Diamond-Blackfan anemia (DBA) is a rare bone marrow failure syndrome accompanied by cardiovascular, skeletal, and urogenital abnormalities. Most of the affected individuals carry mutations in ribosomal proteins, including RPS19, a component of the 40S ribosomal subunit. We developed a transgenic Rps19 mouse model harboring a deletion of conserved R67 that displays a variable phenotype ranging from mild hematopoietic defects to severe anemia and a set of other skeletal, muscular, and cardiac abnormalities with shorter survival. This mouse model exhibited an activation of the p53 signaling pathway in red blood cell committed hematopoietic stem and progenitor cells, affecting erythroid lineage development. Competitive transplantation assays using Rps19 R67∆ bone marrow progenitor cells confirmed that short-term repopulating hematopoietic stem cells (HSCs) and their progenitor lineages were affected, while their differentiation was rescued after deletion of the tumor suppressor Trp53. Rps19 R67∆ mutation leads to pre-ribosomal RNA (pre-rRNA) accumulation coupled with activation of p53, even at relatively immature hematopoietic stages. In conclusion, we present a mouse model that represents a powerful tool for exploring new therapeutic options for the treatment of ribosomal disorders, including DBA.

Introduction: This analysis evaluates the impact of early administration of the growth factor G-CSF during the first therapy cycle in 117 patients with acute myeloid leukemia (AML) treated with venetoclax-based combination therapy. Not only were the clinical characteristics of patients across three groups (de novo AML, N=28; secondary AML [sAML], N=39; salvage treatment [previously treated with HMA for HR-MDS], N=50) taken into account, but also the influence of genetic mutations on hematological parameters, treatment response, and survival. Methods: We retrospectively analyzed AML patients receiving venetoclax-based therapy, stratified by treatment cohort. Associations between G-CSF use, infection rates, and clinical outcomes were assessed using chi-squared tests and Kaplan-Meier survival analysis. Multivariate Cox regression evaluated the impact of G-CSF, infections, and neutrophil recovery on OS. Separately, the influence of recurrent gene mutations on hematologic parameters, complete remission (CR), and OS was analyzed using linear regression, penalized logistic regression, and multivariate Cox models. Statistical significance was set at p < 0.05. Analyses were conducted in R (v4.4.3). Results: Use of G-CSF showed a statistically significant difference between the subgroups (χ² = 11.89; p = 0.0026). G-CSF was most frequently administered in patients in the sAML (89.7%) and salvage (78.0%) groups, while in de novo AML it was administered in only 53.6% of cases. The incidence of severe grade 4 neutropenia also differed significantly between groups (χ² = 8.56; p = 0.0138). The highest relative incidence was observed in patients with de novo AML (28.6%) and sAML (25.6%), while in the salvage group, Gr 4 neutropenia was present in only 6.0% of patients. The incidence of infections was higher in de novo and sAML patients and lower in the salvage group (p = 0.0099). The presence of infection during the first therapy cycle was associated with worse overall survival, although this did not reach statistical significance (p = 0.04). We did not observe statistically significant differences in response among subgroups (p = 0.2615), although the de novo group achieved the highest CR rate (64 %). Survival according to Kaplan-Meier analysis suggests a poorer prognosis for the salvage group. However, G-CSF administration was not associated with a decrease in overall survival (OS). Analysis of the effect of genetic mutations found in 97 patients showed that the presence of certain aberrations can significantly affect both the chance of achieving complete remission (CR) and selected hematological parameters. The most significant positive effect on achieving CR was observed for the IDH2 mutation, which was associated with a statistically significant higher probability of treatment response (OR 6.32; p = 0.0285). The NPM1 mutation also increased the chance of achieving CR (OR 4.62; p = 0.0500) and was associated with a trend toward improved overall survival (HR ~0.65). In contrast, EZH2 mutation indicated a negative impact on treatment response (OR 0.00), and although this effect was not statistically significant for survival. The mutations were further analyzed for their relationship to laboratory parameters such as baseline hemoglobin level (Hb), platelets (Plt), absolute neutrophil count (ANC), and percentage of blasts in peripheral blood. Mutations in EZH2 was associated with a significant decrease in hemoglobin levels (−30.13 g/L; p < 0.001), ASXL1 led to a significant reduction in ANC. Conclusions Based on the available data, we conclude that early administration of G-CSF during first-line treatment of AML is safe even in genetically at-risk patients. These results point to different patterns of toxicity and supportive care between clinical subgroups of AML, with the use of G-CSF not fully corresponding to the incidence of baseline severe neutropenia. The use of G-CSF may promote hematological regeneration in selected patients without fear of accelerating the progression of AML. Administration has no negative effect on survival. IDH2 and NPM1 mutations appear to be favorable in terms of treatment response, while EZH2 and ASXL1 mutations are associated with adverse changes in blood parameters. These results underscore the importance of the patient's genetic profile in predicting clinical course and response to therapy.

