RARα Agonist Research Articles

Targeting CNS myeloid infiltrates provides neuroprotection in a progressive multiple sclerosis model

Demyelination and axonal injury in chronic-progressive Multiple Sclerosis (MS) are presumed to be driven by a neurotoxic bystander effect of meningeal-based myeloid infiltrates. There is an unmet clinical need to attenuate disease progression in such forms of CNS-compartmentalized MS. The failure of systemic immune suppressive treatments has highlighted the need for neuroprotective and repair-inducing strategies. Here, we examined whether direct targeting of CNS myeloid cells and modulating their toxicity may prevent irreversible tissue injury in chronic immune-mediated demyelinating disease. To that end, we utilized the experimental autoimmune encephalomyelitis (EAE) model in Biozzi mice, a clinically relevant MS model. We continuously delivered intracerebroventricularly (ICV) a retinoic acid receptor alpha agonist (RARα), as a potent regulator of myeloid cells, in the chronic phase of EAE. We assessed disease severity and performed pathological evaluations, functional analyses of immune cells, and single-cell RNA sequencing on isolated spinal CD11b+ cells. Although initiating treatment in the chronic phase of the disease, the RARα agonist successfully improved clinical outcomes and prevented axonal loss. ICV RARα agonist treatment inhibited pro-inflammatory pathways and shifted CNS myeloid cells toward neuroprotective phenotypes without affecting peripheral infiltrating myeloid cell phenotypes, or peripheral immunity. The treatment regulated cell-death pathways across multiple myeloid cell populations and suppressed apoptosis, resulting in paradoxically marked increased neuroinflammatory infiltrates, consisting mainly of microglia and CNS / border-associated macrophages. This work establishes the notion of bystander neurotoxicity by CNS immune infiltrates in chronic demyelinating disease. Furthermore, it shows that targeting compartmentalized neuroinflammation by selective regulation of CNS myeloid cell toxicity and survival reduces irreversible tissue injury, and may serve as a novel disease-modifying approach.

Editorial Expression of Concern: HDAC2 phosphorylation-dependent Klf5 deacetylation and RARα acetylation induced by RAR agonist switch the transcription regulatory programs of p21 in VSMCs.

Editorial Expression of Concern: HDAC2 phosphorylation-dependent Klf5 deacetylation and RARα acetylation induced by RAR agonist switch the transcription regulatory programs of p21 in VSMCs.

Unleashing a safe and potent pro-senescence anti-tumor strategy

Inducing senescence in tumor cells can stimulate anti-tumor immune responses. In this issue of Cancer Cell, Colucci etal. demonstrate that the combination of the RAR agonist Adapalene with the chemotherapy drug Docetaxel enhances tumor-suppressing senescence and activates an anti-tumor immune response through natural killer cells.

Discovery of novel RARα agonists using pharmacophore-based virtual screening, molecular docking, and molecular dynamics simulation studies.

Nuclear retinoic acid receptors (RARs) are ligand-dependent transcription factors involved in various biological processes, such as embryogenesis, cell proliferation, differentiation, reproduction, and apoptosis. These receptors are regulated by retinoids, i.e., retinoic acid (RA) and its analogs, as receptor agonists. RAR agonists are promising therapeutic agents for the treatment of serious dermatological disorders, including some malignant conditions. By inducing apoptosis, they are able to inhibit the proliferation of diverse cancer cell lines. Also, RAR agonists have recently been identified as therapeutic options for some neurodegenerative diseases. These features make retinoids very attractive molecules for medical purposes. Synthetic selective RAR agonists have several advantages over endogenous ones, but they suffer poor pharmacokinetic properties. These compounds are normally lipophilic acids with unfavorable drug-like features such as poor oral bioavailability. Recently, highly selective, potent, and less toxic RAR agonists with proper lipophilicity, thus, good oral bioavailability have been developed for some therapeutic applications. In the present study, ligand and structure-based virtual screening technique was exploited to introduce some novel RARα agonists. Pharmacokinetic assessment was also performed in silico to suggest those compounds which have optimized drug-like features. Finally, two compounds with the best in silico pharmacological features are proposed as lead molecules for future development of RARα agonists.

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.

