PML-RARA Fusion Research Articles

The Small Molecule Img-98, a Potent and Selective Inhibitor of the Lysine Demethylase Lsd-1, Effectively Augments the Pro-Differentiation Effects of ATRA in a Pre-Clinical Model of AML

Acute Promyelocytic Leukemia (APL) is a cytogenetically unique subtype of acute myeloid leukemia (AML), characterized by the presence of the t(15;17)-associated PML-RARA fusion gene. This disease is curable in most patients with all-trans-retinoic acid (ATRA) based therapies, which effectively differentiate malignant promyelocytes. In patients with non-APL AML, most patients with die from their disease and ATRA has little activity. Therefore, research strategies that seek to extend the efficacy of ATRA-based treatment in AML are key avenues of investigation. From our previous studies, an epigenetic analysis of primary AML samples revealed that relative to normal CD33+ cells, loss of RARα2 expression in AML is associated with a reduction in H3K4me2 on the RARA2 promoter (a modification that is associated with transcriptional activation). The mono- and di-methyl lysine demethylase LSD1 (KDM1A) is highly expressed in patients with AML, and its overexpression has been implicated in various other tumors. Based on these data we correctly predicted that the use of small-molecule inhibitors targeting LSD1 (LSD1i) could result in epigenetic reprogramming that enhanced or facilitated the execution of the ATRA-induced differentiation program in AML cells.In the current study, we characterized a range of small molecule inhibitors of LSD-1. All the agents tested (RN-1, GSKi, SP2509, TCP, IMG-98 and OG-L002) led to inhibition of LSD-1 in a biochemical assay with varying degrees of potency. From this study, we further characterized the anti-tumor effects of IMG-98 alone and in combination with ATRA. IMG-98 is a novel LSD1 inhibitor relative to drugs of this class with comparatively different specificity, potency, pharmacokinetics, and metabolism. Its greater heavy atom count and chemical complexity contribute to these properties. By fluorine nuclear magnetic resonance (fNMR) and florescent spectrophotometry, the molecule rapidly reacts irreversibly with the FAD co-factor of LSD1 and this polypeptide is necessary to catalyze the reaction. Thermal stability shifts show the inactivated form of the enzyme becomes much more stable suggesting significant structural changes. Treatment with IMG-98 promoted the expression of the cell surface marker CD11b, associated with a differentiated immunophenotype, in both AML cell lines and primary patient material. IMG-98 produced a potent anti-proliferative effect across a range of AML cell lines and also led to growth inhibition of AML blast colony forming ability. In combination studies with ATRA, IMG-98 re-sensitized AML cells to ATRA by reactivating ATRA driven differentiation programs. Post-differentiation apoptosis was more significant for combined therapy (ATRA + IMG-98) than with either agent alone. Heatmap display of unsupervised hierarchical clustering of genes in AML cell lines differentially expressed in response to treatment with combinations of ATRA, IMG-98 or the combination, confirmed that ATRA combined with IMG-98 enhanced the expression of a subset of genes associated with the myeloid differentiation program. Updated studies on mechanisms underpinning mode of action of IMG-98 in this model will be presented.Taken together, these data demonstrate that ATRA combined with pharmacological inhibition of LSD1, may provide a promising treatment for AML by promoting differentiation and subsequent growth inhibition of AML blasts. A closely related molecule to IMG-98 is currently being optimized in late preclinical development, and clinical trials with this compound are anticipated to start in 2016. [Display omitted] DisclosuresRienhoff:Imago: Employment.

Inhibition of the Histone Demethylase LSD1 Combined with Caloric Restriction or IGF1/Insulin Inhibition Leads to Durable Responses in a Preclinical Model of Acute Myeloid Leukemia

Introduction. There is increasing interest in therapeutic modulation of metabolic pathways in cancer. Tumor cells preferentially use aerobic glycolysis to meet their energetic demands. However, glycolysis inhibition alone is unable to bring durable responses because of limited therapeutic index and because of previously underappreciated metabolic adaptability in tumor cells, which can switch to alternative substrate usage when specific nutrients are limiting. The molecular basis of metabolic adaptation is poorly understood. Recently, the histone demethylase LSD1 (Lysine-Specific Demethylase 1) has been implicated in the control of oxidative phosphorylation (OXPHOS) in adipocytes through its interaction with NRF1 (Nuclear Respiratory Factor 1), a master regulator of metabolic gene transcription (1). We hypothesized that LSD1 could regulate metabolic adaptability and be a therapeutic target upon metabolic modulation through Caloric Restriction (CR) in Acute Myeloid Leukaemia (AML) and specifically in APL (Acute Promyelocytic Leukaemia), which we showed to be sensitive to body fatness in the clinic (2).Methods. APLs were generated in mice expressing the PML-RARa fusion under the control of the Cathepsin G promoter (3). Primary leukemias were transplanted into recipients subjected to 30% CR or Standard Diet (SD). We scored the effect of CR alone or in combination with the LSD1 inhibitor IEO368 (4) on mouse survival, Leukemia Initiating Cell (LIC) frequency and epigenomic, transcriptomic and metabolic parameters.Results. Compared to SD controls, CR-fed recipients experienced an initial dramatic decrease in the total leukemic burden accompanied by cell cycle slowdown (“adaptation phase”); this was followed by a delayed disease progression that brought animals to death (“terminal phase”) (median survival 91 vs 51 days, p=0.038). Limiting-dilution transplantation of CR-conditioned leukemias revealed increased frequency of LICs (estimated frequency 1/3064 cells in SD vs 1/947 in CR, p=0.003) and increased aggressiveness (median survival reduced to 49 vs 70.5 days with 5000 cells injected, p<0.0001). Thus, CR limits the expansion of leukemic cells but enriches for cells with increased ability to regrow.RNAseq of leukemic cells purified during the terminal phase (but not earlier) showed that a dramatic transcriptional reprogramming in CR, characterized by upregulation of genes controlling OXPHOS, Krebs cycle and nucleotide and protein biosynthesis, and downregulation of insulin signaling and glucose transporters. Flow cytometry with Mitotracker Red confirmed increased mitochondrial activity. Thus, leukemic cells exposed to CR put in place adaptive transcriptional changes to allow survival in a nutrient/growth factor deprived environment.To investigate the basis of these transcriptional changes, we revised ChIPseq analysis of LSD1 binding in human APL cell lines and found a significant enrichment for i) NRF1 consensus binding motif and ii) promoters of genes encoding for OXPHOS and Krebs cycle enzymes. NRF1 binding to OXPHOS/Krebs enzymes was confirmed on mouse leukemias by ChIPseq. These data suggested that the CR-induced adaptive changes could be mediated by LSD1/NRF1.Strikingly, co-treatment of leukemic mice with CR and our LSD1 inhibitor IEO368 (4) resulted in macroscopic and microscopic eradication of disease (see figure, p=0.0018 compared to SD). In these conditions, leukemic cells completely disappeared in 4/6 mice after 4 weeks. LSD1 inhibition alone was also effective but did not produce bona fide disease eradication. Importantly, some of the features of the CR-LSD1 interaction could be modeled by combining LSD1 and an IGF1/Insulin inhibitor. In vivo, this combination was synergistic and led to durable responses (median survival 121 vs 50 days in untreated controls, p=0.0143, vs 65.5 and 78.5 days with Insulin/IGF1 Inhibitor and IEO368 respectively).Conclusion: the combination of LSD1 inhibition and insulin/IGF1 signaling reduction by pharmacological or dietary intervention appears as a highly effective therapeutic strategy and deserves further investigation. Ongoing preclinical studies will verify its applicability to other models of AML.

