Kasumi-1 Acute Myeloid Leukemia Cells Research Articles

Abstract 1338: Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells

Abstract Human leukocyte antigen-G (HLA-G) suppresses lymphoid and myeloid immune systems by binding ILT2 and ILT4 respectively and activating ITIM signaling. HLA-G is abundantly expressed in placental cytotrophoblasts to block fetal rejection. HLA-G is not expressed in most normal cells but is derepressed in in about 50% of malignancies. We engineered ILT2 and ILT4 to produce activating signals as Chimeric ILT Receptors or CIR. Preliminary results indicated robust killing of Acute Myeloid Leukemia (AML) cells by Natural Killer (NK) cells expressing either ILT2 or ILT4 CIRs. Here, we evaluated the effect of alteration of activating domains in the CIR constructs to produce signals that mimic the effect of IL-18 signaling in CIR-NK cells to enhance cytotoxicity, NK cell growth and persistence. Further, HLA-G expression and CIR-NK cell targeting of primary human cell types were rigorously screened. Activated NK cells derived from peripheral blood (2 to 5 donors) were transduced with γ-retroviruses directing expression of CIR proteins (CIR.X.Y or CIR.Y.X) where X stimulates cytotoxicity through CD3ζ, DAP10 or DAP12, and Y directs coactivation through 4-1BB, fusion of the MyD88 death domain or recruitment of endogenous MyD88 with receptor-derived TIR domains and soluble IL-15. CIR-NK cells were cocultured with GFPffluc-expressing AML and solid tumor target cell lines or normal primary human cells. NK cell proliferation and cytotoxicity of CIR-NK cells was monitored by Incucyte microscopy and supernatants were analyzed for cytokine release by ELISA. At low E:T (1:20 - 1:40) in 7-day cocultures, mock-transduced NK cells displayed innate killing activity against HLA-G+ Molm13 and Kasumi1 AML cells that was augmented by CIR constructs containing direct fusions of MyD88 and DAP10 or DAP12 (mock = 2.06E7 ± 4.53E6 vs. CIR-ILT4.MyD88.DAP10 = 1.03E7 ± 5.46E6) with 7-fold elevation of IFN-γ production. CIR constructs that recruited endogenous MyD88 signaling through the TLR2 TIR domain (4-1BB.DAP10.TLR2) also exhibited enhanced anti-HLA-G activity (9.13E6 ± 5.33E6). Against HLA-G+ HT-1376 bladder cancer cells (E:T = 1:10-1:20), CIR-NK cells also displayed enhanced tumor cell killing with enhanced NK cell growth. Conversely, CIR-enhanced killing was not observed against HLA-G− HCT-116 colorectal cancer cells. The persistence of CIR-NK cell potency after 4 weeks of expansion varied markedly with different activation domains employed. When screened against primary human cells (cardiomyocytes, hepatocytes, hepatic endothelial cells, corneal, colon and lung epithelial cells), no CIR-specific targeting was observed at high E:T ratios (1:1 and 1:5). In conclusion, CIR-NK cells exhibit potent and specific anti-tumor activity against HLA-G+ solid and leukemia cells. Alteration of CIR-NK cell intracellular signaling can optimize the potency and persistence of NK cell anti-tumor activity. Citation Format: MyLinh Duong, Jihyun Park, Raphel Ognar, Simon Wain-Hobson, Henri Bayle. Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1338.

REM-422, a Potent, Selective, Oral Small Molecule mRNA Degrader of the MYB Oncogene, Demonstrates Anti-Tumor Activity in Mouse Xenograft Models of AML

The c-MYB (MYB) oncogenic transcription factor is a key regulator of hematopoietic cell differentiation and proliferation. Recurrent genetic lesions or dysregulation of MYB have been identified in a variety of cancers, including adenoid cystic carcinoma (ACC), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), pediatric low-grade glioma (pLGG), and others. AML is characterized by multiple oncogenic abnormalities and high MYB mRNA expression. Evidence from in vitro and in vivo animal studies suggests that MYB may be a key downstream effector of these oncogenic abnormalities through the direct regulation of genes such as BCL2, MYC and FLT3. Functional genomics studies reveal MYB dependencies in human leukemia cell lines and mouse models of leukemia. Together, these data suggest that MYB is an attractive therapeutic target for AML and other hematological malignancies. Therapeutic agents that directly target MYB have yet to be developed, presumably due to the challenges of targeting this intrinsically disordered transcription factor, which lacks tractable ligand binding pockets. Here, we describe the mechanism of action and biological activity of REM-422, a potent, selective, oral small molecule messenger RNA (mRNA) degrader of MYB. REM-422 was identified through screening and medicinal chemistry efforts directed towards identifying small molecules that induce the inclusion of a normally unused ‘poison exon’ (PE), which contains a premature termination codon, into the MYB pre-mRNA transcript. Poison exon inclusion activates the nonsense-mediated decay pathway and promotes the degradation of MYB mRNA. We demonstrate in a cell-free assay that REM-422 acts by selectively facilitating the binding of the U1 snRNP spliceosome complex, which governs exon usage by recognizing 5‘ splice sites, to oligonucleotides containing the MYB PE 5‘ splice site sequence. REM-422 treatment of human leukemia cell lines promotes poison exon inclusion, leading to degradation of MYB mRNA and subsequent reduction in MYB protein levels (Figure 1A). In a panel of AML cell lines, REM-422 demonstrated preferential anti-proliferative activity in cell lines with high MYB expression, and in those shown to be MYB-dependent through functional genomics studies. To understand the pharmacokinetic (PK) and pharmacodynamic (PD) effects of MYB in vivo, Kasumi-1 AML cells were implanted subcutaneously in CB17 scid immunocompromised mice and REM-422 was dosed by oral gavage after the formation of established tumors. QD PO administration of REM-422 demonstrated dose-dependent effects in tumors on MYB mRNA levels, with induction of the MYB poison exon leading to reductions of MYB functional mRNA. In a 21-day study, REM-422 demonstrated significant anti-tumor activity, including tumor regressions at well tolerated dose levels (Figure 1 left). To further investigate the activity of REM-422, AML patient cells were injected into NOG immunocompromised mice and allowed to engraft. After successful engraftment, mice were dosed by oral gavage once daily with REM-422 at 10 mg/kg or vehicle for 24 days. Flow cytometry analysis of bone marrow and peripheral blood samples demonstrated that REM-422 led to the eradication of human leukemia cells from both compartments (Figure 1 right). Together, these data demonstrate that REM-422 treatment leads to degradation of MYB mRNA in preclinical models of AML and supports the therapeutic potential for REM-422 in AML patients.

Developing a Humanized Murine Model for Co-Transplantation of Acute Myeloid Leukemia

