Doses Of All-trans Retinoic Acid Research Articles

Time to Treatment for Patients with Suspected Acute Promyelocytic Leukemia (APL)

Introduction: Acute promyelocytic leukemia (APL) is a rare subtype of acute myeloid leukemia characterized by dysplastic promyelocytes which cause aberrations in coagulation, including hemorrhage and disseminated intravascular coagulation (DIC), leading to a rapidly fatal course if treatment is not initiated promptly. Early administration of all-trans retinoic acid (ATRA) in patients suspected of having APL is crucial for reducing morbidity and mortality. However, APL still exhibits a high early death rate in part due to failure to rapidly initiate ATRA. Therefore, we conducted a retrospective study to investigate the time it takes for ATRA to be ordered and administered to patients with suspected APL at our tertiary care University hospital. We also examined demographic and clinical variables to identify any opportunities for quality improvement. Methods: We queried our electronic medical record (EMR) for all adult patients without a prior diagnosis of APL from 2017-2021 who received at least one dose of ATRA at Thomas Jefferson University Hospital. Our analysis included 91 patients. We collected a wide range of demographic and clinical data (Table 1). The time of admission was defined as the time the admission order was placed in the EMR, and the time of administration was defined as the time the nurse marked the medication as administered. A simple general linear model was used to evaluate univariate association between each single covariate and each log transformed outcome. Model selection by Akaike information criterion (AIC) was conducted to identify the best model according to goodness of fit criteria. The smaller the AIC value, the better the model fit. The parsimonious models were obtained using backward elimination of non-significant predictors (p-values > 0.05) from the best models according to goodness of fit criteria (AIC). Results: The mean time for interval A (time from hospital admission to ordering ATRA) was 284 minutes, the mean time for interval B (time from ATRA order being placed to ATRA administration) was 104 minutes, and the mean time for interval C (time from admission to ATRA administration) was 388 minutes. Further analysis of the time intervals is presented in Table 2. The results of the demographic and clinical variables (Table 1) show that the majority of patients were transferred from an outside hospital (89%) and admitted overnight (87%). Consequently, ATRA was most often ordered (76%) and administered (70%) overnight. Only 26 (29%) patients were ultimately confirmed to have APL. During the index admission, patients required a mean of 6 units of packed red blood cells and 9 units of platelets. A quarter of patients were noted to have signs or symptoms of bleeding upon admission. The results of the multiple linear regression analysis are shown in Table 2. Only signs or symptoms of bleeding were associated with a statistically significant decrease in time to ATRA. Patients with signs of symptoms of bleeding experienced, on average, 39% less time from admission to ATRA being ordered (95% CI: 0.396-0.946, p=0.030) and 31% shorter time from admission to ATRA administration compared to those without signs or symptoms of bleeding (95% CI: 0.489-0.967, p=0.034). Conclusions: Our study highlights that, even at a large, resource-rich, academic medical center, the time to ATRA administration in patients with suspected APL is longer than optimal. The presence of bleeding was the only variable that significantly decreased the time to ATRA. Earlier identification of bleeding risk (including evolving DIC or thrombocytopenia), increased recognition of possible acute leukemia, and more rapid hematology/oncology consultation could help to decrease the time to ATRA and to avoid bleeding complications before they start. Additionally, we found that most patients (89%) were transferred from an outside hospital. Although we were not able to quantify the time from outside hospital admission to transfer, we suspect that the time was substantial and that there are even greater opportunities to improve the care of these patients by increasing ATRA availability at community hospitals, enhancing the education of all front-line providers regarding the prompt recognition of coagulopathy in the setting of suspected acute leukemia, and improving transfer center acceptance documentation that emphasizes the concern for APL and appropriate next steps in evaluation and treatment.

Randomized phase-III study of low-dose cytarabine and etoposide + /− all-trans retinoic acid in older unfit patients with NPM1-mutated acute myeloid leukemia

