The Investigational Multi-Targeted Kinase Inhibitor TAK-901 Antagonizes Acute Myeloid Leukemia Pathogenesis: Results of Preclinical Studies

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 581-581
Author(s):  
Patrick Griffin ◽  
Steffan T Nawrocki ◽  
Takashi Satou ◽  
Claudia M Espitia ◽  
Kevin R. Kelly ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Mouse Model ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Standard Of Care ◽  
Clonogenic Survival ◽  
Preclinical Studies ◽  
Acute Myeloid

Abstract Abstract 581 The long-term prognosis for the majority of patients diagnosed with acute myeloid leukemia (AML) is very poor due, in part, to pre-existing myelodysplasia, multidrug resistance, and co-existing morbidities that limit therapeutic options. Novel strategies are essential in order to improve clinical outcomes. TAK-901 is an investigational small molecule kinase inhibitor that is currently being evaluated in Phase I trials. In preclinical studies, TAK-901 has demonstrated significant effects against a number of kinases with important roles in cancer including the Aurora kinases, which are key regulators of mitosis and whose overexpression in cancer promotes genetic instability, malignant pathogenesis, and drug resistance. We hypothesized that simultaneously targeting the activity of the Auroras and other oncogenic kinases with TAK-901 would disrupt AML pathogenesis. In order to test our hypothesis, we investigated the efficacy and pharmacodynamic activity of TAK-901 human AML cell lines, primary AML specimens, and an orthotopic bioluminescent disseminated mouse model of AML. TAK-901 potently diminished the viability of a panel of 8 AML cell lines as well as primary cells obtained from patients with AML. Acute exposure to TAK-901 ablated clonogenic survival, triggered the accumulation of polyploid cells, and induced apoptosis. The cytostatic and cytotoxic effects of TAK-901 were associated with significantly increased expression of the cyclin-dependent kinase inhibitor p27, growth arrest and DNA-damage-inducible 45a (GADD45a), and the BH3-only pro-apoptotic protein PUMA. Chromatin immunoprecipitation (ChIP) assays revealed that the elevation in the expression of these genes caused by administration of TAK-901 was due to increased FOXO3a transcriptional activity. The in vivo anti-leukemic activity of TAK-901 was investigated in a disseminated xenograft mouse model of AML established by intravenous injection of luciferase-expressing MV4-11 cells. IVIS Xenogen imaging was utilized to monitor disease burden throughout the study. In this mouse model, administration of TAK-901 was very well-tolerated and significantly more effective than the standard of care drug cytarabine with respect to suppressing disease progression and prolonging overall survival. Analysis of specimens collected from mice demonstrated that TAK-901 inhibited the homing of AML cells to the bone marrow microenvironment and induced AML cell apoptosis in vivo. Our collective findings indicate that TAK-901 is a novel multi-targeted kinase inhibitor that has significant preclinical activity in AML models and warrants further investigation. Disclosures: Satou: Takeda Pharmaceuticals: Employment. Hasegawa:Takeda Pharmaceuticals: Employment. Romanelli:Millennium Pharmaceuticals: Employment. de Jong:Takeda San Diego: Employment. Carew:Millennium Pharmaceuticals: Research Funding.

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 pan-class I phosphatidyl-inositol-3 kinase inhibitor NVP-BKM120 demonstrates anti-leukemic activity in acute myeloid leukemia

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Matteo Allegretti ◽  
Maria Rosaria Ricciardi ◽  
Roberto Licchetta ◽  
Simone Mirabilii ◽  
Stefania Orecchioni ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Phosphatidyl Inositol ◽  
Class I ◽  
Clinical Activity ◽  
Dependent Manner ◽  
Acute Myeloid

Abstract Aberrant activation of the PI3K/Akt/mTOR pathway is a common feature of acute myeloid leukemia (AML) patients contributing to chemoresistance, disease progression and unfavourable outcome. Therefore, inhibition of this pathway may represent a potential therapeutic approach in AML. The aim of this study was to evaluate the pre-clinical activity of NVP-BKM120 (BKM120), a selective pan-class I PI3K inhibitor, on AML cell lines and primary samples. Our results demonstrate that BKM120 abrogates the activity of the PI3K/Akt/mTOR signaling, promoting cell growth arrest and significant apoptosis in a dose- and time-dependent manner in AML cells but not in the normal counterpart. BKM120-induced cytotoxicity is associated with a profound modulation of metabolic behaviour in both cell lines and primary samples. In addition, BKM120 synergizes with the glycolitic inhibitor dichloroacetate enhancing apoptosis induction at lower doses. Finally, in vivo administration of BKM120 to a xenotransplant mouse model of AML significantly inhibited leukemia progression and improved the overall survival of treated mice. Taken together, our findings indicate that BKM120, alone or in combination with other drugs, has a significant anti-leukemic activity supporting its clinical development as a novel therapeutic agent in AML.

