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(R)-2-Hydroxyglutarate Is Sufficient to Promote Leukemogenesis and Its Effects Are Reversible

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Science  29 Mar 2013:
Vol. 339, Issue 6127, pp. 1621-1625
DOI: 10.1126/science.1231677

Focusing on the Right Metabolite

A variety of human cancers, including acute leukemias and brain tumors, have mutations in the genes encoding isocitrate dehydrogenase 1 or 2 (IDH1, IDH2), which cause overproduction of a metabolite called 2-hydroxyglutarate (2HG). Losman et al. (p. 1621, published online 7 February) show that the R- but not the S-enantiomer of 2HG can transform cells and that R-2HG mediates transformation at least in part through effects on protein modifying EglN prolyl hydroxylases. Importantly, the transforming activity of R-2HG was reversible, suggesting that therapeutic strategies focusing on inhibition of R-2HG production or inhibition of EglN prolyl hydroxylases merit further investigation.

Abstract

Mutations in IDH1 and IDH2, the genes coding for isocitrate dehydrogenases 1 and 2, are common in several human cancers, including leukemias, and result in overproduction of the (R)-enantiomer of 2-hydroxyglutarate [(R)-2HG]. Elucidation of the role of IDH mutations and (R)-2HG in leukemogenesis has been hampered by a lack of appropriate cell-based models. Here, we show that a canonical IDH1 mutant, IDH1 R132H, promotes cytokine independence and blocks differentiation in hematopoietic cells. These effects can be recapitulated by (R)-2HG, but not (S)-2HG, despite the fact that (S)-2HG more potently inhibits enzymes, such as the 5′-methylcytosine hydroxylase TET2, that have previously been linked to the pathogenesis of IDH mutant tumors. We provide evidence that this paradox relates to the ability of (S)-2HG, but not (R)-2HG, to inhibit the EglN prolyl hydroxylases. Additionally, we show that transformation by (R)-2HG is reversible.

Acute myeloid leukemia (AML) is caused by somatic genetic mutations that deregulate hematopoietic cell proliferation and differentiation. In many cases of AML, the responsible genetic abnormalities are chromosomal translocations involving key transcription factors, epigenetic regulators, and mediators of cell signaling. However, no translocations are detected in 40% of cases of AML (1). In such cases of normal cytogenetic AML (NC-AML), the pathogenic driver mutations are largely unknown.

Recent genomic sequencing efforts, however, have identified a number of recurrent mutations in NC-AML that might contribute to leukemogenesis, including mutations in isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) (2). IDH1 and IDH2 are key metabolic enzymes that convert isocitrate to α-ketoglutarate [also called 2-oxoglutarate (2OG)], which is an essential cofactor for 2OG-dependent dioxygenases. These enzymes are linked to diverse cellular processes such as adaptation to hypoxia, histone demethylation, and DNA modification (3). Cancer-associated IDH mutants convert 2OG to the (R)-enantiomer of 2-hydroxyglutarate [(R)-2HG] (4, 5). Both the (R)- and (S)-enantiomers of 2HG are structurally similar to 2OG and can inhibit many 2OG-dependent enzymes in vitro and in vivo (68). In the case of the EglN (Egg-laying defective Nine) prolyl hydroxlases that down-regulate the HIF (hypoxia-inducible factor) transcription factor, however, (R)-2HG potentiates EglN activity, whereas (S)-2HG inhibits this same activity (8). Therefore, mutant IDH is widely believed to transform cells by modulating the behavior of specific 2OG-dependent enzymes. Nonetheless, IDH mutations induce a number of metabolic abnormalities in addition to (R)-2HG accumulation (9), and it has not yet been formally proven that (R)-2HG is sufficient to transform cells. Deciphering the pathogenic roles of mutant IDH and (R)-2HG in leukemia has been particularly problematic due to the lack of IDH mutant leukemic cell lines and the lack of robust cell-based assays with which to monitor hematopoietic transformation by mutant IDH and (R)-2HG.

To address this latter deficiency, we stably infected the TF-1 human erythroleukemia cell line with lentiviral vectors encoding hemagglutinin-tagged versions of wild-type (WT) IDH1, a tumor-derived mutant (IDH1 R132H, where R132H denotes Arg132→His132), or an IDH1 R132H variant in which three conserved aspartic acid residues within the IDH1 catalytic domain were replaced with asparagines (R132H/3DN; D, Asp; N, Asn) (Fig. 1A). This leukemic line is unusual insofar as it is cytokine-dependent [granulocyte-macrophage colony-stimulating factor (GM-CSF)] and retains the ability to differentiate in response to erythropoietin (EPO) (10).

