All-trans Retinoic Acid Resistance Research Articles

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.

The Ingenious Multiple Efficacies of the Integrated Gene Fragment of Torque Teno Mini Virus in Orchestrating Promyelocytic Leukemogenesis Via Hijacking Rara

Introduction Torque teno mini virus (TTMV) is a ubiquitous virus which symbiotic with humans, but it has recently been reported to cause acute promyelocytic leukemia (APL) through a unique form of genome fragment integration and forming the TTMV::RARA fusion gene, with 4 cases reported till now. However, the leukemogenesis mechanism of TTMV::RARA-APL has not been explored. Methods We recruited transcriptome sequencing (RNA-seq) data of 7 TTMV::RARA-APL cases, 3 of them also with whole genome sequencing (WGS) data, to analyze the properties of the TTMV integrated sequences. Clinical information was analyzed. In vitro cell experiments were performed to verify the functions of the conserved regulatory sequences and the fusion protein. Results These cases were collected from 4 centers, 5 males and 2 females, aged 3 to 35 (median 9) years, with 6 pediatric cases. Of which 3 pediatric cases were identified from a continuous acute myeloid leukemia (AML) cohort. In this cohort, 3 TTMV::RARA-APL were identified in 205 AML cases under 14 years, but no cases in another 930 AML cases, showing a strong tendency towards pediatrics (p=0.00022). Moreover, we totally identified 85 PML::RARA-APL and 3 TTMV::RARA-APL, and no other X::RAR cases in this cohort, indicating TTMV::RARA is the most common X::RAR fusion besides PML::RARA. All cases manifested typical APL morphological features, and RNA-seq expression analysis showed clustering with PML::RARA-APL cases. Initial effect and remission can be achieved with chemotherapy regimens containing all-trans retinoic acid (ATRA), but early recurrence is prone to occur. RARA-I387T mutation was observed in case #TR4, who relapsed after treatment with ATRA, suggesting that the leukemic cells were responsive to ATRA. However, primary resistance to ATRA and extensive resistance to various agents were observed in case #TR5, who was initially diagnosed with myeloid sarcoma and transformed into APL at relapse. The TTMV integrated sequences of 3 cases with WGS data were 1164bp, 2113bp, and 2859bp (Fig. 1A), respectively, and the common feature was the involvement of the transcription regulatory region and the open reading frame 2 (ORF2). Alignment of TTMV transcription regulatory sequences (Fig. 1B) revealed a highly conserved basal transcription factor region, including the canonical TATA box and GC box. While in the proximal promoter region showing sequence diversity, all 3 cases carried 4 RUNX1 binding elements and 1 octamer motif. In vitro experiments (Fig. 1C) showed that TTMV basal transcription element can initiate basal expression in 293T cells, while RUNX1 can significantly enhance the expression in response to the RUNX1 binding elements. RUNX1 is an important transcription factor protein in the differentiation of myeloid cells, and the 4 RUNX1 binding elements might explain the myelotropic of TTMV and confer an important basis in promyelocytic leukemogenesis. The TTMV-ORF2 coding sequences of all 7 cases were analyzed, with the common region being the N-terminus 58 amino acids, of which 20 were highly conserved (Fig. 1D, 1E). Co-Immunoprecipitation analysis confirmed that the TTMV::RARA protein forms homopolymers and heteropolymers with RXRA (Fig.1F), which is the property shared by all X::RARA proteins. Serine/Threonine protein phosphatase (S/T PPase) assays showed that the fusion protein exhibits strong S/T PPase activity (Fig. 1G). UAS/GAL4 reporter experiment showed that TTMV::RARA protein exhibited a constrained but concentration-dependent response to ATRA compared to RARA (Fig. 1H). However, fusion proteins with ORF2-C32A/C34A mutation and impaired phosphatase activity were significantly more responsive to ATRA (Fig. 1H). This suggests that the S/T PPase activity of the ORF2 coding region plays a role in retarded ATRA response. Conclusions This is the first systematic report on this novelty disease. Data suggests that TTMV::RARA-APL may be more common in children, which may be related to the ubiquity of TTMV and its acquisition early in life. The clinical manifestations and treatment responses are diverse, with overall dismal prognosis. Molecular investigation indicated the multifunctional TTMV integrated molecular machinery in orchestrating RUNX1 enforced myelotropic, aberrant priming expression, homodimer formation, and S/T PPase activity, and confer promyelocytic leukemogenesis via hijacking RARA.

