ATRA Dose Research Articles

All-trans retinoic acid-mediated ADAR1 degradation synergizes with PD-1 blockade to suppress pancreatic cancer.

As a double-stranded RNA-editing enzyme and an interferon-stimulated gene, double-stranded RNA-specific adenosine deaminase (ADAR1) suppresses interferon signaling and contributes to immunotherapy resistance. Suppression of ADAR1 overcomes immunotherapy resistance in preclinical models, but has not yet been translated to clinical settings. By conducting a screening of a subset of the FDA-approved drugs, we found that all-trans retinoic acid (ATRA, also known as tretinoin) caused ADAR1 protein degradation through ubiquitin-proteasome pathways and concomitantly increased PD-L1 expression in pancreatic and breast cancers. In addition, the combination of ATRA and PD-1 blockade reprogrammed the tumor microenvironment and unleashed antitumor immunity and thereby impeded tumor growth in pancreatic cancer mouse models. In a pilot clinical trial, a higher dose of ATRA plus the anti-PD-1 antibody nivolumab prolonged median overall survival in patients with chemotherapy-resistant pancreatic cancer compared to a lower dose of the same regimen. In this study, ATRA was the first drug to be found to cause ADAR1 degradation. We propose translation of a promising 2-pronged antitumor strategy using ATRA and nivolumab to convert immunologically "cold" into "hot" tumors susceptible to immune checkpoint blockade.

Effects of interferon regulatory factor 9 on the biological phenotypes in PML-RARα-induced promyelocytic leukemia

目的研究急性早幼粒细胞白血病（APL）中干扰素调节因子9（IRF9）的表达特征、预后意义和生物学功能，探索IRF9作为潜在治疗靶点的临床转化意义。方法挖掘TCGA公共数据分析IRF9在APL中的表达水平及其表达高低对患者生存的影响；利用Dox诱导的慢病毒载体系统构建可诱导表达PML-RARα（PR）融合基因的U937细胞系，诱导PR融合基因早期表达并加入全反式维甲酸（ATRA）检测IRF9表达变化；构建可诱导表达IRF9的NB4细胞系，进行细胞分化和集落形成实验，初步探讨IRF9诱导表达对NB4白血病细胞生物学功能的影响。结果①TCGA数据库分析显示，在各种类型的AML中，IRF9在APL中表达水平最低且与预后不良相关。②成功构建可诱导表达PR融合基因的U937细胞系；PR表达引起IRF9蛋白水平下调，在NB4细胞系中IRF9表达缺失，ATRA处理后可表达上调。③成功构建可诱导表达IRF9的NB4细胞系，IRF9诱导表达促进NB4细胞的分化，且与较低剂量ATRA有协同促分化作用；IRF9诱导表达显著抑制了NB4细胞的集落形成能力。结论IRF9在APL中低表达且与预后不良相关，提示存在特异性调控机制；上调IRF9表达可发挥抗白血病效应，为其成为临床潜在治疗靶点奠定生物学基础。

Activation of Vitamin D Receptor Pathway Enhances Differentiating Capacity in Acute Myeloid Leukemia with Isocitrate Dehydrogenase Mutations.

Simple SummaryAround 15% of acute myeloid leukemia (AML) patients harbor mutations in isocitrate dehydrogenases (IDH), which lead to the production of the oncometabolite 2-hydroxyglutarate (2-HG). Inhibitors of mutant IDH enzymes and their 2-HG production have been approved by the FDA to be used in patients. However, 60% of IDH mutant AML patients do not respond to these inhibitors or develop mechanisms of resistance, leading to relapse. Among these mechanisms, some produce a 2-HG rebound. Alternative therapies exploiting the 2-HG-dependent molecular effects could therefore be of clinical interest. In this study, we demonstrate that 2-HG specifically activates vitamin D receptor (VDR) in IDH mutant AML cells leading to increased sensitivity to the combination of vitamin D (or VDR agonist) and all-trans retinoic acid and revealing a new therapeutic approach that can be readily applied to AML patients in this subgroup.Relapses and resistance to therapeutic agents are major barriers in the treatment of acute myeloid leukemia (AML) patients. These unfavorable outcomes emphasize the need for new strategies targeting drug-resistant cells. As IDH mutations are present in the preleukemic stem cells and systematically conserved at relapse, targeting IDH mutant cells could be essential to achieve a long-term remission in the IDH mutant AML subgroup. Here, using a panel of human AML cell lines and primary AML patient specimens harboring IDH mutations, we showed that the production of an oncometabolite (R)-2-HG by IDH mutant enzymes induces vitamin D receptor-related transcriptional changes, priming these AML cells to differentiate with pharmacological doses of ATRA and/or VD. This activation occurs in a CEBPα-dependent manner. Accordingly, our findings illuminate potent and cooperative effects of IDH mutations and the vitamin D receptor pathway on differentiation in AML, revealing a novel therapeutic approach easily transferable/immediately applicable to this subgroup of AML patients.

