Cohesin mutations alter DNA damage repair and chromatin structure and create therapeutic vulnerabilities in MDS/AML
Zuzana Tothova, … , Job Dekker, Benjamin L. Ebert
Zuzana Tothova, … , Job Dekker, Benjamin L. Ebert
Published December 22, 2020
Citation Information: JCI Insight. 2021;6(3):e142149. https://doi.org/10.1172/jci.insight.142149.
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Research Article Hematology Oncology

Cohesin mutations alter DNA damage repair and chromatin structure and create therapeutic vulnerabilities in MDS/AML

Abstract

The cohesin complex plays an essential role in chromosome maintenance and transcriptional regulation. Recurrent somatic mutations in the cohesin complex are frequent genetic drivers in cancer, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Here, using genetic dependency screens of stromal antigen 2–mutant (STAG2-mutant) AML, we identified DNA damage repair and replication as genetic dependencies in cohesin-mutant cells. We demonstrated increased levels of DNA damage and sensitivity of cohesin-mutant cells to poly(ADP-ribose) polymerase (PARP) inhibition. We developed a mouse model of MDS in which Stag2 mutations arose as clonal secondary lesions in the background of clonal hematopoiesis driven by tet methylcytosine dioxygenase 2 (Tet2) mutations and demonstrated selective depletion of cohesin-mutant cells with PARP inhibition in vivo. Finally, we demonstrated a shift from STAG2- to STAG1-containing cohesin complexes in cohesin-mutant cells, which was associated with longer DNA loop extrusion, more intermixing of chromatin compartments, and increased interaction with PARP and replication protein A complex. Our findings inform the biology and therapeutic opportunities for cohesin-mutant malignancies.

Authors

Zuzana Tothova, Anne-Laure Valton, Rebecca A. Gorelov, Mounica Vallurupalli, John M. Krill-Burger, Amie Holmes, Catherine C. Landers, J. Erika Haydu, Edyta Malolepsza, Christina Hartigan, Melanie Donahue, Katerina D. Popova, Sebastian Koochaki, Sergey V. Venev, Jeanne Rivera, Edwin Chen, Kasper Lage, Monica Schenone, Alan D. D’Andrea, Steven A. Carr, Elizabeth A. Morgan, Job Dekker, Benjamin L. Ebert

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Figure 1

Identification of DNA replication and damage repair as a dependency in STAG2-mutant cells.

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Identification of DNA replication and damage repair as a dependency in S...
(A) Volcano plot depicting differential dependencies in STAG2-mutant versus WT cells. Composite data for 5 STAG2-mutant cell lines (U937 STAG2-KO2, STAG2-KO3, KOC5, KOD5C, KOG8B) and 6 STAG2-WT cell lines (U937 WT-1, WT-2, NCB1, NCB12, NCB2A, NCC4) are shown. Respective sets of genes representing dependency in STAG2-mutant over WT cells with FDR < 5% are shown in color. (B) Cohesion defect analysis in WT, STAG1-, STAG2-, and double STAG1/STAG2–knockout cells. Mean ± SD is shown for 2 independent biological replicates of STAG2-WT (U937 WT-1, WT-2) and STAG2-knockout cells (U937 STAG2-KO3, STAG2-KO4) transduced with STAG1 or control sgRNAs. For each sample 100 metaphase spreads were scored. *P < 0.0001 (1-way ANOVA). PCS, premature centromere separation; Railroad, railroad chromosomes. (C) Log2 fold change (FC) of protein enrichment after SMC1A IP-MS in WT and STAG2-knockout (KO) cells. Rep1 and Rep2 correspond to different mutant clones. Proteins belonging to the DNA damage repair and replication gene set are highlighted in green. Enrichment P value was determined using a 1-tailed Fisher’s exact test (P = 0.024). U937 WT-1, WT-2, STAG2-KO5, and STAG2-KO6 were used in this experiment. (D) Representative images depicting replication structures of single combed DNA molecules labeled with IdU (red) and CIdU (green) in WT and STAG2-knockout cells. Quantification of replication origin firing (Orig), progressing replication forks (Prog), and stalled replication forks (Stall) in WT and STAG2-mutant cells. Data from 3 WT (U937 WT-1, WT-2, WT-3) and 3 STAG2-KO (U937 STAG2-KO2, STAG2-KO3, STAG2-KO4) cell lines combined. P < 0.05 (unpaired Student’s t test). Original magnification, 400×. (E) Western blotting for γ-H2Ax. U937 WT-1, WT-2, STAG2 KO-1, STAG2 KO-2, STAG2 KO-3, STAG2 KO-4, STAG2 KO-5, and STAG2 KO-6 were used. β-Actin was a loading control. (F) Western blotting for DNA damage checkpoint proteins ATM, phosphorylated ATM (p-ATM), ATR, p-ATR, CHK1, p-CHK1, CHK2, and p-CHK2 in WT and STAG2-mutant cells in the presence and absence of mitomycin C (MMC). Vinculin was a loading control.