The Combined Treatment with the FLT3-Inhibitor AC220 and the Complex I Inhibitor Iacs-010759 Synergistically Depletes Wt- and FLT3-Mutated Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) cells are highly dependent on mitochondrial function for survival 1. We have recently reported a novel oxidative phosphorylation (OXPHOS) inhibitor IACS-010759 that potently inhibits mitochondrial complex I, suppresses OXPHOS and selectively inhibits the growth of AML cells in vitro and in vivo2. In this study, we aimed to identify chemotherapeutic agents that synergistically deplete AML cells when administered in combination with IACS-010759. We performed a high-throughput screening of a drug library (289 anti-cancer compounds) administered either individually or in combination with IACS-010759 on two leukemia cell lines (OCI-AML3, MOLM-13) and three bone marrow stromal cell lines (HS-5, HS-27A, MSC) in both hypoxia (1% O2) and normoxia conditions. Based on the cell viability datasets, we selected top candidates for combinations based on the following criteria: either bliss index > 0.1 (synergy of the combination treatment; red in Fig. 1A&B), or high cytotoxicity to leukemia cells (relative cell viability < 0.5, blue in Fig. 1A&B), as well as low toxicity against normal cells (relative cell viability in normal cells > 0.8, yellow in Fig. 1A&B). Twenty-four compounds satisfied the selection criteria above, either in normoxia or hypoxia, or both. Out of the 24 compounds, 5 agents (Fig. 1C) are known FLT3 (FMS-like tyrosine kinase 3) inhibitors, including AC220 (quizartinib), dovitinib, nintedanib, SGI-1776, and rebastinib, pointing to a molecular target of great potential interest in the design of synergistic drug combinations with IACS-010759. Thus, we investigated more in-depth the synergism between IACS-010759 (10nM) and 13 FLT3 inhibitors, all currently in clinical trials (AC220, sorafenib, gilteritinib, sunitinib, ponatinib, midostaurin, ibrutinib, TP-0903, crenolanib, tandutinib, FF-10101, lestaurtinib, and KW-2449; 0.0128:5x:5000nM), in AML cell lines (FLT3-wt KG-1, U937, OCI-AML2, OCI-AML3; and FLT3-mutant MOLM-13 and MOLM-14). Among the 13 FLT3 inhibitors, only AC220 combined with IACS-010759 showed concentration windows with bliss index higher than 0.1 across different lines. Next, we further characterized the synergism between AC220 and IACS-010759 in AML cell lines (U937 and OCI-AML3) under hypoxic conditions using metabolic flux analysis (MFA) to trace the incorporation of 13C5,15N2-glutamine and 1,2-13C2-glucose and study the metabolic modulation associated with the synergy. Leukemia cells were incubated with unlabeled/labeled medium for 24h and concurrently treated with 5nM IACS-010759 and/or 500nM AC220. While both individual agents modulate glutamine consumption and TCA cycle dynamics, by far the most dramatic metabolic effects on TCA cycle intermediates are observed following administration of the combined treatment. Severe drops in the levels of TCA cycle metabolites, (Fig. 1D) point to a reduced mitochondrial activity following the combined treatment, which is also validated by the increased ratio of oxidized/reduced forms of nicotinamide adenine dinucleotide (NAD/NADH). Interestingly, the total pool of the oncometabolite 2-hydroxyglutarate, while increasing following the individual treatments, significantly dropped to very low levels in response to the combined treatment. The significantly reduced metabolite levels as well as the glucose-derived enrichment fractions of glucose 6-phosphate, fructose bisphosphate, phosphoenolpyruvate and ribose 5-phosphate in the AC220-containing treatment groups (significantly more pronounced in the combined treatment) point to impaired glycolysis /pentose phosphate pathway (Fig. 1E). In turn this results in lower de novo nucleotide biosynthesis (based on the decreased glutamine and glucose incorporation). Similar results were observed in OCI-AML3 cells. Overall, the combinatorial treatment with IACS-010759 and AC220 impaired AML cell metabolism tremendously and to a much greater extent than any of the individual treatments alone. Influx inhibition of both the two main carbon sources, glucose and glutamine, was observed leading to impairment of the TCA cycle and glycolysis for energy production, as well as pentose phosphate pathway and de novo nucleotide biosynthesis. In conclusion, we identified a novel drug combination AC220 and IACS-010759 which synergistically inhibits AML cell growth regardless of FLT3 mutation at least by metabolism disruption. Disclosures Konopleva: Kisoji: Consultancy; Agios: Research Funding; Amgen: Consultancy; Cellectis: Research Funding; Eli Lilly: Research Funding; Rafael Pharmaceutical: Research Funding; Ablynx: Research Funding; Sanofi: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; AbbVie: Consultancy, Research Funding; AstraZeneca: Research Funding; Calithera: Research Funding; Forty-Seven: Consultancy, Research Funding; Ascentage: Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding.

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Targeting Glutamine Metabolism Overcomes Resistance to Targeted Therapies in Refractory Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2020-140736 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 25-26

Author(s):

Lingzhi Li ◽

Changying Jiang ◽

Lucy Jayne Navsaria ◽

Yang Liu ◽

Angela Leeming ◽

...

