Protect Acute Myeloid Leukemia Cells Research Articles

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. TKI resistance is mediated by secondary FLT3‐ITD mutations only in a minority of cases. We hypothesize that the cytokine CCL5 protects AML cells from TKI‐mediated cell death and contributes to treatment resistance. We generated PKC412‐ and sorafenib‐resistant MOLM‐13 cell lines as an in vitro model to study TKI resistance in AML. Increased CCL5 levels were detected in supernatants from PKC412‐resistant cell lines compared to TKI‐sensitive cells. Moreover, CCL5 treatment of TKI‐sensitive cells induced resistance to PKC412. In resistant cell lines with high CCL5 release, we observed a significant downregulation of the CCL5‐receptor CCR5 and CXCR4. In these cell lines, TKI resistance could be partly overcome by addition of the CXCR4‐receptor antagonist plerixafor. Microarray and intracellular flow cytometry analyses revealed increased p‐Akt or p‐Stat5 levels in PKC412‐resistant cell lines releasing high amounts of CCL5. Treatment with the CXCR4 antagonist plerixafor, αCCL5, or CCR5‐targeting siRNA led to a decrease of p‐Akt‐positive cells. Transient transfection of sensitive MOLM‐13 cells with a CCL5‐encoding vector mediated resistance against PKC412 and led to an increase in p‐Akt‐positive and p‐Stat5‐positive cells. Isolated AML blasts from patients treated with PKC412 revealed that CCL5 transcript levels increase significantly at relapse. Taken together, our findings indicate that CCL5 mediates resistance to FLT3‐TKIs in FLT3‐ITD‐mutated AML and could possibly serve as a biomarker to predict drug resistance.

HMG-XY REGULATES DRUG-RESISTANCE OF ACUTE MYELOID LEUKEMIA CELLS THROUGH MAPK PATHWAY

Acute myeloid leukemia (AML) is a malignant, hyperproliferative neoplasm. Since its inception, chemotherapy and hematopoietic stem cell (HSC) transplantation have substantially improved the outcome of AML, but high rate of relapsed and refractory (R/R) AML remain the major obstacles in clinical treatment. As cholesterol metabolism supplies tumor cells with necessary intermediate products and greatly influences the pathogenesis and prognosis of AML. Cholesterol abnormal it, was found between AML CR and R/R patients, and HMGCS1, a critical gene acting in cholesterol synthesis, was supposed to contribute to this phenomenon. Meanwhile, we found that HMGCS1 was distinctively activated in AML R/R and was related with overall survival (OS) rate. To explore the mechanism, we demonstrated that AML cells with HMGCS1 highly expressed have developed endoplasmic reticulum (ER) stress, which promoted the expression of downstream molecules of UPR to protect AML cells. We utilized AML cells and mouse models to confirm that over-expression of HMGCS1 could promote AML cell growth, drug-resistance, while inhibit its’ apoptosis, corresponding to the activation of p-MEK1 and p-ERK1/2. Altogether, for the first time, our study has identified a novel role of HMGCS1 in AML through the MAPK pathway, which might provide an insight into the development of new targeted therapy for R/R AML.

The bone marrow stroma capacity to confer resistance to leukemia cells from Ara-C treatment as a prognostic factor for overall survival in AML.

e18500 Background: The interaction of acute myeloid leukemia (AML) with the bone marrow stroma (BMS) determines a protective microenvironment that favors leukemia development and resistance to chemotherapy. We developed an in vitro platform to study leukemia and stroma interaction, and showed that BMS secretes soluble factors protecting AML cells from Ara-C-induced apoptosis, which correlated with clinical patient outcomes. Methods: BMS from AML patients and healthy donors were cultured to perform chemo-sensitivity studies with Ara-C on THP-1 cells. A Resistance Factor was calculated (RF = IC50 stroma conditioned medium (CM)/IC50 control medium) to classify BMS as “protective” (PS: confers chemo-resistance) or “non-protective” (NPS: does not confer chemo-resistance). Characterization of myofibroblasts in BMS cultures was performed by WB. Quantification of cytokines from primary BMS CM was performed by Luminex. The differential expression of epithelial-mesenquimal-like (EMT-like) and stem cell-markers in THP-1 cells incubated with primary BMS CM was measured by qPCR. Results: We recruited 90 AML patients and 10 healthy BM donors. AML cohort showed 31 patients with PS and 59 patients with NPS. PS patients had a significant poor overall survival (OS) compared with NPS patients (44% versus 70% OS in 2.5 years, median survival 7.3 months versus 20.7 months, HR 2.36). The stroma from healthy BM donors did not confer resistance to THP-1 cells. BMS in both, PS and NPS have activated myofibroblasts and analysis of cytokine expression showed differential expression between groups. THP-1 cells incubated with PS showed significant expression of EMT- and stem cell-markers like Twist, Vimentin, CD44 and CD34 compared to THP-1 cells incubated with NPS and control medium. Conclusions: The capacity of the BMS from AML patients to modulate chemoresistance is a strong prognostic factor for OS, and PS patients have a worst OS compared with NPS patients. Stroma from healthy donors has a NPS phenotype. We propose that the leukemia is capable of educating the stroma to acquire secondarily the capacity to confer resistance to leukemia cells to favor tumor progression and chemoresistance.

