Cultured Acute Myeloid Leukemia Cells Research Articles

Reprogramming of Acute Myeloid Leukemia Patients Cells: Harboring Cancer Mutations Requires Targeting of AML Hierarchy.

Screening of primary patient acute myeloid leukemia (AML) cells is challenging based on intrinsic characteristics of human AML disease and patient-specific conditions required to sustain AML cells in culture. This is further complicated by inter- and intra-patient heterogeneity, and "contaminating" normal cells devoid of molecular AML mutations. Derivation of induced pluripotent stem cells (iPSCs) from human somatic cells has provided approaches for the development of patient-specific models of disease biology and has recently included AML. Although reprogramming patient-derived cancer cells to pluripotency allows for aspects of disease modeling, the major limitation preventing applications and deeper insights using AML-iPSCs is the rarity of success and limited subtypes of AML disease that can be captured by reprogramming to date. Here, we tested and refined methods including de novo, xenografting, naïve versus prime states and prospective isolation for reprogramming AML cells using a total of 22 AML patient samples representing the wide variety of cytogenetic abnormalities. These efforts allowed us to derive genetically matched healthy control (isogenic) lines and capture clones found originally in patients with AML. Using fluorescently activated cell sorting, we revealed that AML reprogramming is linked to the differentiation state of diseased tissue, where use of myeloid marker CD33 compared to the stem cell marker, CD34, reduces reprogramming capture of AML+ clones. Our efforts provide a platform for further optimization of AML-iPSC generation, and a unique library of iPSC derived from patients with AML for detailed cellular and molecular study.

TET2-Mediated mRNA and DNA Oxidation in Leukemia Homing and Immune Evasion

Emerging evidence strongly indicates that dysregulation of chemical modifications in DNA or RNA leads to hematopoietic malignancies, including acute myeloid leukemia (AML). Tet methycytosine dioxygenases 2 (TET2) mutations have been characterized in 15-20% AML and its deficiency promotes leukemogenesis and leukemia stem cell (LSC) self-renewal. However, the underlying cellular/molecular mechanisms have yet to be fully understood. TET2 catalyzes the demethylation of DNA 5-methylcytosine (5mC) and plays a central role in transcriptionally regulating gene expression. Besides DNA methylation, recent studies support a novel function for TET2-mediated demethylation of methyl-5-cytosine (termed as 'm5C' to distinguish from DNA 5mC) in mRNAs. However, it is totally unknown whether and (if so) how TET2-induced mRNA m5C demethylation contributes to leukemogenesis (or tumorigenesis in general). To evaluate the role of Tet2 in de novo leukemogenesis, we have generated various AML models driven by AML1-ETO9a (AE9a, from t(8;21)), MLL-AF9 (MA9, from MLL-r/t(11q23)), PML-RARA (from t(15;17)), and NRAS mutation. Such selection is clinically relevant because TET2 mutation and/or transcriptional suppression have been frequently observed in those AML subtypes (Fig. 1A and B). Notably, Tet2 depletion dramatically promoted primary leukemogenesis in vivo (Fig. 1C). Strikingly, Tet2 depletion had very weak effects on the growth/proliferation of primary AML cells in liquid culture in vitro (Fig. 1D). Collectively, Tet2 deficiency led to fast development of AML and such effect is unlikely due to enhanced cell proliferation. To decipher the underlying mechanism(s), we collected the primary Tet2-/- and Tet2+/+ AML cells for transcriptome-wide RNA-seq and found that Tet2 deficiency significantly increased expression of the genes enriched in the "Hematopoietic Stem Cell" pathway (Fig. 1E). Via flow cytometry, we showed that Tet2 deficiency significantly increased the Lin−c-Kit+Sca-1+ (LSK) primitive cell population (Fig. 1F). Via in vivo limiting dilution assay (LDA), we showed that Tet2 deletion significantly increased LSCs frequency by over 30-fold (Fig. 1G). During leukemogenesis, the homing and/or anchoring of LSC into the BM niche plays a predominant role to facilitate LSC self-renewal in vivo. Via in situ confocal imaging, we observed that there were more Tet2 deficient-AML cells migrating into the BM-derived stromal cells relative to the Tet2+/+ group (Fig. 1H). Further studies revealed that Tetranspanin 13 (Tspan13), a core enriched gene in the "Hematopoietic Stem Cell" pathway, is a functionally essential target of Tet2. Tet2 deficiency stimulated Tspan13 expression in primary AML cells (Fig. 1I) and genetic depletion of Tspan13 could substantially reverse the increased LSC homing (Fig. 1J) and self-renewal ability caused by Tet2 depletion. Tet2 deficiency-mediated upregulation of Tspan13 is not dependent on DNA demethylation or transcriptional regulation, but dependent on mRNA demethylation and post-transcriptional regulation (Fig. 1K). Specifically, Tet2 deficiency led to the accumulation of m5C in Tspan13 mRNA (Fig. 1L), and Y-box binding protein 1 (Ybx1), the m5C "reader", could recognize m5C modification, stabilize Tspan13 transcript, and increase its overall expression. The increased expression of Tspan13 further activated the CXCR4/CXCL12 axis, which pays a dominant role in trafficking LSCs into BM niche. In addition to the BM stromal cells, the leukemia microenvironment contains various immune cells which mediate the anti-tumor immune response. Tet2 deficiency remarkably suppressed various immune-related pathways, especially the signaling and genes which are related to interferon (IFN) responses. Specifically, Tet2 deficiency transcriptionally suppressed the expression of IFN-stimulated genes (ISGs), especially Cxcl10, Oasl2, and Isg1 (Fig. 1M and N), via DNA 5hmC modification. Consequently, Tet2 deficiency in AML cells suppressed the recruitment of CD8+ T cells, but not other types of immune cells, and conferred AML cells resistance to CD8+ T cell-mediated antileukemia immunity (Fig. 1O). Collectively, our studies uncover previously unappreciated roles and underlying molecular mechanisms of TET2 in regulating BM niche programing, LSC homing/self-renewal and immune response in AML pathogenesis, as both RNA and DNA demethylase. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Precision Targeting of Ion Channel Blocker for the Treatment of AML

