scholarly journals Co-inhibition of HDAC and MLL-menin interaction targets MLL-rearranged acute myeloid leukemia cells via disruption of DNA damage checkpoint and DNA repair

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Jing Ye ◽  
Jie Zha ◽  
Yuanfei Shi ◽  
Yin Li ◽  
Delin Yuan ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Clinical Investigation ◽  
Combined Treatment ◽  
Single Agent ◽  
Ros Generation ◽  
Dna Damage Checkpoint ◽  
Acute Myeloid

Abstract While the aberrant translocation of the mixed-lineage leukemia (MLL) gene drives pathogenesis of acute myeloid leukemia (AML), it represents an independent predictor for poor prognosis of adult AML patients. Thus, small molecule inhibitors targeting menin-MLL fusion protein interaction have been emerging for the treatment of MLL-rearranged AML. As both inhibitors of histone deacetylase (HDAC) and menin-MLL interaction target the transcription-regulatory machinery involving epigenetic regulation of chromatin remodeling that governs the expression of genes involved in tumorigenesis, we hypothesized that these two classes of agents might interact to kill MLL-rearranged (MLL-r) AML cells. Here, we report that the combination treatment with subtoxic doses of the HDAC inhibitor chidamide and the menin-MLL interaction inhibitor MI-3 displayed a highly synergistic anti-tumor activity against human MLL-r AML cells in vitro and in vivo, but not those without this genetic aberration. Mechanistically, co-exposure to chidamide and MI-3 led to robust apoptosis in MLL-r AML cells, in association with loss of mitochondrial membrane potential and a sharp increase in ROS generation. Combined treatment also disrupted DNA damage checkpoint at the level of CHK1 and CHK2 kinases, rather than their upstream kinases (ATR and ATM), as well as DNA repair likely via homologous recombination (HR), but not non-homologous end joining (NHEJ). Genome-wide RNAseq revealed gene expression alterations involving several potential signaling pathways (e.g., cell cycle, DNA repair, MAPK, NF-κB) that might account for or contribute to the mechanisms of action underlying anti-leukemia activity of chidamide and MI-3 as a single agent and particularly in combination in MLL-r AML. Collectively, these findings provide a preclinical basis for further clinical investigation of this novel targeted strategy combining HDAC and Menin-MLL interaction inhibitors to improve therapeutic outcomes in a subset of patients with poor-prognostic MLL-r leukemia.

scholarly journals p53 signaling in response to increased DNA damage sensitizes AML1-ETO cells to stress-induced death

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 2190-2199 ◽  
Author(s):  
Ondrej Krejci ◽  
Mark Wunderlich ◽  
Hartmut Geiger ◽  
Fu-Sheng Chou ◽  
David Schleimer ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Genetic Alterations ◽  
P53 Pathway ◽  
P53 Response ◽  
Acute Myeloid

Chromosomal translocation (8;21) is present in 10% to 15% of patients with acute myeloid leukemia. Expression of the AML1-ETO (AE) fusion protein alone is not sufficient to induce leukemia, but the nature of the additional genetic alterations is unknown. It is unclear whether AE facilitates acquisition of these cooperating events. We show that AE down-regulates genes involved in multiple DNA repair pathways, potentially through a mechanism involving direct binding at promoter elements, and increases the mutation frequency in vivo. AE cells display increased DNA damage in vitro and have an activated p53 pathway. This results in increased basal apoptosis and enhanced sensitivity to DNA damaging agents. Intriguingly, microarray data indicate that t(8;21) patient samples exhibit decreased expression of DNA repair genes and increased expression of p53 response genes compared with other acute myeloid leukemia (AML) patient samples. Inhibition of the p53 pathway by RNAi increases the resistance of AE cells to DNA damage. We thus speculate that AML1-ETO may facilitate accumulation of genetic alterations by suppressing endogenous DNA repair. It is possible that the superior outcome of t(8;21) patients is partly due to an activated p53 pathway, and that loss of the p53 response pathway is associated with disease progression.

scholarly journals SIRT6 Inhibition As a Novel Approach for Treating Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5222-5222
Author(s):  
Michele Cea ◽  
Antonia Cagnetta ◽  
Debora Soncini ◽  
Paola Minetto ◽  
Davide Lovera ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Dna Damage ◽  
Dna Repair ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Allogeneic Bone ◽  
A Genome ◽  
Acute Myeloid

