Mediated Topoisomerase II&Beta

Targeting PKCd-mediated topoisomerase IIb overexpression subverts the differentiation block in a retinoic acid-resistant APL cell line

S McNamara1,3, JN Nichol1,3, H Wang2 and WH Miller Jr1

1Division of Experimental Medicine, Department of Oncology, Segal Cancer Comprehensive Centre, Lady Davis Institute for Medical Research, Sir Mortimer B Davis Jewish General Hospital, McGill University, Montre´al, Quebec, Canada and 2Segal Cancer Comprehensive Centre, Lady Davis Institute for Medical Research, Sir Mortimer B Davis Jewish General Hospital, Montre´al, Quebec, Canada

Retinoic acid (RA) relieves the maturation block in t(15:17) therapeutic doses of all-trans RA, a vitamin A derivative that acute promyelocytic leukemia (APL), leading to granulocytic activates RARA and circumvents the differentiation block.4 differentiation. However, RA treatment alone invariably results in RA resistance, both in vivo and in vitro. RA-resistant cell Unfortunately, although treatment with RA alone results in a lines have been shown to serve as useful models for elucida- complete remission, the reprieve is not long-lasting, as RA 5 tion of mechanisms of resistance. Previously, we identified resistance develops in vivo, a phenomenon that can be topoisomerase II beta (TOP2B) as a novel mediator of modeled in vitro.6 RA-resistance in APL cell lines. In this study, we show that Acquired mutation in the PML-RARA oncoprotein is one both TOP2B protein stability and activity are regulated by a source of RA-resistant APL. Studies on the PML-RARA protein in member of the protein kinase C (PRKC) family, PRKC delta (PRKCD). Co-treatment with a pharmacologic inhibitor of RA-resistant patient cells or cell lines established that roughly PRKCD and RA resulted in the induction of an RA responsive 33% possessed a mutation in the ligand-binding domain of the reporter construct, as well as the endogenous RA target genes, RARA portion of the fusion protein, resulting in a dysfunctional CEBPE, CYP26A1 and RIG-I. Furthermore, the co-treatment PML-RARA that is unable to respond to pharmacological doses overcame the differentiation block in RA-resistant cells, as of RA.7–10 However, the mechanisms of acquired resistance assessed by morphological analysis, restoration of promyelo- in the remaining 67% are undefined. The cells continue to cytic leukemia nuclear bodies, induction of CD11c cell surface expression and an increase in nitro-blue-tetrazolium reduction. express wild-type PML-RARA, yet are resistant to RA-induced Cumulatively, our data suggest a model whereby inhibition of differentiation. PRKCD decreases TOP2B protein levels, leading to a loss of In vitro-derived RA-resistant cell lines are useful experimental TOP2B-mediated repressive effects on RA-induced transcrip- models for the study of mechanisms of RA resistance in APL. tion and granulocytic differentiation. Our lab has previously isolated three RA-resistant subclones Leukemia (2010) 24, 729–739; doi:10.1038/leu.2010.27; from the parental RA-sensitive cell line NB4, denoted NB4- published online 4 March 2010 11,12 Keywords: acute promyelocytic leukemia; topoisomerase II beta; MR2, NB4-MR4 and NB4-MR6. Consistent with the pattern retinoic acid; resistance; protein kinase C delta in other models of RA-resistance, one of these resistant subclones, NB4-MR4, contains a mutation in the ligand-binding domain of PML-RARA, whereas the other two retain wild-type PML-RARA expression.12 We have reported that resistance to RA-mediated transcription and differentiation in these cell lines Introduction is associated with enhanced binding of corepressor complexes to PML-RARA.11 We furthered this observation by identifying Acute promyelocytic leukemia (APL) is a subtype of acute topoisomerase II beta (TOP2B) as a novel member of these myelogenous leukemia, representing 5–8% of acute myelo- complexes. In addition, we showed that TOP2B inhibits RA- genous leukemia cases in adults. At the genetic level, APL is induced gene expression and granulocytic differentiation by characterized by a specific chromosomal rearrangement be- negatively modulating RARA transcriptional activity.13 Type II tween the retinoic acid (RA) receptor-a (RARA) and the topoisomerases catalyze changes in the topological state of promyelocytic leukemia (PML) genes.1–3 The resulting chimeric DNA by generating transient breaks in double-strand DNA.14,15 protein, PML-RARA, acts as a dominant negative inhibitor of Owing to their DNA cleavage properties, TOP2s serve as target normal retinoid receptor function. It locates to promoters of antineoplastic agents, including DNA damaging agents.16 normally regulated by RARA, aberrantly recruits co-repressor Two isoforms of TOP2 (A and B) exist in mammals. TOP2B proteins, and thereby inhibits the RARA-mediated gene expres- is a phosphoprotein in which most of the phosphorylation sites sion. On the cellular level, the result is a block in granulocytic have been mapped to the C-terminal domain and are thought differentiation and an accumulation of myeloid progenitors to be mostly serine or threonine residues.17 Sequence analysis arrested at the promyelocyte stage. APL patients are treated with of TOP2 phosphorylation sites revealed 30 possible protein kinase C (PRKC) sites and 40 casein kinase II sites.14 However, Correspondence: Dr WH Miller Jr, Department