[Safety and efficacy of 7-day administration of azacitidine and venetoclax combination therapy (7+7 therapy)].

Acute myelogenous leukemia (AML) has a poor prognosis in patients who are ineligible for intensive chemotherapy. The combination of azacitidine and venetoclax has been shown to have high overall efficiency and remission rates, even in patients ineligible for aggressive chemotherapy. However, myelosuppression is often prolonged after treatment, and infection can also occur. Severe myelosuppression is often addressed by dose titration, but specific dose titration methods have not been clarified. We used the standard induction therapy with azacitidine plus venetoclax, and if blasts decreased to 20% or less, switched to 7+7 therapy to shorten venetoclax to 7 days starting from the next cycle. In the 19 patients we treated (median age 80 years), response rate above MLFS was 100%, CR 57.9%, CRc (CR+CRi) 78.8%, median OS 693 days, median PFS 458 days, and median OS was not reached in previously untreated patients. This indicates that 7+7 is a highly effective and well-tolerated treatment.

Combination therapy with venetoclax and azacitidine for the treatment of myelodysplastic syndromes with DDX41 mutations.

Myelodysplastic syndromes (MDS) patients with DEAD-box helicase 41 (DDX41) mutations have been reported to be treated effectively with lenalidomide; however, there are no randomized studies to prove it. Venetoclax and azacitidine are safe and effective in high-risk MDS/AML. In this study, we evaluated the efficacy of venetoclax and azacitidine combination therapy in eight consecutive MDS patients with DDX41 mutations at our centre from March 2021 to November 2023. We retrospectively analyzed the genetic features and clinical characteristics of these patients. Our findings suggest that MDS patients with DDX41 mutation may benefit from the therapy, for six subjects received this regimen as initial therapy and five of the six subjects achieved complete remission.

Role of plasma EBV-DNA load and EBER status on newly diagnosed peripheral T-cell lymphoma.

PURPOSE: To explore the prognostic and therapeutic role of Epstein-Barr Virus (EBV) on peripheral T-cell lymphoma (PTCL). METHODS: Totally 262 newly diagnosed PTCL patients who were hospitalized from January 2014 to December 2022 were retrospectively enrolled. Molecular analysis included 31 eligible patients. EBV-encoded RNA (EBER) presence in tumor tissue and EBV DNA levels in patients at baseline (DNA1) and after 4 cycles of chemotherapy (DNA4) were assessed. RESULTS: Our findings revealed that the EBER-positive cohort exhibited significant differences compared to counterparts in overall survival (OS, P = 0.047) and progression-free survival (PFS, P = 0.009). Both DNA1 and DNA4 were significantly associated with inferior OS. Multivariate analysis demonstrated that DNA4 independently affected PTCL prognosis for OS (hazard ratio = 5.1617; 95% confidence interval 1.1017-24.1831; P = 0.037). Treatment with the cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) plus azacytidine regimen showed a better OS compared to CHOP or CHOP plus etoposide for patients with partially positive EBER and EBER positive statuses (P = 0.192), although the improvement was not statistically significant. This study delineated the genetic paradigm of PTCL, comparing genetic differences by EBV status and found that EBER partially positive plus positive patients were more likely to have DNMT3A (P = 0.002), RHOAG17V (P = 0.023), and TET2 mutations (P = 0.032). CONCLUSION: EBER, DNA1, and DNA4 emerged as sensitive markers for prognosis. CHOP plus azacytidine might present a preferable option for PTCL patients with DNA methylation due to EBV infection.

Lycocasine A, a Lycopodium Alkaloid from Lycopodiastrum casuarinoides and Its Acid-Sensing Ion Channel 1a Inhibitory Activity.

A novel Lycopodium alkaloid, lycocasine A (1), and seven known Lycopodium alkaloids (2-8), were isolated from Lycopodiastrum casuarinoides. Their structures were determined through NMR, HRESIMS, and X-ray diffraction analysis. Compound 1 features an unprecedented 5/6/6 tricyclic skeleton, highlighted by a 5-aza-tricyclic[6,3,1,02,6]dodecane motif. In bioactivity assays, compound 1 demonstrated weak inhibitory activity against acid-sensing ion channel 1a.

[The Efficacy and Safety of Venetoclax Combined with Azacitidine in the Treatment of Adult Patients with Acute Myeloid Leukemia Who Are Unfit for Intensive Chemotherapy].