MiR-125b-5p-MAPK1-C/EBPα feedback loop regulates all-trans retinoic acid resistance in acute promyelocytic leukemia

BackgroundVarious studies have highlighted the significance of miR-125b-5p in tumour chemotherapy resistance; However, whether miR-125b-5p is associated with all-trans retinoic acid (ATRA) resistance in acute promyelocytic leukemia (APL) has not been reported. MethodsDrug-resistance-related factors in APL were predicted using the DRESIS database. The expression levels of miR-125b-5p in ATRA-sensitive and ATRA-resistant APL cells were determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR). A nitrotetrazolium blue (NBT) reduction assay and flow cytometry (FCM) were used to detect the effect of miR-125b-5p on ATRA resistance in APL cells. An APL xenograft tumour mouse model was established to determine the effect of miR-125b-5p on ATRA resistance. A dual-luciferase gene reporter assay, qRT-PCR, and western blotting verified the regulation by miR-125b-5p of its target gene, MAPK1, and the MAPK1 downstream factor, C/EBPα. An NBT reduction assay and FCM were used to detect the effect of C/EBPα on ATRA resistance in APL cells. Western blotting and qRT-PCR were used to assess the regulation of miR-125b-5p and MAPK1 by C/EBPα. ResultsmiR-125b-5p expression levels were dramatically increased in ATRA-resistant APL cells. Both in vitro and in vivo experiments revealed that miR-125b-5p overexpression enhanced ATRA resistance in APL. miR-125b-5p promoted ATRA resistance by sponging MAPK1. C/EBPα was negatively regulated by miR-125b-5p, which in addition, regulated ATRA resistance in APL cells. C/EBPα also regulated the miR-125b-5p-MAPK1 axis. ConclusionThe findings of this study indicate that the miR-125b-5p-MAPK1-C/EBPα feedback loop regulated ATRA resistance in APL. Thus, miR-125b-5p may be a promising target for treating ATRA resistance in APL.

The Promise of Retinoids in the Treatment of Cancer: Neither Burnt Out Nor Fading Away.

Since the introduction of all-trans retinoic acid (ATRA), acute promyelocytic leukemia (APL) has become a highly curable malignancy, especially in combination with arsenic trioxide (ATO). ATRA's success has deepened our understanding of the role of the RARα pathway in normal hematopoiesis and leukemogenesis, and it has influenced a generation of cancer drug development. Retinoids have also demonstrated some efficacy in a handful of other disease entities, including as a maintenance therapy for neuroblastoma and in the treatment of cutaneous T-cell lymphomas; nevertheless, the promise of retinoids as a differentiating therapy in acute myeloid leukemia (AML) more broadly, and as a cancer preventative, have largely gone unfulfilled. Recent research into the mechanisms of ATRA resistance and the biomarkers of RARα pathway dysregulation in AML have reinvigorated efforts to successfully deploy retinoid therapy in a broader subset of myeloid malignancies. Recent studies have demonstrated that the bone marrow environment is highly protected from exogenous ATRA via local homeostasis controlled by stromal cells expressing CYP26, a key enzyme responsible for ATRA inactivation. Synthetic CYP26-resistant retinoids such as tamibarotene bypass this stromal protection and have shown superior anti-leukemic effects. Furthermore, recent super-enhancer (SE) analysis has identified a novel AML subgroup characterized by high expression of RARα through strong SE levels in the gene locus and increased sensitivity to tamibarotene. Combined with a hypomethylating agent, synthetic retinoids have shown synergistic anti-leukemic effects in non-APL AML preclinical models and are now being studied in phase II and III clinical trials.

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.

UBR5 Is a Hect E3 Ubiquitin Ligase That Regulates Chromatin Bound Nuclear Hormone Receptor Stability