of Oncology, Segal the functions of TOP2B’s phosphorylation sites have not yet Cancer Comprehensive Center, Lady Davis Institute for Medical been characterized and the identification of possible phosphory- Research, Sir Mortimer B. Davis Jewish General Hospital, McGill lation sites responsible for TOP2B stability has remained University, 3755 Coˆte Ste-Catherine Road, Montreál, Quebec, Canada elusive. H3T 1E2. RA treatment leads to an increase of TOP2B protein levels in E-mail: [email protected] 18 3These authors contributed equally to this work. hematopoietic cells during differentiation. Interestingly, this Received 2 November 2009; revised 22 December 2009; accepted correlates with an increase in TOP2B protein stability and 4 January 2010; published online 4 March 2010 phosphorylation levels,18 although the exact mechanism by PKCd regulation of topoisomerase IIb in APL S McNamara et al 730 which RA upregulates and stabilizes TOP2B protein levels in chloramphenicol acetyltransferase, with or without the pTB114 differentiating cells has not been determined. Previously, we plasmid, which contains the full-length TOP2B isoform fused to demonstrated that increased TOP2B levels mediate RA resis- green fluorescent protein in the pEGFP-C3 vector as previously tance by inhibiting RA-induced gene expression and subsequent described.19 Following electroporation, cells were replenished maturation toward granulocytes in APL cell lines. Consistent in media, and grown for 48 h in the absence or presence of RA. with this finding, the RA-resistant NB4-MR2 subline has Chloramphenicol acetyltransferase activity was measured by comparatively higher basal TOP2B protein levels.13 Interest- means of a modified protocol of the organic diffusion method.20 ingly, RA leads to increased levels of TOP2B protein in the RA- The chloramphenicol acetyltransferase counts were normalized sensitive NB4 cell line, suggesting the possibility of a negative with protein concentration to obtain the relative chloramphe- feedback loop. We speculated that RA may activate specific nicol acetyltransferase activity. kinases that lead to increased phosphorylation levels of TOP2B, thereby increasing its stability and decreasing its rate of degradation. In this study, we investigate the mechanism by which TOP2B protein levels are regulated in APL cells. RNA extraction and analysis Total mRNA was isolated using the TRIzol method (Invitrogen, Carlsbad, CA, USA). cDNA was generated from 5 mg total RNA Materials and methods using random primers and SuperScript II reverse transcriptase (Invitrogen). CEBPE and CYP26A1 mRNA levels were assessed Materials by quantitative real-time PCR analysis using Power SYBR green RPMI 1640 and fetal bovine serum were purchased from master mix (Applied Biosystems, FosterCity, CA, USA) with the Wisent (St Bruno, QC, Canada). All-trans RA and 4a-phorbol following primer sets: CEBPE: sense 50-CGGCTGGCCCCTTAC 12-myristate 13-acetate (PMA) were obtained from Sigma- AC-30, antisense 50-AGCCGGTACTCAAGGCTATCTTT-30; and Aldrich (St Louis, MO, USA). Rottlerin was purchased from CYP26A1: sense 50-GACATGCAGGCACTAAAGCAAT-30, anti- Biomol (Plymouth Meeting, PA, USA). The PRKCD pSUPER sense 50-CACTGGCCGTGGTTTCGT-30. cDNA was amplified short hairpin RNA-producing plasmid (Oligoengine, Seattle, for RIGI using a Taqman hybridization probe and Taqman Fast WA, USA) was directed against the following target sequence: Master Mix (Applied Biosystems). DDCt values were normalized 50-AAACTCATGGTTCTTGATGTAGTGG-30. The PRKCD kinase with those obtained from the amplification of endogenous dead (PRKCDKD) construct was kindly provided by Dr Trevor J glyceraldehyde-3-phosphate dehydrogenase mRNA. Quantita- Biden (Garvan Institute of Medical Research, Sydney, Australia). tive real-time PCR was performed on the 7500 Fast Real-time PCR system (Applied Biosystems) using standard parameters and analyzed using relative quantification with dimethylsulfoxide- Cell culture treated NB4 cells as the calibrator. Derivation of the RA-resistant cell line NB4-MR2 from the parental APL cell line NB4 was previously described.11 All cell lines were maintained in RPMI-1640 supplemented with 10% fetal bovine serum. Immunofluorescence NB4 and NB4-MR2 cells were treated for 18 h with either 1 mM RA, 1 mM Rottlerin, or the combination. Cytospin preparations Western blot analysis were fixed with 4% paraformaldehyde and stained with Cell lysates were diluted 1:1 with 2 Â SDS sample buffer. Proteins mouse anti-PML antibody (5E10 antibody from Dr R van Driel, were fractionated by electrophoresis on 10% SDS-polyacrylamide University of Amsterdam, Amsterdam, the Netherlands) as gels and transferred onto nitrocellulose membranes (Bio-Rad previously described.21 Laboratories, Mississauga, ON, Canada). Membranes were probed with antibodies against TOP2B (catalogue no. 611493, BD Biosciences, San Diego, CA, USA), PKCD (catalogue no. 610398, BD Biosciences), PRKCD phospho-threonine 505 (cata- Morphology logue no. 3974S, Cell Signaling, Beverly, MA, USA) and b-actin Morphological changes in NB4 and NB4-MR2 cells were (catalogue no. A5441, Sigma-Aldrich). The resulting signals were evaluated using conventional light-field microscopy of Giemsa– detected using the enhanced chemiluminescence (Amersham Wright (Sigma-Aldrich)-stained cytosmears. Pharmacia, Piscataway, NJ, USA) system.

