Decitabine as epigenetic priming with CLAG induce improved outcome of relapsed or refractory acute myeloid leukemia in children.

BACKGROUND: Decitabine (DAC), a DNA methyltransferase inhibitor, has shown efficacy combined with chemotherapy for relapsed or refractory (R/R) acute myeloid leukemia (AML) in adults, but less is known about its efficacy in children. Accordingly, we conducted a study which involved a priming regimen consisting of DAC with cladribine, cytarabine, and granulocyte-stimulating factor (DAC-CLAG) and compared the efficacy and safety of this regimen with CLAG alone. METHODS: A total of 39 R/R AML children who received the CLAG or DAC-CLAG regimen in Shanghai Children's Hospital were retrospectively enrolled in this non-randomized study. These regimens were studied sequentially over time. Twenty-two patients received CLAG from 2015, while 17 patients were administered epigenetic priming with DAC before CLAG from 2020. Patients were subsequently bridged to stem cell transplantation (SCT) or consolidation chemotherapy. Complete remission (CR) and adverse effects were analyzed by Fisher's exact test, and survival was analyzed by the Kaplan-Meier method. RESULTS: DAC-CLAG conferred a numerically higher CR compared to CLAG (70.59% vs 63.64%; P = 0.740). High CR rates occurred in patients with good cytogenetics (P = 0.029) and prior induction without cladribine (P = 0.099). The 1-year event-free survival (EFS) was 64.71% ± 11.59% and 63.31% ± 10.35% in the DAC-CLAG and CLAG group (P = 0.595), and 1-year overall survival (OS) was 81.45% ± 9.72% and 77.01% ± 9.04%, respectively (P = 0.265). The 1-year OS and EFS after SCT were higher in the DAC-CLAG than in the CLAG cohort (100% vs 92.31% ± 7.39%, P = 0.072; 92.31% ± 7.39% vs 85.71% ± 9.35%, P = 0.158). Univariate analysis revealed that a good prognosis included good cytogenetics (P = 0.002), non-complex karyotype (P = 0.056), CR on reinduction (P < 0.0001), and bridging to SCT (P = 0.0007). Use of a hypomethylating agent (P = 0.049) and bridging to SCT (P = 0.011) were independent prognostic factors. Grade 3/4 hematologic toxicity and infection were the main adverse events. CONCLUSIONS: DAC prior to the CLAG regimen improved remission in pediatric R/R AML, and was feasible and well tolerated. CLAG ± DAC as a salvage therapy prior to SCT induced improved survival.

Promoter hypermethylation as a novel regulator of ANO1 expression and function in prostate cancer bone metastasis.

Despite growing evidence implicating the calcium-activated chloride channel anoctamin1 (ANO1) in cancer metastasis, its direct impact on the metastatic potential of prostate cancer and the possible significance of epigenetic alteration in this process are not fully understood. Here, we show that ANO1 is minimally expressed in LNCap and DU145 prostate cancer cell lines with low metastatic potential but overexpressed in high metastatic PC3 prostate cancer cell line. The treatment of LNCap and DU145 cells with DNMT inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) potentiates ANO1 expression, suggesting that DNA methylation is one of the mechanisms controlling ANO1 expression. Consistent with this notion, hypermethylation was detected at the CpG island of ANO1 promoter region in LNCap and DU145 cells, and 5-Aza-CdR treatment resulted in a drastic demethylation at promoter CpG methylation sites. Upon 5-Aza-CdR treatment, metastatic indexes, such as cell motility, invasion, and metastasis-related gene expression, were significantly altered in LNCap and DU145 cells. These 5-Aza-CdR-induced metastatic hallmarks were, however, almost completely ablated by stable knockdown of ANO1. These in vitro discoveries were further supported by our in vivo observation that ANO1 expression in xenograft mouse models enhances the metastatic dissemination of prostate cancer cells into tibial bone and the development of osteolytic lesions. Collectively, our results help elucidate the critical role of ANO1 expression in prostate cancer bone metastases, which is epigenetically modulated by promoter CpG methylation.

A pharmacodynamic assay to monitor treatment with the hypomethylating cytosine analogs, decitabine and azacitidine.

