Targeting the SUMO Pathway Primes All- trans Retinoic Acid-Induced Differentiation of Nonpromyelocytic Acute Myeloid Leukemias
Abstract
Diabetic cardiomyopathy represents a profoundly distinct and increasingly recognized pathological condition affecting the myocardium, the very muscle of the heart. This severe cardiac complication independently and significantly escalates the inherent risk for the eventual development of congestive heart failure, a debilitating syndrome where the heart is unable to pump sufficient blood to meet the body’s demands. Crucially, this heightened risk is observed irrespective of the co-existence of other common cardiovascular risk factors, such as established coronary artery disease or systemic hypertension. This profound cardiac derangement is a direct and insidious consequence of diabetes mellitus, a complex metabolic disorder that, whether manifesting as type 1 or type 2, is almost invariably characterized by chronic or recurrent episodes of hyperglycemia. This signifies persistently elevated blood glucose levels, a metabolic environment inherently detrimental to various organ systems. Compelling and consistently reproducible evidence derived from numerous preclinical investigations, employing a diverse array of animal models, has unequivocally demonstrated that such sustained hyperglycemia exerts direct and profoundly deleterious effects on myocardial tissue. Notably, this includes the induction of programmed cell death, or apoptosis, within the cardiomyocytes that constitute the fundamental contractile units of the heart muscle. In the realm of recent pharmacological advancements, a novel small molecule compound, specifically designated as ZLN005, has emerged and rapidly garnered considerable scientific interest within the research community. Early reports originating from meticulously conducted studies in a preclinical mouse model of diabetes have indicated that ZLN005 possesses promising antidiabetic efficacy. Its beneficial actions are potentially mediated through the induction of PGC-1α expression, a master transcriptional coactivator widely acknowledged for its critical and multifaceted roles in regulating vital cellular processes, including mitochondrial biogenesis, the formation of new mitochondria, and overall energy metabolism. Building upon these pivotal preliminary findings, the overarching and meticulously defined objective of the current detailed study was to comprehensively investigate whether ZLN005 could indeed confer substantive protective effects on cardiomyocytes directly challenged by high glucose-induced cytotoxicity, a laboratory mimicry of the diabetic milieu. More critically, a significant aim was to meticulously elucidate the intricate underlying molecular mechanisms responsible for any observed cardioprotection, thereby providing a deeper understanding of its therapeutic potential.
To meticulously and rigorously assess the potential therapeutic efficacy of ZLN005 in a controlled environment, our experimental approach strategically leveraged an in vitro model utilizing primary neonatal mouse cardiomyocytes. These delicate and essential cardiac muscle cells were carefully cultivated under stringent laboratory conditions and subsequently exposed to precisely defined glucose concentrations within a controlled in vitro environment designed to simulate both physiological and pathological conditions. Specifically, the cardiomyocytes were incubated for a period of 24 hours in culture media containing either a physiological concentration of glucose, meticulously set at 5.5 millimolar, which served as the healthy control condition, or a pathologically elevated concentration of 33 millimolar glucose. This higher concentration was deliberately designed to effectively mimic the severe hyperglycemic stress characteristic of uncontrolled diabetes. Crucially, these high glucose conditions were applied both in the complete absence of ZLN005 and, concurrently, in the presence of varying concentrations of ZLN005. This systematic experimental design allowed for a direct, precise, and comparative analysis of its protective effects against hyperglycemia-induced cellular damage.
The comprehensive and detailed analysis of our experimental results consistently yielded compelling evidence unequivocally demonstrating ZLN005′s remarkable cardioprotective capabilities. Treatment with ZLN005 within the high glucose environment led to a significant and reproducible amelioration of cardiomyocyte oxidative injury. This finding is particularly significant as it indicates a robust reduction in the pervasive cellular damage primarily caused by an imbalance between the production and neutralization of highly reactive oxygen species, a hallmark of diabetic cellular stress. This profound protective effect was further and robustly substantiated by a demonstrable enhancement in overall cell viability, strongly suggesting that ZLN005 played a critical role in helping to maintain the health, structural integrity, and functional capacity of the cardiomyocytes even under adverse conditions. Most importantly, ZLN005 effectively and substantially mitigated the dire deleterious impact of chronic hyperglycemia on cardiac cells, resulting in a substantial and statistically significant reduction in the rate of apoptosis among the cardiomyocytes exposed to high glucose. This preservation of myocardial cell populations is paramount for maintaining cardiac function. Delving deeper into the intricate molecular underpinnings of these observed protective effects, sophisticated Western blot analysis, a widely recognized laboratory technique specifically employed to accurately quantify the expression levels of various proteins, revealed a critical and insightful observation: high glucose exposure significantly suppressed the endogenous process of autophagy within cardiomyocytes. Autophagy, a fundamental and vital cellular housekeeping mechanism, is intrinsically responsible for the orderly degradation and efficient recycling of damaged cellular components and organelles, essential for maintaining robust cellular health and vigilantly preventing the detrimental accumulation of toxic cellular debris. In stark contrast to the suppressive effect exerted by high glucose, ZLN005 treatment markedly increased the expression levels of several key and well-established autophagy marker proteins. These included ATG5, beclin1, and, notably, a pronounced increase in the LC3 II/LC3 I ratio, all consistently indicative of an enhanced and robust autophagic flux. This beneficial surge in cellular autophagy was concomitantly accompanied by a pronounced and significant increase in the expression of SIRT1, a sirtuin deacetylase widely recognized to play pivotal and multifaceted roles in cellular stress response, metabolic regulation, and promoting cellular longevity. Importantly, SIRT1′s activity is often intimately linked to the precise regulation of autophagy. To definitively establish a direct causal link between SIRT1 activation and ZLN005′s observed protective effects, further corroborating experiments were strategically conducted utilizing EX527, a highly specific and potent pharmacological inhibitor of SIRT1 activity. The administration of EX527 demonstrably weakened, or significantly attenuated, the otherwise robust protective effects of ZLN005 on cardiomyocytes that were subjected to the challenging high glucose conditions. This critical finding strongly and convincingly implicates SIRT1 as a central and indispensable mediator in ZLN005′s intricate mechanism of action.
