Protect Acute Myeloid Leukemia Cells Research Articles

CircFAM193B interaction with PRMT6 regulates AML leukemia stem cells chemoresistance through altering the oxidative metabolism and lipid peroxidation.

Most forms of chemotherapy for acute myeloid leukemia (AML) are often ineffective in eliminating leukemic stem cells (LSCs), as their underlying mechanisms remain unclear. Here, we have identified circFAM193B, which regulates the redox biology of LSCs and is associated with unfavorable outcomes in AML patients. In vitro and in vivo assays suggested that circFAM193B significantly inhibits LSCs chemotherapy resistance and AML progression. Knockdown circFAM193B enhances mitochondrial OXPHOS function and inhibits the accumulation of reactive oxygen species and lipid peroxidation mediated by chemotherapy, which protects AML cells from oxidative stress-induced cell death. Mechanistically, circFAM193B physically interacts with arginine methyltransferase PRMT6 catalytic domain and enhances the transcription efficiency of key lipid peroxidation factor ALOX15 by decreasing H3R2me2a modification. In summary, we have identified circFAM193B was downregulated in LSCs to promote the survival of LSC by modulating energy metabolism and the redox balance in the postchemotherapy persistence of LSC. Our studies provide a conceptual advance and biological insights regarding the drug resistance of LSCs via circRNA mediated PRMT6-deposited methylarginine signaling.

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.

Type 3 Innate Lymphoid Cells Promote Acute Myeloid Leukemia Immune Evasion and Expansion

Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy originated in the bone marrow with poor overall survival rates of less than 30%. The immune system has been shown to play an important role in detecting and eliminating leukemic cells. Recent studies have described a population of immune cells termed Innate lymphoid cells (ILCs). ILCs are known to regulate both innate and adaptive immunity in an antigen-independent manner and are believed to be among the first immune cells to interact with malignantly transformed cells. Natural killer (NK) cells are a subset of ILCs and are known to inhibit AML progression, however the impact of other ILCs is not yet fully understood. Type 3 ILCs (ILC3s) have key roles in maintaining homeostasis in mucosal immunity through their ability to secrete cytokines including IL-17, IL-22, TNFα and GM-CSF (pro-myeloid). An expansion of ILC3s has recently been observed in solid tumors, however the overall functional impact of ILC3s in the setting of AML remains poorly understood and warrants further investigation. We first utilized an MLL-partial tandem duplication and FLT3-internal tandem duplication Flt3-ITD double knock-in mutation murine model of AML that recapitulates two clinically relevant mutations in human AML. Using this AML model, we discovered an expansion of ILC3s in the bone marrow of leukemic mice compared to wild-type control mice (2.364% +/- 0.40% AML vs 0.045% +/- 0.40% WT, n=5, p<0.0001). We also observed an increase in ILC3s in the bone marrow of untreated AML patients relative to age matched donor controls (7.013% +/- 2.6% vs. 2.328% +/- 2.6%). Our published data have previously shown AML blasts secrete aryl hydrocarbon receptor (AHR) ligands, which can bind and activate the transcription factor AHR in ILC precursors and mature ILCs. AHR is an important transcription factor in ILC3 development. Therefore, we hypothesized that the ILC3 increase was due at least in part to AHR-mediated increased ILC3 differentiation. To address this hypothesis, we isolated the ILC precursor (ILCP) from normal human donors and co-cultured with AML cells. By regulating AHR activity in this system, we showed that ILC3s are expanded in AML in part due to AML-mediated AHR activation (28.02% +/- 3.89% without AML, 60% +/- 5.5% with AML and 27% +/- 5% AML+AHR inhibitor, n=15, p<0.001). Next, we sought to determine the direct impact of ILC3s on AML proliferation and differentiation that could contribute to AML relapse. We utilized a colony forming assay (CFU) assay in the presence or absence of ILC3s. Bone marrow from primary untreated AML patients was cultured alone or with ILC3s (Lin-CD94-NKp44+). AML samples co-cultured with ILC3s had significantly increased colony formation relative to AML alone (69.4 CFU AML+ILC3 vs 42.7 CFU AML alone, n=8, p<0.05). Post-culture, we observed significant increases in the transcript of GM-CSF and TNFα in ILC3s co-cultured with AML marrow as compared to ILC3s alone. Furthermore, in our immunocompetent murine AML model, we observed significantly higher production of GM-CSF and TNFα by ILC3s in the bone marrow of leukemic mice relative to nonleukemic controls (GM-CSF: 11.08% +/- 1.6 AML vs 1.128% +/- 1.6 WT, n=10, p<0.0001; TNFα: 5.456% +/- 1.3 AML vs 1.307% +/- 1.3 WT, n=10, p<0.05), suggesting a potential mechanism of ILC3-mediated increased colony formation. Lastly, to determine if ILC3 expansion impacted NK cell immune surveillance, we co-cultured normal donor human ILC3s with AML cells, separated by transwell, for 48 hours before subjecting the AML cells to an NK cell cytotoxicity assay. We observed significantly reduced NK cell killing of AML cells when co-cultured with ILC3s compared to controls, suggesting ILC3s protect AML cells from NK cell-mediated killing (24.3% killing +/- 3.81% AML alone vs 14.57% +/- 1.35% AML with ILC3, n=18, p<0.01). Collectively, these data support a model in which AML-mediated AHR activation promotes ILC3 expansion to both support AML proliferation and protect AML blasts from immune surveillance. Future studies will focus in exploring how to target these interactions to design effective therapeutic approaches and improve patient clinical outcomes.

