A Study on Preclinical Efficacy, Underlying Mechanisms, and Sensitivity Markers of a Novel Hypomethylating Agent Ntx-301 in Acute Myeloid Leukemia

Abstract Introduction: While intensive induction chemotherapy has been standard-or-care for patients with acute myeloid leukemia (AML), intensive regimens have often been limited in elderly patients with comorbidities. As an alternative option, less intensive hypomethylating agents (HMAs), decitabine (DAC) and azacitidine (AZA), are currently being used in these unfit patients. However, their low response rates and adverse events when administered alone suggest a need to develop novel HMAs that elicit enhanced efficacy and reduced mortality. A recent study demonstrated pharmacological improvements of a novel 4′-thio-modified analog, 5-aza-4′-thio-2′-deoxycytidine (hereafter NTX-301), including enhanced chemical stability and incorporation into DNA and improved preclinical efficacy (Thottassery, 2014). However, the mechanism of action (MoA) of NTX-301 has not yet been understood. Herein, we aim to thoroughly investigate the preclinical efficacy and MoAs of NTX-301 in AML through comparative analysis with DAC and AZA. To this end, we used in vitro and in vivo preclinical models of AML and performed multiomics-based analyses. Results: We comprehensively examined viability of 200 cancer cell lines (CCLs) upon treatment with NTX-301. Consistent with the current use of HMAs as therapeutics for hematologic malignancies, this sensitivity profiling displayed the most remarkable potency of NTX-301 toward blood CCLs (OR=3.97, p=0.0003). In addition, phenotypic assays revealed that the anti-leukemic activity of NTX-301 was superior to that of DAC, which is attributed to more effective actions in inducing apoptosis, cell cycle arrest, and differentiation. Supporting the in vitro results, orally administrated NTX-301 led to more potent tumor regression, better tolerability, and survival benefits over DAC and AZA in both systemic (1.5-2.0 mg/kg, qdx5 then 2 days off, 5 days on, and 9 days off, for 3 cycles) and subcutaneous (0.2-1.5 mg/kg) xenograft models. To assess the underlying MoAs, we interrogated global alterations at the transcriptome and methylome levels upon treatment with NTX-301 in three AML cell lines using RNA sequencing and methylation array. Methylome analyses revealed that NTX-301-induced demethylation patterns were distinguished from DAC; DAC triggered stronger global demethylation than NTX-301 did, whereas NTX-301 derived rather selective demethylation, preferentially in early-replicating regions, H3K27ac-marked regions, and non-CpG islands. In transcriptome analyses compared with DAC, NTX-301 more markedly elicited a transcriptional reversal toward a normal myeloid-like signature by increasing a differentiation signature and suppressing a leukemic stem cell signature. NTX-301 also mediated more pronounced activation of DNA damage response and the p53 pathway, which are characterized by marked induction of pH2AX and pChk1 and increased stability of p53, respectively. Given the synthetic lethality of p53 activation and BCL2 inhibition (Rongqing, 2017), stronger p53 stabilization by NTX-301 may confer more benefits in combination with venetoclax. Indeed, the combination of NTX-301 + venetoclax produced a more synergistic combination index compared with DAC + venetoclax. Strikingly, the combined NTX-301 (0.5-2.0 mg/kg) + venetoclax (50 mg/kg) achieved complete tumor remission, no notable toxicity, and prolonged survival benefits over AZA (2.5 mg/kg) + venetoclax (50 mg/kg) in preclinical models of AML. By integrating sensitivity profiles and multiomics data of 200 CCLs, we interrogated molecular determinants associated with sensitivity to NTX-301. Intriguingly, when comparing methylomes between sensitive and resistant CCLs, we found a significantly biased global hypermethylation trend toward sensitive CCLs. A combinatorial set of the most significantly biased 352 differentially methylated regions (FDR<0.05) showed potential as a predictive sensitivity marker for NTX-301, exhibiting a significant correlation (r=0.69, p<0.0001) with sensitivity to NTX-301. Conclusions: Our study demonstrated an improved therapeutic index of NTX-301 over traditional HMAs, providing a rationale for further clinical development of the agent as a single-agent or in combination with other agents. We also believe that our study for MoAs and biomarkers will improve our understanding of NTX-301. Disclosures Lim: Pinotbio: Research Funding. Yoo: Pinotbio: Research Funding. Cho: Pinotbio: Research Funding. Choi: Pinotbio: Research Funding. Jung: Pinotbio: Research Funding. Jung: Pinotbio: Current Employment. Lee: Pinotbio: Current Employment. Chun: Pinotbio: Current Employment. Go: Pinotbio: Current Employment. Lee: Pinotbio: Current Employment. Choi: Pinotbio: Research Funding.