The aim of this randomized clinical trial was to evaluate the impact of all-trans retinoic acid (ATRA) in combination with non-intensive chemotherapy in older unfit patients (> 60 years) with newly diagnosed NPM1-mutated acute myeloid leukemia. Patients were randomized (1:1) to low-dose chemotherapy with or without open-label ATRA 45 mg/m2, days 8–28; the dose of ATRA was reduced to 45 mg/m2, days 8–10 and 15 mg/m2, days 11–28 after 75 patients due to toxicity. Up to 6 cycles of cytarabine 20 mg/day s.c., bid, days 1–7 and etoposide 100 mg/day, p.o. or i.v., days 1–3 with (ATRA) or without ATRA (CONTROL) were intended. The primary endpoint was overall survival (OS). Between May 2011 and September 2016, 144 patients (median age, 77 years; range, 64–92 years) were randomized (72, CONTROL; 72, ATRA). Baseline characteristics were balanced between the two study arms. The median number of treatment cycles was 2 in ATRA and 2.5 in CONTROL. OS was significantly shorter in the ATRA compared to the CONTROL arm (p = 0.023; median OS: 5 months versus 9.2 months, 2-years OS rate: 7% versus 10%, respectively). Rates of CR/CRi were not different between treatment arms; infections were more common in ATRA beyond treatment cycle one. The addition of ATRA to low-dose cytarabine plus etoposide in an older, unfit patient population was not beneficial, but rather led to an inferior outcome.The clinical trial is registered at clinicaltrialsregister.eu (EudraCT Number: 2010-023409-37, first posted 14/12/2010).

Early death (ED) in elderly acute promyelocytic leukemia (APL) patients treated with lower induction doses of all-trans retinoic acid (ATRA).

e19012 Background: With the introduction of the differentiating agent ATRA, APL has progressed from being one of the most life-threatening diseases to becoming the most curable subtype of adult AML. However, despite this advancement, death within the first 30 days of treatment amongst elderly patients remains a critical issue with ED rates reportedly as high as 20-50%. There is limited data looking at the elderly population due to often being excluded from clinical trials given age associated with being prominent negative risk factor. Pharmacokinetic studies comparing 25 mg/m2/day and 45 mg/m2/day doses in adult patients have shown similar peak concentrations and mean area under the curve. Notably, low dose ATRA has been offered in pediatric patients in order to reduce incidence of differentiation syndrome. Differentiation syndrome is a key contributor to ED in elderly APL patients causing treatment failure. We propose that lower doses of ATRA in the elderly reduces the impact differentiation syndrome has on induction mortality amongst elderly APL patients and leads to a lower rate of ED. Methods: Fifteen APL patients > 60 years of age were treated. Thirteen received low doses of ATRA (equal to or less than 25mg/m2/day) and two received standard doses of ATRA (45mg/m2/day). Arsenic trioxide was also used in addition to cytoreductive agents, such as cytarabine, hydroxyurea, and idarubicin as needed during induction. Dose reductions and regimens were based on age, comorbidities, and overall clinical judgement. During the course of treatment, further dose reductions and pauses in therapy were made based on clinical course. Results: A total of 15 elderly APL patients were treated between May 2017 and November 2020 at cancer centers within Georgia and South Carolina. The age range was 65-88 years old with a median age of 71 years old. 9/15 (60%) were female and 2/15 (13%) were classified as high risk. 13/15 were Caucasian and the remaining 2 were African American. Amongst the 13 APL patients who received lower doses of ATRA, one ED was noted. This occurred in 81-year-old Caucasian male with low risk APL who had multiple significant comorbidities and elected to halt treatment and pursue hospice. Amongst the 2 low risk APL patients who received standard doses of ATRA, one ED was noted due to differentiation syndrome. This patient also had many significant comorbidities. Conclusions: The ED rate in elderly APL patients treated with low doses of ATRA during induction was noted to be 1/13 (7.7%) which is significantly lower than what has been reported in literature. [Table: see text]

Research Progress of Combined Application of All-trans Retinoic Acid and Bone Morphogenetic Protein in Bone Metabolism

All-trans retinoic acid (ATRA) can both promote and inhibit osteogenic differentiation of cells, which is closely related to the concentration, dose and cell type. Low dose of ATRA inhibited the function of osteoclasts and promoted the differentiation of osteoblasts, thus achieving bone remodeling: Bone formation is achieved through the coordination of osteoblasts and osteoclasts. ATRA can be combined with various factors to produce different effects, among which, when combined with bone morphogenetic protein (BMP), the bone formation effect is significant. Compared with homologous dimer, BMP heterodimer can repair the bone defect area more effectively with a smaller dose, and promote the formation of bone tissue. Reduce the probability of secondary lesions such as swelling in the operative area and excessive osteogenesis, and greatly reduce the economic burden of patients. In addition, in vitro experiments showed that ATRA and BMP2/7 combined with mouse embryonic osteoblast precursor cells (MC3T3-E1) had synergistic effect on osteogenesis. This article reviews the recent progress in the studies on the combined effects of ATRA and BMP in bone metabolism.