Dexamethasone enhances venetoclax-induced apoptosis in acute myeloid leukemia cells.

Acute myeloid leukemia (AML) therapies have been significantly improved by the development of medicines that can target BCL-2. On the other hand, non-recurrent alterations in oncogenic pathways and gene expression patterns have already been linked to therapeutic resistance to venetoclax therapy. Bone marrow mesenchymal stromal cells (BM-MSCs) support leukemic cells in preventing chemotherapy-induced apoptosis by mitochondrial transfer in leukemic microenvironment. In this study, we investigated the enhancement of the antitumor effect of BCL-2 inhibitor venetoclax by dexamethasone. In particular, dexamethasone had no significant effect on the viability of AML cells, but dexamethasone combined with venetoclax could significantly increase the apoptosis of AML cells induced by venetoclax. When AML cells were co-cultured with BM-MSCs, dexamethasone combined with venetoclax showed additional anti-tumor effect compared to venetoclax alone. Venetoclax increased reactive oxygen species level in co-cultured AML cells, contributed to transfer more mitochondria from BM-MSCs to AML cells and protect AML cells from apoptosis. Dexamethasone combined with venetoclax induced more apoptosis, but dexamethasone reduced the venetoclax-induced reactive oxygen species level in AML cells and reduced the transfer of mitochondria from BM-MSCs to AML cells. This may lead to a diminished protective effect of BM-MSCs on AML cells. Together, our findings indicated that venetoclax in combination with dexamethasone could be a promising therapy in AML.

Immune-related lncRNAs pairs prognostic score model for prediction of survival in acute myeloid leukemia patients

Acute myeloid leukemia (AML) is one of the most common malignant and aggressive hematologic tumors, and risk stratification is indispensable to ensure proper treatment. But immune-related long noncoding RNAs (ir-lncRNAs) pairs prognostic risk models used to stratify AML have yet to be reported. In this study, we established a prognostic risk model based on eight ir-lncRNAs pairs using LASSO-penalized Cox regression analysis and successfully validated the model in an independent cohort. According to risk scores, patients were divided into a high-risk group and a low-risk group. High-risk patients presented more tumor mutation frequency and higher expression of human leukocyte antigen (HLA)-related genes and immune checkpoint molecules. Gene Set Enrichment Analysis (GSEA) indicated that the transforming growth factors β (TGFβ) pathway was activated in the high-risk group; meanwhile, we found that TGFβ1 mRNA levels were significantly elevated in AML patients and correlated with poor prognosis, which is closely related to drug resistance. Consistently, in vitro studies found that exogenous TGFβ1 can protect AML cells from chemotherapy-induced apoptosis. Collectively, we developed an ir-lncRNA prognostic model that helps predict the prognosis of AML patients and provides valuable information about their response to immune checkpoint inhibitors, and we found that increased TGFβ1 levels resulting in chemoresistance may be one of the leading causes of treatment failure in high-risk AML patients.

Lycorine induces apoptosis of acute myeloid leukemia cells and inhibits triglyceride production via binding and targeting FABP5.

Acute myeloid leukemia (AML) is the most common hematopoietic malignancy with abnormal lipid metabolism. However, currently available information on the involvement of the alterations in lipid metabolism in AML development is limited. In this study, we demonstrate that FABP5 expression facilitates AML cell viability, protects AML cells from apoptosis, and maintains triglyceride production. Our bioinformatics analysis revealed that FABP5 expression was upregulated and correlated with unfavorable overall survival of AML patients. FABP5 expression may be used to distinguish normal and AML with high accuracy. FABP5-based risk score was an independent risk factor for AML patients. AML patients with highly expressed FABP5 predicted resistance to drugs. In vitro study showed that FABP5 expression was remarkably elevated in primary AML blasts and an AML cell line. Silencing FABP5 expression attenuated AML cell viability, reduced triglyceride production and lipid droplet accumulation, and induced apoptosis. We utilized AutoDock online tool to identify lycorine as an FABP5 inhibitor by binding FABP5 at amino acid residues Ile54, Thr56, Thr63, and Arg109. Lycorine treatment downregulated the expression levels of FABP5 and its target PPARγ, impaired AML cell viability, triggered apoptosis, and reduced triglyceride production in AML cells. These results demonstrate that FABP5 is critical for AML cell survival and highlight a novel metabolic vulnerability for AML.

