Eradication of acute promyelocytic leukemia ... - Semantic Scholar

ARTICLES

Eradication of acute promyelocytic leukemia-initiating cells through PML-RARA degradation

© 2009 Nature America, Inc. All rights reserved.

Rihab Nasr1,7,8, Marie-Claude Guillemin1,8, Omar Ferhi1, Hassan Soilihi1, Laurent Peres1, Caroline Berthier1, Philippe Rousselot2, Macarena Robledo-Sarmiento1, Vale´rie Lallemand-Breitenbach1, Bernard Gourmel1, Dominique Vitoux1, Pier Paolo Pandolfi3, Ce´cile Rochette-Egly4, Jun Zhu1,5 & Hugues de The´1,5,6 Retinoic acid and arsenic trioxide target the protein stability and transcriptional repression activity of the fusion oncoprotein PML-RARA, resulting in regression of acute promyelocytic leukemia (APL). Phenotypically, retinoic acid induces differentiation of APL cells. Here we show that retinoic acid also triggers growth arrest of leukemia-initiating cells (LICs) ex vivo and their clearance in PML-RARA mouse APL in vivo. Retinoic acid treatment of mouse APLs expressing the fusion protein PLZF-RARA triggers full differentiation, but not LIC loss or disease remission, establishing that differentiation and LIC loss can be uncoupled. Although retinoic acid and arsenic synergize to clear LICs through cooperative PML-RARA degradation, this combination does not enhance differentiation. A cyclic AMP (cAMP)-dependent phosphorylation site in PML-RARA is crucial for retinoic acid–induced PML-RARA degradation and LIC clearance. Moreover, activation of cAMP signaling enhances LIC loss by retinoic acid, identifying cAMP as another potential APL therapy. Thus, whereas transcriptional activation of PML-RARA is likely to control differentiation, its catabolism triggers LIC eradication and long-term remission of mouse APL. Therapy-triggered degradation of oncoproteins could be a general strategy to eradicate cancer stem cells.

APL is characterized by a specific t(15;17) translocation that encodes a fusion of the promyelocytic leukemia (PML) and retinoic acid receptor-a (RARA) proteins. PML-RARA is a transcriptional repressor with both gain-of-function and dominant-negative properties, resulting in transcriptional repression of retinoic acid and non–retinoic acid target genes1,2 and culminating in differentiation arrest. Gene silencing involves enhanced recruitment of nuclear receptor co-repressors, the polycomb complex or Daxx onto PML-RARA or its obligatory retinoid X receptor-a (RXRA) partner, resulting in changes in chromatin organization and DNA methylation3–9. APL is a unique model in cancer biology in that two therapeutic agents, retinoic acid and arsenic trioxide, both target PML-RARA10,11. In vivo, both drugs induce leukemia differentiation to varying extents, making APL the only cancer that can be treated through differentiation therapy12. High plasma concentrations of retinoic acid are essential for prolonged clinical responses in patients3,13. At the molecular level, retinoic acid binds PML-RARA and turns it into a transcriptional activator, but also triggers its degradation3,14,15. PML-RARA degradation depends on PML-RARA cleavage by differentiation-activated proteases14,16 and on recruitment of proteasomes by the retinoic acid–bound RARA moiety of PML-RARA15,17.

Arsenic activates kinases targeting PML-RARA, or its RXRA partner, and induces PML-RARA degradation through specific SUMOylation and subsequent ubiquitination of its PML moiety5,18–22. The respective contributions of drug-induced differentiation, apoptosis, transcriptional activation and PML-RARA degradation to APL eradication have been a matter of debate. The molecular pathogenesis of APL and the bases for its response to therapy have been studied in PML-RARA transgenic mice, which recapitulate responses to therapy in individuals with APL23–26. In particular, the combination of retinoic acid and arsenic triggers prolonged remission in mouse models and humans. However, although the two drugs synergize to effect APL eradication in vivo, this does not seem to result from enhanced differentiation23,24,27–29. Retinoic acid–induced APL differentiation and PML-RARA transactivation are enhanced by cAMP signaling, and many retinoic acid– resistant APLs differentiate after exposure to retinoic acid and cAMP1,30,31. At the molecular level, cAMP activates protein kinase A (PKA), which dissociates RARA from the SMRT co-repressor, allowing transcriptional activation by retinoic acid metabolites through RXRA1,32. RARA phosphorylation by cAMP-activated PKA on Ser369, within the ligand binding domain, also allows recruitment

1Universite ´ de Paris 7/CNRS UMR 7151, Equipe labellise´e N111 Ligue Nationale Contre le Cancer, Service de Biochimie, Hoˆpital St. Louis, 1, Av. C. Vellefaux, 75475 Paris CEDEX 10, France. 2UFR de Me´decine Universite´ de Paris 5 & Hematology, Hoˆpital Mignot, 177 rue de Versailles, 78157 Le Chesnay, France. 3Department of Pathology, Beth Israel Deaconess Medical Center, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. 4Institut de Ge ´ ne´tique et de Biologie Mole´culaire et Cellulaire, Department of Functional Genomics, 1 rue Laurent Fries, BP 10142, Illkirch F-67400, France. 5CNRS laboratoire associe´ MPC, Shanghai Institute of Hematology, Rui Jin Hospital, 197 Rui Jin Road, 200025 Shanghai, China. 6Institut Universitaire de France. 7Current address: Department of Internal Medicine, American University of Beirut, Bliss Street, P.O. Box 11-0236, Beirut, Lebanon. 8These authors contributed equally to this work. Correspondence should be addressed to H.d.T. ([email protected]).

Received 24 July 2008; accepted 10 October 2008; published online 23 November 2008; corrected after print 7 January 2009; doi:10.1038/nm.1891

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ARTICLES of the cdk7–cyclin H subcomplex of transcription factor IIH and phosphorylation-induced activation of the ligand-independent AF1 transactivating domain33. The importance of these events for the APL response to therapy is unknown. Recent evidence has indicated that not all cancer cells are identical: only a minority have the ability to regenerate new tumors and, hence, control transplantability and metastasis development34. PML-RARA promotes immortalization of myeloid progenitors ex vivo and in vivo3,5,35, but the effect of retinoic acid or arsenic on APL LICs has never been analyzed in vivo. Here we show that PML-RARA degradation is tightly regulated by a PKA phosphorylation site in PML-RARA and that PML-RARA degradation triggers LIC loss, which—rather than differentiation—is responsible for eradicating APL in mice.

presence of remaining LICs in the inoculate. Yet comparison with marrow inoculates from untreated mice indicated that LIC abundance was greatly reduced (Fig. 1e,f). Because this reflects in part our inoculation of a smaller number of APL cells (assessed by PML-RARA DNA), we treated mice for 2 d with high doses of retinoic acid (40 mg) and compared time to death in recipients inoculated with similar numbers of treated or untreated APL cells. A highly significant survival difference was observed, indicating that only 1% of LICs remain after a 2-d, 40-mg treatment with retinoic acid (Fig. 1e,f and Supplementary Fig. 2b). We cannot formally exclude the possibility that retinoic acid induces transient cell-cycle arrest of LICs, delaying their regrowth in secondary recipients, but the normal size of secondary colonies derived from retinoic acid–treated methylcellulose ex vivo strongly argues against this hypothesis.

