Myelodysplastic Syndrome Cell Line Research Articles

A new therapeutic perspective: Erastin inhibits tumor progression by driving ferroptosis in myelodysplastic syndromes.

Ferroptosis is a recently identified and evolutionarily conserved form of programmed cell death. This process is initiated by an imbalance in iron metabolism, leading to an overload of ferrous ions. These ions promote lipid peroxidation in the cell membrane through the Fenton reaction. As the cell's antioxidant defenses become overwhelmed, a fatal buildup of reactive oxygen species (ROS) occurs, resulting in the rupture of the plasma membrane. Ferroptosis is implicated in conditions such as ischemia-reperfusion injuries and a range of cancers. In our research, we explored ferroptosis in myelodysplastic syndromes (MDS) by measuring iron levels, transferrin receptor expression, and glutathione peroxidase 4 (GPX4) mRNA. Our findings revealed that MDS patients had significantly higher Fe2+ levels in CD33+ cells and increased transferrin receptor mRNA compared to healthy individuals. GPX4 expression was also higher in MDS but not statistically significant. To investigate potential treatments for myeloid hematological diseases through ferroptosis induction, we treated the myelodysplastic syndrome cell line (SKM-1) and two myeloid leukemia cell lines (KG-1 and K562) with erastin, an iron transfer inducer. We observed that erastin treatment led to glutathione depletion, reduced GPX4 activity, and increased ROS, culminating in cell death by ferroptosis. Furthermore, combining erastin with azacitidine demonstrated a synergistic effect on MDS and leukemia cell lines, suggesting a promising approach for treating these hematological conditions with this drug combination. Our experiments confirm erastin's ability to induce ferroptosis in MDS and highlight its potential synergistic use with azacitidine for treatment.

Elesclomol-copper synergizes with imidazole ketone erastin by promoting cuproptosis and ferroptosis in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) encompass a collection of clonal hematopoietic malignancies distinguished by the depletion of peripheral blood cells. The treatment of MDS is hindered by the advanced age of patients, with a restricted repertoire of drugs currently accessible for therapeutic intervention. In this study, we found that ES-Cu strongly inhibited the viability of MDS cell lines and activated cuproptosis in a copper-dependent manner. Importantly, ferroptosis inducer IKE synergistically enhanced ES-Cu-mediated cytotoxicity both in vitro and in vivo. Of note, the combination of IKE and ES-Cu intensively impaired mitochondrial homeostasis with increased mitochondrial ROS, MMP hyperpolarized, down-regulated iron-sulfur proteins and declined oxygen consumption rate. Additionally, ES-Cu/IKE treatment could enhance the lipoylation-dependent oligomerization of the DLAT. To elucidate the specific order of events in the synergistic cell death, inhibitors of ferroptosis and cuproptosis were utilized to further characterize the basis of cell death. Cell viability assays showed that the glutathione and its precursor N-acetylcysteine could significantly rescue the cell death under either mono or combination treatment, demonstrating that GSH acts at the crossing point in the regulation network of cuproptosis and ferroptosis. Significantly, the reconstitution of xCT expression and knockdown of FDX1 cells have been found to contribute to the tolerance of mono treatment but have little recovery impact on the combined treatment. Collectively, these findings suggest that a synergistic interaction leading to the induction of multiple programmed cell death pathways could be a promising approach to enhance the effectiveness of therapy for MDS.

Investigation of NK cell function against two target hematological cell line using radioactive chromium assay

NK (Natural killer) cells are a special population of peripheral blood lymphocytes that kill virus-infected cells as well as tumor cells. For testing NK cell function, the classic gold standard assay has been used for a long time, determining the activity from target tumor cells using radioactive chromium in cell cultures for 4h. In this study two hematological cell lines K562 and MDS where used and target and results showed different sensitivity to killing by NK cells separated from healthy volunteers. Results have been shown that MDS release significantly more radioactive chromium indicating higher degree of necrosis during cell culture. In addition, K562 cell line is better target for NK killing in all different E:T ratio in comparison to MDS cell line previously described. Based on this, it is suggested that K562 cells be continues used in the future as better target for investigation NK killing.

Distinct Role of STAT3 and STAT5 Factors in MDS and AML

Introduction: Myelodysplastic Syndrome (MDS) refers to a heterogeneous group of clonal hematologic disorders characterized by hematopoietic dysfunctions and impaired differentiation in the hematopoietic stem and progenitor cells compartment. MDS is commonly referred to as a pre-leukemic stage due to the increased risk of progression to Acute Myeloid Leukemia (AML). AML is an aggressive hematologic malignancy characterized by the accumulation of immature myeloblasts in the bone marrow and the peripheral blood. Signal transducers and activators of transcription 3 and 5 (STAT3 and STAT5) are important regulators of several cellular processes including cell proliferation and survival. Abnormal STAT3 and STAT5 signaling has been implicated in various solid and hematologic malignancies, including MDS and AML, which renders them appealing targets for the development of novel therapeutic strategies. Materials: STAT3, STAT5A or STAT5B knock-downs have been generated in two AML (MOLM-13 and Kasumi-1) and two MDS (MDS92 and MDS-L, gift of Prof. Kaoru Tohyama) cell lines, using shRNAs through lenti-viral delivery. The 4 biological replicates with the highest levels of knock-downs for STAT5A, STAT5B or STAT3 have been selected for each cell line and mRNA sequencing has been performed. To define the genome-wide binding of STAT5A, STAT5B and STAT3 in the two conditions, CUT&Tag has been performed in wild type MOLM-13, Kasumi-1 and MDS-L cells. Additionally, ATAC sequencing has been carried out in the same cell lines to investigate the chromatin conformation changes between the two conditions. Furthermore, STAT3, STAT5A and STAT5B knock-downs have been conducted using shRNAs through lenti-viral delivery in CD34+ enriched cells, isolated from bone marrow mononuclear cells of 14 high-risk MDS patients and 14 secondary (MDS-related) AML patients. The samples have been processed for mRNA-Sequencing to model the leukemic transformation in primary human cells. Results: To elucidate the role of STAT3 and STAT5 in MDS to AML transformation, changes in STAT3 and STAT5 target gene networks have been identified. mRNA sequencing data from STAT3, STAT5A or STAT5B knock-downs in MDS and AML cell lines revealed a distinct role of the three factors in each state. The differential binding of each factor, explored through CUT&Tag, and the gene expression profiles in MDS and AML reciprocally reveal a different function of each factor, including pathways involved in cell cycle and apoptosis. Also, the direct and indirect target genes of STAT3, STAT5A and STAT5B in each condition, as well as a possible interplay between STAT3 and STAT5 activation in AML has been uncovered. The exploration of the chromatin accessibility landscapes between the MDS and AML conditions through ATAC sequencing has provided an insight into the chromatin conformation states, unveiling potential leukemia promoting networks, as well as the involvement of STAT3 and STAT5 pathways in the malignant transformation. Furthermore, a potential cross talk of STAT3 and STAT5 factors with the IKAROS family members, has been linked to AML transformation, as significant differential regulation has been detected between MDS and AML conditions after knock-down of STAT5A and STAT5B. The results are currently validated in primary hematopoietic cells of MDS and AML patients after STAT3, STAT5A or STAT5B knock-downs. Conclusions: Our results show a different role between the two STAT5 factors (STAT5A and STAT5B) in MDS and AML conditions and delineate the role of STAT5A, STAT5B and STAT3 factors and their target gene networks in leukemic transformation, thus providing novel targets for therapeutic management of MSD and AML. Funding: This project is funded by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 813091 (ARCH)

