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High-throughput drug screening identifies the ATR-CHK1 pathway as a therapeutic vulnerability of CALR mutated
Ruochen Jia1,2, Leon Kutzner2, Anna Koren2
1Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria.
Abstract:
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 ATR-CHK1 pathway. The selective inhibitory effect of these compounds was validated in a co-culture assay of CALR mutated and wild-type cells. Of the tested compounds, CHK1 inhibitors potently depleted CALR mutated cells, allowing wild-type cell dominance in the co-culture over time. Neither CALR deficient cells nor JAK2V617F mutated cells showed hypersensitivity to ATR-CHK1 inhibition, thus suggesting specificity for 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. This was accompanied by S-phase cell cycle arrest due to incomplete DNA replication. Transcriptomic and phosphoproteomic analyses revealed a replication stress signature caused by oncogenic CALR, suggesting an intrinsic vulnerability to CHK1 perturbation. This study reveals the ATR-CHK1 pathway as a potential therapeutic target in CALR mutated hematopoietic cells.
Insights
Targeting the ATR-CHK1 pathway with CHK1 inhibitors selectively eliminates calreticulin (CALR) mutated cells. This identifies a potential targeted therapy for CALR-driven myeloproliferative neoplasms.
Area of Science:
- Hematology
- Molecular Biology
- Oncology
Background:
- Calreticulin (CALR) mutations are common drivers in essential thrombocythemia and primary myelofibrosis.
- Identifying targeted therapies for CALR-mutated myeloproliferative neoplasms (MPNs) is crucial.
Purpose of the Study:
- To discover small molecules that selectively inhibit the growth of CALR-mutated cells.
- To explore the ATR-CHK1 pathway as a potential therapeutic target in CALR-mutated MPNs.
Main Methods:
- High-throughput drug screening of 89,172 compounds using isogenic cell lines with CALR mutations.
- Validation of selective inhibition in co-culture assays with CALR-mutated and wild-type cells.
- Analysis of replication stress markers (γH2AX, RPA2 phosphorylation) and cell cycle progression.
Main Results:
- Compounds targeting the ATR-CHK1 pathway demonstrated synthetic lethality with CALR mutations.
- CHK1 inhibitors potently depleted CALR-mutated cells, leading to wild-type cell dominance.
- Oncogenic CALR creates a replication stress signature, rendering cells vulnerable to CHK1 inhibition.
- Specificity was observed for CALR mutations, not for CALR deficiency or JAK2V617F mutations.
Conclusions:
- The ATR-CHK1 pathway is a potential therapeutic target for CALR-mutated hematopoietic cells.
- CHK1 inhibitors show promise for selectively treating CALR-driven MPNs.
- Understanding CALR-induced replication stress informs targeted therapy development.
