Pediatric Acute Myeloid Leukemia Cells Research Articles

Constitutively Synergistic Multiagent Drug Formulations Targeting MERTK, FLT3, and BCL-2 for Treatment of AML.

Although high-dose, multiagent chemotherapy has improved leukemia survival rates, treatment outcomes remain poor in high-risk subsets, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in infants. The development of new, more effective therapies for these patients is therefore an urgent, unmet clinical need. The dual MERTK/FLT3 inhibitor MRX-2843 and BCL-2 family protein inhibitors were screened in high-throughput against a panel of AML and MLL-rearranged precursor B-cell ALL (infant ALL) cell lines. A neural network model was built to correlate ratiometric drug synergy and target gene expression. Drugs were loaded into liposomal nanocarriers to assess primary AML cell responses. MRX-2843 synergized with venetoclax to reduce AML cell density in vitro. A neural network classifier based on drug exposure and target gene expression predicted drug synergy and growth inhibition in AML with high accuracy. Combination monovalent liposomal drug formulations delivered defined drug ratios intracellularly and recapitulated synergistic drug activity. The magnitude and frequency of synergistic responses were both maintained and improved following drug formulation in a genotypically diverse set of primary AML bone marrow specimens. We developed a nanoscale combination drug formulation that exploits ectopic expression of MERTK tyrosine kinase and dependency on BCL-2 family proteins for leukemia cell survival in pediatric AML and infant ALL cells. We demonstrate ratiometric drug delivery and synergistic cell killing in AML, a result achieved by a systematic, generalizable approach of combination drug screening and nanoscale formulation that may be extended to other drug pairs or diseases in the future.

Abstract 3729: Targeting the writers of enhancers as a new therapeutic strategy in pediatric AML

Abstract Introduction: Children with acute myeloid leukemia (AML) urgently need new targeted therapeutics to improve their outcomes. Super-enhancers (SEs) are extensive chromatin regions denoted by abundant histone 3 lysine 27 acetylation (H3K27ac), which are associated with high-level expression of cell-identity genes, including oncogenes. SE-regulated genes have unique sensitivity to transcriptional inhibitors, allowing for cancer type-specific approaches. Thus, targeting CBP/EP300 (the highly homologous enzymes responsible for catalyzing H3K27 acetylation) may be a specific strategy to target lineage-specifying oncogenes in AML. However, to date, the high degree of homology between EP300 and CBP has limited chemical strategies to tissue-specific disruption. Gene editing studies have shown that CBP, and not EP300, is essential for maintaining normal hematopoiesis, while EP300 regulates AML growth and myeloid differentiation. We hypothesized that selectively targeting EP300 would suppress AML growth while sparing effects on normal myeloid differentiation. Methods: We studied publicly available expression and CRISPR screening databases for EP300 and CBP data in AML. We treated pediatric AML (pAML) cell lines and patient samples with the EP300/CBP HAT inhibitor A485 and the EP300-selective PROTAC [PROteolysis TArgeting Chimera]) JQAD1, and measured effects on cell growth by CellTiter-Glo, and apoptosis, differentiation, and cell cycle changes by flow cytometry. We treated pAML cells with compounds for 2 hours and performed mass spectrometry on HPLC-purified H3 proteins. Results: pAML patient samples have higher EP300 expression than normal bone marrow samples in the TARGET dataset, and AML cell lines have enhanced dependency on EP300 over CBP by CRISPR knockout analysis. Treatment of pAML cell lines with A485 slowed proliferation and induced apoptosis, cell cycle arrest, and differentiation. In contrast, equimolar concentrations of JQAD1 had more marked antileukemic effects in both pAML cell lines and primary samples. Immunoblotting analysis demonstrated reduced H3K27ac in pAML cells treated with both JQAD1 and A485, but EP300 degradation only with treatment with JQAD1, consistent with findings in other cancer models. Studies on the effects of EP300 degradation versus combined EP300/CBP inhibition on global and SE-regulated gene expression are ongoing. Middle-down mass spectrometry recapitulates decreased H3K27 acetylation with both agents. JQAD1 combined with the BCL2 inhibitor Venetoclax has a synergistic effect on cell line and patient sample growth, with coordinate induction of apoptosis and differentiation. A four-arm study of this combination in a pAML patient-derived xenograft model is ongoing. Conclusion: Selective degradation of EP300 using the PROTAC JQAD1 is a new strategy that demonstrates preclinical efficacy in pAML as a single agent and in combination with BCL2 inhibitors. Citation Format: Joanna S. Yi, Kevin Duong, Abiola Obawemimo, Tingjian Wang, Faith Joseph, Nicolas L. Young, Stephen Mack, Jun Qi, Adam Durbin. Targeting the writers of enhancers as a new therapeutic strategy in pediatric AML. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3729.

Compound K: A systematic review of its anticancer properties and probable mechanisms.