All-Trans Retinoic Acid Reduces Matrix Metalloproteinase-2 and Increases E-Cadherin Levels in BeWo Choriocarcinoma Cells

Background: Choriocarcinoma is a malignant trophoblastic tumor that can degrade the uterine basement membrane to facilitate local and distant metastasis. Matrix metalloproteinase-2 (MMP-2) is a vital proteinase produced by trophoblasts and can cleave type IV collagen in the basement membrane of the uterine epithelium. In addition, E-cadherin controls cell adhesion, which is associated with tumorprogression, and loss of E-cadherin function is associated with metastasis. Vitamin A analogues, including all-trans retinoic acid (ATRA), exhibit anticancer properties, including metastasis inhibition.This study aimed to investigate the ability of ATRA to prevent trophoblast invasion and metastasis in choriocarcinoma by assessing MMP-2 and E-cadherin activity. Methods: This study administered various doses of ATRA to the BeWo (American Type Culture Collection CCL-98) choriocarcinoma cell line and assessed MMP-2 and E-cadherin activity through flow cytometry. The research was performed at the Biomedical Laboratory of the Faculty of Medicine of Universitas Brawijaya Malang, East Java, Indonesia. Results: BeWo cells were exposed to 0, 50, 100, 200, 400, or 800 μg/mL ATRA. MMP-2 was downregulated after ATRA treatment. The greatest reduction in MMP-2 level was observed after 200 μg/mL ATRA treatment, but this result was not statistically significant (P = 0.550). However, ATRA treatment significantly reduced the number of BeWo cells with low E-cadherin levels (P = 0.012). Conclusion: ATRA downregulates MMP-2 and upregulates E-cadherin. Further in vitro and animal studies are required to evaluate the ability of ATRA to inhibit metastasis. Bangladesh Journal of Medical Science Vol. 22 No. 02 April’23 Page : 336-340

Retinoic acid receptor regulation of decision-making for cell differentiation.

All-trans retinoic acid (ATRA) activation of retinoic acid receptors (RARs) is crucial to an organism's proper development as established by findings for mouse foetuses from dams fed a vitamin A-deficient diet. ATRA influences decision-making by embryonic stem (ES) cells for differentiation including lineage fate. From studies of knockout mice, RARα and RARγ regulate haematopoiesis whereby active RARα modulates the frequency of decision-making for myeloid differentiation, but is not essential for myelopoiesis, and active RARγ supports stem cell self-renewal and maintenance. From studies of zebrafish embryo development, active RARγ plays a negative role in stem cell decision-making for differentiation whereby, in the absence of exogenous ATRA, selective agonism of RARγ disrupted stem cell decision-making for differentiation patterning for development. From transactivation studies, 0.24nM ATRA transactivated RARγ and 19.3nM (80-fold more) was needed to transactivate RARα. Therefore, the dose of ATRA that cells are exposed to in vivo, from gradients created by cells that synthesize and metabolize, is important to RARγ versus RARα and RARγ activation and balancing of the involvements in modulating stem cell maintenance versus decision-making for differentiation. RARγ activation favours stemness whereas concomitant or temporal activation of RARγ and RARα favours differentiation. Crosstalk with signalling events that are provoked by membrane receptors is also important.

AML-173 Pseudotumor Cerebri (PTC) in Patients With Acute Leukemia (AL)

Pseudotumor cerebri (PTC) is characterized by headache, vision changes, and fundus papilledema. Primary PTC is often seen in obese women (childbearing age) and secondary with medications such as all-trans retinoic acid (ATRA). We analyze clinical presentation, and outcomes of PTC in patients with acute leukemia (AL) treated at a general hospital. Single-center retrospective study. We reviewed cases of AL patients with PTC at Memorial Hermann Hospital, Texas Medical Center between January 2013 and April 2022. Three (2.3%) of 127 patients (AML=85, APL=29, ALL= 13) had PTC. Fit non-APL patients received induction chemotherapy (IC) with purine analogs-cytarabine-anthracycline. Unfit patients received a hypomethylating agent and venetoclax. APL patients received IC with arsenic trioxide and ATRA. Patients suspected of having APL were started on ATRA, and discontinued with a negative PML-RARA FISH test. We describe the incidence, risk factors, clinical presentation, and outcomes of AL patients with PTC. Mean age was 42 years. Mean BMI was 42. All were white women. Two had AML, and one Philadelphia chromosome-positive ALL. AML patients received 3 doses of ATRA discontinued after negative PML-RARA FISH. One AML patient had PTC at presentation. The other developed PTC 3 months later. One AML patient had PTC 30 years before being treated for 6 months with acetazolamide; PTC recurred 3 months after her diagnosis. All patients had headaches, blurry vision, and papilledema with brain MRI showing optic sheath distension, globe flattening, and papilla cupping, all features suggestive of PTC. In two, the opening lumbar puncture pressures were elevated at 30 and 35 cm H2O. Cerebrospinal fluid cytology and flow cytometry were negative for leukemia. Two patients (AML=1, ALL=1) received intrathecal (IT) chemotherapy for CNS prophylaxis. All patient's symptoms improved with acetazolamide. LP done for IT chemotherapy helped to alleviate symptoms. No patient with APL (29 patients) developed PTC during treatment with ATRA. PTC is a rare diagnosis that can complicate the clinical course of AL patients. In addition to ATRA-induced PTC, clinicians should consider primary PTC, seen most often in morbidly obese women of childbearing age.