Pyruvate Dehydrogenase Inhibition Leads to Decreased Glycolysis, Increased Reliance on Gluconeogenesis and Alternative Sources of Acetyl-CoA in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive disease characterized by poor outcomes and therapy resistance. Devimistat is a novel agent that inhibits pyruvate dehydrogenase complex (PDH). A phase III clinical trial in AML patients combining devimistat and chemotherapy was terminated for futility, suggesting AML cells were able to circumvent the metabolic inhibition of devimistat. The means by which AML cells resist PDH inhibition is unknown. AML cell lines treated with devimistat or deleted for the essential PDH subunit, PDHA, showed a decrease in glycolysis and decreased glucose uptake due to a reduction of the glucose transporter GLUT1 and hexokinase II. Both devimistat-treated and PDHA knockout cells displayed increased sensitivity to 2-deoxyglucose, demonstrating reliance on residual glycolysis. The rate limiting gluconeogenic enzyme phosphoenolpyruvate carboxykinase 2 (PCK2) was significantly upregulated in devimistat-treated cells, and its inhibition increased sensitivity to devimistat. The gluconeogenic amino acids glutamine and asparagine protected AML cells from devimistat. Non-glycolytic sources of acetyl-CoA were also important with fatty acid oxidation, ATP citrate lyase (ACLY) and acyl-CoA synthetase short chain family member 2 (ACSS2) contributing to resistance. Finally, devimistat reduced fatty acid synthase (FASN) activity. Taken together, this suggests that AML cells compensate for PDH and glycolysis inhibition by gluconeogenesis for maintenance of essential glycolytic intermediates and fatty acid oxidation, ACLY and ACSS2 for non-glycolytic production of acetyl-CoA. Strategies to target these escape pathways should be explored in AML.

CircZBTB46 Protects Acute Myeloid Leukemia Cells from Ferroptotic Cell Death by Upregulating SCD.

Circular RNAs (circRNAs) have been shown to be closely linked to the tumorigenesis and treatment response of hematological malignancies. However, the biological functions and clinical implications of circRNAs in acute myeloid leukemia (AML) remain largely unknown. CircRNA microarray datasets were analyzed to screen differentially expressed circRNAs in AML patients. It was found that circZBTB46 was significantly upregulated in AML patients and AML cells. Moreover, the expression of circZBTB46 was associated with the stages of AML patients and showed high sensitivity and specificity for diagnosing AML. Silencing of circZBTB46 inhibited AML cell proliferation and induced cell cycle arrest. Importantly, the depletion of circZBTB46 notably increased ferroptosis and enhanced RSL3-induced ferroptosis in AML cells. Mechanistically, circZBTB46 upregulated the expression of stearoyl-CoA desaturase 1 (SCD) possibly by acting as a miRNA sponge. Finally, the circZBTB46 knockdown repressed the tumor growth of AML in vivo. In conclusion, circZBTB46 protects AML cells from ferroptosis and promotes the proliferation by upregulating SCD, thus suggesting that circZBTB46 may be a potential therapeutic target for AML.

Mesenchymal Stromal Cells Protect Acute Myeloid Leukemia Cells from Venetoclax-Induced Apoptosis through Contact-Dependent Mechanisms

Acute myeloid leukemia (AML) patients have a poor prognosis with standard treatments due to a high relapse rate. Recently, Venetoclax (VEN), the first B-cell lymphoma 2 (BCL-2) selective inhibitor to enter the clinic, has shown a breaking-through effect in combination with demethylating agents in older patients with treatment-naïve AML. However, resistance mechanisms were described, hampering the complete remission of VEN-treated patients. It has been proposed that the bone marrow microenvironment in general and Mesenchymal stromal cells (MSCs), in particular, favor a protective niche that sustains leukemic cell viability and plays a crucial role indetermining drug sensitivity in several hematological disorders, including AML. A previous study demonstrated that VEN, as well as other drugs commonly used in AML therapy, showed a reduced effect in the presence of an MSC-conditioned medium, suggesting that MSC-dependent release of inflammatory signals and other soluble factors could be involved in drug resistance. Our study aims to further investigate the mechanisms of MSC-mediated VEN resistance in AML. We found that primary AML cells isolated from patients at diagnosis and treated with VEN showed different apoptotic rates, indicating an AML cell-specific resistance/sensitivity to the drug. In co-culture experiments, primary MSCs, isolated from healthy donors or AML patients, protected VEN-sensitive AML cells from apoptosis, confirming the critical role of MSCs in drug resistance. To deeply investigate this issue, we performed co-culture experiments using the AML cell line MV4-11, which we demonstrated as sensitive to VEN in a dose-dependent manner. We found that MSCs significantly reduced MV4-11 apoptosis after VEN exposure. The addiction of transwells to the co-culture system abrogated the MSC-protective effect on VEN-induced apoptosis, suggesting the establishment in our setting of a complex interplay between AML cells and MSCs, not only mediated by soluble factors as already demonstrated but also involving direct cell-contact. Furthermore, when we cultured MV4-11 cells in direct contact with MSCs, we were able to distinguish, among leukemic cells, a cell population that remained floating in the supernatant and a cell population that kept adherent to MSCs and could be collected by trypsin detachment only. Interestingly, MSCs protected the adherent but not the floating cells from VEN-induced apoptosis, suggesting a central role of a close adhesion in the MSC-driven resistance to VEN. To gain insight into this mechanism, we sorted adherent vs. floating cells and analyzed and compared Gene Expression Profiling (GEP). Interestingly, the principal component analysis suggested that the floating cell GEP was more similar to the GEP of MV4-11 cells cultured without MSCs compared to adherent cell GEP. We found 159 differential expressed genes (DEGs) in adherent vs. floating MV4-11 cells (FC| ≥ 2 and P ≤ 0.05, according to Transcriptome Analysis Console software guidelines). Enrichment analysis revealed that DEGs were involved in cell adhesion migration and proliferation pathways. Among DEGs, we found that CD90/Thy-1, an adhesion molecule whose expression correlated with unfavorable karyotypes and shorter survival in AML, was significantly up-regulated in adherent MV4-11 cells. Flow cytometry analysis confirmed that the adherent compared to the floating MV4-11 cells, showed a higher expression of CD90 as well as CXCR4 as expected. Our study demonstrated that MSCs protect AML cells from VEN-induced apoptosis through a complex mechanism involving tight adhesion-mediated pathways. Further clarification of these interactions can shed light on putative targets to counteract MSC-mediated drug resistance to VEN treatment.

