Acute Myeloid Leukemia Cell Lines OCI-AML3 Research Articles

P459: NUC-7738 REGULATES BETA-CATENIN SIGNALLING RESULTING IN REDUCED PROLIFERATION AND SELF-RENEWAL OF AML CELLS

Background: NUC-7738, a phosphoramidate transformation of 3’deoxyadenosine (3’dA), is specifically designed to generate the active anti-cancer metabolite 3’-deoxyadenosine triphosphate (3’-dATP) directly in cells, bypassing key cancer resistance mechanisms of transport, activation and breakdown. NUC-7738 is currently in a Phase I/II clinical study to assess safety and determine the recommended dose in patients with advanced solid tumors and lymphoma. We have recently shown in cell lines a potential therapeutic role for NUC-7738 in patients with acute myeloid leukemia (AML), whereby NUC-7738 induced myeloid cell differentiation and mitochondrial-mediated apoptosis. AML leukemia stem cells (LSCs) are required for the initiation and maintenance of the disease. Activation of the Wnt/β-catenin pathway is required for the survival and development of LSCs and therefore, targeting β-catenin is a potential therapeutic strategy. Aims: The aim of this study was to determine whether NUC-7738 regulates β-catenin and the expression of its target genes in AML cells Methods: AML cell lines KG1a, OCI-AML3, HL-60 and U937 were treated with NUC-7738 for 48 or 72 hours. LSCs were determined by the expression of surface markers CD34, CD38 and CD123 via flow cytometry. Self-renewal was assessed by performing the colony forming assay using MethoCult Enriched media. Cell proliferation was determined by the Alamar Blue assay. Immunoblotting was performed for β-catenin, PI3K-p110, phosphorylated AktSer473 and phosphorylated GSK3βSer9 Results: NUC-7738 reduced PI3K-p110, phosphorylated AktSer473 and phosphorylated GSK3βSer9 resulting in reduced β-catenin expression. NUC-7738 decreased the percentage of CD34+ CD38- CD123+ (LSCs) from 15% to 7% and significantly reduced the total number and size of leukemic colonies. NUC-7738 inhibited cell proliferation in U937, OCI-AML3 and HL-60 cells, with IC50’s of 1.55 µM, 7 µM and 16 µM, respectively. Summary/Conclusion: Self-renewal pathways, including Wnt/β-catenin, are key for the initiation and long-term maintenance of LSCs. Through the ability to reduce PI3K-p110, phosphorylated AktSer473, and phosphorylated GSK3βSer9 and suppress β-catenin signalling, NUC-7738 exploits a key developmental process of AML, supressing the expansion and survival of LSCs. These results indicate that targeting LSCs with NUC-7738 may offer a successful therapeutic strategy for treating patients with leukemia.

Abstract P026: NUC-7738 alters oxidative phosphorylation and causes terminal differentiation in acute myeloid leukemia cells

Abstract Background NUC-7738, a phosphoramidate transformation of 3’deoxyadenosine (3’dA), is specifically designed to generate the active anti-cancer metabolite 3’-deoxyadenosine monophosphate (3’-dAMP) directly in cells, bypassing key cancer resistance mechanisms of transport, activation and breakdown. NUC-7738 is currently in a Phase I clinical study to assess safety and determine the recommended dose in patients with advanced solid tumors and lymphoma. Acute myeloid leukemia (AML) cells exhibit impaired differentiation, uncontrolled proliferation and are highly dependent on mitochondrial metabolic processes for survival and chemoresistance. The aim of this study was to determine the effect of NUC-7738 on oxidative phosphorylation (OXPHOS) proteins and resulting changes in mitochondria morphology and activation of the intrinsic apoptotic pathway. Methods AML cell lines OCI-AML3 and HL-60 were treated with NUC-7738 for 48 hours. Cell differentiation was determined by morphologic modification with Giemsa stain and assessing the expression of surface markers CD11b and CD163 by flow cytometry. Immunoblotting was performed for Mitochondrial Electron Transport Chain Complex I subunit NDUFB8, Complex II subunit SDHB, Complex III subunit UQCRC2, Complex IV subunit COX II and ATP synthase subunit ATP5A. Mitochondria morphology was assessed by confocal microscopy with MitoTracker-Deep Red CMXRos dye. Annexin-V, DAPI, Bcl-2, cytochrome c, and cleaved-caspase 7 were assessed by flow cytometry to determine intrinsic apoptosis. Results NUC-7738 increased the proportion of CD11b+ and CD163+ cells in a dose-dependent manner from 6.1% to 28.7% and 9.6% to 26.9%, respectively. CD11b positivity was also increased in HL-60 cells from 14.5% to 31.1%. NUC-7738 induced morphological differentiation as indicated by decreased nucleo-cytoplasmic ratio, appearance of multi-lobed nuclei and vacuolization of the cytoplasm in OCI-AML3 cells. Decreased nucleo-cytoplasmic ratio was also evident in HL-60 cells. NUC-7738 decreased the protein expression of all 5 complexes of oxidative phosphorylation in OCI-AML3 cells at 48 hours and this metabolic disruption was also evident from distinct changes in mitochondria morphology. NUC-7738 activated the intrinsic apoptotic pathway, causing sequestration of BCL-2 and release of cytochrome c, resulting in a decrease in the percentage of live OCI-AML3 cells from 76% to 37% at 48 hours. Conclusion Through the ability of NUC-7738 to inhibit OXPHOS complexes with consequent terminal differentiation and activation of mitochondrial-mediated apoptosis, it exploits the mitochondrial characteristics of certain cancer cells, including their greater dependence on OXPHOS for survival than normal cells. NUC-7738 offers a potential treatment option for patients with a variety of malignancies. Citation Format: Akbar M. Shahid, In Hwa Um, Oliver J. Read, David J. Harrison. NUC-7738 alters oxidative phosphorylation and causes terminal differentiation in acute myeloid leukemia cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P026.

Scalable Enrichment of Immunomodulatory Human Acute Myeloid Leukemia Cell Line-Derived Extracellular Vesicles.

Acute myeloid leukemia (AML) cells can secrete trophic factors, including extracellular vesicles (EVs), instructing the stromal leukemic niche. Here, we introduce a scalable workflow for purification of immunomodulatory AML-EVs to compare their phenotype and function to the parental AML cells and their secreted soluble factors. AML cell lines HL-60, KG-1, OCI-AML3, and MOLM-14 released EVs with a peak diameter of approximately 80 nm in serum-free particle-reduced medium. We enriched EVs >100x using tangential flow filtration (TFF) and separated AML-derived soluble factors and cells in parallel. EVs were characterized by electron microscopy, immunoblotting, and flow cytometry, confirming the double-membrane morphology, purity and identity. AML-EVs showed significant enrichment of immune response and leukemia-related pathways in tandem mass-tag proteomics and a significant dose-dependent inhibition of T cell proliferation, which was not observed with AML cells or their soluble factors. Furthermore, AML-EVs dose-dependently reduced NK cell lysis of third-party K-562 leukemia targets. This emphasizes the peculiar role of AML-EVs in leukemia immune escape and indicates novel EV-based targets for therapeutic interventions.

Anti-Leukemic Properties of Histamine in Monocytic Leukemia: Role of NOX2

We aimed at defining effects of histamine dihydrochloride (HDC) on the maturation and expansion of monocytic acute myeloid leukemia (AML) cells in vitro and in vivo with focus on the role of the myeloid NADPH oxidase (NOX2), which is targeted by HDC acting via H2-type histamine receptors (H2R). The human myelomonocytic/monocytic AML cell lines PLB-985 and OCI-AML3 were found to express NOX2 and H2R and responded to HDC with increased expression of differentiation markers and reduced cell cycle proliferation in vitro . These effects were absent in NOX2-/- (NOX2 -KO) PLB-985 cells. Microarray analysis revealed that HDC regulated the expression of genes involved in cell differentiation and cell cycle progression in NOX2+ but not in NOX2 -KO AML cells. In a xenograft mouse model using immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (NOG) mice, systemic treatment with HDC reduced the expansion of engrafted monocytic AML cells in bone marrow but did not exert anti-leukemic activity against engrafted NOX2 -KO cells. Primary leukemic cells recovered from patients with monocytic forms of AML co-expressed NOX2 and H2R and commonly upregulated the expression of the maturation markers HLA-DR, FPR1 and FPR2 after exposure to HDC. Results from a phase IV trial supported the clinical benefit of treatment with HDC, used in conjunction with low-dose IL-2, in preventing relapse among patients with monocytic forms of AML. These results imply that HDC targets NOX2 to exert anti-leukemic activity against monocytic AML cells and point towards NOX2 as a conceivable target in AML therapy. DisclosuresGrauers Wiktorin:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer. Aurelius:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer. Thoren:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer. Hellstrand:Immune Pharmaceuticals: Patents & Royalties: Filed or pending patents protecting the use of NOX2 inhibitors in cancer. Martner:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer.

