myc induces mirna-mediated apoptosis in response to hdac … · 2016. 1. 20. · fbs. p493-6 cells...

Tumor and Stem Cell Biology

Myc Induces miRNA-Mediated Apoptosis inResponse to HDAC Inhibition in HematologicMalignanciesClare M. Adams1, Scott W. Hiebert2, and Christine M. Eischen1

Abstract

Alterations in the expression or functionof histone deacetylases(HDAC) contribute to the development and progression ofhematologic malignancies. Consequently, the development andimplementation of HDAC inhibitors has proven to be therapeu-tically beneficial, particularly for hematologic malignancies.However, the molecular mechanisms by which HDAC inhibition(HDACi) induces tumor cell death remain unresolved. Here, weinvestigated the effects of HDACi inMyc-driven B-cell lymphomaand five other hematopoietic malignancies. We determined thatMyc-mediated transcriptional repression of the miR-15 and let-7families in malignant cells was relieved upon HDACi, and Myc

was required for their upregulation. ThemiR-15 and let-7 familiesthen targeted and downregulated the antiapoptotic genes Bcl-2and Bcl-xL, respectively, to induce HDACi-mediated apoptosis.Notably, Myc also transcriptionally upregulated these miRNAin untransformed cells, indicating that this Myc-inducedmiRNA-mediated apoptotic pathway is suppressed in malignantcells, but becomes reactivated upon HDACi. Taken together,our results reveal a previously unknown mechanism by whichMyc induces apoptosis independent of the p53 pathwayand as a response to HDACi in malignant hematopoietic cells.Cancer Res; 76(3); 1–13. �2015 AACR.

IntroductionAberrant expression or function of histone deacetylases

(HDAC) has been implicated in hematopoietic malignancies(1). Although HDAC inhibition (HDACi) induces tumor celldeath while leaving normal cells relatively unaffected, theunderlying mechanisms behind this remain unclear. Changesin expression of survival genes were observed following HDACi(1); however, whether these alterations resulted from hyper-acetylation of promoters or altered expression of transcription-al mediators was not determined. HDACi also affects DNAreplication, likely from the inability to deacetylate histones atreplication forks (2, 3).

Myc, an oncogenic transcription factor, is dysregulated in mosthematopoietic malignancies (4). However, untransformed cellsundergo apoptosis to counter hyper-proliferative signals fromMyc dysregulation. Specifically, Myc overexpression activates thep53 tumor-suppressor pathway, eliciting apoptosis (5, 6). Myc-induced apoptosis alsooccurs independent of p53 throughdown-regulation of anti-apoptotic Bcl-2 and Bcl-xL proteins, by anindirect and unclear mechanism (7–9). Both pathways becomeinactivated during tumorigenesis (10).

Myc transcriptionally activates or represses numerous genes,regulating many cellular processes (11). Myc represses protein-coding genes by recruiting HDACs and by binding and inhibitingthe transcriptional activatorMiz-1 (11, 12).Myc also regulates theexpression of noncoding RNA, including miRNA that bindmRNA, typically inhibiting translation (13). In malignant cells,Myc repressesmanymiRNAwhile specifically upregulating others(13, 14). Although Myc-mediated transcriptional activation hasbeen extensively studied, mechanisms of Myc-mediated repres-sion and their contribution to tumorigenesis are less understood.

Here, we describe a previously unknown miRNA-mediatedmechanism of Myc-induced apoptosis. We determined that cel-lular transformation status dictates whetherMyc transcriptionallyactivates or represses the miR-15 and let-7 families that targetantiapoptotic Bcl-2 and Bcl-xL, respectively. This apoptotic mech-anism was inactivated in transformed hematopoietic cells, butreactivated by HDACi. Our data reveal a general mechanismunderlyingHDACi-mediatedmalignant hematopoietic cell deathand provide new insight into Myc-induced apoptosis.

Materials and MethodsCell lines, transfection, infection, and vectors

Daudi, Ramos, Raji, Su-DHL-6, andNIH3T3 cells were culturedas described by the American Type Culture Collection. OCI-Ly-19and OCI-Ly-3 cells were cultured in RPMI-1640 containing 10%FBS. P493-6 cells from Dr. Dirk Eick (Helmholtz-Zentrum-Muenchen, Munich, Germany) were cultured as described (15).Tetracycline (0.1 mg/mL; Sigma) was added to cultures of P493-6cells for 24 hours to turn off MYC expression. Cell lines wereobtained between 2001 and 2007 and immediately frozen, socells were cultured for less than 6 months. Primary murine pre-Bcultures were generated and infected with retrovirus as previouslydescribed (5; details in Supplementary Experimental Procedures).

1Department of Pathology, Microbiology and Immunology,VanderbiltUniversity Medical Center, Nashville, Tennessee. 2Department of Bio-chemistry,Vanderbilt University Medical Center, Nashville, Tennessee.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Christine M. Eischen, Vanderbilt University MedicalCenter, 1161 21st Avenue South, Nashville, TN 37232. Phone: 615-343-2303; Fax:615-343-1633; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-1751

�2015 American Association for Cancer Research.

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Em-myc lymphoma cells were previously isolated and maintainedas published (16). Wild-type or p53�/� murine embryonicfibroblasts (MEF) were generated and cultured as described(6). Fibroblasts were transfected using Lipofectamine 2000(Invitrogen). Retroviral infections of fibroblasts were per-formed as previously reported (6). Vector/retrovirus details arein Supplementary Experimental Procedures.

HDAC inhibition and cell growth and apoptosis assaysIn vitro experiments utilized 1 mmol/L 4-hydroxytamoxifen

(4-OHT) or vehicle (EtOH), and 5 nmol/L depsipeptide (Cel-gene), 10 mmol/L RGFP966 (Repligen), or vehicle (DMSO).Cell number and/or viability were determined by Trypan-BlueDye exclusion (triplicate) and proliferation by MTT (Sigma;570 nm), MTS (Promega; 490 nm), or Alamar Blue (Invitrogen)assays (quadruplicate). Apoptosis was evaluated by flow cyto-metry following propidium iodide (sub-G1 DNA) or AnnexinV/7-AAD staining.