AML-250 Mitochondrial Respiration Regulates GPX4 Inhibition-Induced Ferroptosis in Acute Myeloid Leukemia

Ferroptosis, a form of non-apoptotic cell death regulated by iron-dependent lipid peroxidation, has drawn extensive attention as a potential anti-cancer strategy. However, it remains to be explored in hematologic malignancies. We here investigate the molecular mechanisms of ferroptosis in acute myeloid leukemia (AML) and its therapeutic potential of co-targeting mitochondrial respiration. Analyses of publicly available databases (TCGA, DepMap) showed that AML patients with higher mRNA expression of major anti-ferroptotic genes had significantly shorter survival and that leukemia cells are one of the cell types that are highly dependent on GPX4 among other cancers. This suggests the potential prognostic impact and relevance in therapeutics of this pathway in AML. Indeed, pharmacological inhibition or genetic knockdown of a well-established anti-ferroptosis regulator GPX4 induced profound ferroptosis, evidenced by dependency on iron and lipid peroxidation. Importantly, the induced cell death was agonistic of TP53 mutational status. As a potentially AML-specific mechanism that is unusual in solid tumor models, we found that GPX4 inhibition-induced ferroptosis was efficiently blocked by the mitochondria-targeted ubiquinone MitoQ in AML cells. Ubiquinone is an endogenous antioxidant protecting cells from lipid peroxidation, and in the mitochondria, the respiratory chain is essential for the recycling of ubiquinone. Thus, we hypothesized that mitochondria protect AML cells from ferroptosis by activating the ubiquinone cycle. Consistently, mitochondrial DNA-depleted HL60 Rho0 cells, which lack mitochondrial respiration, were more sensitive to ML210 compared to the parental cells. To translate this finding to a pharmacological approach, we utilized the imipridone ONC201, which hyperactivates mitochondrial protease ClpP to degrade mitochondrial proteins, including the respiratory chain complex, and exerts cancer-selective lethality (Ishizawa et al. Cancer Cell 2019). Indeed, the combination of ONC201 and ML210 resulted in synergistic anti-leukemia effects in primary AML cells. The combinatorial effect was also validated by utilizing genetically engineered AML cells (genetic knockdown of GPX4 or overexpression of hyperactivated mutant ClpP). GPX4 inhibition induces ferroptosis involving mitochondrial redox machinery in AML. Combinatorial targeting of mitochondrial respiration with GPX4 inhibition exerts synergistic anti-leukemia effects. Further studies are in progress to assess the molecular mechanisms and the in-vivo efficacy of the combinatorial treatments.

Exosomes from bone marrow mesenchymal stem cells decrease chemosensitivity of acute myeloid leukemia cells via delivering miR-10a

Bone marrow mesenchymal stem cells (BMSCs) are an integral part of the acute myeloid leukemia (AML) bone marrow microenvironment and contribute to AML progression. In this study, we explored the communication between BMSCs and AML cells via exosomes. The AML cells co-cultured with BMSCs-Exos were found to have lower chemosensitivity exposed to cytarabine, suggesting that BMSCs-Exos could protect AML cells from cytarabine. Interestingly, miR-10a was elevated in BMSCs-Exos derived from AML (AML-BMSCs-Exos) compared with that from healthy donor. The expression levels of miR-10a in AML cells was significantly up-regulated after co-culture with BMSCs-Exos. Furthermore, the up-regulated miR-10a was an crucial factor contributing to the chemoresistance of leukemia cells. Down-regulation of miR-10a substantially increase chemosensitivity of AML cells treated with BMSCs-Exos. Chemosensitivity of AML cells was also decreased through down-regulating RPRD1A by miR-10a that ultimately lead to the stimulation of the Wnt/β-catenin signaling pathway. Collectively, our findings demonstrated that AML-BMSCs could deliver miR-10a to AML cells via exosomes, which could target RPRD1A and activate Wnt/β-catenin signaling pathway that subsequently decreased chemosensitivity of AML cells.

