All-trans Retinoic Acid Treatment Research Articles

All-trans retinoic acid inhibits epithelial-to-mesenchymal transition (EMT) through the down-regulation of IL-6 in endometriosis.

Endometriosis (EMs) is a benign, but potential metastatic, gynecological disease. Our current study aims to examine whether all-trans retinoic acid (ATRA) inhibits the epithelial-to-mesenchymal transition (EMT) of endometriotic stromal stem cells, as well as to explore the mechanisms involved, especially the role of IL-6 played in. Cell clonogenic capacity was examined by the low-density clonogenicity assay. Cell differentiation capacity was assessed by in vitro differentiation. The level of IL-6 was measured by the ELISA assay. Migration and invasion abilities were measured using the transwell assay. Western blot and RT-qPCR were performed to detect EMT-related genes and proteins. Large endometriotic stromal colony forming units (CFUs) could be regarded as the enrichment sets of endometriotic stromal stem cells. They maintained a higher potential for self-renewal, proliferation, invasion, and EMT, along with up-regulated IL-6. After ATRA treatment, the expression of IL-6 was significantly reduced, accompanied by a decrease in the migration, invasion, and EMT of large endometriotic stromal CFUs. In addition, the inhibition of ATRA was mediated by IL-6. Our study showed that one of the therapeutic effects of ATRA on EMs through its modulation in EMT of large endometriotic stromal CFUs. ATRA may be a promising therapeutic strategy aimed at IL-6 for the stem-cell treatment of EMs.

All-trans retinoic acid alters the expression of adipogenic genes during the differentiation of bovine intramuscular and subcutaneous adipocytes

The present study was designed to determine the influence of all-trans retinoic acid (ATRA) on adipogenesis-related gene regulation in bovine intramuscular (IM) and subcutaneous (SC) adipose cells during differentiation. Bovine IM and SC adipocytes were isolated from three 19-mo-old, crossbred steers. Adipogenic differentiation was induced upon cultured IM and SC preadipocytes with various doses (0, 0.001, 0.01, 0.1, 1 µM) of ATRA. After 96 h of incubation, cells were harvested and used to measure the gene expression of CCAAT/Enhancer binding protein β (C/EBPβ), peroxisome proliferator-activated receptor (PPAR) γ, glucose transporter 4 (GLUT4), stearoyl CoA desaturase (SCD), and Smad transcription factor 3 (Smad3) relative to the quantity of ribosomal protein subunit 9 (RPS 9). Retinoic acid receptor (RAR) antagonist also tested to identify the effect of ATRA on PPARγ -RAR related gene expression in IM cells. The addition of ATRA to bovine IM decreased (p < 0.05) expression of PPARγ. The expression of PPARγ was also tended to be downregulated (p < 0.1) in high levels (10 μM) of ATRA treatment in SC cells. The treatment of RAR antagonist increased the expression of PPARγ in IM cells. Expression of C/EBPβ decreased (p < 0.05) in SC, but no change was observed in IM (p > 0.05). Increasing levels of ATRA may block adipogenic differentiation via transcriptional regulation of PPARγ. The efficacy of ATRA treatment in adipose cells may vary depending on the location.

Quantitative and qualitative composition of the innate immune response are equally important in wound healing after myocardial infarction

Abstract Background Emergency hematopoiesis (EH) serves as the foundation of monocyte-derived and macrophage (Mφ) driven efferocytosis and ventricular remodeling after myocardial infarction (MI). Excessive myelopoiesis, however, can stipulate maladaptive wound healing and its therapeutic reduction may be a novel approach to preserve cardiac function. All-trans retinoic acid (ATRA) is a pleiotropic modulator of EH and innate immunity shielding hematopoietic stem cells from activation and driving survival and differentiation of myeloid cells. Purpose This study aimed to investigate this intriguing interplay of ATRA in wound healing after MI. Methods MI was induced by permanent coronary ligation in C57BL/6 mice and treated with daily injections of either ATRA (30mg/kg) or DMSO (vehicle) up to five days, starting 24h after ligation. Flow cytometry (FACS) was used for cell cycle analysis and immunophenotyping of leukocytes in bone marrow (BM), blood and heart. Immunohistochemistry (IH), masson trichrome (MT) staining and echocardiography evaluated inflammatory-fibrotic and functional development. Cytokine expression was analyzed by qPCR in bulk infarct and isolated, polarized Mφ-populations of BM-derived and cardiac resident origin. Results On day 2 after MI, EH was significantly reduced in ATRA-treated mice as compared to vehicle controls by means of cell cycle activity (n=6–13 per group; p&amp;lt;0,01) and myeloid cells in BM, blood and infarct tissue (n=5–13; p&amp;lt;0,05). Consequently, mRNA-expression of key inflammatory cytokines, IL-1β and TNFα, was diminished in the infarct tissue in this early phase (n=5–12; p&amp;lt;0,05). These changes, however, failed to preserve cardiac function and ventricular remodeling, 21 days after MI (n=10–11; not significant). By qPCR, non-canonical activation of recruited ATRA-primed monocyte-derived Mφ, was found to propagate a pro-inflammatory phenotype with higher expression of MMP2 and MMP9 in sorted cardiac Mφ (n=4–5; p&amp;lt;0,001). Furthermore, prominent IL-1β-expression in M2-polarized BM-derived Mφ indicated an impaired anti-inflammatory phenotype after ATRA treatment (n=4–6; p&amp;lt;0,05). Strikingly, these changes also occurred in remote myocardium where IH revealed a 2-fold increase of CD11b - positive myeloid cells accompanied by increased expression of TNFα and TGFβ (n=9; p&amp;lt;0,001). MT-staining, performed 21 days after MI, demonstrated an almost 3-fold increase in collagen deposition in remote myocardium of ATRA treated mice in contrast to vehicle controls (n=4–6; p&amp;lt;0,0001). Conclusion Despite a beneficial reduction of EH after MI, short-term treatment with ATRA induced profound and persisting changes in the cytokine expression of monocyte-derived Mφ, which significantly altered their function and thus prevented improvements in cardiac function. Our data provide evidence that quantitative and qualitative changes in innate immunity are equally important for cardiac remodeling after MI. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