Retinoic acid protects against lipopolysaccharide-induced ferroptotic liver injury and iron disorders by regulating Nrf2/HO-1 and RARβ signaling

Acute liver injury (ALI) can progress to severe liver diseases, making its prevention and treatment a focus of research. Retinoic acid (RA) has been shown to have anti-oxidative and iron-regulatory effects on organs. In this study, we investigated the effect of RA on lipopolysaccharide (LPS)-induced ALI in both in vivo and in vitro experiments. We found that RA significantly reduced LPS-induced serum iron and red blood cell-associated disorders, as well as decreased serum ALT and AST levels. RA also reversed the accumulation of non-heme iron and labile iron in LPS-induced mice and hepatocytes by increasing the expression of FTL/H and Fpn. Furthermore, RA inhibited tissue reactive oxygen species (ROS) and malondialdehyde (MDA) production and improved the expression of Nrf2/HO-1/GPX4 in mice and Nrf2 signaling in hepatocytes. In vitro experiments employing RAR agonists and antagonists have revealed that retinoic acid (RA) can effectively inhibit cell ferroptosis induced by lipopolysaccharide (LPS), erastin, and RSL3. The mechanism underlying this inhibition may involve the activation of retinoic acid receptors beta (RARβ) and gamma (RARγ). Knocking down the RARβ gene in Hepatocytes cells significantly diminished the RA's protective effect, indicating that the anti-ferroptotic role of RA was partially mediated by RARβ signaling. Overall, our study demonstrated that RA inhibited ferroptosis-induced liver damage by regulating Nrf2/HO-1/GPX4 and RARβ signaling.

5-Azacytidine- and retinoic-acid-induced reprogramming of DCCs into dormancy suppresses metastasis via restored TGF-β-SMAD4 signaling

Disseminated cancer cells (DCCs) in secondary organs can remain dormant for years to decades before reactivating into overt metastasis. Microenvironmental signals leading to cancer cell chromatin remodeling and transcriptional reprogramming appear to control onset and escape from dormancy. Here, we reveal that the therapeutic combination of the DNA methylation inhibitor 5-azacytidine (AZA) and the retinoic acid receptor ligands all-trans retinoic acid (atRA) or AM80, an RARα-specific agonist, promotes stable dormancy in cancer cells. Treatment of head and neck squamous cell carcinoma (HNSCC) or breast cancer cells with AZA+atRA induces a SMAD2/3/4-dependent transcriptional program that restores transforming growth factor β (TGF-β)-signaling and anti-proliferative function. Significantly, either combination, AZA+atRA or AZA+AM80, strongly suppresses HNSCC lung metastasis formation by inducing and maintaining solitary DCCs in a SMAD4+/NR2F1+ non-proliferative state. Notably, SMAD4 knockdown is sufficient to drive resistance to AZA+atRA-induced dormancy. We conclude that therapeutic doses of AZA and RAR agonists may induce and/or maintain dormancy and significantly limit metastasis development.

All-Trans Retinoic Acid-Responsive LGR6 Is Transiently Expressed during Myogenic Differentiation and Is Required for Myoblast Differentiation and Fusion.

All-trans retinoic acid (ATRA) promotes myoblast differentiation into myotubes. Leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6) is a candidate ATRA-responsive gene; however, its role in skeletal muscles remains unclear. Here, we demonstrated that during the differentiation of murine C2C12 myoblasts into myotubes, Lgr6 mRNA expression transiently increased before the increase in the expression of the mRNAs encoding myogenic regulatory factors, such as myogenin, myomaker, and myomerger. The loss of LGR6 decreased the differentiation and fusion indices. The exogenous expression of LGR6 up to 3 and 24 h after the induction of differentiation increased and decreased the mRNA levels of myogenin, myomaker, and myomerger, respectively. Lgr6 mRNA was transiently expressed after myogenic differentiation in the presence of a retinoic acid receptor α (RARα) agonist and an RARγ agonist in addition to ATRA, but not in the absence of ATRA. Furthermore, a proteasome inhibitor or Znfr3 knockdown increased exogenous LGR6 expression. The loss of LGR6 attenuated the Wnt/β-catenin signaling activity induced by Wnt3a alone or in combination with Wnt3a and R-spondin 2. These results indicate that LGR6 promotes myogenic differentiation and that ATRA is required for the transient expression of LGR6 during differentiation. Furthermore, LGR6 expression appeared to be downregulated by the ubiquitin-proteasome system involving ZNRF3.

Effects of retinoic acid receptor α modulators on developmental ethanol-induced neurodegeneration and neuroinflammation.