Humanized mouse models and primary disease xenografts have produced significant translational insight into the biology of the bone marrow (BM) niche and aided drug discovery in acute myeloid leukemia (AML). To enable the study of cell-cell interactions between hematopoietic stem and progenitor cells (HSPCs) and AML as an emerging source of drug resistance, we describe two complementary approaches to humanized AML xenotransplantation models. One strategy utilizes the sequential transplantation of humanized hCD34+ cells followed by human AML cells. Recipient NBSGW mice (NOD.Cg-Kit W-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ), known for high rates of medullary engraftment, were conditioned with 15mg/kg busulfan followed by injection with either granulocyte colony-stimulating (G-CSF)-mobilized or BM-derived human CD34+ HSPCs. Flow cytometric analysis of the BM following injection of hCD34+ cells revealed efficient BM engraftment by both G-CSF- and BM- derived hCD34+ cells, demonstrated by BM human chimerism (hCD45%) of 63.7±10.0% by 8-weeks post-injection. Further BM hCD45 subtype analyses revealed B-cell (hCD45+ hCD19+) dominant repopulation (78.3±4.48%), followed by myeloid cell (hCD45+ hCD33+) repopulation (18.12±4.19%). T-cell repopulation was predictably minimal (0.008±0.01%). At 9- and 15- weeks post hCD34+ cells injection, GFP-expressing MOLM-14 AML cells (MOLM-14-GFP; 1E5, 1E6, or 2E6 cells) were injected. Flow cytometric analyses of MOLM-14-GFP BM chimerism up to 21 days post-injection showed complete absence of MOLM-14-GFP cells in the BM with no significant changes in overall hCD45 chimerism and hCD45 subtype composition compared to a PBS-injected control. These observations are consistent with a potential role for residual healthy hematopoietic cells in suppressing AML engraftment in the BM, previously shown in congenic studies. To test this hypothesis, we developed a second humanized AML model designed to investigate immunotherapeutic approaches for AML, where AML blasts are engrafted first, then followed by a human immune cell challenge. FA-AML1 or Kasumi-1 AML cells were injected into NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) at 8 weeks of age. Injected mice were monitored for AML engraftment by HLA-DR expression in peripheral blood (PB). Engraftment occurred 6-12 weeks post-injection. After AML engraftment, we reconstituted a human immune system in the mice with a one-time injection of peripheral blood mononuclear cells (PBMCs), confirmed by flow cytometry for CD3+ T-cells (14.7±9.2%), CD33+ myeloid cells, and CD19+ B-cells. AML burden in the PB decreased 5-10% upon PBMC engraftment. In this model, a PB burden of <10% AML engraftment confers durable leukemic remission, whereas at >60% AML engraftment, leukemic cells persist in the periphery for over 100 days. Flow cytometric analysis demonstrated immune cell numbers peak in week 1 post-injection (30%), then decrease by week 4 to less than 1% in both the BM and PB, but persist at this low level for over 100 days. Altogether, these models confirm the ability of human hematopoietic cells to actively suppress leukemic engraftment in the BM microenvironment. More broadly, we demonstrate the development of two complementary humanized AML co-transplantation models that can attain dynamic ranges of human immune cell and AML cell engraftment to facilitate preclinical therapeutic development studies.

Spatially Confined T-Cell Mediated Anti-AML Tumor Immunity By Dual Recognition Antibodies (CD123/CD200)

Definitive cure of acute myeloid leukemia (AML) remains an unmet need. While recombinant antibody-based and CAR-T cell mediated immunotherapies bear great potential in targeting chemotherapy-resistant leukemic stem cells, these approaches are largely limited by the lack of specific targets uniquely expressed by leukemic cells. Here, we propose a combinatorial strategy using a two-target approach which may have improved on-target and reduced off-target effects. The well-known stem cell marker interleukin-3 receptor α (CD123) is expressed on most AML cells. CD200 recently emerged as a cell surface marker mediating immuno-evasion on high-risk AML cells. We engineered the "Prodrug-activating chain Exchange"(PACE) TriFAB system (Dickopf S et al. Biological Chemistry, 2022) to yield a combinatorial anti-AML directed T-cell response. PACE-TriFAB educts are complementary Knob-into-hole IgG derived prodrugs in which one half is exchanged by a dummy containing an interface destabilizing mutation in the CH3 domain. The CH2 domain of the main body is replaced by a CD3 FAB light or heavy chain, respectively. With the hinge region being replaced by a flexible linker and therefore lacking disulfide bonds, a chain exchange between two educts is enabled when in close proximity on the cell surface. This leads to an activated anti CD3 domain and a dummy-dummy by-product. To demonstrate the T-cell activating potential, we co-cultured fresh PBMCs from healthy donors together with the CD200+/CD123+ AML cell line Kasumi-1 and checked T-cell activation by flow cytometry (E:T ratio 5:1, prodrug conc. 20 nM, 24 h stimulation). We observed a marked increase in CD69, CD25 and CD137 expression on CD8+ cells in the case when the CD123 and CD200 prodrugs were applicated simultaneously together with target cells, but not when single prodrugs alone or without target cells were analyzed (Median fluorescence intensity for CD25, CD69 and CD137: No construct: 450, 8.900 & 2.100; prodrug CD200: 460, 8.700 & 2.100; prodrug CD123: 460, 9.600 & 2.000; both prodrugs: 6.500, 344.300 & 43.100; results confirmed in repeat experiments) For the evaluation of the specific cytotoxic effect of the construct we transduced Kasumi-1 cells with an mCherry lentiviral vector and imaged the cells co-cultured with PBMCs every 2 h over a time span of 60 h (E:T ratio 5:1, prodrug conc. 10 nM). Proliferation of the Kasumi-1-mCherry cells was observed when no prodrug or only a single prodrug was added. However, application of both prodrugs together led to a decrease in mCherry positive cell count (log2 fold changes to starting point: No PBMCs 1,2; no construct 0,9; prodrug CD200 0,9; prodrug CD123 0,4; both prodrugs -2,1; n=3). Not only could we show that this elimination of tumor cells was dose dependent but also it was constrained by the E:T ratio (pivot point from killing to proliferation at 0,1 nM and E:T of 1:2 n=3). Similar results were obtained using patient derived xenograft (PDX) AML cells. In contrast to Kasumi-1 AML cells, specific killing of patient-derived PDX cells could be documented with lower E:T ratios and lower prodrug concentrations (pivot point from killing to proliferation at 0,05 nM and E:T of 1:5) We also started to analyze the anti-leukemic effect in vivo. Luciferase-expressing CD200+/CD123+ AML PDX cells were injected into NSG mice. On day +15, PBMC from a healthy individual were transplanted, followed by additional applications of the antibodies on days +16, +18, +21, +23, +25 & +29. Tumor burden was analyzed by bioluminescence imaging (log10 fold change [P/sec] 2 weeks after PBMC injection: Control 1,9; treated -0,4; group size = 3). In conclusion the combination of anti CD200 and anti CD123 as prodrugs of an on-target activating trispecific T-cell engager kills AML cells in vitro and in vivo and has the potential to eliminate AML LSC while minimizing the risk for severe on-target off-leukemia toxicity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SYK Inhibitors, Entospletinib and Lanraplenib, Show Potent Anti-Leukemic Activity in Combination with Targeted Agents