Qualitative and Quantitative Correlation of PML-Rara Fusion Transcript from Peripheral Blood and Bone Marrow Samples By Quantitative Real-Time PCR in Patients with Acute Promyelocytic Leukemia

Introduction: Acute promyelocytic leukemia (APL) is biologically distinct subtype of acute myeloid leukemia characterized by PML-RARA fusion transcripts, and its survival has significantly improved with all-trans retinoic acid (ATRA). Monitoring PML-RARA fusion transcripts with quantitative real-time polymerase chain reaction (RT-PCR) is the standard of care for the evaluation of response in patients with acute promyelocytic leukemia. We sought to determine the correlation between peripheral blood (PB) and bone marrow (BM) samples in patients treated on our frontline studies.Methods: We correlated results from BM and PB samples obtained from patients with newly diagnosed APL treated on three consecutive prospective clinical trials of combination of arsenic trioxide (ATO) and ATRA with or without gemtuzumab ozogamycin (ID01-014; NCT01409161; and NCT00413166) at our institution. Qualitative RT-PCR was performed on reverse-transcribed RNA from PB and BM samples for the short and long isoforms of PML-RARA, and the percent ratios of PML-RARA to ABL1 transcript levels were calculated. The sensitivity of detection with RT-PCR was 1 in 100,000. For this analysis, we selected samples with the time interval of collection within 1 day. Qualitative correlation was assessed using Kendall tau rank correlation coefficient test. Spearman's correlation coefficient was computed for quantitative variables to measure the extent of the association between samples. P values were two-sided and a p value of <0.05 was considered as statistically significant.Results: From July 2002 to May 2015, 184 patients were enrolled in the clinical trials. RT-PCR for PML-RARA was performed in 2077 samples including 1261 BM samples and 816 PB samples. In total, 584 samples (292 sample pairs) from BM and PB were identified within 1-day time interval. PML-RARA levels from PB samples with RT-PCR were strongly qualitatively and quantitatively correlated with those from BM samples (r= 0.831, p<0.001; τ= 0.792, p<0.001, respectively). Minimal qualitative discrepancy was observed in 13 samples from 11 patients: 8 BM samples from 8 patients with PML-RARA/ABL1 ≤0.01% (4 short form; 4 long form), with undetectable PML-RARA from concurrent PB; and 5 PB samples from 4 patients with PML-RARA/ABL1 ≤0.01% (2 short form; 3 long form), with undetectable PML-RARA from concurrent BM samples. Of 8 BM samples, 4 BM samples were obtained after 2 cycles, 3 cycles, 3 cycles, and 4 cycles of ATRA + ATO, respectively; 4, 1 month, 9 months, 9 months, and 10 months after completion of ATO + ATRA. The discrepancy of 4 BM samples during ATRA + ATO was resolved with undetectable PML-RARA level from peripheral blood samples and bone marrow samples after 1 additional cycle of ATO + ATRA. Of 4 BM samples after completion of ATO + ATRA, previous PML-RARA showed undetectable PML-RARA level, and repeat PML-RARA showed undetectable PML-RARA level from bone marrow and peripheral blood samples without intervention. Of 5 PB samples from 4 patients, 3 PB samples from 2 patients were obtained after 3 cycles, 3 cycles, and 4 cycles of ATRA. One patient had persistent PML-RARA/ABL1 <0.01% (short form) from peripheral blood samples with undetectable PML-RARA from concurrent BM samples after 3 cycles and 4 cycles of ATO + ATRA. Two PB samples from 2 patients were obtained 1 month after completion of ATO + ATRA.Conclusions: Strong correlation between PB and BM results indicate that PML-RARA fusion transcript levels in patients with APL can be monitored using PB samples. [Display omitted] DisclosuresVerstovsek:Incyte Corporation: Research Funding. Estrov:incyte: Consultancy, Research Funding. Cortes:Novartis: Consultancy, Research Funding; Teva: Research Funding; BerGenBio AS: Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy.

