Combination Of All-trans Retinoic Acid Research Articles

Resveratrol Can Differentiate Human Melanoma Stem-like Cells from Spheroids Treated With All-trans Retinoic Acid.

Stem-like cancer cells are believed to be the leading cause of therapy resistance in malignant melanoma (MM). All-trans retinoic acid (ATRA) differentiation therapy is considered a promising approach to eradicate stem-like cancer cells, but some melanoma cells are resistant to ATRA. This study aimed to examine whether resveratrol (RS), a natural polyphenol compound, could improve the response of MM stem-like cells to ATRA and explore the possible underlying mechanisms. MM stem-like cells were established from a spheroid model of A375 human MM cell line. The response to RES alone and in combination with ATRA, was examined through analysis of cancer stemness, cell viability, and protein expression. The stem-like cells showed resistance to the anticancer drug docetaxel; however, the combination of RES and ATRA augmented the effects of docetaxel. Accordingly, these combinatorial effects were associated with significant inhibition of the expression levels of stemness markers CD133, OCT4, CD271, and ABCG2. The tested combinations also led to a significant increase in melanocyte differentiation marker SOX9, while efficiently suppressing the dedifferentiation marker SOX10. Notably, RES alone effectively up-regulated retinoic acid receptor beta (RARβ) expression and down-regulated crucial mediators like DNMT1, polycomb-group proteins EZH2, and BMI-1, which mechanistically explain how RES enhanced the differentiation-inducing effects of ATRA. The resistance of MM stem-like cells to ATRA can be attenuated by RES and combined applications of ATRA and RES provide a promising strategy for MM treatment.

Toxicity and efficacy study of a combination of two retinoic acids in an ApoE knockout mouse model of atherosclerosis.

Atherosclerosis is a major contributor to cardiovascular disease, characterized by inflammation and lipid accumulation in arterial walls, leading to plaque formation. Elevated low-density lipoprotein cholesterol is a primary risk factor for atherosclerosis. All-trans retinoic acid (ATRA), a metabolite of vitamin A, has demonstrated anti-inflammatory effects and potential in regulating vascular injury. 9-cisretinoic acid (9cRA) is an active metabolite of vitamin A and activates the retinoid X receptor. This study investigates whether potassium retinoate (PA9RA), a synthetic combination of ATRA and 9cRA, offers superior efficacy in treating atherosclerosis compared to established treatments such as clopidogrel and atorvastatin. Male ApoE-/- mice were fed a Western-type diet and treated with PA9RA, clopidogrel, or atorvastatin for 10 weeks. The body weight, organ weight, serum biochemistry, and histopathology, including atherosclerotic lesion area and liver steatosis were assessed. PA9RA treatment led to a significant reduction in body weight and inguinal fat, with the 45 mg/kg/day dose showing marked efficacy in decreasing atherosclerotic lesion size and ameliorating liver steatosis. Histopathological evaluation revealed decreased foam cell formation and improved liver histology in PA9RA-treated groups compared to controls. Notable side effects included epidermal hyperplasia and gastric hyperplasia at high doses of PA9RA. PA9RA exhibits superior efficacy over clopidogrel and atorvastatin in ameliorating atherosclerosis and fatty liver in ApoE-/- mice. This study highlights PA9RA's potential as a promising therapeutic agent for atherosclerosis. Further research is needed to elucidate its mechanisms of action and assess long-term safety and efficacy.

In Vivo Chemical Screening in Zebrafish Embryos Identified FDA-Approved Drugs That Induce Differentiation of Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is characterized by the abnormal proliferation and differentiation arrest of myeloid progenitor cells. The clinical treatment of AML remains challenging. Promoting AML cell differentiation is a valid strategy, but effective differentiation drugs are lacking for most types of AML. In this study, we generated Tg(drl:hoxa9) zebrafish, in which hoxa9 overexpression was driven in hematopoietic cells and myeloid differentiation arrest was exhibited. Using Tg(drl:hoxa9) embryos, we performed chemical screening and identified four FDA-approved drugs, ethacrynic acid, khellin, oxcarbazepine, and alendronate, that efficiently restored myeloid differentiation. The four drugs also induced AML cell differentiation, with ethacrynic acid being the most effective. By an RNA-seq analysis, we found that during differentiation, ethacrynic acid activated the IL-17 and MAPK signaling pathways, which are known to promote granulopoiesis. Furthermore, we found that ethacrynic acid enhanced all-trans retinoic acid (ATRA)-induced differentiation, and both types of signaling converged on the IL-17/MAPK pathways. Inhibiting the IL-17/MAPK pathways impaired ethacrynic acid and ATRA-induced differentiation. In addition, we showed that ethacrynic acid is less toxic to embryogenesis and less disruptive to normal hematopoiesis than ATRA. Thus, the combination of ethacrynic acid and ATRA may have broader clinical applications. In conclusion, through zebrafish-aided screening, our study identified four drugs that can be repurposed to induce AML differentiation, thus providing new agents for AML therapy.

Acute Promyelocytic Leukemia: Review of Complications Related to All-Trans Retinoic Acid and Arsenic Trioxide Therapy.

