Contributions
Abstract: S823
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 11:30 - 11:45
Location: Room A6
Background
Loss of function mutations of Enhancer of Zeste (Drosophila) homolog 2 (EZH2), the enzymatic component of Polycomb Repressive Complex 2 (PRC2), are recurrently found in T cell - Acute Lymphoblastic Leukaemia (T-ALL) and deregulation of PRC2 has been strongly associated with the pathogenesis of T-ALL. Moreover, PRC2 mutations are independent predictors of poor survival across all T-ALL subtypes and EZH2 mutations alone are associated with a 60% risk of relapse in paediatric T-ALL, emphasizing the need for identification of drugs that selectively target PRC2 deficient leukemic cells.
Aims
Our aim was to identify specific synthetic lethal vulnerabilities induced by EZH2 deficiency, that can be exploited using drugs targeting other dependency pathways, potentially resulting in a clinically meaningful therapeutic strategy in T-ALL.
Methods
We used double-nicking CRISPR Cas9 to generate isogenic Jurkat T-ALL cells, with and without EZH2-inactivating mutations, and performed a cell-based drug screening using 220 well-characterized compounds, enriched for drugs targeting epigenetic, cell cycle and DNA repair machinery.
Results
Strikingly, we observed that EZH2-knock out (KO) cells are hypersensitive to checkpoint kinase 1 (CHK1) inhibition by the clinical-grade small molecule MK8776, showing an IC50 reduction of over 2-fold. To determine the specificity of this observation, we tested two different EZH2-KO cells against other CHK1 inhibitors (LY2603618 and CHIR-124) obtaining a similar sensitisation to CHK1 inhibition. Moreover, when we transduced the cells with shRNAs that specifically targeted CHK1 expression, we observed a significant reduction of cell growth in EZH2-KO cells compared to the parental control. Furthermore, pharmacological inhibition of EZH2 methyltransferase activity by its potent inhibitor GSK126 in other human T-ALL cell lines resulted in sensitization to CHK1 inhibition by MK8776. Notably, MK8776 resulted in apoptosis of EZH2-deficient cells as determined by Annexin V-PI staining.
To investigate the mechanism, gene expression of two EZH2-KO Jurkat clones was compared to their isogenic parental control by RNA-seq. EZH2-KO cells showed a marked upregulation of genes associated with human ETP-ALL including LYL1, MYCN, KIT and HHEX, a finding that could be recapitulated with GSK126 treatment. Gene set enrichment analysis (GSEA) showed significant changes of expression in MYC target genes. Importantly, MYCN protein was markedly upregulated in both EZH2-KO and GSK126 treated cells. We speculated that increased MYCN expression induces high levels of replication stress and increased dependency on CHK1 for cell survival. In fact, EZH2 deficient cells showed a significant reduction in replication fork speed, compared with WT control, as determined by DNA fiber assay. In line with this observation, EZH2-KO cells exhibited markedly elevated levels of the ssDNA binding protein RPA and MK8776 treatment resulted in higher yH2AX expression in these cells.
Conclusion
Our findings suggest that specific inhibition of the CHK1 pathway can preferentially target PRC2-deficient leukemic cells, providing a potentially less toxic and more effective treatment for this high-risk subgroup of patients.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): Screening, T-ALL, Drug sensitivity, EZH2
Abstract: S823
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 11:30 - 11:45
Location: Room A6
Background
Loss of function mutations of Enhancer of Zeste (Drosophila) homolog 2 (EZH2), the enzymatic component of Polycomb Repressive Complex 2 (PRC2), are recurrently found in T cell - Acute Lymphoblastic Leukaemia (T-ALL) and deregulation of PRC2 has been strongly associated with the pathogenesis of T-ALL. Moreover, PRC2 mutations are independent predictors of poor survival across all T-ALL subtypes and EZH2 mutations alone are associated with a 60% risk of relapse in paediatric T-ALL, emphasizing the need for identification of drugs that selectively target PRC2 deficient leukemic cells.
Aims
Our aim was to identify specific synthetic lethal vulnerabilities induced by EZH2 deficiency, that can be exploited using drugs targeting other dependency pathways, potentially resulting in a clinically meaningful therapeutic strategy in T-ALL.
Methods
We used double-nicking CRISPR Cas9 to generate isogenic Jurkat T-ALL cells, with and without EZH2-inactivating mutations, and performed a cell-based drug screening using 220 well-characterized compounds, enriched for drugs targeting epigenetic, cell cycle and DNA repair machinery.
Results
Strikingly, we observed that EZH2-knock out (KO) cells are hypersensitive to checkpoint kinase 1 (CHK1) inhibition by the clinical-grade small molecule MK8776, showing an IC50 reduction of over 2-fold. To determine the specificity of this observation, we tested two different EZH2-KO cells against other CHK1 inhibitors (LY2603618 and CHIR-124) obtaining a similar sensitisation to CHK1 inhibition. Moreover, when we transduced the cells with shRNAs that specifically targeted CHK1 expression, we observed a significant reduction of cell growth in EZH2-KO cells compared to the parental control. Furthermore, pharmacological inhibition of EZH2 methyltransferase activity by its potent inhibitor GSK126 in other human T-ALL cell lines resulted in sensitization to CHK1 inhibition by MK8776. Notably, MK8776 resulted in apoptosis of EZH2-deficient cells as determined by Annexin V-PI staining.
To investigate the mechanism, gene expression of two EZH2-KO Jurkat clones was compared to their isogenic parental control by RNA-seq. EZH2-KO cells showed a marked upregulation of genes associated with human ETP-ALL including LYL1, MYCN, KIT and HHEX, a finding that could be recapitulated with GSK126 treatment. Gene set enrichment analysis (GSEA) showed significant changes of expression in MYC target genes. Importantly, MYCN protein was markedly upregulated in both EZH2-KO and GSK126 treated cells. We speculated that increased MYCN expression induces high levels of replication stress and increased dependency on CHK1 for cell survival. In fact, EZH2 deficient cells showed a significant reduction in replication fork speed, compared with WT control, as determined by DNA fiber assay. In line with this observation, EZH2-KO cells exhibited markedly elevated levels of the ssDNA binding protein RPA and MK8776 treatment resulted in higher yH2AX expression in these cells.
Conclusion
Our findings suggest that specific inhibition of the CHK1 pathway can preferentially target PRC2-deficient leukemic cells, providing a potentially less toxic and more effective treatment for this high-risk subgroup of patients.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): Screening, T-ALL, Drug sensitivity, EZH2