Homoharringtonine enhances cytarabine-induced apoptosis in acute myeloid leukaemia by regulating the p38 MAPK/H2AX/Mcl-1 axis.

Acute myeloid leukaemia (AML) is a fatal haematopoietic malignancy and is treated with the conventional combination of cytarabine (Ara-C) and daunorubicin (Dau). The survival rate of AML patients is lower due to the cardiotoxicity of daunorubicin. Clinically, homoharringtonine (HHT) plus Ara-C has been reported to be equally effective as Dau plus Ara-C in some types of AML patients with less toxic effects. We utilized the clinical use of homoharringtonine in combination with Ara-C to test its combination mechanism. We found that the insensitivity of AML cells to cytarabine-induced apoptosis is associated with increased Mcl-1 stability and p38 inactivation. HHT downregulates Mcl-1, phosphorylates H2AX and induces apoptosis by activating p38 MAPK. Inactivation of p38 through inhibitors and siRNA blocks apoptosis, H2AX phosphorylation and Mcl-1 reduction. HHT enhances Ara-C activation of the p38 MAPK signalling pathway, overcoming Ara-C tolerance to cell apoptosis by regulating the p38/H2AX/Mcl-1 axis. The optimal ratio of HHT to Ara-C for synergistic lethality in AML cells is 1:4 (M/M). HHT synergistically induces apoptosis in combination with Ara-C in vitro and prolongs the survival of xenografts. We provide a new mechanism for AML treatment by regulating the p38 MAPK/H2AX/Mcl-1 axis to improve cytarabine therapy.

Targeting Metabolic Dependencies Fueling the TCA Cycle to Circumvent Therapy Resistance in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is one of the most prevalent blood cancers, characterized by a dismal survival rate. This poor outcome is largely attributed to AML cells that persist despite treatment and eventually result in relapse. Relapse-initiating cells exhibit diverse resistance mechanisms, encompassing genetic factors and, more recently discovered, nongenetic factors such as metabolic adaptations. Leukemic stem cells (LSC) rely on mitochondrial metabolism for their survival, whereas hematopoietic stem cells primarily depend on glycolysis. Furthermore, following treatments such as cytarabine, a standard in AML treatment for over four decades, drug-persisting leukemic cells exhibit an enhanced reliance on mitochondrial metabolism. In this issue of Cancer Research, two studies investigated dependencies of AML cells on two respiratory substrates, α-ketoglutarate and lactate-derived pyruvate, that support mitochondrial oxidative phosphorylation (OXPHOS) following treatment with the imipridone ONC-213 and the BET inhibitor INCB054329, respectively. Targeting lactate utilization by interfering with monocarboxylate transporter 1 (MCT1 or SLC16A1) or lactate dehydrogenase effectively sensitized cells to BET inhibition in vitro and in vivo. In addition, ONC-213 affected αKGDH, a pivotal NADH-producing enzyme of the TCA cycle, to induce a mitochondrial stress response through ATF4 activation that diminished the expression of the antiapoptotic protein MCL1, consequently promoting apoptosis of AML cells. In summary, targeting these mitochondrial dependencies might be a promising strategy to kill therapy-naïve and treatment-resistant OXPHOS-reliant LSCs and to delay or prevent relapse. See related articles by Monteith et al., p. 1101 and Su et al., p. 1084.

A Novel Approach for Glioblastoma Treatment by Combining Apoptosis Inducers (TMZ, MTX, and Cytarabine) with E.V.A. (Eltanexor, Venetoclax, and A1210477) Inhibiting XPO1, Bcl-2, and Mcl-1.