Selective Abrogation of STAT5 Expression Impairs the Proliferation of Acute Myeloid Leukemia Cells with Various Cytogenetic Alterations

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5024-5024
Author(s):  
Youngsoo Kim ◽  
Tianyuan Zhou ◽  
Shuling Guo ◽  
Andy Siwkowski ◽  
Donna Witchell ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Proliferative Activity ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Significant Inhibition ◽  
Acute Myeloid

Abstract STAT5 is a key common downstream mediator of multiple signaling pathways which are often dysregulated in various hematologic malignancies, including acute myeloid leukemia (AML). Due to the heterogeneity and high relapse rate of the disease, the treatment options for AML are currently limited. Although the approach of treating the disease by inhibiting upstream kinases such as FLT3 within these signaling pathways appeared promising, the clinical efficacy of these drugs as mono-therapy have been disappointing. We hypothesized that this lack of efficacy might be due to the residual STAT5 activity that is present even in the presence of these inhibitors in vivo. Therefore, abrogating the expression of the final regulator of these pathways, STAT5, might be a much more efficient way of blocking signaling, thus inhibiting the proliferation and survival of AML cells. In this study, we first investigated the role of STAT5 in the proliferation of AML cells by selectively suppressing the expression of the gene using 2nd-Generation antisense oligonucleotides (ASOs). Suppression of STAT5 following ASO treatment (>80% over control ASO) led to a significant inhibition of cell proliferation (50~70% over control ASO), a decrease in colony formation, and a modest induction of apoptosis in a range of AML lines including KG-1α, MV-4-11, and MOLM-13. STAT5 ASO treatment was highly specific for the STAT5 target and produced predictable effects on gene expression, as demonstrated by the downregulation of Pim-1 and cyclin D1, well-known STAT5 regulated genes. No changes in the expression levels of Bcl-XL, STAT1, and STAT3 were observed. Furthermore, relative anti-proliferative activity within the various AML lines correlated well with the relative levels of STAT5 activity. Interestingly, there was a strong correlation between the extent of STAT5/Pim-1 downregulation and the degree of anti-proliferation, suggesting a possible role of Pim-1 as a downstream effector of STAT5 ASO anti-proliferative activity. Studies comparing the relative effects of the STAT5 ASO inhibitor with the potent multi kinase inhibitor CEP701 in various AML cell lines demonstrated potent anti-proliferative activity for the STAT5 inhibitor in the cell lines including KG-1α that display resistance to the multi kinase inhibitor. Taken together, these results suggest that a STAT5 ASO therapeutic approach may have utility for the treatment of AML and related hematologic disorders.

scholarly journals An Advanced Preclinical Mouse Model for Acute Myeloid Leukemia Using Patients' Cells of Various Genetic Subgroups and In Vivo Bioluminescence Imaging

PLoS ONE ◽  
2015 ◽  
Vol 10 (3) ◽  
pp. e0120925 ◽  
Author(s):  
Binje Vick ◽  
Maja Rothenberg ◽  
Nadine Sandhöfer ◽  
Michela Carlet ◽  
Cornelia Finkenzeller ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Mouse Model ◽  
Myeloid Leukemia ◽  
Bioluminescence Imaging ◽  
In Vivo Bioluminescence Imaging ◽  
Acute Myeloid

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

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.

