IDH1, Histone Methylation, and So Forth

Cancer Cell

Previews IDH1, Histone Methylation, and So Forth Virginie Penard-Lacronique1,2,3,4,* and Olivier A. Bernard1,2,3,4,* 1INSERM

U1170, 94805 Villejuif, France Labellise´e Ligue Nationale Contre le Cancer, Paris, France 3Universite ´ Paris Sud, Universite´ Paris-Saclay, 94720 Le Kremlin-Biceˆtre, France 4Gustave Roussy, Universite ´ Paris-Saclay, 94805 Villejuif, France *Correspondence: [email protected] (V.P.-L.), [email protected] (O.A.B.) http://dx.doi.org/10.1016/j.ccell.2016.07.008 2Equipe

IDH mutants cause aberrant DNA and histone methylation and contribute to hematological and neuronal malignancies. In this issue of Cancer Cell, Inoue et al. describe a potential specific effect of IDH1 mutations that reduces Atm expression via inhibition of H3K9 demethylases, which may represent a first step toward cellular transformation. The identification of somatically mutated genes is not always enough to understand the mechanisms underlying cellular transformation. Isocitrate dehydrogenase (IDH) enzymes belong to the tricarboxylic acid (TCA) cycle and synthesize alphaKetoglutarate (aKG), an essential cofactor of many biochemical reactions, including those catalyzed by enzymes of the dioxygenase family. IDH1 is mutated in various human tumors, including myeloid and neuronal malignancies. In glioma, IDH1 mutation is associated with chromosomes 1 and 19 abnormalities, for unclear reasons, but perhaps due to altered DNA maintenance. IDH1 mutant proteins (essentially R132 substitution mutants) acquire the ability to catalyze the reduction of aKG to 2-hydroxyglutarate (2HG). Mutant IDH2 proteins (essentially R140 or R172 substitution mutants) have similar properties. Among many consequences of 2HG accumulation, competition with aKG results in the inhibition of virtually all enzymes of the dioxygenase family (for review, see Losman and Kaelin, 2013). 2HG has pleiotropic consequences and also genome-wide effects on epigenetic marks (Figure 1, top panel). Indeed, detailed analyses of human samples established TET enzymes as important enzymes inhibited by this oncometabolite, resulting in loss of hydroxymethylated cytosine and hypermethylation phenotypes in both acute myeloid leukemia (AML) and glioma. Many other consequences of 2HG accumulation have been described, including inhibition of ALKBH, a dioxygenase involved in DNA maintenance (Wang et al., 2015), and hyper-succinylation, resulting in mitochondrial dysfunction (Li

et al., 2015). The inhibition of histone demethylases has been experimentally demonstrated, but the precise consequences have not been determined. Inoue et al. now identify the Atm gene to be downregulated due to histone methylation and closing of the chromatin structure as a consequence of expressing an IDH1 mutant (Inoue et al., 2016). Inoue et al. elegantly used CyTOF mass cytometry to identify differentially expressed proteins in the hematopoietic system of Idh1 (R132H) knockin (KI) mice, with respect to wild-type mice. They identified ATM as underexpressed in progenitor fractions, including those containing long-term hematopoietic stem cells (LT-HSCs). Further analyses revealed reduced numbers and dysfunction of LT-HSCs, together with a decrease of ATM expression, in aged (7–10 months) KI mice but not in young (3–4 months) animals. In addition, this low ATM expression is associated with an increase in DNA damage marks 53BP1 and gH2AX foci in mutant LT-HSCs, both spontaneously and after irradiation, which might trigger the development of myeloid malignancies in these mice. The authors identified the decreased ATM expression as being due to accumulation of methylated H3K9, a transcriptional repression epigenetic mark and closed chromatin structure at the Atm promoter. Low expression of ATM and of other DNAdamage repair-related genes are also specifically associated with IDH1 mutations in human AML. These features are not observed in TET-deficient conditions in mice or in humans. The results presented by Inoue et al. warrant further investigations. LT-HSCs

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have a specific way of dealing with DNA damage that differs from committed progenitors (Mohrin et al., 2010). Indeed, due to their essentially quiescent status, LT-HSCs use non-homologous endjoining mechanism, an error-prone DNA repair mechanism, low ATM levels would decrease the repair accuracy. In addition, DNA damage has been suggested to stimulate differentiation (Santos et al., 2014), potentially accounting for the loss of LT-HSC numbers and functions in Idh1 KI mice. Although these observations need to be replicated in humans, they suggest that transformation can be triggered by accumulating mutations in a specific juncture (LT-HSCs expressing low level of ATM) that is lost once the cells differentiate and proliferate. Oxidized cytosine forms, generated by TET enzymes, have been associated with DNA damage and repair. TET deficiencies are also described to lead to DNA repair defects, but at more mature hematopoietic states, indicating that IDH1 and TET mutations might both affect DNA repair in different ways. It will be important to reproduce these observations in a neuronal context and to find out whether the IDH1 mutant cells are sensitive to DNA repair inhibitors, a ‘‘synthetic lethality’’ therapeutic opportunity. In AML, this would allow targeting mutant LT-HSCs. Defects in DNA maintenance may also have consequences upon IDH1 inhibitor treatment currently being tested in clinical settings; because the chromatin is closed, ATM expression may not be normally resumed after treatment restoring demethylase activity. One wonders why, specifically, ATM is downregulated in IDH1 mutant LT-HSCs.