RESULTS Differentiation by retinoic acid spares clonogenic cells Transduction of PML-RARA DNA into mouse hematopoietic progenitor cells allows them to be replated indefinitely in methylcellulose, with only 1% of the transformed cells being clonogenic and yielding promyelocytic colonies5. Growth in methylcellulose containing retinoic acid led to 8% of the original number of colonies in untreated cultures as well as to terminal granulocytic differentiation of those colonies that developed (Fig. 1a). Unexpectedly, when retinoic acid– treated cultures, or even individual colonies, were replated in methylcellulose without retinoic acid, many dense promyelocytic colonies developed that could be serially replated, qualitatively similar to the untreated parental cells (Fig. 1a). Retinoic acid treatment nevertheless resulted in quantitative differences in the frequency of clonogenic cells, which dropped from 1% to 0.3% (Fig. 1b). In untreated starting cultures, PML-RARA protein levels were comparable to those found in individuals with APL or transgenic mice (data not shown). Human APLs expressing the promyelocytic leukemia zinc finger (PLZF)-RARA fusion protein are resistant to retinoic acid therapy3. Unexpectedly, hematopoietic progenitor cells transformed ex vivo with PLZF-RARA behaved exactly as PML-RARA–transformed cells with respect to retinoic acid–induced differentiation, gene activation and growth inhibition of clonogenic cells (Supplementary Fig. 1 online). Such complete differentiation of PLZF-RARA–transformed cells suggests that differentiation is unlikely to be the sole determinant for clinical response of APL to retinoic acid. Together, these ex vivo experiments establish that retinoic acid triggers two distinct and uncoupled events: induction of terminal differentiation of promyelocytes and transient growth arrest of clonogenic progenitors (Fig. 1b).

Low-dose retinoic acid triggers differentiation but not LIC loss Apart from PML-RARA mutations, low plasma retinoic acid concentrations, linked to retinoic acid–induced catabolism, often account for resistance to retinoic acid3,13. At doses of 10 mg, plasma retinoic acid levels reached 10 7 M after 3 d, and 9-cis-retinoic acid, the main retinoic acid metabolite that activates RXRs, reached 2  10 8 M. Treatment with 1.5 mg of retinoic acid triggered full differentiation after 3 d, with plasma retinoic acid at 1.5  10 8 M (Fig. 2a). However, on day 7, promyelocytes were still present in the marrow, spleen size never normalized and normal hematopoiesis was not restored (Fig. 2a,b). Transplantation of marrow from mice treated with 1.5 mg retinoic acid into secondary recipients did not significantly affect time to death (Fig. 2c). Thus, immediate differentiation can be uncoupled from the decrease in LIC frequency, and the latter is likely to be responsible for retinoic acid–induced clinical remission.

Retinoic acid decreases APL LIC frequency in vivo Ex vivo transformation may not fully reflect APL pathogenesis. We thus turned to an APL transplantation model derived from mice expressing the Tg(PML-RARA)935Kog human transgene (referred to here as MRP8–PML-RARA transgenic mice)23,31. To allow for rapid and reproducible APL development, we injected 107 APL blasts intravenously into syngeneic recipients and then treated wellestablished APLs with retinoic acid for up to 7 d before analyzing therapy response. Retinoic acid treatment led to the rapid in vivo differentiation of APL cells, followed by their replacement by normal marrow (Fig. 1c–f)23–26. Retinoic acid also normalized spleen weight and blood counts (Fig. 1d), although relapses (assessed by luciferase imaging; Supplementary Fig. 2a online) always occurred weeks later23,24. When we inoculated this morphologically normal retinoic acid–treated day 7 marrow into secondary recipients and monitored their survival, we found that they developed APL, demonstrating the

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Retinoic acid differentiates PLZF-RARA APL without LIC loss Mice transgenic for both PLZF-RARA and RARA-PLZF (referred to here as PLZF-RARA–RARA-PLZF transgenic mice) develop an APLlike disease36. We established a transplantation model of these APL cells in nude mice and, unexpectedly, found that retinoic acid induced terminal granulocytic differentiation (Fig. 2d). However, in contrast to our observations in PML-RARA APLs, restoration of normal hematopoiesis in PLZF-RARA–RARA-PLZF mice was undetectable by flow cytometry (Fig. 2d), May-Gru¨nwald-Giemsa (MGG) staining or ZBTB16-RARA (PLZF is now known as ZBTB16) DNA clearance (Fig. 2). Spleen weight remained elevated, even 2 weeks after treatment, and blood counts increased markedly, reminiscent of the retinoic acid syndrome3 (Fig. 2e,f). Transplantation of retinoic acid–treated (granulocytic) or untreated (blastic) marrow into secondary recipients consistently resulted in the same kinetics of leukemia development (Fig. 2g,h). Retinoic acid treatment prolonged survival of primary recipients37 (Supplementary Fig. 3a online). Notably, in a human subject with t(11;17) APL, retinoic acid similarly led to a significant decrease in marrow blast counts, which was immediately reversed after withdrawal of retinoic acid (Supplementary Fig. 3b). This situation exemplifies the complete dissociation between terminal retinoic acid–induced differentiation and the eradication of LIC or APL regression, indicating that retinoic acid–induced differentiation is not the basis for APL remission. LIC loss by retinoic acid and arsenic requires proteolysis Arsenic triggers some differentiation and APL regression in mice23,24,31 (Fig. 3a), although it does not seem to be as efficient in humans, possibly because of a different delivery route (intraperitoneal

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ARTICLES Lin+

a

22 68

PML-RARA virus

5FU

7 3

1 × 104 cells

Marrow GR-1

5d 104 ± 39 colonies

Lin– 16 12 65 7

CD11b

GR-1

RA

b 1×

24 ± 14 colonies

La 10 4 ce

rge

lls

co

lon

ies

CD11b

RA 10 19 8 70 3 4

10

c

es oni col cells all Sm olated + is

53 ± 25 colonies

APL RA 10

Normal

Differentiated cells 99.7%

10 53 7 30 9 38 20 33

RA 10

3 63 31 3

Normal

8 57 35 1

Clonogenic progenitors 0.3%

GR-1

Bone marrow

Untreated

Day 7

CD11b

e

60

4

10 APL cell inoculate

0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0 Day 7

3 –1 WBC (10 cells µl )

Spleen weight (g)

d

50 40 30 20 10

105 106 107

0 Untreated RA 10

Normal

Untreated

RA 10

Normal

10

P = 0.008

15 20 25 30 35 Time to death in primary transplants (d)

120 100 80 60

RA 40

40 Untreated

20 0

P = 0.0001

PML-RARA DNA (%)

f

Clonogenic progenitors 1% RA-treated culture

CD11b

APL untreated

Promyelocytes 89%

M

GR-1

1×

Granulocytes 10%

8 ± 4 colonies

ls cel

CD11b

PML-RARA DNA (%)

© 2009 Nature America, Inc. All rights reserved.

