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mice from cerebral malaria–susceptible and resistant strains. This information is currently unknown. In a large study of infected children including many with cerebral malaria, however, malaria severity was found to be unrelated to the amount of carbon monoxide–bound hemoglobin in the blood10. Third, there is a polymorphism in the HO-1 gene promoter that is predicted to increase HO-1 gene tran-scription. Contrary to what would have been predicted from the Pamplona et al. study, this polymorphism is seen at a higher frequency in individuals with cerebral malaria, compared with individuals with uncomplicated malaria11. Fourth, high HO-1 activity in host tissue mac-rophages would be expected to increase iron

levels in the blood. In human cerebral malaria, however, high blood iron levels are associated with longer time to recover consciousness after antimalarial therapy12. These reports are not readily reconciled with the data of Pamplona et al.1. Although the protective effect of HO-1 in experimental cerebral malaria is an intrigu-ing finding with potential therapeutic implica-tions, the role of free heme in the human disease requires further study.

COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturemedicine/.

1. Pamplona, A. et al. Nat. Med. 13, 703–710 (2007).

2. Hunt, N.H. & Grau, G.E. Trends Immunol. 24, 491–499 (2003).

3. Belnoue, E. et al. J. Immunol. 169, 6369–6375 (2002).

4. Gramaglia, I. et al. Nat. Med. 12, 1417–1422 (2006).

5. Kossodo, S. et al. Immunology 91, 536–540 (1997).6. Stocker, R. & Perrella, M.A. Circulation 114, 2178–

2189 (2006).7. Abraham, N.G. et al. Proc. Natl. Acad. Sci. USA 92,

6798–6802 (1995).8. Clark, I.A., Awburn, M.M., Harper, C.G., Liomba, N.G.

& Molyneux, M.E. Malar. J. [online] 2, 41 (2003).9. Hvidberg, V. et al. Blood 106, 2572–2579 (2005).10. Cunnington, A.J., Kendrick, S.F., Wamola, B., Lowe,

B. & Newton, C.R. Am. J. Trop. Med. Hyg. 71, 43–47 (2004).

11. Takeda, M. et al. Jpn. J. Infect. Dis. 58, 268–271 (2005).

12. Gordeuk, V.R. et al. Blood 85, 3297–3301 (1995).13. Koury, M.J. & Ponka, P. Annu. Rev. Nutr. 24, 105–131

(2004).

Linking calcineurin activity to leukemogenesisMartin R Müller & Anjana Rao

Aberrant activation of the protein phosphatase calcineurin is found in various cancer types. Inhibition of calcineurin leads to a rapid clearance of leukemic cells and significantly improves animal survival in two mouse models of acute T lymphoblastic leukemia.

The authors are in the Department of Pathology,

Harvard Medical School and the CBR Institute for

Biomedical Research, 200 Longwood Avenue,

Boston, Massachusetts 02115, USA.

e-mail: arao@cbr.med.harvard.edu

Mitogenic signals regulate cell proliferation through phosphorylation and dephosphory-lation of intracellular proteins. Deregulation of these cascades can lead to uncontrolled cell division and cancer. Work over the last three decades has convincingly implicated the oncogenic variants of several protein kinases in cancer development and metas-tasis, but there is much less evidence that protein phosphatases have a role. In this issue, Medyouf et al. provide compelling evidence that sustained activation of the calcium-dependent phosphatase calcineu-rin contributes to the pathogenesis of acute T lymphoblastic leukemia (T-ALL)1.

The signaling pathway leading to calci-neurin activation begins with an influx of calcium ions in the cytoplasm (Fig. 1a). The calcium sensor protein calmodulin regu-lates calcineurin, which dephosphorylates and activates many intracellular proteins. Members of the nuclear factor of activated T cells (NFAT) family of transcription fac-tors are prominent targets of calcineurin,

and they have a key role in orchestrating diverse developmental programs, including those of the immune, central nervous and cardiovascular systems2.