MiceFor the in vivo lymphoma experiments, 10- to 12-week-old

C57Bl/6 (The Jackson Laboratory) mice were subcutaneouslyinjected (one flank) with 4 � 106 Em-myc lymphoma cells aspreviously described (17). Once tumors reached 200 mm3, Dep-sipeptide (2 mg/kg) or vehicle (DMSO) was intraperitoneallyinjected. Mice were sacrificed at intervals for tumor evaluation.Studies complied with state and federal guidelines andwere approved by the Vanderbilt Institutional Animal Care andUse Committee. Normal B cells were purified from spleensof mice with the IMag Mouse B-Lymphocyte Enrichment Set(BD Biosciences).

Human tissueNormal human B cells were purified from leukoreduction

filters (Red Cross) and deidentified fresh spleens using the IMagHuman B-Lymphocyte Enrichment Set (BDBiosciences). Deiden-tified fresh spleen and frozen lymph nodes were obtained fromthe Cooperative Human Tissue Network, following an Institu-tional Review Board approval as nonhuman subject research(#150139).

Western blottingWhole-cell protein lysates were prepared and Western blotted

as reported (6, 18). Fibroblasts were lysed 48 hours aftertransfection.

AntibodiesWestern blotting antibodies were as follows: Bcl-2, Bcl-xL (BD

Biosciences); Mcl-1 (Rockland); Bim (22–40; Calbiochem);cleaved caspase-3 (Cell Signaling Technology); Myc, H3K9K14ac(Millipore); H3K56ac, H4K5ac, H3, H4 (Abcam); Bax (N-20),Miz-1 (H-190, Santa Cruz Biotechnology); and b-actin (Sigma).qChIP antibodies: Myc (N-262) and isotype controls (Santa CruzBiotechnology), RNA polymerase-II (Ser2-phosphorylated;Abcam), and H3K9K14ac (Millipore).

Quantitative chromatin immunoprecipitationQuantitative chromatin immunoprecipitation (qChIP) was

performed as previously described (19). Primer sequences andantibodies in Supplementary Experimental Procedures.

Quantitative real-time PCRRNA was isolated, cDNA was generated, and Sybr Green (SA-

Biosciences) and TaqMan MicroRNA Assays (Applied Bio-sciences) were used for quantitative real-time PCR (qRT-PCR;triplicate) to measure mRNA and miRNA, respectively, aspreviously described (16, 20). mRNA and miRNA expressionswere normalized to b-actin and RNU6b levels, respectively, andpresented as 2�DDCt. Primer sequences in Supplementary Exper-imental Procedures.

Luciferase assaysNIH3T3 cells were transfectedwith luciferase reporters, b-galac-

tosidase control plasmid, 50 nmol/L miR-15 or miR-195 miR-IDIAN miRNA mimics or control RNA (Dharmacon/Thermo-ScientificBio), and/or 200 nmol/L miScript Target Protectors(Qiagen). Luciferase and b-galactosidase activity was measuredas previously described (21).

Statistical analysisStudent t tests were used to statistically evaluate the data in

Figs. 1A and C, 2D–G, 3D and E, 4B–G, 5A–E, 6E, 7A, C, E, and F.Wilcoxon rank-sum tests determined statistical significancefor Figs. 2B and C, 3B, and 4A.

ResultsHDAC inhibition decreases Bcl-2 and Bcl-xL expression,inducing apoptosis in multiple hematopoietic malignancies

To evaluate the molecular events following HDACi, B-celllymphomas from Em-myc transgenic mice (Myc-driven B-celllymphoma model; ref. 22) and human Burkitt lymphoma lineswere treated with HDAC inhibitors. Depsipeptide (class-IHDACi), RGFP966 (HDAC3i; ref. 23), RGFP233 (HDAC1/2i),RGFP963 (HDAC1/2/3i), and Panobinostat (pan-HDACi) alldecreased cell expansion and number (Fig. 1A; SupplementaryFig. S1A and S1B). Depsipeptide also reduced cell expansion innineothermalignanthumanhematopoietic lines, including acutemyeloblastic leukemia (Kasumi), chronic myelogenous leukemia(K562), acute T-cell leukemia (Jurkat, Loucy), cutaneous T-celllymphoma (Hut-78, MyLa), diffuse large B-cell lymphoma (Su-DHL-6, OCI-Ly-19), and multiple myeloma (H929; Supplemen-tary Fig. S1C). Furthermore, HDACi decreased cell viability (Fig.1A; Supplementary Fig. S1B) and increased caspase-3 cleavage(Fig. 1B), characteristics of apoptosis.

To identify the molecular determinants of HDACi-mediatedapoptosis, we assessed expression of crucial prosurvival proteins.Comparedwith vehicle control-treated cells,HDACi ofmurine andhuman B-cell lymphoma cells decreased protein expression ofantiapoptotic Bcl-2 and Bcl-xL, but not Mcl-1 (Fig. 1B; Supplemen-tary Fig. S1D). Moreover, depsipeptide treatment also decreasedBcl-2 and Bcl-xL proteins in nine other malignant hematopoieticcell lines (Supplementary Fig. S1D). Decreased Bcl-2 and Bcl-xLmRNA (Fig. 1C) may explain the reduction in protein. InhibitingBcl-2 and/or Bcl-xL reportedly kills malignant hematopoietic cells,including lines we evaluated (24). Thus, HDACi specificallydecreased Bcl-2 and Bcl-xL expression, inducing apoptosis.

HDAC inhibition reveals posttranscriptional regulationofBcl-2and Bcl-xL

HDACi-induced effects on histone acetylation were evaluatedto gain insight into themechanism responsible for decreasing Bcl-2 and Bcl-xL. Western blotting showed increased global histone

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acetylation marks associated with active transcription in murineand human lymphoma cells treated with depsipeptide orRGFP966 (Fig. 2A; Supplementary Fig. S2A). Analogous resultswere obtained in the other nine malignant hematopoietic celllines tested (Supplementary Fig. S2B).