OBJECTIVE: To observe the clinical efficacy and safety of venetoclax (VEN) combined with azacitidine (AZA) in the treatment of adult acute myeloid leukemia (AML) patients who are unfit for intensive chemotherapy. METHODS: The clinical data of 21 adult patients with unfit AML who were treated with VEN combined with AZA in the Second Hospital of Shanxi Medical University from January 2021 to May 2022 were collected, and the efficacy and safety were analyzed retrospectively. RESULTS: After one course of treatment with VEN and AZA, 16 out of 21 unfit AML patients reached complete remission (CR)/CR with incomplete hematologic recovery (CRi), 2 patients reached partial remission (PR), the overall response rate (ORR) was 85.7%. Among the 16 patients with CR/CRi, 13 achieved minimal residual disease (MRD) negativity. Among the 11 patients with adverse prognosis, 8 achieved CR/CRi. By the deadline of follow-up, the median overall suivival (OS) of the entire cohort was not reached, with 1-year OS rate of 61.7%. The main adverse events of VEN combined with AZA were myelosuppression, gastrointestinal reactions and infections. There were 13 cases of leukopenia, 7 cases of neutropenia, 7 cases of anemia, 4 cases of thrombocytopenia, and these hematologic adverse events were all grade 3-4. There were 11 cases with gastrointestinal reactions and 7 cases with infections. The above adverse events were controllable and tolerable. No tumor lysis syndrome or infection related death occurred. CONCLUSION: VEN combined with AZA can quickly achieve deep remission in adult patients with unfit AML, and it shows a good safety profile.

A Case of Relapsed/Refractory CD56-Positive Acute Promyelocytic Leukemia, in Which Complete Molecular Remission Was Achieved Following Combination Therapy with Venetoclax and Azacitidine.

An 84-year-old woman was diagnosed as having acute promyelocytic leukemia(APL)in July Year X-3. The test for promyelocytic leukemia- retinoic acid receptor alpha(PML-RARA)mRNA was positive, while that for CD56 was negative. Since her white blood cell( WBC) count was <3,000/µL, with a count of APL cells of <1,000/µL, she was started on monotherapy with all-trans retinoic acid(ATRA). In September Year X-3, complete hematological remission(CHR)was confirmed. she refused to provide consent for receiving consolidation therapy. In February Year X-2, hematological relapse occurred. She was started on re-induction therapy with arsenite(ATO), and in June Year X-2, complete molecular remission(CMR)was achieved. She was started on post-remission therapy with ATO. In August Year X-1, she developed molecular relapse and was started on tamibarotene(Am80). In October Year X-1, hematological relapse was detected, and the test for CD56 was positive. She was started on combined venetoclax(VEN)+azacitidine(AZA)(VEN+AZA). After completion of 1 course of treatment, CMR was achieved, but she developed hematological relapse after 5 courses of treatment. She died of gastrointestinal hemorrhage. This is considered a valuable case for accumulating information on the treatment of CD56-positive APL resistant to ATRA and ATO.

Differential Gene Regulatory Network Analysis between Azacitidine-Sensitive and -Resistant Cell Lines.

Azacitidine, a DNA methylation inhibitor, is employed for the treatment of acute myeloid leukemia (AML). However, drug resistance remains a major challenge for effective azacitidine chemotherapy, though several studies have attempted to uncover the mechanisms of azacitidine resistance. With the aim to identify the mechanisms underlying acquired azacitidine resistance in cancer cell lines, we developed a computational strategy that can identify differentially regulated gene networks between drug-sensitive and -resistant cell lines by extending the existing method, differentially coexpressed gene sets (DiffCoEx). The technique specifically focuses on cell line-specific gene network analysis. We applied our method to gene networks specific to azacitidine sensitivity and identified differentially regulated gene networks between azacitidine-sensitive and -resistant cell lines. The molecular interplay between the metallothionein gene family, C19orf33, ELF3, GRB7, IL18, NRN1, and RBM47 were identified as differentially regulated gene network in drug resistant cell lines. The biological mechanisms associated with azacitidine and AML for the markers in the identified networks were verified through the literature. Our results suggest that controlling the identified genes (e.g., the metallothionein gene family) and "cellular response"-related pathways ("cellular response to zinc ion", "cellular response to copper ion", and "cellular response to cadmium ion", where the enriched functional-related genes are MT2A, MT1F, MT1G, and MT1E) may provide crucial clues to address azacitidine resistance in patients with AML. We expect that our strategy will be a useful tool to uncover patient-specific molecular interplay that provides crucial clues for precision medicine in not only gastric cancer but also complex diseases.

Reduced dose azacitidine plus venetoclax as maintenance therapy in acute myeloid leukaemia following intensive or low-intensity induction: a single-centre, single-arm, phase 2 trial.

