All-trans retinoic acid (ATRA), a bioactive vitamin A derivative, regulates cellular differentiation by activating retinoic acid receptors (RARs). While ATRA has revolutionised acute promyelocytic leukaemia (APL) treatment, its efficacy in non-APL acute myeloid leukaemia (AML) remains limited due to intrinsic resistance mechanisms, including aberrant epigenetic states and signalling pathways. This review summarises recent mechanistic and translational advances in ATRA-based combination therapies that target differentiation blockade. We focus on the interplay between retinoic acid signalling, chromatin regulation, cell cycle control, and apoptosis, with a particular emphasis on AML. Epigenetic regulators, including the lysine-specific demethylase LSD1 (KDM1A) and the acetyltransferase GCN5 (KAT2A), together with dysregulated cell cycle control and oncogenic signalling, including Fms-like tyrosine kinase 3 (FLT3) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), sustain proliferation and block differentiation. Targeting these pathways with specific inhibitors synergises with ATRA to restore myeloid maturation, induce cell cycle arrest, and promote apoptosis. Future studies should identify predictive biomarkers for patient stratification to translate these synergistic concepts into clinical benefit.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by diverse genetic abnormalities. The standard of care remains to be chemotherapy and stem cell transplantation. In acute promyelocytic leukaemia (APL), differentiation therapy with all-trans retinoic acid (ATRA) has significantly improved outcomes. Despite this, the success of ATRA has yet to be transferred to non-APL AML. Exploring combinations to enhance the efficacy of ATRA in non-APL AML remains a key focus. To investigate the therapeutic effect of ATRA in combination with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors in non-APL AML. Non-APL AML cell lines and primary patient samples were treated with ATRA and CDK4/6 inhibitors. Key outcomes included differentiation, proliferation, cell viability and colony-forming capacity. Combination synergy was evaluated, and gene expression analysis identified pathways associated with therapeutic effects. The combination demonstrated dose-dependent effects, enhancing differentiation and reducing proliferation, cell viability and colony-forming capacity. A synergistic effect was observed across AML cell lines. Gene expression profiling revealed the co-regulation of differentiation-associated genes, unveiling the mechanisms driving therapeutic synergy. Combination of CDK4/6 inhibitors with ATRA shows potential for differentiation-based AML treatment. This approach offers a promising avenue for improved outcomes in non-APL AML.

Supplementary Table S2. List of genes with expression regulated by selamectin treatment

All-trans-retinoic acid (ATRA)-based differentiation therapy of acute promyelocytic leukemia (APL) represents one of the most clinically effective examples of precision medicine and the first example of targeted oncoprotein degradation. The success of ATRA in APL, however, remains to be translated to non-APL acute myeloid leukemia (AML). We previously showed that aberrant histone modifications, including histone H3 lysine 4 (H3K4) and lysine 27 (H3K27) methylation, were associated with this lack of response and that epigenetic therapy with small molecule inhibitors of the H3K4 demethylase LSD1/KDM1A could reprogram AML cells to respond to ATRA. Serving as the enzymatic component of Polycomb Repressive Complex 2, EZH2/KMT6A methyltransferase plays a critical role in normal hematopoiesis by affecting the balance between self-renewal and differentiation. The canonical function of EZH2 is methylation of H3K27, although important non-canonical roles have recently been described. EZH2 mutation or deregulated expression has been conclusively demonstrated in the pathogenesis of AML and response to treatment, thus making it an attractive therapeutic target. In this study, we therefore investigated whether inhibition of EZH2 might also improve the response of non-APL AML cells to ATRA-based therapy. We focused on GSK-343, a pyridone-containing S-adenosyl-L-methionine cofactor-competitive EZH2 inhibitor that is representative of its class, and HKMTI-1-005, a substrate-competitive dual inhibitor targeting EZH2 and the closely related G9A/GLP H3K9 methyltransferases. We found that treatment with HKMTI-1-005 phenocopied EZH2 knockdown and was more effective in inducing differentiation than GSK-343, despite the efficacy of GSK-343 in terms of abolishing H3K27 trimethylation. Furthermore, transcriptomic analysis revealed that in contrast to treatment with GSK-343, HKMTI-1-005 upregulated the expression of differentiation pathway genes with and without ATRA, while downregulating genes associated with a hematopoietic stem cell phenotype. These results pointed to a non-canonical role for EZH2, which was supported by the finding that EZH2 associates with the master regulator of myeloid differentiation, RARα, in an ATRA-dependent manner that was enhanced by HKMTI-1-005, possibly playing a role in co-regulator complex exchange during transcriptional activation. In summary, our results strongly suggest that addition of HKMTI-1-005 to ATRA is a new therapeutic approach against AML that warrants further investigation.