Histone acetylation is typically associated with gene activation,yet Bcl-2 and Bcl-xL mRNA decreased after HDACi. To investigatethis, we examined precision global run-on transcription coupledwith massively parallel sequencing (PRO-seq; ref. 25) data forBCL-2 and BCL-XL loci from Daudi cells treated for 4 hourswith depsipeptide or vehicle control (S.W. Hiebert, unpublisheddata). This analysis showed no significant expression changes(increased or decreased) at these loci (Supplementary Fig. S2C),suggesting a posttranscriptional mechanism is likely responsiblefor the observed changes in Bcl-2 and Bcl-xL mRNA.

Therefore, we assessed expression of the miR-15 family (miR-15a, -16-1, -195, -497) and let-7a, as these miRNAs posttran-scriptionally target and negatively regulate Bcl-2 and Bcl-xL

expression, respectively, and induce apoptosis in multiple celltypes (Supplementary Fig. S3; ref. 26). Consistent with previousreports (14, 27), miR-15 family and let-7a levels were decreasedin human diffuse large B-cell lymphoma and Burkitt lympho-ma cell lines compared with purified B cells and normal lymphnodes (Fig. 2B). Similarly, B-cell lymphomas from Em-myctransgenic mice also had reduced levels of these miRNA com-pared with levels in precancerous Em-myc splenocytes andpurified B cells (Fig. 2C).

Notably, HDACi increased miR-15a and miR-195 (represen-tative miR-15 family members) and let-7a in lymphomacells (Fig. 2D; Supplementary Fig. S4A and S4B). miR-31 levelswere unaffected, demonstrating not all miRNAs were upregulatedwith HDACi. All other malignant hematopoietic lines evaluatedshowed similar results (Supplementary Fig. S4C), indicatingthis HDACi-induced effect is not cell-type specific. Furthermore,with depsipeptide dose escalation, miRNA levels increased, indi-cating a dose response to HDACi (Supplementary Fig. S4D).

Figure 1.HDACi reduces Bcl-2 and Bcl-xLexpression and induces apoptosis.Cells remained untreated (UT) orreceived depsipeptide (Depsi),RGFP966 (966), or vehicle (DMSO)control. A, following drugadministration, proliferation (AlamarBlue; quadruplicate), cell number(triplicate), and viability (triplicate)were determined at the indicatedintervals in murine (Em-myc, EM330)and human (Daudi) lymphoma cells.B andC, protein andmRNA levelswereevaluated by Western blot (B) andqRT-PCR (triplicate; C), respectively.CC3, cleaved caspase-3. mRNAexpression was normalized to b-Actin.Error bars are SD for A (� , P

To evaluate whether the miR-15 family and let-7a were beingtranscribed upon HDACi, we measured primary miRNA tran-scripts of the miR-15a/16-1 andmiR-195/497 clusters and let-7a.Following HDACi, these pri-miRNA transcripts increased (Fig.2E). We next performed qChIP for RNA polymerase-II phosphor-ylated on serine 2 (RNAPII-p-Ser2), which is indicative of activetranscriptional elongation. HDACi resulted in RNAPII-p-Ser2enrichment at miR-15 family and let-7a promoters in lymphomacells (Fig. 2F). Enrichment was not observed at regions upstreamof these promoters or in vehicle control–treated cells. In addition,there was more RNAPII-p-Ser2 enrichment at miR-15 family andlet-7a promoters in depsipeptide-treated lymphoma comparedwith normal lymphocytes (Supplementary Fig. S4E), indicatingincreased transcription following HDACi in lymphoma. Thus,HDACi in lymphoma activates transcription of themiR-15 familyand let-7a.

To determine whether theHDACi-induced increase in themiR-15 family or let-7a was responsible for lymphoma cell death, weretrovirally expressed the miRNA in two lymphoma lines. Lym-phomas expressing the miR-15a/16-1 or let-7a clusters showedreduced Bcl-2 or Bcl-xL protein, cell expansion, and viability, andhad increased caspase-3 cleavage, sub-G1 DNA, and Annexin Vpositivity (Fig. 2G; Supplementary Fig. S4F–S4H). Thus, increasedlevels of the miR-15 family or let-7a are sufficient to induceapoptosis in lymphomas.

In vivo HDAC inhibition increases miR-15 family and let-7alevels, inducing lymphoma cell death

To extend our investigations in vivo, Em-myc lymphoma cellswere subcutaneously injected into C57Bl/6 mice. Once tumorsreached 200 mm3, mice were administered depsipeptide or vehi-cle control, and tumors were harvested at intervals. Analogous toour in vitro results, HDACi increased active histone acetylationmarks (Fig. 3A) and levels of the miR-15 family and let-7a(Fig. 3B), and decreased Bcl-2 and Bcl-xL proteins (Fig. 3C).Apoptosis was evident by caspase-3 cleavage, Annexin V positiv-ity, and sub-G1 DNA (Fig. 3C–E). These data confirm HDACiactivates miR-15 family and let-7a transcription that adverselyaffect the expression of crucial prosurvival proteins, inducinglymphoma cell apoptosis in vivo.

Myc transcriptionally upregulates the miR-15 family and let-7ain untransformed cells

Myc transcriptionally activates specificmiRNAwhile repressingothers in cancer cells (13, 14). To determine whether Myc medi-ated the repression of the miR-15 family and let-7a and/or theirinduction following HDACi, we evaluated untransformed andtransformed cells with altered Myc levels. Unexpectedly, in con-trast with transformed cells,Myc-overexpressing precancerous Em-myc spleens had increased miR-15 family and let-7a transcriptscompared with wild-type littermate spleens (Fig. 4A). miR-31levels were analogous between genotypes, indicating Myc over-expression selectively increased specific miRNA in nontrans-formed cells.