Cancer Cell

Previews 2HG 2HG

2HG

Mutant IDH1

2HG

2HG

2HG

Unbalance metabolism

2HG 2HG

Inhibits dioxygenases

DNA methylation

Histone methylation

DNA maintenance

Response to oxygen tension

Other

Dysregulation

Gene expression Up (e.g., PDGFRA) Down (e.g., Atm)

Chromatin structure

Accumulation of somatic mutations

Open (e.g., PDGFRA) Close (e.g., Atm)

LT-HSC

Selection

AML Figure 1. Hypothetic Scheme of Development of Acute Myeloblastic Leukemia in Idh1 Knockin Mice IDH1 mutations are present in long-term hematopoietic stem cells (or occur in these cells in humans) and synthetize 2HG. Excess 2HG dysregulates many cellular processes by its mere accumulation and by inhibition of dioxygenases. As far as epigenetics is concerned, it alters the control of methylation (loss of hydroxmethylcytosine; accumulation of cytosine and histone methylation), eventually affecting chromatin structure and gene expression. Note that links have been shown in different models and tissues. Methylated H3K9 on the Atm promoter is associated with closed chromatin structure, decreased expression of the gene, and abnormal DNA repair in LT-HSCs. Dysregulating all these processes facilitates selection of the best-fitted clones and triggers cellular transformation.

Obviously, inactivation of other tumor suppressors would also be effective in triggering cellular transformation. Besides DNA repair, ATM functionally interacts with many intracellular processes (Shiloh and Ziv, 2013), including chromatin structure, nucleic acid and cellular metabolism, redox and mitochondrial homeostasis, and cell-cycle and intracellular signaling,

and has an important role in neuronal biology. Whether these activities are affected by the drop in ATM expression, which is even more pronounced at the protein than at the RNA level, will have to be determined, as well as whether they play a role in cellular selection (see below). These observations also fit very well with the current data indicating that

IDH1 mutations affect chromatin structure. DNA methylation induced by mutant IDH1 may deregulate chromatin organization and favor open chromatin structure, leading to oncogenic overexpression (Flavahan et al., 2016); conversely, H3K9 methylation would close the chromatin structure, leading to repression of tumor suppressor (Inoue et al., 2016). How these modifications of chromatin structure would affect DNA repair is currently a matter of intensive investigation. Deregulation of chromatin organization, however, sets the conditions for cellular transformation, as schematized in Figure 1. KDM4 demethylases are responsible for H3K9 demethylation. They have been shown to target the H3K4me3-positive region, but it is not known how they are specifically recruited to the Atm promoter in normal condition. The recruitment of TET enzymes to DNA is likely a combination of interactions with each other, interactions with CpG, and recruitment by linage-specific transcription factors such as SPI1. As suggested from published analyses (including ours; Scourzic et al., 2016) of mutants affecting the control of DNA (de)methylation, it is possible that even a modest deregulation of the control or a decrease in (de)methylation activity allows an epigenetic drift and selection of the best-fitted epigenetic clones. This would allow progressive clonal dominance of the mutant clone(s) in the hematopoietic system, followed by AML development.

ACKNOWLEDGMENTS The authors want to thank Thomas Mercher and Filippo Rosseli for discussion and acknowledge the funding of INCa-DGOS-INSERM 6043 and INCa PLBIO.

REFERENCES Flavahan, W.A., Drier, Y., Liau, B.B., Gillespie, S.M., Venteicher, A.S., Stemmer-Rachamimov, A.O., Suva`, M.L., and Bernstein, B.E. (2016). Nature 529, 110–114. Inoue, S., Li, W.Y., Tseng, A., Beerman, I., Elia, A.J., Bendall, S.C., Lemonnier, F.o., Kron, K.J., Cescon, D.W., Hao, Z., et al. (2016). Cancer Cell 30, this issue, 337–348. Li, F., He, X., Ye, D., Lin, Y., Yu, H., Yao, C., Huang, L., Zhang, J., Wang, F., Xu, S., et al. (2015). Mol. Cell 60, 661–675. Losman, J.A., and Kaelin, W.G., Jr. (2013). Genes Dev. 27, 836–852.

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Previews Mohrin, M., Bourke, E., Alexander, D., Warr, M.R., Barry-Holson, K., Le Beau, M.M., Morrison, C.G., and Passegue´, E. (2010). Cell Stem Cell 7, 174–185. Santos, M.A., Faryabi, R.B., Ergen, A.V., Day, A.M., Malhowski, A., Canela, A., Onozawa, M., Lee, J.E.,

Callen, E., Gutierrez-Martinez, P., et al. (2014). Nature 514, 107–111.