Keyword(s):

Cell Viability ◽

B Cell ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

Tca Cycle ◽

Glutamine Metabolism ◽

Metabolomic Profiling ◽

The Tca Cycle ◽

Mantle Cell

Background: Mantle cell lymphoma (MCL) is an incurable B cell non-Hodgkin's lymphoma characterized by high refractory occurrence following drug treatment. Despite the encouraging initial MCL tumor response to ibrutinib (IBN), relapse occurs only after few months of treatment due to multiple resistance mechanisms. Thus, the novel therapeutic strategies targeting resistant mechanisms are crucial. Our group has recently shown that among the highly proliferative MCL population, a subpopulation of IBN-R cells exhibits increased OXPHOS activity that is fueled by increased glutaminolysis and rely more on mitochondrial respiration for their grow and survival. The aim of this work was to uncover potential targets responsible for the upregulation of OXPHOS pathway in the refractory/relapsed (R/R) MCL by using multiple biochemical and biological strategies. We focused the present study on glutaminase (GLS), the enzyme that converts glutamine to glutamate, a precursor of α-ketoglutarate (α-KG) that links glutamate to the TCA cycle. Incorporation of α-KG into the TCA cycle is a major anaplerotic step in proliferating cells and is critical for the maintenance of TCA cycle function. To further demonstrate the reliance of OXPHOS on glutamine anaplerosis, we have further tested the combinatory effects of targeting GLS and OXPHOS using their respective inhibitors, CB-839 and IACS-010759, on tumor killing activity in R/R MCL. Methods:Primary MCL cells from patient leukapheresis or whole blood specimens, as well as established MCL cell lines were used as experimental models of MCL. Metabolomic profiling was used to determine intracellular metabolite fluxes and levels. Cell Titer Glo assay was used to measure cell proliferation/viability after treatment with inhibitors. Annexin V and propidium iodide were used to measure cell apoptosis and cell cycle arrestviaflow cytometry analysis. Magnetic microbeads-based B-cell isolation method were used for the purification of malignant B cells from patient samples. Western blot analysis was used to evaluate protein level expression. Patient-derived Xenograft (PDX) mouse model created from patients with MCL was used to evaluate the in vivo anti-tumor activity and potential clinical value of GLS and OXPHOS inhibitors. Results:Our recent metabolomic profiling studies have demonstrated that glutaminolysis and OXPHOS are upregulated in IBN-R MCL, manifested by increased glutamine uptake in the ibrutinib-resistant MCL cell lines (p=0.03).Inhibition of glutamine metabolism with the allosteric GLS1-selective inhibitor BPTES resulted in inhibition of cell viability (0.2381uM-9.98uM), indicating that MCL cells are dependent on glutamine metabolism for their proliferation. To corroborate with the above finding, we also presented evidence that GLS1 is highly increased in IBN-R and CART-R MCL patient samples and cell lines confirmed by immunoblotting. Inhibiting of GLS would lead to significant reduction in OXPHOS, mitochondria membrane potential and ATP production, as either single drug or in combination with other targeting agents. To identify a clinical actionable GLS inhibitor for the treatment of MCL, we chose a GLS1 specific inhibitor CB-839 (Selleckchem), currently under several phase II and III clinical trials investigation on solid tumors. Inhibiting GLS1 with CB-839 leads to the decreased cell viability in MCL (0.5626nM-308.4nM). Of note, the treatment with CB-839 to MCL cell lines induces S phase reduction in both Jeko-1 (17.23%) and Z-138 (14.01%), as well as induces significant apoptosis (p=0.013 and p=0.002 in Jeko-1 and Z-138 cells). GLS inhibition will be further explored in the context of mitochondria defect or hypoxia, where OXPHOS maybe deficient. Importantly, while CB-839 is continuing its validation in several solid tumor models, this is the first study providing data on its efficacy in preclinical models of MCL. Conclusion:In conclusion, we report that glutaminolysis and OXPHOS are upregulated in IBN-R MCL that could be partially due to high expression of GLS1. Our preliminary results revealed that the new GLS inhibitor, GCB-839, may present a clinical potential for a new indication and warrants more in-depth investigation. Deciphering the mechanisms involved in MCL metabolic heterogeneity and adaptability during drug resistance development would be crucial to identify key actors enabling MCL cells to escape from therapy. Disclosures Wang: Acerta Pharma:Research Funding;Molecular Templates:Research Funding;InnoCare:Consultancy;Oncternal:Consultancy, Research Funding;Celgene:Consultancy, Other: Travel, accommodation, expenses, Research Funding;Targeted Oncology:Honoraria;MoreHealth:Consultancy;Kite Pharma:Consultancy, Other: Travel, accommodation, expenses, Research Funding;Lu Daopei Medical Group:Honoraria;OMI:Honoraria, Other: Travel, accommodation, expenses;Verastem:Research Funding;Nobel Insights:Consultancy;BioInvent:Research Funding;Guidepoint Global:Consultancy;AstraZeneca:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;Pharmacyclics:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;Janssen:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;Juno:Consultancy, Research Funding;Dava Oncology:Honoraria;Loxo Oncology:Consultancy, Research Funding;Pulse Biosciences:Consultancy;OncLive:Honoraria;Beijing Medical Award Foundation:Honoraria;VelosBio:Research Funding.

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Differential Effects of Combined ATR/WEE1 Inhibition in Cancer Cells

Cancers ◽

10.3390/cancers13153790 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3790

Author(s):

Gro Elise Rødland ◽

Sissel Hauge ◽

Grete Hasvold ◽

Lilli T. E. Bay ◽

Tine T. H. Raabe ◽

...