The survival rate of patients with acute myeloid leukemia (AML) remains modest and thus, there is a clear need for advanced treatments. Here we describe two drug leads for AML that inhibit cellular sodium release without requiring drug internalization. EDC1 and EDC8 are two very potent extracellular antibody drug conjugates that selectively inhibit the all-important Na,K-ATPase, an ion pump required by cells to export excess sodium and to maintain ion gradients needed to import and export many important nutrients and waste products. We first demonstrate that dysadherin (a known metastatic cancer marker), is expressed on the surface many types of AML cell lines and can be used to direct Na,K-ATPase inhibitors using EDC1. We next demonstrate that ATRA inducible AML cells types are selectively destroyed with Na,K-ATPase inhibitors targeted to CD38 using EDC8 and ATRA.Since dysadherin expression is a known to be correlated to metastasis and stemness but yet to be studied in AML, we first analyzed a number of leukemia cell lines and AML patient samples for dysadherin expression by analyzing their surface expression and/or their sensitivity to EDC1. We observed that many cultured AML cells express dysadherin and are sensitive to EDC1 with EC50 values ranged from 0.02 to 1.0 nanoMolar and cell viability after 72 hours of exposure ranging from zero to 10%. For the three primary AML patient specimens analyzed, dysadherin expression was seen. Interestingly, in two of these three samples, the majority of intense staining was found in the CD123+ CD34+ population.Since CD38 expression and inducible expression can now be diagnosed in patients with AML, we analyzed if ATRA was required to sensitize AML to EDC8. Using multiple leukemic cell lines (HL60, U937, MV411, KG1, K562, and OCI/AML3) known to express CD38 with or without requiring ATRA, we analyzed EDC8 sensitivity before and after ATRA addition. We found that CD38 expression was required for EDC8 sensitivity and once expressed, EDC8 displayed potent activity with few viable cells detectable.Further studies analyzing dysadherin and CD38 expression in patient samples are needed to better understand the AML cell populations that may be affected by these EDCs but these results show the potential of this new type of antibody drug conjugate technology for the treatment of AML. EDCs that inhibit a required membrane bound ion pump with the precision of antibodies without requiring internalization may provide a new tool for doctors in their fight against AML. DisclosuresMarshall:CENTROSE: Employment. Malavasi:Jaansen Pharmaceuticals: Research Funding. Prudent:CENTROSE: Employment, Equity Ownership.

