Primary Acute Leukemia Cells Research Articles

Minnelide exhibits antileukemic activity by targeting the Ars2/miR-190a-3p axis

BackgroundThe identification of a novel and effective strategy for the clinical treatment of acute leukemia (AL) is a long-term goal. Minnelide, a water-soluble prodrug of triptolide, has recently been evaluated in phase I and II clinical trials in patients with multiple cancers and has shown promise as an antileukemic agent. However, the molecular mechanism underlying minnelide's antileukemic activity remains unclear. PurposeTo explore the molecular mechanisms by which minnelide exhibits antileukemic activity. MethodsAL cells, primary human leukemia cells, and a xenograft mouse model were treated with triptolide and minnelide. The molecular mechanism was elucidated using western blotting, immunoprecipitation, flow cytometry, GSEA and liquid chromatography-mass spectrometry analysis. ResultsMinnelide was highly effective in inhibiting leukemogenesis and improving survival in two complementary AL mouse models. Triptolide, an active form of minnelide, causes cell cycle arrest in G1 phase and induces apoptosis in both human AL cell lines and primary AL cells. Mechanistically, we identified Ars2 as a new chemotherapeutic target of minnelide for AL treatment. We found that triptolide directly targeted Ars2, resulting in the downregulation of miR-190a-3p, which led to the disturbance of PTEN/Akt signaling and culminated in G1 cell cycle arrest and apoptosis. ConclusionsOur findings demonstrate that targeting Ars2/miR-190a-3p signaling using minnelide could represent a novel chemotherapeutic strategy for AL treatment and support the evaluation of minnelide for the treatment of AL in clinical trials.

S252: M2-POLARIZED MACROPHAGES CONTROL LEUKEMIC STEM CELL FATE BY PROMOTING METABOLIC REPROGRAMMING

Background: Being a crucial part of the tumor microenvironment (TME) in solid tumors, tumor-associated macrophages are often associated with poor prognosis (Bruni et al. Nat Rev Cancer 2020). Yet, in acute myeloid leukemia (AML) the role of macrophages remains unclear. Aims: Here, we evaluated the impact of M2 macrophages in AML using a patient derived xenograft (PDX) model. Methods: To do so, we first injected 1x105 peripheral blood (PB) derived M2 macrophages into NSGS mice and next transplanted notoriously difficult to engraft primary Acute Promyelocytic Leukemia (APL) cells (1 x 106 cells - n=7 different patient samples) via the retro-orbital vein. Results: As a result, mice with co-injected human M2-macrophages developed full-blown leukemia with increased spleen weight in comparison with the control. Perhaps even more strikingly, ex vivo culture of APL and other favorable AML subtypes such as inv (16) or NPM1 mutant leukemic cells on M2-macrophages for 48h was sufficient to “train” these cells to engraft and induce fatal leukemia. Maintenance of self-renewal was shown in a secondary transplant assay and an enhanced frequency of leukemic stem cells was assessed by in vivo LTC-IC assays (LSC frequency: Control: 1/6x106 cells vs M2 pre-culture: 1/7.2x104 cells). To better understand the biological changes induced on leukemic blast when exposed to M2 macrophages, we performed an RNA sequencing analysis comparing AML/APL samples at diagnosis to cells that were “trained” (48h) on M2-macrophages or on MS5 mesenchymal bone marrow stromal cells, as a control. Gene ontology and gene set enrichment analysis on the genes up regulated upon M2 co-culture were significantly enriched for oxidative phosphorylation (OxPhos) signatures. Evaluation of functional respiration using seahorse measurements, confirmed the increase of oxygen consumption rate (OCR, basal and maximum) in primary AML/APL cells (n=7) after exposure to M2 macrophages compared to MS5. The increase of basal and maximum OCR suggested enhanced mitochondrial metabolism, which prompted us to determine whether macrophages, similar to MSC cells, can transfer mitochondria to primary AML cells (van der Vlist et al. Neuron 2022). Flow cytometry analysis revealed an efficient mitochondrial transfer from M2 macrophages to leukemic blasts, which was more efficient compared to mitochondrial exchange from MS5 cells. Treatment with the CPT1A inhibitor Etomoxir (50 µM), prevented the gain in functional respiration and enhanced proliferation of AML cells when those were co-cultured on M2-macrophages, while no changes were observed for MS5 co-cultures. These results suggest increased fatty acid oxidation to drive the OXPHO-like state in AML. Summary/Conclusion: Overall, we revealed that M2 macrophages can support leukemic growth of favorable AML subtypes that are commonly difficult to engraft in PDX models. Even an in vitro exposure to M2 macrophages suffices to alter the biology of AML cells transforming a non-engraftable cell into a cell with high leukemic potential. In vitro we show that AML blast cells cultured on M2 macrophage adapt a more OxPhos-like state linked to intrinsic changes associated with increased uptake of mitochondria. In summary, our study uncovers how the TME can contribute to leukemic transformation which provides alternative avenues for therapeutic interventions.