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The Novel Fluoropyrimidine FdUMP[10] Is Highly Active Against Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v116.21.3302.3302 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3302-3302

Author(s):

Timothy Pardee ◽

Evan Gomes ◽

Jamie Jennings-Gee ◽

David L. Caudell ◽

William Gmeiner

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Single Agent ◽

The Novel ◽

Highly Active ◽

Ic50 Values ◽

Acute Myeloid

Abstract Abstract 3302 Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy that leads to marrow failure and death. This disease affects approximately 12,000 people per year in the United States, causing 9,000 deaths. Despite decades of research, therapy remains essentially unchanged and outcomes are poor. In patients over the age of 60 less then 10% of patients survive 5 years from diagnosis. There is a desperate need for the identification of new active agents with favorable toxicity profiles. The novel polymeric fluoropyrimidine (FP) FdUMP[10] is an oligodeoxynucleotide pro-drug of the thymidylate synthase (TS)-inhibitory FP metabolite 5-fluoro-2'-deoxyuridine-5`-O-monophosphate (FdUMP). The observation that this compound was highly active against several leukemia lines in the NCI 60 cell line screen prompted us to evaluate its activity in several preclinical models of AML. In vitro, FdUMP[10] exhibited remarkable activity against 3 human acute leukemia cell lines, HL60, Jurkat and THP-1, with IC50 values of 3.378 nM (95% CI 2.984 to 3.825), 5.438 nM (4.609 to 6.417) and 4.093 nM (3.413 to 4.907) respectively. We next tested its efficacy against a more genetically defined murine model of AML driven by expression of MLL-ENL. FdUMP[10] exhibited even greater activity against all murine lines tested. The IC50 values of FdUMP[10] against two MLL-ENL driven murine AML cell lines were 214 pM (95%CI 178.9 to 255.9) and 292.3 pM (251.8 to 339.4). The IC50 values observed for FdUMP[10] for all the murine lines tested were lower than both Ara-C (30-40 nM) and doxorubicin (2-4 nM). We then determined the cytotoxic mechanism for FdUMP[10] in vitro. Upon treatment with FdUMP[10] both the human and murine cell lines undergo extensive apoptosis as indicated by Annexin V and propidium iodide staining. Treated cells developed γH2AX foci, rapid and complete TS inhibition and display trapped Topoisomerase I (Topo I) cleavage complexes. FdUMP[10]-mediated induction of apoptosis was p53 independent as murine AML cells that had p53 knocked down by RNAi demonstrated resistance to both Ara-C and doxorubicin, but not to FdUMP[10]. We next tested the efficacy of FdUMP[10] in vivo. The MLL-ENL driven murine AML model results in blasts that can be transplanted into sublethally irradiated, immunocompetent, syngeneic recipients. The recipients develop a fatal and therapy-resistant AML. Lines were generated that expressed a luciferase reporter. Animals were imaged 6–7 days after injection of the leukemias to ensure engraftment and then began treatment with either the combination of Ara-C plus doxorubicin, single-agent FdUMP[10], or observation. Studies were performed using 2 doses of FdUMP[10] at 150 or 300 mg/kg injected on days 1 and 3 and compared to animals treated with 100 mg/kg Ara-C and 3mg/kg doxorubicin injected on days 1 through 5. Both treatments resulted in a statistically significant survival advantage over observation. A preliminary toxicology study compared FdUMP[10], 150 mg/kg daily, to 5-fluorouracil (5 FU), 150 mg/kg daily, or the combination of Ara-C at 100 mg/kg plus doxorubicin at 3 mg/kg daily. All groups were treated for 3, 4 or 5 days. On day 6 animals were sacrificed and organs harvested, sectioned, and stained. Slides were then reviewed by a veterinary pathologist. Tissues most affected were the small intestine, colon, and the bone marrow. The 5FU-treated animals had severe villous blunting and fusion with crypt necrosis in both large and small intestine. In contrast, FdUMP[10]-treated animals had only mild crypt epithelial apoptosis with mitoses. The 5 FU and Ara-C plus doxorubicin groups had a severe pan-cytopenia in the marrow compared to FdUMP[10] treated animals that showed only minimal to mild apoptosis. These data support the assertion that FdUMP[10] has lower toxicity then either Ara-C plus doxorubicin or identically dosed 5 FU. In summary FdUMP[10] exhibited remarkable activity against AML cells in vitro and in vivo. Additionally, FdUMP[10] had decreased toxicity compared to treatment with either single agent 5 FU or combination treatment with Ara-C plus doxorubicin. Disclosures: Gmeiner: Salzburg Therapeutics: Equity Ownership.

