Decitabine Research Articles

Novel Gene Mutations Associated with Disease-Free Survival in Elderly Patients with Acute Myeloid Leukemia Receiving First-Line Induction-Consolidation with or without Azacitidine Post-Remission Treatment

Background: The achievement of complete remission (CR) is an important milestone for patients with acute myeloid leukemia (AML) undergoing curative-intent therapy. However, patients who achieve CR with induction therapy for AML will relapse within months. Therefore, research is focused on prolonging leukemia-free survival (DFS). We have recently reported on the randomized phase III study to compare the efficacy and safety of azacitidine (AZA) versus best supportive care (BSC) for treatment of AML in elderly patients who achieved first CR after a homogeneous intensive induction and consolidation phase (QoLESS AZA-AMLE, Oliva et al. Cancers 2023). At 2 and 5 years, DFS was 6.0 (95% CI:0.2-11.7) months in the BSC arm vs. 10.8 (95% CI:1.9-19.6 p=0.23) months in the AZA arm. We report on the ancillary translational study (QOL-ONE Trans-2) to evaluate biological changes in bone marrow samples through Next Generation Sequencing (NGS). Objective: In patients enrolled in the trial who reached CR and were randomized to receive AZA or BSC, the endpoint of the present study was to evaluate the effect modification by gene mutations at AML diagnosis on the relationship between treatment allocation and relapse (disease-free survival, DFS). Methods: The study was performed on available biological samples collected at baseline, randomization, and 6-month post remission. DNA was subjected to high throughput NGS using genes commonly mutated in myeloid malignancies and prepared with a home-set of 350 genes for Illumina (University of Kyoto, Japan) for all subjects with available vials. Only variants with high-quality reads, were considered. A Variant Allele Frequency (VAF) ≥ 4% was considered an appropriate threshold for minimal burden of clonality to be reported. Results: Samples were available for 24 patients, 12 males, of median age 71 (65-74) years, 11 allocated in AZA arm and 13 in BSC arm. At diagnosis, mutations were detected in all patients with a range of 5 to 17 (median 10) simultaneous mutations, the most frequent being DNMT3A (42%), NPM1 (33%) and TET2 (33%). The most frequently mutated genes at diagnosis are represented in the figure. Only FANCA gene, mutated in 4 patients was associated with a hazard risk (HR) 4.96 (95% CI 1.34 - 18.35; p=0.02) of relapse and, interestingly, HLA-A, which was mutated in 7 patients, was associated with a HR 0.277 (95% CI 0.08 - 0.95; p=0.049). Patients with HLA-A mutation had a significantly longer survival (Figure 2). Three HLA-A mutated patients were allocated in 5-Aza arm and 4 in BSC arm. In a multivariate COX model, the effect of baseline HLA-A mutation on DFS is independent of the allocation arm (P=0.041). HLA Class I and Class II alleles were assessed based on the Luminex xMAP technology: for all cases, the HLA-SNPs are located on chromosome 6 in exon 2, 3 and 4 within protein-codifying sequences. Conclusions: HLA gene mutations are not usually investigated for diagnostic or prognostic purposes in AML. The extensive 350-gene panel used in this study included HLA genes and has revealed a possible favorable role of HLA-A polimorphisms for the prognosis of elderly patients with AML undergoing intensive chemotherapy. Further specific HLA NGS analyses are undergoing to determine whether this concerns genuine somatic mutations. The present finding is worthy of further investigation to confirm its value in prognosis and its therapeutic implications.

CEBPA-Driven Expression of the Transcriptionally Inactive deltaTP73 Isoform Phenocopies TP53mutated Poor Risk and Drug-Resistant Acute Myeloid Leukemia

