Cytarabine Resistance Research Articles

Abstract 5845: Cytarabine resistance leads to a unique mutational signature

Abstract The prognosis of recurrent T-cell acute lymphoblastic leukemia (T-ALL) is poor, mainly due to chemotherapy resistance. The mechanism(s) leading to chemoresistance at relapse are incompletely understood. We previously characterized a mouse strain that is hypomorphic for the DNA replication factor Mcm2. Mcm2hypo mice develop T-ALL due to acquired DNA copy number variations (CNV) involving tumor suppressor or oncogenes. We hypothesize that treatment of Mcm2hypo cells with specific chemotherapy agents may reveal CNV that lead to chemoresistance. We treated two Mcm2hypo T-ALL cell lines (designated 2883 and 2869) and an Mcm2 WT T-ALL cell line (designated 7298) with cytarabine (ARAC), an active agent often incorporated in T-ALL treatment regimens. After one year of treatment with gradually increasing ARAC concentrations, we obtained ARAC resistant cell lines (designated with the suffix CR) that tolerate drug concentrations up to 10,000X that of parental cell lines. These samples were characterized by sparse WGS (for CNV assessment), RNA-Seq and WES. Both the 2883CR and 2869CR cell lines had bi-allelic mutations involving Dck, which is the rate-limiting enzyme in the cytidine salvage pathway, and is thought to be required for metabolism of ARAC. In both cases, one allele was deleted while the second allele had a splice site/region mutation leading to partial intron retention and resultant frameshift. The 7298CR cell line had homozygous deletion of Dck. Western blotting showed absence of WT Dck protein in all three cell lines. Further analysis of WES data revealed thousands of acquired single nucleotide variants (SNV) in both Dck mutant Mcm2hypo cell lines. These mutations occurred in a specific trinucleotide context (C>T in a GCG context, T>C in a GTC context, T>G mutations in GTC and GTT context, and C>G in a GCC context) generating a mutational signature that is not present in the COSMIC database. In addition, the parental 2883 and 2869 cell lines showed fewer mutations (ie, hundreds vs thousands) in a similar context (C>T in a GCG context, T>C in a GTC context, T>G mutations in GTC and GTT context, but not the C>G in GCC context). A GEMINI (Genotoxic Mutational Signature Identified After Clonal Expansion In Vitro) assay of untreated 2883CR and 2869CR cells demonstrated that ongoing ARAC exposure was not required for the mutational signature. We hypothesize that the basic signature (C>T in a GCG context, T>C in a GTC context, T>G mutations in GTC and GTT context) can be produced by Mcm2 deficiency, and this signature is amplified and modified (to include a C>G in GCC context) by Dck inactivation. In summary, we identified three mechanisms responsible for Dck inactivation associated with ARAC resistance: copy number loss, splice site mutations, and splice region mutations. Moreover, Dck inactivation leads to a specific mutation signature. These findings add to emerging data that exposure to specific chemotherapy agents can lead to specific mutational signatures. Citation Format: Dengchao Cao, Mianmian Yin, Zhenhua Zhang, Taimour Baslan, Jack Zhu, Ryan Bertoli, Paul Meltzer, Peter Aplan. Cytarabine resistance leads to a unique mutational signature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5845.

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.

SOX11 is a novel binding partner and endogenous inhibitor of SAMHD1 ara-CTPase activity in mantle cell lymphoma