THIS DYSPNEA FEELS DIFFERENT: A CASE OF DIFFERENTIATION SYNDROME

TOPIC: Pulmonary Manifestations of Systemic Disease TYPE: Medical Student/Resident Case Reports INTRODUCTION: Acute Promyelocytic Leukemia Differentiation Syndrome (DS) also known as Retinoic Acid Syndrome (RAS) is a potentially lethal complication of All-Trans Retinoic Acid (ATRA) treatment. Symptoms are initially nonspecific from dyspnea, weight gain, fever and weakness. Major findings seen in DS includes bilateral infiltrates on chest imaging with possible pleural and pericardial effusion. Patients respond favorably to steroids in the form of Dexamethasone while ATRA is being held. CASE PRESENTATION: A 70-year-old male presented to the hospital with dyspnea and body aches, with initial diagnosis of pneumonia and subsequently started on antibiotics. Chest X-Ray showed right lower lobe infiltrate. Labs also showed Pancytopenia which prompted Flow Cytometry and Bone marrow biopsy revealing Acute Promyelocytic Leukemia. Patient was started on chemotherapeutic agents including Arsenic Trioxide and Tretinoin. The following day, patient's dyspnea worsening requiring BiPAP, his CT chest revealed new bilateral infiltrates with pleural and pericardial effusions. His labs showed worsening leukocytosis of 61, increased creatinine to 1.6. Due to the symptoms mentioned above as well as a high clinical suspicion, patient was started on Dexamethasone. Patient's respiratory status drastically improved over the next 2-3 days. Patient was subsequently discharged to continue Tretinoin outpatient. DISCUSSION: Differentiation Syndrome has unknown epidemiology, however, there have been varying reports that it has an incidence between 2-48% of patients treated with ATRA. The pathogenesis of DS is unclear however the most widely accepted theory is massive cytokine release by blast cells leading to a systematic inflammatory response syndrome and increased vascular permeability leading to both hypotension and organ hypoperfusion. There is currently no universally accepted diagnostic criteria for DS, however common findings are dyspnea, unexplained fever, weight gain >5 kg and hypotension. Lab findings in DS are nonspecific and not pathognomonic, leukocytosis is often seen in approximately half the patient. The presence of 3 of the previous findings warrants empiric therapy with Dexamethasone. Prospective and retrospective studies show a mortality of 1% in patients with DS who are treated with steroids. CONCLUSIONS: Differentiation Syndrome is a potentially fatal complication of ATRA therapy with no universally accepted diagnostic criteria. A high clinical suspicion is required to diagnose and treat DS for a favorable outcome. REFERENCE #1: Stahl M, Tallman MS. Differentiation syndrome in acute promyelocytic leukaemia. Br J Haematol. 2019 Oct;187(2):157-162. doi: 10.1111/bjh.16151. Epub 2019 Aug 13. PMID: 31410848. DISCLOSURES: No relevant relationships by Sreevastav teja Kalangi, source=Web Response Owner/Founder relationship with Palaniandy K. Kogulan, M.D., PLLC Please note: since 2010 Added 05/13/2021 by Palaniandy Kogulan, source=Web Response, value=Salary No relevant relationships by Mohamed Mohamed, source=Web Response