In conclusion, the cumulative, consistent, and compelling findings derived from this comprehensive in vitro investigation strongly suggest that ZLN005 actively suppresses high glucose-induced cardiomyocyte injury through a dual and intricately interconnected molecular mechanism. This involves the robust promotion of SIRT1 expression, which subsequently leads to the crucial activation and enhancement of cellular autophagy. These compelling results collectively position ZLN005 as an extraordinarily promising therapeutic candidate for the prevention or potentially the treatment of diabetic cardiomyopathy, offering a novel and precisely targeted pharmacological strategy. This approach aims to safeguard myocardial cells from the profoundly damaging effects of chronic hyperglycemia by judiciously leveraging and enhancing critical endogenous cellular survival pathways, thereby fostering cardiac resilience and function in the diabetic milieu.
Introduction
Acute Myeloid Leukemias (AML) are recognized as a highly heterogeneous group of severe hematological malignancies, representing a significant challenge in oncology. These aggressive cancers originate through the progressive acquisition of distinct oncogenic mutations within hematopoietic stem or progenitor cells residing in the bone marrow. Instead of following their normal developmental trajectory and differentiating into mature, functional blood constituents, these leukemic cells become pathologically blocked at various intermediate differentiation stages. This blockage is coupled with uncontrolled proliferation, leading to the rapid accumulation of immature blasts that infiltrate and progressively overtake the bone marrow, ultimately leading to the onset and progression of the disease. With the notable exception of the Acute Promyelocytic Leukemia (APL) subtype, the standard treatment regimen for most AMLs has remained largely unchanged over the past four decades, a testament to the persistent difficulty in effectively combating this cancer. This conventional approach generally consists of intensive chemotherapy protocols, typically involving the administration of one anthracycline, such as daunorubicin or idarubicin, in combination with the nucleoside analog cytarabine, often referred to as Ara-C. Despite the intensity of these treatments, relapses are unfortunately frequent, affecting a significant proportion of patients (ranging from 40% to 70%, depending on various prognostic factors). Furthermore, the overall survival rates for AML patients remain disturbingly low, underscoring the urgent and critical need for the development and introduction of novel, more effective therapeutic interventions.
Differentiation therapies have emerged as a conceptually powerful and highly promising strategy for the treatment of AML. This innovative approach is founded on the fundamental idea that the restoration of normal differentiation processes within leukemic cells is intrinsically associated with a crucial arrest of cell division, which is then often followed by the inevitable death of these cells due to the naturally limited lifespan characteristic of terminally differentiated cells. This therapeutic strategy has proved to be particularly efficient and curative in the context of Acute Promyelocytic Leukemia, a distinct AML subtype. APL is specifically characterized by the expression of unique oncogenic fusion proteins, most notably PML-RARα, which aberrantly engage the retinoic acid receptor alpha (RARα). RARα itself belongs to the nuclear receptor family, a class of ligand-activated transcription factors. The highly successful APL therapy is predominantly based on the administration of pharmacological doses of its natural ligand, all-trans-retinoic acid (ATRA), frequently used in potent combination with arsenic trioxide. ATRA exerts its therapeutic effects by leading to the rapid degradation of the oncogenic RARα fusion protein and, simultaneously, by activating the wild-type RARα. This dual action initiates and powerfully activates a specific transcriptional program within the leukemic cells, which in turn drives their terminal differentiation, induces cell cycle arrest, and ultimately leads to the programmed death of these malignant cells. Interestingly, ATRA also demonstrates, to varying degrees, the capacity to induce the in vitro differentiation of certain non-APL AML cell lines and primary cells obtained from a substantial number of non-APL AML cases. This includes AMLs characterized by mutations in the NPM1, IDH1/IDH2, or FLT3-ITD genes, or those overexpressing the transcription factor EVI-1, indicating some inherent sensitivity. However, despite these promising in vitro observations, the clinical trials conducted so far have, unfortunately, failed to consistently demonstrate a significant efficacy of ATRA on non-APL AML patients, even when used in combination with other conventional chemotherapeutic drugs. This persistent lack of clinical effect has been widely attributed to the inability of ATRA to consistently induce the full and sustained expression of critical genes intimately involved in differentiation, cell cycle arrest, or apoptosis in these non-APL subtypes. Intriguingly, recent advancements have shown that targeting various epigenetic enzymes has appeared as a promising new avenue to restore the ability of ATRA to activate RARα target genes and thus potentiate ATRA-induced differentiation of non-APL AMLs. For instance, the inhibition of histone deacetylases (HDAC) with agents such as valproic acid was shown to significantly favor ATRA-induced differentiation of non-APL AML cells in preclinical models. Nevertheless, the subsequent clinical trials conducted with the combination of ATRA and valproic acid have, similarly, shown only limited therapeutic effects in a subset of the treated patients. More recently, the inhibition of the histone demethylase LSD1/KDM1A was also shown to strongly sensitize AML cells to ATRA in various preclinical models, primarily via profound transcriptional reprogramming. Currently, Phase I/II clinical trials are actively ongoing to rigorously determine the clinical efficacy and safety of the combination between ATRA and LSD1 inhibitors, offering a new ray of hope.
SUMO, an acronym for Small Ubiquitin-like Modifier, represents a group of three distinct ubiquitin-related polypeptidic post-translational modifiers, specifically SUMO-1, SUMO-2, and SUMO-3. These modifiers are covalently and reversibly conjugated to numerous intracellular proteins, playing crucial roles in regulating their function, cellular localization, and ultimate fate. The intricate process of SUMO conjugation involves a unique E1 SUMO-activating enzyme, a singular E2-conjugating enzyme known as Ubc9, and a diverse array of several E3 SUMO ligases, each contributing to the specificity and efficiency of the modification. Conversely, the deconjugation or removal of SUMO is precisely ensured by a family of dedicated deSUMOylases, particularly members of the SENP family, maintaining the dynamic balance of SUMOylation. Accumulating and compelling evidence increasingly links the deregulation or dysregulation of the SUMO pathway to the initiation and progression of various cancers, including malignant hematological conditions such as lymphomas and multiple myeloma, highlighting its broad oncogenic potential. In the specific context of AMLs, the SUMO pathway has been shown to be absolutely essential for efficient differentiation therapy of APLs through the potent ATRA plus arsenic trioxide combination treatment. Specifically, arsenic trioxide induces the rapid and profound SUMOylation of the PML-RARα oncoprotein, a critical event that initiates its targeted elimination by the sophisticated ubiquitin-proteasome system, leading to the therapeutic degradation of the oncogenic driver. In addition to this, our prior research has definitively demonstrated that the precise inhibition of the SUMO pathway, often induced by genotoxics-mediated reactive oxygen species, is critically essential for fast and efficient cell death of chemosensitive non-APL AML cells when subjected to conventional antracyclins or Ara-C treatment, underscoring its role in chemotherapy response.