Fig. 1

IDH1 R132H leukemic transformation assays. (A and B) Immunoblot (A) and liquid chromatography–mass spectrometry (LC-MS) (B) analysis of TF-1 cells infected with lentiviruses encoding green fluorescent protein (GFP) alone (empty) or GFP and the indicated IDH1 variants. Mean LC-MS values of triplicate experiments are shown. (C) Proliferation of TF-1 cells described in (A) and (B) under cytokine-poor conditions. Mean values of duplicate experiments ± SD (error bars) are shown. (D and E) Erythroid differentiation of parental TF-1 cells and derivatives described in (A) and (B), as determined by fluorescence activated cell sorting (FACS) for glycophorin A (D) or fetal hemoglobin (E) after 8 days of EPO treatment. Numbers in the FACS plots indicate the percentage of cells in the given quadrant. Log fluorescence intensity is plotted on the x axis; side scatter (SSC) is plotted on the y axis. (F) Differentiation of parental SCF ER-Hoxb8 cells and derivatives expressing the indicated IDH1 variants, as determined by dual staining for CD11b/Mac1 and Gr1 3 days after withdrawal of β-estradiol. FACS plots show representative results of three independent experiments.

As expected, 2HG levels were dramatically increased in cells producing IDH1 R132H, but not in cells producing WT IDH1 or the catalytically inactive R132H/3DN variant (Fig. 1B). In multiple independent experiments, TF-1 cells expressing IDH1 R132H became cytokine-independent 12 to 16 days (four passages) after infection (Fig. 1C). In contrast, parental TF-1 cells spontaneously became cytokine-independent, but with a much longer and more variable latency. Furthermore, after 10 passages in culture, IDH1 R132H-expressing TF-1 cells, in contrast to the control cells, no longer differentiated in response to EPO (Fig. 1, D and E, and fig. S1). Thus, expression of mutant IDH in TF-1 cells promotes two hallmarks of leukemic transformation: growth factor independence and impaired differentiation. Of note, IDH1 R132H impaired the fitness of TF-1 cells grown in the presence of GM-CSF, despite conferring a proliferative advantage to TF-1 cells in the absence of GM-CSF (Fig. 1C and fig. S2). Consistent with this observation, IDH1 R132H expression was attenuated in TF-1 cells after repeated passaging in the presence of GM-CSF (fig. S3).

To confirm that the inhibitory effect of mutant IDH on hematopoietic differentiation is not restricted to TF-1 cells, we stably infected stem cell factor (SCF) estrogen receptor (ER)–Hoxb8 cells, which are granulocyte-macrophage progenitor cells derived from primary murine bone marrow cells immortalized with a conditional oncogene (11), with viruses encoding WT IDH1 or IDH1 R132H (fig. S4). In the presence of estrogen, these cells express a functional ER-Hoxb8 fusion protein that promotes their survival and proliferation. Upon estrogen withdrawal, the cells differentiate and up-regulate expression of the monocytic markers CD11b/Mac1 and Gr1. Expression of IDH1 R132H in SCF ER-Hoxb8 cells, however, blunted their differentiation in response to estrogen withdrawal (Fig. 1F).

To investigate whether the effects of IDH1 R132H on TF-1 cells are mediated by (R)-2HG, we treated TF-1 cells with vehicle [dimethyl sulfoxide (DMSO)] or cell membrane–permeable [trifluoromethyl benzyl (TFMB)–esterified] versions of either (R)-2HG or (S)-2HG. Measurement of intracellular 2HG levels in treated TF-1 cells confirmed that the esterified 2HG enantiomers were equally cell-permeable (Fig. 2A). TF-1 cells passaged in the presence of TFMB–(R)-2HG became growth factor–independent and no longer differentiated in response to EPO (Fig. 2, B and E). Promotion of growth factor independence and loss of EPO responsiveness by TFMB–(R)-2HG were dose-dependent (Fig. 2B and fig. S5) and passage-dependent (Fig. 2C and fig. S5). In contrast, TFMB–(S)-2HG did not promote cytokine independence or block differentiation at any concentration or time point tested (Fig. 2, D and E). Likewise, treatment of SCF ER-Hoxb8 cells with TFMB–(R)-2HG, but not TFMB–(S)-2HG, impaired their differentiation in response to estrogen withdrawal, as determined by decreased expression of Gr1 and persistent expression of the stem cell markers CD34 and c-kit (Fig. 2, F and G, and fig. S6). Thus, (R)-2HG, but not (S)-2HG, promotes leukemic transformation. Of note, absolute quantification of (R)-2HG levels in TF-1 cells transformed by expression of IDH1 R132H or by treatment with TFMB–(R)-2HG confirmed that the intracellular 2HG concentrations achieved in both settings approximate those found in IDH mutant neoplasms in patients (low millimolar values) (fig. S7) (12, 13). The accumulation of 2HG to these high levels in cells treated with 250 to 500 μM TFMB–(R)-2HG suggests that the free 2HG liberated from the ester by intracellular esterases disappears more slowly than TFMB–(R)-2HG enters the cells.

