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Activation of a promyelocytic leukemia–tumor protein 53 axis underlies acute promyelocytic leukemia cure

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© 2014 Nature America, Inc. All rights reserved.

Julien Ablain1–3,8, Kim Rice1–3, Hassane Soilihi1–3, Aurélien de Reynies4, Saverio Minucci5,6 & Hugues de Thé1–3,7 Acute promyelocytic leukemia (APL) is driven by the promyelocytic leukemia (PML)–retinoic acid receptor- (PML-RARA) fusion protein, which interferes with nuclear receptor signaling and PML nuclear body (NB) assembly. APL is the only malignancy definitively cured by targeted therapies: retinoic acid (RA) and/or arsenic trioxide, which both trigger PML-RARA degradation through nonoverlapping pathways. Yet, the cellular and molecular determinants of treatment efficacy remain disputed. We demonstrate that a functional Pml–transformation-related protein 53 (Trp53) axis is required to eradicate leukemia-initiating cells in a mouse model of APL. Upon RA-induced PML-RARA degradation, normal Pml elicits NB reformation and induces a Trp53 response exhibiting features of senescence but not apoptosis, ultimately abrogating APL-initiating activity. Apart from triggering PML-RARA degradation, arsenic trioxide also targets normal PML to enhance NB reformation, which may explain its clinical potency, alone or with RA. This Pml-Trp53 checkpoint initiated by therapy-triggered NB restoration is specific for PML-RARA–driven APL, but not the RA-resistant promyelocytic leukemia zinc finger (PLZF)-RARA variant. Yet, as NB biogenesis is druggable, it could be therapeutically exploited in non-APL malignancies. APL is driven by the PML-RARA fusion gene, which encodes a transcriptional repressor that antagonizes myeloid differentiation and promotes APL-initiating cell self-renewal1,2. Transcriptional deregulation stems from dimerization-induced enhanced co-repressor binding, presence of repressor domains in the PML moiety of the fusion, relaxed DNA binding site specificity and binding to SPI1, the polycomb complex and retinoid X receptor α. PML-RARA also disrupts PML NBs, domains implicated in stem cell self-renewal and apoptosis, at least in part by modulating tumor protein p53 (TP53) signaling3,4. That PML-RARA transgenic mice develop APL, as well as the recent demonstration of lack of additional recurrent genetic alterations in many patients, implies that APL is primarily, if not entirely, driven by PML-RARA1,5. APL is the only malignancy definitively cured by targeted therapies1,6,7. RA induces terminal APL cell differentiation, presumably by reversing PML-RARA–mediated transcriptional inhibition2. Yet, alone, RA rarely eradicates APL in patients8. In contrast to RA, arsenic trioxide (arsenic) prompts APL cell apoptosis ex vivo and delayed differentiation in vivo9. Arsenic does not directly act on RARA-dependent transcription but profoundly alters PML NB biogenesis through direct binding to, and oxidation of, PML and PML-RARA3,10–13. Notably, arsenic alone definitively cures 70% de novo APLs14,15. Thus, differentiation through reversal of PML-RARA–enforced transcriptional silencing is unlikely to be the sole basis of APL eradication.

Both RA and arsenic target PML-RARA stability, RA via its RARA moiety and arsenic via the PML one, suggesting that PML-RARA degradation may be critical for response to therapy16,17. By analyzing retinoids that activate transcription but fail to degrade PML-RARA, we demonstrated that PML-RARA loss is indeed required for APL clearance18, a proposal that may explain the marked synergy between RA and arsenic for APL eradication in mice or patients1,6,7,19–22. Yet, how PML-RARA loss abolishes APL-initiating cell self-renewal remains unknown. The rare cases of APL driven by PLZF-RARA are clinically resistant to RA16. Similar to PML-RARA, PLZF-RARA is degraded upon exposure to RA23,24, which raises the issue of why PML-RARA degradation leads to cure whereas that of PLZF-RARA does not1. Here, we demonstrate that therapy-induced PML-RARA catabolism elicits loss of APL-initiating cell self-renewal through Pml NB reformation and Trp53 activation. This PML-TP53 checkpoint may have therapeutic value in other malignancies. RESULTS Uncoupling APL differentiation and loss of cell self-renewal To define the cellular and molecular events associated with RA therapy, we analyzed the effect of RA in two transplantable models of APL that are known to exhibit distinct sensitivities to RA and are derived from transgenic mice expressing either PML-RARA19 (referred to as

1Université

Paris Diderot, Sorbonne Paris Cité, Hôpital St. Louis, Paris, France. 2INSERM UMR 944, Equipe Labellisée par la Ligue Nationale contre le Cancer, Institut Universitaire d’Hématologie, Hôpital St. Louis, Paris, France. 3CNRS UMR 7212, Hôpital St. Louis, Paris, France. 4Programme Cartes d’Identité des Tumeurs, Ligue Nationale contre le Cancer, Paris, France. 5Department of Experimental Oncology, European Institute of Oncology, Milan, Italy. 6Department of Biosciences, University of Milan, Milan, Italy. 7Assistance Publique Hôpitaux de Paris, Service de Biochimie, Hôpital St. Louis, Paris, France. 8Present address: Stem Cell Program and Division of Hematology/Oncology, Children’s Hospital and Dana-Farber Cancer Institute, Boston, Massachusetts, USA. Correspondence should be addressed to H.d.T. ([email protected]). Received 13 September 2013; accepted 4 December 2013; published online 12 January 2014; doi:10.1038/nm.3441

nature medicine  VOLUME 20 | NUMBER 2 | FEBRUARY 2014

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Figure 1  Dose-response analysis of the effect of RA on PML-RARA and PLZF-RARA APL mice demonstrating uncoupling of blast differentiation and survival benefit. (a) General strategies of APL investigation in vivo. (b) RA-induced differentiation of PML-RARA APL cells, as assessed by MayGrünwald-Giemsa (MGG) staining (scale bar, 20 µm) and FACS analysis after 3-d treatment in vivo (RA low, 1.5 mg; RA intermediate (int), 10 mg; RA high, 50 mg or 100 mg pellets). Numbers indicate the percentage of cells in each quadrant. (c) Survival after 7-d treatment (black line) in the two APL models examined (n ≥ 7 for each model). (d) Survival of secondary recipients transplanted with APL bone marrow cells from 3-d–treated primary APL mice in both models (PML-RARA, n ≥ 14 and PLZF-RARA, n ≥ 8). (e) Same as b with PLZF-RARA–driven APL cells.

