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.

钙网蛋白 (CALR) 突变是原发性血小板增多症和原发性骨髓纤维化中第二常见的驱动突变。为了确定针对CALR突变的骨髓增生性肿瘤的潜在靶向疗法，我们使用高通量药物筛选来寻找选择性抑制CALR突变细胞生长的小分子。我们使用携带CALR突变的同基因细胞系研究了 89172 种化合物，并确定了靶向 ATR-CHK1 途径的化合物的合成致死率。这些化合物的选择性抑制作用在CALR突变和野生型细胞的共培养测定中得到验证。在测试的化合物中，CHK1 抑制剂有效地耗尽了 CALR突变细胞，使野生型细胞随着时间的推移在共培养物中占优势。CALR缺陷细胞和JAK2V617F突变细胞均未表现出对 ATR-CHK1 抑制的超敏反应，因此表明突变 CALR 对致癌激活具有特异性。CHK1 抑制剂在CALR突变细胞中诱导复制应激，通过升高的 γH2AX 泛核染色和 RPA2 过度磷酸化揭示。由于 DNA 复制不完全，这伴随着 S 期细胞周期停滞。转录组学和磷酸蛋白质组学分析揭示了由致癌 CALR 引起的复制压力特征，表明对 CHK1 扰动的内在脆弱性。这项研究揭示了 ATR-CHK1 通路作为CALR的潜在治疗靶点 变异的造血细胞。