Fig. 2

(R)-2HG is sufficient to promote leukemogenesis. (A) LC-MS analysis of TF-1 cells after treatment for 3 hours with DMSO (-) or 250 μM of the indicated esterified (TFMB) 2HG. Mean values of triplicate experiments are shown. (B to D) Proliferation of TF-1 cells under cytokine-poor conditions in the presence of the indicated amounts of TFMB–(R)-2HG (B and C) or TFMB–(S)-2HG (D). Cells in (B) and (D) were passaged 10 times before GM-CSF withdrawal. Cells treated with 250 μM TFMB–(R)-2HG are also included in (D) for comparison. Mean values of duplicate experiments ± SD (error bars) are shown. (E) Differentiation of TF-1 cells, as determined by glycophorin A FACS, after 8 days of EPO treatment following pretreatment for 10 passages with DMSO or 500 μM TFMB-2HG (R or S enantiomer). Representative results of three independent experiments are shown. (F and G) Differentiation of SCF ER-Hoxb8 cells, as determined by dual staining for CD11b/Mac1 and Gr1 (F) or staining for CD34 (G), 3 days after withdrawal of β-estradiol following pretreatment with DMSO or 500 μM TFMB-2HG (R or S) for 20 passages. Representative results of two independent experiments are shown.

To explore how (R)-2HG promotes leukemic transformation, we next infected parental TF-1 cells with a pool of lentiviral short hairpin RNA (shRNA) vectors targeting all the known 2OG-dependent dioxygenases (four to eight shRNAs per enzyme) (table S1), cultured the cells in the absence of GM-CSF, and monitored the abundance of the individual shRNA vectors by next-generation DNA sequencing. We reasoned that the enzymes targeted by shRNAs that were substantially enriched over time (because, for example, those shRNAs confer cytokine independence) would contribute to transformation by (R)-2HG if these enzymes were also inhibited by (R)-2HG at concentrations observed in leukemic cells. One of the top-scoring enzymes from this screen was TET2 (Ten Eleven Translocation 2), which is mutationally inactivated in a subset of AML and has recently been suggested to be a pathogenic target of mutant IDH (Fig. 3A) (14, 15). In contrast, the TET2 paralog TET1 did not score in our screen (Fig. 3A).

Fig. 3

Opposing roles of TET2 and EglN1 in TF-1 cell transformation. (A) Relative enrichment and depletion of shRNAs targeting TET2 or TET1 in TF-1 cells infected with a pool of ~800 lentiviral shRNA vectors targeting 2OG-dependent dioxygenases (four to eight shRNAs per gene) and then grown under cytokine-poor conditions for 10 days. Mean values of triplicate experiments are shown. (B and C) Proliferation under cytokine-poor conditions (B) and differentiation after 8 days of EPO (C) of TF-1 cells expressing a nontargeting shRNA (shControl) or shRNAs targeting TET2 or TET1, as indicated. (D) Proliferation under cytokine-poor conditions of TF-1 cells expressing a TET2 shRNA after pretreatment for 6 days with DMSO, 250 μM TFMB–(R)-2HG, or TFMB–(S)-2HG. (E) Immunoblot of TF-1 cells stably expressing the indicated IDH1 variants: untreated (-), treated with vehicle (V), or treated with 100 μM to 1 mM DMOG (as indicated by the thickness of the wedge) for 3 hours before cell lysis. (F and G) Proliferation under cytokine-poor conditions (F) and differentiation after 8 days of EPO (G) of TF-1 cells expressing either IDH1 R132H or an shRNA targeting TET2, and either a nontargeting shRNA (shControl) or shRNAs targeting EglN1. Growth curves show mean values of duplicate experiments ± SD (error bars). FACS plots show representative results of three independent experiments.