Ethanol exposure in neonatal mice induces acute neurodegeneration followed by long-lasting glial activation and GABAergic cell deficits along with behavioral abnormalities, providing a third trimester model of fetal alcohol spectrum disorders (FASD). Retinoic acid (RA), the active form of vitamin A, regulates transcription of RA-responsive genes and plays essential roles in the development of embryos and their CNS. Ethanol has been shown to disturb RA metabolism and signaling in the developing brain, which may be a cause of ethanol toxicity leading to FASD. Using an agonist and an antagonist specific to RA receptor α (RARα), we studied how RA/RARα signaling affects acute and long-lasting neurodegeneration and activation of phagocytic cells and astrocytes caused by ethanol administered to neonatal mice. We found that an RARα antagonist (BT382) administered 30 min before ethanol injection into postnatal day 7 (P7) mice partially blocked acute neurodegeneration as well as elevation of CD68-positive phagocytic cells in the same brain area. While an RARα agonist (BT75) did not affect acute neurodegeneration, BT75 given either before or after ethanol administration ameliorated long-lasting astrocyte activation and GABAergic cell deficits in certain brain regions. Our studies using Nkx2.1-Cre;Ai9 mice, in which major GABAergic neurons and their progenitors in the cortex and the hippocampus are labeled with constitutively expressed tdTomato fluorescent protein, indicate that the long-lasting GABAergic cell deficits are mainly caused by P7 ethanol-induced initial neurodegeneration. However, the partial reduction of prolonged GABAergic cell deficits and glial activation by post-ethanol BT75 treatment suggests that, in addition to the initial cell death, there may be delayed cell death or disturbed development of GABAergic cells, which is partially rescued by BT75. Since RARα agonists including BT75 have been shown to exert anti-inflammatory effects, BT75 may rescue GABAergic cell deficits by reducing glial activation/neuroinflammation.

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.

Anti-inflammatory Action of BT75, a Novel RARα Agonist, in Cultured Microglia and in an Experimental Mouse Model of Alzheimer's Disease.

BT75, a boron-containing retinoid, is a novel retinoic acid receptor (RAR)α agonist synthesized by our group. Previous studies indicated that activation of retinoic acid (RA) signaling may attenuate progression of Alzheimer's disease (AD). Presently, we aimed to examine the anti-inflammatory effect of BT75 and explore the possible mechanism using cultured cells and an AD mouse model. Pretreatment with BT75 (1-25 µM) suppressed the release of nitric oxide (NO) and IL-1β in the culture medium of mouse microglial SIM-A9 cells activated by LPS. BMS195614, an RARα antagonist, partially blocked the inhibition of NO production by BT75. Moreover, BT75 attenuated phospho-Akt and phospho-NF-κB p65 expression augmented by LPS. In addition, BT75 elevated arginase 1, IL-10, and CD206, and inhibited inducible nitric oxide synthase (iNOS) and IL-6 formation in LPS-treated SIM-A9 cells, suggesting the promotion of M1-M2 microglial phenotypic polarization. C57BL/6 mice were injected intracerebroventricularly (icv) with streptozotocin (STZ) (3mg/kg) to provide an AD-like mouse model. BT75 (5mg/kg) or the vehicle was intraperitoneally (ip) injected to icv-STZ mice once a day for 3 weeks. Immunohistochemical analyses indicated that GFAP-positive cells and rod or amoeboid-like Iba1-positive cells, which increased in the hippocampal fimbria of icv-STZ mice, were reduced by BT75 treatment. Western blot results showed that BT75 decreased levels of neuronal nitric oxide synthase (nNOS), GFAP, and phosphorylated Tau, and increased levels of synaptophysin in the hippocampus of icv-STZ mice. BT75 may attenuate neuroinflammation by affecting the Akt/NF-κB pathway and microglial M1-M2 polarization in LPS-stimulated SIM-A9 cells. BT75 also reduced AD-like pathology including glial activation in the icv-STZ mice. Thus, BT75 may be a promising anti-inflammatory and neuroprotective agent worthy of further AD studies.

Initial Results from SELECT-AML-1, a Phase 2 Study of Tamibarotene in Combination with Venetoclax and Azacitidine in RARA-Positive Newly Diagnosed AML Patients Ineligible for Standard Induction Chemotherapy