Acute promyelocytic leukemia (APML) is driven by the oncogenic PML-RARA fusion, which is unique in that it is degraded by two small molecules, all-trans retinoic acid (ATRA) and arsenic trioxide. ATRA-induced degradation of retinoic acid receptor (RARA) is necessary for sustained anti-tumor effect (Ablain et al, JEM, 2013). More generally, nuclear hormone receptors (NRs) are degraded through a ligand-dependent manner of an unknown mechanism. Here we demonstrate that the HECT E3 ubiquitin ligase UBR5 is a ligand-dependent regulator of RARA and other NRs, solve its structure, and show it binds to NRs on chromatin, regulating transcription by degradation. We performed CRISPR-Cas9 screens on RARA and its partner, retinoid X receptor alpha (RXRA), to identify the ubiquitin ligase responsible for ATRA-dependent degradation. We found that UBR5 sgRNAs rescued ATRA-induced degradation. Moreover, we found that RARA and RXRA bind to UBR5 only in the presence of ATRA using co-immunoprecipitation experiments. NRs undergo a stereotypic structural change in the ligand binding domain (LBD) following ligand binding, revealing a conserved hydrophobic cleft that allows for nuclear co-activator (NCOA) binding through LxxLL peptide motifs, which are also present in UBR5. We performed mutagenesis screens on the RARA LBD and identified key residues within the cleft that, when altered, prevented ATRA-induced degradation and UBR5-RARA binding. Mutation of UBR5 LxxLL motifs also led to deficient RARA binding. We solved a 1.7Å crystal structure of RARA bound to a UBR5 LxxLL peptide, confirming ATRA dependent UBR5 binding in the cleft. To understand UBR5/RARA interactions in the context of full-length UBR5, we solved its structure via cryo-electron microscopy and performed a detailed characterization of its functional domains and binding sites. We confirmed the HECT domain is needed for degradation through a UBR5 CRISPR tiling screen and showed specific loss of function of the HECT domain through targeted sgRNAs led to a rescue of ATRA induced RARA degradation. RARA and PML-RARA are tightly chromatin associated, especially in the context of APML. To examine whether UBR5 binds RARA on chromatin, we performed chromatin-immunoprecipitation and sequencing (ChIP-seq) with anti-RARA and anti-UBR5 antibodies in an APML line (NB4) with and without ATRA treatment. Consistent with ATRA-induced degradation, we found significant loss of RARA peaks after ATRA treatment. RARA binding co-localized with UBR5 binding, suggesting UBR5 associates with RARA on chromatin. RNA-seq analysis of NB4 cells with or without UBR5 KD revealed significant alterations in transcript expression, implying that UBR5 regulates RARA-dependent transcription by degradation and therefore potentially downstream cellular behavior. To test this, we knocked down UBR5 in NB4s, treated with ATRA, and found that UBR5 KD led to increased ATRA resistance. More broadly, we found that UBR5 KD rescues ligand-induced degradation of other NRs including the glucocorticoid (GR), estrogen, vitamin-D, progesterone receptors and liver-X-receptor alpha. Through similar experiments we showed that UBR5 binds to GR in the presence of dexamethasone and recognizes and competes with NCOAs for a similar degron. ChIP and RNA-seq in A549 cells following dexamethasone treatment revealed that UBR5 also co-localizes with GR and regulates its transcription, suggesting UBR5-mediated degradation may be a general regulator of multiple NRs and their transcriptional activities. Our findings reveal a novel molecular mechanism regulating nuclear hormone receptors: ligand dependent E3 ligase binding on chromatin, leading to degradation and transcriptional regulation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting METTL14 and RNA methylation to treat osteosarcoma.

Targeting METTL14 and RNA methylation to treat osteosarcoma.

METTL14-mediated epitranscriptome modification of MN1 mRNA promote tumorigenicity and all-trans-retinoic acid resistance in osteosarcoma.

SummaryBackgroundOsteosarcoma (OS) is the most common primary malignant bone tumor in adolescents. The molecular mechanism behind OS progression and metastasis remains poorly understood, which limits the effectiveness of current therapies. RNA N6-methyladenosine (m6A) modification plays a critical role in influencing RNA fate. However, the biological significance of m6A modification and its potential regulatory mechanisms in the development of OS remain unclear.MethodsLiquid chromatography-tandem mass spectrometry (LC-MS/MS), dot blotting, and colorimetric ELISA were used to detect m6A levels. Western blotting, quantitative real-time PCR (RT-qPCR) and immunohistochemistry (IHC) were used to investigate METTL14 expression levels. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq) were used to screen the target genes of METTL14. RNA pull-down and RNA immunoprecipitation (RIP) assays were conducted to explore the specific binding of target genes and relevant m6A “readers”. RNA stability and polysome analysis assays were used to detect the half-lives and translation efficiencies of the downstream genes of METTL14. IHC and clinical data were applied to explore the clinical correlations of METTL14 and its downstream target genes with the prognosis of OS.FindingsWe observed the abundance of m6A modifications in OS and revealed that METTL14 plays an oncogenic role in facilitating OS progression. MeRIP-seq and RNA-seq revealed that MN1 is a downstream gene of METTL14. MN1 contributes to tumor progression and all-trans-retinoic acid (ATRA) chemotherapy resistance in OS. Mechanistically, MN1 is methylated by METTL14, specifically in the coding sequence (CDS) regions, and this modification is recognized by the specific m6A reader insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) to prevent MN1 mRNA degradation and promote it translation efficiency. IHC showed that MN1 expression was positively correlated with METTL14 and IGF2BP2 expression in OS tissues. The METTL14-IGF2BP2-MN1 panel demonstrated more promising prognostic value for OS patients than any of these molecules individually.InterpretationOur study revealed that METTL14 contributes to OS progression and ATRA resistance as an m6A RNA methylase by regulating the stability and translation efficiency of MN1 and thus provides both an underlying biomarker panel for prognosis prediction in OS patients.FundingThis work was supported by the National Natural Science Foundation of China (Grants 81972510 and 81772864).