All-Trans Retinoic Acid Enhances the Anti-Leukemia Effect of Venetoclax on Acute Myeloid Leukemia Cells

Objective: Acute myeloid leukemia (AML) is a malignant disease of the hematopoietic system in which arrested maturation, excessive proliferation and inhibition of apoptosis have been observed in the process of hematopoietic stem cell differentiation. Some patients, especially elderly patients, do not tolerate cytotoxic chemotherapy, and some patients are resistant to chemotherapeutic drugs, consequently molecular targeting therapy has brought new hope for AML treatment. Venetoclax (ABT-199) is a selective inhibitor of the antiapoptotic protein Bcl-2. Increased expression of Bcl-2 is an important factor in the inhibition of apoptosis in leukemia cells. The use of a single drug is often limited because of resistance, and a better curative effect can be obtained in combination with other drugs. All-trans retinoic acid (all-trans-retinoic acid, ATRA) was first used by Chinese doctor to treat acute promyelocytic leukemia (APL). Many studies have shown that ATRA can inhibit the expression of Bcl-2 in AML cells and promote apoptosis. We aimed to test for the first time whether the combination of ATRA and venetoclax would produce a stronger anti-AML effect. Methods: The effects of ATRA and ABT-199 on the ABT-199-resistant AML cell lines OCI-AML3 and THP-1 and the sensitive cell lines MV-4-11 and MOLM-13 were observed in vitro. The following methods were used in this study. 1. CCK8 assay was used to detect cell proliferation. 2. Annexin V/FITC and PI double staining were used to detect apoptosis rate by flow cytometry. 3. Flow cytometry was used to detect the expression of CD11b. 4. PI single staining and flow cytometry were used to study the cell cycle. 5. Western blotting was used to detect the expression of Bcl-2, Bax, Mcl-1, PARP, caspase3, GSK-3β and Cyclin-D1 proteins in OCI-AML3 cells. Results:1. Compared with the control group and the ATRA or ABT-199 single drug group, cell proliferation was significantly inhibited in the combined drug group, the apoptosis rate was significantly increased, and the cell cycle was blocked in G1 phase. 2. The expression of CD11b in the ATRA or ABT-199 group and combination group was significantly higher than that in the control group. 3. In OCI-AML3 cells, compared with the ABT-199 drug group, the expression of Bcl-2, Mcl-1 and Cyclin-D1 decreased, and the expression of Bax, cleaved-PARP, cleaved caspase3 and p-GSK-3β expression increased in the combined treatment group. Conclusion: All-trans retinoic acid (ATRA) can enhance the anti-leukemia effect of ABT-199 on AML cell lines by inhibiting cell proliferation as seen by the cell cycle arrest of AML cells in G1 phase, promoting apoptosis and inducing differentiation. ATRA can reverse the drug resistance of AML cells to ABT-199 by inhibiting the expression of Bcl-2 and Mcl-1. Our observations may provide new strategies and theoretical support for the clinical treatment of AML. Key Wor ds :AML, Venetoclax, ATRA, Apoptosis Disclosures No relevant conflicts of interest to declare.

PLZF regulates apoptosis of leukemia cells by regulating AKT/Foxo3a pathway.

To explore the regulatory role of PLZF in the malignant phenotype of non-APL acute myeloid leukemia (AML) and its underlying mechanism. The expression level of PLZF in AML cell lines KG-1a, HL-60, OCI-AML3, THP-1 and K562 was detected by quantitative Polymerase Chain Reaction (qPCR) and Western blot, respectively. Subsequently, THP-1 cells were divided into mock group (no treatment), scramble group (transfection with scramble shRNA) and shPLZF group (transfection with shPLZF). THP-1 cell line stably expressing shPLZF was constructed, followed by determination of its transfection efficiency by qPCR and Western blot, respectively. The proliferation and colony formation of THP-1 cells were accessed using CCK-8 (cell counting kit-8) assay and colony formation assay, respectively. The apoptotic rate in THP-1 cells was determined using flow cytometry. Protein levels of apoptosis-related genes in THP-1 cells were detected by Western blot. Finally, protein levels of AKT, Foxo3a, pAKT and pFoxo3a were detected by Western blot as well. Both mRNA and protein levels of PLZF were relatively high in THP-1 cells, and were selected for the following experiments. After construction of THP-1 cell line stably expressing shPLZF, proliferative rate and colony formation abilities increased in the shPLZF group compared with the mock group and the scramble group. We found a decreased apoptotic rate, downregulated Bax and upregulated Bcl-2 in the shPLZF group than those of the mock group and scramble group. Phosphorylation levels of AKT and Foxo3a increased after interference with PLZF, whereas no significant changes in total levels of AKT and Foxo3a were observed. PLZF inhibits the malignant phenotype of AML by regulating the AKT/Foxo3a pathway.

Mebendazole Exerts Potent Anti-Leukemic Effects By Downregulating Protein Levels of Hedgehog Transcription Factors GLI1 and GLI2

The relevance of the Hedgehog signaling pathway in the pathophysiology of acute myeloid leukemia (AML) has been demonstrated by us and others. Inhibition of the downstream Hedgehog transcription factors GLI1 and GLI2 results in strong anti-leukemic effects. Therefore, Hedgehog pathway inhibitors represent a promising therapeutic approach in AML. Mebendazole is an anthelmintic drug commonly used for the treatment of various parasitic worm infections. Recently, mebendazole has been shown to exhibit strong anti-tumor effects in different cancer entities including AML. In the work presented here, we investigated the effect of mebendazole on expression and activity of GLI transcription factors and its anti-leukemic activity.To determine the effect of mebendazole on GLI transcription factors, we treated the AML cell lines MV4-11, MOLM-13, THP-1 and OCI-AML3 with different concentrations of mebendazole and analyzed its impact on GLI1 and GLI2 protein- and mRNA levels. Furthermore, GLI reporter assays (Cignal GLI Reporter (luc) Kit, Qiagen) were performed to determine the effect of mebendazole on the GLI1 and -2 transcriptional activity. Mebendazole strongly inhibited GLI1 and GLI2 signaling activity in a dose-dependent manner. Exemplarily, treatment with 500 nM mebendazole reduced the GLI1 and -2 transcriptional activity in all cell lines tested by 54.8 % (± 9.6) after 24h and 73.2 % (± 11.6) after 48h. We could demonstrate by Western Blotting that GLI1 and -2 protein levels were clearly reduced 24h and 48h after mebendazole exposure, whereas GLI1 and -2 mRNA levels did not decrease. These data suggest that mebendazole may increase degradation of GLI proteins via the proteasome pathway. Therefore, we evaluated the influence of the 26s proteasome inhibitor bortezomib on GLI levels after mebendazole treatment. Inhibiting the 26s proteasome with 2 nM, 5 nM and 10 nM of bortezomib increased GLI signaling activity by 13.6 % (± 8.0), 84.6 % (± 39.2) and 137.1 % (± 37.9), respectively. Furthermore, 10 nM bortezomib abolished the effect of mebendazole on GLI protein levels. Taken together, mebendazole increased the proteasomal degradation of GLI1 and GLI2. These observations were extended to samples from AML patients. After mebendazole treatment for 24h or 48h all analyzed patients had reductions of GLI1 protein levels as confirmed by Western blotting (n=4), whereas GLI1 and GLI2 mRNA levels were not changed (n=7), indicating that proteasomal degradation was operational in primary blasts as well.Evaluating the anti-leukemic effects of mebendazole, we also investigated its combination with the small molecule GLI inhibitor GANT61. We treated the AML cell lines MV4-11, MOLM-13, THP-1 and OCI-AML3 with combinations of mebendazole and GANT61 and analyzed cell proliferation, apoptosis and colony formation. Mebendazole treatment alone already resulted in decreased proliferation and colony forming capacity as well as increased apoptosis rates in a dose-dependent manner. The combination of mebendazole with the GLI inhibitor GANT61 synergistically increased the anti-proliferative effects of mebendazole on all 4 AML cell lines tested. Additionally, GANT61 further increased the effect of mebendazole on colony formation significantly. Incubation with 100 nM, 200 nM and 500 nM mebendazole inhibited the proliferation of primary blasts from AML patients by 15.1 % (± 7.5), 31.6 % (± 16.8) and 66.0 % (± 17.4), respectively (n=8). Moreover, the combination with GANT61 significantly increased these anti-proliferative effects.This work indicates that mebendazole exerts profound anti-leukemic effects by decreasing GLI1 and GLI2 intracellular levels by promoting its proteasomal degradation. Combining mebendazole with GLI1 and GLI2 inhibitors such as GANT61 enhances this effect considerably. These observations may lead to the introduction of novel treatment strategies in AML. DisclosuresStamm:Amgen Research (Munich) GmbH / Amgen Inc.: Patents & Royalties; Astellas GmbH: Other: Travel Grant. Wellbrock:Amgen Research (Munich) GmbH: Patents & Royalties. Fiedler:GSO: Other: support for meeting attendance; Gilead: Other: support for meeting attendance; Amgen: Other: support for meetíng attendance; Pfizer: Research Funding; Amgen: Research Funding; Amgen: Patents & Royalties; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees, support for meeting attendance; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Other: support for meeting attendance; JAZZ Pharmaceuticals: Other: support for meeting attendance; Teva: Other: support for meeting attendance.