Investigating Collateral Sensitivity of AML

Acute Myeloid Leukemia (AML) has a historically low 5-year survival rate compared to other types of cancers. There are many subtypes of AML and despite it being a heterogeneous disease, is often treated with a one-size-fits-all approach. Furthermore, relapse after initial successful treatment is common. This project is investigating the collateral sensitivity of AML by developing chemotherapy resistant cultured AML cells and then treating them with secondary chemotherapies to determine if the combination has a synergistic effect. Cabazitaxel is a chemotherapy drug used clinically to treat AML. Experiments using MOLM-13 cells in GI50 assays with Cabazitaxel were conducted to determine initial sensitivity. Currently, cells are being micro-dosed with increasing concentrations of Cabazitaxel, starting with their GI10 value. Cells are continuously cultured in in either Cabazitaxel or DMSO, as a control. GI50 assays will be re-run when treated cells resume growth rates of untreated cells in order to determine the extent of resistance. Once cell lines that are stable and non-reactive to the addition of the initial chemotherapy, a matrix of secondary chemotherapies will be applied to the resistant cell lines. Secondary treatments that successfully trigger apoptosis in the majority of resistant cells would show promise in collateral sensitivity and fighting chemotherapy resistance. By determining the collateral sensitivity of AML lines it will allow future research to be done on the effectiveness of secondary treatments for patients that have AML recurrence that is found to be resistant to in initial treatment.

Apolipoprotein C2 - CD36 Promotes Leukemia Growth and Presents a Targetable Axis in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a devastating hematologic malignancy that affects the hematopoietic stem cells. The 5-year overall survival (OS) of patients with AML is less than 30%, highlighting the urgent need to identify new therapeutic targets. Here, we analyze gene expression datasets for genes that are differentially overexpressed in AML cells compared with healthy hematopoietic cells. We report that apolipoprotein C2 (APOC2) mRNA is significantly overexpressed in AML, particularly in patients with mixed-lineage leukemia rearrangements. By multivariate analysis, high APOC2 expression in leukemia blasts is significantly associated with decreased OS (HR: 2.51; 95% CI, 1.03-6.07; P = 0.04). APOC2 is a small secreted apolipoprotein that constitutes chylomicrons, very-low-density lipoproteins, and high-density lipoproteins with other apolipoproteins. APOC2 activates lipoprotein lipase and contributes to lipid metabolism. By gain and loss of function approaches in cultured AML cells, we demonstrate that APOC2 promotes leukemia growth via CD36-mediated LYN-ERK signaling activation. Knockdown or pharmacological inhibition of either APOC2 or CD36 reduces cell proliferation, induces apoptosis in vitro, and delays leukemia progression in mice. Altogether, this study establishes APOC2-CD36 axis as a potential therapeutic target in AML.

The long non‐coding RNA LOC285758 promotes invasion of acute myeloid leukemia cells by down‐regulating miR‐204‐5p

Acute myeloid leukemia (AML) is the second most common type of leukemia worldwide. It was previously reported that expression of the long noncoding RNA LOC285758 is positively associated with AML cell proliferation, but the underlying mechanisms have not previously been reported. Here, we report that LOC285758 expression is higher in clinical AML blood samples and cultured AML cells. miR‐204‐5p was confirmed to be a target gene of LOC285758 by bioinformatics analysis and luciferase assay. LOC285758 overexpression promoted AML cell viability and invasion abilities, which were effectively inhibited by miR‐204‐5p overexpression; moreover, miR‐204‐5p overexpression also regulated the expression of E‐cadherin, N‐cadherin and Twist1. The data also showed that increased LOC285758 expression could effectively suppress the earlier effects of miR‐204‐5p on AML cells. Our findings suggest that targeting of miR‐204‐5p by LOC285758 promotes the cell viability and invasion of AML cells, and thus LOC285758 may have potential as a therapeutic target for AML treatment.

DNA-PK Inhibitor, M3814, as a New Combination Partner of Mylotarg in the Treatment of Acute Myeloid Leukemia.