Genetic Ablation of Nuclear Receptor Interacting Protein 1 (NRIP1) Sensitizes Acute Myeloid Leukemia Cells to Retinoic Acids

The success of differentiation therapy is limited to acute promyelocytic leukemia (APL), and approaches to overcome the differentiation block in non-M3 AML have been unsuccessful. Nuclear hormone receptors (NHR) belong to ligand-inducible transcription factors that govern many cellular functions like differentiation, metabolism, and development. Retinoic Acid Receptor Alpha (RXRA) is a class of NHR that, when activated by all-trans retinoic acid (ATRA), successfully alleviates differentiation block in APL.To identify the NHRs/cofactors that could mediate or prevent differentiation in AML, we examined the differentially expressed NHRs and cofactors between ATRA sensitive (ATs) (NB4 and HL60) vs. ATRA resistant (ATr) AML cell lines (KG1a, Hel, K562, MV4-11, and OCI-AML3). Nuclear Receptor Interacting Protein 1 (NRIP1), a corepressor known to prevent transactivation of ligand-activated NHRs preferentially, was one of the top upregulated targets in the ATr cell lines (3.5 fold increase in RNA expression, figure 1a ). Immunoblot analysis also showed a significant increase in NRIP1 protein expression in the ATr than ATs cell lines (Figure 1b). Further, probing for NRIP1 expression in the publicly available TCGA and MILE AML study cohorts showed decreased NRIP1 expression in the APL cohort compared to other AML subtypes. Methylation profile from CCLE database of the NRIP1 promoter in AML cell lines showed ATs cell lines to be highly methylated compared to the ATr cell lines, suggesting the involvement of NRIP1 in mediating differentiation block in non-M3 AML (Figure 1c). To further dissect the role of NRIP1 in mediating this differentiation block, we carried out experiments in the AML cell line KG1a (having primitive blast features, high expression of NRIP1, and unresponsive to ATRA). Using CRISPR-cas9, we developed an NRIP1 knock-out (KO) cell line (Figure 1d). NRIP1 KO cell line showed a significant reduction in proliferation rate (Doubling time 26.2 vs. 36.5Hrs p<0.05). Further, cell cycle analysis revealed that NRIP1 KO leads to increased accumulation of cells in the G0 phase than in the S-phase (Figure 1e & f). We next assessed the sensitivity of the NRIP1 WT/KO cells to retinoic acids ATRA and bexarotene. Cells were treated with 1µM ATRA / bexarotene or in combination for 72 hours and evaluated for differentiation using CD11b marker by flow cytometry. NRIP1 KO alone leads to a marginal increase in basal CD11b expression compared to the WT cells (Mean CD11b expression 2.03% Vs 0.91%). ATRA treatment further increased the CD11b expression to 3.8% in KO cells compared to 1.6% in the WT cells. A similar increase in CD11b expression was observed in bexarotene-treated cells (3.7% Vs 1.24%). Combination of ATRA with bexarotene showed a 3-fold increase in CD11b expression in the KO cells compared to the WT (23.9% Vs 7.2%, Figure 1g). NRIP1 KO diminishes its repressive action on ligand-activated RARA (ATRA activated) and RXRA (Bexarotene-activated), thereby allowing synergistic differentiation induction by retinoic acids in AML cells.This study suggests a potential mechanism of differentiation inhibition mediated by corepressor NRIP1 in AML cells unresponsive to retinoic acids. Further in-depth analyses of molecular pathways governed by NRIP1 during ligand activation of NHRs are warranted to design differentiation therapies for AML. [Display omitted] DisclosuresMathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.

Relapse Analysis and Resistance Mutations of PML-Rara Fusion Gene in Acute Promyelocytic Leukemia Patients Treated with All-Trans Retinoic Acid and Arsenic

