Retinoic Acid Receptor Alpha Gene Research Articles

Mutations and laboratory diagnosis in acute promyelocytic leukemia

Introduction: acute Promyelocytic Leukemia (APL) is a unique subtype of acute myeloid leukemia (AML) characterized by proliferation and accumulation of abnormal promyelocytes in the bone marrow. It arises from a balanced translocation between chromosomes 15 and 17, involving the retinoic acid receptor alpha (RARA) gene on chromosome 17 and the promyelocytic leukemia (PML) gene. It has a higher incidence in young adults. Objective: to establish the mutations associated with acute promyelocytic leukemia and the techniques that aid in its clinical diagnosis. Method: a systematic review of 19 scientific articles published in the databases of PubMed, Scopus, Google Scholar and the Virtual Library of the University of Granada was carried out. The data collected focused on acute promyelocytic leukemia, acquired, secondary or somatic mutations and laboratory diagnosis. Results: of a total of 1730 patients 67.7% had the t(15;17) mutation, of 682 patients 26.7% had the FLT3-ITD mutation and of 175 patients 16% had the FLT3-D835 mutation. Laboratory diagnosis is based on morphological evaluation of promyelocytes, hemostasis tests, biochemical tests Immunophenotyping and molecular cytogenetics. Conclusions: the mutation associated with APL is the promyelocytic leukemia/retinoic acid receptor alpha (PML-RARA) gene, the same that presents a t(15;17), secondary mutations or somatic mutations such as (FLT3-ITD or FLT3-D835) were also known. Multiparametric flow cytometry is one of the most widely used techniques for the diagnosis of APL, allowing the determination of cell morphology and immunophenotypes

Abstract 266: The effects of retinoid agents and retinoic acid receptor genotype on proliferation, differentiation, and stem cells in colorectal cancer

Abstract Understanding how to therapeutically target colonic stem cells (SCs) is important because SC overpopulation drives CRC growth and development. Our goal is to therapeutically sensitize cancer SCs (CSCs) to the differentiation-inducing effects of retinoid agents. We previously found that ALDH specifically marks normal and malignant colonic SCs. We also found that retinoic acid (RA) receptors and other RA signaling components are selectively expressed in ALDH+ SCs, indicating that RA signaling occurs through ALDH+ SCs. Because all-trans retinoic acid (ATRA) is a highly effective treatment for acute promyelocytic leukemia, which is caused by a genetic translocation involving retinoic acid receptor alpha (RARA), we conjecture that the response of RA agents may be effective against solid tumors depending on their pathway genotype. Our bioinformatics analysis on mutations and overexpression of RA signaling component genes in CRC showed that most of the RA pathway genes are altered in human CRCs. Therefore, we hypothesize that the ability of retinoid agents and retinoid metabolism blocking agents to induce differentiation of CSCs depends on the RA pathway genotype of CRC cells.Testing the effect of various retinoid drugs on cell proliferation shows that RA pathway genotype affects the response of RA agents in CRC cell lines. HCT116 and SW480 CRC cell lines, which have RA receptor mutations, displayed resistance to ATRA. In contrast, HT29 CRC cells that have wild-type RA receptors are sensitive to ATRA. All three cell lines however showed similar dose responses to the ATRA metabolizing enzyme, CYP26A1 inhibitor Liarozole. This finding indicates that regardless of RA receptor mutations, inhibition of ATRA metabolism can increase intracellular RA levels in a similar manner on all three cell lines to induce anti-proliferative effects. We used CRISPR-Cas9 gene editing to knock out the RARA gene in HT29 cells and evaluated its response to ATRA compared to HT29 cells. The RARA knockout derived lines showed reduced sensitivity to ATRA treatment and a slower rate of cell division compared to HT29 control cells. Additionally, Nanostring mRNA profiling analysis shows that ATRA treatment of HT29 cells increases expression of RARA, CYP26A1, and KRT20 (differentiation marker); and decreased decreases expression of CSC markers LGR5 and ALDH1A1. Altogether, these findings indicate that upregulated RA signaling can reduce cell proliferation, increase differentiation, and decrease CSC numbers in CRCs. Overall, our findings indicate that colon SCs are regulated by RA signaling mechanisms and dysregulation of RA signaling contributes to the SC overpopulation that drives CRC growth. Thus, therapeutically sensitizing CSCs to differentiation-inducing effects of retinoid agents should provide insight into designing new SC-targeted therapies for CRC. Citation Format: Victoria O. Hunsu, Caroline O. Facey, Bruce M. Boman. The effects of retinoid agents and retinoic acid receptor genotype on proliferation, differentiation, and stem cells in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 266.