We confirmed that TET2 knockdown, but not TET1 knockdown, recapitulated the ability of IDH1 R132H and (R)-2HG to promote cytokine independence and block differentiation of TF-1 cells, suggesting that TET2 inhibition contributes to transformation by mutant IDH (Fig. 3, B and C, and fig. S8). However, this finding created a paradox because (S)-2HG is a more potent inhibitor of TET2 than is (R)-2HG (6, 8), and yet TFMB–(R)-2HG, but not TFMB–(S)-2HG, promoted TF-1 cell transformation (Fig. 2). Moreover, TFMB–(S)-2HG, but not TFMB–(R)-2HG, antagonized transformation induced by TET2 knockdown (Fig. 3D). We reasoned that this conundrum might relate to the fact that (R)-2HG and (S)-2HG have opposing effects on EglN activity, with (S)-2HG serving as an inhibitor and (R)-2HG, at least in some cellular contexts, serving as an agonist (8). Moreover, we found that TF-1 cells expressing IDH1 R132H were relatively resistant to up-regulation of HIF1α by the 2OG competitive antagonist dimethyloxalylglycine (DMOG), suggesting that (R)-2HG acts as an EglN agonist in these cells as well (Fig. 3E). We therefore considered the possibility that inhibition of EglN1 by (S)-2HG prevents (S)-2HG from transforming TF-1 cells. Indeed, we found that knockdown of EglN1 with multiple independent shRNAs abrogated the growth factor independence and restored the differentiation of TF-1 cells transformed by expression of IDH1 R132H or by knockdown of TET2 (Fig. 3, F and G, and figs. S9 and S10).

To determine whether the oncogenic effects of (R)-2HG are reversible, we first confirmed that a minimum of four passages in TFMB–(R)-2HG were required to render TF-1 cells cytokine-independent, provided that TFMB–(R)-2HG exposure was maintained during the cytokine withdrawal period (Fig. 4A). Next, late-passage TF-1 cells treated with TFMB–(R)-2HG were passaged out of TFMB–(R)-2HG for variable periods before removal of GM-CSF (Fig. 4B). Interestingly, the amount of time required for reversion of growth factor independence was influenced by the intensity (duration times dose) of TFMB–(R)-2HG exposure (Fig. 4, A and B). In contrast, removal of TFMB–(R)-2HG rapidly restored the ability of TF-1 cells to differentiate in response to EPO, even after long-term passage in the presence of TFMB–(R)-2HG (Fig. 4C). Of note, late-passage TF-1 cells that expressed lower levels of mutant IDH1 (fig. S3B) and produced lower levels of (R)-2HG (fig. S7) spontaneously regained the ability to differentiate in response to EPO but retained their abiity to proliferate independent of growth factors (fig. S11).

Fig. 4

Transformation by (R)-2HG is reversible. (A) Proliferation of TF-1 cells under cytokine-poor conditions after being passaged four times in the presence of DMSO or 250 μM TFMB–(R)-2HG. At the time of GM-CSF withdrawal, TFMB–(R)-2HG was either maintained [Continuous (R)] or removed [Out of (R) on day 0]. (B) Proliferation of TF-1 cells under cytokine-poor conditions after being passaged 20 times in the presence of DMSO or 250 μM TFMB–(R)-2HG and then undergoing wash-out of TFMB–(R)-2HG for the periods indicated. (C) Differentiation of TF-1 cells after being passaged 20 times in the presence of 500 μM TFMB–(R)-2HG or DMSO. At the time of EPO administration, TFMB–(R)-2HG was either maintained [In (R)] or removed [Out of (R) on day 0 of EPO]. (D to F) Differentiation of TF-1 cells (D and E) and SCF ER-Hoxb8 cells (F) transformed with IDH1 R132H (D and F) or TFMB–(R)-2HG (E) and then passaged five times in the presence of DMSO or a small-molecule inhibitor of IDH1 R132H (IDHi; 1 μM). (G and H) Proliferation under cytokine-poor conditions of early passage (p15) TF-1 cells transformed with IDH1 R132H (G) or TFMB–(R)-2HG (H) and then passaged five times in the presence of DMSO or a small-molecule inhibitor of IDH1 R132H (IDHi; 1 μM) before growth-factor withdrawal. Growth curves show mean values of duplicate experiments ± SD (error bars). FACS plots show representative results of three independent experiments.

In a complementary experiment, we found that a tool compound (AGI-5198) that specifically blocks 2HG production by IDH1 R132H (fig. S12) (16) restored the ability of IDH1 R132H-expressing TF-1 cells (Fig. 4D) and SCF ER-Hoxb8 cells (Fig. 4F) to differentiate in vitro and abrogated the growth factor independence of IDH1 R132H-expressing TF-1 cells (Fig. 4G and fig. S13). These effects were on-target because they were not observed in cells transformed with TFMB–(R)-2HG, rather than IDH1 R132H (Fig. 4, E and H).