Tumor Associated Macrophages Promoted Fatal Patient Derived Acute Promyelocytic Leukemia In Vivo

With immune therapies on the rise, an in-depth understanding of the immunological changes in leukemic bone marrow (BM) niches becomes indispensable. Being an crucial part of the tumor microenvironment (TME) in solid tumours, tumour-associated macrophages are often associated with poor prognosis (Bruni et al. 2020). Yet, in acute myeloid leukaemia (AML) the role of macrophages has not been thoroughly studied. The expression of the M2-markers CD163 and CD206 in the AML BM cell population predicted poor clinical outcome. We identified that this expression emerges from a more mature (CD45 midSSC highHLA-DR +CD14 +CD16 +/-) myeloid cell population (hereafter called AML-associated macrophages - AAM) and not from the leukemic blasts. By employing flow cytometry analysis (FACS) we noted a decrease in the expression of the M1-marker (CD80) and an increase of the M2-markers CD163/CD206on AAM (n=70) compared to healthy donors (HD, n=10). Unsupervised clustering based on the CD163/CD206 levels detected on AAM generated 4 distinct clusters, whereby patients within the CD163 low/CD206 low cluster displayed better overall survival than the other clusters.In vitro, the co-culture of HD-derived M1 macrophages and AML primary/cell lines reduced AML growth via apoptosis induction and cell cycle arrest, while M2-macrophages promoted AML survival and phagocytosis/drug-resistance when treated with FLT3/BCL2 inhibitors. Primary AML cells were also able to repolarize M1- into M2-macrophages, suggesting that leukemic cells actively remodel their microenvironment. Next, we evaluated the impact of M2-macrophages on leukemogenesis in a patient derived xenograft (PDX) model, using the notoriously difficult to engraft primary Acute Promyelocytic Leukaemia (APL) cells (n=7 patient samples). Intra-BM injection of M2-macrophages and retro-orbital transplant of primary APL cells induced full-blown APL in NSGS mice. More strikingly, ex vivo culture of APL cells on M2-macrophages (48h) was sufficient to “train” these cells to engraft and induce fatal APL. Maintenance of self-renewal was shown in a secondary transplant and an enhanced frequency of leukemic stem cells was assessed by in vivo LTC-IC assays.To identify the biological changes acquired by leukemic blasts, we performed RNA sequencing comparing AML/APL samples at diagnosis to cells that were “trained” (48 h) on M2-macrophages or on MS5 mesenchymal BM stromal cells. Gene ontology and gene set enrichment analysis on the genes up-regulated upon M2 co-culture were significantly enriched for cell migration, cell cycle progression and oxidative phosphorylation (OXPHOS) signatures. In line with our RNAseq data, we noted improved in vivo homing of primary APL cells to the BM within 18 h post-transplant upon ex vivo M2 co-culture compared to diagnosis (n=7 APL blasts). Concurrently, we detected increased levels of surface protein expression Integrin-α4 (CD49d) and -α5 (CD49e) on APL/AML blast cells after M2 exposure. The CD49d expression remained high in primary and secondary transplants. Using seahorse measurements, we confirmed the increased respiration capacity (basal and maximum) of primary AML/APL cells (n=7) after exposure to M2 macrophages compared to MS5.FACS analysis revealed that M2-macrophages were able to transfer more mitochondria than MS5 cells to primary AML cells, which could underlie the observed increase in OXPHOS mitochondrial metabolism. Treatment with Etomoxir (50 µM), prevented the gain in functional respiration when AML blast were co-cultured on M2-macrophages, while no changes were observed for MS5 co-cultures, suggesting increased fatty acid oxidation to drive the OXPHO-like state. Finally, we noted that training on M2 macrophages significantly increased colony formation and endowed the cells with long term proliferation in liquid cultures for over 30 days.Overall, we reveal that the frequency of M2-macrophages is up-regulated in a subgroup of AML patients representing a group with poor prognosis. M2 macrophages can support leukemic growth and therapy-resistance, and support fatal APL in PDX models. Even an in vitro exposure to M2 macrophages suffices to alter adhesion, homing and metabolic characteristics of leukemic blasts to allow efficient engraftment and fatal leukemogenesis. Our study uncovers how the TME can contribute to leukemic transformation which provides alternative avenues for therapeutic interventions. DisclosuresSilveira: BMS/Celgene: Research Funding; Servier/Agios: Research Funding; Abbvie: Speakers Bureau; Astellas: Speakers Bureau. Quek: BMS/Celgene: Research Funding; Servier/Agios: Research Funding. Mota: Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Technopharma: Speakers Bureau; Bristol Myer Squibb: Speakers Bureau; Bayer: Speakers Bureau; Pfizer: Speakers Bureau; AstraZeneca: Speakers Bureau; Astellas: Speakers Bureau; Ipsen: Speakers Bureau; Amgen: Speakers Bureau.