Panax ginseng is a common natural product, which is well-known to have a wide range of pharmacological activities in cancer. Its metabolite, compound K (CK), has been reported to have anticancer activity. We aimed to systematically review the literature for evidence of anticancer effects of CK. We conducted a systematic search in eight databases. We included all in vitro and in vivo studies investigating the anticancer effects of CK with no restrictions. Quality assessment was applied by ToxRTool. Fifty-four articles were included in our study. The purity of CK in our included studies was at least 95%. The in vitro studies reported that CK had a potential anticancer activity on several cell lines including human lung cancer cell lines (A549, PC-9), nasopharyngeal carcinoma cell line (Hk-1), liver cancer cell line (BEL 7402), and pediatric acute myeloid leukemia cell lines (Kasumi-1, MV4-11). The in vivo studies reported a significant decrease in tumor volume in mice treated with CK. CK is a potential supplementary treatment in cancer chemotherapies. The safety and further clinical trials of CK should be explored for future drug development.

Tumor-suppressing effects of miR-381-3p in pediatric acute myeloid leukemia via ROCK1 downregulation.

MicroRNA (miR)-381-3p is the newly discovered tumor-associated miRNA, which is frequently associated with diverse human malignancies; but, it is still unknown about its effect on acute myeloid leukemia (AML) in children. This work focused on exploring miR-381-3p's effect on childhood AML and identifying the possible mechanisms facilitating new treatment development. Using qRT-PCR analysis, miR-381-3p expression remarkably reduced in pediatric AML patients and AML cell lines (HL-60 and U937). Following transfection of miR-381-3p mimic or inhibitor into HL-60 and U937 cells, we conducted MTT assay to evaluate cell proliferation, flow cytometry (FCM) to measured cell apoptosis and cell cycle, whereas Transwell assays to detect cell invasion and migration. Our results demonstrated that miR-381-3p overexpression remarkably repressed cell growth, invasion and migration; additionally, miR-381-3p overexpression resulted in arrest of cell cycle and enhanced cell apoptosis. In contrast, miR-381-3p knockdown led to an opposite effect. Moreover, we predicted miR-381's target gene and validated it by luciferase reporter assay and TargetScan, separately. We identified miR-381-3p's binding site in ROCK1 3'-UTR. As revealed by Western-blot (WB) assay, miR-381-3p overexpression notably suppressed ROCK1 level. Moreover, restoring ROCK1 expression abolished miR-381-3p's inhibition on cell proliferation, invasion and migration. Data in this work indicated the role of miR-381-3p as the tumor suppressor within pediatric AML by targeting ROCK1. Therefore, miR-381-3p might serve as a potential therapeutic target for the treatment of pediatric AML.

Identification of Circular RNA Circ_0003256 as a Novel Player in Pediatric Acute Myeloid Leukemia

Aberrant expression of circular RNAs (circRNAs) is tightly associated with the pathogenesis of human cancers, including pediatric acute myeloid leukemia (AML). In this report, we sought to define the precise action of circ_0003256 in the pathogenesis of pediatric AML. Circ_0003256, microRNA (miR)-582-3p, and protein kinase cAMP-activated catalytic subunit beta (PRKACB) were quantified by quantitative real-time polymerase chain reaction and Western blot. Cell proliferation, cycle distribution, and apoptosis were estimated by MTT, 5-ethynyl-2'-deoxyuridine, and flow cytometry assays, respectively. Direct relationships among circ_0003256, miR-582-3p, and PRKACB were verified by a dual-luciferase reporter and RNA pull-down assays. Our data indicated that circ_0003256 was highly expressed in pediatric AML patients and cells. Suppression of circ_0003256 hindered cell proliferation and promoted apoptosis in THP-1 and MV4-11 cells. Mechanistically, circ_0003256 contained functional binding sites for miR-582-3p, and circ_0003256 suppression influenced cell behaviors by upregulating miR-582-3p. MiR-582-3p directly targeted and inhibited PRKACB and the inhibition of PRKACB phenocopied miR-582-3p overexpression in regulating cell functional behaviors. Moreover, circ_0003256 involved the posttranscriptional regulation of PRKACB through miR-582-3p. Our findings identify that suppression of circ_0003256 impedes the malignant behaviors of pediatric AML cells by regulating PRKACB expression by competing for shared miR-582-3p.