Keyword(s):

Lung Cancer ◽

Cell Viability ◽

Cancer Treatment ◽

Cancer Cells ◽

Cell Lines ◽

Cancer Cell ◽

Combined Treatment ◽

Cancer Cell Lines ◽

Variable Extent ◽

Synergistic Induction

Inhibitors of WEE1 and ATR kinases are considered promising for cancer treatment, either as monotherapy or in combination with chemo- or radiotherapy. Here, we addressed whether simultaneous inhibition of WEE1 and ATR might be advantageous. Effects of the WEE1 inhibitor MK1775 and ATR inhibitor VE822 were investigated in U2OS osteosarcoma cells and in four lung cancer cell lines, H460, A549, H1975, and SW900, with different sensitivities to the WEE1 inhibitor. Despite the differences in cytotoxic effects, the WEE1 inhibitor reduced the inhibitory phosphorylation of CDK, leading to increased CDK activity accompanied by ATR activation in all cell lines. However, combining ATR inhibition with WEE1 inhibition could not fully compensate for cell resistance to the WEE1 inhibitor and reduced cell viability to a variable extent. The decreased cell viability upon the combined treatment correlated with a synergistic induction of DNA damage in S-phase in U2OS cells but not in the lung cancer cells. Moreover, less synergy was found between ATR and WEE1 inhibitors upon co-treatment with radiation, suggesting that single inhibitors may be preferable together with radiotherapy. Altogether, our results support that combining WEE1 and ATR inhibitors may be beneficial for cancer treatment in some cases, but also highlight that the effects vary between cancer cell lines.

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P04.20 The role of YAP in Glioblastoma cell lines

Neuro-Oncology ◽

10.1093/neuonc/noab180.074 ◽

2021 ◽

Vol 23 (Supplement_2) ◽

pp. ii22-ii23

Author(s):

G Casati ◽

L Giunti ◽

A Iorio ◽

A Marturano ◽

I Sardi

Keyword(s):

Cell Viability ◽

Cell Lines ◽

Western Blotting ◽

Cytotoxic Effect ◽

Target Genes ◽

Hippo Pathway ◽

Combined Treatment ◽

Set Up ◽

The Hippo Pathway

Abstract BACKGROUND Glioblastoma (GBM) is a primary human malignant brain tumor, the most common in adults. Several studies have highlighted the Hippo-pathway as a cancer signalling network. The Hippo pathway is an evolutionarily conserved signal cascade, which is involved in the control of organ growth. Dysregulations among this pathway have been found in lung, ovarian, liver and colorectal cancer. The key downstream effector of the Hippo-pathway is the Yes-associated protein (YAP); in the nucleus, its function as transcription co-activator is to interact with transcription factors, resulting in the expression of target genes involved in pro-proliferating and anti-apoptotic programs. MATERIAL AND METHODS Using western blotting analysis, we determined the nuclear expression of YAP on three GBM cell lines (U87MG, T98G and A172). To investigate which inhibitors against the Hippo-pathway were the most efficient, we performed a cytotoxic assay: we treated all the three cell lines with different inhibitors such as Verteporfin (VP), Cytochalasin D (CIT), Latrunculin A (LAT), Dobutamine (DOB) and Y27632. Afterwards, we performed a treatment using Doxorubicin (DOX) combined with the inhibitors, evaluating its cytotoxic effect on our cell lines, through cell viability experiments. More western blotting experiments were performed to investigate the oncogenic role of YAP at nucleus level. Furthermore, preliminary experiments have been conducted in order to investigate the apoptosis, senescence and autophagy modulation due to the Hippo-pathway. RESULTS We showed our cell lines express nuclear YAP. We assessed the efficiency of the main inhibitors against Hippo-pathway, proving that VP, LAT A and CIT show a strong cytostatic effect, linked to time increase; plus we saw a cytotoxic effect on T98G. The association of DOX with selected inhibitors is able to reduce cell viability and nuclear YAP expression rate in all three GBM lines. Finally, preliminary experiments were set up to assess how and if the mechanisms of apoptosis, autophagy and senescence were affected by the Hippo-pathway. The combination of DOX with inhibitors promotes resistance to apoptosis. CONCLUSION Our results show that nuclear YAP is present in all tumor lines, thus confirming that this molecular pathway is functioning in GBM lines. Nuclear YAP is more highly expressed after DOX administration. Moreover, the combined treatment (DOX with Hippo-pathway inhibitors) reduces both cell proliferation and viability, and increases the rate of apoptosis. Preliminary experiments on senescence and autophagy were used to determine the best Hippo-pathway inhibitor. These data demonstrate that the Hippo-pathway plays a crucial role in GBM proliferation and resistance to apoptosis. Inhibiting this pathway and in particular the transcription factor YAP, in association with DOX, might be an excellent therapeutic target.

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The Novel Dihydroorotate Dehydrogenase (DHODH) Inhibitor PTC299 Inhibit De Novo Pyrimidine Synthesis with Broad Anti-Leukemic Activity Against Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2020-139940 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 8-9

Author(s):

Sujan Piya ◽

Marla Weetall ◽

Josephine Sheedy ◽

Balmiki Ray ◽

Huaxian Ma ◽

...

Keyword(s):