Abstract Currently available therapeutics against Acute Myeloid Leukemia (AML) have improved patient outcome. However, resistance develops even to novel therapies and patient overall survival remains low, especially for patients who are not eligible for allogeneic bone marrow transplantation. Therefore, there is an urgent need to overcome the biologic mechanisms underlying drug resistance in AML, to enhance the efficacy of existing treatments and to facilitate the design of novel approaches. Recently, our group has demonstrated that SIRT6, a NAD+-dependent histone deacetylase involved in genome maintenance, is frequently up-regulated in Multiple Myeloma and its targeting induces cancer cell killing (Cea M. et al, Blood 2016). Furthermore, gene expression analyses performed by our groups show that SIRT6 is also up-regulated in AML and confers poor prognosis in a series of 200 primary AML cases from our Hematology Clinic. Thus, these data suggested a role for SIRT6 in AML biology. High SIRT6 expression was typically observed in AML cell lines characterized by constitutive DNA damage and intense replicative stress. Likewise, primary AML cases exhibiting an intermediate-to-high chromosome instability (CIN) gene expression signature were also those with the highest SIRT6 expression, and worst prognosis. Subsequent studies demonstrated that SIRT6 silencing or its chemical inhibition, as observed in Multiple Myeloma exacerbates DNA damage in response to genotoxic agents, sensitizing AML cells to cytarabine (ARA-C) and idarubicin in vitro. Overall, enhancing genotoxic stress while concomitantly blocking DNA double-strand breaks (DSBs) repair response, may represent an innovative strategy to increase chemosensitivity of AML cells. Mechanistic studies revealed that SIRT6 acts as a genome guardian in AML cells by binding DNA damage sites and activating DNA-PKcs and CtIP by deacetylation, which in turn promotes DNA repair. Overall our data suggest that genomic instability is present in all hematologic malignancies including AML. Strategies aimed to shift the balance towards high DNA damage and reduced DNA repair by SIRT6 inhibition can decrease AML growth and may benefit patients with otherwise unfavorable outcomes. Disclosures Gobbi: Gilead: Honoraria; Takeda: Consultancy; Janssen: Consultancy, Honoraria; Roche: Honoraria; Celgene: Consultancy; Mundipharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

scholarly journals Targeting acute myeloid leukemia dependency on VCP-mediated DNA repair through a selective second-generation small-molecule inhibitor

2021 ◽  
Vol 13 (587) ◽  
pp. eabg1168
Author(s):  
Blandine Roux ◽  
Camille Vaganay ◽  
Jesse D. Vargas ◽  
Gabriela Alexe ◽  
Chaima Benaksas ◽  
...  
Keyword(s):  
Dna Repair ◽  
Acute Myeloid Leukemia ◽  
Cancer Cells ◽  
Myeloid Leukemia ◽  
Second Generation ◽  
Single Agent ◽  
Dominant Negative ◽  
Aaa Atpase ◽  
Chemical Inhibition ◽  
Acute Myeloid

The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry–based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9–driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.

scholarly journals A Novel Synthetic Lethal Approach Targeting SIRT6 in Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1375-1375 ◽  
Author(s):  
Michele Cea ◽  
Antonia Cagnetta ◽  
Davide Lovera ◽  
Raffaella Grasso ◽  
Nicoletta Colombo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Acute Myeloid Leukemia ◽  
Genomic Instability ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Mrna Level ◽  
Mechanistic Studies ◽  
A Genome ◽  
Acute Myeloid