We next assessed primarymurine pre-B cells retrovirally expres-sing MycER, a 4-OHT–inducible Myc (28). Upon MycER activa-tion with 4-OHT, miR-15 family and let-7a levels significantlyincreased compared with vehicle control-treated pre-B cells (Fig.4B). This increasewas analogous to that ofmiR-20a, awell-knownMyc-induced miRNA (13). Levels of miR-31 were unaffected.Similar results were obtained following MycER activation in

untransformed fibroblasts (Supplementary Fig. S5A), indicatingthis effect also occurs in nonhematopoietic cells. Addition of 4-OHT to non–MycER-expressing pre-B cells or fibroblasts had noeffect on miRNA levels (Supplementary Fig. S5B). Therefore, Mycdoes not repress, but instead induces themiR-15 and let-7 familiesin untransformed cells.

To determine whether Myc was transcriptionally activating themiR-15 and let-7 families in untransformed cells, we utilized aMycER mutant lacking the Myc-Box-II domain (MycDMBII-ER)essential for Myc-mediated transcriptional activation (11). Levelsof the miR-15 family and let-7a were not induced followingMycDMBII-ER activation in wild-type MEFs, but were when tran-scriptionally competent MycER was activated (Fig. 4C). Whenprimary transcripts of the miR-15a/16-1 and miR-195/497 clus-ters and let-7a were evaluated, MycER, but not MycDMBII-ER,induced their expression in MEFs and pre-B cells (Fig. 4D and E),indicating Myc-mediated transcription was necessary to upregu-late these miRNA.

To assess whether Myc was at the miRNA promoters andwhether active transcriptionwas occurring, qChIPwas performed.qChIP for Myc in MycER-expressing MEFs revealed enrichment atthe promoter regions of the miR-15a/16-1 and miR-195/497clusters and let-7a following 4-OHT–induced MycER activation(Fig. 4F). Importantly, RNAPII-p-Ser2 andH3K9K14ac, indicatorsof active transcription, were also enriched (Fig. 4F). No enrich-ment was observed at regions upstream of the miRNA promotersor in vehicle control–treated cells. Similar qChIP results wereobtained in vivo when nontransformed precancerous Em-myctransgenic spleens were compared with nontransgenic litter-mate-matched spleens (Fig. 4G), further demonstrating that Myctranscriptionally upregulates these miRNA families in untrans-formed cells.

Myc is required for HDACi-induced miR-15 family and let-7atranscriptional upregulation

To determine the role of Myc in repressing the miR-15 andlet-7 families in malignant cells, qChIP was performed on Myc-overexpressing murine and human lymphoma cells. Myc wasenriched at the promoters of both miR-15 family clusters andlet-7a in both cell lines, but not at upstream regions (Fig. 5A).ENCODE MYC ChIP-seq data (29) also showed MYC at thesepromoters in hematopoietic and nonhematopoietic malignan-cies and nontransformed human cells (Supplementary Fig.S6A). Importantly, Myc was enriched at these same loci inboth the presence and absence of HDACi (Fig. 5A). Consistentwith Myc-mediated repression of these loci in lymphoma, therewas significantly more enrichment of Myc at the promoterregions of the miR-15a/16-1 and miR-195/497 clusters andlet-7a in lymphoma cells compared with normal lymphocytes(Supplementary Fig. S6B).

Myc transcriptionally activates CAD and represses p21 (11),consistent with the increase in RNAPII-p-Ser2 and H3K9K14acat CAD and the lack of enrichment at p21 we observed inthe lymphoma cells (Fig. 5B). Neither RNAPII-p-Ser2 norH3K9K14ac enrichment was detected at promoters of eithermiR-15 family cluster or let-7a in the lymphomas (Fig. 5B).Collectively, the data indicate Myc activates miR-15 familyand let-7a transcription in untransformed cells, whereas itappears to repress their transcription in transformed cells.Moreover, HDACi of B-cell lymphoma induced the miR-15family and let-7a to levels similar to those in nontransformed

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miRNA Mediate HDAC Inhibition–Induced Apoptosis

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precancerous B cells overexpressing Myc (Supplementary Fig.S6C). Furthermore, the derepression of the miR-15 family andlet-7a detected in lymphomas following 6 hours of HDACi didnot appear to be due to changes in Myc protein, as Myc levelswere similar for at least 12 hours after HDACi (SupplementaryFig. S6D). Together, our data suggest HDACi converts Myc froma transcriptional repressor into a transcriptional activator inlymphoma.

To test whether Myc was required for HDACi-induced upregu-lation of the miR-15 family and let-7a, we utilized the human B-cell lymphoma line, P493-6, that expresses a tetracycline-regula-table MYC (15). With tetracycline present, MYC levels weresignificantly reduced and HDACi failed to increase miR-15a,miR-195, or let-7a levels (Fig. 5C).OnlywhenMYCwas expresseddidHDACi increase their expression. Therefore, Mycwas requiredto mediate the HDACi-induced upregulation of the miR-15family and let-7a. In addition, irrespective of HDACi, when MYCexpression was off, levels of the miR-15 and let-7 families wereslightly increased compared with when MYC was expressed,providing additional evidence that MYC represses these miRNAin lymphoma.

To further assess the requirements of MYC on miR-15 familyand let-7a expression, we performed qChIP with P493-6 cells.When Myc was expressed, it was enriched at the miR-15 familyand let-7a promoters (Fig. 5D). Upon HDACi, enrichment ofRNAPII-p-Ser2 and H3K9K14ac (marks of active transcription)at miR-15 family and let-7a promoters was only observed inMYC-expressing cells (Fig. 5E). When MYC was not expressed,a slight enrichment of RNAPII-p-Ser2 and H3K9K14ac wasdetected at the miR-15 family and let-7a promoters, regardlessof HDACi (Fig. 5E). These data show that MYC mediated therepression of the miR-15 family and let-7a and was requiredfor their HDACi-induced transcriptional upregulation.