Varying Responses of PML-Rara with Different Genetic Mutations to Arsenic Trioxide

Resistance to arsenic and/or ATRA is a challenging problem in the clinical management of acute promyelocytic leukemia (APL). Acquired genetic mutations in the PML moiety of the PML-RARA fusion gene have been found to exist virtually in APL patients at relapse. While C212/C213 mutations in the PML B2 domain of PML-RARA were shown to be capable of abolishing the effect of arsenic on target proteins in vitro, A216V and L218P mutations in the PML portion of PML-RARA were detectable in two APL patients resistant to arsenic therapy. More recently, we identified a panel of point mutations (including previously reported A216V and, newly identified S214L, A216T, L217F and S220G) from nine of 13 APL patients with arsenic resistance. These findings indicated the existence of a mutational hotspot (S214-S220) within the PML-RARA oncoprotein among APL patients at relapse. Whether each of the identified point mutations plays a role and functions distinctly in the mechanism underlying the resistance to arsenic therapy is of particular interest.Here, we performed in vitro functional analyses in both the hematopoietic irrelevant and relevant cells, and a retrospective analysis of APL patients to investigate the effect of PML-RARA mutations on mediating the resistance to arsenic trioxide. Among the five point mutations in the PML part of PML-RARA identified in patients with relapsed APL, we found that A216V, S214L and A216T mutations could attenuate the negative regulation of arsenic on PML-RARA, which resulted in the retention of oncoproteins. In contrast, L217F and S220G mutations function weakly in this context. Furthermore, we demonstrated that high concentration of As2O3could overcome the primary arsenic resistance driven by the indicated point mutations in PML-RARA. In addition to providing more evidence to reinforce the correlation of genetic mutations in PML-RARA with arsenic efficacy, we provide a novel insight into the functional difference of acquired mutations of PML-RARA both in vitro and in the clinical setting. Our findings may help predict the prognosis and select more effective strategies during APL therapy. DisclosuresNo relevant conflicts of interest to declare.

Rapid diagnosis of acute promyelocytic leukemia with the PML-RARA fusion gene using a combination of droplet-reverse transcription-polymerase chain reaction and instant-quality fluorescence in situ hybridization

BackgroundAcute promyelocytic leukemia (APL) with the PML-RARA fusion gene can be effectively cured using molecular-targeted therapies, which require both detection and quantification of the PML-RARA fusion gene. Here, we developed a rapid assay for identifying and measuring the PML-RARA fusion gene in patients with APL using droplet-reverse transcription-polymerase chain reaction (droplet-RT-PCR) and instant quality-fluorescence in situ hybridization (IQ-FISH). MethodsRNA for droplet-RT-PCR and fixed-cell suspensions for IQ-FISH were prepared from five patients with APL and three controls. We evaluated the amplification efficiency and reaction time with droplet-RT-PCR and signal clarity and hybridization time with IQ-FISH. ResultsThe reaction using droplet-RT-PCR was completed in 26min. The PML-RARA fusion gene was detected in all samples from the five patients. IQ-FISH yielded clear signals after 1h of hybridization. There were no significant differences in signal clarity or positive signal ratios between IQ-FISH and conventional FISH. ConclusionsSimultaneous droplet-RT-PCR and IQ-FISH, in addition to morphological examination of blood smears, can be used to diagnose patients as having APL within 4h based on molecular/cytogenetic results. Rapid diagnosis can allow effective therapies to be started promptly.

Abstract C133: Aresenic Trioxide as targeted therapy for cancers harboring TRIM33-RET -fusion proteins

Abstract Chromosomal rearrangements are genomic events that can result in oncogenic gene fusions that are important drivers of tumerogenesis in multiple human cancers. Gene re-arrangements involving RET proto-oncogene were initially reported in papillary thyroid carcinoma. Recent genomic sequencing analysis have identified several rearrangements involving RET gene in other solid cancer types including lung cancers. The fusion protein resulting from the rearrangements usually contain the intact tyrosine kinase domain of RET fused to a partner protein which provides a constitutive dimerization domain. One such rearrangement involved Tripartite repeat motif containing protein 33 (TRIM33) where exons 1-14 of TRIM33 was fused with exon 12-19 of RET which was in frame. Rearrangements involving TRIM33 and RET have been reported in both thyroid cancer and lung cancer. Although these cancers respond to RET inhibitors, the responses are not durable, demonstrating the need for additional treatment approaches to these cancers. Arsenic, an ancient drug originally used in traditional Chinese medicine, shows remarkable anticancer activity in patients with acute promyelocytic leukemia (APL) and is now part of the standard therapy of this disease. Several studies have given new insight into the mechanism of action and specificity of AS2O3 in treatment of APL. AS2O3 promotes degradation of fusion protein, PML-RAR α, a fusion protein containing PML zinc finger protein and retinoic acid receptor alpha. The protein degradation is triggered by arsenic binding to cysteine residues in RBCC domain of PML and PML-RARα. Interestingly PML and TRIM33 both belong to same family of TRIM proteins and have highly conserved RBCC domains, suggesting that arsenic may also induced degradation of TRIM33-fusion proteins. To evaluate the effect of arsenic trioxide on TRIM33-RET fusion proteins, we first synthesized the in-frame fusion gene, and demonstrated that expression of TRIM33-RET fusion protein in primary cell lines leads to transformation. Cells transformed by TRIM33-RET fusion were highly sensitive to arsenic treatment, compared with similar cells expressing other gene fusions. Acute treatment with arsenic induced ubiquitiylation and degradation of TRIM33-RET fusion protein. The detailed molecular mechanism of arsenic-induced degradation of the TRIM33-RET fusion and possible role of such treatment in treating cancers with such rearrangements will be presented and discussed. These data suggest that arsenic trioxide, which is now used to target PML-RARa fusion protein in APML, may also be effective in treatment of solid-tumors driven by TRIM33-containing fusion proteins. Citation Format: Atul Kulkarni, Jeremy Tang, Kim Hirshfield, Lorna Rodriguez, Shridar Ganesan. Aresenic Trioxide as targeted therapy for cancers harboring TRIM33-RET -fusion proteins. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C133.