RARA-positive AML is a novel genomically-defined subset with an actionable target for treatment with tamibarotene, an oral and selective RARα agonist (McKeown, Cancer Discovery 2017). Approximately 30% of non-APL AML patients are RARA-positive based on overexpression of the RARA gene in peripheral blood blasts as determined by an RT-PCR based assay (de Botton, ESH 2019). In patients with newly diagnosed (ND) AML ineligible for standard induction chemotherapy, tamibarotene/azacitidine led to a CR/CRi rate of 61% in RARA-positive patients with a rapid onset of response. The combination was well-tolerated with no increase in myelosuppression compared to azacitidine (aza) alone (de Botton, ASH 2020). Responses were observed irrespective of mutations or cytogenetic risk. Approximately one-third of ND AML patients ineligible for standard induction therapy do not respond to venetoclax (ven) and nearly all eventually relapse (DiNardo, NEJM 2020). Translational data suggest that RARA-positive patients are enriched for monocytic features based on a positive Monocytic Expression Score (MES) that may be associated with ven resistance (Fiore, ASH 2020), suggesting that the blood-based RARA biomarker test selects for patients who may respond to tamibarotene and who may also be less likely to respond to ven/aza alone. SELECT-AML-1 (NCT04905407) is a Phase 2, open-label, multi-center study in the US and France comparing the activity of tamibarotene/ven/aza to ven/aza in treatment-naive RARA-positive ND AML patients ineligible for standard induction therapy. The 3-part study includes a safety lead-in of the tamibarotene/ven/aza combination, a randomized efficacy study versus ven/aza, and a salvage arm for ven/aza only patients with primary endpoints of safety, CR/CRi rate, and overall response rate, respectively. Following the safety lead-in, patients will be randomized 1:1 to receive tamibarotene/ven/aza or ven/aza. In the salvage arm, patients randomized to ven/aza only who experience progressive disease, treatment failure, or relapse, will have tamibarotene added to their ven/aza regimen. Patients currently enrolled in the safety lead-in are treated with starting doses of aza at 75 mg/m2 IV/SC daily on Days 1-7, ven on Days 1-28 per the ven USPI/SMPC, followed by tamibarotene at 6 mg twice per day PO on Days 8-28 of each 28-day cycle. Enrollment is ongoing. As of 22 June 2022, six patients have enrolled to date, including four who are response-evaluable. Baseline characteristics of the response-evaluable patients include 75% male; median age 57 (55-74); median bone marrow blasts 55% (39-100%); cytogenetically normal (n=2), inversion 16 (n=1), del 5q and -7 (n=1); and molecular genetic testing normal (n=3), mutations of IDH2, BCOR, SRSF2, CSF3R, RUNX1 (n=1). The MES was high in 3 of 4 (75%) response evaluable patients consistent with a prior report in RARA-positive AML (Fiore, ASH 2020). All 4 response-evaluable patients achieved a best response of CR/CRi (2 CR, 2 CRi). The onset of response was rapid with all patients achieving an initial response after Cycle 1 (2 CR, 1 CRi, 1 MLFS). Median duration of treatment was 68 days (21-110). Two patients discontinued, both in remission, one due to death from neutropenic sepsis and the other by physician decision. The adverse event (AE) profile of the triplet was consistent with reports of ven/aza in AML. AEs experienced by 2 or more patients (all grades/causality) included febrile neutropenia, pneumonia, anxiety, cough, diarrhea, fatigue, decreased appetite, myalgia, decreased neutrophil count, and decreased platelet count. The only serious AE (all causality) occurring in more than one patient was febrile neutropenia. A high response rate was observed in early results of tamibarotene/ven/aza in RARA-positive ND AML patients ineligible for standard induction chemotherapy with 100% (4 of 4) response evaluable patients achieving CR/CRi, 3 of whom may have features of ven-resistance based on high MES. The triplet demonstrated an initial safety profile similar to ven/aza. Data from the safety lead-in will inform triplet dosing for the randomized portion of the study.

AML-108 SELECT-AML-1 Trial in Progress: A Phase 2 Study of Tamibarotene in Combination With Venetoclax and Azacitidine in Previously Untreated Adult Patients Selected for RARA-Positive AML who are Ineligible for Standard Induction Therapy