AbstractSterile alpha motif and histidine-aspartate (HD) domain–containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate triphosphohydrolase with ara-CTPase activity that confers cytarabine (ara-C) resistance in several hematological malignancies. Targeting SAMHD1’s ara-CTPase activity has recently been demonstrated to enhance ara-C efficacy in acute myeloid leukemia. Here, we identify the transcription factor SRY-related HMG-box containing protein 11 (SOX11) as a novel direct binding partner and first known endogenous inhibitor of SAMHD1. SOX11 is aberrantly expressed not only in mantle cell lymphoma (MCL), but also in some Burkitt lymphomas. Coimmunoprecipitation of SOX11 followed by mass spectrometry in MCL cell lines identified SAMHD1 as the top SOX11 interaction partner, which was validated by proximity ligation assay. In vitro, SAMHD1 bound to the HMG box of SOX11 with low-micromolar affinity. In situ crosslinking studies further indicated that SOX11-SAMHD1 binding resulted in a reduced tetramerization of SAMHD1. Functionally, expression of SOX11 inhibited SAMHD1 ara-CTPase activity in a dose-dependent manner resulting in ara-C sensitization in cell lines and in a SOX11-inducible mouse model of MCL. In SOX11-negative MCL, SOX11-mediated ara-CTPase inhibition could be mimicked by adding the recently identified SAMHD1 inhibitor hydroxyurea. Taken together, our results identify SOX11 as a novel SAMHD1 interaction partner and its first known endogenous inhibitor with potentially important implications for clinical therapy stratification.

SOX11 is a novel binding partner and endogenous inhibitor of SAMHD1 ara-CTPase activity in mantle cell lymphoma.

The sterile alpha motif and histidine-aspartate (HD) domain containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate triphosphohydrolase with ara-CTPase activity that confers cytarabine (ara-C) resistance in several haematological malignancies. Targeting SAMHD1's ara-CTPase activity has recently been demonstrated to enhance ara-C efficacy in acute myeloid leukemia. Here, we identify the transcription factor SRY-related HMG-box containing protein 11 (SOX11) as a novel direct binding partner and first known endogenous inhibitor of SAMHD1. SOX11 is aberrantly expressed not only in mantle cell lymphoma (MCL), but also in some Burkitt lymphomas. Co-immunoprecipitation of SOX11 followed by mass spectrometry in MCL cell lines identified SAMHD1 as the top SOX11 interaction partner which was validated by proximity ligation assay. In vitro, SAMHD1 bound to the HMG box of SOX11 with low-micromolar affinity. In situ crosslinking studies further indicated that SOX11-SAMHD1 binding resulted in a reduced tetramerization of SAMHD1. Functionally, expression of SOX11 inhibited SAMHD1 ara-CTPase activity in a dose-dependent manner resulting in ara-C sensitization in cell lines and in a SOX11-inducible mouse model of MCL. In SOX11-negative MCL, SOX11-mediated ara-CTPase inhibition could be mimicked by adding the recently identified SAMHD1 inhibitor hydroxyurea. Taken together, our results identify SOX11 as a novel SAMHD1 interaction partner and its first known endogenous inhibitor with potentially important implications for clinical therapy stratification.

GNL3L exhibits pro-tumor activities via NF-κB pathway as a poor prognostic factor in acute myeloid leukemia.

Acute myeloid leukemia (AML) is the leukemia with the worst prognosis, and current knowledge of AML pathogenesis and available therapies for AML remain limited. 40% of AML patients exhibit elevated nuclear factor kappa B (NF-κB) activity, which provides a compelling rationale for targeting the NF-κB pathway in AML. Guanine nucleotide-binding protein-like 3-like protein (GNL3L) is a recently identified pro-oncogene that promotes NF-κB activation in a variety of malignancies. For the first time, we comprehensively examined GNL3L expression in AML, reporting GNL3L as a poor prognostic factor in three independent AML cohorts. GNL3L enhanced RELA activity, activated NF-κB, promoted AML cell proliferation, resisted apoptosis, and encouraged cytarabine resistance in AML. In conclusion, these data suggest a role for GNL3L in the malignant process of AML and as a promising therapeutic target.

Prediction of miRNA‑mRNA network regulating the migration ability of cytarabine‑resistant HL60 cells.