Autophagy Promotes the Survival and Therapy Resistance of Human Acute Myeloid Leukemia Cells Under Hypoxia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2236-2236 ◽  
Author(s):  
Dirkje W Hanekamp ◽  
Megan K Johnson ◽  
Scott Portwood ◽  
Joshua Acklin ◽  
Eunice S. Wang
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Tyrosine Kinase Inhibitor ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Single Agent ◽  
Xenograft Models ◽  
Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy occurring primarily in older adults. Despite high remission rates following upfront therapy, the disease eventually recurs in most patients, and overall cure rates remain only 20-30%. Preclinical studies have recently demonstrated that the marrow microenvironment in acute leukemic hosts to be intrinsically hypoxic, with AML progression associated with further hypoxia. Moreover, human AML cells and primary AML colonies cultured under hypoxia are markedly less sensitive to cytarabine chemotherapy than normoxic cells. We hypothesized that AML cells may respond to hypoxic stress and mediate chemoresistance in part by invoking autophagy, a highly regulated catabolic process by which cells evade apoptosis by degrading damaged cellular components. To test our hypothesis, we investigated the effects of two known autophagy inhibitors (bafilomycin A1 (Baf) and chloroquine (CQ)) on the sensitivity of human AML cells to various therapeutic agents under differing oxygen levels. Methods: We treated HEL (FLT-3 wildtype) and MV4-11 (FLT-3 ITD mutant) AML cells with autophagy inhibitors (Baf and CQ) alone and in combination with a chemotherapeutic drug (cytarabine (AraC), doxorubicin (Dox), decitabine (Dac)) or a tyrosine kinase inhibitor (sorafenib, SFN) under normoxic (21% O2) or hypoxic (1% O2) conditions. Apoptosis /cell death and proliferation were measured by flow cytometry for Annexin-PI and MTT assays, respectively. Autophagy was assessed by flow cytometry using Cyto-ID Green Dye (Enzo Life Sciences), fluorescent microscropy for acridine orange dye accumulation, and western blot analysis. Results: Autophagy in human ALL and AML cell lines was significantly increased following 24-72 hours of hypoxia (1% O2) as compared with normoxia and was a relatively late response to prolonged low oxygen levels (> 24 hours). Treatment with cytotoxic agents (AraC or Dox) or hypomethylating agent (Dac) resulted in a dose-dependent increases in the number of autophagic vesicles in AML cells consistent with autophagy induction. Low-doses of Baf which selectively inhibits the vacuolar H+ ATPase to prevent lysosomal acidification, and CQ, which blocks lysosome-autophagosome fusion by raising the pH of lysosomes and endosomes, both resulted in buildup of autophagic vesicles by flow cytometry consistent with inhibition of autophagic flux in human AML cells. Combination treatment with an autophagy inhibitor (Baf, CQ) and cytotoxic chemotherapy (AraC, Dox) significantly enhanced apoptosis and cell death over single agent therapy. Treatment with Baf combined with hypomethylating therapy (Dac) synergistically improved the anti-leukemic effects as compared with monotherapy (CI 0.09-0.31)(see Figure). The addition of Baf also improved cell death induced by sorafenib (SFN) on FLT-3 ITD mutant human AML cells (MV4;11) (CI 0.36-0.9) (see Figure). Single agent Baf or CQ treatment resulted in significantly higher levels of apoptosis and cell death in AML cells under hypoxia. The anti-tumor activity of almost all combination regimens was consistently improved under hypoxic versus normoxic culture conditions. In vivo CQ treatment (25-50 mg/kg i.p. daily) in preclinical human AML xenograft models significantly inhibited systemic leukemia progression as a single agent. Further experiments investigating the in vivo effects of CQ combined with other chemotherapeutic agents in preclinical AML xenograft models are ongoing. Conclusions: Our data suggest that human AML cells preferentially induce autophagy to promote survival under chronic hypoxia and following cytotoxic, hypomethylating, and FLT-3 tyrosine kinase inhibitor therapy. Strategies targeting autophagy therefore may have the potential to improve therapeutic responses and overcome chemoresistance of AML cells within the hypoxic bone marrow microenvironment. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

The BTK Inhibitor Ibrutinib Blocks SDF1/CXCR4 Mediated Migration of Acute Myeloid Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 915-915
Author(s):  
Stuart A Rushworth ◽  
Lyubov Zaitseva ◽  
Megan Y Murray ◽  
Matthew J Lawes ◽  
David J MacEwan ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Cell Trafficking ◽  
Acute Myeloid