The interplay between chromatin remodelers and pioneer transcription factors (TFs) regulates cis-regulatory element accessibility to maintain cell identity and transcriptional fidelity. We investigated the impact of imitation of switch (ISWI) chromatin remodelers, key regulators of nucleosome spacing, on macrophage differentiation and activation, focusing on SMARCA5, the sole ISWI ATPase in myeloid cells. Conditional Smarca5 deletion in bone marrow-derived macrophages disrupted nucleosome phasing near sites bound by PU.1, a pioneer TF essential for myeloid identity, without altering PU.1 occupancy. However, SMARCA5 loss increased accessibility at motifs bound by C/EBPβ, a weak pioneer TF, enabling binding to regulatory regions active in non-hematopoietic lineages and causing lineage-inappropriate transcription. These changes also increased accessibility at sites bound by stimulus-induced TFs, leading to macrophage hyperactivation and mis-expression of stimulus-inappropriate genes. Thus, SMARCA5-dependent nucleosome phasing restrains C/EBPβ and stimulus-induced TF binding, ensuring transcriptional fidelity during macrophage lineage specification and activation, with likely similar roles in other immune cell types.

IntroductionProgressing myelodysplastic syndrome (MDS) into acute myeloid leukemia (AML) is an indication for hypomethylating therapy (HMA, 5-Azacytidine (AZA)) and a BCL2 inhibitor (Venetoclax, VEN) for intensive chemotherapy ineligible patients. Mouse models that engraft primary AML samples may further advance VEN + AZA resistance research.MethodsWe generated a set of transplantable murine PDX models from MDS/AML patients who developed resistance to VEN + AZA and compared the differences in hematopoiesis of the PDX models with primary bone marrow samples at the genetic level. PDX were created in NSGS mice via intraosseal injection of luciferase-encoding Lentivirus-infected MDS/AML primary cells from patient bone marrow. We validated the resistance of PDX-leukemia to VEN and AZA and further tested candidate agents that inhibit the growth of VEN/AZA-resistant AML.Results and discussionTransplantable PDX models for MDS/AML arise with 31 % frequency. The lower frequency of transplantable PDX models is not related to peritransplant lethality of the graft, but rather to the loss of the ability of short-term proliferation of leukemic progenitors after 10 weeks of engraftment. There exist subtle genetic and cytological changes between primary and PDX-AML samples however, the PDX models retain therapy resistance observed in patients. Based on in vitro testing and in vivo validation in PDX models, Panobinostat and Dinaciclib are very promising candidate agents that overcome dual VEN + AZA resistance.

Disruption of redox metabolism is a hallmark of drug-resistant cancer cells, representing a major obstacle to the effective treatment of acute myeloid leukemia (AML). While recent studies have highlighted the importance of redox balance in AML therapy, the specific contribution of protein redox signaling to resistance remains poorly understood. Defining these mechanisms could uncover therapeutic vulnerabilities of resistant AML cells and guide the development of novel combination strategies. Here, we performed comprehensive mass spectrometry-based redox and quantitative proteomic profiling of AML cell lines and patient samples sensitive or resistant to the hypomethylating agent azacitidine (AZA). We demonstrate that AZA disrupts redox homeostasis, which inactivates the glyoxalase system and DNA damage response, and thereby induces cell death. In contrast, AZA resistance is associated with a redox reset characterized by elevated glutathione levels and diminished protein S-glutathionylation. Importantly, AZA failed to induce oxidation of proteins in these pathways in resistant cells and patient-derived AML samples. Pharmacological inhibition of glutathione synthesis restored protein S-glutathionylation and resensitized resistant AML cells to AZA.