Beyond its role in protein modification, SUMO is increasingly viewed and investigated as a significant epigenetic mark highly enriched at gene promoters, indicating a direct role in transcriptional regulation. This post-translational modification regulates gene expression through the intricate modification of numerous transcription factors and co-regulators, various histone-modifying enzymes, essential RNA polymerases, and even directly on histones themselves, thereby influencing chromatin structure and accessibility. Although occasionally associated with transcriptional activation, SUMOylation at gene promoters is predominantly recognized for its role in limiting or actively repressing transcription. In particular, SUMOylation profoundly facilitates the recruitment of specialized SUMO-interacting motif (SIM)-containing co-repressors onto gene promoters, leading to a condensed chromatin state and transcriptional silencing. Within the framework of the current study, we present compelling evidence demonstrating that SUMOylation actively participates in the epigenetic silencing of key ATRA-responsive genes in non-APL AMLs. Crucially, we show that the targeted inhibition of this SUMO pathway effectively activates the inherent pro-differentiating and anti-leukemic effects of ATRA in these challenging cancers. This groundbreaking finding profoundly opens new and promising perspectives in the effective treatment of this particularly poor-prognosis cancer, offering a novel therapeutic strategy to sensitize patients to an existing drug.
Methods
Cell lines and primary AML patient cell culture:
The U937, HL60, and THP1 cell lines, which are commonly utilized models in leukemia research, underwent rigorous authentication processes performed by the American Type Culture Collection (ATCC) using Short-Tandem-Repeat analysis, ensuring their genetic identity and integrity. The MOLM14 cell line was obtained directly from the ATCC, maintaining a consistent and verified source. To ensure the reliability of experimental outcomes, all cell lines were regularly and meticulously tested to confirm their negative status for mycoplasmas, which are common and problematic contaminants in cell cultures. These cells were consistently cultured in RPMI medium, a standard cell culture medium, supplemented with 10% fetal bovine serum (FBS), a vital source of growth factors and nutrients, and fortified with streptomycin/penicillin, to prevent bacterial contamination. Cultures were maintained at a physiological temperature of 37°C in a humidified atmosphere containing 5% carbon dioxide, mimicking in vivo conditions. After initial thawing, cells were routinely passaged at a density of 0.3 x 10^6 cells per milliliter every 2-3 days, with a strict limit of no more than 10 passages to minimize the accumulation of genetic drift and maintain cellular characteristics. U937 cells exhibiting resistance to Ara-C, a commonly used chemotherapeutic drug, were specifically generated for this study by progressively culturing the parental U937 cells for a period of 2 months in the continuous presence of increasing concentrations of Ara-C, ultimately reaching a concentration of 0.1 µM.
Patient bone marrow aspirates, representing primary AML cells, were ethically collected only after obtaining comprehensive written informed consents from all participating patients. These collection procedures strictly adhered to the rigorous ethical guidelines outlined in the Declaration of Helsinki, which governs human experimentation, and received explicit approval from the institutional review board (Ethical Committee « Sud Méditerranée 1 », reference 2013-A00260-45, HemoDiag collection). Upon collection, fresh leukocytes were meticulously purified from the bone marrow aspirates using density-based centrifugation, employing Histopaque 1077 from SIGMA, a method designed to separate cells based on their density. The purified cells were then carefully resuspended at a precise concentration of 10^6 cells per milliliter in IMDM (SIGMA), a rich basal medium, which was comprehensively complemented with a specific cocktail of essential components. This included 1.5 mg/ml bovine serum albumin, 4.4 µg/ml insulin (SIGMA), 60 µg/ml transferrin (SIGMA), 5% streptomycin and penicillin to maintain sterility, 5% FBS for growth support, 5 µM ß-mercaptoethanol, 1 mM pyruvate, 1xMEM non-essential amino acids (Life Technologies), 10 ng/ml IL-3 (PeproTech), 40 ng/ml SCF (PeproTech), and 10 ng/ml TPO (PeproTech). This specialized medium was formulated to optimally support the viability and short-term culture of primary AML cells.
Pharmacological inhibitors, reagents, and antibodies:
All-trans-retinoic acid (ATRA), the primary retinoid under investigation, was acquired from Sigma. It was meticulously resuspended at a high stock concentration of 100 mM in dimethyl sulfoxide (DMSO) to ensure solubility and then stored at -20°C for a maximum period of 2 weeks to preserve its chemical stability and biological activity. Anacardic acid, another pharmacological inhibitor used in the study, was obtained from Santa Cruz Biotechnologies, while 2-D08, also a pharmacological inhibitor, was sourced from Merck-Millipore. For immunodetection purposes, specific antibodies were required. The anti-SUMO-1- (clone 21C7) and anti-SUMO-2 (clone 8A2) hybridomas, which produce antibodies against distinct SUMO isoforms, were generously provided by the Developmental Studies Hybridoma Bank, a valuable resource for research antibodies. The anti-H3K4Me3 antiserum, used to detect a specific histone methylation mark indicative of active transcription, was purchased from Abcam. The Ubc9 antibody, targeting the unique E2 SUMO-conjugating enzyme, was acquired from Santa Cruz. The meticulous selection and careful handling of these high-quality reagents and antibodies were paramount to ensuring the specificity, accuracy, and reproducibility of all biochemical and molecular assays conducted throughout the study.