Adjuvant treatment for Glioblastoma Grade 4 with Temozolomide (TMZ) inevitably fails due to therapeutic resistance, necessitating new approaches. Apoptosis induction in GB cells is inefficient, due to an excess of anti-apoptotic XPO1/Bcl-2-family proteins. We assessed TMZ, Methotrexate (MTX), and Cytarabine (Ara-C) (apoptosis inducers) combined with XPO1/Bcl-2/Mcl-1-inhibitors (apoptosis rescue) in GB cell lines and primary GB stem-like cells (GSCs). Using CellTiter-Glo® and Caspase-3 activity assays, we generated dose-response curves and analyzed the gene and protein regulation of anti-apoptotic proteins via PCR and Western blots. Optimal drug combinations were examined for their impact on the cell cycle and apoptosis induction via FACS analysis, paralleled by the assessment of potential toxicity in healthy mouse brain slices. Ara-C and MTX proved to be 150- to 10,000-fold more potent in inducing apoptosis than TMZ. In response to inhibitors Eltanexor (XPO1; E), Venetoclax (Bcl-2; V), and A1210477 (Mcl-1; A), genes encoding for the corresponding proteins were upregulated in a compensatory manner. TMZ, MTX, and Ara-C combined with E, V, and A evidenced highly lethal effects when combined. As no significant cell death induction in mouse brain slices was observed, we conclude that this drug combination is effective in vitro and expected to have low side effects in vivo.

Polyamine Inhibitor SAM486A Augments Cytarabine Cytotoxicity in Methylthioadenosine Phosphorylase-deficient Leukemia Cells.

BACKGROUND/AIM: Methionine metabolism contributes to supplying sulfur-containing amino acids, controlling the methyl group transfer reaction, and producing polyamines in cancer cells. Polyamines play important roles in various cellular functions. Methylthioadenosine phosphorylase (MTAP), the key enzyme of the methionine salvage pathway, is reported to be deficient in 15-62% of cases of hematological malignancies. MTAP-deficient cancer cells accumulate polyamines, resulting in enhanced cell proliferation. The aim of this study was to investigate the combined effects of the polyamine synthesis inhibitor SAM486A and the anticancer antimetabolite cytarabine in MTAP-deficient leukemic cells in vitro. MATERIALS AND METHODS: The leukemia cell line U937 and the subline, U937/MTAP(-), in which MTAP was knocked down by shRNA, were used. The experiments were performed in media supplemented with 20% methionine (low methionine), which was the minimum concentration for maintaining cellular viability. RESULTS: The knockdown efficiency test confirmed a 70% suppression of the expression of the MTAP gene in U937/MTAP(-) cells. Even in the media with low methionine, the intracellular methionine concentration was not reduced in U937/MTAP(-) cells, suggesting that the minimum supply of methionine was sufficient to maintain intracellular levels of methionine. Both U937/MTAP(+) and U937/MTAP(-) cells were comparably sensitive to anticancer drugs (cytarabine, methotrexate, clofarabine and 6-thioguanine). The combination of SAM486A and cytarabine was demonstrated to have synergistic cytotoxicity in U937/MTAP(-) cells with regard to cell growth inhibition and apoptosis induction, but not in U937/MTAP(+) cells. Mechanistically, SAM486A altered the intracellular polyamine concentrations and reduced the antiapoptotic proteins. CONCLUSION: Methionine metabolism and polyamine synthesis can be attractive therapeutic targets in leukemia.

Identification of several African swine fever virus replication inhibitors by screening of a library of FDA-approved drugs.

African swine fever (ASF) caused by African swine fever virus (ASFV) is a highly infectious and lethal swine disease. Currently, there is only one novel approved vaccine and no antiviral drugs for ASFV. In the study, a high-throughput screening of an FDA-approved drug library was performed to identify several drugs against ASFV infection in primary porcine alveolar macrophages. Triapine and cytarabine hydrochloride were identified as ASFV infection inhibitors in a dose-dependent manner. The two drugs executed their antiviral activity during the replication stage of ASFV. Furthermore, molecular docking studies showed that triapine might interact with the active center Fe2+ in the small subunit of ASFV ribonucleotide reductase while cytarabine hydrochloride metabolite might interact with three residues (Arg589, Lys593, and Lys631) of ASFV DNA polymerase to block new DNA chain extension. Taken together, our results suggest that triapine and cytarabine hydrochloride displayed significant antiviral activity against ASFV in vitro.