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Anti-Leukemic Activity of Daratumumab in Acute Myeloid Leukemia Cells and Patient-Derived Xenografts

Blood ◽

10.1182/blood.v124.21.2312.2312 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2312-2312 ◽

Cited By ~ 8

Author(s):

Cedric Dos Santos ◽

Shan Xiaochuan ◽

Zhou Chenghui ◽

Georges Habineza Ndikuyeze ◽

Joshua Glover ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Research Funding ◽

Cell Killing ◽

Nsg Mice ◽

Cd38 Expression ◽

Acute Myeloid

Abstract Daratumumab is a human antibody that binds to CD38 on the cell surface and induces cell killing by multiple mechanisms including complement mediated cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP) and apoptosis. In pre-clinical and clinical studies, daratumumab has been shown to effectively kill multiple myeloma (MM) cells and to enhance the potency of other treatments against MM. The purpose of the study was to investigate in vitro and in vivo efficacy of daratumumab against 9 acute myeloid leukemia (AML) cell lines and patient-derived samples. First, we evaluated the expression of CD38, complement inhibitory proteins (CIP) CD46, CD55, CD59, and FcgR1 (CD64) on AML cell lines (n=9), AML patient cells (n=10) and healthy donor bone marrow using flow cytometry. CD38 enumeration showed a substantial variation between cell lines (12,827±19, 320 molecules/cell) and between AML patients (11,560±8, 175 molecules/cell), while CD38 expression was more consistent in bone marrow (BM) from healthy donors (1,176±355 molecules/cell). The daratumumab-induced apoptosis observed in cell lines (MOLM-13, MOLM-16, MV-4-11, NB4) in vitro was not correlated with CD38 expression levels. Daratumumab induced minimal ADCC (5-20%) and low levels of (2-5%) CDC mediated cell killing in 6 AML cell lines tested. We did not observe a direct correlation between CD38 expression and ADCC, CDC, nor between CDC and CIP expression. Interestingly, treatment of two human Acute Promyelocytic Leukemia (M3) cell lines HL-60 and NB-4 with all-trans retinoic acid (ATRA) induced a 10-30-fold increase in CD38 expression, suggesting that ATRA could be used in combination with daratumumab. While we, and others, have shown that pre-incubation of primary AML cells with anti-CD38 antibodies inhibits engraftment in NSG mice, we aimed at evaluating the anti-leukemic activity of daratumumab in a therapeutic xenograft model using 3 different AML patients. NSG mice (10/group/patient) were transplanted with T cell-depleted AML cells and BM aspirates were collected 4-6 weeks later to assess leukemia burden in each mouse prior to treatment. Animals were untreated (Ctrl) or received daratumumab (10 mg/kg), or IgG1 isotype once a week for five weeks. We assessed AML burden (% huCD45+ CD33+) in BM, spleen (SPL) and peripheral blood (PB) within 5 days after the last treatment. First, we evaluated an AML (#3406, FLT3-ITD, see figure) with high expression of CD38 (13,445 molecules/cell) and low CD64 (489/cell) was evaluated. Daratumumab significantly reduced leukemia burden in SPL and PB, but had no effect in BM. The same daratumumab-induced reduction in peripheral blasts and lack of effect in BM was observed in 2 other AML patient xenografts (#7577, M1 IDH mutant/FLT3-ITD with 6,529 CD38 molecules/cell; #8096, M2 with 335 CD38 molecules/cell). Interestingly, we observed that daratumumab treatment led to a drastic reduction in CD38 surface expression in AML blasts including in BM, indicating that daratumumab efficiently targeted CD38 in bone marrow blasts. Our results suggest that the bone marrow microenvironment can impair the anti-leukemic activity of daratumumab observed in other tissues. Ongoing xenograft studies are testing whether induction with chemotherapy (Ara-C+doxorubicin), or with other agents disrupting the bone marrow microenvironment, can enhance the anti-leukemic activity of daratumumab. Figure 1: Effect of daratumumab treatment on AML 3406 leukemia burden: Figure 1:. Effect of daratumumab treatment on AML 3406 leukemia burden: Disclosures Dos Santos: Janssen R&D: Research Funding. Xiaochuan:Janssen R&D: Research Funding. Doshi:Janssen R&D: Employment. Sasser:Janssen R&D: Employment. Danet-Desnoyers:Janssen R&D: Research Funding.