GR-1

RA

–6

Untreated culture

100 80 RA 10

60 40

P = 0.04

Untreated

20 0

RA 40

Untreated

0 20 40 Time to death in secondary transplants (d)

NS

Day 2

Untreated

RA 40

0

P = 0.000057

20 40 Time to death in secondary transplants (d)

Day 7

Figure 1 Retinoic acid (RA) triggers APL cell differentiation and loss of clonogenic precursors ex vivo and in vivo. (a) Ex vivo methylcellulose-cultured, PML-RARA–transformed primary hematopoietic progenitors (top) were cultured for 1 week with 10 6 M RA (bottom right) and subsequently without RA (bottom left). Numbers of colonies (mean ± s.d. of five independent experiments) and representative flow cytometry and MGG stains of cells recovered at various steps are shown. Scale bar, 10 mm. 5FU, 5-fluorouracil. (b) Cell composition of untreated and RA-treated cultures. (c–e) RA decreases LIC abundance in PML-RARA APL in vivo. (c) Left, bone marrow MGG staining of a normal mouse and an APL mouse untreated or treated with 10 mg of RA (RA 10) after 7 d; right, flow cytometry of the corresponding marrow cells using two myeloid differentiation markers, CD11b (Mac) and GR-1. (d) Spleen weight (mean ± s.d. of four experiments) and white blood cell counts (WBC; mean ± s.d. of seven experiments) after 1 week of treatment with 10-mg RA pellets. (e) Survival of mice inoculated with increasing amounts of APL cells (mean ± s.d. of two independent experiments, see also Supplementary Fig. 2b). (f) Bone marrow cells from RA-treated and untreated mice were injected into secondary syngeneic recipients. Abundance of PML-RARA DNA as determined by quantitative real-time PCR, and time to death of secondary recipients (mean ± s.d. of three independent experiments) on day 2 with 40 mg of RA (RA 40) or day 7 with 10 mg of RA. NS, not significant.

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28 34 Untreated 26 12

RA 10

Bone marrow

Day 3

Day 7

0.3

Day 3 Day 7

0.25 0.2 0.15 0.1 RA 1.5

RA 10

RA 1.5

40

2 68 10 20

3 63 31 3

RA 10

PLZF-RARA untreated

Control

20

Day 12 Day 7

0 Day 7

0

10

20

30

40 Untreated

Time to death in secondary transplants (d)

12 51 21 16

RA 10

2 87 6 5

C

Control

80 60

9 38 20 33

d

RA 10

on t R rol A 1. 5 R A 10

0.35

PML-RARA DNA (%)

Spleen weight (g)

100 P = 0.05

4 81 9 6

Day 7

c

120

0.4

RA 10

Bone marrow

P = 0.017

0.45

3 67 7 23

Day 3

CD11b

b

RA 1.5

GR-1

RA 1.5

GR-1

PML-RARA untreated

a

As expected from their nonoverlapping pathways11,15,18,20,21, retinoic acid- and arsenic-triggered PML-RARA degradation were synergistic and proteasome dependent in ex vivo–treated mouse APL cells (Fig. 3). To address the role of PML-RARA degradation in LIC clearance, we treated mice with retinoic acid and arsenic in the presence or absence of the proteasome inhibitor bortezomib (Fig. 3f). Although bortezomib induced some differentiation and APL regression on its own, when combined with retinoic acid and arsenic, it consistently delayed APL clearance, blocked restoration of normal hematopoiesis on day 6 and sharply antagonized loss of LICs on day 3 (Fig. 3f–h). Bortezomib also significantly blocked recovery of normal hematopoiesis by retinoic acid alone (Supplementary Fig. 4 online). Bortezomib may also modulate other pathways in this complex biological system, but these results strongly suggest that PML-RARA degradation is a major molecular determinant of LIC clearance.

P = 0.04

0.75

0.55 0.45 0.35 0.25 0.15 0.05 Day 7

g

90 80 70 60 50 40 30 20 10 0 Untreated Day 7

120

3 –1 WBC (10 cells µl )

0.65

f

Untreated RA 10 Normal

GR-1

P = 0.05

0.85

ZBTB16-RARA DNA (%)

0.95

e Spleen weight (g)

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versus intravenous, respectively) and/or pharmacokinetics. In contrast to retinoic acid alone, the retinoic acid–arsenic association triggers rapid and complete remission in most mouse models of APL and most individuals with APL23,24,27,28. Follow-up studies in mice and with human subjects from two independent clinical trials suggests that they are actually cured, even in the absence of chemotherapy12,23 (Z. Chen (Shanghai Institute of Hematology) and E. Estey (Washington University), personal communications). In mice, retinoic acid and arsenic did not synergize for differentiation on day 3 (Fig. 3a), but they synergized markedly for both disease regression and LIC loss on day 3 or 6 (Fig. 3a–d). The retinoic acid–arsenic synergy for LIC eradication on day 3 was shown with a similar abundance of PML-RARA–positive cells in the bone marrow of primary recipients treated with retinoic acid alone or retinoic acid and arsenic, implying that this combination specifically targets APL LICs.

RA 10

100 CD11b

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h

60

RA 10

40 Untreated

20

0 Day 12

Untreated

0 20 40 60 Day 10 Time to death in secondary transplants (d)

RA 10

P = 0.02

Figure 2 Full differentiation without APL clearance in PLZF-RARA APL or PML-RARA APL treated with low-dose retinoic acid (RA). (a) MGG-stained bone marrow (left) and flow cytometry (right) of PML-RARA APL cells from mice treated with high (RA 10) or low (RA 1.5) doses of RA for the indicated times. Arrows indicate remaining APL blasts. Scale bar, 10 mm. (b) Spleen weight and PML-RARA DNA content of mice after 7 d of treatment, as indicated (mean ± s.d. of three experiments). (c) Time to death in secondary recipients inoculated with bone marrow cells treated or not treated with RA for 7 d (mean ± s.d. of three independent experiments). (d) Bone marrow staining (left) and flow cytometry analysis (right) of PLZF-RARA mouse APLs treated for 12 days with 10 mg of RA. Terminal granulocytic differentiation was observed. Arrow indicates the absence of normal hematopoiesis restoration. (e,f) Spleen weight (e) and white blood cell counts (WBC; f) of untreated or RA-treated PLZF-RARA APL after 7 d (mean ± s.d. of three experiments). (g) Abundance of ZBTB16RARA DNA by quantitative PCR after 12 d of RA treatment. (h) Time to death of secondary recipients injected with 3  106 bone marrow cells collected from primary recipients after 10 d of treatment with or without RA (mean ± s.d. of four independent experiments).

