Acute Promyelocytic Leukemia Patient Samples Research Articles

Phase separation of PML/RARα and BRD4 coassembled microspeckles governs transcriptional dysregulation in acute promyelocytic leukemia

In acute promyelocytic leukemia (APL), the promyelocytic leukemia-retinoic acid receptor alpha (PML/RARα) fusion protein destroys PML nuclear bodies (NBs), leading to the formation of microspeckles. However, our understanding, largely learned from morphological observations, lacks insight into the mechanisms behind PML/RARα-mediated microspeckle formation and its role in APL leukemogenesis. This study presents evidence uncovering liquid-liquid phase separation (LLPS) as a key mechanism in the formation of PML/RARα-mediated microspeckles. This process is facilitated by the intrinsically disordered region containing a large portion of PML and a smaller segment of RARα. We demonstrate the coassembly of bromodomain-containing protein 4 (BRD4) within PML/RARα-mediated condensates, differing from wild-type PML-formed NBs. In the absence of PML/RARα, PML NBs and BRD4 puncta exist as two independent phases, but the presence of PML/RARα disrupts PML NBs and redistributes PML and BRD4 into a distinct phase, forming PML/RARα-assembled microspeckles. Genome-wide profiling reveals a PML/RARα-induced BRD4 redistribution across the genome, with preferential binding to super-enhancers and broad-promoters (SEBPs). Mechanistically, BRD4 is recruited by PML/RARα into nuclear condensates, facilitating BRD4 chromatin binding to exert transcriptional activation essential for APL survival. Perturbing LLPS through chemical inhibition (1, 6-hexanediol) significantly reduces chromatin co-occupancy of PML/RARα and BRD4, attenuating their target gene activation. Finally, a series of experimental validations in primary APL patient samples confirm that PML/RARα forms microspeckles through condensates, recruits BRD4 to coassemble condensates, and co-occupies SEBP regions. Our findings elucidate the biophysical, pathological, and transcriptional dynamics of PML/RARα-assembled microspeckles, underscoring the importance of BRD4 in mediating transcriptional activation that enables PML/RARα to initiate APL.

Long Noncoding RNA Profiling Reveals an Abundant Crnde That Inhibits Granulocytic Differentiation in APL

Long noncoding RNAs (lncRNAs) emerge as important factors involved in the regulation of gene expression during normal tissue development. Abnormally expressed lncRNAs lead to the initiation of cancers, and also promote tumor cell proliferation and metastasis in various kinds of cancers. In acute promyelocytic leukemia (APL), it is general accepted that PML/RARα fusion protein acts as a transcriptional repressor, which disrupts the differentiation program, and arrests cells at the promyelocytic stage of myeloid differentiation. However, a significant amount of evidence has demonstrated that PML/RARα is not sufficient to cause APL and additional cooperating events are required for the development of APL. As the key regulators of cancers, the specific expression patterns and functions of lncRNAs involved in APL leukemogenesis are not clear. In our study, we aimed to identify APL-specific lncRNAs and explore the important biological roles of these dysregulated lncRNAs in APL leukemogenesis. To discover the unique dysregulated lncRNAs in APL, we first established a lncRNA identification pipeline by analyzing RNA-seq data of 171 AML patient samples from the TCGA database and totally obtained 3,892 lncRNAs. We further analyzed the differentially expressed lncRNAs in 16 APL patient samples compared with 155 non-APL AML patient samples and identified a total of 414 APL-specific lncRNAs. These unique lncRNAs in APL contained 219 APL-specific upregulated lncRNAs, such as CRNDE, PVT1, MEG3, etc., and 195 APL-specific downregulated lncRNAs, such as HOTAIRM1, NEAT1, HOXA-AS2, etc., which may serve as novel biomarkers for APL. We then evaluated the expression of these dysregulated lncRNAs in response to all- trans retinoic acid (ATRA), which is the first-line drug for APL therapy that efficiently induces APL cells differentiation. We analyzed our RNA-seq data of APL cell line NB4 treated with or without ATRA and performed gene set enrichment analysis (GSEA) to evaluate the potential association of APL-specific lncRNAs with the induction of ATRA treatment. We noticed that highly expressed lncRNAs in APL were significantly enriched in ATRA-downregulated lncRNAs dataset, and the APL-specific downregulated lncRNAs were enriched in ATRA-upregulated lncRNAs dataset. These results suggested that APL-dysregulated lncRNAs could also participate in ATRA-induced granulocytic differentiation. To further identify the possible functional roles of APL-specific dysregulated lncRNAs, we selected one of the APL-specific overexpressed lncRNA, CRNDE, which is involved in the pathogenesis of some solid tumors. We found that the highly expressed CRNDE could be repressed by ATRA in a time dependent manner. Furthermore, functional analysis was performed to identify the potential roles of CRNDE in the leukemogenesis and the differentiation therapy of APL. We designed CRNDE-specific shRNA to downregulate its expression in NB4 cells, and found that silencing of CRNDE could promote differentiation of NB4 cells and enhance ATRA-induced differentiation. Further RNA-seq analysis to explore the mechanism revealed that knockdown of CRNDE could affect the expression of a number of myeloid differentiation-associated genes and signaling pathways. Collectively, our studies have systematically identified a distinct set of lncRNAs in APL, which may serve as novel biomarkers for APL. The further functional analysis of CRNDE reveals its essential roles involved in the leukemogenesis and ATRA-induced differentiation of APL, which provides new insights into the underlying mechanisms of dysregulated lncRNAs in APL. DisclosuresNo relevant conflicts of interest to declare.