Despite significant advances in the treatment of acute myeloid leukemia (AML) the long-term prognosis remains relatively poor and there is an urgent need for improved therapies with increased potency and tumor selectivity. Mylotarg is the first AML-targeting drug from a new generation of antibody drug conjugate (ADC) therapies aiming at the acute leukemia cell compartment with increased specificity. This agent targets leukemia cells for apoptosis with a cytotoxic payload, calicheamicin, carried by a CD33-specific antibody. Calicheamicin induces DNA double strand breaks (DSB) which, if left unrepaired, lead to cell cycle arrest and apoptosis in cancer cells. However, repair of DSB by the non-homologous end joining pathway driven by DNA-dependent protein kinase (DNA-PK) can reduce the efficacy of calicheamicin. M3814 is a novel, potent and selective inhibitor of DNA-PK. This compound effectively blocks DSB repair, strongly potentiates the antitumor activity of ionizing radiation and DSB-inducing chemotherapeutics and is currently under clinical investigation. Suppressing DSB repair with M3814 synergistically enhanced the apoptotic activity of calicheamicin in cultured AML cells. Combination of M3814 with Mylotarg in two AML xenograft models, MV4-11 and HL-60, demonstrated increased efficacy and significantly improved survival benefit without elevated body weight loss. Our results support a new application for pharmacological DNA-PK inhibitors as enhancers of Mylotarg and a potential new combination treatment option for AML patients.

Diminished AHR Signaling Drives Human Acute Myeloid Leukemia Stem Cell Maintenance.

Eliminating leukemic stem cells (LSC) is a sought after therapeutic paradigm for the treatment of acute myeloid leukemia (AML). While repression of aryl hydrocarbon receptor (AHR) signaling has been shown to promote short-term maintenance of primitive AML cells in culture, no work to date has examined whether altered AHR signaling plays a pathologic role in human AML or whether it contributes at all to endogenous LSC function. Here, we show AHR signaling is repressed in human AML blasts and preferentially downregulated in LSC-enriched populations within leukemias. A core set of AHR targets are uniquely repressed in LSCs across diverse genetic AML subtypes. In vitro and in vivo administration of the specific AHR agonist FICZ significantly impaired leukemic growth, promoted differentiation, and repressed self-renewal. Furthermore, LSCs suppressed a set of FICZ-responsive AHR target genes that function as tumor suppressors and promoters of differentiation. FICZ stimulation did not impair normal hematopoietic stem and progenitor (HSPC) function, and failed to upregulate a prominent LSC-specific AHR target in HSPCs, suggesting that differential mechanisms govern FICZ-induced AHR signaling manifestations in HSCs versus LSCs. Altogether, this work highlights AHR signaling suppression as a key LSC-regulating control mechanism and provides proof of concept in a preclinical model that FICZ-mediated AHR pathway activation enacts unique transcriptional programs in AML that identify it as a novel chemotherapeutic approach to selectively target human LSCs. SIGNIFICANCE: The AHR pathway is suppressed in leukemic stem cells (LSC), therefore activating AHR signaling is a potential therapeutic option to target LSCs and to treat acute myeloid leukemia.

Abstract 269: DNA-PK inhibitor, M3814, as a new combination partner of Mylotarg in the treatment of acute myeloid leukemia

Abstract Despite significant advances in the development of new treatments for acute myeloid leukemia (AML) the therapeutic success remains relatively poor. There remains an urgent need for improved therapies with increased tumor potency and selectivity. Mylotarg is the first AML drug from a new generation of antibody drug conjugate (ADC) therapies targeting the acute leukemia cell compartment with increased specificity. This agent targets leukemia cells for apoptosis with a cytotoxic warhead, calicheamicin, carried by a CD33-specific antibody. Calicheamicin induces DNA double strand breaks (DSB) which, if left unrepaired, lead to cell cycle arrest and apoptosis in cancer cells. However, repair of DSBs by the non-homologous end joining pathway driven by DNA-PK (DNA-dependent protein kinase) can reduce the efficacy of calicheamicin. M3814 is a novel, potent and selective inhibitor of DNA-PK protein kinase. This compound effectively blocks DSB repair, strongly potentiates the antitumor activity of ionizing radiation and DSB-inducing chemotherapeutics and is currently under clinical investigation. Suppressing DSB repair with M3814 synergistically enhanced the apoptotic activity of calicheamicin in cultured AML cells. Combination of M3814 with Mylotarg in two AML xenograft models, MV4-11 and HL-60, demonstrated increased efficacy and significantly improved survival benefit without elevated body weight loss. Our results support a new application for pharmacological DNA-PK inhibitors as enhancers of Mylotarg efficacy and a potential new combination treatment option for AML patients. Citation Format: Astrid Zimmermann, Michael Carr, Frank T. Zenke, Andree Blaukat, Lyubomir T. Vassilev. DNA-PK inhibitor, M3814, as a new combination partner of Mylotarg in the treatment of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 269.

