PML-RARα Fusion Protein Research Articles

Halogenated retinoid derivatives as dual RARα and RXRα modulators for treating acute promyelocytic leukemia cells

Acute promyelocytic leukemia (APL), a distinctive subtype of acute myeloid leukemia (AML), is characterized by the t(15; 17) translocation forming the PML-RARα fusion protein. Recent studies have revealed a crucial role of retinoid X receptor α (RXRα) in PML-RARα′s tumorigenesis. This necessitates the development of dual RARα and RXRα targeting compounds for treating APL. Here, we developed a pair of brominated retinoid isomers, 5a and 5b, exhibiting RARα agonistic selectivity among the RAR subtypes and RXRα partial agonistic activities. In the treatment of APL cells, low doses (RARα activation range) of 5a and 5b degrade PML-RARα and strongly induce differentiation, while higher doses (RXRα activation range) induce G2/M arrest and apoptosis in both all-trans retinoic acid (ATRA)-sensitive and resistant cells. We replaced the bromine in 5a with chlorine or iodine to obtain compounds 7 or 8a. Interestingly, the chlorinated compound 7 tends to activate RXRα and induce G2/M arrest and apoptosis, while the iodinated compound 8a tends to activate RARα and induce differentiation. Together, our work underscores several advantages and characteristics of halogens in the rational design of RARα and RXRα ligands, offering three promising drug candidates for treating both ATRA-sensitive and resistant APL.

Arsenic trioxide and p97 inhibitor synergize against acute myeloid leukemia by targeting nascent polypeptides and activating the ZAKα–JNK pathway

Arsenic trioxide (ATO) has exhibited remarkable efficacy in treating acute promyelocytic leukemia (APL), primarily through promoting the degradation of the PML-RARα fusion protein. However, ATO alone fails to confer any survival benefit to non-APL acute myeloid leukemia (AML) patients and exhibits limited efficacy when used in combination with other agents. Here, we explored the general toxicity mechanisms of ATO in APL and potential drugs that could be combined with ATO to exhibit synergistic lethal effects on other AML. We demonstrated that PML-RARα degradation and ROS upregulation were insufficient to cause APL cell death. Based on the protein synthesis of different AML cells and their sensitivity to ATO, we established a correlation between ATO-induced cell death and protein synthesis. Our findings indicated that ATO induced cell death by damaging nascent polypeptides and causing ribosome stalling, accompanied by the activation of the ZAKα-JNK pathway. Furthermore, ATO-induced stress activated the GCN2–ATF4 pathway, and ribosome-associated quality control cleared damaged proteins with the assistance of p97. Importantly, our data revealed that inhibiting p97 enhanced the effectiveness of ATO in killing AML cells. These explorations paved the way for identifying optimal synthetic lethal drugs to enhance ATO treatment on non-APL AML.

ATPR induces acute promyelocytic leukemia cells differentiation and cycle arrest via the lncRNA CONCR/DDX11/PML-RARα signaling axis

Acute promyelocytic leukemia (APL) is a type of acute myeloid leukemia (AML) with a high mortality rate, and the production of PML-RARα fusion protein is the cause of its pathogenesis. Our group has synthesized a novel compound, 4-amino-2-trifluoromethyl-phenyl retinate (ATPR), by structural modification of All-trans retinoic acid (ATRA), which has strong cell differentiation-inducing effects and inhibits the expression of PML-RARα. In this study, acute promyelocytic leukemia NB4 cells before and after ATPR induction were analyzed by whole transcriptome microarray, and the expression of lncRNA CONCR was found to be significantly downregulated. The role of CONCR in ATPR-induced cell differentiation and cycle arrest was explored through overexpression and silencing of CONCR. And then the database was used to predict that CONCR may bind to DEAD/H-Box Helicase 11 (DDX11) protein to further explore the role of CONCR binding to DDX11. The results showed that ATPR could reduce the expression of CONCR, and overexpression of CONCR could reverse the ATPR-induced cell differentiation and cycle blocking effect, and conversely silencing of CONCR could promote this effect. RNA immunoprecipitation (RIP) experiments showed that CONCR could bind to DDX11, the protein expression levels of DDX11 and PML-RARα were elevated after overexpression of CONCR. These results suggest that ATPR can regulate the expression of DDX11 through CONCR to affect the expression of PML-RARα fusion protein, which in turn induces the differentiation and maturation of APL cells.