The clinical manifestations, management, and prognosis of acute promyelocytic leukemia (APL) are distinctive in acute myeloid leukemia (AML). Administration of all-trans retinoic acid (ATRA) and arsenic agents have greatly improved the outcome of APL from the most lethal to the most curable subtype AML. Unfortunately, relapse is still observed. And in the case of combined medication with ATRA and arsenic, the resistance mutation spectrum of PML-RARA is far from being revealed. We retrospectively analyzed 40 APL patients that admitted to our hospital from Jan. 2013 to Jul. 2020. There were 21 males and 19 females, aged 6-65 years (median age 31.5), and follow-up time was 3-210 months (median time 58.85 months).There were 3 cases that developed secondary tumors, although APL remission has been achieved. One developed refractory AML with KMT2A-MLLT3 fusion during the consolidation treatment one year after the diagnosis of APL. One case relapsed with APL 5 years after the initial diagnosis, and she developed refractory AML with RUNX1-MECOM fusion 3.5 years more after enduring multiple relapses and treatment courses. Another case developed AML-M2 3.5 years after the initial diagnosis of APL, and achieved sustained remission again through allogeneic hematopoietic stem cell transplantation (allo-HSCT).A total of 8 cases came to our hospital at the initial onset of APL, and 1 came during remission. All these cases got sustained remission through combined triple therapy (ATRA, arsenic, and chemotherapy), with the follow-up time of 3 to 37 months (median 20.5 months). No PML-RARA resistant mutation was detected in these 9 cases.A total of 28 cases came to our hospital after APL relapse. The time from APL onset to the first relapse was 9-189 months (median 34 months). PML-RARA resistance mutations were detected in 9 cases, including 8 cases that carry single RARA mutations each, and 1 case that relapsed with multiple extramedullary infiltrations carry quadruple mutations (PML S214L-A216T and RARA R276W-H298N) (Figure 1a and b). A total of 4 cases underwent allo-HSCT after APL relapse. One of them relapsed 6 months after allo-HSCT, and the other 3 got sustained remission with the follow-up time of 47, 39, and 52 months, respectively.There were 2 cases relapsed after maintaining sustained remission for more than 5 years. One was a 58-year-old male who achieved sustained remission after 2.5 years of combined triple therapy. However, APL recurred 16 years after the primary diagnosis. He achieved remission again and now underwent the consolidation course of combined triple therapy. Another was a 23-year-old male who achieved sustained remission after combined arsenic and chemotherapy, but APL relapsed 75 months after the primary diagnosis. He was resistant to combined triple therapy and achieved remission through allo-HSCT. We failed to retrieve archived specimens at the first onset of APL for genomic variation comparison with the second onset. Therefore, we could not determine whether these two cases were relapsed from the original APL or developed another APL.Although we have achieved a high remission rate and long-term survival for APL through combined triple therapy, relapse and secondary tumors still occur in some cases. Especially for cases that endure heavy treatment, genomic toxicity may promote the oncogenesis of secondary tumors. Also, APL may recur over 5 years after the primary diagnosis and sustained remission. Whether there were inherited predisposing factors in these cases is worthy of further investigation.The incidence of PML and RARA resistant mutations in cases resistance for ATRA and arsenic is not very high. This may be related to the combination use of chemotherapeutic drugs and suggests underlying resistance mechanisms. Although both ATRA and arsenic were used in most cases, the incidence of ATRA resistance mutations is significantly higher than that of arsenic, which might attribute to the sparse interaction sites of arsenic and PML.A better combination strategy of arsenic, ATRA, with or without chemotherapy is worthy of further investigation to reduce the incidence of secondary tumors and relapse. Adhering to a systematically planed treatment course is essential for acquiring rapid remission and reduction of relapse. Treatment compliance should be paid attention to guaranteed, especially during the oral administration courses outside the hospital. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Biomimetic matrix for the study of neuroblastoma cells: A promising combination of stiffness and retinoic acid

Neuroblastoma is the third most common pediatric cancer composed of malignant immature cells that are usually treated pharmacologically by all trans-retinoic acid (ATRA) but sometimes, they can spontaneously differentiate into benign forms. In that context, biomimetic cell culture models are warranted tools as they can recapitulate many of the biochemical and biophysical cues of normal or pathological microenvironments. Inspired by that challenge, we developed a neuroblastoma culture system based on biomimetic LbL films of physiological biochemical composition and mechanical properties. For that, we used chondroitin sulfate A (CSA) and poly-L-lysine (PLL) that were assembled and mechanically tuned by crosslinking with genipin (GnP), a natural biocompatible crosslinker, in a relevant range of stiffness (30–160 kPa). We then assessed the adhesion, survival, motility, and differentiation of LAN-1 neuroblastoma cells. Remarkably, increasing the stiffness of the LbL films induced neuritogenesis that was strengthened by the combination with ATRA. These results highlight the crucial role of the mechanical cues of the neuroblastoma microenvironment since it can dramatically modulate the effect of pharmacologic drugs. In conclusion, our biomimetic platform offers a promising tool to help fundamental understanding and pharmacological screening of neuroblastoma differentiation and may assist the design of translational biomaterials to support neuronal regeneration. Statement of significanceNeuroblastoma is one of the most common pediatric tumor commonly treated by the administration of all-trans-retinoic acid (ATRA). Unfortunately, advanced neuroblastoma often develop ATRA resistance. Accordingly, in the field of pharmacological investigations on neuroblastoma, there is a tremendous need of physiologically relevant cell culture systems that can mimic normal or pathological extracellular matrices. In that context, we developed a promising matrix-like cell culture model that provides new insights on the crucial role of mechanical properties of the microenvironment upon the success of ATRA treatment on the neuroblastoma maturation. We were able to control adhesion, survival, motility, and differentiation of neuroblastoma cells. More broadly, we believe that our system will help the design of in vitro pharmacological screening strategy.