Selenium Deficiency and Chronic Anemia in an Outpatient Hematology Clinic

Background Selenium (Se) is an essential micronutrient with roles in reproduction, metabolism, immunity, and the cellular cycle, including DNA synthesis and prevention of cell death. Its role in malignancy has also been studied, specifically for esophageal, gastric, lung, prostate cancer. Studies have shown benefit with Se supplementation for those malignancies. There is evidence that low Se levels are independently associated with worse outcomes regarding treatment response and long-term survival for Non-Hodgkin's Lymphoma (NHL) and cutaneous T-cell lymphoma, and additional studies on its association in acute myeloid leukemia (AML), follicular lymphoma (FL), and Hodgkin's lymphoma (HL) have revealed worsening outcomes as well. However, Se was not predicative of treatment response or survival for AML, FL and HL, therefore it's role in the development of hematological malignancies is not clear. Previously, the role of Se in myelodysplastic syndrome (MDS) was investigated, indicating the importance of Se for apoptosis in MDS cell lines. There is evidence that a causal relationship with decreased Se levels and chronic anemia might exist; however, there is a paucity of data regarding its role in the development of chronic anemia and hematological disorders. This descriptive single center study describes Se deficiency in patients with chronic anemia. Objectives Primary endpoint was to determine if there was evidence of Se deficiency in patients with chronic anemia in an academic hematology outpatient clinic. Methods We herein present a descriptive study on 18 patients with hematological disorders and selenium deficiency. Upon initial detection of Se deficiency, levels of hemoglobin (Hb), mean corpuscular volume (MCV), Copper (Cu), Zinc (Zn) and iron (Fe) closest to time of detection were noted if tested. Average values with standard deviation were calculated and association with malignancies were noted. Results Of the 18 patients, 13 had pre-malignant or malignant conditions, including 7/13 who had bone marrow failure/MDS, 5/13 patients with lymphoid disorders, and 1/13 with plasma cell disorders. The 5 remaining patients had chronic gastrointestinal disorders. Averages on detection of Se deficiency were noted to be: Hb (9.9 ± 2.0), MCV (96.0 ± 11.2), Se (100.5 ± 11.6), Cu (95.8 ± 28.8), Zn (70.1 ± 21.5) and Fe (81.6 ± 56.7). No association was noted between Se and other trace elements. 72% (13/18) of patients with Se deficiency also had malignant conditions. Of those with malignancy, 54% were noted to have bone marrow failure. Discussion Currently there are no clinical guidelines that recommend following levels of Se routinely in patients with chronic anemia. Our data might indicate a possible association between chronic anemia and Se deficiency. Given the high number of MDS patients in our study, there might be a link between Se deficiency and MDS. Further investigation is warranted into the role of Se in the development of chronic anemia disorders, as supplementation with Se and routine follow-up may be warranted and can improve clinical outcomes.

Safety and Efficacy of Selinexor Sequential Azacytidine in Newly Diagnosed Patients with Myelodysplastic Syndromes EB1 or EB2: A Single-Center, Single-Arm, Phase Ib/II Trial

Background: Azacytidine is one of the hypomethylating agents (HMAs) for the treatment of higher-risk myelodysplastic syndromes (MDS). However, clinical outcomes of patients treated with azacytidine monotherapy were far from satisfactory with overall response rate (ORR) of 41%-59% and overall survival (OS) of 15-18 months. Selinexor is an oral, first-in-class selective inhibitor of nuclear export compound that inhibits exportin 1 (XPO1). Preclinical studies have shown that inhibition of XPO1 causes nuclear accumulation of p53 and disruption of NF-κB signaling, both relevant targets for myelodysplastic syndromes. Prior study reported an ORR of 26% and mOS of 8.7 months with selinexor in HMAs-refractory MDS patients. We therefore aimed to assess the safety and efficacy of selinexor sequential azacytidine in newly diagnosed patients with MDS-EB1 or EB2. Methods: The effectiveness of simultaneous and sequential treatment of azacytidine and selinexor was measured by cell death rate, cell cycle and colony formation rate in vitro. Western Blot was performed to detect the sublocation of p53. MOLM-13-luciferase xenograft murine model was established to verify the synergetic effect in vivo. Based on the experiments, we conducted a single-center, single-arm, phase Ib/II trial, which included newly diagnosed patients 18 years or older with MDS-EB1 or EB2. Eligible patients received 2-4 weeks long cycles of oral selinexor (40mg qw for 4 weeks; 40mg biw for 2 weeks; 60mg qw for 4 weeks). The primary endpoints were to find the recommended phase 2 dose (RP2D) and maximum tolerated dose (MTD). The secondary endpoint was ORR including complete remission (CR), partial remission (PR), marrow complete remission (mCR), and hematological improvement (HI). This trial was registered with the Chinese Clinical Trial Registry (#ChiCTR2200065167). Results: First we treated three AML cell lines: AML-OCI2, THP1, MV4-11 and three MDS cell lines: SKM-1, MOLM-13, MDSL with azacitidine and selinexor simultaneously or sequentially. Sequential treatment showed stronger synergetic effect (Figure 1A) and reduced colony formation by 90% in SKM-1 and MOLM-13 (Figure 1B). Cell cycle arrest were induced and the percentage of Annexin V cells obviously increased with sequential treatment (Figure 1C). Sequential treatment decreased leukemia burden and prolonged survival of MOLM-13-luciferase xenograft murine model (Figure 1D, E). Mechanistically, pretreatment of azacitidine on SKM-1 and MOLM-13 upregulated the expression level of p53, and selinexor led to massive nuclear accumulation of p53, resulted in better vulnerability lethal to MDS cells (Figure 1F). Secondly, 15 patients were enrolled in our trial with a median age of 57. 26.7% of patients were MDS-EB1, 73.3% were EB2. 93.3% of patients were high or very high risk by the Revised International Prognositic Scoring System (IPSS-R) (see the Table). At Phase Ib, the following selinexor dosages were administered: 40mg qw (n=3), 40mg biw (n=3) and 60mg qw (n=3). One patient in the 60mg qw group discontinued treatment because of nausea, vomiting, fatigue and Herpes simplex virus infection. Subsequently, in order to find RP2D, an extension cohort of 3 additional patients were treated with 40mg qw and 40mg biw, respectively. Therefore, 14 patients were evaluable for efficacy assessment. ORR was 78.6% in 11 patients including CR in 3 patients (21.4%); mCR+HI in 1 patient (7.1%); and mCR in 7 patients (50.0%) (see the swimmer plot). Especially, among the TP53 mutated patients (n=3), one achieved CR, the other two achieved mCR. The common adverse events were neutropenia (93.3%), anemia (86.7%), thrombocytopenia (80.0%), fatigue (53.3%), nausea (26.7%), dizziness (26.7%), vomiting (20.0%) and decreased appetite (20.0%). There were no treatment related deaths. Conclusion: We verified the activity of selinexor sequential azacytidine in MDS both in vitro and in vivo. The treatment showed with encouraging efficacy in newly diagnosed patients with MDS-EB1 or EB2. Adverse events were manageable with supportive care implementation. Enrollment is ongoing to assess the efficacy and safety of this sequential therapy, which may be a new treatment option for EB subjects.