Hypomethylating therapies using decitabine or azacitidine are actively investigated to treat acute myeloid leukemia, myelodysplastic syndromes, as maintenance therapy after allogenic stem cell transplant and hemoglobinopathies. The therapeutic mechanism is to de-repress genes that have been turned off through oncogenesis or development via methylation. The therapy can be non-cytotoxic at low dosage, sparing healthy stem cells and operating on committed precursors. Because the methods of determining maximum tolerated dose are not well suited to this paradigm, and because the mechanism of action, which is depletion of DNA methylase 1 (DNMT1), is complex and dependent on passing through a cell cycle, a pharmacodynamic assay that measures DNMT1 can inform clinical trials aimed at establishing and improving therapy. Herein, we provide an assay that measures DNMT1 relative levels in circulating T cells of peripheral blood.

Biomimetic MDSCs membrane coated black phosphorus nanosheets system for photothermal therapy/photodynamic therapy synergized chemotherapy of cancer.

Photothermal therapy is favored by cancer researchers due to its advantages such as controllable initiation, direct killing and immune promotion. However, the low enrichment efficiency of photosensitizer in tumor site and the limited effect of single use limits the further development of photothermal therapy. Herein, a photo-responsive multifunctional nanosystem was designed for cancer therapy, in which myeloid-derived suppressor cell (MDSC) membrane vesicle encapsulated decitabine-loaded black phosphorous (BP) nanosheets (BP@ Decitabine @MDSCs, named BDM). The BDM demonstrated excellent biosafety and biochemical characteristics, providing a suitable microenvironment for cancer cell killing. First, the BDM achieves the ability to be highly enriched at tumor sites by inheriting the ability of MDSCs to actively target tumor microenvironment. And then, BP nanosheets achieves hyperthermia and induces mitochondrial damage by its photothermal and photodynamic properties, which enhancing anti-tumor immunity mediated by immunogenic cell death (ICD). Meanwhile, intra-tumoral release of decitabine induced G2/M cell cycle arrest, further promoting tumor cell apoptosis. In vivo, the BMD showed significant inhibition of tumor growth with down-regulation of PCNA expression and increased expression of high mobility group B1 (HMGB1), calreticulin (CRT) and caspase 3. Flow cytometry revealed significantly decreased infiltration of MDSCs and M2-macrophages along with an increased proportion of CD4+, CD8+ T cells as well as CD103+ DCs, suggesting a potentiated anti-tumor immune response. In summary, BDM realizes photothermal therapy/photodynamic therapy synergized chemotherapy for cancer.

Epigenetic Activation of Cytochrome P450 1A2 Sensitizes Hepatocellular Carcinoma Cells to Sorafenib.

Cytochrome P450 1A2 (CYP1A2) is a known tumor suppressor in hepatocellular carcinoma (HCC), but its expression is repressed in HCC and the underlying mechanism is unclear. In this study, we investigated the epigenetic mechanisms of CYP1A2 repression and potential therapeutic implications. In HCC tumor tissues, the methylation rates of CYP1A2 CpG island (CGI) and DNA methyltransferase (DNMT) 3A protein levels were significantly higher, and there was a clear negative correlation between DNMT3A and CYP1A2 protein expression. Knockdown of DNMT3A by siRNA significantly increased CYP1A2 expression in HCC cells. Additionally, treating HCC cells with decitabine (DAC) resulted in a dose-dependent upregulation of CYP1A2 expression by reducing the methylation level of CYP1A2 CGI. Furthermore, we observed a decreased enrichment of H3K27Ac in the promoter region of CYP1A2 in HCC tissues. Treatment with the trichostatin A (TSA) restored CYP1A2 expression in HCC cells by increasing H3K27Ac levels in the CYP1A2 promoter region. Importantly, combination treatment of sorafenib with DAC or TSA resulted in a leftward shift of the dose-response curve, lower IC50 values, and reduced colony numbers in HCC cells. Our findings suggest that hypermethylation of the CGI at the promoter, mediated by the high expression of DNMT3A, and hypoacetylation of H3K27 in the CYP1A2 promoter region, leads to CYP1A2 repression in HCC. Epigenetic drugs DAC and TSA increase HCC cell sensitivity to sorafenib by restoring CYP1A2 expression. Our study provides new insights into the epigenetic regulation of CYP1A2 in HCC and highlights the potential of epigenetic drugs as a therapeutic approach for HCC. SIGNIFICANCE STATEMENT: This study marks the first exploration of the epigenetic mechanisms underlying cytochrome P450 (CYP) 1A2 suppression in hepatocellular carcinoma (HCC). Our findings reveal that heightened DNA methyltransferase expression induces hypermethylation of the CpG island at the promoter, coupled with diminished H3K27Ac levels, resulting in the repression of CYP1A2 in HCC. The use of epigenetic drugs such as decitabine and trichostatin A emerges as a novel therapeutic avenue, demonstrating their potential to restore CYP1A2 expression and enhance sorafenib sensitivity in HCC cells.