All-trans Retinoic Acid has Limited Therapeutic Effects on Cognition and Hippocampal Protein Expression After Controlled Cortical Impact

Traumatic brain injury (TBI) is known to impair synaptic function, and subsequently contribute to observed cognitive deficits. Retinoic Acid (RA) signaling modulates expression of synaptic plasticity proteins and is involved in hippocampal learning and memory. All trans-retinoic acid (ATRA), a metabolite of Vitamin A, has been identified as a potential pharmacotherapeutic for other neurological disorders due to this role. This study conducted an ATRA dose response to determine its therapeutic effects on cognitive behaviors and expression of hippocampal markers of synaptic plasticity and RA signaling proteins after experimental TBI. Under isoflurane anesthesia, adult male Sprague Dawley rats received either controlled cortical impact (CCI, 2.5 mm deformation, 4 m/s) or control surgery. Animals received daily intraperitoneal injection of 0.5, 1, 5, or 10 mg/kg of ATRA or vehicle for 2 weeks. Animals underwent motor and spatial learning and memory testing. Hippocampal expression of synaptic plasticity proteins neurogranin (Ng), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluA1 sub-unit, as well as RA signaling proteins STRA6, ADLH1a1, CYP26A1 and CYP26B1 were evaluated by western blot at 2-weeks post-injury. ATRA treatment significantly recovered Ng synaptic protein expression, while having no effect on motor performance, spatial learning, and memory, and GluA1 expression after TBI. RA signaling protein expression is unchanged 2 weeks after TBI.Overall, ATRA administration after TBI showed limited therapeutic benefits compared to the vehicle.

All-trans retinoic acid increases the pathogenicity of the H9N2 influenza virus in mice

BackgroundThe H9N2 virus can infect not only birds but also humans. The pathogenicity of H9N2 virus infection is determined by an excessive immune response in the lung. All-trans retinoic acid (ATRA), the active metabolite of vitamin A, plays an important regulatory role and has been widely used in the clinical practice. This study was aimed to investigate whether ATRA could regulate the immune response to H9N2 virus infection in the lungs of mice, thereby reducing the pathogenicity of the H9N2 virus in mice.MethodsMice were infected intranasally with H9N2 virus, and injected intraperitoneally with 0.2 mL of ATRA at low (1 mg/kg), medium (5 or 10 mg/kg), or high therapeutic dose (20 mg/kg), and toxic dose (40, 60, or 80 mg/kg), once per day for 10 days. Clinical signs, survival rates, and lung gross pathology were compared between the ATRA-treated H9N2-infected group, the ATRA group, and the H9N2-infected group, to investigate the effect of different doses of ATRA on the pathogenicity of H9N2 virus. Additionally, the viral load and cytokine concentration of lungs were measured at 3, 5, 7, and 9 days after infection, to investigate the potential mechanism of ATRA in affecting the pathogenicity of the H9N2 virus. Expression levels of cellular retinoic acid-binding protein 1 (CRABP1), cellular retinoic acid-binding protein 2 (CRABP2), and Retinoic acid-inducible gene-I (RIG-I) were detected using Western blotting.ResultsThe ATRA-treated H9N2-infected mice showed more severe clinical signs compared with the H9N2-infected group. The medium and high therapeutic doses of ATRA reduced the survival rates, aggravated lung tissue damage, decreased the expression of interferon beta (IFN-β), and increased the concentrations of interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and C-C motif chemokine ligand 2 (CCL2) in the lungs of the H9N2-infected mice. At the same time, the expression patterns of CRABP1, CRABP2, and RIG-I were changed in mice infected by H9N2 and treated with different concentrations of ATRA.ConclusionsOur findings suggest that the therapeutic dose of ATRA can increase the pathogenicity of the H9N2 virus. Therefore, the consequences of those infected by influenza virus would be more severe after ATRA treatment.