Ubiquitin Ligase SCF-FBXO11 Controls a Network of RNA-Binding Proteins and Splicing in MDS

The F-Box protein FBXO11 is a substrate receptor of Skp-Cullin-F-Box (SCF) ubiquitin ligase complexes. Loss-of-function mutations in FBXO11 are associated with diffuse large B-cell lymphoma, and FBXO11 has been shown to regulate epigenetic drivers of erythroid cell maturation. In our prior study, we found that deletion of FBXO11 improved cell survival under cytokine deprivation in an MDS cell line. Furthermore, we had observed a striking decrease in FBXO11 protein levels in AML patient samples. However, how FBXO11 contributes to myeloid malignancies is unclear. Here, using RNA-sequencing, proteomics, and CRISPR-Cas9 screening, we reveal broad ubiquitylation of RNA-binding proteins (RBPs) and alternative splicing as a mechanism by which deregulation of FBXO11 enhances malignant cell function. In addition, we report the presence of mutations in the uncharacterized N-terminal extension of FBXO11 in both lymphoid and myeloid malignancies. We first observed that heterozygous deletion of FBXO11 increases colony-forming ability of MDS-AML cell lines in vitro whilea2-fold increase in expression results in decreased cell growth and apoptosis. In a transplant model with a retrovirally-driven Runx1 mutation, heterozygous deletion of Fbxo11 exacerbated neutropenia and shortened overall survival. However, depletion of FBXO11 in healthy human and murine HSPCs significantly impaired myeloid progenitor colony-formation, indicating a unique requirement for FBXO11 in myelopoiesis. Based on its tumor suppressive activity, we evaluated whole-exome sequencing data from patient samples of both lymphoid and myeloid malignancies for FBXO11 mutations. This analysis uncovered rare mutations in FBXO11 in myeloid malignancies and revealed a substantial cluster of previously unidentified mutations in the Q-rich, N-terminal extension of FBXO11. We found that this uncharacterized, extended protein isoform is predominantly expressed in both normal and malignant hematopoietic cells. In colony-forming assays, expression of the long isoform and several of the N-terminal mutations inhibited CD34+ progenitor function. On a cellular level, lack of this N-terminal extension in the short isoform resulted in aberrant subcellular localization of FBXO11 within the nucleus. To elucidate the mechanism of FBXO11 in myeloid malignancy, we performed immunoprecipitation of FBXO11 followed by proteomics to identify endogenous FBXO11 complexes in MDS-AML cells. We integrated these data with ubiquitin proteomics of WT and FBXO11-KO MDS cells to identify candidate substrates of SCF-FBXO11-mediated ubiquitylation. FBXO11 complexes are significantly enriched for RNA-binding proteins affecting mRNA splicing, a process commonly deregulated in MDS. FBXO11-KO MDS cells displayed broad depletion of ubiquitylation in a network of RBPs. As expected, targeting FBXO11 did not affect the level of transcripts of interacting proteins; however, there were variable changes in total protein levels indicative of post-transcriptional regulation. We then applied a focused CRISPR-Cas9-KO screen of candidate SCF-FBXO11 substrates in MDS-AML cells depleted of FBXO11. We observed significant bi-directional effects in colony-forming ability mediated by several of the FBXO11-regulated RBPs including TRIM28, HNRNPU, NPM1, and SYNCRIP. Though FBXO11 is significantly decreased in MDS compared to healthy controls, we saw no difference in FBXO11 expression between splicing factor WT and mutant samples, suggesting that the function of FBXO11 in regulating mRNA splicing in MDS is independent of these mutations. Compared to healthy controls, FBXO11-low MDS samples exhibited a greater number of alternative splicing events, predominantly in skipped or mutually-exclusive exons. To experimentally test whether FBXO11 indeed affects RNA splicing, we utilized a bichromatic splicing reporter assay in MDS-AML cells which showed that depletion of FBXO11 is sufficient to alter splicing of the reading frame in the cassette exon, read out as a shift from GFP to RFP. Finally, STRING protein network analysis revealed that the alternative splicing events occurring in FBXO11-low MDS patient samples affect key nodes in protein translation and metabolism. Collectively, our data support a model whereby the change in FBXO11-mediated ubiquitylation of RBPs drives alternative exon usage that exacerbates malignant cell function in MDS.

Preclinical Activity of Novel TGF Beta Receptor I Kinase Inhibitors IOA-359 and IOA-360 for Treatment of Anemia in MDS/AML

Background: Overactivation of the Transforming growth factor beta (TGF-β) superfamily has been associated with bone marrow failure in MDS. TGF-β binds to set of receptors that include the TGF-β receptor I kinase (also known as ALK5), that in turn phosphorylates and activates the downstream SMAD2/3 proteins. Activation of SMAD2/3 transcription factors has been shown to occur in MDS and is associated with anemia. Thus, we wanted to evaluate the preclinical efficacy of novel, clinic-ready, TGF-b receptor I kinase small molecule inhibitors IOA-359 and IOA-360 in MDS models. Results: Using the TREEspot™ SCAN Max Kinase Panel of 403 kinases, IOA-359 and IOA-360 were found to be highly selective for ALK5. Both, IOA-359 and IOA-360 have a blocking activity against ALK5 similar to clinical stage ALK5 inhibitors. To evaluate the efficacy of IOA-359 and IOA-360, we first used HEK 293- Blue-TGF-β cells that stably overexpresses a SMAD inducible secreted embryonic alkaline phosphatase (SEAP) reporter. Both IOA-359 and IOA-360 were able to significantly inhibit TGF-β driven reporter at nanomolar doses ( Fig1A). Next, we showed that both ALK5 inhibitors were able to block TGF-β-mediated SMAD2/3 downstream phosphorylation/activation in a dose dependent manner in MDS and leukemic cell lines. Functionally, the ALK5 inhibitors did not lead to any increase in viability or proliferation in leukemic cell lines. We next tested the efficacy of the novel ALK5 inhibitors in primary patient samples from MDS and AML (N=8). Treatment with both ALK5 inhibitors led to increase in erythroid colony formation in majority of MDS samples in clonogenic assays. Colonies were picked up and examined for erythroid and myeloid differentiation by FACS analysis. We observed that ALK5 inhibition led to increase in Glycophorin A and CD71 expression suggesting enhanced erythroid differentiation ( Fig1B). Myeloid differentiation effects were modest in comparison. Furthermore, in selected samples, addition of Luspatercept led to greater maturation of erythrocytes. Treatment of healthy CD34 + hematopoietic stem and progenitors did not lead to significant changes in differentiation. Lastly, to evaluate downstream effects of ALK5 inhibition, we performed RNAseq analysis in MDS cells. Inhibition with ALK5 inhibitors led to numerous changes in genes affecting cellular differentiation, validating functional changes observed by us. Conclusion: Our current results support the preclinical in vitro efficacy of ALK5 inhibitors IOA-359 and IOA-360 alone and in combination with Luspartercept, highlighting their potential for further development and clinical testing in MDS/AML.