Decatenation assay Differentiation assays TOP2B enzymatic activity was assayed by measuring the Cells to be used for fluorescence-activated cell sorting analysis decatenation of kinetoplast DNA. A standard assay using 2 mg of differentiation markers and in nitro-blue-tetrazolium reduc- 4 nuclear lysates was carried out according to the manufacturer’s tion assays were seeded at 3 Â 10 cells/ml in 5 ml media in protocol (TopoGEN, Port Orange, FL, USA). Samples were six-well plates. Immunofluorescence staining of the cell surface resolved by electrophoresis on a 1% agarose gel containing myeloid-specific antigen CD11c (PharMingen, Mississauga, ethidium bromide. Reaction products were visualized and ON, Canada) was assessed by flow-assisted cell cytometry and photographed on a ultraviolet transilluminator using the performed according to the antibody manufacturer’s specifica- ChemiGenius2 imaging system (Syngene, Frederick, MD, USA). tions using the FACSCalibur flow cytometer (BD Biosciences). Background staining was controlled using an isotype control PE-conjugated mouse IgG1 (PharMingen). In each sample, Transient transfections viable cells were gated, and expression of CD11c surface NB4 cells (1 Â 107 cells/transfection) were transfected by markers of 1 Â 104 cells was evaluated. Nitro-blue-tetrazolium electroporation with 5 mg of the reporter plasmid bRARE-tk- assays were performed as previously described.22