PDX Models Recapitulate Genetics and Epigenetics of Pediatric T-Cell Leukemia

Patient-derived xenografts (PDX) emerged as important tools to study leukemia biology and potential therapeutic regimens. Here, we compared cellular, molecular and developmental features of PDX-derived blasts with primary pediatric precursor T-cell acute leukemia (T-ALL) cells. The cells were obtained at the times of initial diagnosis and relapse. We analyzed the samples at the genomic level (whole genome sequencing (WGS), whole exome sequencing (WES), multiplex ligation probe amplification (MLPA), targeted sequencing) and the epigenetic level (DNA methylation, Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq)).Altogether 75% of our samples engrafted successfully. Of these 12 were matched pairs of initial diagnosis and relapse as well as corresponding PDX. Of further 12 matched pairs either initial diagnosis (2/12) or relapse (7/12) or neither (3/12) engrafted indicating that relapse samples tend to engraft more successfully (p= 0.1824 (McNemar's test)), which is in agreement with observations in other cancer entities.In 11 out of 12 matched pairs (initial and relapse) of primary and PDX genomic stability of the PDXs was assessed using WGS and WES. Ninety-two % of the mutations with an allele frequencies (AF) >30% were conserved. Of altogether 609 SNVs and InDels (AF>10%) detected in primary samples 68 % (417) were also detected in the corresponding PDXs. In some PDXs, mutations not identified by targeted sequencing in the primary sample (NOTCH1, PHF6, DNA11, USP9X) occured at a high AF (22-59%), which may have been the result of selection of a subclone or of an acquisition of the mutation in the process of PDX generation.Thirty of 35 (86%) large CNA assessed by WGS were preserved in corresponding PDX models indicating that CNAs are likely earlier events in the leukemogenesis that are present at high AF in the primary samples. CNA analysis with MLPA confirmed that PDXs retained 96% (80/83) of the deletions and amplifications of primary leukemia.In 18 out of the matched 22 PDXs all clonal mutations (AF >30%) detected in the primary sample have been preserved in the PDX. Seventeen out of 22 contained more than 1 clone. Fifteen out of these 17 retained the complexity of primary sample with different subclones successfully engrafted.When considering the 11 matched pairs, we observed 4 different engraftment types of initial disease. Type 1 engrafted in a similar way in which they relapsed preserving all or nearly all mutations found in patients' sample (4 samples). Type 2: the relapsing clone that was not detected in initial diagnosis was present in PDX derived of initial diagnosis (PDX_INI) but it was not dominant (3). Type 3: the relapsing clone that was present at initial diagnosis did not engraft into PDX_INI (2). Type 4 preserved the relapsing clone as well as the mutations specific for initial diagnosis (2). Out of 11 PDXs of relapse 9 preserved all or nearly all mutations detected in primary sample. In 2/11 PDXs of relapse one of the clones preexisting in primary relapse was selected for, while the other one was lost.Differential methylation analysis by EPIC array of 21,711 promoters of primary samples and PDXs revealed that the average methylation in primary samples (β=0.43) did not differ from that in PDXs (β=0.45). Principal Component Analysis and unsupervised hierarchical clustering showed that matched pairs, both of the primary and the PDX samples cluster together (ρ=0.9912). Similarly, analysis of chromatin accessibility by ATAC-seq showed that more than 92% of peaks of a height > 20 and more than 75% of the peaks of a height of > 10 were preserved between primary samples and corresponding PDXs.In conclusion, PDX models largely reflect the heterogeneity and the complexity of the primary leukemic sample at the genomic and epigenomic level, although some clonal and particularly subclonal mutations can be lost. Differences in engraftment and various engraftment scenarios of initial diagnosis in contrary to relapse samples suggest that relapse samples are more preselected and stable in engraftment. Altogether, this overall genomic and epigenomic stability of the primary T-ALL in the PDX renders the models as a suitable source for material when primary samples are limiting and also to reduce uncontrollable pre-analytical changes in primary samples. Further, these models are suitable for the preclinical development of new treatment strategies for pediatric T-ALL. DisclosuresSchrappe:Baxalta: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Medac: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; JAZZ Pharma: Consultancy, Research Funding.

Pre-Clinical Evaluation of the Proteasome Inhibitor Ixazomib against Bortezomib-Resistant Leukemia Cells and Primary Acute Leukemia Cells

At present, 20–30% of children with acute leukemia still relapse from current chemotherapy protocols, underscoring the unmet need for new treatment options, such as proteasome inhibition. Ixazomib (IXA) is an orally available proteasome inhibitor, with an improved safety profile compared to Bortezomib (BTZ). The mechanism of action (proteasome subunit inhibition, apoptosis induction) and growth inhibitory potential of IXA vs. BTZ were tested in vitro in human (BTZ-resistant) leukemia cell lines. Ex vivo activity of IXA vs. BTZ was analyzed in 15 acute lymphoblastic leukemia (ALL) and 9 acute myeloid leukemia (AML) primary pediatric patient samples. BTZ demonstrated more potent inhibitory effects on constitutive β5 and immunoproteasome β5i proteasome subunit activity; however, IXA more potently inhibited β1i subunit than BTZ (70% vs. 29% at 2.5 nM). In ALL/AML cell lines, IXA conveyed 50% growth inhibition at low nanomolar concentrations, but was ~10-fold less potent than BTZ. BTZ-resistant cells (150–160 fold) displayed similar (100-fold) cross-resistance to IXA. Finally, IXA and BTZ exhibited anti-leukemic effects for primary ex vivo ALL and AML cells; mean LC50 (nM) for IXA: 24 ± 11 and 30 ± 8, respectively, and mean LC50 for BTZ: 4.5 ± 1 and 11 ± 4, respectively. IXA has overlapping mechanisms of action with BTZ and showed anti-leukemic activity in primary leukemic cells, encouraging further pre-clinical in vivo evaluation.

Acute Myeloid and Lymphoblastic Leukemia Cell Interactions with Endothelial Selectins: Critical Role of PSGL-1, CD44 and CD43.

Acute myeloid and lymphoblastic leukemia are poor prognosis hematologic malignancies, which disseminate from the bone marrow into the blood. Blast interactions with selectins expressed by vascular endothelium promote the development of drug resistance and leukostasis. While the role of selectins in initiating leukemia blast adhesion is established, our knowledge of the involved selectin ligands is incomplete. Using various primary acute leukemia cells and U937 monoblasts, we identified here functional selectin ligands expressed by myeloblasts and lymphoblasts by performing biochemical studies, expression inhibition by RNA interference and flow adhesion assays on recombinant selectins or selectin ligands immunoadsorbed from primary blast cells. Results demonstrate that P-selectin glycoprotein ligand-1 (PSGL-1) is the major P-selectin ligand on myeloblasts, while it is much less frequently expressed and used by lymphoblasts to interact with endothelial selectins. To roll on E-selectin, myeloblasts use PSGL-1, CD44, and CD43 to various extents and the contribution of these ligands varies strongly among patients. In contrast, the interactions of PSGL-1-deficient lymphoblasts with E-selectin are mainly supported by CD43 and/or CD44. By identifying key selectin ligands expressed by acute leukemia blasts, this study offers novel insight into their involvement in mediating acute leukemia cell adhesion with vascular endothelium and may identify novel therapeutic targets.