Function of PML-RARA in Acute Promyelocytic Leukemia.

The transformation of acute promyelocytic leukemia (APL) from the most fatal to the most curable subtype of acute myeloid leukemia (AML), with long-term survival exceeding 90%, has represented one of the most exciting successes in hematology and in oncology. APL is a paradigm for oncoprotein-targeted cure.APL is caused by a 15/17 chromosomal translocation which generates the PML-RARA fusion protein and can be cured by the chemotherapy-free approach based on the combination of two therapies targeting PML-RARA: retinoic acid (RA) and arsenic. PML-RARA is the key driver of APL and acts by deregulating transcriptional control, particularly RAR targets involved in self-renewal or myeloid differentiation, also disrupting PML nuclear bodies. PML-RARA mainly acts as a modulator of the expression of specific target genes: genes whose regulatory elements recruit PML-RARA are not uniformly repressed but also may be upregulated or remain unchanged. RA and arsenic trioxide directly target PML-RARA-mediated transcriptional deregulation and protein stability, removing the differentiation block at promyelocytic stage and inducing clinical remission of APL patients.

Outcomes of Paediatric Acute Promyelocytic Leukaemia in the UK

Acute Promyelocytic Leukaemia (APL) is characterized by unique genetic and molecular abnormalities, clinical features, and treatment strategies. It accounts for 5-10% of paediatric acute myeloid leukaemia (AML) cases. APL is defined by the t(15;17)(q24;q21) chromosomal translocation, leading to the formation of the PML-RARA fusion protein, which plays a crucial role in leukemogenesis. All-trans retinoic acid (ATRA) has revolutionized APL management by targeting the PML-RARA fusion protein and inducing complete remission in the majority of patients. Arsenic trioxide (ATO) has emerged as an effective therapeutic agent, particularly in cases with ATRA resistance or relapse. ATO is now widely recommended as part upfront treatment. However, access to ATO has been limited by funding sources in certain countries. This retrospective national study presents the treatment strategies, complications, and survival outcomes of 50 paediatric patients, aged 1 to 18 years, diagnosed with APL in the UK over an 8-year period, between 1 st November 2014 and 31 st October 2021. There were 21 (42%) high risk (HR) patience and 29 (58%) non-HR patients in the cohort. The majority of patients received a chemotherapy-free regimen comprising ATRA and ATO, with additional agents used selectively. Chemotherapy was usually given to patients in the event of lack of access to ATO or clinical urgency. Availability to ATO varied between countries within the UK. Event-free survival (EFS) and overall survival (OS) were evaluated, with a minimum 2 years follow up from diagnosis and median follow-up of 8 years. Overall, the cohort demonstrated a high OS of 94%, with no significant difference observed between HR and non-HR patients. However, HR patients experienced significantly lower EFS, with all relapses occurring in this group. Relapsed patients received varied treatment regimens, including ATRA, ATO, and chemotherapy, with favourable outcomes. This study contributes valuable insights into the management and outcomes of paediatric APL in the UK, emphasizing the importance of early diagnosis and risk stratification for optimal treatment decisions. Addressing disparities in ATO availability and promoting consistent funding across NHS Trusts will be crucial in further improving patient outcomes and reducing relapse rates in APL. Future research should focus on refining treatment protocols and exploring novel therapeutic strategies to enhance the long-term survival of paediatric APL patients. The study highlights the challenges associated with ATO availability across NHS Trusts, impacting treatment decisions and outcomes for APL patients.

