clonogenic growth
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Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 1-2
Author(s):  
Geethu Emily Thomas ◽  
Grace Egan ◽  
Laura Garcia Prat ◽  
Botham Aaron ◽  
Veronique Voisin ◽  
...  

Abstract Mitochondrial metabolites affect epigenetic marks, but it is largely unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stem cell function in AML. Here, we discovered that the mitochondrial enzyme, Hexokinase 2 (HK2), localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stem cell function. We searched for mitochondrial enzymes moonlighting in the nucleus using 8227 AML cells, a low passage primary AML culture model arranged in a hierarchy with functionally defined stem cells in the CD34+CD38-fraction. By immunoblotting and confocal microscopy, we detected HK2 in the nucleus of 8227 cells with higher expression in the nucleus of stem cells vs bulk cells. HK2 is the first and rate-limiting enzyme in glycolysis and phosphorylates glucose. In contrast, other metabolic enzymes including phosphofructokinase, fumarase, pyruvate kinase 2, glucose phosphate isomerase, enolase1, citrate synthase, aconitase 2, and succinate dehydrogenase were not detected in the nucleus of these cells. We also detected HK2, but not these other metabolic enzymes, in the nucleus of OCI-AML2, U937, NB4 and TEX leukemia as well as 8 of 9 primary AML samples. Next, we tested whether nuclear HK2 was functionally important to maintain stem cell function in AML. We over-expressed HK2 tagged with nuclear localizing signals (PKKKRKV and PAAKRVKLD) in 8227 and NB4 leukemia cells. We confirmed selective over-expression of HK2 in the nucleus of these cells without increasing levels in the cytoplasm or mitochondria. Over-expression of nuclear HK2 increased clonogenic growth and inhibited retinoic acid-mediated cell differentiation without changing basal proliferation. Over expression of HK2 also increased engraftment of 8227 cells into mouse marrow. We evaluated the selective inhibition of nuclear HK2 by over-expressing HK2 with an outer mitochondrial localization signal while knocking down total endogenous HK2 with shRNA targeting the 3'UTR of HK2. Selective depletion of nuclear HK2 reduced clonogenic growth, increased AML differentiation after treatment with retinoic, and decreased the percentage of CD34+CD38- 8227 stem cells without changing basal proliferation. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2(D209A D657A). Nuclear kinase dead HK2 increased clonogenic growth and inhibited differentiation after retinoic acid treatment, demonstrating that HK2 maintains stemness independent of its kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry to identify proteins that interact with nuclear HK2 and identified proteins related to chromatin organization and regulation. Therefore, we examined the impact of nuclear HK2 on chromatin accessibility using ATAC-seq. Over expression of nuclear HK2 enhanced chromatin accessibility, whereas the selective knockdown of nuclear HK2 compacted chromatin. In summary, we discovered that HK2 localizes to nucleus of AML cells and functions independent of its kinase activity to maintain the stem/progenitor state of AML. Thus, we define a new role for mitochondrial enzymes in the regulation of leukemic stemness and differentiation. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Schimmer:Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock .


2020 ◽  
Vol 15 (1) ◽  
Author(s):  
Nikko Brix ◽  
Daniel Samaga ◽  
Roman Hennel ◽  
Katharina Gehr ◽  
Horst Zitzelsberger ◽  
...  

