Treatment Of Acute Myeloid Leukemia Cell Lines Research Articles

Mitochondrial dynamics as a potential therapeutic target in acute myeloid leukemia.

Acute myeloid leukemia (AML) cells are highly dependent on oxidative phosphorylation and the mitochondrial dynamics regulated by fusion-related genes MFN1, MFN2, and OPA1 and fission-related genes DNM1L and MFF. An analysis of previously published gene expression datasets showed that high expression of MFF was significantly associated with poor prognosis in patients with AML. Based on this finding, we investigated the impact of mitochondrial dynamics in AML. Transduction of shRNA against fission-related genes, DNM1L and MFF, inhibited growth and increased the mitochondrial area in AML cell lines. Extracellular flux analysis showed that deletion of mitochondrial dynamic regulators reduced mitochondrial respiration without significantly affecting glycolysis, except in shDNM1L-transfected cells. Immunodeficient NOG mice transplanted with DNM1L- or MFF-knockdown AML cells survived significantly longer than controls. Treatment of AML cell lines with Mdivi-1, which inhibits the DRP1 encoded by DNM1L, inhibited cell proliferation and oxidative phosphorylation. Our results show that mitochondrial dynamics play an important role in AML, and provide novel biological insights. The inhibition of mitochondrial dynamics induces unique mitochondrial alterations, which may be explored as a potential therapeutic target in AML.

Abstract 1674: Inhibition of oncogenic nras signaling in acute myeloid leukemia cell lines through directed transcriptome alterations

Abstract Oncogenic mutations in genes involved in proliferative signaling are hallmarks of malignant transformation. The Ras family of signal transducing proteins are involved in the activation of several proliferative pathways and therefore are found to be oncogenically mutated in a variety of cancer types. The NRAS-directed pathways resulting in Erk1/2 activation are dysregulated in a significant percentage of acute myeloid leukemia (AML), metastatic melanoma, Hodgkin lymphoma, and thyroid cancers. The consequence of oncogenic NRAS signaling is the establishment of a transcriptome commanding constitutive proliferation and survival. It has long been known that phorbol ester treatment of AML cell lines, resulting in activation of protein kinase C (PKC), causes phenotypic changes that include cell cycle arrest, myeloid differentiation, and apoptosis. The paradox is that numerous studies identify PKC as an activator of RAS/ERK signaling. Here we show that phorbol ester treatment of NRAS-driven AML cell lines results in the transcriptional upregulation of genes encoding negative regulators of RAS signaling including DUSP1,2,4,5,6, SPRY2, SPRED1, RASA1, PEA15, RPS6KA1, and RASSF5. Additionally, phorbol ester treatment eventually results to ERK1/2 inactivation through dephosphorylation. Several of these negative regulators, when overexpressed in AML cell lines, dampen proliferation, and induce senescence. We propose that the induction of RAS negative regulators alters the AML transcriptome to a non-proliferative state in part through the transcriptional regulation of genes such as CDKN1A, CDKN2B, ZFP36L1, and MYC. As gene therapy strategies develop, designed to provide negative regulators of proliferative signaling pathways that are repressed in the malignant cell, we imagine the RAS/ERK pathway as a target for transcriptome reprogramming. Citation Format: Michael Roberts, Sophia Kovatsis, Amelia Harper, Megan Cravinho, Kishan Mangru, Sher Bahadur, Keagan Hesse, Katy Meta, Cuong Nguyen. Inhibition of oncogenic nras signaling in acute myeloid leukemia cell lines through directed transcriptome alterations [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 1674.

Abstract A049: Investigation of FHD-609, a potent degrader of BRD9, in preclinical models of acute myeloid leukemia (AML)

Abstract Acute myeloid leukemia (AML) is a complex disease with multiple sub-types, each characterized by unique clinical and molecular features, driving the need to develop targeted therapies which exploit specific vulnerabilities. Bromodomain-containing protein 9 (BRD9) is a component of the ncBAF chromatin remodeling complex, and has been recently indicated as a strong dependency in AML (Weisberg et al 2022). It has been shown that inhibitors of BRD9 induce growth inhibition and expression of apoptotic makers in AML cell lines (Hohmann et al 2016; Zhou et al 2021). It has also been reported that BRD9 is critical to AML cell survival through the maintenance of STAT5 signaling (Del Gaudio et al 2019). Herein, we profile the in vitro anti-proliferative effects of FHD-609, a potent and selective degrader of BRD9, in a panel of 40 AML cell lines representative of a broad range of AML sub-types. We observed that FHD-609 was effective at inhibiting the growth of a subset of AML cell lines, consistent with previous reports in the literature. We further investigated whether treatment of AML cell lines with FHD-609 would induce changes in cell cycle, and found an increase in G1 in the same subset of cell lines that showed cell growth inhibition. Furthermore, treatment with FHD-609 induced apoptosis in these cell lines. To identify the mechanisms of action of FHD-609 in AML, as well as potential predictive biomarkers for AML sensitivity to FHD-609, we performed a range of mechanistic studies including ATACseq, ChIPseq and RNAseq in AML cells treated with FHD-609. We observed significant changes in the chromatin landscape in sensitive AML cell lines, and negligible changes in insensitive cell lines. Further bioinformatics analysis identified a possible biomarker strategy that correlates with the sensitivity of AML cell lines to FHD-609 in vitro. To confirm whether this biomarker strategy would successfully predict response in vivo, we dosed AML CDX and PDX models with FHD-609 based on the biomarker selection strategy. We observed a strong anti-tumor effect in AML CDX models and significantly extended survival of the mice in the AML PDX models. In summary, we have shown that FHD-609 demonstrates strong anti-tumor efficacy in a subtype of AML and have identified a possible biomarker strategy to predict response. Citation Format: Claudia Dominici, David Mayhew, Ammar Adam, Flore Uzan, Victoria Garbitt-Amaral, Oliver Mikse, Brandon Antonakos, Hafiz Ahmad, Salonee Parikh, Mei Yun Lin, Gabriel Sandoval, David Lahr, Hsin-Jung Wu, Mengni Xu, Sean Brennan, Luis M. M. Soares, Jordana Muwanguzi, Huawei Chen, Zhaoxia Yang, Jason T Lowe, Matt Netherton, Laura Zawadzke, Johannes Voigt, Liyue Huang, Sabine Ruppel, Ho Man Chan, Ryan Kruger, David S Milan, Scott Innis, Qianhe Zhou, Steven F Bellon. Investigation of FHD-609, a potent degrader of BRD9, in preclinical models of acute myeloid leukemia (AML) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A049.