BackgroundLuspatercept, an inhibitor of the transforming growth factor beta (TGF-β) pathway, is a novel treatment for anemic patients with lower-risk myelodysplastic syndromes (MDS) with transfusion dependence (TD) who do not respond to erythropoiesis-stimulating agents (ESA) therapy or are not suitable candidates for this treatment. We present real-world experience with luspatercept therapy from two hematology centers in the Czech Republic.MethodsBy January 2024, 54 MDS patients (33 men, 21 women) with a median age of 74 years (range, 55–95) were treated with luspatercept ± ESA at two Charles University hematology centers in Prague and Hradec Králové. According to the WHO 2016 classification, the cohort included 32 MDS-RS-MLD, seven MDS-MLD, two patients with 5q- + ring sideroblasts (RS), 12 RARS-T, and 1 patient with CMML-0 + RS. SF3B1 mutation data were available for 45 patients. All patients were in the IPSS-R and IPSS-M lower-risk groups (except four IPSS-M high). The median follow-up was 17 months (range, 1–54). All patients were transfusion-dependent. Thirty-five (64.8%) patients had a high transfusion burden (HTB) with ≥ 4 transfusion units (TU)/8 weeks, and 19 (35.2%) had a low transfusion burden (LTB) (< 4 TU/8 weeks). The median time between diagnosis and initiation of luspatercept was 27 months (range, 4–156). ESA were used prior to luspatercept in 45 patients, and luspatercept was used as first-line treatment in nine patients. Thirty-one (61%) patients were treated simultaneously with ESA.ResultsOnly patients who received luspatercept for ≥ 8 weeks (51 patients) were assessed. We evaluated the achievement of transfusion independence (TI) lasting 8, 12, 16, and 24 weeks. Thirty-two (62.7%) patients achieved TI for ≥ 8 weeks, 31 (60.7%) for ≥ 12 weeks, 29 (56.8%) for ≥ 16 weeks, and 25 (49%) for ≥ 24 weeks. Hematologic improvement (HI) without TI was achieved in six patients (11.7%). Overall, HI + TI was achieved in 38 patients (74.5%). Epoetin alfa was used simultaneously in 31 patients (60.7%). In 21 (55.2%) of all responding patients, concomitant therapy with epoetin alfa led to an improved response, with 16 reaching TI. Thirteen (25.5%) patients were nonresponders. Eight (21%) patients experienced therapy failure and became transfusion-dependent again. Optimal response required a gradual increase in the luspatercept dose to 1.75 mg/kg in up to 35 patients, with 23 responders (TI + HI). Response rates varied by transfusion burden: 79% in LTB and 50% in HTB reached TI. Of RS+ patients, 70% reached TI, while only one out of five RS− patients achieved TI. Among 39 SF3B1-positive patients, 61.6% achieved TI. In the low and very low IPSS-M groups, 86% of patients responded (TI + HI), compared to 62% in the moderate-low group. Luspatercept was well-tolerated, with no adverse events higher than grade II toxicity.ConclusionWe have demonstrated in real-world clinical practice that luspatercept is a very effective agent, even in an unselected, pretreated, significantly TD MDS population. The effect was particularly high in the IPSS-M low and very low groups. We believe that the relatively high response rate in our patients was influenced by the frequent use of a higher dose (1.75 mg/kg) and especially by adding ESA to luspatercept in poorly responding patients.

