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.

Combination of venetoclax and azacitidine in relapsed/refractory acute B-cell lymphoblastic leukemia: a case series from a single center.

OBJECTIVES: Relapsed/refractory acute B-cell lymphoblastic leukemia (R/R B-ALL) often responds poorly to induction chemotherapy. However, recent research has shown a novel and effective drug treatment for R/R B-ALL. METHODS: A total of eight patients with R/R B-ALL were enrolled in the study from November 2021 to August 2022. All patients received chemotherapy based on a combination regimen of venetoclax and azacitidine. The regimen was as follows venetoclax 100 mg d1, 200 mg d2, 400 mg d3-14, azacitidine 75 mg/m2 d1-7. RESULTS: Five of eight patients achieved very deep and complete remission (CR) with minimal residual disease (MRD) less than 0.1%. One patient achieved partial remission. Two patients did not achieve remission. There were no serious adverse events and all patients were well tolerated. Three patients were eligible for consolidation chemotherapy and were bridged to CAR-T therapy. CONCLUSIONS: The combined regimen of venetoclax and azacitidine may be beneficial for patients with R/R B-ALL.

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.

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.

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.

Single-cell Multiomics Analysis of Myelodysplastic Syndromes and Clinical Response to Hypomethylating Therapy.

Alterations in epigenetic marks, such as DNA methylation, represent a hallmark of cancer that has been successfully exploited for therapy in myeloid malignancies. Hypomethylating agents (HMA), such as azacitidine, have become standard-of-care therapy to treat myelodysplastic syndromes (MDS), myeloid neoplasms that can evolve into acute myeloid leukemia. However, our capacity to identify who will respond to HMAs, and the duration of response, remains limited. To shed light on this question, we have leveraged the unprecedented analytic power of single-cell technologies to simultaneously map the genome and immunoproteome of MDS samples throughout clinical evolution. We were able to chart the architecture and evolution of molecular clones in precious paired bone marrow MDS samples at diagnosis and posttreatment to show that a combined imbalance of specific cell lineages with diverse mutational profiles is associated with the clinical response of patients with MDS to hypomethylating therapy. SIGNIFICANCE: MDS are myeloid clonal hemopathies with a low 5-year survival rate, and approximately half of the cases do not respond to standard HMA therapy. Our innovative single-cell multiomics approach offers valuable biological insights and potential biomarkers associated with the demethylating agent efficacy. It also identifies vulnerabilities that can be targeted using personalized combinations of small drugs and antibodies.

Azacitidine Post-transplant Maintenance Improves Disease Progression in High-Risk Acute Myeloid Leukemia and Myelodysplastic Syndrome.

BACKGROUND: Maintenance after allogeneic hematopoietic cell transplantation (alloHCT) with hypomethylating agents has yielded conflicting results. MATERIALS AND METHODS: We conducted a single center retrospective matched-control analysis with the study group (5-azacitidine [AZA] group) including adults with FLT3-negative acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) who received post-transplant AZA maintenance off clinical trial (n = 93). A matched control group was comprised of contemporaneous AML/MDS patients who did not receive any maintenance (n = 357). Primary endpoint was disease progression. RESULTS: The AZA and control groups had comparable patient and disease characteristics except for older age (median: 61 vs. 57 years, P = .01) and lower hematopoietic comorbidity index (median: 2 vs. 3, P = .04) in the AZA group. The 3-year cumulative incidence of progression in the AZA and control groups was 29% vs. 33% (P = .09). The protective effect of AZA on progression was limited to patients with high-risk AML/MDS (HR = 0.4, 95% CI = 0.2-0.8, P = .009). This led to improved progression-free survival both in high-risk AML and MDS patients with maintenance (HR = 0.2, 95% CI = 0.1-0.6, P = .004 and HR = 0.4, 95% CI = 0.2-0.9, P = .04). CONCLUSION: AZA maintenance was associated with a lower progression rate in patients with high-risk FLT3-negative AML or MDS, and AZA maintenance should be considered for post-alloHCT maintenance in this subset.