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In Vitro and In Vivo Anti-Leukemic Effects of KPT-9274, a Reported PAK4 Allosteric Modulator, in Acute Myeloid Leukemia: Promising Results Justifying Further Development in This Disease

Blood ◽

10.1182/blood.v126.23.2471.2471 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2471-2471

Author(s):

Shaneice Mitchell ◽

Shelley Orwick ◽

Matthew Cannon ◽

Virginia M. Goettl ◽

Taylor D. LaFollette ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Research Funding ◽

Flow Cytometric Analysis ◽

Time Dependent ◽

Inhibition Of Proliferation ◽

Acute Myeloid

Abstract Acute Myeloid Leukemia (AML) is the most common adult acute leukemia and is characterized by numerous driver mutations and/or cytogenetic rearrangements that promote disruption of stem cell/early myeloid progenitor differentiation, apoptosis, and proliferation. Identification of both personalized targets specific to a mutation or genomic abnormality and also global ubiquitous tumor-related targets relevant to AML represents a high priority to improve therapy. p21 protein (Cdc42/Rac)-activated kinase 4 (PAK4) is involved in disease progression for several solid tumors but its expression and contribution to disease pathogenesis in AML has not been examined. Since multiple cellular pathways important in AML (e.g., RAS and Wnt/β-catenin) are regulated by PAKs, we hypothesized PAK4 (and other family members) could represent an attractive pharmacologic target. We first evaluated the expression of PAK4 in AML cell lines and patient blasts (55 patients) using RNA sequencing and Western blot confirmation. This demonstrated PAK4 to be abundantly expressed at the mRNA and protein level in virtually all the analyzed samples. We then tested the in vitro effects of the PAK4 allosteric modulators (PAMs) KPT-9274 (clinical candidate) and KPT-9331 (tool compound) on AML cell lines. These included MV4-11, HL-60, THP-1 and Kasumi-1. Cell lines were treated for 24, 48 and 72 with PAMs KPT-9274 and KPT-9331 at dosages ranging from 1nM to 10uM. Proliferation was measured by MTS assay. All cell lines showed a dose-and time- dependent decrease in cell proliferation with IC50 ranging from 0.14 to 0.28 µM for both compounds. Cell lines with low protein and mRNA expression of PAK4, such as HL-60, were sensitive to PAM treatment suggesting possible alternative targets of these agents. To determine the effect of PAMs on apoptosis, MV4-11 and THP-1 cell lines were treated with KPT-9331 at IC50 concentration (~0.25 µM) and cell death was measured via Annexin-V/PI flow cytometric analysis after 24, 48h and 72h treatment. KPT-9331 induced a time dependent increase in apoptosis in both cell lines. In MV4-11 cells, KPT-9331 caused cell cycle arrest and inhibition of proliferation after 24hrs. We also tested the effect of PAMs in primary AML cells. Patient samples cocultured with a human stromal cell lines were treated with PAMs for 96 hours. IC50 values ranged from 0.14 - 0.19 µM. A dose dependent decrease in proliferation following PAM treatment was observed in all the five analyzed samples irrespective of genetic subtype. PAMs treatment for 48hrs using a whole blood viability assay from normal donors showed no significant cytotoxic effect on T and NK cells, but modest toxicity to normal B cells. Normal hematopoietic colony forming cell assays are being initiated and will be presented. We next utilized a human AML leukemia xenograft model with MV4-11 cells to assess the in vivo activity of KPT-9274. Mice were dosed once daily via oral gavage with KPT-9274 (150 mg/kg) or vehicle control. KPT-9274 dramatically inhibited tumor growth, prevented invasion of MV4-11 cells, and improved overall survival with all mice (n=7) being alive (median not reached) at day 49 of experiment as compared to 1 out of 7 vehicle-treated mice (median survival 36 days) being alive at this time. In summary, KPT-9274 demonstrates promising activity in pre-clinical AML models and warrants further investigation in this disease. Ongoing efforts include validating the specificity of the reported target in AML (versus alternative targets), in vivo exploration in primary human AML xenograft models, and understanding the effects of this compound on normal hematopoiesis and function. Disclosures Baloglu: Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Blum:Gilead Sciences: Research Funding. Byrd:Acerta Pharma BV: Research Funding.