Crosstalk between hnRNP K and SET in ATRA-induced differentiation in acute promyelocytic leukemia.

HnRNP K protein is a heterogeneous nuclear ribonucleoprotein which has been proposed to be involved in the leukemogenesis of acute promyelocytic leukemia (APL), as well as in differentiation induced by all‐trans retinoic acid (ATRA). We previously demonstrated a connection between SET and hnRNP K function in head and neck squamous cell carcinoma (HNSCC) cells related to splicing processing. The objective of this study was to characterize the participation of hnRNP K and SET proteins in ATRA‐induced differentiation in APL. We observed higher (5‐ to 40‐fold) levels of hnRNP K and SET mRNA in APL patients at the diagnosis phase compared with induction and maintenance phases. hnRNP K knockdown using short‐hairpin RNA led to cell death in ATRA‐sensitive NB4 and resistant NB4‐R2 cells by apoptosis with SET cleavage. In addition, hnRNP K knockdown increased granulocytic differentiation in APL cells, mainly in NB4‐R2 with ATRA. hnRNP K knockdown had an effect similar to that of treatment with U0126 (an meiosis‐specific serine/threonine protein kinase/ERK inhibitor), mainly in NB4‐R2 cells. SET knockdown in APL cells revealed that apoptosis induction in cells with hnRNP K knockdown occurred by SET cleavage rather than by reduction in SET protein. Transplantation of NB4‐R2 cells into nude mice confirmed that arsenic trioxide (ATO) combined with U0126 has higher potential against tumor progression when compared to ATO. Therefore, hnRNP K/SET and ERK are potential therapeutic targets for both antineoplastic leukemia therapy and relapsed APL patients with ATRA resistance.

Growth Inhibitory Effects of Ester Derivatives of Menahydroquinone-4, the Reduced Form of Vitamin K2(20), on All-Trans Retinoic Acid-Resistant HL60 Cell Line

The first-choice drug for acute promyelocytic leukemia (APL), all-trans retinoic acid (ATRA), frequently causes drug-resistance and some adverse effects. Thus, an effective and safe agent for ATRA-resistant APL is needed. Menaquinone-4 (MK-4, vitamin K2(20)), used for osteoporosis treatment, does not have serious adverse effects. It has been reported that MK-4 has growth-inhibitory effects on HL60 cells by inducing apoptosis via the activation of Bcl-2 antagonist killer 1 (BAK). However, the effect of MK-4 on ATRA-resistant APL has not been reported. Here, we show that ester derivatives of menahydroquinone-4 (MKH; a reduced form of MK-4), MKH 1,4-bis-N,N-dimethylglycinate (MKH-DMG) and MKH 1,4-bis-hemi-succinate (MKH-SUC), exerted strong growth-inhibitory effects even on ATRA-resistant HL60 (HL-60R) cells compared with ATRA and MK-4. MKH delivery after MKH-SUC treatment was higher than that after MK-4 treatment, and the results indicated apoptosis induced by BAK activation. In contrast, for MKH-DMG, reconversion to MKH was slow and apoptosis was not observed. We suggest that the ester forms, including monoesters of MKH-DMG, exhibit another mechanism independent of apoptosis. In conclusion, the MKH derivatives (MKH-SUC and MKH-DMG) inhibited not only HL60 cells but also HL-60R cells, indicating a potential to overcome ATRA resistance.

Research Advances on Mechanisms of Resistance to All-Trans Retinoic Acid in Acute Promyelocytic Leukemia--Review

Abstract Among myeloid leukemias, the acute promyelocytic leukemia (APL) was found to be specifically sensitive to all-trans retinoic acid (ATRA), almost all APL patients respond to ATRA therapy. The ATRA induces remission of APL patients by stimulating the differentiation of the leukemia cells. However, with the long-term application of ATRA alone, ATRA resistance has become one of the main causes of chemotherapy failure in the patients with acute promyelocytic leukemia. At present, the mechanism of ATRA-resistance is not completely clear, this review discusses the mechanism of drug-resistance in terms of signal pathways, genes, proteins and enzyme.

Research Advances on Mechanisms of Resistance to All-Trans Retinoic Acid in Acute Promyelocytic Leukemia--Review

Abstract Among myeloid leukemias, the acute promyelocytic leukemia (APL) was found to be specifically sensitive to all-trans retinoic acid (ATRA), almost all APL patients respond to ATRA therapy. The ATRA induces remission of APL patients by stimulating the differentiation of the leukemia cells. However, with the long-term application of ATRA alone, ATRA resistance has become one of the main causes of chemotherapy failure in the patients with acute promyelocytic leukemia. At present, the mechanism of ATRA-resistance is not completely clear, this review discusses the mechanism of drug-resistance in terms of signal pathways, genes, proteins and enzyme.