An Insight into the Role of Mesothelin in Pediatric Acute Myeloid Leukemia

Pediatric acute myeloid leukemia (AML) is characterized by the malignant transformation of hematopoietic stem and progenitor cells that lead to the accumulation of immature myeloid cells in the bone marrow. Standard treatment of AML includes intense cytotoxic chemotherapy and hematopoietic stem cell transplants with relatively low cure rates. Targeted therapy options for AML are limited. Mesothelin (MSLN) is a 40‐kDa glycosylphosphatidylinositol (GPI)‐anchored protein that is highly expressed on many tumors, but lacks expression in many normal tissues, making it an excellent target for cancer therapies. More recently, the identification of MSLN expression in a subset (about one‐third) of pediatric AML patients warranted preclinical studies with MSLN‐targeted therapies, including immunotherapy. Recently, our lab utilized bispecific T cell engaging antibodies to recruit T cells to MSLN‐expressing leukemic cells, increasing in vivo survival rates in patient‐derived xenograft models.MSLN has been shown to play a role in promoting proliferation and invasion in mesothelioma, ovarian cancer, and pancreatic adenocarcinoma, mainly through interactions with MUC16/CA125. Current investigative efforts in AML focus on MSLN‐targeted therapies, while the function of MSLN in AML remains unresolved. Previously, we identified a correlation between MSLN expression and extramedullary disease, as well as enhanced leukemic burden in xenograft models. Here, we report that MSLN expression is associated with increased rates of cell proliferation and migration.The proliferation rate of MSLN‐negative AML cell lines, MV4;11 & NOMO‐1‐KO, and two MSLN‐expressing cell lines, MV4;11‐MSLN & NOMO‐1, was evaluated by BrDU proliferation assay or direct measurement of cell numbers over 96 hours. MSLN‐expressing cells proliferated at an increased rate comparative to the non‐expressing cells. Consistent with higher proliferation rate in MSLN‐expressing cells, these cells showed reduced percentage of G1 population and increased population in the S phase of the cell cycle. In a Transwell migration assay, higher number of MV4;11‐MSLN cells migrated towards serum compared to their wild‐type counterpart, indicating that MSLN expression promoted cell migration. Our data suggest that MSLN expression in pediatric AML cells enhances proliferation and migration. Therefore, targeting MSLN may be beneficial to curb AML progression.

Small Molecule Screening of Primary Human Acute Myeloid Leukemia Using Co-culture and Multiplexed FACS Analysis.

Ex vivo culture of primary acute myeloid leukemia (AML) cells is notoriously difficult due to spontaneous differentiation and cell death, which hinders mechanistic and translational studies. To overcome this bottleneck, we have implemented a co-culture system, where the OP9-M2 stromal cells support the growth, but most notably limit the differentiation of primary AML cells, thus allowing for mechanistic studies in vitro. Additionally, the co-culture on OP9-M2 stromal is superior in preserving surface marker expression of primary (adult and pediatric) AML cells in comparison to stroma-free culture. Thus, by combining the co-culture with multicolor, high-throughput FACS, we can evaluate the effect of hundreds of small molecules on multi-parametric processes including: cell survival, stemness (leukemic stem cells), and myeloid differentiation on the primary AML cells at a single-cell level. This method streamlines the identification of potential therapeutic agents, but also facilitates combinatorial screening aiming, for instance, at dissecting the regulatory pathways in a patient-specific manner. Graphic abstract: Schematic representation of the ex vivo small molecule screening of primary human acute myeloid leukemia. Irradiated, sub-confluent OP9-M2 stromal cells are plated in half-area 96 wells plates 4-16 h prior to adding primary AML cells. Compounds are added 36-48 h later and effects on cell number, leukemic stem cell population, and myeloid differentiation are quantifed by FACS after 4 days of treatment.

CircRNF220, not its linear cognate gene RNF220, regulates cell growth and is associated with relapse in pediatric acute myeloid leukemia

BackgroundCircular RNAs (circRNAs) constitute a family of transcripts with unique structures and have been confirmed to be critical in tumorigenesis and to be potential biomarkers or therapeutic targets. However, only a few circRNAs have been functionally characterized in pediatric acute myeloid leukemia (AML).MethodsHere, we investigated the expression pattern of circRNAs in pediatric AML using a circRNA microarray. The characteristics, potential diagnostic value, and prognostic significance of circRNF220 were evaluated. A series of functional experiments were performed to investigate the role of circRNF220 in primary pediatric AML cells. Then we investigated the aberrant transcriptional networks regulated by circRNF220 in primary AML cells by RNA-seq. Furthermore, biotin RNA pulldown assays were implemented to verify the relationship between circRNF220 and miR-30a.ResultsWe identified a circRNA, circRNF220, which was specifically abundant in and accumulated in the peripheral blood and bone marrow of pediatric patients with AML. It could distinguish AML from ALL and other hematological malignancies with high sensitivity and specificity. Significantly, circRNF220 expression independently predicted prognosis, while high expression of circRNF220 was an unfavorable prognostic marker for relapse. Furthermore, we characterized the function of circRNF220 and found that circRNF220 knockdown specifically inhibited proliferation and promoted apoptosis in AML cell lines and primary cells. Mechanistically, circRNF220 may act as an endogenous sponge of miR-30a to sequester miR-30a and inhibit its activity, which increases the expression of its targets MYSM1 and IER2 and implicated in AML relapse.ConclusionsCollectively, these findings demonstrated that circRNF220 could be highly efficient and specific for the accurate diagnosis of pediatric AML, with implications for relapse prediction.