Improving Outcomes in Elderly Acute Promyelocytic Leukemia Patients in the Real World: A Personalized Treatment Approach Along with Expert Support Might Reduce Early Deaths

Background: APL is the most curable leukemia with survival >90% in large co-operative group studies. These excellent outcomes are not seen in elderly patients where early deaths (ED) in trials are 10-18%. Population based registry data shows that ED rate in elderly patients is even higher at 24-50%. The one year relative survival was 37% in patients >60 years in Swedish population data. We report the outcome of patients >60 years from our experience.Methods: We performed a retrospective chart review with IRB approval of 138 patients treated at leukemia treatment hospitals in GA, SC and neighboring states. Newly diagnosed patients were treated using a simplified set of treatment guidelines along with expert support designed to decrease ED. There were no exclusion criteria in this prospective trial. Given the comorbidities in this “real world” elderly population, dose reductions were necessary. We documented the doses of ATRA, ATO, and idarubicin given to patients ≥60 at diagnosis, and then calculated the percentage dose reduction the patient received, if any. For our calculations, standard doses were 45mg/m2/day for ATRA, 0.15mg/kg/dose for ATO, and 12mg/m2/dose for idarubicin. The target dose reduction for patients' ≥60 in the second half of the study was 25mg/m2/day for ATRA and 0.075mg/kg/dose for ATO. In morbidly obese patients, the BSA was capped at 2.0 and dosing weight for ATO at 100 kg.Results: Between 7/2013 and 4/2017, 138 patients were treated at 4 large leukemia centers and 30 community hospitals. Fifty-three patients (38.4%) were >60, with a median age of 69. Male to female ratio was 1:1. Median WBC was 2.3/mm3 (0.3-38); 93% were low risk. 25 (47%) were treated in our institution and 28 (53%) were managed in 18 other centers. Of this group, n=27 were ages 60-69, n=19 were 70-79, and n=7 were >80. 30 (56.6%) patients had scores of > 5 on the age adjusted Charlson Comorbid Index (range 2-12). Dose modifications were necessary given the high comorbidities which are not usually the norm in clinical trials due to exclusion criteria. A total of 53 patients (100%) received ATRA, 43 (81%) received ATO, and 7 (13%) received idarubicin (Ida). For ATRA, n=5 (9%) received an initial dose reduction of 10-24%, n=13 (24.5%) received reduction of 25-49% and n=7 (13%) received a reduction of ≥50%. For ATO, n=2 (4.7%) received a reduction of 10-24%, n=10 (27.9%) received a reduction of 25-49% and n=14 (32.6%) received a dose reduction of ≥50%. Only one patient received 50% dose reduced (Ida). The remaining patients received full doses. For six patients, the dose of ATRA (n=1), arsenic (n=5), and (Ida) (n=1) was not provided. Excluding two patients, one a Jehovah's Witness who refused transfusions and the other we were consulted 12 days after diagnosis with multi-organ failure, there were nine deaths during induction (17.6%). There were 6 late deaths from second cancer (n=1), relapse (n=1) and non APL related deaths (n=4). There were 3 relapses (2 high risk), none of whom received dose reductions and 1 refused consolidation. With a median follow up of 562 days the 1 year survival was 74.5% and overall survival was 70.6%.Conclusions: One year relative survival in elderly APL patients is dismal with population based Swedish and SEER data showing rates <50%. Age and comorbid conditions dictate that a more personalized approach with dose reduction could result in better tolerance and improve outcomes in the elderly. A simplified treatment algorithm along with expert support has the potential to decrease induction mortality (17.6%) and improve population wide survival (1 year 74.5%). [Display omitted] DisclosuresKota:Xcenda: Consultancy; Leukemia Lymphoma Society: Research Funding; Takeda Pharmaceuticals: Consultancy; Novartis: Consultancy; Incyte: Consultancy; Pfizer: Consultancy. Arellano:Cephalon Oncology: Research Funding. Heffner:ADC Therapeutics: Research Funding. Stuart:Novartis: Research Funding; Sunesis: Consultancy, Honoraria, Other: Travel Support, Research Funding; Astellas: Research Funding; ONO: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celator/Jazz: Research Funding; Amgen: Consultancy, Honoraria; Bayer: Research Funding; MedImmune: Research Funding; Seattle Genetics: Research Funding; Cantex: Research Funding; Incyte: Research Funding; Agios: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding. Grunwald:Amgen: Consultancy, Research Funding; Celgene: Consultancy; Alexion: Consultancy; Forma Therapeutics: Research Funding; Incyte: Consultancy, Research Funding; Pfizer: Consultancy; ARIAD: Consultancy; Cardinal Health: Consultancy; Janssen: Research Funding; Genentech: Research Funding.

The Efficacy and Safety of Decitabine in Combination with ATRA, G-CSF and Low Dose Cytarabine(Dlaag Regimen)in Relapsed/Refractory Acute Myeloid Leukemia Not Suitable for Strong Chemotherapy : A Phase 2, Prospective, Single-Arm, Multicenter Study