Dna Damage ◽

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Research Funding ◽

De Novo ◽

Dihydroorotate Dehydrogenase ◽

Nucleotide Synthesis ◽

Inhibition Of Proliferation ◽

Acute Myeloid

Introduction: Acute myeloid leukemia (AML) is characterized by both aberrant proliferation and differentiation arrest at hematopoietic progenitor stages 1,2. AML relies upon de novo nucleotide synthesis to meet a dynamic metabolic landscape and to provide a sufficient supply of nucleotides and other macromolecules 3,4. Hence, we hypothesized that inhibition of de novo nucleotide synthesis would lead to depletion of the nucleotide pool and pyrimidine starvation in leukemic cells compared to their non-malignant counterparts and impact proliferative and differentiation inhibition pathways. PTC299 is an inhibitor of dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme for de novo pyrimidine nucleotide synthesis that is currently in a clinical trial for the treatment of AML. Aim: We investigated the pre-clinical activity of PTC299 against AML in primary AML blasts and cytarabine-resistant cell lines. To confirm that PTC299 effects are due to inhibition of de novo pyrimidine nucleotide synthesis for leukemic growth, we specifically tested the impact of uridine and orotate rescue. In addition, a comprehensive analysis of alteration of metabolic signaling in PI3K/AKT pathways, apoptotic signatures and DNA damage responses were analyzed by Mass cytometry based proteomic analysis (CyTOF) and immunoblotting. The potential clinical relevance of DHODH inhibition was confirmed in an AML-PDX model. Results: The IC50s for all tested cell lines (at 3 day) and primary blasts (at 5-7 day) were in a very low nanomolar range: OCI-AML3 -4.43 nM, HL60 -59.7 nM and primary samples -18-90 nM. Treatment of AML in cytarabine-resistant cells demonstrated that PTC299 induced apoptosis, differentiation, and reduced proliferation with corresponding increase in Annexin V and CD14 positive cells (Fig.1). PTC299-induced apoptosis and inhibition of proliferation was rescued by uridine and orotate. To gain more mechanistic insights, we used an immunoblotting and mass cytometry (CyTOF) based approach to analyze changes in apoptotic and cell signaling proteins in OCI-AML3 cells. Apoptotic pathways were induced (cleaved PARP, cleaved Caspase-3) and DNA damage responses (TP53, γH2AX) and the PI3/AKT pathway were downregulated in response to PTC299. In isogenic cell lines, p53-wildtype cells were sustained and an increased DNA damage response with corresponding increase in apoptosis in comparison to p53-deficient cells was shown. (Fig.2) In a PDX mouse model of human AML, PTC299 treatment improved survival compared to mice treated with vehicle (median survival 40 days vs. 30 days, P=0.0002) (Fig.3). This corresponded with a reduction in the bone marrow burden of leukemia with increased expression of differentiation markers in mice treated with PTC299 (Fig.3). Conclusion: PTC299 is a novel dihydroorotate dehydrogenase (DHODH) inhibitor that triggers differentiation, apoptosis and/or inhibition of proliferation in AML and is being tested in a clinical trials for the treatment of acute myeloid malignancies. Reference: 1. Thomas D, Majeti R. Biology and relevance of human acute myeloid leukemia stem cells. Blood 2017; 129(12): 1577-1585. e-pub ahead of print 2017/02/06; doi: 10.1182/blood-2016-10-696054 2. Quek L, Otto GW, Garnett C, Lhermitte L, Karamitros D, Stoilova B et al. Genetically distinct leukemic stem cells in human CD34- acute myeloid leukemia are arrested at a hemopoietic precursor-like stage. The Journal of experimental medicine 2016; 213(8): 1513-1535. e-pub ahead of print 2016/07/06; doi: 10.1084/jem.20151775 3. Villa E, Ali ES, Sahu U, Ben-Sahra I. Cancer Cells Tune the Signaling Pathways to Empower de Novo Synthesis of Nucleotides. Cancers (Basel) 2019; 11(5). e-pub ahead of print 2019/05/22; doi: 10.3390/cancers11050688 4. DeBerardinis RJ, Chandel NS. Fundamentals of cancer metabolism. Sci Adv 2016; 2(5): e1600200. e-pub ahead of print 2016/07/08; doi: 10.1126/sciadv.1600200 Disclosures Weetall: PTC Therapeutic: Current Employment. Sheedy:PTC therapeutics: Current Employment. Ray:PTC Therapeutics Inc.: Current Employment. Konopleva:Genentech: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Ablynx: Research Funding; Ascentage: Research Funding; Agios: Research Funding; Kisoji: Consultancy; Eli Lilly: Research Funding; AstraZeneca: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; AbbVie: Consultancy, Research Funding; Calithera: Research Funding; Cellectis: Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Sanofi: Research Funding. Andreeff:Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees. Borthakur:BioLine Rx: Consultancy; BioTherix: Consultancy; Nkarta Therapeutics: Consultancy; Treadwell Therapeutics: Consultancy; Xbiotech USA: Research Funding; Polaris: Research Funding; AstraZeneca: Research Funding; BMS: Research Funding; BioLine Rx: Research Funding; Cyclacel: Research Funding; GSK: Research Funding; Jannsen: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Incyte: Research Funding; PTC Therapeutics: Research Funding; FTC Therapeutics: Consultancy; Curio Science LLC: Consultancy; PTC Therapeutics: Consultancy; Argenx: Consultancy; Oncoceutics: Research Funding.

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A simple method to monitor hepatic gluconeogenesis and triglyceride synthesis following oral sugar tolerance test in obese adolescents

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00047.2019 ◽

2019 ◽

Vol 317 (1) ◽

pp. R134-R142 ◽

Cited By ~ 2

Author(s):

Anne-Marie Carreau ◽

Eunsook S. Jin ◽

Yesenia Garcia-Reyes ◽

Haseeb Rahat ◽

Kristen J. Nadeau ◽

...