The hallmark of acute promyelocytic leukemia (APL) is the presence of the characteristic fusion transcript of the promyelocytic leukemia gene with the retinoic acid receptor α gene (PML::RARA). The PML::RARA fusion is a molecular target for all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). Therapies based on ATRA plus ATO have excellent outcomes in terms of complete remission rates, overall survival, and achievement of deep and durable molecular responses with a very low incidence of relapse. However, although the combination of ATRA and ATO has lower hematologic toxicity than standard chemotherapy, its use is associated with a spectrum of distinctive toxicities, such as differentiation syndrome, liver toxicity, QT interval prolongation, and neurotoxicity. Rigorous monitoring of patients' clinical evolution is indispensable for identifying and addressing each complication. The objective is to maintain an equilibrium between treatment-induced adverse events and therapeutic efficacy. This paper focused on non-hematologic complications associated with the combination of ATRA and ATO. Additionally, we discuss late-onset complications of this therapy. In summary, the majority of treatment-related adverse events are manageable, self-limiting, and reversible. More so, there seems to be a lower incidence rate of secondary neoplasms compared to standard chemotherapy. However, further research is required to assess how the ATRA plus ATO regimen affects the emergence of additional comorbidities.

Fucosylation inhibitor 6-alkynylfucose enhances the ATRA-induced differentiation effect on acute promyelocytic leukemia cells

For acute promyelocytic leukemia (APL), differentiation therapy with all-trans retinoic acid (ATRA) is well established. However, the narrow application and tolerance development of ATRA remain to be improved. In this study, we investigated the effects of combinations of glycosylation inhibitors with ATRA to achieve better efficiency than ATRA alone. We found that the combination of fucosylation inhibitor 6-alkynylfucose (6AF) and ATRA had an additional effect on cell differentiation, as revealed by expression changes in two differentiation markers, CD11b and CD11c, and significant morphological changes in NB4 APL and HL-60 acute myeloid leukemia (AML) cells. In AAL lectin blot analyses, ATRA or 6AF alone could decrease fucosylation, while their combination decreased fucosylation more efficiently. To clarify the molecular mechanism for the 6AF effect on ATRA-induced differentiation, we performed microarray analyses using NB4 cells. In a pathway analysis using DAVID software, we found that the C-type lectin receptor (CLR) signaling pathway was enriched with high significance. In real-time PCR analyses using NB4 and HL-60 cells, FcεRIγ, CLEC6A, CLEC7A, CASP1, IL-1β, and EGR3, as components of the CLR pathway, as well as CD45 and AKT3 were upregulated by 6AF in ATRA-induced differentiation. Taken together, the present findings suggest that the CLR signaling pathway is involved in the 6AF effect on ATRA-induced differentiation.

All-trans-retinoic acid and valproic acid: A combinatorial approach for the treatment of nephrotic syndrome

Massive proteinuria, oedema, hypoalbuminemia, and hyperlipidemia are the hallmarks of nephrotic syndrome (NS). Recently, epigenetic pathways in renal diseases have been identified. The present work hypothesizes using the combination of two epigenetic drugs all-trans retinoic acid (ATRA) and valproic acid (VPA) as a prospective treatment method to lessen NS-related glomerulosclerosis, fibrosis, and increased renal function parameters along with attenuating inflammation and improving overall mitochondrial health. To induce NS, doxorubicin (8.5 mg/kg, n = 6) was injected intravenously into female Sprague Dawley rats. After 28 days, ATRA and VPA were orally administered to the rats, alone and in combination at a dose of 5 mg/kg (n = 6) and 200 mg/kg (n = 6) in sesame oil and saline, respectively. Prednisolone (3 mg/kg in saline; n = 6) was used as standard. Following 21-daytreatment period, the rats were sacrificed prior to which 24hrs urine samples were obtained. Blood samples were collected and kidneys were extracted for further analyses. Renal function parameters (proteinuria, BUN, albumin, creatinine), levels of tissue reengineering and fibrosis markers (TGF-β, MMP2 activity), cholesterol and triglyceride levels were significantly improved in the ATRA and VPA combination group as compared to positive control group. Histopathological analyses revealed a reduction in inflammation, glomerulosclerosis and fibrosis. The inflammatory markers, namely TNF-α, IL-1β, IL-6, NF-κB, determined by ELISA were downregulated. Mitochondrial biogenesis markers viz. PGC-1α, TFAM, NRF1, Nrf2, PPAR-γ, KEAP-1, analysed by RT-qPCR were upregulated thereby showing a significant improvement in the combination group as compared to positive control and standard group. The study overall contributes to a novel approach to treating NS and our findings will surely drive additional exploratory preclinical and clinical studies.