RARA-positive AML is a novel, genomically defined subset with an actionable target for treatment with tamibarotene, an oral and selective RARα agonist (McKeown 2017). In a previous trial in RARA-positive newly diagnosed (ND) AML patients ineligible for standard induction therapy, tamibarotene/azacitidine led to a CR/CRi rate of 61% with a rapid onset of response. The combination was generally well tolerated with no increase in myelosuppression compared to azacitidine alone (de Botton 2020). Responses were observed irrespective of mutations or cytogenetic risk. Approximately one-third of ND unfit AML patients do not respond to venetoclax, and nearly all patients eventually relapse (DiNardo 2020). Translational data suggest that RARA positivity, found in approximately 30% of patients in this AML subset (Vigil 2017), enriches for monocytic features reported to be associated with venetoclax resistance (Fiore 2020, Pei 2019). This suggests that the blood-based RARA biomarker selects for patients who may respond to tamibarotene and may be less likely to respond to venetoclax/azacitidine. The primary objectives are to characterize the safety of the combination and to compare the CR/CRi rate of tamibarotene/venetoclax/azacitidine versus venetoclax/azacitidine; the secondary objectives are to compare the CR rate, CR/CRh rate, duration of response, time to response, and overall survival. A phase 2, open-label, multi-center trial comparing the activity of tamibarotene/venetoclax/azacitidine with that of venetoclax/azacitidine in treatment-naive RARA+ unfit AML patients. This 3-part trial includes a safety lead-in, randomized efficacy study, and salvage arm. Following the safety lead-in, patients will be randomized 1:1 to receive tamibarotene/venetoclax/azacitidine or venetoclax/azacitidine. Clinical activity will be characterized by European LeukemiaNet criteria. Response rates and 95% exact binomial confidence intervals will be calculated by treatment group. In the salvage arm, tamibarotene will be added for patients randomized to venetoclax/azacitidine who experience progressive disease, treatment failure, or relapse. Patients will be treated with azacitidine at 75 mg/m2 IV/SC daily on days 1-7, venetoclax on days 1-28 per VENCLEXTA® (venetoclax) USPI, and tamibarotene 6 mg twice per day by mouth on days 8-28 of each 28-day cycle. SELECT-AML-1 (NCT04905407) opened in July 2021 with ongoing enrollment. U.S. and France.

MDS-110 SELECT-MDS-1 Trial in Progress: A Phase 3 Randomized, Double-Blind, Placebo-Controlled Study of Tamibarotene/Azacitidine Versus Placebo/Azacitidine in Newly Diagnosed Adult Patients Selected for RARA-Positive Higher-Risk MDS

A novel genomically defined subset of higher-risk myelodysplastic syndrome (HR-MDS) patients with an actionable target characterized by overexpression of RARA has been identified (McKeown 2017). Approximately 30% of HR-MDS patients are RARA-positive by a blood-based biomarker test (Vigil 2017). Tamibarotene is an oral selective RARα agonist with potential for clinical benefit in RARA-positive HR-MDS patients, irrespective of mutation or cytogenetic risk. Initial clinical data in RARA-positive relapsed/refractory HR-MDS showed myeloid differentiation, improved blood counts, and reduced bone marrow blasts, including one patient who achieved marrow complete remission with hematologic improvement (Jurcic 2017). Tamibarotene/azacitidine led to a CR/CRi rate of 61% with rapid onset of response in RARA-positive newly diagnosed (ND) unfit AML patients, including those with low blast count (≤30%) AML, and a majority of patients achieved or maintained transfusion independence (de Botton 2020). Tamibarotene/azacitidine was generally well tolerated with no increase in myelosuppression compared with azacitidine alone (de Botton 2020). Historical precedent demonstrating similar clinical outcomes in HR-MDS and low blast count AML (Estey 2022) supports further development of tamibarotene/azacitidine in HR-MDS to improve clinical outcomes of standard of care treatment with hypomethylating agents (HMAs). To characterize and compare the CR rate of tamibarotene/azacitidine to placebo/azacitidine in RARA-positive ND HR-MDS patients. Secondary objectives include comparisons of overall response rate, event-free survival, overall survival, transfusion independence, and safety. Global, Phase 3, randomized, double-blind, placebo-controlled trial (NCT04797780). Approximately 190 patients will be randomized 2:1, providing 90% power to detect the difference in CR rates between the experimental and control arms. The included patients will be RARA-positive based on investigational assay and ND with HR-MDS by WHO classification (Arber 2016) with an IPSS-R risk category of very high, high, or intermediate and a blast count >5% at baseline. Patients with prior treatment for MDS with any HMA, chemotherapy, or transplant are excluded. Azacitidine will be administered at 75 mg/m2 IV/SC daily on days 1-7 (or 1-5, 8-9) followed by tamibarotene/placebo at 6 mg BID orally on days 8-28 of each 28-day cycle. Response is assessed per the modified IWG MDS criteria (Cheson 2006).

Retinoic Acid Receptor Activation Reduces Metastatic Prostate Cancer Bone Lesions by Blocking the Endothelial-to-Osteoblast Transition.