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ARTICLES Retinoic acid–cAMP clears LICs in sensitive APLs When we combined cAMP with 1.5 mg of retinoic acid, we noted a marked in vivo synergy for disease regression and restoration of normal hematopoiesis, but not for immediate differentiation, on day 3 (Fig. 4a–e). H89, an inhibitor of PKA, substantially reversed this effect of cAMP (Fig. 4d). When combined with 1.5 mg of retinoic acid, particularly with arsenic, phosphodiesterase inhibitors (PDEIs) consistently decreased the frequency of LICs in secondary

a

RA 10 + As2O3

As2O3

RA 10

As2O3

CD11b

As2O3

Day 3

d

RA 10 + As2O3

RA 10 As2O3

0.25

As2O3

0.15

e

re at R ed A R 10 A 10 As + 2O As 3 2O

nt U

–9

As 5 × 10 M RA 10 –8 M RA 10 –7 M

– – –

– – +

– + –

+ – –

PML-RARA

+ – +

0 5 10 15 20 25 30 35 40 45 50

Secondary transplant (d) + + –

kDa

g

100 75

RA + As MG132

As2O3

RA 10

– +

+ +

PML-RARA PML-RARA

RA 10 + As2O3

RA 10 + As2O3 + bortezomib

Actin

Bone marrow

Bortezomib

Actin

h 42 34 Untreated 15 9

+ –

50

13 38 RA 10 46 3

As2O3

2 86 3 9

8 81 Bortezomib 6 5

11 43 RA 10 + As O 3 84 2 3 RA 10 + As2O3 44 2 + bortezomib 9 4

120 100 80 60 40 20 0

U R nt A re 10 at + ed A Bo s 2O r te R zo 3 A + 10 mi b bo + rte As zo 2 O m 3 ib

GR-1

Day 6

– –

RARA

0 20 40 400 Time to death in secondary transplants (d)

Untreated

RA 10 + As2O3

1 20 40 60 80 Day 3 0 Time to death in secondary transplants (d)

Untreated

0.05 Day 6

3

Spleen weight (g)

RA 10

RA 10

100 10

RA 10 + As2O3

0.35

1,000 As O 2 3

Untreated

0.55 0.45

Light units

D3

Untreated

PML-RARA DNA (%)

RA 10

10,000

RA 10 + As2O3

U nt re at e R d A R A A 10 10 s + 2 O3 As 2O

Primary mouse

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100 90 80 70 60 50 40 30 20 10 0

PML-RARA DNA (%)

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RA 10

1 87 22 60 4 8 10 8 RA 10 + As2O3

GR-1

Day 3

f

3 73 18 6

39 26 28 7 Untreated

Bone marrow

Untreated

transplantation experiments (Fig. 4e) without changing APL cell abundance in the inoculates (data not shown). Thus, cAMP synergizes with low-dose retinoic acid for LIC eradication, but not immediate differentiation. The retinoic acid–cAMP combination differentiates retinoic acid–resistant APLs expressing PML-RARAL902P (Tg(PML-RARAL902P)4048Kog) which is defective for retinoic acid binding1,31 (Fig. 4f,g), presumably through RXRA activation after in vivo

Day 3

CD11b

Figure 3 LIC eradication by retinoic acid (RA) and arsenic depends on active proteolysis. RA 10 + As2O3 (a–e) RA and arsenic synergize for LIC eradication. (a) MGG staining (left) and flow cytometry + bortezomib (right) of cells from mice left untreated or treated with 10 mg of RA or arsenic for 3 d. Normal Bortezomib RA 10 + As2O3 non-myeloid hematopoiesis was restored in marrow treated with RA only (arrow) but not with RA and arsenic. Scale bar, 10 mm. (b) Luciferase imaging of APL mice treated for 3 d (left) and Untreated PML-RARA DNA quantification of the corresponding marrow (right). Scale bar, 1 cm. (c) Survival 50 100 150 Day 3 0 of the secondary transplants of day 3 marrow (left) and corresponding follow-up of luciferase Time to death in secondary transplants (d) activity (right). Representative of three experiments. (d) Day 6 spleen weight (mean ± s.d. of three independent experiments) and survival of secondary day 6 marrow recipients (representative of three independent experiments). The three secondary recipients of RA- and arsenic-treated marrow were negative for PML-RARA DNA after a year. (e) Effects of RA and/or arsenic on PML-RARA protein levels in mouse APL cells after 12 h of ex vivo treatment. Actin served as a control. (f–h) The RA-arsenic synergy for LIC clearance depends on active proteolysis. (f) Bone marrow MGG staining (top) and flow cytometry (bottom) after 6 d of treatments. Arrows indicate lack of normal hematopoiesis recovery when bortezomib was combined with RA-arsenic. (g) Ex vivo PML-RARA degradation in mouse APL cells by RA and arsenic (both 10 7 M) is reversed by the proteasome inhibitor MG132 (PML-RARA is detected with an antibody to RARA; actin served as a control). (h) PML-RARA DNA in the marrow (top) and survival of secondary transplants that received marrow cells from APL mice treated for 3 d (bottom) (representative of two independent experiments).

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ARTICLES isomerization of retinoic acid into 9-cis-retinoic acid1,8,9,32. Despite efficient differentiation, retinoic acid–cAMP did not decrease LIC abundance, nor did it significantly degrade PML-RARAL902P (Fig. 4f–i).

0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05

Light unit

10,000

RA 10

RA 1.5

Day 3

Day 7

Day 3

Day 7

Day 3

Untreated Day 7

Day 7

100 10

39 26

3 68

3 73

2 77

28 7

18 11 RA 1.5

18 6

10 11 RA 1.5 + cAMP

RA 10

cAMP

RA 1.5 + cAMP

Day 3

RA 1.5 + cAMP + H89

Day 7

Day 7

GR-1

Untreated

Bone marrow

M P

M P R A

1. 5

+

cA

10

cA

R A

at ed

R A

at R ed A 1 R R A 0 A 1. c 1.5 5 AM + + cA P cA M R MP P A + 1. H 5 89

1. 5

*

1

c

CD11b

e

Day 3

RA 1.5 + As2O3 + PDEI As2O3 + PDEI RA 1.5 + PDEI PDEI Day 7 RA 1.5 + As2O3 As2O3 RA 1.5 Untreated RA 1.5 + As2O3 + PDEI As2O3 + PDEI RA 1.5 + PDEI PDEI Day 3 RA 1.5 + As2O3 As2O3 RA 1.5 Untreated 160 110 Time to death in secondary transplants (d)

E

F Leu

i

100

PDEI –6 RA 10 M

80 60

cAMP

40

RA 10

+ –

– –

– +

+ +

RA 10 + cAMP

PML-RARA

20

nt

re at

Untreated A 10 10 cA + MP cA M P

at re nt

Day 7 0

20

40

Actin

Time to death in secondary transplants (d)

R

A

R

ed

U

0 Day 7

A

PML-RARA DNA (%)

RA 10 + cAMP

R

h

Pro

RA

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Day 7

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D

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C

Bone marrow

B

Untreated

g

LBD

360

A

f

310

R

60

Spleen weight (g)

10

U

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d

1,000

U nt re

Day 7

Day –1 Day 3 Day 6

b 100,000

U nt re

Spleen weight (g)

a 0.55 0.5

Such uncoupling between efficient differentiation and absence of APL clearance suggests that differentiation reflects transcriptional activation, whereas changes in LIC frequency mirror PML-RARA catabolism.

