Acute Promyelocytic Leukemia Fusion Proteins Research Articles

Pml Nuclear Body Disruption Cooperates in APL Pathogenesis, Impacting DNA Damage Repair Pathways

Acute promyelocytic leukemia (APL) is driven by the oncogene PML-RARA which is generated by fusion of the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARA) genes, and which strongly interferes with downstream signalling and the architecture of multiprotein structures known as PML nuclear bodies (NBs). NB disruption is a diagnostic hallmark of APL, yet the significance of this phenomenon to disease pathogenesis and treatment response remains poorly understood. The majority of APL patients can now be cured with combination therapy with arsenic trioxide (ATO) and ATRA (All Trans-Retinoic Acid), which synergize promoting re-formation of disrupted Pml NBs. To date, the importance of NB disruption has only been studied in vitro. To address this, we generated a knock-in mouse model with targeted NB disruption achieved through mutation of key zinc-binding cysteine residues (C62A/C65A) in the RING domain of Pml. Homozygous PmlC62A/C65A mice are viable, and developmentally normal. At a cellular level, Pml NB disruption was confirmed and treatment with ATO was associated with defective Pml SUMOylation and degradation. A key feature of APL fusion proteins is the capacity to homodimerise (mediated by the fusion partner e.g. PML), which is not a feature of wild-type RARα. This forced homodimerisation of RARα has been shown to be critical for APL pathogenesis. We investigated whether Pml NB disruption could cooperate in vivo with forced RARα homodimerisation (mediated artificially by linking RARα to the dimerisation domain of the NFκB p50 subunit). While no leukemias arose in PmlC62A/C65A mice, p50-RARα mice expressing PmlC62A/C65A presented a doubling in the rate of leukemia development (p<0.0001) compared to PmlWT-p50-RARα, leading to a penetrance comparable to that observed in previously published PML-RARα transgenic models. Moreover, the latency period to onset of leukemia was significantly reduced in the context of NB disruption (p=0.008). ATRA treatment significantly improved the survival of mice transplanted with PmlWT-p50-RARα or Pml-RARα leukemic blasts, but not with PmlC62A/C65A-p50-RARα. These data reveal not only the key role of PML-RARα expression-induced NB disruption in APL development, but also the importance of re-formation of NBs for an effective response to differentiating drug.While formation of the PML-RARA fusion is considered an initiating event in APL pathogenesis, it is insufficient for the full leukemic phenotype. Exome sequencing studies have consistently identified presence of cooperating mutations. Since Pml and Pml NB have established roles in DNA repair and in the maintenance of genomic stability, we speculated that loss of NB integrity could affect these functions. Whole exome sequencing revealed a pattern of higher genomic instability in PmlC62A/C65A-p50-RARα leukemia as compared to PmlWT-p50-RARα, with detection of mutations found in human APL, including Kras, Ptpn11 and Usp9y. Using DNA repair reporter assays, we demonstrated that DNA repair via both non-homologous end joining (NHEJ; p=0.01) and homologous recombination (HR; p=0.006) pathways was less efficient in PmlC62A/C65A primary cells than in PmlWT cells. Importantly, using a PML-RARα-inducible cell line, comparable defects in the NHEJ and HR pathways, which were PML-RARα dependent, were identified. These data were also supported by an increase in sister-chromatid exchange (p<0.0001) and chromosome abnormality (p=0.0002) in the context of PmlC62A/C65A versus PmlWT. Interestingly, the kinetic of repair of ionising radiation (IR)-induced DNA double-strand breaks, assessed by analysis of γH2AX foci formation and clearance, was not affected. None of the DNA repair players analysed (e.g. Blm, Rad51 and 53BP1) failed to form foci in response to IR. However, their basal levels of foci were significantly greater in the presence of PmlC62A/C65A (p<0.04; quantified using Amnis ImageStreamX Mk II imaging flow cytometer). Additionally, we found that Rad51 foci showed a defect in localisation post-IR when PmlC62A/C65A was expressed, with impairment of Rad51 co-localisation and interaction with γH2AX. Altogether, our data therefore highlight the significant contribution of Pml NB to the effectiveness of DNA damage repair processes, and the manner in which their disruption mediated by the PML-RARα oncoprotein can assist APL pathogenesis. DisclosuresHills:TEVA: Honoraria. Grimwade:TEVA: Research Funding.

Extrinsic apoptosis is impeded by direct binding of the APL fusion protein NPM-RAR to TRADD.