Myeloid neoplasm demonstrating a STAT5B-RARA rearrangement and genetic alterations associated with all-trans retinoic acid resistance identified by a custom next-generation sequencing assay.

We describe the case of a patient presenting with several weeks of symptoms related to pancytopenia associated with a maturation arrest at the late promyelocyte/early myelocyte stage of granulocyte differentiation. A diagnosis of acute promyelocytic leukemia was considered, but the morphologic features were atypical for this entity and conventional tests for the presence of a PML-RARA fusion gene were negative. Additional analysis using a custom next-generation sequencing assay revealed a rearrangement producing a STAT5B-RARA fusion gene, which was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and supplementary cytogenetic studies, allowing the diagnosis of a morphologically atypical form of acute promyelocytic leukemia to be made. Analysis of the sequencing data permitted characterization of both chromosomal breakpoints and revealed two additional alterations, a small deletion in RARA exon 9 and a RARA R276W substitution, that have been linked to resistance to all-trans retinoic acid. This case highlights how next-generation sequencing can augment currently standard testing to establish diagnoses in difficult cases, and in doing so help guide selection of therapy.

Arsenic trioxide and all-trans retinoic acid treatment for acute promyelocytic leukaemia in all risk groups (AML17): results of a randomised, controlled, phase 3 trial

Acute promyelocytic leukaemia is a chemotherapy-sensitive subgroup of acute myeloid leukaemia characterised by the presence of the PML-RARA fusion transcript. The present standard of care, chemotherapy and all-trans retinoic acid (ATRA), results in a high proportion of patients being cured. In this study, we compare a chemotherapy-free ATRA and arsenic trioxide treatment regimen with the standard chemotherapy-based regimen (ATRA and idarubicin) in both high-risk and low-risk patients with acute promyelocytic leukaemia. In the randomised, controlled, multicentre, AML17 trial, eligible patients (aged ≥16 years) with acute promyelocytic leukaemia, confirmed by the presence of the PML-RARA transcript and without significant cardiac or pulmonary comorbidities or active malignancy, and who were not pregnant or breastfeeding, were enrolled from 81 UK hospitals and randomised 1:1 to receive treatment with ATRA and arsenic trioxide or ATRA and idarubicin. ATRA was given to participants in both groups in a daily divided oral dose of 45 mg/m(2) until remission, or until day 60, and then in a 2 weeks on-2 weeks off schedule. In the ATRA and idarubicin group, idarubicin was given intravenously at 12 mg/m(2) on days 2, 4, 6, and 8 of course 1, and then at 5 mg/m(2) on days 1-4 of course 2; mitoxantrone at 10 mg/m(2) on days 1-4 of course 3, and idarubicin at 12 mg/m(2) on day 1 of the final (fourth) course. In the ATRA and arsenic trioxide group, arsenic trioxide was given intravenously at 0·3 mg/kg on days 1-5 of each course, and at 0·25 mg/kg twice weekly in weeks 2-8 of course 1 and weeks 2-4 of courses 2-5. High-risk patients (those presenting with a white blood cell count >10 × 10(9) cells per L) could receive an initial dose of the immunoconjugate gemtuzumab ozogamicin (6 mg/m(2) intravenously). Neither maintenance treatment nor CNS prophylaxis was given to patients in either group. All patients were monitored by real-time quantitative PCR. Allocation was by central computer minimisation, stratified by age, performance status, and de-novo versus secondary disease. The primary endpoint was quality of life on the European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30 global health status. All analyses are by intention to treat. This trial is registered with the ISRCTN registry, number ISRCTN55675535. Between May 8, 2009, and Oct 3, 2013, 235 patients were enrolled and randomly assigned to ATRA and idarubicin (n=119) or ATRA and arsenic trioxide (n=116). Participants had a median age of 47 years (range 16-77; IQR 33-58) and included 57 high-risk patients. Quality of life did not differ significantly between the treatment groups (EORTC QLQ-C30 global functioning effect size 2·17 [95% CI -2·79 to 7·12; p=0·39]). Overall, 57 patients in the ATRA and idarubicin group and 40 patients in the ATRA and arsenic trioxide group reported grade 3-4 toxicities. After course 1 of treatment, grade 3-4 alopecia was reported in 23 (23%) of 98 patients in the ATRA and idarubicin group versus 5 (5%) of 95 in the ATRA and arsenic trioxide group, raised liver alanine transaminase in 11 (10%) of 108 versus 27 (25%) of 109, oral toxicity in 22 (19%) of 115 versus one (1%) of 109. After course 2 of treatment, grade 3-4 alopecia was reported in 25 (28%) of 89 patients in the ATRA and idarubicin group versus 2 (3%) of 77 in the ATRA and arsenic trioxide group; no other toxicities reached the 10% level. Patients in the ATRA and arsenic trioxide group had significantly less requirement for most aspects of supportive care than did those in the ATRA and idarubicin group. ATRA and arsenic trioxide is a feasible treatment in low-risk and high-risk patients with acute promyelocytic leukaemia, with a high cure rate and less relapse than, and survival not different to, ATRA and idarubicin, with a low incidence of liver toxicity. However, no improvement in quality of life was seen.