PML-RARA APL mice) or PLZF-RARA plus RARA-PLZF (referred to as PLZF-RARA APL mice), the latter driving an RA-resistant disease25. PML-RARA APL mice were challenged with three RA doses in pellet form (low, 1.5 mg; intermediate, 10 mg; or high, 50 mg or 100 mg). PLZF-RARA–driven APL mice were challenged with the intermediate and high doses only. We examined APL cell differentiation, loss of APL-initiating activity (survival of untreated secondary recipients of bone marrow cells from primary RA-treated APL mice) and survival of APL mice (Fig. 1a). In PML-RARA APL mice, all RA doses similarly yielded terminal granulocytic differentiation, as assessed by cell morphology and expression of surface markers (Fig. 1b); however, survival of treated mice sharply differed by dose (Fig. 1c). The loss of APL-initiating activity was also dose dependent in this model, with only intermediate and high RA doses able to strongly impede APL transplantability (Fig. 1d). Upon RA treatment, cell differentiation levels were comparable in both APL models (Fig. 1b,e); however, the survival of treated mice and the APL-initiating activity sharply differed between models, particularly in response to high RA doses, with PLZF-RARA APL mice being almost completely resistant to treatment (Fig. 1c,d). These findings establish the uncoupling of blast differentiation and tumor eradication suggested by previous studies16,18,19 and provide a model to investigate the cellular and molecular bases of APL clearance. Loss of APL cell self-renewal is linked to cell cycle arrest We next examined the transcriptome of bone marrow cells of APL mice from the two models following treatment for 6 or 12 h with different RA

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doses. We first investigated genes differentially expressed in PML-RARA APL cells regardless of the RA dose. As expected, these genes belonged to myeloid differentiation pathways in mice treated for 6 h, and even more so in mice treated for 12 h (Supplementary Fig. 1a). We next selected genes differentially regulated between high or intermediate and low RA doses. These genes were not linked to differentiation but were strongly associated with cell cycle arrest, especially in mice treated for 12 h (Supplementary Fig. 1a). Although differentiation pathways were induced in both models, albeit with a slight delay in PLZF-RARA–driven APL mice, cell cycle– associated genes were not activated in cells from PLZF-RARA APL mice (Supplementary Fig. 1a). Similarly, an E2F signature, comprising 130 genes previously shown to be regulated by the E2F transcription factor, was downregulated in PML-RARA APL cells only (Fig. 2a). Despite full differentiation, the cell cycle was not significantly modified in leukemic cells from low-dose RA–treated PML-RARA APL mice, whereas high-dose RA treatment triggered cell cycle arrest at 48 h (Fig. 2b). Analysis of a GFP-marked pure APL cell population confirmed cell cycle arrest specifically in PML-RARA APL cells after 48 h and 72 h of high-dose RA treatment (Supplementary Fig. 1b). We did not observe this cell cycle arrest in PLZF-RARA APL cells (data not shown). In these and subsequent experiments, the abundance of GFP-positive APL cells was modestly decreased by RA treatment (Supplementary Fig. 1c), allowing analysis of unsorted APL marrow cells. Thus, RA activates two distinct gene networks, one linked to differentiation and the other associated with cell cycle arrest, with only the latter cosegregating with loss of APL-initiating activity.

VOLUME 20 | NUMBER 2 | FEBRUARY 2014  nature medicine

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High-dose RA induces features of senescence We then focused on the 30 genes most upregulated by high- but not low-dose RA after a 6-h treatment (Fig. 2c). As expected, most of these were not significantly induced in RA-treated PLZF-RARA APL cells. A literature search revealed that 10 of the 30 genes were unconventional primary Trp53 transcriptional targets or displayed Trp53 binding sites (Fig. 2c). Induction of several of these genes was validated by quantitative RT-PCR in independent mouse APL samples (Supplementary Fig. 1d). Several of these Trp53 targets are key drivers of senescence, including serine peptidase inhibitor, clade E, member 1 (Serpine1)26 (Fig. 2c), suggesting that activation of Trp53 signaling underlies the cell cycle arrest triggered by high RA doses and may lead to APL cell senescence. As Trp53 can drive both cell death and senescence, we investigated the fate of APL cells in response to RA treatment in vivo. Myeloid cells may undergo senescence, precipitating their rapid phagocytosis and clearance by bone marrow or liver macrophages27. Liver infiltration by APL cells was reduced by only intermediate- or high-dose RA (Fig. 2d and data not shown). Notably, even high-dose RA failed to clear livers of infiltrating APL cells in PLZF-RARA APL mice after 3 d (data not