Leukemia PKCd regulation of topoisomerase IIb in APL S McNamara et al 731 Results Interestingly, PMA and RA activate PRKCD at different time points, 2 and 24 h, respectively. This correlates with the Activation of PKCD correlates with increased TOP2B observed timing of increased expression of TOP2B by these protein levels two agents. These results suggest that the hyperactive PRKCD A recent study established that protein kinase C delta (PRKCD), may mediate the increased levels of TOP2B in the NB4-MR2 a member of the novel subgroup of the PRKC family of serine/ cell line. threonine kinases, interacted with the TOP2A protein.23 This association led to increased protein expression of TOP2A and was dependent on the kinase activity of PRKCD. PRKCD is the Inhibition of PRKCD leads to decreased TOP2B protein most abundant PRKC isoform found in hematopoietic cells.24 It levels is activated in response to platelet-derived growth factor, PMA To further test our hypothesis that PRKCD regulates TOP2B and RA.25 It is of interest that RA, implicated in TOP2B stability levels in NB4 and NB4-MR2 cells, we tested the effects of and phosphorylation,18 can induce activation of PRKCD.25,26 As PRKCD inhibition on TOP2B protein levels. Treatment of NB4 the two isoforms of TOP2 have 70% amino-acid sequence and NB4-MR2 cells with increasing concentrations of Rottlerin, identity, we therefore hypothesized that PRKCD may be the decreased the levels of TOP2B protein in a dose-dependent kinase responsible for the accumulation of TOP2B protein by manner (Figure 2a). Rottlerin has been reported to be a selective RA, and may also cause the higher endogenous TOP2B levels in PRKCD inhibitor27–29 and has been used in many studies to the NB4-MR2 cell line. implicate PRKCD in a variety of cancers, including hematolo- We asked whether activation of PRKCD by RA correlates with gical malignancies.25,30–33 There have been published reports increased TOP2B levels in both NB4 and NB4-MR2 cell lines. that Rottlerin might have additional effects,34,35 and we there- By western blot analysis we observe that RA induction of TOP2B fore used additional approaches to confirm our initial findings. protein levels and activation of PRKCD, as measured by In accordance with the pharmacological inhibition of PRKCD, phosphorylation at threonine 505 (PRKCD pT505), occur with targeted knockdown of PRKCD in NB4-MR2 cells with short similar timing (Figure 1a). Consistent with previously published hairpin RNA, as well as by expression of a dominant negative data,25 total PRKCD levels are not significantly different in the inhibitory form of PRKCD (PRKCDKD), resulted in reduction of two cell lines, and remained the same before, and after, RA TOP2B levels (Figures 2b and c). In contrast, Go6976, an treatment. Most strikingly, NB4-MR2 cells show substantially inhibitor of the conventional PRKC isoforms (A, B, G),28,29,36 increased basal levels of PRKCD pT505, when compared with had no effect on TOP2B protein levels (Figure 2d). This further NB4. This finding corresponds to the increased basal levels of supports out hypothesis that TOP2B regulation is dependent on TOP2B also seen in this cell line. We then asked whether other the PRKCD isoform. activators of PRKCD would have a similar effect. We found that treatment with the PRKC activator PMA for 2 h increased both PRKCD pT505 and TOP2B levels in both cell lines (Figure 1b). PRKCD modulatesTOP2B catalytic activity On the basis of the evidence that activation of PRKCD regulates TOP2B levels, we next examined the enzymatic consequence NB4 NB4-MR2 of this proposed regulation. Type II topoisomerases alter the RA (h) 0 24 480 24 48 topological state of double-strand DNA by cleaving and religating DNA strands. Previous reports suggested that the TOP2B decatenation activity of TOP2A is enhanced by means of phosphorylation by both PRKCD23 and casein kinase II.37 To monitor the decatenation activity of TOP2B, we assayed its PRKCD pT505 ability to decatenate kinetoplast DNA, a interlocked network of DNA rings made up of several thousand 2.5-kb monomers and a few larger maxicircles.38 We incubated the kinetoplast DNA PRKCD substrate with nuclear extracts from NB4 and NB4-MR2 cells and resolved the products by agarose gel electrophoresis. Here, beta Actin we observed that decatenation activity was substantially enhanced (Figure 2e, lanes 1–4) after treatment with RA. The NB4 NB4-MR2 RA-induced activation is PRKCD-dependent, as inhibiting PRKCD with Rottlerin prevented RA from inducing decatenation µ PMA( M) 0 0.5 1.0 0 0.5 1.0 activity in the cell line. Interestingly, nuclear extracts from TOP2B NB4-MR2 cells possessed higher basal decatenation activity (Figure 2e, lanes 5–8), reflective of their higher TOP2B and PRKCD pT505 activated PRKCD protein levels. Stimulation with RA did not PRKCD enhance this activity, while Rottlerin treatment decreased it below basal levels. beta Actin

Figure 1 Activation of PRKCD correlates with increased TOP2B PRKCD inhibition decreases TOP2B protein stability levels. Threonine phosphorylation at residue 505 of PRKCD (PRKCD We previously determined that there is no signiﬁcant difference pT505) and an increase in TOP2B protein levels occurred after 24 and in basal TOP2B mRNA levels in the NB4-MR2 cell line versus m 48 h 1 M RA (a) and after 2 h PMA treatment (b). Total nuclear protein the NB4 cell line.13 To determine whether the increased TOP2B was separated on a 10% polyacrylamide gel, blotted onto nitrocellulose and subjected to western blotting using TOP2B, PRKCD and protein levels observed in NB4-MR2 cells was due to increased PRKCD pT505 antibodies. b-Actin served as the loading controls. protein stability, TOP2B protein levels were measured by Results are representative of three experiments. immunoblotting after cycloheximide-mediated translation

Leukemia PKCd regulation of topoisomerase IIb in APL S McNamara et al 732 NB4 NB4-MR2 Rottlerin (µM)0 1.0 1.5 2.0 Rottlerin (µM) 0 1.0 1.5 2.0

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Figure 2 PRKCD regulates TOP2B protein levels and catalytic activity. (a) A decrease in TOP2B protein levels with increasing concentrations of Rottlerin was observed in both the NB4 and NB4-MR2 cell lines. Total nuclear protein was subjected to western blotting using a TOP2B antibody. (b) NB4-MR2 cells were transfected with scrambled short hairpin RNA (shRNA) or a shRNA against PRKCD. Total levels of TOP2B and PRKCD protein were analyzed by western blotting. (c) The kinase activity of PRKCD is required for TOP2B protein stability. NB4 and NB4-MR2 cells were transfected with either an empty vector (EV) or a vector expressing a kinase dead PRKCD (PRKC KD). (d) NB4 and NB4-MR2 cells were treated with Go6976 and Rottlerin. To observe a decrease in TOP2B protein levels, total nuclear proteins were subjected to western blotting using TOP2B antibody. b-Actin was used as a loading control for (a–d). (e) TOP2B-mediated decatenation of kinetoplast DNA (kDNA) is PRKC-dependent. TOP2B activity was measured by decatenation of kDNA to nicked open circular and relaxed closed circular DNA. A representative image of a 1% agarose gel (ethidium bromide-stained) is shown.