Abstract Background The clonogenic assay is a versatile and frequently used tool to quantify reproductive cell survival in vitro. Current state-of-the-art analysis relies on plating efficiency-based calculations which assume a linear correlation between the number of cells seeded and the number of colonies counted. The present study was designed to test the validity of this assumption and to evaluate the robustness of clonogenic survival results obtained. Methods A panel of 50 established cancer cell lines was used for comprehensive evaluation of the clonogenic assay procedure and data analysis. We assessed the performance of plating efficiency-based calculations and examined the influence of critical experimental parameters, such as cell density seeded, assay volume, incubation time, as well as the cell line-intrinsic factor of cellular cooperation by auto-/paracrine stimulation. Our findings were integrated into a novel mathematical approach for the analysis of clonogenic survival data. Results For various cell lines, clonogenic growth behavior failed to be adequately described by a constant plating efficiency, since the density of cells seeded severely influenced the extent and the dynamics of clonogenic growth. This strongly impaired the robustness of survival calculations obtained by the current state-of-the-art method using plating efficiency-based normalization. A novel mathematical approach utilizing power regression and interpolation of matched colony numbers at different irradiation doses applied to the same dataset substantially reduced the impact of cell density on survival results. Cellular cooperation was observed to be responsible for the non-linear clonogenic growth behavior of a relevant number of cell lines and the impairment of survival calculations. With 28/50 cell lines of different tumor entities showing moderate to high degrees of cellular cooperation, this phenomenon was found to be unexpectedly common. Conclusions Our study reveals that plating efficiency-based analysis of clonogenic survival data is profoundly compromised by cellular cooperation resulting in strongly underestimated assay-intrinsic errors in a relevant proportion of established cancer cell lines. This severely questions the use of plating efficiency-based calculations in studies aiming to achieve more than semiquantitative results. The novel approach presented here accounts for the phenomenon of cellular cooperation and allows the extraction of clonogenic survival results with clearly improved robustness.


Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3150
Author(s):  
Sascha D. Markowitsch ◽  
Patricia Schupp ◽  
Julia Lauckner ◽  
Olesya Vakhrusheva ◽  
Kimberly S. Slade ◽  
...  

Although innovative therapeutic concepts have led to better treatment of advanced renal cell carcinoma (RCC), efficacy is still limited due to the tumor developing resistance to applied drugs. Artesunate (ART) has demonstrated anti-tumor effects in different tumor entities. This study was designed to investigate the impact of ART (1–100 µM) on the sunitinib-resistant RCC cell lines, Caki-1, 786-O, KTCTL26, and A-498. Therapy-sensitive (parental) and untreated cells served as controls. ART’s impact on tumor cell growth, proliferation, clonogenic growth, apoptosis, necrosis, ferroptosis, and metabolic activity was evaluated. Cell cycle distribution, the expression of cell cycle regulating proteins, p53, and the occurrence of reactive oxygen species (ROS) were investigated. ART significantly increased cytotoxicity and inhibited proliferation and clonogenic growth in both parental and sunitinib-resistant RCC cells. In Caki-1, 786-O, and A-498 cell lines growth inhibition was associated with G0/G1 phase arrest and distinct modulation of cell cycle regulating proteins. KTCTL-26 cells were mainly affected by ART through ROS generation, ferroptosis, and decreased metabolism. p53 exclusively appeared in the KTCTL-26 cells, indicating that p53 might be predictive for ART-dependent ferroptosis. Thus, ART may hold promise for treating selected patients with advanced and even therapy-resistant RCC.


2020 ◽  
Author(s):  
Nikko Brix ◽  
Daniel Samaga ◽  
Roman Hennel ◽  
Katharina Gehr ◽  
Horst Zitzelsberger ◽  
...  