Mutations in TP53 ( TP53mut) are present in roughly 10% of acute myeloid leukemia (AML) patients and represent a unique subgroup of patients with very poor prognosis. While the exact mechanisms of TP53mut-driven leukemogenesis remain elusive, transcriptional changes associated with suppression of the TP53 signaling pathway are considered to confer a dismal prognosis on TP53mut patients. Using molecular profiling of a large cohort of AML patients (n=823) we identify a new poor prognostic subgroup in AML that is TP53 WT but shares strong similarities with TP53mut patients. Patients included in this new group present increased expression of the oncogenic TP73 gene isoform ΔNp73,which lacks the transactivation domain but still binds to chromatin. We find that the transcriptional and metabolic program of ΔNp73high AMLs strongly resembles that of TP53mut AML, being enriched for stemness signatures like “17LSC” and “LSC UP”. In line with TP53mut AML, ΔNp73high/ TP53WT frequently co-occurs with U2AF1, SRSF2, TET2, and RUNX1 mutations . Lentiviral overexpression (OE) of ΔNp73 in healthy CD34 + cells increased cell proliferation and stemness. ΔNp73-OE in TP53WT AML models, imposed drug resistance against several standard-of-care cytotoxic therapies in AML (e.g., AraC, venetoclax (VEN) ± azacytidine (AZA), and FLT3 inhibitors), while drug resistance was not further enhanced upon ΔNp73-OE in TP53mut AML models. These results were validated using ex vivo treated primary AML samples (n=80), whereby ΔNp73 high samples were more resistant to several cytotoxic therapies, including VEN+AZA . Lentiviral ΔNp73-OE in primary APL samples (n=10) allowed efficient engraftment in vivo using xenograft mouse models with colonization of secondary organs and splenomegaly, which was not observed in the empty vector controls. To unravel mechanisms via which ΔNp73controls oncogenic transformation we performed RNA-seq and ChIP-seq to identify direct transcriptional targets of TP53, ΔNp73, and the transcriptionally active isoform TAp73. These studies revealed that ΔNp73 and TP53 compete for the same target genes, whereby ΔNp73-OE resulted in complete loss of TP53 binding and strong downregulation of these loci, including downregulation of known TP53 target genes CDKN1A, BBC3, and GAS6, but also of TP53 itself. Gene set enrichment analyses of molecular programs in ΔNp73-OE cells revealed that these were associated with the terms “LSC UP”, “CEBPA targets”, and “SIGNAL TRANSDUCTION BY TP53”, indicating that ΔNp73 phenocopies TP53mut AML. Chromatin accessibility data on AML patients revealed a differentially accessible region in intron 2 (+24 Kb after the transcription start site, referred to as peak B) of the TP73 gene, with unknown regulatory functions on TP73 expression in AML. To access the potential enhancer functions of this region driving ΔNp73 expression we generated CRISPR/Cas9-KO MOLM13 cells lacking the peak B region (MOLM13-KO). MOLM13-KO cells exhibited reduced ΔNp73 expression, and the ability to up-regulate ΔNp73 expression upon high dose AraC-induced stress was abrogated. Since CEBPA was a transcription factor predicted to bind to Peak B, we investigated whether CEBPA could regulate the expression of the different TP73 isoforms. CEBPA-KD in AML cells resulted in decreased ΔNp73 levels and restored the TAp73 expression, suggesting that CEBPA controls the balance of the distinct TP73 isoforms. Pharmacological inhibition of CEBPA using the FDA-approved drug Guanfacine (GFC), diminished ΔNp73 levels and restored drug sensitivity against several cytotoxic agents in ΔNp73-OE AML cells. Moreover, the combination of GFC+VEN displayed strong synergistic effects in a cohort of ex vivo treated AML blasts (n=20), including patients with TP53mut, which are often resistant to VEN. Here, we describe a new AML patient subgroup that is TP53WT but behaves similarly to TP53mut AML due to the expression of the ΔNp73 isoform that impairs TP53 signaling. This patient subgroup displays a very poor prognosis and is resistant to standard-of-care drugs used for AML patients. We identify a novel enhancer region that drives the expression of oncogenic TP73 isoforms, which is controlled by CEBPA, and inhibition of CEBPA by GFC enhances sensitivity to cytotoxic therapies in TP53mut-like AMLs. Our insightsfurther refine patient stratification and provide alternatives for treatment for the most dismal group of AML patients.

Targeting ADSS2 Enhances BH3 Mimetics Treatment Induced Mitochondrial Apoptosis in AML Cells

Recently, the BH3 mimetic-Bcl2 inhibitor venetoclax (VEN), has been approved by FDA in 2018 for the treatment of patients with AML in combination with a hypomethylating agent (HMA) such as azacytidine (AZA). This synergy is likely due to HMA-induced mitochondrial vulnerability associated with cytoplasmic RNA metabolism. Nonetheless, most AML patients treated with the combination eventually relapse, possibly due to persistence of tumor cells not dependent on antiapoptotic proteins (Bcl2, Mcl1). There is a pressing need to develop targeted therapeutics sensitizing AML cells to mitochondrial apoptosis, particularly for those patients failed from VEN associated treatment. To achieve the goal, we performed a CRISPR/Cas9 Knockout (KO) screen (using Bim peptide-based dynamic ΔBH3 profiling as read-out) targeting genes (n=534) involved in cytosolic nucleotides metabolism pathways to increase mitochondrial vulnerability in a BH3 mimetic-refractory AML cell line (HEL). That search identified ADSS2, which catalyzes the first committed/rate-limiting step of AMP biosynthesis from IMP, emerged as the top hit, exhibiting highest ΔBH3 priming. Subsequent BH3 priming assays validated that ADSS2 KO significantly enhanced sensitivity to BH3 mimetics across various AML cell lines, including MOLM13, SKM1, OCI-AML3, and HEL cells. ADSS2 KO also led to a noteworthy reduction in the IC50 values of both VEN and the Mcl1 inhibitor S63845 in these cell lines. Additionally, the synergistic effect between sgRNA targeting ADSS2 and VEN or S63845 was also confirmed in Cas9 expressing cells from two AML patient-derived-xenograft (PDX) samples. We next transplanted AML cells (form one PDX samples) with or without ADSS2-KO into NGS mice, then treated mice with VEN. Notably, VEN treatment (100 mg/kg, QDx14, PO) significantly reduced tumor burden in ADSS2-KO AML mice compared to WT AML mice. Additionally, ADSS2 KO combined with VEN treatment group displayed a survival advantage compared to single VEN treatment group. Further mechanistic investigations revealed that ADSS2 KO led to a remarkable depletion of AMP rather than ATP, and a robust repression of AMPK activity in HEL cells. To assess whether AMPK inhibition mimics the effect of ADSS2 KO, AMPKα-deficient (Mx1-Cre/Prkaa1 fl/fl/ Prkaa2 fl/fl) MLL-AF9 (MA9) transformed murine AML cells were assessed. Indeed, AMPKα depletion substantially enhanced VEN treatment efficiency ex-vivo. Notably, enforced overexpression of a constitutively active AMPKa2 (T172D) construct in a VEN sensitive cell line SKM1, promoted cell resistance to mitochondrial apoptosis by VEN or S63845. Moreover, T172D expression can also abolish ADSS2-KO mediated mitochondrial vulnerability to VEN or S63845 treatment, indicating the pivotal role of AMPK signaling in mediating ADSS2 function. There are no reported ADSS2 inhibitors. To explore ADSS inhibitor, we conducted a fragment-based virtual screen of 8,000,000 compounds (MolPort) library and requested the top-ranking hits for ADSS2 catalysis evaluation by ELISA. Notably, the top two hits AS104 and AS71 exhibited the most potent inhibitory effects (AS104 IC50=2.43µM, AS71 IC50=3.85µM). NMR-STD analysis was conducted in a cell-free system, which unequivocally confirmed the physical binding of inhibitors and the ADSS2 protein. To further investigate the interactions between AS71 and AS104 with ADSS2 in a cellular context, both compounds were subjected to a cellular thermal shift assay (CETSA). Notably, treatment with AS71 and AS104 led to substantial shifts in ADSS2 protein thermal stability, providing evidence of their interactions with ADSS2 within the cellular environment. In both HEL cells and a primary VEN-resistant AML samples, treatment with AS71 and AS104 significantly increased ΔBH3 priming. Collectively, our observation revealed that targeting ADSS2 is critical for sensitizing AML cells to BH3 mimetics. We are now conducting preclinical assessments of ADSS2 inhibitors.