CCL5 Mediates Target-Kinase Independent Resistance to FLT3 Inhibitors in FLT3-ITD-Positive AML

FLT3-ITD tyrosine kinase inhibitors (TKI) show limited clinical activity in acute myeloid leukemia (AML) due to emerging resistance. However, only in a small number of cases, secondary FLT3 mutations can be identified that mediate the resistance. Therefore, other mechanisms must be active. We hypothesized that soluble factors such as cytokines might protect AML cells from TKI mediated cell death and thus contribute to treatment resistance.For this analysis, we generated PKC412- and sorafenib-resistant MOLM-13 cell lines as an in vitro model to study resistance to FLT3-ITD TKIs in human AML. In two out of five PKC412-resistant cell lines, cytokine arrays, ELISAs and quantitative PCR revealed an up to 5-fold overexpression of CCL5/RANTES in cell supernatants as well as on mRNA-level compared to PKC412 sensitive MOLM-13 cells. Notably, via MTS-assays and flow cytometry analysis, we were able to show that both short- and longterm CCL5-treatment mediated resistance against PKC412 to TKI sensitive MOLM-13 cells. In resistant MOLM-13 cell lines with high CCL5-release, CCL5-treatment downregulated the main CCL5-receptor CCR5, and these cell lines showed the lowest chemotaxis index among PKC412-resistant cell lines when migrating against CCL5 as well as CXCL12/SDF-1α. In addition, regulation of CXCR4, which is present in a complex together with CCR5 seems to be also dependant from the CCL5-level, since in the PKC412-resistant cell lines with high CCL5-release, we found CXCR4 to be downregulated on protein level. Moreover, in an ELISA we could show, that these cells secrete almost no SDF-1α, which is the known CXCR4 ligand. Finally, we analyzed isolatedblasts from patients with FLT3-mutated AML before and after treatment with FLT3 inhibitors for changes in CCL5 mRNA expression. Interestingly, isolated AML blasts revealed increased CCL5 transcripts after PKC412-treatment in three cases tested compared to controls. These observations suggest that CCL5 may be an important factor mediating resistance to FLT3 inhibitors in FLT3-mutated AML and could possibly serve as a biomarker to predict resistance to treatment. Disclosuresvon Bubnoff:BMS: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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.

Tipifarnib enhances eradication of acute myeloid leukemia by altering CXCL12/CXCR4 signaling in AML and by modifying the bone marrow microenvironment

Abstract The prognosis of acute myeloid leukemia (AML) remains poor in part due to the leukemic bone marrow microenvironment. Our lab has found that CXCL12, a chemokine abundant within the leukemic bone marrow microenvironment, induces apoptosis of AML cells expressing CXCR4, the receptor for CXCL12. However, this CXCL12/CXCR4-induced apoptosis is inhibited by differentiating osteoblasts, which protect AML cells from apoptosis in the bone marrow. Tipifarnib is a farnesyltransferase inhibitor shown to increase progression-free survival in AML patients that express high levels of CXCL12. Here, we report that tipifarnib inhibits the CXCL12/CXCR4-directed migration of AML cells via an ERK independent pathway. Furthermore, tipifarnib enhances CXCL12/CXCR4-mediated AML cell apoptosis via a mechanism that alters expression of apoptosis-regulating proteins. In addition, tipifarnib disrupts AML protection by osteoblasts, increasing AML cell apoptosis. Tipifarnib inhibits the osteoblast-mediated protection of AML cells via disrupting COL1A1 and TNAP, proteins essential for extracellular matrix production. In conclusion, tipifarnib alters the bone marrow microenvironment which is predicted to enhance eradication of AML via inhibiting CXCL12/CXCR4 directed cellular migration of AML cells, reducing the protective effects of differentiating osteoblasts by disrupting matrix protection proteins, and increasing CXCL12/CXCR4-mediated apoptosis.

Expression and role of fibroblast activation protein α in acute myeloid leukemia.

Currently, the prognosis of acute myeloid leukemia (AML) is poor. In the AML microenvironment, bone marrow (BM) mesenchymal stem cells (BMMSCs) serve an important role in protecting AML cells from chemotherapy-induced apoptosis. The present study aimed to evaluate the expression of fibroblast activation protein α (FAPα) in BMMSCs and BM biopsy samples via flow cytometry, reverse transcription-quantitative PCR and immunohistochemistry, as well as to identify the correlation between the expression of FAPα in BM with clinical parameters and survival of newly diagnosed patients with AML. Subsequently, the protective effect of FAPα on Cytosine arabinoside (Ara-C)-induced apoptosis in Kasumi-1 cells was investigated via small interfering (si)RNA, and its underlying mechanism was examined by western blotting. The results demonstrated significant differences in FAPα expression in BMMSCs and BM biopsy samples between patients with AML and healthy donors. Furthermore, BMMSCs protected Ara-C-induced Kasumi-1 cells from apoptosis, and knockdown of FAPα using siRNA decreased this protection. It was found that Kasumi-1 cells expressed β-catenin, which could be inhibited by Ara-C, and β-catenin expression was significantly activated when co-cultured with BMMSCs, even in the presence of Ara-C. Knockdown of FAPα with siRNA significantly suppressed the expression of β-catenin. The present results indicated that FAPα serves an important role in the AML BM microenvironment, and that increased expression of FAPα in BM may be a poor prognostic factor in patients with AML. Moreover, the current findings demonstrated that BMMSCs protected AML cells from apoptosis, which was in part contributed by FAPα, and may occur via the β-catenin signaling pathway.