Abstract Background:The clonogenic assay is a versatile and frequently used tool to quantify reproductive cell survival in vitro. Current state-of-the-art analysis relies on plating efficiency-based calculations which assume a linear correlation between the number of cells seeded and the number of colonies counted. The present study was designed to test the validity of this assumption and to evaluate the robustness of clonogenic survival results obtained.Methods:A panel of 50 established cancer cell lines was used for comprehensive evaluation of the clonogenic assay procedure and data analysis. We assessed the performance of plating efficiency-based calculations and examined the influence of critical experimental parameters, such as cell density seeded, assay volume, incubation time, as well as the cell line-intrinsic factor of cellular cooperation by auto‑/paracrine stimulation. Our findings were integrated into a novel mathematical approach for the analysis of clonogenic survival data.Results:For various cell lines, clonogenic growth behavior failed to be adequately described by a constant plating efficiency, since the density of cells seeded severely influenced the extent and the dynamics of clonogenic growth. This strongly impaired the robustness of survival calculations obtained by the current state-of-the-art method using plating efficiency-based normalization. A novel mathematical approach utilizing power regression and interpolation of matched colony numbers at different irradiation doses applied to the same dataset substantially reduced the impact of cell density on survival results. Cellular cooperation was observed to be responsible for the non-linear clonogenic growth behavior of a relevant number of cell lines and the impairment of survival calculations. With 28/50 cell lines of different tumor entities showing moderate to high degrees of cellular cooperation, this phenomenon was found to be unexpectedly common.Conclusions:Our study reveals that plating efficiency-based analysis of clonogenic survival data is profoundly compromised by cellular cooperation resulting in strongly underestimated assay-intrinsic errors in a relevant proportion of established cancer cell lines. This severely questions the use of plating efficiency-based calculations in studies aiming to achieve more than semiquantitative results. The novel approach presented here accounts for the phenomenon of cellular cooperation and allows the extraction of clonogenic survival results with clearly improved robustness.


Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3922-3922
Author(s):  
Matthew Tcheng ◽  
Alessia Roma ◽  
Nawaz Ahmed ◽  
Richard Smith ◽  
Preethi Jayanth ◽  
...  

Targeting cell metabolism has emerged as a viable treatment strategy in acute myeloid leukemia (AML), a malignant hematological disease characterized by poor patient outcomes and limited chemotherapeutic options. Compared to the normal hematopoietic population, leukemia cells exhibit an altered phenotype characterized by increased mitochondrial mass as well as a greater reliance on oxidative phosphorylation and fatty acid oxidation (FAO) for survival. Mitochondrial FAO is a four-reaction process that catabolizes fatty acids to acetyl-CoA, generating reductive equivalents for the electron transport chain (ETC) and anaplerotic intermediates for the TCA cycle. Clinically approved FAO inhibitors, such as trimetazidine and ranolazine, are tissue-specific, targeting FAO in some tissues but affecting additional pathways in others. To better understand the potential clinical utility of targeting FAO, we systematically tested a panel of clinical and pre-clinical FAO inhibitors, reasoning that the most potent FAO inhibitor would lead to a novel anti-AML target. Avocadyne was the most potent anti-AML compound, inducing leukemic cell death (EC50: 2.5 µM) and suppressing clonogenic growth of primary samples, while sparing normal hematopoietic cells. Further, avocadyne (100mg/kg twice weekly for 5 weeks) reduced patient-derived AML cell engraftment in the bone marrow of immune deficient mice. As a component of avocatin-B, a mixture of two fatty alcohols previously determined to accumulate in the mitochondria, we confirmed that avocadyne inhibited long chain FAO using radiolabeled studies and high resolution respirometry. To identify a molecular target, avocadyne treated cells were immunoprecipitated with antibodies against each intramitochondrial enzyme involved in long chain FAO (e.g., very long acyl-CoA dehydrogenase (VLCAD; step 1) and the alpha and beta subunits of the mitochondrial trifunctional protein: HADHA, HADHB; steps 2-4). Immunoblotting and LC/MS analysis confirmed that avocadyne co-eluted with VLCAD, but not with HADHA or HADHB, confirming a direct physical interaction between avocadyne and VLCAD. VLCAD introduces a double bond to a fully saturated long chain fatty acid and reduces electron transfer flavoprotein (ETF)-bound FAD, transferring these electrons to the ETC. Using fluorescence spectrophotometry and respirometry, avocadyne directly inhibited VLCAD activity, resulting in reduced ETF-supported respiration. The activity of MCAD, an acyl-CoA dehydrogenase catalyzing medium chain fats, was not affected, suggesting avocadyne inhibits long chain FAO exclusively. Further, profiling of acyl-carnitines following avocadyne treatment also showed a pattern characteristic of long chain FAO inhibition at VLCAD. To further understand how VLCAD modulated avocadyne sensitivity, avocadyne-resistant and VLCAD knockdown cells were generated. Lentiviral knockdown of VLCAD sensitized leukemic cells to avocadyne-induced FAO inhibition and death. In contrast, cells resistant to avocadyne had increased protein expression of VLCAD but no change in other long chain FAO enzymes. With increased VLCAD-supported ETF respiration, higher concentrations of avocadyne were required to induce cell death, compared to the parental line. These results show that VLCAD expression modulated leukemic sensitivity to avocadyne. Inhibiting FAO at VLCAD triggered an adaptive metabolic switch towards glycolysis, characterized by increased extracellular acidification. This compensatory increase in glycolysis was ultimately insufficient to prevent the depletion of TCA metabolites and ATP, leading to leukemic death. In contrast, following avocadyne treatment, normal umbilical cord blood-derived mononuclear cells increased glycolytic as well as pyruvate dehydrogenase activity and had no decrease in ATP levels, cell viability, or clonogenic growth. Together, these results highlight, for the first time, VLCAD as a novel anti-AML target and further suggest the clinical utility of FAO inhibition as a potential anti-AML strategy. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Schimmer:Medivir Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy; Novartis Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy.


Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2532-2532
Author(s):  
Geethu Emily Thomas ◽  
Grace Egan ◽  
Laura Garcia Prat ◽  
Boaz Nachmias ◽  
Jordan M Chin ◽  
...  

Hematopoietic cells are arranged in a hierarchy where mature blood cells arise from stem and progenitor precursors. AML is also hierarchical with differentiated blasts arising from leukemic stem/progenitor cells. Recent studies show that metabolites can affect epigenetic marks; however, it is unknown whether metabolic enzymes can directly localize to the nucleus to regulate stemness in AML and normal hematopoietic cells. Here, we discovered that the mitochondrial enzyme, Hexokinase 2, localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stemness. Metabolic enzymes that localize to nucleus of stem cells were identified by evaluating stem and bulk fractions of OCI-AML-8227 leukemia cells, which are arranged in a hierarchy with functionally defined stem cells. We separated OCI-AML-8227 cells into CD34+38- and CD34-38+ populations by FACS and prepared nuclear and cytoplasmic lysates. Immunoblotting of the lysates revealed that the metabolic enzyme Hexokinase 2 (HK2) was increased in the nuclear fraction of 8227 stem cells compared to bulk cells. In contrast, other mitochondrial enzymes such as Enolase1, Aconitase2, and Succinate Dehydrogenase A & B, were not detected in the nuclear lysates. HK2 is an outer mitochondrial membrane protein that phosphorylates glucose to glucose-6-phosphate, initiating glycolysis. We confirmed nuclear HK2 in OCI-AML-8227 stem cells by confocal microscopy and also demonstrated nuclear HK2 in AML cell lines (OCI-AML2, NB4, K563, and MV411) and in 7 of 9 primary AML samples. We FACS sorted normal cord blood into populations of stem/progenitor (HSC, MPP, MLP, CMP, GMP and MEP) and differentiated (Monocytes, Granulocytes, B, T, and NK) cells. The localization of HK2 in these cells was analysed and quantified by immunofluorescence. Nuclear HK2 was detected in the stem/progenitor cells and progressively declined to minimal levels as cells matured. Next, we explored mechanisms that regulate nuclear localization of HK2. AKT-mediated phosphorylation of HK2 promoted localization to mitochondria while inhibition of phosphorylation increased its nuclear levels. Moreover, the nuclear import of HK2 was dependent on IPO5, a member of b-importin family that imports protein to the nucleus; CRM1 was responsible for HK2 nuclear export. We tested whether the nuclear localization of HK2 was functionally important to maintain stemness. We overexpressed HK2 tagged with nuclear localizing signals (PKKKRKV or PAAKRVKLD) in 8227 and NB4 leukemia cells. Selective overexpression of HK2 in the nucleus did not alter the rate of proliferation of the cells, however there was enhanced clonogenic growth and inhibition of retinoic acid-mediated cell differentiation. Conversely, we selectively reduced nuclear HK2 by expressing HK2 with an outer mitochondrial localization signal while knocking down endogenous HK2 with shRNA targeting the 3'UTR of HK2. Selective depletion of nuclear HK2 in AML cells did not alter growth rate, but did reduce clonogenic growth and increased differentiation after treatment with retinoic acid. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2(D209A D657A). Nuclear kinase dead HK2 increased clonogenic growth and inhibited differentiation after retinoic acid treatment, demonstrating that HK2 maintains stemness independent of kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry to identify proteins that interact with nuclear HK2. A top hit in our screen was Exonuclease 3'-5' domain containing 2 (EXD2), involved in DNA repair. Of note, DNA damage induces differentiation of AML cells. In 8227 cells, nuclear EXD2 was higher in the stem cell fraction compared to the bulk fraction. Moreover, knockdown of EXD2 reduced AML growth, clonogenic growth and decreased nuclear HK2 levels. Finally, nuclear HK2 overexpression conferred resistance to the PARP inhibitor, olaparib. In summary, we discovered that unphosphorylated HK2 localizes to the nucleus in malignant and normal hematopoietic stem cells. Through mechanisms independent of its kinase function, nuclear HK2 maintains AML cells in their stem/progenitor state potentially by regulating DNA damage and repair. Thus, we define a new role for a mitochondrial enzyme in the regulation of stemness and differentiation. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Schimmer:Medivir Pharmaceuticals: Research Funding; Otsuka Pharmaceuticals: Consultancy; Novartis Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy.