Use of tamibarotene, a potent and selective RARα agonist, in combination with azacitidine in patients with relapsed and refractory AML with RARA gene overexpression

Tamibarotene-based therapy is a novel targeted approach for the treatment of relapsed/refractory (R/R) acute myeloid leukemia (AML) with retinoic acid receptor alpha (RARA) gene overexpression. Approximately, 50% of higher-risk myelodysplastic syndrome (MDS) patients and approximately 30% of AML patients are positive for RARA overexpression using a blood-based biomarker test that measures RARA expression in peripheral blasts. A phase 2 study investigating the activity of tamibarotene in patients with RARA overexpression was conducted in patients with AML and MDS (NCT02807558). In 28 patients with R/R AML and RARA overexpression treated with tamibarotene in combination with azacitidine, the median overall survival was 5.9 months. In 21 response-evaluable patients, the complete remission/complete remission with incomplete hematologic recovery (CR/CRi) rate was 19%, and median time to initial CR/CRi was 1.2 months. The favorable safety profile and preliminary clinical activity support the development of combination therapies with tamibarotene in myeloid malignancies with RARA overexpression.

Targeting RARA overexpression with tamibarotene, a potent and selective RARα agonist, is a novel approach in AML.

A superenhancer at the retinoic acid receptor alpha (RARA) gene is associated with RARA mRNA overexpression in ∼30% of non-acute promyelocytic leukemia acute myeloid leukemia (AML) and in ∼50% of myelodysplastic syndromes (MDS). RARA overexpression is an actionable target for treatment with tamibarotene, an oral potent and selective RARα agonist. Sensitivity to the RARα agonist tamibarotene was demonstrated in RARA-high but not RARA-low preclinical AML models. The combination of oral tamibarotene plus azacitidine was evaluated in a phase 2 clinical study in 51 newly diagnosed unfit patients with AML identified as RARA-positive (n= 22) or RARA-negative (n= 29) for RARA mRNA overexpression in peripheral blasts using a blood-based biomarker test. In 18 response-evaluable RARA-positive patients, complete remission (CR)/CR with incomplete hematologic recovery rate was 61%, CR rate was 50%, and time to initial composite CR was rapid at 1.2 months. Transfusion independence was attained by 72% of RARA-positive patients. In contrast, 28 response-evaluable RARA-negative patients had response rates that were consistent with azacitidine monotherapy. Tamibarotene in combination with azacitidine was well tolerated. The majority of nonhematologic adverse events were low grade and hematologic adverse events were comparable to single-agent azacitidine, demonstrating that there was no additional myelosuppression when tamibarotene was combined with azacitidine. These results support further evaluation of tamibarotene-based treatment strategies in patients with AML or MDS with RARA overexpression to provide a targeted approach with the goal of improving patient outcomes. This trial was registered at www.clinicaltrials.gov as #NCT02807558.

Vitamin A deficiency and vitamin A supplementation affect innate and T cell immune responses to rotavirus A infection in a conventional sow model.

Rotavirus A (RVA) causes ~200,000 diarrheal deaths annually in children <5yrs, mostly in low- and middle-income countries. Risk factors include nutritional status, social factors, breastfeeding status, and immunodeficiency. We evaluated the effects of vitamin A (VA) deficiency/VA supplementation and RVA exposure (anamnestic) on innate and T cell immune responses in RVA seropositive pregnant and lactating sows and passive protection of their piglets post-RVA challenge. Sows were fed VA deficient (VAD) or sufficient (VAS) diets starting at gestation day (GD)30. A subset of VAD sows received VA supplementation from GD|76 (30,000IU/day, VAD+VA). Sows (6 groups) were inoculated with porcine RVA G5P[7] (OSU strain) or Minimal Essential Medium (mock) at GD~90: VAD+RVA; VAS+RVA; VAD+VA+RVA; VAD-mock; VAS-mock; and VAD+VA-mock. Blood, milk, and gut-associated tissues were collected from sows at several time points to examine innate [natural killer (NK), dendritic (DC) cells], T cell responses and changes in genes involved in the gut-mammary gland (MG)-immunological axis trafficking. Clinical signs of RVA were evaluated post inoculation of sows and post-challenge of piglets. We observed decreased frequencies of NK cells, total and MHCII+ plasmacytoid DCs, conventional DCs, CD103+ DCs and CD4+/CD8+ and T regulatory cells (Tregs) and NK cell activity in VAD+RVA sows. Polymeric Ig receptor and retinoic acid receptor alpha (RARα) genes were downregulated in mesenteric lymph nodes and ileum of VAD+RVA sows. Interestingly, RVA-specific IFN-γ producing CD4+/CD8+ T cells were increased in VAD-Mock sows, coinciding with increased IL-22 suggesting inflammation in these sows. VA supplementation to VAD+RVA sows restored frequencies of NK cells and pDCs, and NK activity, but not tissue cDCs and blood Tregs. In conclusion, similar to our recent observations of decreased B cell responses in VAD sows that led to decreased passive immune protection of their piglets, VAD impaired innate and T cell responses in sows, while VA supplementation to VAD sows restored some, but not all responses. Our data reiterate the importance of maintaining adequate VA levels and RVA immunization in pregnant and lactating mothers to achieve optimal immune responses, efficient function of the gut-MG-immune cell-axis and to improve passive protection of their piglets.

How retinoic acid and arsenic transformed acute promyelocytic leukemia therapy.