Keyword(s):

Metabolic Diseases ◽

De Novo ◽

Tca Cycle ◽

Tolerance Test ◽

Serum Glucose ◽

Pentose Phosphate ◽

Invasive Technique ◽

Simple Method ◽

Obese Adolescents ◽

Sugar Tolerance

Hepatic energy metabolism is a key element in many metabolic diseases. Hepatic anaplerosis provides carbons for gluconeogenesis (GNG) and triglyceride (TG) synthesis. We aimed to optimize a protocol that measures hepatic anaplerotic contribution for GNG, TG synthesis, and hepatic pentose phosphate pathway (PPP) activity using a single dose of oral [U−13C3]glycerol paired with an oral sugar tolerance test (OSTT) in a population with significant insulin resistance. The OSTT (75 g glucose + 25 g fructose) was administered to eight obese adolescents with polycystic ovarian syndrome (PCOS) followed by ingestion of [U-13C3]glycerol at t = 180 or t = 210 min. 13C-labeling patterns of serum glucose and TG-glycerol were determined by nuclear magnetic resonance. 13C enrichment in plasma TG-glycerol was detectable and stable from 240 to 390 min with the [U-13C3]glycerol drink at t = 180 min(3.65 ± 2.3 to 4.47 ± 1.4%; P > 0.4), but the enrichment was undetectable at 240 min with the glycerol drink at t = 210 min. The relative contribution from anaplerosis was determined at the end of the OSTT [18.5 ±3.4% ( t = 180 min) vs. 16.0 ± 3.5% ( t = 210 min); P = 0.27]. [U-13C3]glycerol was incorporated into GNG 390 min after the OSTT with an enrichment of 7.5–12.5%. Glucose derived from TCA cycle activity was 0.3–1%, and the PPP activity was 2.8–4.7%. In conclusion, it is possible to obtain relative measurements of hepatic anaplerotic contribution to both GNG and TG esterification following an OSTT in a highly insulin-resistant population using a minimally invasive technique. Tracer administration should be timed to allow enough de novo TG esterification and endogenous glucose release after the sugar drink.

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Some Leukemia Cells Are Dependent on Glutamine as Energy Source

Blood ◽

10.1182/blood.v116.21.4861.4861 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4861-4861

Author(s):

Hiroshi Miwa ◽

Masato Shikami ◽

Norikazu Imai ◽

Kazuto Suganuma ◽

Mineaki Goto ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Warburg Effect ◽

Conflicts Of Interest ◽

Tca Cycle ◽

Ammonia Concentration ◽

Glutamine Metabolism ◽

Leukemia Cells ◽

Glutamine Deprivation ◽

Dependent Cell

Abstract Abstract 4861 In cancer cells, glucose uptake is elevated and glycolysis persists even under aerobic conditions (Warburg effect). Glutamine metabolism is another target for alteration in cancer development. Glutaminolysis (catabolism of glutamine to generate ATP) is known to increase in tumors. We examined the dependency of the leukemia cells (Kasumi-1, THP-1, HL-60 and NB4) on glucose or glutamine by measuring the growth (MTS count) in glucose- or glutamine-deprived condition. Glucose withdrawal greatly suppressed the growth of all 4 cell lines. However, glutamine withdrawal showed different growth suppressive effects among the cell lines (Kasumi-1: 55% of control, THP-1: 60%, HL-60: 39%, NB4: 70%). HL-60 was most sensitive to glutamine deprivation. The growth suppression of HL-60 due to glutamine withdrawal was partially rescued by oxaloacetate (OAA), a TCA cycle metabolite, while the growth of other cell lines was not rescued by OAA. In the course of glutamine catabolism, ammonia is liberated. Although basal level of the ammonia concentration was not so different among each cell line, glycolysis inhibitor (2-deoxyglucose) treatment enhanced the ammonia generation in HL-60 (Kasumi-1: 2.8% increased, THP-1: 1.7%, HL-60: 6.1%, NB4: 2.8%). Glutaminase, an enzyme converting glutamine to glutamate, is most abundantly expressed in HL-60 in western blot analysis. In addition, HL-60 was most sensitive to the treatment with aminooxyacetate, an inhibitor of glutamate-dependent transaminases that convert glutamate into a-ketoglutarate in the glutaminolytic pathway (Kasumi-1: 86% of control, THP-1: 97%, HL-60: 79%, NB4: 83%). Taken together, HL-60 was considered as glutamine dependent cell line. Therapies targeting glutamine metabolism, such as glutamine depletion or use of inhibitor of glutaminolytic pathway, might be effective against some leukemia. Disclosures: No relevant conflicts of interest to declare.

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A Novel STAT3 Inhibitor OPB-31121 Induces Tumor-Specific Growth Inhibition in a Wide Range of Hematopoietic Malignancies without Growth Suppression of Normal Hematopoietic Cells

Blood ◽

10.1182/blood.v118.21.577.577 ◽

2011 ◽

Vol 118 (21) ◽

pp. 577-577 ◽

Cited By ~ 1

Author(s):

Fumihiko Hayakawa ◽

Keiki Sugimoto ◽

Shingo Kurahashi ◽

Takumi Sumida ◽

Tomoki Naoe

Keyword(s):