Cardioprotective effect of all-trans retinoic acid on cisplatin induced cardiotoxicity in rats

used in the treatment of various tumors. Retinoic acid has potent antioxidant effects. In this study, it is aimed to reveal the effects of all-trans retinoic acid (ATRA) on CP induced cardiotoxicity.&#x0D; Materials and Methods: In the study, wistar albino rats were used. Control group received single daily doses of 1 ml/kg saline and ATRA group received single daily doses of ATRA(7,5mg/kg/day) intraperitoneally (i.p.) for 10 days. ATRA+CP group received a single dose of CP(7mg/kg) i.p. on the fourth day of the 10 days of ATRA (7,5mg/kg/day) i.p. treatment. The rats in the CP group received only a single dose of cisplatin(7mg/kg) i.p. given on the 4th of 10 days of treatment. After treatment, the groups were compared based on cardiac histopathology findings.&#x0D; Results: Necrosis, cytoplasmic vacuolization, congestion, hemorrhage and edema were more common in the CP group than the control group). Necrosis and cytoplasmic vacuolization in the all-trans retinoic acid + cisplatin group was observed statistically significantly less frequently than the CP group. &#x0D; Conclusion: This study confirmed that cisplatin therapy had destructive effects on heart tissue, and showed that all-trans retinoic acid treatment could histopathologically prevent cisplatin-induced cardiotoxicity.

All-trans retinoic acid induces differentiation in primary acute myeloid leukemia blasts carrying an inversion of chromosome 16

All-trans retinoic acid (ATRA)-based therapy for acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML), is the most successful example of differentiation therapy. Although ATRA can induce differentiation in some non-APL AML cell lines and primary blasts, clinical results of adding ATRA to standard therapy in non-APL AML patients have been inconsistent, probably due to use of different regimens and lack of diagnostic tools for identifying which patients may be sensitive to ATRA. In this study, we exposed primary blasts obtained from non-APL AML patients to ATRA to test for differentiation potential in vitro. We observed increased expression of differentiation markers, indicating a response to ATRA, in four out of fifteen primary AML samples. Three samples in which CD11b increased in response to ATRA had an inversion of chromosome 16 as well as the CBFB-MYH11 fusion gene, and the fourth sample was from a patient with KMT2A-rearranged, therapy-related AML. In conclusion, we identified a subgroup of non-APL AML patients with inv(16) and CBFB-MYH11 as the most sensitive to ATRA-mediated differentiation in vitro, and our results can help identify patients who may benefit from ATRA treatment.

Regulation of inflammation and COX-2 gene expression in benzo (a) pyrene induced lung carcinogenesis in mice by all trans retinoic acid (ATRA)

AimInflammation provides favourable microenvironment for cancer development. An enhanced COX-2 gene expression is a key inflammatory mediator of cancers and the drug that inhibits it, helps to manage cancer effectively and increases survival rate. The objective is to analyse the inflammatory changes and COX-2 gene expression in benzo (a) pyrene induced mice and to evaluate the regulatory effect of all trans retinoic acid. Materials and methodsThe body and organ weights were recorded in B(a)P induced mice. The haematological parameters and serum inflammatory markers of carcinogenesis were tested. The H & E stained liver and lung tissues were examined for histopathologic changes. The COX-2 gene expression was analysed by RT-PCR and qPCR in lung and liver. Key findingsThe decreased body weight, increased organ weights and the damages in liver and lung were observed in B(a)P induced mice and were prevented significantly upon ATRA treatment. The lowered Hb, RBC and lymphocytes and an enhanced WBC, monocytes and neutrophils observed in B(a)P group were significantly reversed in treated group. A drastic increase in cancer associated inflammatory markers observed in B(a)P induced mice were significantly (P ≤ 0.001) reduced in treated mice. The RT-PCR product density of COX-2 gene was very high in B(a)P group (lung-0.43 ± 0.06; liver-0.39 ± 0.04) significantly lower in treated group (lung-0.12 ± 0.03; liver-0.08 ± 0.03) with a significant difference in RQ values (B(a)P lung-18.46 ± 0.04, liver-12.46 ± 0.08; treated lung-5.93 ± 0.07, liver-2.92 ± 0.10). SignificanceThe ATRA has decreased the inflammatory condition with downregulation of COX-2 gene expression and thereby prevented carcinogenesis during early stage of B(a)P induced cancer development.