Cytarabine is an important medicine for acute myeloid leukemia (AML) treatment, however, drug resistance hinders the treatment of AML. Although microRNA (miRNA or miR) alteration is one of the well-recognized mechanisms underlying drug resistance in AML, few studies have investigated the role and function of miRNAs in the development of cytarabine resistance. In the present study, total RNA was isolated from parental HL60 and cytarabine-resistant HL60 (R-HL60) cells. Subsequently, miRNAs and mRNAs were detected using small RNA sequencing and gene expression array, respectively. Differentially expressed mRNAs (DEMs) and differentially expressed genes (DEGs) with more than two-fold changes between HL60 and R-HL60 cells were screened out. Negatively associated miRNA-mRNA pairs were selected as candidate miRNA-mRNA target pairs according to the miRDB, Targetscan or miRTar databases. Functional enrichment analysis of DEGs included in the candidate miRNA-mRNA pairs was performed. The results indicated that 10 DEGs (CCL2, SOX9, SLC8A1, ICAM1, CXCL10, SIPR2, FGFR1, OVOL2, MITF and CARD10) were simultaneously involved in seven Gene Ontology pathways related to the regulation of migration ability, namely the 'regulation of cell migration', 'regulation of locomotion', 'regulation of cellular component movement', 'cell migration', 'locomotion', 'cell motility', and 'localization of cell'. DEMs predicted to negatively regulate the aforementioned 10 DEGs were paired with DEGs into 16 candidate miRNA-mRNA pairs related to the regulation of migration ability. In addition, migration assays revealed that the migration ability of R-HL60 cells was greater than that of HL60 cells. These findings provide a new perspective for the treatment of cytarabine-resistant AML and advance our understanding of altered migration ability underlying cytarabine resistance development, specifically related to miRNAs.

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.

Metabolic Reprogramming By PRDM16 Drives Cytarabine Resistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) patients with high PRDM16 expression frequently experience induction failure and have a poor prognosis. However, the underlying molecular mechanisms are poorly understood. Here, we show that AML cells with high PRDM16 expression acquire resistance to cytarabine (AraC) through the activation of oxidative phosphorylation (OxPHOS) via regulation of c-MYC. Initially, we established a murine model of AML with high Prdm16 expression. We transduced c-Kit + bone marrow cells from wild-type mice with a retrovirus vector encoding MLL-AF9 (MF9) and the oncogenic short form of Prdm16 (sPrdm16). Using MF9/sPrdm16 cells, we next investigated whether AML cells with high Prdm16 expression exhibited resistance to AraC and/or daunorubicin (DNR), chemotherapeutic agents commonly used for induction chemotherapy. In the cell viability assay, MF9/sPrdm16 cells showed significantly higher IC 50 against AraC, but not against DNR, compared to MF9/control (control) cells, suggesting that high sPrdm16 expression confers relative resistance to AraC on AML cells. Colony-forming assay also confirmed resistant to AraC in MF9/sPrdm16 cells compared to control. Moreover, MF9/sPrdm16 cells were also resistant to AraC in vivo as demonstrated by the shortened survival of mice receiving MF9/sPrdm16 cells. To elucidate the mechanism of AraC resistance, we first examined cell cycle status of MF9/sPrdm16 cells since AraC exerts cytotoxicity in cell cycle dependent manner. To our surprise, MF9/sPrdm16 cells were cycling more rapidly than control cells as demonstrated by decreased G 0 phase and increased S/G 2/M phase. Intriguingly, RNA-sequencing analysis revealed that MF9/sPrdm16 cells displayed a signature of high OxPHOS by gene set enrichment analysis (GSEA). Indeed, oxygen consumption rate, superoxide production from mitochondria, and mitochondrial membrane potential were significantly elevated in MF9/sPrdm16 cells compared to control cells. The inhibition of mitochondrial respiration with metformin, the electron transport chain complex I inhibitor, or tigecycline, the mitochondrial protein synthesis inhibitor, abrogated AraC resistance in MF9/sPrdm16 cells by inducing an energetic shift towards a lower OxPHOS status. These results clearly indicated that sPrdm16 overexpression conferred MF9 cells with AraC resistance byincreasing OxPHOS in mitochondria. Next, we explored the mechanism how sPrdm16 regulates OxPHOS. In addition to elevated OxPHOS signature, RNA-sequencing data also showed strong enrichment of Myc target-signature in MF9/sPrdm16 cells. Western blot analysis showed that sPrdm16 overexpression induced up-regulation of c-Myc and its transcriptional target, Slc1a5, a glutamine transporter. We went on to analyze metabolic changes induced by sPrdm16 overexpression by capillary electrophoresis and mass spectrometry-based metabolome analysis. We found that sPrdm16 induced the accumulation of glutamine, glutamate, α-ketoglutaric acid, succinate, fumarate, and malate in MF9 cells, possibly by activating glutaminolysis via the regulation of c-Myc and Slc1a5 expression, leading to the activation of tricarboxylic acid (TCA) cycle and OxPHOS. We also confirmed the contribution of c-Myc to AraC resistance by overexpressing c-Myc in MF9 cells. As expected, MF9/c-Myc cells mimicked the phenotype of MF9/sPrdm16 cells such as resistance to AraC and high OxPHOS status. Furthermore, 10058-F4, a c-Myc inhibitor, abrogated AraC resistance in MF9/sPrdm16 cells. Next, we asked if the observation in MF9/sPrdm16 cells could be translated into human AML cells. Human AML cell lines, MOLM-13 and THP-1, with sPRDM16 overexpression also showed significantly higher IC 50 against AraC compared to Mock expressed cells. Moreover, RNA-seq data from the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 clinical trial showed that AML blasts with high PRDM16 expression demonstrated a gene expression signature associated with “OxPHOS” and “Myc Targets”. In conclusion, our findings demonstrate that PRDM16 overexpression activates mitochondrial respiration through metabolic reprogramming via c-MYC, thereby conferres AraC resistance on AML cells. These results suggest that targeting mitochondrial respiration might be a novel treatment strategy to overcome chemoresistance in AML patients with high PRDM16 expression.

