Dataset Information

Cooperative treatment effectiveness of VE821 and AUY922 in Ewing’s sarcoma cells

ABSTRACT: Ewing's sarcoma (ES) is the second most frequent bone cancer during childhood and adolescence originating from a translocation between the EWSR1 and the FLI1 gene, which inactivates BRCA-mediated DNA damage response. Both ATR and HSP90 inhibitors are promising new candidates in the field of ES treatment. We show that the HSP90 inhibitor AUY922 alone induced DNA damages, apoptosis and ER stress, while reducing the abundance of DNA repair proteins. The combinations of the ATR inhibitor VE821 and AUY922 (AUY-VE) led to strong apoptosis induction in ES cell lines, originating from a common set of molecular targets and independent of their p53 status, yet based on different molecular mechanisms. P53 wild-type ES cells activated pro-apoptotic gene transcription and underwent mitochondria-mediated apoptosis. P53 null ES cells, however, accumulated higher level of DNA damage, ER stress and autophagy, finally leading to apoptosis. Moreover, AUY-VE treatment compromised ES cell vitality due to impaired PI3K/AKT/mTOR signaling. Unexpectedly, the combinations of AUY922 with the ATM inhibitor, KU55933 failed to kill ES cells and showed protective effects compared to AUY-VE. In summary, we provide mechanistic insights into the effective ES cell killing by ATR and HSP90 inhibitor combinations, which also offers a targetable approach for BRCA-deficient tumors, irrespective of their p53 status and may give rise to new therapeutic strategies.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

DISEASE(S): Peripheral Primitive Neuroectodermal Tumor

SUBMITTER: Joanna Kirkpatrick

LAB HEAD: Zhao-Qi Wang

PROVIDER: PXD021562 | Pride | 2021-09-09

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	190802_EKS_CM_DDA_AUY922VE821_15.raw	Raw
	190802_EKS_CM_DDA_AUY922_5.raw	Raw
	190802_EKS_CM_DDA_VE821_11.raw	Raw
	190802_EKS_CM_DDA_ctrl_1.raw	Raw
	190802_EKS_CM_DIA_AUY922VE821_13.raw	Raw

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Publications

Cooperative treatment effectiveness of ATR and HSP90 inhibition in Ewing's sarcoma cells.

Marx Christian C Schaarschmidt Marc U MU Kirkpatrick Joanna J Marx-Blümel Lisa L Halilovic Melisa M Westermann Martin M Hoelzer Doerte D Meyer Felix B FB Geng Yibo Y Buder Katrin K Schadwinkel Hauke M HM Siniuk Kanstantsin K Becker Sabine S Thierbach René R Beck James F JF Sonnemann Jürgen J Wang Zhao-Qi ZQ

Cell & bioscience 20210320 1

<h4>Introduction</h4>Ewing's sarcoma is an aggressive childhood malignancy whose outcome has not substantially improved over the last two decades. In this study, combination treatments of the HSP90 inhibitor AUY922 with either the ATR inhibitor VE821 or the ATM inhibitor KU55933 were investigated for their effectiveness in Ewing's sarcoma cells.<h4>Methods</h4>Effects were determined in p53 wild-type and p53 null Ewing's sarcoma cell lines by flow cytometric analyses of cell death, mitochondrial ...[more]