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.

Durvalumab and guadecitabine in advanced clear cell renal cell carcinoma: results from the phase Ib/II study BTCRC-GU16-043.

Epigenetic modulation is well established in hematologic malignancies but to a lesser degree in solid tumors. Here we report the results of a phase Ib/II study of guadecitabine and durvalumab in advanced clear cell renal cell carcinoma (ccRCC; NCT03308396). Patients received guadecitabine (starting at 60 mg/m2 subcutaneously on days 1-5 with de-escalation to 45 mg/m2 in case of dose limiting toxicity) with durvalumab (1500 mg intravenously on day 8). The study enrolled 57 patients, 6 in phase Ib with safety being the primary objective and 51in phase II, comprising 2 cohorts: 36 patients in Cohort 1 were treatment naive to checkpoint inhibitors (CPI) with 0-1 prior therapies and 15 patients in Cohort 2 were treated with up to two prior systemic therapies including one CPI. The combination of guadecitabine 45 mg/m2 with durvalumab 1500 mg was deemed safe. The primary objective of overall response rate (ORR) in cohort 1 was 22%. Sixteen patients (44%) experienced stable disease (SD). Secondary objectives included overall survival (OS), duration of response, progression-free survival (PFS), clinical benefit rate, and safety as well as ORR for Cohort 2. Median PFS for cohort 1 and cohort 2 were 14.26 and 3.91 months respectively. Median OS was not reached. In cohort 2, one patient achieved a partial response and 60% achieved SD. Asymptomatic neutropenia was the most common adverse event. Even though the trial did not meet the primary objective in cohort 1, the tolerability and PFS signal in CPI naive patients are worth further investigation.

Successful treatment of a B/T MPAL patient by chemo-free treatment with venetoclax, azacitidine, and blinatumomab.

B/T mixed phenotype acute leukemia (MPAL), which represents only 2-3% of all MPAL cases, is classified as a high-risk leukemia subtype. Adults diagnosed with B/T MPAL have a notably low 3-year survival rate, estimated at 20-40%. The rarity and undercharacterization of B/T MPAL present substantial challenges in identifying an optimal treatment protocol. This report aims to shed light on this issue by presenting a case in which a patient with a complex karyotype was treated using a combination of venetoclax, azacitidine, and blinatumomab. This novel, chemo-free regimen resulted in the patient achieving both hematologic and molecular complete remission, with no severe organ or hematological toxicity observed. Notably, the patient continued to maintain molecular remission for 1 year following the transplantation. Based on these findings, the combination of venetoclax, azacitidine, and blinatumomab could be considered a potential therapeutic approach for B/T MPAL patients, meriting further investigation.

Dismal outcome of refractory or relapsing patients with myelodysplasia-related acute myeloid leukemia partially alleviated by intensive chemotherapy.

BACKGROUND: Acute myeloid leukemia (AML) with myelodysplasia-related characteristics is a heterogeneous subset of AML that has been challenged throughout the history of myeloid malignancies classifications, considered to have similar outcomes as intermediate- or adverse-risk AML depending on the subgroup. However, little is known about the fate of these patients in refractory or relapsed situation (R/R) after first line therapy. METHODS: A large series of R/R AML patients, recorded in the French DATAML registry, have received either intensive chemotherapy (ICT), azacitidine (AZA) as single agent, or best supportive care (BSC). A cohort of 183 patients (median age 63-year-old) with what was called at the time AML-MRC has been explored, and data are reported here. RESULTS: Patient status was refractory for 93, while 90 had relapsed. Respectively, 88, 34, and 61 were included in the three treatment arms. The median OS of the whole cohort was 4.2 months (95%CI: 3.1-5.6) with a mean 1-year overall survival of 24% ± 3.2%. There was no significant survival difference between refractory and relapsed patients. The BSC group had overall a significantly worse outcome (p = 0.0001), and this remained true in both refractory (p = 0.01) and relapsed (p = 0.002) patients. Similar survivals were observed in both groups comparing ICT and AZA. CONCLUSIONS: These data, reporting about an ill-explored population, indicate the poor prognosis of this condition where both ICT and AZA can be proposed. The latter, which was demonstrated here to be a feasible option, should be added to new targeted therapies.