Decitabine combined with cold atmospheric plasma induces pyroptosis via the ROS/Caspase-3/GSDME signaling pathway in Ovcar5 cells.

BACKGROUND: High methylation of the DFNA5 gene results in the absence of GSDME, a key protein that mediates pyroptosis, while decitabine demethylates the DFNA5 gene, resulting in high expression of the GSDME protein. Cold atmospheric plasma (CAP) is a novel anti-cancer method that induces tumor cell death. METHODS: The pyroptosis induced by decitabine in combination with CAP in Ovcar5 cells was evaluated. In particular, mitochondrial membrane potential was estimated by JC-1 staining, dehydrogenase (LDH) release was assessed by ELISA, Annexin V/PI staining was detected by flow cytometry, the cell cycle changes were evaluated using PI staining followed by detection by flow cytometry, and Caspase-9 cleavage, Caspase-3 cleavage and GSDME expression were evaluated by western blot. RESULTS: Decitabine resulted in high expression of the GSDME in Ovcar5 in a concentration-dependent manner and increased tumor cell sensitivity to CAP. CAP induced mitochondrial damage and activated the Caspase-9/Caspase-3 pathway. Therefore, decitabine combined with CAP induced Ovcar5 cell pyroptosis through Caspase-3 mediated GSDME cleavage. Reactive oxygen species (ROS) generated by CAP treatment played an important role in the CAP/decitabine combination-induced production of ROS, activation of Caspase-9/Caspase-3, GSDME cleavage and pyroptosis that ROS scavenger NAC inhibited all these processes. CONCLUSIONS: CAP combined with decitabine induced Caspase-3 activation, which cleaved decitabine-upregulated GSDME and ediated pyroptosis.

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.

DNA hypomethylation ameliorates erosive inflammatory arthritis by modulating interferon regulatory factor-8.

Epigenetic regulation plays a crucial role in the pathogenesis of autoimmune diseases such as inflammatory arthritis. DNA hypomethylating agents, such as decitabine (DAC), have been shown to dampen inflammation and restore immune homeostasis. In the present study, we demonstrate that DAC elicits potent anti-inflammatory effects and attenuates disease symptoms in several animal models of arthritis. Transcriptomic and epigenomic profiling show that DAC-mediated hypomethylation regulates a wide range of cell types in arthritis, altering the differentiation trajectories of anti-inflammatory macrophage populations, regulatory T cells, and tissue-protective synovial fibroblasts (SFs). Mechanistically, DAC-mediated demethylation of intragenic 5'-Cytosine phosphate Guanine-3' (CpG) islands of the transcription factor Irf8 (interferon regulatory factor 8) induced its re-expression and promoted its repressor activity. As a result, DAC restored joint homeostasis by resetting the transcriptomic signature of negative regulators of inflammation in synovial macrophages (MerTK, Trem2, and Cx3cr1), TREGs (Foxp3), and SFs (Pdpn and Fapα). In conclusion, we found that Irf8 is necessary for the inhibitory effect of DAC in murine arthritis and that direct expression of Irf8 is sufficient to significantly mitigate arthritis.

5-Aza-2'-Deoxycytidine Ameliorates Choroidal Neovascularization by Inhibiting the Wnt/ß-Catenin Signaling Pathway.