Enterotoxins A and B produced by Staphylococcus aureus increase cell proliferation, invasion and cytarabine resistance in acute myeloid leukemia cell lines

As in the case of cancer, the risk of infection increases when the host's immune system is not working properly. It has been shown that toxins produced by the bacteria responsible for bacterial infections can alter the properties of cancer cells as well as their sensitivity to chemotherapy agents. Staphylococcus aureus (S. aureus) is one of the most prevalent pathogens in acute myeloid leukemia (AML) patients and it produces several virulence factors, including Staphylococcal enterotoxin A (SEA) and Staphylococcal enterotoxin B (SEB). Cytotoxicity, transwell migration, invasion assays, and various transcriptomic and gene set enrichment (GSE) analyses were used to determine how SEA and SEB alter cell proliferation, migration, invasion, and Cytarabine (Cyt) resistance in AML cell lines. The treatment of AML cell lines with SEA/SEB caused an increase in cell proliferation and Cyt resistance. Toxins enhanced the proclivity of cells to migrate and invade, with around 50% of cells in the presence of SEA and SEB. Transcriptomic and gene set enrichment analyses, and subsequent PCR validations showed dysregulation of immune related genes and genesets. Apparently, this allows AML cells to escape and survive the undesirable environment created by toxins, possibly via the ER stress signaling pathway. Therefore, SEA and SEB can significantly alter the characteristics of AML cancer cells and evaluation of alterations in responsible immune genes and pathways may be crucial for controlling the progression of cancer. In addition, our results suggest that there may be a strong interaction between the immune related pathways and the ER signaling pathway.

Combination of the Kdm-Family Inhibitor, Jib-04, Improves Response to Acute Myeloid Leukemia Cells to Venetoclax

Background: Acute Myeloid Leukemia (AML) is a heterogeneous hematological malignancy with a 5-year survival rate of about 30% in adults with AML. While over the prior several decades, the standard of care treatment for AML changed very little, a number of novel targeted therapies have emerged in recent years. Promising results from clinical trials with the BCL2-specific inhibitor, Venetoclax, in combination with hypomethylating agents (HMA) or low-dose cytarabine for AML treatment have generated enthusiasm in the field. However, data from a phase II trial indicated a 30% failure rate after Venetoclax+HMA treatment of newly diagnosed patients, suggesting additional options are needed. Thus, we sought to explore alternative drug combinations that may enhance the efficacy of Venetoclax. JIB-04 is a potent small molecule inhibitor of multiple members of the histone lysine demethylase (KDM) 4, 5 and 6 families with KDM5A being the most sensitive. Expression levels of histone lysine-specific demethylases are higher in AML compared to normal bone marrow, suggesting they are potential targets for therapeutic intervention. Pharmacological inhibition or genetic knock down of KDM 4, 5 and 6 family members leads to reduced proliferation, cell cycle arrest, and increased apoptosis of AML cells of various subtypes. These preliminary findings compelled us to explore the anti-leukemic activity of JIB-04 alone and in combination with Venetoclax. Methods: We treated AML cell lines, patient AML blasts, and CD34+ cells from healthy donors with JIB-04, Venetoclax, or the combination for 72 hours in vitro. Apoptosis was quantified by AnnexinV/propidium iodide (PI) staining. Cell proliferation of AML cell lines treated with JIB-04 was evaluated using MTT assay and transcriptome profiling was done by RNA-Seq. NSG mice were injected with MOLM-13 cells and treated with JIB-04, Venetoclax or the combination for two weeks. For the knock-down (KD) of DDIT4 MOLM-13 cells were transduced with a shRNA and cells were treated with Venetoclax for 72h and analyzed for apoptosis. Results: Treatment of AML cell lines with low nanomolar doses (IC50 range from 8-60nM) of JIB-04 resulted in inhibition of proliferation and induction of apoptosis. In all AML cell lines tested, including AML cell lines with relative resistance to Venetoclax, the combination of JIB-04 with Venetoclax showed a strong synergistic effect. Moreover, patient AML blasts also showed a synergistic response to the drug combination compared to single-drug treatments (Figure 1a). In contrast, viability of normal CD34+ hematopoietic cells was not affected by JIB-04 treatment even up to a dose of 100nM that leads to apoptosis in AML cells, indicating a broad therapeutic window. The combination treatment of NSG mice injected with MOLM-13 cells resulted in reduced engraftment of huCD45+ cells compared to the single treatments in the blood, bone marrow and spleen. RNA sequencing of AML-OCI3 and MOLM-13 cells treated with JIB-04 revealed regulation of genes related to the mTOR pathway, stress response or amino acid metabolism, indicating possible mechanisms affecting proliferation and apoptosis. DDIT4 (REDD1), a regulator of the mTOR pathway, was downregulated after JIB-04 treatment in both cell lines. KD of DDIT4 in MOLM-13 cells resulted in increased sensitivity to Venetoclax (Figure 1b). Summary/Conclusion: While the epigenetic inhibitor JIB-04 alone shows modest anti-leukemic effect, this study highlights the potential therapeutic benefit of combining JIB-04 and Venetoclax in AML. The analysis of transcriptomic changes after JIB-04 treatment identified the mTOR pathway regulator DDIT4 as a likely mediator of the sensitization of AML cells to Venetoclax. High DDIT4 expression is associated with a worse outcome in AML making it an attractive therapeutic target for AML, especially in combination with Venetoclax. Our result would suggest this drug combination as a possible treatment option for AML patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

VCP inhibition induces an unfolded protein response and apoptosis in human acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a heterogeneous malignancy characterized by the accumulation of undifferentiated white blood cells (blasts) in the bone marrow. Valosin-containing protein (VCP) is an abundant molecular chaperone that extracts ubiquitylated substrates from protein complexes and cellular compartments prior to their degradation by the proteasome. We found that treatment of AML cell lines with the VCP inhibitor CB-5083 leads to an accumulation of ubiquitylated proteins, activation of unfolded protein response (UPR) and apoptosis. Using quantitative mass spectrometry-based proteomics we assessed the effects of VCP inhibition on the cellular ubiquitin-modified proteome. We could further show that CB-5083 decreases the survival of the AML cell lines THP-1 and MV4-11 in a concentration-dependent manner, and acts synergistically with the antimetabolite cytarabine and the BH3-mimetic venetoclax. Finally, we showed that prolonged treatment of AML cells with CB-5083 leads to development of resistance mediated by mutations in VCP. Taken together, inhibition of VCP leads to a lethal unfolded protein response in AML cells and might be a relevant therapeutic strategy for treatment of AML, particularly when combined with other drugs. The toxicity and development of resistance possibly limit the utility of VCP inhibitors and have to be further explored in animal models and clinical trials.

