Translocation In Acute Promyelocytic Leukemia Research Articles

Identification of variant APL translocations PRKAR1A-RARα and ZBTB16-RARα (PLZF-RARα) through the MI-ONCOSEQ platform

The cornerstone of management in patients with acute promyelocytic leukemia (APL) is early diagnosis and prompt initiation of treatment with an all-trans retinoic acid (ATRA)-based regimen. Identification of the t(15;17)(PML-RARA) chromosomal translocation through conventional cytogenetics fluorescence in-situ hybridization (FISH) or detection of the promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) fusion through RT-PCR represent the current standard of care for diagnosing APL. However, about 1–2% of patients with APL have a variant translocation involving other fusion partners with RARα besides PML. These patients present a unique diagnostic and clinical challenge in that conventional cytogenetics in addition to FISH and/or RT-PCR for PML-RARα may fail to identify these clinically relevant genetic lesions leading to an inappropriate diagnosis and treatment. We present two cases of patients who had APL with variant translocations whose bone marrow specimens were sent to the University of Michigan for enrollment in the MI-ONCOSEQ study (HUM00067928) after standard testing failed to identify PML-RARα or t(15;17) despite a phenotypic concern for this diagnosis. In these two patients, whole exome and transcriptome profiling via the MI-ONCOSEQ platform identified a PRKAR1A-RARα fusion in one patient and ZBTB16-RARα fusion in another patient. These cases illustrate the utility of whole exome and transcriptome profiling in diagnosing variant translocations in patients in whom there is a high clinical suspicion for APL based on hematopathology review.

Characterization of Cryptic Rearrangements and Variant Translocations in Acute Promyelocytic Leukemia

AbstractAcute promyelocytic leukemia (APL) is typified by the reciprocal translocation, t(15; 17)(q22; q21), leading to the formation of PML-RARα and RARα-PML fusion genes. We have characterized 7 cases of morphologic APL found to lack the t(15; 17) on conventional cytogenetic assessment. In 6 of 7 cases, cryptic PML-RARα rearrangements were identified by reverse transcriptase-polymerase chain reaction and fluorescent in situ hybridization (FISH); whereas, in the remaining patient, APL was associated with the variant translocation, t(11; 17)(q23; q12-21), leading to the formation of PLZF-RARα and RARα-PLZF fusion genes. In each of the cases with cryptic PML-RARα rearrangements, PML-RARα transcripts were detected in the absence of RARα-PML, consistent with the concept that PML-RARα is the critical oncogenic fusion protein. In 4 of these cases with evaluable metaphase spreads, the occurrence of a nonreciprocal translocation was confirmed by FISH with sole formation of the PML-RARα fusion gene; in 3 cases with morphologically normal chromosomes 15 and 17, RARα was inserted into PML on 15q, whereas in the remaining patient the PML-RARα fusion arose due to insertion of 15q-derived material including PML into RARα on 17q. Immunofluorescence studies were performed using antibodies raised against PML and PIC 1, a ubiquitin-homology domain protein previously identified as an interaction partner of PML. In acute myeloid leukemia (AML) of subtypes other than M3, PIC 1 was localized to the nuclear membrane and colocalized with PML within discrete nuclear bodies. In APL cases with cryptic PML-RARα rearrangements, the characteristic microparticulate pattern of PML staining was detected with partial colocalization with PIC 1, indicative of disruption of the nuclear bodies; whereas in t(11; 17)-associated APL, PML and PIC 1 remained colocalized within discrete nuclear bodies, as observed in non-APL cases. Although deregulation of the putative growth suppressor PML and delocalization of other nuclear body constituents have been advocated to play a key role in the development of t(15; 17)-associated APL, the present study shows that disruption of PML nuclear bodies per se is not a prerequisite for the pathogenesis of APL.