Regulatory Effect of All-Trans Retinoic Acid on the Expression of IL-1β in Macrophages and the Mechanisms Involved

To investigate the regulatory effect of all-trans retinoic acid (ATRA) on the expression interleukin-1β (IL-1β) in macrophages and the mechanisms involved. Macrophages were treated with 1 μmol/L ATRA for 24 h before RNA-Sequence. Differentially expressed genes (DEGs) were screened out and analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, gene ontology (GO) functional analysis, and protein-protein interaction networks (PPI) analysis. After treatment with different doses of ATRA for 24 h, the expression of IL-1β was examined with qRT-PCR and Western blot. The activation of NF-κB signaling and caspase-1 was observed by Western blot and immunofluorescence staining. Compared with the blank control group, a total of 71 DEGs of macrophages were upregulated in the ATRA treatment group. KEGG analysis showed that the up-regulated DEGs were involved in IL-17 signaling pathway, tumor necrosis factor (TNF) signaling pathway, etc. GO analysis indicated that the up-regulated DEGs were involved in the biological processes of the production of IL-1β, response to lipopolysaccharide, etc. PPI analysis revealed that inflammatory cytokines, adhesion molecules, and chemokines were the key genes that ATRA acted on. In vitro experiments showed that ATRA promoted IL-1β expression in macrophages in a concentration-dependent manner. The expression of p-NF-κB, NF-κB, and caspase-1 were significantly increased by ATRA compared with those of the control group ( P<0.05), and p-NF-κB translocated to the cell nucleus in the ATRA group. ATRA may promote the expression of IL-1β by activating NF-κB signaling and caspase-1 in macrophages, this study may provide evidence for the immune regulatory function of ATRA on macrophages.

Clinical Profile, Complications and Outcomes of Patients with Acute Promyelocytic Leukemia: Indian Perspective

Background:The introduction of all-trans-retinoic-acid (ATRA) and arsenic trioxide (ATO) has changed acute promyelocytic leukaemia (APL) from a highly fatal malignancy to a major success in the sphere of haemato-oncology, with long-term survival exceeding 90% in some studies [1]. While literature from the developed world boasts of outstanding outcomes of patients with APL, there is a paucity of data on management strategies, complications and outcomes from the developing world in general and India in particular [2]. With a large population residing in rural areas, high infection rates and limited access to healthcare, we aim to delineate the challenges faced in curing APL in the developing world. MethodsWe retrospective collected data from medical records of all patients diagnosed with APL between January 2016 to December 2020 in the department of Hematology. A presumptive diagnosis of APL was made based on the presence of abnormal promyelocytes in peripheral blood or bone marrow and flow cytometry (CD13, CD33 positive, HLADR, CD34 negative). The diagnosis was confirmed by the presence of PML-RARα fusion gene by RT-PCR. Data on baseline characteristics, therapy, complications and outcome was collected. Overall survival (OS) was defined from the date of diagnosis to death or date of last follow up. Disease free survival (DFS) was defined from the date of first remission to date of first relapse, death or date of last follow up. Results64 patients were treated during the study period. Baseline characteristics, therapy administered and induction outcomes are mentioned in Table 1. 1.Complications:56.25% patients developed differentiation syndrome (DS). The incidence was higher in Sanz high-risk group as compared to Sanz-low risk group (68.8% vs 43.2%, p value 0.044).The incidence of infection at presentation was similar in Sanz high-risk and low-risk groups (55.2% vs 59.4%, p value 0.7). The incidence of new episode of febrile neutropenia during therapy was 62.5% and was higher in Sanz high-risk group as compared to low-risk group (75% vs 50%, p value 0.039). On multivariate analysis, development of infection during induction was a significant predictor of death or relapse (p value 0.02 HR:0.3; 95% CI 0.12-0.84).17.2% patients developed pseudotumor cerebri. All of them were treated with acetazolamide, 6 patients' symptoms persisted despite acetazolamide and the dose of ATRA was lowered to 25mg/m2.3 patients developed thrombosis. One patient had superior sagittal sinus thrombosis at presentation and two patients developed central line related thrombosis during therapy.15.6% patients developed hepatotoxicity of which 3 had an underlying chronic liver disease. The remaining had a normal liver echotexture on ultrasonography and viral markers were normal; the liver dysfunction was attributed to drug induced liver injury.One patient who received AIDA protocol developed PSVT and improved with adenosine. Another patient who received ATRA+ATO+Daunorubicin developed accelerated hypertension and was managed symptomatically.1.Outcomes75% patients attained complete remission after induction. 9 patients died from infection, 6 from bleeding and one from DS.At a median follow up of 26 months, 2 patients belonging to high-risk group had relapsed. They received reinduction with ATRA and ATO; one succumbed to pancreatitis during induction, the other attained CR2 and is on maintenance therapy.The 4-year DFS and OS was 71.25% (95% CI 59.9 to 82.5) and 73.13% (95% CI 62.2 to 84.1) respectively. Patients in Sanz low risk group had a better 4-year DFS (84.9% vs 58.3%, p value 0.026) and OS (84.9% vs 62.5%, p value 0.044) as compared to the Sanz high-risk group. There was a trend towards improved outcomes in high-risk patients with the use of ATRA+ATO+Daunorubicin. ConclusionAPL patients in India have a higher incidence of DS, pseudotumor cerebri, hepatotoxicity, baseline infections & new infections during induction complicating therapy. CR rate, DFS, OS were comparable to other population-based studies. ATRA, ATO and daunorubicin combination is the preferred protocol for treating high-risk patients. REFERENCES1. Iland HJ et al. All-trans-retinoic acid, idarubicin, and IV arsenic trioxide as initial therapy in acute promyelocytic leukemia (APML4). Blood. 2012;120(8):1570-802. Lo-Coco F et al. Retinoic acid and arsenic trioxide for acute promyelocytic leukemia. N Engl J Med. 2013;369(2):111-21. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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.