Role of Arsenic Trioxide in EVI-1 Apoptosis in Patients with Acute Myeloid Leukaemia

Background:High expression of the ecotropic viral integration site-1 (EVI-1) is an independent negative prognostic indicator of survival in leukaemia patients. Zebrafish (Danio rerio) is a vertebrate animal model commonly used to examine haematopoiesis and myeloid malignancies. To clarify the molecular mechanisms of EVI-1, we previously introduced the human EVI-1 gene into embryonic zebrafish through a heat-shock promoter and established the stable germ-line Tg(EVI-1: HSE: EGFP) zebrafish (Shen et al, 2013). Arsenic trioxide (As2O3, ATO) is one of the effective anticancer drugs, especially for patients with leukaemia (Udupa et al, 2017). We thus aimed to explore the anticancer effects of ATO and the underlying functions associated with EVI-1 in an in vivo zebrafish model and in AML cells in vitro.Results:We determined EVI-1 expression in mononuclear cells isolated from the bone marrow and peripheral blood of AML patients and healthy donors by RT-qPCR and Western blot analysis. EVI-1 was highly expressed in primary AML (Fig 1A). Then, EVI-1 expression was detected in five leukaemia cell lines (K562, HL-60, U937, THP-1 and MV4-11) and normal PBMCs. Among these five leukaemia cell lines, THP-1 has the highest EVI-1 expression (Fig 1B and Fig1C).Primary acute monocytic leukaemia cells from one patient with high expression of EVI-1 were treated with ATO. We found ATO could significantly decrease EVI-1 mRNA (Fig 2A). Between the ATO-treated groups and the control group, the expression of EVI-1 were significantly reduced in the THP-1 cell line (Fig 2B). Next, we evaluated the EVI-1 expression in Tg(EVI-1: HSE; EGFP) transgenic zebrafish embryos over dose courses of ATO exposure (Fig 2C). Consistent with the results of our in vitro study, ATO decreased EVI-1 expression in a dose-dependent manner after 72 h (Fig 2C). Taken together, these results indicate that ATO is an inhibitor of EVI-1 expression both in vivo and in vitro.We investigate whether the reduction of THP-1 cells viability is due to apoptosis, THP-1 cells were incubated with 3 µM of ATO for 24 h, 48 h or 72 h. In the light microscopy images, THP-1 cells exhibited typical apoptotic characteristics (Fig 3A). The proportion of apoptotic cells was represented as early apoptotic cells (annexin V+/PI- staining, the lower right quadrant) plus late apoptotic cells (annexin V+/PI+ staining, the upper right quadrant) (Fig 3B). In cytometric analysis, ATO increased the percentage of apoptotic THP-1 cells in a dose- and time-dependent manner. We found that ATO increased the expressions of JNK, p-JNK, p-P53, PUMA, Bax, caspase-9 and caspase-3 (including cleaved caspase-9 and -3) but decreased the expressions of Bcl-2 and Bcl-xl (Fig 3C).To further verify the role of the JNK pathway in ATO-mediated THP-1 cell apoptosis, we examined if the inhibitor of JNK (SP600125) could reverse ATO-induced apoptosis in THP-1 cells. We found SP600125 not only decreased the pro-apoptotic effect of ATO in the THP-1 cell line (Fig 4A and Fig 4B) but also decreased the activation of the JNK-mediated apoptotic signalling pathway (Fig 4C). SP600125 silenced the activation of JNK by completely inhibiting the phosphorylation of JNK but had little effect on EVI-1 expression (Fig 4C).To test whether EVI-1 modulates apoptosis via the JNK signalling pathway, we transiently transfected THP-1 cells with EVI-1 siRNA which significantly reduced EVI-1 expression (Fig 5A). Silencing EVI-1 had a significant effect on the activation of the JNK pathway and the induction of THP-1 cell apoptosis (Fig 5B and Fig 5C).Conclusion:Our study demonstrated that the apoptotic pathway in THP-1 cells induced by ATO is closely associated with the oncogene EVI-1, the pro-apoptotic protein JNK, p-JNK, p-P53, PUMA, Bax, caspase-9 and caspase-3 (including cleaved caspase-9 and cleaved caspase-3), and the anti-apoptotic proteins Bcl-2 and Bcl-xL. ATO can downregulate EVI-1 mRNA and oncoprotein and block the repression of EVI-1 in the JNK pathway. Furthermore, the activated JNK signalling pathway regulated the expression level of apoptosis-associated proteins, including p-P53, PUMA, Bax, Bcl‐xL, Bcl‐2, Bax, caspase-9 and caspase-3(Fig 6). These findings may provide a novel theoretical basis for the development of personalized medical strategies for the treatment of EVI-1 positive AML patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Study of the mechanisms of crocetin-induced differentiation and apoptosis in human acute promyelocytic leukemia cells.

Crocetin, the major carotenoid in saffron, exhibits potent anticancer effects. However, the antileukemic effects of crocetin are still unclear, especially in primary acute promyelocytic leukemia (APL) cells. In the current study, the potential antipromyelocytic leukemia activity of crocetin and the underlying molecular mechanisms were investigated. Crocetin (100 µM), like standard anti-APL drugs, all-trans retinoic acid (ATRA, 10 µM) and As2 O 3 (arsenic trioxide, 50 µM), significantly inhibited proliferation and induced apoptosis in primary APL cells, as well as NB4 and HL60 cells. The effect was associated with the decreased expressions of prosurvival genes Akt and BCL2, the multidrug resistance (MDR) proteins, ABCB1 and ABCC1 and the inhibition of tyrosyl-DNA phosphodiesterase 1 (TDP1), while the expressions of proapoptotic genes CASP3, CASP9, and BAX/BCL2 ratio were significantly increased. In contrast, crocetin at relatively low concentration (10 µM), like ATRA (1 µM) and As 2 O 3 (0.5 µM), induced differentiation of leukemic cells toward granulocytic pattern, and increased the number of differentiated cells expressing CD11b and CD14, while the number of the immature cells expressing CD34 or CD33 was decreased. Furthermore, crocetin suppressed the expression of clinical marker promyelocytic leukemia/retinoic acid receptor-α ( PML/RARα) in NB4 and primary APL cells, and reduced the expression of histone deacetylase 1 ( HDAC1) in all leukemic cells. The results suggested that crocetin can be considered as a candidate for future preclinical and clinical trials of complementary APL treatment.