Introduction: Spleen tyrosine kinase (SYK) acts as a key integrator of cellular signaling from surface contact and receptor tyrosine kinase receptors containing an immunoreceptor tyrosine-based activation motif (ITAM). In acute myeloid leukemia (AML), SYK serves as a relay to an oncogenic transcriptional regulatory network (TRN) linked to NPM1, HOXA9, and MEIS1 helping to suppress myeloid progenitor maturation and promote proliferation. The selective, oral SYK inhibitor, entospletinib (ENTO), has demonstrated clinical activity in HOXA9/MEIS1-driven AML with acceptable tolerability when combined with intensive induction chemotherapy. A global phase 3 trial (AGILITY, NCT05020665) is currently underway, evaluating the combination of ENTO with intensive induction/consolidation chemotherapy in patients with treatment-naive NPM1-mutated (NPM1m) AML with measurable residual disease as the primary endpoint. Lanraplenib (LANRA) is a next-generation SYK inhibitor with similar potency, enhanced selectivity, and more favorable pharmacologic properties that is currently being evaluated in combination with gilteritinib in patients with relapsed or refractory (R/R) FLT3-mutated AML (NCT05028751). Results: ENTO exhibits anti-proliferative activity in NPM1m AML with associated HOXA9/MEIS1 dysregulation in ex vivo drug sensitivity studies. We hypothesized that SYK inhibition could synergize with other targeted therapies to enhance anti-leukemic activity at different nodes in this pathway. To address this hypothesis, the FLT3 internal tandem duplication/MLL-rearranged cell lines, MOLM13 and MV411, were treated with increasing doses of either LANRA or ENTO in combination with a small molecule MLL-Menin inhibitor (SNDX5613). Synergistic anti-proliferative effects were observed across a broad range of concentrations. No combinatorial or single-agent SNDX5613 activity was seen in the KASUMI-1 AML cell line, which lacks an MLL rearrangement. Phenotypic studies using flow cytometry were performed to assess the mechanistic response for SYK inhibition with LANRA in combination with SNDX5613. LANRA was found to increase the percentage of CD11b-positive cells and mean fluorescence intensity, while SNDX5613 led to a marked increase in CD14. In contrast to either single agent, the combination triggered early apoptosis and cell death, suggesting a more complete blockade of the HOXA9/MEIS1 transcriptional program through synergistic inhibition by orthogonal mechanisms. Furthermore, treatment of leukemic cells with either ENTO or LANRA inhibited SYK auto-phosphorylation in a dose-dependent manner. We also explored the potential for synergistic activity with gilteritinib (FLT3 inhibitor) and venetoclax (BCL2 inhibitor) using patient-derived AML isolates ex vivo based on the potential for SYK signaling cross talk with FLT3 itself and survival pathways. We found strong synergistic anti-proliferative activity for the combination of LANRA with either gilteritinib or venetoclax. Patient-derived xenograft (PDX) studies also demonstrated deeper reductions in leukemic burden in the peripheral blood, liver, and bone marrow after 28 days of treatment. Follow-up PDX studies assessing overall survival and pharmacodynamic activity are ongoing using an optimized dosing regimen and schedule. Summary/Conclusion: SYK is a promising therapeutic target for genetically defined subsets of AML. Two highly selective SYK inhibitors are currently undergoing clinical testing in different but complementary clinical settings. Given its central role as a modulator of leukemogenic signaling, SYK inhibition has the potential to synergize with other targeted therapies in AML. In preclinical studies, LANRA-mediated SYK inhibition shows compelling anti-proliferative and pro-apoptotic activity in combination with gilteritinib, venetoclax, and SNDX5613 in genetically defined subsets of AML.

Interferon-γ Induces Distinct mRNA and Protein Profiles in Acute and Chronic Myeloid Leukemia

Leukemia cells promote tumor survival and progression by avoiding host immune surveillance. A key pathway involves indoleamine 2,3-dioxygenase-1 (IDO1), an interferon (IFN)-γ-inducible molecule mediating feedback inhibition and hindering anti-tumor immunity. We have previously shown thattreatment of primary acute myeloid leukemia (AML) cells with IFN-γ in vitro induces functional IDO1 in 50% of unselected cases (‘IFN-γ responders‘), an effect which was abrogated by STAT3 inhibitors. With a median follow-up of 8 years, these IFN-γ responders experienced a shorter event-free survival and overall survival. The current study was undertaken to dissect the intracellular signaling pathways that are activated in leukemia cells in response to IFN-γ.We employed an alpha version of the Vantage 3D™ DNA:RNA:Protein Heme Assay (NanoString Technologies Inc., Seattle, USA) to profile DNA mutations, RNA, protein and phosphoprotein simultaneously. The assay profiles 180 mRNA species involved in hematology/oncology pathways, 38 total and phosphorylated proteins (including JAK-STAT, Src, BTK, FLT3, PI3K-Akt, NF-kB), and 124 single nucleotide variants (SNVs) relevant to hematological malignancies (IDH, FLT3, KRAS, NRAS, DNMT3, JAK3 and others). K562 and TCC-S cells (chronic myeloid leukemia [CML] in blast crisis), KG1a cells (leukemia stem cell [LSC]-like AML) and Kasumi-1 cells [AML with t(8;21)] were stimulated with 100 ng/mL IFN-γ then harvested and lysed after 30m, 1h, 2h, 12h and 24h for processing in parallel workflows (protein/mRNA and DNA SNV assays) prior to overnight hybridization with NanoString reporters. Pooled RNA/DNA/protein samples were analyzed using the nCounter® FLEX platform and the nSolver® software (alpha version 4.0). CCRF-CEM and HUT78 cell lines, with known DNA variants and expression of protein targets represented in the 3D Heme panel, were used as controls.K562, TCC-S and Kasumi-1 cells, but not KG1a cells, up-regulated IDO1 mRNA after IFN-γ challenge. IFN-γ-sensitive CML cells (K562 and TCC-S) expressed high levels of mRNA transcripts for JAK-STAT intermediates and genes implicated in apoptosis, DNA damage and cytokine signaling (Fig. 1). IFN-γ-sensitive t(8;21) AML cells displayed a distinct transcriptomic profile with an up-regulated subset of IFN-inducible genes, including IRF1 , STAT1, BCL3, SOCS1 , PD-L1 , ISG15 , OSM , HLA class I and genes involved in antigen processing (CIITA ). In contrast, the transcriptome of IFN-γ-insensitive LSC-like KG1a cells consisted of high levels of genes implicated in epigenetics and transcription, NF-κB, MYC, B-cell receptor and T-cell receptor signaling, and low expression levels of genes mediating JAK-STAT signaling, apoptosis, DNA damage and cytokine signaling. Overall phosphorylation of intracellular proteins was largely unchanged after IFN-γ stimulation of KG1a cells. In contrast, IFN-γ activation of Kasumi-1 AML cells was associated with increased phosphorylation of Akt(S473), STAT3(Y705) and proline-rich Akt substrate of 40kD (PRAS40) (T246). Similar phosphorylation changes were apparent in TCC-S CML cells, although with lower magnitude. Interestingly, HOXA9 transcription factor, which is commonly dysregulated in acute leukemias, was highly expressed, both as mRNA and protein (r2=0.912; p<0.001), in LSC-like KG1a cells, but not in t(8;21) AML and CML cell lines. SNV analysis identified all expected gene mutations in the control cell lines and TP53(R248Q) in Kasumi-1 cells.The use of artificial neural network algorithms and a public collection of AML transcriptomic data (641 patients; E-MTAB-3444) allowed us to identify enriched functional-related gene groups (‘immune response’, ‘antigen processing and presentation’, ‘MAPK signaling pathway’; DAVID v6.8), the expression of which was predicted by the 3 most up-regulated transcripts in our IFN-γ-sensitive leukemia cells (IRF1, STAT1 and BCL3 ).In conclusion, the combined analysis of SNVs, mRNA and protein expression on the nCounter® platform is a powerful tool to characterize the molecular landscape of leukemias. Our analysis of IFN-γ-stimulated leukemia cells has identified molecular pathways that could be targeted in patients with ‘inflamed’ tumors, in which microenvironmental production of IFN-γ is heightened due to ongoing T-cell responses.Grant support: Roger Counter Foundation, UK; Qatar National Research Fund (#NPRP8-2297-3-494). [Display omitted] DisclosuresMeredith:NanoString Technologies: Employment. Ross:NanoString Technologies: Employment. Demirkan:NanoString Technologies: Employment. Rutella:NanoString Technologies, Seattle, USA: Research Funding.