Abstract 639: Acute promyelocytic leukemia

Abstract Introduction: Acute Promyelocytic Leukemia (APL) accounts for 10%-15% of all acute myeloid leukemia's. The use of All-Trans Retinoic Acid/ Arsenic trioxide (ATRA/ATO) in the treatment regimens of low to intermediate-risk patients has led to remarkable cure rates (~80%). However, treatment guidelines of high-risk patients (WBC count≥ 10 x 109/L) remain unclear and these patients are at an increased risk of relapse. Design: Due to ambiguous treatment guidelines for high-risk APL patients as well as limited options for patients with arsenic resistant disease we sought to analyze clinical outcomes in this subset of patients. We conducted a literature search through Pubmed, EBSCO, and JSTOR to identify original articles that evaluated treatment protocols as well as outcomes for high-risk/refractory APL patients. We also evaluated the literature for clinical data involving patients who had arsenic resistant APL. Only relevant articles from 2000 to 2019 were included. Clinical trials where patients did not receive ATRA/ATO treatment were excluded. High risk patients have an increased mortality and decreased overall survival compared to intermediate and low risk patients. Although early death resulting from bleeding complications remains an important clinical hindrance, cases of arsenic resistant disease are becoming more prevalent. Resistant/refractory APL is a rare occurrence; however, no effective treatment exists to overcome resistance in this subset of patients. Of particular interest is the identification of variant translocations in APL; those that differ from the classical PML-RARA (~95%). 12 non-characteristic rearrangements have so far been described; of these, the ZBTB16-RARA and STAT5b-RARA are the most common variants. Both ZBTB16-RARA & STAT5b-RARA are insensitive to ATRA/ATO combination therapy, leaving these patients with limited options. Clinical outcomes have recently been described in a subset of 13 patients with arsenic resistant disease. Of these 13 patients with arsenic resistant disease, 11 eventually died. Mutations in a particular region of the PML protein have been shown to mediate arsenic resistant disease in both in vitro and in vivo models, suggesting a potential benefit of molecular screening to improve clinical outcomes. Neither ATO nor cytotoxic chemotherapy was successful in inducing remissions in this particular subset of patients. Conclusion: Well-defined treatment guidelines in high-risk APL and the need for alternative agents in patients resistant to ATO therapy are needed to improve clinical efficacy and outcomes. 1. Zhu, H.H.; Qin, Y.Z.; Huang, X.J. Resistance to arsenic therapy in acute promyelocytic leukemia. N Engl J Med 2014, 370, 1864-1866, doi:10.1056/NEJMc1316382. Citation Format: Ravinder Singh Chale, Joaquin J. Jimenez. Acute promyelocytic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 639.

A complex translocation (3;17;15) in acute promyelocytic leukemia confirmed by fluorescence in situ hybridization.

Acute promyelocytic leukemia (APL) is typified by t(15;17)(q22;q21), generating the promyelocytic leukemia (PML) gene at 15q22 with the retinoic acid α-receptor (RARA) gene at 17q21. The PML-RARA fusion gene is believed to play a vital role in leukemogenesis. A sizeable minority of patients with complex variants of APL have been reported. The present study reports the case of a 33-year-old male with APL carrying a potential complex translocation. The initial symptom was bleeding gums. Chromosomal analysis of the bone marrow cells revealed an atypical 17q aberration. Fluorescence in situ hybridization further indicated that 92% of analyzed cells were positive for the PML-RARA fusion gene. The patient experienced complete remission following treatment with arsenic trioxide and chemotherapy. The atypical translocations in acute promyelocytic leukemia require further investigation.

C-Myc functions as a competing endogenous RNA in acute promyelocytic leukemia.

Recent reports have described a new post-transcriptional regulation that RNA transcripts can crosstalk with each other by competing for their common microRNAs. These RNA transcripts termed competing endogenous RNAs (ceRNAs) regulate the distribution of miRNAs on their targets. One corollary from ceRNA interaction is that chromosomal translocation in acute promyelocytic leukemia (APL) would perturb ceRNA regulation due to altered expression of 3′UTRs. In our study, we demonstrate that expression of PML/RARα, the APL-associated fusion oncogene is repressed by c-Myc mRNA transcript independent of protein-coding function but dependent upon microRNA. Attenuation of c-Myc transcript results in PML/RARα-degraded cellular phenotypes in APL cells, but these Myc reduction-associated cell phenotypes are sufficient to abrogate in a microRNA dependent manner. We also show that let-7 microRNA family members promote differentiation of All-Trans-Retinoic Acid (ATRA)-induced NB4 cells and their activities are affected by expression levels of both c-Myc and PML/RARα through altering miRNA targets. These results indicate that c-Myc mRNA represses PML/RARα expression via altering the distribution of let-7 miRNAs on their targets. Our findings reveal a previously unrecognized role of c-Myc as a potential ceRNA for PML/RARα in APL.