Flow cytometry:
To quantitatively assess cellular differentiation markers, cells were prepared for flow cytometric analysis. Initially, cells were carefully washed in phosphate-buffered saline (PBS) containing 2% fetal bovine serum to remove residual culture medium and minimize non-specific antibody binding. Following washing, cells were incubated at a controlled temperature of 4°C for a period of 30 minutes in the presence of specific fluorophore-conjugated antibodies. The antibodies utilized included CD45-Pacific Blue (A74763; Beckman Coulter), a pan-leukocyte marker, CD14-PE (130-091-242; Miltenyi), a marker for monocytes/macrophages and differentiated myeloid cells, CD15-PE-Vio770 (130-100-425; Miltenyi), a marker for granulocytic differentiation, and CD11b-APC (130-109-286; Miltenyi), another key marker for myeloid differentiation. To account for non-specific antibody binding and background fluorescence, matched isotype controls, which are antibodies with the same immunoglobulin class as the primary antibodies but without specificity for a cellular antigen, were used for each treatment condition. After the incubation period, cells underwent further washing steps to remove unbound antibodies. Subsequently, the stained cells were meticulously analyzed using the LSR Fortessa flow cytometer (Becton Dickinson), a sophisticated instrument capable of rapidly acquiring multi-parameter data from individual cells. Data acquisition was managed by the FacsDiva software. For subsequent analysis and interpretation, the raw flow cytometry data were processed using FlowJo software (version 10), a widely used and powerful bioinformatics tool for flow cytometry. For patient samples, which often contain heterogeneous cell populations, Mean Fluorescence Intensities (MFIs) for each differentiation marker were specifically measured on leukemic cells. These leukemic cells were rigorously pre-selected using a CD45/SSC gating strategy, which exploits differences in cell size and granularity, as previously described in the literature. To ensure accurate quantification of specific antibody binding, the MFIs obtained from the matched isotype controls were systematically subtracted from the MFIs of each treatment condition, providing a precise measure of specific marker expression.
Microscopic analyses:
For detailed morphological examination and assessment of cellular differentiation, cell lines or patient samples were prepared for microscopic analyses. A precise volume of cell suspension was meticulously cytospun onto clean microscope slides using a cytocentrifuge (at 1500 rpm for 5 minutes). This process gently adheres the cells to the slide surface while maintaining their morphology. After cytospinning, the slides were carefully dried for 5 minutes. Subsequently, a classical two-step staining procedure, known as May-Grunwald-Giemsa (MGG) staining, was performed to visualize cellular components. First, the slides were stained with May-Grunwald stain for 5 minutes, followed by staining with Giemsa stain at a 1/10 dilution for 15 minutes. This staining combination provides excellent cytological detail, allowing for the differentiation of various cell types and assessment of maturation. Microscopic examinations were then diligently performed using the AxioImager Z2 microscope (Zeiss), a high-quality research microscope equipped for brightfield imaging, allowing for detailed observation and documentation of cellular changes induced by experimental treatments.
Cell viability, cell cycle and proliferation assay:
For proliferation assays conducted on cell lines, cells were meticulously seeded at a starting concentration of 3 x 10^5 cells per milliliter. Subsequently, viable cells were counted at regular intervals over time using the Trypan-blue exclusion method, a common technique that distinguishes viable cells (which exclude the dye) from non-viable cells (which take up the dye due to compromised membranes). Cell counts were performed using an EVE automatic cell counter, ensuring consistency and efficiency. To analyze cell cycle distribution, a critical indicator of cellular proliferation and differentiation, Propidium Iodide (PI) staining was employed. Initially, cells were washed once with PBS to remove residual medium and then fixed with cold 70% ethanol for 10 minutes, a standard method to permeabilize cells for DNA staining. After fixation, cells were washed once more with PBS. To ensure that PI specifically stained DNA and not RNA, 100 µg/mL RNAase A (Sigma) was then added to the cell suspension for 10 minutes at room temperature, which digests cellular RNA. Cells were then washed again with PBS and stained with 50 µg/mL PI (Sigma) for 10 minutes at room temperature. PI, a fluorescent intercalating dye, binds stoichiometrically to double-stranded DNA, allowing for quantitative assessment of DNA content and, consequently, cell cycle phases (G0/G1, S, G2/M). Finally, cells were washed once with PBS and analyzed by flow cytometry, which measures the fluorescence intensity of individual cells, corresponding to their DNA content. For patient cells, given their potential scarcity and heterogeneity, equal numbers of CountBright absolute counting beads (C36950; Life Technologies) were strategically added to each sample. These beads serve as an internal standard, allowing for absolute quantification of cell numbers. Viable cells from patient samples were precisely selected using the CD45/SSC gating strategy, distinguishing leukemic cells from other bone marrow components. Their number was then normalized to the number of beads counted in the same sample, providing a robust and accurate measure of viable cell counts.
Retroviral Infections:
Retroviral constructs designed to express either Ubc9, SENP2, or SENP5 were meticulously generated by inserting the human cDNA sequences corresponding to these genes. This was achieved using the Gateway cloning technology (ThermoFisher Scientific), a highly efficient and standardized method for transferring DNA fragments into various vectors. The cDNAs were cloned into the pMIG retroviral vector, which is specifically designed to coexpress Enhanced Green Fluorescent Protein (EGFP) from the same polycistronic mRNA, serving as a convenient marker for successful viral transduction. To produce the retroviruses, these constructs were cotransfected with gag-pol and VSV-G expression vectors into HEK293T cells, a commonly used cell line for viral packaging, utilizing Lipofectamine-2000 (Invitrogen) as the transfection reagent. Viral supernatants, containing the newly formed retroviral particles, were collected 48 hours post-transfection. These supernatants were then clarified by passing them through a 0.45 µm filter to remove cellular debris, and directly used to infect AML cell lines. For subsequent flow cytometry analyses, only EGFP-positive cells, indicating successful retroviral transduction and expression of the gene of interest, were considered for analysis, ensuring that the observed effects were directly attributable to the introduced gene. Where explicitly indicated, the EGFP-positive cells were further purified using the FACS-Aria cell sorter (Becton Dickinson), allowing for the isolation of a highly enriched population of transduced cells for downstream experiments.
RT-qPCR assays:
For the precise quantification of gene expression at the messenger RNA (mRNA) level, Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) assays were meticulously performed. Total mRNA, representing the complete set of transcribed genes within the cells, was rigorously purified using the GenElute Mammalian Total RNA kit (Sigma), a reliable method for isolating high-quality RNA from mammalian cells. After mRNA purification, samples were subjected to DNase I treatment to enzymatically remove any contaminating genomic DNA, ensuring that only RNA was used for subsequent analysis. Following this critical step, 1 µg of the purified total RNA was used as a template for cDNA synthesis, utilizing the Maxima First Strand cDNA kit (ThermoFisher Scientific). This step reverse transcribes the mRNA into more stable complementary DNA (cDNA), which is suitable for PCR amplification. Subsequently, qPCR assays were accurately conducted using Taq platinum (Invitrogen), a high-fidelity DNA polymerase, and performed on the LightCycler 480 device (Roche), a real-time PCR system. Specific DNA primers, designed to amplify discrete regions of the target genes, were utilized for each reaction (refer to the primer table for specific sequences). To account for variations in RNA input and reverse transcription efficiency across samples, the quantitative data obtained for each target gene were meticulously normalized to the mRNA levels of the housekeeping gene TBP (TATA-box binding protein), which is known for its stable expression across various cell types and conditions.