The Zymogen Form of Caspase-1 Is Required to Fine Tune Excessive Cell-Intrinsic Inflammation in Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is characterized by the expansion of clonally-derived mutant hematopoietic stem and progenitor cells. Many patients do not respond to standard chemotherapy because AML cells acquire mutations that exploit inflammatory and cell death pathways, allowing them to evade cell death. Targeted therapies are urgently needed to block AML cell pro-survival and inflammatory signaling for more effective outcomes. One promising area of interest is Caspase-1 (CASP1), a cysteine-aspartic acid protease responsible for cleaving the precursors of interleukin-1β (IL-1β) and Gasdermin D, which induces pyroptosis, into their active mature peptides. In myeloid malignancies, IL-1β levels are significantly elevated, and pyroptosis has been linked to ineffective hematopoiesis as observed in MDS, thereby implicating CASP1. CASP1 is typically expressed as a zymogen (Pro-CASP1) that can be cleaved into an active enzyme. In this report, we present a novel and unexpected role for Pro-CASP1, which appears to be independent of its proteolytic function, in MDS/AML leukemic cells. Pyroptosis, an inflammatory cell death mechanism, is driven by CASP1-mediated osmotic lysis and IL-1b secretion. Notably, CASP1 expression is significantly higher in AML patients compared to healthy hematopoietic cells, and elevated CASP1 expression in AML patients correlates with shorter overall survival. In leukemic cells, CASP1 primarily exists in its zymogen form, lacking the ability to induce pyroptosis. It is thus unexpected that elevated CASP1 expression, which typically leads to heightened susceptibility to pyroptotic cell death, does not result in pyroptosis in leukemic cells, and the underlying mechanisms for this evasion remain unknown. To investigate Pro-CASP1's significance in AML cells, we used CRISPR-Cas9 to create Pro-CASP1 knockout (Pro-CASP1 KO) human isogenic MDS and AML cell lines (THP1 and MDSL). Pro-CASP1 KO MDS/AML cells showed reduced cell growth and leukemic colony forming potential in methylcellulose compared to isogenic WT control cells. When Pro-CASP1 KO THP1 AML cells were transplanted into immunocompromised mice, the loss of Pro-CASP1 significantly reduced leukemic cell burden and extended overall survival. We next preformed experiments to probe the mechanism of action of Pro-CASP1 in AML cells. Previous studies suggest that Pro-CASP1 can function as a scaffold to elicit downstream signaling without cleaving into its activated form. The functional defect in Pro-CASP1 KO THP1 AML cells was rescued with full-length Pro-CASP1, but not with a scaffolding mutant of CASP1, indicating a specific dependency of leukemic cells on the zymogen form of CASP1. To precisely delineate which domains of Pro-CASP1 are essential for AML, we performed a tiled CRISPR dropout screen in MDSL and THP1 cells. The results showed similar CRISPR-Knockout High Sensitivity (CKHS) domain footprints in both cell lines mapping to the N-terminal CARD and the Inter-Domain Linker (IDL) domains. These results indicate that Pro-CASP1's proteolytic function was found to be dispensable, whereas the CARD domain, a scaffolding region, was crucial for leukemogenesis. We next investigated how loss of Pro-CASP1 affects inflammatory signaling in AML cells. Deletion of Pro-CASP1 in THP1 AML cells led to excessive and constitutive activation of NF-kB as indicated by nuclear translocation and phosphorylation of p65/RelA. Restoring NF-kB regulation using the inhibitor of kappa B (IkB) super-repressor (IkB-SR) was sufficient to reverse the leukemic progenitor defect and cell viability in Pro-CASP1 KO AML cells. These findings suggest that Pro-CASP1 is required for AML cells to finetune canonical NF-kB activation. Conversely, loss of Pro-CASP1 results in excessive NF-kB activation that is incompatible with AML, consistent with previous observations (Ellegast et al., Cancer Discovery 2022). In summary, Pro-CASP1 serves as a critical signaling hub, fine-tuning NF-kB pathways independently of its proteolytic function in leukemic cells. Its loss exposes leukemic cells to functional and therapeutic vulnerabilities.

MDS-MSC Derived Exosomes Promote MDS Cells Malignant Proliferation through RIG-1/Isgs Pathway Via RN7SL1

The interactions between bone marrow stem cells (BM-MSCs) and myelodysplastic syndromes (MDS) cells perform a crucial role in MDS pathogenesis by secreting cytokines ,growth factors, and exosomes. Although BM-MSCs induced growth of MDS cells has been studied, the role of exosomes from MDS-derived MSC (MDS-MSC-EXO) in this action remains unclear. In our study, we investigate the effect of MDS-MSC-EXO on the viability, proliferation, migration, cellular apoptosis of MDS cell lines, such as MDS-L and SKM-1. Our data identified that MDS-MSC-EXO can promote proliferation, migration, and decrease cell apoptosis. Both LR-MDS-MSC and HR-MDS-MSC-derived exosomes increased MDS cell growth.Exosomes mediate local cell-to-cell communication by transferring mRNAs, miRNAs, and proteins. In our study, we found that MDS-MSC-EXO expressed higher unshielded RN7SL1,an endogenous RNA normally shielded by RNA binding proteins SRP9/14, than that from health controls (HC-MSC-EXO). MDS cloned cells expressed higher retinoic acid inducible gene-I (RIG-I) and interferon-stimulated genes (ISGs), such as ISG15, IFIT1 and IFITM1, which are the receptor and effector molecules of RN7SL1. Both MDS-MSC-EXO and exogenous RN7SL1 can promote MDS cloned cells proliferation and stimulate ISGs expression by activating RIG-1. RIG-I inhibitor can block this effect. We demonstrate that MDS-MSC may promote MDS cloned cell proliferation through RIG-ISGs signal activated by exosomes-derived RN7SL1. Overall, our studies provide new insights that MSC-EXO functions the crucial roles in mediating the progression of MDS via RIG-1/ISGs activation, and demonstrate novel targets of RN7SL1 to the pathogenesis of MDS.This study will provide new ideas for MDS treatment in the aspect of microenvironment.