Purpose: Choroidal neovascularization (CNV) can constitute the final pathology of many ocular diseases and result in severe vision loss. Studies have demonstrated that DNA methylation is critical in retinal development, aging, and disorders. The current work investigated the effects and underlying mechanism of 5-Aza-2'-deoxycytidine (5-aza-dC), a suppressor of DNA methylation, in the pathological progression of CNV. Methods: The DNA methylation profiles of retinal pigment epithelial (RPE)/choroidal complexes in normal and laser-induced CNV mice were assessed by Arraystar Mouse RefSeq Promoter Arrays. The CNV area and blood flow density and intensity were observed by optical coherence tomography angiography, and fluorescence leakage was examined by fundus fluorescein angiography in CNV mice with systemic administration of 5-aza-dC. The effects of 5-aza-dC on the biological functions of bEnd.3 cells were estimated by related assays. Notum gene promoter methylation was measured using bisulfite sequencing PCR. Methyltransferases and Wnt signaling-related genes were detected in animal and cell culture experiments by real-time PCR and immunoblot. Results: Methyltransferases were upregulated, but Notum (a secretion inhibitor of Wnt signaling) was downregulated in the RPE/choroidal complexes of mice with experimental CNV. Intraperitoneal injection of 5-aza-dC inactivated the Wnt pathway and ameliorated the lesion area and the intensity and density of blood flow, as well as the degree of leakage in CNV. In vitro, vascular endothelial growth factor A (VEGFA) stimulation promoted methyltransferases expression and suppressed Notum expression, consequently activating Wnt signaling, whereas exogenous 5-aza-dC reversed VEGFA-induced hyperpermeability, proliferation, migration, and tube formation in bEnd.3 cells via demethylation of Notum promoter. Conclusions: We observed that 5-aza-dC attenuates the growth of CNV by inhibiting the Wnt signaling pathway via promoter demethylation of the Wnt antagonist Notum. These findings provide a theoretical basis for methylation-based treatment with the Notum gene as a potential target for CNV treatment.

MC180295 is a highly potent and selective CDK9 inhibitor with preclinical in vitro and in vivo efficacy in cancer.

BACKGROUND: Inhibition of cyclin-dependent kinase 9 (CDK9), a novel epigenetic target in cancer, can reactivate epigenetically silenced genes in cancer by dephosphorylating the SWI/SNF chromatin remodeler BRG1. Here, we characterized the anti-tumor efficacy of MC180295, a newly developed CDK9 inhibitor. METHODS: In this study, we explored the pharmacokinetics of MC180295 in mice and rats, and tested the anti-tumor efficacy of MC180295, and its enantiomers, in multiple cancer cell lines and mouse models. We also combined CDK9 inhibition with a DNA methyltransferase (DNMT) inhibitor, decitabine, in multiple mouse models, and tested MC180295 dependence on T cells. Drug toxicity was measured by checking body weights and complete blood counts. RESULTS: MC180295 had high specificity for CDK9 and high potency against multiple neoplastic cell lines (median IC50 of 171 nM in 46 cell lines representing 6 different malignancies), with the highest potency seen in AML cell lines derived from patients with MLL translocations. MC180295 is a racemic mixture of two enantiomers, MC180379 and MC180380, with MC180380 showing higher potency in a live-cell epigenetic assay. Both MC180295 and MC180380 showed efficacy in in vivo AML and colon cancer xenograft models, and significant synergy with decitabine in both cancer models. Lastly, we found that CDK9 inhibition-mediated anti-tumoral effects were partially dependent on CD8 + T cells in vivo, indicating a significant immune component to the response. CONCLUSIONS: MC180380, an inhibitor of cyclin-dependent kinase 9 (CDK9), is an efficacious anti-cancer agent worth advancing further toward clinical use.

The predictive value of BTG1 for the response of newly diagnosed acute myeloid leukemia to decitabine.

BACKGROUND: Decitabine has been widely used to treat acute myeloid leukemia (AML); however as AML is a heterogeneous disease, not all patients benefit from decitabine. This study aimed to identify markers for predicting the response to decitabine. METHODS: An intersection of in vitro experiments and bioinformatics was performed using a combination of epigenetic and transcriptomic analysis. A tumor-suppressor gene associated with methylation and the response to decitabine was screened. Then the sensitivity and specificity of this marker in predicting the response to decitabine was confirmed in 54 samples from newly diagnosed AML patients treated with decitabine plus IA regimen in a clinical trial (ChiCTR2000037928). RESULTS: In vitro experiments showed that decitabine caused hypomethylation and upregulation of BTG1, while downregulation of BTG1 attenuated the inhibitory effect of decitabine. In newly diagnosed AML patients who received decitabine plus IA regimen, the predictive value of BTG1 to predict complete remission (CR) was assigned with a sensitivity of 86.7% and a specificity of 100.0% when BTG1 expression was < 0.292 (determined using real-time quantitative PCR), with area under the curve (AUC) = 0.933, P = 0.021. The predictive value of BTG1 to predict measurable residual disease (MRD) negativity was assigned with a sensitivity of 100.0% and a specificity of 80.0% when BTG1 expression was < 0.292 (AUC = 0.892, P = 0.012). Patients were divided into low and high BTG1 expression groups according to a cutoff of 0.292, and the CR rate of the low-expression group was significantly higher than that of the high-expression group (97.5% vs. 50%, P < 0.001). CONCLUSIONS: Low expression of BTG1 was associated with CR and MRD negativity in newly diagnosed AML patients treated with a decitabine-containing regimen, suggesting that BTG1 is a potential marker for predicting the response to decitabine in newly diagnosed AML. CLINICAL TRIAL REGISTRATION: ChiCTR2000037928.