Comprehensive Characterization of Cytogenetic and Mutational Analysis of Acute Promyelocytic Leukemia: Is PML-Rara Everything?

Acute promyelocytic leukemia (APL) is characterized by PML-RARA fusion caused by the t(15;17)(q24;q21) translocation. Although PML-RARA fusion explains the dedifferentiation in most of APL patients, it still does not entirely represent the unique cause of all the clinical manifestations of the disease failing to determine the full leukemic phenotype. Up to 40 % of APL patients have an additional chromosomal abnormality other than PML-RARA. Murine studies have reported that additional cytogenetic abnormalities and secondary somatic mutations (for instance FLT3-ITD) might contribute to leukemia progression. Indeed, mice expressing mutant PML-RARA develop definitive leukemia after one year, suggesting that additional hits are required for transformation. Moreover, no distinct genetic signature has been characterized by next generation sequencing (NGS). This confirms that no gene has been reproducibly identified. APL respond to all-trans retinoic acid (ATRA) in the great majority of patients. However, one quarter of APL develop ATRA resistance suggesting that additional secondary chromosomal abnormalities might be evolving resistance. Combination of low dose arsenic, modify certain epigenetics, with ATRA decreased resistance potential and improved response. In the line with other possible factors involved in ATRA resistance, is the broad nature of the targets of ATRA. A molecular core network of ATRA's targets has been clustered in differentiation, growth factors and nuclear receptors possibly cooperating with PML-RARA and additional chromosomal abnormalities. Herein, we aimed to characterize the gene mutations and chromosomal abnormalities playing key roles in cellular differentiation and epigenetic regulation and to correlate the occurrence of these alterations with treatment response and survival outcomes in APL. We took advantage of a large cohort of APL patients (n=145). Median age of the cohort was 50 yrs (19-85); equal gender distribution; median blood counts were: [WBC 6.2 x 109/L (0.4-155); 37% had leukopenia], hemoglobin [9.8 g/dL (2.7-16.2); 32% had anemia] and platelets [29 x 109/L (range of 0-228); 93% had thrombocytopenia]. In terms of karyotype, 15% of the patients carried +8, 7% had complex karyotyping (≥3 cytogenetic abnormalities), 2% had -7/del (7q) or del (12p), 1% had -17/del(17p), and 1 patient had -5. Mutational analysis of 30 genes panel, identified 141 mutations carried by 65% (94/145) of APL patients. The most frequent mutations were observed in FLT3-ITD (61/143; 43%), WT1 (26/139; 23%), and ASXL1 (7/136; 5%) genes. Less frequent mutations were found in 3.7% of CEBPA, KRAS, and NRAS genes as well as in CBL, EZH2, TET2 (3% each) genes. Additionally, we noted that all mutations were recurrent in specific functional pathways and patients carried mutations in more than 1 gene of the same pathway. Of note, cell signaling and proliferation genes (CBL, NRAS, KRAS, KIT, FLT3) were the most frequently mutated (77/141, 55%) and impacted OS (HR: 1.7, P=0.02). Moreover, transcriptional factors which are often mutated in AML (e.g. CEBPA, TP53, NPM1, RUNX1, WT1) as well as major determinants of cell's fate were markedly mutated (38/141, 27%) suggesting that genetic impairment of signaling and transcription might contribute to the lack of differentiation observed in APL phenotypes. Mutations in epigenetic genes and histone methyltransferases (ASXL1, BCORs, DNMT3A, EZH2, IDH1/2, TET2) were also found in 18/141 (13%) while genes regulating cell proliferation and RAS family (CBL, NRAS, KRAS, NPM1) were enriched in 16/132 (12%) of APL cohort. We then analyzed the genetic picture of remission (APLRm, n=131, 90%) and relapsed (APLR, 1sr relapsed to ATRA, n=14, 10%) patients. Acknowledging the low number of APLR, we observed that molecular mutations did not make a key difference in APLRvs. APLRm [except for a complete lack of mutations in epigenetic pathways (0% vs. 13%)]. Contrarily, specific cytogenetic abnormalities were more common in APLR compared to APLRm as the case of +8 (36% vs. 11%; P= .02) and -17/del(17p) (2/14 vs. 0/131; P= .008). In sum, our study demonstrates that PML-RARA might be accompanied by additional acquired chromosomal change with a variety of genetic mutations in key pathways driving cellular differentiation. These molecular/ cytogenetic associations could determine resistance to ATRA and overall APL patients' survival. Disclosures No relevant conflicts of interest to declare.

TRIB3 Stabilizes High TWIST1 Expression to Promote Rapid APL Progression and ATRA Resistance.