Cell Lines ◽

Research Funding ◽

Hematopoietic Cells ◽

Inhibitory Effect ◽

Growth Suppression ◽

Leukemia Cells ◽

Human Leukemia ◽

Stat Proteins ◽

Hematopoietic Malignancies ◽

Wide Range

Abstract Abstract 577 Signal Transduction and Activator of Transcription (STAT) proteins are extracellular ligand-responsive transcription factors that mediate a wide range of biological processes such as cell proliferation, apoptosis, differentiation, development, and immune response. Stimulation with cytokines or growth factors results in the tyrosine phosphorylation of STAT proteins via activation of upstream tyrosine kinases like JAK family kinases and Src family kinases. Activated STAT proteins translocate to the nucleus and regulate gene expression through direct binding to the promoters of responsive genes. STAT3 is widely recognized as being a master regulator of the cellular functions that lead to the cancer phenotype. Constitutive activation of STAT3 is observed in a broad spectrum of human cancers and induces uncontrolled cell proliferation and apoptosis resistance. It has been identified as a promising target for anti-tumor drug, but to date most of the trials to block STAT-signaling were the inhibition of upstream kinases like JAK family kinases, especially in clinical trials. Here, we report a novel STAT3 inhibitor, OPB-31121, that has no inhibitory effect on kinases including JAK family kinases and Src family kinases. In HEL92.1.7 cells with constitutively active mutation of JAK2, OPB-31121 treatment inhibited phosphorylation of STAT3 without inhibition of JAK2 phosphorylation (Figure A). Src-dependent constitutive phosphorylation of STAT3 was also inhibited by OPB-31121 without inhibition of Src in H1650 cells that had active mutantation of EGF receptor. In addition, STAT3 immunoprecipitated from OPB-31121-treated cells was neither phosphorylated by JAK2 nor Lyn, a Src family kinase, in vitro without decrease in auto phosphorylation of upstream kinases, OPB-31121 demonstrated strong growth inhibitory effect (IC50 < 10 nM) in cell lines of a wide range of cancer especially hematopoietic malignancies including myeloma, AML with JAK2 mutation and CML. It is revealed that STAT3 is constitutively activated by oncogenic autocrine of IL-6 pathway or tyrosine kinase signal from oncoprotein in these cell lines. We also demonstrated growth inhibition or reduction of cell lines including HEL92.1.7 (AML with JAK2 mutation, T/C: 16%), KU812 (CML, T/C: 2%), and TCCy/sr (ALL positive for BCR-ABL with T315I mutation, T/C: 5.9%) in NOD/SCID mice. For further analyses, we used human leukemia model mice where clinical samples of human leukemia were transplanted into NOD/SCID/IL2-Rgammac−/− (NOG) mice and could be maintained by serial transplantation. In this system, heterogeneity and hierarchy of differentiation of leukemia cells, if they had, are maintained. OPB-31121 induced significant growth reduction of leukemia cells of BCR-ABL-positive ALL (T/C: 4%, Figure B), CML-BC with T315I mutation in BCR-ABL (T/C: 15.3%), and AML (T/C: 15.9%). Notably, OPB-31121-induced growth reduction was extremely selective for leukemia cells. Normal hematopoietic cells of mice were hardly affected by OPB-31121, whereas, cytarabine showed non-specific growth suppression of both leukemia cells and normal hematopoietic cells (Figure C). The safety of OPB-31121 on normal hematopoietic cells was also confirmed by colony formation assay, where OPB-31121 hardly affected colony formation of human cord blood cells at 100 nM. For further analyses, we transplanted human cord blood cells into NOG mice and investigated the growth inhibitory effect of OPB-31121 on normal hematopoietic cells in vivo. No significant growth suppression of human normal hematopoietic cells was observed in OPB-31121 treated mice (T/C: 99.9%, Figure D). Taken together, we conclude that OPB-31121 holds promise as a non-myelosuppressive therapeutic agent against a wide range of hematopoietic malignancies. This drug is under phase I/II trial in Japan. Disclosures: Hayakawa: Otsuka Pharmaceutical Co. Ltd.: Research Funding. Sugimoto:Otsuka Pharmaceutical Co. Ltd.: Employment. Sumida:otsuka Pharmaceutical Co. Ltd.: Employment. Naoe:Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.

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Preclinical Evaluation of Oncolytic Reovirus in Targeted Therapeutics for High-Risk Pediatric Leukemia

Blood ◽

10.1182/blood.v120.21.3571.3571 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3571-3571

Author(s):

Matthew F. Clarkson ◽

Aru Narendran ◽

Randal N. Johnston

Keyword(s):

Cell Death ◽

High Risk ◽

Cell Viability ◽

Cell Lines ◽

Leukemia Cell ◽

Treatment Strategies ◽

Leukemia Cells ◽

Western Blots ◽

Pediatric Leukemia ◽

Leukemia Cell Lines

Abstract Abstract 3571 Purpose: Leukemia is the most common malignancy in children. Improved treatment strategies in recent decades have yielded substantially enhanced outcomes for children with leukemia, reaching survival rates >80%. However, there remain significant issues with current treatment. Certain subgroups of patients who are resistant to or relapse from current treatments have a dismal prognosis. Furthermore, there are significant late effects of intensive treatments, including secondary cancers, neurocognitive defects, cardiotoxicity, obesity and infertility. For these reasons, novel treatment strategies are urgently needed for high-risk leukemia in children. Reovirus type 3 Dearing is a wild-type double-stranded RNA virus that has shown great promise as a selective oncolytic agent by its ability to replicate in transformed cells but not in normal cells. Although a number of early phase clinical studies have been completed in patients with advanced, refractory solid tumors in adults, systematic evaluation of this agent in the treatment of refractory pediatric leukemia has not been reported. As an initial step towards developing an oncolytics based treatment approach, we report preclinical data with respect to the activity, target validation, target modulation and drug combinability of reovirus in childhood leukemia cells. Experimental Design: A panel of pediatric leukemia cell lines representing high-risk molecular features such as Bcr-Abl, MLL rearranged and mixed lineage was used (n =6). Expression of JAM-A, the cell surface receptor for reovirus, was assessed by flow cytometry. The Ras Activation Assay Kit (EMD Millipore) was used to assess activity of the RAS protein. Western Blots were used to assess the activation (phosphorylation) of the signaling partners downstream of RAS. Cells treated with reovirus, chemotherapy drugs, or both for distinct treatment schedules were assessed for cell viability by the CellTiter-Glo© Luminescent Cell Viability Assay (Promega), and cell death by apoptosis was confirmed by cleavage of PARP. Productive viral infection was assessed by measuring reoviral protein synthesis by Western Blots, and reoviral replication was assessed by virus plaque titration assay. Drug synergies were calculated according to the method of Chou and Talalay. Results: Target validation assays showed the expression of JAM-A, which facilitates effective viral entry into malignant cells, in five of six cell lines. These cell lines also demonstrated differential activation of RAS and downstream kinases, suggesting targeted susceptibility of these cells to reovirus oncolysis. To further test this, we infected cells with reovirus for 1–4 days and assessed cytopathic effects. Using phase contrast microscopy, we observed the virus treated cell lines to demonstrate morphological changes characteristic of cell death following infection. Cell viability assays were used to quantify this effect, and the mechanism of cell death was determined to be apoptotic as evidenced by caspase-dependent cleavage of PARP. Reovirus-induced cell death was correlated with viral protein production and replication. Next, we screened for the ability of reovirus to induce synergistic activity in a panel of conventional and novel targeted therapeutic agents. Our studies showed that, in contrast to the current antileukemic agents, the Bcl-2 inhibitor BH3 mimetic ABT-737 was able to significantly synergize with reovirus in all cell lines tested. Conclusions: In our in vitro studies, oncolytic reovirus as a single agent showed potent oncolytic activity against all pediatric leukemia cell lines tested that express the receptor for reovirus, regardless of the status of the RAS signaling pathway. Further, we found reovirus-induced oncolysis can be enhanced by combination with Bcl-2 inhibition but was unaltered or antagonized by the other drugs indicating a key relationship between the two pathways. As such, our data for the first time, show that pediatric leukemia cells carry the potential to be targeted by reovirus induced oncolysis and the identification of drug synergy and the biomarkers of target modulation provide the basis for further studies to develop this novel therapeutic approach for clinical studies in the near future. Disclosures: No relevant conflicts of interest to declare.