Bone Marrow Stromal Cell Mediated Resistance to Mertk Inhibition in Acute Myeloid Leukemia

MERTK is a receptor tyrosine kinase of the TAM family (TYRO-3, AXL, MERTK) that is aberrantly expressed in 80-100% of pediatric and adult primary acute myeloid leukemia (AML) samples. MERTK inhibition mediated by small molecule tyrosine kinase inhibitors (TKIs), UNC2025 or MRX-2843, altered cell cycle progression and induced apoptosis in leukemia cell cultures. Moreover, treatment with these MERTK TKIs prolonged survival in mouse xenograft models of acute leukemia, but was not curative. In these models, treatment with the TKI effectively reduced peripheral disease burden but was less effective in the bone marrow, suggesting a role for the bone marrow microenvironment in therapeutic resistance.To evaluate the role of the bone marrow stromal niche in mediating resistance to MERTK inhibition by MRX-2843, AML cell lines were cultured in the presence of a fibroblast-like cell line (HS27) and induction of cell death and apoptosis was determined by flow cytometry after treatment with MRX-2843 or vehicle. Co-culture with the HS27 cell line significantly reduced cell death in Kasumi-1 AML cell cultures in response to treatment with 200nM MRX-2843 compared to leukemia cells alone (31.9% versus 61.2%, p<0.05). Similar results were observed in NOMO-1 and OCI-AML5 AML cell cultures. Additionally, cell cycle profiles were determined by flow cytometry in the presence or absence of stromal cells and MRX-2843. Treatment of Kasumi-1 cell cultures with 200nM MRX-2843 in the absence of stromal cells resulted in a significant accumulation of cells in G2 phase relative to vehicle-treated cultures (8.9% versus 14.8%, p<0.01) and was accompanied by an increase in γH2AX expression detected by immunoblot. In contrast, there was no change in cell cycle progression or γH2AX expression in cultures treated with MRX-2843 when co-cultured with the stromal cell line. NOMO-1 cells also accumulated in G2 phase in response to treatment with MRX-2843 and were protected from changes in cell cycle progression by co-culture with HS27 cells. Additionally, MERTK inhibition resulted in induction of polyploidy in NOMO-1 cell cultures, but this effect was abrogated in the presence of stromal cells. Collectively these results indicate abrogation of cell cycle progression in leukemia cells in response to treatment with MRX-2843 and demonstrate protection from this effect mediated by stromal cell interactions.The MERTK ligand, GAS6, mediates resistance to cytotoxic chemotherapy in leukemia cells and is a poor prognostic marker in AML. To evaluate the role of the MERTK ligands GAS6 and PROS1 in resistance to MERTK inhibition, their expression in stromal cells was determined by immunoblot. GAS6, but not PROS1, was expressed in HS27 cells; however, MRX-2843 retained its ability to inhibit MERTK phosphorylation in stromal cell co-cultures. We hypothesized that upregulation of the other TAM kinases, AXL and/or TYRO3, may mediate resistance as they share common ligands. To test this hypothesis, Kasumi-1 cells were treated with UNC4241, a small molecule pan-TAM inhibitor, in the presence of stromal cells. While co-culture protected Kasumi-1 cells from MRX-2843 induced apoptosis, treatment with UNC4241 induced apoptosis in the presence of stromal cells (32.7% versus 20.4% in vehicle control, p<0.01).To further evaluate the interactions between stromal cells and leukemia cells, Kasumi-1 cells were cultured in HS27-conditioned medium in the presence or absence of MRX-2843. Conditioned medium was not sufficient to provide protection from MRX-2843 induced apoptosis (74.0% vs. 69.8% in unconditioned medium, p>0.05). Similarly, addition of 500nM exogenous GAS6 or PROS1 did not protect leukemia cells from apoptosis in the presence of MRX-2843. Together, these results suggest that direct cell-cell contact between leukemia and stromal cells is necessary to provide protection.Together these data support a model whereby direct cell-cell contact with stromal cells in the bone marrow niche protects leukemia cells from MRX-2843 induced apoptosis and cell cycle alterations and suggest that bone marrow mobilizing agents may be effective to overcome resistance to MERTK inhibitors. DisclosuresWang:Meryx: Equity Ownership; University of North Carolina: Patents & Royalties. Frye:University of North Carolina: Patents & Royalties; Meryx: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Earp:Meryx: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. DeRyckere:Meryx: Equity Ownership. Graham:Meryx: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Abstract 260: Bromodomain and extra-terminal motif proteins regulate linear and circular PVT1 in acute myeloid leukemia cells under normoxia and hypoxia

Abstract Bromodomain and extra-terminal motif-protein inhibitors (BETi) have shown an antileukemic effect in several subtypes of acute myeloid leukemia (AML), both in vitro and in vivo (Daniel Gerlach et al. 2018 Oncogene). The effect of BETi is mainly mediated by suppression of bromodomain-containing protein 4 (BRD4) activity, an epigenetic regulator that sustains MYC and c-KIT expression, with consequences on a number of cellular processes, including proliferation and apoptosis (Kazuki Homma et al. 2017 Blood). Moreover recent findings suggested that MYC regulates through a feedback loop, a long-non coding RNA, named PVT1, which is located in the same genomic region (8q24) and is often associated with its pro-tumorigenic role (Alberto L'Abbate et al. 2018 Leukemia). PVT1 also encodes for a circular isoform (circPVT1), with MYC-dependent and independent functions. The aim of the present study is to investigate the role of BETi in the regulation of the circular and linear forms of PVT1 in AML and the functional consequences of their downregulation. OCI-AML3 and KASUMI-1 AML cell lines were treated for 16 hours with BETi under normoxic and hypoxic conditions mimicking the bone marrow microenvironment. circPVT1 and linear PVT1 (PVT1) were quantified by qRT-PCR, using total RNA and di-polisomal fraction. circPVT1 and PVT1 total RNA significantly decreased in both cell lines after treatment with BETi, under normoxia and hypoxia. Notably, KASUMI-1 expressed higher PVT1 levels under hypoxia compared to normoxia. Interestingly, circPVT1 and PVT1 were also detected in the polysomal fraction. This result was confirmed by RNA in situ hybridization (RNA-ISH) of PVT1 isoforms in combination with the S6 ribosomal marker with PVT1 showing mostly a nuclear localization, while circPVT1 colocalizing with ribosomes. In addition, circPVT1 was silenced by small interfering (siRNA) in order to investigate the effects on apoptosis, proliferation, and its downstream targets in OCI-AML3 cells. circPVT1 knockdown slightly but significantly affected cell number, with a modest impact on apoptosis and no changes of MYC transcript. We then evaluated the consequences of circPVT1 silencing on the expression of PKR, which has been previously reported to have a role in the innate immunity and to be inhibited by circRNA (Chu-Xiao Liu et al. 2019 Cell). Of note, an increased PKR expression was detected after siRNA treatment. Our findings show that circPVT1 and PVT1 are regulated by BETi, along with MYC, in AML cell lines under normoxia and hypoxia and that silencing of circPVT1 dampens cell proliferation. Therefore PVT1 and circPVT1 may contribute to AML pathogenesis and progression, and our results suggest that targeting circPVT1 may have therapeutic potentials in AML. Citation Format: Martina Ghetti, Ivan Vannini, Samantha Bruno, Chiara Liverani, Debora Traversa, Lorenzo Montanaro, Roberta Napolitano, Andrea Ghelli Luserna di Rorà, Toni Ibrahim, Clelia Tiziana Storlazzi, Giovanni Martinelli, Giorgia Simonetti. Bromodomain and extra-terminal motif proteins regulate linear and circular PVT1 in acute myeloid leukemia cells under normoxia and hypoxia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 260.

Synthetic lethality of combined AT-101 with idarubicin in acute myeloid leukemia via blockade of DNA repair and activation of intrinsic apoptotic pathway.

Leukemia stem cells (LSCs) are deemed to the mainspring for treatment failure in acute myeloid leukemia (AML). Conventional chemotherapeutic drugs fail to eradicate leukemia stem cells, which becomes the root of drug resistance and disease recurrence. Hence, new therapeutic strategies targeting LSCs are supposed to be critical for patients with AML. Here we report that combination of Bcl-2 inhibitor AT-101 and chemotherapeutic drug idarubicin (IDA) results in synergistic lethality in CD34+CD38- leukemia stem-like cells sorted from KG-1α and Kasumi-1 AML cell lines and primary CD34+ AML cells in vitro while sparing the normal counterparts. In addition, combinatorial treatment also significantly inhibits the growth of patient-derived xenograft (PDX) mouse models generated from FLT3-ITDmut AML patient in vivo. Mechanistically, the synergistic effects of AT-101 with IDA to induce cell death are closely associated with blockage of DNA damage repair and thus activates the intrinsic apoptotic pathway. In summary, these findings suggest that combinatorial therapy with AT-101 and IDA selectively eliminates leukemia stem-like cells both in vitro and in vivo, representing a potent and alternative salvage therapy for the treatment of relapsed and refractory patients with AML.