AHR signaling pathway mediates mitochondrial oxidative phosphorylation which leads to cytarabine resistance.

The aryl hydrocarbon receptor (AHR) has been identified as a significant driver of tumorigenesis. However, its clinical significance in acute myeloid leukemia (AML) remains largely unclear. In this study, RNA-Seq data from AML patients (bone marrow samples from 173 newly diagnosed AML patients) obtained from the TCGA database, and normal human RNA-Seq data (bone marrow samples from 70 healthy individuals) obtained from the GTEX database are downloaded for external validation and complementarity. The data analysis reveals that the AHR signaling pathway is activated in AML patients. Furthermore, there is a correlation between the expressions of AHR and mitochondrial oxidative phosphorylation genes. In vitro experiments show that enhancing AHR expression in AML cells increases mitochondrial oxidative phosphorylation and induces resistance to cytarabine. Conversely, reducing AHR expression in AML cells decreases cytarabine resistance. These findings deepen our understanding of the AHR signaling pathway's involvement in AML.

ULK2 Is a Key Pro-Autophagy Protein That Contributes to the High Chemoresistance and Disease Relapse in FLT3-Mutated Acute Myeloid Leukemia.

We recently demonstrated that a small subset of cells in FLT3-mutated acute myeloid leukemia (AML) cell lines exhibit SORE6 reporter activity and cancer stem-like features including chemoresistance. To study why SORE6+ cells are more chemoresistant than SORE6- cells, we hypothesized that these cells carry higher autophagy, a mechanism linked to chemoresistance. We found that cytarabine (Ara-C) induced a substantially higher protein level of LC3B-II in SORE6+ compared to SORE6- cells. Similar observations were made using a fluorescence signal-based autophagy assay. Furthermore, chloroquine (an autophagy inhibitor) sensitized SORE6+ but not SORE6- cells to Ara-C. To decipher the molecular mechanisms underlying the high autophagic flux in SORE6+ cells, we employed an autophagy oligonucleotide array comparing gene expression between SORE6+ and SORE6- cells before and after Ara-C treatment. ULK2 was the most differentially expressed gene between the two cell subsets. To demonstrate the role of ULK2 in conferring higher chemoresistance in SORE6+ cells, we treated the two cell subsets with a ULK1/2 inhibitor, MRT68921. MRT68921 significantly sensitized SORE6+ but not SORE6- cells to Ara-C. Using our in vitro model for AML relapse, we found that regenerated AML cells contained higher ULK2 expression compared to pretreated cells. Importantly, inhibition of ULK2 using MRT68921 prevented in vitro AML relapse. Lastly, using pretreatment and relapsed AML patient bone marrow samples, we found that ULK2 expression was higher in relapsed AML. To conclude, our results supported the importance of autophagy in the relapse of FLT3-mutated AML and highlighted ULK2 in this context.

Mitocurcumin utilizes oxidative stress to upregulate JNK/p38 signaling and overcomes Cytarabine resistance in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a type of blood cancer that is characterized by the rapid growth of abnormal myeloid cells. The goal of AML treatment is to eliminate the leukemic blasts, which is accomplished through intensive chemotherapy. Cytarabine is a key component of the standard induction chemotherapy regimen for AML. However, despite a high remission rate, 70-80% of AML patients relapse and develop resistance to Cytarabine, leading to poor clinical outcomes. Mitocurcumin (MitoC), a derivative of curcumin that enters mitochondria, leading to a drop in mitochondrial membrane potential and mitophagy induction. Further, it activates oxidative stress-mediated JNK/p38 signaling to induce apoptosis. MitoC demonstrated a preferential ability to kill leukemic cells from AML cell lines and patient-derived leukemic blasts. RNA sequencing data suggests perturbation of DNA damage response and cell proliferation pathways in MitoC-treated AML. Elevated reactive oxygen species (ROS) in MitoC-treated AML cells resulted in significant DNA damage and cell cycle arrest. Further, MitoC treatment resulted in ROS-mediated enhanced levels of p21, which leads to suppression of CHK1, RAD51, Cyclin-D and c-Myc oncoproteins, potentially contributing to Cytarabine resistance. Combinatorial treatment of MitoC and Cytarabine has shown synergism, increased apoptosis, and enhanced DNA damage. Using AML xenografts, a significant reduction of hCD45+ cells was observed in AML mice bone marrow treated with MitoC (mean 0.6%; range0.04%-3.56%) compared to control (mean 38.2%; range10.1%-78%), p = 0.03. The data suggest that MitoC exploits stress-induced leukemic oxidative environment to up-regulate JNK/p38 signaling to lead to apoptosis and can potentially overcome Cytarabine resistance via ROS/p21/CHK1 axis.