nature medicine  VOLUME 20 | NUMBER 2 | FEBRUARY 2014

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Figure 2  RA-induced senescence mirrors the loss of APL-initiating cell self-renewal. (a) E2F activities in APL models 1.05 (PML-RARA untreated (untreat.) n = 4 and RA intermediate or high doses (int/high) n = 6; PLZF-RARA untreat. n = 2 and RA int/high n = 4), as reflected by the arrays. NS, not significant. (b) Cell cycle analysis in bone marrow cells from PML-RARA APL mice treated with high-dose (100 mg or 50 mg) or low-dose (1.5 mg) RA (n = 3). Data are 1.00 expressed as mean ± s.e.m. (c) Fold induction (FI) upon RA treatment (L, low-dose RA (1.5 mg); I, intermediate-dose RA (10 mg); H, high-dose RA (100 mg)) of the top 30 genes activated by high- but not low-dose RA treatment in PML-RARA (P/R) and PLZF-RARA (PZ/R) APL cells ranked relative to induction by high-dose RA (2 mice per condition). Genes involved in senescence (S) or transcriptionally regulated by Trp53 or demonstrating Trp53 binding (Trp53) are PML-RARA PLZF-RARA indicated. (d) Liver sections from PML-RARA APL mice treated with low (1.5 mg) or high (50 mg) RA doses for 3 d. Scale bar, 50 µm. Bottom right, electron microscopy showing an APL cell (white arrowhead) phagocytosed by a hepatocyte (black arrowheads). Scale bar, 200 nm. (e) Effect of caspase inhibition (Z-VAD) on RA-induced loss of APL-initiating activity (n ≥ 8). (f) Loss of Lmnb1 in bone marrow of PML-RARA mice treated with high-dose RA (50 mg) for 72 h, assessed by immunofluorescence (left, scale bar, 10 µm) and western blot (right, Vinculin (Vcl) used as an internal control). (g) Serpine1 expression in bone marrow of PML-RARA APL mice treated for 48 h with low (1.5 mg) or high (50 mg) RA doses. Data are expressed as mean ± s.d. of two independent experiments. (h) Nuclear morphology of GFP-sorted APL blasts treated in vivo for 72 h with low (1.5 mg) or high (50 mg) RA doses. Heterochromatin blebbing (arrowhead). Scale bars, 5 µm. Quantification in Supplementary Figure 1f. (i) SASP as reflected by the arrays (PML-RARA untreat. n = 4 and RA int/high n = 6; PLZF-RARA untreat. n = 2 and RA int/high n = 4). Data are expressed as median, first and third quartiles. Bars represent the maximum and minimum values. R

© 2014 Nature America, Inc. All rights reserved.

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shown). We previously observed massive phagocytosis of nonapoptotic APL cells by bone marrow macrophages in APL mice treated with a combination of RA and arsenic (RA-arsenic)19. We similarly observed phagocytosis of APL cells by hepatocytes or Kupffer cells in mice treated by high- but not low-dose RA (Fig. 2d and data not shown), which explains the rapid restoration of normal liver architecture. Furthermore, we did not detect TUNEL and activated caspase-3 staining in liver-infiltrating APL cells upon treatment with RA (data not shown). The percentage of annexin V–positive bone marrow APL cells was not increased after intermediate- or high-dose RA treatment, which argues against induction of apoptosis (Supplementary Fig. 1e). Notably, loss of clonogenicity elicited by intermediate- or high-dose RA was paradoxically enhanced by treatment with the Z-VAD caspase inhibitor, which demonstrates that caspase-dependent apoptosis does not contribute to APL eradication (Fig. 2e and data not shown). Although we did not observe induction of β-galactosidase activity in cells from PML-RARA APL mice treated with high-dose RA after 72 h, they displayed decreased expression of lamin B1 (Lmnb1), a senescence marker28 (Fig. 2f), and massive induction of the master senescence gene, Serpine1, at 48 h (Fig. 2g). By examining

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PML-RARA degradation activates Trp53 The selective induction of Trp53 targets in cells from APL mice treated with intermediate- or high-dose RA suggested that Trp53 was activated. Indeed, in bone marrow or liver-infiltrating APL cells from these mice, PML-RARA degradation was followed by increased Trp53 levels (Fig. 3a,b). The latter reflects Trp53 stabilization, as Trp53 transcripts were unaffected by RA (Supplementary Fig. 2a). Although RA elicited PLZF-RARA degradation with similar kinetics to that of PML-RARA degradation23,24, it did not affect Trp53 transcription (data not shown) or increase Trp53 protein stability (Fig. 3a). Similar RA-induced TP53 stabilization was observed in primary leukemia cells from the blood of two individuals with APL (Fig. 3c), with one individual also exhibiting cyclin-dependent kinase inhibitor 1A (CDKN1A) induction. The amplitude of TP53 stabilization was modest; however, TP53 activation can occur through

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GFP-sorted APL cells, we found that granulocyte nuclei were larger in mice treated with high-dose RA compared to those treated with low-dose RA (Fig. 2h; quantification in Supplementary Fig. 1f). These also exhibited characteristic cytoplasmic blebbing of nuclear heterochromatin (Fig. 2h) reminiscent of cytoplasmic chromatin described in senescent fibroblasts29. Finally, we observed a senescence-associated secretory phenotype (SASP) by transcriptome analysis in PML-RARA but not PLZF-RARA APL cells from mice treated with high-dose RA (Fig. 2i). Collectively, the E2F shutoff, cell cycle arrest, activation of Trp53 targets, loss of Lmnb1, changes in nuclear architecture, SASP, active phagocytosis and tumor clearance in the absence of apoptotic features, point to a senescence-like program selectively activated in PML-RARA APL cells treated by high-dose RA.