Using HSP90 Inhibitors to Target DNA Repair Proteins in AML

Acute myeloid leukemia (AML) is a disease of proliferative and abnormally differentiated myeloid cells(Döhner, Weisdorf, & Bloomfield, 2015) that is characterized by chromosomal aberrations and gene mutations that influence outcome(De Kouchkovsky & Abdul-Hay, 2016). Despite the approval of targeted therapies, induction therapy with cytarabine (Ara-C) and anthracycline has remained the standard of care for AML(Dombret & Gardin, 2016; Showel & Levis, 2014). Newer chemotherapeutics such as Sapacitabine have shown comparable clinical outcomes to Ara-C in older patients(Burnett et al., 2015). Ara-C is a nucleoside analog which incorporates during DNA replication and stalls replication(Liu et al., 2010) while Sapacitabine induces single stranded nicks in the DNA leading to double stranded breaks which are repaired by Homologous Recombination (HR)(Burnett et al., 2015; Lim & Jamieson, 2014). However, eventually patients develop resistance to chemotherapy leading to poor prognosis after relapse(Wu, Duan, Chen, & Chen, 2017). One aspect of this is due to upregulation of DNA damage repair proteins leading to increased repair of any DNA lesions primarily through HR. We therefore are interested in developing a way to target DNA repair to extend nucleoside analog therapy in AML.HSP90 is a molecular chaperone that is critical for the folding and proper function of multiple key proteins of various DNA repair pathways(Dote, Burgan, Camphausen, & Tofilon, 2006). Relevant to such repair, ATM and Chk1 become recruited to DNA double strand breaks to activate defined downstream signaling cascades that result in cell cycle arrest and DNA repair(Ceccaldi, Rondinelli, & D'Andrea, 2016). Of these,γ-H2AX acts a sensor of DNA damage and repair by forming foci at the sites of damaged DNA that get resolved upon successful repair(Sharma, Singh, & Almasan, 2012). Similarly, the Rad51 helicase plays a key role in HR repair and the presence of Rad51 foci can be used as a marker for successful HR(Gachechiladze, Škarda, Soltermann, & Joerger, 2017). To determine whether inhibiting HSP90 compromised DNA repair, we treated both AML cell lines OCI-AML3 and MV4-11 with HSP90 inhibitor, onalespib. OCI-AML3 cells show a time dependent decrease in DNA damage repair proteins Chk1 and Rad51 by western blotting. Furthermore, primary patient samples treated with onalespib for 24 and 36 hours also show a depletion of Chk1 and Rad51. We then used ionizing radiation (IR) to induce dsbreaks and induce signaling and repair(Ceccaldi et al., 2016). Using immunofluorescence, we found that in OCI-AML3 after exposure to IR there was a significant increase in number of Rad51 foci after 6 hours. The large number of γ-H2AX foci formed at .5 hour after IR decrease by 6 hours post-IR. This suggests that there is a successful HR repair occurring in this AML cell line. When OCI-AML3 cells were pretreated with onalespib, Rad51 foci are no longer present and γ-H2AX foci remained present at 6 hours suggesting unrepaired dsbreaks. Using the colony forming assay we see that there is a slight decrease in viability of OCI-AML3 cells exposed to onalespib however, in MV4-11 cells there is a steep decline which could be due to the differential expression of genes such as FLT3-ITD, a client protein of HSP90. Low doses of cytarabine or sapacitabine modestly decreased colony growth of OCI-AML3 and MV4-11 cells but exposing cells to onalespib for 24 hours sensitized the cells to both cytarabine and sapacitabine. Finally, we have also evaluated effects of onalespib in an in-vivo MOLM13 driven mouse model of AML and have shown that treatment with onalespib extended survival in this mouse model. DisclosuresMims:Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

KMT2E-Mediated Epigenetic Reprogramming Promotes the Sensitivity to All-Trans Retinoic Acid and Increases the Granulocytic Differentiation in AML Cells

Background: The histone-lysine N-methyltransferase 2E (KMT2E) is a member of the KMT2 family that regulates terminal myeloid differentiation and hematopoietic stem cell self-renewal. However, unlike the other members of KMT2 family, KMT2E does not exhibit the intrinsic histone methyl transferase activity, but has been shown to indirectly interact with chromatin by regulating SET7/9 expression and directing the tri-methylation state of the histone H3K4 (H3K4me3). We previously reported that the low expression of KMT2E predicts poorer prognosis in acute promyelocytic leukemia (APL) and that the overexpression of KMT2E contributed to granulocytic differentiation of APL cells in response to all-trans retinoic acid (ATRA). ATRA as a single agent is not effective in cell differentiation of non-APL acute myeloid leukemia (AML) but changes in H3K4 methylation promotes ATRA sensitivity and upregulates myeloid-differentiation-associated genes in AML cells. Therefore, to better understand the role of KMT2E in leukemogenesis and to test whether ATRA was capable to epigenetically mediate the KMT2E function in non-APL AML blasts, we investigated the effect of KMT2E overexpression in the cell viability, tumor growth and signaling response to ATRA in vitro and in vivo.Methods: qPCR analyzes revealed that the AML cell lines OCI-AML3, U937 and THP-1 express low levels of KMT2E in relation to the APL cell line NB4 and then, they were selected for further experiments. OCIAML3, THP1, and U937 were transduced with the pMEG or the pMEG-KMT2E vector, and U937 cells were also submitted to KMT2E gene silencing by a specific shRNA. The transduced cells were subjected to ATRA (1µM) treatment to evaluate proliferation (Ki-67), clonogenicity, cell cycle and apoptosis (annexin V). For dose-response curves (MTT), cells were administrated graduated ATRA concentrations and the IC50 values were calculated. ATRA-induced granulocytic differentiation was determined by immunophenotyping analysis of myeloid markers: CD11b, CD11c, CD14, CD16, CD64, and HLA-DR. Genes (SETD7/9) and protein (H3K4me3) expressions were established by qPCR and Western blot analysis respectively. 8-10 week-old NSG mice received a dorsal s.c. injection of 5 x 106 U937 cells (transduced with empty/KMT2E/shKMT2E) and 7 days after initial tumor growth treatment was initiated by intraperitoneal injection of the vehicle (n=26) or ATRA (5 mg/kg, n=30) for another 7 days. Tumor volume (V) was obtained using the formula (V=W2 x L x 0.52), where W represents the smaller and L the large diameter. The tumor apoptotic index was determined by TUNEL assay.Results:KMT2E overexpression significantly increased cell proliferation and the proportion of G2/M cells in all cell lines (all P<.01) but no differences in clonogenicity were observed. The IC50 value of ATRA for U937 was of 23 µM for cells transduced with empty vector and 11 µM for pMEG-KMT2E cells, while for THP1 empty/KMT2E cells received a dose of 9.93 µM and 1.68 µM respectively. ATRA increased apoptosis in a time-dependent manner in U937 and THP1 cells (all P<.05), but not in OCIAML3 p-MEG-KMT2E cells. Furthermore, ATRA-treated U937 and THP1 cells presented G0/G1-cell cycle arrest (all P<.05) after 72 hours, while no effect was observed in OCI-AML3. ATRA-induced granulocytic differentiation increased the CD11b/CD14 expression in U937 and THP1-KMT2E cells, within 72 hours of treatment compared to controls (all P<.05). ATRA treatment enhanced expression of H3K4me3 in a time-dependent manner which was correlated with enhanced transcript levels of SETD7/9 (all P<.05).In NSG mice, ATRA significantly reduced the tumor burden in U937- KMT2E cells and increased the apoptotic index (P<.01) after 7 days of treatment, albeit the high tumor burden in KMT2E cells treated with the vehicle. As a result the mean±SD of tumor volume for empty/KMT2E cells was 1296±95.6 mm3 and 445±59.6 mm3, respectively (P=.001). Finally, U937 shKMT2E indicated no difference in ATRA sensitivity and tumor volume (840±54.8 mm3 and 550±32.1 mm3 for empty/shKMT2E cells, respectively (P>.05).Conclusion: Our results demonstrated that KMT2E increases the cellular proliferation and ATRA-induced granulocytic differentiation in non-APL AML cell lines. Furthermore, ATRA triggered epigenetic reprogramming resulting in increased levels of H3K4me3, which redirected the biological function of KMT2E. DisclosuresNo relevant conflicts of interest to declare.