Homoharringtonine potentiates the antileukemic activity of arsenic trioxide against acute myeloid leukemia cells

Relapse of minimal residual disease (MRD) is a major problem after conventional chemotherapy in patients with acute myeloid leukemia (AML). The bone marrow stroma can protect AML cells from insults of chemotherapy, partly contributing to AML relapse. Arsenic trioxide (ATO) is the main component of arsenical traditional Chinese medicines and has been widely used for the treatment of hematologic malignancies particularly acute promyelocytic leukemia over the past three decades. ATO acts through a direct arsenic binding to cysteine residues in zinc fingers located in promyelocytic leukemia protein (PML), thus killing the leukemia stem cells (LSCs). Our prior study has demonstrated that adhesion to stroma cells could render AML cells resistant to ATO but the detailed mechanism remains to be explored. Here, we report that the adhesion-induced resistance to ATO is related to the up-regulation of myeloid cell leukemia-1 (Mcl-1). Homoharringtonine (HHT) can potentiate the anti-leukemia effects of ATO on adhered AML cells by suppressing Mcl-1 through glycogen synthase kinase-3β (GSK3β). Furthermore, a potentiating effect of HHT on ATO was also observed in primary AML cells and AML xenografted tumors. Thus, these data indicate that HHT could enhance ATO anti-leukemia activity both in vitro and in vivo.

Leukemia-derived exosomes induced IL-8 production in bone marrow stromal cells to protect the leukemia cells against chemotherapy

AimsThe interplay between bone marrow stromal cells (BMSCs) and acute myeloid leukemia (AML) cells plays a critical role in AML drug resistance by secreting growth factors, cytokines, and extracellular vesicles. As kind of extracellular vesicles, exosomes consist of proteins and RNAs and regulate communication among cells. Main methodsThe BMSCs, HS5 cells, and AML cells were co-cultivated with transwell membranes, and treated with different doses of AML chemotherapy drug, etoposide. Key findingsFindings of our research proved that co-cultivation of BMSCs with AML cells defended AML against cell death triggered via etoposide, without having an impact on cell growth. An increase in the expression of the 70 kDa heat shock proteins (HSP70) as well as lysosomal associated membrane protein 3 (CD63) was observed in the exosomes from BMSC and AML, co-cultivated in conditioned media. Exosome repression in BMSC and AML co-cultivating system rebuilt the sensitivity of the KG1A cells to apoptosis triggered via etoposide, indicating that exosome modulated drug resistance in AML. Our study proved that exosomes arising from KG1A cells could propel BMSCs to generate IL-8, which could regulate the effect of etoposide treatment. Furthermore, IL-8 inhibition by its antibody increased the sensitivity of AML cells to cell death triggered via etoposide. SignificanceOur results suggested that exosomes secreted by AML cells is an essential communicator for the interaction of BMSCs and AML, which can protect AML cells from chemotherapy drug induced apoptosis.

Concomitant Targeting of FLT3 and BTK with CG'806 Overcomes FLT3-Inhibitor Resistance through Inhibition of Autophagy