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Anti-leukemia properties of cadmium nanoparticles in the in vitro and in vivo condition: A chemobiological study

Archives of Medical Science ◽

10.5114/aoms/142465 ◽

2021 ◽

Author(s):

Yudi Miao ◽

Behnam Mahdavi ◽

Mohammad Zangeneh

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Aqueous Extract ◽

Myeloid Leukemia ◽

Antioxidant Properties ◽

Leukemia Cell ◽

Sem Images ◽

Acute Myeloid

IntroductionThe present study investigated the anti-acute myeloid leukemia effects of Ziziphora clinopodides Lam leaf aqueous extract conjugated cadmium nanoparticles.Material and methodsTo synthesize CdNPs, Z. clinopodides aqueous extract was mixed with Cd(NO3)2 .4H2O. The characterization of the biosynthesized cadmium nanoparticles was carried out using many various techniques such as UV-Vis. and FT-IR spectroscopy, XRD, FE-SEM, and EDS.ResultsThe uniform spherical morphology of NPs was proved by FE-SEM images with NPs the average size of 26.78cnm. For investigating the antioxidant properties of Cd(NO3)2, Z. clinopodides, CdNPs, and Daunorubicin, the DPPH test was used. The cadmium nanoparticles inhibited half of the DPPH molecules in a concentration of 196 µg/mL. To survey the cytotoxicity and anti-acute myeloid leukemia effects of Cd(NO3)2, Z. clinopodides, CdNPs, and Daunorubicin, MTT assay was used on the human acute myeloid leukemia cell lines i.e., Murine C1498, 32D-FLT3-ITD, and Human HL-60/vcr. The IC50 of the cadmium nanoparticles was 168, 205, and 210 µg/mL against Murine C1498, 32D-FLT3-ITD, and Human HL-60/vcr cell lines, respectively. In the part of in vivo study, DMBA was used for inducing acute myeloid leukemia in mice. CdNPs similar to daunorubicin ameliorated significantly (p≤0.01) the biochemical, inflammatory, RBC, WBC, platelet, stereological, histopathological, and cellular-molecular parameters compared to the other groups.ConclusionsAs mentioned, the cadmium nanoparticles had significant anti-acute myeloid leukemia effects. After approving the above results in the clinical trial studies, these cadmium nanoparticles can be used as a chemotherapeutic drug to treat acute myeloid leukemia in humans.