Background. Patients with relapsed or refractory acute myeloid leukemia (R/R AML)who are intolerant of strong regimen chemotherapy have poor responsibility to chemotherapy and clinic remission is lower. The DNMT is depleted to reactivate the silencing tumor suppressor gene to exert anti-tumor effects. Vitro experiments confirmed that ATRA has an anti-leukemia effect on non-M3-AML cell lines. We perform the 2 phase, single-arm, multicenter study of Decitabine in Combination with ATRA, G-CSF and low dose cytarabine(DLAAG Regimen)in R/R AML not suitable for strong chemotherapy (NCT03356080). Methods. R/R AML patients aged more than 18 years not suitable for strong chemotherapy were eligible to enroll on this study. The objective was to assess the responses and safety of this therapy. All patients received subcutaneous decitabine injection 0.1-0.2mg/kg on days 1-3 ,8-10,15-17 .ATRA was taken orally at 45mg/m2 on day 4-6 and 15mg/m2on days 7-20. Subcutaneous injection cytarabine 15mg/m2 every 12 hours on days 1-10 and G-CSF 300ug/d on day 0 until disease remission or absolute neutrophils count ≥ 0.5×109/L were administered. The FLT3 inhibitor, arsenious acid, and the JAK-2 inhibitor, rotectinib, were allowed to be combined.All patients received one or two courses of induction treatments. Results. During the period from December 1, 2017 to July 2019, a total of 33 patients were recruited, including 17 women and 16 men. The median age is 64 years (range 46-82). In 33 patients, the overall response rate (ORR = CR + PR) was 36.3%, and the complete response rate (CRR = CR + CRi) was 33.3%. The early treatment-related mortality rate was 6.0%. One died of infection and one died of organ bleeding. Of the 26 patients with karyotype data, 11 received CR. Seven patients (63.6%) in CR showed normal karyotype, 2 patients (18.1%) in CR, and 1 patient (9.0%) in PR showed other moderate karyotypes. One case (9.0%) of the CR group had a poor karyotype. Statistical analysis showed that there was a statistically significant difference between the different karyotype groups (P &lt; 0.05). Only 2 of the 10 FLT3 mutant patients achieved CR (20%). Two patients with abnormal MLL gene did not receive CR. Conclusions DLAAG regimen is effective and tolerated in patients with R/R AML who are not suitable for intensive therapy and could be as a bridging therapy regimen followed by HSCT. Disclosures No relevant conflicts of interest to declare.

All-Trans Retinoic Acid Impairs Platelet Function and Thrombus Formation and Inhibits Protein Kinase CßI/δ Phosphorylation

All-trans retinoic acid (ATRA) is widely used for induction of complete remission in patients with acute promyelocytic leukemia (APL). ATRA also regulates protein kinase C (PKC) activity. Therapeutic use of ATRA reportedly interferes with hemostatic function in APL patients, including effects on coagulation or other vascular cells, although effects of ATRA on platelets remain unclear. This study aims to investigate the effect of therapeutic-relevant doses of ATRA on platelet function. Human platelets were preincubated with ATRA (0-20 μM) for 1 hour at 37°C, followed by analysis of aggregation, granule secretion, receptor expression by flow cytometry, platelet spreading, or clot retraction. Additionally, ATRA (10 mg/kg) was injected intraperitoneally into mice and tail bleeding time and arterial thrombus formation were evaluated. ATRA inhibited platelet aggregation and adenosine triphosphate release induced by collagen (5 μg/mL) or thrombin (0.05 U/mL) in a dose-dependent manner without affecting P-selectin expression or surface levels of glycoprotein (GP) Ibα, GPVI, or αIIbβ3. ATRA-treated platelets demonstrated reduced spreading on immobilized fibrinogen or collagen and reduced thrombin-induced clot retraction together with reduced phosphorylation of Syk and PLCγ2. In addition, ATRA-treated mice displayed significantly impaired hemostasis and arterial thrombus formation in vivo. Further, in platelets stimulated with either collagen-related peptide or thrombin, ATRA selectively inhibited phosphorylation of PKCßI (Ser661) and PKCδ (Thr505), but not PKCα or PKCßII phosphorylation (Thr638/641). In conclusion, ATRA inhibits platelet function and thrombus formation, possibly involving direct or indirect inhibition of PKCßI/δ, indicating that ATRA might be beneficial for the treatment of thrombotic or cardiovascular diseases.

Regulation of in Vitro Adipogenesis by All-trans Retinoic Acid (P21-075-19)

ObjectivesVitamin A, referred to as retinol, is an essential nutrient that affects the cell growth and differentiation including adipogenesis. Although previous studies using supraphysiological doses (over 1 mM) of all-trans retinoic acid (atRA) demonstrated anti-adipogenic activity, effects of atRA at various levels on differentiation of 3T3-L1 preadipocytes have not been extensively investigated. MethodsVarious doses of atRA were supplemented during the differentiation of 3T3-L1 preadipocytes. Lipid accumulation was assessed by Oil-Red-O staining and cellular triglyceride levels. Expression levels of adipogenic markers and cell cycle regulators were measured by quantitative real-time PCR or Western blot analyses. In addition, cell proliferation, cell cycle, and cytotoxicity were analyzed. ResultsOur study showed that the amount of cellular triglyceride and intensities of Oil-Red-O staining were decreased by supplementing atRA (1 and 10 mM) but increased by low concentrations of atRA (0.01 to 100 nM) compared with the control. Also PPARg and FABP4 were gradually over-expressed by atRA up to 1 nM but decreased at over the 1 nM concentrations. Moreover, mitotic clonal expansion (MCE) and consequential growth-arrest were analyzed as important steps in adipogenesis of 3T3-L1 cells. The 1 nM group showed more cell proliferation and thereafter a higher ratio of G0/G1 phase on day 2. Protein levels of S/G2-phase factors were dose-dependently increased by atRA up to 1 nM on day 1, but the factors were highly expressed in higher doses on day 2. G0/G1 markers were higher at the higher doses of atRA on day 1; whereas, they were highly expressed in mild or medium doses on day 2. ConclusionsOur data indicate that atRA controls adipogenesis with accompanied changes in expression of PPARgamma and FABP4, cell proliferation and follow-up growth-arrest. These results indicate that atRA can function both as a negative and positive regulator of adipogenesis depending on dosages, providing a strategy for achieving proper nutritional balance for treatment of obesity. Funding SourcesUSDA-NIFA and KRIBB Research Initiative Program.

Supplementation of All-Trans-Retinoic Acid Below Cytotoxic Levels Promotes Adipogenesis in 3T3-L1 Cells.