Pyrrolidine Dithiocarbamate Enhances the Cytotoxic Effect of Arsenic Trioxide on Acute Promyelocytic Leukemia Cells.

More than 95% patients with acute promyelocytic leukemia (APL) carry the PML-RARα fusion oncoprotein. Arsenic trioxide (ATO) is an efficacious therapeutic agent for APL, and the mechanism involves the binding with PML and degradation of PML-RARα protein. Pyrrolidine dithiocarbamate (PDTC) demonstrates the function of facilitating the cytotoxic effect of ATO. To investigate whether PDTC is potential to enhance the cytotoxic effect of ATO to APL cells by acting on PML-RARα oncoproteins. Inhibitory effects of drugs on cell viability were examined by CCK-8 test, and apoptosis was evaluated by flow cytometry. Western blotting and immunofluorescence assays were used to explore the mechanism. PDTC improved the effect of ATO on inhibiting proliferation of NB4 cells in vitro. Further, PDTC-ATO promoted apoptosis and cell cycle arrest in NB4 cells. The expression of caspase- 3 and Bcl-2 was reduced in PDTC-ATO-treated NB4 cells, while cleaved caspase-3 and Bax was up-regulated. Furthermore, less PML-RARα expression were found in PDTC-ATO-treated NB4 cells than that in NB4 cells treated with ATO singly. Laser confocal microscopy showed that protein colocalization of PML and RARα was disrupted more significantly by PDTC-ATO treatment than that with ATO monotherapy. In conclusion, PDTC enhanced the cytotoxic effect of ATO on APL, and the mechanism was, at least in part, related to the promotion of ATO-induced degradation of PML-RARα fusion protein via forming a complex PDTC-ATO.

AML-503 An Unusual Presentation of Acute Promyelocytic Leukemia (APML): Complex Cytogenetic Isochromosome 17 and WT1 Gene Mutation in the Absence of PML-RARA Fusion Protein

AML-503 An Unusual Presentation of Acute Promyelocytic Leukemia (APML): Complex Cytogenetic Isochromosome 17 and WT1 Gene Mutation in the Absence of PML-RARA Fusion Protein

The clinically relevant CHK1 inhibitor MK-8776 induces the degradation of the oncogenic protein PML-RARα and overcomes ATRA resistance in acute promyelocytic leukemia cells

Acute promyelocytic leukemia (APL) is a hematological disease characterized by the expression of the oncogenic fusion protein PML-RARα. The current treatment approach for APL involves differentiation therapy using all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). However, the development of resistance to therapy, occurrence of differentiation syndrome, and relapses necessitate the exploration of new treatment options that induce differentiation of leukemic blasts with low toxicity. In this study, we investigated the cellular and molecular effects of MK-8776, a specific inhibitor of CHK1, in ATRA-resistant APL cells. Treatment of APL cells with MK-8776 resulted in a decrease in PML-RARα levels, increased expression of CD11b, and increased granulocytic activity consistent with differentiation. Interestingly, we showed that the MK-8776-induced differentiating effect resulted synergic with ATO. We found that the reduction of PML-RARα by MK-8776 was dependent on both proteasome and caspases. Specifically, both caspase-1 and caspase-3 were activated by CHK1 inhibition, with caspase-3 acting upstream of caspase-1. Activation of caspase-3 was necessary to activate caspase-1 and promote PML-RARα degradation. Transcriptomic analysis revealed significant modulation of pathways and upstream regulators involved in the inflammatory response and cell cycle control upon MK-8776 treatment. Overall, the ability of MK-8776 to induce PML-RARα degradation and stimulate differentiation of immature APL cancer cells into more mature forms recapitulates the concept of differentiation therapy. Considering the in vivo tolerability of MK-8776, it will be relevant to evaluate its potential clinical benefit in APL patients resistant to standard ATRA/ATO therapy, as well as in patients with other forms of acute leukemias.