Abstract B032: Outcomes of patients with acute promyelocytic leukemia treated at John Peter Smith Hospital

Abstract Background: Acute Promyelocytic Leukemia (APL) is a subtype of Acute Myeloid Leukemia (AML), accounting for about 10% of all AML cases. Historically, APL was considered to be the subtype of AML with the worst prognosis because most newly diagnosed patients died within a few weeks, and only 35% to 45% of patients achieved long-term survival. The discovery and application of all trans retinoic acid (ATRA) improved complete remission (CR) rate and overall survival (OS) to 70%. Combination of ATRA, a vitamin A derivative, and arsenic trioxide (ATO) as induction therapy for APL resulted in 90-95% CR and >90% 5-year OS. Even though CR and OS has improved, early death (5%-10%) remains the most challenging issue for the disease, especially in resource poor settings. Since APL is rare, oncologists in community may not be fully equipped to treat early complications. John Peter Smith hospital (JPS) is a safety-net hospital serving the needs of underserved population in north Texas. We report outcomes of patients with APL treated at JPS. Methods: Data was obtained from the JPS Oncology Registry. Patients were diagnosed with APL from 1/1/2017 to 12/31/2020 with partial or complete treatment at JPS. We collected non-identifiable patient demographics, diagnosis and initial treatment and complication details. Remission documentation by disappearance of the chromosome abnormality, time from diagnosis to date of death (if applicable) or last activity in medical chart (any phone call with patient, office visit) before 6/12/2023 were obtained. Time to CR and any use of consolidation or maintenance treatment was also collected. Results: 8 APL patients were included- 3 were high-risk patients and 5 were low-risk. Half were male. Age ranged from 22 to 64 years. 5 were under the age of 40 years, 2 were in their 40s and 1 was in 60s at diagnosis. Hispanic:4, Black:3, Asian:1. Induction treatment included ATRA+ATO in 6 patients, while 2 high risk patients received ATRA plus anthracycline-based chemotherapy. Bone marrow morphology post induction was negative in all patients indicating CR (median time to CR was 41.5 days). During induction, treatment complications occurred in all patients including bleeding (75%), clotting (63%), neutropenic fever (50%). Differentiation syndrome (50%) and DIC (50%). All patients underwent ATRA+ATO consolidation treatment for 8-10 months. No patients relapsed. All patients were alive at the time of last medical chart entry (6/12/2023) without active cancer. Conclusions: All patients were in CR post induction treatment. 4 had minimal residual disease post induction. All patients received consolidation with ATRA+ATO, eventually there was no disease detected at molecular level. No patients relapsed. All patients were alive with no active disease at their last follow-up. Excellent outcomes in APL are possible at safety-net setting but will need diligent care to prevent, predict and treat early complications; this may require communication with academic leukemia specialists when transfer to leukemia treatment centers is not possible. Citation Format: Kavya Athipatla, Kari Teigen, Kalyani Narra. Outcomes of patients with acute promyelocytic leukemia treated at John Peter Smith Hospital [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B032.

Epigenetic Modulation Enhances the Therapeutic Potential of All-Trans Retinoic Acid in Acute Myeloid Leukemia

Epigenetic dysregulation has been strongly associated with the development and progression of acute myeloid leukemia (AML). While all-trans retinoic acid (ATRA), a derivative of vitamin A, has demonstrated significant anti-cancer effects in acute promyelocytic leukemia (APL-AML), its clinical efficacy in non-APL AMLs has been limited. This limited response can be attributed, in part, to the epigenetic silencing of genes targeted by or involved in the ATRA pathway, rendering them unresponsive to the drug. Consequently, our research aims to overcome the epigenetic barriers in AML by combining ATRA with small molecules that possess epigenetic modulating properties. To investigate the activation of the retinoic acid pathway in response to ATRA and a library of over 650 small molecules with epigenetic activity, we transduced HEK-293 cells with the p-GreenFire-RARE-Tk-Luc reporter construct. Through the screening process, we have identified 11 compounds that show promise as potential candidates for combination therapy with ATRA in non-APL AML. Notably, when combined with ATRA, two specific compounds, a dual PI3K HDAC inhibitor, and a pan-PKC inhibitor, demonstrated a significant 4-5 fold increase in RARE activity, indicating a synergistic effect. To determine the impact of selected compounds on the functional level, we evaluated the viability, differentiation, and phosphorylation status of ERK1/2, AKT1, MAPK9, p38, and MAPK9 in various cell lines, including HL-60, KG1a, BMNC, and primary MSC and AML cells. The dual PI3K HDAC inhibitor and a pan-PKC inhibitor were noncytotoxic to HL60, BMNC, and primary MSC. Interestingly, dual PI3K HDAC inhibitor with and without ATRA reduced the viability of KG1α by more than 95%. Moreover, dual PI3K HDAC inhibitor and a pan-PKC inhibitor, when combined with ATRA, lead to a 2-3 fold increase in the differentiation of HL60 cells and a 2-4 fold increase in the differentiation of AML cells ex vivo compared to ATRA in combination with the LSD1 inhibitor TCP (treatment control protocol) and untreated cells from AML subtypes M1, M2, and M4. PI3K HDAC inhibitor combined with ATRA significantly reduced p-MAPK and p-p38 levels while increasing the phosphorylation of p53 in HL-60 cells. Furthermore, RNA sequencing and chromatin profiling by Cleavage Under Targets and Tagmentation (CUT&Tag) were performed to gain additional insights into the molecular mechanisms involved. Based on obtained results, the combination of ATRA with specific dual PI3K HDAC and pan-PKC inhibitors holds promise as a potential therapeutic approach for AML. Nevertheless, further investigations, including studies utilizing patient-derived xenograft models, are warranted to validate the efficacy and feasibility of this proposed treatment strategy.