Abstract 3037: Stromal cell activation of MAPK signaling pathways mediates resistance to MERTK inhibition in acute myeloid leukemia

Abstract MERTK is a TAM family (TYRO-3, AXL, MERTK) receptor tyrosine kinase that is aberrantly expressed in &amp;gt;80% of primary acute myeloid leukemia (AML) samples. MERTK inhibition mediated by the small molecule tyrosine kinase inhibitor (TKI), MRX-2843, induced apoptosis in leukemia cell cultures and prolonged survival in mouse xenograft models of AML, but was not curative. In these models, treatment efficiently reduced peripheral disease burden but was less effective in the bone marrow, suggesting a role for the bone marrow microenvironment in therapeutic resistance. To evaluate the role of the bone marrow stromal niche in resistance to MERTK inhibition by MRX-2843, Kasumi-1 and OCI-AML5 AML cell lines were cultured with the fibroblast-like Hs27 cell line and induction of apoptosis and cell death was measured by flow cytometry after treatment with MRX-2843 or vehicle. Co-culture with Hs27 cells significantly reduced AML cell death in response to MRX-2843 compared to leukemia cells alone (e.g. Kasumi-1 31.9% vs 61.2%, p&amp;lt;0.05). To identify parallel signaling pathways that may be activated under cytokine rich bone marrow stromal conditions and mediate resistance to MRX-2843, a unbiased phospho-kinase array was performed to identify targets differentially activated by co-culture and/or MRX-2843. Kasumi-1 cells were treated with MRX-2843 in the presence or absence of stromal co-culture, then cell lysates were incubated with human phospho-kinase arrays and proteins were quantitated by densitometry. Five kinases were differentially inhibited by &amp;gt;1.5-fold in the presence or absence of stromal cells, including numerous MAPK pathway components - p38α, ERK1/2, EGFR, TOR, and HSP27. Changes in ERK1/2 phosphorylation (pERK) were confirmed by immunoblot in Kasumi-1 and OCI-AML5 cell cultures. In the absence of co-culture MRX-2843 potently inhibited pERK at doses as low as 100nM, while in co-culture even a 3-fold higher dose did not fully inhibit pERK, consistent with a potential role in resistance. To determine whether the observed upregulation and persistent activation of ERK1/2 has functional significance, Kasumi-1 cells were treated with MRX-2843 and the MEK1/2 TKI pimasertib in the presence or absence of stromal co-culture. Treatment with MRX-2843 or pimasertib alone did not significantly affect survival of AML cells in co-culture (31.3% MRX-2843, 28.1% pimasertib vs 22.7% vehicle, p=ns), but treatment with a combination of MRX-2843 and pimasertib resulted in significant induction of apoptosis compared to vehicle or single TKIs (56.7%, p&amp;lt;0.05). Similar results were observed in OCI-AML5 cell cultures and in combination with the MEK1/2 inhibitor PD0325901. Together these results indicate a critical role for stromal cell-mediated activation of ERK1/2 in resistance to MRX-2843 and demonstrate the utility of translational strategies targeting both MERTK and MAPK pathways to overcome resistance. Citation Format: Katherine A. Minson, Eleana Vasileiadi, Madeline G. Huey, Xiadong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. Stromal cell activation of MAPK signaling pathways mediates resistance to MERTK inhibition in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3037.

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

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

Bone Marrow Stromal Cell Mediated Resistance to Mertk Inhibition in Acute Leukemia

MERTK is a receptor tyrosine kinase of the TAM family (TYRO-3, AXL, MERTK) that is ectopically expressed in 30-50% of newly diagnosed pediatric acute lymphoblastic leukemia (ALL) patient samples and aberrantly expressed in 80-100% of pediatric and adult primary acute myeloid leukemia (AML) samples. MERTK inhibition mediated by shRNA or a small molecule inhibitor, MRX-2843, decreased colony-forming potential and induced apoptosis in leukemia cell cultures. Moreover, MERTK inhibition prolonged survival in mouse xenograft models of acute leukemia, but was not curative. In these models, treatment with MRX-2843 effectively reduced peripheral disease burden but was less effective in the bone marrow, suggesting a role for the bone marrow microenvironment in therapeutic resistance. Additionally, Gas6, a MERTK ligand, is a poor prognostic factor in AML, mediates increased resistance to cytotoxic chemotherapy in leukemia cells, and is expressed in the bone marrow.To determine the role of Gas6 produced by bone marrow stromal cells in mediating resistance to MERTK inhibition by MRX-2843, acute leukemia cell lines were cultured in the presence of a Gas6-producing fibroblast-like cell line (HS27) or bone marrow derived stromal cells (BMDSCs) from wild type or Gas6 knockout mice and induction of apoptosis and cell death was determined by flow cytometry after treatment with MRX-2843 or vehicle. Co-culture with the HS27 cell line significantly reduced cell death in Kasumi-1 AML cell cultures in response to treatment with 300nM MRX-2843 compared to leukemia cells alone (29.4% versus 60.5%, p=0.002). Similar results were observed in Nomo1 AML and 697 pre-B ALL cell cultures. To evaluate whether soluble factors mediated this protective effect, Kasumi-1 cells were cultured in HS27-conditioned medium in the presence or absence of MRX-2843. Interestingly, conditioned medium was not sufficient to provide protection from MRX-2843 induced apoptosis (86.0% vs. 85.5% in unconditioned medium). To more directly assess the role of Gas6, BMDSCs isolated from wild-type and Gas6 knockout C57Bl/6 mice were co-cultured with 697 leukemia cells and sensitivity to MRX-2843 was determined. BMDSCs from wild-type mice protected 697 leukemia cells from MRX-2843 induced cell death much more effectively than BMDSCs from Gas6 knockout mice (4.3% apoptotic and dead cells versus 72.4%, respectively).To investigate biochemical mechanisms of Gas6-mediated protection, Kasumi-1 AML cells were cultured with 200nM MRX-2843 or vehicle in the presence or absence of HS27 cells and expression and activation of MERTK, AXL, TYRO-3, and downstream signaling effectors STAT5, AKT, and ERK1/2 were determined by immunoblot. AXL was not expressed in Kasumi-1 cells with or without co-culture. Treatment with MRX-2843 mediated robust inhibition of MERTK activation indicated by reduced levels of phosphorylated protein in both the presence and absence of stromal cell co-culture. In contrast, activation of TYRO-3 was increased after treatment with MRX-2843 in leukemia cells co-cultured with HS27 stromal cells. Similarly, in the absence of co-culture MRX-2843 inhibited activation of STAT5, AKT, and ERK1/2. However, in the presence of HS27 cells there was robust activation of STAT5 that was sustained even after treatment with MRX-2843. In contrast, MRX-2843 inhibited activation of AKT and ERK1/2 in HS27 co-cultures, although higher doses were required.Together these data support a model whereby Gas6 produced by stromal cells mediates leukemia cell resistance to MERTK inhibition in the bone marrow by inducing activation of TYRO-3, thereby promoting downstream signaling and cell survival despite MERTK inhibition. Thus, combined treatment with MRX-2843 and a TAM ligand sink (eg MERTK-Fc), a TYRO-3 inhibitor, or a bone marrow mobilizing agent may be particularly effective therapeutic strategies. DisclosuresWang:Meryx, Inc: Equity Ownership, Patents & Royalties: MRX-2843. Frye:Meryx, Inc: Equity Ownership, Patents & Royalties: MRX-2843. Earp:Meryx, Inc: Equity Ownership, Patents & Royalties: targeting MERTK. DeRyckere:Meryx, Inc: Equity Ownership, Patents & Royalties: targeting MERTK. Graham:Meryx, Inc: Equity Ownership, Patents & Royalties: targeting MERTK.

