OGFOD1 enables AML chemo- and nutrient stress resistance by regulating protein synthesis.

The Splicing-Associated Network PAK1-Clk-SRRM1 Is a Critical Vulnerability to Overcome Chemoresistance in Acute Myeloid Leukemia

Modulating mRNA Translation Fidelity As a Novel Mechanism for AML Chemoresistance

Identification of Somatic Mutation Contributing to Chemotherapy Resistance in Acute Myeloid Leukemia

BackgroundBone marrow stromal cells (BMSCs) are known to promote chemoresistance in acute myeloid leukemia (AML) cells. However, the molecular basis for BMSC-associated AML chemoresistance remains largely unexplored.MethodsThe mitochondrial oxidative phosphorylation (OXPHOS) levels of AML cells were measured by a Seahorse XFe24 cell metabolic analyzer. The activity of total or mitochondrial signal transducer and transcription activator 3 (STAT3) in AML cells was explored by flow cytometry and Western blotting. Real-time quantitative PCR, Western blotting and enzyme-linked immunosorbent assay (ELISA) were used to analyze expression of interleukin 6 (IL-6) in the human BMSC line HS-5, and IL-6 was knocked out in HS-5 cells by CRISPR/Cas9 system.ResultsIn this study, we observed that co-culturing with BMSCs heightened OXPHOS levels in AML cells, thus promoting chemoresistance in these cells. HS-5 cell-induced upregulation of OXPHOS is dependent on the activation of STAT3, especially on that of mitochondrial serine phosphorylated STAT3 (pS-STAT3) in AML cells. The relationship among pS-STAT3, OXPHOS, and chemosensitivity of AML cells induced by BMSCs was demonstrated by the STAT3 activator and inhibitor, which upregulated and downregulated the levels of mitochondrial pS-STAT3 and OXPHOS, respectively. Intriguingly, AML cells remodeled HS-5 cells to secrete more IL-6, which augmented mitochondrial OXPHOS in AML cells and stimulated their chemoresistance. IL-6 knockout in HS-5 cells impaired the ability of these cells to activate STAT3, to increase OXPHOS, or to promote chemoresistance in AML cells.ConclusionsBMSCs promoted chemoresistance in AML cells via the activation of the IL-6/STAT3/OXPHOS pathway. These findings exhibit a novel mechanism of chemoresistance in AML cells in the bone marrow microenvironment from a metabolic perspective.

<div>Abstract<p>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. <i>MFN1</i> knockdown impaired the effects of IL6 on mitochondrial function and chemoresistance in AML cells. In an <i>MLL::AF9</i> 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.</p>Implications:<p>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.</p></div>

<div>Abstract<p>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. <i>MFN1</i> knockdown impaired the effects of IL6 on mitochondrial function and chemoresistance in AML cells. In an <i>MLL::AF9</i> 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.</p>Implications:<p>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.</p></div>

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.

IntroductionProgression and chemoresistance of acute myeloid leukemia (AML) contribute to most of the treatment failure. Notch pathway has been proven to be involved in many biological processes and diseases, especially AML. In this study, we aimed to explore genes correlated with Notch1 pathway in AML and determine their roles in the regulation of AML progression and chemoresistance.MethodsTCGA database was used to explore Notch1 associated genes. Kaplan–Meier survival analysis was performed to evaluate the prognostic significance of genes. Quantitative RT-PCR (qRT-PCR) and Western blot were performed to examine the expression of genes. The expression of PRKD2 was up-regulated or knocked down in AML cell lines by lentivirus or siRNAs. CCK-8 and flow cytometry were used to analyze the effect of PRKD2 on cell proliferation and chemoresistance.ResultsBased on TCGA database, PRKD2 was found to be positively correlated with Notch1 expression, cytogenetic risk status and poorer prognosis in AML. Moreover, the expression level of PRKD2 was higher in AML chemo-resistant cells than in chemo-sensitive cells. Functionally, knockdown of PRKD2-induced apoptosis and increased chemosensitivity of AML cells. PRKD2 overexpression promoted proliferation and chemoresistance of AML cells. Furthermore, we found PRKD2 could regulate Notch1 pathway. Besides, high PRKD2 expression was correlated with higher risk group of AML patients which indicated that PRKD2 was an independent prognostic marker for AML.ConclusionTaken together, our results showed that PRKD2 could promote the proliferation and chemoresistance of AML cells by regulating Notch1 pathway. The study broadened our insights into the underlying mechanisms in chemoresistance and proliferation of AML, and provided a new prognostic marker and treatment target for AML.