Abstract Background: Acute Myeloid Leukemia (AML) is an incurable disease characterized by a highly unstable genome, resulting in large-scale changes at diagnosis, as well as further evolution contributing to disease progression. However, the mechanisms whereby tumor cells adapt to genomic instability are largely unknown, but recent observations have correlated these abnormalities with dysfunctional DNA damage repair (DDR) machinery. SIRT6 is an important regulator of cellular stress response and genomic integrity. Here we investigated the role of this NAD+ -dependent deacetylase in regulating ongoing DNA damage observed in AML patients. Methods: SIRT6 mRNA level was determined by RT-qPCR in AML patients (n=100) diagnosed at the Hematology Department of University of Genoa (Italy), compared with normal bone marrow derived CD34+ cells (n=5). Correlation studies with clinical and molecular characteristics of these patients were also performed. A panel of different AML cell lines and primary cells, both sensitive and resistant to conventional and novel anti-AML therapies, was used in the study. The anti-leukemic effect of DNA-damaging agents (DDAs) including idarubicin, Ara-C and fludarabine was evaluated in presence of SIRT6 depletion/inhibition by CTG assay and Annexin-V/propidium iodide staining. Mechanistic studies were performed with Western-blotting, lentivirus-mediated shRNAs and immunofluorescence assay. Analysis of DNA DSB repair was done using chromosomally integrated reporter constructs, followed by cytometer analysis. Results: AML patients were grouped into lower and higher SIRT6 expressers according to its median mRNA level. Patients with lower expression had a higher incidence of FLT3-ITD (p=0.034, Wilcoxon signed rank test). No significant association was observed with respect to mutations of NPM1, nor with WT1 and BAALC expression. SIRT6 expression correlated also with adverse clinical outcome in term of event free and overall survival (p=0.035 and p=0.025, respectively; unpaired t test). Based on these data, we evaluated SIRT6 role in biology of AML. We found higher SIRT6 protein level in AML cell lines carrying FLT3-ITD mutation (MOLM-14 and MV 4-11) compared to cell lines harboring other mutations (OCI-AML3, THP-1, KG, NB4, HL60, Nomo1 and U937). Targeting SIRT6 by specific shRNAs weakly reduced AML cell survival compared with control-scrambled cells, by impairing DNA repair efficiency. Indeed, a restricted effect of SIRT6 impairment on DNA damage proteins (H2AX, RAD51, 53BP1, RPA32) was measured. We next examined the therapeutic relevance of SIRT6 inhibition in AML by testing effects of its depletion in combination with genotoxic agents. Remarkably, SIRT6 depletion conferred increased sensitivity of AML cells to idarubicin, Ara-C and Fludarabine. Overall, enhancing genotoxic stress while concomitantly blocking DNA double-strand breaks (DSBs) repair response, may represents an innovative strategy to increase chemosensitivity of AML cells. Further mechanistic studies revealed that SIRT6 acts as a genome guardian in leukemia cells by binding DNA damage sites and activating DNA-PKcs and CtIP by deacetylation, which in turn promotes DNA repair. Conclusion: Genomic instability is present in all hematologic malignancies including AML. Strategies aimed to shift the balance towards high DNA damage and reduced DNA repair by SIRT6 inhibition can decrease AML growth and may benefit patients with otherwise unfavorable outcomes. Disclosures No relevant conflicts of interest to declare.

Effect of miR-128 in DNA Damage of HL-60 Acute Myeloid Leukemia Cells

2014 ◽  
Vol 15 (5) ◽  
pp. 492-502 ◽  
Author(s):  
Hugo Seca ◽  
Raquel Lima ◽  
Gabriela Almeida ◽  
Manuel Sobrinho-Simoes ◽  
Rui Bergantim ◽  
...  
Keyword(s):  
Dna Damage ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

scholarly journals FLT3 mutated acute myeloid leukemia: 2021 treatment algorithm

2021 ◽  
Vol 11 (5) ◽  
Author(s):  
Naval Daver ◽  
Sangeetha Venugopal ◽  
Farhad Ravandi
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Single Agent ◽  
Prognostic Significance ◽  
Treatment Algorithm ◽  
Molecular Testing ◽  
Prognostic Impact ◽  
Cell Transplant ◽  
Newly Diagnosed ◽  
Acute Myeloid

AbstractApproximately 30% of patients with newly diagnosed acute myeloid leukemia (AML) harbor mutations in the fms-like tyrosine kinase 3 (FLT3) gene. While the adverse prognostic impact of FLT3-ITDmut in AML has been clearly proven, the prognostic significance of FLT3-TKDmut remains speculative. Current guidelines recommend rapid molecular testing for FLT3mut at diagnosis and earlier incorporation of targeted agents to achieve deeper remissions and early consideration for allogeneic stem cell transplant (ASCT). Mounting evidence suggests that FLT3mut can emerge at any timepoint in the disease spectrum emphasizing the need for repetitive mutational testing not only at diagnosis but also at each relapse. The approval of multi-kinase FLT3 inhibitor (FLT3i) midostaurin with induction therapy for newly diagnosed FLT3mut AML, and a more specific, potent FLT3i, gilteritinib as monotherapy for relapsed/refractory (R/R) FLT3mut AML have improved outcomes in patients with FLT3mut AML. Nevertheless, the short duration of remission with single-agent FLT3i’s in R/R FLT3mut AML in the absence of ASCT, limited options in patients refractory to gilteritinib therapy, and diverse primary and secondary mechanisms of resistance to different FLT3i’s remain ongoing challenges that compel the development and rapid implementation of multi-agent combinatorial or sequential therapies for FLT3mut AML.