Acute Promyelocytic Leukemia (APL): A Review of the Classic and Emerging Target Therapies towards Molecular Heterogeneity

The occurrence of severe bleeding syndrome because of the PML-RARα fusion protein is a life-threatening event in APL. This protein destabilizes homeostasis, maturation, remodeling, and tissue regeneration in addition to hampering the maintenance and differentiation of hematopoietic cells into different lineages, fixing cells in the promyelocyte stage. APL is a classic example of how effective targeted therapy is and, therefore, how important the use of such therapy is to the overall survival of patients, which in this case is represented by the use of ATRA/ATO. Despite that, about 10% of cases of APL patients demonstrate resistance to treatment. Facing this scenario, we point out promising target therapies such as those recommended by the NCCN and Leukemia Net. Since this is such a heterogeneous molecular disease, it is of great importance to understand how important combined chemotherapy, target therapy, immune-based therapy, and combined therapies are in the survival of these APL patients.

Targeting Interferon Pathway in TBLR1-RARa-Driven Promyelocytic Leukemia in the Context of ATRA and ATO Unresponsiveness

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) mostly driven by the t(15;17) translocation that yields the PML-RARα (PR) fusion protein. Apart from PML, other partner genes have been uncovered to fuse with RARα forming rare X-RARα fusion genes. Among them, TBLR1-RARα (TR) is a novel oncogene to induce APL identified in our previous studies, which elicited differed response and worse prognosis in contrast to the canonical PR fusion gene. Despite the textbook rationale of all-trans retinoid acid (ATRA) and arsenic trioxide (ATO) turning most APL patients with PR fusion gene from highly fatal to highly curable, TR-driven APL did not yield long-term remission both in murine models and patients, highlighting the necessity to dissect the mechanism of drug unresponsiveness and explore novel targeted therapies. In this study, we first established two doxycycline-inducible leukemia cell lines to directly initiate the expression of TR and PR fusion protein in U937 cells, respectively. Transcriptome sequencing analysis showed that interferon (IFN) pathways were significantly suppressed in TR rather than PR-induced leukemia cells. Then we administered IFNs as well as the standard regimen of ATRA and ATO in TR- and PR-induced APL, comparing therapy responses and biological phenotypes with in vitro and in vivo studies. Main results are summarized as follows concerning TR- in comparison to PR-driven APL. Firstly, ATO was unresponsive in TR-induced leukemia cells, which failed to elicit oncoprotein degradation, apoptosis and loss of self-renewal. Secondly, lower doses of ATRA were inadequate. Although a lower dose of ATRA was sufficient to trigger differentiation, increasing doses of ATRA further promoted the degradation of oncoprotein and reduced the colony formation capacity of TR leukemic cells, which ultimately conferred survival benefits in TR mice. Thirdly, type I IFNs was promising, which induced apoptosis, cooperated with ATRA to boost differentiation, and exhibited potential to reduce self-renewal, reflecting anti-leukemia efficacy. Finally, type I IFNs combining with ATRA displayed survival advantage to TR mice, and murine STING agonist DMXAA, which acted upstream to produce type I IFNs, significantly endowed TR mice with extended survival time. Based on our results, we further propose mechanistic hypotheses of TR-APL in comparison with the classic PR-APL. In TR-driven APL, despite the unresponsiveness of ATO, we may utilize STING-IFN pathway agonist to upregulate PML expression, which may obliterate self-renewal independent of ATO response triggered by initial PML nuclear body disruption observed in PR-driven APL, providing therapeutic implications for APL driven by rare RARα fusion genes. Our study aims to gain further understanding of TR-driven APL and integrate insights into principles underlying leukemogenesis mediated by rare RARα fusion genes, which may benefit such a therapy-resistant population and enable APL to be a bona fide curable leukemia.

The role of adjuvant chemotherapy in the management of acute promyelocytic leukemia differentiation syndrome.