Acute promyelocytic leukemia (APL) is associated with severe coagulopathy leading to rapid morbidity and mortality if left untreated. The definitive diagnosis of APL is made by identifying a balanced reciprocal translocation between chromosomes 15 and 17. This t(15;17) results in a fusion transcript of promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARA) genes and the expression of a functional PML/RARA protein. Detection of a fused PML/RARA genomic DNA sequence using fluorescence in situ hybridization (FISH) or by detection of the PML/RARA fusion transcript via reverse transcriptase polymerase chain reaction (RT-PCR) has revolutionized the diagnosis and monitoring of APL. Once confirmed, APL is cured in over 90% of cases, making it the most curable subtype of acute leukemia today. Patients with low-risk APL are successfully treated using a chemotherapy-free combination of all-trans retinoic acid and arsenic trioxide (ATO). In this review, we explore the work that has gone into the modern-day diagnosis and highly successful treatment of this once devastating leukemia.

Abstract 6067: Studying the anti-proliferating and differentiating effects of retinoids based on pathway genotype of colorectal cancer cell lines

Abstract Background: Our overarching goal is to therapeutically sensitize human colorectal cancer stem cells to differentiation-inducing effects of retinoid agents. Tumorigenesis in colorectal cancer (CRC) results from the acquisition of mutations in a colonic stem cell which transforms it into a cancer stem cell (CSC). There are no curative treatments for advanced CRC due to the resistance of CSCs to chemotherapy and/or radiation. All-trans retinoic acid (ATRA) is known to be a greatly effective treatment for acute promyelocytic leukemia (APL) which is caused by translocation in the retinoic acid receptor alpha (RARA) gene. We conjecture that the response of retinoic acid (RA) agents may have effects on solid tumors depending on their pathway genotype. Results from our bioinformatics analysis on mutations and overexpression of RA signaling component genes in CRC showed that the majority of RA pathway genes are overexpressed in CRC. Therefore, we hypothesize that the ability of retinoid and retinoid metabolism blocking agents to induce differentiating effects in CSCs depends on the RA pathway genotype expressed in CRC cells. Methods: The ability of RA agents (ATRA, 13-cis retinoic acid, 9-cis retinoic acid) and RA metabolism blocking agents (Liarozole and Talarozole) to inhibit proliferation and induce differentiation on CRC cell lines with different mutations in RA signaling were evaluated. Additionally, Nanostring Profiling was used to measure the effects of the agents on mRNA expression of CSC markers, indicators of cell differentiation, and RA receptors. Results: Our results show that the RA pathway genotype affects the response of RA agents in CRC cell lines. Both HCT116 and SW480 CRC cell lines, which are both mutant in RARA and RARA and RXRG respectively displayed resistance to ATRA. HT29 CRC cells express wild- type RA receptors and are sensitive to ATRA. All three cell lines showed similar dose responses to the CYP26A1 inhibitor Liarozole. This indicates that regardless of RA receptor mutations, inhibition of ATRA metabolism increases intracellular RA levels in a similar manner on all 3 cell lines. Nanostring Profiling shows that ATRA treatment of HT29 cells induces: an increase in the expression of RARA, CYP26A1 (ATRA metabolizing enzyme) and KRT20 (differentiation marker); and a decrease in the expression of CSC markers LGR5 and ALDH1A1. This indicates an active RA signaling pathway, induced differentiation, and a decrease in stem cells. Discussion: Overall, our findings indicate that colon SCs are regulated by RA signaling mechanisms and dysregulation of RA signaling contributes to the SC overpopulation that drives CRC growth. Therefore, further investigation on how to therapeutically sensitize human colonic CSCs to the differentiation-inducing effects of retinoid agents should provide insight into designing new SC-targeted therapies for CRC. Citation Format: Victoria O. Hunsu, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. Studying the anti-proliferating and differentiating effects of retinoids based on pathway genotype of colorectal cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6067.

Acute promyelocytic leukemia: from genetic lesions identification to molecularly targeted therapy

Acute promyelocytic leukemia (APL) differs from other types of acute myeloid leukemia both in terms of the spectrum of clinical symptoms, as well as cytogenetic and molecular background. Fast diagnosis of APL enables highly effective targeted therapy initiation and avoiding of serious organ/tissue damage (including fatal bleeding into the central nervous system). In the initial diagnostic process the most important is the rapid identification of the presence of specific cytogenetic and molecular changes involving the retinoic acid receptor alpha ( RARA ) gene located on the 17q21 chromosome. In patients with APL, alongside the most commonly observed translocation t(15;17)(q24;q21) leading to the formation of the PML-RARA fusion, several dozen variant cases have also been identified as a result of other translocations involving RARA gene with different clinical symptomatology and variable sensitivity to the targeted therapy with all-trans retinoic acid and arsenic trioxide. The paper presents the recent data concerning the epidemiology, symptomatology and accurate diagnostics methods useful for early identification of APL and immediate initiation of the molecularly targeted therapy