Eltrombopag and its iron chelating properties in pediatric acute myeloid leukemia.

Pediatric acute myeloid leukemia (AML) represents 20% of total childhood leukemia diagnoses and is characterized by poor prognosis with a long-term survival rate around the 50%, when patients are properly treated. The standard treatment for pediatric AML currently consists in a combination of cytarabine (Ara-C) and antracycline. Iron plays an important role in cancer development and progression. Targeting iron and its metabolism mediators could be a novel therapeutic strategy in cancer.Deferasirox (DFX) inhibits cancer cell proliferation and its use as an antiblastic drug could be suggested. Eltrombopag (ELT), a thrombopoietin receptor agonist used in immunethrombocytopenia, shows anticancer properties related to its emerging iron chelating properties. We compare the anticancer effect of classically used cytarabine with DFX and ELT effects in a pediatric AML cell line, THP-1, in order to identify innovative and more effective therapeutic strategies. ELT and DFX reduce intracellular iron concentration by inhibiting its uptake and by promoting its release. In particular, even though further investigations are needed to better understand the extact underlying action mechanisms, we demonstrated that ELT improves cytarabine antineoplastic activity in pediatric AML cell line.

Atovaquone Demonstrates Single Agent Activity and an Additive Effect in Combination with Cytotoxic Chemotherapy (Cytarabine and Daunorubicin) in AML

Background: To improve outcomes for pediatric acute myeloid leukemia (AML), identifying and targeting mechanisms of therapy resistance is critical. Recently, the antimicrobial drug atovaquone (AQ) was shown to reduce IL-6/STAT3 signaling in vitro and to improve relapse-free survival in adult AML patients who used AQ for prophylaxis after bone marrow transplant (Xiang et al, Blood, 2016). Clinically, this FDA approved drug is well tolerated, even in patients with organ dysfunction. It has established pediatric dosing, and plasma concentrations at standard doses are typically 40-80µM. We performed in vitro and in vivo experiments to evaluate the efficacy of AQ as a single agent and in combination with standard induction chemotherapy (cytarabine, daunorubicin, and etoposide). In order to ensure clinical relevance, all experiments examined cytotoxicity at clinically achievable doses.Methods: AML cell lines were incubated with vehicle control or increasing doses of AQ (1µM - 30µM) for 24, 48, 72, or 96 hours, and then apoptosis was quantified by Annexin-V staining. Primary pediatric AML cells were exposed to 30µM AQ for the same time periods. To reduce spontaneous apoptosis of primary AML cells over prolonged culture, samples were cultured in HS5 stromal cell-conditioned media (CM) or co-cultured with HS5 stromal cells. For in vivo studies, female NSG mice were injected with 5x105 THP-1 cells expressing firefly luciferase (THP-1.ffluc). Mice were randomly assigned to receive AQ 200 mg/kg daily (n=6) or vehicle (n=5) by oral gavage, starting on day 1, and disease burden was monitored by bioluminescent imaging 3 times a week. A cohort of male mice receiving VC or AQ starting the day of injection with THP-1.ffluc cells were followed for survival (n=7 for each group). For in vitro drug combination cytotoxicity assays, 103 THP-1.ffluc cells per well were plated in 96 well plates with media only, single drugs at increasing concentrations, and AQ+chemotherapy combinations (AQ 1-15uM, AraC 0.1-3uM, Dauno 0.03-0.6uM, VP-16 0.3-3uM). After 48 hours, viability was determined by luminescence. Background luminescence of media-only wells was subtracted from all values and means of 4 replicates were used for analysis. All values were normalized to the cells + vehicle wells, which were set to 0 to quantify the percent affected. The Calcusyn method was used to calculate the combination index (CI). An AQ+chemotherapy combination was considered synergistic if CI<0.8, additive if CI>0.8 and <1.2, or antagonistic if CI>1.2.Results: All 7 AML cell lines demonstrated time and dose-dependent induction of apoptosis in response to 1-30µM doses of AQ. IC50s were within the range of typical clinically achievable doses. For AML cell lines, the IC50s (in µM) after 72 hour incubation were: MOLM13 13.7, MV4-11 14.0, THP-1 15.7, NB4 16.9, Kasumi 1 18.6, HL60 33.7, KG1 98.9. AQ was generally less potent against the CML cell line K562 and the precursor B ALL cell line RS4 (72 hour IC50s were 116.6 and 81.4 respectively). All tested primary samples demonstrated AQ induced apoptosis that increased over time. After subtracting spontaneous apoptosis at each timepoint, the apoptosis induced by AQ at 72 hours for 5 patient samples supported by co-culture were 36, 37, 39, 67 and 87%. Annexin-V+% for the same patient samples supported by CM were 19, 13, 51, 47, and 24%. Bioluminescent imaging of female NSG mice xenografted with THP-1.ffluc cells and treated with single agent AQ daily starting on the day of cell injection demonstrated decreased disease burden compared to control mice (for example on day 21, the average radiance of VC treated mice vs AQ treated mice was 2 x 105 vs 6.3 x 104 p/s/cm²/sr; p=.0048 by ANOVA). Single agent AQ prolonged survival of male xenografted mice with all VC mice succumbing to disease at a median of 63 days vs 1/7 AQ treated mice by day 70 (p=.003 by log-rank test). Our drug combination assays found an antagonistic effect in combination with etoposide, and an additive effect with cytarabine and daunorubicin.Conclusions: Atovaquone induced dramatic levels of apoptosis of AML cells at clinically achievable doses in vitro ; reduced disease burden and improved survival in AML xenografts; and had an additive effect with standard chemotherapy drugs for pediatric AML. Our data support further evaluation of atovaquone for pediatric AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