Figure 4 Activation of cAMP signaling synergizes with low-dose retinoic acid (RA) to induce LIC loss in wild-type PML-RARA but not in RA-resistant PMLRARAL902P APL. (a) Day 7 spleen weight (mean ± s.d. of six different experiments). (b) Luciferase quantification of PML-RARA APL mice treated with 10 or 1.5 mg of RA, with or without cAMP, for 1 to 6 d. Asterisk indicates a mouse that died of leukemia progression. Representative of three independent experiments. (c) Flow cytometry on day 3 after treatment, showing similar differentiation for all treatments. (d) Bone marrow MGG staining of mice treated as indicated. PKA inhibitor H89 blocked APL recovery of normal hematopoiesis after treatment with RA-cAMP on day 7. Arrows indicate remaining promyelocytes. Scale bar, 10 mm. (e) Survival of secondary transplant recipients from primary APL mice treated as indicated. In this experiment, cAMP signaling was triggered by a PDEI. Six secondary mice, four inoculated with primary day 3 marrows and two with day 6 marrow, were all negative for PML-RARA DNA after 1 year, as assessed by PCR. Representative of three independent experiments. (f–i) RA-cAMP differentiates RA-resistant PMLRARAL902P APL31 but does not degrade PML-RARA or induce LIC loss. (f) PML-RARAL902P does not bind RA. LBD, ligand binding domain. (g) Left, MGG staining of control and RA-cAMP–treated mice on day 7; right, corresponding spleen weights (mean ± s.d. of six independent experiments). (h) PML-RARA DNA content after 7 d of the indicated treatments (mean ± s.d. of three experiments) and survival of secondary recipients of marrow treated for 7 d (mean ± s.d. of five independent experiments). (i) Analysis of PML-RARA protein after ex vivo treatment with 10 6 M RA and PDEI, alone or together, for 12 h. Actin served as a control.

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Figure 5 Loss of the Ser873 PKA phosphorylation site in PML-RARA results in retinoic acid (RA)-resistant APLs. (a) Mutated version of PML-RARA used to generate transgenic mice. (b) Spleen weight after 6 d of treatment with RA, cAMP and/or arsenic (mean ± s.d. of four independent experiments). (c) Flow cytometry of day 3 cells from corresponding mice, showing RA-induced differentiation. (d) MGG staining on day 6. Scale bar, 10 mm. (e) White blood cell counts (WBC) showing RA-induced leukocytosis, as in PLZF-RARA APLs. (f) PML-RARA DNA abundance on day 10. (g) Survival (mean ± s.d. of three experiments) of secondary recipients after 6 d of treatment. (h) RA-induced PML-RARA phosphorylation in retrovirally transduced mouse progenitor cells expressing PML-RARA (left) or PML-RARAS873A (right). Detection was carried out with an antibody33 recognizing Ser369-phosphorylated RARA (P-(S873); top); output of affinity column was blotted with antibody to RARA (bottom).

Phosphorylation of PML-RARA is essential for LIC loss cAMP-activated PKA phosphorylates RARA on Ser369 (corresponding to PML-RARA Ser873; ref. 33). After exposure to retinoic acid, PML-RARA Ser873 becomes phosphorylated (C.R.-E., unpublished data, and Fig. 5). We generated MRP8–PML-RARAS873A transgenic mice to address the role of this residue in retinoic acid response and retinoic acid–cAMP synergy (Fig. 5). One founder and its offspring eventually yielded two independent APLs with features identical to those previously obtained with PML-RARA5,8,23,31. In a transplantation model, initial retinoic acid–induced differentiation was maintained on day 3, but the mutant APLs all showed a markedly impaired response to retinoic acid on day 7, which most likely reflects ongoing LIC proliferation (Fig. 5). Indeed, the marrow remained leukemic, normal hematopoiesis never reappeared and blood counts rose sharply (Fig. 5d,e). As assessed by PCR, the marrow remained entirely leukemic, whatever the treatment combination, including retinoic acid–arsenic, even when treatments were prolonged after 7 d (Fig. 5f and data not shown). Arsenic-triggered differentiation was maintained, and these APLs regressed with cAMP, consistent with the existence of PML-RARA–independent cAMP growth-suppressive

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pathways32. Synergy between cAMP and retinoic acid or arsenic for differentiation31 was maintained on day 6, possibly reflecting release of SMRT independently of RARA phosphorylation32. However, LIC clearance by the various drug associations was substantially diminished (Fig. 5g), establishing that Ser873 phosphorylation is crucial for retinoic acid–induced LIC loss. Ser873 modulates PML-RARA degradation, not gene activation In principle, retinoic acid resistance of these mutant leukemias could reflect altered retinoic acid–activated transcription and/or PML-RARA degradation. We first analyzed target genes activated in RARAtransduced Rara–/–; Rarb–/–; Rarg–/– mouse embryonic fibroblasts6 and found that the wild-type or phosphorylation mutant similarly activated expression of Rarb and Cyp26a1 in dose-response experiments (Fig. 6a). Rarb and Cyp26a1 (RARA and PML-RARA targets) and Tgm2 (a PML-RARA–specific target) were also normally induced by retinoic acid in PML-RARA or PML-RARAS873A APL cells (Supplementary Table 1 online). Thus, the absence of LIC loss does not reflect defective retinoic acid–dependent transcriptional activation by the mutant receptor, although we cannot rule out

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Figure 6 PKA phosphorylation site in RARA and PML-RARA is dispensable for retinoic acid (RA)-induced activation of target genes but desensitizes PMLRARA to RA-induced degradation. (a) Activation of Rarb and Cyp26a1 gene expression in RARA- or RARAS369A-transduced Rara–/–; Rarb–/–; Rarg–/– mouse embryonic fibroblasts after 6 h with the indicated RA concentrations. (b) PML-RARA degradation in mouse APLs triggered by 15 h of exposure to the indicated compounds ex vivo. Top, PML-RARAS873A is less sensitive to RA-induced degradation than wild-type PML-RARA (arrows; see Supplementary Fig. 5 for a lower RA concentration). Middle, synergistic effects of 10 7 M RA and a PDEI in normal APLs. Bottom, PDEI-arsenic combination results in additive PML-RARA or PML-RARAS873A degradation. (c) A new model of the mechanism of RA, arsenic and cAMP action in APL.