Fms-like tyrosine kinase 3 (FLT3)-targeted therapy represents an important paradigm in the management of patients with highly aggressive FLT3 mutated acute myeloid leukemia (AML). However, clinical efficacy is usually transient and followed by emergence of resistance to FLT3-inhibitors (Borthakur et al., 2011; Cortes et al., 2013; Zhang et al., 2008). Such resistance often results from acquired mutations of TKD, which are frequently identified in D835, Y842 and F691 residues (Smith et al., 2015; Smith et al., 2012; Zhang et al., 2014). It was reported that the FLT3-ITD-targeting drug sorafenib can induce autophagy in human myeloid dendritic cells (Lin et al., 2013). Induction of autophagy has also been reported to play a crucial role in resistance to BCR-ABL targeted imatinib therapy in CML (Hekmatshoar et al., 2018). Additionally, inhibition of autophagy can re-sensitize cancer cells to apoptosis induction (Fitzwalter et al., 2018; Piya et al., 2017), suggesting that inhibition of autophagy may represent a novel therapeutic strategy for overcoming resistance to FLT3-targeted therapy.In the present study, we assessed autophagy levels in leukemia cell lines bearing different FLT3 mutations and in AML patient samples obtained from sorafenib-resistant patients. All tested resistant cell lines bearing TKD or ITD+TKD mutations showed increased basal autophagy levels. Resistant AML patient samples also demonstrated greater autophagy compared to matched pre-treatment samples in FLT3-mutated, but not in FLT3-wild type samples. Upregulation of autophagy was also observed in the bone marrow (BM)-mimetic microenvironment (i.e., hypoxia and the presence of mesenchymal stem cells (MSCs) in vitro. Inhibition of autophagy with chloroquine (CQ) potentiated quizartinib-induced apoptosis and partially abrogated MSC-mediated protection in FLT3-ITD- and/or D835-mutated AML cells by suppressing c-Myc, mTOR/S6K signaling and activating transcription factor 4 (ATF4).We also observed upregulation of BTK activation accompanied by increased autophagy levels in hypoxic/MSC co-culture with leukemic cells and in resistant primary patient samples. Co-targeting BTK and FLT3 with ibrutinib (or BTK siRNA) and quizartinib enhanced leukemic cell killing and abrogates MSC-mediated protection of FLT3 mutated leukemia cells. We further investigated a novel, highly potent small molecule pan-FLT3/pan-BTK kinase inhibitor CG-806 (IC50s 0.8 and 5.0 nM against FLT3-ITD and BTK, respectively) (Aptose, San Diego, CA). CG'806 abolished MSC/hypoxia-mediated protection of AML cells and induced apoptosis in FLT3-mutated cells in vitro. Of note, CG'806, but not quizartinib, exerted profound pro-apoptotic effects in primary AML patient cells harboring ITD+D835 mutations ex vivo. Further evaluation in a PDX leukemia model inoculated with the ITD+D835 mutated primary AML cells showed that CG'806 significantly reduced leukemia cell burden and benefited for mouse survival.Taken together, autophagy is associated with AML resistance to FLT3-targeted therapy, which can be overcome by the pan-FLT3/pan-BTK kinase inhibitor CG-806 through concomitant blockade of FLT3 and BTK. Co-targeting FLT3 and BTK might provide a strategy for preventing/overcoming FLT3 inhibitor resistance in AML patients with FLT3 mutations. Phase I trials of CG'806 are in preparation. DisclosuresZhang:Aptose Biosciences, Inc: Employment. Battula:United Therapeutics Inc.: Patents & Royalties, Research Funding. Konopleva:Stemline Therapeutics: Research Funding. Rice:Aptose Biosciences, Inc: Equity Ownership. Andreeff:Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Astra Zeneca: Research Funding; Jazz Pharma: Consultancy; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Amgen: Consultancy, Research Funding; Celgene: Consultancy.