This study provides mechanistic insights into how RAR agonists suppress prostate cancer-induced bone formation and offers a rationale for developing RAR agonists for prostate cancer bone metastasis therapy. See related commentary by Bhowmick and Bhowmick, p. 2975.

Dissecting super-enhancer heterogeneity: time to re-examine cancer subtypes?

The heterogeneity of transcriptional regulations by super-enhancers (SEs) is poorly understood in human cancers. Herein, we summarize a bioinformatics workflow for genome-wide SE profiling and identification of subtype-specific SEs and regulatory networks. Dissecting SE heterogeneity provides new insights into cancer biology and alternative therapeutic strategies for cancer precision medicine.

S290: ATRA CAN CORRECT DEFECTIVE HIF-1Α/S1P AXIS-MEDIATED CYTOSKELETAL REORGANIZATION IN PROPLATELET FORMATION OF ITP

Background: Bone marrow physiological hypoxia plays a crucial role in haematopoietic stem cell homeostasis. Hypoxia inducible factor (HIF) is central to mediating the cellular response to hypoxia. HIF-1α expression was decreased in the bone marrow of patients with immune thrombocytopenia (ITP), and HIF-1α activation was shown to enhance megakaryopoiesis in mice. Recent studies suggest that “inside-out” signalling by S1P in megakaryocytes (MKs) plays a critical role in proplatelet formation (PPF) (Blood, 2013; J EXP MED, 2012). Our previous data indicated that impaired PPF contributed to the development of thrombocytopenia in ITP. To further explore the underlying mechanism of impaired PPF in ITP, we found that HIF-1α/S1P axis-mediated cytoskeletal reorganization was defective in the PPF of ITP. All-trans retinoic acid (ATRA), which has been shown to be a promising treatment option for ITP patients in our clinical studies (Blood, 2021; Lancet haematology, 2017; Lancet haematology, 2021), could restore cytoskeletal reorganization and correct impaired PPF. Aims: This study aimed to explore the role of hypoxia inducible factor-1α (HIF-1α) in proplatelet formation (PPF) and the underlying mechanisms of ATRA treatment in ITP patients. Methods: Thirty consecutive patients with newly diagnosed ITP and 30 healthy donors were included in our study. MKs were isolated from bone marrow samples. Targeted and untargeted metabolomic profiling through metabolomic analysis was performed to explore the relationship between the metabolome and ITP. Confocal microscopy and transmission electron microscopy were used to observe the PPF and cytoskeleton structure of ITP MKs. An ITP mouse model was established to observe the therapeutic effects of ATRA in the PPF. Results: In the present study, we observed that MKs displayed altered cytoskeletal reorganization and impaired proplatelet formation (PPF) in ITP patients. Targeted and untargeted metabolite profiling revealed a decreased sphingosine-1-phosphate (S1P) level in ITP. Downregulated sphingosine kinase 2 (SPHK2) expression in MKs accounted for the low level of S1P in ITP. S1P is essential for S1P receptor 1 (S1PR1) and Rac1 activation, Src family kinases (SFKs) activity, and subsequent cytoskeletal reorganization and PPF regulation. Moreover, we demonstrated that HIF-1α mediated SPHK2 activation and S1P production. Decreased HIF-1α levels were found in the MKs of patients with ITP, contributing to impaired PPF. We then investigated the effect of ATRA on PPF in ITP patients. ATRA upregulated HIF-1α and SPHK2 expression, increased S1P production and corrected impaired PPF in vitro. In an ITP mouse model, ATRA alleviated thrombocytopenia and restored cytoskeletal reorganization. ATRA corrected impaired PPF by upregulating HIF-1α expression. The exposure of ITP MKs to selective RARα (AM580) or RARγ (BMS961) agonists did not change PPF. However, the treatment of ITP MKs with a selective RARβ agonist (CD2314) significantly increased PPF. Furthermore, we found that the effect of ATRA on enhancing PPF in ITP MKs was reversed in the presence of an RARβ antagonist (CD2665). These data suggest that impaired PPF in ITP MKs is corrected by ATRA in a RARβ-dependent manner. Summary/Conclusion: Together, our data show that the HIF-1α/S1P axis mediates altered cytoskeletal reorganization and impaired PPF in ITP and suggest that ATRA correction of impaired PPF is a potential mechanistic explanation for the clinical efficacy of ATRA in ITP.

