Recent studies have identified mutations in PHF8, an X-linked gene encoding a JmjC domain-containing protein, as a causal factor for X-linked mental retardation (XLMR) and cleft lip/cleft palate. However, the underlying mechanism is unknown. Here we show that PHF8 is a histone demethylase and coactivator for retinoic acid receptor (RAR). Although activities for both H3K4me3/2/1 and H3K9me2/1 demethylation were detected in cellularbased assays, recombinant PHF8 exhibited only H3K9me2/1 demethylase activity in vitro, suggesting that PHF8 is an H3K9me2/1 demethylase whose specificity may be modulated in vivo. Importantly, a mutant PHF8 (phenylalanine at position 279 to serine) identified in the XLMR patients is defective in enzymatic activity, indicating that the loss of histone demethylase activity is causally linked with the onset of disease. In addition, we show that PHF8 binds specifically to H3K4me3/2 peptides via an N-terminal PHD finger domain. Consistent with a role for PHF8 in neuronal differentiation, knockdown of PHF8 in mouse embryonic carcinoma P19 cells impairs RA-induced neuronal differentiation, whereas overexpression of the wild-type but not the F279S mutant PHF8 drives P19 cells toward neuronal differentiation. Furthermore, we show that PHF8 interacts with RARα and functions as a coactivator for RARα. Taken together, our results suggest that histone methylation modulated by PHF8 plays a critical role in neuronal differentiation.

While all-trans retinoic acid (ATRA) treatment in acute promyelocytic leukemia (APL) has been the paradigm of targeted therapy for oncogenic transcription factors, the underlying mechanisms remain largely unknown, and a significant number of patients still relapse and become ATRA resistant. We identified the histone demethylase PHF8 as a coactivator that is specifically recruited by RARα fusions to activate expression of their downstream targets upon ATRA treatment. Forced expression of PHF8 resensitizes ATRA-resistant APL cells, whereas its downregulation confers resistance. ATRA sensitivity depends on the enzymatic activity and phosphorylation status of PHF8, which can be pharmacologically manipulated to resurrect ATRA sensitivity to resistant cells. These findings provide important molecular insights into ATRA response and a promising avenue for overcoming ATRA resistance.

Mutations of isocitrate dehydrogenase 1 (IDH1) are frequently found in certain cancers such as glioma. Different from the wild-type (WT) IDH1, the mutant enzymes catalyze the reduction of α-ketoglutaric acid to d-2-hydroxyglutaric acid (D2HG), leading to cancer initiation. Several 1-hydroxypyridin-2-one compounds were identified to be inhibitors of IDH1(R132H). A total of 61 derivatives were synthesized, and their structure–activity relationships were investigated. Potent IDH1(R132H) inhibitors were identified with Ki values as low as 140 nM, while they possess weak or no activity against WT IDH1. Activities of selected compounds against IDH1(R132C) were found to be correlated with their inhibitory activities against IDH1(R132H), as well as cellular production of D2HG, with R2 of 0.83 and 0.73, respectively. Several inhibitors were found to be permeable through the blood–brain barrier in a cell-based model assay and exhibit potent and selective activity (EC50 = 0.26–1.8 μM) against glioma cells with the IDH1 R132H mutation.

Esophageal cancer is the sixth leading cause of cancer-related deaths worldwide. It has been reported that histone demethylases are involved in the carcinogenesis of certain types of tumors. Here, we studied the role of one of the histone lysine demethylases, plant homeodomain finger protein 8 (PHF8), in the carcinogenesis of esophageal squamous cell carcinoma (ESCC). Using short hairpin RNA via lentiviral infection, we established stable ESCC cell lines with constitutive downregulation of PHF8 expression. Knockdown of PHF8 in ESCC cells resulted in inhibition of cell proliferation and an increase of apoptosis. Moreover, there were reductions of both anchorage-dependent and -independent colony formation. In vitro migration and invasion assays showed that knockdown of PHF8 led to a reduction in the number of migratory and invasive cells. Furthermore, downregulation of PHF8 attenuated the tumorigenicity of ESCC cells in vivo. Taken together, our study revealed the oncogenic features of PHF8 in ESCC, suggesting that PHF8 may be a potential diagnostic marker and therapeutic target for ESCC.

