Chemoresistance Of Acute Myeloid Leukemia Cells Research Articles

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.

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

In Acute Myeloid Leukemia (AML), distinguishing between chemoresistance driver mechanisms and cell plasticity in response to chemotherapy is challenging due to their interconnectedness. To address this issue, we generated an MLL-AF9-driven syngeneic mouse model of AML, which we evolved to develop resistance to a front-line chemotherapy regimen by repetitive exposure to a maximally-tolerated combination of cytarabine and doxorubicin. An RNA sequencing (RNAseq)-based profiling of the chemoresistant (Chemo R) AML subpopulation and its naive (Chemo N) counterpart revealed a significant upregulation in the expression of 326 genes in Chemo R cells which were markedly enriched in gene sets related to splicing/mRNA processing. Notably, single-sample GSEA analysis of relapsed AML samples from the BEAT-AML cohort supported these findings, as they were enriched for the upregulation of genes in our chemoresistance gene signature. A combined in vivo functional screening endeavor using a pool of 2098 shRNAs targeting these upregulated genes and a motif discovery analysis performed on the set of dysregulated transcripts in Chemo R cells identified three candidate hits, Srrm1, Sf3b3, and Prpf8, which scored as chemotherapy-specific dependencies and were found to interact with central regulators of alternative splicing ( Figure 1A). Silencing these three candidate genes in our chemoresistant mouse model and the human AML cell lines, MOLM-14, MV4-11, and U937, evolved to become resistant to the combination of cytarabine and daunorubicin, revealed that the splicing regulator, SRRM1, is a critical vulnerability of chemoresistant leukemic cells. An orthogonal phospho-proteomic approach identified that PAK1 and CLK kinases are regulators of SRRM1 in chemoresistant AML cells, through the phosphorylation of multiple serine and threonine phosphosites in its arginine-rich domain. Human and murine chemoresistant cells were found to be markedly more sensitive to inhibitors of PAKs (FRAX597) and CLKs (TG003 and ML167) compared to their naive counterparts, an effect which was even further enhanced by treatment with cytarabine and daunorubicin. Notably, a whole-exome sequencing pipeline at our institution revealed a clonal variant of uncertain significance (c.1429G>T p.(Ala477Ser)) in PAK1 in an AML patient at relapse. Absence of this variant at diagnosis suggested its association with chemotherapy resistance. Biochemical and cellular assays confirmed that the A477S variant increased PAK1 activation as well as chemotherapy resistance in AML cell lines. This effect could be counteracted by suppressing SRRM1, indicating that PAK1 activation in relapsed AML confers increased vulnerability to SRRM1 suppression. Simultaneous suppression of Clk1 and Pak1 or Clk4 and Pak1 using validated shRNAs and combinations of FRAX597 and TG003, or FRAX597 and ML167 synergized to reduce the growth and the colony-forming capacity of chemoresistant AML cells and sensitized them to chemotherapy. These observations were supported by the RNAseq-based estimate of the inclusion ratios of alternative exons and introns which showed that the combination of PAK and CLK inhibitors phenocopied partially the effects of SRRM1 silencing on splicing perturbations. In preclinical studies, the combined treatment improved overall mouse survival and reduced disease burden in our chemoresistant AML mouse models compared to single-drug treatments ( Figure 1B). Moreover, AML patient cells that were primarily refractory or from a post chemotherapy relapse (n=21) exhibited a higher ex vivo sensitivity to the FRAX597 + TG003 combination in comparison with chemosensitive patients at diagnosis (n=28). Moreover, in NSG-S mice transplanted with matched samples from the same AML patient at diagnosis and relapse post-chemotherapy, combined PAK1 and CLK inhibition more effectively reduced disease progression in animals transplanted with the relapse sample than in those engrafted with the sample taken at diagnosis. In conclusion, our study advances the rationale that combined inhibition of PAK1 and CLK kinases effectively curtails malignant cell splicing adaptation to chemotherapy by affecting SRRM1 function. Combined PAK1 and CLK inhibitors either concomitant with or sequential to chemotherapy may offer greater clinical benefit and significantly improve AML patients' long-term outcomes.

Protective Function of Human Skin Mesenchymal Niches for Acute Myeloid Leukemia-Initiating Stem Cells