Tuspetinib Myeloid Kinase Inhibitor Safety and Efficacy As Monotherapy and Combined with Venetoclax in Phase 1/2 Trial of Patients with Relapsed or Refractory (R/R) Acute Myeloid Leukemia (AML)

INTRODUCTION: Tuspetinib is a potent once daily oral myeloid kinase inhibitor of SYK, FLT3, JAK1/2, RSK1/2, mutant KIT, and TAK1-TAB1 kinases that mediate dysregulated cellular proliferation in AML. In an orthotopic mouse model of FLT3-mutant AML, tuspetinib exhibited greater antitumor activity than gilteritinib, entospletinib, venetoclax (VEN), and azacitidine (AZA) and combined favorably with VEN and AZA individually. Tuspetinib is being evaluated clinically as monotherapy (TUS) and in combination (VEN/TUS) in a global Phase 1/2 trial of patients with R/R AML. AIMS: Study objectives are to assess the safety, tolerability, and pharmacokinetics (PK) of TUS and VEN/TUS, to support selection of a recommended phase 2 dose (RP2D). METHODS: Dose escalation (Part A) and exploration (Part B) of single agent TUS have completed dosing of patients treated at 20 mg to 200 mg once daily. In dose expansion (Part C), 80 mg of TUS is administered continuously as once daily oral monotherapy in 28-day cycles, or in combination with daily VEN with a C1D15 marrow assessment and planned 28-day cycles. Treatment emergent adverse events (TEAE) and clinical responses are evaluated using CTCAE and Revised IWG criteria, respectively. RESULTS: As of May 30, 2023, 100 patients received TUS in Parts A/B (20mg to 200mg; n=77) and in Part C (n=23: TUS 120 mg (n=14) and TUS 80 mg + VEN 400 mg dose equivalent (n=9)). Patients are predominantly Asian (46%) and White (41%) with a median age of 63 (range 18 to 84); 60% FLT3 wild-type (FLT3-WT), 35% FLT3-mutated (FLT3m) of which 18 (51%) received prior FLT3 inhibitors, and 5% FLT3 undetermined. Patients received a mean of 2.6 prior lines of therapy (range 1-6) (Table 1). Sixty percent of patients have received prior venetoclax. In Parts A/B, clinical responses with TUS monotherapy (composite complete remission [CRc; including CR, CRh, CRp and CRi]; see Figure 1 for definitions), were observed at 40 mg (15.4%), 80 mg (29.4%), 120 mg (7.1%), and 160 mg (16.7%) in both FLT3-WT (n=33, CRc:15.2%,) and FLT3-mutated (n=23, CRc:21.7%) patients. Overall response rate (ORR, including CRc and PR) was 14.3% in prior VEN treated patients (n=28), 23.1% in FLT3-mutated patients with previous FLT3i treatment (n=13), and 66.7% in NPM1 and FLT3-mutated patients (n=6). Notably, responses were also seen in patients with TP53 (n=5; CRc:40%) and RAS (n=9; CRc:22.2%) mutations frequently associated with resistance to VEN and standard therapies. TUS was well tolerated, with no dose limiting toxicity (DLT) observed at doses ranging from 20 mg to 160 mg. One patient experienced a DLT (Grade 3 muscular weakness) at 200 mg which demonstrated partial improvement at the time of treatment discontinuation (9 days after AE onset). Related TEAEs of Grade ≥3 were low (10.4%) with only neutrophil count decreased and muscular weakness (n=2 each; 2.6%) reported in more than 1 patient. No treatment-related non-hematologic SAEs, deaths, or discontinuations were observed. PK exposure was linear (20 mg-160 mg). The RP2D of 80 mg was selected for TUS monotherapy. In Part C, all 9 patients receiving VEN/TUS have tolerated treatment to date and remain on study. Eight (89%) patients had prior VEN treatment. Preliminary responses have been observed at the first post-dose bone marrow assessment (C1D15), including patients with prior VEN treatment. No treatment-related SAEs or TEAEs of Grade ≥3 have been reported. Updated enrollment, safety, PK, and response data will be presented at the meeting along with findings on the association between somatic mutations and TUS sensitivity as well as results for the TUS food-effect absorption study. CONCLUSIONS: Tuspetinib was well-tolerated and delivered single agent clinical responses across four dose levels among diverse AML genotypes; with a RP2D of 80 mg chosen for future single agent studies. Although early, the VEN/TUS combination has been well tolerated with preliminary objective responses noted.