Acute Promyelocytic Leukemia (APL) is characterized by the t(15;17) chromosomal translocation resulting in a PML-RARA fusion protein. The all-trans-retinoic acid (ATRA) and Arsenic Trioxide (ATO) only regimens have demonstrated success in treating low- and intermediate-risk patients. However, induction with ATRA/ATO only regimens have been showing increased incidence of differentiation syndrome (DS), a potentially lethal complication, traditionally treated with dexamethasone. We conducted a three-institution retrospective study, aiming to evaluate the role of short-term adjuvant chemotherapy in managing moderate DS for patients with low- or intermediate-risk APL initially treated with ATRA/ATO only protocols. We evaluated the difference in incidence and duration of moderate DS in APL patients who were treated with ATRA/ATO with or without adjuvant chemotherapy. 57 low- or intermediate-risk APL patients were retrospectively identified and included for this study; 36 patients received ATRA/ATO only induction treatment, and 21 patients received ATRA/ATO/adjuvant chemotherapy combination induction therapy. Similar proportions of patients experienced DS in both groups (66.7% vs. 81.0%, P = 0.246). The median duration of DS resolution in patients receiving ATRA/ATO only was 17 days (n = 23), and in patients receiving combination therapy was 8 days (n = 16) (P = 0.0001). The lengths of hospital stay in patients receiving ATRO/ATO only was 38 days (n = 7), and in patients receiving combination therapy was 14 days (n = 17) (P = 0.0007). In conclusion, adding adjuvant chemotherapy to ATRA/ATO only protocol may reduce the duration of DS and the length of hospital stay during APL induction treatment.

Vitamin D Derivatives in Acute Myeloid Leukemia: The Matter of Selecting the Right Targets.

Acute myeloid leukemia (AML) is an aggressive and often fatal hematopoietic malignancy. A very attractive way to treat myeloid leukemia, called “differentiation therapy”, was proposed when in vitro studies showed that some compounds are capable of inducing differentiation of AML cell lines. One of the differentiation-inducing agents, all-trans-retinoic acid (ATRA), which can induce granulocytic differentiation in AML cell lines, has been introduced into clinics to treat patients with acute promyelocytic leukemia (APL) in which a PML-RARA fusion protein is generated by a chromosomal translocation. ATRA has greatly improved the treatment of APL. Since 1,25-dihydroxyvitamin D (1,25D) is capable of inducing monocytic differentiation of leukemic cells, the idea of treating other AMLs with vitamin D analogs was widely accepted. However, early clinical trials in which cancer patients were treated either with 1,25D or with analogs did not lead to conclusive results. Recent results have shown that AML types with certain mutations, such as isocitrate dehydrogenase (IDH) mutations, may be the right targets for differentiation therapy using 1,25D, due to upregulation of vitamin D receptor (VDR) pathway.

In vivo temporal resolution of acute promyelocytic leukemia progression reveals a role of Klf4 in suppressing early leukemic transformation.

Genome organization plays a pivotal role in transcription, but how transcription factors (TFs) rewire the structure of the genome to initiate and maintain the programs that lead to oncogenic transformation remains poorly understood. Acute promyelocytic leukemia (APL) is a fatal subtype of leukemia driven by a chromosomal translocation between the promyelocytic leukemia (PML) and retinoic acid receptor α (RARα) genes. We used primary hematopoietic stem and progenitor cells (HSPCs) and leukemic blasts that express the fusion protein PML-RARα as a paradigm to temporally dissect the dynamic changes in the epigenome, transcriptome, and genome architecture induced during oncogenic transformation. We found that PML-RARα initiates a continuum of topologic alterations, including switches from A to B compartments, transcriptional repression, loss of active histone marks, and gain of repressive histone marks. Our multiomics-integrated analysis identifies Klf4 as an early down-regulated gene in PML-RARα-driven leukemogenesis. Furthermore, we characterized the dynamic alterations in the Klf4 cis-regulatory network during APL progression and demonstrated that ectopic Klf4 overexpression can suppress self-renewal and reverse the differentiation block induced by PML-RARα. Our study provides a comprehensive in vivo temporal dissection of the epigenomic and topological reprogramming induced by an oncogenic TF and illustrates how topological architecture can be used to identify new drivers of malignant transformation.