Preliminary Translational Results from an Ongoing Phase 1 Study of Flotetuzumab, a CD123 x CD3 Dart®, in AML/MDS: Rationale for Combining Flotetuzumab and Anti-PD-1/PD-L1 Immunotherapies

Background:Immunotherapy has transformed cancer treatment; in particular, checkpoint inhibitors (CPI) are now approved for the treatment of several solid tumors and in patients with Hodgkin's disease. In hematological malignancies, including acute myeloid leukemia (AML), the utility of CPIs may be more limited due to less mutations observed in their genomes. Immunologic treatments of hematologic malignancies targeting “shared” epitopes differentially overexpressed by hematologic cancers also represents a rational approach to the immunotherapy of AML.Methods:Translational studies were performed in an ongoing phase 1 study in patients with AML and MDS treated with flotetuzumab (MGD006/S80880), a novel CD123 x CD3 bispecific DART® protein, to assess the potential role of combining PD-1/PD-L1 inhibitors with flotetuzumab.Results:Flotetuzumab has anti-leukemic activity in patients with relapsed/refractory AML (ESMO 2017). Inherent to flotetuzumab's mode of action is redirecting T-cells to lyse AML cells in conjunction with enhancement of T-cell activation, proliferation and cytokine production. Multispectral immunohistochemistry analysis of bone marrow samples from our study showed flotetuzumab in situ with a ~2-fold increase in CD8 T-cells upon treatment; consistent with this, we observe up-regulation of PD-1 on both CD4 and CD8 T-cells along with dose dependent secretion of IFNɣ in patient samples. In line with reports of PD-L1 induction by IFNɣ, we observed PD-L1 upregulation on AML blasts incubated with IFNɣ. Furthermore, circulating levels of IFNɣ secretion in flotetuzumab-treated patients correlated with PD-L1 expression on AML blasts. Notably, primary AML samples with higher levels of PD-L1 on malignant blasts were less susceptible to flotetuzumab-mediated killing in vitro. Furthermore, patients that progressed early (within two-weeks) on flotetuzumab treatment had higher baseline levels of PD-L1 on AML cells. Interestingly, a number of patients with residual disease on flotetuzumab showed higher proportion of PD-L1 positive AML blasts vs. basal levels.Conclusions:Flotetuzumab has shown evidence of activity against CD123+ AML blasts in vivo, associated with an increase in T-cell infiltration and activation in the bone marrow, with enhanced IFNɣ secretion, which in turn may lead to upregulation of PD-1 on T cells and PD-L1 expression by the AML blasts. Synergistic cytotoxicity was observed after treatment of AML cell lines with both flotetuzumab and CPI in vitro. Furthermore, AML cells in patients that progressed on flotetuzumab or with residual disease tend to be PD-L1 positive. Taken together, these data suggest that combination with anti-PD1/PD-L1 therapy may enhance the effect of flotetuzumab in selected patients with relapsed or refractory AML. clinicaltrials.gov NCT02152956 DisclosuresFox:Bristol Myers-Squibb: Consultancy, Research Funding; AstraZeneca/MedImmune: Consultancy, Research Funding; PerkinElmer: Consultancy, Research Funding; Janssen/Johnson and Johnson: Consultancy, Research Funding; Argos: Consultancy; Bayer: Consultancy; Definiens: Consultancy; OncoSec: Consultancy, Research Funding; PrimeVax: Consultancy, Equity Ownership; Peregrine: Consultancy; UbiVac: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Co-Founder and managing Member of LLC; Ventana/Roche: Consultancy; Viralytics: Consultancy, Research Funding. Primo:Vivia Biotech: Employment. Ballesteros:Vivia Biotech: Employment. Sun:Macrogenics Inc: Employment, Equity Ownership. Lelièvre:Institut de recherches international Servier: Employment. Baughman:MacroGenics, Inc.: Employment. La Motte-Mohs:MacroGenics: Employment, Equity Ownership, Patents & Royalties; Sunnybrook Health Sciences Centre: Patents & Royalties. Muth:MacroGenics, Inc.: Employment, Equity Ownership. Moore:MacroGenics, Inc.: Employment, Equity Ownership. Bonvini:MacroGenics, Inc.: Employment, Equity Ownership, Research Funding. Wigginton:MacroGenics: Employment, Equity Ownership. Davidson-Moncada:MacroGenics: Employment, Equity Ownership.

Nettle Tea Inhibits Growth of Acute Myeloid Leukemia Cells In Vitro by Promoting Apoptosis.

Urtica dioica (UD), commonly known as “stinging nettle”, is a herbaceous flowering plant that is a widely used agent in traditional medicine worldwide. Several formulations of UD leaf extract have been reported to exhibit anti-inflammatory and antioxidant properties, with anticancer potential. The current study investigated the possible anticancer properties of nettle tea, prepared from Urtica dioica leaves, on acute myeloid leukemia (AML) cell lines, and deciphered the underlying molecular mechanisms. Treatment of AML cell lines (U-937 and KG-1) with UD aqueous leaf extract resulted in a dose- and time-dependent inhibition of proliferation, an increase in apoptotic hallmarks such as phosphatidylserine flipping to the outer membrane leaflet, and DNA fragmentation as revealed by cell-death ELISA and cell-cycle analysis assays. Apoptosis induction in U937 cells involves alterations in the expression of Bax and Bcl-2 upon exposure to nettle tea. Furthermore, the chemical composition of UD aqueous extract indicated the presence of multiple chemical agents, such as flavonoids and phenolics, mainly patuletin, m/p-hydroxybenzoic acid, and caffeic acid, among others, to which the pro-apoptotic and anti-tumor effects may be attributed.

Malva pseudolavatera leaf extract promotes ROS induction leading to apoptosis in Acute Myeloid Leukemia cells in vitro

Malva pseudolavatera Webb & Berthel is a plant from the Malvaceae family that has long been included in the human diet due to its various curative effects. Many plant leaf extracts from the various species of Malva genus have been reported to possess anti‐cancer properties, however, studies on M. pseudolavatera Webb & Berthel leaves have documented anti‐inflammatory and anti‐oxidant effects with no emphasis on their possible anti‐cancer potential. The present study explores the anti‐cancer properties of Malva pseudolavatera Webb & Berthel leaf extract on Acute Myeloid Leukemia (AML) cell lines in vitro and deciphers the underlying molecular mechanism. Treatment of AML cell lines with M. pseudolavatera methanolic leaf extract showed a dose and time dependent inhibition of proliferation and a dose‐dependent increase in apoptotic hallmarks such as an increase in phosphatidylserine on the outer membrane leaflet, membrane leakage in addition to DNA fragmentation. The pro‐apoptotic effect was induced by ROS induction as well as an upregulation of cleaved PARP, an increase in Bax/Bcl‐2 ratio, and the release of cytochrome‐c from the mitochondria. Major compounds of the extract included methyl linolenate, phytol, γ‐sitosterol and stigmasterol as revealed by Gas Chromatography coupled to Mass Spectrometry.Support or Funding InformationSchool Research and Development Council, Lebanese American University

Malva pseudolavatera Leaf Extract Promotes ROS Induction Leading to Apoptosis in Acute Myeloid Leukemia Cells In Vitro.