SIRT3 Sumoylation Contributes to Chemoresistance in AML

Acute myeloid leukemia (AML) is a clonal disease originated from a rare population of malignant hematopoietic cells, called leukemic stem cells (LSCs), which is not only often resistant to standard chemotherapies, but also the major cause of relapse and eventual death of AML patients. The five-year survival of AML keeps as low as 27% for the last few decades. LSCs possess unique metabolism profiles such as higher rates of oxidative phosphorylation, and dependence on fatty acid oxidation for survival, which is distinct from normal hematopoietic cells, and, as a consequence, relatively low level of reactive oxygen species (ROS), a critical regulator for stemness maintenance. Therefore, targeting mitochondrial metabolism, especially ROS, may be a promising strategy to improve chemotherapy outcome for AML.We have previously found in hepatocarcinoma cells that SUMOylation is one of the important post-translational modifications for a variety of cellular proteins, and is capable of regulating the enzymatic activity of some key mitochondrial enzymes involved in the metabolic control, one example of which is SIRT3, a NAD+-dependent protein deacetylase. SIRT3 is reported to influence cellular metabolism and downregulate ROS generation by deacetylating mitochondrial anti-oxidant enzymes. The targets of SIRT3 include superoxide dismutase 2 (SOD2), manganese superoxide dismutase (MnSOD) and isocitrate dehydrogenase 2 (IDH2), which have been shown closely related to leukemogenesis. Since sophisticated regulation of ROS production is required for the maintenance of LSCs, we reproduced SUMOylation of SIRT3, and investigated its role in the mitochondrial metabolism in AML. In fact, SIRT3 SUMOylation at lysine 288 was also found in AML cells.To reveal the consequences of SIRT3 SUMOylation in AML, we constructed a plasmid expressing SIRT3-K288R that fails to be SUMOylated in AML cells. As a result, AML cells expressing SIRT3-K288R protected AML cells from as shown by apoptotic assays and quantitation of activated caspase 3 via reduction of not only total but also mitochondrial ROS production under chemotherapeutic agent-induced cell death comparing to those transfected with vector or overexpressing wild type SIRT3.To further investigate the role of SIRT3 de-SUMOylation in AML, we examined the influence of mitochondrial metabolism and anti-oxidant enzymes by SIRT3-K288R. SIRT3-K288R significantly downregulated the acetylation of mitochondrial anti-oxidant enzymes, such as SOD2, leading to decreased NADP/NADPH ratio and increased GSH/GSSG ratio. SIRT3 de-SUMOylation enhanced OCR but impaired ECAR under both basic and cytarabine treated conditions. We analyzed 18 primary AML samples to evaluate the correlation among SIRT3 SUMOylation, ROS level and chemoresistance. As we expected, low level of SIRT3 SUMOylation correlates with low cellular ROS level in both bulk AML and CD34+CD38- AML stem cells, and less sensitivity to cytarabine.Furthermore, MV4-11 cells bearing control vector, wild type SIRT3 or SIRT3-K288R were engrafted in NSG mice. Cytarabine was administered to the xenografts to evaluate the chemoresistance in these cell line-derived xenograft (CDX) mouse models. Consistent to the in vitro data, SIRT3-K288R reduced total and mitochondrial ROS in vivo, resulted in enhanced leukemogenesis and impaired survival.Taken together, our study showed that SIRT3 can be SUMOylated in AML. De-SUMOylation enhances SIRT3 deacetylase activity, and contribute to the chemoresistance of AML cells via altered mitochondrial metabolism and reduced ROS generation. Thus, SIRT3 and its de-SUMOylase can be utilized as potential therapeutic targets to improve AML chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2137: Osteoblast-lineage cells protect AML cells from cytarabine-induced apoptosis via a mechanism sensitive to HDACi and reduced cell-cell contact

Abstract The endosteal niche is a component of the bone marrow microenvironment that can serve to protect hematological malignancies such as acute myeloid leukemia (AML) from standard chemotherapies such as cytarabine (Ara-C). Surviving AML cells harbored by this niche can eventually lead to relapse. The endosteal niche is rich in osteoblast lineage cells. U937 or KG1a AML cell lines were cultured with or without osteoblast lineage cells (MC3T3 or W-20-17 cell lines), challenged with doses of 0µM, 0.1µM, 0.5µM, 1µM, 5µM, or 10µM of Ara-C, and assayed for apoptosis via annexin-V staining and flow cytometry. Osteoblast lineage cells (MC3T3 or W-20-17 cell lines) were able to protect AML cells (U937 or KG1a cell lines) from Ara-C-induced apoptosis. Histone deacetylase inhibitors (HDACi) globally alter gene expression within cells. When we pre-treated osteoblast lineage cells (MC3T3) with the HDACi vorinostat (suberoylanilide hydroxamic acid, SAHA), it reduced the ability of the osteoblast lineage cells (MC3T3) to protect AML cells (U937) from Ara-C-induced apoptosis, which we have previously confirmed in the KG1a AML cell line as well. This indicates that osteoblast lineage cell-mediated protection of AML from Ara-C occurs via an HDACi sensitive mechanism. To begin to further explore the mechanism of action, we co-cultured AML cells (KG1a or U937) with and without osteoblast lineage cells (MC3T3) in the presence or absence of a transwell. We found that the presence of the transwell reduced the osteoblast lineage cell-mediated protection, indicating that osteoblast lineage cell-mediated protection of AML from Ara-C is cell contact dependent. Thus, osteoblast lineage cells can protect AML cells from Ara-C induced apoptosis, this protection can be reduced by pre-treatment of the osteoblast lineage cells with the HDACi vorinostat, and osteoblast lineage cell-mediated protection from Ara-C is cell contact dependent. These studies begin to characterize the mechanisms of osteoblast lineage cell-mediated protection of AML from Ara-C. Manipulating the protective properties of osteoblast lineage cells of the endosteal niche may help make AML cells more susceptible to chemotherapeutics. Therefore, developing combination therapies that target the protective mechanisms of osteoblast-lineage cells may help to further deplete the bone marrow microenvironment of AML cells and prevent relapse of disease. Citation Format: Rosalie M. Sterner, Kimberly N. Kremer, Meagan R. Rollins, Amel Dudakovic, Jennifer J. Westendorf, Andre J. van Wijnen, Karen E. Hedin. Osteoblast-lineage cells protect AML cells from cytarabine-induced apoptosis via a mechanism sensitive to HDACi and reduced cell-cell contact [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 2137.