Tryptase is a serine protease that is expressed in leukemic cells of chronic myeloid leukemia (CML) patients and in blasts of acute myeloid leukemia (AML) patients. Tryptase may be useful for diagnosis, assessment of severity of disease (leukemic cell burden), monitoring minimal residual disease and prognosis of AML and CML patients. The main objective of this study was to assess the serum levels of tryptase activity among CML and AML patients and to compare the serum levels of tryptase activity of acute and chronic myeloid leukemia patients with each other and with those of healthy controls. To meet this objective, a hospital-based comparative cross-sectional study was conducted among CML and AML patients from February 2016 up to December 2016. Serum samples were obtained from 24 AML, 60 CML and 35 healthy controls. Fluorogenic assays for serum tryptase activity using aminomethylcoumarin (AMC) peptide derivative were carried out. Statistical analysis was done by using SPSS version 20. Descriptive statistics, Paired Samples T-test, Wilcoxon Signed Rank test and Spearman’s rho test were used to investigate any correlation among different parameters. The minimum level of statistical significance was set at p-value <0.05. Accordingly, the mean and median serum levels of tryptase activity were significantly higher in patients with AML and CML than in the healthy controls (P-value < 0.05). CML patients in chronic phase (CP) and secondary AML patients had significantly higher mean and median serum levels of tryptase activity than CML patients in accelerated/blast phase (AP/BP) and de novo AML patients (p-value < 0.05). These elevated mean and median levels of serum tryptase activities were due to a subset of individuals with elevated serum tryptase levels (41.7 % of AML & 30 % of CML); the remaining leukemic individuals (58.3 % AML & 70 % of CML) had normal serum levels of tryptase activity. Finally, it was concluded that the serum tryptase level might be a useful diagnostic and prognostic marker in a subset of patients with CML and AML. However, further studies that incorporate other protocols such as tryptase immunoassay are warranted to exclude contaminant non-tryptase proteases from the serum samples.

Background: As the most prevalent internal decorations in mammalian mRNA, N6-methyladenosine (m6A) has been reported to be involved in many physiological and pathological processes, including acute myeloid leukemia (AML). METTL3 and METTL14, the well-recognized m6A methyltransferase complex, contribute to AML. METTL16 is a recently identified m6A methyltransferase that has been reported to deposit m6A in a few targets. While, unlike METTL3/14, the biological functions of METTL16 are largely unknown. Here, we explored the function and mechanism of METTL16 in AML pathogenesis and evaluated its therapeutic potential for AML treatment. Methods: We performed CRISPR-Cas9 screen to evaluate the dependency of METTL16 in AML cells. We created METTL16 knockout (KO) cells and conditional KO mice to evaluate its role in leukemogenesis and normal hematopoiesis. We employed bone marrow transplantation (BMT), xenograft, and AML patient-derived xenograft (PDX) models to determine its role in AML development and progression. To identify the targets of METTL16, we performed m6A-seq and RNA-seq, followed m6A-qPCR, CLIP-qPCR, in vitro methyltransferase assays and RNA stability assays. To examine the effect of METTL16 on branched chain amino acid (BCAA) metabolism, we performed metabolic profiling with 13C, 15N-leucine. Results: CRISPR-Cas9 screen showed METTL16 is one of the most essential genes for the survival of AMLs. The AML cells display more robust dependency on METTL16 than METTL3/14. We found METTL16 is highly expressed in AML patients compared to healthy controls. METTL16 KO significantly inhibited AML cell proliferation, promoted cell apoptosis and myeloid differentiation in vitro, which could be totally reversed by forced expression of wild-type METTL16, but not catalytic-dead mutant. METTL16 depletion dramatically inhibited AML progression and prolonged survival of recipient mice in the BMT, xenograft and PDX models. In addition, METTL16 is highly expressed in LSCs contrast to leukemic bulk cells and METTL16 KO significantly attenuates LSC self-renewal in vitro and in vivo. By contrast, the role of METLL16 is largely spared in normal hematopoietic cells. Via integrated analysis of m6A-seq data and RNA-seq data, we identified two bona fide targets of METTL16, BCAT1 and BCAT2, which encode two critical BCAA transaminases in BCAA biosynthesis pathway. METTL16 promotes the expression of BCAT1 and BCAT2 via an m6A dependent manner. Metabolomics with 13C, 15N-leucine tracing showed that METTL16 KO results in suppressed pools of TCA cycle intermediates, some non-essential amino acids and nucleotides. Conclusion: We uncover a tumor-promoting role of METTL16 in AML and LSC self-renewal via reprogramming BCAA metabolism, in which METTL16 functions as an m6A methyltransferase to regulate expression of BCAT1 and BCAT2. Our data suggest that METTL16 is an attractive target for AML therapy. Citation Format: Li Han, Lei Dong, Keith Leung, Zhicong Zhao, Yangchan Li, Ying Qing, Jianhuang Xue, Chao Shen, Zhenhua Chen, Lei Gao, Kitty Wang, Keren Zhou, Wei Li, Brandon Tan, Zheng Zhang, Xi Qin, Rui Su, Xiaolan Deng, Jianjun Chen. METTL16 drives leukemogenesis and maintains leukemia stem cell self-renewal via reprogramming BCAA metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3617.