PML/RARa blocks the differentiation and promotes the proliferation of acute promyelocytic leukemia through activating MYB expression by transcriptional and epigenetic regulation mechanisms.

The promyelocytic leukemia (PML)/retinoic acid receptor-alpha (RARα) onco-fusion protein that is generated from t(15;17) chromosome translocation is crucial for the leukemogenesis of acute promyelocytic leukemia (APL) and is well documented as a transcriptional repressor. To understand the relationship between PML/RARα and the oncogene in the development of APL, we investigate the regulation mechanism of PML/RARα to MYB proto-oncogene and the role of this regulation on the proliferation and differentiation of APL cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays show that MYB expression was significantly higher in PML/RARα positive cell lines. Microarray data verify that the MYB expression was significantly higher in APL patient samples than in normal promyelocyte samples. Further expression analysis from RT-qPCR and microarray data verifies that the expression of MYB is upregulated by PML/RARα. Transcriptional factor binding analysis shows that MYB is directly bound by PML/RARα and its cofactors. Luciferase assays show that PML/RARα transactivated MYB promoter activity through the RARα binding site and the coexistence of CCAAT enhancer binding protein ε. We also find that PML/RARα increases the acetylation level of the promoter region of MYB. Further evidence demonstrates that PML/RARα regulates MYB expression through long-range interaction. Functionally, PML/RARα increases the cell proliferation and blocks the differentiation through activating MYB expression. Collectively, this study uncovers a novel mechanism of PML/RARα-mediated transcriptional activation and enriches our knowledge of the onco-fusion protein-mediated transcription activation.

Profiling and functional analysis of circular RNAs in acute promyelocytic leukemia and their dynamic regulation during all-trans retinoic acid treatment

Circular RNAs (circRNAs) are a novel class of powerful regulators in gene expression and participate in the pathogenesis of many diseases, including cancer. However, little is known about the roles of circRNAs in the development and treatment of acute promyelocytic leukemia (APL). Here we report the expression profiling and function of circRNAs in APL, including their dynamic regulation during all-trans retinoic acid (ATRA)-induced differentiation. We performed two independent ribosomal RNA-minus RNA-sequencing (Ribo-minus RNA-seq) experiments with and without RNase R treatment on APL patient-derived NB4 cells and identified a total of 4313 circRNAs, including 1098 newly identified circRNAs. Detailed analysis showed that circRNAs expressed in APL cells were mostly exon-derived, not by-products during splicing, and could be distinguished from hematopoietic stem cells, neutrophils and lymphocytes. The true presence and stability of circRNAs were verified both in NB4 cells and primary APL patient samples. Moreover, we conducted a time-series analysis of circRNAs on ATRA-treated NB4 cells and uncovered 508 circRNAs with dynamic expression during ATRA treatment, including 246 upregulated and 262 downregulated. Further evidence demonstrated that the majority of circRNAs were regulated independently of their host linear mRNAs. Detailed functional experiments demonstrated that circ-HIPK2, one of the differentially expressed circRNAs, significantly influenced ATRA-induced differentiation of APL cells. Further mechanistic studies revealed that circ-HIPK2 was located in cytoplasm and served as a sponge for differentiation-associated miR-124-3p. Finally, circ-HIPK2 expression in APL patients was significantly lower than that in normal peripheral mononuclear cells and other subtypes of AML, indicating its potential role as an APL biomarker. Our study indicates the biological functions of circRNAs in the development and treatment of APL, and provides a comprehensive circRNA resource for future studies.