Leukemia cutis (LC) or leukemic cell infiltration in skin is one of the common extramedullary manifestations in acute myeloid leukemia (AML) and signifies a poorer prognosis. However, the pathogenesis and maintenance of LC have been understudied, limiting our understanding of its impact on AML progression and relapse. We recently have shown that the AML cells infiltrated in the mouse skin during steady state and after chemotherapy were capable to regenerate AML after serial transplantation (Sandhow et al., J. Exp. Med, 2023). Further characterization of the skin mesenchymal stem cells (MSCs) suggests that skin MSCs could maintain AML-initiating stem cells (LSCs) and protect them from chemotherapy, even to a greater degree than their bone marrow (BM) counterparts. These data suggest the protective role of skin mesenchymal niche in AML LSCs during chemotherapy. However, it remains unexplored whether human skin MSCs exert the same functional impact on human AML cells. We here have explored the role of human skin MSCs in AML maintenance and survival during chemotherapy by RNA sequencing and in vitro co-culture of patient-derived AML cells with primary human skin MSCs in comparison with BM MSCs. We first by prospective characterization identified human skin MSCs with a phenotype of CD45-CD235a-CD31-CD44+CD146- from healthy donors. RNA sequencing and Q-PCR revealed expression of key hematopoietic stem cell (HSC) regulatory genes including THPO, SCF, CXCL12 and LAMA4 in human skin MSCs. Interestingly, while HSC maintenance genes such as THPO and KITLG were significantly upregulated in skin MSCs (p<0.0001 and p<0.05, respectively), HSC negative regulator SPP1 was downregulated (p=0.05), pointing to a possible better hematopoiesis-supportive function of skin MSCs. In line with this, our functional studies indicated that skin MSCs displayed a superior protective effect on human AML cell line THP-1 than their BM cell counterparts during cytarabine (Ara-C) treatment in the in vitro co-culture. There were more residual chemo-resistant AML cells, reflected in the higher number of CD36+ AML cells after treatment with Ara-C in cocultures with skin MSCs, compared to that with BM MSCs (p=0.0065). This result was confirmed by functional assays showing the increased number of cobblestone area-forming cells (p=0.0298) and colony-forming units (p=0.038) within the residual AML cells co-cultured with skin MSCs. To investigate the mechanisms involved in the protective function of human skin MSCs, we have performed RNA-sequencing of the human skin and BM MSCs before and after co-culture with AML cells. This has revealed potential unique molecular pathways between AML and skin MSCs. Furthermore, there were dramatically less altered genes in the skin MSCs compared to BM MSCs after 3 day-coculture with THP1 AML cells (221 vs 1761 altered genes with adjusted p values <0.05). These data indicate that skin MSCs are likely more resistant to AML-remodeling, which could in turn maintain AML cells in quiescence status, thereby protecting AML cells from Ara-C treatment. This speculation is currently under validation. In addition, gene set enrichment analysis showed upregulated genes associated with fatty acid metabolisms and ribosome in skin MSCs compared to BM MSCs, providing molecular evidence for a potential better metabolic support of skin MSCs for AML cells. Taken together, our preliminary data indicate an important role of human skin MSCs in supporting and protecting AML cells during Ara-C treatment. We are further investigating the underlying mechanisms.

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

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.

Acute myeloid leukemia (AML)-derived mesenchymal stem cells induce chemoresistance and epithelial-mesenchymal transition-like program in AML through IL-6/JAK2/STAT3 signaling.

Acute myeloid leukemia (AML) has a high rate of treatment failure due to increased prevalence of therapy resistance. Mesenchymal stem cells (MSCs) in the leukemia microenvironment contribute to chemoresistance in AML, but the specific mechanism remains unclear. The critical role of the epithelial-mesenchymal transition (EMT)-like profile in AML chemoresistance has been gradually recognized. However, there is no research to suggest that the AML-derived bone marrow mesenchymal stem cells (AML-MSCs) induce the EMT program in AML thus far. We isolated AML-MSCs and cocultured them with AML cells. We found that AML-MSCs induced a significant mesenchymal-like morphology in drug-resistant AML cells, but it was scarce in parental AML cells. The AML-MSCs promoted growth of AML cells in the presence or absence of chemotherapeutics in vitro and in vivo. Acute myeloid leukemia MSCs also induced EMT marker expression in AML cells, especially in chemoresistant AML cells. Mechanistically, AML-MSCs secreted abundant interleukin-6 (IL-6) and upregulated IL-6 expression in AML cells. Acute myeloid leukemia cells upregulated IL-6 expression in AML-MSCs in turn. Meanwhile, AML-MSCs activated the JAK2/STAT3 pathway in AML cells. Two JAK/STAT pathway inhibitors counteracted the AML-MSCs induced morphology change and EMT marker expression in AML cells. In conclusion, AML-MSCs not only promote the emergence of chemoresistance but also enhance it once AML acquires chemoresistance. AML-MSCs induce EMT-like features in AML cells; this phenotypic change could be related to chemoresistance progression. AML-MSCs induce the EMT-like program in AML cells through IL-6/JAK2/STAT3 signaling, which provides a therapeutic target to reverse chemoresistance in AML.

Chemoresistance in acute myeloid leukemia: An alternative single-cell RNA sequencing approach.