Efficacy and safety of daratumumab combined with all-trans retinoic acid in relapsed/refractory multiple myeloma

AbstractThe efficacy of daratumumab depends partially on CD38 expression on multiple myeloma (MM) cells. We have previously shown that all-trans retinoic acid (ATRA) upregulates CD38 expression and reverts daratumumab-resistance ex vivo. We therefore evaluated the optimal dose, efficacy, and safety of daratumumab combined with ATRA in patients with daratumumab-refractory MM in a phase 1/2 study (NCT02751255). In part A of the study, 63 patients were treated with daratumumab monotherapy. Fifty patients with daratumumab-refractory MM were subsequently enrolled in part B and treated with daratumumab (reintensified schedule) combined with ATRA until disease progression. The recommended phase 2 dose of ATRA in combination with daratumumab was defined as 45 mg/m2. At this dose, the overall response rate (ORR) was 5%, indicating that the primary endpoint (ORR ≥15%) was not met. However, most patients (66%) achieved at least stable disease. After a median follow-up of 43 months, the median progression-free survival (PFS) for all patients was 2.8 months. Patients who previously achieved at least a partial response or minimal response/stable disease with prior daratumumab monotherapy had a significantly longer PFS compared with patients who immediately progressed during daratumumab as single agent (median PFS 3.4 and 2.8 vs 1.3 months). The median overall survival was 19.1 months. The addition of ATRA did not increase the incidence of adverse events. Flow cytometric analysis revealed that ATRA temporarily increased CD38 expression on immune cell subsets. In conclusion, the addition of ATRA and reintensification of daratumumab had limited activity in patients with daratumumab-refractory MM, which may be explained by the transient upregulation of CD38 expression. This trial was registered at www.clinicaltrials.gov as #NCT02751255.

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 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.

Upregulation of CD38 Expression on Multiple Myeloma Cells By the HDAC Inhibitor Ricolinostat Augments the Efficacy of Daratumumab