Ex Vivo High-Throughput Flow Cytometry Screening Identifies Subsets of Responders to Differentiation Agents in Individual AML Patient Samples

Background:Prognoses for acute promyelocytic leukemia (APL) patients improved drastically upon the introduction of differentiation therapy with all-trans-retinoic acid (ATRA) in combination with conventional chemotherapy. Unfortunately, this therapeutic approach has not translated to other genetic subtypes of acute myeloid leukemia (AML) where patients demonstrate marked heterogeneity to differentiating agents. To provide improved detection of drug-induced differentiation in AML patients, we have developed a high-throughput, flow cytometry-based personalized medicine platform.Methods:Total white blood cells were isolated from each patient sample by red cell lysis, plated in serum-free media in 384-well format and incubated with drugs for 3 days. Viable cells remaining after each drug treatment were identified and quantified using cell surface marker expression, cell membrane integrity, and morphology (FSC/SSC) to determine the compound's efficacy and specificity against the blast population. Changes in cell surface marker expression and shifts in morphology indicative of blast differentiation were also evaluated with each compound. As a control for ex vivo differentiation, two APL patient samples were treated ex vivo with ATRA and we observed the blasts gaining CD66b expression indicating granulocytic differentiation.Results:A refractory AML patient was identified whose leukemic blasts exhibited a strong differentiating response to dexamethasone treatment ex vivo. This resulted in loss of CD34 expression (a marker of immature blast cells), gain of CD163 expression (a marker of monocytic/macrophage maturation) and a significant change in cellular size and granularity. After being enrolled in a clinical trial (REB: 13-6962-C) the patient was treated based on the assay for 1 week (40 mg/day) with dexamethasone. Post-treatment samples from the peripheral blood and bone marrow of the patient exhibited the same morphological and cell surface marker changes predicted by the ex vivo assay. The CD163+ cells in the patient also gained additional markers of myeloid differentiation (CD11b, CD14, CD16). After additional cytarabine and fludarabine treatment, the patient remains in remission 4 months post-treatment.Conclusions:Following this initial study, we have continued to identify subgroups of both AML and Myelodysplastic Syndrome patients where blasts differentiate in response to dexamethasone, calcitriol, ATRA or other known differentiating agents using unique cell surface markers of monocytic and myeloid maturation. Flow cytometry expression changes correlated with changes in morphology as observed by May-Grunwald Giemsa staining. In the patient described above this included an increase in cytoplasm and vacuoles consistent with monocytic/macrophage differentiation, which positively correlates with CD163 expression. We aim to apply our assay towards the identification of subgroups of AML patients who respond to differentiation therapies and develop clinical trials to combine differentiating agents with chemotherapy. This approach has the potential to extend the clinical success of APL differentiation therapy to AML patients. DisclosuresPrashad:Notable Labs: Employment, Equity Ownership. Western:Notable Labs: Consultancy. Biondi:Notable Labs: Employment. Shah:Notable Labs: Employment. Liu:Notable Labs: Employment, Equity Ownership. Nguyen:Notable Labs: Employment, Equity Ownership. Warnock:Notable Labs: Employment, Equity Ownership. Quinzio:Notable Labs: Employment, Equity Ownership. De Silva:Notable Labs: Employment, Equity Ownership. Schimmer:Novartis: Honoraria. Heiser:Notable Labs: Employment, Equity Ownership.

Coordinated Repression of Cyclin-Dependent Kinase Inhibitors By PML/RARa in Acute Promyelocytic Leukemia