In Vitro Apoptotic Effects of Farnesyltransferase blockade in Acute Myeloid Leukemia Cells.

Farnesyltransferase inhibitors (FTIs) are a class of oral anti-cancer drugs currently tested in phase I-II clinical trials for treatment of hematological malignancies. The in vitro effects of various FTIs (alpha-hydroxyfarnesylphosphonic acid, manumycin-A and SCH66336) were tested on CD34+ KG1a cell line and in primary acute myeloid leukemia (AML) cells from 64 patients. By cell viability and clonogeneic methylcellulose assays, FTIs showed a significant inhibitory activity in CD34+ KG1a and primary bone marrow (BM) leukemic cells from 56% of AML patients. FTIs also induced activation of caspase-3 and Fas-independent apoptosis, confirmed by the finding that inhibition of caspase-8 was not associated with the rescue of FTI-treated cells. We concluded that other cellular events induced by FTIs may trigger activation of caspase-3 and subsequent apoptosis, but the expression of proapoptotic molecules, as Bcl-2 and Bcl-XL, and antiapoptotic, as Bcl-X(s), were not modified by FTIs. By contrast, expression of inducible nitric oxide synthase (iNOS) was increased in FTI-treated AML cells. Our results suggest a very complex mechanism of action of FTIs that require more studies for a better clinical use of the drugs alone or in combination in the treatment of hematological malignancies.

Efficient RNA Interference of Primary Leukemic Cells for Loss-of-Function Studies in Xenograft Mouse Models.

RNA interference (RNAi) is a powerful tool for efficient and highly specific gene silencing. Transduction with lentiviral vectors provides stable and long-term gene expression in slowly and non-dividing cells. This chapter describes how to couple these two technologies to efficiently silence specific genes in primary acute myeloid leukemia (AML) cells for subsequent xenotransplantation in immunocompromised mice. This approach could be used for loss-of-function studies aimed at identifying oncogenic targets in AML.

The Novel Technique of Genetically Engineered Patient-Derived Xenografts (GEPDX) Reveals That the X-Linked Inhibitor of Apoptosis Protein (XIAP) Plays an Essential Role for Maintenance and Growth of Patients' Acute Lymphoblastic Leukemia In Vivo

Expression profiling and next generation sequencing have enabled a detailed knowledge on alterations present in tumors from individual patients. In contrast, only limited understanding exists on the role that each alteration plays for the existing tumor. Of direct clinical interest, genes are of special interest which harbor an essential function for tumor maintenance and growth as they represent putative targets for anti-cancer therapy.Characterizing gene functions is demanding regarding both techniques and resources. Questions on gene function are often studied in established tumor cell lines, although establishing cell lines from primary tumors is rarely successful in acute leukemias. Primary acute leukemia cells poorly grow in vitro and established acute leukemias cell lines rely on additional mutations enabling in vitro growth, making them doubtful models to study genes with essential function.To bridge the gap, we aimed at studying gene function in the complex environment of individual tumors. As primary acute leukemia cells are unable to grow in vitro, we used the orthotopic model of patient-derived xenograft (PDX) leukemia and amplified cells in mice.We established a novel technique to manipulate distinct signaling proteins in PDX cells using lentiviruses and knockdown. We expressed small hairpin RNA (shRNA) in the background of micro RNA 30 (miR30) under control of a Pol II promoter and 3 prime of dsRED as molecular marker. This approach closely links expression of the shRNA to the fluorochrome and resulted in a potent and stable knockdown. We expressed a control shRNA targeting Renilla luciferase and several shRNA sequences targeting XIAP. In order to discriminate different derivative cell populations within a single mouse, we co-expressed a second fluorochrome from a second plasmid so that green cells harbored a knockdown of XIAP, while blue cells harbored the control construct, thus allowing in vivo outcompete proliferation assays. We called our new approach "genetically engineered PDX (GEPDX)" models in parallel to genetically engineered mouse models (GEMM).We used our new technology to study the role of XIAP, the X-linked inhibitor of apoptosis for acute lymphoblastic leukemia (ALL), the single most frequent tumor in children. XIAP is frequently and highly overexpressed in hematological malignancies and its up-regulation was shown to be associated with inferior prognosis of patients in different tumors. Nevertheless XIAP's role for tumor maintenance remains unclear.In several preB- and T-cell ALL cell lines, potent and stable knockdown of XIAP did not alter cell proliferation in vitro or upon xeno-transplantation in vivo. Thus expression levels of XIAP seem irrelevant for spontaneous proliferation of established acute leukemia cell lines in vitro and in vivo.We next studied PDX ALL cells growing in mice as model closer related to patients. We generated GEPDX cells from two children with relapse of a B precursor ALL with either knockdown of XIAP or control knockdown together with the appropriate molecular color markers. When blue and green GEPDX cells harboring control or XIAP knockdown, respectively, were co-transplanted into mice at a 1:1 ratio in a competitive outcompete assay, control transfected cells significantly overgrew or even eliminated cells with XIAP knockdown in both samples studied. GEPDX cells with knockdown of XIAP showed a significant and dose-dependent growth disadvantage in vivo compared to control cells indicating that XIAP played an essential role for PDX cells growing in vivo.Thus, our novel technique of genetic engineering in PDX cells revealed an essential role of XIAP for tumor maintenance and growth in patients' tumor cells making XIAP an attractive therapeutic target in ALL. As established ALL cell lines were unable to unravel this important role of XIAP, GEPDX might be superior to cell lines for identifying genes with essential function. GEPDX represent a powerful new tool to characterize the complex environment of individual patients' tumor cells in vivo, the function of the many lesions and alterations described by expression profiling and next generation sequencing. DisclosuresNo relevant conflicts of interest to declare.