Defining the transcriptional control of pediatric AML highlights RARA as a superenhancer-regulated druggable dependency

AbstractSomatic mutations are rare in pediatric acute myeloid leukemia (pAML), indicating that alternate strategies are needed to identify targetable dependencies. We performed the first enhancer mapping of pAML in 22 patient samples. Generally, pAML samples were distinct from adult AML samples, and MLL (KMT2A)–rearranged samples were also distinct from non–KMT2A-rearranged samples. Focusing specifically on superenhancers (SEs), we identified SEs associated with many known leukemia regulators. The retinoic acid receptor alpha (RARA) gene was differentially regulated in our cohort, and a RARA-associated SE was detected in 64% of the study cohort across all cytogenetic and molecular subtypes tested. RARA SE+ pAML cell lines and samples exhibited high RARA messenger RNA levels. These samples were specifically sensitive to the synthetic RARA agonist tamibarotene in vitro, with slowed proliferation, apoptosis induction, differentiation, and upregulated retinoid target gene expression, compared with RARA SE− samples. Tamibarotene prolonged survival and suppressed the leukemia burden of an RARA SE+ pAML patient-derived xenograft mouse model compared with a RARA SE− patient-derived xenograft. Our work shows that examining chromatin regulation can identify new, druggable dependencies in pAML and provides a rationale for a pediatric tamibarotene trial in children with RARA-high AML.

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

Mechanistic effects of arsenic trioxide on acute promyelocytic leukemia and other types of leukemias.

Acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia characterized with a translocation between promyelocytic leukemia gene (PML) on chromosome 15 and retinoic acid receptor alpha gene (RARα) on chromosome 17. Transcription of this fusion gene results in PML/RARα fusion protein blocking expression of critical genes involved in differentiation of myeloid cells through interaction with RAR element. PML/RARα fusion protein prevents normal function of PML and RARα as well as inhibiting apoptosis. Arsenic trioxide (ATO) is an important agent for the treatment of relapsed and newly diagnosed APL. ATO induces apoptosis, autophagy, and partial cellular differentiation as well as inhibiting cell growth and angiogenesis. Recognition of signaling pathways and molecular mechanisms induced by ATO can be effective for discovering novel treatment strategies to target leukemia cells. Also, it can be developed for the treatment of a variety of cancer cells. This review provides a perspective on anticancerous effects of ATO on APL and leukemia cells.

Genetic Predictors of Severe Skin Toxicity in Patients with Stage III Colon Cancer Treated with Cetuximab: NCCTG N0147 (Alliance).

Cetuximab, an EGFR inhibitor used to treat multiple cancer types, including colon cancer, causes severe skin toxicity in 5%-20% of patients, leading to decreased quality of life and treatment delays. Our understanding of which patients have an increased risk of severe toxicities is limited. We conducted a genome-wide association study to identify germline variants predictive of cetuximab-induced severe skin toxicity. Our study included 1,209 patients with stage III colon cancer randomized to receive cetuximab plus 5-fluorouracil and oxaliplatin as part of the NCCTG N0147 (Alliance) clinical trial. Skin toxicity outcomes were collected using the Common Toxicity Criteria for Adverse Events version 3.0. We performed genotyping, evaluating approximately 10 million genetic variants. We used logistic regression to evaluate the association of each genetic variant and severe (grade ≥ 3) skin toxicity, adjusting for age, sex, and genetic ancestry. Genome-wide significance was defined as P < 5 × 10-8. Participants were predominantly middle-aged white men; 20% (n = 243) experienced severe skin toxicity. Two genetic variants in the retinoic acid receptor alpha (RARA) gene were significantly associated with severe skin toxicity [OR, 3.93; 95% confidence interval (CI), 2.47-6.25; P < 7.8 × 10-9]. Functional annotations indicate these variants are in the RARA promoter. Additional significantly associated variants were identified in chromosome 2 intergenic regions. Identified variants could represent a potential target for risk stratification of patients with colon cancer receiving cetuximab. Retinoids have shown promise in the treatment of cetuximab-induced skin toxicity, so follow-up work could evaluate whether individuals with the RARA variant would benefit from retinoid therapy.

Predicting Resistance to the Combination of ATO and ATRA in APL Patients with PML-Rara Fusions, Using a Computational Biology Modeling Approach: Mycare-021-01