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The Salt-Inducible Kinase inhibitor YKL-05-099 suppresses MEF2C function and acute myeloid leukemia progressionin vivo

10.1101/636969 ◽

2019 ◽

Author(s):

Yusuke Tarumoto ◽

Shan Lin ◽

Jinhua Wang ◽

Joseph P. Milazzo ◽

Yali Xu ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Disease Progression ◽

Cell Lines ◽

Myeloid Leukemia ◽

Kinase Inhibitor ◽

Clinical Investigation ◽

Genetic Targeting ◽

Acute Myeloid

AbstractLineage-defining transcription factors (TFs) are compelling targets for leukemia therapy, yet they are among the most challenging proteins to modulate directly with small molecules. We previously used CRISPR screening to identify a Salt-Inducible Kinase 3 (SIK3) requirement for the growth of acute myeloid leukemia (AML) cell lines that overexpress the lineage TF MEF2C. In this context, SIK3 maintains MEF2C function by directly phosphorylating histone deacetylase 4 (HDAC4), a repressive cofactor of MEF2C. Here, we evaluated whether inhibition of SIK3 with the tool compound YKL-05-099 can suppress MEF2C function and attenuate disease progression in animal models of AML. Genetic targeting of SIK3 or MEF2C selectively suppressed the growth of transformed hematopoietic cells underin vitroandin vivoconditions. Similar phenotypes were obtained when exposing cells to YKL-05-099, which caused cell cycle arrest and apoptosis in MEF2C-expressing AML cell lines. An epigenomic analysis revealed that YKL-05-099 rapidly suppressed MEF2C function by altering the phosphorylation state and nuclear localization of HDAC4. Using a gatekeeper allele ofSIK3, we found that the anti-proliferative effects of YKL-05-099 occurred through on-target inhibition of SIK3 kinase activity. Based on these findings, we treated two different mouse models of MLL-AF9 AML with YKL-05-099, which attenuated disease progressionin vivoand extended animal survival at well-tolerated doses. These findings validate SIK3 as a therapeutic target in MEF2C-positive AML and provide a rationale for developing drug-like inhibitors of SIK3 for definitive pre-clinical investigation and for studies in human patients with leukemia.Key PointsAML cells are uniquely sensitive to genetic or chemical inhibition of Salt-Inducible Kinase 3in vitroandin vivo.A SIK inhibitor YKL-05-099 suppresses MEF2C function and AMLin vivo.

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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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The Vitamin E Derivative Gamma Tocotrienol Promotes Anti-Tumor Effects in Acute Myeloid Leukemia Cell Lines

Nutrients ◽

10.3390/nu11112808 ◽

2019 ◽

Vol 11 (11) ◽

pp. 2808 ◽

Cited By ~ 6

Author(s):

Ghanem ◽

Zouein ◽

Mohamad ◽

Hodroj ◽

Haykal ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Vitamin E ◽

Cell Lines ◽

Myeloid Leukemia ◽

Apoptotic Cell ◽

Leukemia Cell ◽

Therapeutic Effects ◽

Apoptotic Pathway ◽

Acute Myeloid

Acute myeloid leukemia (AML) is a blood cancer characterized by the formation of faulty defective myelogenous cells with morphological heterogeneity and cytogenic aberrations leading to a loss of their function. In an attempt to find an effective and safe AML treatment, vitamin E derivatives, including tocopherols were considered as potential anti-tumor compounds. Recently, other isoforms of vitamin E, namely tocotrienols have been proposed as potential potent anti-cancerous agents, displaying promising therapeutic effects in different cancer types. In this study we evaluated the anti-cancerous effects of γ-tocotrienol, on AML cell lines in vitro. For this purpose, AML cell lines incubated with γ-tocotrienol were examined for their viability, cell cycle status, apoptotic cell death, DNA fragmentation, production of reactive oxygen species and expression of proapoptotic proteins. Our results showed that γ-tocotrienol exhibits time and dose-dependent anti-proliferative, pro-apoptotic and antioxidant effects on U937 and KG-1 cell lines, through the upregulation of proteins involved in the intrinsic apoptotic pathway.