H22954, a novel long non-coding RNA down-regulated in AML, inhibits cancer growth in a BCL-2-dependent mechanism.

Long non-coding RNAs (lncRNAs) are important in cancer biology. In this study, we analyzed differentially expressed genes in CD34 + hematopoietic cells and identified a novel lncRNA, H22954, which was down-regulated in acute myeloid leukemia (AML) patients. In cultured AML cells and mouse xenograft models, H22954 expression inhibited cell proliferation and tumor growth, respectively. Bioinformatic analysis and RNA antisense purification assay indicated that H22954 targeted the 3' untranslated region of the BCL2 gene. In luciferase assays, H22954 expression inhibited BCL2 expression. In transfected K562 cells and mouse xenograft tumors, H22954 overexpression reduced BCL-2 protein levels and promoted cell death. In AML patients, H22954 expression inversely correlated with BCL-2 protein levels in bone marrow cells, blast cell numbers and disease prognosis. These results indicate that H22954 is a novel regulator of BCL-2 and that reduced H22954 expression may play an important role in the pathogenesis of AML.

S-Adenosyl Methionine Synthesis Impacts Maintenance of Acute Myeloid Leukemia Cells with Regulation of Transcriptional Elongation

Epigenetic dysregulation is at the heart of Acute Myeloid Leukemia (AML), and its mechanistic understanding holds promise in improving AML dismal prognosis. Modification of chromatin accessibility through post-translational modification of histones alters gene expression activity and is directly dependent on cell metabolism for generation of the modifying groups. Methionine metabolism generates S-adenosyl methionine (SAM), the universal methyl donor in the cell, through the activity of multimeric methionine adenosyl transferase (MAT) enzymes. MAT2A is an obligatory subunit in hematopoietic cells, and we have recently tabled it in a CRISPR drop-out screen [1] as a candidate vulnerability in AML.We have validated the anti-AML effects of MAT2A ablation using shRNA-mediated gene expression knockdown, and could further phenocopy cell line selectivity observed in our CRISPR screen through the use of the MAT2A selective inhibitor FIDAS-5 [2] (IC50 MOLM-13 1.0μM, OCI-AML3 0.7μM, MV4.11 11μM).MAT2A inhibition significantly depleted SAM levels (fold change to DMSO, FC=0.20±0.07; p<0.05, MOLM-13 cells), and resulted in decreased growth and enhanced apoptosis (MOLM-13 FC=1.34±0.21;p<0.05, OCI-AML3 FC=3.0±0.34; p<0.05, MV4.11 FC=1.09±0.19;p=0.52) of cultured AML cells, without an obvious impact on differentiation. Similar results were observed in AML patient samples (n=11), which displayed reduced capacity to initiate colonies in semi-solid medium (FC=0.47±0.21; p<0.0005) and had hindered expansion in medium-term co-cultures on MS-5 stroma, with accumulation of cells with intermediate, but not high, levels of primitive marker CD34. MLL-AF9 transformed primary mouse bone marrow cells were equally affected (FC colony efficiency=0.05±0.03; p<0.005). Significantly, MAT2A inhibition did not affect colony-formation or multi-lineage differentiation of cord blood-derived CD34+ cells in colony-forming progenitor assays (FC=1.01±0.25p=0.97), indicating selective vulnerability of AML cells.We analysed the consequences of MAT2A inhibition on chromatin methyl modifications, and observed a specific reduction in the active elongation mark H3K36me3. The associated transcriptional programme is currently being investigated. Interestingly, contrary to reports in colon cancer cells upon methionine restriction and reduced SAM levels [3], we did not observe loss of H3K4me3 at active promoters, suggesting a selective AML dependence on transcription elongation.In conclusion, we identify SAM synthesis as a novel vulnerability in AML through regulation of transcriptional elongation. Our findings highlight the therapeutic potential of modulating metabolite availability for epigenetic and transcriptional re-programming in leukemia.

