Targeting GLUT1 in acute myeloid leukemia to overcome cytarabine resistance.
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ABSTRACT:
SUBMITTER: Abacka H
PROVIDER: S-EPMC8018118 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Children with Down syndrome (DS) have increased risk for developing AML (DS-AMKL), and they usually experience severe therapy-related toxicities compared to non DS-AMKL. Refractory/relapsed disease has very poor outcome, and patients would benefit from novel, less toxic, therapeutic strategies that overcome resistance. Relapse/resistance are linked to cancer stem cells with high aldehyde dehydrogenase (ALDH) activity. The purpose of the present work was to study less toxic alternative therapeutic agents for relapsed/refractory DS-AMKL.Fourteen AML cell lines including the DS-AMKL CMY and CMK from relapsed/refractory AML were used. Cytarabine (Ara-C), bortezomib (BTZ), disulfiram/copper (DSF/Cu2+) were evaluated for cytotoxicity, depletion of ALDH-positive cells, and resistance. BTZ-resistant CMY and CMK variants were generated by continuous BTZ treatment. Cell viability was assessed using CellTiter-Glo®, ALDH activity by ALDELUORTM, and proteasome inhibition by western blot of ubiquitinated proteins and the Proteasome-Glo™ Chymotrypsin-Like (CT-like) assay, apoptosis by Annexin V Fluos/Propidium iodide staining, and mutations were detected using PCR, cloning and sequencing.Ara-C-resistant AML cell lines were sensitive to BTZ and DSF/Cu2+. The Ara-C-resistant DS-AMKL CMY cells had a high percentage of ALDHbright "stem-like" populations that may underlie Ara-C resistance. One percent of these cells were still resistant to BTZ but sensitive to DSF/Cu2+. To understand the mechanism of BTZ resistance, BTZ resistant (CMY-BR) and (CMK-BR) were generated. A novel mutation PSMB5 Q62P underlied BTZ resistance, and was associated with an overexpression of the ?5 proteasome subunit. BTZ-resistance conferred increased resistance to Ara-C due to G1 arrest in the CMY-BR cells, which protected the cells from S-phase damage by Ara-C. CMY-BR and CMK-BR cells were cross-resistant to CFZ and MG-132 but sensitive to DSF/Cu2+. In this setting, DSF/Cu2+ induced apoptosis and proteasome inhibition independent of CT-like activity inhibition.We provide evidence that DSF/Cu2+ overcomes Ara-C and BTZ resistance in cell lines from DS-AMKL patients. A novel mutation underlying BTZ resistance was detected that may identify BTZ-resistant patients, who may not benefit from treatment with CFZ or Ara-C, but may be responsive to DSF/Cu2+. Our findings support the clinical development of DSF/Cu2+ as a less toxic efficacious treatment approach in patients with relapsed/refractory DS-AMKL.
| S-EPMC5286849 | biostudies-literature
Project description:Reduced expression of the uptake transporter, OCTN1 (SLC22A4), has been reported as a strong predictor of poor event-free and overall survival in multiple cohorts of patients with acute myeloid leukemia (AML) receiving the cytidine nucleoside analog, cytarabine (Ara-C). To further understand the mechanistic basis of interindividual variability in the functional expression of OCTN1 in AML, we hypothesized a mechanistic connection to DNA methylation-based epigenetic repression of SLC22A4. We found increased basal SLC22A4 methylation was associated with decreased Ara-C uptake in AML cell lines. Pre-treatment with hypomethylating agents, 5-azacytidine, or decitabine, restored SLC22A4 mRNA expression, increased cellular uptake of Ara-C, and was associated with increased cellular sensitivity to Ara-C compared with vehicle-treated cells. Additionally, lower SLC22A4 methylation status was associated with distinct clinical advantages in both adult and pediatric patients with AML. These findings suggest a regulatory mechanism is involved in the interindividual variability in response to Ara-C, and provides a basis for the integration of hypomethylating agents into Ara-C-based treatment regimens.