[Platelet Count Doubling after One Course of Demethylated Drug Therapeutic Predicts the Treatment Response and Efficacy of Patients with Myelodysplastic Syndrome].

OBJECTIVE: To investigate the predictive value of platelet doubling (platelet count doubling) after one course of hypomethylating agents (HMA) on the treatment response and efficacy of myelodysplastic syndrome (MDS). METHODS: Clinical and pathological data of 75 patients who received HMA in our hospital from January 2017 to March 2022 were collected and analyzed. All patients were divided into two groups according to whether their platelet count doubled after one course of treatment, including platelet doubling group and non-doubling group, and statistical analysis was performed to compare the treatment response and efficacy between the two groups. In addition, platelet count changes were compared between azacitidine and decitabine therapy. RESULTS: Compared with the non-doubling platelet count group, the ORR of the doubling platelet group was significantly better after 3 courses of treatment (P =0.002), and there was a statistically significant difference in the number of HI between the two groups (P =0.005). In addition, the median survival time (MST) was 26 months in the platelet doubling group and 11 months in the non-doubling group (P =0.001). The overall survival (OS) and 1- and 2-year survival rates of the platelet doubling group were also significantly better than those of the non-doubing group. Multivariate COX analysis showed that platelet count doubling was an independent predictor of OS in MDS patients after 1 course of treatment (P =0.013). There was no significant difference in the response rate of platelet count doubling between MDS patients treated with azacitidine and decitabine (33.3% vs 23.8%, P >0.05). CONCLUSION: Platelet count doubling after one course of treatment can be used as a predictor of response rate and survival of demethylated drug therapy in MDS patients. In addition, there was no significant difference in the response rate of platelets in MDS patients treated with azacitidine or dicetabine.

[Clinical Efficacy of Hypomethylating Agent Therapy in Patients with Chronic Myelomonocytic Leukemia].

OBJECTIVE: To observe the clinical efficacy and safety of hypomethylating agent therapy in chronic myelomonocytic leukemia (CMML). METHODS: From February 2014 to June 2021, the clinical data, efficacy, survival time and safety of CMML patients diagnosed in the Second Hospital of Hebei Medical University and treated with hypomethylating agent therapy were retrospectively analyzed. RESULTS: A total of 25 CMML patients received hypomethylating agent therapy, including 18 cases treated with decitabine (DEC) and 7 cases treated with azacytidine (AZA) as the basic treatment. Among them, 20 patients responded, and 7 patients got complete remission (CR). All patients with CR were treated with DEC as the basic treatment. Five cases of CR occurred in the first 4 courses of treatment. After a median follow-up of 16.4 (9.4-20.5) months, 4 patients with CR progressed to acute myeloid leukemia (AML). The median overall survival (OS) time of 25 CMML patients was 17.4 months (95%CI: 12.437-22.363). According to MD Anderson prognostic scoring system (MDAPS), CMML-specific prognostic scoring system (CPSS), CPSS molecular (CPSS-mol), Mayo molecular model (MMM), risk stratification of patients was performed, and the difference only between different risk stratification of MDAPS and survival time was statistically significant. Common adverse reactions of hypomethylating agent therapy in CMML patients included infection, gastrointestinal reaction, hematological toxicity, skin allergy and liver function damage. All patients' symptoms were improved after corresponding treatment. CONCLUSION: Hypomethylating agent therapy is effective and safe for CMML patients. CR mostly occurs in the first 4 courses of treatment, and hypomethylating agent therapy combined with low-dose chemotherapy can be used for patients who do not respond. Hypomethylating agent therapy can delay the disease, but can't prevent progression.