Chromatin immunoprecipitation assays (ChIPs):
To investigate the association of specific proteins and histone modifications with particular genomic regions, particularly gene promoters, chromatin immunoprecipitation (ChIP) assays were meticulously performed. Initially, 30 x 10^6 cells were carefully collected and subjected to cross-linking with 1% paraformaldehyde for precisely 8 minutes. This step creates covalent bonds between proteins and DNA, preserving their native interactions. The cross-linking reaction was then promptly neutralized by the addition of 125 mM glycine for 10 minutes. Cross-linked cells were subsequently washed with cold PBS and gently resuspended in a specialized cell lysis buffer (composed of 5 mM PIPES pH 7.5, 85 mM KCl, 0.5% NP40, 20 mM N-ethyl maleimide, 1 µg/mL of aprotinin, pepstatin, leupeptin, and 1 mM AEBSF), followed by incubation at 4°C for 10 minutes with gentle rotation. Nuclei were then isolated by centrifugation (5,000 rpm for 10 minutes at 4°C) and carefully resuspended in a nucleus lysis buffer (containing 50 mM Tris-HCl pH 7.5, 1% SDS, 10 mM EDTA, 20 mM N-ethyl maleimide, 1 µg/mL of aprotinin, pepstatin, leupeptin, and 1 mM AEBSF). This suspension was incubated at 4°C for 2.5 hours to ensure complete lysis of nuclear membranes.
Lysates were then subjected to sonication for 30 cycles of 30 seconds each at 4°C using the Bioruptor Pico (Diagenode) under standard conditions. This sonication step fragments the chromatin into smaller, manageable pieces, making it accessible for antibody binding. After sonication, samples were centrifuged (13,000 rpm for 10 minutes at 4°C) to remove insoluble debris, and the clear supernatants, containing the fragmented chromatin, were diluted 10-fold in the immunoprecipitation buffer (comprising 1.1% Triton X100, 50 mM Tris-HCl pH 7.5, 167 mM NaCl, 5 mM N-ethyl maleimide, 1 mM EDTA, 0.01% SDS, 1 µg/mL of aprotinin, pepstatin, leupeptin, and 1 mM AEBSF). To this diluted chromatin, 2 µg of specific antibodies (targeting the protein or histone modification of interest) and Dynabeads Protein G (ThermoFisher Scientific), which bind to antibodies, were added. Immunoprecipitations were performed overnight at 4°C to allow for thorough antibody-antigen binding.
Following immunoprecipitation, the beads, now bound to antibody-chromatin complexes, were subjected to rigorous sequential washing steps. These included washes in low salt buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X100, 0.1% SDS, 1 mM EDTA), high salt buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 1% Triton X100, 0.1% SDS, 1 mM EDTA), LiCl salt buffer (20 mM Tris-HCl pH 7.5, 250 mM LiCl, 1% NP40, 1% deoxycholic acid, 1 mM EDTA), and finally TE buffer (10 mM Tris-HCl pH 7.5, 0.2% Tween20, 1 mM EDTA). These stringent washes are crucial to remove non-specifically bound chromatin. Elution of the immunoprecipitated chromatin was performed in 200 µL of 100 mM NaHCO3, 1% SDS. The protein-DNA crosslinking was subsequently reversed by overnight incubation at 65°C with 280 mM NaCl, followed by 2 hours at 45°C with 35 mM Tris-HCl pH 6.8, 9 mM EDTA, 88 µg/mL RNAse, and 88 µg/mL Proteinase K. This step degrades proteins and liberates the DNA. The immunoprecipitated DNA fragments were then purified using the Nucleospin Gel and PCR Cleanup kit (Macherey-Nagel), ensuring high purity. Both the immunoprecipitated DNA and the input DNA (purified from samples before immunoprecipitation, serving as a positive control for DNA abundance) were then subjected to PCR analysis using the Roche LightCycler 480 device, utilizing appropriate specific primers (refer to Table 1 for primer sequences) to quantify the enrichment of specific genomic regions.
Tumor xenografts:
Animal experiments were conducted in strict accordance with a meticulously reviewed and formally approved protocol by the Institutional Animal Care and Use Committee of Région Midi-Pyrénées (France), ensuring ethical and humane treatment of all animal subjects. Tumors were precisely generated by subcutaneously injecting 2 x 10^6 U937 cells, a human AML cell line, which were suspended in 100 µl of sterile PBS. These injections were carefully administered onto both flanks of NOD-Scid-IL2gRnull (NSG) mice, an immunodeficient strain suitable for xenograft studies (adult males and females, each weighing approximately 25 g; obtained from Charles River Laboratories). This immunodeficient mouse model allows for the sustained growth of human cancer cells. Once the subcutaneous tumors reached a palpable volume of approximately 100 mm^3, signaling established tumor growth, the mice commenced treatment. They received peritumoral injections of either ATRA (at a dose of 2.5 mg/kg/day), or 2-D08 (at a dose of 10 mg/kg/day), or a combination of both drugs, administered every 2 days. Injections of vehicle (DMSO), which was used as the solvent for the drugs, were concurrently administered to control groups to account for any effects of the solvent itself. Tumor sizes were meticulously measured at regular intervals using a digital caliper, and their volumes were precisely calculated using the established formula: V = (π/6) × A × B^2, where A represents the larger diameter of the tumor and B represents the smaller diameter. This formula allows for a consistent and quantitative assessment of tumor growth and response to treatment.