A case of acute promyelocytic lekuemia with absence of PML-RARA fusion protein involving complex cytogenetic isochromosome 17

A case of acute promyelocytic lekuemia with absence of PML-RARA fusion protein involving complex cytogenetic isochromosome 17

STMN1 is highly expressed and contributes to clonogenicity in acute promyelocytic leukemia cells.

Stathmin 1 (STMN1) is a microtubule-destabilizing protein highly expressed in hematological malignancies and involved in proliferation and differentiation. Although a previous study found that the PML-RARα fusion protein, which contributes to the pathophysiology of acute promyelocytic leukemia (APL), positively regulates STMN1 at the transcription and protein activity levels, little is known about the role of STMN1 in APL. In this study, we aimed to investigate the STMN1 expression levels and their associations with laboratory, clinical, and genomic data in APL patients. We also assessed the dynamics of STMN1 expression during myeloid cell differentiation and cell cycle progression, and the cellular effects of STMN1 silencing and pharmacological effects of microtubule-stabilizing drugs on APL cells. We found that STMN1 transcripts were significantly increased in samples from APL patients compared with those of healthy donors (all p < 0.05). However, this had no effect on clinical outcomes. STMN1 expression was associated with proliferation- and metabolism-related gene signatures in APL. Our data confirmed that STMN1 was highly expressed in early hematopoietic progenitors and reduced during cell differentiation, including the ATRA-induced granulocytic differentiation model. STMN1 phosphorylation was predominant in a pool of mitosis-enriched APL cells. In NB4 and NB4-R2 cells, STMN1 knockdown decreased autonomous cell growth (all p < 0.05) but did not impact ATRA-induced apoptosis and differentiation. Finally, treatment with paclitaxel (as a single agent or combined with ATRA) induced microtubule stabilization, resulting in mitotic catastrophe with repercussions for cell viability, even in ATRA-resistant APL cells. This study provides new insights into the STMN1 functions and microtubule dynamics in APL.

Stathmin 1 Is Highly Expressed in Acute Promyelocytic Leukemia and Microtubule Dynamics Is a Potential Target for ATRA-Resistant APL Cells