A PML–PPAR-δ Pathway for Fatty Acid Oxidation Regulates Hematopoietic Stem Cell Maintenance Through the Control of Asymmetric Division.

AbstractAbstract 2327Stem-cell function is an exquisitely regulated process. Thus far, the contribution of metabolic cues to stem-cell function has not been well understood. Here we have established a new assay for the assessment of asymmetric division in hematopoietic stem cells (HSCs), and identify a previously unknown promyelocytic leukemia (PML)–peroxisome proliferator-activated receptor δ (PPAR-δ)–fatty-acid oxidation (FAO) pathway for the maintenance of HSCs and the control of asymmetric cell division.HSCs exist in a quiescent state in the bone marrow niche and are the source of all hematological progenitors and differentiated cells throughout the lifespan of an organism. One of the central tasks of stem-cell biology is to define the modes and mechanisms that regulate the self-renewal and commitment of stem cells, as alterations in this equilibrium have a substantial effect on hematopoietic homeostasis and maintenance. It has been suggested that asymmetric division of HSCs ensures that a fraction of daughter cells retain features of stem cells while replenishing the committed compartment of hematopoietic progenitors. Therefore, identifying the factors that regulate this process would be of great biological and therapeutic relevance.The PML tumor-suppressor gene, originally cloned at the break point of the t(15;17) chromosomal translocation of acute promyelocytic leukemia, has a key role in the maintenance of HSCs. However, how PML exerts its crucial function in the biology and maintenance of HSCs has not been established. In this study, we will present a new metabolic pathway downstream of PML, which can be regarded as a fine-tuning rheostat essential for HSC maintenance and their asymmetric division.Specifically, we find that loss of PPARδ or inhibition of mitochondrial FAO induces loss of HSC maintenance both in vitro and in vivo, whereas treatment with PPARδ agonists improved HSC maintenance. We demonstrate that PML exerts its essential role in HSC maintenance through regulation of PPAR signaling and FAO. Mechanistically, we show that the PML–PPAR δ–FAO pathway controls the asymmetric division of HSCs. Deletion of the Ppard or Pml genes as well as inhibition of FAO results in the symmetric commitment of HSC daughter cells, whereas PPARδ activation increases asymmetric cell division, and rescues the defect exhibited by Pml-deficient HSCs, therefore ensuring the correct maintenance of the HSC population.Thus, our findings identify a metabolic switch for the control of HSC cell fate with potential therapeutic implications. Disclosures:Avigan:Curetech: Research Funding; Celgene: Research Funding; Genzyme: Membership on an entity's Board of Directors or advisory committees.

Molecular methods for genomic analyses of variantPML-RARAor otherRARA-related chromosomal translocations in acute promyelocytic leukemia