The resistance to differentiation therapy and early death caused by fatal bleeding endangers the health of a significant proportion of patients with acute promyelocytic leukemia (APL). This study aims to investigate the molecular mechanisms of all-trans retinoic acid (ATRA) resistance and uncover new potential therapeutic strategies to block the rapid progression of early death. The important role of TWIST1 in APL leukemogenesis was first determined by gain- and loss-of-function assays. We then performed in vivo and in vitro experiments to explore the interaction of TWIST1 and TRIB3 and develop a potential peptide-initiated therapeutic opportunity to protect against early death and induction therapy resistance in patients with APL. We found that the epithelial-mesenchymal transition (EMT)-inducing transcription factor TWIST1 is highly expressed in APL cells and is critical for leukemic cell survival. TWIST1 and TRIB3 were highly coexpressed in APL cells compared with other subtypes of acute myeloid leukemia cells. We subsequently demonstrated that TRIB3 could bind to the WR domain of TWIST1 and contribute to its stabilization by inhibiting its ubiquitination. TRIB3 depletion promoting TWIST1 degradation reverses resistance to induction therapy and improves sensitivity to ATRA. On the basis of a detailed functional screen of synthetic peptides, we discovered a peptide analogous to the TWIST1 WR domain that specifically represses APL cell survival by disrupting the TRIB3/TWIST1 interaction. Our data not only define the essential role of TWIST1 as an EMT-TF in patients with APL but also suggest that disrupting the TRIB3/TWIST1 interaction reverses induction therapy resistance and blocks rapid progression of APL early death.See related commentary by Peeke and Gritsman, p. 6018.

Twist of Fate for Acute Promyelocytic Leukemia: TRIB3-TWIST1 Interaction Promotes Resistance.

While acute promyelocytic leukemia has a good prognosis with all-trans retinoic acid (ATRA) treatment, ATRA resistance is a major obstacle. It is now demonstrated that TRIBBLES 3 (TRIB3) stabilizes TWIST1, leading to ATRA resistance. Peptides that disrupt this interaction lead to the degradation of TWIST1 and overcome ATRA resistance.See related article by Lin et al., p. 6228.

The acetyltransferase GCN5 maintains ATRA-resistance in non-APL AML.

To date, only one subtype of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) can be effectively treated by differentiation therapy utilizing all-trans retinoic acid (ATRA). Non-APL AMLs are resistant to ATRA. Here we demonstrate that the acetyltransferase GCN5 contributes to ATRA resistance in non-APL AML via aberrant acetylation of histone 3 lysine 9 (H3K9ac) residues maintaining the expression of stemness and leukemia associated genes. We show that inhibition of GCN5 unlocks an ATRA-driven therapeutic response. This response is potentiated by coinhibition of the lysine demethylase LSD1, leading to differentiation in most non-APL AML. Induction of differentiation was not correlated to a specific AML subtype, cytogenetic, or mutational status. Our study shows a previously uncharacterized role of GCN5 in maintaining the immature state of leukemic blasts and identifies GCN5 as a therapeutic target in AML. The high efficacy of the combined epigenetic treatment with GCN5 and LSD1 inhibitors may enable the use of ATRA for differentiation therapy of non-APL AML. Furthermore, it supports a strategy of combined targeting of epigenetic factors to improve treatment, a concept potentially applicable for a broad range of malignancies.

The combination of UCN-01 and ATRA triggers differentiation in ATRA resistant acute promyelocytic leukemia cell lines via RAF-1 independent activation of MEK/ERK

With the introduction of arsenic trioxide and all-trans retinoic acid, the prognosis of acute promyelocytic leukemia has greatly improved. However, all-trans retinoic acid resistance is still unresolved in acute promyelocytic leukemia relapsed patients. In this study, the clinical achievable concentration of 7-hydroxystaurosporine synergized with all-trans retinoic acid to induce terminal differentiation in all-trans retinoic acid resistant acute promyelocytic leukemia cell lines. Though 7-hydroxystaurosporine is a PKC inhibitor, PKC might not be involved in the combination-induced differentiation since other PKC selective inhibitors, Gö 6976 and rottlerin failed to cooperate with all-trans retinoic acid to trigger differentiation. The combination significantly enhanced the protein level of CCAAT/enhancer binding protein β and/or PU.1 as well as activated MEK/ERK. U0126 (MEK specific inhibitor) not only suppressed the combination-induced differentiation but also restored the protein level of CCAAT/enhancer binding protein β and/or PU.1. However, RAF-1 inhibitor had no inhibitory effect on MEK activation and the combination-induced differentiation. Therefore, the combination overcame differentiation block via RAF-1 independent MEK/ERK modulation of the protein level of CCAAT/enhancer binding protein β and/or PU.1. These findings may provide a preclinical rationale for the potential role of this combination in the treatment of all-trans retinoic acid resistant acute promyelocytic leukemia patients.