The mutations of splicing factor genes (e.g., SF3B1, SRSF2, U2AF1, ZRSR2) are found in approximately 60% of MDS patients and lead to the accumulation of R-loops and associated DNA damage, resulting in the activation of the ATR pathway in affected cells. The most commonly mutated gene is SF3B1. These are hotspot mutations, mostly missense substitutions, with K700E being the most common variant. SF3B1 mutations are strongly associated with MDS with ring sideroblasts, which has a relatively slower progression. However, the results are heterogeneous, so identifying additional features that may affect the prognosis of MDS patients with SF3B1 mutations could lead to a better understanding of the disease and improved treatment. The aims of the study were to assess the frequency of SF3B1 mutations, the prognostic value of different mutation variants, and the association of SF3B1 mutations with cytogenomic findings and co-mutations with other genes. Bone marrow cells of 386 MDS patients were investigated using a combination of cytogenomic methods (G-banding, I-FISH, mFISH/mBAND, aCGH/SNP) and next-generation sequencing (NGS) with the Archer VariantPlex Myeloid panel (ITD), covering 75 genes associated with myeloid malignancies. SF3B1 mutations were confirmed in 60/386 patients (15.5%). Among these, 29/60 cases (48.3%) had a normal karyotype, 8/60 (13.3%) had isolated del(5q), 9/60 (15.0%) had a complex karyotype, and 14/60 patients (23.3%) had other chromosomal aberrations. In accordance with previously published data, patients with mutated SF3B1 had better overall survival than patients with SF3B1 wildtype (p=0,018). K700E was the most common variant, detected in 34/60 cases (46.7%). The most common genes co-mutated with SF3B1 were TET2 (19 cases), DNMT3A (13 cases), RUNX1 (13 cases), ASXL1 (7 cases), TP53 (6 cases), and JAK2 (6 cases). For further analyses, patients with an SF3B1 mutation were divided into four groups based on cytogenomic and molecular findings: group SF3B1α[other co-mutation than SF3B1β; 30 cases], group SF3B1β[the co-mutation with any gene from BCOR, BCORL1, NRAS, RUNX1, SRSF2, STAG2; 18 cases], group SF3B1del5q [the presence with isolated del(5q) without other co-mutations; 3 cases] and group SF3B1complex [the association with complex karyotype; 9 cases]. Patients included in the SF3B1α had the most favorable IPSS-M prognostic score, while patients in the SF3B1complex category had the worst. Nine of the 60 SF3B1-mutated cases (15%) fell into the IPSS-M very high-risk category. Five of these patients had a complex karyotype (in three cases associated with a TP53 mutation and in one case associated with mutations of NRAS and RUNX1 genes). In two other patients, we detected the K666N variant of SF3B1 mutation, which is associated with increased progression of MDS and distinct RNA splicing. Of the nine patients in this group, six have died. Three are still alive and are undergoing azacitidine treatment at 6.5, 8.5, and 21 months after their diagnosis. Identification of splicing factor gene mutations is an important diagnostic tool for the stratification of MDS patients. According to IPSS-M, SF3B1 mutations have a more favorable prognostic score compared to other splicing gene mutations (e.g., SRSF2 or U2AF1). However, the prognostic significance of individual variants may differ. Other biological factors such as the mutation variant, association with complex karyotypes, and mutations in other genes, may also affect the prognosis of patients with mutated SF3B1. Therefore, a comprehensive view that includes all cytogenomic, molecular, and clinical data is important for accurate diagnosis and personalized treatment of MDS patients. Supported by MH CZ-DRO 0064165

The establishment of long-lasting immunity against pathogens is facilitated by the germinal center (GC) reaction, during which B cells increase their antibody affinity and differentiate into antibody-secreting cells (ASC) and memory cells. These events involve modifications in chromatin packaging that orchestrate the profound restructuring of gene expression networks that determine cell fate. While several chromatin remodelers were implicated in lymphocyte functions, less is known about SMARCA5. Here, using ribosomal pull-down for analyzing translated genes in GC B cells, coupled with functional experiments in mice, we identified SMARCA5 as a key chromatin remodeler in B cells. While the naive B cell compartment remained unaffected following conditional depletion of Smarca5, effective proliferation during B cell activation, immunoglobulin class switching, and as a result GC formation and ASC differentiation were impaired. Single-cell multiomic sequencing analyses revealed that SMARCA5 is crucial for facilitating the transcriptional modifications and genomic accessibility of genes that support B cell activation and differentiation. These findings offer novel insights into the functions of SMARCA5, which can be targeted in various human pathologies.