Bitter Taste Receptors System Is Expressed and Functional in Both HSCs and Leukemic Cells

Acute Myeloid Leukemia (AML) is a clonal disease sprouting from a rare population of leukemic stem cells. Over the past years, increasing interest is gaining the contribution that cell-extrinsic factors have in AML generation and maintenance. In this context, the ability of leukemia cells to detect changes in the microenvironment is important in responsiveness to environmental fluctuations.Bitter taste receptors (T2Rs) are typical G-protein coupled receptors and are normally found on the surface of the tongue. Recent studies showed that T2Rs are widely expressed in various parts of human anatomy and have been shown to be involved in physiology of respiratory system, gastrointestinal tract and endocrine system. thus suggesting a wider function in “sensing microenvironment”. We recently reported that AML cell lines OCI-AML3, THP-1, and AML primary cells expressed fully functional T2Rs subtypes. Gene expression profile analysis showed that after T2Rs activation, leukemic cell lines underwent down-regulation of genes involved in positive regulation of cell proliferation, migration, and cell-cycle. Whereas genes involved in cell adhesion and DNA repair were up-regulated. Functional assays supported these results (Blood 2017 130:3949).In the present work, we further investigated the role of T2Rs in BM microenvironment by extending the analysis to AML primary samples and to normal hematopoietic stem cells (HSCs). Similarly to AML cell lines, T2Rs activation with high dose of agonist induced a reduction of cell viability associated to apoptosis induction, while non-toxic doses reduced cell migration and clonogenic capacity. In addition, T2Rs stimulation with agonist makes AML cell lines more prone to oxidative and metabolic stress. Leukemia cells displayed a quiescent phenotype in response to T2Rs activation suggesting that mitochondrial activity is significantly limited by T2Rs agonist treatment. Since no data are available on the presence and the function of T2Rs on normal hematopoietic stem cell counterpart, we characterized T2Rs expression on CD34+ cell isolated from healthy donor. CD34+ cells express several T2Rs subtype without significant differences compare to AML cells. Their activation with high dose of agonist reduced HSCs viability inducing apoptosis, while non-cytotoxic doses reduced clonogenic capacity and promoted migration.Given the effect of T2Rs activation on crucial AML cell function, we tested the therapeutical potential of T2R agonist with and without conventional chemioterapic agent. Interestingly we observed that T2Rs agonist have a synergistic effect with cytarabine, reducing leukemia cell viability when combined with ARA-C compared to their use as single compound. The combination allowed to reach a high toxicity using lower doses of chemotherapic agent.Overall our results indicate that T2Rs receptor system is expressed and functional in both leukemic cells and HSCs. In particular, in AML cells T2Rs activation is associated with quiescence induction and prevention of migration. T2Rs stimulation modulates HSCs function but their role need to be further deepen. These data may suggest a role for microenvironment “bitter” molecules in regulating normal and leukemic hematopoiesis. DisclosuresCavo:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

The Methylcytosine Dioxygenase TET3 Is Aberrantly Expressed in Acute Myeloid Leukemia and Promotes AML Growth

The epigenetic regulator dioxygenase Ten-eleven translocation 3 (TET3) catalyzes the oxidation of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) and plays an important role in histone modification, thereby regulating the gene expression. Epigenetic aberrations play a key role in the pathophysiology of acute myeloid leukemia (AML), however the reasons for the highly distorted epigenome in AML are poorly known. Though in particular TET2 is widely studied in normal and malignant hematopoiesis, the role of TET3 in human myeloid leukemia is still unknown. First we quantified the expression of TET3 in normal murine and human hematopoiesis by quantitative real-time (qRT)-PCR. Among the Tet genes Tet3 showed highest expression in murine hematopoietic stem cells (HSCs) (2-fold and 3-fold compared to Tet1 and Tet2 respectively, p=0.01, p=0.006, n=3). Within the hematopoietic hierarchy highest expression of Tet3 was found in HSCs and lymphoid committed progenitors (CLPs) compared to committed myeloid progenitors (CMPs) (up to ~3.5-fold, p=0.003, n=3). In humans TET3 was higher expressed in CD34+ bone marrow (BM) cells compared to total BM cells (p=0.024, n=3) and within the lineage positive compartment the TET3 was higher expressed in CD33+ myeloid cells compared to lymphoid cells (CD19+ B cells, p=0.008 and CD3+ T cells, p=0.02, n=3). Our qRT-PCR expression data were in agreement with published microarray data demonstrating that TET3 is significantly higher expressed in myeloid cells compared to lymphoid cells (p<0.001, n=5) (Bagger FO et. al., Nucleic Acids Res. 2013). In AML patients TET3 showed a broad range of expression (ΔCT: -1.4 ± 1.4 SD, n=80), but was aberrantly expressed in majority of the AML patients compared to normal total BM (n=3) and CD33+ BM myeloid cells (n=3) (60% and 81% respectively, cut-off for higher expression: 1.5-fold SD of the median). TET3 was higher expressed in 30% of the cases in cytogenetically normal (CN)-AML (n=50) and in 95% of cases in PML-RARa+ AML (n=21) compared to total BM and CD33+ myeloid cells. The AML cell lines OCI-AML3 (NPM1 mutation+), NB4 (PML-RARa+)and KASUMI-1 (AML1-ETO+) showed higher expression of TET3 compared to other AML cell lines (EOL1, Molm 13, MONO-MAC-6 and THP-1) (p<0.001, n=3). shRNA mediated knockdown (KD) of TET3 in human AML cell lines (OCI-AML3, NB4 and KASUMI-1) adversely affected their cell growth (45%-80% reduction compared to scrambled (scr) control, p<0.001, n=3) and clonogenicity (57%-76% reduction, n=3) in vitro. Furthermore, TET3 KD significantly reduced the leukemic engraftment (median 32±17 and 5.6±6 for scr and shRNA respectively) of NB4 and OCI-AML3 in NSG mice (shRNA n=12; scr n=11, p<0.001). Histopathology showed absence or reduced infiltration of leukemic blast cells in different organs of mice transplanted with shRNA expressing AML cells. In contrast, shRNA mediated KD of TET3 in human normal CD34+ cord blood (CB) cells did not impact cell growth in vitro (n=3). TET3 depletion decreased total 5hmC level in the AML cell line NB4 and in parallel changed expression of genes involved in regulation of cell proliferation, MAP kinase activity and cell migration as determined by microarray gene expression analysis (n=3). Constitutive overexpression of wild-type TET3 in human AML cell lines significantly further enhanced their clonogenicity compared to empty vector control (p<0.05, n=3). Interestingly, overexpression of wild-type TET3 or the TET3-CDconstruct containing only the catalytic domain (CD) inhibited cell growth of human CD34+ CB cells and impaired myeloid differentiation in the CFC assays compared to empty vector (EV) (number of colonies EV=68± 5.7SD, TET3=23±1, TET3-CD=36±10, p<0.001, n=3) whereas erythroid colony formation was not affected. These results were also confirmed by flow cytometry with a decrease in the number of CD11b+CD14+ myeloid cells in the colonies (>70% reduction, n=3). In conclusion, our data indicates that ordered expression levels of TET3 are necessary for normal human HSC proliferation and differentiation. The growth and engraftment potential of AML cells are depending on high TET3 expression levels. DisclosuresAkashi:Shionogi & Co., Ltd: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Celgene: Research Funding; Astellas Pharma: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol Meyers Squibb: Research Funding; Sunitomo Dainippon Pharma: Consultancy. Buske:Celltrion, Inc.: Consultancy, Honoraria.