Quiescent self-renewal of leukemia stem cells (LSCs) and resistance to conventional chemotherapy are the main factors leading to relapse of acute myeloid leukemia (AML). Alpha-enolase (ENO1), a key glycolytic enzyme, has been shown to regulate embryonic stem cell differentiation and promote self-renewal and malignant phenotypes in various cancer stem cells. Here, we sought to test whether and how ENO1 influences LSCs renewal and chemoresistance within the context of AML. We analyzed single-cell RNA sequencing data from bone marrow samples of 8 relapsed/refractory AML patients and 4 healthy controls using bioinformatics and machine learning algorithms. In addition, we compared ENO1 expression levels in the AML cohort with those in 37 control subjects and conducted survival analyses to correlate ENO1 expression with clinical outcomes. Furthermore, we performed functional studies involving ENO1 knockdown and inhibition in AML cell line. We used machine learning to model and infer malignant cells in AML, finding more primitive malignant cells in the non-response (NR) group. The differentiation capacity of LSCs and progenitor malignant cells exhibited an inverse correlation with glycolysis levels. Trajectory analysis indicated delayed myeloid cell differentiation in NR group, with high ENO1-expressing LSCs at the initial stages of differentiation being preserved post-treatment. Simultaneously, ENO1 and stemness-related genes were upregulated and co-expressed in malignant cells during early differentiation. ENO1 level in our AML cohort was significantly higher than the controls, with higher levels in NR compared to those in complete remission. Knockdown of ENO1 in AML cell line resulted in the activation of LSCs, promoting cell differentiation and apoptosis, and inhibited proliferation. ENO1 inhibitor can impede the proliferation of AML cells. Furthermore, survival analyses associated higher ENO1 expression with poorer outcome in AML patients. Our findings underscore the critical role of ENO1 as a plausible driver of LSC self-renewal, a potential target for AML target therapy and a biomarker for AML prognosis.

Chemoresistance is a major burden for the treatment of many cancers, including acute myeloid leukemia (AML). AML is a clonal disease and chemoresistant cells may either evolved from the expansion of a subclone of the primary clone or from the clone gained additional mutations during therapy. We have generated chemoresistant cell lines from AML patient sample derived cell lines, OCI-AML2 and MV4-11, to study the chemoresistant mechanism. Interestingly, we found that both established resistant lines have TP53 mutations (Y220C or R248W) but not in the parental cell lines by Sanger sequencing. However, high resolution sequencing result shows that residual TP53 mutation is present in both parental cell lines. These small population of cells with TP53 mutation was expanded under the selection by chemotherapy and eventually became a dominant clone in chemoresistant lines. TCGA AML patient sample sequencing data also shows, there’re around 3% of patients have residual TP53 mutation reads. However, since the reads are low, these patients are considered TP53 wild type conventionally. Importantly, these patients have worse outcome of survival as compared to the TP53 wild type patients, indicating the residual TP53 mutation may play an important role on chemoresistance in patients. Consistent with TP53 mutation in chemoresistant cells, Gene Set Enrichment Analysis (GSEA) from RNA-seq data shows p53 target genes are repressed in resistant cells. The cyclin-dependent kinase inhibitor p21 is one of p53 target genes and is down regulated in chemoresistant cells. Knocking down of p21 in parental cells increased cell chemoresistance, indicating p21 gene repression contributes to chemoresistance. Data from TCGA also shows patients with lower p21 expression have worse outcome of survival. In chemoresistant cell line, there is reduced p53 recruitment at p21 promoter, leading to failure of p21 activation. The epigenetic drug Romidepsin can elevate histone acetylation at p21 promoter region, reactivates p21 gene expression and induces cell death. Furthermore, GSEA from RNA-seq data shows Romidepsin can reactivate genes that repressed by p53 mutation. Our data indicate that TP53 mutation is directly linked to chemoresistance. Residual TP53 mutation in patients was neglected previously, however, it can be an important driver for clonal evolution and chemoresistance in AML. In addition, epigenetic drug Romidepsin can be a potential therapeutic drug to prevent chemoresistance development especially for patients with TP53 mutation or residual TP53 mutation. Citation Format: Bowen Yan, Yi Qiu. Clonal expansion of TP53 mutated cells is associated with chemoresistance in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3023.