PMID: 33743824

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Project description:Comprehensive Model Description Overview This model simulates the cellular response to DNA damage under conditions of ATM/ATR deficiency—such as in ataxia telangiectasia—and predicts the potential benefits of drug repositioning with Omaveloxolone and spermidine. It integrates several interconnected modules: DNA damage production and repair, ATM/ATR–p53 signaling, NRF2/KEAP1 regulation, spermidine-induced autophagy (with additional positive modulation of ATR signaling), and downstream p53 effectors involved in cell fate decisions. 1. DNA Damage Production and Repair Module Basal DNA Damage Production: DNA damage is continuously generated at a constant rate, representing both endogenous processes and low-level environmental stress. This reaction acts as a source term for the DNA_damage species. Repair Pathways: Several mechanisms work to reduce DNA damage: ATM-Mediated Repair: Active ATM facilitates the direct repair of DNA damage. ATR-Mediated Repair: Active ATR, in cooperation with CHK1, repairs DNA damage. NRF2-Mediated Repair: Active NRF2 triggers antioxidant responses that reduce DNA damage. Autophagy-Mediated Repair: The autophagy pathway, induced by spermidine, repairs DNA damage in a saturable manner (modeled using Michaelis–Menten kinetics). This ensures that even a significant pulse of damage is only partially repaired, leaving a nonzero steady-state level. 2. ATM/ATR–p53 Signaling Module ATM and ATR Activation: DNA damage, along with a positive input from TOPBP1, converts inactive ATM and ATR to their active forms. Under ATM/ATR-deficient conditions (as in ataxia telangiectasia), the activation is severely reduced, resulting in higher levels of unrepaired DNA damage. p53 Activation: Both ATM_active and ATR_active phosphorylate and activate p53. Active p53 then acts as a central integrator of the DNA damage signal. Regulatory Proteins: HDAC4: HDAC4 cycles between inactive and active states. Its active form can attenuate p53 activity. TOPBP1: TOPBP1 is activated in a DNA damage–dependent manner and helps promote ATR activation. CK2: CK2 is a constitutively active kinase that supports TOPBP1 activation. 3. NRF2/KEAP1 Module and Omaveloxolone NRF2 Activation: DNA damage stimulates the conversion of NRF2_inactive to NRF2_active. Active NRF2 enhances antioxidant defenses and promotes DNA repair. KEAP1-Mediated Degradation: Under normal conditions, KEAP1 targets NRF2_inactive for degradation, keeping NRF2 activity low. Modulation by Omaveloxolone: Omaveloxolone is modeled as a constant (boundary) species. It inhibits KEAP1’s activity, thereby reducing NRF2 degradation. This shifts the balance in favor of increased NRF2_active, which boosts the cell’s antioxidant and repair responses. 4. Spermidine-Induced Autophagy and Positive Modulation of ATR Signaling Spermidine is modeled as a constant species and has two major effects: Induction of Autophagy: Spermidine promotes the conversion of Autophagy_inactive to Autophagy_active. The active autophagy machinery repairs DNA damage using a saturable (Michaelis–Menten) mechanism. This pathway helps clear DNA damage but is limited by its maximum capacity. Enhancement of ATR Signaling: Spermidine also positively modulates ATR signaling by enhancing the CK2-mediated activation of TOPBP1. In the model, the effective rate of TOPBP1 activation is increased proportionally to the concentration of spermidine. This is represented by a modulation factor, where the effective rate constant for TOPBP1 activation is multiplied by a term that increases with spermidine concentration. The boosted TOPBP1_active level in turn enhances ATR activation, partially compensating for the loss of ATM/ATR function. 5. Downstream p53 Targets: Cell Fate Decisions Activated p53 regulates cell fate by inducing the production of several downstream effectors: p21: p21 is produced in response to p53 activation and is associated with cell cycle arrest and senescence. Its production is balanced by a degradation reaction. BAX and PUMA: Both BAX and PUMA are pro-apoptotic proteins. They are produced in proportion to p53_active and are subsequently degraded. Their relative levels help determine whether the cell will undergo apoptosis. 6. Reaction Kinetics (Qualitative Overview) Mass-Action and Michaelis–Menten Kinetics: Most reactions in the model are governed by mass-action kinetics. For example, activation and deactivation reactions (such as ATM activation by DNA damage or p53 activation by ATM/ATR) follow simple proportional relationships. Saturable Repair Processes: The autophagy-mediated DNA repair reaction uses Michaelis–Menten kinetics to reflect a saturation effect; when DNA damage is high, the repair rate approaches a maximum value, ensuring that not all damage is cleared immediately. Modulatory Effects: Spermidine increases the effective rate of TOPBP1 activation (and hence ATR activation) via a modulation term that is proportional to its concentration. Omaveloxolone reduces the degradation rate of NRF2 by inhibiting KEAP1. Biological Implications and Model Utility ATM/ATR Deficiency: In ataxia telangiectasia, ATM/ATR functions are diminished, leading to increased DNA damage. The model simulates this scenario and examines how alternative pathways (NRF2-mediated repair, autophagy, and enhanced ATR signaling via spermidine) help mitigate the damage. Drug Repositioning Predictions: The model can be used to predict the outcomes of repositioning drugs: Omaveloxolone is expected to stabilize NRF2, enhancing antioxidant defenses and reducing DNA damage. Spermidine acts dually by promoting autophagy (with a saturable repair capacity) and by boosting ATR signaling via TOPBP1 activation. Cell Fate Outcomes: The downstream p53 targets (p21, BAX, and PUMA) serve as markers for cell cycle arrest/senescence and apoptosis. By simulating the dynamics of these proteins, the model can predict whether a cell is likely to survive, arrest its cycle, or undergo apoptosis based on the integrated response to DNA damage and drug interventions. Conclusion This detailed and comprehensive model integrates multiple layers of the cellular response to DNA damage in ATM/ATR-deficient conditions. It includes upstream damage sensing, multiple repair pathways (including direct kinase-mediated repair, antioxidant responses via NRF2, and autophagy-mediated repair), and downstream effectors that determine cell fate. Omaveloxolone and spermidine are modeled to specifically modulate the NRF2/KEAP1 pathway and ATR signaling (via TOPBP1 activation), respectively. This framework provides a robust platform for exploring drug repositioning strategies and predicting cellular outcomes in diseases such as ataxia telangiectasia.