Background:The anti-CD38 antibody daratumumab is effective and increasingly being utilized to treat multiple myeloma. A clinical observation with daratumumab is that expression of CD38 on pretreatment myeloma cells correlates with efficacy, and patients with higher CD38 expression have a higher chance to respond. Moreover, during daratumumab therapy, there are reduced CD38 expression levels on myeloma cells, favoring immune escape and disease progression. We have recently shown that the histone deacetylase (HDAC) inhibitor panobinostat induces CD38 upregulation and augments the antibody-dependent cellular cytotoxicity (ADCC) of daratumumab (García-Guerrero E, Blood 2017). Here, we investigated the alternative HDAC inhibitor ricolinostat and all-trans retinoic acid (ATRA) to determine whether upregulation of CD38 is a class effect, and to identify the substance that works most synergistically with daratumumab.Methods:The myeloma cell lines MM1.S and OPM2 were treated with titrated doses of ricolinostat (0, 5, 10 µM), ATRA (0, 10, 25 nM) and panobinostat (0, 10, 25 nM). Expression of CD38, B-cell maturation antigen (BCMA) and SLAMF7 was analyzed by flow cytometry after 24, 48 and 72 h of exposure. ADCC against HDAC inhibitor-treated and untreated myeloma cells was analyzed after addition of PBMC at an effector to target ratio of 25:1 in the presence of daratumumab (1, 10, 50 ug/mL) or isotype control.Results:We first treated the myeloma cell line MM1.S with each of the HDAC inhibitors and analyzed their direct cytotoxic antimyeloma effect. We found the strongest effect with ricolinostat where live MM1.S myeloma cells had decreased to 50% and 20% after 48 h of exposure to 5 and 10 µM of the drug, respectively. Consistent with previous work the direct anti-myeloma effect of panobinostat (decrease of live MM1.S myeloma cells to 85% and 50% after 48 h of exposure to 10 and 25 nM, respectively) and ATRA (no cytotoxic effect) was lower compared to ricolinostat.We then analyzed expression of CD38 on residual live i.e. 7-AAD negative MM1.S cells by flow cytometry and observed a 2-fold (5 µM) and 2,5-fold (10 µM) increase in CD38 expression by mean fluorescence intensity (MFI) after treatment with ricolinostat-treated compared to untreated MM1.S cells. The increase in CD38 expression was already detectable after 24 h and further increased with longer exposure to the drug. Off note, withdrawal of ricolinostat resulted in rapid decline of CD38 expression to baseline levels but increased again to the same magnitude within 24 h of re-exposure to the drug. Overall, the increase in CD38 expression was strongest with ricolinostat followed by panobinostat and ATRA, which we confirmed with several myeloma cell lines and primary myeloma cells. We also confirmed that ricolinostat-induced upregulation of CD38 specifically occurred in myeloma cells and not in the lymphoma cell lines Raji and JeKo-1. In contrast to CD38, expression of BCMA and SLAMF7 after treatment of myeloma cells with ricolinostat had decreased.We then treated myeloma cells with ricolinostat for 48 hours to induce upregulation of CD38 and then performed ADCC assays with daratumumab in the presence of ricolinostat. We found, that the combination treatment ricolinostat plus daratumumab eliminated 95,6% of MM1.S cells after 20h; while 87,4% were eliminated with ricolinostat alone, and only 22,6% with daratumumab alone. Preliminary data show that ricolinostat does not affect expression of the complement inhibitor CD55 and induces a lower increase in CD59 expression compared to panobinostat. Experiments to evaluate complement-dependent cytotoxicity (CDC), and to confirm the synergistic antimyeloma effect of ricolinostat and daratumumab in a murine xenograft myeloma model, are ongoing.Conclusions:Our data demonstrate that the HDAC inhibitor ricolinostat induces upregulation of CD38 expression on myeloma cells. The increase in CD38 expression after treatment with ricolinostat is stronger compared to panobinostat and ATRA. Our data suggest that upregulation of CD38 on myeloma cells is a class effect of HDAC inhibitors, and identify ricolinostat as a highly synergistic combination partner for daratumumab, due to its considerable intrinsic antimyeloma activity. Our data suggest that combination treatment with ricolinostat and daratumumab could increase response rates and extend duration of responses of daratumumab therapy. 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.

Rate of Differentiation Syndrome in Patients Based on Timing of Initial All-Trans Retinoic Acid Administration

Rate of Differentiation Syndrome in Patients Based on Timing of Initial All-Trans Retinoic Acid Administration

Combinatorial Use of Bexarotene Plus ATRA for the Treatment of Acute Myeloid Leukemia

