Shuo Dong

The Histone Demethylase PHF8 Governs Retinoic Acid Response in Acute Promyelocytic Leukemia

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.

Stat3 Isoforms, α and β, Demonstrate Distinct Intracellular Dynamics with Prolonged Nuclear Retention of Stat3β Mapping to Its Unique C-terminal End

Two isoforms of Stat3 (signal transducer and activator of transcription 3) are expressed in cells, α (p92) and β (p83), both derived from a single gene by alternative mRNA splicing. The 55-residue C-terminal transactivation domain of Stat3α is deleted in Stat3β and replaced by seven unique C-terminal residues (CT7) whose function remains uncertain. We subcloned the open reading frames of Stat3α and Stat3β into the C terminus of green fluorescent protein (GFP). Fluorescent microscopic analysis of HEK293T cells transiently transfected with GFP-Stat3α or GFP-Stat3β revealed similar kinetics and cytokine concentration dependence of nuclear accumulation; these findings were confirmed by high throughput microscope analysis of murine embryonic fibroblasts that lacked endogenous Stat3 but stably expressed either GFP-Stat3α or GFP-Stat3β. However, although time to half-maximal cytoplasmic reaccumulation after cytokine withdrawal was 15 min for GFP-Stat3α, it was >180 min for GFP-Stat3β. Furthermore, although the intranuclear mobility of GFP-Stat3α was rapid and increased with cytokine stimulation, the intranuclear mobility of GFP-Stat3β in unstimulated cells was slower than that of GFP-Stat3α in unstimulated cells and was slowed further following cytokine stimulation. Deletion of the unique CT7 domain from Stat3β eliminated prolonged nuclear retention but did not alter its intranuclear mobility. Thus, Stat3α and Stat3β have distinct intracellular dynamics, with Stat3β exhibiting prolonged nuclear retention and reduced intranuclear mobility especially following ligand stimulation. Prolonged nuclear retention, but not reduced intranuclear mobility, mapped to the CT7 domain of Stat3β.

Transforming activity of AML1-ETO is independent of CBFβ and ETO interaction but requires formation of homo-oligomeric complexes

Although both heterodimeric subunits of core binding factors (AML1/RUNX1 and CBFβ) 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 CBFβ for wild-type AML1 function and its direct involvement in chromosomal translocation, we demonstrate that both the expression and interaction with CBFβ 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 homo-oligomerization 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.

Reduced Intranuclear Mobility of APL Fusion Proteins Accompanies Their Mislocalization and Results in Sequestration and Decreased Mobility of Retinoid X Receptor α

ABSTRACT Acute promyelocytic leukemia (APL) cells contain one of five chimeric retinoic acid α-receptor (RARα) genes (X-RARα) created by chromosomal translocations or deletion; each generates a fusion protein thought to transcriptionally repress RARα 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-RARα proteins was distinct from that of RARα and established which portion(s) of each X-RARα protein—X, RAR, or both—contributed to its altered localization. Using FRAP, we demonstrated that the intranuclear mobility of each X-RARα was reduced compared to that of RARα. In addition, the mobility of each X-RARα was reduced further by ligand addition, in contrast to RARα, which showed no change in mobility when ligand was added. Both the reduced baseline mobility of X-RARα and the ligand-induced slowing of X-RARα could be attributed to the protein interaction domain contained within X. RXRα aberrantly colocalized within each X-RARα; colocalization of RXRα with promyelocytic leukemia (PML)-RARα resulted in reduced mobility of RXRα. Thus, X-RARα may interfere with RARα through its aberrant nuclear dynamics, resulting in spatial and temporal sequestration of RXRα and perhaps other nuclear receptor coregulators critical for myeloid differentiation.

Physical and functional interaction of Runt-related protein 1 with hypoxia-inducible factor-1α

Angiogenesis and hematopoiesis are closely linked and interactive with each other, but few studies were given to identify possible links between angiogenesis-promoting proteins and hematopoiesis-related transcription factors. Here we investigated the potential relationship of oxygen-sensitive α-subunit of angiogenesis-related hypoxia-inducible factor-1α (HIF-1α) with Runt-related protein 1 (Runx1, also known as acute myeloid leukemia-1, AML-1), an important hematopoietic transcription factor. The results demonstrated that Runx1 and HIF-1α proteins directly interacted with each other to a degree, in which Runt homology domain of Runx1 was mainly involved. Leukemia-related abnormal Runx1 fusion protein AML1-ETO, which fuses the N-terminal 177 amino acid residues of the Runx1 protein in frame to ETO (eight-twenty-one) protein, also interacted with HIF-1α protein with greater ability than Runx1 itself. More intriguingly, Runx1 overexpression inhibited DNA-binding and transcriptional activity of HIF-1 protein with reduced expression of HIF-1-targeted genes such as vascular endothelial growth factor, while silence of Runx1 expression by specific small interfering RNA significantly increased transcriptional activity of HIF-1 protein, suggesting that Runx1 inhibited transcription-dependent function of HIF-1. Vice versa, HIF-1α increased DNA-binding ability and transcriptional activity of Runx1 protein. All these data would shed new insight to understanding Runx1 and HIF-1α-related hematopoietic cell differentiation and angiogenesis.