Statistical analyses:
All statistical analyses of the experimental data meticulously generated throughout the entirety of this comprehensive study were rigorously performed utilizing the Prism 5 software, a widely recognized, highly respected, and robust platform specifically designed for advanced scientific data analysis. To rigorously assess and determine statistical significance for experiments involving various cell lines, the two-tailed paired Student’s t-test was specifically utilized. This particular statistical test is highly appropriate for comparing the mean values of two related groups, such as observations made before and after a treatment in the same cell population. For experiments involving precious patient samples, which often inherently exhibit greater biological variability and may not strictly meet all the assumptions of parametric statistical tests, the Wilcoxon matched-pairs signed rank test was judiciously employed. This non-parametric alternative is particularly suitable for paired data, providing robust comparisons without relying on assumptions of data distribution. For the comprehensive analysis of data derived from the in vivo tumor xenograft experiments, the Mann-Whitney test, another powerful non-parametric test, was consistently used to compare differences between two independent groups of animal subjects. In all statistical analyses systematically conducted across the study, a conventional and widely accepted predetermined threshold of P < 0.05 was consistently utilized to define statistical significance. This critical criterion means that any observed difference yielding a p-value less than 0.05 was considered to be highly unlikely to have occurred solely by random chance alone, thereby strongly suggesting a genuine and biologically meaningful effect of the experimental intervention or treatment. The precise levels of statistical significance were clearly denoted in the results as follows: an asterisk (*) indicated a p-value of <0.05; two asterisks (**) indicated a p-value of <0.01; three asterisks (***) indicated a p-value of <0.001; and four asterisks (****) indicated an even higher level of significance with a p-value of <0.0001. Conversely, a designation of “ns” was used to explicitly indicate a non-significant statistical difference between the compared groups. This systematic, transparent, and clear statistical approach ensured that all conclusions drawn from the extensive experimental findings were not only robust and statistically sound but also reliably interpretable, lending strong scientific credibility to the study’s outcomes.
Results
The SUMO pathway represses ATRA-induced differentiation of non-APL AML cells.
To comprehensively assess the crucial role of SUMOylation, a significant post-translational modification, in the context of all-trans retinoic acid (ATRA)-induced differentiation of non-APL Acute Myeloid Leukemias, our initial experimental approach involved utilizing two well-established and extensively characterized non-APL AML cell lines: U937 and HL-60. These cell lines serve as valuable in vitro models for studying AML biology. U937 cells belong to the M4 subtype, while HL60 cells are classified as M2 subtype, according to the French-American-British (FAB) classification system, which encompasses eight distinct AML subtypes. Both of these cell lines possess the inherent capacity to differentiate in vitro in the presence of ATRA, although their efficiency in undergoing this differentiation process is typically observed to be low when ATRA is administered as a single agent. For the purpose of inhibiting the SUMO pathway, we employed two commercially available and distinct pharmacological inhibitors. The first, 2-D08, is known to specifically act as an inhibitor of the E2 SUMO-conjugating enzyme, Ubc9. The second, anacardic acid (AA), functions as an inhibitor of the SUMO-activating enzyme Uba2/Aos1. Both inhibitors were carefully utilized at concentrations that were empirically determined to lead to a moderate degree of hypoSUMOylation of cellular proteins, ensuring a controlled and partial inhibition of the SUMO pathway. This judicious partial inhibition of the SUMO pathway consistently resulted in a statistically significant and readily observable increase in ATRA-induced differentiation in both U937 cells and HL60 cells. The extent of differentiation was rigorously assessed by monitoring the enhanced expression of specific differentiation markers: CD15 in U937 cells and CD11b in HL60 cells, both of which are indicative of myeloid maturation. Furthermore, the combination of ATRA with 2-D08 also demonstrated a compelling capacity to increase the differentiation of THP1 cells, another non-APL AML cell line, and MOLM14 cells, as well as remarkably, in Ara-C-resistant U937 cells that had been specifically developed in our laboratory, highlighting the potential to overcome drug resistance. The synergistic combination of ATRA and SUMOylation inhibitors not only enhanced marker expression but also significantly increased the number of U937, HL-60, and THP1 cells exhibiting clear morphological changes typical of terminal myeloid differentiation. These morphological hallmarks included pronounced nuclear lobulation and the distinct appearance of numerous cytosolic granules, characteristic features of mature myeloid cells. Collectively, these robust and consistent data strongly suggest that the SUMO pathway intrinsically limits the differentiating effects of ATRA on various non-APL AML cell lines. Moreover, these findings compellingly indicate that targeted inhibition of the SUMO pathway could substantially favor and potentiate their ATRA-induced differentiation, including in cases where resistance to conventional chemotherapeutics, such as Ara-C, which are commonly used in AML treatment, has developed.
SUMOylation limits ATRA-induced expression of myeloid differentiation-associated genes
Following our initial observations, we then logically extended our investigation to determine if the pervasive repressive action of SUMOylation on ATRA-induced differentiation could be intrinsically linked to its well-characterized and documented ability to repress or limit gene expression at the transcriptional level. Our experiments revealed that the direct inhibition of SUMOylation, specifically achieved with the pharmacological agent 2-D08, was sufficient on its own to increase the basal expression of various ATRA-responsive genes intimately associated with myeloid differentiation. These critically important genes included RARA, CEBPA, TNFSF10, ITGAX, ITGAM, and IL1B. This compelling finding strongly suggests that even in the absence of ATRA, the inhibition of SUMOylation might serve to “prime” cells for differentiation by increasing the foundational, basal expression levels of these master genes that are fundamentally involved in myeloid differentiation. In addition to influencing basal expression, 2-D08 also robustly increased the ATRA-induced expression of most of these same genes, demonstrating a synergistic effect. To precisely determine if SUMOylation exerts its control over the expression of these genes at the level of chromatin, the highly organized structure of DNA within the nucleus, we meticulously assayed the presence of the active transcription-associated histone mark H3K4me3 on their respective gene promoters. An observable increase in the presence of this active chromatin mark on the promoters of RARA, ITGAX, CEBPA, and TNFSF10 genes consistently correlated with the observed higher levels of their corresponding mRNA expression. This strong correlation provides robust evidence, strongly suggesting that SUMOylation actively represses the induction of ATRA-responsive genes at the fundamental level of chromatin structure and accessibility, thereby directly influencing their transcriptional state and ultimately limiting differentiation.
Inhibition of SUMOylation potentiates the anti-leukemic effects of ATRA.
The process of cellular differentiation is intricately linked to fundamental changes in cellular behavior, particularly with regard to proliferation and lifespan. Differentiated cells typically cease to proliferate and possess a significantly shorter lifespan compared to their undifferentiated counterparts. Consistently with this biological principle, our experiments revealed that ATRA and 2-D08 synergized powerfully to block the uncontrolled proliferation of both U937 cells and their Ara-C-resistant variants when cultured in vitro. This significant anti-proliferative effect correlated directly with a notable accumulation of cells in the G0/G1 phase of the cell cycle, indicating a prominent cell cycle arrest. Furthermore, this was associated with a strong activation of the CDKN1A gene, which encodes the cyclin-dependent kinase inhibitor p21CIP1, a key protein involved in enforcing cell cycle arrest. These findings strongly suggest that the inhibition of SUMOylation considerably increases the anti-proliferative effects of ATRA in vitro by inducing a robust and sustained cell cycle arrest. To extend these findings and determine if this beneficial effect would also translate to an in vivo context, we conducted a study using immunodeficient mice. These mice were subcutaneously xenografted with U937 cells to establish tumors and subsequently received systemic treatment with ATRA, 2-D08, or a combination of both drugs after successful tumor engraftment. Our results unequivocally demonstrated that only the strategic combination of ATRA and 2-D08 induced a significant and measurable reduction in tumor growth in vivo, whereas ATRA and 2-D08 administered as single agents showed only slight, if any, discernible effects on tumor progression. Thus, these compelling in vivo data confirm that the combined therapeutic approach of ATRA with an inhibitor of SUMOylation can not only effectively promote non-APL AML cell differentiation but also exert potent anti-proliferative effects both in vitro and within a living organism.