Correlation between peripheral blood and bone marrow samples for detection of PML-RARA fusion transcripts by quantitative PCR in patients with acute promyelocytic leukemia

Correlation between peripheral blood and bone marrow samples for detection of PML-RARA fusion transcripts by quantitative PCR in patients with acute promyelocytic leukemia

Acute promyelocytic leukemia with isochromosome 17q and cryptic PML-RARA successfully treated with all-trans retinoic acid and arsenic trioxide

Acute promyelocytic leukemia (APL) is a subtype of acute leukemia that is characterized by typical morphology, bleeding events and distinct chromosomal aberrations, usually the t(15;17)(q22;q21) translocation. Approximately 9% of APL patients harbor other translocations involving chromosome 17, such as the t(11;17)(q23;q21), t(5;17)(q35;q12-21), t(11;17)(q13;q21), and der(17). All-trans retinoic acid (ATRA) and arsenic trioxide (ATO) have specific targeted activities against the PML-RARA fusion protein. The combination of ATRA and ATO is reportedly superior to chemotherapy and ATRA as induction therapy for APL. The clinical significance of non-t(15:17) APL-related aberrations is controversial, with conflicting reports regarding sensitivity to modern, targeted therapy. Isochromosome 17q (iso(17q)) is rarely associated with APL and usually occurs concurrently with the t(15:17) translocation. No published data is available regarding the efficacy of ATO-based therapy for APL patients who harbor iso(17q). We report on an APL patient with iso(17q) as the sole cytogenetic aberration and a cryptic PML-RARA transcript, who was treated with ATRA and ATO after failure of chemotherapy and achieved complete remission. To our knowledge, this is the first published report of APL associated with iso(17q) as the sole cytogenetic aberration, which was successfully treated with an ATO containing regimen.

APL-The Age-Old Harlequin: Do We See it All?

Dear Sir, Acute promyelocytic leukemia (APL) is a unique type of acute myeloid leukemia characterized by distinct cytomorphological features, immunophenotype, molecular rearrangement t(15;17) involving the PML and RARa genes and a very good response to all-trans retinoic acid (ATRA) [1]. However in rarity of cases, no evidence for rearrangement of RARa is found indicating that alternative mechanisms could mediate the differentiation block that typifies this disease. Here we describe a case of APL, with classical morphological and immunophenotypic features, which was of interest because of presence of isochromosome(17) along with the absence of rearrangement of RARa. APL hence always proves to be a harlequin that takes various molecular and morphological masquerades. An 11-year-old male child presented at our centre with high grade fever and cervical lymphadenopathy for 2 months duration. Baseline laboratory investigations showed TLC of 14,800/lL with 85 % abnormal promyelocytes with hemoglobin of 7.7 gm% and platelet count of 20,000/lL (Fig. 1). On flow cytometry, abnormal promyelocytes were positive for cMPO, CD117, CD13, CD33, CD9, CD64 and CD65. These cells were negative for CD34, CD2, cCD79a, CD3, CD11b, CD18, CD14, CD38, CD56, CD4 and HLA-DR. A diagnosis of APL was made and genetic studies performed. RT-PCR was negative for PML–RARa fusion transcript. Metaphase karyotyping revealed 45–48,XY,i(17)(q10), -6, ?8, -14, -21, ?21[cp10] (Fig. 2). ATRA based combination chemotherapy with Daunomycin was given. Patient was in morphological remission at day 28 of induction and at the end of first consolidation cycle. After completion of consolidation phase, patient received maintenance therapy and is doing well at a follow up of 4 months. APL can have varied clinical and morphogenetic presentation. Besides the conventional reciprocal translocation t(15;17)(q22;q21) disrupting the PML and RARa genes, other chromosomal abnormalities such as t(11;17) (q23;q21), t(5;17)(q35;q12–21), t(11;17)(q13;q21) and der(17) are also observed in APL whereby RARa is fused to the PLZF, NPM, NuMA, and STAT5b genes, respectively. An isochromosome of the long arm of the derivative chromosome 17 is rarely observed in APL patients. However our case is further rare as no evidence for rearrangement of RARa was found. Only 3 such cases have been reported previously in literature [2–4]. Two of these three patients responded poorly to therapy and died within a year [2, 4]. The prognosis of third cases is not known [3]. Our case responded to standard doses of ATRA and BM was in morphological remission at day 45. Consolidation and maintenance was uneventful and is doing well on maintenance. The present case hence highlights the importance of immune-morphological and genetic basis of APL. These cases can still be managed by ATRA which shows that differentiation block by any molecular mechanism in APL can still be overcome by its therapeutic doses as in our case. Management and prognosis of such patients remains unknown as there is not much literature on management of such patients. The lack of independent prognostic value of additional chromosomal abnormalities in APL does not support the use of alternative therapeutic strategies when such abnormalities are found [5]. ATRA however remains A. Misra S. Soni A. Dutta R. Kumar A. Chopra (&) Laboratory Oncology, BRA IRCH, AIIMS, New Delhi, India e-mail: chopraanita2005@gmail.com

High-Risk Microgranular Acute Promyelocytic Leukemia with a Five-Way Complex Translocation Involving PML-RARA.

Acute promyelocytic leukemia (APL) is classically characterized by chromosomal translocation (15;17), resulting in the PML-RARA fusion protein leading to disease. Here, we present a case of a 50-year-old man who presented with signs and symptoms of acute leukemia with concern for APL. Therapy was immediately initiated with all-trans retinoic acid. The morphology of his leukemic blasts was consistent with the hypogranular variant of APL. Subsequent FISH and cytogenetic analysis revealed a unique translocation involving five chromosomal regions: 9q34, 17q21, 15q24, 12q13, and 15q26.1. Molecular testing demonstrated PML/RARA fusion transcripts. Treatment with conventional chemotherapy was added and he went into a complete remission. Given his elevated white blood cell count at presentation, intrathecal chemotherapy for central nervous system prophylaxis was also given. The patient remains on maintenance therapy and remains in remission. This is the first such report of a 5-way chromosomal translocation leading to APL. Similar to APL with chromosomal translocations other than classical t(15;17) which result in the typical PML-RARA fusion, our patient responded promptly to an ATRA-containing regimen and remains in complete remission.