Our previous study demonstrated that myc, mitochondrial oxidative phosphorylation, mTOR, and stemness are independently responsible for chemoresistance in acute myeloid leukemia (AML) cells. This study aimed to identify potential mechanisms of chemoresistance of the "7+3" induction in AML by using a single-cell RNA sequencing (scRNA-seq) approach. In the present study, 13 untreated patients with de novo AML were enrolled and stratified into two groups: complete remission (CR; n=8) and non-CR (n=5). Single-cell RNA sequencing was used to analyze genetic profiles of 28,950 AML cells from these patients; results were validated using a previously published bulk RNA-seq dataset. Our study results showed chemoresistant AML cells had premature accumulation during early hematopoiesis. Hematopoietic stem cell-like cells from the non-CR group expressed more leukemic stem cell markers (CD9, CD82, IL3RA, and IL1RAP) than those from the CR group. Chemoresistant progenitor cells had impaired myeloid differentiation owing to early arrest of hematopoiesis. Notably, AML cells analyzed by scRNA-seq and bulk RNA-seq harbored a comparable myeloid lineage cell fraction, which internally validated our results. Using the TCGA database, our analysis demonstrated that patients with AML with higher expression of chemoresistant genetic markers (IL3RA and IL1RAP) had a worse overall survival (p<0.01 for IL3RA; p<0.05 for IL1RAP). In conclusion, AML cells responsive and resistant to the "7+3" induction were derived from a diverse cancerous hematopoietic stem cell population, as indicated by the specific genetic biomarkers obtained using scRNA-seq approach. Furthermore, arrest of hematopoiesis was shown to occur earlier in chemoresistant AML cells, furthering the current understanding of chemoresistance in AML.

DNA damage to bone marrow stromal cells by antileukemia drugs induces chemoresistance in acute myeloid leukemia via paracrine FGF10–FGFR2 signaling

Chemo-resistance remains a major challenge in the current treatment of acute myeloid leukemia (AML). The bone marrow microenvironment (BMM) plays a complex role in protecting leukemia cells from chemotherapeutics, and the mechanisms involved are not fully understood. Anti-leukemia drugs kill AML cells directly but also damage the BMM. Here, we determined anti-leukemia drugs induce DNA damage in bone marrow stromal cells (BMSCs), resulting in resistance of AML cell lines to adriamycin and idarubicin killing. Damaged BMSCs induced an inflammatory microenvironment through NF-κB; suppressing NF-κB with small molecule inhibitor Bay11-7082 attenuated the pro-survival effects of BMSCs on AML cell lines. Furthermore, we used an ex vivo functional screen of 507 chemokines and cytokines to identify 44 proteins secreted from damaged BMSCs. Fibroblast growth factor-10 (FGF10) was most strongly associated with chemo-resistance in AML cell lines. Additionally, expression of FGF10 and its receptors, FGFR1 and FGFR2, was increased in AML patients after chemotherapy. FGFR1 and FGFR2 were also widely expressed by AML cell lines. FGF10-induced FGFR2 activation in AML cell lines operates by increasing P38 MAPK, AKT, ERK1/2, and STAT3 phosphorylation. FGFR2 inhibition with small molecules, or gene silencing of FGFR2 inhibited proliferation and reverses drug-resistance of AML cells by inhibiting P38 MAPK, AKT, and ERK1/2 signaling pathways. Finally, release of FGF10 was mediated by β-catenin signaling in damaged BMSCs. Our data indicate FGF10-FGFR2 signaling acts as an effector of damaged BMSC-mediated chemo-resistance in AML cells, and FGFR2 inhibition can reverse stromal protection and AML cell chemo-resistance in the BMM.

Trial in Progress: Tagraxofusp-Based Therapy to Eradicate Measurable Residual Disease of Acute Myelogenous Leukemia Prior to Allogeneic Hematopoietic Cell Transplantation