Myc transcriptional activity is required to suppress Bcl-2 andBcl-xL expression

In untransformed cells, Myc suppressed Bcl-2 and Bcl-xLexpression, inducing apoptosis through an indirect, unresolvedmechanism (7, 8), purportedly through binding Miz-1 (9).However, Bcl-2 and Bcl-xL proteins are overexpressed in themajority of Em-myc B-cell lymphomas and human lymphomas(8). Evaluation of Em-myc B-cell lymphomas compared withnormal B cells and spleens from precancerous Em-myc miceconfirmed these results (Fig. 6A). In contrast, precancerous Em-myc spleens with increased Myc had reduced Bcl-2 and Bcl-xLproteins compared with nontransgenic littermate-matchedspleens (Fig. 6B). Mcl-1 and Bax (proapoptotic Bcl-2 familymember) were unaffected. Similarly, in MycER-expressing pri-

mary pre–B cells and MEFs, decreases in Bcl-2 and Bcl-xL weredetected following MycER activation (Fig. 6C). No change inMcl-1 or Bax expression was detected, whereas Bim, a proa-poptotic Bcl-2 family member upregulated by Myc (30), wasincreased upon MycER activation (Fig. 6C). Therefore, withelevated Myc, nontransformed cells decrease Bcl-2 and Bcl-xLexpression, whereas their expression is increased in trans-formed cells.

Myc associates with the transcription factor Miz-1 through amotif requiring valine 394, reportedly suppressing Bcl-2 (9). Toinvestigate this, MycER harboring a point mutation (V394D)disrupting the Myc:Miz-1 interaction (MycV394D-ER; Supple-mentary Fig. S7A; ref. 31) was expressed in wild-type MEFs.Following MycV394D-ER activation with 4-OHT, Bcl-2 andBcl-xL were suppressed equivalently to cells expressing wild-typeMycER, ruling out a Miz-1–mediated mechanism (Fig. 6D).Analogous data were obtained using p53-null MEFs (Supplemen-tary Fig. S7B), consistent with prior results in p53-null myeloidandB cells (7, 8). These data reveal aMiz-1– andp53-independentmechanism for Myc-mediated suppression of Bcl-2 and Bcl-xLexpression. Moreover, MycV394D-ER effectively induced miR-15family and let-7a expression (Fig. 6E), further supporting thismiRNA-mediated mechanism of Myc-induced suppression ofBcl-2 and Bcl-xL. In addition, evaluation of ChIP-seq data forMiz-1 in four human and six murine cell lines/tissues (32–35)showed Miz-1 enrichment at promoters of known Miz-1–regu-lated genes (e.g., VAMP4; Supplementary Fig. S7C). However,Miz-1 was not enriched at the miR-15 family or let-7a promotersin any of the cell lines/tissues evaluated, indicatingMiz-1 does nottranscriptionally regulate these miRNAs.

To determine whether Myc transcriptional activity isrequired to decrease Bcl-2 and Bcl-xL expression in normalcells, wild-type MEFs expressing MycER or the transcription-ally inactive MycDMBII-ER mutant were evaluated. Following4-OHT addition, decreased Bcl-2 and Bcl-xL and increased Bimexpression were only observed in cells expressing wild-typeMycER and not MycDMBII-ER (Fig. 6F). Again, Mcl-1 and Baxexpression were unaffected. Therefore, Myc-mediated transcrip-tional activity is necessary for Bcl-2 and Bcl-xL downregulation.

Myc transcriptionally induces themiR-15 family and let-7a thatthen target Bcl-2 and Bcl-xL

We reasoned that defining the Myc-induced mechanism ofBcl-2 and Bcl-xL downregulation in normal cells would provideinsight into the mechanism behind their downregulationfollowing HDACi. To test whether the reduction in Bcl-2 andBcl-xL was a direct consequence of Myc-induced upregulation ofthe miR-15 family and let-7a in untransformed cells, luciferase

Figure 2.HDACi reverses Myc-mediated repression of the miR-15 family and let-7a. Lymphoma cells were treated with depsipeptide (Depsi), RGFP966 (966), or vehicle(DMSO) control. A, Western blots in murine (EM330) and human (Daudi) lymphoma cells were performed. B and C, mature miRNA levels were determined byqRT-PCR (triplicate) in human diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma , two normal human lymph nodes, and purified B cells from humanperipheral blood (PB) and spleen (Sp; B), and six murine Em-myc lymphomas, two precancerous Em-myc spleens, and purified murine splenic B cells (C). D and E,lymphoma cells were treated with depsipeptide, RGFP966 (966), or vehicle (DMSO) for the time indicated. Mature miRNA (D) or pri-miRNA (E) levels weredetermined by qRT-PCR (triplicate) in Em-myc (EM330) and Daudi lymphoma cells. Small RNA, RNU6b, was used for qRT-PCR normalization for B–E. F, aftertreatmentwith depsipeptide or vehicle (DMSO) for 4 hours, ChIPwith anti–RNAPII-phosphorylated-Serine2 (RNAPII-p-Ser2) or isotype control (IgG)was performedfollowed by qRT-PCR (triplicate) for the indicated promoters or upstream regions (up; negative controls) in murine (EM330) and human (Daudi) lymphoma cells.Values are relative to their respective IgG control and inputDNA.G, EM330 lymphoma cellswere infectedwith either empty retrovirus (vector) or retrovirus encodingthe miR-15a/16-1 or let-7a/7f miRNA clusters (14). Western blotting (left) was performed; U, uninfected; CC3, cleaved caspase-3. Viability (center; triplicate) and thepercentage of cells with sub-G1 DNA content (right; triplicate) were determined at the indicated intervals. Error bars are SEM for B–F. B and C, � , P < 0.01, lymphomaversus mean of all normals; D–F, � , P < 0.001, depsipeptide or 966 versus DMSO. Error bars are SD for G, � , P < 0.03, compared with empty vector.