Malva pseudolavatera Webb & Berthel. is a plant from the Malvaceae family that has long been included in the human diet due to its various curative effects. Many plant leaf extracts from the various species of Malva genus have been reported to possess anti-cancer properties, however, studies on M. pseudolavatera Webb & Berthel. leaves have documented anti-inflammatory and anti-oxidant effects with no emphasis on their possible anti-cancer potential. The present study explores the anti-cancer properties of Malva pseudolavatera Webb & Berthel. leaf extract on acute myeloid leukemia (AML) cell lines in vitro and deciphers the underlying molecular mechanism. Treatment of AML cell lines with M. pseudolavatera methanolic leaf extract showed a dose- and time-dependent inhibition of proliferation and a dose-dependent increase in apoptotic hallmarks such as an increase in phosphatidylserine on the outer membrane leaflet and membrane leakage in addition to DNA fragmentation. The pro-apoptotic effect was induced by reactive oxygen species (ROS) as well as an upregulation of cleaved poly(ADP-ribose) polymerase (PARP), increase in Bax/Bcl-2 ratio, andrelease of cytochrome-c from the mitochondria. Major compounds of the extract included methyl linolenate, phytol, γ-sitosterol, and stigmasterol as revealed by gas chromatography coupled with mass spectrometry, and amino acids, amino acid derivatives, tiliroside, 13-hydroxyperoxyoctadecadienoic, and quercitrin as detected by liquid chromatography coupled to mass spectrometry.

Mitophagy Plays a Key Role in the Anti-Leukemic Activity of Autophagy Inhibitors Under Hypoxia in Acute Myeloid Leukemia

Background: Leukemia stem cells (LSCs) and acute myeloid leukemia (AML) blasts persisting in the bone marrow (BM) after chemotherapy are key drivers of AML relapse and chemotherapy refractoriness. The hypoxic microenvironment of the BM is known to protect these cells, however the mechanisms behind this chemoresistance are not well understood. We have previously shown that AML cell lines cultured under hypoxia upregulate autophagy. Blocking autophagy with lysosome-based autophagy inhibitor Bafilomycin A1 (Baf A1) was shown to target LSCs in colony formation (CF) assays and in vivo serial transplants in NSG mice. Another autophagy inhibitor chloroquine (CQ) also demonstrated anti-LSC effects in CF assays. The clinical development of Baf A1 and CQ has been limited due to poor pharmacokinetics and toxicity. Lys05 is a promising CQ derivative with increased potency and therapeutic potential. Recent studies have shown that LSCs have an increased reliance on oxidative phosphorylation (OXPHOS) as well as mitophagy, the autophagic degradation of damaged mitochondria. Hypoxia has also been demonstrated to increase mitophagy in certain cell types through upregulation of BNIP3. We hypothesized that AML cells cultured under hypoxia would have an increased reliance on mitophagy for mitochondrial homeostasis and survival. Treatment with autophagy inhibitors would be expected to block mitophagy and induce AML cell death specifically under hypoxia. Methods: Human AML cells (MOLM13) were cultured under hypoxia (1% O2) or normoxia (21% O2). NSG mice were inoculated with luciferase expressing MOLM13-BLIV & treated with vehicle (DMSO) or Lys05 (40 mg/kg) IP for 18 days. Tumor burden was assessed by bioluminescence. CF assays were established with AML patient cells in the presence of vehicle or Lys05 in MethoCult under normoxia/hypoxia and counted on day 13. Annexin V-FITC/PI flow cytometry was used to measure apoptosis. OXPHOS was assessed with a Seahorse XFe96 using the Mitochondrial Stress Test. Mitochondrial mass was measured by flow cytometry using MITO-ID Green. BNIP3 & PINK1 protein expression was visualized via western blot. Results: Treatment with Lys05 decreased in vivo tumor burden significantly in NSG mice systemically engrafted with human AML cells. We also saw a marked decrease in the number of CF-units in primary AML patient samples treated with Lys05 which was further enhanced under hypoxia. Treatment of AML cell lines with CQ and Lys05 also enhanced apoptosis under hypoxia as compared to normoxia. Baf A1, however, showed equal amounts of apoptosis in normoxia and hypoxia (Fig. 1A). While Baf A1, CQ, and Lys05 all inhibit autophagy through deacidification of the lysosome, Baf A1 has been shown to induce additional effects on mitochondria, inducing uncoupling of OXPHOS and depolarization. We therefore examined the autophagy inhibitors' effect on mitochondrial function. At 24 hours, Baf A1 caused a significant decrease in basal and maximal respiration under both hypoxia and normoxia. CQ and Lys05, however, only showed a significant decrease in OXPHOS under hypoxia, with no effect on mitochondrial function under normoxia. We postulated that this effect arose from increased mitophagy under hypoxia. BNIP3 expression levels were enhanced under hypoxia in MOLM13 cells at 24- 48 hours. If mitophagy is constitutively occurring, blocking this process would cause an increase in the number of mitochondria. As expected, the number of mitochondria increased when treated with Lys05 or CQ under hypoxia, but not under normoxia, suggesting that mitophagy is occurring only under hypoxic conditions. Baf A1 caused an increase in mitochondria under both hypoxia and normoxia, suggesting that Baf A1 can both induce mitophagy and block mitochondrial degradation. We further confirmed this was due to mitophagy by assessing expression of the mitophagy protein PINK1. In normoxia, Baf A1 showed an almost 2-fold increase in PINK1 compared to the vehicle whereas CQ did not (Fig. 1B). Conclusion: We have identified a class of autophagy inhibitors that displays enhanced efficacy in AML cells under hypoxic conditions that reflect the BM microenvironment. This is due in part to their ability to target AML cellular reliance on mitophagy. These results provide the rationale for the further clinical development of Lys05 or other lysosome-based autophagy inhibitors as a novel means of targeting minimal residual disease in AML therapy. Disclosures Guzman: Samus Therapeutics: Patents & Royalties: intellectual rights to the PU-FITC assay; SeqRx: Consultancy; Cellectis: Research Funding. Wang:Pfizer: Other: Advisory role, Speakers Bureau; Stemline: Other: Advisory role, Speakers Bureau; Daiichi: Other: Advisory role; Amgen: Other: Advisory role; Agios: Other: Advisory role; Abbvie: Other: Advisory role; Kite: Other: Advisory role; Jazz: Other: Advisory role; Astellas: Other: Advisory role, Speakers Bureau; celyad: Other: Advisory role.