BACKGROUND:Cell cycle progression and leukemia development are tightly regulated processes in which even a small imbalance in the expression of cell cycle regulatory molecules and microRNAs (miRNAs) can lead to an increased risk of cancer/leukemia development. Here, we focus on the study of a ubiquitous, multifunctional, and oncogenic miRNA-hsa-miR-155-5p (miR-155, MIR155HG), which is overexpressed in malignancies including chronic lymphocytic leukemia (CLL). Nonetheless, the precise mechanism of how miR-155 regulates the cell cycle in leukemic cells remains the subject of extensive research. METHODS:We edited the CLL cell line MEC-1 by CRISPR/Cas9 to introduce a short deletion within the MIR155HG gene. To describe changes at the transcriptome and miRNome level in miR-155-deficient cells, we performed mRNA-seq/miRNA-seq and validated changes by qRT-PCR. Flow cytometry was used to measure cell cycle kinetics. A WST-1 assay, hemocytometer, and Annexin V/PI staining assessed cell viability and proliferation. RESULTS:The limited but phenotypically robust miR-155 modification impaired cell proliferation, cell cycle, and cell ploidy. This was accompanied by overexpression of the negative cell cycle regulator p21/CDKN1A and Cyclin D1 (CCND1). We confirmed the overexpression of canonical miR-155 targets such as PU.1, FOS, SHIP-1, TP53INP1 and revealed new potential targets (FCRL5, ISG15, and MX1). CONCLUSIONS:We demonstrate that miR-155 deficiency impairs cell proliferation, cell cycle, transcriptome, and miRNome via deregulation of the MIR155HG/TP53INP1/CDKN1A/CCND1 axis. Our CLL model is valuable for further studies to manipulate miRNA levels to revert highly aggressive leukemic cells to nearly benign or non-leukemic types.

Introduction: Myelodysplastic syndromes (MDS) are difficult-to-treat myeloid malignancies characterized by dysplasia, peripheral cytopenia, and progression to acute myeloid leukemia (AML). The standard treatment for high-risk MDS is inhibition of DNA methyltransferase with 5-azacytidine (AZA). Pevonedistat (PEVO) is a small molecule inhibitor of the activating enzyme NEDD8 that downregulates Cullin ring ligases (CRLs), which interferes with the trafficking and degradation of proteins in the proteasome and leads to the accumulation of CRL substrates. One of the CRL substrates is Nuclear factor erythroid 2-related factor 2 (NRF2) which represents a first line of antioxidant defense and has been previously implicated in drug resistance. The combination of AZA with PEVO was compared to AZA arm in patients with MDS in phase 2, multicenter, global, randomized, controlled, open-label PANTHER trial (NCT02610777, L. Ades 2022). AZA+PEVO arm was as safe as AZA alone and provided benefits in the OS and ORR among patients with higher-risk MDS. However, the primary endpoint, EFS, was not met, which may also implicate the emergence of therapeutic resistance. In the present study, we investigated the role of the NRF2 antioxidant pathway in the development of resistance to PEVO. Methods: We derived PEVO-resistant (PEVO-R) clone from AZA-resistant cells (PEVO-S) developed in our lab (Minarik et al., 2022) by sequential treatment of the human OCI-M2 MDS/AML cell line with PEVO and analyzed them using whole exome sequencing, transcriptomic and proteomic analyses. We applied a quantitative mass spectrometry-based proteomic approach to identify protein targets of NRF2-driven redox changes in PEVO-S and PEVO-R clones. Autophagy was determined using flow cytometry and immunodetection. Results: DNA sequencing revealed mutation of the PEVO target such as Ubiquitin activating enzyme 3 UBA3Leu227Valat the site of interaction with NEDD8, which resulted in loss of response to PEVO. The transcriptomic analysis identified 14,484 protein-coding genes, of which 6,097 were significantly differentially expressed in PEVO-treated PEVO-S compared to untreated control. Resistance to PEVO displayed 2,241 differentially expressed genes in comparison to PEVO-S. We identified 148 out of 353 (42%) overexpressed NRF2-targets in PEVO-treated PEVO-S in contrast to 48 genes in PEVO-R. Neither NRF2 nor its upstream regulator Kelch-like associated protein 1 (KEAP1) were differentially expressed, instead PEVO induced expression of sequestosome-1 (SQSTM1), providing a positive feedback loop for NRF2 activation. We also found higher accumulation of NRF2 in the nucleus of PEVO-R cells in comparison to PEVO-S cells. The proteome of PEVO-R cells was significantly more oxidized compared to PEVO-S. Out of 6,367 identified and quantified cysteine peptides the PEVO-R cells displayed 275 significantly more oxidized cysteines compared to PEVO-S. Moreover, PEVO-R cells had significantly oxidized CYS289 and CYS290 of SQSTM1. The S-acylation of those cysteines was linked to autophagy promotion. Indeed PEVO-R cells had markedly active autophagy and were sensitive to autophagy inhibitors in comparison to PEVO-S. Conclusions: Our data suggest that PEVO induces accumulation of SQSTM1, thereby activating autophagy and NRF2, and placing cells in a transient state that protects them from oxidative damage associated with PEVO administration. This allows mutagenesis to occur which results in complete loss of PEVO responsiveness possibly via mutating UBA. Thus, the SQSTM1-KEAP1-NRF2 pathway appears to be a major checkpoint during the treatment of MDS with PEVO and could stay behind the loss of therapeutic efficacy in MDS patients. Grants: AZV (NU21-08-00312), GAUK (273223), GAČR (24-10353S), EXCELES (LX22NPO5102).

