Dataset Information

Calreticulin mutated hematopoietic cells are vulnerable to ATR-CHK1 pathway inhibition

ABSTRACT: Disease frameshift mutations of calreticulin (CALR) are the second most prevalent driver mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF). To identify potential targeted therapies for CALR mutated myeloproliferative neoplasms, we aimed to search for small molecule drugs that selectively inhibit the growth of CALR mutated cells using high-throughput drug screening. We investigated 89,172 compounds using isogenic cell lines and identified several hits targeting the ATR-CHK1 pathway. The selective inhibitory effect of these candidate compounds was validated in a co-culture assay of CALR mutated and wild type cells. Of the tested hit compounds, CHK1 inhibitors potently depleted CALR mutated cells, allowing CALR wild type cell dominance in the co-culture over time. Neither CALR deficient cells nor JAK2V617F mutated cells showed hypersensitivity to the drug treatment, suggesting that the vulnerability to ATR-CHK1 inhibition is specifically caused by the oncogenic activation by the mutant CALR. CHK1 inhibitors induced replication stress in CALR mutated cells revealed by elevated pan-nuclear staining for γH2AX and hyperphosphorylation of RPA2. They also promoted S-phase cell cycle arrest and blocked the completion of DNA replication in CALR mutated cells. Transcriptomic and phosphoproteomic analyses revealed a replication stress signature caused by the oncogenic CALR mutation, suggesting an intrinsic vulnerability of these cells to CHK1 perturbation. This study indicates that ATR-CHK1 pathway is a potential therapeutic target in CALR mutated hematopoietic cells.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Blood Cell, Blood

DISEASE(S): Myeloproliferative Neoplasm

SUBMITTER: Andre Mueller

LAB HEAD: Robert Kralovics

PROVIDER: PXD026803 | Pride | 2021-09-07

REPOSITORIES: Pride

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Publications

High-throughput drug screening identifies the ATR-CHK1 pathway as a therapeutic vulnerability of CALR mutated hematopoietic cells.

Jia Ruochen R Kutzner Leon L Koren Anna A Runggatscher Kathrin K Májek Peter P Müller André C AC Schuster Michael M Bock Christoph C Loizou Joanna I JI Kubicek Stefan S Kralovics Robert R

Blood cancer journal 20210731 7

Mutations of calreticulin (CALR) are the second most prevalent driver mutations in essential thrombocythemia and primary myelofibrosis. To identify potential targeted therapies for CALR mutated myeloproliferative neoplasms, we searched for small molecules that selectively inhibit the growth of CALR mutated cells using high-throughput drug screening. We investigated 89 172 compounds using isogenic cell lines carrying CALR mutations and identified synthetic lethality with compounds targeting the A ...[more]

PMID: 34333533

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Project description:The cell division cycle is tightly controlled in normal cells. Cancer cells are characterized by deregulation in cell division cycle, which is associated with increased DNA replication and elevated cellular proliferation. Recently, genetic studies indicate that cyclin dependent kinases CDK2, CDK4 and CDK6 are not essential for the mammalian cell cycle, instead, they are only required for the proliferation of specific cell types.Thus, targeting the activities of CDK2, CDK4 and CDK6 may be a promising approach for cancer treatment. Rocaglamides (Roc) (= Flavaglines), derived from the traditional Chinese medicinal plant Aglaia, are a group of naturally occurring herbal chemicals characterized by a cyclopenta[b]benzofurans skeleton. A number of Roc derivatives have been found to have potent inhibitory effects on tumor growth with IC50 concentrations at the nM rages. Roc-mediated inhibition of proliferation was first found to be associated with inhibition of protein synthesis in 1998. In this study, using the representative Roc-A compound we revealed a novel mechanistic function of Roc. We show that Roc-A induces rapid activation of ATM (ataxiatelangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related) kinases. Activated ATM and ATR in turn activate the downstream checkpoint kinases CHK1 and CHK2. The latter then phosphorylate Cdc25A that leads to a phosphorylation-mediated degradation of Cdc25A. Usually, ATR and ATM are activated in response to DNA damage. However, in this study we show that Roc-A does not directly induce DNA breaks. To elucidate the molecular mechanism by which Roc-A activates ATM and ATR we performed a time-resolved microarray analysis to compare Roc-A-inducible genes in malignant vs. normal T lymphocytes. Based on the dynamic gene responses we found that Roc-A stimulates in Jurkat, but not in normal T lymphocytes, a set of genes responsive to DNA damage and nucleotide excision repair. This finding provides a hint of the molecular bases for Roc-induced activation of ATM and ATR. This finding also revealed a new scope for cancer treatment using Roc-A. The isolation time points for Roc A treated D6 T-cells were recorded at t=[1,2,3,4,6,8,10,14,20,28,36] hours after stimulation. Post-stimulation time points for Jurkat cells are t=[0.5,1,2,3,4,5,6,7,8,10,12,14,16,20,24,28,32,36]. Unstimulated control time points were taken at t=[0,4,10,16,24,36] hours for Jurkat cells at t=[0,6,14,24,36] hours.

Dataset Information

Calreticulin mutated hematopoietic cells are vulnerable to ATR-CHK1 pathway inhibition

Publications

High-throughput drug screening identifies the ATR-CHK1 pathway as a therapeutic vulnerability of CALR mutated hematopoietic cells.

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