Abstract 1297: Casein kinase II (CK2) as a potential therapeutic target in acute myeloid leukemia (AML)

Abstract Casein Kinase II (CK2) is a pro-oncogenic kinase that is overexpressed in a majority of human malignancies, including leukemia. One third of AML cases without detectable high risk molecular alteration have high expression of CK2 which is strongly associated with poor outcome. Previously published data showed that CK2 directly phosphorylates several transcription factors including Ikaros (encoded by IKZF1) resulting in reduced DNA-binding affinity and loss of Ikaros function as a tumor suppressor. Here we present the evidence demonstrating role of Ikaros and CK2 in regulation of apoptosis in AML. We show that CK2 inhibitor, CX4945 has strong anti-leukemic effect in AML and one of the novel mechanisms of action involves restoration of Ikaros function as a transcriptional regulator of genes involved in apoptosis. Results: Treatment of AML cell lines and primary cells with CK2 inhibitor, CX4945 show cytotoxicity (IC50 3-5uM), increased apoptosis, cell cycle arrest and poor colony formation. Treatment of murine xenografts of AML with CX4945 showed significant anti-tumor effect. Further studies dissecting the mechanism of action of CX4945 are as follows: Genome-wide binding studies using chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) demonstrated that treatment of AML cell line, U937 with CX4945 at IC50 concentration enhances binding affinity of Ikaros transcription factor at the promoter regions of several target genes. Among the significantly affected genes (more than two fold change) are anti-apoptotic genes such as BCL2A1 (B Cell Lymphoma 2 related protein A1) and Bcl-xL (B Cell Lymphoma extra-Large). Dysregulation of apoptotic pathway is hallmark of cancer cells. These findings were confirmed qChIP where CX4945 treated AML cells showed increased binding of Ikaros to the BCL2A1 and Bcl-xL promoters. We used gain-of-function and loss-of-function experiments to determine how Ikaros regulates BCL2A1 and Bcl-xL genes in AML. Ikaros overexpression using lentiviral transduction results in reduced expression of BCL2A1 and Bcl-xL both at mRNA and protein level. Similar results were seen with silencing of CK2 using CK2 SiRNA as well as CX4945. Increased expression of BCL2A1 and Bcl-xL was noted after Ikaros silencing using SiRNA which did not revert with CX4945 treatment.In conclusion, (1) Ikaros represses transcription of anti-apoptotic genes BCL2A1and Bcl-xL, (2) CK2 directly phosphorylates tumor suppressor Ikaros and inhibits its function as transcriptional regulator, (3) Inhibition of CK2 enhances Ikaros-mediated repression of BCL2A1 and Bcl-xL genes resulting in increased apoptosis, (4) CX4945 has strong anti-leukemia effect in AML xenograft models. CK2 is a promising therapeutic target in AML. Our results unveal a novel mechanism of action of CK2 inhibitor, CX4945 in AML which involves restoration of Ikaros mediated regulation of apoptosis. Citation Format: Badhauria Preeti, Soumya Iyer, Elanora Dovat, Pavan Kumar Dhanyam Raju, Jonathan Payne, Chunhua Song, Yali Ding, Claxton David, Sharma Arati, Chandrika Gowda. Casein kinase II (CK2) as a potential therapeutic target in acute myeloid leukemia (AML) [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 1297.

The Bcl-2 inhibitor venetoclax inhibits Nrf2 antioxidant pathway activation induced by hypomethylating agents in AML.

Induction of reactive oxygen species (ROS), an important process for the cytotoxicity of various acute myeloid leukemia (AML) therapies including hypomethylating agents (HMAs), concurrently activates the NF-E2-related factor 2 (Nrf2) antioxidant response pathway which in turn results in induction of antioxidant enzymes that neutralize ROS. In this study, we demonstrated that Nrf2 inhibition is an additional mechanism responsible for the marked antileukemic activity in AML seen with the combination of HMAs and venetoclax (ABT-199). HMA and venetoclax combined treatment augmented mitochondrial ROS induction and apoptosis compared with treatment HMA alone. Treatment of AML cell lines as well as primary AML cells with venetoclax disrupted HMA decitabine-increased nuclear translocation of Nrf2 and induction of downstream antioxidant enzymes including heme oxygenase-1 and NADP-quinone oxidoreductase-1. Venetoclax treatment also leads to dissociation of B-cell lymphoma 2 from the Nrf2/Keap-1 complex and targets Nrf2 to ubiquitination and proteasomal degradation. Thus, our results here demonstrated an undiscovered mechanism underlying synergistic effect of decitabine and venetoclax in AML cells, elucidating for impressive results in antileukemic activity against AML in preclinical and early clinical studies by combined treatment of these drugs.

Alvocidib Potentiates the Activity of Azacytidine in an MCL-1-Dependent Fashion

Despite significant efforts, the clinical mechanism of action of hypomethylating agents such as 5-azacytidine (5-aza) is still poorly understood. 5-aza is currently indicated for the treatment of patients with myelodysplastic syndrome (MDS). While 5-aza has achieved good single-agent activity in acute myeloid leukemia (AML), complete response rates remain low when used as a single agent. In a recent report aimed at identifying rational therapeutic combinations with 5-aza, Bogenberger and colleagues identified multiple BCL-2 family member/BH3-containing therapeutic targets, which synergize with 5-aza when inhibited genetically or pharmacologically. The CDK9 inhibitor, alvocidib, has achieved significant improvement in complete response rates of newly diagnosed AML patients when administered before cytarabine and mitoxantrone (FLAM regimen) in a randomized multi-center Phase 2 trial when compared to 7+3 standard of care treatment. Recent reports suggest that the transcriptional repression of key anti-apoptotic proteins (eg., MCL-1) mediated by alvocidib's CDK9 inhibition, drive the pro-apoptotic activity of alvocidib in the FLAM regimen. We, therefore, hypothesized that alvocidib and 5-aza would synergize therapeutically in the treatment of AML by means of transcriptional repression of MCL-1 and sensitization to 5-aza. In this report, we demonstrate that treatment of AML cell lines with alvocidib inhibits both mRNA and protein expression of MCL-1 in a time and concentration-dependent fashion. Pre-treatment of cells with alvocidib, to repress MCL-1 expression prior to 5-aza treatment, reduced the 5-aza cell viability EC50 more than 2.5-fold, from 1.8 µM to 0.6 µM in MV4-11 cells. The alvocidib/5-aza combination also resulted in synergistic increases in caspase activity relative to either single agent within the combination, at multiple dose levels. Therefore, following reports suggesting inhibition of BCL-2 family members including MCL-1, sensitizes cells to 5-aza, our data suggest that the alvocidib/5-aza combination may constitute a viable therapeutic regimen. We also conclude that a CDK9 inhibitor/5-aza combination may be an effective clinical approach for the treatment of AML. DisclosuresKim:Tolero Pharmaceuticals: Employment. Soh:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Lee:Tolero Pharmaceuticals: Employment. Peterson:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Weitman:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment.