Abstract 4823: Alvocidib enhances the efficacy of cytarabine and daunorubicin (7+3) in non-clinical models of acute myeloid leukemia

Abstract The ‘7+3' induction regimen has been the standard of care for frontline acute myeloid leukemia (AML) for more than 30 years. There are still significant unmet needs for frontline patients, particularly those with high-risk features. Alvocidib, a potent CDK9 inhibitor, has shown clinical activity in multiple studies of frontline and relapsed/refractory AML. In some clinical studies, alvocidib has been administered as part of the ACM (alvocidib, cytarabine, and mitoxantrone) regimen. The ACM regimen has demonstrated greater clinical responses compared to 7+3 in frontline intermediate and high-risk AML patients. The ACM regimen is now being evaluated in a multi-institution, Phase II study using a MCL-1 dependency biomarker to select patients likely to be sensitive to the alvocidib-containing regimen. Alvocidib activity is primarily driven by its inhibition of CDK9/RNA-polymerase II and the subsequent suppression of key anti-apoptotic proteins, including MCL-1. Alvocidib induces apoptosis and potentiates the activity of apoptosis-inducing agents, such as cytarabine and mitoxantrone. The synergy of the ACM regimen led us to hypothesize that alvocidib may enhance the anti-leukemia activity of the 7+3 regimen in preclinical models as well. Cell viability and apoptosis measurements were made following treatment using the CellTiter-Glo and Caspase-Glo assays according to manufacturer protocol. MCL-1 mRNA expression and the expression of additional markers was determined using RT-qPCR. Protein expression was determined using standard immunoblotting techniques. Alvocidib and the 7+3 regimen were also tested in vivo in the MV4-11 AML xenograft model. Here, it was observed that the 72-hour viability assays yielded single agent IC50 values of alvocidib, cytarabine, or daunorubicin in cultured AML cells ranging from 2.2 nM to 567 nM. In apoptosis assays, a modest induction was observed with cytarabine, daunorubicin, or alvocidib. However, the three-drug combination more than doubled the induction of apoptosis compared to any of the agents alone. We also observed that MCL-1 was suppressed in the combination treatment. In a xenograft study, 21.1 and 48.5% tumor growth inhibition (TGI) was observed following either single agent treatment of daunorubicin (0.5 mg/kg) or cytarabine (60 mg/kg), respectively. Treatment of alvocidib (1.25 mg/kg) yielded 60.0% TGI. Combination treatment, however, resulted in tumor shrinkage, yielding 116.2% TGI. While we have reported previously that alvocidib synergizes with cytarabine and mitoxantrone preclinically in the ACM regimen, our data here demonstrates that alvocidib potentiates the activity of cytarabine and daunorubicin as well. Our results here provide additional scientific rationale for an investigational Phase Ib clinical study employing alvocidib in combination with 7+3 in frontline AML patients, which is currently ongoing. Citation Format: Wontak Kim, Clifford J. Whatcott, Adam Siddiqui-Jain, David J. Bearss, Steven L. Warner. Alvocidib enhances the efficacy of cytarabine and daunorubicin (7+3) in non-clinical models of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4823.

A Mitochondrial-targeted purine-based HSP90 antagonist for leukemia therapy.

Reprogramming of mitochondrial functions sustains tumor growth and may provide therapeutic opportunities. Here, we targeted the protein folding environment in mitochondria by coupling a purine-based inhibitor of the molecular chaperone Heat Shock Protein-90 (Hsp90), PU-H71 to the mitochondrial-targeting moiety, triphenylphosphonium (TPP). Binding of PU-H71-TPP to ADP-Hsp90, Hsp90 co-chaperone complex or mitochondrial Hsp90 homolog, TRAP1 involved hydrogen bonds, π-π stacking, cation-π contacts and hydrophobic interactions with the surrounding amino acids in the active site. PU-H71-TPP selectively accumulated in mitochondria of tumor cells (17-fold increase in mitochondria/cytosol ratio), whereas unmodified PU-H71 showed minimal mitochondrial localization. Treatment of tumor cells with PU-H71-TPP dissipated mitochondrial membrane potential, inhibited oxidative phosphorylation in sensitive cell types, and reduced ATP production, resulting in apoptosis and tumor cell killing. Unmodified PU-H71 had no effect. Bioinformatics analysis identified a “mitochondrial Hsp90” signature in Acute Myeloid Leukemia (AML), which correlates with worse disease outcome. Accordingly, inhibition of mitochondrial Hsp90s killed primary and cultured AML cells, with minimal effects on normal peripheral blood mononuclear cells. These data demonstrate that directing Hsp90 inhibitors with different chemical scaffolds to mitochondria is feasible and confers improved anticancer activity. A potential “addiction” to mitochondrial Hsp90s may provide a new therapeutic target in AML.