An Open-Label, Multicenter, Single-Arm, Phase I Dose Escalation Study of OP-2100, an Orally Bioavailable Prodrug of Decitabine, for Patients with Relapsed or Refractory Higher-Risk Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia

CONCLUSIONS Hypomethylating agents (HMA) are commonly used to treat high-risk myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML).Decitabine (DEC), one of the HMAs, is S phase-specifically incorporated into DNA to exert its effect, and sustained plasma concentration may increase its efficacy (Saunthararajah et al. J Clin Invest. 2015). Therefore, we developed OP-2100, an orally bioavailable prodrug of DEC. It showed equal efficacy to conventional HMAs in various blood cancer mouse models and exhibited a favorable safety profile in healthy mice (Watanabe et al. Blood. 2020; Ureshino et al. Mol Cancer Ther. 2021). In addition, it exhibited a sustained-release pharmacokinetic (PK) profile when orally administered to monkeys. In a phase I study, OP-2100 monotherapy showed the expected PK profile on patients with relapsed or refractory higher-risk MDS and CMML, and we reported the preliminary result. We designed a single-arm, multicenter phase I study for Japanese patients with MDS or CMML with intermediate-risk, high-risk, or very high-risk by Revised International Prognostic Scoring System (IPSS-R) and relapsed, refractory, or intolerant to standard therapy. The primary objective was to determine the maximum tolerated dose following the incidence of dose-limiting toxicity (DLT) in the first cycle, whereas the secondary objectives were to evaluate the safety, PK, and efficacy. Treatment-emergent adverse events (TEAEs) and response to therapy were assessed per NCI CTCAE v5.0 and IWG 2018 response criteria, respectively. Oral administration of OP-2100 as a single agent was continued for the patients daily on days 1-5 of each 28-day cycle until meeting discontinuation criteria, including disease progression. The dose level was defined as 30, 50, 80, and 120 mg/day. The initial dose of 50 mg/day was determined based on the highest non-severely toxic dose of OP-2100 in monkeys and the PK of DACOGEN TM in humans. This study was implemented in the 3+3 design according to DLT incidence. The plasma concentration of DEC and OP-2100 was measured on days 1 and 5. Furthermore, the LINE-1 methylation, a common HMA pharmacodynamics marker, was measured. This study is registered in the Japan Registry for Clinical Trials (jRCT2071220035). In total 3 patients with a history of azacitidine treatment for MDS were registered in the 50-mg initial cohort. These patients had been categorized as intermediate to high-risk in the IPSS-R, respectively. Table 1 shows patient characteristics. Out of 3, 2 patients did not experience DLT, while the 3rd one suffered from Grade 4 neutropenia, which was confirmed as DLT. As of April 30, the most common TEAEs were cytopenia of leukopenia, neutropenia, and thrombocytopenia, all of which were Grade 3 or 4. Other adverse events of Grade 3 or 4 include anemia, lymphopenia, COVID-19, and fall. In terms of efficacy, 2 patients had stable disease, while one had progressive, according to the IWG2018 response criteria. However, the 2 patients with stable disease still received administration up to 9 and 5 cycles, respectively (Figure 1). The PK profile of DEC after OP-2100 administration indicated sustained release, with an area under the curve (AUC) similar to that of DACOGEN TM (5-day regimen, 1-h continuous intravenous administration at 20 mg/m 2) and C max approximately 1/5. The LINE-1 methylation exhibited the similar behavior as conventional HMAs. The PK profile of DEC released from the prodrug after 50 mg of OP-2100 oral administration in 3 patients showed a similar AUC to that of the 5-day regimen of DACOGEN TM, and then C max showed a low with sustained plasma concentration. Among the 3 patients, OP-2100 could be administered for at least 5 cycles in 2 patients. To determine maximum tolerated dose, enrollment for this study is ongoing.