BACKGROUND: Patients with acute myeloid leukaemia have high rates of relapse, especially if they are unable to complete standard consolidation strategies or allogeneic haematopoietic stem-cell transplantation (HSCT). The phase 3 QUAZAR AML-001 study showed an overall survival benefit with oral azacitidine maintenance. The BCL2 inhibitor venetoclax is highly active in acute myeloid leukaemia and synergistic with azacitidine. We aimed to evaluate the efficacy and safety of low dose azacitidine plus venetoclax as maintenance therapy in acute myeloid leukaemia. METHODS: We performed a single-centre, single-arm, phase 2 study at the University of Texas MD Anderson Cancer Center in the USA. Eligible patients were adults (aged ≥18 years) with a WHO 2016 diagnosis of acute myeloid leukaemia in complete remission or complete remission with incomplete blood count recovery following intensive or low-intensity induction and not immediately eligible for HSCT. Eastern Cooperative Oncology Group performance status had to be 3 or less. Patients were assigned to maintenance therapy with azacitidine 50 mg/m2 intravenously or subcutaneously for 5 days and venetoclax 400 mg orally for 7 days or 14 days. The primary outcome was relapse-free survival. The study was closed early due to slow accrual. All patients were included in the efficacy and safety analyses. This trial is registered with ClinicalTrials.gov (NCT04062266). FINDINGS: Between Sept 26, 2019, and Oct 26, 2022, 35 patients were enrolled, of whom 25 (71%) were assigned to cohort 1 following intensive induction and ten (29%) to cohort 2 following low-intensity induction. Of 35 patients, 18 (51%) were male and 17 (49%) were female. The median age was 55 years (IQR 41-62). The median number of cycles given was 9 (IQR 2-22) and median follow-up time was 23·3 months (IQR 9·0-30·0). The median relapse-free survival was not reached (95% CI 20·2 to not calculable) in the full cohort, not reached (29·1 to not calculable) in cohort 1, and 30·3 months (16·5 to not calculable) in cohort 2. The 2-year relapse-free survival was 65% (95% CI 50-85) in the full cohort, 71% (53-94) in cohort 1, and 52% (27-100) in cohort 2. The most common grade 3-4 treatment-emergent adverse events were thrombocytopenia (n=6), lung infection (n=4), leukopenia (n=4), and neutropenia (n=3). No deaths occurred during maintenance therapy. INTERPRETATION: Low dose azacitidine plus venetoclax is a feasible maintenance strategy in acute myeloid leukaemia following intensive and low-intensity induction. FUNDING: University of Texas MD Anderson Cancer Center, MDS/AML Moon Shot, Genentech.

Gilteritinib combined with venetoclax and azacitidine for relapsed acute myeloid leukemia cocurrent with pure red cell aplasia after allogeneic hematopoietic stem cell transplantation: a case report.

Pure red cell aplasia (PRCA) is a rare bone marrow (BM) disorder characterized by ineffective erythropoiesis, reduced reticulocyte count, normocytic anemia, and the absence of erythroid precursors. Here, we present a rare instance of PRCA occurring after ABO-matched allo-HSCT in a refractory/relapsed acute myeloid leukemia (R/R AML) patient. In this case, the patient received a combination treatment of Gilteritinib, Venetoclax, and Azacitidine. Remarkably, this treatment not only reduced myeloblasts but also facilitated the restoration of erythroid hematopoiesis.

Venetoclax plus cytarabine and azacitidine in relapsed/refractory AML: An open-label, single-arm, phase 2 study.

BACKGROUND: The outcome of relapsed/refractory (R/R) acute myeloid leukemia (AML) remains extremely poor. Venetoclax (VEN)-based regimens have shown promise in treating R/R AML. OBJECTIVE: This phase 2 study aimed to systematically evaluate the efficacy and safety of the VAA regimen (VEN plus Cytarabine and Azacitidine) in R/R AML patients. METHODS: Thirty R/R AML patients were enrolled. The study adopted a stepwise ramp-up of VEN dosing, starting with 100 mg on day 1, escalating to 200 mg on day 2, and reaching 400 mg from day 3 to day 9. Cytarabine (10 mg/m2, q12h) was administered intravenously twice daily from days 1 to 10, and Azacitidine (75 mg/m2) was administered via subcutaneous injection once daily from days 1-7. The primary efficacy endpoint was the composite complete remission rate (CRc), including complete response (CR) and complete response with incomplete blood count recovery (CRi). Secondary endpoints included overall survival (OS), duration of response (DOR), and safety analysis. RESULTS: The CRc rate was 63.3% (19/30), with CR in 36.7% of patients and CRi in 26.7%. Notably, 14 (73.7%) of 19 patients achieving CRc showed undetectable measurable residual disease by flow cytometry. With a median follow-up of 10.7 months, the median OS had not been reached, and the median DOR was 18.3 months. The most common grade 3-4 adverse events (AEs) were neutropenia (100%), anemia (96.7%), thrombocytopenia (90.0%), and leukopenia (90.0%). Infections, with pneumonia being the most prevalent (43.3%), were observed, including one fatal case of Pseudomonas aeruginosa septicemia. There were no treatment-related deaths. CONCLUSION: The VAA regimen is an effective and safe option for patients with R/R AML, demonstrating a high CRc rate and manageable safety profile.