ESAM Is a Novel Human Hematopoietic Stem Cell Marker Associated with a Subset of Human Leukemias

Murine hematopoietic stem cells (HSCs) can be isolated with high efficiency as Lineage- Sca-1+ c-kitHigh (LSK) CD34-/Low CD150+ CD48- cells. In humans, however, the same method is not useful because of critical differences between murine and human HSC phenotypes. Such discrepancy has hampered the translation of findings in mice into a human preclinical or clinical context. Therefore, the identification of common HSC antigens between the two species would be a significant advance with respect to translational studies of HSC biology. We previously identified endothelial cell-selective adhesion molecule (ESAM) as a novel maker for HSCs in mice (Blood, 2009). We also found that ESAM is functionally important for murine HSCs to reconstitute hematopoiesis after 5-FU treatment (J Immunol, 2012). In the present study, we have extended our research of ESAM to human HSCs and leukemia.We first examined whether ESAM expression showed potential as a marker of human HSCs. In addition to adult BM, the majority of CD34+ CD38- cells in cord blood (CB) and G-CSF mobilized peripheral blood expressed ESAM. The addition of anti-CD90 and CD45RA antibodies divides the adult BM CD34+ CD38- fraction into three subpopulations, namely HSCs, multipotent progenitors (MPPs), and multi-lymphoid progenitors (MLPs). We found that HSCs expressed high levels of ESAM whereas MPPs expressed lower levels and many MLPs lost ESAM expression. Functional assessment for ESAM-/Low and ESAMHigh cells in the CD34+ CD38- fractionconfirmed that high ESAM expression distinguishes progenitors that are more primitive and multipotent.We also identified a subset of CD34+ CD38- cells in adult BM and CB that expressed extremely high levels of ESAM, namely ESAMBright cells. Gene expression profiles of the CD34+ CD38- ESAMHigh and CD34+ CD38- ESAMBright populations showed that the former cells expressed HSC-related genes whereas the latter showed more endothelial-related profiles. Indeed, the CD34+ CD38- ESAMBright cells produced CD31+ endothelial cells, but not CD45+ hematopoietic cells, in co-culture with MS5 stromal cells. These results suggest that the CD34+ CD38- fraction, which is conventionally considered the human HSC fraction, also contains a substantial number of non-hematopoietic progenitors. Thus, the inclusion of ESAM provides a more accurate estimation of HSC numbers.Since some of HSC-related antigens are useful for determining leukemia lineage and have utility as prognostic indicators, we determined whether ESAM might also be a valuable addition to this antigen panel. First, we examined human leukemia cell lines. Tested myeloid leukemia lines including KG-1a, HL60, THP1, U937 and Kasumi were uniformly negative for ESAM expression. Jurkat and MOLT4, lymphoid lineage lines were also negative. On the other hand, HEL, an erythroid leukemia cell line, and CMK, a megakaryocytic leukemia cell line, exhibited high expression of ESAM. Additionally, K562 cells, which originated from CML that subsequently transformed into acute erythro-leukemia, also express ESAM. We then evaluated ESAM expression on primary acute leukemia cells, which were isolated from patients upon diagnosis. Interestingly, while all of ALL cases were virtually negative for ESAM, more than half of AML cases were ESAM-positive. Notably, the ESAM expression pattern on AML cases substantially differs even in the same FAB classification.We inferred that AML cells might change their ESAM expression levels according to cell intrinsic features and/or the surrounding environment in vivo. Therefore, we inoculated ESAM- KG-1a cells into NOD/SCID mice and harvested reconstituted KG-1a (rKG-1a) cells after the inoculation. They were then cultured in vitro and inoculated again into NOD/SCID mice. FACS analyses revealed that, although parental KG-1a cells were ESAM-negative, rKG-1a cells expressed a substantial amount of ESAM. Notably, rKG-1a cells were more aggressive and killed the recipient mice in a shorter period. This observation indicates that leukemia cells change their surface phenotype according to the environment, and that ESAM expression may be related to the acquisition of a more aggressive phenotype.In conclusion, we demonstrate that ESAM is a reliable marker of HSCs in humans as well as in mice. Additionally, ESAM is expressed on some of human acute leukemia cells and might be useful for lineage determination and as prognostic indicator. DisclosuresYokota:SHIONOGI & CO., LTD.: Research Funding. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Alpha-bisabolol, not a matter for cancer therapy. Commentary: "Research on the immunosuppressive activity of ingredients contained in sunscreens".

Alpha-bisabolol, not a matter for cancer therapy. Commentary: "Research on the immunosuppressive activity of ingredients contained in sunscreens".