RNAi prodrugs decrease elevated mRNA levels of Polo-like kinase 1 in ex vivo cultured primary cells from pediatric acute myeloid leukemia patients.

Polo-like kinase 1 (Plk1) is an important regulator of the cell cycle and it is frequently overexpressed in cancer cells. Several small molecule inhibitors have been developed to target Plk1 and some of them have reached clinical trials in adults with acute myeloid leukemia (AML). Pediatric AML patients have a poor prognosis and survivors suffer from long-term side effects. As adult AML cells have an elevated expression of Plk1, AML is a disease candidate for Plk1 inhibition. However, the relative success of clinical trials have been hampered by adverse reactions. Herein, PLK1-targeting RNA interference (RNAi) prodrugs that enter cells without a transfection reagent are used to target PLK1 selectively in primary cells from pediatric AML patients. We show that PLK1 and PLK4 mRNA expression are significantly higher in pediatric AML patients when compared to healthy donors and that PLK1 is downregulated by on average 50% using RNAi prodrugs without a significant effect on other PLK family members. In addition, the RNAi prodrug-induced decrease in PLK1 can be used to potentiate the effect of cytarabine. In summary, PLK1-targeting RNAi prodrugs can decrease the elevated levels of PLK1 in primary cells from pediatric AML patients and sensitize pediatric AML cells to chemotherapeutics.

Circular RNA circ_0005774 contributes to proliferation and suppresses apoptosis of acute myeloid leukemia cells via circ_0005774/miR-192–5p/ULK1 ceRNA pathway

Circular RNAs (circRNAs) and microRNAs (miRNAs) have been emerging as new players in acute myeloid leukemia (AML). Hsa_circ_0005774 (circ_0005774) is an upregulated circRNA in pediatric AML, while its role is uncovered. Thus, we intended to measure the function and mechanism of circ_0005774 in AML leukemogenesis. Real time-quantitative PCR revealed that circ_0005774 was highly expressed in blood of pediatric AML patients and AML cells (HL-60 and NB4), accompanied with downregulated miRNA-192–5p (miR-192–5p) which was a crucial tumor-associated and leukemia-related miRNA. Circ_0005774 was abundant in miRNA response element according to CSCD software, and miR-192–5p was identified as a target of circ_0005774, as evidenced by RNA immunoprecipitation and dual-luciferase reporter assays. Cell viability assay, flow cytometry and western blotting were performed to measure cell functions. Accordingly, blocking circ_0005774 and/or overexpressing miR-192–5p could enhance apoptosis rate of HL-60 and NB4 cells, but suppress cell viability and cell cycle entrance, accompanied with depression of proliferation markers including proliferating cell nuclear antigen (PCNA), CyclinD1 and B cell lymphoma 2 (Bcl-2). Meanwhile, depleting miR-192–5p counteracted the role of circ_0005774 knockdown in AML cells. Uncoordinated 51-like kinase 1 (ULK1) was previously demonstrated to be associated with diagnosis, prognosis and therapeutic strategy for AML, and restoring ULK1 could abrogate miR-192–5p overexpression-induced effects in HL-60 and NB4 cells. Notably, ULK1 was a downstream target of miR-192–5p and indirectly modulated by circ_0005774. In conclusion, circ_0005774 knockdown repressed cell proliferation and promoted apoptosis of AML cells partially through regulating miR-192–5p/ULK1 axis.

MiR-451 Promotes Cell Apoptosis and Inhibits Autophagy in Pediatric Acute Myeloid Leukemia by Targeting HMGB1.