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

ABSTRACTObjectivesAlthough 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.MethodsAML 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.ResultsCo-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.ConclusionsIn summary, ART enhanced the sensitivity of AML/AraC-resistant cells to AraC by modulating the JAK/STAT3 pathway.

Enterotoxins A and B produced by Staphylococcus aureus increase cell proliferation, invasion and cytarabine resistance in acute myeloid leukemia cell lines

As in the case of cancer, the risk of infection increases when the host's immune system is not working properly. It has been shown that toxins produced by the bacteria responsible for bacterial infections can alter the properties of cancer cells as well as their sensitivity to chemotherapy agents. Staphylococcus aureus (S. aureus) is one of the most prevalent pathogens in acute myeloid leukemia (AML) patients and it produces several virulence factors, including Staphylococcal enterotoxin A (SEA) and Staphylococcal enterotoxin B (SEB). Cytotoxicity, transwell migration, invasion assays, and various transcriptomic and gene set enrichment (GSE) analyses were used to determine how SEA and SEB alter cell proliferation, migration, invasion, and Cytarabine (Cyt) resistance in AML cell lines. The treatment of AML cell lines with SEA/SEB caused an increase in cell proliferation and Cyt resistance. Toxins enhanced the proclivity of cells to migrate and invade, with around 50% of cells in the presence of SEA and SEB. Transcriptomic and gene set enrichment analyses, and subsequent PCR validations showed dysregulation of immune related genes and genesets. Apparently, this allows AML cells to escape and survive the undesirable environment created by toxins, possibly via the ER stress signaling pathway. Therefore, SEA and SEB can significantly alter the characteristics of AML cancer cells and evaluation of alterations in responsible immune genes and pathways may be crucial for controlling the progression of cancer. In addition, our results suggest that there may be a strong interaction between the immune related pathways and the ER signaling pathway.

POSTER: AML-332 Mitocurcumin Utilizes Oxidative Stress to Upregulate JNK/p38 Signaling and Overcomes Cytarabine Resistance in Acute Myeloid Leukemia

POSTER: AML-332 Mitocurcumin Utilizes Oxidative Stress to Upregulate JNK/p38 Signaling and Overcomes Cytarabine Resistance in Acute Myeloid Leukemia

AML-332 Mitocurcumin Utilizes Oxidative Stress to Upregulate JNK/p38 Signaling and Overcomes Cytarabine Resistance in Acute Myeloid Leukemia

AML-332 Mitocurcumin Utilizes Oxidative Stress to Upregulate JNK/p38 Signaling and Overcomes Cytarabine Resistance in Acute Myeloid Leukemia

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.