Lenalidomide derivatives and proteolysis-targeting chimeras for controlling neosubstrate degradation

Lenalidomide, an immunomodulatory drug (IMiD), is commonly used as a first-line therapy in many haematological cancers, such as multiple myeloma (MM) and 5q myelodysplastic syndromes (5q MDS), and it functions as a molecular glue for the protein degradation of neosubstrates by CRL4CRBN. Proteolysis-targeting chimeras (PROTACs) using IMiDs with a target protein binder also induce the degradation of target proteins. The targeted protein degradation (TPD) of neosubstrates is crucial for IMiD therapy. However, current IMiDs and IMiD-based PROTACs also break down neosubstrates involved in embryonic development and disease progression. Here, we show that 6-position modifications of lenalidomide are essential for controlling neosubstrate selectivity; 6-fluoro lenalidomide induced the selective degradation of IKZF1, IKZF3, and CK1α, which are involved in anti-haematological cancer activity, and showed stronger anti-proliferative effects on MM and 5q MDS cell lines than lenalidomide. PROTACs using these lenalidomide derivatives for BET proteins induce the selective degradation of BET proteins with the same neosubstrate selectivity. PROTACs also exert anti-proliferative effects in all examined cell lines. Thus, 6-position-modified lenalidomide is a key molecule for selective TPD using thalidomide derivatives and PROTACs.

Acute myeloid leukemia cells and MSC-derived exosomes inhibiting transformation in myelodysplastic syndrome

AimsTo investigate the mechanism of exosomes' role in the transformation of MDS to AML.MethodsExosomes in culture supernatants of MDS and AML cell lines, were extracted by ultrafiltration and identified in three ways: morphology, size, and exosome protein surface markers. Exosomes from AML cell lines were then co-cultured with MDS cell lines and their impacts on MDS cell microenvironment, proliferation, differentiation, cell cycle, and apoptosis were analyzed by CCK-8 assay and flow cytometry. Furthermore, exosomes from MSC were extracted for further authentication.ResultsThe transmission electron microscopy, nanoparticle tracking analysis, Western blotting, and flow cytometry methods all verify that ultrafiltration is a reliable method to extract exosomes in the culture medium. Exosomes from AML cell lines inhibit the proliferation of MDS cell lines, block cell cycle progression, and promote apoptosis and cell differentiation. It also leads to increased secretion of tumor necrosis factor-α (TNF-α) and reactive oxygen species (ROS) in MDS cell lines. In addition, MSC-derived exosomes were found to inhibit the proliferation of MDS cell lines, arrest cell cycle progression, promote apoptosis, and inhibit differentiation.ConclusionUltrafiltration is a proper methodology in extracting exosomes. The exosomes of AML origin and MSC origin may play a role in MDS leukemia transformation via targeting TNF-α/ROS-Caspase3 pathway.

A new pathway for the transformation of AML in MDS: APA mechanism regulated by NUDT21 and RUNX1.

We have identified that NUDT21 plays a vital role in MDS transformations, while the transcription factor RUNX1 is essential for normal hematopoiesis, which is a high expression in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), and we aim to explore the linkage between the two genes and new pathways for MDS transformation to AML. Prediction of RUNX1 expression levels and its relationship with NUDT21 in AML and MDS patients was performed using bioinformatics techniques and validated in patients. Using lentiviral packaging technology, NUDT21 knockdown and overexpression models were developed in AML and MDS cell lines. These models were validated using quantitative polymerase chain reaction (qPCR) and western blotting. The cell cycle, apoptosis, differentiation, and cytokines were examined by flow cytometry, CCK-8 analyzed proliferation, and the intracellular localization of NUDT21 and RUNX1 was examined by immunofluorescence. mRNA transcriptome sequencing was performed on THP-1, MUTZ-1, and Dapars analyzed SKM-1 cell lines and the sequencing data to observe the knockdown effect of NUDT21 on RUNX1. qPCR and western blot revealed a positive correlation between NUDT21 and RUNX1; both were located in the nucleus. Overexpression of NUDT21 reduced apoptosis, promoted cell proliferation, and possibly increased the invasive ability of cells. It also altered the APA site in the RUNX1 3'-UTRs region. NUDT21 regulates RUNX1 gene expression and promotes AML transformation in MDS through an APA mechanism.

All-trans retinoic acid enhances the cytotoxic effect of decitabine on myelodysplastic syndromes and acute myeloid leukaemia by activating the RARα-Nrf2 complex

BackgroundDecitabine (DAC) is used as the first-line therapy in patients with higher-risk myelodysplastic syndromes (HR-MDS) and elderly acute myeloid leukaemia (AML) patients unsuitable for intensive chemotherapy. However, the clinical outcomes of patients treated with DAC as a monotherapy are far from satisfactory. Adding all-trans retinoic acid (ATRA) to DAC reportedly benefitted MDS and elderly AML patients. However, the underlying mechanisms remain unclear and need further explorations from laboratory experiments.MethodsWe used MDS and AML cell lines and primary cells to evaluate the combined effects of DAC and ATRA as well as the underlying mechanisms. We used the MOLM-13-luciferase murine xenograft model to verify the enhanced cytotoxic effect of the drug combination.ResultsThe combination treatment reduced the viability of MDS/AML cells in vitro, delayed leukaemia progress, and extended survival in murine xenograft models compared to non- and mono-drug treated models. DAC application as a single agent induced Nrf2 activation and downstream antioxidative response, and restrained reactive oxygen species (ROS) generation, thus leading to DAC resistance. The addition of ATRA blocked Nrf2 activation by activating the RARα-Nrf2 complex, leading to ROS accumulation and ROS-dependent cytotoxicity.ConclusionsThese results demonstrate that combining DAC and ATRA has potential for the clinical treatment of HR-MDS/AML and merits further exploration.