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Project description:The standard therapeutic approaches for acute myeloid leukemia (AML) continue to be based on anthracyclines and cytarabine. However, the prognosis for AML remains poor, especially for patients with high-risk disease. During the past decade, promising novel agents that target DNA replication and repair, as well as cell cycling and apoptosis, have been developed and are being actively investigated in AML. Among these agents is flavopiridol, which interferes with key steps of the cell cycle and effectively promotes cell death, and voreloxin, an intercalating agent that also targets topoisomerase II. Also under clinical study in AML are oligonucleotide antisense constructs, which suppress the translation of proteins essential for leukemic blast survival and proliferation, and agents that target antiapoptotic cascades. In summary, it is hoped that novel therapies such as these will augment and/or supplant our current cytarabine- and anthracycline-based approaches, overcome active drug-resistance pathways, and eventually improve outcomes for patients with AML.
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Project description:Cytarabine (AraC) represents the most effective single agent treatment for AML. Nevertheless, overriding AraC resistance in AML remains an unmet medical need. Here we show that the CHK1 inhibitor (CHK1i) GDC-0575 enhances AraC-mediated killing of AML cells both in vitro and in vivo, thus abrogating any potential chemoresistance mechanisms involving DNA repair. Importantly, this combination of drugs does not affect normal long-term hematopoietic stem/progenitors. Moreover, the addition of CHK1i to AraC does not generate de novo mutations and in patients' samples where AraC is mutagenic, addition of CHK1i appears to eliminate the generation of mutant clones. Finally, we observe that persistent residual leukemic cells are quiescent and can become responsive to the treatment when forced into cycle via granulocyte colony-stimulating factor (G-CSF) administration. This drug combination (AraC+CHK1i+G-CSF) will open the doors for a more efficient treatment of AML in the clinic.
| S-EPMC5698422 | biostudies-literature
Project description:In chronic myeloid leukemia, resistance against BCR-ABL1 tyrosine kinase inhibitors can develop because of BCR-ABL1 mutations, activation of additional pro-oncogenic pathways, and stem cell resistance. Drug combinations covering a broad range of targets may overcome resistance. CDDO-Me (bardoxolone methyl) is a drug that inhibits the survival of leukemic cells by targeting different pro-survival molecules, including STAT3. We found that CDDO-Me inhibits proliferation and survival of tyrosine kinase inhibitor-resistant BCR-ABL1+ cell lines and primary leukemic cells, including cells harboring BCR-ABL1T315I or T315I+ compound mutations. Furthermore, CDDO-Me was found to block growth and survival of CD34+/CD38- leukemic stem cells (LSC). Moreover, CDDO-Me was found to produce synergistic growth-inhibitory effects when combined with BCR-ABL1 tyrosine kinase inhibitors. These drug-combinations were found to block multiple signaling cascades and molecules, including STAT3 and STAT5. Furthermore, combined targeting of STAT3 and STAT5 by shRNA and STAT5-targeting drugs also resulted in synergistic growth-inhibition, pointing to a new efficient concept of combinatorial STAT3 and STAT5 inhibition. However, CDDO-Me was also found to increase the expression of heme-oxygenase-1, a heat-shock-protein that triggers drug resistance and cell survival. We therefore combined CDDO-Me with the heme-oxygenase-1 inhibitor SMA-ZnPP, which also resulted in synergistic growth-inhibitory effects. Moreover, SMA-ZnPP was found to sensitize BCR-ABL1+ cells against the combination 'CDDO-Me+ tyrosine kinase inhibitor'. Together, combined targeting of STAT3, STAT5, and heme-oxygenase-1 overcomes resistance in BCR-ABL1+ cells, including stem cells and highly resistant sub-clones expressing BCR-ABL1T315I or T315I-compound mutations. Whether such drug-combinations are effective in tyrosine kinase inhibitor-resistant patients with chronic myeloid leukemia remains to be elucidated.