There are strong hints that the calcineu-rin-NFAT signaling pathway is involved in the pathogenesis of other cancer types and in tumor metastasis (Fig. 1b)3–7. NFAT family members are overexpressed in a number of tumors, including lymphomas3 and breast carcinomas4. In pancreatic cancer, NFAT2 can upregulate c-myc expression and pro-c-myc expression and pro-c-mycmote cell proliferation5. What’s more, NFAT2 can induce the expression of different cell survival factors, rendering lymphoma cells more resistant to apoptosis6. Additionally, calcineurin and NFAT are crucially involved in angiogenesis7.

In careful and thorough experiments, Medyouf et al. investigated the role of cal-cineurin activity in the pathogenesis of T-ALL using two different mouse models1. In one model, they retrovirally transduced bone marrow cells ex vivo with a construct encoding the activating intracellular domain of Notch1 (ref. 8), and used the transduced cells to reconstitute the hematopoietic sys-tem of lethally irradiated mice. T-ALL devel-oped in all recipients within several weeks. In a second model, they used transgenic mice expressing a leukemia-associated TEL-JAK2

fusion protein that constitutively activates the JAK-STAT signaling pathway9. These mice developed fatal T-ALL at 4 to 22 weeks of age. Both mouse models are strongly rele vant to human T-ALL: more than half of all individuals with T-ALL have activat-ing mutations in the NOTCH1 gene10, and a majority of individuals with T-ALL have activating mutations of the JAK-STAT path-way11. In both mouse models, the authors found constitutive dephosphorylation of NFAT proteins in leukemic cells, indicating aberrant activation of the calcineurin-NFAT pathway in vivo1. The study also suggested that signals from the tumor microenviron-ment activated calcineurin: shortly after cells were cultured in vitro, NFAT reverted to its phosphorylated form, indicating that calci-neurin activity had greatly decreased.

To demonstrate that calcineurin activa-tion was essential for leukemia development in vivo, Medyouf et al.1 treated mice trans-planted with leukemic cells with the calci-neurin inhibitors cyclosporine or FK506 (ref. 2). This led to rapid disease remission: normal hematopoiesis was restored and treated mice showed significantly increased survival compared with untreated controls1. Conversely, when the authors introduced a constitutively active form of calcineurin into leukemic cells that were then injected

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into mice, the mice developed leukemias that were considerably more aggressive and showed accelerated disease progression. The authors also documented sustained calci-neurin activation in fresh biopsies from human B and T cell lymphomas, as well as in tumor cells from additional animal models for lymphoid malignancies: a xeno-graft model of a human B-cell lymphoma, and T-ALL induced by loss of function of the transcriptional regulator IKAROS. The study thus shows for the first time that aber-rant calcineurin activity can contribute to the leukemic phenotype in vivo, and iden-tifies the calcineurin-NFAT pathway as a potential molecular target in the treatment of T-ALL.

The calcineurin inhibitor cyclosporine has been used in the past to treat hemato-logic malignancies due to its known inhibi-tory effects on multidrug exporters, whose expression level correlates with prognosis. In one clinical trial, adding cyclosporine to a standard chemotherapy regimen signifi-cantly prolonged the duration of remission and survival in patients with high-risk acute myeloid leukemia12, although subsequent trials showed less convincing results.

Given these data, it is tempting to specu-late that calcineurin inhibitors such as cyclo-sporine and FK506 might be useful in the therapy of T-ALL and other malignancies. Inhibition of calcineurin, however, has con-siderable organ toxicity, and its long-term application can lead to the development of lymphoproliferative disorders (Fig. 1c). These considerations place substantial restrictions on the use of calcineurin inhi-bition in tumor therapy. Instead, it would be extremely valuable to identify the down-stream targets of calcineurin—NFAT and others—that are responsible for leukemia induction. Inhibiting these could be clini-cally more useful than inhibiting calcineu-rin itself.