Galectin-9 has non-apoptotic cytotoxic activity toward acute myeloid leukemia independent of cytarabine resistance

Acute myeloid leukemia (AML) is a malignancy still associated with poor survival rates, among others, due to frequent occurrence of therapy-resistant relapse after standard-of-care treatment with cytarabine (AraC). AraC triggers apoptotic cell death, a type of cell death to which AML cells often become resistant. Therefore, therapeutic options that trigger an alternate type of cell death are of particular interest. We previously identified that the glycan-binding protein Galectin-9 (Gal-9) has tumor-selective and non-apoptotic cytotoxicity towards various types of cancer, which depended on autophagy inhibition. Thus, Gal-9 could be of therapeutic interest for (AraC-resistant) AML. In the current study, treatment with Gal-9 was cytotoxic for AML cells, including for CD34+ patient-derived AML stem cells, but not for healthy cord blood-derived CD34+ stem cells. This Gal-9-mediated cytotoxicity did not rely on apoptosis but was negatively associated with autophagic flux. Importantly, both AraC-sensitive and -resistant AML cell lines, as well as AML patient samples, were sensitive to single-agent treatment with Gal-9. Additionally, Gal-9 potentiated the cytotoxic effect of DNA demethylase inhibitor Azacytidine (Aza), a drug that is clinically used for patients that are not eligible for intensive AraC treatment. Thus, Gal-9 is a potential therapeutic agent for the treatment of AML, including AraC-resistant AML, by inducing caspase-independent cell death.

Prolactin receptor signaling induces acquisition of chemoresistance and reduces clonogenicity in acute myeloid leukemia

BackgroundDevelopment of precision medicine requires the identification of easily detectable and druggable biomarkers. Despite recent targeted drug approvals, prognosis of acute myeloid leukemia (AML) patients needs to be greatly improved, as relapse and refractory disease are still difficult to manage. Thus, new therapeutic approaches are needed. Based on in silico-generated preliminary data and the literature, the role of the prolactin (PRL)-mediated signaling was interrogated in AML.MethodsProtein expression and cell viability were determined by flow cytometry. Repopulation capacity was studied in murine xenotransplantation assays. Gene expression was measured by qPCR and luciferase-reporters. SA-β-Gal staining was used as a senescence marker.ResultsThe prolactin receptor (PRLR) was upregulated in AML cells, as compared to their healthy counterpart. The genetic and molecular inhibition of this receptor reduced the colony-forming potential. Disruption of the PRLR signaling, either using a mutant PRL or a dominant-negative isoform of PRLR, reduced the leukemia burden in vivo, in xenotransplantation assays. The expression levels of PRLR directly correlated with resistance to cytarabine. Indeed, acquired cytarabine resistance was accompanied with the induction of PRLR surface expression. The signaling associated to PRLR in AML was mainly mediated by Stat5, in contrast to the residual function of Stat3. In concordance, Stat5 mRNA was significantly overexpressed at mRNA levels in relapse AML samples. A senescence-like phenotype, measured by SA-β-gal staining, was induced upon enforced expression of PRLR in AML cells, partially dependent on ATR. Similar to the previously described chemoresistance-induced senescence in AML, no cell cycle arrest was observed. Additionally, the therapeutic potential of PRLR in AML was genetically validated.ConclusionsThese results support the role of PRLR as a therapeutic target for AML and the further development of drug discovery programs searching for specific PRLR inhibitors.