Exosome-shuttled FTO from BM-MSCs contributes to cancer malignancy and chemoresistance in acute myeloid leukemia by inducing m6A-demethylation: A nano-based investigation.

Although bone marrow mesenchymal stem cells (BM-MSCs)-derived exosomes have been reported to be closely associated with acute myeloid leukemia (AML) progression and chemo-resistance, but its detailed functions and molecular mechanisms have not been fully delineated. Besides, serum RNA m6A demethylase fat mass and obesity-associated protein (FTO)-containing exosomes are deemed as important indicators for cancer progression, and this study aimed to investigate the role of BM-MSCs-derived FTO-exosomes in regulating the malignant phenotypes of AML cells. Here, we verified that BM-MSCs-derived exosomes delivered FTO to promote cancer aggressiveness, stem cell properties and Cytosine arabinoside (Ara-C)-chemoresistance in AML cells, and the underlying mechanisms were also uncovered. Our data suggested that BM-MSCs-derived FTO-exo demethylated m6A modifications in the m6A-modified LncRNA GLCC1 to facilitate its combination with the RNA-binding protein Hu antigen R (HuR), which further increased the stability and expression levels of LncRNA GLCC1. In addition, LncRNA GLCC1 was verified as an oncogene to facilitate cell proliferation and enhanced Ara-C-chemoresistance in AML cells. Further experiments confirmed that demethylated LncRNA GLCC1 served as scaffold to facilitate the formation of the IGF2 mRNA binding protein 1 (IGF2BP1)-c-Myc complex, which led to the activation of the downstream tumor-promoting c-Myc-associated signal pathways. Moreover, our rescuing experiments validated that the promoting effects of BM-MSCs-derived FTO-exo on cancer aggressiveness and drug resistance in AML cells were abrogated by silencing LncRNA GLCC1 and c-Myc. Thus, the present firstly investigated the functions and underlying mechanisms by which BM-MSCs-derived FTO-exo enhanced cancer aggressiveness and chemo-resistance in AML by modulating the LncRNA GLCC1-IGF2BP1-c-Myc signal pathway, and our work provided novel biomarkers for the diagnosis, treatment and therapy of AML in clinic.

n-Tuples on Scaffold Diversity Inspired by Drug Hybridisation to Enhance Drugability: Application to Cytarabine.

A mathematical concept, n-tuples are originally applied to medicinal chemistry, especially with the creation of scaffold diversity inspired by the hybridisation of different commercial drugs with cytarabine, a synthetic arabinonucleoside derived from two marine natural products, spongouridine and spongothymidine. The new methodology explores the virtual chemical-factorial combination of different commercial drugs (immunosuppressant, antibiotic, antiemetic, anti-inflammatory, and anticancer) with the anticancer drug cytarabine. Real chemical combinations were designed and synthesised for 8-duples, obtaining a small representative library of interesting organic molecules to be biologically tested as proof of concept. The synthesised library contains classical molecular properties regarding the Lipinski rules and/or beyond rules of five (bRo5) and is represented by the covalent combination of the anticancer drug cytarabine with ibuprofen, flurbiprofen, folic acid, sulfasalazine, ciprofloxacin, bortezomib, and methotrexate. The insertion of specific nomenclature could be implemented into artificial intelligence algorithms in order to enhance the efficiency of drug-hunting programs. The novel methodology has proven useful for the straightforward synthesis of most of the theoretically proposed duples and, in principle, could be extended to any other central drug.