TO THE EDITOR: We read an interesting paper by Palta et al. in a recent issue of the Korean Journal of Hematology titled, variant of acute promyelocytic leukemia with tuberculosis: a case report and review of literature [1]. We would like to add some comments to their article and suggest additional molecular methods to confirm variant translocations in acute promyelocytic leukemia (APL). Apart from its characteristic morphology, APL is strongly correlated with presence of the PML-RARA fusion gene due to a t(15;17) translocation. Patients displaying cryptic PML-RARA gene rearrangements or other translocations involving RARA are very rare. Since RARA generates chimeric fusion genes with various partners such as ZBTB16 at 11q23, NUMA1 at 11q13, NPM1 at 5q35, FIP1L1 at 4q12, and STAT5b at 17q11.2 (Fig. 1), confirmatory molecular methods to detect gene rearrangements are necessary when unique or unusual chromosomal translocations are found in APL cases. Palta et al. reported a case of APL accompanied by tuberculosis where cytogenetic study showed a variant chromosomal translocation, t(11;17)(q23;q21). First, although they describe this case as APL with t(11;17)(q23;q21) through bone marrow (BM), immunophenotyping, and cytogenetic studies, we noted an absence of results based on molecular methods. To demonstrate the existence of the ZBTB16-RARA fusion gene, a single specific reverse transcriptase-polymerase chain reaction (RT-PCR) with a specific primer to detect the gene rearrangement or multiplex RT-PCR (e.g. HemaVision) to detect ZBTB16-RARA could have provided an accurate molecular diagnostic result. Second, the authors' reference to a paper by Kang et al. was puzzling despite the fact that it was cited as a similar case with chromosomal translocations at a similar location, in cases of AML with chromosomal translocation t(11;17) [2]. In the first case, cytogenetic analysis from BM aspirate showed t(11;17)(q23;q21) in 18 out of 20 cells while the MLL probe in fluorescence in situ hybridization (FISH) showed split-out signals in 98.5% of interphase cells. The second case also showed t(11;17)(q23;q25) in the chromosome study while the FISH assay showed split-out signals in 79% of interphase cells. However, normal fluorescence patterns were found in 200 cells in additional FISH assays when a dual-color single-fusion PML-RARA probe was used for both cases, which led to the conclusion that the MLL gene was involved in the 11q23 breakpoint and RARA was not involved in the 17q breakpoint. Hence, the authors' citation of this study to explain the ZBTB16-RARA fusion gene appears inadequate, and a molecular study could unambiguously identify the involvement of ZBTB16 at 11q23 in their case. Fig. 1 Various RARA partner genes in acute promyelocytic leukemias. We suggest the authors conduct RT-PCR analysis of the ZBTB16-RARA fusion gene if any remnant RNA or cDNA samples from their APL patient are available. However, if only genomic DNA is available, analyzing the genomic breakpoints by multiplex long distance-polymerase chain reaction (LD-PCR) would be helpful. LD-PCR or long distance inverse (LDI)-PCR can identify known or unknown partner genes, monitor minimal residual disease (MRD), and identify multi-way chromosomal translocations such as three-way or four-way translocations [3]. We have recently used LD-PCR to confirm APL cases showing unusual karyotypes or discrepant results between conventional cytogenetics and molecular studies. More precisely, we identified a novel PML-ADAMTS17-RARA rearrangement and confirmed the genomic fusion breakpoints of cryptic PML-RARA rearrangement in 2 APL patients [3-5]. In conclusion, detection of leukemia-associated fusion genes is important due to their direct relevance to diagnosis, MRD assessment, and treatment response. Since APL patients with ZBTB16-RARA are resistant to all-trans-retinoic acid (ATRA), molecular methods with higher resolution than chromosome studies must be conducted to detect fusion genes and facilitate selection of the most appropriate treatment. We believe that molecular techniques such as FISH, RT-PCR, and LD-PCR should be used in addition to morphology and cytogenetic studies in APL diagnosis and evaluation so that atypical PML-RARA or RARA variant gene rearrangements are not missed.

Acute Promyelocytic Leukemia With Complex Translocation t(5;17;15)(q35;q21;q22)

The t(15;17)(q22;q21), resulting in PML-RARA fusion gene, is a characteristic chromosomal translocation in acute promyelocytic leukemia (APL). We report a pediatric APL case with a 3-way translocation: t(5;17;15)(q35;q21;q22). Complete blood cell counts of a 12-year-old girl, of pale appearance, showed pancytopenia with increased blasts. Morphology and immunophenotype of the leukemic cells were compatible with APL. Karyotype analysis showed t(5;17;15)(q35;q21;q22) and add(7)(q32). We detected the PML-RARA fusion gene by both reverse transcriptase-polymerase chain reaction and fluorescent in situ hybridization analysis. The patient underwent successful treatment with cytarabine with all-trans retinoic acid and anthracycline-based therapy.