Roles of the bone marrow niche in hematopoiesis, leukemogenesis, and chemotherapy resistance in acute myeloid leukemia

ABSTRACTObjectives: To summarize the effects of the bone marrow niche on hematopoiesis and leukemogenesis and discuss the chemotherapy resistance that can arise from interactions between the niche and leukemia stem cells.Methods: We review the major roles of the bone marrow niche in cell proliferation, adhesion and drug resistance. The signaling pathways and major molecular participants in the niche are discussed. We also address potential niche-targeting strategies for the treatment of acute myeloid leukemia (AML).Results: The bone marrow niche supports normal hematopoiesis and affects acute myeloid leukemia (AML) initiation, progression and chemotherapy resistance.Discussion: AML is a group of heterogeneous malignant diseases characterized by the excessive proliferation of hematopoietic stem and/or progenitor cells. Even with intensive chemotherapy regimens and stem cell transplantation, the overall survival rate for AML is poor. The bone marrow niches of malignant cells are remodeled into a leukemia-permissive environment, and these reformed niches protect AML cells from chemotherapy-induced cell death. Inhibiting the cellular and molecular interactions between the niche and leukemia cells is a promising direction for targeted therapies for AML treatment.Conclusions: Interactions between leukemia cells and the bone marrow niche influence hematopoiesis, leukemogenesis, and chemotherapy resistance in AML and require ongoing study. Understanding the mechanisms that underlie these interactions will help identify rational niche-targeting therapies to improve treatment outcomes in AML patients.

2559 Role of tissue non-specific alkaline phosphatase (TNAP) in promoting the survival of acute myeloid leukemia (AML) cells within the bone marrow microenvironment

OBJECTIVES/SPECIFIC AIMS: Treatment of acute myeloid leukemia (AML) is challenging, as apoptosis-resistant AML cells often persist within the bone marrow microenvironment despite chemotherapy. The overall goal of our laboratory is to identify and ultimately target the bone marrow factors that protect AML cells. METHODS/STUDY POPULATION: Using cell cultures, we previously reported that SDF-1 (CXCL12), an abundant bone marrow chemokine, induces apoptosis of isolated CXCR4+ AML cells, including freshly isolated bone marrow-derived AML cells from approximately one-third of AML patients. However, co-culture of AML cells with differentiating osteoblasts protected AML cells from apoptosis. RESULTS/ANTICIPATED RESULTS: Histone deacetylase inhibitors (HDACi) abrogated the ability of osteoblasts to protect AML cells and altered expression of matrix mineralization genes including tissue nonspecific alkaline phosphatase (TNAP). A different drug, cyclosporine A (CSA), similarly inhibited osteoblast-mediated protection of AML cells and reduced TNAP expression. Specifically targeting osteoblast TNAP via siRNA was sufficient to prevent osteoblasts from protecting AML cells in co-cultures. In addition, we are targeting TNAP enzymatically. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results indicate that targeting TNAP may be useful in AML treatment to render the bone marrow microenvironment more hostile to leukemic cell survival.

Protein Kinase C Epsilon Is a Key Regulator of Mitochondrial Redox Homeostasis in Acute Myeloid Leukemia.

Purpose: The intracellular redox environment of acute myeloid leukemia (AML) cells is often highly oxidized compared to healthy hematopoietic progenitors and this is purported to contribute to disease pathogenesis. However, the redox regulators that allow AML cell survival in this oxidized environment remain largely unknown.Experimental Design: Utilizing several chemical and genetically-encoded redox sensing probes across multiple human and mouse models of AML, we evaluated the role of the serine/threonine kinase PKC-epsilon (PKCε) in intracellular redox biology, cell survival and disease progression.Results: We show that RNA interference-mediated inhibition of PKCε significantly reduces patient-derived AML cell survival as well as disease onset in a genetically engineered mouse model (GEMM) of AML driven by MLL-AF9. We also show that PKCε inhibition induces multiple reactive oxygen species (ROS) and that neutralization of mitochondrial ROS with chemical antioxidants or co-expression of the mitochondrial ROS-buffering enzymes SOD2 and CAT, mitigates the anti-leukemia effects of PKCε inhibition. Moreover, direct inhibition of SOD2 increases mitochondrial ROS and significantly impedes AML progression in vivo Furthermore, we report that PKCε over-expression protects AML cells from otherwise-lethal doses of mitochondrial ROS-inducing agents. Proteomic analysis reveals that PKCε may control mitochondrial ROS by controlling the expression of regulatory proteins of redox homeostasis, electron transport chain flux, as well as outer mitochondrial membrane potential and transport.Conclusions: This study uncovers a previously unrecognized role for PKCε in supporting AML cell survival and disease progression by regulating mitochondrial ROS biology and positions mitochondrial redox regulators as potential therapeutic targets in AML. Clin Cancer Res; 24(3); 608-18. ©2017 AACR.