inhibition. Although NB4 cells demonstrated a time-dependent inhibited, compared with only a 20% decrease in the untreated degradation of TOP2B protein levels starting at 8 h (Figure 3a, sample (Figure 3b, lane 4 vs lane 8). This considerable decrease lanes 1–4), no detectable degradation was observed in NB4- in TOP2B protein levels at 24 h suggests that PRKCD contributes MR2 cells until 24 h (Figure 3b, lanes 1–4). to the increased TOP2B stability in the NB4-MR2 cell line. Of We next sought to examine whether a hyperactive PRKCD note, because of the increased levels of TOP2B in the NB4-MR2 may be responsible for the increased stability of the TOP2B cell line, a higher concentration of Rottlerin (2 mM) was required protein in the NB4-MR2 cell line. Thus, we measured the rate of for downregulation of TOP2B. Despite decreasing TOP2B TOP2B degradation after PRKCD inhibition by treating NB4 and protein levels in both cell lines at 24 h, Rottlerin treatment NB4-MR2 cells with either the vehicle, dimethylsulfoxide, or results in an increase in TOP2B transcript at this same time point Rottlerin. Twenty-four hours after treatment, the cells were (Figure 3c, ﬁrst column). In addition, actinomycin D time course incubated with cycloheximide, and, at the indicated times, cell assays following the 24 h dimethylsulfoxide or Rottlerin treat- lysates were harvested. Western blot analysis shows increased ment were carried out to rule out variability in TOP2B mRNA TOP2B protein degradation on PRKCD inhibition in both cell stability (Figure 3c). No appreciable decrease in mRNA stability lines. In the NB4 cell line, we observe a signiﬁcant decrease of due to treatment with Rottlerin was observed. Surprisingly, TOP2B after inhibition of PRKCD, as compared with dimethyl- chemical inhibition of PRKCD resulted in an increased TOP2B sulfoxide treatment (Figure 3a, lanes 5–8). Importantly, in the mRNA expression in both the NB4 and NB4-MR2 cell lines, NB4-MR2 cell line, at 24 h cycloheximide treatment, we while simultaneously decreasing the amount of TOP2B protein, observed an 80% decrease in TOP2B levels when PRKCD is suggesting a possible positive feedback mechanism.

Leukemia PKCd regulation of topoisomerase IIb in APL S McNamara et al 733 CHX Rottlerin 24 hrs + CHX NB4

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NB4 NB4-MR2 2.5 2.5 DMSO *** DMSO 2.0 Rottlerin 2.0 Rottlerin * 1.5 1.5

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0.5 0.5 Relative TOP2B mRNA levels TOP2B Relative Relative TOP2B mRNA levels TOP2B Relative 0.0 0.0 0h 2h 4h 6h 0h 2h 4h 6h Actinomycin D Actinomycin D

Figure 3 TOP2B half-life levels are affected by PRKCD. (a, b) NB4 and NB4-MR2 cells were treated with either dimethylsulfoxide or Rottlerin for 24 h, followed by treatment with cycloheximide (CHX) for 0, 8, 16 and 24 h. To observe a decrease in TOP2B protein levels, total nuclear proteins were subjected to western blotting using a TOP2B antibody. Densitometry was performed on the western blot analysis, using b-actin as a loading control. Densitometry results were plotted and shown in the graph. Results are representative of three experiments. (c) NB4 cells (top panel) or NB4-MR2 cells (lower panel) were pretreated for 24 h with either dimethylsulfoxide or Rottlerin. Accumulation of TOP2B mRNA was analyzed after actinomycin D treatment for the indicated times. Results shown are representative of three experiments. Error bars represent s.d. Asterisks indicate signiﬁcant differences between dimethylsulfoxide-treated cells and Rottlerin-treated cells with no actinomycin D treatment (*Po0.05; ***Po0.001).

Inhibition of PRKCD relievesTOP2B-mediated of the RA target genes, CEBPE, CYP26A1 and RIG-I. Increased repressive effects on RA target genes mRNA induction of all three genes was observed in both cell We have previously shown that TOP2B overexpression efﬁ- lines with the combination treatment as compared with RA or ciently represses RA-induced gene expression.13 To test whether Rottlerin alone (Figure 4b). PRKCD inhibition could alleviate the repressive effects of increased TOP2B levels on gene expression, we performed transient co-transfections with an RARE-reporter gene and a Inhibition of PRKCD restores sensitivity to RA-induced TOP2B overexpression vector (pTB114) in NB4 cells. RA differentiation in the NB4-MR2 cell line treatment, in the absence of TOP2B overexpression, strongly Our data that PRKCD inhibition overrides the transcriptional induces RARE reporter gene transcription. Upon TOP2B over- repression exerted by TOP2B on RA-target genes led to the expression, we observed a repression of RA-induced RARE hypothesis that this inhibition would then be able to abolish the reporter gene activation (Figure 4a). However, the addition of differentiation block in the NB4-MR2 cell line. In normal cells, Rottlerin restored induction of the RARE reporter gene by RA. PML locates to punctuate structures known as PML nuclear To conﬁrm that PRKCD inhibition overcomes repression of bodies, whereas in APL cells PML-RARA expression disrupts RA responsive genes, we treated NB4 and NB4-MR2 cells with these structures and disperses PML into nuclear micro- Rottlerin, RA and the combination and measured mRNA levels speckles. Treatment with RA reverses this abnormal nuclear