Abstract Introduction Despite recent significant progress in the understanding of the biology of acute myeloid leukemia (AML) the clinical outcomes for the majority of patients diagnosed with AML presently remain poor. Consequently, there is an urgent need to identify pharmacological strategies in AML, which are not only effective but can be tolerated by the older, less well patient. Recently our group and others have shown that there is high Bruton’s Tyrosine Kinase (BTK) phosphorylation and RNA expression in AML. Moreover, our recent study described for the first time that ibrutinib and BTK-targeted RNA interference reduced factor-induced proliferation of both AML cell lines and primary AML blasts, as well as reducing AML blast adhesion to bone marrow stromal cells. Inhibition of BTK has been shown to regulate chronic lymphocytic leukemia, mantle cell lymphoma and multiple myeloma cell migration by inhibiting SDF1 (stromal derived factor 1) induced CXCR4 regulated cell trafficking. Here we report that in human AML ibrutinib in addition functions in a similar way to inhibit SDF1/CXCR4-mediated AML migration at concentrations achievable in vivo. Methods To investigate the role of BTK in regulating AML migration we used both pharmacological inhibitor ibrutinib and genetic knockdown using a lentivirus mediated BTK targeted miRNA in primary AML blasts and AML cell lines. We examined migration of AML blasts and AML cells to SDF-1 using Transwell permeable plates with 8.0µM pores. Western blotting was used to examine the role of SDF-1 in regulating BTK, AKT and MAPK activation in primary AML blasts. Results We initially examined the expression of CXCR4 in human AML cell lines and found that 4/4 cell lines were positive for CXCR4 expression. Next we examined the effects of ibrutinib on the migration of the AML cell lines U937, MV4-11, HL60 and THP-1 in response to SDF1. We found that ibrutinib can inhibit the migration of all AML cell lines tested. We tested the in-vitro activity of ibrutinib on SDF-1 induced migration in a spectrum of primary AML blasts from a wide age spectrum of adult patients and across a range of WHO AML subclasses and found that ibrutinib significantly inhibits primary AML blast migration (n=12). Next we found that ibrutinib can inhibit SDF-1 induced BTK phosphorylation and downstream MAPK and AKT signalling in primary AML blast. Finally to eliminate the problems associated with off target ibrutinib activity we evaluated migration of AML cells lines using genetic inhibition of BTK. The introduction of BTK-specific miRNA dramatically inhibited the expression of BTK in THP-1 and HL60 and reduced SDF1 mediated migration confirming that BTK is involved in regulating AML migration in response to SDF1. Conclusions These results reported here provide a molecular mechanistic rationale for clinically evaluating BTK inhibition in AML patients and suggests that in some AML patients the blasts count may initially rise in response to ibrutinib therapy, analgous to similar clinical observations in CLL. Disclosures No relevant conflicts of interest to declare.

scholarly journals The cytoplasmic NPM mutant induces myeloproliferation in a transgenic mouse model

Blood ◽  
2010 ◽  
Vol 115 (16) ◽  
pp. 3341-3345 ◽  
Author(s):  
Ke Cheng ◽  
Paolo Sportoletti ◽  
Keisuke Ito ◽  
John G. Clohessy ◽  
Julie Teruya-Feldstein ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Mouse Model ◽  
Transgenic Mice ◽  
Transgenic Mouse ◽  
Myeloid Leukemia ◽  
Gene Mutations ◽  
Transgenic Mouse Model ◽  
Acute Myeloid

Abstract Although NPM1 gene mutations leading to aberrant cytoplasmic expression of nucleophosmin (NPMc+) are the most frequent genetic lesions in acute myeloid leukemia, there is yet no experimental model demonstrating their oncogenicity in vivo. We report the generation and characterization of a transgenic mouse model expressing the most frequent human NPMc+ mutation driven by the myeloid-specific human MRP8 promoter (hMRP8-NPMc+). In parallel, we generated a similar wild-type NPM trans-genic model (hMRP8-NPM). Interestingly, hMRP8-NPMc+ transgenic mice developed myeloproliferation in bone marrow and spleen, whereas nontransgenic littermates and hMRP8-NPM transgenic mice remained disease free. These findings provide the first in vivo evidence indicating that NPMc+ confers a proliferative advantage in the myeloid lineage. No spontaneous acute myeloid leukemia was found in hMPR8-NPMc+ or hMRP8-NPM mice. This model will also aid in the development of therapeutic regimens that specifically target NPMc+.

scholarly journals Epigenetic silencing of UBXN8 contributes to leukemogenesis in t(8;21) acute myeloid leukemia

2021 ◽  
Author(s):  
Erna Yang ◽  
Wei Guan ◽  
Desheng Gong ◽  
Jieying Li ◽  
Caixia Han ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Fusion Protein ◽  
Cell Lines ◽  
Promoter Region ◽  
Myeloid Leukemia ◽  
Specific Inhibitor ◽  
Epigenetic Silencing ◽  
Cycle Arrest ◽  
Acute Myeloid