A subset of acute promyelocytic leukemia (APL) cases has been characterized by the t(5;17)(q35;q21) translocation variant, which fuses nucleophosmin (NPM) to retinoic acid receptor α (RARA). The resultant NPM-RAR fusion protein blocks myeloid differentiation and leads to a leukemic phenotype similar to that caused by the t(15;17)(q22;q21) PML-RAR fusion. The contribution of the N-terminal 117 amino acids of NPM contained within NPM-RAR has not been well studied. As a molecular chaperone, NPM interacts with a variety of proteins implicated in leukemogenesis. Therefore, a proteomic analysis was conducted to identify novel NPM-RAR-associated proteins. TNF receptor type I-associated DEATH domain protein (TRADD) was identified as a relevant binding partner for NPM-RAR. This interaction was validated by coprecipitation and colocalization analysis. Biologic assessment found that NPM-RAR expression impaired TNF-induced signaling through TRADD, blunting TNF-mediated activation of caspase-3 (CASP3) and caspase-8 (CASP8), to ultimately block apoptosis. This study identifies a novel mechanism through which NPM-RAR affects leukemogenesis.

Discovery of Critical Functions of Histone Demethylase, PHF8, in Mediating ATRA Response in APL

Abstract 226 Introduction:Transcription deregulation plays a key role in acute leukemogenesis, which is mostly initiated by chimeric transcription factors such as PML–RARa that accounts for almost 100% of acute promyelocytic leukemia (APL). While APL is uniquely sensitive to all trans retinoic acid (ATRA) treatment and has been the paradigm of differentiation/epigenetic therapies, the underlying mechanisms remain largely unknown and are major interests in the field with a potential of extending the success to other hematological malignancies. Previously, others and we have shown that aberrant recruitment of histone deacetylases and polycomb repressive complexes by oligomeric PML-RARa are key for suppression of its downstream targets and promote cellular transformation. ATRA treatment disrupts their bindings and results in de-repression of target genes. However, the identification of the co-activator complex responsible for gene activation upon ATRA treatment remains a major hurdle that significantly hinders the progress of understanding mechanisms underlying the epigenetic therapy. Results and Discussion:Given the critical functions of JmjC-domains containing histone demethylases in mediating transcriptional regulation, we performed a systematic screening for differential interaction between JmjC-histone demethylases and PML–RARa upon ATRA treatment. We identified a highly specific interaction between PML–RARa and Plant Homeodomain Finger 8 (PHF8). To assess its effect on histone methylation, we detected specific reduction of H3K9me2 and enhancement of H3K4me3 in PHF8 transfected cells, consistent with its function as a transcriptional activator. This was further supported by results from chromatin immuno-precipitation (ChIP) assays in human APL cell line (NB4) showing that PHF8 differentially bound and mediated the same histone modifications in RARb promoter and activated its expression. To investigate the functional significance of this interaction, PHF8 was expressed in human NB4 cells harboring PML-RARa or murine primary bone marrow cells transformed by APL fusion proteins. Induction of PHF8 expression significantly decreased their in vitro transformation capacity in the presence of physiological concentrations of ATRA. Conversely, specific down-modulation of PHF8 expression by shRNAs reduced ATRA sensitivity of these cells, suggesting a critical function of PHF8 in mediating ATRA response. We hypothesized that PHF8 may be able to sensitize ATRA resistant cells to the treatment. Hence, we induced expression of PHF8 in the ATRA resistant variant of NB4 line, NB4-MR2. We were able to demonstrate that PHF8 sensitized NB4-MR2 cells to ATRA treatment in vitro. In contrast, NB4-MR2 cells expressing PHF8-F279S, a catalytically inactive mutant could not be sensitized to ATRA treatment, indicating that the enzymatic activity is critical for mediating the ATRA response. Most importantly, NB4-MR2 cells expressing wild type PHF8 were also sensitive to ATRA treatment in vivo and failed to induce leukemia in NOD/SCID mice, which would otherwise succumb to leukemia in a very brief latency.To gain further insights into the molecular regulation of PHF8 in ATRA response, we characterized the potential functions of CDK1-mediated phosphorylation of PHF8. It is known that in ATRA treated leukemic cells Cyclin A translocates into the nucleus where it interacts with CDK1. Activated CDK1 induces phosphorylation of PHF8 at two serine (S33)/ threonine (T84) phosphorylation sites. Our results showed that PHF8 in its phosphorylated form had a much higher binding affinity to PML-RARa. Consistently, ChIP analyses revealed that the binding of PHF8 to the RARb promoter and the resultant activation were significantly augmented when PHF8 was constitutively phosphorylated. Moreover, inhibition of PHF8 dephosphorylation by Okadaic Acid, sensitized NB4-MR2 cells to ATRA treatment. Conclusions:We discovered a novel function of the histone demethylase PHF8 in mediating therapeutic response in both ATRA sensitive and ATRA resistant cells. This function of PHF8 is critically dependent on two phosphorylation sites as well as its histone modification activity. Thus, therapeutic interventions such as phosphatase inhibitors that enhance the PHF8 activity might become useful tools for development of new epigenetic therapies sensitizing leukemic cells to ATRA treatment. Disclosures:No relevant conflicts of interest to declare.