GATA Factor-Dependent Positive-Feedback Circuit Controls Acute Myeloid Leukemia Cell Proliferation: Mechanisms and Targets for Therapeutic Intervention

Abstract Recent studies have established a relationship between GATA-2, a master regulator of hematopoiesis, and acute myeloid leukemia (AML). Loss-of-function mutations and elevated GATA2 expression are implicated in AML. While inhibitory GATA2 mutations occur in patients with primary immunodeficiency, which progresses to AML, GATA2 can be overexpressed in AML patients with poor prognosis. Many questions remain unanswered regarding mechanisms by which GATA2 deregulation results in AML. We reported that the Ras-p38α pathway induces multi-site GATA-2 phosphorylation in proerythroblasts, and Ser192 is required for the phosphorylation. Because RAS is mutated in 20% of AML patients, and elevated GATA2 expression correlates with poor prognosis, we tested whether this pathway functions in AML. GATA-2 was phosphorylated in the steady-state in AML cell lines, and oncogenic Ras(G12V) expression induced GATA-2 hyperphosphorylation in a p38/ERK-dependent manner. GATA-2 contains a sequence that conforms to the MAPK docking site termed "DEF motif". Mutation of S192 or the DEF motif abrogated Ras(G12V)-mediated GATA-2 hyperphosphorylation. We analyzed function using a Mouse Aortic Endothelial (MAE) cell line, in which GATA-2 and Ras(G12V) synergistically induce expression of specific GATA-2 target genes, e.g. Hdc. The DEF mutation abrogated GATA-2 hyperphosphorylation by Ras(G12V) and attenuated GATA-2 activity to induce Hdc expression in the presence of Ras(G12V) in MAE cells (50% of control, p &lt; 0.05). GATA-2 hyperphosphorylation was also induced by expression of constitutively-active (ca) p38α and caMEK-1. caMEK-1-induced hyperphosphorylation was suppressed by the p38 inhibitor, SB203580 (p &lt; 0.05) while the MEK inhibitor U0126 did not influence cap38α-induced hyperphosphorylation. The phosphatase inhibitor okadaic acid induced GATA-2 hyperphosphorylation, and p38 was more important than ERK in this context. These data support a model in which a hierarchical network of kinases controls GATA-2 phosphorylation and function in AML. Phospho-proteomics is being used to establish interconnectivity between network components and temporal aspects of the multi-step signaling mechanism. ChIP-Seq analysis in Kasumi-3 AML cells revealed GATA-2 occupancy at IL1B and CXCL2. IL1B and CXCR2, encoding the CXCL2 receptor, are implicated in AML. GATA-2 downregulation decreased expression of these genes (IL1B: 81.5% decrease, p &lt; 0.001, CXCL2: 73% decrease, p &lt; 0.001). Inhibition of ERK or p38 attenuated GATA-2 phosphorylation, decreased GATA-2 occupancy at GATA2, IL1B, and CXCL2, and lowered mRNA expression of these genes. To determine the consequences of the reduced occupancy, FAIRE was used to quantitate accessibility at the occupancy sites. Under conditions of reduced GATA-2 occupancy, accessibility decreased 50-65% at the GATA2 -77 enhancer and GATA-2 occupancy sites of IL1B and CXCL2 (p &lt; 0.05). IL-1β and CXCL2 stimulated GATA-2 phosphorylation via p38 and ERK activation in Kasumi-1 and primary AML cells and increased GATA-2 chromatin occupancy and target gene expression. These results reveal a Ras-p38/ERK-GATA-2-IL-1β/CXCL2 axis that constitutes a positive-feedback circuit. We tested whether this circuit impacts Kasumi-1 cell proliferation. GATA2 downregulation reduced the proliferative rate (p &lt; 0.001), which was partially rescued with recombinant CXCL2 (p &lt; 0.01). To determine whether the signal-dependent mechanism can be extrapolated to human AML patients, we analyzed AML datasets from TCGA and GEO. GATA2 and CXCL2 expression was elevated in AML (p &lt; 0.01). GATA2, IL1B, and CXCL2 mRNA levels correlated in M5 AML (GATA2 vs. IL1B: p=0.0015, GATA2 vs. CXCL2: p=0.0046, IL1B vs. CXCL2: p=0.000001). Among the patients with normal karyotype AML, high GATA2 and CXCL2 expression correlated with significantly reduced survival (p=0.003). In aggregate, our results establish a Ras-p38/ERK-GATA-2-IL-1β/CXCL2 axis in AML. It is attractive to propose that the GATA-2-dependent positive-feedback circuit is deployed upon ectopic elevation of GATA-2 activity in certain AML contexts and may harbor potential therapeutic targets. Given the profound capacity of this mechanism to alter GATA-2 activity, it is crucial to consider the GATA-2 activation state, rather than mRNA or total GATA-2 protein levels in clinical scenarios. Disclosures No relevant conflicts of interest to declare.

Oroxylin A, a natural anticancer flavonoid compound, induces differentiation of t(8;21)-positive Kasumi-1 and primary acute myeloid leukemia cells.

AML1/ETO fusion gene is one of disease-causing genes of t(8;21)-positive acute myeloid leukemia (AML). Oroxylin A (OA) has showed anticancer effects on other cancer cells. Here, studies were conducted to determine the antileukemia effect of OA on t(8;21)-positive AML cells in vitro and in vivo. The effects of OA on cell viability of t(8;21)-positive Kasumi-1 and primary AML cells were analyzed by MTT assay. Cell differentiation was examined by NBT reduction assay, flow cytometry analysis for CD11b/CD14, and Giemsa stain. Protein expressions were determined by Western blots. Immunofluorescence assay was used to verify the effect of OA on HDAC-1 expression in vivo. Immunohistochemical staining was applied to evaluate leukemic infiltration of AML-bearing NOD/SCID mice. OA enhanced NBT reduction activity and CD11b/CD14 expression of AML1/ETO-positive AML cells markedly. Results of Giemsa staining also demonstrated that OA could induce the morphologic changes with reduction of nuclear/cytoplasmic ratios, suggesting the cell differentiation induced by OA. Further study showed that OA decreased the expression of fusion protein AML1/ETO and down-regulated HDAC-1 protein levels in vitro and in vivo. Moreover, OA increased the expression of differentiation-related proteins C/EBPα and P21. Acetylation levels of histones were also advanced obviously after treatment of OA. In vivo study indicated that OA could prolong the survival of AML-bearing NOD/SCID mice and reduce leukocytic infiltration of the spleen. All these results suggested that OA might be a novel candidate agent for differentiation therapy for AML1/ETO-positive AML and the mechanism required further investigation.