FLT3, NPM1 and CEBPA Mutations in 195 Children with Acute Myeloid Leukemia in Argentina

Background: Mutations of FLT3, NPM1 and CEBPA are found in 25 to 35% of adult-AML. These mutations correlate with outcome, especially in AML with normal karyotype. There are few reports concerning the incidence and prognostic significance of these mutations in childhood-AML and there is no data from Argentina.Objectives: To describe the prevalence of FLT3, NPM1 and CEBPA mutations and to analyze the prognostic impact in the outcome in our setting.Methods: Samples from 195 children treated with AML protocols were retrospectively analyzed. The mean age at diagnosis was 6.8 [0.0-17.9] years, including 65 patients younger than 2 years of age. FAB subtypes were M2: 18%, M3: 15%, M4: 12%, M5: 34%, M6: 3%, M7: 10%, while 16 cases (8%) disclosed an ambiguous lineage immunophenotype. Genetic abnormalities of AML cases were characterized by cytogenetic analysis (97%) and/or RT-PCR for AML1-ETO, CBFB-MYH11, PML-RARA, MLL-AF4, MLL-AF9, MLL-ENL and MLL-AF10 fusion transcripts (95%). The distribution of the genetic abnormalities was: AML1-ETO: 11%, PML-RARA: 15%, CBFB-MYH11: 6%, MLL/11q23: 23%, other abnormalities: 25% and normal karyotype: 16%. Detection of NPM1 and CEBPA mutations was performed by Gene-scanning; FLT3-ITD and FLT3-TKD were studied by RT-PCR and RFLP respectively. Positive cases were further characterized by sequencing analysis.Results: The prevalences of the studied mutations were: FLT3-ITD: 10.3%, FLT3-TKD: 8.2%, NPM1mut: 4.6% and CEBPAmut: 2.1%. Within the group of AML with normal karyotype the incidences were: FLT3-ITD: 12.5%, FLT3-TKD: 6.3%, NPM1mut: 25.0% and CEBPAmut: 12.5%. The mean age for each subgroup was: FLT3-ITD: 14 years, FLT3-TKD: 9 years, NPM1mut: 12 years and CEBPAmut: 12 years. Simultaneous presence of FLT3-ITD and NPM1 mutations was detected in 2 cases while 1 patient disclosed both FLT3-TKD and CEBPAmut. FLT3-ITD and FLT3-TKD showed significant association with the presence of PML-RARA (p<0.00001 and p=0.055 respectively). Eight out of nine patients with NPM1mut and 4/4 patients with CEBPAmut were AML with normal karyotype. The FAB subtypes more frequently observed for each subgroup were: FLT3-ITDmut: M3 (n:10/20; p<0.00001), FLT3-TKDmut: M5 (n:8/16; p=n.s.), NPM1mut: M2 (n:4/9; p=0.062) and CEBPAmut: M2 (n:3/4; p=0.019). The mean ages of patients with FLT3-ITDmut, NPM1mut and CEBPAmut were significantly higher (p<0.00001, p=0.006 and p=0.033, respectively). FLT3-TKD was the only mutation detected in 5/45 (11%) of patients younger than 1 year of age. The five-years leukemia-free survival probabilities (pLFS) and standard error (SE) were: Total AML: 49 (4)%, FLT3-ITDmut:68 (12)%, FLT3-TKDmut:46 (17)%, NPM1mut: 75 (15)%, CEBPAmut: 100 (0)% and NPM1mut/CEBPAmut/FLT3-ITDneg: 83 (15)% (p<0.00001). The pLFS (SE) of patients with normal karyotype and FLT3-ITDneg and NPM1mut or CEBPAmut was 88 (12)% (p=0.066).Conclusions: This is the first report of the frequencies of FLT3, NPM1 and CEBPA mutations in childhood AML in our country. The incidences of NPM1mut and CEBPAmut were significantly higher in AML with normal karyotype. Our data confirm the favorable prognosis of AML with NPM1mut/FLT3-ITDneg and CEBPAmut/FLT3-ITDneg genotypes, especially in cases with normal karyotype. The present results support the notion that this group should be considered as a new AML subset with better outcome. This group of AML patients with better outcome could be included in the standard risk group, thus avoiding intensive treatments and related toxicity. DisclosuresNo relevant conflicts of interest to declare.

The Role of Histone Demethylases in the Transcription Regulation of HOX Genes in PML-RARa+ AML Patients