Impact of Arsenic Trioxide in the Treatment of Higher Risk Acute Promyelocytic Leukemia

INTRODUCTION: Acute promyelocytic leukemia (APL) accounts for 5-8% of all cases of acute myeloid leukemia (AML). The combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) without chemotherapy is currently the reference treatment of standard APL (ie with baseline white blood count (WBC)<10 G/L), curing about 90% of the patients. However, the prognosis of high-risk APL (ie with WBC>10 G/L) remains more challenging, with higher rates of early death and relapse. Here we compared the French practices for the treatment of high-risk APL patients whether patients were treated with ATRA-Chemo or ATRA-ATO according to physician decision, and evaluate the response rates, overall survival (OS) and leukemia-free survival (LFS) in the real-life settings. PATIENTS AND METHODS: ATO (in combination with ATRA) became accessible in France for the first line treatment of standard risk APL in 2012, but some patients with high-risk APL also received the same combination (generally with some form of cytoreductive chemotherapy) from that date. We retrospectively analyzed cases of high-risk APL diagnosed between 2010 and 2021 in 12 French centers, constituting a cohort of 135 patients with diagnostic of APL confirmed by cytogenetic, FISH and molecular biology assays. RESULTS: Among the 135 patients with WBC>10 G/L, 88 (65%) were classified as APL variant according to FAB classification. Median age was 46 years (range 18-89) and 62 % were male. Median diagnostic WBC was 39.1 G/L (range 10-270) and median platelet count was 27 G/L (range 5-344). At diagnosis, 112 patients (83%) had hemorrhagic manifestations and disseminated intravascular coagulation (DIC) was observed in 124 patients (92%). Pulmonary and cerebral leucostasis were reported in 10 (7%) and 14 (10%) patients, respectively. Eighty-five patients received corticosteroid prophylaxis (81 (95%) with Dexamethasone and 5 (5%) with Prednisolone). Induction therapy consisted in ATRA-ATO for 50 patients (38%) while 85 patients (62%) were treated with ATRA combined with chemotherapy (anthracycline and cytarabin) but without ATO. All patients treated with ATO were cytoreduced: 7 with Hydroxyurea (14%), 17 with Idarubicin (34%) and 26 with both (52%). 29 patients treated without ATO during induction were cytoreduced with Hydroxurea (34%). Most patients experienced one or more adverse events during induction, including sepsis (49 in the ATO group versus 71 in the non ATO group, 98% versus 83.5%, p=0.01), differentiation syndrome (20 in the ATO group versus 27 in the non ATO group, 40% versus 31.7%, p=0.33), transaminase increased (14 in ATO group versus 11 in the non ATO group, 28% versus 12.9%, p=0.03), and bleeding (7 in the ATO group versus 13 in the non ATO group, 14% versus 15.3%, p=0.8). Following induction, 110 patients (81%) achieved complete remission (CR): 45 in the ATO group and 65 in the non ATO group (90% versus 76.4%, p=0.052). One patient (receiving ATRA with chemotherapy) was refractory, and 24 patients experienced early death (5 in the ATO group and 19 in the non ATO group, 10% versus 22.3%, p=0.069) mostly due to hemorrhage or sepsis. Median time between diagnosis and early death was 4.5 days (0-42). Relapse was observed in 6 (5.5 %) patients (5 patients treated without ATO and 1 patient with ATO during induction). After a median follow-up of 34.6 months (0-121.1), OS at 3 years was significantly higher for the ATO group (89.9% (81.8-98.7) versus 75.1% (66.3-84.9) for the non ATO group, p= 0.035, Figure). LFS at 3 years was significantly higher for the ATO group (87.6% (78.7-97.4) versus 71.2% (62-81.7) for the non ATO group, p=0.028). CONCLUSIONS: The survival outcomes were significantly poorer in high-risk APL patients treated without ATO during induction, regardless of the cytoreduction strategy. The toxicity profile of ATO was acceptable. Combining ATO and ATRA limits the use of cytotoxic chemotherapy, which could reduce myelosuppression and long-term complications such as cardiotoxicity and secondary myeloid neoplasms. Early disease-related mortality, due to haemorrhagic or infectious complications, remains the major issue for these patients but tend to be reduced in those receiving ATRA-ATO based regiment. This retrospective study shows that ATO-ATRA and limited chemotherapy could be a better approach than ATRA and standard intensive chemotherapy in terms of early deaths, LFS and OS.