Nicotinamide Phosphoribosyltransferase Inhibitors Induce Apoptosis of AML Stem Cells through Dysregulation of Lipid Metabolism

Current chemotherapeutic regimens for acute myeloid leukemia (AML) often fail to eliminate leukemic stem cells (LSCs) which contribute to disease relapse. A key step towards the development of more effective therapies is the identification of vulnerabilities that are unique to LSCs. Here, we sought to identify LSC-specific metabolic dependencies by performing a flow cytometry-based screen of 110 metabolically-focused drugs against a primary human AML sample. This sample harbored distinct subsets defined by CD34 and CD38 expression, and LSC activity assayed by xenotransplantation was restricted to the CD34+CD38- fraction. Through this screen, we found that inhibitors of nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes the rate-limiting step in the NAD+ salvage pathway, preferentially depleted CD34+CD38- cells, implicating NAMPT inhibitors as potential anti-LSC agents. To evaluate the therapeutic potential of NAMPT inhibitors, we focused on KPT-9274, a small-molecule NAMPT inhibitor currently under clinical development for other cancer types. Treatment with KPT-9274 depleted the CD34+CD38- fraction across multiple primary human AML samples through induction of apoptosis. The preferential sensitivity of CD34+CD38- cells to NAMPT inhibition correlated with a lower basal level of intracellular NAD+ and greater dependency on NAMPT activity for NAD+ generation relative to the other fractions. In contrast, normal CD34+ HSPCs were largely resistant to the cytotoxic effects of KPT-9274 due to their capacity to utilize the Preiss-Handler pathway for NAD+ generation. Consistent with the in vitro findings, KPT-9274 treatment significantly reduced LSC activity as determined by secondary engraftment potential in 2 of 3 patient-derived xenograft (PDX) models of human AML and had minimal impact on normal HSC activity in mice engrafted cord blood cells. To gain mechanistic insights into how NAMPT inhibition induces cell death, we performed transcriptomic analysis of sorted CD34+CD38- cells treated with KPT-9274. This analysis revealed a striking upregulation of genes involved in cholesterol and lipid synthesis including the stearoyl-CoA desaturase (SCD) gene. The upregulated genes were highly enriched for known targets of the sterol regulatory element binding protein (SREBP) transcription factors. Functional studies demonstrated that this transcriptional response was protective against the cytotoxic effect of NAMPT inhibition in AML cells. To uncover the metabolic basis of this protective effect, we performed global metabolomic profiling of AML cells treated with KPT-9274 and observed a decrease in the ratio of monounsaturated fatty acids (MUFAs) to saturated fatty acids (SFAs) upon drug treatment. This drop in MUFA:SFA ratio reflected a reduction in SCD activity which catalyzes the desaturation of SFAs to MUFAs in a NADPH-dependent reaction. Since depletion of intracellular MUFAs could trigger apoptosis, we hypothesized that the SREBP response might protect against cell death through upregulation of SCD activity and consequent increase in MUFA synthesis. In line with this hypothesis, we found that exogenous oleic acid, a MUFA, completely rescued cell death induced by KPT-9274, while treatment with SCD inhibitors sensitized AML cells to the cytotoxic effects of NAMPT inhibition. To explore the translational application of our findings, we tested whether dipyridamole (DP), a clinically approved anti-platelet agent with inhibitory activity against SREBP signaling, can be repurposed to enhance the anti-leukemic effects of KPT-9274. We showed that treatment with DP, at non-toxic concentrations, potentiated the cytotoxicity of KPT-9274 against AML cells in vitro. Importantly, in vivo combination treatment with KPT-9274 and DP effectively targeted LSC activity in a PDX model that was refractory to KPT-9274 as single agent. In summary, our findings demonstrate that LSCs are preferentially dependent on NAMPT activity for survival over non-LSCs and normal HSCs. We further uncovered that NAMPT inhibition results in dysregulation of lipid homeostasis and induces a lipogenic response coordinated by SREBPs that protects AML cells against NAD+ depletion. These findings offer insights into drug combination strategies to enhance the efficacy of NAMPT inhibitors and provide the rationale for testing NAMPT inhibitors in the treatment of AML in clinical trials. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Wang:Trilium therapeutics: Patents & Royalties: There is an existing license agreement between TTI and University Health Network and J.C.Y.W. may be entitled to receive financial benefits further to this license and in accordance with UHN's intellectual property policies. .

Overexpression of CD200 Is a Stem Cell-Specific Mechanism of Immune Escape in AML

