Targeting IFN Pathway And IRFs In APL:Phenotypes And Treatment Strategies

Background:Acute promyelocytic leukemia(APL)is a distinct hematopoietic malignancy of acute myeloid leukemia(AML).More than 98%of APL cases are driven by the t(15;17)(q22;q21)chromosomal translocation that generates the PML-RARa oncoprotein,eliciting differentiation block at the promyelocytic stage and self-renewal enhancement of myeloid progenitors.For decades,the advent of all-trans retinoic acid(ATRA)synergized with arsenic trioxide(As2O3)has turned most APL from highly fatal to highly curable,while there are still patients refractory to the classic combination regimen or relapsing after remission.In addition to those with point mutation in PML and RARa moiety,patients with rare RAR? fusion genes may also be unresponsive or resistant to the standard treatment of ATRA and ATO.TBLRl-RAR?(TR)is the tenth fusion gene of APL identified in our lab.In our previous studies,we found similar to PML-RAR?,TR could self-associate into homodimers and form heterodimers with RXR?.Also,TR exhibited diminished transcriptional activity by recruiting more transcriptional corepressors than RAR?.Our previous in vitro studies have verified that pharmacologic doses of ATRA would trigger the degradation of TR protein with abrogated homodimerization,incomplete corepressor dissociation and full differentiation.Then we successfully established a novel transplantable murine leukemia model of TR APL.We found the expression of TR in mouse hematopoietic progenitors induces blockade of differentiation with enhanced proliferative capacity in vitro,with in vivo serial transplantation assay confirming the oncogenic role of TR fusion gene.Purpose:Despite the in vitro sensitivity to ATRA-induced cell differentiation,neither a dose of 2.5mg/kg ATRA monotherapy nor combination with As2O3 confers survival benefit to TR mice,consistent with poor clinical outcome of APL patients with TR fusion gene.Given the poor clinical outcome of TR patients,unveiling the unresponsiveness of TR-induced APL to RA and ATO treatment as well as exploring potential therapeutic strategies are of great necessity.In our study,we intend to find the answer of two core questions.First,why does the paradigmatic combination regimen of ATRA and ATO fail to improve the prognosis of TR-induced APL?How to explain the discrepancy between the in vitro and in vivo studies?Second,Furthermore,is there any other potential treatment option for TR-induced APL?With attempts to explore the molecular pathway in the pathogenesis of TR-induced APL,we compared the difference between TR and PR in several aspects.,Our study aims to fill the gap in understanding TR-induced APL and lead to insights into the principles underlying leukemogenesis mediated by rare RARa fusion genes,which may help prolong the survival of these patients and enable APL to be a bona fide curable leukemia.Methods:In order to identify the molecular pathway directly and specifically regulated by TR fusion gene,we established two Tet-on inducible lentiviral model to induce the expression of TR and PR fusion gene in U937 cells.The advantages of our model are listed as follows.(1)Reversible,flexible and reproducible(2)High inducibility and low off-target effect(3)Extremely tight regulation and low background.We used doxycycline to induce the expression of fusion gene at early stage and characterize phenotypes of TR compared with PR through a series of assays.Based on a transcriptome analysis through RNA-seq,as well as epigenetic signature inspired by ChIP-seq,we explored a core pathway which may play a key role in the pathogenesis of TR-induced APL.Then we utilized a series of in vitro and in vivo assays to confirm and compare the drug response between TR and PR-induced APL.Results:Analysis based on RNA-seq showed that the interferon(IFN)pathway was significantly down-regulated in U937-TR rather than U937-PR.At the same time,motif analysis results of ChIP-seq showed that the expression of TR induced a significant enrichment of interferon regulatory factor(IRF),implying the core role of IFN pathway in TR-induced APL.Hence,we used Type I IFNs and IFNy as well as the standard regimen of ATRA and ATO to treat TR-induced APL,and compared the drug response and phenotype of TR with PR in multiple aspects.Main results are listed as follows.Firstly,in terms of oncoprotein degradation,ATRA can degrade both TR and PR fusion protein,while ATO can only degrade PR fusion protein rather than TR fusion protein.On the contrary,both Type ? IFNs and IFNy can up-regulate both PML and PR fusion protein,rather than TBLR1 and TR fusion protein.The upregulation of PR fusion protein induced by IFN suggests that IFN therapy should be used with caution in PR-induced APL,since the accumulation of fusion protein may aggravate the progression of leukemia.In contrast,in TR-induced APL,IFN does not increase the expression of TR fusion protein,suggesting that IFN treatment in may be relatively safe in TR APL.Moreover,IFN can also act as a tumor suppressor by up-regulating wild-type PML in TR-induced APL.Secondly,with regard to leukemia phenotype,single drug of both Type ? IFNs and IFNy fail to induce cell differentiation.However,in TR-induced APL,IFN and 100nM ATRA can synergize to promote differentiation and achieve a terminal differentiation level similar to a higher dose 1 ?M ATRA.ATO can cause apoptosis of PR leukemia cells rather than TR leukemia cells.On the contrary,Type ? IFNs