Improving Prediction of Early Death in Acute Promyelocytic Leukemia: External Validation Study from Italian Single Center Experience

Background: Despite significant advancements in treatment efficacy of acute promyelocytic leukemia (APL), early death (ED) - mainly related to major hemorrhagic and thrombotic events occurring within 30 days from diagnosis - remains a prominent hurdle to therapeutic success. In this regard, Österroos et al. very recently developed a score which stratifies the risk of ED of patients (pts) with APL in three categories (low [score 0-2], high [score 3-4], very high [score 5-7]) according to age (<50 years, 50-59 years, 60-69 years and ≥70 years), white blood cell count (<3.0x10 9/L, 3.0-5.0x10 9/L and >5.0x10 9/L), and platelet count (≥30x10 9/L and <30x10 9/L), all of which are readily available, real-world variables. However, the presence of specific comorbidities, or diseases characteristics, such as the presence of FLT3-ITD mutation, CD2 expression and bcr3 PML::RARA transcript were not evaluated, even if they could potentially further improve the risk stratification ( Österroos et al. Haematologica 2022;107(7):1528-1537). Aims: The aims of this study were to integrate the score proposed by Österroos et al. and evaluate the role of pts comorbidities and disease biological data in modifying risk stratification. Methods: Data were retrospectively collected from 127 consecutive pts diagnosed at our Center from January 2000 to May 2023. Patients had been treated according to the AIDA protocol (n = 76, 60%) or with the combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) (n = 39, 31%) ( Lo-Coco et al. N Engl J Med 2013;369:111-121); 10 pts received ATRA; only 2 pts did not receive any treatment. Results: Table 1 summarizes the main clinical characteristics of the 127 pts included in this study. Overall, ED rate was 11% (n = 14, 12 died within 7 days from diagnosis); the cause of death was a hemorrhagic event in 11 pts. The score identified low- (n = 79, 64%), high- (n = 39, 31%) and very high-risk (n = 6, 5%) categories; ED rates for each category were 3.8%, 20.5% and 0%, respectively. Despite no ED events were registered in our small very-high risk cohort (n = 6), a third of these pts (n = 2) experienced cerebral hemorrhage at disease onset and eventually survived. We confirmed that the score proposed by Österroos et al. was better at predicting ED risk than the Sanz score, with the Area Under the Receiver Operating Characteristic (AUROC) curve of 0.73 (95% CI 0.60-0.87) vs 0.66 (95% CI 0.51-0.81). The AUROC of the reference study cohort was 0.77 (95% CI 0.72-0.83). The presence of specific biological characteristics of the disease, such as FLT3-ITD mutation, bcr3 PML::RARA transcript or CD2 expression were not associated with an increased risk of ED (Table 2). Similarly, the presence of comorbidities (i.e. cardiovascular diseases, hypertension, chronic kidney disease, diabetes mellitus, dyslipidemia, and smoking) were not associated with an increased risk of ED (data not shown). By univariate analysis the presence of fever at diagnosis and male sex showed a trend toward an increased risk of ED (OR 3.77, 95% CI 0.88-19.26, P = 0.08 and OR 3.13, 95% CI 0.92-14.36, P = 0.09, respectively). Conclusions: Our data support the use of the score developed by Ö sterroos et al. to better predict the risk of ED in APL and to select the pts who may benefit from more aggressive supportive care. The presence of fever at diagnosis and male sex seems to be associated with a further increase in the risk of ED but this association needs to be confirmed in a larger study. The integration of biological characteristics of disease and comorbidities does not improve the risk stratification of ED.

Fatty Acid Binding Protein FABP5 Inhibition Activates Retinoic Acid Signaling and Induces Differentiation in Acute Myeloid Leukemia