Bone marrow stromal-cell line HS-5 affects apoptosis of acute myeloid leukemia cells HL-60 through GLI1 activation

The Hedgehog (HH) pathway regulates cell proliferation and survival and contributes to tumorigenesis. However, not much is known about the role of the HH-signaling system in the bone marrow microenvironment during the development of Acute Myeloid Leukemia (AML). The aim of this study was to investigate the anti-leukemic effects of the HH-signaling pathway and molecular mechanisms of apoptosis induction in AML cells, by blocking the hedgehog signaling pathway during co-culture of AML cells with Bone-Marrow Stromal Cells (BMSCs). It was found that the HS-5 line of BMSCs protected AML cells from spontaneous apoptosis via up-regulation of transcription factors GLI1, BCL-2 and BCL-XL. These results indicate that the up-regulation of the transcription factors involved activation of the hedgehog pathway. Thus, targeting a microenvironment-related signaling pathway may be a novel approach to AML therapy.

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.

Abstract 5930: Osteoblasts protect AML cells from cytarabine-induced death

Abstract The hematological malignancy acute myeloid leukemia (AML) interacts closely with osteoblasts within the protective bone marrow microenvironment. The bone marrow microenvironment protects tumor cells from chemotherapies, which can prevent sufficient eradication of tumor cells. To study the role of osteoblasts in the bone marrow microenvironment, our lab utilized a co-culture model of osteoblasts (MC3T3 osteoblast cell line) and acute myelogenous leukemia cells (KG1a AML cell line or AML patient samples from bone marrow aspirates). Osteoblasts were cultured with AML cells, or AML cells were cultured alone; AML cells were challenged with the standard chemotherapeutic agent cytarabine (Ara-C) at doses of 0µM, 0.1µM, 0.5µM, 1µM, 5µM, or 10µM in the presence or absence of osteoblasts; and AML cells were assayed by flow cytometry to assess cell death via annexin-V staining. Our lab has previously found that differentiating osteoblasts protect AML cells from an apoptosis inducing agent naturally present in the bone marrow. We now show that osteoblasts are also capable of protecting AML cells from the standard chemotherapeutic cytarabine. In addition, we have found that treatment of osteoblasts with the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) prevents these treated osteoblasts from protecting AML cells from cytarabine treatment, which is consistent with our lab’s previous findings that HDACi treatment inhibits protection of AML cells from an apoptosis inducing agent naturally present in the bone marrow. We have preliminary data that indicates that TAZS89A over-expression, a constitutively active form of TAZ, which is a transcriptional modulator that regulates osteoblast differentiation, may be sufficient to inhibit osteoblast mediated protection of AML cells from cytarabine. This finding would be consistent with the HDACi manipulated Nherf1-protein phosphatase 1α-TAZ signaling pathway that we have previously found to be sufficient to inhibit protection of AML cells from an apoptosis inducing agent naturally present in the bone marrow. Overall, these studies have delivered insights into the role of osteoblasts in protecting AML cells from chemotherapy in the bone marrow microenvironment and begun the characterization of mechanisms and targets responsible for the protective effects of osteoblasts. Manipulating differentiating osteoblasts within the bone marrow microenvironment therapeutically could aid in more complete destruction of the tumor cell burden and improve patient survival. Citation Format: Rosalie M. Sterner, Kimberly N. Kremer, Amel Dudakovic, Jennifer J. Westendorf, Andre J. van Wijnen, Karen E. Hedin. Osteoblasts protect AML cells from cytarabine-induced death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5930. doi:10.1158/1538-7445.AM2017-5930