Genetic modulation of the SUMO pathway affects ATRA-induced differentiation of non-APL AML cells.
To definitively rule out the possibility of any confounding off-target effects associated with the pharmacological inhibitors 2-D08 and anacardic acid, we judiciously resorted to precise genetic manipulation of the SUMO pathway. This approach aimed to independently confirm the indispensable role of SUMOylation on ATRA-induced differentiation in non-APL AMLs. In a crucial first step, we successfully overexpressed either the SENP-2 or the SENP-5 deSUMOylase in U937 cells. These enzymes are responsible for removing SUMO from target proteins, thereby counteracting SUMOylation. This genetic manipulation led to a significant and reproducible increase in the ATRA-induced differentiation of U937 cells, as rigorously assayed by the enhanced expression of the CD11b differentiation marker. Furthermore, this genetic deSUMOylation also correlated directly with a notable reduction in their proliferation, reinforcing the anti-leukemic effects. Overexpression of SENP-2 similarly increased ATRA-induced differentiation of HL-60 cells, demonstrating consistency across different cell lines. Interestingly, this genetic enhancement of deSUMOylation in HL60 cells strongly decreased SUMOylation, particularly by SUMO-2, of chromatin-bound proteins, as meticulously analyzed by ChIP-qPCR on the promoter region of the RARA gene. This reduction in promoter-associated SUMOylation was directly associated with stronger and more robust expression of RARA, as well as other ATRA-responsive genes such as CEBPA, ITGAM, and IL1B. Although SENP-2-expressing HL60 cells did not appear overtly more differentiated than control cells in the complete absence of ATRA, they consistently showed a higher basal expression of these crucial differentiation-associated genes. This further supported the compelling idea that even partial inhibition or reversal of SUMOylation could effectively “prime” AML cells, making them more receptive and sensitive to ATRA-induced differentiation. In a second, complementary step, we intentionally increased global SUMOylation levels in THP1 cells through the precise overexpression of the SUMO E2-conjugating enzyme Ubc9. This genetic intervention, designed to enhance SUMOylation, led to a massive and profound decrease in ATRA-induced differentiation, as comprehensively assayed by the significantly reduced expression of the CD14 differentiation marker and evident morphological changes, including the lack of pseudopod formation and reduced cytosolic vesicles and granules. Moreover, Ubc9 overexpression strongly decreased both the basal expression and the ATRA-induced upregulation of genes intimately involved in myeloid differentiation. Altogether, these compelling genetic data rigorously confirmed the critical and pervasive repressive role of the SUMO pathway on ATRA-induced differentiation of non-APL AMLs, providing robust mechanistic validation for our pharmacological findings.
Inhibition of SUMOylation potentiates the pro-differentiating and anti-proliferative activities of ATRA on primary AML cells.
To further bridge the gap between in vitro cell line studies and potential clinical relevance, we finally investigated whether inhibiting SUMOylation could also significantly favor the in vitro differentiation of primary AML cells directly obtained from bone marrow aspirates of patients at the time of diagnosis. These primary cells often exhibit greater physiological relevance compared to established cell lines. Our initial assessment revealed that ATRA alone did not significantly induce the differentiation of these primary AML cells, as assayed by CD15 expression, consistent with clinical observations of limited ATRA efficacy in non-APL AML. Individual treatment with 2-D08 or anacardic acid alone showed a slight, albeit statistically non-significant, pro-differentiating trend. In stark contrast, combining either 2-D08 or anacardic acid with ATRA consistently and significantly increased CD15 expression compared to cells treated with ATRA alone, unequivocally demonstrating a synergistic effect. While some patient cells exhibited greater sensitivity to the differentiating effects of the ATRA plus SUMOylation inhibitor combination than others, this differential sensitivity did not correlate with any unique FAB subtype or shared cytogenetic/genetic abnormalities tested at diagnosis, suggesting broader applicability across heterogeneous AMLs. Beyond marker expression, inhibitors of SUMOylation also substantially increased the number of cells showing characteristic morphological changes typical of differentiation, such as nuclear lobulation, significant cytosol enlargement, or the appearance of cytosolic granules, as observed on cells from the two patients meticulously tested. Interestingly, and of significant clinical importance, 2-D08 and anacardic acid also powerfully potentiated ATRA-induced differentiation of primary cells obtained from one patient who was notably unresponsive to initial induction chemotherapy, as well as from two out of three patients who were at relapse, highlighting their potential utility in refractory settings. Importantly, both inhibitors consistently increased the anti-leukemic activity of ATRA on the primary AML cells taken at both diagnosis and relapse, demonstrating broad efficacy. Altogether, these comprehensive data, derived from patient samples, definitively confirmed that SUMOylation inhibitors potentiate ATRA-induced differentiation across different non-APL AML subtypes and strongly indicate a novel and promising therapeutic approach, particularly valuable in cases of conventional chemotherapy failure.