Although both heterodimeric subunits of core binding factors (AML1/RUNX1 and CBF␤) essential for normal hematopoiesis are frequently mutated to form different chimeric fusion proteins in acute leukemia, the underlying molecular mechanisms and structural domains required for cellular transformation remain largely unknown. Despite the critical role of CBF␤ for wild-type AML1 function and its direct involvement in chromosomal translocation, we demonstrate that both the expression and interaction with CBF␤ are superfluous for AML1-ETO (AE)-mediated transformation of primary hematopoietic cells. Similarly, the hetero-oligomeric interaction with transcriptional repressor ETO family proteins and the highly conserved NHR1 domain in AE fusion are also dispensable for transforming activity. In contrast, AE-mediated transformation is critically dependent on the DNA binding and homo-oligomeric properties of the fusion. Abolishment of homooligomerization by a small-molecule inhibitor could specifically suppress AML1 fusion-mediated transformation of primary hematopoietic cells. Together, these results not only identify the essential molecular components but also potential avenues for therapeutic targeting of AE-mediated leukemogenesis.A ML1/RUNX1 and CBF␤ are 2 critical transcription factors essential for generation of hematopoietic stem cells (HSCs) (1, 2). Mice deficient in AML1 or CBF␤ have almost identical phenotypes; they completely lack definitive hematopoiesis and die at approximately embryonic day 12.5. In acute myeloid leukemia in which leukemic stem cells have been functionally identified, AML1 and CBF␤ represent the most commonly mutated targets (1, 2). t(8;21) resulting in AML1-ETO (AE) fusion can be found in up to 40% of AML-M2; and inv(16) leading to CBF␤-SHMMC fusion constitutes approximately 30% of AML-M4. AML1 is also fused to TEL as a result of t(12;21) in approximately 25% of childhood leukemia, the most common form of childhood cancers. Although animal modeling clearly indicates that AML1 fusions per se are not sufficient for induction of full-blown leukemia, they function to enhance self-renewal and expand targeted HSCs and early progenitors for cooperative secondary mutations to take place (3-5).While enhanced self-renewal has emerged as a critical feature associated with various oncogenic transcription factors involved in acute leukemia (6-8), much less is known about the underlying molecular mechanisms. It is clear that most, if not all, of the transcription factors do not work as monomers but need to complex with various proteins for full activity and specificity, although the molecular composition of the associated transcriptional complexes required for self-renewal remains largely unknown. At the molecular level, AE encodes the DNA-binding runt homology domain (RHD) fused in-frame with almost the entire transcriptional repressor protein ETO containing 4 different Nervy homology regions (NHRs) (Fig. 1A), suggesting a gain of transcriptional repressor function by the oncogenic fusion (9). A well-reco...

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Acute promyelocytic leukemia (APL) cells contain one of five chimeric retinoic acid ␣-receptor (RAR␣) genes (X-RAR␣) created by chromosomal translocations or deletion; each generates a fusion protein thought to transcriptionally repress RAR␣ target genes and block myeloid differentiation by an incompletely understood mechanism. To gain spatiotemporal insight into these oncogenic processes, we employed fluorescence microscopy and fluorescence recovery after photobleaching (FRAP). Fluorescence microscopy demonstrated that the intracellular localization of each of the X-RAR␣ proteins was distinct from that of RAR␣ and established which portion(s) of each X-RAR␣ protein-X, RAR, or both-contributed to its altered localization. Using FRAP, we demonstrated that the intranuclear mobility of each X-RAR␣ was reduced compared to that of RAR␣. In addition, the mobility of each X-RAR␣ was reduced further by ligand addition, in contrast to RAR␣, which showed no change in mobility when ligand was added. Both the reduced baseline mobility of X-RAR␣ and the ligand-induced slowing of X-RAR␣ could be attributed to the protein interaction domain contained within X. RXR␣ aberrantly colocalized within each X-RAR␣; colocalization of RXR␣ with promyelocytic leukemia (PML)-RAR␣ resulted in reduced mobility of RXR␣. Thus, X-RAR␣ may interfere with RAR␣ through its aberrant nuclear dynamics, resulting in spatial and temporal sequestration of RXR␣ and perhaps other nuclear receptor coregulators critical for myeloid differentiation.Acute promyelocytic leukemia (APL) accounts for 10% of cases of acute myeloid leukemia. In over 95% of APL cases, there is a chromosomal translocation within leukemic cells involving the promyelocytic leukemia (PML) gene at 15q22 and the retinoic acid ␣-receptor (RAR␣) gene at 17q21, resulting in the PML-RAR␣ gene and protein product (7, 35). Four variant translocations or deletions occur in the remaining cases of APL, each involving the RAR␣ locus on chromosome 17 including t(11;17)(q23;q21), t(11;17)(q13; q21), t(5;17)(q35; q21), and del(17) (21, 35). These chromosomal abnormalities produce fusions of RAR␣ with PLZF, NuMA, NPM, and STAT5b respectively (1,4,9,10,21). It is important to identify APL patients with these alternative chromosomal abnormalities since they may not respond as well as PML-RAR␣-positive APL patients to treatment regiments with all-trans-retinoic acid (ATRA) and other conventional chemotherapies for APL (17).Nuclear hormone receptors (NRs) comprise a large family of ligand-dependent transcription factors that bind to hormone response elements of target genes and regulate their transcription (3). Type I nuclear hormone receptors such as estrogen receptor (ER), androgen receptor (AR), and glucocorticoid receptor (GR) bind to their response elements as homodimers, whereas type II nuclear hormone receptors, including RAR, thyroid hormone receptor (TR) and vitamin D receptor (VDR), bind to their response elements as heterodimers with retinoid X receptors (RXRs). Both type I and II receptors can...

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