Manipulating the Bone Marrow Microenvironment to Prevent Survival of AML Cells

The bone marrow microenvironment acts as a protective niche for malignant cells. Within the bone marrow microenvironment is the endosteal niche, which is the area where the ossified surface of the bone and marrow cavity interact. The endosteal niche provides the opportunity for hematological malignancies such as acute myeloid leukemia (AML) of the bone marrow to interact with osteoblasts from the bone. To study the interaction between AML cells and osteoblasts, our lab developed a co‐culture model of osteoblasts (MC3T3 osteoblast cell line) and AML cells (KG1a cell line or patient samples from bone marrow aspirates). AML cells expressing high levels of CXCR4 were cultured alone or with osteoblasts and then challenged with SDF‐1, the ligand to CXCR4 and a chemokine readily abundant in the bone marrow microenvironment that our lab has previously shown actually induces apoptosis in AML cells expressing high levels of CXCR4 and that osteoblasts are able to protect AML cells from SDF‐1 induced apoptosis. Our lab now shows that treating osteoblasts with cyclosporine (CSA) prevents their ability to effectively protect AML cells from SDF‐1 induced cell death as assayed by annexin‐V staining of AML cells by flow cytometry. CSA treatment of osteoblasts decreased protein levels of tissue non‐specific alkaline phosphatase (ALPL), a phosphatase necessary for osteoblast mineralization, compared to vehicle control. As such, osteoblasts were treated with control siRNA or ALPL siRNA. CXCR4 expressing AML cells were then co‐cultured with the transfected osteoblasts and challenged with SDF‐1. siRNA knockdown of ALPL in osteoblasts was sufficient to prevent protection of AML cells from the apoptotic stimuli. These studies indicate that osteoblast differentiation plays a role in mediating the protection of AML cells. Therefore, targeting specific differentiation factors in osteoblasts may help to make to bone marrow microenvironment a more hostile environment for AML cells and aid in better malignancy eradication.Support or Funding InformationThis study was supported by NIH R21 CA194217 (to K.E.H.). R.M.S. is supported by the Mayo Clinic Medical Scientist Training Program Robert L. Howell Physician‐Scientist Scholarship.

Microrna-494 Activation Suppresses Bone Marrow Stromal Cell-Mediated Drug Resistance in Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is not sensitive to chemotherapy partially because of the protection of AML cells by mesenchymal stromal cells (MSCs). Our previous studies found that MSCs protected AML cells from apoptosis through the c-Myc-dependent pathway. However, the mechanism by which MSCs regulate c-Myc in AML cells is still unknown. To elucidate the mechanism, we performed microRNA array analysis of AML cell lines and validated by TaqMan realtime PCR. The results showed that the expression of microRNA-494 (miR-494) in AML cells after coculture with MSCs was down-regulated. Reporter gene analysis confirmed miR-494 as one of the regulators of c-Myc. In the coculture system, activation of miR-494 in AML cells suppressed proliferation and induced apoptosis of AML cells in vitro. After addition of mitoxantrone to the coculture system, the proliferation of AML cells with miR-494 activation was suppressed more than that of control cells. After subcutaneous injection of AML cell lines in combination with MSC, tumor growth was suppressed in mice injected with miR-494-overexpressing AML cells. The rate of tumor formation was even lower after mitoxantrone treatment in the miR-494 overexpressing group. Moreover, miR-494 activation resulted in a decrease of leukemic cell counts in peripheral blood and bone marrow and prolonged survival in mice injected with miR-494-overexpressing AML cellls and MSCs compared to the control mice. Our results indicate that miR-494 suppresses drug resistance in AML cells by down-regulating c-Myc through interaction with MSCs and that miR-494 therefore is a potential therapeutic target. DisclosuresNo relevant conflicts of interest to declare.

Therapeutic targeting of necroptosis by Smac mimetic bypasses apoptosis resistance in acute myeloid leukemia cells.

Resistance to apoptosis, for example due to overexpression of Inhibitor of Apoptosis (IAP) proteins, is associated with poor prognosis in acute myeloid leukemia (AML). Here, we identify that Smac mimetics such as BV6, which antagonizes IAP proteins, elicit necroptosis in AML cells, in which apoptosis is inhibited pharmacologically by caspase inhibitors or genetically by caspase-8 knockdown. Importantly, BV6 triggers necroptosis also in apoptosis-resistant patient-derived AML blasts, underlining the clinical relevance of our findings. Mechanistically, we show that BV6-induced cell death depends on key components of necroptosis signaling such as RIP1, RIP3 and MLKL, since pharmacological or genetic inhibition of these proteins significantly protects AML cells from BV6-mediated cell death, whereas PGAM5 is dispensable. Interestingly, we identify constitutive tumor necrosis factor-alpha (TNFα) secretion and an autocrine/paracrine TNFα loop as critical mediators of BV6-induced necroptosis in AML cell lines and patient-derived blasts, as the TNFα-blocking antibody Enbrel or tumor necrosis factor-alpha receptor 1 (TNFR1) knockdown significantly rescue cell death. Notably, AML cells exhibit high basal levels of TNFα compared to non-malignant CD34+ cells, which is further increased by BV6. In conclusion, this is the first report showing that Smac mimetics circumvent apoptosis resistance in AML cells by inducing necroptosis in a TNFα-dependent manner, which has important implications for the development of new strategies to overcome treatment resistance in AML.