Supplementary Table S1 and Fig S1 and S2. Supplementary Table 1: qPCR primers used in this study; Supplementary Figure S1: Structures of compounds screened in Figure 1A and 1B; Supplementary Figure S2: Proximity ligation assay (PLA) analyzing SIN3A-MAD interactions

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

Supplementary Figure S3. Effect of ATRA-TCP treatment on AML cell lines.

Figure S4. ATRA-TCP induces the expression of a RA-responsive gene signature in patient responders.

Supplementary Table S3. Ingenuity Canonical Pathways modulated by selamectin treatment

Supplementary Figure S2. Differences in gene expression in patient samples between responders and non-responders.

All-trans-retinoic acid (ATRA) plays critical regulatory roles in normal haematopoiesis and the pathogenesis of adult and pediatric acute myeloid leukemia (AML). While ATRA can inhibit growth and stimulates myeloid differentiation via RARα, it is equally potent in causing expansion of haematopoietic stem cells via RARγ. RARG mRNA is expressed in AML patients and normal stem/progenitor cells but not in more mature myeloid cells. Changes in RARγ expression are paralleled by a reciprocal change in expression of RARG 3'-targeting miRNAs (mIR-24, mIR-30a, mIR-331). RARγ protein and RARG mRNA are also expressed in cell lines derived from primary AML samples but not in ATRA-responsive AML cell lines. Lastly, expression of mIR-30a in TEX cells promotes differentiation and inhibits proliferation. Our results suggest that miRNA-mediated down-regulation of RARγ expression in the myeloid lineage switches ATRA responsiveness from RARγ-mediated pro-proliferation to RARα-mediated pro-differentiation and that the combinatorial use of RARα and RARγ selective agonists and antagonists, respectively, could be effective in retinoid-based differentiation therapy of AML.

Animal research undoubtedly provides scientists with virtually unlimited data but inflicts pain and suffering on animals. Currently, legislators and scientists alike are promoting alternative in vitro approaches allowing for an accurate evaluation of processes occurring in the body without animal sacrifice. Historically, one of the most infamous animal tests is the Draize test, mainly performed on rabbits. Even though this test was considered the gold standard for around 50 years, the Draize test fails to mimic human response mainly due to human and rabbit eye physiological differences. Therefore, many alternative assays were developed to evaluate ocular toxicity and drug effectiveness accurately. Here we review recent achievements in tissue engineering of in vitro 2D, 2.5D, 3D, organoid and organ-on-chip ocular models, as well as in vivo and ex vivo models in terms of their advantages and limitations.

Aberrant activity of the phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR [PAM]) pathway, as well as suppressed retinoic acid signalling, contribute to enhanced proliferation and the differentiation blockade of immature myeloid cells in acute myeloid leukaemia (AML). Inhibition of the PAM pathway was shown to affect especially mixed-lineage leukaemia-rearranged AML. Here, we sought to test a combined strategy using small molecule inhibitors against members of the PAM signalling pathway in conjunction with all-trans retinoic acid (ATRA) to target a larger group of different AML subtypes. We find that ATRA treatment in combination with inhibition of PI3K (ZSTK474), mTOR (WYE132) or PI3K/mTOR (BEZ235, dactolisib) drastically reduces protein levels of the proto-oncogene MYC. In combination with BEZ235, ATRA treatment led to almost complete eradication of cellular MYC, G1 arrest, loss of clonal capacity and terminal granulocytic differentiation. We demonstrate that PAM inhibitor/ATRA treatment targets MYC via independent mechanisms. While inhibition of the PAM pathway causes MYC phosphorylation at threonine 58 via glycogen synthase kinase 3 beta and subsequent degradation, ATRA reduces its expression. Here, we present an approach using a combination of known drugs to synergistically reduce aberrant MYC levels, thereby effectively blocking proliferation and enabling differentiation in various AML subtypes.

The retinoids are a group of compounds including vitamin A and its active metabolite all-trans-retinoic acid (ATRA). Retinoids regulate a variety of physiological functions in multiple organ systems, are essential for normal immune competence, and are involved in the regulation of cell growth and differentiation. Vitamin A derivatives have held promise in cancer treatment and ATRA is used in differentiation therapy of acute promyelocytic leukemia (APL). ATRA and other retinoids have also been successfully applied in a variety of dermatological conditions such as skin cancer, psoriasis, acne, and ichthyosis. Moreover, modulation of retinoic acid receptors and retinoid X (or rexinoid) receptors function may affect dermal cells. The studies using complex genetic models with various combinations of retinoic acid receptors (RARs) and retinoid X (or rexinoid) receptors (RXRs) indicate that retinoic acid and its derivatives have therapeutic potential for a variety of serious dermatological disorders including some malignant conditions. Here, we provide a synopsis of the main advances in understanding the role of ATRA and its receptors in dermatology.