Leukemic transformation by the APL fusion protein PRKAR1A-RARα critically depends on recruitment of RXRα

AbstractPRKAR1A (R1A)–retinoic acid receptor-α (R1A-RARα) is the sixth RARα–containing fusion protein in acute promyelocytic leukemia (APL). Using the murine bone-marrow retroviral transduction/transformation assay, we showed that R1A-RARα fusion protein could transform bone-marrow progenitor/stem cells. In gel-shift assays, R1A-RARα was able to bind to a panel of retinoic acid response elements both as a homodimer and as a heterodimer with RXRα, and demonstrated distinct DNA-binding characteristics compared with wild-type RARα/RXRα or other X-RARα chimeric proteins. The ratio of R1A-RARα to RXRα proteins affected the retinoic acid response element interaction pattern of R1A-RARα/RXRα complexes. Studies comparing R1A-RARα with R1A-RARα(ΔRIIa) demonstrated that the RIIa protein interaction domain located within R1A was responsible for R1A-RARα homodimeric DNA binding and interaction with wild-type R1A protein. However, the RIIa domain was not required for R1A-RARα–mediated transformation because its deletion in R1A-RARα(ΔRIIa) did not compromise its transformation capability. In contrast, introduction of point mutations within the RARα portion of either R1A-RARα or R1A-RARα(ΔRIIa), previously demonstrated to eliminate RXRα interaction or treatment of transduced cells with RXRα shRNA or a RXRα agonist, reduced transformation capability. Thus, leukemic transformation by APL fusion protein PRKAR1A-RARα is critically dependent on RXRα, which suggests RXRα is a promising target for APL.

Evidence of functional interaction between NuMA-RARα and RXRα in an in vivo model of acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by reciprocal balanced chromosomal translocations involving retinoic acid receptor-alpha (RARalpha). RARalpha heterodimerizes with the retinoid X receptor-alpha (RXRalpha) and transcriptionally regulates myeloid differentiation in response to ATRA (all-trans retinoic acid). Several lines of evidence suggest that APL fusion proteins interact with RXRalpha. To elucidate the role of RXRalpha in APL, we conditionally knocked out RXRalpha in the hCG-NuMA-RARalpha APL mouse model. Phenotype analysis of NuMA-RARalpha+ mice demonstrated that these mice developed a myeloproliferative disease-like myeloid leukemia within 4 months of birth. While hemizygous and homozygous RXRalpha conditional knockout mice were phenotypically normal as late as 12 months of age, we observed that the leukemic phenotype in NuMA-RARalpha+ mice was dependent on the presence of functional RXRalpha. Bone marrow promyelocyte counts were significantly reduced in NuMA-RARalpha+ mice with RXRalpha knocked down. Significant differences in the accumulations of Gr-1+ and Mac-1+ cells were also seen. We further observed that genes previously identified to be cooperating events in APL were also regulated in an RXRalpha-dependent manner. We therefore propose that the APL fusion protein NuMA-RARalpha cooperates with RXRalpha in the development of leukemia in hCG-NuMA-RARalpha transgenic mice and suggest a novel role for RXRalpha in the pathogenesis of APL.

Comparative Analysis of Downstream Genetic Targets of the Variant Acute Promyelocytic Leukemia Fusion Proteins NPM-RARA and NuMA-RARA.