Background Numerous studies convincingly demonstrate that measurable residual disease (MRD) in AML at time of allogeneic cell transplantation (alloHCT) is a powerful, independent predictor of subsequent relapse. Attempts to eradicate AML MRD pre-transplant with consolidation chemotherapy have been unsuccessful, with reported MRD conversion rates as low as 20% (Appelbaum et al., Blood 2019). Tagraxofusp is an IL-3 truncated diphtheria toxin recombinant protein that binds CD123 with high affinity. Preliminary data using tagraxofusp as consolidation therapy in AML patients has demonstrated good tolerability (Lane et al., Blood 2017). Pathways of resistance to tagraxofusp have been identified preclinically as loss of the intracellular target for diphtheria toxin, diphthamide, through methylation of an enzyme involved in diphthamide synthesis. Treatment with a hypomethylating agent has the potential to restore expression of diphthamide and increase tagraxofusp sensitivity in AML cells (Togami et al., J Clin Invest 2019). Design This is a single-arm, open-label, multi-center phase Ib/II clinical trial to evaluate the safety and effectiveness of tagraxofusp +/- azacitidine in AML patients who are in first or second remission with detectable MRD and are planned to undergo alloHCT. MRD will be measured by multi-dimensional flow cytometry with sensitivity of 0.02% (Hematologics). Patients will be enrolled in a modified 3+3 dose escalation design with two cohorts: cohort A) tagraxofusp 12 mcg/kg IV qday on days 1-5; and cohort B) azacitidine 75 mg/m2 subq qday on days 1-5 plus tagraxofusp 12 mcg/kg qday on days 8-12. Patients achieving MRD-negativity after cycle 1 will proceed to alloHCT. If a patient remains MRD-positive, an additional cycle of investigational therapy will be administered. All patients will proceed to alloHCT after no more than 2 cycles of investigational therapy. Both cohorts contain a dose escalation and a dose expansion phase. Cohort B will open if cohort A is not efficacious. Up to 44 participants will be enrolled in this trial at University of California Los Angeles and University of California San Francisco (two constituent sites of the University of California Hematologic Malignancies Consortium). Objectives The primary objective is to estimate the rate of conversion from MRD-positive pre-investigational therapy to MRD-negative after 1 or 2 cycles of investigational therapy. Key secondary objectives include to determine the safety of tagraxofusp +/- azacitidine in this patient population and to evaluate if adverse effects of this investigational therapy lead to delays in alloHCT. Exploratory objectives will involve use of single cell RNA sequencing to investigate if response to investigational therapy is predicted by leukemia-specific biomarkers and to evaluate if putative cell surface markers (inferred from RNA detection) are associated with MRD persistence. Main Outcomes The primary endpoint is MRD status after 1 or 2 cycles of investigational therapy. Key secondary endpoints include dose-limiting toxicities (any grade 4 or 5 non-hematologic toxicity or any grade 3 or greater hematologic toxicity at day 42) and number of days between investigational therapy and alloHCT. Conclusions There is an unmet need to improve outcomes in AML with detectable MRD pre-transplant. The strategy in this study is to employ tagraxofusp as an alternative mechanism of action to eradicate chemo-resistant AML cells responsible for subsequent relapse. In order to mitigate any potential resistance the residual leukemia population may have against tagraxofusp, azacitidine will be added to optimize the likelihood for MRD negativity and the possibility for improved outcomes after alloHCT. This study was sponsored by the ASH CRTI. References Appelbaum J, Loeb A, Gardner K, et al. Additional Cytotoxic Chemotherapy Is Unlikely to Eliminate Measurable Residual Acute Myeloid Leukemia (AML). Blood. 2019;134(Supplement_1):260-260. Lane AA, Sweet KL, Wang ES, et al. Results from Ongoing Phase 1/2 Trial of SL-401 As Consolidation Therapy in Patients with Acute Myeloid Leukemia (AML) in Remission with Minimal Residual Disease (MRD). Blood. 2017;130(Supplement 1):2583-2583. Togami K, Pastika T, Stephansky J, et al. DNA methyltransferase inhibition overcomes diphthamide pathway deficiencies underlying CD123-targeted treatment resistance. J Clin Invest. 2019;129(11):5005-5019. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Tumor-suppressive MEG3 induces microRNA-493-5p expression to reduce arabinocytosine chemoresistance of acute myeloid leukemia cells by downregulating the METTL3/MYC axis

BackgroundChemoresistance serves as a huge obstacle for acute myeloid leukemia (AML) patients. To counteract the chemoresistance in AML cells, we discussed the role of maternally expressed gene 3 (MEG3) in arabinocytosine (AraC) chemoresistance in AML cells.MethodsMEG3, microRNA (miR)-493-5p, methyltransferase-like 3 (METTL3) and MYC expression in AML cells was determined and then their interactions were also analyzed. Then, the viability and apoptosis of AML cells were determined through loss- and gain- function assay. The level of m6A modification in AML cells was examined. AML mouse models were also established to validate the potential roles of MEG3.ResultsMEG3 and miR-493-5p were downregulated in AML cells, and they were lower in resistant cells than in parental cells. MEG3 led to elevated expression of miR-493-5p which targeted METTL3. METTL3 increased expression of MYC by promoting its m6A levels. Overexpression of MEG3 and miR-493-5p or knockdown of METTL3 inhibited HL-60 and Molm13 cell proliferation and promoted their apoptosis. Overexpressed MEG3 induced heightened sensitivity of AML cells to AraC. However, the suppression of miR-493-5p reversed the effects of overexpressed MEG3 on AML cells.ConclusionsCollectively, MEG3 could upregulate miR-493-5p expression and suppress the METTL3/MYC axis through MYC m6A methylation, by which MEG3 promoted the chemosensitivity of AML cells.

Histone methyltransferase EZH2 epigenetically affects CCNA1 expression in acute myeloid leukemia

Cyclin A1 (CCNA1) is an alternative A-type cyclin that is expressed in acute myeloid leukemia (AML). However, its functions in AML cell chemoresistance, an important cause for mortality, are incompletely understood. The purpose of this study was to expound the role and potential mechanism of CCNA1 in AML cell chemoresistance. Upregulation of CCNA1 promoted resistance of AML cells to PKC412, AC220, and AraC. Mechanistically, it was confirmed that CCNA1 transcription was negatively regulated by forkhead box A2 (FOXA2), and the downregulation of FOXA2 promoted chemoresistance in AML cells. Moreover, the promoter sequence of CCNA1 has a significant H3K27me3 modification. Enhancer of zeste homolog 2 (EZH2) enhanced H3K27me3 modification of CCNA1 promoter to inhibit CCNA1 expression, thus promoting sensitivity of AML cells to drugs. Taken together, these findings lead to deeper insights into the mechanism of AML cell chemo-sensitivity by inhibiting CCNA1 at the transcriptional level.