FIP1L1/RARA with breakpoint at FIP1L1 intron 13: a variant translocation in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia that is characterized by three distinct features: i) accumulation in the bone marrow (BM) of tumor cells with promyelocytic phenotype; ii) association with specific translocations which involve chromosome 17 at the

Rara haploinsufficiency modestly influences the phenotype of acute promyelocytic leukemia in mice

AbstractRARA (retinoic acid receptor alpha) haploinsufficiency is an invariable consequence of t(15;17)(q22;q21) translocations in acute promyelocytic leukemia (APL). Retinoids and RARA activity have been implicated in hematopoietic self-renewal and neutrophil maturation. We and others therefore predicted that RARA haploinsufficiency would contribute to APL pathogenesis. To test this hypothesis, we crossed Rara+/− mice with mice expressing PML (promyelocytic leukemia)-RARA from the cathepsin G locus (mCG-PR). We found that Rara haploinsufficiency cooperated with PML-RARA, but only modestly influenced the preleukemic and leukemic phenotype. Bone marrow from mCG-PR+/− × Rara+/− mice had decreased numbers of mature myeloid cells, increased ex vivo myeloid cell proliferation, and increased competitive advantage after transplantation. Rara haploinsufficiency did not alter mCG-PR–dependent leukemic latency or penetrance, but did influence the distribution of leukemic cells; leukemia in mCG-PR+/− × Rara+/− mice presented more commonly with low to normal white blood cell counts and with myeloid infiltration of lymph nodes. APL cells from these mice were responsive to all-trans retinoic acid and had virtually no differences in expression profiling compared with tumors arising in mCG-PR+/− × Rara+/+ mice. These data show that Rara haploinsufficiency (like Pml haploinsufficiency and RARA-PML) can cooperate with PML-RARA to influence the pathogenesis of APL in mice, but that PML-RARA is the t(15;17) disease-initiating mutation.

The Role of PML in the Nervous System

The promyeloctic leukemia protein PML is a tumor suppressor that was originally identified due to its involvement in the (15;17) translocation of acute promyelocytic leukemia. While the majority of early research has focused upon the role of PML in the pathogenesis of leukemia, more recent evidence has identified important roles for PML in tissues outside the hemopoietic system, including the central nervous system (CNS). Here, we review recent literature on the role of PML in the CNS, with particular focus on the processes of neurodevelopment and neurodegeneration, and propose new lines of investigation.

Impact of additional chromosomal abnormalities in patients with acute promyelocytic leukemia: 10-year results of the Japan Adult Leukemia Study Group APL97 study

Impact of additional chromosomal abnormalities in patients with acute promyelocytic leukemia: 10-year results of the Japan Adult Leukemia Study Group APL97 study

Relapsed APL patient with variant NPM–RARα fusion responded to arsenic trioxide-based therapy and achieved long-term survival

The t(5;17)/NPM-RARalpha is the second variant chromosomal translocation in acute promyelocytic leukemia (APL) to be characterized and also the second most plentiful variant translocation. So far, there is a lack of information on the effectiveness of arsenic trioxide (ATO) in relapsed APL with variant RARalpha chimera including t(5;17)/NPM-RARalpha. We report here a long-term survived APL patient with variant NPM-RARalpha fusion who relapsed four times and each time responded well to ATO or ATO-based re-induction therapy. The patient had received a total of more than 3,500 mg of ATO, but showed no obvious arsenic-related toxicities. This case illustrates the long-term efficiency and safety of ATO-based therapy not only in newly diagnosed APL, but also in relapsed APL including those with variant translocations.

PML Controls PTEN Localization and Function in APL through a Novel Deubiquitination Pathway