Repressing HIF-1α-Induced HDAC9 Contributes to the Synergistic Effect of Venetoclax and Menin Inhibitor in KMT2Ar AML

Background: MLL-rearranged acute myeloid leukemia (MLLr-AML) is one aggressive subtype of AML, and is conferred one extremely poor prognosis under the current therapeutic strategy. Up to now, it still lacked one effective treatment to maintain the long-term survival for patients with MLLr-AML, while the developments of targeted therapy brought hopes. At present, the star drug BCL-2 inhibitor Venetoclax combined with Azacitidine (AZA) or low-dose Cytarabine (LDAC) has become the first-line scheme for elderly AML, which is not beneficial for MLLr-AML patients. So we are committed to finding a more suitable partner for VEN because MLLr-AML is very sensitive to VEN in vitro studies. The MLL-menin complex is crucial for maintaining MLLr-AML, and the research and application of MLL-menin inhibitors (MEN1i) is currently one of the hotspots in the AML field. Therefore in this study, we aimed to uncover the synergistic effect and mechanism of MEN1i combined with Venetoclax in treating MLLr-AML. Methods: Cell culture and transfection, Cell proliferation, apoptosis and Cell cycle assay, Quantitative RT-PCR, and Western blotting analysis were used to explore biological phenotypes. RNA-seq profiling was performed for DMSO, VEN, MEN1i, and combination of VEN and MEN1i groups in three MLLr-AML cell lines MV4-11, MOLM13, THP-1. Bioinformatic tools were used for differential analysis. Results: MLLr-AML cells were sensitive to VEN or MEN1i monotherapy, and the combination of two drugs had a strong synergistic effect in vitro and in vivo. Furthermore, we found that combination of VEN and MEN1i significantly enhanced apoptotic induction to influence the MLLr-AML survival. To uncover the synergetic mechanism of VEN and MEN1i, we displayed RNA-Sequencing for DMSO, VEN, MEN1i, and combination of VEN and MEN1i groups in three MLLr-AML cell lines MV4-11, MOLM13, THP-1. Finally we found that repressing HIF-1α-induced HDAC9 contributes to the synergistic effect of Venetoclax and MEN1i in MLLr-AML. Conclusion: VEN combined with MEN1i had a strong synergistic effect in the treatment of MLLr-AML. Repressing HIF-1α-induced HDAC9 contributes to the synergistic effect of Venetoclax and MEN1i in MLLr-AML.

Evaluation of Expression Profile of Patients with Acute Myeloid Leukemia in Response to Azacitidine with Biological System Approach.

Acute myeloid leukemia (AML) is a prevalent type of leukemia that is associated with high rates of chemoresistance, including resistance to Azacitidine (AZA). Understanding the molecular mechanisms of chemoresistance can lead to the development of novel therapeutic approaches. In this study, we aimed to identify dysregulated miRNAs and their target genes involved in chemoresistance to AZA in AML patients. We analyzed expression profiles from two GEO datasets (GSE16625 and GSE77750) using the "Limma" package in R. We identified 29 differentially expressed miRNAs between AML patients treated with AZA and healthy individuals. MultiMiR package of R was used to predict target genes of identified miRNAs, and functional enrichment analysis was performed using FunRich software. Protein-protein interaction networks were constructed using STRING and visualized using Cytoscape. MiR-582 and miR- 597 were the most up- and down-regulated miRNAs, respectively. Functional enrichment analysis revealed that metal ion binding, regulation of translation, and proteoglycan syndecan-mediated signaling events were the most enriched pathways. The tumor necrosis factor (TNF) gene was identified as a hub gene in the protein-protein interaction network. Our study identified dysregulated miRNAs and their target genes in response to AZA treatment in AML patients. These findings provide insights into the molecular mechanisms of chemoresistance and suggest potential therapeutic targets for the treatment of AML. Further experimental validation of the identified miRNAs and their targets is warranted.

Clinical Outcomes in Patients With Refractory Anemia With Excess Blasts (RAEB) Who Receive Hypomethylating Agents (HMAs)

BackgroundWe sought to understand the clinical effectiveness associated with use of hypomethylating agents (HMAs) azacitidine (AZA) and decitabine (DEC) for patients with refractory anemia with excess blasts (RAEB; an established proxy for higher-risk myelodysplastic syndromes/neoplasms) in contemporary and representative real-world settings. Patients and methodsWe used the Surveillance, Epidemiology and End Results (SEER)-Medicare database, a linkage of cancer registry and Medicare claims data, to identify patients aged ≥ 66 years diagnosed with RAEB, between 2009 and 2017 in the United States, and who received AZA or DEC as first-line therapy. Outcomes measured were overall survival (OS), event-free survival (EFS), and incidence of progression-related acute myeloid leukemia (AML). ResultsOf 973 eligible patients, 738 (75.8%) received AZA and 235 (24.2%) received DEC; 6.4% received hematopoietic cell transplantation during follow-up. In the overall population, median OS was 13.9 months (95% confidence interval [CI]: 12.9-15.0), median EFS was 5.2 months (95% CI: 4.9-5.7), and 38.0% of patients progressed to AML. Incidences of AML progression and death were 25.6% and 29.9%, respectively, at Year 1, and 34.3% and 44.8%, respectively, at Year 2. There were no significant differences in clinical benefits between AZA and DEC. ConclusionMedian OS with both HMAs remained significantly shorter than in the AZA-001 clinical trial, highlighting how patient outcomes vary between clinical and real-world settings. Further research is required to understand why these disparities exist.