Inappropriate cell proliferation during oncogenesis is often attributed to dysregulation of components of the cell cycle machinery, which is strictly controlled by the activity of cyclins, cyclin-dependent kinases (CDKs) and cyclin-dependent kinases inhibitors (CKI). In our previous study, we have reported that the oncogenic fusion protein PML/RARa directly targets and represses the expression of CDKN2D, one of the CKI member, in acute promyelocytic leukemia (APL). Since different members of CKIs can mediate separate pathways to negatively regulate CDKs so as to control the cell cycle progression, thus implicating that multiple components of CKIs might be affected during the malignant transformation. However, the transcription regulation of the overall machinery of CKIs in APL remains unclear. Here, we report that PML/RARa coordinately represses multiple members of CKIs by directly binding to their regulatory regions. To identify potential CKIs that were dys-regulated in APL cells, we first studied the chromatin status of all the CKIs in APL by chromatin immunoprecipitation combined with next generation sequencing (ChIP-seq) using antibodies against histone modification markers (H3K4me3, H3K4me1 and H3K27me3). We found that the regulatory regions of CDKN2C, CDKN2D and CDKN1A were accessible in APL, suggesting that these CKIs were dynamically regulated in APL. We then performed ChIP-seq using antibody against PML/RARa and found that these three CKIs were occupied with PML/RARa binding, indicating that PML/RARa coordinately regulated CKIs in APL. To further study the role of PML/RARa in transcription regulation of CKIs, we used luciferase reporter assays to detect the effect of PML/RARα on CKIs transcription regulation and found that PML/RARα directly inhibited the promoter activity of CDKN2C and CDKN2D. Furthermore, we demonstrated that ATRA treatment increased the expression of CDKN2C, CDKN2D and CDKN1A through releasing the PML/RARα binding on chromatin in NB4 cells. Ectopic expression of these three CKIs could induce both cell cycle arrest and differentiation. Finally, the finding on the transcriptional regulation of CDKN1A, CDKN2C and CDKN2D in NB4 cells was validated in primary APL patient samples. Collectively, this study emphasizes that PML/RARa represses the CKI machinery and leads to cell cycle progression and differentiation block of APL. The coordinated regulation of multi-factors involved in the same pathway may shed new lights on the study of pathogenesis and target therapy of APL. DisclosuresNo relevant conflicts of interest to declare.

Repression of CDKN2C caused by PML/RARα binding promotes the proliferation and differentiation block in acute promyelocytic leukemia.

Inappropriate cell proliferation during oncogenesis is often accompanied by inactivation of components involved in the cell cycle machinery. Here, we report that cyclin-dependent kinase inhibitor 2C (CDKN2C) as a member of the cyclin-dependent kinase inhibitors is a target of the PML/RARα oncofusion protein in leukemogenesis of acute promyelocytic leukemia (APL).We found that CDKN2C was markedly downregulated in APL blasts compared with normal promyelocytes. Chromatin immunoprecipitation combined with quantitative polymerase chain reaction demonstrated that PML/RARα directly bound to the CDKN2C promoter in the APL patient-derived cell line NB4. Luciferase assays indicated that PML/RARα inhibited the CDKN2C promoter activity in a dose-dependent manner. Furthermore, all-trans retinoic acid treatment induced CDKN2C expression by releasing the PML/RARα binding on chromatin in NB4 cells. Functional studies showed that ectopic expression of CDKN2C induced a cell cycle arrest at the G0/G1 phase and a partial differentiation in NB4 cells. Finally, the transcriptional regulation of CDKN2C was validated in primary APL patient samples. Collectively, this study highlights the importance of CDKN2C inactivation in the abnormal cell cycle progression and differentiation block of APL cells and may provide new insights into the study of pathogenesis and targeted therapy of APL.

Human DMTF1β antagonizes DMTF1α regulation of the p14ARF tumor suppressor and promotes cellular proliferation

The human DMTF1 (DMP1) transcription factor, a DNA binding protein that interacts with cyclin D, is a positive regulator of the p14ARF (ARF) tumor suppressor. Our earlier studies have shown that three differentially spliced human DMP1 mRNAs, α, β and γ, arise from the human gene. We now show that DMP1α, β and γ isoforms differentially regulate ARF expression and promote distinct cellular functions. In contrast to DMP1α, DMP1β and γ did not activate the ARF promoter, whereas only β resulted in a dose-dependent inhibition of DMP1α-induced transactivation of the ARF promoter. Ectopic expression of DMP1β reduced endogenous ARF mRNA levels in human fibroblasts. The DMP1β- and γ-isoforms share domains necessary for the inhibitory function of the β-isoform. That DMP1β may interact with DMP1α to antagonize its function was shown in DNA binding assays and in cells by the close proximity of DMP1α/β in the nucleus. Cells stably expressing DMP1β, as well as shRNA targeting all DMP1 isoforms, disrupted cellular growth arrest induced by serum deprivation or in PMA-derived macrophages in the presence or absence of cellular p53. DMP1 mRNA levels in acute myeloid leukemia samples, as compared to granulocytes, were reduced. Treatment of acute promyelocytic leukemia patient samples with all-trans retinoic acid promoted differentiation to granulocytes and restored DMP1 transcripts to normal granulocyte levels. Our findings imply that DMP1α- and β-ratios are tightly regulated in hematopoietic cells and DMP1β antagonizes DMP1α transcriptional regulation of ARF resulting in the alteration of cellular control with a gain in proliferation.