The Hypomethylating Agent Decitabine Prior to Chemotherapy Improves the Therapy Efficacy in Refractory/Relapsed Acute Myeloid Leukemia Patients

Introduction. Most acute myeloid leukemia (AML) patients with adverse prognosis either fail to achieve complete remission (CR) or relapse after short-term remission, new strategies are needed to improve therapeutic effect in these patients. Gene silencing via DNA methylation are frequent and reversible in high-risk AML, it provides the possibility to treat AML patients with DNA-hypomethylating agent. Decitabine alone has limited anti-leukemia effect and minimal toxicity. Studies demonstrated that decitabine prior to chemotherapy was likely to increase efficacy in AML. In this study, we investigated the effect of decitabine on susceptibility to cytotoxic drugs in chemoresistant AML cells. Efficacy and safety of decitabine prior to HAA regimen was evaluated in refractory, relapsed and high-risk AML patients.Methods. HL60, HL60/ADR, Kasumi-1, and primary AML cells were pre-treated with 1.0 μM decitabine for 72 hours, Sensitivities to harringtonine, adriamycin, cytarabine and the combination was determined by MTT, apoptosis was analyzed by flow cytometry. Protein expression was detected by western blotting. In clinic, Twenty-three patients were enrolled in decitabine prior to HAA regimen, and twenty-four patients in HAA alone. CR ratio was used to evaluate efficacy. Durations of neutrocytopenia or thrombcytopemia and infection incidence were observed to assess the safety. All of patients were followed up to 36 monthes, overall survival (OS) and disease-free survival (DFS) were calculated.Result. Decitabine increased sensitivity and apoptosis induced by harringtonine in HL60/ADR, as well as adriamycin and cytarabine in HL60/ADR and Kasumi-1 cells. But no change in sensitivity to each drug was observed in HL60, and sensitivity to harringtonine in Kasumi-1. The similar changes in sensitivity to adriamycin and cytarabine were also observed in primary AML cells from refractory patients (n=6). Cytotoxic effect of HAA was alsoincreaed by decitabine in HL60/ADR and Kasumi-1 cells (p=0.004, 0.018). Western blotting showed that decitabine decreased expression of DNMT1, activation of p53 and inhibition of c-Myc, Survivin and Bcl-2. In clinic, 16 (69.6%) patients in decitabine plus HAA regimen responded to the first course of induction therapy: 14 CR (60.9%) and 2 PR (8.7%). 1 in 2 patients with PR achieved CR after second course induction. It brought the overall CR was 65.2%. Otherwise, 10 (45.8%) patients in HAA regimen alone responded to the first induction: 7 CR (29.2%) and 4 PR (16.7%), 3 in 4 patients with PR achieved CR after the second induction, and overall CR reached 41.7%. The first-cycle CR ratio in decitabine plus HAA was higher than that in HAA alone (p=0.041). Patients continued to receive chemotherapy alternatively combined with decitabine. The median OS was 14 months in chemotherapy plus decitabine, 6 months in chemotherapy alone, and median DFS were 18 and 5 months in the former and latter. No significant difference was observed in the time of neutrophil (p=0.832, 0.631) or platelet recovery (p=0.798, 0.544) after induction and intensification therapy. The incidences of infection were also similar (p=0.724, 0.697). The clinic data indicated that decitabine plus HAA regimenit was efficacy and well-tolerated to treat refractory AML patients.Conclusion. Our results demonstrated that decitabine increased cytotoxic effect against chemoresistant AML cells. Combination of decitabine and HAA was safe and efficacy to treat refractory/relapsed AML. These findings support clinic protocols based on decitabine prior to chemotherapy to overcome resistance and improve therapeutic efficacy in AML patients. DisclosuresCarter:PrismBiolab: Research Funding.

SPARC contributes to Leukemia Growth and Aggressive Disease in Acute Myeloid Leukemia (AML)

Abstract 773Altered expression of SPARC, [secreted protein, acidic, cysteine-rich (osteonectin)] a gene encoding a protein that modulates cell-matrix interactions, has been reported in cancer and leukemia, but its precise contribution to malignant transformation is unknown. We previously reported that SPARC was among the most upregulated genes in the gene-expression profile (GEP) associated with IDH2-R172 mutations (mut; Marcucci et al. JCO 2010;28:2348) and among the most downregulated genes associated with NPM1 mut (Becker et al. JCO 2010;28:596) in cytogenetically normal (CN) AML. Both mutations have strong but opposite impact (IDH2-R172 mut, adverse; NPM1 mut, favorable) on clinical outcome. Thus, we hypothesized that SPARC may act as an oncogene and contribute to AML aggressiveness. To determine functional and mechanistic activities of SPARC in AML, we conducted gain- and loss-of-function experiments in cell lines and primary blasts collected from patients under the OSU Leukemia Tissue Bank protocol. In vitro, ectopic expression and knockdown of SPARC in THP-1 and Kasumi-1 AML cells led to increase (2.5-fold P=0.03) and decrease (45%, P=0.03) in colony forming ability, respectively. Clonogenic assays in SPARC- vs empty vector (EV)-infected AML blasts also showed 2-fold increase in colony-formation (P=.01) supporting a role of SPARC overexpression in promoting leukemia growth. The leukemogenic activity of the SPARC protein was likely mediated by its binding to αvβ3 integrin and in turn by the activation of the kinase ILK. This induced GSK3β phosphorylation (p) and in turn activation of β-catenin, previously reported to contribute to AML. Indeed, while ILK knockdown abolished SPARC-induced p-GSK3β, ectopic SPARC expression led to a 4-fold increased p-GSK3β, measured by p-GSK3β (Ser-9) immunoblot assay, and to stabilization of β-catenin, measured by p-β-catenin(Ser-552) and p-β-catenin(Ser-675) immunoblot assays in AML blasts. As a result, we observed increase in translocation of β-catenin to the nucleus in SPARC overexpressing cells by confocal microscopy. Nuclear β-catenin led to increased activity of the TCF/LEF transcription factors as shown by a TCF/LEF luciferase reporter assay (5.8-fold, P<.001) and the expression of MYC, a TCF/LEF target gene. In contrast, SPARC knockdown decreased MYC expression (50%; P=0.05) in AML blasts. In vivo, mice engrafted with THP-1 cells overexpressing SPARC exhibit more aggressive disease, significant splenomegaly (P=.008), and hepatomegaly with increased number and size of hepatic granulocytic sarcoma-like lesions when compared with mice engrafted with THP-1-EV cells.The results in the preclinical models prompted us to evaluate whether higher SPARC expression associated with aggressive human disease. Thus, SPARC expression levels were measured by NanoString nCounter assay in a cohort of CN-AML patients (pts; n=362; age, 18–83 years) comprehensively characterized for mutations in the NPM1, FLT3, CEBPA, WT1, TET2, MLL, IDH1, IDH2, RUNX1, ASXL1 and DNMT3A genes, and treated with cytarabine-anthracycline based regimens. Higher SPARC expressers were more likely classified in the European Leukemia Net (ELN) Intermediate-I (CEBPA wt, NPM1 mut with FLT3-ITD, or NPM1 wt) than the ELN Favorable Genetic Group (CEBPA mut and/or NPM1 mut without FLT3-ITD; P<.001). Higher expression of SPARC associated with lower complete remission (CR) rates (P<.001; OR .75), shorter disease-free (DFS; P<.001; HR 1.17) and overall (OS; P<.001; HR 1.16) survival. In multivariable analyses, higher SPARC expression remained associated with worse CR rates (P<.001), once adjusted for RUNX1, white blood count (WBC) and age group (<60 vs >60 years); shorter DFS (P=.07), once adjusted for FLT3-ITD, RUNX1, WBC and age group; and shorter OS (P<.001), once adjusted for FLT3-ITD, WT1, ASXL1 and age group. Gene and microRNA (miR)-expression profiling associated SPARC with CD34, BAALC and MN1 among the most upregulated and the HOX genes among the most downregulated genes, and with the hematopoietic stem cell-specific miR-126 and miR-130a among the most upregulated miRs.In conclusion, high SPARC expression contributes to aggressive AML growth via β-catenin activation-mediated mechanisms. SPARC overexpression independently predicted worse outcome in CN-AML pts, and may represent a potentially novel prognostic marker and therapeutic target in AML. Disclosures:No relevant conflicts of interest to declare.