Introduction. Accumulation of undifferentiated immature blood cells is a hallmark of myeloid neoplasia (MN), and indicates decoupling of terminal-differentiation from proliferation. Therefore, reinstating to a programmed state of differentiation (lineage-maturation) is a treatment approach, exemplified by all-trans retinoic acid (ATRA) treatment of acute promyelocytic leukemias that contain t(15;17) (q24;q21). This translocation inactivates retinoic acid receptor alpha (RARA) due to its fusion with promyelocytic leukemia PML resulting in PML-RARA. This fusion gene responsible for cellular transformation confers sensitivity to treatment with ATRA. However, ATRA's therapeutic-success in APLs has not extended to other AML phenotypes. We and others have shown that RA is a ligand, not just for RARs, but also pro-survival peroxisome proliferator-activated receptor β/δ (PPARβ/δ), a member of the nuclear hormone receptor superfamily transcription factors that regulate several metabolic pathways. RA is transported by two carrier proteins, cellular retinoid-binding protein CRABP2, or fatty-acid-binding protein 5 (FABP5), depending on cellular context. These carriers preferentially deliver RA to RARs versus PPARβ/δ respectively. Somatic lesions such as loss of function TET2 or Tp53 mutation, NPM1 and ASXL1 mutations, are associated with upregulated FABP5 and downregulated CRABP2. In AML cells with these mutations, therefore, the high FABP5 can be expected to deliver RA to PPARβ/δ instead of RAR, to potentially contribute to decoupling of differentiation from proliferation. We therefore investigated this possibility and its treatment implications. Methods. Overexpression, gene knockdown/knockout (ko/kd) of key regulators of RAR and PPARβ/δ were generated in specific genotypes of AMLs. Small molecule inhibitors of FABP5 were used to further interrogate the pathways in a translationally relevant manner, both in vitro, in both malignant and normal cells, and in pre-clinical in vivo models. Results. Gene expression analysis of AMLs in the Beat AML and TCGA databases demonstrated upregulation of pro-survival PPARβ/δ target genes including FABP5 and downregulation of differentiation promoting RAR pathway genes including CRABP2in AMLs 1. In particular, FABP5 was highly expressed in AMLs containing TET2 NPM1, TP53 and ASXL1 mutations. AML cell lines OCIAML3 (NPM1 ins/DNMT3a R882c), OCIAML5 (ASXL1 Y591*/TET2 S825*), THP1 (TP53 del) and isogenic CRISPR engineered K562 (TET2 +/+/TET2 -/-) demonstrated 4-100 fold higher FABP5 expression compared to normal myeloid precursors. These models did not differentiate in response to pharmacologic concentrations of ATRA. To test if FABP5 is necessary and sufficient to maintain the undifferentiated state of these cells in the presence of ATRA, we genetically inactivated FABP5 either using RNAi (kd) or CRISPR (ko). FABP5 knockdown or knockout cells created sensitivity to ATRA, resulting in terminal-differentiation as observed by flow cytometry for terminal-differentiation markers ( See Figure). FABP5 was then inhibited using highly specific small molecule FABP5 inhibitors with a 56-fold higher affinity compared to ATRA 1: combination of iFABP5 and ATRA induced terminal-differentiation with significant upregulation of the monocyte lineage markers CD14 and granulocyte lineage marker CD11b measured by flow cytometry at 72h. Toinvestigatewhether knocking down FABP5 and ATRA treatment affects gene expression changes we performed transcriptomic profiling of OCIMAL3 Scr, shFABP5 cells treated with or without ATRA using whole exome RNA sequencing. Treatment of FABP5 kd OCIMAL3 cells with ATRA resulted in the upregulation of RAR pathways genes and downregulation of proliferation promoting genes. Conclusion. In sum, recoupling of lineage-maturation to proliferation can be achieved in non-APL AMLs that highly express the RA carrier FABP5, by inhibiting FABP5 using small molecules. This candidate treatment modality is distinct from cytotoxic chemotherapy, and importantly, can spare normal hematopoiesis.

Additional cytogenetic abnormalities in patients with newly diagnosed acute promyelocytic leukemia predict inferior event-free survival.

The innovative combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) has established a new chapter of curative approach in acute promyelocytic leukemia (APL). The disease characteristics and prognostic influence of additional cytogenetic abnormalities (ACA) in APL with modern therapeutic strategy need to be elucidated. In the present study, we retrospectively investigated disease features and prognostic power of ACA in 171 APL patients treated with ATRA-ATO-containing regimens. Patients with ACA had markedly decreased hemoglobin levels than that without ACA (p = 0.021). Risk stratification in the ACA group was significantly worse than that in the non-ACA group (p = 0.032). With a median follow-up period of 62.0 months, worse event-free survival (EFS) was demonstrated in patients harboring ACA. Multivariate analysis showed that ACA was an independent adverse factor for EFS (p = 0.033). By further subgroup analysis, in CD34 and CD56 negative APL, patients harboring ACA had inferior EFS (p = 0.017; p = 0.037). To sum up, ACA remains the independent prognostic value for EFS, we should build risk-adapted therapeutic strategies in the long-term management of APL when such abnormalities are detected.

Targeting the retinoic acid signaling pathway as a modern precision therapy against cancers.

Retinoic acid (RA) is a vital metabolite derived from vitamin A. RA plays a prominent role during development, which helps in embryological advancement and cellular differentiation. Mechanistically, RA binds to its definite nuclear receptors including the retinoic acid receptor and retinoid X receptor, thus triggering gene transcription and further consequences in gene regulation. This functional heterodimer activation later results in gene activation/inactivation. Several reports have been published related to the detailed embryonic and developmental role of retinoic acids and as an anti-cancer drug for specific cancers, including acute promyelocytic leukemia, breast cancer, and prostate cancer. Nonetheless, the other side of all-trans retinoic acid (ATRA) has not been explored widely yet. In this review, we focused on the role of the RA pathway and its downstream gene activation in relation to cancer progression. Furthermore, we explored the ways of targeting the retinoic acid pathway by focusing on the dual role of aldehyde dehydrogenase (ALDH) family enzymes. Combination strategies by combining RA targets with ALDH-specific targets make the tumor cells sensitive to the treatment and improve the progression-free survival of the patients. In addition to the genomic effects of ATRA, we also highlighted the role of ATRA in non-canonical mechanisms as an immune checkpoint inhibitor, thus targeting the immune oncological perspective of cancer treatments in the current era. The role of ATRA in activating independent mechanisms is also explained in this review. This review also highlights the current clinical trials of ATRA in combination with other chemotherapeutic drugs and explains the future directional insights related to ATRA usage.