Acute promyelocytic leukemia (APL) is characterized by accumulation of abnormal promyelocytes in the bone marrow and peripheral blood, and sensitivity to treatment with all-trans retinoic acid. APL cases have a balanced chromosomal translocation involving retinoic acid receptor alpha (RARA) on chromosome 17. The resulting fusion proteins (X-RARA) are aberrant transcription factors and block ATRA-induced neutrophil differentiation. Loss of RARA signalling impairs granulopoiesis, but is not sufficient to cause a leukemic phenotype. We elucidated the identities of additional signalling pathways, which can potentially cooperate with X-RARA in APL, that are commonly modulated by multiple X-RARA. We used the U-937 hematopoetic cell line retrovirally transduced with NPM-RARA (Kamel-Reid et al, 2003), and NuMA-RARA, in addition to NB4 cells (expressing PML-RARA) to determine the common genes and pathways deregulated in APL. Gene expression analysis was carried out on RNA harvested in triplicate from control and X-RARA expressing cell lines, using the Affymetrix U133Plus2 array platform. Gene expression and pathways analysis of array data was carried out using a suite of analysis tools. Array data were validated in an independent sample set by real-time quantitative PCR. We observed a total of 311 genes deregulated at least 2-fold by NuMA-RARA (192 up-regulated, 119 down-regulated), 393 genes deregulated by NPM-RARA (292 up-regulated, 101 down-regulated), and 2056 genes deregulated by PML-RARA (1097 up-regulated, 959 down-regulated). A total of 65 genes, in 5 major interaction networks, were commonly deregulated by all three X-RARA (42/65 up-regulated, 23/65 down-regulated). The majority of these genes are involved in cellular signalling (14 genes, p-value 1.57E-07–7.77E-3), transcription (13 genes, p-value 5.68E-7–3.91E-3), cell proliferation (25 genes, p-value 9.73E-7–7.77E-3), apoptosis (26 genes, p-value 9.96E-7–7.71E-3), and cell movement (17 genes, p-value 1.43E-6–7.60E-3). Genes involved in the CEBPA interaction network (GFI1, TRIB2, ELA2), as well as other genes that we anticipated to be deregulated in APL including ID1, MMP9, and JUN were found through this analysis. NF-kB (p-value 2.10E-3), AHR (p-value 2.12E-3), IL-6 (p-value 5.40E-3), and G-protein coupled receptor (p-value 7.45E-3) signalling were among the top canonical pathways determined to be altered by X-RARA. Over-expression of a number of NF-kB downstream transcriptional targets, including VEGF, IL8, MMP9, cIAP2, and TNFAIP3, were also observed in multiple X-RARA expressing cell lines. In addition, in vitro results were compared to NuMA-RARA gene targets identified in primary bone marrow cultures derived from the hCG-NuMA-RARA transgenic mouse model (Sukhai et al, 2004). We observed that pathways involved in cell signalling, cell death, gene expression, proliferation, and cell cycle were significantly deregulated in both mouse and human datasets, indicating that these pathways may be important cooperating events in APL. Our data represent the first comparison of the genetic profiles of the variant fusion proteins NPM-RARA and NuMA-RARA in a haematopoietic cell system. Our studies are a significant step in identifying key targets that cooperate with X-RARA in the development of APL.

Aberrant Chromatin Remodeling by Retinoic Acid Receptor α Fusion Proteins Assessed at the Single-Cell Level

Acute promyelocytic leukemia (APL) is characterized by specific chromosomal translocations, which generate fusion proteins such as promyelocytic leukemia (PML)-retinoic acid receptor (RAR)alpha and promyelocytic leukemia zinc finger (PLZF)-RARalpha (X-RARalpha). In this study, we have applied lac operator array systems to study the effects of X-RARalpha versus wild-type RARalpha on large-scale chromatin structure. The targeting of these enhanced cyan fluorescent protein-lac repressor-tagged RARalpha-containing proteins to the gene-amplification chromosomal region by lac operator repeats led to local chromatin condensation, recruitment of nuclear receptor corepressor, and histone deacetylase complex. The addition of retinoic acid (RA) induced large-scale chromatin decondensation in cells expressing RARalpha; however, cells expressing X-RARalpha, especially PML-RARalpha, demonstrated insensitive response to this effect of all-trans retinoic acid (ATRA). Although we did not reveal differences in RA-dependent colocalization of either silencing mediator for retinoid and thyroid or steroid receptor coactivator (SRC)-1 with RARalpha versus X-RARalpha, the hormone-independent association between SRC-1 and X-RARalpha on the array has been identified. Rather, compared with cells expressing RARalpha, fluorescence recovery after photobleaching of live transfected cells, demonstrated decreased mobility of SRC-1 on the X-RARalpha-bound chromatin. Thus, the impaired ability of APL fusion proteins to activate gene transcription in response to ATRA corresponds to their reduced ability to remodel chromatin, which may link to their ability to impair the mobility of key nuclear receptor coregulators.

Physical and Functional Association of PLZF with Histone Methyl Transferase and Histone Demethylase.