EZH2 Inhibition Mediates Proptosis Via DHX9 and S100A9 in Myelodysplastic Syndromes

Myelodysplastic Syndromes (MDS) is considered as an archetypal epigenetic disorder due to the aberrant epigenome, frequent epigenetic regulator mutations and the most responsive disease to DNA methylation inhibitors. Among numerous epigenetic regulators, Enhancer of zeste 2 homologue 2 (EZH2) occupies an important position owing to its involvement in multiple epigenetic modifications. In MDS, EZH2 mutation frequency is 5%-10%. Given that the EZH2 gene is located at 7q36.1, abnormalities of chromosome 7q disturb EZH2 as well. To investigate the expression and effects of EZH2 in MDS patients without EZH2 mutation and chromosome 7 abnormalities, qPCR was performed. We found that EZH2 transcript level was significantly higher in MDS patients and its expression was highest in very high risk group, followed by the high risk group. The survival time of EZH2 high expression was shorter than the low expression group. Multivariable Cox regression showed that EZH2 expression was an independent prognostic factor. Next, we treated MDS cell lines MDS-L and SKM-1 with EZH2 inhibitor DZNep and found that DZNep inhibited cell proliferation in a dose- and time-dependent manner. To determine which pattern of cell death played the most important role, we pretreated different cell death inhibitors for 1 hour before adding DZNep. Compared with the group without cell death inhibitors, pyroptosis inhibitor (VX765) was the most effective in restoring cell viability. Besides, both DZNep and knockdown of EZH2 could trigger pyroptosis. To elucidate the possible mechanism of pyroptosis induced by EZH2 inhibition, we searched for RNA-seq data from tumor cells treated with EZH2 inhibitors by using the GEO database. We overlapped 53 genes by analyzing GSE151576, GSE80239 and GSE119088, of which DHX9 was the highest expressed in BM and attracted attention.Meanwhile, down-regulation of DHX9 was observed after pharmacological inhibition or EZH2 knockdown. There was a positive correlation between EZH2 and DHX9 in MDS patients. Furthermore, DHX9 expression was increased in MDS patients in contrast to control, and its overexpression was connected with shorter OS. Knockdown of DHX9 with two shRNAs induced pyroptosis. CUT&RUN and qPCR assay results validated that EZH2 and H3K4me3 occupancy at DHX9 promoter. Moreover, with RNA sequencing we found DHX9 can regulate S100A9 and confirmed by western blot. Overexpression of S100A9 could cause pyroptosis of MDS cell lines, which could be reversed by a S100A9 inhibitor. DHX9 and S100A9 are negatively correlated in MDS patients. Finally, we established xenograft mouse models by intravenously injecting luciferase-labeled SKM-1 cells. Intraperitoneal injection of 2mg/kg DZNep daily could significantly reduce the tumor burden and prolong the survival time of mice. We further confirmed that inhibition of EZH2 regulated DHX9 and S100A9 and induced pyroptosis in mouse bone marrow cells. In conclusion, we found that EZH2 expression in de novo MDS patients without EZH2 mutation and chromosome 7 abnormality was higher than that of the control group, and its high expression was correlated with inferior prognosis. EZH2 suppression inhibited tumor growth and induced pyroptosis both in vitro and in vivo. Inhibition of EZH2 triggered pyroptosis by reducing its binding in the DHX9 promoter and upregulating S100A9. In this present study, we reveal a novel molecular mechanism by which EZH2 inhibition induced pyroptosis and provide a basis for the treatment of MDS with EZH2 inhibitor. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mitotic Perturbation Is a Key Mechanism of Action of Decitabine in Myeloid Tumor Treatment

Background: Decitabine (DAC) is one of the first generation of epigenetic drugs used clinically to treat myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Historically, DAC was considered as a cytotoxic agent when it was first developed in the 1960s. Subsequent studies in 1980s showed that DAC can induce DNMT1 degradation and DNA hypomethylation at non-cytotoxic concentration. Since then, DNA demethylation has been considered as the main mechanism of action of DAC. The DAC-induced DNA hypomethylation was shown to induce upregulation of tumor suppressor genes or endogenous retroviruses, which could contribute to the antitumor effect of DAC. Recently, DAC was also shown to be effective even in MDS and AML with TP53 mutations. However, it remains unclear why myeloid tumors, especially those with TP53 mutations, are particularly susceptible to DAC treatment. Methods: Cancer Dependency Map (DepMap) database was used to evaluate in vitro sensitivity of human cancer cell lines to DAC. To identify essential genes that regulate sensitivity of myeloid tumors to DAC, we performed a genome-wide CRISPR-dCas9 activation screen using murine MDS/AML cells expressing ASXL1 and SETBP1 mutants (cSAM cells). After transduction of mouse genome-wide CRISPRa-v2 library, cSAM cells were cultured with DMSO or DAC for 1 week. We extracted genomic DNA from the cells at the start and end of our screen and quantified the relative representation of each sgRNA. To assess the effect of DAC on mitosis, we established the time-lapse system optimized for non-adherent MDS-L-2007 cells (a human secondary AML (sAML) cell line derived from an MDS cell line, MDS-92). Results: We first explored DepMap database and found that myeloid tumors are susceptible to DAC at clinically achievable concentration, while most solid tumor cell lines are not. The CRISPR/dCas9-activation screen revealed that genes involved in chromosome segregation, including Cdk1, Cdc20, Cdca8 and Dsn1, were significantly enriched in DAC-resistant cells, indicating that overexpression of mitotic regulators conferred resistance to DAC in cSAM cells. We also found that DAC strongly induced aneuploidy and polyploidy (>4n) in myeloid tumors, especially in those with TP53 mutations and sAML cells. Furthermore, the time-lapse imaging revealed that DAC treatment prolonged the duration of metaphase and increased multipolar mitosis and abscission failure in MDS-L-2007 cells (Figure). Thus, DAC perturbs mitosis and has cytotoxic effects at low concentrations in myeloid tumors. Next, we assessed the role of DNMT1 in the DAC-induced mitotic defects in myeloid tumors. To our surprise, the DAC-induced aneuploidy and apoptosis were markedly attenuated in DNMT1-depleted myeloid tumors. In contrast, overexpression of Dnmt1 increased sensitivity of myeloid tumors to DAC. These results indicate that DAC perturbs mitosis not through DNMT1 degradation and subsequent DNA demethylation, but through DNMT1 itself in myeloid tumors. In line with this, chromatin-bound DNMT1 protein was retained even with DAC, whereas DAC induced rapid degradation of DNMT1 in the nucleoplasm fraction. Importantly, the Dnmt1-C1229A mutant, which loses the ability to induce DNMT1-DNA covalent bond formation, did not enhance the DAC-driven mitotic defects. In addition, DAC treatment increased frequency of cohesion loss between sister chromatids in MDS-L-2007 cells, as evidenced by the absence of centromere connection of sister chromatids at metaphase. Moreover, the in vitroXenopus cell-free system which reproduces replication-dependent DNA methylation maintenance revealed that addition of 5-aza-dCTP strongly promoted chromatin binding of DNMT1, while it inhibited that of cohesion complex members (SMC3 and SCC1). Taken together, these results strongly indicate that the aberrant DNMT1 retention on chromatin induced by DAC leads to cohesion loss and mitotic defects in myeloid tumors. Conclusion: These findings challenge the current assumption that DAC inhibits leukemogenesis by inducing DNA hypomethylation. Rather, the anti-leukemia effect of DAC is likely mediated mainly by the non-epigenetic mechanism: mitotic perturbation through aberrant DNMT1-DNA covalent bonds and cohesion loss of sister chromatids. Our study also suggests that DAC will be most effective on tumors with impaired mitotic fidelity, such as AML and MDS with TP53 mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The M6A Methyltransferase METTL14 Promotes Myelodysplastic Syndromes Development Via PI3K/Akt Signaling Pathway