Homeobox genes (HOX) encode transcription factors that are frequently deregulated in leukemias. Our previous findings described that HOX gene expression differs among genetically characterized subtypes of pediatric AML with PML-RARa+ patients having the lowest overall HOX gene expression. We observed that HOX gene expression positively correlated with expression of histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX and negatively with DNA methyltransferase DNMT3b. Interestingly, it has been shown that JMJD3 is a direct target of PML-RARa protein (Martens, JH et al, 2010, Cancer Cell). These findings led us to postulate the hypothesis that reduced levels of HOX genes in PML-RARa+ AML can be caused by the suppressed expression of histone demethylases, such as JMJD3 and UTX, resulting in increased H3K27 methylation and transcription inhibition.We chose PML-RARa+ NB4 cell line to study the role of PML-RARa fusion gene in the regulation of HOX gene expression. To inhibit the effect of PML-RARa we used all-trans retinoic acid (ATRA; 1 uM, 10 uM) which was described to release the block caused by this fusion protein. Expression of particular HOX genes (e.g., HOXA1, HOXA3, HOXA5, HOXA7) together with that of JMJD3 and UTX assessed by qPCR was significantly elevated after ATRA treatment, while gene expression of DNMT3b was decreased.To test whether the reduction in HOX gene expression is directly related to the levels of JMJD3 and UTX, we cultured NB4 cells with a specific inhibitor of these histone demethylases, GSK-J4 (1 uM, 10 uM), in combination with ATRA. This co-treatment led to inhibition of JMJD3 and UTX proteins, followed by significant reduction of HOX genes expression (e.g., HOXA1, HOXA3, HOXA5, HOXA7). This result supports our hypothesis that HOX genes expression is directly related to JMJD3/UTX activity.To determine the effect of ATRA and GSK-J4 on histone marks we have isolated histones by acid extraction and detected the levels of histones by western blot in NB4 ATRA or GSK-J4/ATRA treated cells. We observed that the level of repressive histone methylation mark (trimethylated H3K27; H3K27me3) was decreased after ATRA treatment (activation of JMJD3/UTX) and increased after GSK-J4/ATRA co-treatment (inhibition of JMJD3/UTX). The opposite effect was observed in active histone methylation marks where di- and tri-methylated H3K4 (H3K4me2, H3K4me3) increased after ATRA treatment and decreased after GSK-J4/ATRA co-treatment. H3K9 dimethylated (another repressive histone methylation mark) levels did not change.Next, to investigate the histone code directly in particular HOX genes regions we performed chromatin immunoprecipitation (ChIP) assays. We studied the presence of H3K27me3 and H3K4me2 in 5´UTR genomic region of particular HOX genes (HOXA1, HOXA2, HOXA3, HOXA5, HOXA7) in cells treated with ATRA alone or in the combination with GSK-J4. Preliminary results showed reduction in repressive marks (H3K27me3) upon ATRA treatment, whereas addition of GSK-J4 prevented this decrease. Accordingly, we observed that ATRA/GSK-J4 co-treatment reduced active histone mark H3K4me2.To evaluate the role of DNA methylation in observed expression changes after ATRA treatment we performed bisulfite sequencing of particular promoter sites of HOX genes (e.g., HOXA7, HOXA5). Although we detected decreased DNMT3b gene expression after ATRA treatment there was no change in DNA methylation of CpGs in studied regions.Our results demonstrate that changes in chromatin activity correspond with changes in HOX gene expression. Moreover, ChIP data show direct binding of the modified histones and HOX 5´UTR sites. Our data implicate histone demethylases in regulation of HOX gene expression in PML-RARa+ leukemic blasts. DNA methylation in these particular HOX genes is not involved in the regulation. Elucidating the mechanism of regulation of HOX genes expression can help to understand their role in the leukemogenic process.Supported by GACR P304/12/2214 and GAUK 568213. DisclosuresNo relevant conflicts of interest to declare.

Whole Genome Bisulfite Sequencing of Purified Mouse Promyelocytes Reveals Differentially Methylated Regions in Cells Expressing PML-Rara

Acute promyelocytic leukemia (APL) is an AML subtype that is characterized by aberrant expansion of immature myeloid progenitors and precursors that are arrested at the promyelocyte stage. Almost all APL cases are characterized by the t(15;17)(q22;q11.2) translocation that creates the PML-RARA fusion oncogene. Human APL cells are known to have a canonical expression signature and a specific methylation phenotype that is unique to this form of AML. Our laboratory previously created a mouse model of APL by expressing a human PML-RARA cDNA from the mouse Cathepsin G (Ctsg) locus (Ctsg-PML-RARA), which activates human PML- RARA expression in early myeloid progenitor cells, with peak expression in promyelocytes. After a long latent period (6-12 months), ~60% of these mice develop a clonal, APL-like myeloid malignancy. The long latent period is probably due to the requirement for cooperating mutations that synergize with PML-RARA to accelerate the disease.Human APL samples have a unique gene expression signature that distinguishes them from all other subtypes of AML. We evaluated RNA-Seq data derived from Poly A+ enriched cDNAs obtained from purified promyelocytes derived from 3 young (6 week old) WT and 3 Ctsg-PML-RARA mice. We identified 779 annotated genes that are significantly dysregulated in murine promyelocytes expressing PML-RARA with a log2 fold change >= 2 and P<0.05. Some of these genes included Spib/Pu.1, Pou2af1, Jak2, Runx1, and many others. We also identified a set of 24,018 RNAs in promyelocytes that were defined as novel transcripts. This set contains 7,413 lncRNAs with an FPKM value of >= 2. Differential expression analysis yielded 56 dysregulated lncRNA regions in PML-RARA expressing promyelocytes.To explore the association between gene dysregulation and DNA methylation in promyelocytes, we carried out whole-genome bisulfite sequencing using DNA derived from the purified promyelocytes of a 6 week old Ctsg-PML-RARA mouse, and a WT littermate. We generated a total of approximately 800 million sequencing reads, of which 78% mapped uniquely to the reference genome (mm9); we were able to map ~19 million CpGs with at least 10x coverage. Differential methylation analysis performed on ~4.5 million 1 Kb windows spanning the entire genome identified 17,633 differentially methylated regions with a mean difference of >= 25% and a q-value of < 0.01, the vast majority of which (17,264, 98%) were hypomethylated in the Ctsg-PML-RARA promyelocytes. These windows overlap several known genes, including Runx1, Jak2, Dnmt3a, Gata2, and the Hoxa and Hoxb gene clusters. Using more strict criteria (> 50% mean methylation difference), we identified 87 differentially methylated regions of at least 2 Kb in size. Of these 87 distinct regions, 74 (85%) were hypomethylated in PML-RARA promyelocytes, and 13 were hypermethylated; examples of both as shown in Figure 1. These data strongly suggest that PML-RARA has at least two distinct mechanisms by which it can modify DNA methylation. In regions where CpGs are hypomethylated, PML-RARA may be blocking the normal methylation of CpGs by the de novo DNA methyltransferases Dnmt3a and/or Dnmt3b. In contrast, PML-RARA may be directing de novo methyltransferases to act on the hypermethylated regions. Regardless, these data, when coupled with comprehensive chromatin accessibility mapping and complete RNA sequencing data, should provide new insights into the mechanisms used by PML-RARA to alter gene expression and initiate APL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In Vivo Analysis of PML-RARA in a Humanized Mouse Model