The promyelocytic leukemia zinc finger protein (PLZF) is a transcription factor fused to RARα in the t(11;17) translocation associated with retinoic acid resistant acute promyelocytic leukemia (APL). As a result of this chromosomal abnormality, two oncogenic proteins are produced, PLZF-RARα and RARα-PLZF. Wild type PLZF is expressed in CD34+ progenitor cells and declines during differentiation. PLZF is a tumor suppressor that causes cell cycle arrest, downregulating genes such as cyclinA2 and c-myc. We previously showed that transcriptional repression by PLZF is mediated by the recruitment of histone deacetylases to target genes, this being critical for its ability to control growth and affect RAR target genes. We now show that PLZF alters the methylation state of histones in its target genes. A biotinylated form of PLZF co-purified in cells along with a histone methyl transferase (HMT) activity for native histones. Using mutant histone H3 tail peptides, we showed that this activity methylated histone H3 on lysine 9 (H3K9me). Tagged forms of PLZF as well as endogenous PLZF co-precipitated in vivo with G9a histone methyl transferase, an enzyme that can mono and dimethylate H3K9 in euchromatin subject to gene repression. The interaction of PLZF with G9a required the presence of the N-terminal BTB/POZ domain as well as a second, more C-terminal, repression domain of PLZF. Given the newly found role of active histone demethylation in gene control we also tested the interaction of PLZF with LSD1, an enzyme associated with gene repression that demethylates H3K4. As in the case of G9a, the interaction of PLZF with LSD1 required both repression domains, suggesting, that these proteins may be part of a multi-protein complex containing multiple contact points with PLZF. Expression of G9a or LSD1 augmented transcriptional repression mediated by PLZF on reporter genes, indicating a functional interaction between histone methylation modifiers and PLZF. To determine the ability of PLZF to affect chromatin methylation in vivo, a Gal4-PLZF fusion protein was expressed in cells containing a chromatin-embedded Gal4-tk-Luciferase reporter gene. In the presence of PLZF, a chromatin immunoprecipitation experiment showed an increase in H3K9 methylation of the target gene while H3K4 methylation decreased, consistent with the ability of PLZF to interact with LSD1 and G9a. Lastly we compared the ability of the histone modifying proteins to interact with the APL fusion proteins PLZF-RARα, PML-RARα and NPM-RARα. Co-precipitation experiments showed a robust interaction between PLZF-RARα and G9a and LSD1 while the PML-RARα and NPM-RARα fusions bound these proteins significantly less avidly. Collectively all these data indicate that specific histone methylation is an important mode of action of PLZF in gene repression. The retinoic acid resistance of t(11;17)-APL may be related to its ability to interact with HMTs and histone demethylases. Hence therapeutic targeting of HMTs and histone demethylases might be considered as a novel mode of therapy in APL and other hematological malignancies.

Aberrant Chromatin Remodeling by APL Fusion Proteins Assessed at the Single-Cell Level: Contribution of Reduced Mobility of Nuclear Coregulators.

Acute promyelocytic leukemia (APL) is notable for its specific t(15;17) chromosomal translocation-generated fusion protein, PML-RARα , and a dramatic clinical response to all-trans retinoic acid (ATRA) therapy. To explore the hypothesis that aberrant chromatin remodeling plays a role in PML-RARα-induced leukemogenesis and its reversal by ATRA at the single-cell level, we subcloned the open reading frames of wild type RARα , PML-RARα and PLZF-RARα into the amino-terminus of CFP-LacR. Immunoblot analysis, DNA binding studies, reporter assays and fluorescent microscopic analysis demonstrated that each CFP-LacR tagged construct encoded proteins of the expected size and that each gained the ability to bind to a Lac operator-containing reporter construct and activate transcription in an ATRA-dependent manner; addition of the tag did not interfere with their RARE binding ability or their cellular localization and distribution. Fluorescent microscopic examination of chromosomally integrated Lac operator arrays within the CHO cell line, A03_1, following expression of CFP-LacR-tagged proteins demonstrated that each of the tagged proteins localized to tightly packed arrays of less than half the volume of arrays within A03_1 cells transfected with CFP-LacR alone (p<0.001 for each). Addition of ATRA (10−7 M) normalized the array volume in cells expressing CFP-LacR-RARα but not in cells expressing CFP-LacR-PML-RARα or CFP-LacR-PLZF-RARα . To determine directly whether or not the Lac operon array volume correlated with recruitment of nuclear receptor co-regulators, we imaged A03_1 cells co-expressing CFP-LacR-RARα , CFP-PML-RARα or CFP-LacR-PLZF-RARα and YFP-SMRT or YFP-SRC-1. In the absence of ATRA, YFP-SMRT bound almost exclusively to the arrays within cells expressing CFP-LacR-RARα , CFP-PML-RARα or CFP-LacR-PLZF-RARα , while YFP-SRC-1 did not; rather, CFP-SRC-1 distributed throughout the nucleus in each case. Surprisingly, the concentration of ATRA required to induce dissociation of YFP-SMRT from the arrays and recruitment of YFP-SRC-1 to the arrays (10−7 M) was similar in cells expressing each construct. In contrast, while the mobility of YFP-SRC-1 localized on the CFP-LacR-RARα-bound arrays assessed by its recovery half-life (t1/2) was 17.80 ± 4.31 s, its mobility on the CFP-LacR-PML-RARα-bound arrays or the CFF-LacR-PLZF-RARα-bound arrays was reduced by 47% (t1/2=26.07 ± 8.35 s; p<0.001) and 45% (t1/2=25.80 ± 9.89 s; p<0.001), respectively, indicating that YFP-SRC-1 bound more tightly to both PML-RARα and to PLZF-RARα than to wild type RARα . In addition, while the mobility of YFP-SMRT localized on the CFP-LacR-RARα-bound arrays assessed by its t1/2 (13.57 ± 4.53 s) was similar to the mobility of YFP-SMRT localized on the CFP-LacR-PML-RARα-bound arrays (t1/2=13.50 ± 4.21 s), the mobility of YFP-SMRT on the CFF-LacR-PLZF-RARα-bound arrays was reduced by 68% (t1/2=22.76 ± 7.87 s; p< 0.001) indicating that YFP-SMRT bound more tightly to PLZF-RARα than to either wild type RARα or PML-RARα . Thus, the impaired ability of APL fusion proteins to activate gene transcription in response to ATRA corresponds to their reduced ability to remodel chromatin; aberrant chromatin remodeling by APL fusion proteins at the single-cell level did not correlate with their altered ATRA concentration-dependent associations with key nuclear receptor coregulators rather with their ability to impair the nuclear mobility of these coregulators.