8-Hydroxydaidzein Induces Apoptosis and Inhibits AML-Associated Gene Expression in U-937 Cells: Potential Phytochemical for AML Treatment.

BACKGROUND: 8-hydroxydaidzein (8-OHD) is a compound derived from daidzein, known for its anti-inflammatory and anti-proliferative properties in K562 human chronic myeloid leukemia (CML) cells. However, its effects on acute myeloid leukemia (AML) cells have not been fully understood. METHOD: To investigate its potential anti-AML mechanism, we employed an integrated in vitro-in silico approach. RESULTS: Our findings demonstrate that 8-OHD suppresses the expression of CDK6 and CCND2 proteins and induces cell apoptosis in U-937 cells by activating Caspase-7 and cleaving PARP-1. Microarray analysis revealed that 8-OHD downregulates differentially expressed genes (DEGs) associated with rRNA processing and ribosome biogenesis pathways. Moreover, AML-target genes, including CCND2, MYC, NPM1, FLT3, and TERT, were downregulated by 8-OHD. Additionally, molecular docking software predicted that 8-OHD has the potential to interact with CDK6, FLT3, and TERT proteins, thereby reducing their activity and inhibiting cell proliferation. Notably, we discovered a synergic pharmacological interaction between 8-OHD and cytarabine (Ara-C). CONCLUSIONS: Overall, this study provides insights into the therapeutic applications of 8-OHD in treating AML and elucidates its underlying mechanisms of action.

DUSP1 Signaling Pathway Regulates Cytarabine Sensitivity in Acute Myeloid Leukemia.

Objectives: Dual specificity phosphatase 1 (DUSP1) is high-expressed in various cancers and plays an important role in the cellular response to agents that damage DNA. We aimed to investigate the expressions and mechanisms of DUSP1 signaling pathway regulating cytarabine (Ara-C) resistance in acute myeloid leukemia (AML). Methods: Immunohistochemistry was performed on bone marrow biopsy specimens from AML and controls to explore the expression of DUSP1. Western blot and Q-PCR were used to detect the protein and mRNA expression levels. MTT assay was used to detect the proliferation of cells. Cell apoptosis was detected by flow cytometry. The immune protein-protein interaction (PPI) network of DUSP1 was analyzed in the platform of Pathway Commons, and immune infiltration analysis was used to study the immune microenvironment of AML. Results: We found that the expression levels of DUSP1 in AML patients exceeded that in controls. Survival analysis in public datasets showed that AML patients with higher levels of DUSP1 had poor clinical outcomes. Further public data analysis indicated that DUSP1 was overexpressed in NRAS mutated AML. DUSP1 knockdown by siRNA could sensitize AML cells to Ara-C treatments. The phosphorylation level of mitogen-activated protein kinase (MAPK) pathway was significantly elevated in DUSP1 down-regulated NRAS G13D mutated AML cells. The PPI analysis showed DUSP1 correlated with immune gene CREB1 and CXCL8 in NRAS mutated AML. We also revealed a correlation between tumor-infiltrating immune cells in RAS mutated AML microenvironment. Conclusion: Our findings suggest that DUSP1 signaling pathways may regulate Ara-C sensitivity in AML.

Artesunate reverses cytarabine resistance in acute myeloid leukemia by blocking the JAK/STAT3 signaling.