Delayed Hematopoietic Recovery After Autologous Stem Cell Transplantation In Patients Receiving Arsenic Trioxide-Based Therapy For Acute Promyelocytic Leukemia

Introduction Arsenic trioxide (ATO)-based therapy for acute promyelocytic leukemia (APL) is highly effective and has been incorporated into standard therapeutic algorithms for patients with newly diagnosed and relapsed disease. Therapy for patients with high-risk or relapsed APL also includes high-dose chemotherapy with autologous stem cell transplantation (autoSCT). Both treatments have independently been demonstrated to improve long-term disease-free survival; however, the use of ATO prior to autoSCT has not been well studied. We sought to determine whether use of ATO prior to autologous stem cell collection impacts hematopoietic recovery following autoSCT. Methods Clinical histories of 31 consecutive patients undergoing autoSCT for APL at our institution since 1994 were reviewed. Viable CD34-positive cell recovery from cryo-preserved autograft products was assessed by pre- and post-thaw 7-amino-actinomycin D (7-AAD) staining. Student's t-tests and chi-squared tests were used for statistical analyses. Results A total of 32 autoSCTs were performed in this population. Twenty-four transplants (75.0%) were performed for patients in first complete remission (CR1) versus 8 transplants (25.0%) for patients in second complete remission (CR2). Seven of the 32 transplants (21.8%) were performed for patients who received ATO therapy prior to autologous stem cell collection. One patient received ATO with initial induction and consolidation therapy, 1 patient received ATO with initial consolidation therapy only, and 5 patients received ATO with re-induction therapy for relapsed disease. A significantly higher proportion of patients who received ATO were transplanted in CR2 (71.5% vs 12.0%, p<0.01). There were otherwise no significant differences in clinical characteristics or risk features between the ATO pre-treated and the ATO-naïve transplant recipients, including: median age at diagnosis (45 years vs 44 years, p=0.62), median age at transplant (46 years vs 45 years, p=0.36), white blood cell count >10,000/uL at presentation (28.6% vs 45.8%, p=0.42), mean CD34-positive cells collected (94.5 x106/kg vs 69.9x106/kg, p=0.35), or mean CD34-positive cells infused (41.4x106/kg vs 39.7x106/kg, p=0.90). Four cases (12.5%) of delayed hematopoietic recovery were identified, defined as requiring >16 days after stem cell infusion to recover an absolute neutrophil count (ANC) >1,000/uL. Three of 7 patients (43%) who received prior ATO had delayed hematopoietic recovery, compared with 1 of 24 patients (4.2%) not previously treated with ATO (p<0.01). Compared to ATO-naïve patients, ATO-treated patients experienced significantly longer time to ANC recovery (mean 19.7 days vs 10.4 days, p<0.01; median 14 days vs 10 days). In addition, ATO-treated patients required significantly longer transplant hospitalizations (37.2 days vs 24.6 days, p<0.01). Platelet recovery, defined as a sustained platelet count >50,000/uL, was also significantly delayed in the ATO-treated group (198.8 days vs 36.5 days, p<0.01; median 81 vs 18 days). The only significant predictor of delayed hematopoietic recovery was prior treatment with ATO (75.0% vs 14.2%, p<0.01). Of 7 assessable APL patient samples, the mean viable CD34-positive cell recovery was 39 +/- 25.1%. This was significantly lower than the viable CD34-recovery (71.3 +/- 27.3%, p=0.02) evaluated in a random selection of 11 other cryo-preserved autologous stem cell products from patients with lymphoma (n=2) and multiple myeloma (n=9). Samples from 2 ATO-treated APL patients who experienced delayed hematopoietic recovery exhibited viable CD34-positive cell recoveries of only 3% and 26%. Conclusions There is an association between ATO exposure and delayed hematopoietic recovery after autoSCT for APL, but a causal relationship is not yet established. Because of the increasing frequency with which ATO is being used in the front-line treatment of APL, larger studies are warranted to validate these findings and to determine the potential mechanism of delayed hematopoietic recovery after autoSCT. Lower-than-expected viable CD34-positive cell recovery from APL patients suggests a possible detrimental contribution of prior ATO exposure. Our institution is now prospectively examining pre- and post-thaw CD34-positive cell recovery, viability, and colony-forming assays prior to autoSCT in patients who have previously received ATO. Disclosures: No relevant conflicts of interest to declare.