Shiloh, Y., and Ziv, Y. (2013). Nat. Rev. Mol. Cell Biol. 14, 197–210.

Scourzic, L., Couronne´, L., Pedersen, M.T., Della Valle, V., Diop, M., Mylonas, E., Calvo, J., Mouly, E., Lopez, C.K., Martin, N., et al. (2016). Leukemia 30, 1388–1398.

Wang, P., Wu, J., Ma, S., Zhang, L., Yao, J., Hoadley, K.A., Wilkerson, M.D., Perou, C.M., Guan, K.L., Ye, D., and Xiong, Y. (2015). Cell Rep. 13, 2353– 2361.

Sticking It to Cancer with Molecular Glue for SHP2 Hao Ran,1 Ryouhei Tsutsumi,1 Toshiyuki Araki,1 and Benjamin G. Neel1,* 1Laura and Isaac Perlmutter Cancer Center, New York University Langone Medical Center, New York, NY 10016, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ccell.2016.07.010

Much effort has been expended to develop inhibitors against protein-tyrosine phosphatases (PTPs), nearly all of it unsuccessful. A recent report, describing a highly specific, orally bioavailable inhibitor of the PTP oncoprotein SHP2 with in vivo activity, suggests that allostery might provide a way forward for PTP inhibitor development.

Tyrosine phosphorylation, which is controlled by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs), regulates cell survival, proliferation, migration, and differentiation. Not surprisingly, aberrant tyrosine phosphorylation often causes disease: for example, activating mutations/amplifications of PTKs occur in multiple malignancies. PTK inhibitors (e.g., Imatinib [BCR-ABL, others], Erlotinib [EGFR], Lapatinib [HER2], and Sunitinib [VEGFR, others]) are mainstays of ‘‘precision oncology’’ and are among the bestselling drugs worldwide (Wu et al., 2015). Individual PTPs can have positive (signal-enhancing) or negative (signalinhibiting) roles, and several are implicated in cancer (Labbe´ et al., 2012). However, the best-validated PTP oncogene is PTPN11, encoding SHP2 (Neel et al., 2009). SHP2, one of two SH2 domain-containing PTPs (the other is SHP1, encoded by PTPN6), features two SH2 domains (N-SH2/C-SH2), a catalytic (PTP) domain, and a C-terminal tail with two tyrosine phosphorylation sites. SHP2 toggles between closed (inactive) and open (active) states (Barford and Neel, 1998). In the closed state, the N-SH2 is wedged into the PTP domain, blocking substrate access.

Phosphotyrosyl (pTyr) peptide binding to the N-SH2 disrupts auto-inhibition, activating the enzyme (Figure 1A). SHP2 binding sites are found in receptor tyrosine kinases (RTKs) and scaffolding adapters (GAB, IRS, FRS proteins), so this ‘‘molecular switch’’ ensures that SHP2 is activated only at proper cellular locales. In growth factor and cytokine signaling, SHP2 acts upstream of RAS to dephosphorylate one or more still hotly debated substrate(s) and enables full activation of the ERK/ MAP kinase pathway (Figure 1B). In addition, the C-terminal tyrosines of SHP2 undergo phosphorylation in response to most agonists. Tyrosylphosphorylated SHP2 recruits GRB2/ SOS, contributing to RAS activation (Neel et al., 2009). Furthermore, SHP2 binds immune-inhibitory receptors, including PD-1 (Pardoll, 2012), and, often in concert with SHP1, inhibits signaling from activating immunoreceptors (e.g., TCR). Malignancies hyper-activate SHP2 in two ways (Neel et al., 2009). In approximately one-third of juvenile myelomyelogenous leukemia (JMML) and less frequently (1%–10%) in acute leukemias, neuroblastomas, and carcinomas, somatic mutations—usually affecting the N-SH2 domain—disrupt auto-inhibition.

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Analogous, but less-activating, PTPN11 mutations can occur in the germline, and cause 50% of Noonan syndrome (NS) cases (Roberts et al., 2013). Constitutively active fusion-PTKs (e.g., BCR/ ABL) or amplification/overexpression of growth factors, RTKs, or scaffolding adapters also drive inappropriate SHP2 activation. Clearly, a specific inhibitor for SHP2 might have therapeutic utility, but developing PTP inhibitors presents unique challenges (He et al., 2013). Substantial binding energy is contributed by the substrate phosphate residue, which is then targeted by the highly reactive catalytic cysteine. Therefore, catalytic inhibitors must mimic phosphotyrosine and have low reactivity. Unfortunately, conventional screens for PTP inhibitors have typically recovered reactive, polar, low-affinity, and/or crossreactive compounds. A few SHP2 inhibitors have been reported to have substantial in vitro potency, PTP selectivity, and beneficial effects in animal models. However, collectively these molecules have poor bioavailability and/or troublesome pharmacophores for further drug development. In addition, none have been profiled extensively for off-target effects against other enzyme families. Furthermore, where