Efficacy and safety of azacitidine in pediatric patients with newly diagnosed advanced myelodysplastic syndromes before hematopoietic stem cell transplantation in the AZA-JMML-001 trial.

Here we report efficacy, pharmacokinetics, and safety data obtained in treatment-naive, pediatric patients with newly diagnosed advanced MDS receiving azacitidine in the AZA-JMML-001 study. The primary endpoint was response rate (proportion of patients with complete response [CR], partial response [PR], or marrow CR, sustained for ≥4 weeks). Of the 10 patients enrolled, one had an unconfirmed marrow CR and none had confirmed responses after three cycles; the study was therefore closed after stage 1. Azacitidine was well tolerated. The lack of efficacy of azacitidine in pediatric patients with newly diagnosed advanced MDS highlights the need for effective new treatments in these patients.

Epigenetic remodeling to improve the efficacy of immunotherapy in human glioblastoma: pre-clinical evidence for development of new immunotherapy approaches.

BACKGROUND: Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor, that is refractory to standard treatment and to immunotherapy with immune-checkpoint inhibitors (ICI). Noteworthy, melanoma brain metastases (MM-BM), that share the same niche as GBM, frequently respond to current ICI therapies. Epigenetic modifications regulate GBM cellular proliferation, invasion, and prognosis and may negatively regulate the cross-talk between malignant cells and immune cells in the tumor milieu, likely contributing to limit the efficacy of ICI therapy of GBM. Thus, manipulating the tumor epigenome can be considered a therapeutic opportunity in GBM. METHODS: Microarray transcriptional and methylation profiles, followed by gene set enrichment and IPA analyses, were performed to study the differences in the constitutive expression profiles of GBM vs MM-BM cells, compared to the extracranial MM cells and to investigate the modulatory effects of the DNA hypomethylating agent (DHA) guadecitabine among the different tumor cells. The prognostic relevance of DHA-modulated genes was tested by Cox analysis in a TCGA GBM patients' cohort. RESULTS: The most striking differences between GBM and MM-BM cells were found to be the enrichment of biological processes associated with tumor growth, invasion, and extravasation with the inhibition of MHC class II antigen processing/presentation in GBM cells. Treatment with guadecitabine reduced these biological differences, shaping GBM cells towards a more immunogenic phenotype. Indeed, in GBM cells, promoter hypomethylation by guadecitabine led to the up-regulation of genes mainly associated with activation, proliferation, and migration of T and B cells and with MHC class II antigen processing/presentation. Among DHA-modulated genes in GBM, 7.6% showed a significant prognostic relevance. Moreover, a large set of immune-related upstream-regulators (URs) were commonly modulated by DHA in GBM, MM-BM, and MM cells: DHA-activated URs enriched for biological processes mainly involved in the regulation of cytokines and chemokines production, inflammatory response, and in Type I/II/III IFN-mediated signaling; conversely, DHA-inhibited URs were involved in metabolic and proliferative pathways. CONCLUSIONS: Epigenetic remodeling by guadecitabine represents a promising strategy to increase the efficacy of cancer immunotherapy of GBM, supporting the rationale to develop new epigenetic-based immunotherapeutic approaches for the treatment of this still highly deadly disease.

A 2-week treatment with 5-azacytidine improved the hypercontractility state in prostate from obese mice: Role of the nitric oxide-cyclic guanosine monophosphate signalling pathway.