and IFNy monotherapy can prompt the apoptosis of TR leukemia cells.When it comes to serial colony formation assay,higher doses of ATRA and Type ? IFNs can reduce the colony forming ability and self-renewal capacity of TR murine leukemia cells,while IFN y and ATO seemed to increase the number or size of colonies at an earlier or later stage.Finally,we used IFNs combined with ATRA to treat TR mice.Both 15mg/kg and 25mg/kg ATRA can significantly prolong the survival of mice,implying a dose-dependent efficacy of ATRA.ATRA combining with IFNy group showed no survival benefit,consistent with in vitro colony formation results.Although compared with DMSO group,the combination of Type ? IFNs and ATRA can prolong the survival of TR mice,this combining regimen elicited no advantages over single-agent ATRA.On the one hand,the administered doses of Type ? IFNs in our experiment may be too low to exert its bona fide anti-tumor efficacy.On the other hand,the development of TR leukemia may not be necessarily dependent on the activation of Type ? IFNs pathway.Conclusions:First,ATO is 'unresponsive' in TR-induced APL.ATO failed to elicit the degradation of TR fusion gene,promote apoptosis and induce loss of self-renewal.The resistance of ATO may be related to the absence of PML NBs destruction in TR-APL,thus ATO cannot subsequently activate p53 and reduce self-renewal by restoring PML NBs.Second,ATRA is 'inadequate' in TR-induced APL.Although low-dose ATRA is sufficient to promote cell differentiation,increasing the dose of ATRA can promote the degradation of oncoprotein and reduce the colony formation capacity of TR mouse cells,which ultimately brings survival benefits.In TR-induced APL,the mechanism by which high-dose retinoic acid reduces self-renewal capacity is obviously independent of the reorganization of PML NBs in PR.Third,type ? interferon is promising' in TR-induced APL.On the one hand,IFN treatment does not upregulate the expression of TR fusion protein,thus may not aggravate the progression of leukemia.On the other hand,IFN treatment induces cell apoptosis,cooperates with ATRA to promote differentiation,and exhibits the potential to reduce self-renewal,reflecting anti-leukemia efficacy.This may be associated with IFN-induced upregulation of PML,which usually acts as a tumor suppressor to elicit anti-tumor efficacy.Background:Interferon regulatory factors(IRFs)are a type of transcription factors involved in the regulation of innate immunity and adaptive immune response.There are nine known members of IRF family in humans,numerically designated IRF1 to IRF9.All IRFs share a conserved N-terminal with a highly homologous DNA binding domain(DBD),which mainly recognizes and binds target genes containing the interferon stimulus response element(ISRE)in the promoter sequence.The C-terminus of genes contains different types of IRF-association domains(IAD),which mediate the interaction of specific IRF with proteins of other family members.Compared with other members of the IRFs family,IRF9 has once been called'the forgotten interferon regulatory factor' due to limited research.In the Type ? IFNs pathway,IRF9 is known to form an ISGF3 complex with STAT1 and STAT2 and activate downstream interferon-stimulating genes(ISGs).Abnormalities in IRFs-dependent pathway can lead to either activation or inhibition,which are both related to tumor development and inflammation response,suggesting the roles of IRFs as potential therapeutic targets.Some IRFs are involved in regulating the development of leukocytes,and are therefore closely related to leukemogenesis.Furthermore,in acute promyelocytic leukemia(APL)with RARa fusion genes,there are crosstalks between IFN pathway and retinoic acid(RA)pathway.Studies have found the coexistence of interferon response elements and retinoic acid response elements in the same promoter region of certain genes,which can act as both IFN target genes and RARa target genes.Whether IRF9 is directly involved in the transcriptional regulation of the RA pathway has not been elucidated yet.Purpose:Our study intends to decipher the role of IRF9 in APL and its regulatory relationship with RARa as well as exploring potential therapeutic targets in APL.Methods:We utilized lentiviral inducible system to induce the expression of IRF9 in NB4 promyelocytic leukemia cell line.In vitro studies focused on the leukemia phenotypes upon IRF9 overexpression.Based on bioinformatic methods,we predicted the potential RARa sequence in the proximal promoter region of IRF9 and perfomed dual dual luciferase reporter assays to identify the regulation relationship between IRF9 and RAR? fusion genes.Results:IRF9 is down-regulated in APL,with lowest expression among all AML subtypes.Based on transcriptome sequencing analysis of our previously constructed TBLR1-RAR?(TR)and PML-RAR?(PR)inducible expression models,we found IRF9 can be down-regulated by both TR and PR fusion genes,and this down-regulation can be rescued by ATRA.Overexpression of IRF9 can promote the differentiation of NB4 cells and has a synergistic effect with lower doses of ATRA.IRF9 expression can also reduce the colony formation capacity of NB4 cells.Results from dual-luciferase reporter assays failed to find evidence to determine that IRF9 was directly regulated by RARa or RARa fusion genes.However,we cannot rule out the possibility that RAR?fusion genes may repress IRF9 promoter activity via the distal regulatory region.And,IRF9 may not be a direct target of RAR?.