Background: Acute myeloid leukemia (AML) stem cells (LSC), the likely source of relapsed disease, are capable of surviving current standard chemotherapy. Therefore, novel therapeutic approaches specifically engineered to eradicate LSCs are critical for curing AML. We previously introduced a novel bioinformatics approach that harnessed publically available AML gene expression datasets and identified CD200 as significantly over-expressed in LSCs when compared to paired blast cells, as well as when compared to their normal hematopoietic stem cell (HSC) counterparts (Fig 1A; Herbrich et al Blood. 2018; 130:3962). CD200 can identify AML cells with LSC activity in vivo (Ho et al Blood. 2016; 128:1705). Functionally, CD200 has been shown to have an immunosuppressive effect on macrophages (Hoek et al Science. 2000; 290:1768) and NK cells (Coles et al Leukemia. 2012; 26:2148), and correlates with a high prevalence of FOXP3+ regulatory T cells (Coles et al Leukemia. 2012; 26:2146). Additionally, CD200 has been implicated as a poor prognostic marker in AML (Damiani et al Oncotarget. 2015; 6:30212). To date, we have screened 40 primary AML patient samples by flow cytometry, 95% of which are positive for CD200. Methods: To study the functional role of CD200 in AML, we generated a CD200 overexpression model in the human OCI-AML3 cell line (with no basal expression) and characterized changes in proliferation, survival, and gene expression. To examine the immune function of CD200 in AML in vitro, we performed a series of mixed lymphocyte reactions with isolated effector immune cells and target isogenic AML cell lines to assess immune cell-mediated apoptosis, proliferation, and cytokine secretion. To understand the contribution of CD200 immune protection in a physiological setting, we developed a peripheral blood mononuclear cell (PBMC)-humanized mouse in which we tracked the engraftment and overall survival of the CD200+/- OCI-AML3 cells. Lastly, the utility of CD200-blockade using a fully humanized anti-CD200 monoclonal antibody (CD200-IgG1) was evaluated both in vitro and in vivo. Results: In vitro, CD200+ AML significantly inhibited the secretion of inflammatory cytokines and cytotoxic enzymes from healthy PBMCs; a phenomenon that could be largely reversed by blocking the CD200/CD200R interaction with the CD200 antibody (Fig 1B). In vivo, OCI-AML3 CD200+/- cells showed no difference in engraftment, progression, and overall survival in immunodeficient NSG mice (Fig 1C). However, when mice were humanized using healthy PBMCs, CD200+ leukemia progressed rapidly, escaping T cell-mediated elimination, compared to CD200- control leukemic cells (Fig 1D). Cytokine production in PBMC-humanized mice was significantly compromised in those with CD200-expressing leukemia. Transcriptome analysis revealed that T cells from humanized mice exposed to CD200 expressing disease were metabolically quiescent. In humanized mice, CD200-IgG1 therapy eliminated CD200+ AML disease (Fig 1E). The novel CD200-IgG1 antibody also induced potent, specific NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) and macrophage-mediated antibody dependent cellular phagocytosis (ADCP; Fig 1F). Conclusion: We have identified CD200 as a putative stem cell-specific immunomodulatory target that aids in establishing an immunosuppressive microenvironment by significantly suppressing cytokine secretion in response to AML. In a PBMC-humanized mouse model, the presence of cell-surface CD200 was sufficient to protect AML cells from immune-mediated clearance and could be reversed using a blocking anti-CD200 mAb. These findings indicate a utility of CD200 as a novel immune checkpoint target for the development of therapeutic strategies in AML. Disclosures Konopleva: Calithera: Research Funding; Kisoji: Consultancy; AbbVie: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Ablynx: Research Funding; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; AstraZeneca: Research Funding; Sanofi: Research Funding; Agios: Research Funding; Forty-Seven: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Ascentage: Research Funding.

A Functional Profiling of Microenvironmental Factors and Small Molecules Reveals Monocyte Chemoattractant Protein-1 Mediates Drug Resistance in Acute Myeloid Leukemia

Background: The treatment of acute myeloid leukemia (AML) patients is a formidable challenge, due to disease heterogeneity and the ability to acquire secondary mutations in response to treatment. As a result, persistence and expansion of residual AML clones prevents complete remission. Emerging evidence suggests that extrinsic factors from the microenvironment protect AML cells from therapy, promote disease progression and drug resistance. Previous work from our lab highlights that higher levels of proinflammatory cytokines in the microenvironment, promote AML progression. This emphasizes the rationale to implement targeted therapies that block extrinsic signaling along with tumor intrinsic changes to overcome drug resistance in AML. Methods and Results: To identify factors and pathways that promote drug sensitivity and resistance in AML, we quantified the levels of 41 cytokines and growth factors from the plasma of 350 newly diagnosed AML patient samples using a multiplex luminex assay. The same cohort were analyzed for drug response to 122 small molecule inhibitors using an ex vivo functional drug sensitivity assay. These data were integrated with the gene expression and reactome pathway data to identify differentially regulated pathways and markers for drug response. Data integration revealed that protein synthesis pathways were significantly enriched in venetoclax, (Bcl-2 inhibitor) resistant AML samples, whereas, cytokine and immune pathways such as aberrant activation of monocyte chemoattractant protein-1 (MCP-1; p= 4.9e-07) were correlated with resistance to trametinib, a MEK inhibitor. In contrast, trametinib sensitivity was associated with activation of IL-1 and TLR signaling, suggesting specific extrinsic pathways may drive specific drug response. As a proof-of-concept, we dissected the role of MCP-1 activation in mediating trametinib resistance in AML. For this, we treated primary AML cells with MCP-1 in the presence of trametinib that led to a 4-fold increase in IC50 (0.4 vs 2.4 µM). To model drug response in vitro, we generated trametinib resistant AML cell lines MOLM13 and MV4;11 (FLT3-ITD mutated) and OCI-AML2 (DNMT3A mutated) by culturing cells in trametinib continuously over 4 months. All the trametinib resistant AML cell lines showed 2-10-fold increase in MCP-1 levels in whole cell lysate and in the conditioned media compared to the parental cells. Further, a long-term exposure of AML cells to MCP-1 in the presence of trametinib conferred a growth advantage with a 4-fold increase in cell numbers in comparison to the cells cultured with trametinib alone (n=4/condition). To delineate the molecular pathways driven by MCP-1 in trametinib resistance, we performed global phosphoproteomics using AML cell lines. We identified that MCP-1 activated various pro-survival pathways such as ERK, JNK, SRC, P70, and PKC kinases as well as cell cycle regulatory proteins as early as 5 mins in trametinib resistant cells. Targeting MCP-1 receptor-CCR2 by pharmacological inhibition, significantly reduced viability of trametinib resistant AML cells by 3-fold. The treatment with CCR2 inhibitor or trametinib individually reduced p-JNK levels by 3-fold in AML cells. A combined treatment with CCR2 inhibitor and trametinib, showed a 7-fold reduction in p-JNK levels. These data reinforced the role of MCP-1 in conferring trametinib resistance and that blockade of MCP-1 signaling re-sensitizes AML cells to trametinib. Conclusion: We show that MCP-1 augments trametinib resistance in AML by triggering novel signaling pathways. These effects can be reversed by blockade of MCP-1-CCR2 axis. Such combination treatment strategy incorporating extrinsic pathways together with the intrinsic ones would aid in re-sensitizing AML cells to therapy and represent an attractive new strategy. Our functional screen identifies that these extrinsic signals modulating drug response are unique to specific targeted therapy and should be considered when designing new therapies and clinical trials. We also created a large resource with 350 primary AML samples correlating microenvironment factors and gene expression with response to 122 small molecules, many of those which are FDA approved. This resource will help identify new drug response pathways in the context of microenvironment to design novel treatment regimens for preclinical and clinical testing and to improve AML outcomes. Disclosures Tyner: Syros: Research Funding; Agios: Research Funding; Incyte: Research Funding; Seattle Genetics: Research Funding; Takeda: Research Funding; Petra: Research Funding; Janssen: Research Funding; Constellation: Research Funding; Aptose: Research Funding; Genentech: Research Funding; Gilead: Research Funding; Array: Research Funding; AstraZeneca: Research Funding.