Vitamin A, referred to as retinol, is an essential nutrient that affects the cell growth and differentiation including adipogenesis. Although previous studies using supraphysiological doses (over 1 μM) of all-trans-retinoic acid (atRA) demonstrated antiadipogenic activity, effects of atRA at various levels on differentiation of 3T3-L1 preadipocytes have not been extensively investigated. Our study showed that the amount of cellular triacylglycerol (TAG) and intensities of Oil-Red-O staining were decreased by supplementing atRA (1 and 10 μM) but increased by low concentrations of atRA (0.01 to 100 nM) compared with the control. Also PPARγ and FABP4 were gradually overexpressed by atRA up to 1 nM but decreased at over 1 nM concentrations. Moreover, mitotic clonal expansion (MCE) and consequential growth-arrest were analyzed as important steps in adipogenesis of 3T3-L1 cells. The 1 nM group showed more cell proliferation and thereafter a higher ratio of the G0/G1 phase on Day 2. Protein levels of S/G2-phase factors were dose dependently increased by atRA up to 1 nM on Day 1, but the factors were highly expressed in higher doses on Day 2. G0/G1 markers were higher at the higher doses of atRA on Day 1; whereas, they were highly expressed in mild or medium doses on Day 2. These data indicate that atRA controls adipogenesis with accompanied changes in cell proliferation and follow-up growth-arrest. These results indicate that atRA can function both as a negative and positive regulator of adipogenesis depending on dosages, providing a strategy for achieving proper nutritional balance for treatment of obesity.

A Refined Regimen for Maximally Eliminating Early Life-Threatening Complications for Patients with High-Risk Acute Promyelocytic Leukemia

Life-threatening complications, such as severe bleeding and/or differentiation syndrome at admission and/or along with induction treatment, among high-risk patients with acute promyelocytic leukemia (APL) are a worldwide puzzle towards the cure of the disease. Taking the rationale that high WBC count, at least in part, may cause cytokine storm related symptoms, we designed this refined regimen with low dose mitoxantrone and ATRA plus arsenic trioxide to determine the safety and efficiency of this WBC reduction, prevention of differentiation syndrome, and supportive care centered approach.In total there were 50 patients with high risk APL (WBC>10x109) from 2003-2017 were enrolled with a medium follow-up of 39 months (15-72 months). Our treatment strategy and detailed protocol are: 1) WBC reduction: it started with ATRA (25mg/m2/day) and arsenic trioxide (0.15mg/kg/day) based double induction. The advantage of decreased dose of ATRA can help to lower the happening of leukocytosis which will trigger the cytokine releasing related syndrome. Low dose mitoxantrone (3mg/m2) was added from day 2-5 plus hydroxyurea (1.5g, p.o. q6h) started from day 1. The criteria for withdrawal hydroxyurea is once both of the following standard are met: i) the WBC count has continuously decreased for three days after reached the peak value; ii) WBC < 10x109/L. 2) Prevention of Differentiation Syndrome (DS): Dexamethasone (5mg/day) was added once the diagnosis was confirmed at day 1. The criteria to taper dexamethasone are the same as to stop hydroxyurea. We require to withdraw dexamethasone within one week once start the taper process. In general, our goal is to prevent the occurrence of both leukopenia and leukocytosis and maintain the WBC count between 2-20x109. 3) Supportive care: We maintain the PLT count between 20-30x109 and fibrinogen >1.5g/L which helped to decrease the occurrence of myelo-suppression, DS and severe bleeding. 4) CNS Prophylaxis: The Intrathecal injection was given after the CR was achieved. Unless there was evidence of CNS infiltration, we gave intrathecal injection after correcting the coagulation abnormality.The consolidation began at four weeks after the end of induction. It contained four weeks of an on and off schedule of arsenic trioxide (0.15mg/kg/day), in total four cycles, and two weeks of an on and off schedule of ATRA (25mg/m2/day), in total seven cycles. A total of eight intrathecal injections were given on the first and last day of arsenic trioxide. Two death was observed through our strategy during the induction. All patients reached hCR by the end of induction. Relapse occurred in four out of 50 patients, and there was no treatment-related mortality. All the relapsed patients entered CR again after using the same protocol. Grade 3-4 adverse events were observed among 12% of all the cases. Three-year probability of overall survival (pOS) was found to be 92%. Our strategy focus of reducing WBC count sheds new light on maximally eliminating early mortality of high-risk APL patients. DisclosuresNo relevant conflicts of interest to declare.

Mechanism of cleft palate in C57BL/6N mice induced by retinoic acid

Objective: To investigate the mechanism of cleft palate in mice induced by excessive all-trans retinoic acid (atRA). Methods: The pregnant mice were randomly divided into atRA-treated group (n=27) and control group (n=27). atRA-treated group was given by gavage a single dose of atRA (100 mg/kg) at 8: 00 AM on gestation day 10 (GD10) and the control group was given by gavage the isopyknic corn oil. At GD13-GD15, the fetal mice palate development was observed by HE staining. The mouse embryonic palatal mesenchymal cell proliferation was detected by 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry. The expressions of Smad2, phospho-Smad2 (p-Smad2), Smad4 and Smad7 in mouse embryonic palatal mesenchyme were analyzed by Western blotting. Results: At GD13-GD15, compared with the control, the ratio of BrdU-positive cells in the palatal mesenchyme of atRA-treated fetuses decreased significantly (P<0.05), especially at GD14, atRA inhibition rate was (65.4±1.7)%. Moreover, atRA decreased the levels of p-Smad2 and Smad4 in embryonic palate mesenchymal cells, whereas the expression of Smad7 was significantly increased at GD14 and GD15. Conclusions: atRA may lead to cleft palate by inhibiting the activation of Smad signaling pathway and affecting the proliferation of palatal mesenchymal cells.