To date, only one subtype of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) can be effectively treated by differentiation therapy utilizing all-trans retinoic acid (ATRA). Non-APL AMLs are resistant to ATRA. Here we demonstrate that the acetyltransferase GCN5 contributes to ATRA resistance in non-APL AML via aberrant acetylation of histone 3 lysine 9 (H3K9ac) residues maintaining the expression of stemness and leukemia associated genes. We show that inhibition of GCN5 unlocks an ATRA-driven therapeutic response. This response is potentiated by coinhibition of the lysine demethylase LSD1, leading to differentiation in most non-APL AML. Induction of differentiation was not correlated to a specific AML subtype, cytogenetic, or mutational status. Our study shows a previously uncharacterized role of GCN5 in maintaining the immature state of leukemic blasts and identifies GCN5 as a therapeutic target in AML. The high efficacy of the combined epigenetic treatment with GCN5 and LSD1 inhibitors may enable the use of ATRA for differentiation therapy of non-APL AML. Furthermore, it supports a strategy of combined targeting of epigenetic factors to improve treatment, a concept potentially applicable for a broad range of malignancies.

A precision medicine approach is appealing for use in AML due to ease of access to tumor samples and the significant variability in the patients' response to treatment. Attempts to establish a precision medicine platform for AML, however, have been unsuccessful, at least in part due to the use of small compound panels and having relatively slow turn over rates, which restricts the scope of treatment and delays its onset. For this pilot study, we evaluated a cohort of 12 patients with refractory AML using an ex vivo drug sensitivity testing (DST) platform. Purified AML blasts were screened with a panel of 215 FDA-approved compounds and treatment response was evaluated after 72h of exposure. Drug sensitivity scoring was reported to the treating physician, and patients were then treated with either DST- or non-DST guided therapy. We observed survival benefit of DST-guided therapy as compared to the survival of patients treated according to physician recommendation. Three out of four DST-treated patients displayed treatment response, while all of the non-DST-guided patients progressed during treatment. DST rapidly and effectively provides personalized treatment recommendations for patients with refractory AML.

Alterations to the gene encoding the EZH2 (KMT6A) methyltransferase, including both gain-of-function and loss-of-function, have been linked to a variety of haematological malignancies and solid tumours, suggesting a complex, context-dependent role of this methyltransferase. The successful implementation of molecularly targeted therapies against EZH2 requires a greater understanding of the potential mechanisms by which EZH2 contributes to cancer. One aspect of this effort is the mapping of EZH2 partner proteins and cellular targets. To this end we performed affinity-purification mass spectrometry in the FAB-M2 HL-60 acute myeloid leukaemia (AML) cell line before and after all-trans retinoic acid-induced differentiation. These studies identified new EZH2 interaction partners and potential non-histone substrates for EZH2-mediated methylation. Our results suggest that EZH2 is involved in the regulation of translation through interactions with a number of RNA binding proteins and by methylating key components of protein synthesis such as eEF1A1. Given that deregulated mRNA translation is a frequent feature of cancer and that eEF1A1 is highly expressed in many human tumours, these findings present new possibilities for the therapeutic targeting of EZH2 in AML.

Here we report the case of a 30-year-old woman with relapsed acute myeloid leukemia (AML) who was treated with all-trans retinoic acid (ATRA) as part of investigational therapy (NCT02273102). The patient died from rapid disease progression following eight days of continuous treatment with ATRA. Karyotype analysis and RNA-Seq revealed the presence of a novel t(4;15)(q31;q22) reciprocal translocation involving the TMEM154 and RASGRF1 genes. Analysis of primary cells from the patient revealed the expression of TMEM154-RASGRF1 mRNA and the resulting fusion protein, but no expression of the reciprocal RASGRF1-TMEM154 fusion. Consistent with the response of the patient to ATRA therapy, we observed a rapid proliferation of t(4;15) primary cells following ATRA treatment ex vivo. Preliminary characterization of the retinoid response of t(4;15) AML revealed that in stark contrast to non-t(4;15) AML, these cells proliferate in response to specific agonists of RARα and RARγ. Furthermore, we observed an increase in the levels of nuclear RARγ upon ATRA treatment. In summary, the identification of the novel t(4;15)(q31;q22) reciprocal translocation opens new avenues in the study of retinoid resistance and provides potential for a new biomarker for therapy of AML.