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Figure 3  A key role for Trp53 activation in RA-induced loss of APL-initiating cell self-renewal in vivo. (a) Western blot of PML-RARA, PLZFRARA, Trp53 and Actb in bone marrow of mice treated with 10 mg (intermediate dose, I) or 100 mg (high dose, H) RA. Ø, untreated. (b) Immunohistochemical analysis of Trp53 in livers of APL mice treated with 10 mg RA for 24 h (scale bar, 20 µm). (c) Western blot of TP53 and CDKN1A expression in APL blasts from the blood of two RA-treated (12-h treatment) hyperleukemic patients. (d) Colonies formed by PML-RARA–transformed mouse hematopoietic progenitors stably expressing scrambled shRNA (ctrl) or shRNAs against Trp53 (shTrp53 #1 and shTrp53 #2) and grown in methylcellulose for 7 d in the presence of 1 × 10−7 M RA. Relative colony numbers are shown in Supplementary Figure 2c. Data are expressed as the mean of three independent experiments ± s.d. (e) Percentages of GFP-positive APL cells in the bone marrow (BM) of mice treated with 10 mg RA for 6 d. Pooled data from two independent experiments. Horizontal lines represent s.e.m. (f) MGG staining of bone marrow cells from Trp53+/+ or Trp53−/− APL mice treated with 10 mg RA for 3 d (scale bar, 20 µm). (g) Spleen weights of Trp53+/+ or Trp53−/− APL mice treated with 10 mg for 3 or 6 d (n ≥ 3). Data are expressed as mean ± s.d. (h) Survival of Trp53+/+ or Trp53−/− APL mice treated for 7 d with 10 mg RA; quantification in Supplementary Figure 2f (n = 6). (i) Survival of secondary recipients of bone marrow blasts from Trp53+/+ or Trp53−/− APL mice treated for 3 d with 10 mg RA (Trp53+/+ n = 12, Trp53−/− n = 10).

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© 2014 Nature America, Inc. All rights reserved.

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post-translational modifications, with minor changes in protein abundance. Thus, in PML-RARA APL mice or individuals with APL, RA-triggered loss of APL-initiating activity is accompanied by Trp53 activation. Trp53 deficiency blunts RA effect on APL cell self-renewal shRNA-mediated Trp53 depletion (Supplementary Fig. 2b) increased intrinsic clonogenicity but also impaired response to RA of PML-RARA retrovirally transformed mouse primary hematopoietic progenitors grown in methylcellulose (Fig. 3d and Supplementary Fig. 2c). Thus, ex vivo, Trp53 contributes to APL cell response to RA. Similarly, in vivo, shRNA-mediated Trp53 depletion resulted in slightly higher APL cell basal growth rates (Supplementary Fig. 2d,e) and greatly reduced APL clearance in response to RA (Fig. 3e). Silencing of Trp53 also led to shorter survival following 7-d RA treatment and impaired RA-induced APL-initiating cell clearance (Supplementary Fig. 2d,e). We then compared the in vivo response to RA of Trp53+/+ or Trp53−/−PML-RARA–driven APLs30. Although differentiation and tumor regression were identical in both models (Fig. 3f,g), and despite comparable PML-RARA degradation (Supplementary Fig. 2g), we observed even greater differences in RA’s effect on survival and APLinitiating activity between Trp53+/+ and Trp53−/− PML-RARA–driven APL mice compared to in vivo shRNA silencing experiments (Fig. 3h,i; quantification in Supplementary Fig. 2f). We could not determine the effects of high-dose RA in this model owing to occurrence of sudden death in Trp53−/− APL mice, reminiscent of RA syndrome1. Collectively, these data establish that, downstream of PML-RARA degradation, Trp53 activation is essential for full RA-induced APL elimination.

VOLUME 20 | NUMBER 2 | FEBRUARY 2014  nature medicine

articles Figure 4  Loss of Pml impedes RA-triggered APL clearance. (a) Immunofluorescence analysis Ctrl shPml #1 shPml #2 Pml+/+ Pml–/– RA high Untreated Bone marrow 6 h of Pml in bone marrow cells from APL mice –8 0.7 1,000 9 P = 1 × 10 treated with high-dose RA (100 mg) for 6 h. P = 1 × 10–4 8 P = 0.01 0.6 P = 3 × 10–4 Three mice per condition (scale bar, 5 µm); 7 P = 2 × 10–8 0.5 –3 quantification of NBs (mean ± s.d.) in the 100 6 P = 2 × 10 0.4 5 graph (n ≥ 34). (b) Numbers of colonies formed Pml DAPI 4 0.3 by PML-RARA–transformed mouse hematopoietic 10 3 0.2 progenitors stably expressing scrambled shRNA 2 0.1 1 (ctrl) or shRNAs against Pml (shPml #1 and 0 1 0 shPml #2), and grown in methylcellulose for 7 d 3d in the presence of 1 × 10−7 (10−7) M RA. Relative colony numbers (mean of three independent experiments ± s.d.) are shown –/– 3d 3d Pml APL 7d in Supplementary Figure 3b. (c) Spleen weights 100 100 100 P = 2 × 10–8 of Pml+/+ and Pml−/− APL mice treated for 3 d –5 80 80 80 –6 –6 P = 8 × 10 P = 5 × 10 P = 4 × 10 with RA int (10) or RA high (100) (n = 3). 60 60 60 Mean ± s.d. (d) Survival of Pml+/+ or Pml−/− APL 40 40 40 mice treated for 7 d with 10 mg (int) or 100 mg 20 20 20 –4 (high) RA. Log-rank test for the comparison n≥8 P = 5 × 10 n=6 n = 12 0 0 0 between Pml+/+ and Pml−/− APL mice in each 0 20 40 60 80 0 20 40 60 80 10 20 30 40 50 treatment condition; quantification in Time to death (d) Time to death of secondary Time to death of secondary recipients (d) recipients (d) Supplementary Figure 3d (n = 6). (e) Survival +/+ of secondary recipients of bone marrow cells Untreated RA int RA high Untreated Pml : Ctrl Pml-l Pml-l K160R from Pml+/+ or Pml−/− APL mice treated for 3 d Untreated RA int RA high RA-treated Pml–/– : with 10 mg (int) or 100 mg (high) RA. Log-rank test results for the comparison between Pml+/+ and Pml−/− APL mice in each treatment condition (n ≥ 8). (f) Survival of secondary recipients of bone marrow cells from primary mice inoculated with Pml−/− APL cells stably expressing Pml-I or Pml-I K160R and treated for 3 d with 10 mg RA (n = 12).