Retinoic Acid Metabolites and Isomers have Differential Effects on Lipid Metabolism

All‐trans retinoic acid (atRA) and 13‐cisRA, and their metabolites 4‐oxo‐atRA and 4‐oxo‐13‐cisRA are found in circulation in humans as endogenous compound and following clinical doses of atRA and 13‐cisRA. Of these four compounds, only atRA is believed to be biologically important due to its high affinity towards RARs and activation of PPARδ. 13‐cisRA and 4‐oxo‐atRA also activate RARs but less is known about the 4‐oxo‐13‐cisRA. The activation of PPARs by 13‐cisRA and 4‐oxo‐metabolites has not been studied. The aim of this study was to determine whether atRA, 4‐oxo‐atRA, 13‐cisRA and 4‐oxo‐13‐cisRA have biological effects via RAR and other non‐RAR mediated pathways in human hepatocytes, HepG2 cells and adipocytes. At 1 μM concentration atRA, 4‐oxo‐atRA, 13‐cisRA and 4‐oxo‐13‐cisRA each significantly increased the mRNA levels of RARβ demonstrating that all four compounds activate RARs. atRA treatment increased expression of PPARδ targeted genes PGC1α, PGC1β and NRF1 and fatty acid oxidation, but had no effect on fatty acid and triglyceride synthesis genes SREBP1, MTTP, DGAT2 and PPARγ. In contrast, 13‐cisRA, 4‐oxo‐atRA, and 4‐oxo‐13‐cisRA had no effect on PPARδ targeted genes PGC1α, PGC1β and NRF1 and fatty acid oxidation, but significantly increased the expression of SREBP1, MTTP, DGAT2 and PPARγ. This suggests that 13‐cisRA, 4‐oxo‐atRA, and 4‐oxo‐13‐cisRA increase triglyceride synthesis and secretion. In addition, 4‐oxo‐atRA antagonized the induction of PGC1α, PGC1β and NRF1 and fatty acid oxidation by atRA demonstrating an independent activity of the 4‐oxo‐atRA when compared to atRA. Collectively, these results indicate that atRA promotes lipid catabolism via PPARδ, and 13‐cisRA and 4‐oxo‐RAs increase triglyceride synthesis, possibly via SREBP1 and PPARγ. Our findings regarding 13‐cisRA and its main circulatory metabolite 4‐oxo‐13‐cisRA explain the clinical data of increased incidence of hypertriglyceridemia and hypercholesterolemia following 13‐cisRA dosing. In addition, our data supports the mouse findings that atRA increases fatty acid oxidation in vivo. This study demonstrates that the 13‐cisRA isomer and 4‐oxo‐metabolites of RA found in circulation regulate lipid metabolism independent of atRA and RAR activation, and may play an important role in vivo in modulating lipid homeostasis in human tissues.Support or Funding InformationNIH R01 GM111772, R01 GM081569, R01 GM081569‐S1 and P30 DK035816

The Combination of ATRA and Dasatinib for Differentiation Therapy in Acute Myeloid Leukemias with IDH Mutations

Acute myeloid leukemia (AML) is characterized by the accumulation of malignant blasts with impaired differentiation programs due to recurrent mutations, amongst which IDH mutations occur in 15% of AML patients. Here, we show both in vitro as well as in a xenografted mouse model,that clinically achievable doses of ATRA are sufficient to achieve a terminal granulocytic differentiation in primary AML samples and in AML cell lines harboring IDH1-R132H mutation. There is no effect at this concentration on the WT controls. This is associated with reduction of both proliferation and colony formation, and further leads to apoptosis, thereby improving overall survival of mutant xenografted mice.We further showed, through transcriptomic and western blot analysis, that specific ATRA sensitivity is due to overexpression and activation of C/EBPα in the presence of IDH1-R132H mutation. This primes blasts into myeloid differentiation.Moreover, IDH1 R132H mutation also reduces LYN activation, and thus, also sensitizes to clinically achievable doses of dasatinib, a LYN inhibitor. As ATRA induces a brief LYN activation, which transiently reduces ATRA activity, its combination with dasatinib synergistically increases differentiation. In vivo, the combination of ATRA and dasatinib reduces tumor growth of mutant xenografted mice.The combination ATRA and dasatinib might also be considered for other IDH mutations that produce 2-hydroxyglutarate, since treatment with the mutant-specific oncometabolite (eg. 2-hydroxyglutarate) sensitizes AML cells to ATRA and dasatinib-induced differentiation.Finally, ATRA also reduces BCL2 expression specifically in the presence of IDH1 R132H mutation. Since it has been shown that IDH mutations increase BCL2 dependence in leukemic cells, our results identified a subgroup of patients that is likely to respond to pharmacologic concentrations of ATRA.To conclude, our data provide the preclinical rationale for investigating the use of the combination ATRA and dasatinib in a subgroup of patients who carry IDH1 R132H mutation, in clinical trials. The addition of a BCL2 inhibitor such as ABT-199 would also be considered. DisclosuresOff Label Use: ATRA and dasatinib for treatment of non APL AML. Recher:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Chugai: Research Funding.

Is There Still a Role for Low-Dose All-Transretinoic Acid in the Treatment of Acute Promyelocytic Leukemia in the Arsenic Trioxide Era?