Systems Analysis Reveals Regulators of Apoptosis, Cell Cycle, Signal Transduction and Transcription as Novel Direct Targets of the Acute Promyelocytic Leukemia Fusion Protein NuMA-RAR α

We used the hCG-NuMA-RARα transgenic model of acute promyelocytic leukemia (APL) (Sukhai et al, 2004) to determine the downstream genetic targets of NuMA-RARα, one of five APL-associated fusion proteins (X-RARα). X-RARα retain the C-terminal domains of retinoic acid receptor (RAR) α, and are thought to interfere with retinoid signaling pathways and thus inhibit neutrophil differentiation. However, X-RARα have a wider range of DNA binding specificities compared to RARα, and may deregulate novel downstream targets that play critical roles in APL. Our group previously identified PPARγ signaling as being one such pathway deregulated by X-RARα (Kamel-Reid et al, 2003). We sought to identify additional, nonretinoid signaling, target genes of NuMA-RARα by using a combined experimental and in silico approach. Affymetrix oligonucleotide array analysis (mouse 430A arrays) was performed on RNA harvested from bone marrow cultures established from wild-type and transgenic mice. Genes deemed to be significantly deregulated and of biological relevance were validated by quantitative real-time RT-PCR. In our analysis, we identified 260 significantly over-expressed and 278 significantly under-expressed genes in transgenic bone marrow cultures, compared to wild-type. As NuMA-RARα is an aberrant transcriptional repressor, we focused on under-expressed genes: 251/278 were putative direct targets of NuMA-RARα. 82/251 were retinoid response genes; 169/251 were novel targets, external to retinoid signaling pathways. 150/251 had known function. Genes involved in regulation of apoptosis, cell cycle, signal transduction and transcription were over-represented within this dataset, in comparison to their frequency within the mouse genome. NuMA-RARα deregulated a number of myeloid transcription factors, including PU.1 (11.5-fold under-expression compared to wild-type), members of the C/EBP (3.5–5.0 fold under-expression) and GATA families (8.5–11.0-fold over-expression), as well as c-Myb and AML1. Binding sites for these transcription factors were overrepresented within the promoters of NuMA-RARα-deregulated genes. NuMA-RARα also deregulated a set of genes involved in cell cycle regulation and DNA damage response, including Gadd45α (1.9-fold under-expressed) and Dusp1 (3.8-fold under-expressed), and components of signal transduction pathways, including Jak2 (20.0-fold under-expressed). By mapping the chromosomal addresses of deregulated genes onto the murine genome, we determined that changes in chromosome copy number may not be responsible for deregulation. Finally, our comparison with other, previously published, microarray analyses indicated that NuMA-RARα shared some common target genes with other leukemia fusion genes, and that gene deregulation caused by NuMA-RARα could give rise to a molecular phenotype reminiscent of hematopoietic stem cells. Several genes identified as candidate downstream targets in these analyses were also previously identified as putative secondary events in APL mouse models. NuMA-RARα therefore has specific effects on the transcriptome, distinct from retinoid signaling, and from other leukemia fusion genes. Future functional studies are required to determine the cooperative relationship between these novel target genes and NuMA-RARα.