Safety and efficacy of Compound Huangdai Tablets combined with all-trans retinoic acid for treatment of acute promyelocytic leukemia: Clinical evidence and potential mechanisms

ObjectiveTo evaluate the safety and efficacy of Compound Huangdai Tablets (Realgar-Indigo Naturalis formula, RIF) combined with all-trans retinoic acid (ATRA) to treat acute promyelocytic leukemia (APL). MethodsThis study was registered in PROSPERO (CRD42018108118). The relevant literatures on RIF treatment of APL were systematically searched in the following databases: China National Knowledge Infrastructure, Wanfang, VIP Medical Information System, Chinese Biomedical Database, EMBASE, Cochrane Library, and PubMed. The quality of the included studies was evaluated and Review Manager 5.3 software and Stata 13.0 software were used to perform the Meta-analysis. In addition, this study used the method of network pharmacology to conduct a preliminary exploration of the mechanism of RIF on APL. ResultsThe study included 12 studies involving 775 APL patients. The Meta-analysis showed that there was no significant difference (P ＞ 0.05) between the RIF group and the arsenic trioxide (ATO) group for primary outcomes, secondary outcomes apart from liver dysfunction. The incidence of liver dysfunction (P = 0.006) in the RIF group were significantly lower than those in the ATO group. In addition, the cost of maintenance therapy in the RIF group was significantly lower (P ＜ 0.05) than the ATO group. Besides, the active ingredients in RIF mainly act on targets proteins such as ACHE, NCOA2, RXRA, and then play a role in the treatment of APL through regulating multiple molecular mechanisms, such as TP53 regulates transcription of cell cycle genes, nuclear receptor transcription pathway. ConclusionThere was no significant difference in efficacy of oral RIF combined with ATRA compared with intravenous ATO combined with ATRA for the treatment of APL. The oral RIF exposed patients to less risk, offered more convenience and had lower prices. RIF can treat APL by multi-target and multi-pathway interventions that inducing apoptosis of APL cells and inhibiting the proliferation of APL cells, and so on. Therefore, oral RIF in the treatment of APL is worthy of further research and development.

The component of the m6A writer complex VIRMA is implicated in aggressive tumor phenotype, DNA damage response and cisplatin resistance in germ cell tumors

BackgroundGerm cell tumors (GCTs) are developmental cancers, tightly linked to embryogenesis and germ cell development. The recent and expanding field of RNA modifications is being increasingly implicated in such molecular events, as well as in tumor progression and resistance to therapy, but still rarely explored in GCTs. In this work, and as a follow-up of our recent study on this topic in TGCT tissue samples, we aim to investigate the role of N6-methyladenosine (m6A), the most abundant of such modifications in mRNA, in in vitro and in vivo models representative of such tumors.MethodsFour cell lines representative of GCTs (three testicular and one mediastinal), including an isogenic cisplatin resistant subline, were used. CRISPR/Cas9-mediated knockdown of VIRMA was established and the chorioallantoic membrane assay was used to study its phenotypic effect in vivo.ResultsWe demonstrated the differential expression of the various m6A writers, readers and erasers in GCT cell lines representative of the major classes of these tumors, seminomas and non-seminomas, and we evidenced changes occurring upon differentiation with all-trans retinoic acid treatment. We showed differential expression also among cells sensitive and resistant to cisplatin treatment, implicating these players in acquisition of cisplatin resistant phenotype. Knockdown of VIRMA led to disruption of the remaining methyltransferase complex and decrease in m6A abundance, as well as overall reduced tumor aggressiveness (with decreased cell viability, tumor cell proliferation, migration, and invasion) and increased sensitivity to cisplatin treatment, both in vitro and confirmed in vivo. Enhanced response to cisplatin after VIRMA knockdown was related to significant increase in DNA damage (with higher γH2AX and GADD45B levels) and downregulation of XLF and MRE11.ConclusionsVIRMA has an oncogenic role in GCTs confirming our previous tissue-based study and is further involved in response to cisplatin by interfering with DNA repair. These data contribute to our better understanding of the emergence of cisplatin resistance in GCTs and support recent attempts to therapeutically target elements of the m6A writer complex.

All-trans retinoic acid impairs glucose-stimulated insulin secretion by activating the RXR/SREBP-1c/UCP2 pathway.

Diabetes is often associated with vitamin A disorders. All-trans retinoic acid (ATRA) is the main active constituent of vitamin A. We aimed to investigate whether ATRA influences diabetic progression and its mechanisms using both Goto-Kazizazi (GK) rats and INS-1 cells. Rat experiments demonstrated that ATRA treatment worsened diabetes symptoms, as evidenced by an increase in fasting blood glucose (FBG) levels and impairment of glucose homeostasis. Importantly, ATRA impaired glucose-stimulated insulin secretion (GSIS) and increased the expression of sterol regulatory element-binding protein 1c (SREBP-1c) and uncoupling protein 2 (UCP2) in the rat pancreas. Data from INS-1 cells also showed that ATRA upregulated SREBP-1c and UCP2 expression and impaired GSIS at 23 mM glucose. Srebp-1c or Ucp2 silencing attenuated GSIS impairment by reversing the ATRA-induced increase in UCP2 expression and decrease in ATP content. ATRA and the retinoid X receptor (RXR) agonists 9-cis RA and LG100268 induced the gene expression of Srebp-1c, which was almost completely abolished by the RXR antagonist HX531. RXRα-LBD luciferase reporter plasmid experiments also demonstrated that ATRA concentration-dependently activated RXRα, the EC50 of which was 1.37 μM, which was lower than the ATRA concentration in the pancreas of GK rats treated with a high dose of ATRA (approximately 3 μM), inferring that ATRA can upregulate Srebp-1c expression in the pancreas by activating RXR. In conclusion, ATRA impaired GSIS partly by activating the RXR/SREBP-1c/UCP2 pathway, thus worsening diabetic symptoms. The results highlight the roles of ATRA in diabetic progression and establish new strategies for diabetes treatment.