Phase 1 Study of CB-839, a First-in-Class, Orally Administered Small Molecule Inhibitor of Glutaminase in Patients with Relapsed/Refractory Leukemia

Background: Malignant cells alter metabolism in order to enable their highly anabolic state. In addition to a massive increase in glycolysis, neoplastic cells frequently depend on glutamine to feed the TCA cycle and provide key building blocks for cell growth and proliferation. CB-839 is a first-in-class potent and selective inhibitor of the glutaminase (GLS), the first step in glutamine metabolism, and has broad in vitro and in vivo anti-tumor activity in solid and heme malignancies. Acute myeloid leukemia (AML) cells are particularly dependent on glutamine, and treatment of AML cell lines and primary AML cells with CB-839 results in growth arrest and/or cell death (Padre et al., ASH, 2014 and Jacque et al., Blood, 2015). CB-839 causes a decrease in glutamate and glutamate-derived metabolites, such as TCA cycle intermediates and glutathione, crippling the proliferative capacity of these cells and in some cases inducing apoptosis.Methods: CX-839-003 is an ongoing Phase 1 (Ph1) trial of CB-839 in patients with relapsed/refractory hematologic malignancies. This study includes a dose escalation cohort to identify a recommended Phase 2 dose (RP2D), followed by expansion cohorts of patients (pts) with relapsed/refractory AML (IDH wt and mutant), acute lymphocytic leukemia, and IDH-mutant myelodysplastic syndrome (MDS). Pharmacokinetics (PK) is being monitored on Days 1 and 15.Results: Safety data are available for a total of 20 patients (18 AML, 1 ALL, and 1 mixed-lineage leukemia) that have enrolled across the dose escalation and expansion cohorts. These patients have received a median of 3 prior lines of systemic induction therapy, with 5 pts (25%) having received prior allogeneic bone marrow transplantation. Patients' median age was 75 (range 35 to 84). Dose levels from 100-1000 mg TID were explored during the dose escalation. CB-839 has been well tolerated to date, with no DLTs occurring on study. One patient discontinued due to an adverse event (AE) of elevated transaminases, and 5 pts (25%) had G3/4 AEs considered possibly related to study drug. Across three Ph1 studies for CB-839 in solid tumors and heme malignancies, reversible asymptomatic elevations in transaminases have been the primary G3 AE. Although initially CB-839 was given three times daily (TID) without food, the drug is now being given twice daily (BID) with meals based on PK and safety data generated across these 3 studies. Liver abnormalities occurred primarily on the TID schedule in 6/59 (10.2%) pts, but in only one of 60 pts (1.7%) receiving the BID regimen. Pts are currently being enrolled in expansion cohorts at the RP2D, 600 mg BID. Inhibition of GLS activity was evaluated across all 3 Ph1 studies and established clear exposure-dependent inhibition of the target in peripheral blood 4 hr after the first dose of CB-839, with >90% inhibition being maintained for most patients at the RP2D. Preliminary evidence of clinical activity has been noted, including reductions in blast counts in the bone marrow and/or periphery in 2 pts, one of whom achieved a CRi and remains on study for more than one year. 5 (31%) of 16 evaluable pts have remained on study for at least 4 cycles (range 4-17). Additional data from >8 additional patients have recently started on the "BID with meals" regimen will be presented. Correlative studies on clinical samples will also be presented.Conclusions: CB-839 has been well tolerated at and above doses that produced robust inhibition of GLS in blood platelets and in tumors. Dosing BID with food has mitigated the frequency and severity of LFT elevations, which has been the primary safety signal to date. Reductions in marrow and peripheral blast counts in a heavily pretreated acute leukemia population, with a significant number of patients tolerating therapy and remaining on study for a prolonged period, provide the rationale for continued evaluation of CB-839 as a single agent, and in combination with azacitidine, in AML patients. DisclosuresWang:Immunogen: Research Funding. Orford:Calithera Biosciences: Employment, Equity Ownership. Bennett:Calithera Biosciences: Employment, Equity Ownership. Flinn:Cephalon, Inc; Teva Pharmaceutical Industries Ltd; Genentech, inc; Gilead: Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

Abstract 3513: Inhibition of LSD1 for the treatment of cancer

Abstract Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. LSD1 mediated H3K4 demethylation can result in a repressive chromatin environment that silences gene expression and has been shown to play a role in hematopoietic differentiation. LSD1 is also overexpressed in multiple tumor types. These studies implicate LSD1 as a key regulator of the epigenome that modulates gene expression through post-translational modification of histones and its presence in transcriptional complexes. The current study describes the anti-tumor effects of a novel, irreversible, GSK LSD1 inhibitor (GSK2879552) in acute myeloid leukemia (AML) and small cell lung cancer (SCLC). GSK2879552 is a potent, selective, mechanism-based inhibitor of LSD1. Screening of over 150 cancer cell lines revealed that AML and SCLC cells have a unique requirement for LSD1. While GSK2879552 treatment did not affect the global levels of H3K4me1 or H3K4me2, local changes in these histone marks were observed near transcriptional start sites of genes whose expression increased with LSD1 inhibition. Treatment of AML cell lines with GSK2879552 increased cell surface expression of CD11b and CD86, markers associated with a differentiated immunophenotype. Six days of GSK2879552 treatment resulted in potent anti-proliferative growth effects in 19 of 25 AML cell lines representing a range of AML subtypes. Treating for longer time periods revealed sensitivity in all AML cell lines. AML blast colony forming ability was also inhibited in 4 of 5 bone marrow samples derived from primary AML patient samples. The effects of LSD1 inhibition were further characterized in vivo using a mouse model of AML induced by transduction of mouse hematopoietic progenitor cells with a retrovirus encoding MLL-AF9 and GFP. Primary AML cells were transplanted into a cohort of secondary recipient mice and were treated upon engraftment. After 17 days of treatment, control mice had 80% GFP+ cells in the bone marrow whereas treated mice had only 2.8% GFP positive cells (p<0.012), and the treated animals survived weeks beyond control mice. Growth inhibition was also observed in a subset of SCLC cell lines. GSK2879552 treatment of mice engrafted with SCLC lines resulted in greater than 80% tumor growth inhibition. Studies using patient derived primary SCLC showed similar efficacy demonstrating the growth inhibition of SCLC with an LSD1 inhibitor extended beyond cell lines. Together, these data demonstrate that pharmacological inhibition of LSD1 may provide a promising treatment for AML and SCLC. A Phase I clinical trial using GSK2879552 was initiated in March, 2014. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed by the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Citation Format: Kimberly Smitheman, Monica Cusan, Yan Liu, Michael Butticello, Melissa Pappalardi, James Foley, Kelly Federowicz, Glenn Van Aller, Jiri Kasparec, Xinrong Tian, Dominic Suarez, Jess Schneck, Jeff Carson, Patrick McDevitt, Thau Ho, Charles McHugh, William Miller, Scott Armstrong, Christine Hann, Neil Johnson, Ryan G. Kruger, Helai P. Mohammad, Shekhar Kamat. Inhibition of LSD1 for the treatment of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3513. doi:10.1158/1538-7445.AM2015-3513

Inhibition of NF-κB Activation Augments the Effect of 5-Azacytidine On Acute Myeloid Leukemia Cell Line