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PML is required for APL clearance PML is a master gene of senescence, controlling both E2F and TP53 activities through multiple mechanisms, including recruitment of TP53 and its regulators within PML NBs3,31,32. Using an antibody to murine Pml that does not detect PML-RARA, we found that NB reformation started as early as 6 h after high-dose RA treatment in vivo (Fig. 4a) and mirrored the dose dependency of PML-RARA degradation (data not shown), suggesting that NB reformation could be implicated in activating Trp53 signaling. We therefore silenced Pml by shRNA in PML-RARA–transformed progenitors grown ex vivo (Supplementary Fig. 3a), which decreased RA-induced loss of clonogenicity tenfold (Fig. 4b and Supplementary Fig. 3b). Thus, the nonrearranged Pml allele is a key contributor to RA-induced loss of clonogenicity. We then analyzed the response to treatment of Pml+/+ or Pml−/− APL mice. RA-triggered regression of Pml−/− APLs was impaired, indicated by the sustained high spleen weight of treated mice (Fig. 4c). The modest difference in spleen weights between treated and untreated Pml−/− animals probably reflects some inhibition of tumor growth due to the differentiating effect of RA. Indeed, we observed comparable cell differentiation upon RA treatment in Pml+/+ and Pml−/− APL mice (Supplementary Fig. 3c). The survival of Pml−/− APL mice treated with intermediate- or high-dose RA was substantially shortened compared to that of Pml+/+ APLs (Fig. 4d; quantification in Supplementary Fig. 3d), and RA did not impinge on APLinitiating activity in Pml−/− APL mice as it did in Pml+/+ APL mice (Fig. 4e). The greater effect of Pml absence than of Trp53 absence on RA response is consistent with the hypothesis that Pml acts upstream of Trp53 through both Trp53-dependent and Trp53-independent pathways32,33. Unlike inactivation of Trp53, Pml inactivation only modestly activated basal cell growth ex vivo or in vivo, suggesting that Pml does not control the basal antiproliferative effect of Trp53 in our system. A key role of functional NB reformation was supported by addback experiments in Pml−/− APL cells, using Pml-I or Pml-I K160R,

nature medicine  VOLUME 20 | NUMBER 2 | FEBRUARY 2014

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a sumoylation-defective mutant previously shown to allow NB formation but to impair recruitment of partner proteins into NBs13. Notably, Pml-I, but not Pml-I K160R, partially reversed the resistance to RA of Pml−/− APL cells (Fig. 4f), suggesting that recruitment of partner proteins into NBs contributes to RA-induced loss of APL-initiating activity. The limited rescue reflects the likely need for the cooperation of several isoforms for PML-induced senescence34,35. Pml-dependent activation of a specific Trp53 signature Whereas RA-induced PML-RARA degradation was identical in Pml−/− and Pml+/+ APL cells (Supplementary Fig. 3e), Trp53 was stabilized in only Pml+/+ APL cells (Fig. 5a), consistent with the idea that NB reformation drives Trp53 activation. To identify putative downstream Pml-Trp53 targets implicated in APL eradication, we profiled Pml−/− and Pml+/+ APL cells after 6- or 12-h treatment with intermediate- or high-dose RA in vivo. Genes differentially expressed between Pml−/− and Pml+/+ APL cells in the absence of treatment were excluded, and we focused on genes regulated by RA in Pml+/+ but not Pml−/− APL cells. As anticipated, pathway analysis identified proliferation arrest and Trp53 signatures (Supplementary Fig. 4a), in addition to cell death signatures. The most differentially expressed genes upon RA treatment were primarily upregulated in Pml+/+ APL cells (20 of 21) (Fig. 5b) and comprised many Trp53 targets, including senescence (Serpine1) or cell fate (Nr4a1, Vdr and Lif) master genes. We subsequently validated the regulation of these genes in independent samples from 6- or 12-h RA-treated Pml−/− and Pml+/+ APL cells (Supplementary Fig. 4b). The senescence driver, Serpine1, was more strongly upregulated in RA-treated PML-RARA– than PLZF-RARA–transformed cells (Fig. 5c), making it an attractive candidate for mediating Pml-Trp53– triggered loss of APL-initiating activity. Indeed, overexpression of Serpine1 in primary mouse progenitors transformed with either PML-RARA or the MLL-ENL fusion, found in acute leukemias, sharply decreased clonogenicity in methylcellulose cultures (data not shown). Collectively, these data establish that

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DISCUSSION Although the curative activity of combined RA and arsenic in APL is well established1,6,7, the cellular and molecular bases of the only definitive cancer cure by targeted therapies remained unclear. This study demonstrates that upon PML-RARA degradation, a Pml NB– driven, Trp53-enforced program with features of senescence is a key contributor to APL eradication. PML-RARA blunts Trp53 response to DNA damage through Trp53 deacetylation39, but it was never envisioned that therapyinduced PML-RARA loss could reactivate Trp53 and drive therapy response. Transcriptional reactivation of PML-RARA–silenced genes is believed to initiate RA-triggered APL cell differentiation18, although PML-RARA loss alone may suffice for leukemia maturation (J. Halftermeyer, unpublished data). Differentiation has a transient debulking effect but appears insufficient to cure disease, and a distinct program dependent on Pml NBs and Trp53 entails loss of APLinitiating cell self-renewal and definitive tumor clearance. Previous