PML was discovered as a gene frequently involved in the t(15:17) translocation of acute promyelocytic leukemia (APL). Since then, consequences of PML-loss of function in APL and other solid tumors have been extensively studied. Numerous pathways influenced by this tumor suppressor, including the PI3K pathway, have been identified but their importance for the pathogenesis APL is still unclear. We have now discovered that PTEN, the main tumor suppressor opposing PI3K signaling, is aberrantly delocalized to the cytoplasm in APL, but not in AML. Moreover, we show that drugs such as arsenic trioxide and retinoic acid, currently used for the treatment of APL and known to target the oncogenic PML-RARalpha fusion protein, rescue the nuclear localization of PTEN. This discovery has critical biological implications since nuclear localization of PTEN is central to its tumor suppressive activity. Utilizing an APL cell line, genetically engineered cells and transgenic mice, we have delineated the mechanism by which PML sustains nuclear PTEN. PML opposes the activity of a novel PTEN-deubiquitinating enzyme, HAUSP, towards PTEN through a framework involving the adaptor protein DAXX (death domain-associated protein). We demonstrate that PML-RARalpha translocation results in overactive HAUSP and as a consequence, nuclear-excluded PTEN. In support of this notion, we show that HAUSP is overexpressed in human cancer, (e.g. in prostate cancer) which is in turn associated with a nuclear exclusion phenotype of PTEN. Overall our results describe a novel role for PML in the maintenance of PTEN function that is perturbed by oncogenic cues in human leukemia and solid tumors. Significantly, our findings open new avenues for the design of targeted therapies focused on potentiating the activity of the PML-PTEN tumor suppressive network.

Proteome Analysis of the Proteins Associated with All-Trans Retinoic Acid Resistance in Acute Promyelocytic Leukemia Cells

Although 90% patients with untreated acute promyelocytic leukemia(APL) obtain complete remission because of the usage of all-trans retinoic acid(ATRA), patients with ATRA-resistance are increased gradually. ATRA-resistance has become one of the main causes which affect the long-term therapeutic efficacy of APL. The mechanisms of ATRA-resistance are complex, which probably involve the metabolism of ATRA, abnormal expression of cellular retinoic acid binding protein(CRABP) and P-glycoprotein(P-gp), mutation of RARα and aberration translocation of APL. However, in these previous researches, it was one or a few proteins but not the entirety proteins that were emphasized on the mechanisms of ATRA-resistance. Comparative proteomics can analyze the entire protein expression in cells in whole and has the superiority in screening the drug-resistance proteins differentially expressed. In order to investigate the mechanisms of ATRA-resistance in APL in whole, we compared and analyzed the protein expression profiles between MR2 cells(APL cell line with ATRA-resistance) and NB4 cells(APL cell line with ATRA-sensitiveness) by comparative proteomics. After the total proteins of MR2 cells and NB4 cells were extracted respectively, they were separated by two-dimensional electrophoresis(2-DE). The differences in proteome profile between MR2 cells and NB4 cells analyzed by ImageMaster™ 2D Platinum software. The average protein spots in 2-DE maps of MR2 and NB4 cells were 1160±51 and 1068±33 respectively. 8 protein spots were selected to be identified by Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS), in which the quantity of the protein differentially expressed was more than two times(≥2 or ≤0.5) between MR2 and NB4 cells' 2-DE map. They were all successfully identified and their definite information was obtained. Among them, 6 proteins were probably involved in the mechanisms of ATRA-resistance in APL and they were Cofilin-1, Elongation factor 1-beta (EF-1β), Tropomyosin isoform(TM), High mobility group protein B1(HMGB1), Ran-specific GTPase-activating protein (RanGAP1) and Galectin-1. Moreover, so far there was no related report on the roles of HMGB1, RanGAP1 and Galectin-1 in the mechanisms of ATRA-resistance in APL. These differential proteins identified provide the new clues for us to further elucidate the mechanisms of ATRA-resistance from multiple factor.

PML Protects HIPK2 and p300 from SCF-Mediated Ubiquitin-Dependent Degradation To Activate Transcription.