Determination of cellular uptake and intracellular levels of Cenersen (Aezea®, EL625), a p53 antisense oligonucleotide in acute myeloid leukemia cells

TP53 encodes for tumor protein p53. The suppression of p53 protein results in interruption of DNA repair mechanisms in dividing malignant cells thereby increasing the DNA damage and activating p53-independent mechanisms of apoptosis. This ultimately may translate into enhanced cytotoxic effects of standard chemotherapy. Based on this rationale, Cenersen, a phosphorothioate oligonucleotide antisense to p53-mRNA was synthesized and tested in clinical trials for patients with acute myeloid leukemia (AML). An important component of Cenersen clinical development is to develop a sensitive and specific method to quantify plasma and intracellular levels of Cenersen in different biologic matrices in order to determine tissue and intracellular distribution of the parent compound and its metabolites. Ultimately, this will allow us to determine pharmacokinetic and pharmacodynamic relationship for dose–effect correlation and design effective regimen to be rapidly translate into the clinic. An ELISA-based assay was adapted for assay development and validation of Cenersen in mouse plasma and cell lysate. Cellular uptake of Cenersen was studied in MV4-11 and KASUMI-1 AML cell lines. Real-time RT-PCR was used to measure P53-mRNA expression changes in treated cells. The assay had a limit of quantification of 35pmol/L in mouse plasma. Within-day and between-day precision of <15% and accuracy nearly 100% were observed in a linear range of 10–2000pmol/L (R2=0.99) in AML cell lysate. The selectivity of this assay examined as cross-reactivity with its 3′N-1, 3′N-2-metabolites, was 16.8% and 0.4%, respectively, and with its mismatch and the scramble oligonucleotides was 0.06% and 0.4%, respectively. Cenersen was stable in mouse plasma up to 8h at 37°C. When exposed to 0.1–1μmol/L Cenersen, MV4-11 and KASUMI-1 cells showed intracellular concentration in the range of 9.97–45.34nmol/mg protein and 0.1–2.1nmol/mg protein, respectively. Successful downregulation of p53-mRNA expression was observed in Cenersen treated cells. This ELISA-based assay was applicable to plasma and intracellular concentration measurement of Cenersen. Assessment of achievable concentration of Cenersen in different biologic matrices will be useful to elucidate the biological and clinical activity of this promising drug and define its recommended dose in future clinical trials.

Abstract 543: Stroma-mediated chemotherapy resistance in acute myeloid leukemia cells

Abstract Overall objective: Acute myeloid leukemia (AML) is a devastating malignancy with a relapse rate near 50% in children, despite very toxic chemotherapy. Accumulating evidence shows that the bone marrow stromal environment protects a subset of leukemia cells and allows them to survive chemotherapy, eventually leading to recurrence. Furthermore, activation of signal transducer and activator of transcription 3 (Stat3) contributes to chemotherapy resistance. Indeed, Stat3 is activated in AML cells by cytokines and adhesion pathways in the stromal environment, leading to increased expression of pro-survival genes. Our goal is to determine the effect of stromal cytokines and adhesion pathways on stroma-mediated chemotherapy resistance in AML cells, which could lead to potential new therapies for AML. Methods, Results &amp; Conclusions: We used two human bone marrow stromal cell lines, HS-5 and HS-27, for our experiments. First, we examined the ability of stroma-conditioned medium to activate Stat3 in AML cells. Kasumi-1 AML cells were treated with HS-5 or HS-27 conditioned medium, or control medium, for 15 minutes, then the level of phosphorylated Stat3 was measured by flow cytometry. Conditioned medium from HS-5, but not HS-27 cells, strongly induced phosphorylation of Stat3 in AML cells at a level comparable to that induced by G-CSF (100 ng/mL). These results suggest that HS-5 cells secrete factors that induce Stat3 activation, but HS-27 cells do not. Next, we performed co-culture experiments to determine the ability of each stromal cell line to confer resistance to chemotherapy. Human AML cell lines (NB-4 and THP-1) were cultured alone or co-cultured with PKH-26-labeled HS-5 or HS-27 cells, then treated with etoposide (30 nM – 10 uM) or mitoxantrone (3 nM – 300 nM) for 48 hours. Cells were then harvested and labeled with annexin V-FITC. The percentage of apoptotic AML cells (FITC positive and PKH-26 negative) was determined by FACS. Both HS-5 (p&amp;lt;0.001) and HS-27 (p&amp;lt;0.05) cells protected NB-4 and THP-1 cells from etoposide-induced apoptosis (apoptosis rate at the 1 uM dose: 69.4±3.0% THP-1 alone vs 48.2±1.1% HS-5 co-culture vs 59.3±3.4% HS-27 co-culture). HS-5 cells also provided protection for AML cells against mitoxantrone-induced apoptosis (apoptosis rate at the 300 nM dose: 85.1±0.8% THP-1 alone vs 57.7±5.6% HS-5 co-culture; p&amp;lt;0.05). In contrast, co-culture with HS-27 cells did not reduce mitoxantrone-induced apoptosis (apoptosis rate: 77.7±2.4%). The results with NB-4 were similar to THP-1 cells. These findings suggest that 1) HS-5 and HS-27 stromal cells confer resistance to various chemoagents by different mechanisms, which are consistent among human AML cell lines; and 2) resistance to individual chemotherapy agents may depend on different mechanisms, such as secreted factors vs adhesion pathways. Understanding these mechanisms will be very valuable in targeting therapies to overcome stroma-mediated chemotherapy resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 543. doi:10.1158/1538-7445.AM2011-543

Assaying pharmacodynamic endpoints with targeted therapy: Flavopiridol and 17AAG induced dephosphorylation of histone H1.5 in acute myeloid leukemia

Histone H1 is commonly used to assay kinase activity in vitro. As many promising targeted therapies affect kinase activity of specific enzymes involved in cancer transformation, H1 phosphorylation can serve as potential pharmacodynamic marker for drug activity within the cell. In this study we utilized a phosphoproteomic workflow to characterize histone H1 phosphorylation changes associated with two targeted therapies in the Kasumi-1 acute myeloid leukemia cell line. The phosphoproteomic workflow was first validated with standard casein phosphoproteins and then applied to the direct analysis of histone H1 from Kasumi-1 nuclear lysates. Ten H1 phosphorylation sites were identified on the H1 variants, H1.2, H1.3, H1.4, H1.5 and H1.x. LC MS profiling of intact H1s demonstrated global dephosphorylation of H1.5 associated with therapy by the cyclin-dependent kinase inhibitor, flavopiridol and the Heat Shock Protein 90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin. In contrast, independent treatments with a nucleotide analog, proteosome inhibitor and histone deacetylase inhibitor did not exhibit decreased H1.5 phosphorylation. The data presented herein demonstrate that potential of histones to assess the cellular response of reagents that have direct and indirect effects on kinase activity that alters histone phosphorylation. As such, this approach may be a highly informative marker for response to targeted therapies influencing histone phosphorylation.

Vectors selected from adeno-associated viral display peptide libraries for leukemia cell–targeted cytotoxic gene therapy

For acute myeloid leukemia (AML), gene therapy may be used to treat patients refractory to conventional chemotherapy. However, availability of vectors sufficiently and specifically transducing this cell type is very limited. Here we report the selection of capsid-modified adeno-associated viral (AAV) vectors targeting Kasumi-1 AML cells by screening random AAV displayed peptide libraries. The peptide inserts of the enriched capsid mutants share a common sequence motif. The same motif was selected in an independent library screening on HL-60 AML cells. Recombinant targeted vectors displaying the selected peptides transduced the target leukemia cells they have been selected on up to 500-fold more efficiently compared to AAV vectors with control peptide inserts. One of the selected clones (NQVGSWS) also efficiently transduced all members of a panel of four other AML cell lines. Binding and blocking experiments showed that NQVGSWS binding to leukemia cells is independent of the wild-type AAV-2 receptor heparin sulfate proteoglycan. Transduction assays on a panel of hematopoietic and nonhematopoietic cell lines showed that the NQVGSWS capsid was able to overcome resistance to AAV transduction, especially in hematopoietic cancer cells, whereas normal peripheral blood mononuclear cells and CD34(+) hematopoietic progenitor cells were not transduced. Consequently, recombinant targeted NQVGSWS AAV vectors harboring a suicide gene conferred selective killing to Kasumi-1 cells, but not to control cells. This suggests that the AAV mutant selected here may be used as a tool to target therapeutic genes to AML cells.