The limited contribution of DNA methylation to PML-RARα induced leukemia

The limited contribution of DNA methylation to PML-RARα induced leukemia

PU.1 is linking the glycolytic enzyme HK3 in neutrophil differentiation and survival of APL cells

AbstractThe transcription factor PU.1 is a master regulator of myeloid differentiation and function. On the other hand, only scarce information is available on PU.1-regulated genes involved in cell survival. We now identified the glycolytic enzyme hexokinase 3 (HK3), a gene with cytoprotective functions, as transcriptional target of PU.1. Interestingly, HK3 expression is highly associated with the myeloid lineage and was significantly decreased in acute myeloid leukemia patients compared with normal granulocytes. Moreover, HK3 expression was significantly lower in acute promyelocytic leukemia (APL) compared with non-APL patient samples. In line with the observations in primary APL patient samples, we observed significantly higher HK3 expression during neutrophil differentiation of APL cell lines. Moreover, knocking down PU.1 impaired HK3 induction during neutrophil differentiation. In vivo binding of PU.1 and PML-RARA to the HK3 promoter was found, and PML-RARA attenuated PU.1 activation of the HK3 promoter. Next, inhibiting HK3 in APL cell lines resulted in significantly reduced neutrophil differentiation and viability compared with control cells. Our findings strongly suggest that HK3 is: (1) directly activated by PU.1, (2) repressed by PML-RARA, and (3) functionally involved in neutrophil differentiation and cell viability of APL cells.

The significance of low PU.1 expression in patients with acute promyelocytic leukemia

BackgroundAlthough the importance of the hematopoietic transcription factor PU.1 in acute myeloid leukemia (AML) has been demonstrated, the expression of PU.1 in acute promyelocytic leukemia (APL) patient samples awaits further investigation. The current study used APL patient samples to assess the expression pattern of PU.1 in the initiation and progression of APL.FindingsWe used real-time RT-PCR to compare PU.1 expression between de novo APL patient samples and normal blood specimens, and the results indicated that PU.1 expression was significantly lower in newly diagnosed APL patient samples as compared to normal hematopoietic cells. Further evidence showed a significant inverse correlation between the expression level of PML-RARα and that of PU.1. In addition, we analyzed the correlation between PML-RARα and PU.1 expression in a large population of AML patients retrieved from the expression profiles. The results showed that PU.1 expression was lower in patients with APL than other AML subtypes and there was also a trend towards increasing PU.1 expression from AML-M0 to AML-M5, with the exception of AML-M3 (APL). These observations suggested that PU.1 expression was reduced by PML-RARα in APL patients. Furthermore, we measured PU.1 expression in APL-initiating cells isolated from de novo APL patients by side population cell analysis and found that suppression of PU.1 expression occurred concurrently with PML-RARα expression, indicating the pivotal role of PU.1 in APL initiation.ConclusionOur findings provide evidence that low PU.1 expression in APL patients is required for disease initiation and progression.

PML/RARα -Regulated MicroRNA 181b Targets the Tumor Suppressor RASSF1A in Acute Promyelocytic Leukemia