OBJECTIVES: Although cytarabine (AraC) has greatly contributed to improving the prognosis of patients with acute myeloid leukemia (AML), many patients developed drug resistance and eventually succumbed to AML. Thus, resistance to AraC is a major obstacle to improve the efficacy of chemotherapy in AML. Hence, this study aimed to demonstrate that artesunate (ART) can reliably induce cell death in vitro and block AraC resistance. METHODS: AML cell lines resistant to AraC were first constructed by repeated dosing for 5 months. Further, we analyzed whether ART intervention affected the sensitivity of AraC-resistant cells to AraC by cell function experiments, mainly including CCK-8 to assess cell viability, flow cytometry to examine apoptosis, and Western blotting to measure the Janus kinase (JAK)/signal transducers and activators of transcription 3 (STAT3) pathway protein expression. Furthermore, whether JAK/STAT3 pathway knockdown has a blocking effect on the efficacy of ART was also assessed. RESULTS: Co-treatment of ART and AraC increased the sensitivity of AML cells to AraC. Also, it effectively reversed the resistance of AML cells to AraC that is shown by the significantly reduced proliferation and increased apoptosis rates. ART intervention suppressed the activation of the JAK/STAT3 signaling pathway in AraC-resistant AML cells, suggesting that the function of ART in reversing AraC resistance is indeed dependent on the JAK/STAT3 signaling pathway. CONCLUSIONS: In summary, ART enhanced the sensitivity of AML/AraC-resistant cells to AraC by modulating the JAK/STAT3 pathway.

A novel anticancer quinolone, (R)-WAC-224, has anti-leukemia activities against acute myeloid leukemia.

Approximately 60%-80% of patients who achieve complete remission eventually relapse after conventional chemotherapy and have poor prognoses despite the recent advances of novel anticancer agents. Continuing development of more effective novel treatments for acute myeloid leukemia (AML) is necessary. We developed (R)-WAC-224 (R-WAC), which is an anticancer quinolone, targeting topoisomerase II. This study evaluated the anti-leukemia potential of R-WAC or racemic WAC-224 (WAC) in vitro and in vivo. R-WAC significantly inhibited the human AML cell line proliferation (MV4-11, HL60, and KG1a), which was comparable to daunorubicin and cytarabine, not affected by P-glycoprotein overexpression. WAC did neither increase serum troponin-T nor decrease the crypt numbers in the small intestine, indicating WAC was less toxic than doxorubicin. R-WAC monotherapy demonstrated prolonged survival in the AML mice model and inhibited tumor growth in the MV4-11 xenograft mice model. Moreover, the combination of R-WAC and cytarabine demonstrated more active anti-leukemia effects than daunorubicin and cytarabine. Finally, R-WAC inhibited the colony-forming abilities using primary AML cells. These results indicate that R-WAC is a promising therapeutic agent for AML.

Interleukin-6 Facilitates Acute Myeloid Leukemia Chemoresistance via Mitofusin 1-Mediated Mitochondrial Fusion.

Acute myeloid leukemia (AML), an aggressive hematopoietic malignancy, exhibits poor prognosis and a high recurrence rate largely because of primary and secondary drug resistance. Elevated serum IL6 levels have been observed in patients with AML and are associated with chemoresistance. Chemoresistant AML cells are highly dependent on oxidative phosphorylation (OXPHOS), and mitochondrial network remodeling is essential for mitochondrial function. However, IL6-mediated regulation of mitochondrial remodeling and its effectiveness as a therapeutic target remain unclear. We aimed to determine the mechanisms through which IL6 facilitates the development of chemoresistance in AML cells. IL6 upregulated mitofusin 1 (MFN1)-mediated mitochondrial fusion, promoted OXPHOS, and induced chemoresistance in AML cells. MFN1 knockdown impaired the effects of IL6 on mitochondrial function and chemoresistance in AML cells. In an MLL::AF9 fusion gene-induced AML mouse model, IL6 reduced chemosensitivity to cytarabine (Ara-C), a commonly used antileukemia drug, accompanied by increased MFN1 expression, mitochondrial fusion, and OXPHOS status. In contrast, anti-IL6 antibodies downregulated MFN1 expression, suppressed mitochondrial fusion and OXPHOS, enhanced the curative effects of Ara-C, and prolonged overall survival. In conclusion, IL6 upregulated MFN1-mediated mitochondrial fusion in AML, which facilitated mitochondrial respiration, in turn, inducing chemoresistance. Thus, targeting IL6 may have therapeutic implications in overcoming IL6-mediated chemoresistance in AML. IMPLICATIONS: IL6 treatment induces MFN1-mediated mitochondrial fusion, promotes OXPHOS, and confers chemoresistance in AML cells. Targeting IL6 regulation in mitochondria is a promising therapeutic strategy to enhance the chemosensitivity of AML.

