas
confirmed by Immunohistochemistry(IHC) by showing the nuclearlocalization ofkey components of NF-κ complex (c-Rel and p50)inBPDCN-derived cell line and BPDCN cases, in contrastto normal pDCs, which presented acytoplasmic staining. Thiswas associated tosensitivity toNF-κBinhibition andthus suggestive of anovel therapeutic strategy in BPDCN(Sapienzaet al., 2014).
Chapter 3
C l in ica l management of BPDCN
Given BPDCNsrarity and onlyrecentrecognitionas a distinct clinico-pathologicalen- tity, no standardizedtherapeutic approach has been establishedfor BPDCN andthe op-timaltherapyremainsto be defined. Current practice considers acute myeloidleukemia- like oracutelymphoblastic leukemia (ALL)-like regimens acceptableforinduction treatment.
Patients usually respond well toinitial chemotherapy, with completeresponse rates of 47% to 86%. However, the disease oftenrelapses, andtherelapsed cases aretypically resistant tothepreviously used chemotherapeutic agents(Fachetti, 2008, Feuillard et al., 2002). Outcomes are morefavorable incases thatlack cutaneous disease at presentation, although a comparison of cutaneous and non-cutaneouscases might be confounded by differences intreatment regimens. Conversely, patients with isolated cutaneouslesions survive longer(Alayedet al., 2013, Shi andWang, 2014).
These short-lived responses, with second remissions that are difficult to achieve, underscorethe need to consider hematopoieticcell transplantation earlyinthe disease course (Kharfan-Dabaja et al., 2013) as longlastingremissions have been documented in sporadic cases, usually occurring in young patients who have been treated with acute leukemia-type inductiontherapy,followed byallogeneic stem cell transplantationinfirst completeremission (Fachetti, 2008).
53
Durable remissions have been observed even inelderly patients, followed by allogeneic stem cell transplantfrom matchedrelated or unrelated donors after h igh-dose chemotherapy; in a retrospective study on 39 patients with BPDCN who underwent allogeneic stem cell transplantation(allo-SCT), n = 34, or autologous stem cell trans- plantation(auto-SCT), n = 5. The 3-year cumulative incidence of relapse, disease-free survival, and overall survival ofthese patients was 32%, 33%, and 41%, respectively (Roos-Weil et al., 2013).
It is noteworthy thatalthoughthe disease isrecognizedamongacute myeloid leukemias and acute myeloid leukemia-like regimes are often adopted, recent reports have suggested a potential role forALL-like protocols(Pagano et al., 2013,Piccaluga et al., 2012, Pileri et al., 2012).Recently, in a case report study, it has been demon-stratedthat using a combined ALL-like andAML-liketreatment protocolsfollowed by cord blood stem cell transplanthave achieved a complete remission (10 months since diagnosis)(Ramanathan et al., 2013).
Since AML, ALL andlymphomatypetherapies actually are usedfor BPDCN treatment,thereforethesetherapies areexplained inthefollowing section.
3 .1 Convent iona l AML type therapy
Traditionally AML treatmentinvolvestwo phases: 1) remission inductiontherapy, 2) consolidation, or post-remission, therapy andthen maintenancetherapy.Remission in- duction is designed to bringaboutcompleteremission,andconsolidation therapy is designed to kill any remaining leukemia cells. AML is usuallytreated with two or threeanti-cancer drugs(combination chemotherapy): theinitialtreatmentapplied to AML consists ofcytosine arabinoside (ara-C or Cytarabine) (is a nucleoside analogue thatincorporatesDNA andinduces apoptosis) combinedwith anthracycline (DNA and RNA intercalatingthatprevents DNA replication andtranscription). Consolidationther- apy includes one to twocourses of high-dose chemotherapy using onlyara-C, ora hematopoietic cell (stemcell or bonemarrow) transplant(Roboz, 2012).
Most patients will experience arelapse oftheir disease withoutthisadditional therapy. Approximately 70%-80% of patients<60 years of age will achieve complete remission, but most ultimately relapse andoverall survival is only40%-45% at 5 years (Roboz, 2011).
3 .2 Convent iona l ALL type therapy
Glucocorticoids(GCs) particularly prednisone and dexamethasone were amongthefirst drugs used inthetreatment of acutelymphoblasticleukemia(ALL) andhave remained essential components of therapy. The most commonly used steroids aredexamethasone and prednisone. ALL hasa higherincidencerate inchildrenthan inadults with a medianage of 39 years. Treatment usuallyincludes multi-agent chemotherapy with induction, consolidation, and maintenance.
Although a significant number of ALL patients respond well to GCs,some
Although a significant number of ALL patients respond well to GCs,some