PB1921: A RANDOMIZED, DOUBLE-BLIND, PHASE 3 STUDY OF TAMIBAROTENE/AZACITIDINE VERSUS PLACEBO/AZACITIDINE IN NEWLY DIAGNOSED ADULT PATIENTS SELECTED FOR RARA-POSITIVE HR-MDS (SELECT-MDS-1)

Background: A novel genomically defined subset of higher-risk MDS (HR-MDS) patients with an actionable target characterized by overexpression of RARA has been identified (McKeown 2017). Approximately 30% of HR-MDS patients are RARA-positive (RARA+) by a blood-based biomarker test (Vigil, 2017). This RARA positivity enables biologically targeted therapy with tamibarotene (formerly SY-1425), an oral selective RARα agonist with potential to provide clinical benefit for HR-MDS patients, irrespective of mutation or cytogenetic risk. Initial Phase 2 clinical data in RARA+ relapsed/refractory HR-MDS patients treated with single agent tamibarotene showed myeloid differentiation, improved blood counts, and reduced bone marrow blasts, including one patient who achieved marrow complete remission with hematologic improvement (Jurcic 2017). Tamibarotene in combination with azacitidine (aza) demonstrated high complete response rates with rapid onset of response in RARA+ newly diagnosed (ND) unfit AML patients, including those with low blast count (≤ 30%) AML, and a majority of patients achieved or maintained transfusion independence (de Botton 2020). Tamibarotene plus aza was generally well tolerated with no increase in myelosuppression compared to single agent aza (de Botton 2020). HR-MDS and AML have common cytogenetic abnormalities and gene mutations present in the blast populations in each condition. The historical precedent demonstrating similar clinical outcomes among patients with HR-MDS and AML, particularly low blast count AML (Estey 2021), supports further development of the combination of tamibarotene plus aza in the HR-MDS population, with the opportunity to improve upon the clinical outcomes of standard of care treatment with hypomethylating agents (HMAs). Aims: This is a global Phase 3, multi-center, randomized, double-blind, placebo-controlled study comparing the activity of tamibarotene plus aza, to placebo plus aza in RARA+ patients with ND HR-MDS. The primary objective is to compare the complete remission rate of tamibarotene plus aza vs. placebo plus aza, with secondary objectives to compare objective response rate, event-free survival, overall survival, transfusion independence rate, time to and duration of initial and complete responses, and safety. Methods: Patients must be RARA+ based on the investigational biomarker test, ND with HR-MDS by WHO classification (Arber 2016), classified by IPSS-R risk category as very high, high, or intermediate risk with a bone marrow blast count > 5% at study entry. Patients suitable for allogeneic HSCT at the time of screening, or who received prior treatment for MDS with any HMA, chemotherapy or allogeneic HSCT are excluded. Aza will be administered at 75 mg/m2 IV/SC daily on days 1-7 (or days 1-5, 8-9) followed by tamibarotene/placebo at 6 mg BID orally days 8-28 of each 28-day cycle. Response will be assessed per the modified IWG MDS criteria (Cheson 2006). Results: Approximately 190 patients will be randomized 2:1 and stratified by IPSS-R risk group and geographic region, providing 90% power to detect the difference in CR rates between the experimental and control arms, respectively, with one-sided alpha of 0.025. Summary/Conclusion: The SELECT-MDS-1 trial opened to enrollment in February 2021, recruitment is ongoing, with sites located in North America, Israel, and Europe (NCT04797780).

P492: TAMIBAROTENE IN COMBINATION WITH VENETOCLAX AND AZACITIDINE IN PREVIOUSLY UNTREATED ADULT PATIENTS SELECTED FOR RARA-POSITIVE AML WHO ARE INELIGIBLE FOR STANDARD INDUCTION THERAPY (SELECT AML-1)