AbstractThe formation of the RUNX1-RUNX1T1 fusion protein, resulting from the t(8;21) translocation, is considered to be one of the initiating events of t(8;21) acute myeloid leukemia (AML). However, the mechanisms of the oncogenic mechanism of RUNX1-RUNX1T1 remain unclear. In this study, we found that RUNX1-RUNX1T1 triggers the heterochromatic silencing of UBXN8 by recognizing the RUNX1-binding sites and recruiting chromatin-remodeling enzymes to the UBXN8 promoter region. Decitabine, a specific inhibitor of DNA methylation, upregulated the expression of UBXN8 in RUNX1-RUNX1T1+ AML cell lines. Overexpression of UBXN8 inhibited the proliferation and colony-forming ability of and promoted cell cycle arrest in t(8;21) AML cell lines. Enhancing UBXN8 levels can significantly inhibit tumor proliferation and promote the differentiation of RUNX1-RUNX1T1+ cells in vivo. In conclusion, our results indicated that epigenetic silencing of UBXN8 via methylation of its promoter region mediated by the RUNX1-RUNX1T1 fusion protein contributes to the leukemogenesis of t(8;21) AML and that UBXN8 targeting may be a potential therapeutic strategy for t(8;21) AML.

scholarly journals Inhibiting Mitochondrial Complex II Exposes a Novel Metabolic Vulnerability in Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1300-1300
Author(s):  
Alessia Roma ◽  
Matthew Tcheng ◽  
Nawaz Ahmed ◽  
Sarah Walker ◽  
Preethi Jayanth ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Succinate Dehydrogenase ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Tca Cycle ◽  
Cell Fates ◽  
Reductive Carboxylation ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a hematological malignancy, characterized by an increased reliance on mitochondria-related energetic pathways including oxidative phosphorylation (OXPHOS). Consistent with this, the electron transport chain (ETC), a component of OXPHOS has been demonstrated to be a suitable anti-leukemia target, with ETC complex I inhibitors currently in clinical development. Relative to its counterparts, complex II (CII) is unique in that it directly links the ETC to the tricarboxylic acid (TCA) cycle through succinate dehydrogenase (SDH) activity. Moreover, it is the only ETC complex with elevated activity in AML, relative to normal hematopoietic samples, with indirect inhibition selectively targeting AML cells. However, direct CII inhibition in AML has not been previously investigated, nor have the mechanisms underlying the divergent fates of AML and normal cells upon CII inhibition. A genetic approach was first used to assess the effects of CII impairment on AML growth in vitro and in vivo. Using lentiviral mediated shRNA we generated AML cell lines lacking succinate dehydrogenase assembly factor 1 (Sdhaf1). Sdhaf1 knockdown suppressed CII activity, cell proliferation and clonogenic growth across all three cell lines and delayed leukemia growth in vivo. To recapitulate these effects through a pharmacological approach, we aimed to identify a novel CII inhibitor, since currently available inhibitors are only effective at high doses and are neurotoxic. Through an in silico structural screen and molecular docking study, shikonin was identified as a small molecule that selectively binds to CII. Shikonin inhibited CII activity in the AML cells lines and patient-derived samples, and selectively killed AML cells (EC 50: 1.0μM ± 0.04) while sparing normal progenitors. In murine engraftment models, shikonin (2.0-3.0 mg/kg, 3x/week for 5 weeks) significantly reduced engraftment of patient-derived AML cells but had no effect on normal hematopoiesis. To further characterize the mechanisms governing the divergent cell fates of CII inhibition, we performed stable isotope metabolic tracing using 13C 6- glucose and 13C 5, 15N 2-glutamine in patient-derived AML cells and normal mobilized peripheral blood mononuclear cells (MNCs). Both pharmacological and genetic loss of CII resulted in TCA cycle truncation by impairing oxidative metabolism of both glucose and glutamine. In Sdhaf1 knockdown and primary AML cells, this led to a depletion in steady state levels of TCA metabolites proceeding SDH. Inhibition of CII most notably suppressed levels of aspartate, a nucleotide precursor whose levels dictate the proliferative capacity of a cell under ETC dysfunction. Remarkably, MNCs maintained aspartate levels despite inhibition of CII, which was attributed to reductive carboxylation of glutamine, an alternate metabolic pathway that can regenerate TCA intermediates when OXPHOS is impaired. In contrast, while reductive carboxylation was also active in AML cells after CII inhibition, this activity was insufficient to maintain aspartate levels and resulted in metabolite depletion and cell death. Thus, loss of CII activity results in diverse cell fates whereby normal haematopoietic, but not AML cells sufficiently use reductive carboxylation of glutamine to overcome TCA cycle truncation, sustain aspartate levels and avert cell death. This is further evident through modulation of glutamine entry into the TCA cycle, where supplementation of cell-permeable α-ketoglutarate abrogated shikonin-mediated cell death while concomitant treatment with the glutaminase inhibitor CB-839, sensitized cells. Together, these results expose reductive carboxylation to support aspartate biosynthesis, as a novel metabolic vulnerability in AML that can be pharmacologically targeted through CII inhibition for clinical benefit. Disclosures Minden: Astellas: Consultancy. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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