Leukemia PKCd regulation of topoisomerase IIb in APL S McNamara et al 734 30000 ***

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Figure 4 Inhibition of PRKCD relieves TOP2B repressive effects on RA target genes. (a) Transiently transfected NB4 cells were electroporated with empty vector, bRARE-tk-chloramphenicol acetyltransferase and the TOP2B vector (pTB114) alone or in combination. Twenty-four hours after transfection, the cells were treated with RA, Rottlerin or the combination for an additional 24 h. Results shown are representative of three experiments. Error bars represent s.d. Asterisks indicate signiﬁcant differences between RA-treated cells with and without overexpression of TOP2B and between TOP2B overexpressing cells treated with RA or the combination of RA and Rottlerin (***Po0.001). mRNA expression was analyzed in response to treatments with 1 mM RA, 1.5 mM Rottlerin or the combination for CEBPE, Cyp26A1 (24 h) and RIG-I (72 h) in NB4 cells (b) or NB4- MR2 cells (c). Results shown are representative of three experiments. Error bars represent s.d.. Asterisks indicate signiﬁcant differences between RA-treated cells and RA plus Rottlerin-treated cells (***Po0.001).

localization.39 We first assessed PML staining by immuno- 40,6-diamidino-2-phenylindole to confirm nuclear staining (data fluorescence in NB4 and NB4-MR2 cells treated for 18 h either not shown). In the untreated NB4 and NB4-MR2 cells, with RA, Rottlerin or the combination (Figure 5a). Cells immunofluorescence showed a diffuse PML nuclear staining. were stained with anti-PML antibodies and counterstained with We saw that RA alone and the combination of RA and Rottlerin

Leukemia PKCd regulation of topoisomerase IIb in APL S McNamara et al 735 are both sufficient to cause larger PML nuclear bodies to form in only in the presence of both RA and Rottlerin do we observe the RA-sensitive NB4 cells. However, in the NB4-MR2 cell line, a robust increase in CD11c expression levels in the NB4-MR2 only the combination treatment led to the re-establishment of cell line. PML nuclear bodies. As a final measure of the ability of PRKCD inhibition to To extend and confirm these results, we performed morpho- overcome the differentiation block in APL cells, we performed a logical analysis of both cell lines treated for 5 days. Figure 5b nitro-blue-tetrazolium reduction analysis. Rottlerin alone had demonstrates that the NB4-MR2 cells only differentiate after little discernible effect on differentiation in either cell line. exposure to both RA and Rottlerin. The granulocytic maturation Again, the effect of the combination is greater than either agent pattern was similar for all the differentiated cells, with alone; RA and Rottlerin co-treatment leads to significant nitro- polylobular nuclei, chromatin condensation and a decreased blue-tetrazolium reduction within 5 days (Figure 5d). This is nuclear:cytoplasmic ratio. consistent with the immunofluorescence, morphology and Next, we examined expression levels of a cell surface marker CD11c data, where only a moderate increase in terminal associated with granulocytic differentiation (Figure 5c) after differentiation was observed in the NB4-MR2 cell line in 5-day treatment. As expected, RA alone is enough to stimulate response to 10À6 M RA after 5 days. significant CD11c expression in NB4 cells, whereas the same The published report establishing that RA induces PRKCD treatment in NB4-MR2 cells generates only a modest increase in activation25 also presents data that inhibition of PRKCD by expression. In addition, with Rottlerin alone there is a slight Rottlerin reversed RA induction of cd11b cell surface expression increase in CD11c expression in both cell lines. However, in the NB4 cell line, suggesting an opposite effect on

Figure 5 Inhibition of PRKCD restores RA sensitivity in the NB4-MR2 cell line. (a) NB4 and MB4-MR2 cells were treated for 12 h with 1 mM RA, 1 mM Rottlerin or the combination and stained for PML and detected with fluorescein isothiocyanate (FITC)-anti-mouse antibodies by immunofluorescence. 40,6-diamidino-2-phenylindole (DAPI) was used to control for nuclear staining (not shown). (b) Morphological analysis of representative NB4 and NB4-MR2 cells treated with Rottlerin and RA for 5 days. Cells were stained with Giemsa–Wright and were viewed at Â 100 magnification. (c) Cytofluorimetric analysis of surface marker expression. Percentages of NB4 and NB4-MR2 cells expressing CD11c in response to 5-day exposure to 1 mM RA and Rottlerin, or the combination. Results are representative of one of three experiments performed in triplicate. (d) Results of nitro-blue-tetrazolium reduction assay performed on NB4 and NB4-MR2 cells treated with RA, Rottlerin or the combination for 5 days. Asterisks indicate significant differences between RA-treated cells and RA plus Rottlerin-treated cells (***Po0.001).