We have previously reported that about 80% of acute myeloid leukaemia (AML) samples tested at diagnosis constitutively expressed cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). The present study compared CTLA-4 expression and function of leukaemic cells from AML patients at diagnosis with those from AML patients resistant to conventional chemotherapy. We also explored the possibility of targeting CTLA-4 for apoptosis induction in chemoresistant AML cells. AML cells either from untreated patients (n = 15) or in chemoresistant phase (n = 10) were analysed for CTLA-4 protein and transcript expression by flow cytometry and reverse transcription-polymerase chain reaction respectively. CTLA-4 expression was similar in untreated and in chemoresistant samples and was not associated with patients' clinical features. In chemoresistant AML cells, CTLA-4 transduced an apoptotic signal on engagement with its recombinant ligands r-CD80 and r-CD86, which induced an average of 71% and 62% apoptotic cells, respectively, at highest concentration. Apoptosis was equally induced in untreated leukaemic cells accompanied by cleavage of procaspase-8 and -3. Thus, this study provides the first evidence that killing of leukaemic cells from AML patients may be obtained by the engagement of CTLA-4 with its ligands, opening the way to a novel potential therapeutic approach based on triggering the CTLA-4 molecule to circumvent chemoresistance in AML.

Acute myeloid leukemia (AML), caused by abnormal proliferation and accumulation of hematopoietic progenitor cells, is one of the most common malignancies in adults. We reported here DYRK1A expression level was reduced in the bone marrow of adult AML patients, comparing to normal controls. Overexpression of DYRK1A inhibited the proliferation of AML cell lines by increasing the proportion of cells undergoing G0/G1 phase. We reasoned that the proliferative inhibition was due to downregulation of c-Myc by DYRK1A, through mediating its degradation. Moreover, overexpression of c-Myc markedly reversed AML cell growth inhibition induced by DYRK1A. DYRK1A also had significantly lower expression in relapsed/refractory AML patients, comparing to newly-diagnosed AML patients, which indicated the role of DYRK1A in chemoresistance of AML. Our study provided functional evidences for DYRK1A as a potential tumor suppressor in AML.

Resistance to chemotherapy and subsequent relapse is the most challenging issue in the treatment of patients with Acute Myeloid Leukemia (AML). However, the underlying mechanisms still remain incompletely understood. Here we report that loss of the histone methyltransferase EZH2 and subsequent reduction of H3K27 trimethylation contribute to chemoresistance in AML. In Myelodysplastic Syndrome (MDS) and Myeloproliferative Neoplasms (MPN) EZH2 is often inactivated due to mutations which is associated with poor prognosis. By use of quantitative PCR and immunohistochemistry we show that a decrease of EZH2 mRNA and protein also correlated with a poor prognosis of AML patients indicating a tumor suppressor role of EZH2 in AML. EZH2 is located on chromosome 7q36.1 and it is not yet fully clear whether EZH2 expression is affected in MDS and AML patients with del(7)/del(7q) who are largely refractory to chemotherapy and have a poor prognosis. We found EZH2 levels to be reduced in del(7)/del(7q) AML patients as determined by Western Blot. Notably, the reduction of EZH2 protein levels via treatment with H3K27 methyltransferase inhibitors or lentiviral knockdown was sufficient to induce chemoresistance of Normal Karyotype (NK)- AML blasts and cell lines in vitro and in a xenograft mouse model. Furthermore, we observed that EZH2 loss occurred during the acquisition of drug resistance in a Tyrosine Kinase Inhibitor- and Cytarabine (AraC)- resistant AML cell line. Pharmacological inhibition of CDK1 and treatment with the proteasome inhibitor Bortezomib, respectively, increased EZH2 protein and restored drug sensitivity. Functionally, the loss of EZH2 directly induced upregulation of HOX genes, suggesting a stem-cell-like signature to be associated with the resistance phenotype, which could be reverted by Bortezomib treatment. To evaluate the potential of Bortezomib to affect EZH2 levels in patient blasts we treated primary NK-AML blasts collected at diagnosis ex vivo with Bortezomib. In almost all samples Bortezomib treatment induced cytotoxic effects. In 5 out of 10 patients the EZH2 protein level could be increased by Bortezomib treatment. We furthermore examined the sensitivity of patient samples to AraC, Bortezomib or combined treatment. Notably, for those patients with increased EZH2 levels after Bortezomib exposure we found a significantly decreased cell survival for the combined treatment compared to single-agent treatment. Our data strongly suggest that restoration of EZH2 protein levels e.g. via proteasome inhibitors and thereby restoration of EZH2 function might be a novel promising approach to increase therapy response in AML. Citation Format: Stefanie Göllner, Shuchi Agrawal-Singh, Tino Schenk, Hans-Ulrich Klein, Christian Rohde, Tim Sauer, Mads Lerdrup, Sigal Tavor, Friedrich Stölzel, Gerhard Ehninger, Gabriele Köhler, Martin Dugas, Arthur Zelent, Christian Thiede, Wolfgang E. Berdel, Klaus Hansen, Carsten Müller-Tidow. Loss of the histone methyltransferase EZH2 induces chemoresistance in acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4430. doi:10.1158/1538-7445.AM2015-4430