Background: RARA-positive (RARA+) AML is a novel genomically defined patient subset with an actionable biological target for treatment with tamibarotene, an oral and selective RARα agonist (McKeown 2017). RARA+ patients can be selected by a blood-based biomarker test, with approximately 30% of newly diagnosed (ND) AML patients being RARA+ (Vigil 2017). As a biologically targeted agent for patients with RARA overexpression, tamibarotene has the potential to provide benefit irrespective of mutation or cytogenetic risk classification. In RARA+ ND AML patients ineligible for standard induction therapy, tamibarotene plus azacitidine (aza) led to a CR/CRi rate of 61% and a rapid onset of response (de Botton 2020). Approximately one-third of patients with ND unfit AML do not respond to front-line standard of care venetoclax (ven)/aza (DiNardo 2020). Translational data suggest RARA positivity enriches for monocytic features reported to be associated with ven resistance (Fiore 2020, Pei 2020). This data suggests the RARA biomarker selects for patients who may respond to tamibarotene and may be less likely to respond to ven/aza. Given that tamibarotene plus aza has been generally well tolerated, with no increase in myelosuppression compared to single agent aza (de Botton 2020), it is anticipated that tamibarotene can be administered safely in combination with ven/aza. Aims: This is a Phase 2, open-label, multi-center study in the U.S. and France comparing the clinical activity of tamibarotene/ven/aza to ven/aza in treatment-naive RARA+ AML patients ineligible for standard induction chemotherapy. The primary objectives are to characterize the safety of the combination and to compare the CR/CRi rate of tamibarotene/ven/aza vs. ven/aza, with secondary objectives to compare CR rate, CR/CRh rate, duration of response, and time to response. The overall response rate using tamibarotene/ven/aza following ven/aza treatment failure will be explored. Clinical activity will be characterized by ELN criteria (Dohner 2017). Methods: This 3-part trial includes a safety lead-in, randomized efficacy study, and salvage arm. Following the safety lead-in, approximately 80 patients will be randomized 1:1 to receive tamibarotene/ven/aza or ven/aza. In the salvage arm, tamibarotene will be added for study patients randomized to ven/aza who experience progressive disease, relapse, or treatment failure. Patients will be treated with aza at 75 mg/m2 IV/SC daily on days 1-7, ven on days 1-28 per VENCLEXA USPI, followed by tamibarotene at 6 mg twice per day by mouth on days 8-28 of each 28-day cycle. Results: Response rates and 95% exact binomial confidence intervals will be calculated by treatment group. Summary/Conclusion: The SELECT AML-1 trial (NCT04905407) opened in July 2021 with ongoing enrollment.

Tamibarotene in combination with venetoclax and azacitidine in previously untreated adult patients selected for RARA-positive AML who are ineligible for standard induction therapy (SELECT AML-1).

TPS7065 Background: RARA-positive (RARA+) AML is a novel genomically defined patient subset with an actionable biological target for treatment with tamibarotene, an oral and selective RARα agonist (McKeown 2017). RARA+ patients can be selected by a blood-based biomarker test, with approximately 30% of newly diagnosed (ND) AML patients being RARA+ (Vigil 2017). As a biologically targeted agent for patients with RARA overexpression, tamibarotene has the potential to provide benefit irrespective of mutation or cytogenetic risk classification. In RARA+ ND AML patients ineligible for standard induction therapy, tamibarotene plus azacitidine (aza) led to a CR/CRi rate of 61% and a rapid onset of response (de Botton 2020). Approximately one-third of patients with ND unfit AML do not respond to front-line standard of care venetoclax (ven)/aza (DiNardo 2020). Translational data suggest RARA positivity enriches for monocytic features reported to be associated with ven resistance (Fiore 2020, Pei 2020). This data suggests the RARA biomarker selects for patients who may respond to tamibarotene and may be less likely to respond to ven/aza. Given that tamibarotene plus aza has been generally well tolerated, with no increase in myelosuppression compared to single agent aza (de Botton 2020), it is anticipated that tamibarotene can be administered safely in combination with ven/aza. Methods: This is a Phase 2, open-label, multi-center study in the U.S. and France comparing the clinical activity of tamibarotene/ven/aza to ven/aza in treatment-naive RARA+ AML patients ineligible for standard induction chemotherapy. The primary objectives are to characterize the safety of the combination and to compare the CR/CRi rate of tamibarotene/ven/aza vs. ven/aza, with secondary objectives to compare CR rate, CR/CRh rate, duration of response, and time to response. The overall response rate using tamibarotene/ven/aza following ven/aza treatment failure will be explored. Clinical activity will be characterized by ELN criteria (Dohner 2017). This 3-part trial includes a safety lead-in, randomized efficacy study, and salvage arm. Following the safety lead-in, approximately 80 patients will be randomized 1:1 to receive tamibarotene/ven/aza or ven/aza. Response rates and 95% exact binomial confidence intervals will be calculated by treatment group. In the salvage arm, tamibarotene will be added for study patients randomized to ven/aza who experience progressive disease, relapse, or treatment failure. Patients will be treated with aza at 75 mg/m2 IV/SC daily on days 1-7, ven on days 1-28 per VENCLEXA USPI, followed by tamibarotene at 6 mg twice per day by mouth on days 8-28 of each 28-day cycle. The SELECT AML-1 trial opened in July 2021 with ongoing enrollment. Clinical trial information: NCT04905407.