the possibility that the latter fails to regulate an unidentified subset of crucial PML-RARA target genes. In contrast, we consistently observed that PML-RARAS873A was less sensitive to retinoic acid–induced proteolysis (Fig. 6b). PDEIs induced baseline degradation (additive with that triggered by arsenic) for both PML-RARA and its S873A mutant (Fig. 6b). However, PDEIs strongly synergized with retinoic acid to enhance PML-RARA degradation in a Ser873-dependent manner, as previously suggested38 (Fig. 6b and Supplementary Fig. 5 online). Thus, PML-RARA phosphorylation at Ser873 regulates retinoic acid–triggered degradation of the fusion protein, most likely by enhancing proteasome docking onto ligand-activated RARA15,17. DISCUSSION It was previously thought that the therapeutic benefit of retinoic acid in APL relied on the induction of terminal cell differentiation through retinoic acid–triggered reactivation of crucial targets repressed by PML-RARA3. This model was called into question by the fact that arsenic, despite being an excellent APL therapy27,39, only modestly induces differentiation in vivo, does not do so ex vivo and does not activate most PML-RARA targets23,29. Here, we establish that APL drugs trigger two separable biological events: differentiation and LIC clearance, with only the latter being associated with remission of mouse APL (Fig. 6c). Ex vivo, retinoic acid triggers transient growth arrest of most LICs and terminal granulocytic differentiation of colonies that develop (Fig. 1a,b). Such heterogeneity in LIC response may reflect hierarchical differences in the originally transduced progenitors. A study of human stem cells transduced by PML-RARA found that retinoic acid actually increases LIC abundance, without any differentiation35. However, APL is not a stem cell disease40, and PML-RARA overexpression in stem cells may not accurately recapitulate oncogene-triggered changes of fate for other hematopoietic progenitors. Elucidation of the actual cellular basis for LIC clearance, whether it is loss of self-renewal or apoptosis, awaits purification of these cells. However, the fact that agents degrading PML-RARA (retinoic acid, arsenic and cAMP) all cooperate

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for LIC loss and APL eradication23,24,27,28,31 strongly suggests that even minute amounts of PML-RARA allow LIC self-renewal in vivo. How could PML-RARA degradation control LIC fate? Given the overlap between retinoic acid and arsenic targets in APL cells only39,41, a subset of target genes may be regulated through PML-RARA degradation. Alternatively, PML-RARA may behave as a nuclear anchor that sequesters master proteins, as shown for Daxx and PML18,42. Daxx sequestration is dependent on PML Lys160 SUMOylation18, and, notably, the latter is essential for PML-RARA–mediated transformation5. Although, as in Bcr-Abl–transformed cells, degradation of PML by arsenic may contribute to abrogation of LIC self-renewal43, arsenic is unlikely to act solely through the normal PML proteins, as it is ineffective in PLZF-RARA APLs24 (data not shown). In several settings (1.5 mg of retinoic acid, bortezomib and APLs expressing PML-RARAL902P or PML-RARAS873A), retinoic acid– dependent transcriptional activation and differentiation are maintained, whereas PML-RARA degradation and LIC clearance are impeded1,31 (Figs. 2–5). These observations could suggest that LIC proliferation is dependent on the absolute level of PML-RARA, whereas differentiation of the bulk of APL cells primarily relies on transcriptional activation of PML-RARA target genes by retinoic acid. The molecular basis for the different behavior of these two cell populations remains to be understood. Accordingly, higher retinoic acid concentrations are required for complete PML-RARA catabolism and LIC clearance than for in vivo differentiation and gene activation1,15,31 (Figs. 1 and 6). In this respect, liposomal retinoic acid, which maintains higher and more prolonged plasma levels than do 10-mg retinoic acid pellets (10 6 M versus 10 7 M, respectively), cures most APL mice and even some humans with APL as a single agent25,44. The suboptimal retinoic acid and arsenic doses used in our study may therefore have facilitated the synergies between retinoic acid and arsenic and between retinoic acid and cAMP. Thus, RARA agonists resistant to Cyp26a1 catabolism should be better drugs than retinoic acid and, conversely, a low-dose retinoic acid induction regimen, while initially retaining the ability to fully differentiate APL cells45, would be expected to yield poor long-term outcome. That

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ARTICLES retinoic acid fully differentiates PLZF-RARA APLs, but fails to clear LICs, was unexpected but consistent with some clinical observations3 (Supplementary Fig. 3b). Our experiments therefore do not support the proposal that retinoic acid resistance reflects impaired PLZFRARA target gene activation and differentiation, both dependent on a repression domain in PLZF3. Because retinoic acid degrades PLZFRARA24,46, retinoic acid resistance may reflect either a high basal level of PLZF-RARA protein expression or the influence of the reciprocal RARA-PLZF fusion, which may control progenitor fate through PLZF target genes36,47. Retinoic acid resistance of PML-RARAS873A APL implies that basal, cAMP-induced or retinoic acid–induced phosphorylation of Ser873 is crucial for the response of APL to retinoic acid (Fig. 5), although PML-RARA phosphorylation does not account for all of the effects of cAMP on APL cells. For example, cAMP-induced release of SMRT from RARA, which is independent of Ser369 (ref. 32), could contribute to PDEI-induced degradation of PML-RARAS873A, as well as to enhanced differentiation. In any case, we establish the in vivo relevance of retinoic acid–cAMP cross-talk1,30–32,38 and show that it targets APL LICs, at least in part, through PML-RARA phosphorylation and accelerated catabolism (as defective AF1 phosphorylation33 could also contribute to retinoic acid resistance). We thus identify the first biological system, to our knowledge, in which phosphorylation controls nuclear receptor signaling in vivo and identify cAMP signaling as another candidate for targeted APL therapy. Given that the RARs have been implicated in stem cell maintenance48,49 and that the retinoic acid–cAMP association blocks clonogenic growth of acute myeloid leukemias that do not express PML-RARA32, cAMP could enhance retinoic acid sensitivity in other malignancies, such as acute myeloid leukemia or neuroblastoma50. Our results provide insight into the very high rates of complete clinical remission induced by frontline combined retinoic acid–arsenic treatments in individuals with APL27,28 and suggest the further investigation of PDEIs, which were indeed beneficial for an index case31. At a fundamental level, our studies show that differentiation is not the primary basis for the therapeutic efficacy of retinoic acid or arsenic in clearing APL. In cancers where acquisition of stem cell features is conferred by oncogene expression, oncoprotein degradation could be a new general therapeutic strategy. METHODS Cell culture and viruses. We obtained retroviruses by transient transfection of Plat-E cells with various pMSCV vectors. We infected lineage-depleted mouse hematopoietic cells collected from 5-fluorouracil–treated mice with these retroviruses5. After spinoculation, we cultured transduced cells in methylcellulose medium (Stem Cell Technologies) supplemented with 100 ng ml–1 stem cell factor and 10 ng ml–1 each of interleukin (IL)-3, IL-6 and granulocytemacrophage colony-stimulating factor (AbCys), with G418 for selection. After 1 week, we recovered neomycin-selected cells from methylcellulose and either analyzed them or replated them at 10,000 cells per well in the presence or absence of 1 mM retinoic acid (Sigma-Aldrich). Immortalized mouse embryonic fibroblasts from which the three RARs were excised6,32 were transduced with RARA derivatives. We cultured cells in DMEM containing 10% FBS and treated them overnight with retinoic acid before gene expression analysis. We collected APL or bone marrow cells from femurs and tibiae of leukemic mice by flushing marrow with RPMI through a 26-gauge needle. We then cultured APL cells in complete RPMI medium supplemented with IL-3, IL-6 and stem cell factor, as well as retinoic acid, arsenic, MG132 and/or the phosphodiesterase inhibitor piclamilast8. In vivo mouse studies. PML-RARA and PML-RARAL902P APLs were previously described5,23,31. A similar transplantation model was established in nude mice, using APLs from PLZF-RARA–RARA-PLZF mice36. For secondary