Abstract 1369Dehydroxymethylepoxyquinomycin (DHMEQ) is a unique NF-kB inhibitor that is a 5-dehydroxymethyl derivative of novel compound epoxyquinomicin C and is currently under development for clinical use. We have shown that DHMEQ specifically binds to NF-kB having transactivation domain and inhibits NF-kB activation at the level of the nuclear translocation and the DNA binding. Constitutive activation of NF-kB has turned out to be a hallmark of various types of cancers originated from hematopoietic and other organs. We showed the effectiveness of DHMEQ in hematological malignancies with constitutive activation of NF-kB including multiple myeloma, chronic lymphocytic leukemia, adult T-cell leukemia, pleural effusion lymphoma and Hodgkin lmphoma. However, significance of NF-kB as a molecular target in acute myeloid leukemia (AML) is not well understood.Recently 5-azacytidine is widely used for the treatment of myelodysplastic syndrome and other cancers. 5-Azacytidine is a nucleoside-based DNA methyltransferase inhibitor that induces demethylation and gene reactivation. Therefore it is possible that 5-azacytidine induces molecules required for NF-kB activation. However, the effect of 5-azacytidine on NF-kB induction, which may antagonize the anti-cancer effect is poorly understood. In this study we examined the constitutive NF-kB activation and the effectiveness of DHMEQ on AML cell lines, HL-60, ML-2, HEL, K562, CMK and Meg0-1. We also examined the induction of NF-kB by 5-azacytidine and evaluated the combined treatment of AML cell lines by 5-azacytidine and DHMEQ.AML cell lines except for K562 showed constitutive activation of NF-κB consisting of RelA and p50 subcomponent. The NF-κB binding activity observed in AML cell lines were relatively modest compared to that observed in Jurkat cells treated with tumor necrosis factor (TNF). Treatment of AML cell lines with constitutive activation of NF-κB by DHMEQ inhibited NF-κB and significantly reduced their viability. Treatment with 5-azacytidine also significantly reduced the viability of AML cell lines, however K562 showed resistance against the treatment. Addition of DHMEQ enhanced the effect of 5-azacytidine on cell viability of HL-60 and CMK cells. The treatment with 5-azacytidine enhanced NF-kB activation although the induction was modest and DHMEQ abolished both constitutive and induced NF-kB activation in HL-60 and CMK cells.The results indicate that NF-κB is a molecular target of AML cells and DHMEQ is a potential compound for the treatment of AML. Although the NF-κB activity is relatively low compared to that in lymphoid malignancies, the constitutive activation of NF-κB in AML cell lines appears to play a significant role for their survival. The results also indicate that 5-azacytidine can reduce viability of AML cells and combined use of an NF-κB inhibitor augments the effect of 5-azacytidine by inhibiting constitutive and induced NF-κB. Since NF-κB cooperates with various transcription factors and supports the survival of cells, interruption of the crosstalk between NF-kB and molecules induced by 5-azacytidine might also be responsible for the combined effect of DHMEQ and 5-azacytidine. Disclosures:No relevant conflicts of interest to declare.

SGI-110, a Novel Hypomethylating Agent, Induces the WNT Inhibitor Secreted Frizzled Related Protein-2 (SFRP2), and Down Regulates β-Catenin in Acute Myeloid Leukemia (AML) Cells

Abstract 1290 Introduction:SGI-110 (Astex Pharmaceuticals, Inc.) is a dinucleotide hypomethylating agent whose active metabolite is decitabine (DAC). This drug demonstrates superior pharmacokinetics relative to the parent drug as a result of resistance to modification by cytidine deaminase, and is being investigated in myeloid malignancy in the phase I/II setting. We and others have demonstrated that WNT inhibitory genes including SFRP2 are epigenetically silenced in AML and that exposure to DNA methyltransferase inhibitors such as 5-Azacitidine (AZA) and DAC can re-express these genes and down-regulate β-catenin signaling in AML cell lines. We hypothesized that treatment with SGI-110 would have a similar effect upon the epigenetically silenced WNT inhibitor SFRP2 and further would down-regulate β-catenin signaling in AML cells in vitro. Methods:The AML cell lines HL60 and U937 were cultured in vitro using standard techniques and treated with phosphate buffered saline, 0.1, 1 or 5 μM SGI-110, 2μM AZA or 0.5μM DAC. Results presented are pooled data from a minimum of three biological replicates. Samples were harvested on day 5 and viable cells, DNA, RNA and protein obtained. β-catenin levels and cellular localization were quantified using imaging flow cytometry (ImageStream), DNA was extracted and bisulfite converted for analysis of gene specific and global DNA methylation by pyrosequencing (LINE-1, SFRP2), RNA was converted to cDNA for analysis by RT-PCR, and protein was obtained to confirm ImageStream results by Western blot. Nuclear translocation of β-catenin, indicative of its signaling activity, was assessed in individual cells by ImageStream using a similarity score: a log-transformed Pearson’s correlation coefficient between the digitized images of immunostained β-catenin and a nuclear stain (DAPI). Shifts in the population (n=5,000) distributions of this similarity score were assessed by a resolution metric (Fishers discriminant ratio, Rd). Results:Treatment of AML cell lines with 5μM SGI-110 was toxic, and in line with previous experiments in AML cell lines, above the IC90. Treatment at the lowest dose of SGI-110 had minimal effects upon viability, methylation, and mRNA and protein expression in both cell lines tested. Treatment with SGI-110 at the 1μM dose resulted in reductions in LINE-1 methylation in HL60 cells by 21% (from 82% to 61%), compared to 8% with AZA (to 74%) and 20% with DAC (to 62%). In U937 cells, LINE-1 methylation decreased by 40% (from 67% to 27%) after SGI-110 treatment compared to a 25% reduction with AZA (to 42%) and a 30% reduction with DAC (to 36%). SFRP2 methylation in HL60 and U937 decreased from 86 and 88% at baseline to 66 and 60% with SGI-110 at the 1μM dose, compared to 68% with AZA and to 61% with DAC. Expression of SFRP2 mRNA was observed following treatment with 1μM SGI-110 and with DAC, but was limited following AZA treatment. ImageStream analysis of total cellular β-catenin in HL-60 and U937 cells demonstrated 2.4-fold and 1.2-fold reductions in total β-catenin following 1μM SGI-110 treatment. These results were similar to those seen with DAC (1.8-fold and 1.3-fold in HL-60 and U937 cells respectively). AZA treatment appeared to have a greater effect on total β-catenin in U937 cells (1.3-fold reduction) than in HL-60 cells (0.84-fold reduction). Western blot confirmed reductions in β-catenin protein. We also observed decreased nuclear translocation of β-catenin after treatment of HL-60 and U937 cells with 1 μM SGI-110 (Rd = −0.58 and −0.21 respectively; the negative sign indicates a change in cellular distribution from the nucleus to the cytoplasm). Changes were comparable to those observed with DAC (Rd = −0.75 and −0.26 in HL-60 and U937 cells respectively). AZA treatment of U937 cells resulted in a shift in cellular distribution (Rd = −0.20) similar to that for DAC and SGI-110 but had no effect on β-catenin distribution in HL-60 cells (Rd= 0.00). Conclusions:SGI-110 is a novel DNMT inhibitor which demonstrates robust effects on LINE-1 methylation, SFRP2 mRNA expression, and β-catenin level and localization consistent with epigenetically mediated re-expression of the WNT inhibitor SFRP2. Both upregulated β-catenin signaling and SFRP2 methylation have been demonstrated to correlate with inferior survival in patients with myeloid malignancies. Re-expression of epigenetically silenced WNT inhibitory genes such as SFRP2 may abrogate β-catenin signaling in AML cells. Disclosures:Karpf:Astex Pharmaceuticals: Research Funding. Griffiths:Celgene: Honoraria; Astex Pharmaceuticals: Research Funding.