Tumor-associated IDH mutations cause many metabolic abnormalities in addition to the accumulation of (R)-2HG (9). However, our studies indicate that (R)-2HG is sufficient to confer the two hallmarks of leukemia: enhanced proliferation and impaired differentiation. (R)-2HG can inhibit many 2OG-dependent enzymes, including various histone demethylases and the 5′-methylcytosine hydroxylase TET2 (68). We found that down-regulation of TET2, like high levels of (R)-2HG, promotes cytokine independence and blocks differentiation of TF-1 cells. This finding is consistent with the hypothesis that leukemic transformation by mutant IDH is linked to inhibition of TET2 by (R)-2HG, thus accounting for the observation that IDH and TET2 mutations both occur in acute leukemia but are mutually exclusive (15).

At intermediate (R)-2HG concentrations, TF-1 cells remain cytokine-independent but can once again differentiate. Together with the behavior of TF-1 cells treated with different doses of (R)-2HG and their responses to (R)-2HG withdrawal, this observation strongly indicates that higher levels of (R)-2HG are needed to block differentiation than to confer cytokine independence. The differentiation phenotype may require more profound TET2 inhibition than the growth phenotype. Interestingly, targeted expression of IDH1 R132H in the murine hematopoietic stem cell (HSC) compartment causes gradual expansion of long-term HSCs without any evidence of impaired myeloid differentiation (17). In contrast, mice lacking TET2 exhibit both expansion of their HSC compartment and aberrant myeloid differentiation (18).

Although TET2 appears to be an important target of (R)-2HG, we have not formally proven that TET2 inhibition is necessary, in addition to being sufficient, for transformation by mutant IDH. Inhibition of other 2OG-dependent enzymes by (R)-2HG may enhance or suppress leukemic transformation by mutant IDH. With respect to the latter, the observation that the levels of mutant IDH and (R)-2HG fell over time in TF-1 cells grown under growth factor–rich conditions suggests that high levels of (R)-2HG may be deleterious in certain cellular contexts, perhaps due to inhibition of other 2OG-dependent enzymes that promote cell growth and survival. These considerations might be relevant to the clinical differences between IDH mutant and TET2 mutant myeloid disorders (19).

Leukemic transformation by 2HG is specific to the (R)-enantiomer produced by mutant IDH and not the (S)-enantiomer, even though (S)-2HG is a more potent inhibitor of all of the 2OG-dependent enzymes tested to date, including TET2 (68). This conundrum appears to be explained by the differential effects of the two enantiomers on EglN1, with (R)-2HG serving as an agonist and (S)-2HG as an antagonist (8), as well as by our observation that the loss of EglN1 activity blocks transformation by mutant IDH or the loss of TET2. Several studies have indicated that the canonical EglN1 target, HIF, can inhibit HSC and leukemic cell proliferation (2026). Interestingly, people living at high altitude—and, hence, presumed to have lower basal EglN1 activity due to chronic hypoxia—appear to have a lower risk of leukemia (27, 28).

The hypothesis that mutant IDH transforms cells by affecting epigenetic marks has raised fears that these changes will not be reversible on a therapeutically relevant time scale. Our studies suggest that the effects of (R)-2HG are reversible, offering hope that compounds that block (R)-2HG production by IDH mutants will benefit patients. Moreover, the relatively rapid reversion of the (R)-2HG transformation phenotypes suggests that the relevant epigenetic marks are more dynamic than previously suspected or that these phenotypes reflect noncanonical functions of enzymes such as TET2. In addition, our findings suggest that pharmacological inhibition of EglN1 might also be useful for the treatment of leukemias that harbor IDH or TET2 mutations.

Supplementary Materials

www.sciencemag.org/cgi/content/full/science.1231677/DC1

Materials and Methods

Figs. S1 to S13

Table S1

References (2931)

References and Notes

  1. Acknowledgments: We thank K. Yen, M. Su, and Agios Pharmaceuticals for the IDH1 inhibitor and for sharing unpublished data; R. Bejar and A. Mullally for their valuable input; A. Lane and D. Sykes for assistance with the SCF ER-Hoxb8 cells; M. Paktinat and R. Mathieu for assistance with flow cytometry; and members of the Kaelin and Ebert lab for technical help and valuable discussion. This work was supported by the NIH (W.G.K. and J.-A.L.) and HHMI (W.G.K.). W.G.K. is a HHMI Investigator and a paid Scientific Advisor for Agios, a biotechnology company developing IDH inhibitors. W.G.K. is also a paid consultant for and owns equity in Fibrogen, Inc., a biotechnology company developing EglN inhibitors.
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