Aim: All-trans retinoic acid (ATRA) results in high response rates in acute promyelocytic leukemia (APL), but has not had a significant impact on non-APL forms of acute myeloid leukemia (AML), even though retinoic acid receptors have been implicated in hematopoietic maturation and self-renewal. RXRs bind as heterodimers with RARs, and are expressed at higher levels in AML cells than are RARs. We sought to determine whether natural RXR ligands exist in primary leukemia cells, whether RXR ligands might augment or inhibit leukemic cell growth, and whether RXRs might be rational targets for AML therapeutics.Background: Retinoid receptors are nuclear hormone receptors, induced during terminal myeloid maturation with highest expressed in M4 and M5 AML. Retinoids serve as intracellular messengers and are activating ligands for the retinoic acid receptors (RAR) and retinoid X receptors (RXR).Methods: We developed an in vivo reporter assay that measures intracellular, activating nuclear receptor ligands in mouse bone marrow cells. We generated transgenic mice with GFP regulated by a UAS promoter. The yeast Gal4 transcription factor recognizes UAS promoter sequences. When a fusion of the Gal4-DNA binding domain (DBD) and a retinoid receptor ligand-binding domain (LBD) is expressed in UAS-GFP bone marrow cells, and an activating ligand is present, the cells express GFP. Leukemia was generated in bone marrow cells from these mice and from Mx1-Cre x Rxrαfl/fl x Rxrβfl/fl mice using MSCV-MLL-AF9 retrovirus.Results: We found that intermediate levels of RXRA ligands are present in mouse MLL-AF9 leukemia cells in vivo. To determine if activation of RXRA is required for MLL-AF9 cell growth, we transduced MSCV-MLL-AF9 retrovirus into bone marrow cells from Mx1-Cre+ x Rxrαfl/fl x Rxrβfl/fl mice and transplanted these into recipient mice. Mx1-Cre is “leaky.” Without external activation of Mx1-Cre (i.e. without pIpC treatment) we found that all subsequent leukemias were associated with deleted (“floxed out”) Rxra and Rxrb alleles, suggesting that the deleted allele was positively selected for. In secondary transplants, we found that MLL-AF9 leukemias with deleted Rxrα and Rxrβ were associated with shorter survival when compared with MLL-AF9 leukemias with wild type Rxrα and Rxrβ, suggesting that naturally occurring RXR ligands suppressed leukemic growth. To determine if pharmacologic activation of retinoid receptors could inhibit MLL-AF9 cell growth, we treated MLL-AF9 cells ex vivo with ATRA and bexarotene. We found that ATRA modestly inhibited growth, and this effect reached a plateau at 62 nM (this may partially explain the limited clinical efficacy of ATRA in non-APL AML) (see Figure). In contrast, the EC50 of bexarotene was 125 nM and the EC50 decreased to 54 nM when bexarotene was combined with 62 nM ATRA. Higher doses of ATRA decreased the bexarotene EC50 further to 43 nM and resulted in near complete culture collapse. Finally, to determine whether combination bexarotene and ATRA inhibited cell growth by inducing differentiation or apoptosis, we examined cytomorphology, cell division by Celltrace Violet staining (similar to CFSE), and Annexin V. Unexpectedly, we observed no evidence of differentiation, but rather increased Annexin V associated with ATRA and bexarotene-induced growth arrest.Conclusion: RXRs act as tumor suppressors in MLL-AF9 murine leukemia cells in vivo, and the activation of RXR/RAR heterodimers with bexarotene and all-trans retinoic acid leads to synergistic apoptosis and growth arrest. Additional experiments are now being performed to test the efficacy of this dug combination in vivo and assess their potential use in elderly relapsed/refractory AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

All-trans retinoic acid regulating angiopoietins-1 and alleviating extracellular matrix accumulation in interstitial fibrosis rats

All-trans retinoic acid (ATRA) is one of essentially active metabolite of vitamin A, and plays an important role in diverse physiological processes, such as cellular growth and function. Renal interstitial fibrosis (RIF) is a common pathological characteristic of chronic renal disease causing end-stage renal disease currently lacking effective treatment. Low level of Angiopoietins-1 (Angpt-1) is associated with extracellular matrix accumulation and fibrosis diseases. This study was performed to assess the association of ATRA with Angpt-1 in RIF disease. Rats were divided into three groups: group of sham (SHO group), group of unilateral ureteral obstruction group (UUO group), UUO mice administrated daily at the dose of ATRA (ATRA group). Masson-staining was used to detect the histologic lesion. Immunohistochemistry and Western-blot were applied to determine the targeted proteins. RIF score was significantly increased in UUO rats when compared with that of SHO group, and the fibrosis score was notably reduced in ATRA group. Transforming growth factor-β1 (TGF-β1), collagen IV (Col-IV) and fibronectin (FN) expressions in UUO group were significantly up-regulated, whereas Angpt-1 expression was significantly down-regulated compared with the SHO group. ATRA treatment reduced TGF-β1, Col-IV and FN expressions and improved Angpt-1 expression compared with the UUO group. The protein expression of Angpt-1 in kidney tissue of UUO group was negatively correlated with RIF index and protein expressions of Col-IV, FN and TGF-β1. In conclusion, low expression of Angpt-1 was associated with the RIF disease and ATRA treatment can increase the Angpt-1 and alleviate the RIF lesion in UUO rats.