The Pml gene is the target of t(15;17) chromosome translocation in acute promyelocytic leukemia. PML protein is known to localize in discrete nuclear speckles, named PML nuclear bodies (NBs). In NBs, PML interacts with several transcription factors, such as p53 and AML1, and their co-activators, such as HIPK2 and p300. PML activates transcription of their target genes. PML is thought to stabilize transcription factor complex and function as a mediator in transcription activation, but little is known about the molecular mechanism by which PML activates transcription. To clarify the role of PML in transcription regulation, we purified the PML complex and identified a novel F-box protein (FBP), Skp1, and Cullin1 (Cul1) in the PML complex by LC/MS/MS analysis. FBPs form SCF ubiquitin ligase complexes with Skp1, Cul1 and ROC1 and mediate recognition of specific substrates for ubiquitination. We found that the FBP that we identified here also forms a SCF complex with Skp1, Cul1 and ROC1. To identify substrates for the SCF complex, we tested several proteins that could bind to PML, and found that the FBP promotes degradation of HIPK2 and p300. These degradations were inhibited in the presence of a proteasome inhibitor, MG132. The FBP stimulated ubiquitination of HIPK2. These results suggest that the SCF promotes degradation of these proteins by the ubiquitin-proteasome pathway. The fact that the SCF is a part of the PML complex suggests that PML plays a role in the SCF-mediated degradation of HIPK2 and p300 by the ubiquitin-proteasome pathway. In order to clarify the role of PML in degradation of HIPK2 and p300, we tested effects of PML on the degradation and found that PML inhibited the SCF-mediated degradation of HIPK2 and p300 without inhibition of ubiquitination. To clarify roles of HIPK2, PML IV and the FBP in p53-dependent transcription, we performed reporter analysis using the MDM2 promoter in H1299 cells. Since the FBP promotes degradation of HIPK2, we initially thought that the FBP might inhibit activation of p53-dependent transcription by HIPK2 and PML IV. However, the FBP, HIPK2 and PML synergistically stimulated the p53-dependent transcriptional activation. Taken together our data suggest that the SCF-induced ubiquitination of transcription co-activators HIPK2 and p300 plays a critical role in transcriptional regulation, and that PML stimulates transcription by protecting HIPK2 and p300 from ubiquitin-dependent degradation.

From dissection of disease pathogenesis to elucidation of mechanisms of targeted therapies: leukemia research in the genomic era

Leukemia is a group of heterozygous diseases of hematopoietic stem/progenitor cells that involves dynamic change in the genome. Dissection of genetic abnormalities critical to leukemia initiation provides insights into the elusive leukemogenesis, identifies distinct subsets of leukemia and predicts prognosis individually, and can also provide rational therapeutic targets for curative approaches. The past three decades have seen tremendous advances in the analysis of genotype-phenotype connection of leukemia, and in the identification of molecular biomarkers for leukemia subtypes. Intriguingly, differentiation therapy, targeted therapy and chemotherapy have turned several subtypes of leukemia from highly fatal to highly curable. The use of all-trans retinoic acid and arsenic trioxide, which trigger degradation of PML-RARalpha, the causative fusion protein generated by t (15;17) translocation in acute promyelocytic leukemia (APL), has led to a dramatic improvement of APL clinical outcome. Imatinib mesylate/ Gleevec/STI571, which inhibits the tyrosine kinase activity of BCR-ABL oncoprotein, has now become the new gold standard for the treatment of chronic myeloid leukemia. Optimal use of chemotherapeutic agents together with a stringent application of prognostic factors for risk-directed therapy in clinical trials has resulted in a steady improvement in the treatment outcome of acute lymphoblastic leukemia. Hence, the pace of progress extrapolates to a prediction of leukemia control in the twenty-first century.

A Case of Relapsed Variant Acute Promyelcytic Leukemia with t(11;17)(q23q21), PLZF/RARa Fusion Gene, with Durable Molecular Remission Following an Induction and Consolidation Regimen That Included ATRA with Low Dose G-CSF and Valproic Acid as a Histone Deacetylase Inhibitor.