Real-life data of azacitidine-venetoclax combination in acute myeloid leukemia patients: a single center experience

Aims: To evaluate real-life data on the efficacy and safety of Venetoclax (Ven) therapy used in combination with hypomethylating agent (HMA) in patients with acute myeloid leukemia (AML).
 Methods: The records of newly diagnosed, relapsed or refractory (RR) AML patients over 18 years of age who were planned to be treated with Azacitidine (AZA) combined with Ven because they were not suitable for intensive chemotherapy and patients who received AZA combined with Ven maintenance therapy after achieving remission were retrospectively analyzed. The standard protocol for patients is subcutaneous or intravenous AZA 75 mg/m2 on days 1-7/ every 28 days + oral Ven treatment 100-400 mg/day for 28 days. The treatment response rates, survival times, and side effect profiles of 18 newly diagnosed patients, 12 RR patients, and 4 patients receiving AZA+Ven as maintenance treatment between January 2021 and March 2022 were evaluated.
 Results: It was found that 8 of the 34 patients (23.5%) who were examined in the present study died before the first response could be evaluated. When the response rates were evaluated, complete response (CR) or complete remission with incomplete blood count recovery (CRi) (CR+CRi) was found to be 61% in the group receiving AZA+Ven in the first line, and CR+CRi was 50% in the group receiving AZA+Ven because of RR AML. In the group receiving AZA+Ven in the first line, the average Overall Survival (OS) was 8.00 months (95% CI: 1.58-14.41), and 7.00 months in the RR group (95% CI: 1.78-12, 21). All patients in the group receiving AZA+Ven for maintenance purposes were alive and the median follow-up period was 12.50±6.02 months in this group (Mean±SD). The most common side effect was neutropenia, and the most common cause of death was disease progression.
 Conclusion: In AML patients ineligible for intensive treatment due to advanced age or comorbidities, real-life data of AZA+Ven therapy with effective CR+CRi rates and a manageable spectrum of side effects promise hope.

Laser boosting the influx of calcium ions to enhance gasdermin e-dependent pyroptosis driven by a dual-layer polydopamine nanoagonist

As a recently revealed form of programmed cell death (PCD), pyroptosis can evoke a powerful inflammatory response and promote systemic immunity via a gasdermin E (GSDME)-dependent pathway. However, pyroptosis inevitably suffers from setbacks arising from the downregulated expression of GSDME and deficient caspase-3 activity in solid tumors. Herein, we designed a laser-boosted dual-layer polydopamine (PDA) hybrid nanoplatform (DMP@P) that codelivered decitabine (DCT) and mitoxantrone (MIT) to achieve high-efficiency pyroptosis. After reaching the tumor region, the PDA cloak is decomposed by the weakly acidic tumor environment, and the DCT released from the mesoporous polydopamine (MPDA) core upregulates GSDME expression by inhibiting DNA methylation. Moreover, near-infrared (NIR) laser irradiation causes a sharp influx of calcium ions (Ca2+), leading to cytochrome c release and cell dysfunction, which synergistically boosts caspase-3 activation by MIT. Activated caspase-3 cleaves upregulated GSDME by splitting the C-terminal region to generate the GSDME N-terminus (GSDME-N), and then GSDME-N aggregates to form pore-forming domains. Finally, inflammatory cytokines and antigens are rapidly released through the pores, which initiates extensive cell pyroptosis and induces an intense antitumor immune response to inhibit tumor proliferation and suppress metastasis. Overall, this nanoplatform with intrinsic biocompatibility provides inspiration for pyroptosis-induced cancer therapy.

Refractory patients with favorable/intermediate-risk acute myeloid leukemia benefit from azacytidine maintenance therapy following allogeneic hematopoietic stem cell transplantation.

Recurrence following allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the major cause of treatment failure in patients with myeloid malignancy. Azacytidine (AZA) maintenance is a promising therapy to prevent relapse and improve survival. We conducted a prospective, one-arm study involving 78 patients with myeloid malignancy at a high risk of recurrence who were enrolled between September 2019 and April 2022. Furthermore, 102 matched historical controls were selected using propensity score matching. With a median follow-up time of 19.6 (3.5-91.7) months, AZA maintenance therapy significantly improved relapse-free survival (RFS; log-rank test, p=0.01). The AZA and control groups had a 1-year RFS of 87.7% (95% confidence interval [CI], 0.80-0.96) and 72.2% (95% CI, 0.64-0.82), respectively, with a hazard ratio (HR) of 0.21 (95% CI, 0.09-0. 47; p<0.01). There were no grade 4 adverse effects or deaths related to AZA. Refractory patients with favorable/intermediate-risk acute myeloid leukemia (AML) benefited more from AZA maintenance therapy than those with adverse-risk AML according to the European Leukemia Net guidelines (RFS in favorable/intermediate-risk AML, HR=0.29, 95% CI, 0.11-0.79; RFS in adverse-risk AML, HR=0.57, 95% CI, 0.21-1.6; p for interaction=0.03). Our findings suggest that AZA maintenance therapy following allo-HSCT was safe and could reduce the incidence of relapse, particularly for refractory patients with favorable/intermediate-risk AML.