DNA methylation landscape reveals GNAS as a decitabine-responsive marker in patients with acute myeloid leukemia.

BACKGROUND: The demethylation agent decitabine (DAC) is a pivotal non-intensive alternative treatment for acute myeloid leukemia (AML). However, patient responses to DAC are highly variable, and predictive biomarkers are warranted. Herein, the DNA methylation landscape of patients treated with a DAC-based combination regimen was compared with that of patients treated with standard chemotherapy to develop a molecular approach for predicting clinical response to DAC. METHODS: Twenty-five non-M3 AML patients were enrolled and subjected to DNA methylation sequencing and profiling to identify differentially methylated regions (DMRs) and genes of interest. Moreover, the effects of a DAC-based regimen on apoptosis and gene expression were explored using Kasumi-1 and K562 cells. RESULTS: Overall, we identified 541 DMRs that were specifically responsive to DAC, among which 172 DMRs showed hypomethylation patterns upon treatment and were aligned with the promoter regions of 182 genes. In particular, GNAS was identified as a critical DAC-responsive gene, with in vitro GNAS downregulation leading to reduced cell apoptosis induced by DAC and cytarabine combo treatment. CONCLUSIONS: We found that GNAS is a DAC-sensitive gene in AML and may serve as a prognostic biomarker to assess the responsiveness of patients with AML to DAC-based therapy.

The potential of epigenetic therapy to target the 3D epigenome in endocrine-resistant breast cancer.

Three-dimensional (3D) epigenome remodeling is an important mechanism of gene deregulation in cancer. However, its potential as a target to counteract therapy resistance remains largely unaddressed. Here, we show that epigenetic therapy with decitabine (5-Aza-mC) suppresses tumor growth in xenograft models of pre-clinical metastatic estrogen receptor positive (ER+) breast tumor. Decitabine-induced genome-wide DNA hypomethylation results in large-scale 3D epigenome deregulation, including de-compaction of higher-order chromatin structure and loss of boundary insulation of topologically associated domains. Significant DNA hypomethylation associates with ectopic activation of ER-enhancers, gain in ER binding, creation of new 3D enhancer-promoter interactions and concordant up-regulation of ER-mediated transcription pathways. Importantly, long-term withdrawal of epigenetic therapy partially restores methylation at ER-enhancer elements, resulting in a loss of ectopic 3D enhancer-promoter interactions and associated gene repression. Our study illustrates the potential of epigenetic therapy to target ER+ endocrine-resistant breast cancer by DNA methylation-dependent rewiring of 3D chromatin interactions, which are associated with the suppression of tumor growth.

Epigenetic therapy: Research progress of decitabine in the treatment of solid tumors.

Decitabine's early successful therapeutic outcomes in hematologic malignancies have led to regulatory approvals from the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for addressing myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). These approvals have sparked keen interest in exploring the potential of decitabine for treating solid tumors. Continuous preclinical and clinical trials have proved that low doses of decitabine also bring benefits in treating solid tumors, and various proposed mechanisms attempt to explain the potential efficacy. It is important to note that the application of decitabine in solid tumors is still considered investigational. This article reviews the application mechanism and current status of decitabine in the treatment of solid tumors.

Synergistic cytotoxicity of decitabine and YM155 in leukemia cells through upregulation of SLC35F2 and suppression of MCL1 and survivin expression.