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Structure-Based Drug Design of c-Kit Inhibitors for Use in the Treatment of Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v108.11.1906.1906 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1906-1906

Author(s):

David S. Maxwell ◽

Ashutosh Pal ◽

Zhenghong Peng ◽

Alexandr Shavrin ◽

Stefan Faderl ◽

...

Keyword(s):

Crystal Structure ◽

Acute Myeloid Leukemia ◽

Drug Design ◽

Cell Lines ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Kinase Assay ◽

Structure Based Drug Design ◽

Acute Myeloid

Abstract Inhibitors of c-Kit kinase have shown clinical relevance in various myeloid disorders, including acute myeloid leukemia (AML). Research in our lab has been oriented towards structure-based drug design of c-Kit inhibitors based on the available crystal structure. We describe the design, synthesis, and preliminary results from the in-vitro testing of several c-Kit kinase inhibitors in both enzymatic and cell-based assays. The design resulted from in-silico screening of several targeted libraries via docking to the crystal structure of c-Kit, followed by aggressive post-filtering by several criteria to significantly bias synthesis efforts towards candidate compounds with best chance for success. This led to 128 structures built from 8 common structural cores, from which 2 cores were initially selected based on the synthetic feasibility. Five compounds were initially synthesized, and were immediately followed by 60 compounds with variations to probe local structure-activity relationships. The initial set of compounds, designated APCKxxx, was tested in a c-Kit kinase assay; two compounds were found to have an IC50 in the high nM to low uM range. These compounds have been tested in a MTT-based assay using OCIM2 and OCI/AML3 cell lines. In the c-Kit expressing OCI/AML3 cell line, all five compounds possessed an EC50 < 500 nM and two had and EC50 ~100 nM. Our most recent results show that these compounds also show efficacy in some imatinib-resistant cell lines. We will discuss these results and our strategies for the second generation of compounds that are optimized for better activity, selectivity, and ADME properties.

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Aberrant Expression of the Homeobox Gene CDX2 in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v108.11.8.8 ◽

2006 ◽

Vol 108 (11) ◽

pp. 8-8 ◽

Cited By ~ 1

Author(s):

Claudia Scholl ◽

Dimple Bansal ◽

Konstanze Dohner ◽

Karina Eiwen ◽

Benjamin H. Lee ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Gene Copy ◽

Coding Region ◽

Aberrant Expression ◽

Cdx2 Expression ◽

Frequent Event ◽

Acute Myeloid

Abstract The caudal-type homeobox transcription factor 2 (CDX2) plays an important role in embryonic development and regulates the proliferation and differentiation of intestinal epithelial cells in the adult. Ectopic expression of CDX2 in the hematopoietic compartment was previously identified as the key pathogenetic event in a single patient with acute myeloid leukemia (AML) and t(12;13)(p13;q12). Using real-time quantitative PCR, we detected aberrant CDX2 expression in 153 (90%) of 170 patients with AML, in patients with high-risk myelodysplastic syndrome or advanced-stage chronic myeloid leukemia, and in several AML cell lines, but not in bone marrow derived from normal individuals. Expression of CDX2 was monoallelic in the majority of cases with informative single-nucleotide polymorphisms in the CDX2 coding region, but was not related to mutations in the CDX2 coding region or in the predicted CDX2 promoter sequence, gene-specific hypomethylation of the CDX2 promoter, or increased CDX2 gene copy numbers. Stable knockdown of CDX2 expression by lentivirus-mediated RNA interference inhibited the proliferation of various human AML cell lines exhibiting CDX2 transcript levels that were in the range of those observed in most primary AML samples, and strongly reduced their clonogenic potential in vitro. Primary murine hematopoietic progenitor cells transduced with Cdx2 acquired serial replating activity in vitro, could be continuously propagated in liquid culture, generated a fully penetrant and transplantable AML in vivo, and displayed dysregulated expression of Hox family members. Together, these results (i) demonstrate that aberrant expression of CDX2 is a frequent event in myeloid leukemogenesis, (ii) suggest a role for CDX2 as part of a common effector pathway that increases the proliferative capacity and promotes the self-renewal potential of hematopoietic progenitors, and (iii) support the unifying hypothesis that CDX2 is responsible, at least in part, for abnormalities in HOX gene expression in AML.