Synergistic interactions of cytarabine-adavosertib in leukemic cell lines proliferation and metabolomic endpoints.

Drug synergy allows reduced dosing, side effects and tolerance. Optimization of drug synergy chemotherapy is fundamental in acute lymphocytic leukemia and other cancers. This study aimed to analyze the pharmacodynamic synergy between the anti-metabolite cytarabine and WEE1 inhibitor adavosertib on acute leukemia cell lines CCRF-CEM and Jurkat. In both cell lines analysis of concentration-inhibition curves of adavosertib-cytarabine combinations and synergy matrixes supported mutually synergistic drug interactions. Overall mean ( ± SD) synergy scores were higher in Jurkat than CCRF-CEM: Jurkat, ZIP 22.51 ± 1.1, Bliss 22.49 ± 1.1, HSA 23.44 ± 1.0, Loewe 14.16 ± 1.2; and, CCRF-CEM, ZIP 9.17 ± 1.9, Bliss 8.13 ± 2.1, HSA 11.48 ± 1.9 and Loewe 4.99 ± 1.8. Jurkat also surpassed CCRF-CEM in high-degree synergistic adavosertib-cytarabine interactions with mean across-models synergy values of ∼89.1% ± 2.9 for 63 nM cytarabine-97 nM adavosertib (91.4% inhibition synergy barometer). Combination sensitivity scores scatter plots confirmed combination's synergy efficacy. This combined approach permitted identification and prioritization of 63 nM cytarabine-97 nM adavosertib for multiple endpoints analysis. This combination did not affect PBMC viability, while exhibiting Jurkat selective synergy. Immunoblots also revealed Jurkat selective synergistically increased γH2AX phosphorylation, while CDC2 phosphorylation effects were attributed to adavosertib's WEE1 inhibition. In conclusion, the high synergistic efficacy combination of cytarabine (63 nM) and adavosertib (97 nM) was associated with remarkable alterations in metabolites related to the Krebs cycle in Jurkat. The metabolic pathways and processes are related to gluconeogenesis, amino acids, nucleotides, glutathione, electron transport and Warburg effect. All above relate to cell survival, apoptosis, and cancer progression. Our findings could pave the way for novel biomarkers in treatment, diagnosis, and prognosis of leukemia and other cancers.

Posttranslational splicing modifications as a key mechanism in cytarabine resistance in acute myeloid leukemia.

Despite the approval of several drugs for AML, cytarabine is still widely used as a therapeutic approach. However, 85% of patients show resistance and only 10% overcome the disease. Using RNA-seq and phosphoproteomics, we show that RNA splicing and serine-arginine-rich (SR) proteins phosphorylation were altered during cytarabine resistance. Moreover, phosphorylation of SR proteins at diagnosis were significantly lower in responder than non-responder patients, pointing to their utility to predict response. These changes correlated with altered transcriptomic profiles of SR protein target genes. Notably, splicing inhibitors were therapeutically effective in treating sensitive and resistant AML cells as monotherapy or combination with other approved drugs. H3B-8800 and venetoclax combination showed the best efficacy in vitro, demonstrating synergistic effects in patient samples and no toxicity in healthy hematopoietic progenitors. Our results establish that RNA splicing inhibition, alone or combined with venetoclax, could be useful for the treatment of newly diagnosed or relapsed/refractory AML.