Low-dose all-transretinoic acid (LD-ATRA) has shown similar peak plasma concentrations and a mean area under the concentration time curve in comparison with standard doses of ATRA. We evaluated the efficacy of LD-ATRA plus anthracycline-based chemotherapy in patients with newly diagnosed acute promyelocytic leukemia (APL). Patients diagnosed with APL during the period of 2002 to 2014 were included. They received ATRA 25 mg/m(2) plus anthracycline (doxorubicin or mitoxantrone) as induction chemotherapy, followed by 3 consolidations with LD-ATRA and anthracycline and maintenance therapy with intermittent LD-ATRA and oral chemotherapy for 2 years. Twenty-two patients with a median age of 28 years (range, 18-55 years) were included; 17 (77%) were in the low-risk group. Complete remission occurred in 86%, and the early death rate was 9%. At a median follow-up of 32 months (range, 4-126 months) disease-free survival (DFS) was 75% and overall survival (OS) was 86%, with a relapse rate of 27% for the entire follow-up period. LD-ATRA plus anthracycline is safe and effective in achieving CR of APL. The early death rate is similar to that of treatment with standard doses, but it appears to be inferior in preventing relapses.

Evaluating Packed Red Blood Cell (RBC) Product Utilization on Hematologic Malignancy Service (HMS): A Quality Improvement Initiative

Dual differentiation therapy with arsenic trioxide and tretinoin (all-trans-retinoic acid; ATRA) has demonstrated non-inferiority compared to chemotherapy for low and intermediate risk acute promyelocytic leukemia. As clinical experience grows with this regimen, less commonly reported toxicities may begin to surface. At our center, we have treated ten patients thus far with dual differentiation therapy; five (50%) have developed pseudotumor cerebri (PTC). One of these 5 patients was managed with temporary discontinuation of ATRA and initiation of acetazolamide. In the remaining 4 patients, topiramate was substituted after acetazolamide failure, in order to relieve symptoms and permit ATRA dose re-escalation. All patients treated with this approach are in complete remission. Given our experience with dual differentiation therapy, we recommend close monitoring for signs and symptoms of PTC. In patients with inadequate symptom control with ATRA dose reduction and acetazolamide, topiramate should be considered.

Inhibition of Cytochrome P450 and Multidrug Resistance Proteins Potentiates the Efficacy of All-Trans Retinoic Acid in Pancreatic Cancer In Vitro and In Vivo

Objectives: All-trans retinoic acid (atRA) potently induces differentiation and apoptosis in pancreatic cancer. However, the clinical use of retinoids is limited by retinoid resistance or development of toxicity at high doses. We tested the hypothesis that blocking atRA degradation and elimination from the cell would potentiate its effectiveness in pancreatic cancer therapy. Methods: In vitro, AsPc-1 and HPAF cells were co-treated with atRA and inhibitors of either multidrug resistance (MDR: verapamil, LY335979, and quinidine) or cytochrome P450 (CYP450s: troleandomycin, clotrimazole and liarozole). In addition, cells were co treated with atRA and antisense oligonucleotides against MRP, Pgp, CYP26 and CYP3A4. Proliferation and apoptosis were investigated. In vivo, AsPc-1 xenografts were treated with atRA, verapamil, and troleandomycin alone or in combination. Results: The anti-proliferative effect of atRA on AsPc-1 and HPAF cells was markedly potentiated by the inhibition of MDR and CYP450 or by antisense oligonucleotides to reduce their production. The combination also enhanced atRA-induced apoptosis. Coadministration of inhibitors of MDR and CYP450 also potentiated the inhibitory effect of atRA on growth of xenografts. Conclusions: Co-treatment of pancreatic cancer with low nontoxic doses of atRA combined with MDR blockade and inhibition of CYP450 is effective suppressing tumor growth, suggesting a novel clinical application.

Improved Outcome with ATRA-Arsenic Trioxide Compared to ATRA-Chemotherapy in Non-High Risk Acute Promyelocytic Leukemia – Updated Results of the Italian-German APL0406 Trial on the Extended Final Series

▪Background: We recently showed that the combination of ATRA and arsenic trioxide (ATO) is at least not inferior and possibly superior to standard ATRA and chemotherapy (CHT) in the front-line management of low/intermediate risk APL (Italian-German APL 0406 trial; Lo-Coco et al., NEJM 2013). We report herein on the extended and final series of 276 patients (162 were in the previous report) with the last case being enrolled into the study in January 2013.Methods: The APL0406 study was a prospective, open-label, randomized intergroup trial conducted by the Italian GIMEMA and the German SAL and AMLSG study groups. Eligible patients were adults aged 18-<71 years with newly diagnosed, genetically confirmed, non-high-risk (WBC≤10x109/L) APL. Patients were randomized to receive the ATO+ATRA combination originally developed by the MD Anderson Cancer Center group, or the Italian AIDA2000 protocol (Estey et al., Blood 2006 and Lo-Coco et al., Blood 2010). Patients in the ATRA-ATO arm received ATO 0.15/kg plus ATRA 45 mg/m2 daily until CR, then ATO 5 days/week, 4 weeks on 4 weeks off, for a total of 4 courses and ATRA 2 weeks on and 2 weeks off for a total of 7 courses. Patients in the ATRA-CHT arm received the standard AIDA (ATRA+Idarubicin) induction followed by 3 cycles of anthracycline-based consolidation together with ATRA and low dose CHT and ATRA for maintenance. The primary study objective was EFS at 2 years.Results: A total of 254 patients were evaluable for response to induction. CR was achieved in 122/122 (100%) in the ATRA-ATO versus 128/132 (97%) in the ATRA-CHT arm (P=0.12). Four patients died during induction in the ATRA-CHT arm. After a median follow-up of 36 months (range 1-75 months), the 2-year EFS was 98% and 84.9% in the ATRA-ATO and ATRA-CHT groups respectively (P= 0.0002), The 2-year cumulative incidence of relapse (CIR) rate was 1.1% and 9.4%, respectively (P=0.005) and, finally, the 2-year overall survival (OS) rate was 99.1% vs. 94.4% (P=0.01) for ATRA-ATO vs ATRA-CHT, respectively.Conclusions: The data on this extended cohort demonstrate a significantly augmented survival benefit coupled to a higher antileukemic efficacy provided by ATRA-ATO as compared to ATRA-CHT, in low/intermediate risk APL. These results further support ATRA-ATO as the new standard of care in this clinical setting. [Display omitted] DisclosuresPlatzbecker:Teva: Honoraria. Off Label Use: Presentation includes off-label use of arsenic trioxide (ATO) in front-line management of APL. ATO is currently approved in treatment of relapsed APL in the US and Europe. Breccia:Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Fiedler:TEVA: Travel reimbursement for meeting attendance Other. Lübbert:Cephalon / TEVA: Travel support Other. Link:TEVA: Consultancy, Speakers Bureau. Radsak:Celgene: Research Funding. Döhner:TEVA: Research Funding. Schlenk:TEVA: Research Funding, Speakers Bureau. Lo-Coco:TEVA: Honoraria; Lundbeck: Honoraria.