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Project description:Background: Acute myeloid leukemia (AML) is a heterogeneous disease that frequently relapses after standard chemotherapy. Therefore, there is a need for the development of novel chemotherapeutic agents that could treat AML effectively. Radotinib, an oral BCR-ABL tyrosine kinase inhibitor, was developed as a drug for the treatment of chronic myeloid leukemia. Previously, we reported that radotinib exerts increased cytotoxic effects towards AML cells. However, little is known about the effects of combining radotinib with Ara-C, a conventional chemotherapeutic agent for AML, with respect to cell death in AML cells. Therefore, we investigated combination effects of radotinib and Ara-C on AML in this study.Methods: Synergistic anti-cancer effects of radotinib and Ara-C in AML cells including HL60, HEL92.1.7, THP-1 and bone marrow cells from AML patients have been examined. Diverse cell biological assays such as cell viability assay, Annexin V-positive cells, caspase-3 activity, cell cycle distribution, and related signaling pathway have been performed.Results: The combination of radotinib and Ara-C was found to induce AML cell apoptosis, which involved the mitochondrial pathway. In brief, combined radotinib and Ara-C significantly induced Annexin V-positive cells, cytosolic cytochrome C, and the pro-apoptotic protein Bax in AML cells including HL60, HEL92.1.7, and THP-1. In addition, mitochondrial membrane potential and Bcl-xl protein were markedly decreased by radotinib and Ara-C. Moreover, this combination induced caspase-3 activity. Cleaved caspase-3, 7, and 9 levels were also increased by combined radotinib and Ara-C. Additionally, radotinib and Ara-C co-treatment induced G0/G1 arrest via the induction of CDKIs such as p21 and p27 and the inhibition of CDK2 and cyclin E. Thus, radotinib/Ara-C induces mitochondrial-dependent apoptosis and G0/G1 arrest via the regulation of the CDKI-CDK-cyclin cascade in AML cells. In addition, our results showed that combined treatment with radotinib and Ara-C inhibits AML cell growth, including tumor volumes and weights in vivo. Also, the combination of radotinib and Ara-C can sensitize cells to chemotherapeutic agents such as daunorubicin or idarubicin in AML cells.Conclusions: Therefore, our results can be concluded that radotinib in combination with Ara-C possesses a strong anti-AML activity.
| S-EPMC7718665 | biostudies-literature
Project description:Human clinical trials suggest that inhibition of enzymes in the DNA base excision repair (BER) pathway, such as PARP1 and APE1, can be useful in anticancer strategies when combined with certain DNA-damaging agents or tumor-specific genetic deficiencies. There is also evidence suggesting that inhibition of the BER enzyme 8-oxoguanine DNA glycosylase-1 (OGG1), which initiates repair of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-dG), could be useful in treating certain cancers. Specifically, in acute myeloid leukemia (AML), both the RUNX1-RUNX1T1 fusion and the CBFB-MYH11 subtypes have lower levels of OGG1 expression, which correlate with increased therapeutic-induced cell cytotoxicity and good prognosis for improved, relapse-free survival compared with other AML patients. Here we present data demonstrating that AML cell lines deficient in OGG1 have enhanced sensitivity to cytarabine (cytosine arabinoside [Ara-C]) relative to OGG1-proficient cells. This enhanced cytotoxicity correlated with endogenous oxidatively-induced DNA damage and Ara-C-induced DNA strand breaks, with a large proportion of these breaks occurring at common fragile sites. This lethality was highly specific for Ara-C treatment of AML cells deficient in OGG1, with no other replication stress-inducing agents showing a correlation between cell killing and low OGG1 levels. The mechanism for this preferential toxicity was addressed using in vitro replication assays in which DNA polymerase δ was shown to insert Ara-C opposite 8-oxo-dG, resulting in termination of DNA synthesis. Overall, these data suggest that incorporation of Ara-C opposite unrepaired 8-oxo-dG may be the fundamental mechanism conferring selective toxicity and therapeutic effectiveness in OGG1-deficient AML cells.