In certain high-risk situations or upon relapse of T-ALL, an allogeneic bone-mar-row or stem-cell transplant is usually per-formed13. According to current thinking, transplantation therapies have two ben-eficial effects: the direct antitumor effect of the chemotherapy regimen and the so-called graft-versus-leukemia effect resulting from the immune response mounted by graft lymphocytes against leukemic cells14. The unwanted side effect is graft-versus-host disease, in which graft lymphocytes attack normal host tissues. To control the graft-versus-host response, combinations of dif-ferent immunosuppressive drugs including calcineurin inhibitors are routinely admin-

istered in clinical practice. If sustained cal-cineurin activity is crucial for survival of leukemic cells, as suggested by the studies of Medyouf et al.1, it may be that part of the therapeutic effect observed after stem-cell

Cyclosporine,FK506

Ca2+

Ca2+

Ca2+

IP3

Calcineurin

NFAT

Endoplasmicreticulum P

POthertargets

NFAT

Othertargets

PLC-γ

VEGF

VEGFRRTK

a

b

c

NFAT?

c-myc, CD40 ligand, BLyS

ProliferationResistance to apoptosis

AngiogenesisMigration of tumor cells

Proliferation

NFAT?

Cox-2, VEGF

Cyclosporine, FK506Beneficialeffects

Adverseeffects

Antitumor effects(various mechanisms)

Control of graft versus hostdisease

NeurotoxicityNephrotoxicity

Gastrointestinal toxicityHypertension

Lymphomas and other tumors

Suppression of graft versus leukemiaeffect K

im C

aesa

r

transplantation is due to exposure to calci-neurin inhibitors.

Because of the high toxicity of classical immunosuppressive drug regimens consist-ing of calcineurin inhibitors and the folic acid

Figure 1 Potential role of the calcineurin-NFAT pathway in the pathogenesis of cancer. (a) Receptor tyrosine kinases (RTKs), such as the receptor (VEGFR) for vascular endothelial growth factor (VEGF), activate phospholipase Cγ, which generates inositol-1,4,5-triphosphate (IP3). IP3 induces calcium release from intracellular calcium stores, including the endoplasmic reticulum. Once in the cytoplasm, calcium binds the ubiquitous calcium sensor calmodulin, which regulates a variety of intracellular proteins, among them the phosphatase calcineurin. Activated calcineurin dephosphorylates and regulates NFAT transcription factors and other intracellular targets. Cyclosporine and FK506 are two well characterized inhibitors of calcineurin activity. (b) Proposed mechanisms for involvement of NFAT transcription factors in tumor induction. Other transcriptional regulators that might cooperate with NFAT are indicated by a question mark. The NFAT target genes involved in each biological response are shown. BLyS, B-lymphocyte stimulator; Cox-2, cyclooxygenase-2. (c) Beneficial and adverse effects of treatment with calcineurin inhibitors.

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metabolism inhibitor methotrexate, there has been considerable interest in alternative drugs that operate by different mechanisms. In consequence, there is a wide array of data from subjects with T-ALL and other hemato-logic malignancies, enrolled in several large clinical trials, who have received bone mar-row or stem cell transplants with different degrees of exposure to calcineurin inhibi-tors13. In light of the Medyouf et al. study1, a careful retrospective analysis of these data is clearly warranted. Correlating graft mis-match, the degree of exposure to calcineurin

inhibitors and the overall response to treat-ment would allow immediate insights into the actual clinical potential of targeting the calcineurin-NFAT signaling pathway in the treatment of T-ALL.

COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompanying the full-text HTML version of the paper at http://www.nature.com/naturemedicine/.

1. Medyouf, H. et al. Nat. Med. 13, 736–741 (2007).2. Hogan, P. et al. Genes Dev. 17, 2205–2232 (2003).3. Marafioti, T. et al. Br. J. Haematol. 128, 333–342

(2005).4. Jauliac, S. et al. Nat. Cell Biol. 4, 540–544 (2002).5. Buchholz, M. et al. EMBO J. 25, 3714–3724

(2006).6. Fu, L. et al. Blood 107, 4540–4548 (2006).7. Hernandez, G. et al. J. Exp. Med. 193, 607–619

(2001).8. Aster, J.C. et al. Mol. Cell. Biol. 20, 7505–7515

(2000).9. Carron, C. et al. Blood 95, 3891–3899 (2000).10. Weng, A.P. et al. Science 306, 269–271 (2004).11. Benekli, M. et al. Blood 101, 2940–2954 (2003).12. List, A.F. et al. Blood 98, 3212–3220 (2001).13. Gökbuget, N. & Hoelzer, D. Hematology 2006, 133–

141 (2006).14. Passweg, J.R. et al. Bone Marrow Transplant. 21,

153–158 (1998).