Background: Acute promyelocytic leukemia (APL) is a biologically and clinically distinct subtype of acute myeloid leukemia (AML) with unique molecular pathogenesis, clinical manifestations, and treatment. APL is cytogenetically characterized by a balanced translocation t(15;17) (q24;q21), which involves the retinoic acid receptor alpha (RARA) gene on chromosome 17 and the promyelocytic leukemia (PML) gene on chromosome 15 that results in a PML-RARA fusion gene (PMID: 30575821). The PML-RARA fusion gene is the most critical event involved in the pathogenesis of APL, reported in 99% of APL patients (PMID: 32182684). The fusion confers a selective sensitivity to the targeted drugs, arsenic trioxide (ATO) and all-trans-retinoic acid (ATRA), with response rates over 90% (PMID: 31635329). However, the mechanism of resistance in the minority of non-responders is not well understood. This study used the Cellworks Omics Biology Model (CBM) to predict response to the combination of ATO-ATRA in patients harboring the PML-RARA fusion and identify mechanisms of resistance. Methods: Outcomes of 30 APL patients treated with ATRA or ATRA plus ATO were compared with outcomes predicted by CBM (Table 1). Genomic data from 6 publications (Table 2) derived from whole exome sequencing (WES), targeted next-generation sequencing (NGS), copy number variation (CNV) and/or karyotype data were used. All data was anonymized, de-identified and exempt from IRB review. The available genomic data for each profile was entered into the CBM which generates a patient-specific disease protein network model using PubMed and other online resources. The CBM predicts the patient-specific biomarker and phenotype response of a personalized diseased cell to drug agents, radiation and cell signaling. Disease biomarkers that are unique to each patient were identified within the protein network models. ATO and ATRA were simulated on all 30 patient cases. The treatment impact was assessed by quantitatively measuring the drug's effect on a cell growth score which is a composite of the quantified values for cell proliferation, survival, and apoptosis, along with the simulated impact on each patient-specific disease biomarker score. Each patient-specific model was also digitally screened to identify response to ATO and ATRA. Results: The CBM correctly predicted the response to ATO and ATRA in 28 of 30 cases. The overall prediction accuracy was 93% with a PPV of 100%, NPV of 60%, sensitivity of 93%, and specificity of 100%. In 2 of 30 patients who did not respond to ATO and ATRA, the CBM identified clinically relevant deletions to EZH2, KMT2E, and HIPK2 genes. All three genes are located on chromosome 7 and these non-responders had monosomy 7. Conclusions: The Cellworks Omics Biology Model predicted response to ATO and ATRA in APL patients harboring PML-RARA fusions. Predicting non-response to ATO and ATRA in patients with PML-RARA fusion up-front could prevent ineffective treatment, avoid unnecessary adverse events and reduce treatment costs. Additionally, computational modeling can identify new mechanisms of resistance and suggest alternative regimens for non-responding patients by targeting the patient-specific disease biomarkers unique to each. Disclosures Howard: Servier: Consultancy, Other: Speaker; Boston Scientific: Consultancy; Sanofi: Consultancy, Other: Speaker; EUSA Pharma: Consultancy; Cellworks: Consultancy. Nair:Cellworks Research India Private Limited: Current Employment. Grover:Cellworks Research India Private Limited: Current Employment. Tyagi:Cellworks Research India Private Limited: Current Employment. Kumari:Cellworks Research India Private Limited: Current Employment. Prasad:Cellworks Research India Private Limited: Current Employment. Mitra:Cellworks Research India Private Limited: Current Employment. Lala:Cellworks Research India Private Limited: Current Employment. Azam:Cellworks Research India Private Limited: Current Employment. Gupta:Cellworks Research India Private Limited: Current Employment. Mohapatra:Cellworks Research India Private Limited: Current Employment. G:Cellworks Research India Private Limited: Current Employment. Mundkur:Cellworks Group Inc.: Current Employment. Macpherson:Cellworks Group Inc.: Current Employment. Kapoor:Cellworks Research India Private Limited: Current Employment.

Abstract A55: Defining the transcriptional regulation of pediatric AML as a new strategy to find potential druggable vulnerabilities

Abstract Introduction: Nearly 35% of children with acute myeloid leukemia (AML) die, despite aggressive but toxic and nonspecific therapies. New approaches to find and drug pediatric AML targets are clearly needed. Super enhancers (SEs), large regions of highly active chromatin, define cell state and cell identity by regulating oncogenes in many cancers. Recent enhancer profiling of 66 adult AML patient samples revealed SE-defined, prognostically relevant subgroups. An SE was at the retinoic acid receptor alpha (RARA) gene locus in 59% of the samples, which were sensitive to the RARA agonist tamibarotene. We are delineating the transcriptional regulation of pediatric AML (pAML) by SE analysis, which has already elucidated deeper insights into pediatric leukemogenesis, as typified by RARA regulation. Methods: Four AML/APL cell lines and 19 pAML primary samples were enhancer profiled by H3K27Ac chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq). SEs were detected and assigned to genes using the rank ordering of super-enhancers (ROSE) algorithm. Tamibarotene treatment of cell lines and patient samples were assessed for gene and protein expression changes and phenotypic differences. Results: The primary pAML sample cohort encompassed the diverse pAML cytogenetic subtypes, with an over-representation of KMT2A rearrangements (n=9, 47%). The number of unique enhancer regions was nearly saturated in the 19 samples. Median SE size was 3,780bp, much larger than the 511bp of typical enhancers. When SE regions across all samples were clustered together, a RARA SE was seen in two of the ten clusters. Some of the highly correlated, SE-associated genes in these clusters encode proteins involved in inflammation. Eleven of the 19 samples (58%) contained a RARA SE, crossing multiple cytogenetic subtypes. Tamibarotene treatment of RARA SE+ pAML cell lines and patient samples reduced cell viability and increased apoptosis (detected by annexin V+ and activated caspase 3/7), with no effect in RARA SE- pAML samples. In the RARA SE+ samples, tamibarotene increased CD38 (a myeloid differentiation marker usually suppressed by ligand-unbound RARA) and DHRS3 (another RARA target gene used as a pharmacodynamic biomarker in the adult tamibarotene phase II trial). Conclusion: We have profiled the enhancer landscapes of 19 primary pAML samples, the largest dataset of its kind, and have seen a high frequency of a RARA SE in pAML. Tamibarotene has antiproliferative, proapoptotic, and prodifferentiation effects in RARA SE+ pAML. We are confirming these results in a RARA SE+ patient-derived xenograft mouse model, evaluating combinations, and interrogating other SE-regulated genes interacting with RARA that may predict degree of response or resistance to tamibarotene. Our studies confirm that studying the transcriptional regulation of pAML samples through SE analysis can identify druggable targets and also lay the preclinical foundation for a biomarker-defined tamibarotene trial in pediatric AML. Citation Format: Monika Perez, Alfred Daramola, Oscar Sias-Garcia, Helen Wei, Nikitha Cherayil, Charles Y. Lin, Joanna S. Yi. Defining the transcriptional regulation of pediatric AML as a new strategy to find potential druggable vulnerabilities [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A55.