Comparative Efficacy of Venetoclax-Based Combination Therapies and Other Therapies in Treatment-Naive Patients With Acute Myeloid Leukemia Ineligible for Intensive Chemotherapy: A Network Meta-Analysis

ObjectivesThis network meta-analysis (NMA) assessed the efficacy of venetoclax (VEN) + azacitidine (AZA) and VEN + low-dose cytarabine (LDAC) compared with AZA, LDAC, and decitabine monotherapies and best supportive care (BSC) in adults with untreated acute myeloid leukemia ineligible for intensive chemotherapy. MethodsA systematic literature review and feasibility assessment was conducted to select phase III randomized controlled trials for inclusion in the NMA. Complete remission + complete remission with incomplete blood count recovery and overall survival (OS) were compared using a Bayesian fixed-effects NMA. Treatments were ranked using surface under the cumulative ranking curves (SUCRAs) with higher values indicating a higher likelihood of being effective. ResultsA total of 1140 patients across 5 trials were included. VEN + LDAC (SUCRA 91.4%) and VEN + AZA (87.5%) were the highest ranked treatments for complete remission + complete remission with incomplete blood count recovery. VEN + LDAC was associated significantly higher response rates versus AZA (odds ratio 5.64), LDAC (6.39), and BSC (23.28). VEN + AZA was also associated significantly higher response rates than AZA (5.06), LDAC (5.74), and BSC (20.68). In terms of OS, VEN + AZA (SUCRA: 95.2%) and VEN + LDAC (75.9%) were the highest ranked treatments. VEN + AZA was associated with significant improvements in OS compared with AZA (hazard ratio 0.66), LDAC (0.57), and BSC (0.37), and VEN + LDAC was associated with significant improvements in OS compared with LDAC (0.70) and BSC (0.46). ConclusionsVEN + AZA and VEN + LDAC demonstrated improved efficacy compared with alternative therapies among treatment-naive patients with acute myeloid leukemia ineligible for intensive chemotherapy.

Venetoclax with Hypomethylating Agents in Newly Diagnosed Acute Myeloid Leukemia: A Systematic Review and Meta-Analysis of Survival Data from Real-World Studies.

In recent years, the association of venetoclax (VEN) with hypomethylating agents (HMAs) significantly improved the outcome of patients with newly diagnosed acute myeloid leukemia (AML) who were unfit for intensive chemotherapy and became the standard of care after the publication of the pivotal RCT VIALE-A. However, it is still not clear to what extent the results observed in the VIALE-A apply to a real-world setting. For this reason, we carried out a systematic review and meta-analysis of real-world studies on newly diagnosed patients with AML, ineligible for intensive induction chemotherapy, receiving first-line VEN+HMA. We then compared their results in term of survival with those from the VIALE-A. Kaplan-Meier curves were extracted from all included studies and individual survival data was reconstructed. We then estimated a pooled survival curve and compared it with the results of the VIALE-A using the log-rank test. We also conducted a secondary analysis including only studies considering VEN plus azacytidine (AZA) as treatment, as this was the schedule originally used in the VIALE-A. Nineteen real-world studies met the inclusion criteria and were included in the systematic review. Most of them reported a worse survival than the VIALE-A. The pooled survival curve was similar to that reported in the VIALE-A during the first three months of treatment but diverged thereafter (p-value = 0.0001). The pooled median survival among the real-world studies was 9.37 months (95%CI 8.81-10.5), substantially lower than that reported in the VIALE-A (14.7 months; 95%CI 11.9-18.7). Results slightly increased when the analysis was restricted to the studies using VEN+AZA as treatment (median survival: 11.5 months; 95%CI 10.2-14.8). Survival of newly diagnosed AML patients treated with VEN+HMAs in a real-world setting seems to be lower than previously reported in the VIALE-A, while the effect of VEN+AZA is more in line with expected results. Future studies are needed to evaluate whether this apparent discrepancy is due to the different characteristics of enrolled patients or to a non-optimal adherence to therapy, and whether alternative regimens can provide better results in terms of safety and effectiveness.

Antileishmanial potentials of azacitidine and along with meglumine antimoniate on Leishmania major: In silico prediction and in vitro analysis.

This study aimed to investigate the in vitro and in silico antileishmanial activity of azacitidine (AZA) on Leishmania major promastigotes and amastigotes. The in silico method was used to evaluate the possibility of the interaction of AZA into the binding pocket of inducible nitric oxide synthase (iNOS), a leading defensive oxidative metabolite. Following that, in vitro anti-promastigote, and anti-amastigote activity of AZA was determined using an MTT assay and a macrophage model, respectively. Cytotoxic effects of AZA and meglumine antimoniate (MA) were also assessed by MTT assay on murine macrophages. All experiments were performed in triplicate. The results showed that AZA interacted with Ser133, Gln134, and Lys13 amino acids of iNOS, and the molecular docking score was obtained at -241.053 kcal/mol. AZA in combination with MA significantly (P<0.001) inhibited the growth rate of nonclinical promastigote (IC50 247.6±7.3 μM) and 8.5-fold higher of clinical intramacrophage amastigote stage (29.8±5.3 μM), compared to the untreated group. A significant upsurge of Th1 subsets and transcription genes and a meaningful decline in Th2 cytokines subclasses at the equivalent concentrations of AZA and MA was observed (P<0.001). The apoptosis effect of AZA along with MA was significantly induced on L. major in a dose-dependent manner (P<0.001). The present study demonstrated that AZA possesses antileishmanial activity in in vitro and in silico models. However, AZA combined with MA was more effective than AZA alone in inhibiting the growth rate of promastigotes and amastigotes of L. major. This study indicates that AZA in combination with MA demonstrated a potent antileishmanial mechanism, promoting immune response and enhancing an immunomodulatory role toward the Th1 pathway. This experimental study is a basic study for applying more knowledge about the mechanisms of AZA along with MA in animal models in the future.

