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

Ruochen Jia,Leon Kutzner,Christoph Bock,Joanna I Loizou,Anna Koren,Michael Schuster,Kathrin Runggatscher,Peter Májek,Stefan Kubicek,Robert Kralovics,André C Müller

doi:10.1038/s41408-021-00531-2

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.

Highlights

Classical myeloproliferative neoplasms (MPNs) are a group of clonal hematopoietic diseases that are driven by somatic mutations acquired by hematopoietic stem/progenitor cells
ATR and checkpoint kinase 1 (CHK1) inhibitors selectively target CALR mutated cells To discover novel drug candidates for CALR mutated MPN, we screened a library of 89,172 compounds against UT-7/Tpo CALR wild type and mutated cell lines
We showed tion of γ-H2AX induced by MK8776 in CD34+ cells derived from a that CHK1 inhibitor at the concentration used in this assay did not CALR patient, whereas γ-H2AX remained at the basal level kill most of the wild-type cells (Supplementary figure 3)

Summary

Introduction

Classical myeloproliferative neoplasms (MPNs) are a group of clonal hematopoietic diseases that are driven by somatic mutations acquired by hematopoietic stem/progenitor cells. Three phenotypic driver mutations have been uncovered in MPNs including mutations in JAK2, MPL, and CALR genes, which collectively are genetic determinants of more than 95% of MPN cases [1,2,3,4,5,6,7,8]. Recent research revealed additional molecular features of cells with mutations in CALR, such as an elevated unfolded protein response and abnormalities in calcium signaling [19, 20]. None of these features have been exploited sufficiently to develop novel drug treatments. Our group demonstrated the possibility of targeting the glycan-binding pocket of mutant CALR protein by small molecules as a chemotherapeutic approach [21]

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Conclusion