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.

Retinoic Acid-Induced Gene G(RIG-G) as a Novel Monitoring Biomarker in Leukemia and Its Clinical Applications

Retinoic acid inducible gene G (RIG-G) is an inducible gene produced during the treatment of acute promyelocytic leukemia with all-trans retinoic acid (ATRA). However, it is unclear the expression level of RIG-G gene in the peripheral blood of healthy subjects and patients with acute promyelocytic leukemia (APL or AML-M3). In the present study, we established the TaqMan-MGB fluorescent probe qPCR (real-time polymerase chain reaction) method for the first time to detect the expression of RIG-G gene in APL. Twenty APL patients were selected, and their RIG-G expression levels were quantified to assess the correlation between the expression of peripheral blood and bone marrow samples. U test was used to analyze the expression level of RIG-G in the peripheral blood of 40 normal specimens and 20 APL patients to observe the prognostic monitoring effect of RIG-G gene in the ATRA treatment process. ROC (receiver operating characteristic curve) was used to analyze and test the diagnostic efficiency of RIG-G gene for APL patients. There is a strong positive correlation between the expression of RIG-G in peripheral blood and bone marrow of APL patients. The expression level of RIG-G in peripheral blood of APL patients is significantly lower than that in healthy controls (p < 0.001). The changes in the expression level of RIG-G in peripheral blood changed indicates the remission and recurrence of APL patients after ATRA treatment, and the ROC curve shows that it has a better diagnostic power for APL. In summary, the TaqMan-MGB real-time PCR method we have established has successfully run. The detection of RIG-G gene expression in peripheral blood can effectively monitor the disease changes of APL patients and avoid harmful bone marrow puncture injury.

Determinants of response to daratumumab in Epstein-Barr virus-positive natural killer and T-cell lymphoma

BackgroundThe potential therapeutic efficacy of daratumumab in natural killer T-cell lymphoma (NKTL) was highlighted when its off-label usage produced sustained remission in a patient with highly refractory disease. This is...

Cytokine Profiles in Acute Promyelocytic Leukemia Patients at Diagnosis and during the Treatment with Acid All-Trans Retinoic and Anthracyclines

INTRODUCTION:Acute promyelocytic leukemia (APL) cells have been shown to secrete IL-1β, TNFα and IL-6 in vitro and particularly the first one has a relevant role in the APL-associated coagulopathy. All-trans retinoic acid (ATRA) treatment decreased levels of IL-8 and increased of IL-1β and G-CSF secreted in the medium. However, the serial analysis of cytokine levels in the plasma of patients with APL during ATRA treatment has never been performed. Considering the importance of inflammation in the pathogenesis of APL-associated coagulopathy, we have analyzed plasma levels of IL-8, IL-1β, IL-6, IL-10, TNF and IL-12p70 in 19 Brazilian patients with APL treated with ATRA and anthracyclines according to the International Consortium on APL protocol.METHODS:We retrospectively analyzed the levels of pro- and anti-inflammatory cytokines in previously frozen plasma samples of 19 APL patients, enrolled in the IC-APL study. Samples were collected at diagnosis (D0) and in the 3rd (D3) and 7th (D7) days of treatment with ATRA and anthracyclines. The control group consisted of 11 healthy adult volunteers. IL-8, IL-1β, IL-6, IL-10, TNF and IL-12p70 were measured using cytometric bead array (CBA-Human Inflammatory Cytokine Kit, BD Biosciences) . The results were analyzed using FCAP Array software (BD Biosciences). The cytokine levels were correlated with white blood cell count (WBC) as well as with the frequency of death during induction (early death) and overall survival (OS). Statistical analysis was performed by using either Mann-Whitney (for comparison of two groups) or ANOVA (comparison of three or more groups) test. Fisher's exact test was used to associate cytokine levels and white blood cells (WBC) count. Overall survival analysis was considered from the date of diagnosis to date of death or last contact. Overall survival curves were prepared by the Kaplan-Meier method and compared by the log-rank test. Significance was set at p <0.05. RESULTSIL-1β, IL-10, TNF and IL-12p70 cytokines were not detected in the plasma from all APL patients and healthy controls, except for the presence of low levels of IL-10 in 3 APL patients at D0 (median concentration: 0 pg/mL; range 0 to 4.5 pg/mL). Plasma IL-8 (median 25.9 pg/mL; range 2.6 to 285.5 pg/mL) and IL-6 levels (median 5.0 pg/mL; range 0 to 580.1 pg/mL) at D0 were higher in APL patients compared with healthy controls (undetected in all samples). ATRA significantly decreased IL-8 plasma levels at D7 in 84.2% of APL patients (p<0.005, D0 vs D7) whereas the plasma concentrations of IL-6 did not change with treatment. There were no correlation between the plasma levels of IL-8 and high (WBC > 104) or low (WBC < 104) risk groups of APL patients. Furthermore, IL-8 and IL-6 levels were not associated with early mortality with IL-6 and IL-8 plasma concentrations of 0.8 vs. 6.8 and 5.1 vs. 34.1 in patients who died or not during induction. Moreover, there was no difference in the overall survival in patients with IL-6 levels above or below the median value of the group (p=0.538; 53.2 vs. 75.2 months; 95% confidence interval = 32.9 to 73.5). The same was observed for IL-8 (p=0.59; 61.5 vs. 65.0 months; 95% confidence interval = 45.6 to 77.4).CONCLUSIONS:APL patients exhibit increased cytokine plasma levels of the pro-inflammatory cytokines IL-8 and IL-6 but these parameters were not associated with known prognostic factors or with disease outcome. DisclosuresNo relevant conflicts of interest to declare.