Untargeted Metabolomics Identifies Glutamine Metabolism As a Driver of Chemoresistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is one of the most challenging cancers to treat. Induction chemotherapy (iCT) remains the standard of care, but the incidence of refractory and relapsed AML is high. Unraveling the molecular regulation of this chemoresistance is critical to provide new treatment options for patients. Cellular metabolism plays a central role in the control of cell fate, and a dysregulated metabolism is now widely accepted as a hallmark of many cancers. We hypothesized that chemoresistance in AML arises at the time of maximal iCT response, with the residual cells manifesting distinctive metabolic features that enable their survival under the extreme stress of chemotherapy.To test this hypothesis, we used a mouse model of aggressive, MLL-AF9-driven AML that allows real-time monitoring of leukemic burden through bioluminescence imaging, and therefore identification of the moment of maximal response to iCT. We isolated GFP-expressing AML cells from bone marrow of mice receiving treatment with vehicle or iCT (cytarabine for 5 days and doxorubicin for the first 3 days), both at the moment of maximal response (3 days after last dose of iCT) and after relapse (10 days after last dose). We then developed a platform for untargeted metabolomics analysis of freshly sorted cells, which allows us to measure the levels of more than 670 metabolites using 500,000 cells per sample. Amongst the top hits were multiple metabolites involved in glutamine metabolism, including glutamine, glutamate, aspartate and pyroglutamate. All of these metabolites were low in vehicle-treated cells, very high in the residual cells at the moment of maximal response to iCT, and low again in cells after relapse, suggesting a dynamic role for glutamine metabolism in the immediate stress response to iCT. Analysis of the transcriptomic profile of the cells (RNAseq) showed that expression of the majority of genes encoding for enzymes involved in glutamine metabolism did not differ between groups. In contrast, mRNA levels of several glutamine transporters were increased in chemoresistant AML cells. To confirm that glutamine metabolism plays a functional role in protecting AML cells from chemotherapy, we treated AML cells in culture with iCT in combination with 6-diazo-5-oxo-L-norleucine (DON), a glutamine analog and antagonist that inhibits all glutamine-dependent enzymes. At several concentrations synergism between DON and iCT was observed, proving that activation of glutamine metabolism confers chemoresistance in AML cells.These results highlight the power of using untargeted metabolomics to uncover novel chemoprotective metabolic pathways, and underscore the uniqueness of our approach as glutamine metabolism would not have been picked up through transcriptomics analysis alone. In addition, we identified several currently unknown metabolites, of which levels differed significantly in chemoresistant AML cells, that will be further investigated in future studies. Taken together, our findings provide insight into the metabolic programs that determine chemoresistance in vivo and indicate that targeting glutamine metabolism may provide a basis for overcoming chemoresistance in AML. DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Stromal Cells Induce ALDH+ Stem Cell Phenotype and Chemotherapy Resistance in AML Cells through TGF-β1 Mediated Signaling: Reversal By TGFβ Inhibitor Galunisertib