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transplantations, we injected 3  106 bone marrow cells intravenously into three syngeneic recipient mice per primary recipient. We used two mice for each treatment modality and time point (days 3 and 6). We repeated mouse experiments two to six times. We administered retinoic acid by subcutaneous implantation of release pellets containing 10 or 1.5 mg of retinoic acid (Innovative Research of America). We administered cAMP by subcutaneous implantation of Alzet pumps31. We administered arsenic and piclamilast (200 mg d–1) by daily intraperitoneal injections18. We administered bortezomib (10 mg d–1) and H89 (60 mg d–1) using Alzet pumps. We measured plasma retinoic acid concentrations as previously described31. In vivo imaging was done in a Xenogen IVIS100 facility. Animal handling was done according to the guidelines of institutional animal care committees, using protocols approved by the Comite´ Re´gional d’Ethique Expe´rimentation Animale n14. We paraffinembedded and stained tissues as previously described31. We flushed, cytospinned and colored bone marrow cells with May Grunwald Giemsa (MGG) as previously described31. PML-RARAS873A transgenic mice were generated on an FVB background using the human MRP8 expression cassette5,8. Seven founders were obtained that had Mendelian transgene transmission and presented with PML-RARA expression similar to that of our previous PML-RARA transgenic mice5,8. Several lines developed mild splenomegaly, and one line developed APLs in the F0 founder and one F1. Resequencing of the transgene in these APLs did not reveal any additional genetic changes in PML-RARAS873A. Real-time PCR. We isolated total RNA from the bone marrow using an RNeasy kit (Qiagen). We synthesized first-strand cDNA with SuperScript II reverse transcriptase (Invitrogen). We did quantitative real-time PCR with a LightCycler system (Roche). We purchased probes and primers for TaqMan assays for Rarb, Cyp26a1 and Tgm2 from Applied Biosystems. We used the Ywhaz gene as an endogenous control to calibrate the amount of mRNA target in different samples. We quantified PML-RARA and ZBTB16-RARA DNAs by real-time PCR using a 7500 Fast Real-Time PCR system (Applied Biosystems) with either TaqMan (for PML-RARA) or SYBR Green (for ZBTB16-RARA). The gene encoding 18S RNA was used as an internal control. The primers used were: PML, 5¢-GCTGCCGTGCGCACCGAT-3¢ and 5¢-GGGGCTAGGCGGTCC ATC-3¢; ZBTB16, 5¢-TACAGGAAGCTGCACAGTGG-3¢ and 5¢-TTGTAGA TGCGGGGTAGAGG-3¢. Protein analyses. We resolved cell lysates by 7% SDS-PAGE and transferred them onto nitrocellulose membranes. We carried out detection with the chemiluminescent substrate SuperSignal West Pico (Pierce Biotechnology), using rabbit polyclonal antibodies to RARA (RP115) and with actin serving as a loading control. For the detection of phosphorylated PML-RARA, we first applied cell extracts to PhosphoProtein affinity purification columns (Qiagen). After being washed, column eluates containing protein peaks were concentrated, resolved by SDS-PAGE and analyzed by immunoblotting using antibodies to RARA phosphorylated at the PKA site33, or RP115 as a control for the total amount of PML-RARA (Fig. 5h). For flow cytometry analysis, we first blocked cellular Fc receptors with normal rat IgG. We then carried out immunophenotypic analysis using fluorochrome-conjugated monoclonal antibodies to CD11b and Gr-1 (clone RB6-8-C5, eBioscience). Staining was done at 4 1C for 20 min. We washed the cells twice and resuspended them in HBSS with 2% FBS and 0.5 mg ml–1 propidium iodide. We gated dead cells out by high propidium iodide staining and forward light scatter. Statistics. Two-sided t tests were used to validate the significance of the observed differences, which were considered statistically significant when P o 0.05. Note: Supplementary information is available on the Nature Medicine website. ACKNOWLEDGMENTS R.N. was supported by the Lady Tata Foundation (London). This work was supported by the ARECA, EPITRON (an integrated project funded by the European Union under the sixth framework program (LSHC-CT-2005-518417)) and INCa/Canceropole programs. We thank J. Godet and the Comite´ des Yvelines de la Ligue contre le Cancer for their continuous support of this project; M. Pla and the animal housing facility; C. Leboeuf, L. Legre`s and A. Janin for facilitation

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ARTICLES of the pathological analysis of the mice; M. Kawatika, M. Giovanini and F. Riaucoux for the derivation of MRP8-PML-RARAS873A transgenic mice; P. Chambon for the antibody to RARA; S. Kogan for the APLs and the MSCVluciferase vector; B. Arnulf for bortezomib; H. Tenor (Altana/Nycomed) for piclamilast; O. Hermine and F. Valensi for help with the t(11;17) human data; the Treilles and IPSEN foundations for providing the setting where this work was first presented and developed; and A. Saib, J.C. Gluckman and F. Sigaux for critical reading of the manuscript. COMPETING INTERESTS STATEMENT The authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturemedicine/.