The Novel BET Inhibitor PLX51107 Has In Vitro and In Vivo Activity Against Acute Myeloid Leukemia

Background:Acute myeloid leukemia (AML) has very poor long-term survival with traditional therapies. AML has a diverse pathogenesis and likely represents multiple different diseases. Various epigenetic effector proteins are altered in AML by mutation, over-expression, or compartmental displacement and these changes maintain transcriptional programs important for leukemogenesis. The bromodomain and extra-terminal domain (BET) proteins, including BRD2, BRD3 and BRD4, play roles in many cellular functions important to leukemogenesis, such as super-enhancer function, transcriptional elongation, histone acetylation and cell cycle progression. In particular, AML cells depend on BRD4 for expression of the pro-survival proteins MYC and BCL2. BRD4 has therefore become an attractive target for novel therapeutics. PLX51107 is a novel BET inhibitor with a unique binding mode in the acetylated lysine binding pocket of BRD4 that differentiates it from other compounds under investigation. Our group has previously shown this compound to have antineoplastic activity in models of aggressive B cell malignancies. We have now investigated the anti-leukemic properties of PLX51107 in both in vitro and in vivo models of AML.Results:PLX51107 treatment potently reduced viability and proliferation of the human AML cell lines MV4-11, MOLM-13, OCI-AML3, and Kasumi-1, with IC50 of 0.17, 1.8, 0.2 and 0.2 μM, respectively. We then evaluated the in vitro activity of PLX51007 in primary human AML samples. PLX51107 inhibited the proliferation of primary human AML cells co-cultured with HS5 stromal cells. For nearly all samples tested (n=9), the IC50 of PLX51007 was less than 1 μM (average = 0.41 μM, range 0.039 - 1.5 μM). Notably, PLX51107 showed efficacy across a broad range of AML risk groups, including samples with adverse risk features such as 11q23 abnormalities and FLT3-ITD mutations. In comparison, for the same AML samples, the average IC50 for JQ1 was 0.71 μM (range 0.02 - 3.3 μM) and for cytarabine was 3.5 μM (range 0.33 to >10 μM). Furthermore, PLX51107 treatment reduced the clonogenicity of primary AML cells. Following incubation of AML cells in 1 μM PLX51107, there was significantly decreased colony formation (p<0.05) in drug-free, cytokine-supplemented methylcellulose media.We next examined the efficacy of PLX51107 in vivo, utilizing luciferase labeled MV4-11 AML cells xenotransplanted into NOD / SCID / IL2rgnull (NSG) immunodeficient mice. Daily oral dosing with 20 mg/kg PLX51107 resulted in prolonged survival (median 47 days) compared to vehicle treated control animals (median 30 days, p< 0.001). Weekly measurement of bioluminescence showed decreased disease burden in PLX51107 treated mice. In addition, human peripheral blood CD45 / CD33 double positive cells were significantly decreased in treated animals. Histologic analysis conducted at day 16 showed decreased leukemic burden in the bone marrow of the PLX51107 treated animals. In addition, examination of tissues from moribund mice at time of euthanasia demonstrated fewer leukemia cells in the spleen, liver and bone marrow.Conclusions:Collectively, our results show pre-clinical activity of PLX51107 in AML, supporting further development of this compound in clinical trials for relapsed or refractory myeloid malignancies. We are currently working to define downstream targets of PLX51107 action and developing patient derived AML xenografts to further characterize the in vivo effects of PLX51107. DisclosuresWalker:Gilead Sciences: Research Funding. Bhatnagar:Karyopharm: Research Funding.

Opposing Effects of PD-1/PD-L1/L2 Engagement and IFN-γ/TNF-α in the Treatment of AML w/ Anti-CD33 Chimeric Antigen Receptor-Modified T Cells

Chimeric antigen receptor-modified T lymphocytes (CART cells) have shown benefit as an adjuvant immunotherapy in the treatment of B cell malignancies. This success of re-targeted T cells has not been extended to other hematologic malignancies. We have developed an immunotherapeutic approach to treat acute myeloid leukemia (AML) using CAR T cells re-directed against the myeloid-specific antigen CD33 (CART-33). CART-33 cells are potent and specific in eliminating AML cells in vitro and in vivo. Despite this, CART-33 cells have shown poor in vivo expansion and persistence in NOD-SCID IL2rγ (-/-) (NSG) AML xenograft models. To address the reason for this, we assessed the impact of AML-expressed programmed death ligands 1 & 2 (PD-L1/2) on CART-33 cell activity. PD-L1 inhibits T cell functions upon binding PD-1, which is upregulated with T cell activation. Less is known about PD-L2’s effect. Interferon-gamma (IFN-γ), a primary effector cytokine secreted by CD4+ and CD8+ effector T cells, is a known potent inducer of PD-L1 on AML blasts. Using AML cell lines U937, Oci-AML3, CMK, and MV4-11 we show that IFN-γ, TNF-α, and activated CART-33 supernatant can induce up-regulation of PD-L1 and PD-L2 on AML. IFN-γ and TNF-α synergize strongly in up-regulating PD-1 ligands on AML. The kinetics and induction of PD-L2 are distinct from that of PD-L1. Although PD-L1 is well documented to suppress T cell function via ligation of T cell expressed PD-1, induction of PD-L1/L2 had no effect on the cytolytic activity of CART-33 cells against AML in short term (<48 h) cultures. Paradoxically, 24 hr pre-treatment of AML with either IFN-γ or CART-33 supernatant increased AML susceptibility to killing by CART-33 cells despite elevated expression of PD-L1/L2 by AML. Our results highlight the regulatory complexity of AML cytolysis by re-targeted T lymphocytes, and argue that tumor-expressed PD-L1 and PD-L2 impacts the sustainability, but not short-term killing activity, of adoptively transferred CAR T cells in the treatment of AML. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2731: Impact of hypomethylating agents on hTERT expression and synergistic effect in combination with imetelstat, a telomerase inhibitor, in AML cell lines

Abstract The catalytic subunit of human telomerase (hTERT) is highly expressed in acute myeloid leukemia (AML). Transcriptional factors and epigenetic modifications regulate this expression. Studies suggest hypermethylation promotes hTERT overexpression. Imetelstat is a competitive telomerase inhibitor. Decitabine (DAC) and azacitidine (AZA) are DNA methyltransferase inhibitors (DNMTIs) used in the treatment of AML. Combining imetelstat and DNMTI may be an improved treatment option for AML. We investigated effects of DAC and AZA on hTERT expression and cell viability in AML cell lines OCI-AML3 and OCI-AML5, and evaluated growth inhibition when combined with imetelstat. OCI-AML3 had higher baseline expression of hTERT compared with OCI-AML5. Dose-dependent downregulation of hTERT expression was observed with DAC and AZA treatments. OCI-AML5 was more sensitive to DAC (1 μM, 46% viability) than OCI-AML3 (1 μM, 95% viability) whereas sensitivity to AZA was similar in both cell lines (1 μM, 29% vs 33% viability). After removal of the drugs, growth inhibition was not sustained and cell proliferation rebounded. Single-agent imetelstat reduced both hTERT expression and telomerase activity but resulted in only limited growth inhibition in these cell lines. It was hypothesized that combining DNMTI with imetelstat may exert more potent growth inhibition. To test this, both cell lines were treated with DAC or AZA for 72 hours followed by different concentrations of imetelstat for 2 to 4 weeks. Dose-dependent synergistic activity was demonstrated for DAC followed by imetelstat: OCI-AML3 cell viability changed from 95% with DAC to 61% and 10% after 2 and 4 weeks of imetelstat, respectively; OCI-AML5 cell viability changed from 46% with DAC to 6% and 1% after 2 and 4 weeks of imetelstat, respectively. In contrast, no enhanced activity was observed in either cell line with AZA followed by imetelstat, but imetelstat was able to slow the growth rebound after AZA removal. Additional studies are ongoing to further understand the mechanism of action of this drug combination in AML and to evaluate different combination sequences of DNMTI with imetelstat for optimal anticancer activity. In summary, DAC and AZA reduced hTERT expression and differentially inhibited cell viability in AML cell lines. Although at the same concentration AZA was more active than DAC in OCI-AML3 and OCI-AML5 lines, the sustainability of growth inhibition after washout differed. Growth rebounded after removal of AZA or DAC treatment. Dose-dependent synergistic activity demonstrated by DAC followed by imetelstat was not seen with AZA followed by imetelstat. Distinctly different mechanisms of action of DAC and AZA may underlie their differential activity when combined with imetelstat, warranting further investigation. Citation Format: Joshua Rusbuldt, Jacqueline Bussolari, Aleksandra Rizo, Fei Huang. Impact of hypomethylating agents on hTERT expression and synergistic effect in combination with imetelstat, a telomerase inhibitor, in AML cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2731.