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BGB324 Inhibits BCR-ABL TKI-Resistant Chronic Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.1569.1569 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1569-1569

Author(s):

Isabel Ben Batalla ◽

Robert Erdmann ◽

Heather Jørgensen ◽

Rebecca Mitchell ◽

Thomas Ernst ◽

...

Keyword(s):

Cell Proliferation ◽

Tyrosine Kinase ◽

Cell Lines ◽

Therapeutic Target ◽

Research Funding ◽

Combined Treatment ◽

K562 Cells ◽

Resistant Cell ◽

Additive Effect ◽

Significant Prolongation

Abstract Resistance and CML stem cell persistence preclude cure for the majority of patients treated with tyrosine kinase inhibitor (TKI) therapies. We demonstrated that the receptor tyrosine kinase (RTK) Axl of the Tyro-3, Axl, Mer (TAM) family is expressed by TKI-sensitive and -resistant CML cells (Erdmann R. et al. ASH 2013 and 2014). We have shown that blockade of the Gas6-Axl axis by the small molecule Axl inhibitor BGB324 (BerGenBio) represents a therapeutic target in AML. We are currently investigating BGB324 in a Phase 1b trial in refractory AML patients and in those non-eligible for intensive chemotherapy (BGBC003, NCT02488408). We hypothesised that Axl represents a tractable therapeutic target even in the most resistant forms of CML. Upon treatment with imatinib KCL-22 and K562 cells showed upregulation of Axl at the protein level indicating that Axl might be involved in resistance towards TKIs in CML. Consistently, Axl levels were higher in MNCs of TKI-resistant patients compared to -sensitive patients after 6 months of treatment (n=17/20, 1±0.4 vs. 0.16±0.03; *p<0.05). Upon combined treatment of KCL-22 and K562 cells with BGB324 and imatinib we detected an additive effect of growth inhibition (KCL-22 cells; n=3, viability 66.0±0.5% BGB324, 52.0±1.1% imatinib, 42.3±1.5% combo; combo vs. IM *p<0.01 and combo vs. BGB324 *(p<0.0001), and not shown). Analysis of intracellular signal transduction in these cell lines indicated that Axl induces phosphorylation of Stat5 by BCR-ABL independent pathways because we detected an additive effect of inhibition of Stat5 phosphorylation when combining imatinib and BGB324. We could not detect an additive inhibitory effect on phosphorylation of Erk and Akt. Consistently, combined BCR-ABL and Axl blockade by means of imatinib and shRNA respectively, demonstrated an additive effect in reducing cell viability in KCL-22 and K562 cells (KCL-22 cells; n=3, viability 84.5±0.8% shControl+imatinib, 74.5±2.6% shAxl, 50.4±0.9% shAxl+imatinib; shAxl+IM vs. IM *(p<0.0001) and vs. shAxl *(p<0.001) and not shown). We next investigated Axl activation in TKI insensitive BCR-ABL+ cell lines. In addition we tested a novel Ponatinib-resistant cell line KCL-22 PonR generated by subcloning parental KCL-22 in increasing concentrations of ponatinib. BCR-ABL is unmutated in these cells; oncoprotein kinase activity is switched off but cell death is not induced with 2mM ponatinib. We found that Axl phosphorylation was higher in the TKI-resistant cell lines BaF3/T315I, KCL-22 T315I and KCL-22 PonR when compared to the parental cell lines (n=3, 139±3.8% KCL-22 T315I, 214±1.3% KCL-22 PonR with respect to KCL-22 WT, *p<0.001 for both comparisons; 169±8.7% with respect to BaF3/p210, *p<0.005). Treatment with BGB324 inhibited cell proliferation with an IC50of 726, 3178 and 2720nM for BaF3/T315I, KCL-22 T315I and KCL-22 PonR, respectively. BGB324 could induce apoptosis and reduce proliferation in these cell lines. Furthermore, BGB324 blocked growth of colonies and induced apoptosis of T315I-mutated and pan-TKI-resistant (including ponatinib) primary CML MNCs. The finding that BGB324 inhibits TKI-resistant CML was further corroborated with KCL-22 T315I mutated and KCL-22 PonR xenograft models. In both models we observed a significant tumor growth reduction upon treatment with 25 mg/kg BGB324 twice daily compared to placebo leading to a 34% and 58% reduction in tumor volume, p=0.0044, p=0.0021 for KCL-22 T315I and KCL-22 PonR, respectively). Cell proliferation was quantified by pHH3 analysis indicating a significant reduction in KCL-22 T315I and KCL-22 PonR tumors (n=8, 242.8±10.9 vs. 182.2±8.1, *p<0.0001; n=9, 259.5±9.3 vs. 213.2±6.8; *p<0.0001, respectively). Furthermore, we observed a significant decrease of Axl, Erk and Stat5 phosphorylation after treatment with BGB324 for 8 days. We also investigated the therapeutic effect of BGB324 in a systemic model, by transplantation of KCL-22 PonR into sublethally irradiated NSG mice. In this model, treatment with 25 mg/kg BGB324 twice daily resulted in significant prolongation of overall survival (median OS 36 days (control) vs 43 days (BGB324), n=5 *p<0.05). In summary, our data highlight the advantage to be gained from inhibition of Axl even in the most resistant CML cells, and support the need for human clinical trials of the novel inhibitor BGB324 alone and in combination with TKIs. Disclosures Schafhausen: Novartis: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Hochhaus:Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding. Holyoake:Novartis: Research Funding; BMS: Research Funding. Loges:BerGenBio: Honoraria, Other: travel support, Research Funding.