The impact of differential binding of wild-type RARα, PML-, PLZF- and NPM-RARα fusion proteins towards transcriptional co-activator, RIP-140, on retinoic acid responses in acute promyelocytic leukemia

Retinoic acid receptor (RA) heterodimer (RAR/RXR) activities have been shown to be repressed by transcriptional co-repressor, SMRT/N-CoR, in the absence of the ligand while upon all-trans retionic acid (ATRA) treatment, SMRT/N-CoR is dissociated from RARα leading to gene expression by the recruitment of transcriptional co-activators to the transcriptional complex. The difference in response to ATRA therapy between acute promyelocytic leukemia (APL) patients with PML-RARα fusion and PLZF-RARα fusion has recently been found to be partially due to the strong association of the transcriptional co-repressor, SMRT/N-CoR, with PLZF domain. We demonstrate that SMRT association, as with PML-RARα, can be released from NPM-RARα at pharmacological concentration of ATRA (10−6 M). Moreover, we show for the first time that the interaction between the transcriptional co-activator, RIP-140, and PML-, PLZF- or NPM-RARα fusion proteins can be positively stimulated by ATRA although they are less sensitive as compared with the wild-type RARα. Our results suggest that the dissociation of transcriptional co-repressors, SMRT/N-CoR, and recruitment of co-activators, eg RIP-140, to APL-associated fusion proteins constitute a common molecular mechanism in APL and underlie the responsiveness of the disease to RA therapy.

Variant-type PML-RARα fusion transcript in acute promyelocytic leukemia: Use of a cryptic coding sequence from intron 2 of the RARα gene and identification of a new clinical subtype resistant to retinoic acid therapy

The physiologic actions of retinoic acids (RAs) are mediated through RA receptors (RARs) and retinoid X receptors (RXRs). The RARα gene has drawn particular attention because it is the common target in all chromosomal translocations in acute promyelocytic leukemia (APL), a unique model in cancer research that responds to the effect of RA. In the great majority of patients with APL, RARα is fused to the PML gene as a result of the t(15;17) translocation. Three distinct types of PML-RARα transcripts, long (L), short (S), and variant (V), were identified. The V-type is characterized by truncation of exon 6 of PML and in some cases by the insertion of a variable “spacer” sequence between the truncated PML and RARα mRNA fusion partners, although the precise mechanisms underlying formation of the V-type transcript remain unclear. To get further insights into the molecular basis of the t(15;17), we sequenced the entire genomic DNA region of RARα. Of note, all previously reported “spacer” sequences in V-type transcripts were found in intron 2 of the RARα gene and most of these sequences were flanked by gt splice donor sites. In most cases, these “cryptic” coding sequences maintained the ORF of the chimeric transcript. Interestingly, two cases with a relatively long spacer sequence showed APL cellular and clinical resistance to RA treatment. In these cases, the aberrant V-type PML-RARα protein displayed increased affinity to the nuclear corepressor protein SMRT, providing further evidence that RA exerts the therapeutic effect on APL through modulation of the RAR–corepressor interaction. Finally, among patients with the L- or S-type PML-RARα fusion transcript, some consensus motifs were identified at the hotspots of the chromosome 17q breakpoints within intron 2 of RARα, strengthening the importance of this intron in the molecular pathogenesis of APL.

Oncogenic Features of PHF8 Histone Demethylase in Esophageal Squamous Cell Carcinoma

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.

Inhibition of cancer-associated mutant isocitrate dehydrogenases: synthesis, structure-activity relationship, and selective antitumor activity.

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.

Essential role for the dimerization domain of NuMA-RARα in its oncogenic activities and localization to NuMA sites within the nucleus

Nuclear mitotic apparatus protein-retinoic acid receptor α (NuMA-RARα) is the fourth of five fusion proteins identified in acute promyelocytic leukemia (APL) patients. The molecular basis for its oncogenic activity has not been delineated. In gel-shift assays, NuMA-RARα bound to retinoic acid response elements (RAREs) both as a homodimer and as a heterodimer with RXRα. The binding profile of NuMA-RARα to a panel of RAREs was very similar to PML-RARα and PLZF-RARα. In transient transfection assays using HepG2 cells, NuMA-RARα inhibited wild-type RARα transcriptional activity, while it augmented STAT3 transcriptional activity. In GST-pull down experiments, NuMA-RARα formed a complex with the corepressor SMRT, was released from the NuMA-RARα/SMRT complexes by all-trans retinoic acid (ATRA) at 10−7–10−6 M and became associated with the coactivator TRAM-1 at 10−8 M ATRA. Studies comparing NuMA-RARα with NuMA-RARα(ΔCC) demonstrated that the dimerization or α-helical coiled-coil domain of NuMA was required for homodimer formation, transcriptional repression of wild-type RARα, transcriptional activation of STAT3, and stability of the NuMA-RARα/SMRT complex. Confocal fluorescent microscopy of HeLa cells was performed following transient expression of cyan fluorescent protein (CFP)-tagged proteins and incubation of cells with or without ATRA. Within the nucleus, CFP-NuMA-RARα exhibited a speckled pattern identical to that observed in cells transfected with CFP-NuMA. Furthermore, CFP-NuMA-RARα colocalized with yellow fluorescent protein-tagged (YFP)-NuMA. In contrast, CFP-NuMA-RARα(ΔCC) exhibited a diffuse granular pattern within the nucleus, similar to RARα. These results indicate that the dimerization domain of NuMA-RARα is critical for each of the known oncogenic activities of NuMA fusion proteins as well as its sequestration to nuclear sites normally occupied by NuMA and is distinct from RARα.