The NPM-RAR fusion protein associated with the t(5;17) variant of APL does not interact with PML

The PML protein localizes to regions of the nucleus known as nuclear bodies or PODs. However, in t(15;17) Acute Promyelocytic Leukemia (APL) blasts, PML is found in a micro-punctate pattern. In order to test the hypothesis that delocalization of PML from PODs is necessary for APL, we investigated the interaction of the t(5;17) APL fusion protein NPM-RAR with PML. NPM-RAR localizes diffusely throughout the nucleoplasm. NPM-RAR does not alter the localization of PML in transfected HeLa cells, and does not associate with PML in vitro. These studies suggest that NPM-RAR does not interact with PML.

Reduced intranuclear mobility of APL fusion proteins accompanies their mislocalization and results in sequestration and decreased mobility of retinoid X receptor alpha.

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

Leukemia: beneficial actions of retinoids and rexinoids

Acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia, is the prototype of a cancer that can be cured by differentiation therapy using combined retinoic acid (RA) and chemotherapy. Acute promyelocytic leukemia is caused by chromosomal translocations, which in the large majority of cases generate the prototypic promyelocytic leukemia–retinoic-acid receptor α (PML–RARα) an oncogenic fusion protein formed from the retinoic-acid receptor α and the so-called PML protein. The fusion protein leads to the deregulation of wild type PML and RARα function, thus inducing the differentiation block and an altered survival capacity of promyelocytes of affected patients. A plethora of studies have revealed molecular details that account for the oncogenic properties of acute promyelocytic leukemia fusion proteins and the events that contribute to the therapy-induced differentiation and apoptosis of patients’ blasts. Illustrating the beneficial mechanisms of action of retinoids for acute promyelocytic leukemia patients this review goes on to discuss a plethora of recently recognized molecular paradigms by which retinoids and rexinoids, alone or in combination with other compounds, regulate growth, differentiation and apoptosis also in non-acute promyelocytic leukemia cells, highlighting their potential as drugs for cancer therapy and prevention.

Cross-talk Between Retinoic Acid and STAT3 Signaling Pathways in Acute Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) is a form of acute myelogenous leukemia characterized by chromosomal alterations involving the retinoic acid receptor-α (RARα) gene that generate unique chimeric proteins (N-RARα) and by clinical responsiveness to all-trans-retinoic acid (ATRA) treatment. APL cells are notable for differentiation block and resistance to apoptosis. While increasing evidence suggests that N-RARα fusion proteins interfere with normal RARα transcription function at retinoic acid response elements (RAREs) resulting in inhibition of normal myeloid differentiation, the mechanism for apoptosis resistance remains unexplained. Recently, we and others have reported that APL-fusion proteins can augment STAT3 transcriptional activity. Constitutive STAT3 activation has been observed in a number of hematopoietic and non-hematopoietic malignancies where it contributes to apoptosis resistance. In this review, we summarize a series of recent observations concerning cross talk between the retinoic acid and STAT3 signaling pathways in APL cells. These findings support the hypothesis that apoptosis resistance in APL may be mediated through the effects of APL fusion proteins on STAT3 signaling and suggest that targeting of STAT3 may be a useful adjunctive treatment strategy in APL.

Molecular targets of arsenic trioxide in malignant cells.

Arsenic trioxide (As2O3; ATO) has considerable efficacy in the treatment of relapsed acute promyelocytic leukemia (APL), inducing partial differentiation and promoting apoptosis of malignant promyelocytes. Although initial studies focused on the role of the characteristic APL fusion protein, PML-RARalpha, in mediating response to ATO, subsequent investigations have revealed that ATO acts on numerous intracellular targets. ATO broadly affects signal transduction pathways and causes a wide range of alterations leading to apoptosis. Key mediators of sensitivity to ATO-induced apoptosis include intracellular glutathione and hydrogen peroxide (H2O2). The loss of inner mitochondrial membrane potential is also an important step in ATO-mediated cell killing. Cellular and physiologic pathways affected by ATO provide some clues as to the mechanisms for the biologic effects of ATO. Recent research has shown that hematologic cancers other than APL and solid tumors derived from several tissue types may be responsive to monotherapy or combination therapy with ATO. A better understanding of the mechanisms of action of ATO may help guide the use of ATO for the treatment of a wide variety of malignancies and allow its potential in cancer therapy to be fully realized.