Effects of itraconazole plus doxorubicin on proliferation and apoptosis in acute myeloid leukemia cells

To explore the effects of itraconazole (ITC) plus adriamycin (ADM) on proliferation and apoptosis of acute myeloid leukemia cells in vitro. The growth inhibition effects of different concentrations of ITC, ADM or ITC (2, 6, 15 µmol/L) plus ADM (0.30, 0.75 µmol/L) were detected by CCK8 assay in KG1α and primary adult acute leukemia cells. Different concentrations of ITC for 7, 14 and 21 days were applied for observing the effect on colony formation. After 48 h treatments with 6 µmol/L ITC, 0.75 µmol/L ADM or ITC (6 µmol/L) plus ADM (0.75 µmol/L), the morphological changes of cells were observed by Wright staining. Flow cytometry was used to detect cell apoptotic rate, mitochondrial membrane potential and cell cycle arrest. And the expression levels of Sonic Hedgehog (Shh) signal pathway-related proteins Shh and glima-associated oncogene homdog1 (Gli1) were determined by Western blot. ITC and ADM inhibited the proliferation of KG1α and primary adult acute leukemia cells and ITC reduced the colony-formation ability of KG1α cells both in dose-dependent manners. Compared with control or single drug group, the changes of cell morphology were more apparent in combined group. Weeb coefficient test revealed a synergistic effect (D ≤ 70%C) of 0.75 µmol/L ADM plus 6 µmol/L ITC. When KG1α cells were treated with 6 µmol/L ITC plus 0.75 µmol/L ADM, the apoptotic rate was 31.72% ± 1.58%. And it was significantly higher than that in control, ITC and ADM groups (4.17% ± 0.74%, 4.33% ± 1.12%, 9.53% ± 1.15%, P < 0.01). Detection of mitochondrial membrane potential showed that low red-fluorescent cells (P3) of combined group were obviously higher than that in control, ADM and ITC single drug groups (18.80% ± 0.96% vs 5.00% ± 0.38%, 9.70% ± 0.43%, 7.10% ± 0.77%, P < 0.01) . KG1α cells were obviously arrested in G2 phase in combined group and it showed no statistical significance compared with control or single drug group. Western blot showed that the expression levels of Shh and Gli1 decreased with rising concentration of ITC. ITC plus ADM can obviously inhibit cell proliferation and increase KG1α cell apoptotic rate, mitochondrial damage and G2 phase retardation. And ITC inhibits the Shh signal pathways.

Molecular targeting of the oncoprotein PLK1 in pediatric acute myeloid leukemia: RO3280, a novel PLK1 inhibitor, induces apoptosis in leukemia cells.

Polo-like kinase 1 (PLK1) is highly expressed in many cancers and therefore a biomarker of transformation and potential target for the development of cancer-specific small molecule drugs. RO3280 was recently identified as a novel PLK1 inhibitor; however its therapeutic effects in leukemia treatment are still unknown. We found that the PLK1 protein was highly expressed in leukemia cell lines as well as 73.3% (11/15) of pediatric acute myeloid leukemia (AML) samples. PLK1 mRNA expression was significantly higher in AML samples compared with control samples (82.95 ± 110.28 vs. 6.36 ± 6.35; p < 0.001). Kaplan-Meier survival analysis revealed that shorter survival time correlated with high tumor PLK1 expression (p = 0.002). The 50% inhibitory concentration (IC50) of RO3280 for acute leukemia cells was between 74 and 797 nM. The IC50 of RO3280 in primary acute lymphocytic leukemia (ALL) and AML cells was between 35.49 and 110.76 nM and 52.80 and 147.50 nM, respectively. RO3280 induced apoptosis and cell cycle disorder in leukemia cells. RO3280 treatment regulated several apoptosis-associated genes. The regulation of DCC, CDKN1A, BTK, and SOCS2 was verified by western blot. These results provide insights into the potential use of RO3280 for AML therapy; however, the underlying mechanisms remain to be determined.

Structural Biology of Mutant IDH2 Inhibition

A number of human cancers harbor somatic point mutations in the genes encoding isocitrate dehydrogenases- 1 and -2 (IDH1, IDH2)[1]. These mutations alter residues in the enzyme active sites and confer a gain-of-function in cancer cells, resulting in the accumulation and secretion of the oncometabolite R (-)-2-hydroxyglutarate (2HG). 2HG is a potent inhibitor of DNA methylating enzymes such as TET2[2]. This suggests a connection between cancer related IDH mutations and aberrant epigenetics. As such, IDH represents an important new druggable target in the pursuit of novel cancer therapies. We have developed a small molecule, AGI-6780, that potently and selectively inhibits the tumor-associated mutant IDH2/R140Q. A crystal structure of AGI-6780 complexed with IDH2/R140Q revealed that the inhibitor binds in an allosteric manner at the dimer interface[3]. While structures of IDH1 and IDH2 were known, this is the first ever structure of an inhibited IDH protein and shows a novel conformation of IDH2. The results of steady-state enzymology analysis were consistent with allostery and slow-tight binding by AGI-6780. Treatment with AGI-6780 induced differentiation of TF-1 erythroleukemia and primary human acute myelogenous leukemia (AML) cells in vitro. These data provide proof-of- concept that inhibitors targeting mutant IDH2/R140Q could have potential applications as a differentiation therapy for cancer.

Inhibitory effect of PD98059 on MAPK signaling pathway in acute lymphocytic leukemia cells

This study was purposed to investigate the effect of blocking Ras/Erk signaling pathway on expression of important transcription factor c-fos, c-jun and TAK1 gene in primary acute lymphocytic leukemia (ALL) cells. The best effective concentration and effect time of PD98059 were screened; the expression levels of c-fos, c-jun and TAK1 in primary cultured cells of normal persons, primary cultured ALL cells and primary cultured ALL cells treated by PD98059 were detected by SYBR GreenI real-time quantitative-PCR. The results showed that before treatment by PD98059 the expression levels of c-fos and TAK1 mRNA were significantly up-regulated in primary cultured ALL cells as compared with primary cultured cells of normal persons (P = 0.014 and P = 0.017 respectively). After treatment by PD98059, the expression levels of c-fos, c-jun mRNA decreased in all 7 serum samples, while expression of TAK1 was down-regulated in 5 samples, and up-regulated in 2 samples. After treatment with PD98059, there was no statistical difference of c-fos, c-jun and TAK1 expression levels in primary cultured ALL cells and primary cultured normal cells. It is concluded that the c-fos and TAK1 activity of primary cultured ALL cells increases, and blocking the Ras/Erk signaling pathway of ALL cells can lead to obvious decrease of important transcription factors c-fos, c-jun, TAK1 genes expression.