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Deregulated Apoptotic Pathways Point to Effectiveness of IAP Inhibitor Therapy in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.1275.1275 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1275-1275

Author(s):

Sonja C Lück ◽

Annika C Russ ◽

Konstanze Döhner ◽

Ursula Botzenhardt ◽

Domagoj Vucic ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Cell Line ◽

Cell Lines ◽

Myeloid Leukemia ◽

Gene Set Enrichment Analysis ◽

Core Binding Factor ◽

Binding Factor ◽

Acute Myeloid

Abstract Abstract 1275 Poster Board I-297 Core binding factor (CBF) leukemias, characterized by translocations t(8;21) or inv(16)/t(16;16) targeting the core binding factor, constitute acute myeloid leukemia (AML) subgroups with favorable prognosis. However, 40-50% of patients relapse, and the current classification system does not fully reflect the heterogeneity existing within the cytogenetic subgroups. Therefore, illuminating the biological mechanisms underlying these differences is important for an optimization of therapy. Previously, gene expression profiling (GEP) revealed two distinct CBF leukemia subgroups displaying significant outcome differences (Bullinger et al., Blood 2007). In order to further characterize these GEP defined CBF subgroups, we again used gene expression profiles to identify cell line models similar to the respective CBF cohorts. Treatment of these cell lines with cytarabine (araC) revealed a differential response to the drug as expected based on the expression patterns reflecting the CBF subgroups. In accordance, the cell lines resembling the inferior outcome CBF cohort (ME-1, MONO-MAC-1, OCI-AML2) were less sensitive to araC than those modeling the good prognostic subgroup (Kasumi-1, HEL, MV4-11). A previous gene set enrichment analysis had identified the pathways Caspase cascade in apoptosis and Role of mitochondria in apoptotic signaling among the most significant differentially regulated BioCarta pathways distinguishing the two CBF leukemia subgroups. Thus, we concluded that those pathways might be interesting targets for specific intervention, as deregulated apoptosis underlying the distinct subgroups should also result in a subgroup specific sensitivity to apoptotic stimuli. Therefore, we treated our model cell lines with the Smac mimetic BV6, which antagonizes inhibitor of apoptosis (IAP) proteins that are differentially expressed among our CBF cohorts. In general, sensitivity to BV6 treatment was higher in the cell lines corresponding to the subgroup with good outcome. Time-course experiments with the CBF leukemia cell line Kasumi-1 suggested a role for caspases in this response. Interestingly, combination treatment of araC and BV6 in Kasumi-1 showed a synergistic effect of these drugs, with the underlying mechanisms being currently further investigated. Based on the promising sensitivity to BV6 treatment in some cell lines, we next treated mononuclear cells (mostly leukemic blasts) derived from newly diagnosed AML patients with BV6 in vitro to evaluate BV6 potency in primary leukemia samples. Interestingly, in vitro BV6 treatment also discriminated AML cases into two distinct populations. Most patient samples were sensitive to BV6 monotherapy, but about one-third of cases were resistant even at higher BV6 dosage. GEP of BV6 sensitive patients (at 24h following either BV6 or DMSO treatment) provided insights into BV6-induced pathway alterations in the primary AML patient samples, which included apoptosis-related pathways. In contrast to the BV6 sensitive patients, GEP analyses of BV6 resistant cases revealed no differential regulation of apoptosis-related pathways in this cohort. These results provide evidence that targeting deregulated apoptosis pathways by Smac mimetics might represent a promising new therapeutic approach in AML and that GEP might be used to predict response to therapy, thereby enabling novel individual risk-adapted therapeutic approaches. Disclosures Vucic: Genentech, Inc.: Employment. Deshayes:Genentech, Inc.: Employment.

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