Molecular Targets of Arsenic Trioxide in Malignant Cells

After taking all of the CME courses in this supplement the reader will be able to: Describe the basic biology of various leukemias, multiple myeloma, and myelodysplastic syndrome (MDS). Discuss new targeted treatment strategies for hematologic malignancies. Understand the rationale for the use of nontraditional cytotoxic agents such as arsenic trioxide in the treatment of hematologic malignancies. Examine the role of arsenic trioxide and other novel agents in early-versus accelerated-stage hematologic disease. Discuss the preclinical and clinical efficacy of arsenic trioxide and various agents in treating acute promyelocytic leukemia, MDS, and multiple myeloma. Access CME test online and receive one hour category 1 credit at CME.TheOncologist.com Arsenic trioxide (As2O3; ATO) has considerable efficacy in the treatment of relapsed acute promyelocytic leukemia (APL), inducing partial differentiation and promoting apoptosis of malignant promyelocytes. Although initial studies focused on the role of the characteristic APL fusion protein, PML-RARα, in mediating response to ATO, subsequent investigations have revealed that ATO acts on numerous intracellular targets. ATO broadly affects signal transduction pathways and causes a wide range of alterations leading to apoptosis. Key mediators of sensitivity to ATO-induced apoptosis include intracellular glutathione and hydrogen peroxide (H2O2). The loss of inner mitochondrial membrane potential is also an important step in ATO-mediated cell killing. Cellular and physiologic pathways affected by ATO provide some clues as to the mechanisms for the biologic effects of ATO. Recent research has shown that hematologic cancers other than APL and solid tumors derived from several tissue types may be responsive to monotherapy or combination therapy with ATO. A better understanding of the mechanisms of action of ATO may help guide the use of ATO for the treatment of a wide variety of malignancies and allow its potential in cancer therapy to be fully realized.

Targeting aberrant transcriptional repression in leukemia: a therapeutic reality?

Targeting aberrant transcriptional repression in leukemia: a therapeutic reality?

The t(5;17) acute promyelocytic leukemia fusion protein NPM-RAR interacts with co-repressor and co-activator proteins and exhibits both positive and negative transcriptional properties

The t(5;17) variant of acute promyelocytic leukemia (APL) fuses the genes for nucleophosmin (NPM) and the retinoic acid receptor alpha (RAR). Two NPM-RAR molecules are expressed as a result of alternative RNA splicing. Both contain RAR sequences that encode the DNA binding, heterodimerization, and ligand activation domains of RAR. This study was designed to test the ability of these fusion proteins to act as transcriptional activators of retinoic acid responsive promoters. The NPM-RAR fusion proteins bind to retinoic acid response element sequences as either homodimers or as heterodimers with RXR. Transcription of retinoic acid–inducible promoters is activated by the fusion proteins in the presence of retinoic acid. The level of transactivation induced by the NPM-RAR fusions differs from the level of transactivation induced by wild-type RAR in both a promoter and cell specific fashion, and more closely parallels the pattern of activation of the PML-RAR fusion than wild-type RAR. In addition, NPM-RAR decreases basal transcription from some promoters and acts in a dominant-negative fashion when co-transfected with wild-type RAR. Both NPM-RAR and PML-RAR interact with the co-repressor protein SMRTe in a manner that is less sensitive than RAR to dissociation by retinoic acid. Retinoic acid induces binding of the co-activator protein RAC3. These data indicate that the NPM-RAR fusion proteins can modulate expression of retinoid-responsive genes in a positive or negative manner, depending on context of the promoter, and lend support to the hypothesis that aberrant transcriptional activation underlies the APL phenotype.

The t(5;17) acute promyelocytic leukemia fusion protein NPM-RAR interacts with co-repressor and co-activator proteins and exhibits both positive and negative transcriptional properties

AbstractThe t(5;17) variant of acute promyelocytic leukemia (APL) fuses the genes for nucleophosmin (NPM) and the retinoic acid receptor alpha (RARα). Two NPM-RAR molecules are expressed as a result of alternative RNA splicing. Both contain RARα sequences that encode the DNA binding, heterodimerization, and ligand activation domains of RARα. This study was designed to test the ability of these fusion proteins to act as transcriptional activators of retinoic acid responsive promoters. The NPM-RAR fusion proteins bind to retinoic acid response element sequences as either homodimers or as heterodimers with RXR. Transcription of retinoic acid–inducible promoters is activated by the fusion proteins in the presence of retinoic acid. The level of transactivation induced by the NPM-RAR fusions differs from the level of transactivation induced by wild-type RARα in both a promoter and cell specific fashion, and more closely parallels the pattern of activation of the PML-RAR fusion than wild-type RARα. In addition, NPM-RAR decreases basal transcription from some promoters and acts in a dominant-negative fashion when co-transfected with wild-type RARα. Both NPM-RAR and PML-RAR interact with the co-repressor protein SMRTe in a manner that is less sensitive than RARα to dissociation by retinoic acid. Retinoic acid induces binding of the co-activator protein RAC3. These data indicate that the NPM-RAR fusion proteins can modulate expression of retinoid-responsive genes in a positive or negative manner, depending on context of the promoter, and lend support to the hypothesis that aberrant transcriptional activation underlies the APL phenotype.