| S-EPMC7980395 | biostudies-literature
Project description:BACKGROUND:Variation in terms of outcome and toxic side effects of treatment exists among acute myeloid leukemia (AML) patients on chemotherapy with cytarabine (Ara-C) and daunorubicin (Dnr). Candidate Ara-C metabolizing gene expression in primary AML cells is proposed to account for this variation. METHODS:Ex vivo Ara-C sensitivity was determined in primary AML samples using MTT assay. mRNA expression of candidate Ara-C metabolizing genes were evaluated by RQPCR analysis. Global gene expression profiling was carried out for identifying differentially expressed genes between exvivo Ara-C sensitive and resistant samples. RESULTS:Wide interindividual variations in ex vivo Ara-C cytotoxicity were observed among samples from patients with AML and were stratified into sensitive, intermediately sensitive and resistant, based on IC50 values obtained by MTT assay. RNA expression of deoxycytidine kinase (DCK), human equilibrative nucleoside transporter-1 (ENT1) and ribonucleotide reductase M1 (RRM1) were significantly higher and cytidine deaminase (CDA) was significantly lower in ex vivo Ara-C sensitive samples. Higher DCK and RRM1 expression in AML patient's blast correlated with better DFS. Ara-C resistance index (RI), a mathematically derived quotient was proposed based on candidate gene expression pattern. Ara-C ex vivo sensitive samples were found to have significantly lower RI compared with resistant as well as samples from patients presenting with relapse. Patients with low RI supposedly highly sensitive to Ara-C were found to have higher incidence of induction death (p = 0.002; RR: 4.35 [95% CI: 1.69-11.22]). Global gene expression profiling undertaken to find out additional contributors of Ara-C resistance identified many apoptosis as well as metabolic pathway genes to be differentially expressed between Ara-C resistant and sensitive samples. CONCLUSION:This study highlights the importance of evaluating expression of candidate Ara-C metabolizing genes in predicting ex vivo drug response as well as treatment outcome. RI could be a predictor of ex vivo Ara-C response irrespective of cytogenetic and molecular risk groups and a potential biomarker for AML treatment outcome and toxicity. Original submitted 22 December 2014; Revision submitted 9 April 2015.
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Project description:Drugs targeting metabolism have formed the backbone of therapy for some cancers. We sought to identify new such targets in acute myeloid leukemia (AML). The one-carbon folate pathway, specifically methylenetetrahydrofolate dehydrogenase-cyclohydrolase 2 (MTHFD2), emerged as a top candidate in our analyses. MTHFD2 is the most differentially expressed metabolic enzyme in cancer versus normal cells. Knockdown of MTHFD2 in AML cells decreased growth, induced differentiation, and impaired colony formation in primary AML blasts. In human xenograft and MLL-AF9 mouse leukemia models, MTHFD2 suppression decreased leukemia burden and prolonged survival. Based upon primary patient AML data and functional genomic screening, we determined that FLT3-ITD is a biomarker of response to MTHFD2 suppression. Mechanistically, MYC regulates the expression of MTHFD2, and MTHFD2 knockdown suppresses the TCA cycle. This study supports the therapeutic targeting of MTHFD2 in AML.
| S-EPMC4925018 | biostudies-literature
Project description:Nucleosides and their analogues constitute an essential family of anticancer drugs. DNA has been the presumptive target of the front-line prodrug for acute myeloid leukemia (AML), cytarabine (ara-C), since the 1980s. Here, the biomolecular targeting of ara-C was evaluated in primary white blood cells using the ara-C mimic "AzC" and azide-alkyne "click" reactions. Fluorescent staining and microscopy revealed that metabolic incorporation of AzC into primary white blood cells was unexpectedly enhanced by the DNA polymerase inhibitor aphidicholine. According to RNaseH digestion and pull-down-and-release experiments, AzC was incorporated into short RNA fragments bound to DNA in peripheral blood monocytes (PBMCs) collected from all six healthy human donors tested. Samples from 22 AML patients (French-American-British classes M4 and M5) exhibited much more heterogeneity, with 27% incorporating AzC into RNA and 55% into DNA. The overall survival of AML patients whose samples incorporated AzC into RNA was approximately 3-fold higher as compared to that of the DNA cohort (p ≤ 0.056, χ2 = 3.65). These results suggest that the RNA primers of DNA synthesis are clinically favorable targets of ara-C, and that variable incorporation of nucleoside drugs into DNA versus RNA may enable future patient stratification into treatment-specific subgroups.
| S-EPMC7737216 | biostudies-literature