MiR-451 plays a tumor suppressive role in a variety of cancers. However, the function of miR-451 in acute myeloid leukemia (AML) has not been fully understood. Herein, we focused on the effect of miR-451 in pediatric AML and its regulatory mechanism. MiR-451 and high mobility group box 1 (HMGB1) levels were tested in bone marrow of pediatric AML patients and healthy controls, and in AML cells and HS-5 cells by qRT-PCR and Western blot analysis. HL-60 and THP-1 cells were treated with miR-451 mimics, pcDNA-HMGB1, and corresponding controls. The changes in apoptosis and autophagy were evaluated in miR-451 overexpressed AML cells with MTT and flow cytometry. The interaction between miR-451 and HMGB1 was determined by dual-luciferase reporter assay, qRT-PCR, and Western blot. After cells were co-transfected with pcDNA-HMGB1 and pc-DNA-ctrl, we investigated apoptosis and autophagy in miR-451 overexpressed cells perturbed by exogenous HMGB1 through MTT, flow cytometry, and Western blot. miR-451's role in drug sensitivity was further measured. Pediatric AML bone marrow and cell lines presented low expression of miR-451 coupled with high expression of HMGB1. HMGB1 was determined to be a functional target of miR-451. MiR-451 overexpression remarkably enhanced apoptosis and reduced autophagy in both AML cell lines, which was reversed by pcDNA-HMGB1 transfection. Additionally, exogenous miR-451 significantly enhanced the sensitivity of HL-60 cells to the chemotherapy drug As2O3. MiR-451 exerted a tumor suppressive effect in enhancing cell death and reducing autophagy of AML cells by targeting HMGB1. MiR-451 might be considered a candidate target for treating pediatric AML.

Engineered type 1 regulatory T cells designed for clinical use kill primary pediatric acute myeloid leukemia cells.

Type 1 regulatory (Tr1) T cells induced by enforced expression of interleukin-10 (LV-10) are being developed as a novel treatment for chemotherapy-resistant myeloid leukemias. In vivo, LV-10 cells do not cause graft-versus-host disease while mediating graft-versus-leukemia effect against adult acute myeloid leukemia (AML). Since pediatric AML (pAML) and adult AML are different on a genetic and epigenetic level, we investigate herein whether LV-10 cells also efficiently kill pAML cells. We show that the majority of primary pAML are killed by LV-10 cells, with different levels of sensitivity to killing. Transcriptionally, pAML sensitive to LV-10 killing expressed a myeloid maturation signature. Overlaying the signatures of sensitive and resistant pAML onto the public NCI TARGET pAML dataset revealed that sensitive pAML clustered with M5 monocytic pAML and pAML with MLL rearrangement. Resistant pAML clustered with myelomonocytic leukemias and those bearing the core binding factor translocations inv(16) or t(8;21)(RUNX1- RUNX1T1). Furthermore, resistant pAML upregulated the membrane glycoprotein CD200, which binds to the inhibitory receptor CD200R1 on LV-10 cells. In order to examine if CD200 expression on target cells can impair LV-10 cell function, we overexpressed CD200 in myeloid leukemia cell lines ordinarily sensitive to LV-10 killing. Indeed, LV-10 cells degranulated less and killed fewer CD200-overexpressing cells compared to controls, indicating that pAML can utilize CD200 expression for immune evasion. Altogether, the majority of pAML are killed by LV-10 cells in vitro, supporting further LV-10 cell development as an innovative cell therapy for pAML.

Clinical Grade Production of Wilms' Tumor-1 Loaded Cord Blood-Derived Dendritic Cells to Prevent Relapse in Pediatric AML After Cord Blood Transplantation.

Hematopoietic cell transplantation (HCT) is a last resort, potentially curative treatment option for pediatric patients with refractory acute myeloid leukemia (AML). Cord blood transplantation (CBT) results in less relapses and less graft-versus-host disease when compared to other sources. Nevertheless, still more than half of the children die from relapses. We therefore designed a strategy to prevent relapses by inducing anti-AML immunity after CBT, using a CB-derived dendritic cell (CBDC) vaccine generated from CD34+ CB cells from the same graft. We here describe the optimization and validation of good manufacturing practice (GMP)-grade production of the CBDC vaccine. We show the feasibility of expanding low amounts of CD34+ cells in a closed bag system to sufficient DCs per patient for at least three rounds of vaccinations. The CBDCs showed upregulated costimulatory molecules after maturation and showed enhanced CCR7-dependent migration toward CCL19 in a trans-well migrations assay. CBDCs expressed Wilms’ tumor 1 (WT1) protein after electroporation with WT1-mRNA, but were not as potent as CBDCs loaded with synthetic long peptides (peptivator). The WT1-peptivator loaded CBDCs were able to stimulate T-cells both in a mixed lymphocyte reaction as well as in an antigen-specific (autologous) setting. The autologous stimulated T-cells lysed not only the WT1+ cell line, but most importantly, also primary pediatric AML cells. Altogether, we provide a GMP-protocol of a highly mature CBDC vaccine, loaded with WT1 peptivator and able to stimulate autologous T-cells in an antigen-specific manner. Finally, these T-cells lysed primary pediatric AML demonstrating the competence of the CBDC vaccine strategy.