ATRA sensitized the response of hepatocellular carcinoma to Sorafenib by downregulation of p21-activated kinase 1

BackgroundSorafenib resistance greatly reduces the efficacy of treatments in advanced hepatocellular carcinoma (HCC) patients, but the underlying mechanisms are not thoroughly understood. All-trans retinoic acid (ATRA), an anti-leukaemia agent, has attracted considerable attention due to its role in sensitizing cells to other anticancer treatments. We aimed to investigate the combined effect of ATRA and Sorafenib on HCC and the underlying mechanisms.MethodsCCK-8, cell sphere formation, trans-well migration, and wound-healing assays were used to analyse the biological behaviours of HCC cells in vitro. Western blotting and qRT-PCR analysis were conducted to measure the expression of p21 activated kinase 1 (PAK1) and phospho-p21 activated kinase 1 (pPAK1). Xenograft models were established to confirm the synergistic effects of ATRA and Sorafenib in vivo. TUNEL assays and immunohistochemistry were utilized to determine apoptosis, proliferation, PAK1 and pPAK1 levels in tumour tissues.ResultsWe observed that PAK1 was overexpressed in HCC, and its expression was negatively correlated with the survival of patients. PAK1 promoted the proliferation, self-renewal and epithelial-mesenchymal transition of HCC cells. Correlation analysis indicated that the IC50 of Sorafenib was positively correlated with the level of pPAK1 in HCC cell lines. ATRA inhibited the progression of HCC and sensitized HCC response to Sorafenib by downregulation of PAK1, as shown by the calculated coefficient of drug interaction and the data obtained from xenograft models.ConclusionsOur findings indicated that instead of treatment with Sorafenib alone, the combination of ATRA and Sorafenib provides a more effective treatment for HCC patients.2_mPhFeBDQdvWNSurq11yxVideo

ATO Increases ROS Production and Apoptosis of Cells by Enhancing Calpain-Mediated Degradation of the Cancer Survival Protein TG2.

Transglutaminase 2 (TG2) is a critical cancer cell survival factor that activates several signalling pathways to foster drug resistance, cancer stem cell survival, metastasis, inflammation, epithelial-mesenchymal transition, and angiogenesis. All-trans retinoic acid (ATRA) and chemotherapy have been the standard treatments for acute promyelocytic leukaemia (APL), but clinical studies have shown that arsenic trioxide (ATO), alone or in combination with ATRA, can improve outcomes. ATO exerts cytotoxic effects in a variety of ways by inducing oxidative stress, genotoxicity, altered signal transduction, and/or epigenetic modification. In the present study, we showed that ATO increased ROS production and apoptosis ratios in ATRA-differentiated NB4 leukaemia cells, and that these responses were enhanced when TG2 was deleted. The combined ATRA + ATO treatment also increased the amount of nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor, an adaptive regulator of the cellular oxidative stress response, and calpain proteolytic activity, resulting in TG2 degradation and the reduced survival of WT leukaemia cells. We further showed that the induced TG2 protein expression was degraded in the MCF-7 epithelial cell line and primary peripheral blood mononuclear cells upon ATO treatment, thereby sensitising these cell types to apoptotic signals.

Combined Application of Salinomycin and ATRA Induces Apoptosis and Differentiation of Acute Myeloid Leukemia Cells by Inhibiting WNT/β-Catenin Pathway.

All-trans retinoic acid (ATRA) is only effective in acute promyelocytic leukemia (APL), but not in other subtype of acute myeloid leukemia (AML). Salinomycin targets tumor cells rather than non-tumorigenic cells, and WNT/β-catenin pathway inhibition is one of the mechanisms of its anti-tumor activity. There is a crosstalk between RA and WNT/β-catenin pathway. Here, we investigate the effect of the combination of salinomycin and ATRA (S+RA) in non-APL AML cells. Apoptosis was evaluated by cell viability and Annexin-V assay. Cell differentiation was analyzed by CD11c expression and morphology. To explore the underlying mechanisms, Western blot analysis and mitochondrial transmembrane potentials (ΔΨm) were used. S+RA induced differentiation and apoptosis in AML cell lines and AML primary cells. S+RA inhibited the β-catenin signal pathway as determined by the decreased protein levels of β-catenin, the low-density lipoprotein receptor-related proteins 6 (LRP6), and its downstream proteins such as survivin, c-Myc, caspase-3/7, cdc25A and cyclinD1 and reduced phosphorylation level of GSK3β S9. S+RA also increased the protein levels of CCAAT/enhancer-binding proteins (C/EBPs) and PU.1 and collapsed Δψm. The above molecular and cellular changes induced by S+RA were inhibited by β-catenin specific activator and promoted by β-catenin specific inhibitor. S+RA induced differentiation by β-catenin-inhibition-mediated up-regulation of C/EBPs and PU.1 and suppression of c-Myc. S+RA triggered apoptosis through β-catenin-inhibition-regulated ΔΨm collapse and caspase-3/7 activation. Taken together, our findings may provide novel therapeutic strategies for AML patients by targeting the WNT/β-catenin pathway.

Chemotherapy-free treatment for acute promyelocytic leukemia: the pediatric view of a revolutionary tale.