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assays were performed in MycER-expressing fibroblasts withreporters harboring wild-type or mutated miRNA-binding sitesin the Bcl-2 or Bcl-xL 30-untranslated region (30-UTR). FollowingMycER activation, luciferase activity decreased in cells contain-ing the reporter with wild-type miR-15 family or let-7a–bindingsites in the Bcl-2 or Bcl-xL 30-UTR, respectively (Fig. 7A). Lucif-erase activity remained unchanged in cells containing reporterswith mutated miR-15 family or let-7a–binding sites or cellsexpressing the transcriptionally impaired MycDMBII-ER mutant(Fig. 7A). A reporter containing the miR-17 family binding siteof the p21 30-UTR served as a positive control, as p21 is avalidated target of the Myc-regulated miR-17 family (13). Thesedata indicate a novel mechanism where Myc transcriptionallyupregulates the miR-15 family and let-7a, which then target the30-UTR of Bcl-2 and Bcl-xL, respectively, leading to their down-regulation in untransformed cells.

To further validate this mechanism, wild-type MEFs weretransfected with modified RNA molecules (Target Protectors)designed to block the miR-15 family or let-7a from bindingtheir specific target sites in the Bcl-2 or Bcl-xL 30-UTR, respec-tively. Bcl-2 and Bcl-xL proteins increased in wild-type MEFstransfected with Bcl-2 or Bcl-xL Target Protectors, respectively,as endogenous miR-15 family members and let-7a were unableto bind and inhibit their expression (Fig. 7B). Combining theBcl-2 Target Protector with miR-15a overexpression, whichalone decreased Bcl-2 protein, rescued Bcl-2 protein expression(Fig. 7B). Then, MycER-expressing fibroblasts were transfectedwith luciferase reporters, as described above, together with theBcl-2 or Bcl-xL Target Protectors. In the presence of Bcl-2 orBcl-xL Target Protectors, little, if any, decrease in luciferaseactivity was detected following MycER activation (Fig. 7C).Next, MEFs expressing MycV394D-ER (unable to interact withMiz-1) were transfected with Bcl-2 or Bcl-xL Target Protectors.MycV394D-ER activation decreased Bcl-2 and Bcl-xL proteinexpression in the absence of any Target Protector (Fig. 7D).However, levels of Bcl-2 or Bcl-xL were maintained when TargetProtectors blocked the miR-15 or let-7 family binding sites,respectively (Fig. 7D). Therefore, downregulation of Bcl-2 andBcl-xL upon Myc activation in untransformed cells was due toinduction of the miR-15 family and let-7a that bind the 30-UTRof Bcl-2 and Bcl-xL, respectively.

We then tested whether targeting of Bcl-2 and Bcl-xL by themiR-15 family and let-7a contributes to Myc-induced apoptosis,independent of p53. MycV394D-ER was activated in p53-nullMEFs under reduced serum conditions with or without TargetProtectors. MycV394D-ER–activated MEFs containing Target Pro-tectors had increased cell expansion and reduced cleaved caspase-3 and Annexin V positivity (Fig. 7D–F). Thus, Myc induces theexpression of miR-15 family and let-7a independent of p53,leading to apoptosis. Collectively, the data reveal a novel mech-anismwherebyMyc upregulates themiR-15 family and let-7a thattarget Bcl-2 and Bcl-xL in untransformed cells to trigger apoptosis,and that this mechanism is reactivated in lymphomas followingHDACi.

DiscussionAlthough selective killing of tumor cells by HDACi is being

clinically tested and multiple effects have been noted, such asaltered expression of apoptotic genes andDNAdamage (1–3), themechanism for its effects remains incompletely understood.Here,

Figure 3.In vivo, HDACi increases miR-15 family and let-7a levels, inducing lymphomacell death. C57Bl/6 mice with subcutaneous Em-myc lymphoma tumors(EM330) that reached 200 mm3 were treated with depsipeptide (Depsi) orvehicle (DMSO) control (n¼4/group). Tumorswere harvested 24 hours later,and levels of the histone acetylation marks (A) and indicated proteins (C)were determined by Western blot. B, miRNA levels were assessed by qRT-PCR (triplicate), and RNU6b was used for qRT-PCR normalization. As apositive control, cultured EM330 lymphoma cells (in vitro) were treated withvehicle (DMSO;�) or depsipeptide (þ). Apoptosis was measured by cleavedcaspase-3 (CC3), Annexin V positivity (triplicate; D), and propidium iodidestaining of sub-G1 (apoptotic) DNA (triplicate; E). Error bars are SEM for B (� ,P

Figure 4.Myc transcriptional activity is necessary to inducethemiR-15 family and let-7a in nontransformed cells.MaturemiRNA (A–C) and pri-miRNA (D and E) levelswere determined by qRT-PCR (triplicate) and arenormalized to RNU6b levels. A, precanceroussplenocytes from Em-myc mice and wild-type (WT)nontransgenic littermates were evaluated (n ¼ 4/group). B–E, MycER or MycDMBII-ER was activatedwith 4-OHTor vehicle (EtOH) control at the indicatedintervals in primary pre-B cells (B and E) andMEFs (Cand D). F and G, following ChIP with anti-Myc, anti–RNAPII-phosphorylated-Serine2 (RNAPII-p-Ser2),anti-H3K9K14ac, or isotype controls (IgG), qRT-PCRfor the indicated promoters or regions upstream (up)that Myc does not bind (negative controls) wasperformed (triplicate). F, MycER-expressing p53�/�

MEFs received vehicle (EtOH; �) or 4-OHT (þ)for 4 hours to induce MycER. G, splenocytes fromwild-type (nontransgenic; Tg�) or precancerousEm-myc transgenic (Tgþ) littermate mice. Values forqChIP are relative to their respective IgG control andinput DNA. Error bars, SEM; � , P < 0.001;Em-myc versus mean of all WT spleens (A); 4-OHTversus EtOH (B, E, and F); MycER versus MycDMBII-ER, and (G) Em-myc (þ) versus wild-type (�)(C and D).

Adams et al.