Activity of the Hypoxia-Activated Prodrug, TH-302, in Human Acute Myeloid Leukemia Models

Abstract 3611Recent evidence has demonstrated the bone marrow (BM) microenvironment, the principal site of acute myeloid leukemia (AML) initiation and expansion, is characterized by intrinsically low oxygen tension. Theoretically, such microenvironmental changes may lead to the selective outgrowth of AML clones which are “better adapted” to survive within a severely hypoxic microenvironment and/or may confer resistance to chemotherapeutic agents, similar to solid tumor cells. We report here that human AML cells (HL60, ML-2) cultured under chronic hypoxic conditions mimicking the marrow microenvironment (1% O2, 72 hours) exhibited reduced sensitivity to cytarabine-induced apoptosis as compared with normoxic cells, as determined by flow cytometric analysis, western blot analysis, and cell viability assays. Similar results were noted in primary AML samples treated with cytarabine under normoxic and hypoxic conditions in colony formation assays (n=3 samples, p=0.01). In order to improve upon chemotherapy outcomes, we investigated the effects of TH-302, a hypoxia-activated bromo-isophosphoramidate mustard prodrug, which is currently undergoing clinical trial evaluation in multiple tumor types. Treatment of AML cell lines (HL60, HEL) and primary AML samples with TH-302 (at doses ranging from 0.1– 5 mM, p values ranging from <0.05–0.0001) resulted in dose- and hypoxic-dependent inhibition of AML proliferation and apoptosis. In vivo TH-302 treatment significantly decreased disease burden, as measured by total animal bioluminescence, and prolonged overall survival in two systemic human AML xenograft models (HEL-luciferase, HL60-luciferase) (Figure 1). Immunohistochemical studies demonstrated that TH-302 treatment reduced numbers of hypoxic (pimonidazole-positive) cells within the leukemic marrow microenvironment. Because prior data in animal models has shown that AML progression within the marrow is associated with expansion of hypoxic BM areas, we examined the effects of TH-302 treatment on systemic AML growth when initiated early (prior to AML inoculation) or late (several days following AML engraftment) in the disease process. TH-302 was equally effective at both time points. Although anti-vascular therapy has been shown to enhance tumor hypoxia in other cancer types, we noted no synergistic or additive in vivo effects when TH-302 therapy was combined with sorafenib, an inhibitor of vascular endothelial growth factor receptors (VEGFR), in our models. TH-302 therapy administered for two weeks in non-leukemic and leukemia-engrafted mice was not associated with hematologic toxicities. In summary, our results demonstrate the anti-leukemic activity of TH-302 in preclinical AML models and suggest that the efficacy of this and other drugs for AML therapy may be uniquely affected by the BM microenvironment. Further clinical development of TH-302 and other hypoxia-targeted drugs for AML therapy are warranted. Based on our data, higher TH-302 doses and/or chronic drug administration may be needed for optimal in vivo anti-leukemic activity. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Gene Expression Analysis of Bexarotene Differentiated Acute Myeloid Leukemias Identifies Known and Novel Gene Targets.

Despite advances in understanding the molecular pathogenesis of acute myeloid leukemia (AML), therapy for relapsed disease remains inadequate with high mortalities. Clinicians at the University of Pennsylvania have demonstrated that the FDA approved retinoid X receptor (RXR) agonist bexarotene (Targretin™) stimulates leukemic cell differentiation in a subset patents with relapsed AML leading to clinical responses. This underscores the importance of identifying the mechanism by which bexarotene induces differentiation in AML in order to enhance the efficacy of this therapeutic approach. To understand the role of bexarotene and RXR receptors in leukemic cell differentiation, we initially utilized a pharmacogenetic approach to study the effects of bexarotene on AML cell lines using combinations of bexarotene with other differentiation induction agents. These studies demonstrate that bexarotene induces myeloid differentiation in MOLM14, HL60, THP-1, and NB4 cell lines but not in the myeloblastic cell line KG1a. Combination treatment of AML cell lines with bexarotene in combination with all trans retinoic acid (ATRA) enhanced differentiation suggesting that the mechanism of action for bexarotene is through RARα (retinoic acid receptor)/RXRα heterodimer stimulation. Consistent with this, differentiation induced by the drug combination was effectively blocked by the RAR antagonist, LG100815 and partially blocked by the RXR antagonist, LG101208. In contrast, bexarotene does not cooperate with valproic acid, theophylline, the PPARγ agonist rosiglitazone, or the LXR agonist T0901317. Preliminary data from quantitative RT-PCR and Affymetrix microarray analysis of bexarotene responsive AML cell lines at 3, 6, 12, and 96 hours post treatment has identified a subset of genes potentially regulated by bexarotene. CEBPε, a transcription factor known to play a critical role in granulopoiesis and PIM-1, a known oncogenic transcription factor, were among the genes that were significantly upregulated after bexarotene treatment of AML cells. Analysis of the functional role of C/EBPε in retinoid induced differentiation will be presented. Overall, this data supports the hypothesis that bexarotene, like ATRA, induces myeloid differentiation through activation of a RAR/RXR heterodimeric partner. However, other data suggests the presence of RAR independent pathways of signaling. LG100268, a pure RXR agonist induced myeloid differentiaton although not as robustly as bexarotene. Analysis of RAR and RXR mRNA expression in AML cell lines demonstrates that bexarotene does not induce expression of RARβ or p21, known targets induced by ATRA during myeloid differentiation. Chromatin immunoprecipitation assays demonstrate RXRα occupancy at RARβ and p21 promoter regions containing retinoid response elements (RARE). However, expression of these genes does not correlate with bexarotene-induced differentiation. This data suggests that although their expression has been linked to ATRA responsiveness, induction of RARβ and p21 expression is not necessary for retinoid induced myeloid differentiation. In summary, bexarotene induces myeloid differentiation through RAR dependent and independent pathways. Further analysis of the signaling events necessary for induction of myeloid differentiation by bexarotene may allow for improved selection of patients with AML who will respond to bexarotene.