The formation of hematopoietic cells relies on the chromatin remodeling activities of ISWI ATPase SMARCA5 (SNF2H) and its complexes. The Smarca5 null and conditional alleles have been used to study its functions in embryonic and organ development in mice. These mouse model phenotypes vary from embryonic lethality of constitutive knockout to less severe phenotypes observed in tissue-specific Smarca5 deletions, e.g., in the hematopoietic system. Here we show that, in a gene dosage-dependent manner, the hypomorphic allele of SMARCA5 ( S5 tg ) can rescue not only the developmental arrest in hematopoiesis in the hCD2iCre model but also the lethal phenotypes associated with constitutive Smarca5 deletion or Vav1iCre-driven conditional knockout in hematopoietic progenitor cells. Interestingly, the latter model also provided evidence for the role of SMARCA5 expression level in hematopoietic stem cells, as the Vav1iCre S5 tg animals accumulate stem and progenitor cells. Furthermore, their hematopoietic stem cells exhibited impaired lymphoid lineage entry and differentiation. This observation contrasts with the myeloid lineage which is developing without significant disturbances. Our findings indicate that animals with low expression of SMARCA5 exhibit normal embryonic development with altered lymphoid entry within the hematopoietic stem cell compartment.

Concrete is the common extensively utilized man-made material in the world. Concrete plays an important role in our Infrastructure development that shows our status worldwide among the other nations. One of the difficulties that have arisen since the introduction of concrete structures in the building business is how to increase concrete strength while still being efficient and cost-effective. The use of novel materials in concrete will boost the strength of the concrete in a more significant way; one such development is High Strength Concrete.