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Anti-Leukemic Effects of Venetoclax on Philadelphia Chromosome Positive Leukemia Cells

Blood ◽

10.1182/blood.v128.22.5428.5428 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5428-5428 ◽

Cited By ~ 1

Author(s):

Seiichi Okabe ◽

Tetsuzo Tauchi ◽

Yuko Tanaka ◽

Kazuma Ohyashiki

Keyword(s):

Research Funding ◽

Combined Treatment ◽

Philadelphia Chromosome ◽

K562 Cells ◽

Leukemia Cells ◽

Apoptotic Cells ◽

Dependent Manner ◽

T315i Mutation ◽

Mutant Cells ◽

Dose Dependent

Abstract Introduction: Chronic myeloid leukemia (CML) is characterized by the t(9:22) translocation known as the Philadelphia chromosome (Ph). Although ABL tyrosine kinase inhibitors (ABL TKI) such as imatinib, dasatinib and nilotinib have improved CML treatment, such therapies cannot cure patients with Philadelphia chromosome (Ph)-positive leukemia because of leukemia stem cells. Moreover, some patients develop BCR-ABL point mutations including T315I and become resistant to ABL TKI therapy. These leukemia stem cells are contained within a niche in the bone marrow and are often impervious to current treatments. Therefore, new approach against BCR-ABL mutant cells and LSCs may improve the outcome of Ph-positive leukemia patients. B cell lymphoma 2 (BCL-2) protein families are key regulator of apoptosis and highly promising targets for the development of anti-cancer treatment. Venetoclax, also known as ABT-199 is a selective, orally bioavailable BCL-2 inhibitor. Venetoclax is investigated in a pivotal phase 3 clinical trial against hematological malignancies such as chronic lymphocytic leukemia (CLL) and approved for the treatment of patients with CLL. Materials and methods: In this study, we investigated whether venetoclax could suppress Ph-positive leukemia cells including T315I mutation and primary samples. Results: BCL-2 expression was found in Ph-positive leukemia cells including primary samples, however, BCL-2 expression was reduced in K562 cells. We found 72 h venetoclax treatment inhibited the growth of Ba/F3 BCR-ABL and KCL-22 cells in a dose dependent manner. However, venetoclax activity was reduced in K562 cells. We examined the intracellular signaling after treatment of venetoclax. Phosphorylation of BCR-ABL and Crk-L was not reduced. However, activity of caspase 3, poly (ADP-ribose) polymerase (PARP) was increased. We next investigated the efficacy between ABL TKI and venetoclax by using these cell line. Combined treatment of Ba/F3 BCR-ABL cells with imatinib and venetoclax caused significantly more cytotoxicity than each drug alone. Apoptotic cells were also increased. Phosphorylation of BCR-ABL, Crk-L was reduced and cleaved caspase 3 and PARP activity was increased after imatinib and venetoclax treatment. We investigated the venetoclax activity against T315I positive cells. Venetoclax potently induced cell growth inhibition of Ba/F3 T315I mutant cells in a dose dependent manner. Combined treatment of Ba/F3 T315I mutant cells with ponatinib and venetoclax caused significantly more cytotoxicity than each drug alone. Apoptotic cells were also increased. Phosphorylation of BCR-ABL, Crk-L was reduced and cleaved PARP was increased after ponatinib and venetoclax treatment. To assess the activity of ponatinib and venetoclax, we examined tumor formation in mice model. We injected subcutaneously 1×107 Ba/F3 T315I mutant cells in nude mice. A dose of 20 mg/kg/day p.o of ponatinib and 50 mg/kg/day p.o of venetoclax inhibited tumor growth and reduced tumor volume compared with control mice. In the immunohistochemical analysis, we found that tumors in mice treated with ponatinib and venetoclax exhibited an increase in apoptotic cells. We also found that co-treatment with ponatinib and venetoclax increased mouse survival. The treatments were well tolerated with no animal health concerns observed. We also found that the treatment of venetoclax exhibits cell growth inhibition against CD34 positive CML samples. Conclusion: The results of our study indicate that the BCL-2 inhibitor venetoclax may be a powerful strategy against ABL TKI resistant cells including T315I mutation and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells. Disclosures Tauchi: Pfizer Inc.: Research Funding. Ohyashiki:Novartis International AG,: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding.

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