Figure 5.Myc is required for HDACi-induced transcriptional upregulation of the miR-15 family and let-7a. A, B, D, and E, following ChIP with anti-Myc, anti–RNAPII-phosphorylated-Serine2 (RNAPII-p-Ser2), anti-H3K9K14ac, or isotype controls (IgG), qRT-PCR for the indicated promoters or regions upstream (up) that Myc doesnot bind (negative controls) was performed (triplicate). A, murine (EM330) and human (Daudi) lymphoma cells treated with depsipeptide (Depsi) or vehicle(DMSO) for 4 hours. B,p21 (Myc repression target) andCAD (Myc activation target)were controls (11). Values for qChIP are relative to their respective IgG control andinput DNA. C, human P493-6 lymphoma cells containing tetracycline-regulatable MYC exposed to tetracycline (þ; MYC Off) for 24 hours or not (�; MYC On) wereWestern blotted. These cells were also treated for 12 hours with depsipeptide or vehicle (DMSO) control. miRNA were measured by qRT-PCR (triplicate)and normalized to RNU6b levels. D and E, before qChIP analyses, P493-6 cells received tetracycline for 24 hours (MYC Off) or not (MYC On) prior totreatment with depsipeptide or vehicle (DMSO) for 4 hours. Values for qChIP are relative to their respective IgG control and input DNA. Error bars, SEM;� , P < 0.0002 (B), � , P < 0.01 (E), both RNAPII-p-Ser2 and H3K9K14ac versus IgG (B), depsipeptide versus DMSO (C).






we show HDACi switches Myc from a repressor to an activator ofmiRNA that control the expression of Bcl-2 and Bcl-xL, signifi-cantly contributing to HDACi-mediated tumor cell death. Weidentified a mechanism of HDACi-induced apoptosis that occursin Myc-driven B-cell malignancies and likely contributes to otherhuman cancers. Our data provide evidence of a novel Myc-inducedmiRNA-mediatedmechanismof apoptosis that is presentin nontransformed cells, repressed in malignant cells, and reacti-vated in tumor cells upon HDACi.

It was previously postulated that HDACi kills myeloid leuke-mia cells through changes in expression of extrinsic apoptoticpathway proteins (36). Others have reported that HDACi causesglobal changes in gene expression that alter the apoptotic thresh-old in favor of cancer cell killing (37). Specifically, expressionprofiling showed that HDACi altered mRNA levels of pro- andantiapoptotic Bcl-2 family members, yielding a proapoptoticsignature in malignant cells (37). However, measuring stablepools of individual or multiple mRNA as an indirect readout oftranscription would miss the posttranscriptional regulation ofgene expression. Our data reveal that increased miRNA transcrip-tion, rather than direct transcriptional repression, leads to down-regulation of antiapoptotic Bcl-2 and Bcl-xL gene expression.Likewise, other groups have reported HDACi-mediated changesin miRNA expression (38–41). For example, HDACi of a breastcancer line changed the expression of 27 miRNA within 5 hours,including let-7a (38). However, they reported downregulation of

let-7a, whereas we detected increased let-7a upon HDACi; thisdiscrepancymaybedue todifferences in the cell types evaluatedorthe HDAC inhibitors used. Importantly, our results indicate thatHDACi-induced changes in Bcl-2 and Bcl-xL were mediated byMyc. Myc is overexpressed and/or dysregulated in most humanmalignancies and is essential in cancers driven by other onco-genes, such as mutant Ras (4, 42). Thus, our data, providing amolecular link between Myc, miRNA, and Bcl-2 and Bcl-xL, yieldnew insights into the transforming action ofMyc and suggest howthis pathway can be targeted therapeutically. Moreover, our worksuggests that the expression of these miRNA may be usefulbiomarkers for HDACi sensitivity.

Due to previous reports that Myc repressed miRNA in malig-nant cells (14, 27), we were initially surprised whenMyc inducedthe expression of the miR-15 family and let-7a in untransformedcells. However, Myc overexpression drives cancer cell prolifera-tion, but triggers apoptosis in untransformed cells (4). Mycinduces apoptosis by activating the p53 pathway and simulta-neously downregulating Bcl-2 and Bcl-xL mRNA expressionthrough an indirect mechanism (7, 8), reportedly involvingMiz-1 (9). Myc expression did not change the half-life of Bcl-2(7); therefore, we suspected a transcriptional or posttranscrip-tional mechanism. The miR-15 family and let-7a were known totarget Bcl-2 and Bcl-xL, respectively, contributing to apoptosis(26), sowe investigatedwhether a connection betweenMyc, thesemiRNA, andBcl-2 andBcl-xL downregulation existed. Indeed,Myc

Figure 6.Myc transcriptional activity regulatesBcl-2 and Bcl-xL expressionindependent of Miz-1 and p53.A, Em-myc lymphomas (n ¼ 11) andprecancerous Em-myc purified B cells(n ¼ 2) and spleens (n ¼ 3) wereWestern blotted. B, precanceroussplenocytes from Em-mycmice (n¼ 5)and wild-type (WT) nontransgeniclittermates (n ¼ 5) were Westernblotted. C, D, and F, at intervalsfollowing addition of 4-OHT, WTMycER-expressing MEFs and primarypre-B cells (C) and MycV394D-ER- (D) and MycDMBII-ER–expressing(F) WT MEFs were harvested andWestern blotted. E,miRNA levelsweredetermined by qRT-PCR (triplicate;normalized toRNU6b levels) followingMycV394D-ER activation with 4-OHTor vehicle (EtOH). Error bars, SEM;� , P < 0.001, 4-OHT versus EtOH.


Adams et al.




suppressed the expression of Bcl-2 and Bcl-xL independent of itsinteraction with Miz-1 and of p53, which can itself transcrip-tionally repress Bcl-2 and Bcl-xL expression (43, 44). Transcrip-tionally competent Myc was required for the reduction in Bcl-2and Bcl-xL and the induction of the miR-15 and let-7 families.Moreover, open chromatin and activated RNA polymerase-IIwere observed at the miRNA promoters in Myc-overexpressinguntransformed cells. Luciferase reporter assays confirmed thatMyc induced the miR-15 family and let-7a, which directlytargeted Bcl-2 and Bcl-xL 30-UTRs, respectively. By inhibitingthe miR-15 family from binding Bcl-2 and let-7a from bindingBcl-xL, the decrease in Bcl-2 and Bcl-xL following Myc activationwas blocked. Combined, our data provide strong evidence thatin untransformed cells, Myc induces the miR-15 family andlet-7a that then target Bcl-2 and Bcl-xL, respectively, triggeringapoptosis. These results reveal a novel miRNA-mediated mech-anism of tumor suppression that is activated in normal cellsupon Myc dysregulation.