Leukemia PKCd regulation of topoisomerase IIb in APL S McNamara et al 736

Figure 5 Continued.

differentiation than was found here. We therefore undertook first to report that increased levels of TOP2B protein can mediate additional experiments to show that the combination of RA and resistance to RA in APL cell lines.13 That observation is now Rottlerin induced differentiation in both the NB4 and NB4-MR2 furthered by this report demonstrating that a hyperactive PRKCD cell lines. The combination treatment was more active in regard is responsible for increased TOP2B levels in the RA-resistant cell to the differentiation induction than either agent alone, as line, NB4-MR2, and hence, ultimately contributes to RA evaluated by all four criteria tested. To our knowledge, these resistance. data are the first to define PRKCD as a negative regulator of RA- Both of the human topoisomerase II isoforms, TOP2A and induced gene regulation and differentiation toward granulo- TOP2B, are phosphorylated at several sites, primarily in the cytes. We thus conclude that the hyperactive PRKCD in the C-terminal domain.40–42 Some specific phosphorylation sites NB4-MR2 cell line leads to enhanced TOP2B levels, which and their functional consequences have been mapped for blocks the differentiation pathway toward granulocytes. TOP2A,43–46 and a few, but not all, of the phosphoacceptor sites are conserved between the A and B isoforms. However, little is currently known about the ramifications of TOP2B Discussion phosphorylation. Potential functional roles for site-specific phosphorylation have been hinted at by several groups The amount of an active protein available to carry out defined demonstrating hyperphosphorylation of TOP2B in doxorubicin- physiological functions is a major player in governing cell resistant HL-60 cells47 and phosphorylation during RA-induced growth and proliferation. Our studies begin to uncover the differentiation of wild-type HL-60 cells, which correlated with a mechanisms and ramifications of TOP2B protein regulation. We slower degradation rate of TOP2B protein.18 The latter show that activation of PRKCD, by RA and PMA, leads to observation is now made more concrete by the research increased levels of TOP2B protein and conversely, inhibition of contained herein showing increased protein degradation on PRKCD correlates with decreased levels of TOP2B. We were the PRKCD inhibition.