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A role for Pml in the synergy between RA and arsenic We and others have demonstrated marked synergy between RA and arsenic for the eradication of APL16,19,22. This was previously attributed to synergistic PML-RARA degradation by nonoverlapping biochemical pathways1. Our identification of a key role for PML NB reformation in APL eradication and the fact that arsenic also targets normal PML to trigger NB assembly and partner protein recruitment in non-APL cells10–13,36 raised the issue of PML’s contribution to the therapeutic efficacy of the RA-arsenic combination. RA unexpectedly induced expression of Pml in APL cells in vivo (Fig. 6a,b), presumably by activating interferon signaling37,38. Moreover, NB reformation was more complete in APL blasts from mice treated with RA-arsenic for 6 h when compared to those treated with RA alone (Fig. 6c), which probably reflects the dual effects of increased PML-RARA loss and direct Pml targeting by arsenic. We then compared the effect of the RA-arsenic combination in Pml+/+ and Pml−/− APL cells. In Pml+/+ APL cells, 3-d RA-arsenic treatment triggered marked tumor regression and complete loss of clonogenicity (Fig. 6d,e), as previously demonstrated16,19. Notably, in Pml−/− APL cells, RA-arsenic yielded only modest tumor regression and did not affect clonogenicity (Fig. 6d,e). The combined treatment elicited enhanced activation of previously identified Trp53 targets in Pml+/+ APL cells only (Fig. 6f). Hence, in addition to increasing RA-induced PML-RARA degradation, arsenic may independently cooperate with RA to cure APL by binding PML, thereby accelerating NB reformation and potentiating the activation of downstream TP53 signaling (Fig. 6g). Thus, PML NBs have a key role in the therapeutic response of APL cells.

+/+

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Figure 5  Identification of a Pml-dependent Trp53 signature. (a) Western blot analysis of PML-RARA and Trp53 in bone marrow cells of RA-treated Pml+/+ or Pml−/− APL mice treated with 10 mg (intermediate dose, I) or 100 mg (high dose, H) RA. Expo, exposure. (b) The 20 most differentially upregulated genes in response to RA in Pml+/+ versus Pml−/− APL mice. Trp53 targets are indicated, as well as genes implicated in senescence and/or proliferation (S/P). Independent validation given in Supplementary Figure 4b. (c) Induction of Serpine1 in retrovirally transformed hematopoietic progenitors grown in methylcellulose and treated overnight with 1 × 10 −7 (10−7) M RA. (d) Revisited model of RA therapeutic activity.

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studies highlighted the role of Trp53 inhibition in aberrant selfrenewal and leukemia development40; however, to our knowledge, this is the first implication of Trp53 as a key effector of targeted therapies. Phagocytosis of APL cells in the bone marrow19 or liver may reflect the Trp53-dependent attraction of immune cells by senescent cells41. Pml NBs are not disrupted in PLZF-RARA–driven APL cells23, which may explain why PLZF-RARA degradation does not activate Trp53 signaling and marginally affects self-renewal. Additionally, the presence of the reciprocal RARA-PLZF fusion, which interferes with PLZF signaling42 and facilitates APL development25, may also contribute to RA resistance. Our findings match many clinical observations in individuals with APL. Apart from PML-RARA mutations, TP53 or PML mutations have been detected in several of the rare patients resistant to therapy 43,44 (J. Lehman-Che, unpublished data, and P. Koeffler and V. Madan, National University of Singapore, personal communication). In mouse APLs, full PML-RARA degradation24 and APL clearance require high concentrations of RA. Similarly, individuals with APL durably respond only to high RA doses24,45, and some cures have been observed with single-agent liposomal RA, which sustains high intracellular concentrations46. That arsenic has a dual role, triggering PML-RARA degradation and independently enforcing NB targeting of normal PML protein10–13,36, may explain its clinical potency. Notably, among the Pml-Trp53–dependent genes identified is Serpine1, the key regulator of thrombin activity, whose strong upregulation may account for the currently unexplained RA-triggered reversion of coagulation disorder47. Previous studies demonstrated Pml control over Trp53 in fibro­ blasts48–50. We establish that Pml, Trp53 and Serpine1, three master drivers of senescence, are sequentially activated by APL therapy in vivo, which suggests that APL may constitute the first example of a senescence-driven leukemia cure51. The APL cell lines NB4 and UF-1, which are both TP53 mutant, only exhibited differentiation programs

VOLUME 20 | NUMBER 2 | FEBRUARY 2014  nature medicine

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Figure 6  Role of Pml in the efficacy of the RA-arsenic combination. (a,b) Transcriptional induction (n = 4, mean ± s.d.) (a) and western blot analysis (b) of Pml and PML-RARA in bone marrow cells of APL mice treated with 10 mg RA (RA int), 100 mg RA (RA high) or 10 mg RA and arsenic (RA int/As) for 6 h. (c) Immunofluorescence analysis of murine Pml in bone marrow cells from untreated, RA int– or RA int/arsenic–treated APL mice (scale bar, 5 µm). Images show a progressive shift from a microspeckled pattern to fully NB-associated Pml. Quantification is shown in the graph (n ≥ 29). Data are expressed as mean ± s.d. (d) Spleen weights of Pml+/+ or Pml−/− APL mice treated with RA int or the RA int/arsenic combination for 3 d (n = 3). Data are expressed as mean ± s.d. (e) Survival of secondary recipients of bone marrow cells from Pml+/+ or Pml−/− APL mice treated with RA int/arsenic for 3 d. Mice implanted with Pml+/+ APL cells from RA/arsenic-treated primary donors never developed APL (n = 6). (f) Trp53 target gene induction in Pml+/+ or Pml−/− APL mice treated with RA int or RA int/arsenic for 6 h. (g) Model by which targeting normal Pml contributes to APL cure by the RA-arsenic combination.