Bone marrow (BM) microenvironment in acute myeloid leukemia (AML) consists of various cell types including mesenchymal stromal cells (MSCs), osteoprogenitor cells, osteoblasts, osteocytes, endothelial, immune and perivascular reticular cells that support AML cell growth and protect them from chemotherapy. We have recently reported that AML cells alter the BM microenvironment by inducing osteogenic differentiation in MSCs to gain growth advantage in the bone marrow (Battula et al, JCI Insight, 2017). However the stroma-induced functional alterations in AML cells are not well understood. Stromal cells induce chemo-resistance in AML cells and as chemo-resistance and high tumorigenic potential are the characteristics of leukemia stem cells, we hypothesize that stromal cells can induce stem cell characteristics in AML cells. To test our hypothesis we co-cultured AML cell lines including OCI-AML3 and HL60 cells with BM-derived MSCs for 3 or 5 days and determined aldehyde dehydrogenase (ALDH) enzyme activity (a well-established marker of hematopoietic and leukemia stem cells) and gene expression by RNA sequencing. We found that the percentage of ALDH+ cells in OCI-AML3 increased from 7% to 89.1% in 3 days and from 23% to 92.3% in 5 days of co-culture with MSCs. Similarly, ALDHwas increased in HL60 cells from 7% to 68% in 3 days and from 20.7% to 67.9% in 5 days of co-culture with MSCs suggesting that stromal cells can induce certain stem cell features in AML cells.Next, to better determine the mechanism, OCI-AML3 cells were co-cultured with BM-MSCs (isolated from 3 different donors) for 5 days, the OCI-AML3 cells were FACS sorted and gene expression was analyzed by RNA sequencing. Using R statistics we identified differentially expressed genes in OCI-AML3 cells and found that gene sets associated with stem cell function including ALDH2, Wnt5b, Wnt5a were up-regulated whereas inflammatory and innate immune response genes were down-regulated in OCI-AML3 cells co-cultured with MSCs compared to cells cultured alone. These genes were validated by qRT-PCR assay. Ingenuity pathway analysis® (IPA) revealed a TGFβ1 activated gene signature in OCI-AML3 cells co-cultured with MSCs compared to cells culture alone. To investigate the role of TGFβ1 in the stroma-induced stem cell phenotype in AML cells, OCI- AML3 and HL60 cells were treated with recombinant TGFβ1 (5ng/ml) for 3 or 5 days. Indeed, treatment with TGFβ1 induced ALDH positive cells from 27.7% to 89.5% in 3 days and from 3.4% to 92.5% in 5 days (OCI-AML3). Similarly, in HL60 cells treated with TGFβ1, ALDH+ cells increased from 9.8% to 25.1 in 3 days suggesting that TGFβ1 induces a stem cell phenotype in AML cells. Next, we measured levels of TGFβ1 in cell culture supernatants from MSCs by ELISA and found that MSCs secrete TGFβ1 up to 1ng/ml into the surrounding medium. In addition, to study the role of TGFβ1 in stroma-induced stem cell activity in AML cells, we treated AML cells with or without TGFβ1 type-1 receptor kinase inhibitor Galunisertib (LY2157299 monohydrate from Eli Lilly®) in the presence or absence of MSCs. Strikingly, treatment of galunisertib at 1µM completely inhibited MSCs-induced ALDH activity in OCI-AML3 and HL60 cells in 5 day co-culture setting. To determine if galunisertib sensitizes AML cells to chemotherapy in the presence of MSCs, we treated OCI-AML3 with galunisertib (1µM) or cytarabine (1µM) or in combination for 72hrs in the presence or absence of MSCs. As expected presence of MSCs inhibited cytarabine induced cell killing (positive for annexin-V and DAPI) from 47.1% to 28.3% cells. In turn, the combination of cytarabine and galunisertib negated this affect and induced cell killing in OCI-AML3 cells from 28% to 54% even in the presence of MSCs suggesting complete loss of MSC-mediated chemo-protection in OCI-AML3 cells upon inhibition of TGF-β signaling. In-vivo testing of galunisertib in combination with cytarabine and doxorubicin in AML PDX models in NSG mice is currently ongoing.Collectively, data indicate that stromal cells induce chemo-resistance in AML cell by inducing stem cells phenotype through TGF-beta mediated signaling (Fig 1). TGF-β inhibition by galunisertib, an agent in early clinical trials in solid tumors inhibits stem cell phenotype and BM microenvironment-induced chemo-resistance and facilitates cell killing of AML cells. [Display omitted] DisclosuresBattula:United Therapeutics Corporation: Research Funding.

Epigenetic Drugs Selectively Target a Population of AML Cells Which Are Positive for CD123 Cell Surface Marker and Are Chemoresistant

Acute Myeloid leukemia (AML) is the most common acute leukemia in adults. While Cytarabine (Ara-C) based chemotherapy has been a standard for initial treatment, over 50% of patients will eventually relapse within a period of time. Chemoresistance remains a major burden for treatment of relapse patient. Recent studies suggest that AML relapse is caused by the survival of drug resistant leukemia stem cell (LSC) population. Since LSCs are in low frequency and remain quiescent in patients, it is difficult to study the chemoresistant mechanism on this population of cells. It has been previously shown that cell lines that derived from patient sample might be considered as attractive models aimed to better characterize LSC biological properties and their chemotherapy resistance mechanisms. Multiple LSC cell surface markers has been identified in patient sample and cell lines. However, it remains to be determined whether subpopulations with different LSC markers has similar level of chemoresistance.To better understand the mechanism of drug resistance and whether it is correlated to the expression of certain group of LSC surface markers, we collected various human AML cell lines and examined correlations of their IC50 of Ara-C and percentage of individual LSC surface markers in these cell lines. We found that CD34 surface marker is not correlated to chemoresistance. Other LSC surface markers, such as CD13, CD44, CD117, CD96 and CD45RA are also not correlated to chemoresistance. Interestingly, CD47 (R square = 0.6660, P value = 0.0476) and CD 123 (R square = 0.7421, P value = 0.0275) are positively correlated to chemoresistance. In order to rule out heterogeneous genetic background contribute to chemoresistance of cell lines, we generated a drug resistant cell line from an drug sensitive AML cell line, OCI-AML2, by gradually increased the Ara-C concentration in culture media. During the selection, the percentage of cells with CD47 and CD123 gradually increased. When the resistant cell line is established, the percent of CD123 positive cells are over 90 percent. Through cell tracking experiments, we found that resistant cells grow at least two cycles slower than sensitive cells in a 5 day tracking assay. RNAseq analysis shows that genes related to cell cycle regulator and resistance of apoptosis changed significantly. However, genes that related to stemness, such as Oct4, Nanog, KLF4, Myc, FLT3, are no significant changed in drug resistant cells compare with the sensitive line, although Sox2 increased 50% and ALDH1A3 increased 3 folds. Therefore, chemoresistant cells may represent cells in a slow growing, de-differentiate stage, however, it may not bare all characteristics of a LSC.We further investigate the role of epigenetic factors in regulating the survival of chemoresistant leukemia cells. Our data shows epigenetic drugs that targeting histone deacetylases, bromodomain proteins and PRC1 complex are effective to induce apoptosis and cell death of chemoresistant cells, therefore they may be potential second line of chemotherapy drug for chemoresistant patients. Histone deacetylase inhibitor Romidepsin efficiently cleared chemoresistant AML blast in xenographed NSG mice. Furthermore, we found Romidepsin has synergetic effect when combine with Ara-C. Interestingly, Romedepsin preferentially target CD123 positive, slow growing cells while Ara-C mainly target fast growing, CD123 negative cells. Therefore, combine treatment with Romedepsin and Ara-C may prevent the development of chemoresistance in AML cells. In summary, we identify CD123 and CD47cell surface markers may potentially serve as biomarker for chemresistance AML cells. Our study also sheds light on a new mechanism of drug resistance in leukemia, and provide a rationale to develop and test epigenetic-targeted therapies in leukemia, especially in drug resistant relapse patients. DisclosuresNo relevant conflicts of interest to declare.

MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin.

Anthracyclines remain a cornerstone of induction chemotherapy for acute myeloid leukemia (AML). Refractory or relapsed disease due to chemotherapy resistance is a major obstacle in AML management. MicroRNAs (miRNAs) have been observed to be involved in chemoresistance. We previously observed that miR-15a-5p was overexpressed in a subgroup of chemoresistant cytogenetically normal AML patients compared with chemosensitive patients treated with daunorubicin and cytarabine. MiR-15a-5p overexpression in AML cells reduced apoptosis induced by both drugs in vitro. This study aimed to elucidate the mechanisms by which miR-15a-5p contributes to daunorubicin resistance. We showed that daunorubicin induced autophagy in myeloid cell lines. The inhibition of autophagy reduced cell sensitivity to daunorubicin. The overexpression of miR-15a-5p decreased daunorubicin-induced autophagy. Conversely, the downregulation of miR-15a-5p increased daunorubicin-induced autophagy. We found that miR-15a-5p targeted four genes involved in autophagy, namely ATG9a, ATG14, GABARAPL1 and SMPD1. Daunorubicin increased the expression of these four genes, and miR-15a-5p counteracted this regulation. Inhibition experiments with the four target genes showed the functional effect of miR-15a-5p on autophagy. In summary, our results indicated that miR-15a-5p induces chemoresistance in AML cells through the abrogation of daunorubicin-induced autophagy, suggesting that miR-15a-5p could be a promising therapeutic target for chemoresistant AML patients.

CircNPM1 strengthens Adriamycin resistance in acute myeloid leukemia by mediating the miR-345-5p/FZD5 pathway

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with poor long-term outcomes. Numerous studies claim that circular RNAs (circRNAs) are important regulators in AML progression. This study intended to explore the role of circNPM1 in AML development and drug chemoresistance. The expression of circNPM1 and miR-345-5p was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cellular activities, including cell growth, apoptosis, cell cycle, migration and invasion, were monitored using colony formation assay, flow cytometry assay and transwell assay, respectively. The relationship between miR-345-5p and circNPM1 or Frizzled-5 (FZD5) was predicted by the bioinformatics tool starBase and validated by dual-luciferase reporter assay or RNA immunoprecipitation (RIP) assay. CircNPM1 was abundantly expressed in serum samples from AML patients and AML cell lines. CircNPM1 silence or miR-345-5p restoration repressed colony formation, cell migration and invasion, contributed to cell apoptosis and cell cycle arrest, and weakened Adriamycin (ADM) resistance of AML cells. MiR-345-5p was a target of circNPM1 and was downregulated in AML serum and cells. MiR-345-5p deficiency reversed the effects of circNPM1 silence. Further, FZD5 was targeted by miR-345-5p, and circNPM1 regulated FZD5 expression by adsorbing miR-345-5p. FZD5 overexpression could block the function of miR-345-5p restoration. CircNPM1 might be a vital regulator for ADM chemoresistance in AML cells, which partly depended on the role of the miR-345-5p/FZD5 axis. Our study presents the view that circNPM1 degradation may be a key strategy in AML resistance therapy.

Stromal cells promote chemoresistance of acute myeloid leukemia cells via activation of the IL-6/STAT3/OXPHOS axis.

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.

PRKD2 Promotes Progression and Chemoresistance of AML via Regulating Notch1 Pathway.

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.