A Novel Path for ATRA Differentiation Therapy in Acute Myeloid Leukemia with Isocitrate Dehydrogenase Mutations

Acute myeloid leukemia (AML) is characterized by accumulation of malignant blasts with impaired differentiation programs due to recurrent mutations, among which IDH mutations occur in 15% of AML patients. These mutations lead to a block in erythroid commitment while they may also bias hematopoietic differentiation to myeloid lineage. Interestingly, Lyn tyrosine kinase is required for erythroid differentiation and we have observed a reduction of Lyn expression in the presence of IDH1-R132H mutation. It is also a negative regulator of ATRA-induced granulocytic differentiation. Accordingly, we hypothesized that IDH mutations may sensitize AML cells to ATRA-induced differentiation.Here, we report that clinically achievable doses of ATRA are sufficient to trigger differentiation specifically on AML cell lines, primary patient samples and xenograft mice models carrying IDH1 mutation as observed by an increase in CD11b expression, granulocytic enzyme activity and morphologic changes in May-Grunwald-Giemsa staining. We also showed that ATRA-induced terminal granulocytic differentiation increases apoptosis while decreases proliferation and colony formation specifically in IDH1 mutant cells. Moreover, inhibition of IDH1-R132H activity reduced ATRA-sensitivity while increasing expression of IDH mutation correlated with highest ATRA sensitivity. Furthermore, treatment with a cell-permeable form of the oncometabolite specifically produced by the mutant (eg. 2-HydroxyGlutarate) sensitized AML cells to ATRA-induced differentiation. Finally, because ATRA-induced differentiation triggers a transient increase of Lyn activation, its association with Lyn inhibitors synergistically increased ATRA-induced differentiation of IDH mutant blasts.In summary, our results showed that IDH mutations by producing 2-HG sensitized leukemic blasts to ATRA and that this synergizes with Lyn inhibition. Since 2HG concentration reaches millimolar in AML patient serum and is 100-fold higher in IDH mutated patients than in non-mutated ones, we would predict a strong efficacy and specificity of ATRA. Furthermore, as IDH mutations are systematically conserved at relapse, this therapeutic strategy might be promising to achieve a long-term remission specifically for this AML patient subgroup. DisclosuresNo relevant conflicts of interest to declare.

High pseudotumor cerebri incidence in tretinoin and arsenic treated acute promyelocytic leukemia and the role of topiramate after acetazolamide failure

Dual differentiation therapy with arsenic trioxide and tretinoin (all-trans-retinoic acid; ATRA) for the management of low and intermediate risk acute promyelocytic leukemia has recently been recommended by the National Comprehensive Cancer Network. Some less common toxicities of the combination may have yet to be fully realized. Of ten patients we have treated thus far, five (50%) have developed pseudotumor cerebri. In one patient, temporary discontinuation of ATRA and initiation of acetazolamide controlled symptoms. In four patients, topiramate was substituted for acetazolamide to relieve symptoms and allow ATRA dose re-escalation. We conclude that providers should monitor for pseudotumor cerebri and consider topiramate if acetazolamide fails.

TBLR1 Fuses To Retinoid Acid Receptor Alpha In a Variant t(3;17)(q26;q21) Translocation Of Acute Promyelocytic Leukemia