The hypomethylation agent decitabine (DAC), in combination with other apoptosis inducers, is considered a potential modality for cancer treatment. We investigated the mechanism underlying the combined cytotoxicity of DAC and YM155 in acute myeloid leukemia (AML) cells because of increasing evidence that YM155 induces apoptosis in cancer cells. Co-administration of DAC and YM155 resulted in synergistic cytotoxicity in AML U937 cells, which was characterized by the induction of apoptosis, NOXA-dependent degradation of MCL1 and survivin, and depolarization of mitochondria. Restoration of MCL1 or survivin expression attenuated DAC/YM155-induced U937 cell death. DAC initiated AKT and p38 MAPK phosphorylation in a Ca2+/ROS-dependent manner, thereby promoting autophagy-mediated degradation of ß-TrCP mRNA, leading to increased Sp1 expression. DAC-induced Sp1 expression associated with Ten-eleven-translocation (TET) dioxygenases and p300 was used to upregulate the expression of SLC35F2. Simultaneously, the activation of p38 MAPK induced by DAC, promoted CREB-mediated NOXA expression, resulting in survivin and MCL1 degradation. The synergistic cytotoxicity of DAC and YM155 in U937 cells was dependent on elevated SLC35F2 expression. Additionally, YM155 facilitated DAC-induced degradation of MCL1 and survivin. A similar mechanism explained DAC/YM155-mediated cytotoxicity in AML HL-60 cells. Our data demonstrated that the synergistic cytotoxicity of DAC and YM155 in AML cell lines U937 and HL-60 is dependent on AKT- and p38 MAPK-mediated upregulation of SLC35F2 and p38 MAPK-mediated degradation of survivin and MCL1. This indicates that a treatment regimen that amalgamates YM155 and DAC may be beneficial for AML.

Fast proliferating and slowly migrating non-small cell lung cancer cells are vulnerable to decitabine and retinoic acid combinatorial treatment.

Non-small cell lung cancer (NSCLC) patients are often elderly or unfit and thus cannot tolerate standard aggressive therapy regimes. In our study, we test the efficacy of the DNA-hypomethylating agent decitabine (DAC) in combination with all-trans retinoic acid (ATRA), which has been shown to possess little systemic adverse effects. Screening a broad panel of 56 NSCLC cell lines uncovered a decrease in cell viability after the combination treatment in 77% of the cell lines. Transcriptomics, proteomics, proliferation and migration profiling revealed that fast proliferating and slowly migrating cell lines were more sensitive to the drug combination. The comparison of mutational profiles found oncogenic KRAS mutations only in sensitive cells. Additionally, different cell lines showed a heterogeneous gene expression response to the treatment pointing to diverse mechanisms of action. Silencing KRAS, RIG-I or RARB partially reversed the sensitivity of KRAS-mutant NCI-H460 cells. To study resistance, we generated two NCI-H460 cell populations resistant to ATRA and DAC, which migrated faster and proliferated slower than the parental sensitive cells and showed signs of senescence. In summary, this comprehensive dataset uncovers a broad sensitivity of NSCLC cells to the combinatorial treatment with DAC and ATRA and indicates that migration and proliferation capacities correlate with and could thus serve as determinants for drug sensitivity in NSCLC.

Hypomethylating agent decitabine sensitizes diffuse large B-cell lymphoma to venetoclax.

Despite recent advances in the therapy of diffuse large B-cell lymphoma (DLBCL), many patients are still not cured. Therefore, new therapeutic strategies are needed. The anti-apoptotic B-cell lymphoma 2 (BCL2) gene is commonly dysregulated in DLBCL due to various mechanisms such as chromosomal translocation t(14;18)(q32;q21) and copy number alterations; however, targeting BCL-2 with the selective inhibitor, venetoclax, led to response in only a minority of patients. Thus, we sought to identify a rational combination partner of venetoclax to improve its activity against DLBCL cells. Utilizing a functional assay, dynamic BH3 profiling, we found that the DNA hypomethylating agent decitabine increased mitochondrial apoptotic priming and BCL-2 dependence in DLBCL cells. RNA-sequencing analysis revealed that decitabine suppressed the pro-survival PI3K-AKT pathway and altered the mitochondria membrane composition in DLBCL cell lines. Additionally, it induced a DNA damage response and increased BAX and BAK activities. The combination of decitabine and venetoclax synergistically suppressed proliferation of DLBCL cells both in vitro and in vivo in a DLBCL cell line-derived xenograft mouse model. Our study suggests that decitabine plus venetoclax is a promising combination to explore clinically in DLBCL.

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

BACKGROUND: We 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 METHODS: We 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). RESULTS: Of 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. CONCLUSION: Median 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.