Leukemia PKCd regulation of topoisomerase IIb in APL S McNamara et al 737 There are examples of phosphorylation increasing the that cumulatively our data support the model where TOP2B is a metabolic stability, and consequently the activity, of a protein, more active mediator of resistance, as inhibition of TOP2B, as has been shown for the transcription factors p5348 and either directly13 or indirectly by PRKCD, re-establishes sensitivity c-fos.49 Our assessment of decatenation ability confirms that to RA-induced differentiation. TOP2B catalytic activity is considerably enhanced after activa- TOP2B protein levels can have an impact on both sensitivity tion of PRKCD and abrogated after inhibition of PRKCD. These and resistance to cancer therapeutics. Thus, identifying the results suggest that TOP2B be introduced into a family of mechanisms by which TOP2B levels are regulated may assist in proteins whose functional activity is managed through phos- the treatment of leukemia as well as the multitude of cancers phorylation-dependent stability. However, no increase in total that respond to TOP2 poisons. Our findings that RA resistance in TOP2B phosphorylation after RA treatment, or conversely, no APL cells can be overcome by targeting both the PRKCD and RA decrease with PRKCD inhibition, was observed in 32P-labeling pathways may provide a basis for the rational design of novel experiments (data not shown). We speculate that this might be therapies for not only RA-resistant APL, but other more common due to changes in only a few of the many possible TOP2B leukemias that have an increased TOP2B expression. phosphoacceptor sites, which would not affect the overall phosphorylation signal of the protein. Our ongoing studies aim to identify the specific TOP2B residues of PRKCD-mediated Conflict of interest phosphorylation in order to characterize their functional role. RA has been shown to directly bind the different PKC The authors declare no conflict of interest. isoforms, including PRKCD.50 Given the late timing of PRKCD activation after RA treatment, it is reasonable to hypothesize that Acknowledgements RA is not acting directly on PRKCD and that another kinase might lie upstream in the pathway. Several MAP kinases have We are grateful to Bjo¨rn D Kuhl and Filippa Pettersson for critical been previously shown to be involved in the response to reading of the paper. This work was supported by a grant from RA.51,52 In particular, the p38 MAP kinase is phosphorylated 53 the Canadian Institutes of Health Research. WH Miller, Jr, is a and activated by RA treatment in NB4 cells and PRKCD Chercheur National of Fonds de la Recherche en Sante´ du phosphorylation at Thr505 was recently shown to be dependent 54 Que´bec. J Nichol was supported by a student fellowship from the upon p38. However, RA activates many complex signaling Cole Foundation. cascades that may also lie upstream of PRKCD and TOP2B. Indeed, future directions will aim to elucidate more clearly the entire signaling pathway leading to TOP2B regulation. References We previously found that TOP2B is overexpressed in the RA- resistant APL cell line, NB4-MR2,13 and TOP2B has also been 55 1 de The H, Lavau C, Marchio A, Chomienne C, Degos L, Dejean A. found to be overexpressed in acute myeloid leukemia and The PML-RAR alpha fusion mRNA generated by the t(15;17) 56 lymphoma patients. However, mechanistic data on the causes translocation in acute promyelocytic leukemia encodes a of this upregulation have been lacking. Given that TOP2B is a functionally altered RAR. Cell 1991; 66: 675–684. target of many anticancer agents, its increased expression can 2 Kakizuka A, Miller Jr WH, Umesono K, Warrell Jr RP, Frankel SR, enhance the cytotoxic activity of these agents in tumor cells.57–60 Murty VV et al. Chromosomal translocation t(15;17) in human In particular, RA-resistant APL cells with excess TOP2B protein acute promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor, PML. Cell 1991; 66: 663–674. may respond better to treatment with anthracyclines. This 3 Pandolfi PP, Grignani F, Alcalay M, Mencarelli A, Biondi A, hypothesis is supported by data from the clinic; when RA is LoCoco F et al. Structure and origin of the acute promyelocytic co-administered with anthracycline-based therapy, APL patients leukemia myl/RAR alpha cDNA and characterization of its have improved remission and survival rates.61 On the other retinoid-binding and transactivation properties. Oncogene 1991; hand, in the context of leukemic differentiation, increased levels 6: 1285–1292. of TOP2B protein has an inhibitory effect on RA-induced 4 Fenaux P, Le Deley MC, Castaigne S, Archimbaud E, Chomienne 17 C, Link H et al. Effect of all transretinoic acid in newly diagnosed differentiation and apoptosis. acute promyelocytic leukemia. Results of a multicenter rando- It is also of general interest to mention that other RA targets mized trial. European APL 91 Group. Blood 1993; 82: 3241–3249. are regulated in a similar manner as TOP2B in cancer cells: the 5 Cornic M, Chomienne C. Induction of retinoid resistance by all- transcription factor SOX9,62 the myeloid transcription factor trans retinoic acid in acute promyelocytic leukemia after remis- CEBPE,63 the growth inhibitory protein IGFBP-364 and the sion. Leuk Lymphoma 1995; 18: 249–257. translation repressor 4E-BP265 are all regulated by RA. 6 Duprez E, Ruchaud S, Houge G, Martin-Thouvenin V, Valensi F, Kastner P et al. A retinoid acid ‘resistant’ t(15;17) acute Specifically, the basal level of expression of these proteins is promyelocytic leukemia cell line: isolation, morphological, low, but inducible in RA-sensitive cells, whereas in some immunological, and molecular features. Leukemia 1992; 6: RA-resistant cell lines, the basal level of expression is 1281–1287. constitutively high and is only minimally further induced upon 7 Imaizumi M, Suzuki H, Yoshinari M, Sato A, Saito T, Sugawara A RA treatment. It had been shown that transcriptional activation et al. Mutations in the E-domain of RAR portion of the PML/RAR by nuclear receptors requires a signal-dependent exchange of chimeric gene may confer clinical resistance to all-trans retinoic 66,67 acid in acute promyelocytic leukemia. Blood 1998; 92: 374–382. co-activators and corepressors. The deregulated TOP2B 8 Ding W, Li YP, Nobile LM, Grills G, Carrera I, Paietta E et al. levels may negatively affect RA signaling at the level of Leukemic cellular retinoic acid resistance and missense mutations coregulator cycling, either by preventing efficient dissociation in the PML-RARalpha fusion gene after relapse of acute promye- and/or degradation of the corepressor complex or by inhibiting locytic leukemia from treatment with all-trans retinoic acid and recruitment of the co-activator complexes, even in the presence intensive chemotherapy. Blood 1998; 92: 1172–1183. of pharmacological doses of RA. A similar mechanism has 9 Marasca R, Zucchini P, Galimberti S, Leonardi G, Vaccari P, 68 Donelli A et al. Missense mutations in the PML/RARalpha ligand recently been proposed for another repressor of RA signaling. binding domain in ATRA-resistant As(2)O(3) sensitive relapsed The possibility does exist that increased TOP2B levels are mere acute promyelocytic leukemia. Haematologica 1999; 84: bystanders to the development of resistance. However, we feel 963–968.

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