AML-446 Time to Resolution of Myelosuppression and Associated Hospitalization in Patients With Newly-Diagnosed Acute Myeloid Leukemia (AML) Treated With Ivosidenib (IVO) + Azacitidine (AZA) Compared to Placebo (PBO) + AZA

AML-446 Time to Resolution of Myelosuppression and Associated Hospitalization in Patients With Newly-Diagnosed Acute Myeloid Leukemia (AML) Treated With Ivosidenib (IVO) + Azacitidine (AZA) Compared to Placebo (PBO) + AZA

POSTER: AML-446 Time to Resolution of Myelosuppression and Associated Hospitalization in Patients With Newly-Diagnosed Acute Myeloid Leukemia (AML) Treated With Ivosidenib (IVO) + Azacitidine (AZA) Compared to Placebo (PBO) + AZA

POSTER: AML-446 Time to Resolution of Myelosuppression and Associated Hospitalization in Patients With Newly-Diagnosed Acute Myeloid Leukemia (AML) Treated With Ivosidenib (IVO) + Azacitidine (AZA) Compared to Placebo (PBO) + AZA

AML-454 Real-World Use of Azacitidine (AZA) and Venetoclax (VEN) in Acute Myeloid Leukemia (AML) in Frontline and Relapse/Refractory (R/R) Settings: Multicenter Study From French AURAML Group

AML-454 Real-World Use of Azacitidine (AZA) and Venetoclax (VEN) in Acute Myeloid Leukemia (AML) in Frontline and Relapse/Refractory (R/R) Settings: Multicenter Study From French AURAML Group

POSTER: AML-454 Real-World Use of Azacitidine (AZA) and Venetoclax (VEN) in Acute Myeloid Leukemia (AML) in Frontline and Relapse/Refractory (R/R) Settings: Multicenter Study From French AURAML Group

POSTER: AML-454 Real-World Use of Azacitidine (AZA) and Venetoclax (VEN) in Acute Myeloid Leukemia (AML) in Frontline and Relapse/Refractory (R/R) Settings: Multicenter Study From French AURAML Group

Decitabine disrupts EBV genomic epiallele DNA methylation patterns around CTCF binding sites to increase chromatin accessibility and lytic transcription in gastric cancer.

Epstein-Barr virus (EBV) latency is controlled by epigenetic silencing by DNA methylation [5-methyl cytosine (5mC)], histone modifications, and chromatin looping. However, how they dictate the transcriptional program in EBV-associated gastric cancers remains incompletely understood. EBV-associated gastric cancer displays a 5mC hypermethylated phenotype. A potential treatment for this cancer subtype is the DNA hypomethylating agent, which induces EBV lytic reactivation and targets hypermethylation of the cellular DNA. In this study, we identified a heterogeneous pool of EBV epialleles within two tumor-derived gastric cancer cell lines that are disrupted with a hypomethylating agent. Stochastic DNA methylation patterning at critical regulatory regions may be an underlying mechanism for spontaneous reactivation. Our results highlight the critical role of epigenetic modulation on EBV latency and life cycle, which is maintained through the interaction between 5mC and the host protein CCCTC-binding factor.

Acquired resistance to venetoclax plus azacitidine in acute myeloid leukemia: In vitro models and mechanisms

The combination of venetoclax (VEN) and azacitidine (AZA) has become the standard of care for acute myeloid leukemia (AML) patients who are ≥ 75 years or unfit for intensive chemotherapy. Though initially promising, resistance to the combination therapy is an issue and VEN + AZA-relapsed/refractory patients have dismal outcomes. To better understand the mechanisms of resistance, we developed VEN + AZA-resistant AML cell lines, MV4-11/VEN + AZA-R and ML-2/VEN + AZA-R, which show > 300-fold persistent resistance compared to the parental lines. We demonstrate that these cells have unique metabolic profiles, including significantly increased levels of cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP), changes in fatty acid and amino acid metabolism and increased utilization and reliance on glycolysis. Furthermore, fatty acid transporter CD36 is increased in the resistant cells compared to the parental cells. Inhibition of glycolysis with 2-Deoxy-D-glucose re-sensitized the resistant cells to VEN + AZA. In addition, the VEN + AZA-R cells have increased levels of the antiapoptotic protein Mcl-1 and decreased levels of the pro-apoptotic protein Bax. Overexpression of Mcl-1 or knockdown of Bax result in resistance to VEN + AZA. Our results provide insight into the molecular mechanisms contributing to VEN + AZA resistance and assist in the development of novel therapeutics to overcome this resistance in AML patients.