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Published online at http://www.nature.com/naturemedicine/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/ 1. Kamashev, D.E., Vitoux, D. & de The´, H. PML/RARA-RXR oligomers mediate retinoidand rexinoid- /cAMP in APL cell differentiation. J. Exp. Med. 199, 1163–1174 (2004). 2. van Wageningen, S. et al. Gene transactivation without direct DNA binding defines a novel gain-of-function for PML-RAR{alpha}. Blood 111, 1634–1643 (2008). 3. Melnick, A. & Licht, J.D. Deconstructing a disease: RARalpha, its fusion partners, and their roles in the pathogenesis of acute promyelocytic leukemia. Blood 93, 3167–3215 (1999). 4. Di Croce, L. et al. Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor. Science 295, 1079–1082 (2002). 5. Zhu, J. et al. A sumoylation site in PML/RARA is essential for leukemic transformation. Cancer Cell 7, 143–153 (2005). 6. Zhou, J. et al. Dimerization-induced corepressor binding and relaxed DNA-binding specificity are critical for PML/RARA-induced immortalization. Proc. Natl. Acad. Sci. USA 103, 9238–9243 (2006). 7. Villa, R. et al. Role of the polycomb repressive complex 2 in acute promyelocytic leukemia. Cancer Cell 11, 513–525 (2007). 8. Zhu, J. et al. RXR is an essential component of the oncogenic PML/RARA complex in vivo. Cancer Cell 12, 23–35 (2007). 9. Zeisig, B.B. et al. Recruitment of RXR by homotetrameric RARalpha fusion proteins is essential for transformation. Cancer Cell 12, 36–51 (2007). 10. Quignon, F., Chen, Z. & de The´, H. Retinoic acid and arsenic: towards oncogene targeted treatments of acute promyelocytic leukaemia. Biochim. Biophys. Acta 1333, M53–M61 (1997). 11. Zhu, J., Lallemand-Breitenbach, V. & de The, H. Pathways of retinoic acid- or arsenic trioxide-induced PML/RARalpha catabolism, role of oncogene degradation in disease remission. Oncogene 20, 7257–7265 (2001). 12. Wang, Z.Y. & Chen, Z. Acute promyelocytic leukemia: from highly fatal to highly curable. Blood 111, 2505–2515 (2008). 13. Muindi, J. et al. Continuous treatment with all-trans retinoic acid causes a progressive reduction in plasma drug concentrations: implications for relapse and retinoid ‘‘resistance’’ in patients with acute promyelocytic leukemia. Blood 79, 299–303 (1992). 14. Nervi, C. et al. Caspases mediate retinoic acid-induced degradation of the acute promyelocytic leukemia PML/RARalpha fusion protein. Blood 92, 2244–2251 (1998). 15. Zhu, J. et al. Retinoic acid induces proteasome-dependent degradation of retinoic acid receptor alpha (RAR alpha) and oncogenic RAR alpha fusion proteins. Proc. Natl. Acad. Sci. USA 96, 14807–14812 (1999). 16. Lane, A.A. & Ley, T.J. Neutrophil elastase cleaves PML-RARalpha and is important for the development of acute promyelocytic leukemia in mice. Cell 115, 305–318 (2003). 17. vom Baur, E. et al. Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1. EMBO J. 15, 110–124 (1996). 18. Lallemand-Breitenbach, V. et al. Role of promyelocytic leukemia (PML) sumolation in nuclear body formation, 11S proteasome recruitment, and As(2)O(3)-induced PML or PML/retinoic acid receptor alpha degradation. J. Exp. Med. 193, 1361–1372 (2001). 19. Mann, K.K. et al. Arsenic trioxide inhibits nuclear receptor function via SEK1/JNKmediated RXRalpha phosphorylation. J. Clin. Invest. 115, 2924–2933 (2005). 20. Zhu, J. et al. Arsenic-induced PML targeting onto nuclear bodies: implications for the treatment of acute promyelocytic leukemia. Proc. Natl. Acad. Sci. USA 94, 3978–3983 (1997). 21. Lallemand-Breitenbach, V. et al. Arsenic degrades PML or PML-RARalpha through a SUMO-triggered RNF4/ubiquitin-mediated pathway. Nat. Cell Biol. 10, 547–555 (2008). 22. Hayakawa, F. & Privalsky, M.L. Phosphorylation of PML by mitogen-activated protein kinases plays a key role in arsenic trioxide-mediated apoptosis. Cancer Cell 5, 389–401 (2004). 23. Lallemand-Breitenbach, V. et al. Retinoic acid and arsenic synergize to eradicate leukemic cells in a mouse model of acute promyelocytic leukemia. J. Exp. Med. 189, 1043–1052 (1999).

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24. Rego, E.M., He, L.Z., Warrell, R.P., Jr., Wang, Z.G. & Pandolfi, P.P. Retinoic acid (RA) and As2O3 treatment in transgenic models of acute promyelocytic leukemia (APL) unravel the distinct nature of the leukemogenic process induced by the PML-RARalpha and PLZF-RARalpha oncoproteins. Proc. Natl. Acad. Sci. USA 97, 10173–10178 (2000). 25. Westervelt, P. et al. Adaptive immunity cooperates with liposomal all-trans-retinoic acid (ATRA) to facilitate long-term molecular remissions in mice with acute promyelocytic leukemia. Proc. Natl. Acad. Sci. USA 99, 9468–9473 (2002). 26. Lallemand-Breitenbach, V., Zhu, J., Kogan, S., Chen, Z. & de The, H. Opinion: how patients have benefited from mouse models of acute promyelocytic leukaemia. Nat. Rev. Cancer 5, 821–827 (2005). 27. Shen, Z.X. et al. All-trans retinoic acid/As2O3 combination yields a high quality remission and survival in newly diagnosed acute promyelocytic leukemia. Proc. Natl. Acad. Sci. USA 101, 5328–5335 (2004). 28. 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VOLUME 14

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NATURE MEDICINE

E r r at u m a n d C o r r i g e n d u m

Erratum: BMP type I receptor inhibition reduces heterotopic ossification Paul B Yu, Donna Y Deng, Carol S Lai, Charles C Hong, Gregory D Cuny, Mary L Bouxsein, Deborah W Hong, Patrick M McManus, Takenobu Katagiri, Chetana Sachidanandan, Nobuhiro Kamiya, Tomokazu Fukuda, Yuji Mishina, Randall T Peterson & Kenneth D Bloch Nat. Med. 14, 1363–1369 (2008); published online 30 November 2008; corrected after print 4 December 2008 In the version of this article initially published, the title included a misspelling—‘heterotropic’ should have been ‘heterotopic’. Additionally, the fourth and fifth sentences of the abstract were incorrectly worded and have been corrected to state more clearly the role of Ad.Cre. These changes do not affect the scientific content of the text. The errors have been corrected in the HTML and PDF versions of the article.

Corrigendum: Eradication of acute promyelocytic leukemia-initiating cells through PML-RARA degradation

© 2009 Nature America, Inc. All rights reserved.

Rihab Nasr, Marie-Claude Guillemin, Omar Ferhi, Hassan Soilihi, Laurent Peres, Caroline Berthier, Philippe Rousselot, Macarena RobledoSarmiento, Valérie Lallemand-Breitenbach, Bernard Gourmel, Dominique Vitoux, Pier Paolo Pandolfi, Cécile Rochette-Egly, Jun Zhu & Hugues de Thé Nat. Med. 14, 1333–1342 (2008); published online 23 November 2008; corrected after print 7 January 2009 In the version of this article initially published, the x-axis title of the right-hand graph in the ‘Day 2’ part of Figure 1f was incorrect. The title should read ‘Time to death in secondary transplants (d)’. The error has been corrected in the HTML and PDF versions of the article.

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