Targeting Galectin As A Strategy To Improve BH3 Mimetic Efficacy For The Therapy Of Acute Myeloid Leukemia

Background: Galectins are a family of glycan binding proteins that regulate cell survival by mechanisms including apoptosis, cell cycle, and mRNA processing. Galectin-3 (LGALS3) is unique among galectins by having an N terminal region of roughly 130 amino acids that allows for multimerization and binding to other proteins independent of carbohydrate binding. In addition to promoting BCL2 gene expression and mitochondrial integrity, LGALS3 (along with LGALS1) positively regulates RAS signaling and thus the stability of proteins dependent on ERK phosphorylation such as MCL-1. The pro-survival functions of LGALS3 and other galectins suggest that their targeting could be therapeutic for cancers including AML. Indeed, galectin 3 (LGALS3) expression is a predictor of poor prognosis in acute myeloid leukemia (AML), as reported by Cheng and colleagues (Blood 2013). Current strategies to eradicate leukemia cells involve use of agents targeting BCL2 family members such a BH3 mimetics ABT-7373 and ABT-199. MCL-1 has been implicated as a resistance factor against BH3 mimetic killing and unfortunately an effective anti-MCL-1 drug is currently not available for use in the clinic. Alternatively, a strategy to target MCL-1 indirectly by suppressing Galectin 3 could improve BH3 mimetic killing of leukemia cells. In the current study we examine the effect of Galectin 3 suppression by shRNA or with a broad Galectin inhibitor on BH3 mimetic induced apoptosis in AML cell lines OCI-AML3 and THP1. Study Design: GCS-100 (kind gift from George Tidmarsh; La Jolla Pharmaceutical, San Diego, CA), is now in a Phase II clinical trial for chronic kidney disease. The drug was used in combination with BH3 mimetic ABT-737 (which targets BCL2 and BCL-XL at low dose). Galectin 3 was suppressed in both AML cell lines using lentiviral shRNA. Effect of GCS-100/ABT-737 combination was tested in an in vitro model of the tumor microenvironment using mesenchymal stem cells (MSC). Cell growth and induction of apoptosis was measured by flow cytometry. Analysis of protein expression and signaling was assessed by immunoblot analysis. Level of gene expression of various BCL2 family members was measured by qRT-PCR. Results: As a single agent GCS-100 suppresses the growth of AML cell lines OCI-AML3, THP-1, and HL60. The drug potently suppresses phosphorylation of AKT and ERK and reduces protein expression of BCL2 and MCL-1. We found that GCS-100 potently synergized with ABT-737 to kill OCI-AML3 cells: while 1 uM ABT-737 or 125 ug/mL GCS-100 reduced total viable cells by w 30% and induced apoptosis in 80% and > 60% reduction of viable cells, respectively, despite the presence of MSC when treated with 250 ug/mL GCS-100 for 72 hours. In addition, GCS-100 was found to block adhesion of OCI-AML3 cells to MSC suggesting that GCS-100 could be effective preventing leukemia cell contact with MSC. Conclusions: Our findings suggest that GCS-100 can induce apoptosis in AML cells as a single agent or in combination with the BH3 mimetic ABT-737. The agent is effective even in the presence of MSC suggesting it could be efficacious in the leukemia niche. These findings suggest GCS-100 could be effective for AML therapy.

GCS-100 Induces Apoptosis of Acute Myeloid Leukemia Cells By Disrupting Galectin-Mediated Survival Signaling

Galectins are a family of b-galactoside binding proteins with effects on cell adhesion, apoptosis, cell cycle, and mRNA processing. Galectin-3 (LGALS3) is unique among galectins by having an N terminal region of roughly 130 amino acids that allows for multimerization and binding to other proteins independent of carbohydrate binding. In addition to promoting BCL2 gene expression and mitochondrial integrity, LGALS3 (along with LGALS1) positively regulates RAS signaling and thus stabilizes survival proteins dependent on ERK phosphorylation such as MCL-1. The pro-survival functions of LGALS3 and other galectins suggest that their targeting could be therapeutic for cancers including AML. Indeed, LGALS3 expression is a predictor of poor prognosis in acute myeloid leukemia (AML), as reported by Cheng and colleagues (Blood 2013) for patients with non-M3 AML and CN-AML. The modified pectin GCS-100 (La Jolla Pharmaceutical, San Diego, CA), now in a Phase II clinical trial for chronic kidney disease, binds and blocks the function of LGALS3. We report that GCS-100 suppresses the growth of AML cell lines OCI-AML3, THP-1, and HL60 in vitro as a single agent, at doses under the 250 ug/mL (i.e., within clinically-achievable concentrations). Short-term treatment of cells (i.e., < 6 hr) potently suppressed phosphorylation of AKT and ERK and reduced expression of BCL2 and MCL-1. Because LGALS3 positively regulates anti-apoptotic BCL2 family members, the Raz group has suggested targeting galectins to enhance efficacy of BH3 mimetic drugs (Harazano et al Cancer Metastasis Review 2013). We found that GCS-100 potently synergized with ABT-737 to kill OCI-AML3 cells: while 1 uM ABT-737 or 125 ug/mL GCS-100 reduced total viable cells by ~ 30% and induced apoptosis in < 20% of cells after 48 hr as single agents, their combination at those doses and time point reduced viable cells by ~ 94% and induced apoptosis in ~ 70% of cells. Suppression of LGALS3 by lentiviral shRNA reduced BCL2 gene expression as determined by qRT-PCR and augmented killing with ABT-737. Lentiviral suppression of LGALS3 protected cells from GCS-100 at doses of 250 ug/mL but reduction of the galectin failed to protect cells from higher doses of the drug (i.e., 500 ug/mL). This result suggests other galectins are likely inhibited at higher doses of the agent. We used gene expression profiling (GEP) on Illumina HT12v4 human whole-genome arrays to assess more broadly the molecular effects of inhibiting galectins in AML cell lines OCI-AML3 and THP-1 treated with 250 ug/mL or 500 ug/ml GCS-100 for 24 hr. Data were analyzed by Gene Set Enrichment Analysis (GSEA) using gene sets from the Molecular Signatures Database (www.broadinstitute.org/gsea/msigdb/). GSEA suggested that GCS-100 promotes differentiation and inhibits genes associated with proliferation. Multiple upregulated gene sets suggest that there may be a release of a differentiation block as a result of GCS-100 treatment. Furthermore, two gene sets suggest that GCS-100 behaves similar to a GSK3 inhibitor: Known pathways regulated by GSK3 in hematopoietic stem cells are mTOR and Wnt/beta Catenin. Inhibition of Wnt/beta Catenin can release a differentiation block. Consistent with GCS-100 promoting cell differentiation, lentiviral shRNA reduced LGALS3 protein > 90% in THP-1 cells and increased CD11b expression, suggesting increased differentiation, compared to cells with control shRNA. GCS-100 was tested in an in vitro model of the bone marrow microenvironment using BM-derived mesenchymal stromal cell (MSC). MSC can protect leukemia cells from a variety of clinically relevant chemotherapy drugs including AraC. GCS-100 was effective at killing AML cells despite the presence of MSC. Both THP-1 and OCI-AML3 cells exhibited > 80% and > 60% reduction of viable cells, respectively, despite the presence of MSC when treated with 250 ug/mL GCS-100 for 72 hours. In addition, GCS-100 was found to block adhesion of OCI-AML3 cells to MSC suggesting that GCS-100 could be effective in mobilizing AML cells. In summary, our findings suggest that GCS-100 can induce apoptosis in AML cells as a single agent or in combination with the BH3 mimetic ABT-737. The agent is effective even in the presence of MSC suggesting it could be efficacious in the leukemia niche. These findings suggest GCS-100 could be effective for AML therapy. DisclosuresRolke:La Jolla Pharmaceutical Company: Employment. Tidmarsh:La Jolla Pharmaceutical Company: Employment.