Discussion
Differentiation therapies, particularly those utilizing all-trans retinoic acid (ATRA), have profoundly transformed the landscape of Acute Promyelocytic Leukemia (APL), converting it from a historically fatal disease into a highly curable malignancy. However, despite this remarkable success, the efficacy of ATRA in other, more common Acute Myeloid Leukemia (AML) subtypes has unfortunately remained profoundly disappointing in clinical trials. Our extensive and rigorous work definitively demonstrates that SUMOylation, a critical post-translational modification, acts as a significant repressor of ATRA-induced differentiation in non-APL AMLs. From a mechanistic perspective, we show that the SUMO pathway intrinsically limits the expression of genes that are absolutely critical for proper myeloid differentiation, such as RARA and CEBPA. Furthermore, it also represses the expression of genes required for essential cell cycle arrest, including CDKN1A, and key genes involved in retinoid-induced apoptosis, such as IL1B and TNFSF10. Crucially, our findings indicate that the inhibition of SUMOylation leads to a measurable increase in the presence of H3K4me3, a well-established histone mark of active transcription, on the promoters of these critical genes. This epigenetic modulation subsequently enhances both their basal expression and their ATRA-induced expression, rendering the cells more susceptible to differentiation. This robust evidence strongly suggests that strategically targeting SUMOylation could effectively “prime” AML cells, making them more responsive to ATRA-induced differentiation, notably by augmenting the constitutive expression of critical regulators of myeloid differentiation. The inherent ability of retinoids to induce cell cycle arrest, primarily through the precise transcriptional regulation of various cell-cycle regulators, is universally recognized as a critical component of their therapeutic effects. In addition, other molecules known to induce cell cycle arrest, such as the histone deacetylase (HDAC) inhibitor valproic acid, have also been shown to enhance ATRA-induced differentiation of non-APL AMLs, indicating a shared mechanism of action. Thus, the induction of cell cycle arrest, mediated by the synergistic combination of ATRA with SUMO inhibitors, appears to play an exceedingly important role in the observed pro-differentiating and anti-leukemic effects of these drugs.
To date, a vast and continuously expanding proteomic landscape reveals that more than 6000 SUMOylated proteins have been identified. Among this extensive repertoire are numerous transcription factors and crucial co-regulators, some of which play absolutely key and indispensable roles in orchestrating myeloid differentiation. This is notably the case for CEBPα and CEBPε. While their SUMOylation has been shown to repress and activate their respective transactivation capacities, this context-dependent regulation highlights the complexity of SUMOylation’s impact on gene expression. The all-trans retinoic acid receptor (RARα) itself also undergoes dynamic and reversible SUMOylation/deSUMOylation cycles, which are unequivocally essential for its ATRA-induced activation and subsequent transcriptional activity. The SUMOylation of these and other crucial transcription factors could indeed play a significant part in the widespread epigenetic silencing of ATRA-responsive genes that we have meticulously uncovered in non-APL AMLs. However, the transcriptional repression of differentiation-associated genes by the SUMO pathway might not solely result from the SUMOylation of single, isolated transcription factors. Instead, it is increasingly understood to stem from the coordinated SUMOylation of multiple proteins that are intricately bound to critical regulatory elements within the promoter and enhancer regions of these genes. Supporting this latter and more comprehensive possibility is the emerging concept of “group SUMOylation,” which originally gained traction from detailed studies on DNA repair regulation. This concept implies that SUMO can effectively exert its function wherever it is conjugated within a larger multiprotein complex comprising several SUMOylatable proteins, leading to a collective regulatory effect. The SUMOylation of such promoter-bound protein complexes is thought to favor, through specific SUMO/SIM (SUMO-interacting motif) interactions, the recruitment of potent transcriptional repressors. These repressors include well-known complexes such as the N-Cor/HDAC complex and the CoRest/LSD1 complex, both of which are associated with chromatin condensation and gene silencing. Therefore, the strategic inhibition of SUMO conjugation would logically lead to a measurable decrease in their recruitment onto the chromatin, which would, in fine, facilitate and enhance both the basal and the ATRA-induced expression of genes critically involved in myeloid differentiation, thereby promoting a more differentiated and less malignant cellular state.
Our comprehensive work compellingly indicates that combining ATRA with pharmacological inhibitors of the SUMO pathway profoundly potentiates the inherent pro-differentiating, anti-proliferative, and pro-apoptotic effects of ATRA on non-APL AMLs. This enhanced efficacy extends remarkably even to those AMLs that have developed resistance to the genotoxic chemotherapeutics currently utilized in clinical practice, highlighting a significant therapeutic advantage. Considering the inherent multiplicity and critical nature of essential cellular pathways that are meticulously controlled by SUMOylation, a complete and indiscriminate inhibition of SUMOylation would likely prove to be too profoundly toxic in human patients, potentially leading to unacceptable side effects due to its ubiquitous cellular roles. However, similar to the experiments conducted with anacardic acid in our previous work, we observed no general systemic toxicity of 2-D08 in preclinical mouse models. Both 2-D08 and anacardic acid are characterized as poorly efficient inhibitors of SUMOylation, inducing only a slight and controlled decrease in overall SUMO conjugation at the specific doses we employed. This partial and controlled inhibition likely explains their remarkably low systemic toxicity, allowing for a therapeutic window. Furthermore, hemizygous mice expressing approximately 50% of Ubc9, and consequently showing slightly reduced global SUMOylation activity, are fully viable and exhibit no overt phenotype, providing additional evidence that a limited reduction in cellular SUMOylation is not detrimental to essential physiological functions. This compelling evidence collectively suggests that a controlled and partial inhibition of the SUMO pathway could thus, when used in strategic combination with ATRA, preferentially target leukemic cells without overly affecting the vital functions of normal, healthy cells. In our in vivo experiments, the ATRA plus 2-D08 combination only managed to reduce AML growth in vivo but did not achieve a complete blockage or eradication of the tumors. Such a seemingly limited effect might, at first glance, appear to have no immediate clinical relevance. However, it is crucial to consider that 2-D08, much like anacardic acid, is characterized by a poor bioavailability, primarily linked to its hydrophobic nature. This limited systemic exposure and distribution could very well explain the relatively modest effects observed in vivo, despite strong in vitro efficacy. Therefore, the development of novel, more soluble SUMOylation inhibitors, or a significant improvement of the pharmacological properties and bioavailability of the existing ones, is unequivocally necessary. ML-792 has been recently described as a promising novel inhibitor of SUMOylation, demonstrating potent activity. However, its comprehensive pharmacological properties and in vivo bioavailability have not yet been thoroughly tested. Should these properties prove to be superior to those of 2-D08 and anacardic acid, this would undoubtedly permit a more in-depth and definitive assessment of the true therapeutic benefit of the ATRA plus SUMOylation inhibitor association in preclinical models and, ultimately, pave the way for its eventual clinical use in human patients. In conclusion, our comprehensive work strongly and consistently suggests that strategically targeting the SUMO pathway represents an exceptionally promising novel strategy to significantly enhance the clinical efficacy of ATRA in non-APL AML, thereby offering a crucial opportunity to profoundly improve the treatment outcomes for this challenging and poor-prognosis cancer.