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.

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

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

LSD1 Inhibition Leads to Differentiation in Hoxa9/Meis1- but Not in MN1-Induced Acute Myeloid Leukemia

Lysine specific demethylase 1 (LSD1) represents a promising epigenetic target in the treatment of acute myeloid leukemia (AML) and inhibition of LSD1 has been demonstrated to facilitate a response of AML cells to all- trans retinoic acid (ATRA). We have previously shown that LSD1 depletion or inhibition using tranylcypromine or its derivatives induces granulocytic differentiation, reduces leukemia-initiating cell (LIC) frequency and leads to a survival benefit in Hoxa9/Meis1 (H/M)-driven AML. We have now modeled the effect of pharmacological or genetic inactivation of LSD1 in another murine leukemia model based on the retroviral overexpression of MN1.We transformed lineage-depleted bone marrow cells from conditional LSD1 knock-out (KO) mice with MN1 and induced the LSD1 KO by treatment with 4-hydroxy-tamoxifen (4-OHT). As a control we compared the results observed in this system with results from cells transformed with the combination of Hoxa9 and Meis1 (H/M). We analyzed short-term proliferation based on XTT assays and differentiation based on cell morphology as well as surface marker expression. We observed a 61% reduction in proliferation in H/M cells, while we only found 28% reduction in proliferation in MN1 cells. LSD1 KO induced marked granulocytic differentiation in H/M cells, but we did not observe any signs of granulocytic differentiation in MN1 cells. While LSD1 KO led to a reduction in the expression of c-Kit and upregulation of Ly-6G in H/M cells, c-Kit expression remained unchanged in MN1 cells. There were also no changes in Gr1 and Mac1, but MN1 cells acquired Sca1 and FcεR1. LSD1 inhibition has been shown to enhance the sensitivity of leukemic cells towards a treatment with ATRA. We therefore now compared the influence of LSD1 KO on the effect of ATRA treatment in H/M and MN1 cells. We saw a 95 % reduction in proliferation in H/M cells with LSD1 KO upon treatment with 0.1 µM of ATRA. In MN1 cells with LSD1 KO we only observed a reduction in proliferation of 28 % at this concentration.We previously found a differentiating effect of irreversible LSD1 inhibitors in H/M transformed cells. We therefore also tested different potent LSD1 inhibitors (derivatives of tranylcypromine) in MN1 cells (trans-N-((2-methoxypyridin-3-yl)methyl)-2-phenylcyclopropan-1-amine (ORY86, AW69), (1S,2R)-2-phenyl-N-(piperidin-4-ylmethyl) cyclopropanamine (AW84), GSK-LSD1). Again we observed no signs of granulocytic differentiation, but an acquisition of the surface markers Sca1 and FcεR1 without concomitant changes in the expression of c-Kit, Gr1 and Mac1. Treatment with AW69 leads to a strong reduction in LIC frequency in the H/M model system. We now investigated if pre-treatment with AW69 in vitro for 96 h also affected the ability of MN1 cells to induce leukemia in mice. In this experiment we observed no change in leukemia development upon transplantation of 20.000 cells per mouse. However while 2 of 3 mice transplanted with control treated cells developed leukemia upon transplantation of 2.000 cells per mouse, no leukemia development was seen at this cell dose with AW69-treated cells. We therefore cannot exclude a moderate effect on LIC frequency in the MN1 model.In order to test if our findings can be transferred to human AML we characterized the expression of MN1, HOXA9 and MEIS1 in 6 primary human AML cell cultures. We then treated these cells with GSK-LSD1 and assessed the expression of CD86, a known differentiation marker induced by LSD1 inhibition. In these experiments we observed a particularly strong upregulation of CD86 in samples expressing higher levels of MEIS1.In summary, our results show that while pharmacological or genetic inactivation of LSD1 potently induces differentiation in H/M-induced AML, it is not sufficient to induce differentiation in MN1-driven AML. This finding could be relevant for selecting patients for treatment with LSD1 inhibitors and warrants further molecular investigations. DisclosuresBug:Astellas: Other: Travel Funding; Janssen: Other: Travel Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Jazz Pharmaceuticals: Other: Travel Funding; Celgene: Honoraria, Other: Travel funding. Lübbert:Celgene: Other: Travel Funding; Janssen-Cilag: Other: Travel Funding, Research Funding; Ratiopharm: Other: Study drug valproic acid. Berg:Astellas: Other: Travel Funding; Riemser: Consultancy; Alexion: Other: Travel Funding; Celgene: Other: Travel Funding; Abbvie: Other: Travel Funding.