Chemotherapeutic drugs elicit stemness and metabolic alteration to mediate acquired drug-resistant phenotype in acute myeloid leukemia cell lines

Chemotherapy resistance leading to disease relapse is a significant barrier in treating acute myeloid leukemia (AML). Metabolic adaptations have been shown to contribute to therapy resistance. However, little is known about whether specific therapies cause specific metabolic changes. We established cytarabine-resistant (AraC-R) and Arsenic trioxide-resistant (ATO-R) AML cell lines, displaying distinct cell surface expression and cytogenetic abnormalities. Transcriptomic analysis revealed a significant difference in the expression profiles of ATO-R and AraC-R cells. Geneset enrichment analysis showed AraC-R cells rely on OXPHOS, while ATO-R cells on glycolysis. ATO-R cells were also enriched for stemness gene signatures, whereas AraC-R cells were not. The mito stress and glycolytic stress tests confirmed these findings. The distinct metabolic adaptation of AraC-R cells increased sensitivity to the OXPHOS inhibitor venetoclax. Cytarabine resistance was circumvented in AraC-R cells by combining Ven and AraC. In vivo, ATO-R cells showed increased repopulating potential, leading to aggressive leukemia compared to the parental and AraC-R. Overall, our study shows that different therapies can cause different metabolic changes and that these metabolic dependencies can be used to target chemotherapy-resistant AML.

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.

Targeting Cholesterol Homeostasis Pathway to Overcome Acquired Cytarabine Resistance in Acute Myeloid Leukemia

The current backbone of Acute Myeloid Leukemia (AML)therapy is treatment with the cytidine analogue Ara-C and the anthracycline daunorubicin. Despite a high rate of initial remissions, a substantial fraction of patients with AML relapse and acquire resistance to Ara-C. In acquired resistance, there is an initial therapy response, but resistance develops over time. To uncover the acquired drug resistant mechanisms, we utilized two cytarabine (Ara-C) resistant cell lines - THP1 and U937 developed from the parental respective cell lines (kind gift from Dr. Martin Michaelis, University of Kent). In-vitro cytotoxicity assay confirmed the cytarabine resistance in THP1 cell line (THP1 Ara-C R IC50: 1457µM; THP1 parental IC50: 56 µM) and U937 cell line (U937 Ara-C R IC50: 1578.3 µM and U937 parental IC50: 0.14 µM). THP1-AraC-R was marginally cross resistant to both DNR (IC50 Parent -0.19µM, AraC 0.26µM) and ATO (IC50 Parent -2.1µM, AraC >4µM). To identify the factors associated with Ara-C drug resistance, we subjected both the cell lines along with their parental counterparts for transcriptomic profiling. Several protein coding genes were differentially expressed in both the cell lines. A total of 845 genes were upregulated and 268 genes were downregulated in THP1 Ara-C R cell line while 344 genes were upregulated, and 647 genes were downregulated in U937 Ara-C R cell line. To unravel the pathways associated with Ara-C resistance, the differentially expressed genes were subjected to Gene Set Enrichment Analysis (GSEA). The pathway enrichment showed Cholesterol Homeostasis pathway to be highly enriched in THP1 (NES: 2.17 & FDR q-value: 0) and U937 (NES:1.25 & FDR q-value:0.13) Ara-C resistant cell lines in comparison to the respective parental cell lines. To address the role of Cholesterol Homeostasis pathway in mediating chemoresistance in these two Ara-C resistant AML cell lines (THP1, U937) we screened few selected pharmacological inhibitors targeting different genes in the Cholesterol Homeostasis pathway [HMG Co-A reductase (HMGCR) the rate limiting step in Cholesterol metabolism, Squalene synthase (SQS), an intermediate enzyme in the pathway of Cholesterol synthesis and HMG Co-A synthase (HMGCS1) catalyzes the condensation of acetyl Co-A with aceto-acetyl Co-A to form HMGCo-A ]. Among all the inhibitors screened, Hymeglusin, an inhibitor of HMGCS1, re-sensitized only the THP1 Ara-C resistant cell with good synergy (combinatorial Index for THP1 Ara-C cell line 0.68 and 0.2 with Ara-C). These results demonstrate the dependence of leukemic cells on the Cholesterol Homeostasis pathway to mediate cytarabine resistance and pharmacological inhibition of this pathway can result in the reversal of cytarabine resistance.