TGF-β1 and CXCL12 modulate proliferation and chemotherapy sensitivity of acute myeloid leukemia cells co-cultured with multipotent mesenchymal stromal cells

ABSTRACTObjectives: Multipotent mesenchymal stromal cells (MSCs) play a central role within the bone marrow (BM) niche, supporting hematopoiesis via soluble factors like cytokines and chemokines. In our study, we sought to investigate the effect of blocking transforming growth factor beta 1 (TGF-β1) and C-X-C motif chemokine 12 (CXCL12) receptor CXCR4 on acute myeloid leukemia (AML) cells in an MSC co-culture system.Methods: Human MSCs were obtained by BM aspirates and their phenotype and functional properties were confirmed in vitro. Co-cultures of AML cells on MSCs were initiated and compared to those on mouse fibroblasts (MS-5) and liquid cultures. Additionally, the effect of blocking CXCR4 and TGF-β1 on AML cells was tested with and without the addition of cytarabine.Results: MSCs from BM showed a typical phenotype and differentiation pattern. Co-culture of AML cells on MSCs resulted in a significantly higher proliferation capacity than on MS-5 or liquid culture. Blockade of TGF-β1 increased AML cell proliferation and chemosensibility, while the CXCR4 antagonist plerixafor showed anti-proliferative effects and did not change cytarabine-induced cell death compared to control.Discussion: Human MSCs are potent feeder cells, able to maintain AML cells in long-term culture. This favorable co-existence seems to be due in part to molecules important for communication within the niche. Blockade of TGF-β1 and CXCL12 was associated with different effects on AML cell proliferation and chemotherapy resistance.Conclusion: These findings suggest a strong supporting affinity between MSCs and AML cells within the leukemic niche, where TGF-β1 and CXCL12 pathways play an important role.

Histone deacetylase inhibitors reduce differentiating osteoblast-mediated protection of acute myeloid leukemia cells from cytarabine.

The bone marrow microenvironment protects acute myeloid leukemia (AML) cells during chemotherapy and is a major factor in relapse. Here, we examined which type(s) of bone marrow cells are responsible for the relapse of AML following treatment with cytarabine (Ara-C), and we identified a means to inhibit this protection. To determine the protective cell type(s), AML cells were treated with Ara-C, and AML cell survival in the presence or absence of osteoblast lineage cells was assessed. Cultured AML cells and patient bone marrow isolates were each significantly protected from Ara-C-induced apoptosis by co-culture with differentiating osteoblasts. Moreover, pretreating differentiating osteoblasts with the histone deacetylase inhibitors (HDACi) vorinostat and panobinostat abrogated the ability of the differentiating osteoblasts to protect AML cells. Together, our results indicate that differentiating osteoblasts have the potential to promote residual AML in the bone marrow following standard chemotherapy and act via a mechanism requiring HDACi-sensitive gene expression. Using HDACi to target the leukemic microenvironment in combination with Ara-C could potentially improve treatment of AML. Moreover, other strategies for manipulating bone marrow osteoblasts may also help eradicate AML cells and reduce relapse.

Disulfiram/copper selectively eradicates AML leukemia stem cells in vitro and in vivo by simultaneous induction of ROS-JNK and inhibition of NF-\u03baB and Nrf2

Acute myeloid leukemia (AML) is a heterogeneous malignancy. Despite the advances in past decades, the clinical outcomes of AML patients remain poor. Leukemia stem cells (LSCs) is the major cause of the recurrence of AML even after aggressive treatment making, promoting development of LSC-targeted agents is an urgent clinical need. Although the antitumor activity of disulfiram (DS), an approved anti-alcoholism drug, has been demonstrated in multiple types of tumors including hematological malignancies such as AML, it remains unknown whether this agent would also be able to target cancer stem cells like LSCs. Here, we report the in vitro and in vivo activity of DS in combination with copper (Cu) against CD34+/CD38+ leukemia stem-like cells sorted from KG1α and Kasumi-1 AML cell lines, as well as primary CD34+ AML samples. DS plus Cu (DS/Cu) displayed marked inhibition of proliferation, induction of apoptosis, and suppression of colony formation in cultured AML cells while sparing the normal counterparts. DS/Cu also significantly inhibited the growth of human CD34+/CD38+ leukemic cell-derived xenografts in NOD/SCID mice. Mechanistically, DS/Cu-induced cytotoxicity was closely associated with activation of the stress-related ROS-JNK pathway as well as simultaneous inactivation of the pro-survival Nrf2 and nuclear factor-κB pathways. In summary, our findings indicate that DS/Cu selectively targets leukemia stem-like cells both in vitro and in vivo, thus suggesting a promising LSC-targeted activity of this repurposed agent for treatment of relapsed and refractory AML.