Rara Is a Druggable Super-Enhancer Regulated Dependency in Pediatric AML

Introduction: New approaches to find and then drug pediatric acute myeloid leukemia (AML)-specific targets are clearly needed to help the nearly 35% of patients who still die from the disease. While RARA is a known druggable target in acute promyelocytic leukemia (APL), the utility of using retinoic acid agonists in non-APL AML has not proven consistently beneficial. Super enhancers (SEs), large regions of highly active chromatin, define cell state and cell identity by regulating oncogenes in many cancers. Recent enhancer profiling of 66 adult non-APL AML patient samples revealed SE-defined, prognostically relevant subgroups. An SE was detected at the retinoic acid receptor alpha (RARA) gene locus in 59% of the samples, which were sensitive to the second-generation retinoic acid agonist tamibarotene which has led to a phase II clinical trial. This study confirms that characterization of the chromatin-defined dependencies in specific cancers can pinpoint targets which can be drugged. We are delineating the transcriptional regulation of pediatric AML (pAML) by SE analysis, which has already elucidated deeper insights into pediatric leukemogenesis, as typified by strong RARA dependence in a majority of pAML. Methods: Three AML cell lines and 19 pAML primary samples were enhancer profiled by H3K27Ac chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq). SEs were detected and assigned to genes using the rank ordering of super-enhancers (ROSE) algorithm. Tamibarotene treatment of cell lines and patient samples were assessed for gene and protein expression changes and phenotypic differences. For in vivo assessment, 200,000 cells of a RARA SE+ pAML patient sample were injected into each NSGS mouse by tail-vein injection. One week after injection, tamibarotene (6mg/kg) or vehicle treatment was initiated by gavage (n=7 each arm). Peripheral blood monitoring of leukemia burden was determined flow cytometry. Results: The primary pAML sample cohort encompassed the diverse pAML cytogenetic subtypes (Fig 1a), with an overrepresentation of KMT2A rearrangements (n=9, 47%). The number of unique enhancer regions was nearly saturated in the 19 samples. Median SE size was 3,780bp, much larger than the 511bp of typical enhancers. When SE regions across all samples were clustered together, a RARA SE was seen in two of the ten clusters (Fig 1b). Eleven of the 19 samples (58%) contained a RARA SE, crossing multiple cytogenetic subtypes (Fig 1c). Tamibarotene treatment of RARA SE+ pAML cell lines and patient samples suppressed proliferation and increased apoptosis (detected by annexin V+), with minimal effect in Kasumi, a pAML cell line without a RARA SE (Fig 2a). In the RARA SE+ cell lines and samples only, tamibarotene increased CD38 (a myeloid differentiation marker usually suppressed by ligand-unbound RARA) (Fig 2b). High RARA mRNA levels confirmed the SE assignments in both cell lines and patient samples (Fig 2c). Tamibarotene induced the transcription of DHRS3 (another RARA target gene used as a pharmacodynamic biomarker in the adult tamibarotene phase II trial) (Fig 2d). Tamibarotene suppressed colony formation ability in RARA SE+ cell lines. An ongoing RARA SE+ patient-derived pAML xenograft confirmed tamibarotene markedly suppressed disease progression (Fig 3), with vehicle mice requiring euthanasia 42 days after tail-vein injection for significant disease burden, while the tamibarotene treated mice continued to be well-appearing at the same timepoint. Conclusion: We have profiled the enhancer landscapes of 19 primary pAML samples, the largest dataset of its kind, and seen a high frequency of a RARA SE in pAML. Tamibarotene has anti-proliferative, proapoptotic, and on-target pro-differentiation effects in RARA SE+ pAML in vitro and marked anti-leukemia activity in vivo. Given these positive findings, we are evaluating combinations of other AML-active agents with tamibarotene. Additionally, as there is a range of sensitivity to tamibarotene in RARA SE+ samples, we are also interrogating other SE-regulated genes interacting with RARA that may predict degree of response or resistance to tamibarotene. Our studies confirm that studying the transcriptional regulation of pAML samples through SE analysis can identify druggable targets and also lay the preclinical foundation for a biomarker-defined tamibarotene trial in pediatric AML. Disclosures Wei: NHI NHLBI Grant: Other: received funding . Lin:Syros Pharmaceuticals: Equity Ownership, Patents &amp; Royalties.