Pro-Survival Effects of TGF-β1 Are Associated with Molecular Signaling Changes of ERK, FLI-1, and CD44 in AML Cells

Transforming growth factor β (TGF-β) is a key regulator of cell growth and survival. We have previously reported the pro-survival role of TGF-β1 and the anti-leukemic efficacy of TGF-β neutralizing antibody in the bone marrow (BM) microenvironment (Xu et al., Br J Haematol.2008, Tabe et al. PLoS One. 2013). FLI-1 is a transcription factor that plays a pivotal role in the regulation of extracellular matrix genes under negative regulation by TGF-β1–dependent acetylation. We reported that patients with acute myeloid leukemia (AML) expressing a low level of FLI-1 achieved high rates of remission, but their remission duration and survival were curtailed (Kornblau et al., Blood, 2011). Moreover, treatment with rhTGF-β1 induced expression of matrix metalloproteinase-1 (MMP-1) mRNA in AML cells, which inversely correlated with FLI-1 expression (Tabe et al. ASH 2012), concordant with a previous report that FLI-1 downregulates the expression of MMP-1 promoter activity.In this study, we investigated the molecular mechanisms by which TGF-β induces AML cell survival. In AML cell lines NB4, U937, and OCI-AML3, rhTGF-β1 (2ng/mL) induced the TGF-β downstream targets plasminogen activator inhibitor-1 (PAI-1, mRNA) and Smad2 phosphorylation (p-Smad2). These effects were reversed by anti–TGF-β neutralizing antibody 1D11 but not by the isotype control antibody 13C4. Treatment with rhTGF-β1 significantly inhibited serum starvation–induced apoptosis and promoted G0/G1 cell cycle arrest in NB4 and U937 cells but not in OCI-AML3 cells (subG1% vs controls at 72 h: NB4, 18.9 ± 1.7 vs 12.8 ± 1.3; U937, 51.9 ± 2.8 vs 29.1 ± 2.4; OCI-AML3, 8.8 ± 0.5 vs 7.7 ± 0.5; G0/G1 % vs controls at 72 h:,NB4, 39.2 ± 3.5 vs 55.6 ± 5.6; U937, 16.0 ± 1.8 vs 37.6 ± 2.1; OCI-AML3, 68.0 ± 4.2 vs 55.1 ± 9.9). These changes were effectively reversed by 1D11, indicating that rhTGF-β1 facilitated pro-survival effects along with transition into a quiescent G0 state in NB4 and U937 cells but not in OCI-AML3 cells.To investigate the differences in TGF-β downstream signaling activation in these cells, we focused on ERK phosphorylation and FLI-1 expression. TGF-β is known to activate ERK downstream of the Ras pathway, which cross-talks with Smad signaling and associates with FLI-1 activation. In U937 and NB4 cells, after rhTGF-β1 treatment, ERK phosphorylation was upregulated in the early phase (0.5-3 h) and then downregulated (24 h), and FLI-1 expression was downregulated (mRNA and protein, 24 h). Protein phosphatase 2A (PP2A), a modulator of TGF-β and a negative regulator of p-ERK, was downregulated by rhTGF-β1. ERK is known to participate in multiple feedback loops that include its upstream regulator Ras, which is important for the reduction of ERK activity at a late stage after stimulation. Indeed, p-Raf, a direct upstream regulator of p-ERK, demonstrated concordant expression changes with p-ERK after rhTGF-β1 treatment. No changes in p-ERK and FLI-1 expression were detected in OCI-AML3 cells. rhTGF-β1 induced significant upregulation of anti-apoptotic Bcl-2 in NB4 and U937 but not in OCI-AML3 cells.We next investigated the rhTGF-β1–induced expression of CD44 and its variant CD44v. CD44 is recognized as a marker of cancer stem cells, and CD44v regulates redox status in stem-like cancer cells. In several types of cancer cells, CD44 is known to mediate cell migration via interaction with TGF-β type I receptor and close association with ERK signaling. Treatment with rhTGF-β1 increased both total and variant CD44 mRNA in NB4 cells but caused no change in OCI-AML cells (fold change of CD44/CD44v mRNA compared to controls: NB4, 3.0/3.7; OCI-AML3, 1.2/0.9). These changes were inversely correlated with FLI-1 expression in NB4 cells.Taken together, these results show that TGF-β–induced ERK activation and its subsequent negative feedback on the Ras/Raf pathway are associated with FLI-1 downregulation and other responses, including CD44 upregulation, in AML in a cell-dependent manner (Figure 1). These molecular changes might contribute to maintenance of the stem cell–like properties, support survival of AML cells, and play pathologic roles in the TGF-β–abundant BM microenvironment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

DOT1L As a Therapeutic Target for the Treatment of DNMT3A-Mutant Acute Myeloid Leukemia

Mutations of the DNA methyltransferase, DNMT3A, occur in approximately 20% of adult patients with acute myeloid leukemia (AML), and portend a poor prognosis. The most common of these mutations results in a dominant negative loss of function. Our lab observed that upon conditional inactivation of Dnmt3a in the murine hematopoietic system, Dnmt3a–/– hematopoietic stem cells (HSCs) expanded dramatically while their differentiation was inhibited, consistent with a pre-leukemic state. The likely mechanism by which Dnmt3a loss contributes to leukemogenesis is altered DNA methylation and the attendant gene expression changes, however our current understanding is incomplete.In analyses of gene expression data, we observed that murine Dnmt3a–/– HSCs markedly overexpress the histone 3, lysine 79 (H3K79) methyltransferase, Dot1l. This is of interest given the known functional interplay between DNA methylation and histone modifications. Additionally, DOT1L plays a critical role in leukemia with MLL-rearrangements, lesions that essentially never occur concomitantly with DNMT3A mutations in AML. The mutual exclusion of these lesions combined with the observed overexpression of Dot1l in our murine model, led us to postulate that MLL-rearrangements and DNMT3A mutations are distinct epigenetic aberrations that converge on a common mechanism resulting in dysregulated gene expression, specifically mediated by H3K79 methylation (H3K79me). Therefore, in the pathogenesis of DNMT3A-mutant AML, like in MLL-rearranged leukemia, DOT1L-induced H3K79me may play a central role, and may represent a viable therapeutic target.Throughout the genome of normal HSCs, expansive regions with low DNA methylation (canyons) exist. These canyons span conserved domains frequently containing transcription factors. In our Dnmt3a-/- model, canyon borders, particularly flanking genes frequently dysregulated in human leukemia such as HOX genes, are highly prone to DNA methylation loss when Dnmt3a is deleted, resulting in canyon expansion. However, not all canyons expand with Dnmt3a loss. We found a close association between canyon behavior and the associated histone marks, with expanding canyons characterized by a lack of the repressive histone mark, H3K27me. To determine if in Dnmt3a-mediated malignant hematopoiesis, H3K79me also correlates with altered DNA methylation, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) for H3K79 di-methylation (H3K79me2) and aligned these data with whole genome DNA methylation data. This revealed that H3K79me2 specifically coats canyons that lose methylation with Dnmt3a loss, including the HoxA and HoxB clusters, but is not present at canyons without methylation loss. This strong correlation between H3K79me and DNA hypomethylation with Dnmt3a loss suggests a functional interaction. To examine whether this also occurred in human samples with DNMT3A mutations, we analyzed TCGA data, which confirmed many canyon borders as regions with marked DNA methylation loss. Further, many canyon-associated genes, including HOX genes are significantly changed in human DNMT3A-mutant AML.To explore the role of H3K79me, and specifically of DOT1L in human DNMT3A-mutant AML, we utilized the DNMT3A-mutant human AML cell lines OCIAML2 and OCIAML3. These cell lines were found to have increased total H3K79me compared to DNMT3A-wild type controls, consistent with the increased Dot1l expression in Dnmt3a–/– HSCs. We then tested the in vitro efficacy of two selective DOT1L inhibitors, SYC-522 (Anglin. J Med Chem. 2011) and the Epizyme compound, EPZ004777 (Daigle. Cancer Cell. 2011), against DNMT3A-mutant cells. Both compounds led to a dose- and time-dependent inhibition of proliferation and induction of apoptosis in the DNMT3A-mutant cell lines at concentrations comparable to those used for MLL-rearranged cell lines. With treatment, DNMT3A-mutant cells also had evidence of induction of differentiation with increased expression of the mature monocyte marker, CD14. Importantly, oncogenic HOX genes overexpressed in DNMT3A-mutant AML were repressed in a time-dependent fashion with DOT1L inhibitor treatment.In conclusion, our data suggest that DOT1L may be a novel, immediately actionable therapeutic target for the treatment of DNMT3A-mutant AML. DisclosuresRau:Epizyme: Honoraria.