Role of Wnt/β-Catenin Signalling in Acute Myeloid Leukemia (AML) Cell Response to Chemotherapy

Background: Growing evidences from both preclinical and clinical investigations reveal the critical role of Wnt signalling for the development of many cancers and for their response to chemotherapy. Although recent studies suggest that aberrant Wnt signalling can be involved in the neoplastic myeloid cell growth, the contribution of the Wnt/β-catenin pathway to AML survival and chemoresistance is still unclear.Aims: In this study, we investigated the contribution of WNT/β-CATENIN signalling to AML survival and chemoresistance. For this purpose we tested different modulators of Wnt/β-Catenin pathway for their ability to influence AML cells proliferation and response to Cytarabine (Ara-C) or Idarubicin treatment.Methods: AML primary blast cells(30 samples) or AML cell lines cultured alone or in presence of human bone marrow mesenchymal stromal cells (hBM-MSCs), were treated with with Cytarabine (Ara-C) or Idarubicin, in presence or absence of Wnt modulators, including ligands (Wnt3a, Wnt5a/5b), Porcupine inhibitors (IWP-2), LRP6 inhibitors (Niclosamide), or antagonists of TCF/β-catenin (PKF118-310, PNU-74654).Results:In silico analysis showed the enrichment of Wnt signalling components in AML samples. Western Blot and flow cytometry showed the presence of total β-catenin only in about 2/3 of primary samples analyzed, while . β-catenin positive samples had different degree of activation of the pathway, as revealed by the expression of active forms of β-catenin, including (Ser675)β-catenin and non-phospho-(Ser33/37/Thr41) β-catenin. Notably, we found that active forms of β-catenin increased in AML samples in co-culture with hBM-MSCs, thus suggesting that Wnt signalling could be involved in the crosstalk between bone marrow stroma and AML cells. The addition of Wnt or pharmacological inhibitors, such as IWP-2, PNU-74654 and Niclosamide, to the culture medium of β-catenin-positive AML samples, either cultured alone or in co-culture with hBM-MSCs, reduced AML cell proliferation with slight effect on cell death. When associated to Idarubicin, all Wnt inhibitors except IWP-2 synergycally induced a dramatic cell death in AML cells in both culture conditions. However, when Idarubicin was replaced by Ara-C the synergism was observed only with Niclosamide and PKF. Cell death was mainly due to apoptosis, as shown by Annexin-V staining.Conclusion: Overall our data show that Wnt inhibitors reduce proliferation and chemoresistance of AML cells in culture or co-culture with bone marrow stroma cells. Wnt/β-catenin signalling may represent a potential therapeutic strategy to improve AML treatment, overcoming bone marrow stromal-mediated anti-apoptotic and chemoresistance effects. DisclosuresBonifacio:Ariad Pharmaceuticals: Consultancy; Pfizer: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Amgen: Consultancy.

The role of VDR and BIM in potentiation of cytarabine-induced cell death in human AML blasts.

Acute Myeloid Leukemia (AML) has grave prognosis due to aggressive nature of the disease, the toxicity of standard treatment, and overall low cure rates. We recently showed that AML cells in established culture treated with cytarabine (AraC) and a differentiation agent combination show enhancement of AraC cytotoxicity. Here we elucidate molecular changes which underlie this observation with focus on AML blasts in primary culture. The cells were treated with AraC at concentrations achievable in clinical settings, and followed by the addition of Doxercalciferol, a vitamin D2 derivative (D2), together with Carnosic acid (CA), a plant-derived antioxidant. Importantly, although AraC is also toxic to normal bone marrow cell population, the enhanced cell kill by D2/CA was limited to malignant blasts. This enhancement of cell death was associated with activation of the monocytic differentiation program as shown by molecular markers, and the increased expression of vitamin D receptor (VDR). Apoptosis elicited by this treatment is caspase-dependent, and the optimal blast killing required the increased expression of the apoptosis regulator Bim. These data suggest that testing of this regimen in the clinic is warranted.

Development of FF-10101, a Novel Irreversible FLT3 Inhibitor, Which Overcomes Drug Resistance Mutations

Development of FF-10101, a Novel Irreversible FLT3 Inhibitor, Which Overcomes Drug Resistance Mutations