upon treatment with RA38,52. We observed rapid Trp53 stabilization, activation of Pml-dependent Trp53 targets and loss of self-renewal in murine transgenic-derived APL cells grown ex vivo. Thus, inactivation of TP53 in human APLs allowed establishment of cell lines but precluded determination of the basis for therapy response. Our data suggest that partner protein recruitment into NBs contributes to the PMLdependent loss of APL-initiating cell self-renewal. Association of Trp53 to NBs, together with a number of its modifying enzymes (Crebbp, Hipk2 and Usp7), was proposed to facilitate Trp53 activation31,49. Yet, in individuals with APL, despite RA-induced TP53 stabilization, we could not detect changes in TP53 acetylation or in the phosphorylation of Ser15 or Ser16, modifications that are important for TP53 activity and were previously proposed to be PML dependent. Identification of a small subset of Pml-regulated Trp53 target genes should facilitate elucidation of the molecular basis for Pml-dependent Trp53 activation. Arsenic has a biphasic effect, first enhancing NB formation and partner recruitment and only later enforcing PML degradation10. NB formation by arsenic can be augmented and prolonged via PML transcriptional activation by interferons, promoting maximal recruitment and post-translational modification of partner proteins10,37,53. This interferon-arsenic combination markedly alters leukemia-initiating activity in adult T cell leukemia54,55 or murine chronic myelogenous leukemia56. Therefore, this NB-initiated PML-TP53 checkpoint may obliterate self-renewal even when NBs are not disrupted, providing therapeutic rationales for other malignancies. Methods Methods and any associated references are available in the online version of the paper. Accession codes. Microarray data were deposited in the Gene Expression Omnibus database with accession code GSE51723.

nature medicine  VOLUME 20 | NUMBER 2 | FEBRUARY 2014

Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments The laboratory of H.d.T. is supported by the Ligue Nationale contre le Cancer, the Cartes d’Identité des Tumeurs program, the Institut National de la Santé et de la Recherché Médicale (INSERM), the Centre National de la Recherché Scientifique (CNRS), University Paris Diderot, Institut Universitaire de France, Institut National du Cancer, Fondation Association pour la Recherche contre le Cancer (ARC) (Prix Griffuel) and the European Research Council (senior grant 268729 – STEMAPL). J.A. was supported by a fellowship from Ecole Polytechnique and Fondation ARC, K.R. by a fellowship from the Lady Tata and ARC Foundations. S.M. is supported by grants from EPIGEN and the Italian Association for Cancer Research (AIRC). We thank A. Janin, F. Bouhidel and P. Bertheau for assistance with mouse pathology; P. Chambon (Institut de Génétique et de Biologie Moléculaire et Cellulaire) for RARA-specific antibody; S. Lowe (Memorial Sloan-Kettering Cancer Center) for shRNA vectors and Pml-specific antibody; L. Peres and S. Gressens for technical help; M. Pla for the animal facility; N. Setterblad for imaging; E. Del Neyri and V. Dessirier for imaging statistical analysis; and I. Pallavicini and A. Marinelli for mouse work in Milan. We thank E. Raffoux, C. Bailly, P. Fenaux and N. Boissel (Hôpital St. Louis) for providing the patients’ blood samples. We thank R. Ohki and L. Attardi for sharing unpublished p53 ChIP-Seq data. We thank all members of the laboratory of H.d.T. for helpful discussions, J. Godet for continuous support and V. Lallemand-Breitenbach, U. Sahin, S. Benhenda, F. Sigaux and J.C. Gluckman for critical reading of the manuscript. AUTHOR CONTRIBUTIONS J.A., H.S. and K.R. performed the experiments, J.A., A.d.R. and H.d.T. analyzed the bioinformatic data, S.M. provided reagents and discussed results and J.A., K.R. and H.d.T. analyzed the experiments and wrote the manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html.

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Patient samples. Peripheral blood was obtained from two individuals with APL treated at our institution. The first one gave blood prior to and 12 h after in vivo treatment with RA. APL-containing blood was obtained from another subject and treated ex vivo for 12 h with RA 10−6 M (Sigma). Both patients were included in the French cooperative APL 2006 trial, sponsored by Assistance Publique– Hôpitaux de Paris, approved by the Comité de Protection des Personnes Ile-deFrance X. Patients enrolled in this trial gave written informed consent, including for blood and marrow samples to be used in correlative studies. Mouse in vivo experiments. PML-RARA and PLZF-RARA APL cells were transplanted as reported16,19. RA and arsenic (5 µg per g body weight) were administered as described19. The RA dose (1.5, 10, 50 and 100) refers to the drug content (in mg) of subcutaneously implanted 21-d-release RA pellets (Innovative Research of America). Z-VAD was administered daily by intraperitoneal injection (200 µg per mouse). Doxorubicin was administered once by intraperitoneal injection (200 µg per mouse). For secondary transplantation, 1 × 106 bone marrow cells were injected intravenously in two syngeneic secondary recipients per treated primary mouse. Sample size for all mouse experiments was determined in line with our previous studies after obtaining advice from a statistician16,19. All murine experiments were repeated at least three times. Animals were randomly assigned to specific treatment groups, and analysis was performed by a nonblinded investigator. FACS analyses were performed as described16. Cell cycle profiles were assessed using propidium iodide. Animal handling was performed using protocols approved by the Comité Régional d’Ethique Expérimentation Animale no. 4 (Paris Nord). FVB/N mice (7 weeks old) were purchased from Janvier, and 129/SV and Swiss nude mice (7 weeks) were purchased from Charles River. Protein analyses. Immunofluorescence and western blot analyses were performed as reported16. PML-RARA was detected with rabbit polyclonal antibodies to RARA (RP115, 1:2,500, provided by P. Chambon), Trp53 by mouse polyclonal antibodies (HR231, 1:1,000, Santa Cruz), TP53 by mouse monoclonal antibodies (DO-1, 1:2,000, Santa Cruz), Pml by a murine monoclonal antibody provided by S. Lowe (1:1,000), CDKN1A by polyclonal rabbit antibodies (12D1, 1:1,000, Cell Signaling), Actin, beta (Actb) and ACTB by rabbit polyclonal antibodies (20-33, 1:5,000, Sigma), Serpine1 by rabbit polyclonal antibodies (ab28207, 1:2,000, Abcam), Vinculin (Vcl) and VCL by mouse monoclonal antibodies (7F9, 1:1,000, Santa Cruz) and Lmnb1 by goat polyclonal antibodies (M-20, 1:500, Santa Cruz). The CM-5 rabbit polyclonal antibody to Trp53 (1:250, Novo Castra) was used for immunohistochemistry. shRNAs targeting Pml in a pCMV-turboGFP vector were purchased from Sigma. Sequences of sh1 targeting Trp53 (inserted in MLS vector), and sh2 (pTRIP vector) are available upon request. Ex vivo transformation and culture of murine hematopoietic progenitors. Lin− murine hematopoietic progenitor cells from female C57/BL6 mice were retrovirally transduced with a pMSCV–PML-RARA vector. Transformed cells were grown in methylcellulose supplemented with cytokines and stem cell factor, and serially replated every week. Ex vivo culture of APL cells extracted from the bone marrow of leukemic mice was performed in RPMI medium supplemented with interleukin-3 (IL-3), IL-6 and stem cell factor in the presence or absence of RA at the indicated concentrations. Retroviral or lentiviral transductions of PML-RARA–transformed cells with vectors expressing shRNAs against Trp53 or Pml were performed by spinoculation. Cells were then grown for 1 week in methylcellulose and sorted for GFP positivity before analysis. RT-PCR. Total RNA was isolated from whole-bone-marrow-cell extracts using the RNeasy kit (Qiagen). First-strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen). Quantitative RT-PCR was performed using