Background: Hypomethylation therapy with 5-azacytidine (AZA) represents the treatment of choice for patients with high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) with dysplasia unsuitable for transplantation and also represents consolidation in AML with oral AZA. AZA inhibits tumor cell proliferation and induces cell death through inhibition of DNA methylation. While AZA often leads to a hematologic response under well-tolerated therapy, the main concern is the emergence of resistance leading to loss of response. In this work, we reveal the relationship between AZA resistance and cellular oxidation/reduction (redox) homeostasis. We previously noted that AZA regulates cell death/survival pathways through modulation of redox homeostasis and oxidative modification of proteins and activates key factors of the redox signaling pathway involving Kelch like associated protein 1 (KEAP1) and nuclear factor erythroid 2-related factor 2 (NRF2). Aims: Our aim was to identify the AZA-specific oxidized protein cysteines that would be related to AZA resistance and to investigate how specifically the KEAP1-NRF2 pathway is involved in this process. Methods: To identify AZA-associated key oxidative modifications of protein cysteine residues we used a mass spectrometry-based quantitative proteomic approach and corroborated these data with functional and transcriptomic analyses. The study was performed on MDS/AML clones of the OCI-M2 cell line that were prepared using AZA selection (AZA-R). Total cellular redox state and glutathione (GSH) levels were measured by flow cytometry. Results: AZA resistance is marked by a significantly higher oxidation state and higher GSH compared to the parental state. Thus, AZA resistant cells are less sensitive to oxidizing agents such as diamide. While in parental cells AZA has the ability to rapidly increase the oxidative state of the cells, this does not occur in AZA-R. Upon GSH depletion, we achieve a significant increase in sensitivity to AZA. We next performed a proteomic analysis to identify specific protein targets of AZA mediated oxidation and found a redox change in 15% of proteins (403 out of 2’643), many of them involved in biological processes such as AZA metabolism and cell survival/death pathways. Among proteins with the significantly altered redox state was the Sequestosome-1 (SQSTM1)-KEAP1-NRF2 antioxidant pathway. NRF2 is a transcription factor that responds to redox stress by inducing transcription of antioxidant enzymes. However, in contrast to the parental cells AZA does not stimulate the NRF2 downstream target program in AZA-R cells. Therefore, we inhibited KEAP1, which acts as a ubiquitin ligase for NRF2, and this treatment led to a change in the redox balance in AZA-R cells and restoration of sensitivity to AZA. To validate the identified mechanism under in vivo conditions, we transplanted AZA-R cells into immunodeficient CDX mice and used different KEAP1 inhibitors to demonstrate prolonged event-free survival and overall survival of MDS/AML mice after AZA treatment. Next, we studied the KEAP1-NRF2 pathway and, in particular, the redox modifications identified in model systems in myeloblasts from MDS/AML patients. Despite heterogeneous results, we found encouraging agreements with in vitro systems that are subject to validation. Summary/Conclusion: We revealed that the mechanism of AZA resistance involves redox reset and modulation of the KEAP1-NRF2 cellular antioxidant response pathway. Complete sensitization of AZA-resistant cells represents a previously unconsidered and tempting way to enhance the efficacy of AZA therapy. This opens the way for further studies to modulate the KEAP1-NRF2 pathway to block AZA resistance in patients.Keywords: Proteomics, Azacitidine, Myelodysplastic syndrome, Drug resistance

Chromatin remodeling complexes are required for many distinct nuclear processes such as transcription, DNA replication, and DNA repair. However, the contribution of these complexes to the development of complex tissues within an organism is poorly characterized. Imitation switch (ISWI) proteins are among the most evolutionarily conserved ATP-dependent chromatin remodeling factors and are represented by yeast Isw1/Isw2, and their vertebrate counterparts Snf2h (Smarca5) and Snf2l (Smarca1). In this study, we focused on the role of the Snf2h gene during the development of the mammalian retina. We show that Snf2h is expressed in both retinal progenitors and post-mitotic retinal cells. Using Snf2h conditional knockout mice (Snf2h cKO), we found that when Snf2h is deleted, the laminar structure of the adult retina is not retained, the overall thickness of the retina is significantly reduced compared with controls, and the outer nuclear layer (ONL) is completely missing. The depletion of Snf2h did not influence the ability of retinal progenitors to generate all the differentiated retinal cell types. Instead, the Snf2h function is critical for the proliferation of retinal progenitor cells. Cells lacking Snf2h have a defective S-phase, leading to the entire cell division process impairments. Although all retinal cell types appear to be specified in the absence of the Snf2h function, cell-cycle defects and concomitantly increased apoptosis in Snf2h cKO result in abnormal retina lamination, complete destruction of the photoreceptor layer, and consequently, a physiologically non-functional retina.