The addition of all-trans retinoic acid (ATRA) to the standard anthracycline-base chemotherapy has revolutionized the treatment of acute promyelocytic leukemia (APL) over the last decades, becoming a model for precision medicine. The protocols based on the combination of ATRA and chemotherapy allowed obtaining excellent response rates both for children and adults. However, the persistence of anthracycline chemotherapy as a backbone was a matter of concern for both acute and long-term complications. Efforts in reducing anthracycline cumulative dose or even eliminating anthracycline have been pursued in more recent pediatric protocols thanks to the introduction of arsenic trioxide (ATO). The impressive results of the ATRA/ATO combinations led to the introduction of protocols completely chemotherapy-free for standard-risk adult patients as the standard of care, whereas pediatric chemo-free protocols are still currently under evaluation. In this Review, we will critically retrace the history of this unique revolution in precision medicine, discussing the peculiar advantages for pediatric patients with APL.

All-Trans Retinoic Acid Promotes a Tumor Suppressive OTUD6B-β-TrCP-SNAIL Axis in Esophageal Squamous Cell Carcinoma and Enhances Immunotherapy.

β-TrCP is an E3 ubiquitin ligase that plays important roles in multiple human cancers including esophageal squamous cell carcinoma (ESCC). Analysis of ESCC patient samples reveal that only protein level but not transcript level of β-TrCP associated with patient prognosis, suggesting regulators of β-TrCP protein stability play an essential role in ESCC progression and may be novel targets to develop ESCC therapies. Although β-TrCP stability is known to be mediated by the ubiquitin-proteasome system, it is unclear which enzymes play a major role to determine β-TrCP stability in the context of ESCC. In this study, OTUD6B is identified as a potent deubiquitinase of β-TrCP that suppress ESCC progression through the OTUD6B-β-TrCP-SNAIL axis. Low OTUD6B expression is associated with a poor prognosis of ESCC patients. Importantly, all-trans retinoic acid (ATRA) is found to promote OTUD6B translation and thus suppress ESCC tumor growth and enhance the response of ESCC tumors to anti-PD-1 immunotherapies. These findings demonstrate that OTUD6B is a crucial deubiquitinase of β-TrCP in ESCC and suggest combination of ATRA and anti-PD-1 immune checkpoint inhibitor may benefit a cohort of ESCC patients.

Treating low- and intermediate-risk acute promyelocytic leukemia with and without chemotherapy: A comparison in a tertiary care center.

Acute promyelocytic leukemia (APL) comprises approximately 10% of acute myeloid leukemia (AML) cases. Both options of treatment (ATRA-ATO and ATRA-chemotherapy) were discussed with patients with low- and intermediate-risk APL, pros and cons explained in details, and treatment regimen selected after getting informed written consent. Total 71 patients were included in the study; among these patients, 3 were negative for both FISH for t (15,17) and RT-PCR for promyelocytic leukemia retinoic acid receptor alpha, and 36 patients with APL had white blood cell count at diagnosis >10 × 109/l. Total 30 patients with newly diagnosed as low- and intermediate-risk-APL fulfilled all inclusion criteria, treated and followed for a minimum period of 2 years up to June, 2016. Fifteen patients liked to be treated with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), while rest of the 15 patients preferred treatment with ATRA and chemotherapy. Combination of ATRA and ATO is equally effective, less toxic, and more feasible in comparison to ATRA and chemotherapy for patients with low- and intermediate-risk APL and is a viable option for this subset of patients, especially in countries with limited resources.

ERβ1 Sensitizes and ERβ2 Desensitizes ERα-Positive Breast Cancer Cells to the Inhibitory Effects of Tamoxifen, Fulvestrant and Their Combination with All-Trans Retinoic Acid.

Adjuvant endocrine therapy (AET) is the treatment of choice for early-stage estrogen receptor alpha (ERα)-positive breast cancer (BC). However, almost 40% of tamoxifen-treated cases display no response or a partial response to AET, thus increasing the need for new treatment options and strong predictors of the therapeutic response of patients at high risk of relapse. In addition to ERα, BC research has focused on ERβ1 and ERβ2 (isoforms of ERβ), the second ER isotype. At present, the impact of ERβ isoforms on ERα-positive BC prognosis and treatment remains elusive. In the present study, we established clones of MCF7 cells constitutively expressing human ERβ1 or ERβ2 and investigated their role in the response of MCF7 cells to antiestrogens [4-hydroxytamoxifen (OHΤ) and fulvestrant (ICI182,780)] and retinoids [all-trans retinoic acid (ATRA)]. We show that, compared to MCF7 cells, MCF7-ERβ1 and MCF7-ERβ2 cells were sensitized and desensitized, respectively, to the antiproliferative effect of the antiestrogens, ATRA and their combination and to the cytocidal effect of the combination of OHT and ATRA. Analysis of the global transcriptional changes upon OHT-ATRA combinatorial treatment revealed uniquely regulated genes associated with anticancer effects in MCF7-ERβ1 cells and cancer-promoting effects in MCF7-ERβ2 cells. Our data are favorable to ERβ1 being a marker of responsiveness and ERβ2 being a marker of resistance of MCF7 cells to antiestrogens alone and in combination with ATRA.