Prospective Evaluation of Thrombo-Hemorrhagic Events and Hypercoagulation Markers in Patients with Acute Promyelocytic Leukemia (APL) Receiving All-Trans Retinoic Acid (ATRA) Plus Chemotherapy

Introduction: Patients with APL typically present with a life-threatening thrombo-hemorrhagic syndrome (THS), due to the consumption of coagulation factors and platelets, secondary to clotting activation. Presently, all- trans retinoic acid (ATRA) has greatly improved the management of APL, but has not significantly changed the rate of early hemorrhagic deaths and may actually promote thrombosis.Characterizing the coagulopathy and identifying predictive markers of bleeding/clotting complications remain critical issues. Aim: In newly diagnosed APL patients, in the first month after diagnosis, we wanted prospectively to: 1. register thrombo/hemorrhagic events; 2. evaluate, the profiles of circulating hypercoagulation markers and the molecular expression of Tissue Factor (TF), before and during ATRA therapy; and 3. verify whether changes in the biomarker profiles correlate with the occurrence of thrombo-hemorragic events. Methods: Sixty consecutive APL patients receiving ATRA+Idarubicin for remission induction (GIMEMA protocol AIDA2000) were enrolled from January 2005 to March 2017. Blood samples from a subgroup of 30 patients (F/M= 11/19) were obtained at the onset (T0), and on days 7, 14 and 28 (T7, T14, T28) of remission induction therapy. Plasma levels of activated factor VII-antithrombin complex (FVIIa-AT), thrombin-antithrombin complex (TAT), and D-dimer were measured by ELISA; TF mRNA levels in peripheral blood mononuclear cells (PBMC) was evaluated in 16 APL patients by real time-polymerase chain reaction. Results: At enrolment (T0), before starting ATRA, 13.3% of APL patients presented with early major hemorrhages, including 3 (5%) fatal intracranial bleeding and 5 (8.3%) non-fatal major bleeding (i.e. gengivorrhagia, gastrointestinal bleeding, epistaxis, menorrhagia). At T0, the prevalence of thrombosis in the total group of 60 APL patients was 6.6%, including 1 fatal pulmonary embolism (PE), 2 non-fatal venous thrombosis (1 PE, 1 lower limb deep vein thrombosis [DVT]), and 1 non-fatal arterial thrombosis (acute myocardial infarction [AMI]). During ATRA therapy, 8.3% of APL patients developed major bleeding: including 2 fatal intracranial hemorrhage after 3 days of ATRA treatment and 3 non-fatal major bleeding (i.e. gengivorrhagia, epistaxis, menorrhagia) after 7, 10 and 17 days of ATRA. Two more patients developed non-fatal thrombosis (i.e. 1 AMI and 1 DVT) after 9 and 20 days of ATRA treatment, respectively. The laboratory sub-study of 30 subjects showed that, at T0, before starting therapy, the plasma levels of FVIIa-AT, TAT and D-dimer were significantly higher in APL patients compared to controls (p<0.05). During induction therapy with ATRA, the levels of TAT and D-dimer significantly (p<0.05) and progressively decreased at T7 and T14, reaching the lowest value at 28 days. Differently, FVIIa-AT levels were unchanged at T7 and T14, and dropped significantly only at T28 (p<0.05 vs T0). In addition, in APL PBMC from 16 patients, the levels of TF mRNA, initially elevated compared to healthy control PBMC (p<0.05), during induction therapy were significantly decreased by 68% and 90%, at T7 and T28, respectively. In these 16 APL patients, a statistically significant correlation (p<0.05) was found between decrease in TF mRNA and FVIIa-AT levels during ATRA therapy. Of the 30 patients included in the laboratory sub-study, 3 had non-fatal thrombosis at diagnosis, no major hemorrhages were recorded. The predictive value of THS of thrombotic markers could not be calculated. Conclusions: Our data show a significant rate (31%) of severe (fatal and non-fatal) thrombo/hemorrhagic events in our cohort of APL patients in the first 3 weeks of diagnosis, with 4 fatal events (6.6%) occurring early, before starting ATRA. Laboratory data show for the first time that, while TAT and D-dimer confirm to be sensitive to ATRA downregulation, differently FVIIa-AT levels persist at high level over time, showing the perseverance of TF-dependent clotting activation. Future large prospective clinical trials should include FVIIa-AT to evaluate persistent clotting activation during ATRA. DisclosuresFalanga:Advisory Board: Bayer, Janssen Speakers' bureau: Rovi, BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Rambaldi:Novartis, Roche/Genentech, Amgen, Italfarmaco: Consultancy; Novartis, Amgen, Celgene, Sanofi: Other: Travel, Accomodations, Expenses. Lo Coco:Lundbeck: Honoraria, Speakers Bureau; TEVA: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Immunomodulatory agents as potential therapeutic or preventive strategies for COVID-19.