A-Type Proanthocyanidins Prevent Engraftment Of Primary Acute Myelogenous Leukemia Cells In Mice and Exhibit Potentially Novel Anti-Leukemia Mechanisms

Acute myelogenous leukemia (AML) is often a fatal disease where after strong induction therapy most patients relapse and die. AML originates and is maintained by leukemia stem cells (LSCs). Failure to eliminate LSCs by chemotherapy is likely to result in disease relapse. Therefore, it is a priority to identify new therapies that eliminate blasts while ablating LSCs and preventing a relapse.We have found that a unique class of compounds in cranberries (Vaccinium macrocarponAit.), known as A-type proanthocyanidins (A-PACs), were effective against several leukemia cell lines and primary AML samples in vitro. A-PACs consist of monomeric epicatechin units attached to one another by a carbon-carbon bond and a distinctive ether bond that differentiates these compounds from other proanthocyanidins found in nature. Moreover, A-PACs possess ortho-hydroxyl phenolic groups that have the potential to bind to iron and alter redox status.Preliminary work showed that pre-treatment with antioxidants or holo-transferrin (iron-saturated transferrin) partially protected AML cells from A-PAC induced cell death (p<0.01). A-PACs were also found to selectively ablate leukemia stem and progenitor cells, with minimal effects on normal hematopoetic stem cells. Furthermore, AML engraftment of cells treated ex vivo with 62.5 µg/ml A-PACs was decreased (90.6%, n=3, p<0.001), while normal CD34+ cells retained engraftment capability in immunodeficient mice. It was also found that a fraction of A-PACs of up to 7 degree of polymerization was more effective than individual A-PACs. This information prompted us to investigate the in vivo anti-leukemia effects of A-PACs in xenotransplanted mice with primary AML samples, and to further investigate the mechanisms associated with these compounds.Primary AML cells were injected in sub-lethally irradiated NOD/SCID mice. Four weeks after injections, when human leukemia cells have engrafted, intraperitoneal injections of cytarabine (AraC) at 60 mg/kg were given to the mice for 1 week everyday or A-PACs (100 mg/kg dose every 3 days for A-PACs) and vehicle control (1% DMSO in PBS every 3 days) were injected for 2.5 weeks. Mice were sacrificed and leukemia engraftment evaluated using anti-human CD45 and CD33. Moreover, primary cells treated with A-PACs were assessed for effects on iron metabolism, ROS, and survival pathways either by gene expression analysis, flow cytometry or mass spectrometry.Administration of A-PACs to NOD-SCID mice bearing AML tumors reduced tumor burden. Mice that were treated with the vehicle control had engraftment of AML primary cells equivalent to 16.1% (95% CI: -6.0, 38.37; n=4), whereas the mice treated with the A-PACs and AraC showed a level of engraftment of 4.9% (95% CI: 2, 8; n=5) and 5.8% (95% CI: -1.1, 12.7; n=5), respectively. No significant changes in hemoglobin or weight were found between the different treatment groups. Moreover, qPCR analysis of sensitive leukemia cell lines treated with A-PACs showed changes in gene expression of several iron metabolism genes in sensitive leukemia cell lines (up-regulation of ferritin and transferrin receptors 1 and down-regulation of ferroportin) and several ROS-relevant genes (down-regulation of nuclear factor erythroid-2-related factor 2 and glutamate-cysteine ligase regulatory subunit). Mass spectrometry also confirmed that A-PACs bind iron.The results indicate that A-PACs not only target primary AML cells in vitro but are also effective in vivo. Secondary transplants are also being performed to determine the effects on LSC activity. Some of the anti-leukemia mechanisms under investigation include effects related to iron metabolism, ROS or inhibition of survival pathways. Understanding the unique structure and biological effects of A-PACs may provide novel information about pathways involved in the survival of LSCs and provide crucial information in preparation for clinical trials and/or optimal combination drug therapies. Disclosures:Rivella:Novartis: Consultancy; Bayer: Consultancy; Isis: Consultancy, Research Funding; Merganser: Equity Ownership, Research Funding; Biomarin: Consultancy; Alexion: Consultancy; Imago: Consultancy.

Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells.

Differences in proliferative capacity of primary human acute myelogenous leukaemia cells are associated with altered gene expression profiles and can be used for subclassification of patients

Proliferative capacity of acute myelogenous leukaemia (AML) blasts is important for leukaemogenesis, and we have investigated whether proliferative capacity of primary human AML cells could be used for subclassification of patients. In vitro proliferative capacity of AML cells derived from two independent groups was investigated. Cells were cultured under highly standardized conditions and proliferation assayed by (3) H-thymidine incorporation after seven days culture. Patients were subclassified by clustering models, and gene expression profile was examined by microarray analyses. Based on proliferative capacity of the AML cells, three different patient clusters were identified: (i) autocrine proliferation that was increased by exogenous cytokines; (ii) detectable proliferation only in presence of exogenous cytokines; and (iii) low or undetectable proliferation even in presence of exogenous cytokines. Patients with highest proliferative capacity cells had no favourable prognostic impact by NPM-1 mutation. Analysis of gene expression profiles showed that the most proliferative cells generally had altered expression of genes involved in regulation of transcription/RNA functions, whereas patients with high proliferative capacity and internal tandem duplications (ITDs) in the FLT3 cytokine receptor gene had altered expression of several molecules involved in cytoplasmic signal transduction. In vitro proliferative capacity of primary human AML cells was considerably variable between patients and could be used to identify biologically distinct patient subsets.