Abstract 1361In the acute promyelocytic leukemia (APL) bearing the translocation t(15;17), all-trans-retinoic acid (ATRA) treatment induces granulocytic maturation and complete remission of leukemia. Several factors are involved in the formation of the leukemic phenotype. Latest studies identified microRNAs as critical players in this network. In a micro array based microRNA screen we could identify miR-181b as downregulated in the APL cell line NB4 by treatment with pharmacological doses of ATRA. Additionally, we showed the transcriptional induction of miR-181b by the APL-associated PML-RARα oncogene which is released after treatment with ATRA. The overexpression of miR-181b by microRNA mimics leads to an inhibition of ATRA induced granulocytic differentiation. Furthermore, the downregulation of miR-181b by locked nucleic acids (LNAs) causes the induction of granulocytic transcription factor C/EBPβ. In a PML/RARα knock in mouse model we could show the enhanced expression of miR-181b. Also, APL patient samples show a significant enhanced expression of miR-181b in comparison to AML patient samples with normal karyotype. In addition, the miR-181b expression was strongly reduced in APL patient samples after ATRA treatment. In a microRNA target search we identified the novel ATRA regulated tumor suppressor RASSF1A as a putative target of miR-181b. In functional studies we showed that enforced expression of miR-181b reduces the protein level of RASSF1A by binding to the 3’UTR of RASSF1A mRNA. Accordingly, RASSF1A protein was enriched after knock down of miR-181b. The role of RASSF1A in ATRA induced differentiation was verified by knock down of RASSF1A protein by specific siRNA: Here we could show the reduction of ATRA induced CD11b expression. In conclusion, we identified miR-181b as a new player in the PML/RARα associated APL. Moreover, we firstly described the miR-181b target RASSF1A as a crucial factor in the ATRA activated granulocytic differentiation program in APL. Our data reveal the importance of deregulated miRNA biogenesis in cancer and may provide novel biomarkers and therapeutic targets in myeloid leukemia. Disclosures:No relevant conflicts of interest to declare.

Identification of Recurrent Submicroscopic Deletions of chromosome1q31.3 As Possible Molecular Prognostic Markers in Acute Promyelocytic Leukemia (APL)

Abstract 420▪▪This icon denotes a clinically relevant abstract Introduction:The characteristic hallmark of acute promyelocytic leukemia (APL) is a balanced reciprocal chromosomal translocation between chromosomes 15 and 17 leading to a fusion product consisting of the promyelocytic leukemia gene (PML) and the retinoic acid receptor alpha (RARA). The PML-RARA fusion product is necessary but not sufficient for the generation of leukemia and it is hypothesized that additional genomic lesions play a role in the pathogenesis of APL. Therefore, we have performed a high density SNP array analysis on 101 APL patient samples to identify new copy number alterations (CNAs) which may be relevant for the biology and prognosis of APL. Patients and Methods: Molecular and clinical outcome analyses were carried out retrospectively on patients diagnosed with APL, whose samples were referred to the molecular laboratory of the Department of Hematology and Oncology of the Medical Faculty Mannheim, University of Heidelberg, Germany between 1997 and 2010. 500 ng of genomic DNA from leukemic blasts per sample were processed according to the Genome Wide Human SNP 6.0 Array protocol (Affymetrix, Santa Clara, CA). The CNAG 3.3 software was used to perform allele-specific copy number analysis with anonymous references. CNAs of special interest were validated to be acquired in leukemia cells by performing allele-specific copy number analysis in matched pair SNP 6.0 array analysis, quantitative real time PCR and direct sequencing of genomic DNA from initial diagnosis and molecular remission samples. Results: We identified 279 acquired CNAs consisting of 185 heterozygous deletions, 87 amplifications and 7 regions of copy number neutral loss of heterozygosity (CNLOH). Besides common chromosomal aberrations such as trisomy 8 or duplications of the long arm of chromosome 8, deletions of 7q or isochromosome ider(17)(q10)t(15;17), numerous novel recurrent micro-deletions were discovered. The most common was a somatically acquired ∼100 kilobase deletion of chromosome 1q31.3 in 13 of 101 (13%) patients. These deletions encompassed the coding regions for the microRNAs mir181a1/b1. In univariate analyses of overall survival (OS) and relapse free survival (RFS) using Logrank tests, we found that patients carrying 2 or more CNAs as compared to 0 or 1 CNA as detected by SNP array had a significantly increased risk of death (p=0.016) and relapse (p=0.019). Patients carrying the recurrent deletion of chromosome 1q31.3 as compared to patients not carrying this deletion had a significantly increased risk of relapse (p=0.005), a markedly higher number of CNAs (median 8 vs. 2, p < 0.0001) and significantly higher white blood cell counts (WBC) at initial diagnosis (median 2550/μl vs. 16900/μl, p=0.009). We performed a multivariable analysis using Cox proportional hazards models to evaluate power of CNAs detected by SNP-array and deletions of chr. 1q31.3 as possible independent prognostic markers in APL as compared to age, WBC, platelet count (PC) and FLT3 mutational status. For the full model of OS only age and the number of CNAs detected by SNP-A met a 0.10 level of entry into the model and confirmed that the group of patients with 2 or more CNAs represent a subgroup with inferior outcome (“2 or more lesions”: hazard ratio = 5.942, p = 0.0015, age: hazard ratio = 1.08, p < 0.0001). Of note, for the analysis of RFS the presence of a chr. 1q31.3 deletion was the only effect, which met the entry level into the model of RFS and therefore was identified as a new strong predictor for an increased risk of relapse (“presence of del1q31.3”: hazard ratio = 28.9, p = 0.0031). Conclusion: The profiles of submicroscopic CNAs in APL patients are heterogeneic and may serve as strong independent prognostic markers for disease risk definition. Recurrent submicroscopic deletions of chr. 1q31.3 in leukemia cells of APL patients were associated with an increased number and characteristic pattern of further newly identified CNAs, unfavorable laboratory parameters and a higher risk of relapse. The number of CNAs was shown to be predictive for early death rate and overall survival. The further pursuit of these new potential molecular markers is highly warranted as they could refine the current risk stratification of APL by identifying new subgroups of patients, who could possibly gain from adapted treatment strategies. Disclosures:No relevant conflicts of interest to declare.