Introduction : Stathmin 1 is a microtubule destabilizer protein involved in the proliferation and differentiation of early hematopoietic progenitors. Aberrant Stathmin 1 expression has been reported in hematological malignancies. Stathmin 1 silencing reduces cell proliferation and clonogenicity of leukemia cell lines. A previous study identified that the PML-RARα fusion protein, which is necessary to acute promyelocytic leukemia (APL) genesis, positively regulates Stathmin 1 at transcription and protein activity levels. However, Stathmin 1 expression and its impact on prognosis and leukemia cell biology have rarely been studied in APL. Objectives : To evaluate Stathmin 1 expression and its association with laboratory and clinical parameters in a cohort of APL patients. To investigate Stathmin 1 expression and activity upon all-trans retinoic acid (ATRA) treatment and during cell cycle in the PML-RARα cell lines. Finally, to phenocopy the effects of Stathmin 1 inhibition using the pharmacological microtubule stabilizing agent paclitaxel. Methods : Bone marrow samples from 121 APL patients enrolled in the International Consortium on Acute Promyelocytic Leukemia (IC-APL) at diagnosis and 22 healthy donors were included. Patients were treated with ATRA and chemotherapy as described elsewhere. Patients were dichotomized according to Stathmin 1 expression using the median as cut-off. The PML-RARα cell lines, NB4 (ATRA-sensible) and NB4-R2 (ATRA-resistant) were used for functional assays. Cell cycle was synchronized by double thymidine block. For dose-response curves (MTT), PML-RARα cells were treated with graded concentrations of paclitaxel and IC50 values were calculated using a nonlinear regression analysis. For apoptosis assay (annexin V/PI), PML-RARα cells were treated with ATRA (1 µM) and/or IC50 paclitaxel dose for 72 hours. Gene and protein expressions were evaluated by qPCR and Western blot, and microtubule networks by confocal microscopy. For statistical analyses, Fisher's exact test or Chi-square test were used to compare categorical variables. Mann-Whitney test, t Student test or ANOVA test and Bonferroni post-test were used to compare continuous variables. Log-rank test and Cox regression were used for survival analyzes. P -value <0.05 was considerate statistically significant. Results : Stathmin 1 transcripts were significantly increased in bone marrow samples from APL patients compared with healthy donors (median 1.21 [range 0.02-4.62] versus (vs.) 0.66 [0.01-1.71]; p <0.01). Stathmin 1 levels did not impact on disease-free survival, overall survival and complete remission rates of APL patients. No relevant association between Stathmin 1 expression and clinical and laboratory parameters were observed. In NB4 cells, but not in NB4-R2, ATRA induced granulocytic differentiation, associated with a prominent reduction of Stathmin 1 gene and protein expression (p <0.05). Paradoxically, ATRA-induced granulocytic differentiation strongly reduced α-tubulin acetylation (microtubule stability marker) in NB4 cells. Using thymidine-synchronized cells, we observed that Stathmin 1 phosphorylation at serine 16 (an inhibitory site) was predominant in pool of mitosis-enriched cells, and its phosphorylation was marked reduced in pool of G1-enriched cells. NB4 and NB4-R2 cells presented high sensibility to paclitaxel treatment (IC50 of 3.6 nM and 1.8 nM, respectively). In NB4-R2 cells, but not in NB4 cells, paclitaxel increased the number of apoptotic cells after 72 hours of ATRA treatment; increased annexin V and caspase 3 cleavage (p <0.05). Paclitaxel treatment increased acetyl-α-tubulin, y-H2AX and CDKN1A levels. Conclusions : Stathmin 1 is highly expressed, but does not impact clinical outcomes in APL patients. Stathmin 1 reduces during ATRA-induced cell differentiation. Stathmin 1 activity is reduced during mitosis (as demonstrate by increased p-Stathmin 1S16) and increases post-mitosis (reduced p-Stathmin 1S16), indicating that this protein participates in microtubule dynamics and cell cycle progression in PML-RARα cells. Paclitaxel was an efficient pharmacological agent in inducing microtubule stabilization, reducing cell viability, increasing mitotic catastrophe as monotherapy or in combination with ATRA, in PML-RARα cells, including ATRA-resistant cells. Our study provides new insights on Stathmin 1 and microtubule dynamics in APL. DisclosuresBittencourt:Janssen, Takeda: Honoraria.

Multi-Omics and Machine Learning Reveal Context-Specific Gene Regulatory Activities of PML-RARA in Acute Promyelocytic Leukemia

The PML-RARA fusion protein is the hallmark driver of Acute Promyelocytic Leukemia (APL) and disrupts retinoic acid signaling, leading to wide-scale gene expression changes and uncontrolled proliferation of myeloid precursor cells. While known to be recruited to binding sites across the genome, its impact on gene regulation and expression is under-explored. Using integrated multi-omics datasets, we characterize the influence of PML-RARA binding on gene expression and regulation in an inducible cell line model and APL patient ex vivo samples. We find that genes whose regulatory elements recruit PML-RARA are not uniformly transcriptionally repressed, as commonly suggested, but also may be upregulated or remain unchanged. We apply a novel computational machine learning strategy to deconvolute the complex, local transcription factor binding site environment at PML-RARA bound positions to reveal distinct signatures that modulate how PML-RARA directs the transcriptional response.

Microfluidic-Based Detection of AML-Specific Biomarkers Using the Example of Promyelocyte Leukemia.