IntroductionThe majority of acute promyelocytic leukemia (APL) cases are characterized by PML-RARα fusion gene. Although PML-RARα fusion gene can be detected in more than 98% of APL cases, RARα is also found to be fused with other partner genes, which are also related to ATRA-dependent transcriptional activity and cell differentiation. In this study, we identified a novel RARα fusion gene, TBLR1-RARα, in a rare case of APL with a t(3;17)(q26;q21),t(7;17)(q12;q21) complex chromosomal rearrangement. The structure, pathogenesis and response to drug therapy of the novel fusion gene were investigated to illustrate the characteristics, pathogenesis and the therapeutic effect in this variant APL. MethodsTo identify and amplify the novel chimeric fusion transcript, 5' RACE and RT-PCR was performed. The TBLR1-RARα expression vector was constructed and transfected into 293T cell line by Lipofectamine2000 reagent. When the transfected 293T cell line was treated with or without ATRA, the expression level and the subcellular localization of TBLR1-RARα were investigated by Western blot and immunofluorescence analysis, and then coimmunoprecipitation and immunofluorescence analysis were performed to investigate the formation of homodimer and the recruitment of the corepressors by TBLR1-RARα. Dual-luciferase assay was used to clarify the transcriptional activity of TBLR1-RARα. Then, a lentiviral vector of TBLR1-RARα was constructed and infected the HL-60 cell line. The HL-60 cells which highly expressed TBLR1-RARα were sorted by flow cytometry. Colony formation assay and flow cytometry analysis were performed to detect the differentiation status in the TBLR1-RARα highly expressed HL-60 cells. ResultsIn our study, the novel TBLR1-RARα fusion gene was cloned from an APL patient who demonstrated the typical clinical features of APL, such as bleeding tendency, leukocytosis, hypergranular promyelocytes accumulated in the bone marrow, coagulopathy. However, RT-PCR analysis and FISH studies failed to detect PML-RARα fusion gene in this case, while karyotype analysis revealed a rare complex translocation, t(3;17)(q26;q21),t(7;17)(q12;q21). When treated with ATRA, As2O3 and chemotherapeutic drugs, this patient achieved complete remission. After three courses of consolidation therapies, the patient relapsed with leukocytosis and the bone marrow karyotype analysis displayed a recurring chromosomal rearrangement, 46,xy,t(3;17)(q26;q21),t(7;17)(q12;q21),5q+,6q-,10q+,11p-, which was more complex in comparison with the karyotype at diagnosis.There have been nine RARα fusion genes reported so far. Like other RARα fusion genes, TBLR1-RARα contains the exon 3 and the 3' sequence of RARα. TBLR1-RARα oncoprotein contains the LisH domain from TBLR1 and the B-F domains from RARα. TBLR1-RARα diffusely locates in nucleus and cytoplasm. Like other RARα fusion protein, TBLR1-RARα can form homodimer and recruit corepressors to inhibit the transcription of the RARα target gene. TBLR1-RARα inhibits the RARα transcriptional activation in a dominant-negative manner and the transcriptional inhibition can be rescued by overexpression of wild-type RARα. In the presence of pharmacological doses of ATRA, TBLR1-RARα could be degraded and its homodimerization was abrogated. Moreover, when treated with ATRA, TBLR1-RARα could mediate the dissociation and degradation of transcriptional corepressors and consequently transactivated transcription of RARα target genes and induced cell differentiation in a dose- and time- dependent manner. Finally, TBLR1-RARα was also detected in another two cases of APL with t(3;17) chromosomal translocation. ConclusionsIn this study, we discovered a novel RARα fusion gene TBLR1-RARα from a APL patient with t(3;17) chromosomal translocation, and investigated its function, pathogenesis and response to drug treatment. Unlike wild-type RARα, TBLR1-RARα can form homodimer, recruit more corepressors to inhibit the transcription of RARα target gene and play a role in APL pathogenesis. The transcriptional inhibition of TBLR1-RARα can be rescued by overexpression of RARα and ATRA treatment and finally leads to cell differentiation. Disclosures:Wang:Bristol Myers Squibb: Consultancy; Novartis: Consultancy.

Abstract PD01-05: A Combination of HDAC Inhibitor Entinostat (MS-275), All Trans Retinoic Acid (ATRA) and Chemotherapy Drug(s) Causes Regression of Established Xenografts of Triple Negative Breast Cancer

Abstract Triple negative breast cancer (TNBC) is a subgroup of breast cancer that rapidly develops resistance to chemotherapy. Currently, TNBC patients are treated with anthracycline and taxane-based regimens. These treatments result in some improvements in the clinical outcomes; however, they are associated with poor prognosis. Also, endocrine therapies are ineffective since the tumors do not express the estrogen receptor. Recent studies have shown that histone deacetylase (HDAC) inhibitors could reverse the epigenetic profile of some genes, including ER, EGFR, and RAR≥2. We have recently shown that treatments that combine the HDAC inhibitor, Entinostat (MS-275), with the aromatase inhibitor, Letrozole, result in regression of xenografts of MDA-MB-231 breast cancer cells. Since RAR≥2 is re-expressed as well, we reasoned that combining epigenetic therapy using MS-275, with differentiation therapy using a RAR≥2 agonist (ATRA) will provide an effective combination of drugs against TNBC. We also examined possible potentiation of anticancer activity of these agents by low nontoxic doses of chemotherapy (doxorubicin and paclitaxel). In vitro, human TNBC cell lines, MDA-MB-231, HCC 1143, SUM159, SUM149 and BT20 responded significantly better to the triple combination compared to the drugs used singly or in combination with MS-275. Upon treatment, re-expression of the silenced RAR≥2 and downstream effectors was observed in the cell lines in vitro and in tumor xenografts of MDA-MB-231 cells. Subcutaneous tumor growth in immunodeficient mice was strongly inhibited (10/14 significant regression) using a combination of MS-275, ATRA and low dose Doxorubicin, compared to the drugs administered singly or in combination with MS-275. No significant weight loss was observed in any of the groups. This combination was then used to treat tumor xenografts of SUM159, another TNBC cell line, and gave a promising result. Furthermore, combination of MS-275, ATRA, and a clinical dose of Paclitaxel can effectively inhibit growth of MDA-MB-231 and SUM159 cells. These results suggest that combination of HDAC inhibitor and RAR≥2 agonist with low dose chemotherapy has the potential to be an effective treatment against TNBC. This study provides encouraging new information for a nontoxic, and novel combination of drugs which could be effective against TNBC. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD01-05.