Activities and Mechanism Based Combinations Of Omacetaxine In Acute Myeloid Leukemia

Omacetaxine, an inhibitor of translation, was recently granted accelerated approval for the treatment of chronic myeloid leukemia (CML). Omacetaxine blocks translation elongation by competing with the incoming aminoacyl-tRNAs for binding to the A-site cleft in the peptidyl-transferase center. Our previous studies showed that by transiently inhibiting translation, omacetaxine reduced the expression of the key, short-lived oncoproteins Bcr/Abl and Mcl-1, leading to cell death in the CML cells. This action sensitized the cells to the Abl kinase inhibitor and killed the CML cells synergistically. Further, as omacetaxine acts in a different mechanism than the Abl kinase inhibitors, it overcame resistance to TKI that was associated with kinase domain mutations. These studies paved the foundation for the clinical development of omacetaxine in CML. We also demonstrated that omacetaxine was active in chronic lymphocytic leukemia by translational inhibition of Mcl-1 expression. In contrast to normal tissues, the fact that the leukemia cells are critically dependent on the oncogene activity for survival provided a biologic context for a positive therapeutic index. As the biological features of acute myeloid leukemia (AML) rely largely on the overexpressed oncoproteins or constitutively activated kinases, we hypothesized that omacetaxine would have therapeutic benefit in AML either alone or in mechanism based combinations.To test this hypothesis, first, we compared omacetaxine to AC220, a potent FLT3 inhibitor, in AML cell lines OCI-AML3 and MV4-11. OCI-AML3 cells harbor the signature mutation of NPM1, whereas MV4-11 is a widely used model for the internal tandem duplications of FLT3 (FLT3-ITD), a common FLT3 mutation that constitutively activates the receptor tyrosine kinase. AC220 was selectively toxic to the MV4-11 cells, but had no effect on the viability of OCI-AML3. This is consistent with the biological context of MV4-11, but not OCI-AML3, that is addicted to the sustained activity of FLT3 for survival. In contrast, omacetaxine induced apoptosis in both cell lines with IC50s less than 100 nM. Protein synthesis was inhibited in both lines, measured by the incorporation of tritiated leucine. Apoptosis was induced rapidly within 24 h by omacetaxine, whereas AC220 required 72 h to kill the leukemia cells. These results indicated a common dependence on the continued protein synthesis in the AML lines, suggesting a potentially broad application of omacetaxine in AML patients with diverse genetic backgrounds. Over-expression of the anti-apoptotic protein Mcl-1 is associated with AML disease maintenance and resistant to therapy. Both Mcl-1 and FLT3 turn-over rapidly and are vulnerable targets of transient translation inhibition. Immunoblots showed that omacetaxine reduced the levels of both FLT3 and Mcl-1 in the MV4-11 cells. This activity augmented the effect of AC220 on FLT3 kinase, and induced synergistic apoptosis. Same synergistic combination was observed with omacetaxine and sunitinib, an inhibitor of FLT3, KIT and PDGF-R. Dose reduction index derived from these analyses showed that omacetaxine greatly potentiated the activity of both AC220 and sunitinib, resulting in profound apoptosis.Both Bcl-2 and Mcl-1 are pro-survival proteins that regulate apoptosis by interacting with the BH3 motifs of their pro-apoptotic partners. BH3 mimetics, such as ABT-199, bind with high affinity to Bcl-2 and block this interaction, but not to Mcl-1. Resistance to BH3 mimetics in AML cells is associated with upregulation of Mcl-1. Since ABT-199 inhibits Bcl-2 but spares Mcl-1, and omacetaxine reduces Mcl-1 without affecting Bcl-2 expression, we hypothesized that their combination would target the two parallel arms of apoptosis control and kill the AML cells synergistically. Indeed, omacetaxine reduced Mcl-1 in the OCI-AML3 cells, leading to loss of mitochondrial membrane potential and apoptosis. ABT-199 blocked Bcl-2 function and also induced the intrinsic pathway of apoptosis. Their combination induced greater mitochondrial damage and apoptosis than either drug alone. The median effect analysis showed that they potentiate each other and exhibited strong synergy. Taken together, these results demonstrated that omacetaxine is active in AML cells alone and in mechanism based combinations. These actions provide rationale that warrants investigation in the clinic. Disclosures:No relevant conflicts of interest to declare.

The Synergistic Effect of DNA Methylation Inhibitor 5-Azacytidine and Bcl-2 Signaling Blockade ABT-737 Combination Treatment Induce Apoptosis In AML

AbstractAbstract 3311Bcl-2 family proteins are key regulators of apoptosis. Aberrations in Bcl2 levels are known to promote tumorigenesis and chemoresistance. Thus strategies to target Bcl2 will likely provide effective therapies for malignancies such as acute myeloid leukemia (AML). ABT-737 is a small molecule BH3 mimetic that binds tightly to a hydrophobic cleft on Bcl-2 and Bcl-XL and exerts its proapoptotic function by preventing antiapoptotic Bcl-2 family members from sequestering activating BH3 proteins (Oltersdorf et al., Nature 2005). We have reported that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induces the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway (Konopleva et al., Cancer Cell 2006). The ABT-737 related clinical compound, ABT-263, is undergoing Phase I/II studies in chronic lymphocytic leukemia, with initial signs of clinical activity. However, the main side effect is thrombocytopenia resulting from inhibition of Bcl-XL. Hence, combinations of ABT-737 with non-cytotoxic agents are desirable to take full advantage of ABT236's unique spectrum of biophysical and preclinical activities.In this project, we studied pharmacologic interactions between ABT-737 and the DNA methyltransferase inhibitor 5-azacytidine (5-azaC). 5-azaC is a cytidine analog with clinical activity in myelodysplastic syndromes (MDS) and in AML. Since recent studies indicate that 5-azaC induce DNA damage in p53-dependent fashion, we tested the hypothesis that the pro-apoptotic effects of 5-azaC/ABT-737 combination are related to non-redundant activation of BH3-only proteins and mitochondrial apoptosis in AML cells with wt p53.In vitro, 5-azaC and ABT-737 in combination for 72 hrs induced growth inhibition and apoptosis in AML cell lines OCI-AML3, MOLM-13 and U937 in a highly synergistic, dose-dependent fashion, with combination indices (CIs) ranging from 0.1 to 0.22. These effects were observed even at low concentrations (5-azaC 100nM and ABT-737 10nM, at 10:1 ratio). In contrast, no synergistic apoptosis was seen in p53-null HL-60 cells. Likewise, ABT-737/5-azaC induced apoptosis in a synergistic fashion in OCI-AML3 cells infected with vector control shRNA (CI=0.04) but failed to induce cell death in OCI-AML3 p53 shRNA cells, indicating critical p53-dependent mechanisms of cell death. In primary AML samples sensitive to ABT-737 alone (n=3), synergistic and additive effects were seen. The combined effects of 5-azaC and ABT-737 were further investigated in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice injected with cells from a patient with primary refractory AML. Seven days after leukemia transplantation, mice were treated with vehicle, ABT-737 (intraperitoneally (IP), 75mg/kg/day for 10 days), 5-azacytidine (IP, 4mg/kg/day for 5 days) or with the combination. Engraftment of patient leukemia cells was analyzed by the immunohistochemical detection of CD45-positive cells in bone marrow and spleen seven weeks after transplantation. Both, ABT-737 and 5azaC each exerted anti-leukemic effects, as evidenced by significant reduction in leukemia cells in bone marrow and spleen (10% and 3% CD45+ cells detectable in 1/3 mice in ABT-737 and 5-azaC groups, respectively), no CD45+ AML cells were detected in organs of 3/3 mice treated with the combination. No overt hemorrhage was detected in the animals.In summary, the combination of 5-azaC and ABT-737 induces synergistic cells death in AML cell lines and in a subset of primary AML samples in a p53-dependent fashion. The mechanisms of this pharmacologic interactions including the p53-dependent upregulation of BH3-only proteins, described by us for ABT-737/MDM2 inhibitor combinations, are currently under investigation. Results suggest that this therapeutic strategy can be successfully utilized in AML patients with low mutation rate and unimpaired signaling of p53. Disclosures:No relevant conflicts of interest to declare.