The Distinctive Metabolic Environment of the Bone Marrow Niche Drives Leukemia Chemoresistance

Chemoresistance is the difference between remission and cure in cancer patients and is particularly evident in acute myeloid leukemia (AML) where complete remissions are common, but few are cured. Genetic analysis in patients points to residual founder clones persisting through therapy and ultimately causing relapse. However, most of these genetic alterations are currently not amenable to targeted therapies. We hypothesized that residual chemoresistant cells must pass through extreme metabolic challenges when under selection from chemotherapy and surrounded by massive cell death. Our goal was to define the distinctive metabolic profile of AML cells as they pass through that time of extreme selection pressure and determine whether metabolic pathway manipulation would reveal an exploitable vulnerability for therapy. We used in vivo imaging to define the kinetics of leukemic growth, response to chemotherapy and relapse in the mouse. We reasoned that the moment of maximal response immediately following chemotherapy represents a time when the metabolic milieu is maximally hostile and therefore a distinctive selection moment that the cells ultimately causing relapse must pass through. We captured cells at that time and conducted unbiased metabolic profiling, which revealed a metabolite profile that was distinct from AML cells during pre-chemo growth or late post-chemo relapse. Metabolite enrichment analysis revealed hyperactivation of glutamine metabolism during the extreme selection period, with chemoresistant cells showing high levels of glutamine, glutamate and aspartate. Inhibition of glutamine metabolism using the glutamine analog 6-diazo-5-oxo-L-norleucine specifically during the window of maximal selection pressure reduced the number of chemoresistant cells and improved survival in mouse models of AML. Unexpectedly, we found that residual AML cells were not sensitive to inhibition of the enzyme glutaminase, a target currently under clinical investigation for AML. In vivo metabolic tracer analysis instead revealed that chemoresistant AML cells mainly depend on glutamine nitrogen to fuel pyrimidine synthesis. Aspartate, another essential metabolite for pyrimidine synthesis, can be made from glutamine through the Krebs cycle. Interestingly, we detected almost no glutamine carbon entering the Krebs cycle in vivo even though labeled aspartate was found, indicating a different aspartate source. We found that aspartate levels in peripheral blood plasma are very low compared to those in bone marrow plasma, and explored the possibility of a local bone marrow source. Single cell RNA sequencing of bone marrow stromal cells (BMSC) revealed that CXCL12-positive BMSC express high levels of aspartate synthesis genes and transporters, which further increased in the presence of AML cells. In vitro co-cultures showed that BMSC convert glutamine to aspartate, and transfer this to AML cells. Genetic inhibition of aspartate synthesis in BMSC sensitized AML cells to chemotherapy, showing the importance of this metabolic crosstalk. We propose that induction of a timed metabolic collapse targeting AML cells both directly and indirectly through the bone marrow niche can prevent development of chemoresistance and improve treatment outcomes. Figure Disclosures Scadden: Novartis: Other: Sponsored research; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Equity Ownership; Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fog Pharma: Consultancy; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Mitochondrial Oxphos As Survival Mechanism of Minimal Residual AML Cells after Induction Chemotherapy : Survival Benefit By Complex I Inhibition with Iacs-010759

Mitochondrial Oxphos As Survival Mechanism of Minimal Residual AML Cells after Induction Chemotherapy : Survival Benefit By Complex I Inhibition with Iacs-010759

Targeting Myeloperoxidase Disrupts Mitochondrial Redox Balance and Overcomes Cytarabine Resistance in Human Acute Myeloid Leukemia.

Chemotherapies alter cellular redox balance and reactive oxygen species (ROS) content. Recent studies have reported that chemoresistant cells have an increased oxidative state in hematologic malignancies. In this study, we demonstrated that chemoresistant acute myeloid leukemia (AML) cells had a lower level of mitochondrial and cytosolic ROS in response to cytarabine (AraC) and overexpressed myeloperoxidase (MPO), a heme protein that converts hydrogen peroxide to hypochlorous acid (HOCl), compared with sensitive AML cells. High MPO-expressing AML cells were less sensitive to AraC in vitro and in vivo. They also produced higher levels of HOCl and exhibited an increased rate of mitochondrial oxygen consumption when compared with low MPO-expressing AML cells. Targeting MPO expression or enzyme activity sensitized AML cells to AraC treatment by triggering oxidative damage and sustaining oxidative stress, particularly in high MPO-expressing AML cells. This sensitization stemmed from mitochondrial superoxide accumulation, which impaired oxidative phosphorylation and cellular energetic balance, driving apoptotic death and selective eradication of chemoresistant AML cells in vitro and in vivo. Altogether, this study uncovers a noncanonical function of MPO enzyme in maintaining redox balance and mitochondrial energetic metabolism, therefore affecting downstream pathways involved in AML chemoresistance. SIGNIFICANCE: These findings demonstrate the role of myeloperoxidase in the regulation of ROS levels and sensitivity of AML cells to cytarabine, an essential chemotherapeutic backbone in the therapy of AML.