45P - Global and sex-specific epigenome-wide association studies for the identification of the main methylated loci related to smoking in a mediterranean population

BackgroundTobacco use has been reported to be the main cause of 90% of male and 80% of female lung cancers, as well as a relevant risk factor for other cancers such as oropharynx, larynx, esophagus, stomach, liver, pancreas, kidney, bladder, and colorectum. Smoking directly affects DNA methylation and although several genes have been reported to be differentially methylated in smokers, gender differences and population-specific differences remain to be further investigated. Our aim was to undertake global and sex-specific genome-wide epigenomic studies (EWAS) in a Mediterranean population to identify the main smoking DNA-methylated genes. MethodsWe analyzed 88 participants in the PREDIMED PLUS-Valencia study paired by age and sex (44 males and 44 females). We included current smokers, former and non-smokers according to the WHO classification. We performed genome-wide DNA methylation using Illumina 850K methylation EPIC arrays. Differential methylation (M-values) was statistically analyzed with Partek, including multivariate adjustement. ResultsIn the whole sample, the top-ranked differentially methylated CpG sites (blood) were: cg05575921 in the AHRR (aryl hydrocarbon receptor repressor) gene (P=2.73E-14), cg21566642 (P=1.51E-12); cg01940273 (P=2.10E-10); cg03636183 in the F2RL3 (coagulation factor II thrombin receptor-like 3) gene (P=6.90E-09); cg23576855 in the AHRR (9.05E-08); and cg17739917 in the RARA (retinoic acid receptor, alpha) gene (P=1.90E-07), replicating previous finding of the hypomethylation in the AHRR and F2RL3 sites in smokers. In the sex-specific analyses, we detected for both men and women similar hypomethylation in the cg05575921-AHRR site, but some heterogeneity for other loci including the BRCA1 gene. ConclusionsSmoking has consistent effect of on the methylation of the AHRR gene, but some gender differences for other genes have been detected. This may be related with further gender-smoking differences for some cancers. Clinical trial identification2011-005398-22. Legal entity responsible for the studyInstituto de Salud Carlos III (ISCIII), Ministerio de Ciencia, Innovación y Universidades. FundingInstituto de Salud Carlos III (ISCIII), Ministerio de Ciencia, Innovación y Universidades. DisclosureAll authors have declared no conflicts of interest.

Autophagy: New Insights into Mechanisms of Action and Resistance of Treatment in Acute Promyelocytic leukemia.

Autophagy is one of the main cellular catabolic pathways controlling a variety of physiological processes, including those involved in self-renewal, differentiation and death. While acute promyelocytic leukemia (APL) cells manifest low levels of expression of autophagy genes associated with reduced autophagy activity, the introduction of all-trans retinoid acid (ATRA)—a differentiating agent currently used in clinical settings—restores autophagy in these cells. ATRA-induced autophagy is involved in granulocytes differentiation through a mechanism that involves among others the degradation of the PML-RARα oncoprotein. Arsenic trioxide (ATO) is another anti-cancer agent that promotes autophagy-dependent clearance of promyelocytic leukemia retinoic acid receptor alpha gene (PML-RARα) in APL cells. Hence, enhancing autophagy may have therapeutic benefits in maturation-resistant APL cells. However, the role of autophagy in response to APL therapy is not so simple, because some autophagy proteins have been shown to play a pro-survival role upon ATRA and ATO treatment, and both agents can activate ETosis, a type of cell death mediated by the release of neutrophil extracellular traps (ETs). This review highlights recent findings on the impact of autophagy on the mechanisms of action of ATRA and ATO in APL cells. We also discuss the potential role of autophagy in the development of resistance to treatment, and of differentiation syndrome in APL.