scholarly journals DNA damage in acute myeloid leukemia patients of Northern Mexico

2017 ◽  
Author(s):  
Martha I. Dávila-Rodríguez ◽  
Elva I. Cortés-Gutiérrez ◽  
Roberto Hernández-Valdés ◽  
Karla Guzmán-Cortés ◽  
Rosa E. De León-Cantú ◽  
...  
Keyword(s):  
Dna Damage ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Fluorescent In Situ Hybridization ◽  
Control Group ◽  
Repair Pathway ◽  
Blood Leukocytes ◽  
Northern Mexico ◽  
Acute Myeloid

The purpose of this study was to evaluate DNA damage in the whole genome of peripheral blood leukocytes from patients with acute myeloid leukemia (AML) compared with a control group using DNA breakage detection-fluorescent in situ hybridization (DBD-FISH). Our results suggest that the DNA damage detected in patients with newly diagnosed AML was similar to that observed for the controls; this might be explained by the stimulation of a repair pathway by the pathogenesis itself. These findings indicate that inhibiting the repair pathway could be proposed to enhance the efficacy of chemotherapy.

Octadecyloxyethyl Adefovir Exhibits Potent in vitro and in vivo Cytotoxic Activity and Has Synergistic Effects with Ara-C in Acute Myeloid Leukemia

Chemotherapy ◽  
2018 ◽  
Vol 63 (4) ◽  
pp. 225-237 ◽  
Author(s):  
Haytham Khoury ◽  
Ruijuan He ◽  
Aaron Schimmer ◽  
James R. Beadle ◽  
Karl Y. Hostetler ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Acute Myeloid Leukemia ◽  
Cell Cycle Arrest ◽  
Clinical Benefit ◽  
Myeloid Leukemia ◽  
Mouse Bone Marrow ◽  
Medical Needs ◽  
Cycle Arrest ◽  
Acute Myeloid

Acute myeloid leukemia (AML) continues to be a deadly disease, with only 50–70% of patients achieving complete remission and less than 30% of adults having sustained long-term remissions. In order to address these unmet medical needs, we carried out a high-throughput screen of an in-house library of on- and off-patent drugs with the OCI/AML-2 cell line. Through this screen, we discovered adefovir dipi­voxil (adefovir-DP) as being active against human AML. In addition to adefovir-DP, there are second-generation formulations of adefovir, including octadecyloxyethyl adefovir (ODE-adefovir) and hexadecyloxypropyl adefovir (HDP-adefovir), which were designed to overcome the pharmacokinetic problems of the parent compound adefovir. Given the known clinical benefit of nucleoside analogs for the treatment of AML, we undertook studies to evaluate the potential benefit of adefovir-based molecules. In AML cell lines and patient samples, adefovir-DP and ODE-adefovir were highly potent, whereas HDP-adefovir was significantly less active. Interestingly, ODE-adefovir was remarkably less toxic than adefovir-DP towards normal hematopoietic cells. In addition, ODE-adefovir at a dose of 15 mg/kg/day showed potent activity against human AML in a NOD/SCID mouse model, with a reduction of human leukemia in mouse bone marrow of > 40% in all mice tested within 20 days of treatment. Based on its chemical structure, we hypothesized that the cytotoxicity of ODE-adefovir toward AML was through cell cycle arrest and DNA damage. Indeed, ODE-adefovir treatment induced cell cycle arrest in the S phase and increased levels of pH2Ax, indicating the induction of DNA damage. Furthermore, there was an increase in phospho-p53, transactivation of proapoptotic genes and activation of the intrinsic apoptotic pathway. Subsequent investigation unveiled strong synergism between ODE-adefovir and ara-C, making their coadministration of potential clinical benefit. Expression of MRP4, a nucleoside transporter, appeared to influence the response of AML cells to ODE-adefovir, as its inhibition potentiated ODE-adefovir killing. Taken together, our findings indicate that clinical development of ODE-adefovir or related compounds for the treatment of AML is warranted.

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

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.

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

Blood ◽  
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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