Aldehyde dehydrogenase 3a2 protects AML cells from oxidative death and the synthetic lethality of ferroptosis inducers

AbstractMetabolic alterations in cancer represent convergent effects of oncogenic mutations. We hypothesized that a metabolism-restricted genetic screen, comparing normal primary mouse hematopoietic cells and their malignant counterparts in an ex vivo system mimicking the bone marrow microenvironment, would define distinctive vulnerabilities in acute myeloid leukemia (AML). Leukemic cells, but not their normal myeloid counterparts, depended on the aldehyde dehydrogenase 3a2 (Aldh3a2) enzyme that oxidizes long-chain aliphatic aldehydes to prevent cellular oxidative damage. Aldehydes are by-products of increased oxidative phosphorylation and nucleotide synthesis in cancer and are generated from lipid peroxides underlying the non–caspase-dependent form of cell death, ferroptosis. Leukemic cell dependence on Aldh3a2 was seen across multiple mouse and human myeloid leukemias. Aldh3a2 inhibition was synthetically lethal with glutathione peroxidase-4 (GPX4) inhibition; GPX4 inhibition is a known trigger of ferroptosis that by itself minimally affects AML cells. Inhibiting Aldh3a2 provides a therapeutic opportunity and a unique synthetic lethality to exploit the distinctive metabolic state of malignant cells.

TNFAIP8 promotes AML chemoresistance by activating ERK signaling pathway through interaction with Rac1

BackgroundChemoresistance is emerging as a major barrier to successful treatment in acute myeloid leukemia (AML), and evasion of apoptosis is among the fundamental underlying mechanisms. Therefore, unraveling molecular networks that drive this process constitutes an urgent unmet need. Herein, we aim to characterize the role and molecular mechanism of the tumor necrosis factor ɑ-induced protein 8 (TNFAIP8), a novel anti-apoptotic molecule, in AML chemoresistance.MethodsThe expression levels of TNFAIP8 were assessed in AML patients and cell lines by RT-qPCR and western blots. The transcriptional regulation of TNFAIP8 was analyzed with luciferase reporter assay and ChIP followed by RT-qPCR. Functional experiments were conducted to evaluate the effects of TNFAIP8 on apoptosis, drug sensitivity and proliferation of AML cells. Potential effects of TNFAIP8 on the activation of extracellular signal-regulated kinase (ERK) pathway were detected by western blots. CoIP and P21-activated kinase (PAK) pull-down assay were performed to ascertain the upstream target. The overall effects of TNFAIP8 on AML were examined in murine models.ResultsUpregulated TNFAIP8 expression was first confirmed in human AML patients and cell lines. E74 like ETS transcription factor 1 (ELF1) was then identified to contribute to its aberrant expression. Through manipulating TNFAIP8 expression, we described its role in protecting AML cells from apoptosis induced by chemotherapeutic agents and in promoting drug resistance. Notably, the leukemia-promoting action of TNFAIP8 was mediated by sustaining activity of the ERK signaling pathway, through an interaction with Rac family small GTPase 1 (Rac1). In addition, in vivo experiments confirmed that TNFAIP8 suppression lowered leukemia infiltration and improved survival.ConclusionOur data provide a molecular basis for the role of TNFAIP8 in chemoresistance and progression of AML and highlight the unique function of TNFAIP8 as an attractive therapeutic target.