PB1768 PREDICTIVE FACTORS OF SEVERE BLEEDING IN ACUTE PROMYELOCYTIC LEUKEMIA

Background:Acute promyelocytic leukemia (APL) is identified by the predomination of abnormal promyelocytes in the bone marrow (BM) and a specific chromosomal translocation – t(15;17) – resulting in a fusion transcript between the promyelocytic (PML) gene on chromosome 15 and the retinoic acid receptor alpha (RARα) gene on chromosome 17; this transcript is known as PML–RARα. The t(15;17) chromosomal translocation can cause hyperexpression of tissue factor (TF) in cells of patients with APL and render the patient hypercoagulable.Aims:The objective of the current study was to assure more appropriate therapeutic strategies for reducing severe hemorrhaging by assessing the recovery of abnormal coagulation indexes in patients with acute promyelocytic leukemia (APL) during induction therapy.Methods:Retrospective analyses of newly diagnosed with APL were performed during initial treatment. A total of 23 patients newly diagnosed with APL were treated at the Hematology department of Emergency Hospital (Semey, Kazakhstan), between April 2008 and January 2019. This cohort included 9 males and 14 females with a median age of 41 years (range: 12–75). The APL diagnostic criteria were based on the World Health Organization Classification of Tumors–Pathology and Genetics of Tumors of Hematopoietic and Lymphoid Tissues (2008) and French–American–British classification systems (1976) criteria. A molecular diagnosis was confirmed by identification of t(15;17) in a cytogenetic analysis and/or positive detection of PML–RARα using either fluorescence in situ hybridization or reverse transcription‐polymerase chain reaction. The immune phenotype diagnosis of APL was established by positivity for CD33, CD9, CD13, and CD117, and low expression of HLA‐DR and CD34. Early death (ED) was defined as death due to any cause within 30 days of diagnosiResults:Hemorrhage was the principal cause of death during the induction period. The values of white blood cell count, lactate dehydrogenase, prothrombin time (PT), fibrinogen (Fbg), hemoglobin, and bone marrow leukemic promyelocytes were significantly different in the high‐risk group compared to the low/intermediate‐risk groups. There were significant differences in the white blood cell count, bone marrow leukemic promyelocytes, platelet (PLT) count, and the levels of lactate dehydrogenase, d‐dimer, PT, and Fbg, as well as in FLT3‐ITD mutations between patients with major bleeding and those with minor bleeding. Hemostatic variables significantly improved over time during induction therapy. The recovery times of the PLT, PT, and Fbg values were significantly slower in patients with major bleeding than in those with minor bleeding. Particularly, the PLT level in patients with major bleeding was not similar to that in the minor bleeding group until after 4 weeks of treatment. Hemorrhages were the most common cause of induction death in this study. High‐risk patients were more prone to serious clinical bleeding symptoms. Patients with major bleeding had more rapid proliferation characteristics and an increased incidence of FLT3‐ITD mutations compared to patients with minor bleeding. Hemostatic variables recovered significantly more slowly in patients with major bleeding than in those with minor bleeding. Active induction therapy and blood product infusion are efficient in preventing severe bleedingSummary/Conclusion:Our results suggested that low PLT count might be the leading cause of fatal bleeding in patients newly diagnosed with APL.

The novel dihydroorotate dehydrogenase (DHODH) inhibitor BAY 2402234 triggers differentiation and is effective in the treatment of myeloid malignancies

Acute myeloid leukemia (AML) is a devastating disease, with the majority of patients dying within a year of diagnosis. For patients with relapsed/refractory AML, the prognosis is particularly poor with currently available treatments. Although genetically heterogeneous, AML subtypes share a common differentiation arrest at hematopoietic progenitor stages. Overcoming this differentiation arrest has the potential to improve the long-term survival of patients, as is the case in acute promyelocytic leukemia (APL), which is characterized by a chromosomal translocation involving the retinoic acid receptor alpha gene. Treatment of APL with all-trans retinoic acid (ATRA) induces terminal differentiation and apoptosis of leukemic promyelocytes, resulting in cure rates of over 80%. Unfortunately, similarly efficacious differentiation therapies have, to date, been lacking outside of APL. Inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pyrimidine synthesis pathway, was recently reported to induce differentiation of diverse AML subtypes. In this report we describe the discovery and characterization of BAY 2402234 - a novel, potent, selective and orally bioavailable DHODH inhibitor that shows monotherapy efficacy and differentiation induction across multiple AML subtypes. Herein, we present the preclinical data that led to initiation of a phase I evaluation of this inhibitor in myeloid malignancies.

Acute Promyelocytic Leukemia: A History over 60 Years-From the Most Malignant to the most Curable Form of Acute Leukemia.

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) that is cytogenetically characterized by a balanced reciprocal translocation between chromosomes 15 and 17, which results in the fusion of the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARα) genes. Because patients with APL present a tendency for severe bleeding, often resulting in an early fatal course, APL was historically considered to be one of the most fatal forms of acute leukemia. However, therapeutic advances, including anthracycline- and cytarabine-based chemotherapy, have significantly improved the outcomes of APL patients. Due to the further introduction of all-trans retinoic acid (ATRA) and—more recently—the development of arsenic trioxide (ATO)-containing regimens, APL is currently the most curable form of AML in adults. Treatment with these new agents has introduced the concept of cure through targeted therapy. With the advent of revolutionary ATRA-ATO combination therapies, chemotherapy can now be safely omitted from the treatment of low-risk APL patients. In this article, we review the six-decade history of APL, from its initial characterization to the era of chemotherapy-free ATRA-ATO, a model of cancer-targeted therapy.