Cancers ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 276 ◽  
Author(s):  
Marlena Beyreis ◽  
Martin Gaisberger ◽  
Martin Jakab ◽  
Daniel Neureiter ◽  
Katharina Helm ◽  
...  

Biliary tract cancer is a devastating disease with limited therapeutic options. The involvement of cancer stem cells in biliary tract cancer is likely. Napabucasin is a previously described cancer stem cell inhibitor that is currently being used in clinical trials. However, data regarding napabucasin and biliary tract cancer are not available yet. We tested the general cytotoxic effect of napabucasin on a comprehensive biliary tract cancer in vitro model, using resazurin assay and Annexin V/7-AAD staining. The effect of napabucasin on functional cancer stem cell characteristics was analyzed via soft agar assay, aldehyde-dehydrogenase-1 assay, measurement of surface CD326 expression, and measurement of clonogenic growth. The evaluation of the effect of napabucasin on cancer stem cell protein and gene expression was performed using Western blot and reverse transcription-PCR-based human cancer stem cell array. Napabucasin showed a concentration- and cell line-dependent cytotoxic effect, and increased the apoptotic and necrotic cell fractions. Treatment with napabucasin significantly reduced the formation of tumor spheres and clonogenic growth, as well as CD326 surface expression. Expression of cancer stem cell markers were reduced following napabucasin treatment on the protein and mRNA levels. Our study provides first data regarding napabucasin as a promising substance for the treatment of biliary tract cancer.


Pancreas ◽  
2019 ◽  
Vol 48 (1) ◽  
pp. 94-98 ◽  
Author(s):  
Joey H. Li ◽  
Ross H. McMillan ◽  
Asma Begum ◽  
Christian B. Gocke ◽  
William Matsui

2018 ◽  
Vol 15 (4) ◽  
pp. 239-248 ◽  
Author(s):  
DIPANJAN BASU ◽  
CLÁUDIA M. SALGADO ◽  
BRUCE BAUER ◽  
YASMIN KHAKOO ◽  
JANKI R. PATEL ◽  
...  

2017 ◽  
Vol 39 (2) ◽  
pp. 242-251 ◽  
Author(s):  
Xiangning Bu ◽  
Jiro Kato ◽  
Julie A Hong ◽  
Maria J Merino ◽  
David S Schrump ◽  
...  

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