A microfluidic assay for the detection of promyelocytic leukemia (PML)-retinoic acid receptor α (RARα) fusion protein was developed. This microfluidic-based system can be used for rapid personalized differential diagnosis of acute promyelocyte leukemia (APL) with the aim of early initiation of individualized therapy. The fusion protein PML-RARα occurs in 95% of acute promyelocytic leukemia cases and is considered as diagnostically relevant. The fusion protein is formed as a result of translocation t(15,17) and is detected in the laboratory by fluorescence in situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR). Diagnostic methods require many laboratory steps with specialized staff. The developed microfluidic assay includes a sandwich enzyme-linked immunosorbent assay (ELISA) system for PML-RARα on surface of magnetic microparticles in a microfluidic chip. A rapid detection of PML-RARα in cell lysates is achieved in less than one hour. A biotinylated PML-antibody on the surface of magnetic streptavidin coated microparticles is used as capture antibody. The bound translocation product is detected by a RARα antibody conjugated with horseradish peroxidase and the substrate QuantaRed. The analysis is performed in microfluidic channels which involves automated liquid processing with stringent washing and short incubation times. The results of the developed assay show that cell lysates of PML-RARα-positive cells (NB-4) can be clearly distinguished from PML-RARα-negative cells (HL-60, MV4-11).

Transcriptional and Metabolic Dissection of ATRA-Induced Granulocytic Differentiation in NB4 Acute Promyelocytic Leukemia Cells.

Acute promyelocytic leukemia (APL) is a hematological disease characterized by a balanced reciprocal translocation that leads to the synthesis of the oncogenic fusion protein PML-RARα. APL is mainly managed by a differentiation therapy based on the administration of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). However, therapy resistance, differentiation syndrome, and relapses require the development of new low-toxicity therapies based on the induction of blasts differentiation. In keeping with this, we reasoned that a better understanding of the molecular mechanisms pivotal for ATRA-driven differentiation could definitely bolster the identification of new therapeutic strategies in APL patients. We thus performed an in-depth high-throughput transcriptional profile analysis and metabolic characterization of a well-established APL experimental model based on NB4 cells that represent an unevaluable tool to dissect the complex mechanism associated with ATRA-induced granulocytic differentiation. Pathway-reconstruction analysis using genome-wide transcriptional data has allowed us to identify the activation/inhibition of several cancer signaling pathways (e.g., inflammation, immune cell response, DNA repair, and cell proliferation) and master regulators (e.g., transcription factors, epigenetic regulators, and ligand-dependent nuclear receptors). Furthermore, we provide evidence of the regulation of a considerable set of metabolic genes involved in cancer metabolic reprogramming. Consistently, we found that ATRA treatment of NB4 cells drives the activation of aerobic glycolysis pathway and the reduction of OXPHOS-dependent ATP production. Overall, this study represents an important resource in understanding the molecular “portfolio” pivotal for APL differentiation, which can be explored for developing new therapeutic strategies.

Acute promyelocytic leukemia (APL): a review of the literature.

Acute Promyelocytic Leukemia (APL) is characterized by a block in differentiation where leukemic cells are halted at the promyelocyte stage. A characteristic balanced chromosomal translocation between chromosomes 15 and 17 t (15;17) (q24; q21) is seen in 95% of cases — the translocation results in the formation of the PML-RARA fusion protein. The introduction of retinoic acid (RA) and arsenic trioxide (ATO) has been responsible for initially remarkable cure rates. However, relapsed APL, particularly in the high-risk subset of patients, remains an important clinical problem. In addition, despite the success of ATRA & ATO, many clinicians still elect to use cytotoxic chemotherapy in the treatment of APL. Patients who become resistant to ATO have an increased risk of mortality. The probability of relapse is significantly higher in the high-risk subset of patients undergoing treatment for APL; overall approximately 10-20% of APL patients relapse regardless of their risk stratification. Furthermore, 20-25% of patients undergoing treatment will develop differentiation syndrome, a common side effect of differentiation agents. Recent evidence using in vitro models has shown that mutations in the B2 domain of the PML protein, mediate arsenic resistance. Alternative agents and approaches considering these clinical outcomes are needed to address ATO resistance as well as the relapse rate in high risk APL.