Background:Acute promyelocytic leukemia (APL) accounts for 10% of acute myeloid leukemias. Greater than 95% of cases are associated with the t(15;17)(q22;q21) translocation with the PML-RARa fusion product which is associated with increased affinity for nuclear co-repressor protein complex and recruitment of histone deacetylase. This leads to an alteration in chromatin conformation and inhibition of transcription and differentiation in promyelocytes in the presence of physiologic levels of retinoic acid. Pharmacologic doses of retinoic acid however are usually required to overcome this repression of transcription and leads to promyelocyte differentiaton and improvement in clinical outcomes in classic APL. A variant translocation in APL, t(11;17) (q23;q21) where the 3′ end of the RARa gene is fused to the 5′ end of a gene called PLZF (promyelocytic leukemia zinc finger), accounts for about 1% of cases. This disease phenotype is associated with refractoriness to pharmacologic doses of retinoic acid.Case Report;52 year-old male presented with fever and rapidly progressive leukocytosis. His HCT was 24%, WBC count was 75,000/mcl and platelet count was 109,000/mcl. He required endotracheal intubation and mechanical ventilation with leukapharesis on his second day of admission due to leukostasis. He had been diagnosed with APL variant with t(11;17) (q23;q21) two years prior to this admission and had induction with Ara-c + daunorubicin (7+3), with an additional cycle for consolidation. He was then placed on maintenance with 6-MP + MTX for 24 months. Bone marrow and peripheral blood analysis confirmed disease relapse. Flow cytometric analysis revealed immature myeloid population CD34−, CD15−, CD13+, CD56+, HLA-DR-, with partial expression of CD33. FISH analyses on peripheral blood was negative for t(15;17) and positive for the variant t(11;17)(q23q21). He was started on induction therapy with Idarubicin 12mg/m2/day for three consecutive days with ATRA 45mg/m2/day (via NGT) + G-CSF 5mcg/kg/day. His Day 21 bone marrow examination revealed complete hematologic response. Consolidation/ maintanance with sodium valproate 500mg po BID (to maintain serum levels of 50–100mcg/ml), ATRA 45mg/m2/day (via NGT) + G-CSF 5mcg/kg/day (with G-CSF dose reductions for leukocytosis with a final stable dose of 30mcg/day). He subsequently achieved a complete cytogenetic remission and relapsed after 18 months on this regimen.Conclusion:Induction of promyelocyte differentiation and reversal of retinoic acid refractoriness in variant APL with t(11;17) (q23;q21) can be achieved with G-CSF priming and the addition of valproic acid s a HDAC inhibitor.

Acute Monocytic Leukemia with t(11;17)(q23;q21) Involving a Rearrangement of Mixed Lineage Leukemia Gene

A case of acute monocytic leukemia (AMoL) by French-American-British (FAB) classification in a 63-year-old male showed the abnormal karyotype 46,XY,t(11;17)(q23;q21), previously reported as a variant translocation in acute promyelocytic leukemia (APL). Fluorescence in situ hybridization (FISH) analysis identified a mixed lineage leukemia (MLL) gene rearrangement, but not visible disruptions of promyelocytic leukemia (PML) or retinoic acid receptor alpha (RARA) genes. We suggest that a certain gene proximal to RARA was rearranged in this case onto a gene close to MLL on chromosome 11q. Now, a few cases of AMoL with a similar translocation have been reported in the literature, and these cases emphasize the importance of cytogenetic and FISH studies in addition to morphology, cytochemistry, and immunophenotype in classifying acute myeloid leukemia (AML).

Pro-proliferative function of the long isoform of PML-RARα involved in acute promyelocytic leukemia

The promyelocytic leukemia (PML) gene codes for a tumor suppressor protein that is associated with distinct subnuclear macromolecular structures called the PML bodies. The PML gene is frequently involved in the t(15;17) chromosomal translocation of acute promyelocytic leukemia (APL). The translocation results in a fusion gene product, PML-RARalpha, in which the PML gene fuses to the retinoic acid receptor alpha (RARalpha) gene. PML-RARalpha has been shown to promote transcriptional repression of genes involved in myeloid terminal differentiation and to disrupt the architecture of PML bodies, a phenotype reversed by treatment with all trans retinoic acid (ATRA). However, there are several alternatively spliced isoforms of PML-RARalpha. Here, we addressed the differences between the short and the long isoforms of PML-RARalpha (L and S) since both are associated with APL. We demonstrate that PML-RARalphaL, but not PML-RARalphaS, can directly promote cell growth by transcriptionally activating the pro-proliferative gene, c-fos, in response to mitogenic stimulation. The activity of the PML-RARalphaL is completely sensitive to ATRA. We further show that this activation is not via direct recruitment of the protein to the c-fos promoter but indirectly by altering the chromosomal environment of the c-fos gene, thereby rendering it more accessible to the signal induced transcriptional activators. Our results suggest that in addition to antagonizing the PML-tumor suppressor or the PML-pro-apoptotic activity, PML-RARalpha proteins can also directly promote cell growth by activating c-fos.