Currently, the COVID-19 pandemic, caused by the novel SARS-CoV-2 coronavirus, represents the greatest global health threat. Most people infected by the virus present mild to moderate respiratory symptoms and recover with supportive treatments. However, certain susceptible hosts develop an acute respiratory distress syndrome (ARDS), associated with an inflammatory "cytokine storm", leading to lung damage. Despite the current availability of different COVID-19 vaccines, the new emerging SARS-CoV-2 genetic variants represent a major concern worldwide, due to their increased transmissibility and rapid spread. Indeed, it seems that some mutations or combinations of mutations might confer selective advantages to the virus, such as the ability to evade the host immune responses elicited by COVID-19 vaccines. Several therapeutic approaches have been investigated but, to date, a unique and fully effective therapeutic protocol has not yet been achieved. In addition, steroid-based therapies, aimed to reduce inflammation in patients with severe COVID-19 disease, may increase the risk of opportunistic infections, increasing the hospitalization time and mortality rate of these patients. Hence, there is an unmet need to develop more effective therapeutic options. Here, we discuss the potential use of natural immunomodulators such as Thymosin α1 (Tα1), all-trans retinoic acid (ATRA), and lactoferrin (LF), as adjunctive or preventive treatment of severe COVID-19 disease. These agents are considered to be multifunctional molecules because of their ability to enhance antiviral host immunity and restore the immune balance, depending on the host immune status. Furthermore, they are able to exert a broad-spectrum antimicrobial activity by means of direct interactions with cellular or molecular targets of pathogens or indirectly by increasing the host immune response. Thus, due to the aforementioned properties, these agents might have a great potential in a clinical setting, not only to counteract SARS-CoV-2 infection, but also to prevent opportunistic infections in critically ill COVID-19 patients.

Regulatory Action of all trans Retinoic Acid on Metastasis Induced lung Cell Metabolic Changes during Implantation of B16F10 Cancer Cells in C57BL6 Mice

The changes that occur during metastasis lodging is under intense research now to develop preventive new drugs to fight against the deadly metastasis. The molecular drug, all trans Retinoic Acid (ATRA) has regulatory effects on signal mediated metabolism. In this study, we have analyzed the metastasis facilitating metabolic changes in mice lung when a highly metastatic melanoma cell line (B16F10) having potency to lodge in lung was implanted via tail vein injection into C57BL/6 mice (1×106 cells/ml in PBS). One group of implanted mice were treated with 0.60 mg of ATRA per Kg body weight daily for 21 days. The alteration of protein, enzymatic and non-enzymatic antioxidants (SOD, Catalase, GPX, GSH) levels and the lipid profile with cholesterol level were evaluated in the lung tissues. The ATRA treatment caused 62.16% inhibition on metastatic nodule formation. Compared to normal mice, the cancer control mice showed an increased (p≤ 0.01**) total protein, LPO and NO and a decreased antioxidant. In ATRA treated group, all these levels were reverted to near normal levels with a high significance (p≤ 0.01**) difference from untreated cancer mice. The lipid profile and cholesterol level also were altered in cancer and were normalized in ATRA treated group with high significance (p≤ 0.01**). All these results implies that the metabolic changes induced in the lung tissue during metastatic lodging of melanoma cells were prevented and regularized by the ATRA treatment in vivo which give a scope of anti-metastatic therapy using ATRA.