Inhibition of Methyltransferase DOT1L Sensitizes to Sorafenib Treatment AML Cells Irrespective of MLL-Rearrangements: A Novel Therapeutic Strategy for Pediatric AML.

Pediatric acute myeloid leukemia (AML) is an aggressive malignancy with poor prognosis for which there are few effective targeted approaches, despite the numerous genetic alterations, including MLL gene rearrangements (MLL-r). The histone methyltransferase DOT1L is involved in supporting the proliferation of MLL-r cells, for which a target inhibitor, Pinometostat, has been evaluated in a clinical trial recruiting pediatric MLL-r leukemic patients. However, modest clinical effects have been observed. Recent studies have reported that additional leukemia subtypes lacking MLL-r are sensitive to DOT1L inhibition. Here, we report that targeting DOT1L with Pinometostat sensitizes pediatric AML cells to further treatment with the multi-kinase inhibitor Sorafenib, irrespectively of MLL-r. DOT1L pharmacologic inhibition induces AML cell differentiation and modulates the expression of genes with relevant roles in cancer development. Such modifications in the transcriptional program increase the apoptosis and growth suppression of both AML cell lines and primary pediatric AML cells with diverse genotypes. Through ChIP-seq analysis, we identified the genes regulated by DOT1L irrespective of MLL-r, including the Sorafenib target BRAF, providing mechanistic insights into the drug combination activity. Our results highlight a novel therapeutic strategy for pediatric AML patients.

Silencing of lncRNA UCA1 curbs proliferation and accelerates apoptosis by repressing SIRT1 signals by targeting miR-204 in pediatric AML.

The long noncoding RNA urothelial carcinoma-associated 1 (UCA1) has been reported to sustain the proliferation of acute myeloid leukemia (AML) cells through downregulating cell cycle regulators p27kip1 . Yet, the foundational mechanism of UCA1 in AML pathologies remains unclear. Herein, we found an escalation of UCA1 expression and suppression of miR-204 expression in pediatric AML patients and cells. UCA1 silencing suppressed cell proliferative abilities, promoted apoptotic rates, decreased Ki67, and increased cleaved caspase-3 in AML cells. Moreover, UCA1 sponged miR-204 and suppressed its expression. UCA1 overexpression inversed the miR-204 suppressed proliferation and promoted apoptosis. UCA1 also boosted the expression of SIRT1, a miR-204 target, via the sponging interaction. Furthermore, miR-204 inhibited inducible nitric oxide synthase and cyclooxygenase-2 expression, while UCA1 overexpression inversed the inhibitory effects in AML cells. Our findings concluded that UCA1 downregulation repressed cell proliferation and promoted apoptosis through inactivating SIRT1 signals by upregulating miR-204 in pediatric AML.

MiR-100 regulates cell viability and apoptosis by targeting ATM in pediatric acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common pediatric malignancy and a major cause of morbidity and mortality in children. miR-100 is associated with progression of various diseases including AML. The aim of this study was to explore the underlying molecule mechanisms of miR-100 involved in AML. The expressions of miR-100 and ataxia telangiectasia mutated (ATM) in pediatric AML patients and cell lines were monitored using qRT-PCR and western blot assays. MTT assay was carried to evaluate cell viability. Cell apoptosis was measured by flow cytometry. The binding sites between miR-100 and ATM were predicted by mirtarbase database. Luciferase reporter assay was used to confirm the relationship between miR-100 and ATM. miR-100 expression was highly expressed in bone marrow of AML patients and cell lines. Moreover, Knockdown of miR-100 led to the inhibition of viability and promotion of apoptosis in Kasumi-1 and MV-4-11 cells. miR-100 harbored the 3′UTR of ATM. Meanwhile, the expression of ATM was downregulated in bone marrow of AML patients and AML cell lines. Subsequently, a negative correlation between miR-100 and ATM in bone marrow of AML patients was also observed. Furthermore, ectopic expression of ATM repressed cell viability while enhanced apoptosis. Notably, loss of ATM attenuated the effect of miR-100 depletion on cell viability and apoptosis in AML cells. miR-100 participates in cell viability and apoptosis by targeting ATM in pediatric AML.