Overexpression of Nrf2 Promotes OGG1 Expression By Activating AKT Signaling Pathway to Mediate Cytarabine Resistance in Acute Myeloid Leukemia Cells

Background：Nuclear factor E2 related factor 2 (Nrf2) exerts a vital function in drug resistance of many tumors. Base excision repair (BER) pathway gene 8-hydroxyguanine DNA glycosidase (OGG1) is a key component of tumor drug resistance. Nevertheless, the potential molecular mechanism of its mediating drug resistance in acute myeloid leukemia (AML) remains unclear. Methods: Firstly, isolation of mononuclear cells from normal healthy donors and AML patients. Drug-resistant AML cell lines were cultured by increasing drug concentration, and AML cell lines with up-regulation and down-regulation of Nrf2 were constructed by lentivirus transfection. The possible mechanism of drug resistance of leukemic cells mediated by overexpression of Nrf2 was investigated by CCK-8 method, immunofluorescence, immunocytochemistry, immunohistochemistry, flow cytometry, Chromatin immunoprecipitation, Western blot, xenograft mouse model and so on. Results: In clinical samples, we found that the expression of Nrf2 and OGG1 were higher in patients with relapse AML. At the cellular level, we confirmed that up-regulation of Nrf2 increased the expression of OGG1, decreased the sensitivity of leukemic cells to cytarabine, and activated the AKT signaling pathway. However, after down-regulation of Nrf2, the expression of OGG1 decreased, the sensitivity of leukemic cells to cytarabine increased and the expression of phosphorylated AKT decreased. MK-2206, an inhibitor of AKT pathway, can inhibit the activation of AKT signaling pathway, decrease the expression of OGG1 and increase the expression of apoptotic protein. In the case of overexpression of Nrf2, the use of OGG1 inhibitors can increase the sensitivity of AML cells to cytarabine. In vivo, we found that the expression of OGG1 decreased after down-regulation of Nrf2, which had a certain protective effect on xenografted tumor mice. Conclusion: overexpression of Nrf2 may promote the expression of OGG1 and mediate drug resistance in AML cells by activating AKT signal pathway. In order to prevent the progression of AML cells, blocking AKT signal pathway with MK-2206 or targeting OGG1 inhibitors may be a potential therapeutic strategy for AML resistance.

The sWT1+ - Transcript Confers Cytarabine Resistance Via Upregulation of HOXA3 and GATA2 Axis

The Wilms tumor gene (WT1) encodes for a zinc finger transcription factor which manifests as both tumor suppressive and oncogenic activities. Due to 3 translation start sites and alternative splicing at exon 5 and a lysine, threonine, and serine (KTS) motif, WT1 generates 12 major transcripts. WT1 expression is upregulated in ~90% of leukemia samples and is associated with chemoresistance and disease relapse. However, which WT1 transcripts are associated with chemoresistant has not been defined. We characterized that sWT1+/-, but not other transcripts decreased cell growth, downregulated CD71 expression, and conferred cytarabine resistance. Leukemia blast cells with low CD71 expression are relatively resistant to cytarabine, suggesting that CD71 could be used as a biomarker for cytarabine treatment. Subsequent RNAseq analysis revealed two transcription factors HOXA3 and GATA2 to be uniquely upregulated in sWT1+/-, but not in other transcripts. Consistent with the sWT1 +/- phenotype, HOXA3 or GATA2 overexpression decreased cell growth, reduced CD71 expression, and conferred cytarabine resistance. Real time-PCR analysis showed that overexpression of WT1 and GATA2 upregulates each other and HOXA3 expression; whereas overexpression of HOXA3 upregulates GATA2 expression. Furthermore, relapsed leukemia samples express a relatively higher level of HOXA3 and GATA2 compared to de novo AML samples in the BeatAML cohort. GATA2 and WT1 are also coexpressed in AML samples (TCGA AML and BeatAML). Interestingly, WT1 and GATA2 mutations are mutual exclusive in primary AML and MDS patient samples (TCGA AML, BeatAML, and MDS (MSKCC+ IWG, IPSSM, NEJM)). These data indicates sWT1+-, HOXA3, and GATA2 forms a transcript complex that regulate cytarabine sensitivity. As such, we have identified transcription factors and a biomarker for cytarabine sensitivity in AML.