Chloroquine enhances the efficacy of chemotherapy drugs against acute myeloid leukemia by inactivating the autophagy pathway.

Current therapy for acute myeloid leukemia (AML) is largely hindered by the development of drug resistance of commonly used chemotherapy drugs, including cytarabine, daunorubicin, and idarubicin. In this study, we investigated the molecular mechanisms underlying the chemotherapy drug resistance and potential strategy to improve the efficacy of these drugs against AML. By analyzing data from ex vivo drug-response and multi-omics profiling public data for AML, we identified autophagy activation as a potential target in chemotherapy-resistant patients. In THP-1 and MV-4-11 cell lines, knockdown of autophagy-regulated genes ATG5 or MAP1LC3B significantly enhanced AML cell sensitivity to the chemotherapy drugs cytarabine, daunorubicin, and idarubicin. In silico screening, we found that chloroquine phosphate mimicked autophagy inactivation. We showed that chloroquine phosphate dose-dependently down-regulated the autophagy pathway in MV-4-11 cells. Furthermore, chloroquine phosphate exerted a synergistic antitumor effect with the chemotherapy drugs in vitro and in vivo. These results highlight autophagy activation as a drug resistance mechanism and the combination therapy of chloroquine phosphate and chemotherapy drugs can enhance anti-AML efficacy.

The m6A modulator-mediated cytarabine sensitivity and immune cell infiltration signature in acute myeloid leukemia.

PURPOSE: The study aims to investigate the impact of m6A modulators on drug resistance and the immune microenvironment in acute myeloid leukemia (AML). The emergence of drug resistance is a significant factor that contributes to relapse and refractory AML, leading to a poor prognosis. METHODS: The AML transcriptome data were retrieved from the TCGA database. The "oncoPredict" R package was utilized to assess the sensitivity of each sample to cytarabine (Ara-C) and classify them into distinct groups. Differential expression analysis was performed to identify m6A modulators differentially expressed between the two groups. Select Random Forest (RF) to build a predictive model. Model performance was evaluated using calibration curve, clinical decision curve, and clinical impact curve. The impacts of METTL3 on Ara-C sensitivity and immune microenvironment in AML were examined using GO, KEGG, CIBERSORT, and GSEA analyses. RESULTS: Seventeen out of 26 m6A modulators exhibited differential expression between the Ara-C-sensitive and resistant groups, with a high degree of correlation. We selected the 5 genes with the highest scores in the RF model to build a reliable and accurate prediction model. METTL3 plays a vital role in m6A modification, and further analysis shows its impact on the sensitivity of AML cells to Ara-C through its interaction with 7 types of immune-infiltrating cells and autophagy. CONCLUSION: This study utilizes m6A modulators to develop a prediction model for the sensitivity of AML patients to Ara-C, which can assist in treating AML drug resistance by targeting mRNA methylation.

Smart and Generalizable Cytarabine Derivative-Triggered Nanoparticles for Synergistic Therapy of Relapsed/Refractory Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is rapidly progressed hematologic malignancy with relapsed and refractory characteristics. Cytarabine combined with the BCL2 inhibitor venetoclax showed impressive response rates in the treatment of relapsed/refractory acute myeloid leukemia (R/R AML), while it requires complicated administration regimens and brings added toxicity. In this work, we synthesized a mercaptopropionic acid-substituted derivative of Ara-C (Ara-SH) and used it as the trigger to fabricate a smart cytarabine and venetoclax-coloaded nanoparticle (AV-NP) through self-assembly. The AV-NP characterized with redox-responsive drug release, rapid uptake by leukemia cells, and long retention in circulation had the potential to accumulate in leukemia-enriched sites. It generated a remarkable synergistic effect with higher antileukemia activity in vitro and better safety in the hematologic system compared with free drugs and significantly improved the therapeutic effect on orthotopic AML mice in vivo. These similar results were also confirmed in primary cells from R/R-AML patients. Besides, the AV-NP has the superiority of facile fabrication and generalizability, rendering it easy for clinical translation.