Our data indicate that Myc transcriptionally activates the miR-15 family and let-7a in untransformed cells, while transcription-ally repressing them in transformed cells. In lymphoma cells, Mycwas present at miR-15 family and let-7a promoters, which wereclosed and transcriptionally inactive, indicating that Myc was

likely mediating this repression, as had been reported (14). Ourdata from multiple hematopoietic malignancies indicate HDACscontributed to the repression of both miR-15 family clusters andlet-7a, as repression was relieved following HDACi, resulting intranscriptional upregulation of these miRNA, which requiredMyc. The HDAC3-selective inhibitor RGFP966 induced miR-15a and let-7a to an equal extent as the class-I HDAC inhibitordepsipeptide and the more specific HDAC1/2/3 inhibitorRGFP963 did, suggesting HDAC3 may be the primary HDACinvolved in mediating the repression. Consistent with theseresults, others have reported HDAC3 is specifically involved withrepression of miR-29a/b/c and miR-15a/16-1 in B-cell and man-tle-cell lymphoma lines, respectively (41, 45). Moreover, Myc isreported to recruit HDAC3 to the promoters of protein-codinggenes and miRNA to repress their expression (12, 41, 45). How-ever, RGFP233, the HDAC1/2 inhibitor, also increased levels ofmiR-15a and let-7a, indicating that they may also contribute tothe repression. Although how Myc and HDAC interactions con-tribute to tumorigenesis remains unresolved, our data suggest thatMyc, together with HDACs, alters the epigenome, leading torepression of miRNA and possibly other genes whose expressionresults in cancer cell apoptosis. HDACi relieves the transcriptionalrepression of the miR-15 and let-7 families in malignant

Figure 7.Myc induces themiR-15 family and let-7a that then targetBcl-2 andBcl-xL. A andC, luciferase expression vectors containing the 30-UTRofBcl-2orBcl-xLwith thewild-type (WT) or a mutated (Mut) miRNA-binding site were transfected into fibroblasts expressing the 4-OHT–inducible MycER or MycDMBII-ER. An expression vectorcontaining the miR-17 family binding site in the wild-type p21 30-UTR was a positive control (13). Luciferase activity was measured (triplicate)48 hours following vehicle (EtOH) control or 4-OHT addition to activateMycER. A b-galactosidase reporter plasmidwas cotransfected for normalization. B–F,miR-15family and let-7a miRNA-binding sites in the Bcl-2 and Bcl-xL 30-UTR were blocked with site-specific small molecules (TP; Target Protectors). B, wild-typeMEFs transfected with either Bcl-2 or Bcl-xL Target Protectors and/or miR-15a mimic were Western blotted. UT, untransfected cells. D–F, p53

�/� MEFs, with orwithout Bcl-2 and/or Bcl-xL Target Protectors, expressing the 4-OHT–inducible MycV394D-ERwereWestern blotted (D), subjected toMTT assay (E; quadruplicate),or analyzed for Annexin V positivity (triplicate) by flow cytometry at intervals following addition of 4-OHT. CC3, cleaved caspase-3. Error bars, SEM(A and C, � , P < 0.009, 4-OHT vs. EtOH) and SD (E, � , P < 0.02; F, � , P < 0.0001; both TP vs. control).






hematopoietic cells, resulting in transcription of these miRNA,which targeted Bcl-2 and Bcl-xL, killing the cancer cells.

Furthermore, hematopoietic cell lines with either mutant orwild-type p53 showed analogous results, indicating HDACi-induced effects are independent of p53 status. Given the p53pathway is inactivated and Myc is dysregulated in most humancancers (4, 46), our results identify a new potentially therapeuticavenue to induce apoptosis that capitalizes on Myc and is inde-pendent of p53. Of note, overexpression of Bcl-2 and/or Bcl-xLprotected from HDACi-induced cell death (47–50), supportingour conclusion that miRNA targeting these genes leads to apo-ptosis. Our studies have identified a novel mechanism of Myc-induced apoptosis that capitalizes on miRNA to suppress theexpression of crucial prosurvival proteins. While this mechanismis inactivated in malignancies through epigenetic alterationsinvolving HDACs, we have shown that it can be reactivated byHDACi. Our current study provides a novel mechanism thatunderlies HDACi-mediated cell death and offers new insightsthat should aid in improving cancer therapies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: C.M. Adams, S.W. Hiebert, C.M. EischenDevelopment of methodology: C.M. Adams

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C.M. AdamsAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C.M. Adams, S.W. Hiebert, C.M. EischenWriting, review, and/or revision of themanuscript:C.M. Adams, S.W.Hiebert,C.M. EischenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.M. EischenStudy supervision: C.M. Eischen

AcknowledgmentsThe authors thank Pankaj Acharya, Pia Arrate, Robert Duszynski, and

Christina Wells for technical assistance, Repligen for RGFP compounds, Dr.Josh Mendell for miRNA retroviruses, the ENCODE Consortium and produc-tion laboratories, Eischen lab members for helpful discussions, and Dr. BillTansey for critically reading the article.

Grant SupportThis work was supported by F31CA165728 (to C.M. Adams), T32GM08554

(C.M. Adams), R01CA178030 (S.W. Hiebert), R01CA148950 (C.M. Eischen),and the NCI Cancer Center Support Grant P30CA068485 utilizing the VAN-TAGE core.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 29, 2015; revised September 18, 2015; accepted October 12,2015; published OnlineFirst December 16, 2015.

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Published OnlineFirst December 16, 2015.Cancer Res Clare M. Adams, Scott W. Hiebert and Christine M. Eischen HDAC Inhibition in Hematologic MalignanciesMyc Induces miRNA-Mediated Apoptosis in Response to

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