Background: RNA N6-methyladenosine (m6A) plays a critical role in normal and malignant hematopoiesis. The m6A methyltransferases METTL3 and METTL14 were reported as oncogenes in acute myeloid leukemia. Epigenetic dysregulation is a prominent biological feature of myelodysplastic syndromes (MDS), however, whether RNA methylation dysregulation exists in MDS, and what's the functions of m6A methyltransferases are largely unknown. Methods: The global m6A modification level of RNA in bone marrow was detected in MDS patients by colorimetric method, and the expression level of METTL14 in bone marrow of MDS patients was detected by q-PCR. Both gain- and loss-of-function experiments were performed to study the role of METTL14 in MDS. M6A sequencing and RNA sequencing were examined to explore the potential signaling pathways and target genes that mediated METTL14 function in MDS. Results: We detected obvious RNA m6A dysregulation in bone marrow of MDS patients compared with normal controls, and found that the up-regulation of the global m6A modification is closely related to higher-risk category and worse survival of MDS patients. By analyzing the MDS public database (GSE_58831) and q-PCR data of MDS patients in our center, we found that among 23 classical m6A related genes, m6A methyltransferase METTL14 was the key factor causing the m6A dysregulation in MDS: high expression of METTL14 was associated with higher disease risk stratification, more bone marrow blasts, poorer prognosis, and higher response rates to hypomethylating agents (HMAs). In order to explore the functions of METTL14 in MDS, we performed gain- and loss-of-function experiments in MDS cell line MDSL in vitro. It was observed that the expression of METTL14 promoted the cell proliferation and clone formation ability of MDS cells by inducing cell apoptosis and differentiation. More interestingly, we found that HMAs down-regulated the level of global m6A modification by inhibiting METTL14 expression, and the expression of METTL14 could increase the sensitivity of MDS cells to HMAs in MDS cells. To search for upstream regulatory genes of METTL14, we analyzed the ChIPBase online database and identified ELF1 might be the potential upstream transcription factor. The relationship between ELF1 and METTL14 was further confirmed by ELF1-overexpressed and ELF1-knowndowned MDSL cell lines. To explore the relationship between gene mutations and METTL14, we analyzed the correlations between next generation sequencing (NGS) data and METTL14 expression level in MDS patients in our center, and found that the METTL14 expression level was significantly increased in MDS patients carrying TP53 gene mutations (P<0.05). The increased METTL14 and ELF1 expression were all observed in MDS cell lines with introduction of TP53 missense mutations (M237I and Y220C) or TP53 knockdown. Mechanistically, m6A sequencing and RNA-seq sequencing revealed METTL14 promoted MDS development via PI3K/Akt signaling pathway, and the potential target genes involved in PI3K/Akt signaling pathway were ANGPT1, CDK6, FGFR2, GNG11, IGF1R et al. Conclusion： Herein, we reported RNA m6A methylation dysregulation did exist in MDS, and m6A methyltransferase METTL14 was an important regulatory factor. Higher METTL14 expression was associated with higher-risk stratification, poorer survival, and better response to HMAs in MDS patients. METTL14 was required for the cell growth and response to HMAs in MDS cells. TP53 mutations were significantly correlated with the up-regulation of METTL14 expression level, and TP53 mutations could induce METTL14 overexpression by regulating transcription factor ELF1. Mechanistically, METTL14 exerts its oncogenic role via PI3K/Akt signaling pathway. Our findings highlighted that METTL14 could potentially be a novel therapeutic target in MDS especially those with TP53 mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Human NGFR+/VCAM-1+/Mcam+ Bone Marrow-Derived Stromal Cells (NVML) Provide Enhanced Support for Normal Hematopoiesis and Are Disrupted in Myelodysplastic Syndrome

The bone marrow microenvironment (BMME) is indispensable for support of normal and malignant hematopoiesis. Mesenchymal stem/stromal cells (MSCs) give rise to multiple non-hematopoietic components that play a pivotal role in myelodysplastic syndromes (MDS). However, therapeutic targeting of MSCs has been challenging, in part due to the limited definition of the heterogeneity of MSC populations from human bone marrow. Using bone marrow from normal healthy controls obtained through aspirates rather than surgical specimens (normal bone marrow; NBM), we successfully identified two novel distinctive subsets of human MSCs by combining three MSC cell surface markers: CD271/ Nerve Growth Factor Receptor (NGFR), CD106/ Vascular Cell Adhesion Molecule-1 (VCAM-1) and CD146/ Melanoma Cell Adhesion Molecule (MCAM). We first isolated three BM-derived non-endothelial lineage (CD45/CD235ab/CD34-CD31-) cell populations from NBM: CD271+/CD146- (NGFR+/Lineage- cells: NLCs), CD271+/CD146+/CD106+ (NGFR+/VCAM1+/MCAM+/Lineage- NVML cells), and CD271+/CD146+/CD106- (NGFR+/MCAM+/Lineage-; NML cells). Gene profiling by RNA-seq revealed that NLCs and NVML cells represent distinct populations, whereas NML cells are a transitional population. Transcriptional analysis suggested that NVML cells, compared to NLCs, have stem cell characteristics, including quiescence and multipotentiality, and express higher levels of key genes critical for the support of hematopoietic stem and progenitor cells (HSPCs), including CXCL12 (SDF1A), Kit ligand (KITL) and Angiopoietin-1 (ANGPT1). This signature was compatible with BM-derived Leptin receptor+ MSCs with high capacity of supporting HSPCs based on previously reported single cell RNA-seq analysis in mice (Baryawno et al 2019, Cell). Consistent with their transcriptome, functional analysis showed that NVML cells had higher colony forming units (CFU-F) and tri-lineage differentiation capacity compared to NLCs. Normal NLCs or NVML cells were also co-cultured with NBM or human MDS cell line (MDS-L) in the absence of cytokines/chemokines to evaluate the ability of these novel human MSC populations to provide a supportive niche for HSPCs. NVML cells provided increased support for normal hematopoietic progenitors compared to baseline and NLCs (fold increase over baseline 1.78±0.78(NLCs, p<0.05) vs 2.71±1.71(NVML cells, p<0.001), NLCs vs NVML cells, p<0.05). In contrast, NLCs showed increased ability of supporting MDS-L (fold increase over baseline 2.35±1.35 (NLCs, p<0.01) vs 1.5±0.5 (NVML cells, p=0.26), NLCs vs NVML cells, p=0.053). We next profiled the transcriptome of NLCs and NVML cells from 17 patients with MDS in comparison with age-matched healthy controls (n=8, >50 years old). In spite of mutational heterogeneity in this MDS patient cohort, principal component analysis (PCA) revealed a distinctive shift of MDS-derived NVML cells compared to controls. For upregulated transcripts, pathways and gene ontology analyses of differentially expressed genes in NVML cells from MDS compared to controls identified signals related to DNA/RNA processing, while most of the down-regulated transcripts identified cytokines/chemokines critical for HSPCs. When the transcriptional profiles of NVML cells from MDS patients were compared to age-matched NVML controls, critical genes for HSPC support highly expressed in normal NVML (e.g. SDF1A, ANGPT1) were significantly down-regulated. Moreover, the transcriptional signature of NVML cells from MDS patients was similar to that of normal NLCs. Together these data suggest that, in spite of mutational heterogeneity in the hematopoietic clone, MDS may differentially impact these novel human marrow MSC subsets, decreasing normal HSPC support by disrupting NVML cells, which acquired the transcriptional signature of MDS-supportive normal NLCs. These data highlight a novel paradigm to enrich key human niche cells that provide support for HSPCs. Moreover, our data suggest that these human MSC cell subsets are differentially disrupted in MDS. Mutation specific effects are also likely to impact MDS-dependent disruption of the BMME. Therefore, our novel strategy to enrich critical human BM MSC subsets that differentially support normal and malignant hematopoiesis represents a new paradigm to accelerate therapeutic targeting of the BMME in MDS and other hematologic malignancies.