MicroRNA-494 Activation Suppresses Bone Marrow Stromal Cell-Mediated Drug Resistance in Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is not sensitive to chemotherapy partially because of the protection of AML cells by mesenchymal stromal cells (MSCs). Our previous studies found that MSCs protected AML cells from apoptosis through the c-Myc-dependent pathway. However, the mechanism by which MSCs regulate c-Myc in AML cells is still unknown. To elucidate the mechanism, we performed microRNA array analysis of AML cell lines and validated by TaqMan realtime PCR. The results showed that the expression of microRNA-494 (miR-494) in AML cells after coculture with MSCs was downregulated. Reporter gene analysis confirmed miR-494 as one of the regulators of c-Myc. In the coculture system, activation of miR-494 in AML cells suppressed proliferation and induced apoptosis of AML cells in vitro. After addition of mitoxantrone to the coculture system, the proliferation of AML cells with miR-494 activation was suppressed more than that of control cells. After subcutaneous injection of AML cell lines in combination with MSC, tumor growth was suppressed in mice injected with miR-494-overexpressing AML cells. The rate of tumor formation was even lower after mitoxantrone treatment in the miR-494 overexpressing group. Moreover, miR-494 activation resulted in a decrease of leukemic cell counts in peripheral blood (PB) and bone marrow, and prolonged survival in mice injected with miR-494-overexpressing AML cellls and MSCs compared to the control mice. Our results indicate that miR-494 suppresses drug resistance in AML cells by downregulating c-Myc through interaction with MSCs and that miR-494 therefore is a potential therapeutic target. J. Cell. Physiol. 232: 1387-1395, 2017. © 2016 Wiley Periodicals, Inc.

The CDK9 Inhibitor Dinaciclib Exerts Potent Apoptotic and Antitumor Effects in Preclinical Models of MLL-Rearranged Acute Myeloid Leukemia.

Translocations of the mixed lineage leukemia (MLL) gene occur in 60% to 80% of all infant acute leukemias and are markers of poor prognosis. MLL-AF9 and other MLL fusion proteins aberrantly recruit epigenetic regulatory proteins, including histone deacetylases (HDAC), histone methyltransferases, bromodomain-containing proteins, and transcription elongation factors to mediate chromatin remodeling and regulate tumorigenic gene expression programs. We conducted a small-molecule inhibitor screen to test the ability of candidate pharmacologic agents targeting epigenetic and transcriptional regulatory proteins to induce apoptosis in leukemic cells derived from genetically engineered mouse models of MLL-AF9-driven acute myeloid leukemia (AML). We found that the CDK inhibitor dinaciclib and HDAC inhibitor panobinostat were the most potent inducers of apoptosis in short-term in vitro assays. Treatment of MLL-rearranged leukemic cells with dinaciclib resulted in rapidly decreased expression of the prosurvival protein Mcl-1, and accordingly, overexpression of Mcl-1 protected AML cells from dinaciclib-induced apoptosis. Administration of dinaciclib to mice bearing MLL-AF9-driven human and mouse leukemias elicited potent antitumor responses and significantly prolonged survival. Collectively, these studies highlight a new therapeutic approach to potentially overcome the resistance of MLL-rearranged AML to conventional chemotherapies and prompt further clinical evaluation of CDK inhibitors in AML patients harboring MLL fusion proteins.

Acute myeloid leukemia in the vascular niche

The greatest challenge in treating acute myeloid leukemia (AML) is refractory disease. With approximately 60–80% of AML patients dying of relapsed disease, there is an urgent need to define and target mechanisms of drug resistance. Unfortunately, targeting cell-intrinsic resistance has failed to improve clinical outcomes in AML. Emerging data show that cell-extrinsic factors in the bone marrow microenvironment protect and support AML cells. The vascular niche, in particular, regulates AML cell survival and cell cycling by both paracrine secretion and adhesive contact with endothelial cells. Moreover, AML cells can functionally integrate within vascular endothelia, undergo quiescence, and resist cytotoxic chemotherapy. Together, these findings support the notion of blood vessels as sanctuary sites for AML. Therefore, vascular targeting agents may serve to remit AML. Several early phase clinical trials have tested anti-angiogenic agents, leukemia mobilizing agents, and vascular disrupting agents in AML patients. In general, these agents can be safely administered to AML patients and cardiovascular side effects were reported. Response rates to vascular targeting agents in AML have been modest; however, a majority of vascular targeting trials in AML are monotherapy in design and indiscriminate in patient recruitment. When considering the chemosensitizing effects of targeting the microenvironment, there is a strong rationale to build upon these early phase clinical trials and initiate phase IB/II trials of combination therapy where vascular targeting agents are positioned as priming agents for cytotoxic chemotherapy.