Deregulation of Transcription Factors GATA-1, GATA-2 and C/EBPa in Acute Promyelocytic Leukemia.

Acute promyelocytic leukemia (APL) accounts for ~10% of all acute myelogenous leukemia (AML) cases, and is characterized by accumulation of abnormal promyelocytes in the bone marrow. APL is uniquely associated with balanced chromosomal translocations involving the retinoic acid receptor alpha (RARA) locus. A functional chimeric protein, X-RARA, is created, whose N-terminus is derived from the partner (“X”) gene, and retains its oligomerization domain. The C-terminus is derived from RARA. X-RARA contributes to the APL phenotype by interfering with granulocyte differentiation, through acquisition of novel transactivating activity and/or interference with the normal functions of RARA and X. Several studies have indicated that defective retinoid signaling, though sufficient to block myeloid differentiation, cannot induce a leukemia. Thus, pathways external to retinoid signaling must be deregulated by X-RARA in order to give rise to APL. We previously reported whole genome gene expression analysis of the hCG-NuMA-RARA transgenic mouse model (Sukhai et al, Blood, Blood , 2006: a2247), and that the NuMA-RARA fusion protein deregulated a wide array of hematopoietic and myeloid transcription factors in leukemic cells derived from transgenic mice. Here, we report that the deregulated expression of this transcription factor set (specifically, Gata-1, Gata-2, C/ebpa and Pu.1) accurately distinguished leukemic TM mice from WT animals, and was dependent upon both the presence of functional RXRA (Sukhai et al, Oncogene, 2008) and transgene dosage. We thus formulated the hypothesis that NuMA-RARA initiated the deregulation of a range of transcription factors, which in turn were responsible for deregulating pathways within the cell. In order to determine whether this transcription factor signature was unique to APL, we focused on GATA-1, GATA-2, PU.1 and C/EBPA, and examined their expression in a range of AML cell lines. Strikingly, the over-expression of GATA-1 was unique to APL cell lines and the OCI/AML4 cell line. GATA-2 was over-expressed in most cell systems tested, suggesting that its up-regulation may play a general role in leukemogenesis. C/EBPA was under-expressed in NB4 cells specifically, while PU.1 was not significantly deregulated in any cell system tested. Having identified that APL cell lines recapitulated our observation of deregulated myeloid transcription factor expression in transgenic mice, we sought to extend these studies to human patients. We analyzed the expression of GATA- 1, GATA-2, PU.1 and C/EBPA in a series of 12 APL patients, 10 AML patients with a range of diagnoses, and 12 normal BM samples. We observed specific up-regulation of GATA-1 (2.0–20.0-fold change, 7/12 patients) and GATA-2 (2.0–25.0-fold change, 9/12 patients) and under-expression of C/EBPA (0.05–0.5-fold change, 11/12 patients), in APL, but not AML, patient samples, in comparison to normal BM. The deregulated expression of these three transcription factors could accurately distinguish APL patient samples from AML and normal BM in both principle component analysis and hierarchical clustering analysis. We therefore report herein that NuMA-RARA deregulated a wide array of myeloid transcription factors, suggesting that a state of globally deregulated myeloid transcription exists in APL cells. Furthermore, a specific subset of transcription factors, GATA-1, GATA-2 and C/EBPA, can be used to specifically classify APL patient samples. These deregulated transcription factors may therefore serve as potential therapeutic targets and a means of distinguishing APL from other forms of acute myelogenous leukemia.