Mis-Splicing Due to Somatic U2AF2 Mutations in Myeloid Neoplasms

Background Recent genetic studies have identified frequent mutations affecting a number of genes encoding RNA splicing factors (SDs) in myelodysplastic syndromes (MDS) and related myeloid neoplasms (MNs). Among these, most frequently mutation are SF3B1, SRSF2, U2AF1, and ZRSR2, whose functions have been intensively studied. By contrast, U2AF2, another SF, is also recurrently mutated in MNs. U2AF2 is a component of the U2 auxiliary factor that forms a heterodimer with U2AF1 for the recognition of the 3’ splice site (3'SS). U2AF2 contains a sequence-specific RNA-binding region with two RNA recognition motifs and identifies polypyrimidine (Py) tract signals of nascent transcripts. However, compared to other splicing factor mutations, U2AF2 mutations are much rare, preventing the detailed analysis of their role in leukemogenesis. Methods To characterize the role of U2AF2 mutations, we systematically analyzed mutation spectrum in 6,369 with different MNs using targeted-capture sequencing. We also performed RNA sequencing of cKit(+) bone marrow cells from 6 U2AF2 mutated MDS cases along with 52 MDS patients without common splicing factor mutations and 25 healthy individuals. THP1 and HL60 leukemia cells with exogenous expression of wildtype or mutant U2AF2 were established to evaluate the splicing response. We also used clustered regularly interspaced short palindromic repeats /CRISPR-associated protein-9 nuclease (CRISPR/cas9) to introduce the p.190_195del mutation to U2AF2 in K562 and MOLM-13 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant U2AF2. Results In total, U2AF2 mutations were found in 34 (0.53%) of 6,369 MN cases, of which 29 had the recurrent p.190_195del mutation, the median age of U2AF2 mutations was 55 years (range 31-79) and 55% were diagnosed with MDS, 31% with CMML and 7% with AML. U2AF2-mutated patients showed a male predominance (90%). The most frequently co-mutated gene was ASXL1 with 52%, followed by SETBP1 (35%) STAG2 (21%),RUNX1 (21%), CSF3R, NRAS, DNMT3A, NF1, PTPN11, ETV6, and STAT3. To investigate the effects of the U2AF2 p.190_195del mutation on RNA splicing, we analyzed transcriptome sequencing, followed by the rMATS bioinformatics pipeline to determine alternative splicing (AS) events . Various types of AS events were identified, including skipped exons (SEs), alternative 5’ ss exons (A5SSs), alternative 3’ ss exons (A3SSs), retained introns (RIs), and mutually exclusive exons (MXEs). Among these, the skipping exons are the most frequent in both U2AF2-mutant primary MDS cases and cell lines. To explore these alternative splicing results in more detail, we next examined differentially skipped exons. Unsupervised uniform manifold approximation and projection (UMAP) analysis based on the standardized splicing ratio of skipping exons segregated U2AF2-mutated patients from other MDS cases without common splicing factor mutations and healthy individuals. To prioritize mutant U2AF2-induced alterative splicing events, we intersected significant skipping exons across 2 datasets: MDS patient samples with and without U2AF2 p.190_195del mutations, and MDS patient samples with U2AF2 p.190_195del mutations and healthy individuals. The differential skipping exon usages and differential gene expression profiles enables both consensus sequence analysis and pathway/gene-set enrichment analysis. Conclusions Our study revealed that the U2AF2 p.190_195del regulated aberrant alternative splicing facilitated MDS progression through perturbations in splice isoforms. A better understanding of the U2AF2 p.190_195del and its critical specific changes will provide novel insights into disease pathophysiology and potentially inform future treatment decisions.

Whole-Genome DNA Methylation Sequencing Reveals Epigenetic Changes in Myelodysplastic Syndromes.

Epigenetic dysregulation of cancer-associated genes has been identified to contribute to the pathogenesis of myelodysplastic syndromes (MDS). However, few studies have elucidated the whole-genome DNA methylation in the initiation pathogenesis of MDS. Reduced representation bisulfite sequencing was performed in five de novo MDS patients and four controls to investigate epigenetic alterations in MDS pathogenesis. The mean global methylation in five MDS patients showed no significant difference compared with the four controls. In depth, a total of 1,459 differentially methylated fragments, including 759 hypermethylated and 700 hypomethylated fragments, were identified between MDS patients and controls. Targeted bisulfite sequencing further identified that hypermethylation of DLEU7, FOXR1, LEP, and PANX2 were frequent events in an additional cohort of MDS patients. Subsequently, LEP hypermethylation was confirmed by real-time quantitative methylation-specific PCR in an expanded cohort of larger MDS patients. In clinics, LEP hypermethylation tended to be associated with lower bone marrow blasts and was significantly correlated with U2AF1 mutation. Survival analysis indicated that LEP hypermethylation was associated with a markedly longer survival time but was not an independent prognostic biomarker in MDS patients. Functional studies revealed pro-proliferative and anti-apoptotic effects of leptin in the MDS cell line SKM-1, and it was significantly associated with cell growth and death as well as the Toll-like receptor and NF-kappa B signaling pathways. Collectively, our findings demonstrated that whole-genome DNA methylation analysis identified novel epigenetic alterations such as DLEU7, FOXR1, LEP, and PANX2 methylations as frequent events in MDS. Moreover, LEP might play a role in MDS pathogenesis, and LEP hypermethylation was associated with longer survival but not as an independent prognostic biomarker in MDS.