Benign prostatic hyperplasia (BPH) is characterised by increases in prostate volume and contraction. Downregulation of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signalling pathway contributes to prostate dysfunctions. Previous studies in cancer cells or vessels have shown that the epigenetic mechanisms control the gene and protein expression of the enzymes involved in the production of NO and cGMP. This study is aimed to evaluate the effect of a 2-week treatment of 5-azacytidine (5-AZA), a DNA-methyltransferase inhibitor, in the prostate function of mice fed with a high-fat diet. Functional, histological, biochemical and molecular assays were carried out. Obese mice presented greater prostate weight, α-actin expression and contractile response induced by the α-1adrenoceptors agonist. The relaxation induced by the NO-donor and the protein expression of endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) were significantly decreased in the prostate of obese mice. The treatment with 5-AZA reverted the higher expression of α-actin, reduced the hypercontractility state of the prostate and increased the expression of eNOS and sGC and intraprostatic levels of cGMP. When prostates from obese mice treated with 5-AZA were incubated in vitro with inhibitors of the NOS or sGC, the inhibitory effect of 5-AZA was reverted, therefore, showing the involvement of NO and cGMP. In conclusion, our study paves the way to develop or repurpose therapies that recover the expression of eNOS and sGC and, hence, to improve prostate function in BPH.

Flow cytometry of DNMT1 as a biomarker of hypomethylating therapies.

The 5-azacytidine (AZA) and decitabine (DEC) are noncytotoxic, differentiation-inducing therapies approved for treatment of myelodysplastic syndrome, acute myeloid leukemias (AML), and under evaluation as maintenance therapy for AML postallogeneic hematopoietic stem cell transplant and to treat hemoglobinapathies. Malignant cell cytoreduction is thought to occur by S-phase specific depletion of the key epigenetic regulator, DNA methyltransferase 1 (DNMT1) that, in the case of cancers, thereby releases terminal-differentiation programs. DNMT1-targeting can also elevate expression of immune function genes (HLA-DR, MICA, MICB) to stimulate graft versus leukemia effects. In vivo, there is a large inter-individual variability in DEC and 5-AZA activity because of pharmacogenetic factors, and an assay to quantify the molecular pharmacodynamic effect of DNMT1-depletion is a logical step toward individualized or personalized therapy. We developed and analytically validated a flow cytometric assay for DNMT1 epitope levels in blood and bone marrow cell subpopulations defined by immunophenotype and cell cycle state. Wild type (WT) and DNMT1 knock out (DKO) HC116 cells were used to select and optimize a highly specific DNMT1 monoclonal antibody. Methodologic validation of the assay consisted of cytometry and matching immunoblots of HC116-WT and -DKO cells and peripheral blood mononuclear cells; flow cytometry of H116-WT treated with DEC, and patient samples before and after treatment with 5-AZA. Analysis of patient samples demonstrated assay reproducibility, variation in patient DNMT1 levels prior to treatment, and DNMT1 depletion posttherapy. A flow-cytometry assay has been developed that in the research setting of clinical trials can inform studies of DEC or 5-AZA treatment to achieve targeted molecular pharmacodynamic effects and better understand treatment-resistance/failure.

Antileukemic effect of azacitidine, a DNA methyltransferase inhibitor, on cell lines of myeloid leukemia associated with Down syndrome.

Myeloid leukemia associated with Down syndrome (ML-DS) responds well to chemotherapy and has a favorable prognosis, but the clinical outcome of patients with refractory or relapsed ML-DS is dismal. We recently reported a case of relapsed ML-DS with an effective response to a DNA methyltransferase inhibitor, azacitidine (AZA). However, the efficacy of AZA for refractory or relapsed ML-DS remains uncertain. Here, we investigated the effects and mechanism of action of AZA on three ML-DS cell lines derived from relapsed cases. AZA inhibited the proliferation of all examined ML-DS cell lines to the same extent as that of AZA-sensitive acute myeloid leukemia non-Down syndrome cell lines. Transient low-dose AZA treatment exerted durable antileukemic effects on ML-DS cells. The inhibitory effect included cell cycle arrest, apoptosis, and reduction of aldehyde dehydrogenase activity. Comprehensive differential gene expression analysis showed that AZA induced megakaryocytic differentiation in all ML-DS cell lines examined. Furthermore, AZA induced activation of type I interferon-stimulated genes, primarily involved in antiproliferation signaling, without stimulation of the interferon receptor-mediated autocrine system. Activation of the type I interferon pathway by stimulation with interferon-α exerted antiproliferative effects on ML-DS cells, suggesting that AZA exerts its antileukemic effects on ML-DS cells at least partially through the type I interferon pathway. Moreover, the effect of AZA on normal hematopoiesis did not differ significantly between individuals with non-Down syndrome and Down syndrome. In summary, this study suggests that AZA is a potentially effective treatment option for ML-DS disease control, including relapsed cases, and has reduced side effects.