Epigenetic drug screen identifies the histone deacetylase inhibitor NSC3852 as a potential novel drug for the treatment of pediatric acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease regarding morphology, immunophenotyping, genetic abnormalities, and clinical behavior. The overall survival rate of pediatric AML is 60% to 70%, and has not significantly improved over the past two decades. Children with Down syndrome (DS) are at risk of developing acute megakaryoblastic leukemia (AMKL), which can be preceded by a transient myeloproliferative disorder during the neonatal period. Intensification of current treatment protocols is not feasible due to already high treatment-related morbidity and mortality. Instead, more targeted therapies with less severe side effects are highly needed. To identify potential novel therapeutic targets for myeloid disorders in children, including DS-AMKL and non-DS-AML, we performed an unbiased compound screen of 80 small molecules targeting epigenetic regulators in three pediatric AML cell lines that are representative for different subtypes of pediatric AML. Three candidate compounds were validated and further evaluated in normal myeloid precursor cells during neutrophil differentiation and in (pre-)leukemic pediatric patient cells. Candidate drugs LMK235, NSC3852, and bromosporine were effective in all tested pediatric AML cell lines with antiproliferative, proapoptotic, and differentiation effects. Out of these three compounds, the pan-histone deacetylase inhibitor NSC3852 specifically induced growth arrest and apoptosis in pediatric AML cells, without disrupting normal neutrophil differentiation. NSC3852 is a potential candidate drug for further preclinical testing in pediatric AML and DS-AMKL.

Identifying Combination Therapies Targeting Apoptosis Pathways in Pediatric Acute Myeloid Leukemia (CAuSAL Study)

Leukemia is the most frequently (30%) occurring type of pediatric cancer. Of these, approximately 80% are acute lymphoblastic leukemia (ALL) with acute myeloid leukemia (AML) cases making up the remaining 20%. Management of ALL has improved dramatically over the years with around 90% of patients surviving 5 years or more. Unfortunately, children with AML do not have such a promising prognosis with only 60% surviving 5 years or longer, plus a high risk of relapse, the side effects of the harsh chemotherapy regimen and a risk of not responding to induction therapy, provide a bleak outlook. Recently, Bolouri et al., (2017) identified the need for age-specific therapies for AML patients.To work towards age-specific treatment regimens we used the TARGET AML transcriptomic data, (https://ocg.cancer.gov/programs/target/data-matrix) and identified several therapeutically targetable pathways, in particular anomalies in apoptotic pathways amongst cytogenetically normal relapsed AML patients. Based on these apoptotic pathways we screened 80 apoptosis-inducing drugs from a compound library obtained from Selleckchem. The effect of three doses (0.01 µM, 0.1 µM and 1 µM) over 72 hrs of each drug was assayed using CellTox™ Green reagent (Promega) to investigate the cytotoxic potential of the compounds against three pediatric AML cell lines (Kasumi-1, MV4-11 and CMK) representative of a spectrum of cytogenetic and mutational anomalies. The cell lines showed varying responses in a time and dose-dependent manner with mutual and unique effects being highlighted, six compounds, including Navitoclax and TP-0903, overlapped in their effect on cell viability between the three cell lines. The cell lines also showed unique responses to many compounds, including Sunitinib and Obatoclax in the MV4-11 cell line. Based on these findings a dose of 0.1 µM was taken forward for the combination screen.To carry out the combination screen we used R to compile an algorithm, with the capability of determining all possible pairwise combinations for a given number of compounds. Several inputs were accommodated, including: the number of compounds to be used; the number of compounds in a well and the number of times the algorithm should run to get the best solution, with the best solution being the least number of wells to reduce redundant repeats of pairs. The output detailed which compounds should be in which well. With an input of 80 apoptotic-inducing compounds into the algorithm; the output was as follows: 3,160 possible pairwise combinations, 1,197 pairs used once, 827 pairs used twice, and so on with the largest number of repeats being 148 pairs used eight times. This output enabled us to cross validate our data, by analysing wells containing the successful combinations to corroborate them as ‘hits’ or as containing possible antagonistic pairings if they were deemed unsuccessful. Using the output of the algorithm, 160 wells were needed for all combinations to enable our Multiplex Screening for Interacting Compounds for pediatric AML (MuSICAL). CellTox™ Green was used to determine cytotoxicity up to 72 hours and the same three cell lines used as for the single agent screen. The MuSICAL screen identified some wells in common amongst the three cell lines (6.25%), although many of the positive wells (12.5%) were unique to each of the cell lines. Well 158, which included Nutlin-3, Embelin and Poziotinib, was common for the MV4-11 and the CMK cell lines, with the individual compounds in this well having no substantial effect on cell viability as single agents, but a combination of 2 or more compounds induced substantial cell death. The 10 drugs in well 158 were deconvoluted and screened, resulting in 45 pairwise combinations. Several pairwise combinations, at 0.1 µM each, induced cell death in the MV4-11 and CMK cell lines. The most effective pairwise combination was the combination of ABT-737, a Bcl-2 inhibitor and Purvalanol A, a CDK inhibitor, in the MV4-11 cell line. The effect of this combination was also a ‘hit’ in Well 6 validating the therapeutic potential. For the CMK cell line, the most effective combination was Poziotinib, a pan-HER inhibitor and SU9516, another CDK inhibitor.In conclusion, the MuSICAL screen has identified multiple apoptosis-inducing drug combinations across the 3 cell lines highlighting the potential for personalised treatment of pediatric AML with different cytogenetic and mutational backgrounds. DisclosuresNo relevant conflicts of interest to declare.