doi:10.1038/nm.3441

TaqMan Fast Universal PCR Master Mix and 7500 Fast Real-Time PCR System (Applied Biosystems). Probes and primers for TaqMan assays for Serpine1, Dusp5 and Egr1 genes were purchased from Applied Biosystems. Ywhaz was used as endogenous control to calibrate the amount of target mRNA. Bioinformatics analysis of expression arrays. Samples were hybridized on Affymetrix Mouse Gene 1.0 ST Arrays and log2 measures were obtained using RMA normalization. Supplementary Figure 1a shows enrichment analysis of Gene Ontology (GO) terms that involved 3 steps: (i) we built ten lists of nonredundant murine gene symbols deregulated in treated APL models compared with controls under various conditions (see the last ten columns), and symbols were mapped to human orthologs using the Homole Gene system; (ii) we obtained the lists of human proteins corresponding to GO terms (either directly or via their specialized terms), proteins were mapped to nonredundant HUGO gene symbols using the BioMart system and lists were restricted to orthologs measured on the MG 1.0 ST array; and (iii), hypergeometric tests were performed to assess whether the ten ‘deregulation’ lists significantly overlapped with the ‘GO’ lists. For Figure 2a,i, E2F and SASP signatures were based on reports from the literature57,58. The mean intensity (log2) of the genes comprised in these two signatures was calculated from the expression measures for untreated or intermediate-dose– (10 mg) or high-dose (100 mg))-treated APL mice and reported as fold induction relative to untreated APLs. For Figure 2c, known TP53 targets or genes implicated in senescence. For each condition (model/RA dose/time), we calculated the fold induction between treatment and control based on averaged expression measures of replicate samples. Probe sets related to a gene symbol and not induced upon low-dose (1.5 mg) RA treatment (fold induction (FI) < 1.8) were ranked according to decreasing fold induction upon high-dose (100 mg) RA treatment in the PML-RARA model. For Figure 5b and Supplementary Figure 4a,b, each gene was assigned a score of 1 if it was differentially regulated in Pml+/+ and Pml−/− APL mice upon treatment but not in untreated conditions, i.e., if it fulfilled the following criteria: P value (expression untreated Pml+/+ APL mice = expression untreated Pml−/− APL mice) > 0.05 and ((FI (Pml+/+) > α and FI (Pml+/+)/FI (Pml−/−) > α and FI (Pml−/−) > 1/α) or (FI (Pml+/+) < 1/α and FI (Pml+/+)/FI (Pml−/−) < 1/α and FI (Pml−/−) < α)), where α was arbitrarily set at 1.25. Six treatment conditions were considered (6- or 12-h RA int (10), RA high (100) and RA int/arsenic) and genes were assigned an agglomerate score (i.e., the sum of the scores obtained in the six conditions) ranging between 0 and 6. Enrichment analyses of GO terms comprising 20 genes or more and of a TP53 response pathway constructed from a literature report59 were performed on the list of genes with an agglomerate score of 4 or more, as described above for the previous array analysis. To study individual genes, a shorter list of genes was generated with more stringent criteria (α = 1.5). Genes with scores of 4 or more were selected and sorted according to decreasing fold induction upon RA int (10 mg) in Pml+/+ APL mice. Annotations from Figure 2c and Figure 5b are derived from literature search and analysis of TP53 binding sites in non-APL cells (R. Ohki, National Cancer Center Research Institute, Tokyo, personal communication). Statistical analyses. For the other experimental data, when necessary, log-rank tests (for Kaplan-Meier curves) or bilateral Student’s t-tests were used to assess the statistical significance of observed differences. 57. Bracken, A.P., Ciro, M., Cocito, A. & Helin, K. E2F target genes: unraveling the biology. Trends Biochem. Sci. 29, 409–417 (2004). 58. Coppé, J.P. et al. Senescence-associated secretory phenotypes reveal cellnonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol. 6, 2853–2868 (2008). 59. Ragazzon, B. et al. Transcriptome analysis reveals that p53 and β-catenin alterations occur in a group of aggressive adrenocortical cancers. Cancer Res. 70, 8276–8281 (2010).

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