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML) characterized by the formation of a PML-RARa fusion protein, which leads to the accumulation of abnormal promyelocytes. Xenograft mouse models with human leukemic cells have advantages for analyzing the human leukemias in vivo, especially for genetic analyses. However, human primary APL cells are difficult to engraft even in very severely immunodeficient mice, such as NOD/shi-SCID IL2Rg-/- (NOG) mice. In order to understand the mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model using the transplantation of PML-RARA-transduced CD34+ cells from human cord blood into NOG mice. The expression of PML-RARa in the CD34+ cells disrupted the nuclear bodies in vitro. The clonogenic assay showed that PML-RARa inhibited the total colony formation, but favored the growth of myeloid colonies. When CD34+ cells with PML-RARA were transplanted, they proliferated in the NOG mice for more than three to four months after transplantation (in 24 out of the 34 mice). All 16 mice with more than 3,000 PML-RARA-transduced CD34+ cells were engrafted, while the engraftment was only detected in eight out of 18 mice when the cell density used for transplantation was less than 3,000 cells. These cells possessed abundant azurophilic abnormal granules in the cytoplasm, and some of them had bundles of Auer rods. They expressed CD13, CD33 and CD117, but not HLA-DR or CD34. In addition, the gene expression analysis revealed that these cells and human primary APL were clustered together among various types of AML, suggesting that these induced APL cells well recapitulated human primary APL. Similar to human primary APL, the induced APL cells possessed the ability for myeloid differentiation after treatment with all-trans retinoic acid in vitro and in vivo, and a very low potential for re-transplantation, which was similarly observed in both unsorted induced APL cells and the CD34- fraction. When human cord blood was fractionated before the PML-RARA transduction, the CD34+/CD38+ cells and common myeloid progenitors (CMP) in the CD34+/CD38+ cells led to the efficient development of APL in vivo. These findings demonstrate that CMP is a target for PML-RARA in APL, whereas the resultant CD34- APL cells may share the ability to maintain the leukemia. DisclosuresNo relevant conflicts of interest to declare.

Homeobox gene expression in acute myeloid leukemia is linked to typical underlying molecular aberrations.

BackgroundAlthough distinct patterns of homeobox (HOX) gene expression have been described in defined cytogenetic and molecular subsets of patients with acute myeloid leukemia (AML), it is unknown whether these patterns are the direct result of transcriptional alterations or rather represent the differentiation stage of the leukemic cell.MethodTo address this question, we used qPCR to analyze mRNA expression of HOXA and HOXB genes in bone marrow (BM) samples of 46 patients with AML and sorted subpopulations of healthy BM cells. These various stages of myeloid differentiation represent matched counterparts of morphological subgroups of AML. To further study the transcriptional alterations of HOX genes in hematopoiesis, we also analyzed gene expression of epigenetic modifiers in the subpopluations of healthy BM and leukemic cells.ResultsUnsupervised hierarchical clustering divided the AMLs into five clusters characterized by the presence of prevalent molecular genetic aberrations. Notably, the impact of genotype on HOX gene expression was significantly more pronounced than that of the differentiation stage of the blasts. This driving role of molecular aberrations was best exemplified by the repressive effect of the PML-RARa fusion gene on HOX gene expression, regardless of the presence of the FLT3/ITD mutation. Furthermore, HOX gene expression was positively correlated with mRNA levels of histone demethylases (JMJD3 and UTX) and negatively correlated with gene expression of DNA methyltranferases. No such relationships were observed in subpopulations of healthy BM cells.ConclusionOur results demonstrate that specific molecular genetic aberrations, rather than differentiation per se, underlie the observed differences in HOX gene expression in AML. Moreover, the observed correlations between epigenetic modifiers and HOX ex pression that are specific to malignant hematopoiesis, suggest their potential causal relationships.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-014-0094-0) contains supplementary material, which is available to authorized users.

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.

The PML domain of PML-RARα blocks senescence to promote leukemia.

In most acute promyelocytic leukemia (APL) cases, translocons produce a promyelocytic leukemia protein-retinoic acid receptor α (PML-RARα) fusion gene. Although expression of the human PML fusion in mice promotes leukemia, its efficiency is rather low. Unexpectedly, we find that simply replacing the human PML fusion with its mouse counterpart results in a murine PML-RARα (mPR) hybrid protein that is transformed into a significantly more leukemogenic oncoprotein. Using this more potent isoform, we show that mPR promotes immortalization by preventing cellular senescence, impeding up-regulation of both the p21 and p19(ARF) cell-cycle regulators. This induction coincides with a loss of the cancer-associated ATRX/Daxx-histone H3.3 predisposition complex and suggests inhibition of senescence as a targetable mechanism in APL therapy.

MIR125B1 represses the degradation of the PML-RARA oncoprotein by an autophagy-lysosomal pathway in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by the t(15;17)-associated PML-RARA fusion gene. We have previously found that MIR125B1 is highly expressed in patients with APL and may be associated with disease pathogenesis; however, the mechanism by which MIR125B1 exerts its oncogenic potential has not been fully elucidated. Here, we demonstrated that MIR125B1 abundance correlates with the PML-RARA status. MIR125B1 overexpression enhanced PML-RARA expression and inhibited the ATRA-induced degradation of the PML-RARA oncoprotein. RNA-seq analysis revealed a direct link between the PML-RARA degradation pathway and MIR125B1-arrested differentiation. We further demonstrated that the MIR125B1-mediated blockade of PML-RARA proteolysis was regulated via an autophagy-lysosomal pathway, contributing to the inhibition of APL differentiation. Furthermore, we identified DRAM2 (DNA-damage regulated autophagy modulator 2), a critical regulator of autophagy, as a novel target that was at least partly responsible for the function of MIR125B1 involved in autophagy. Importantly, the knockdown phenotypes for DRAM2 are similar to the effects of overexpressing MIR125B1 as impairment of PML-RARA degradation, inhibition of autophagy, and myeloid cell differentiation arrest. These effects of MIR125B1 and its target DRAM2 were further confirmed in an APL mouse model. Thus, MIR125B1 dysregulation may interfere with the effectiveness of ATRA-mediated differentiation through an autophagy-dependent pathway, representing a novel potential APL therapeutic target.