Retinaldehyde: more than meets the eyeBéatrice Desvergne

Retinaldehyde, an intermediate metabolite between vitamin A and retinoic acid, is present at biologically active concentrations in fat tissue, where it antagonizes PPAR-γ activity, inhibiting adipogenesis and improving insulin γ activity, inhibiting adipogenesis and improving insulin γsensitivity.

Béatrice Desvergne is at the University of

Lausanne, Center of Integrative Genomics,

Lausanne, Switzerland.

e-mail: beatrice.desvergne@unil.ch

The identification of nuclear receptors defined a new paradigm: small lipophilic hormones specifically bind their cognate receptor and the complex directly regulates the expression of multiple targets. The genes controlled by these activated nuclear recep-tor transcription factors are diverse, and they act on energy balance, lipid metabo-lism and glucose homeostasis, to name a few. Deregulation of these genes is at the heart of the metabolic syndrome, which features a collection of risk factors, such as obesity, hypertension, dyslipidemia and insulin resis-tance; these conditions increase the chance of developing heart disease, stroke and diabetes. Ziouzenkova et al. now report that retinalde-hyde, a vitamin A metabolite that was previ-ously thought active only in the visual system, regulates adipogenesis and possibly improves insulin sensitivity1.

Vitamin A is a fat-soluble vitamin provided by the diet, for example by milk, liver and eggs, but also in form of some carotenoids such as found in carrots or red pepper. Vitamin A deficiency mainly occurs under malnutri-tion and is a major cause of blindness, weak immune system and developmental problems in developing countries.

All-trans-retinoic acid, the major vitamin A metabolite, regulates downstream genes by directly activating its cognate nuclear hor-mone receptors, the retinoic acid receptors (RARs), which associate with a partner, the retinoid X receptor (RXR)2. RXR also forms heterodimers with several metabolic regula-tors, including peroxisome proliferator-acti-vated receptors (PPARs). Still in question is whether the vitamin A metabolite 9-cis-reti-noic acid is formed in the cell, acting as a spe-cific ligand for RXR to regulate genes involved in metabolism. The major form of vitamin A, retinol, travels through the bloodstream to reach peripheral cells, where it is taken up by cells in a receptor-mediated process (Fig. 1). In the cytoplasm, retinol is metabolized into retinoic acid, which is considered the major biologically active product of retinol metabo-lism. Retinaldehyde is an intermediate in this reaction. It plays a key role in the retina for night vision but had no known functions out-side the eye.

Adipogenesis involves two main events: first, the recruitment and proliferation of adi-pocyte precursor cells, called preadipocytes, and second, the subsequent differentiation of preadipocytes into mature fat cells3. This dif-ferentiation is itself a complex process during which early and late stages are defined by the response to key hormonal, nutritional, para-crine and neuronal signals. White adipocytes are capable of storing large amount of triglyc-erides reflecting excess energy and of releasing

lipids in times of need. In contrast, brown adi-pose tissue dissipates energy by using stored lipids to generate heat.

Vitamin A status can influence the develop-ment and function of adipose tissues in whole animals. For example, low vitamin A status favors increased fat deposition4. Retinoic acid also influences adipocyte differentiation in cell culture, mainly inhibiting adipogenesis if given at an early stage of differentiation5. In general, it was thought that retinaldehyde primarily served as the required intermediary step for generating retinoic acid from vitamin A. The work by Ziouzenkova et al. shows that retinaldehyde is a naturally occurring signal-ing molecule in fat tissue, with its own distinct effects independent of retinoic acid formation and with important metabolic properties1.

In a cell-based culture system, Ziouzenkova et al. found that retinaldehyde as well as retin oic acid inhibits adipogenesis when administered at early stage of development1. While retinoic acid had no effect on adipocyte differentia-tion at later stages, retinaldehyde maintained its antiadipogenic activity and decreased the secretion of adipose-derived signaling mole-cules, such as adiponectin. These results show that retinaldehyde acts independently of reti-noic acid on adipocyte differentiation.

Does retinaldehyde regulate adipogenesis in response to obesity in the animal? The authors measured retinaldehyde in lean and obese mice and found that lean mice have more retinalde-hyde activity. This seems to be achieved by the

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