EWS/FLI Confers Tumor Cell Synthetic Lethality to CDK12 Inhibition in Ewing Sarcoma

Iniguez, Amanda Balboni; Stolte, Björn; Wang, Emily Jue; Conway, Amy Saur; Alexe, Gabriela; Dharia, Neekesh V.; Kwiatkowski, Nicholas; Zhang, Tinghu; Abraham, Brian J.; Mora, Jaume; Kalev, Peter; Leggett, Alan L.; Chowdhury, Dipanjan; Benes, Cyril H.; Young, Richard A.; Gray, Nathanael S.; Stegmaier, Kimberly

doi:10.1016/j.ccell.2017.12.009

Cited by 134 publications

(129 citation statements)

References 44 publications

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“…Except for MV4-11 cells, the cell lines tested exhibited a low sensitivity to PARPi, with an IC50 in the micromolar range (Figure S7A), an order of magnitude higher than what is reported for other cancer cells in vitro, such as a BRCA1/2-deficient breast cancer cell line (Farmer et al, 2005). We examined cell viability after four days of treatment, and used two different methods to assess possible synergistic effects between PRMT5i and PARPi: the Bliss synergy method (He et al, 2018; Iniguez et al, 2018) (Figure 7B), and the Chou-Talalay method (Chou, 2010) (Figure S7B). We observed positive Bliss scores for all cell lines tested (Figure 7B), indicating a synergistic effect.…”

Section: Resultsmentioning

confidence: 99%

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PRMT5 Regulates DNA Repair by Controlling the Alternative Splicing of Histone-Modifying Enzymes

et al. 2018

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Protein arginine methyltransferase 5 (PRMT5) is overexpressed in many cancer types and is a promising therapeutic target for several of them, including leukemia and lymphoma. However, we and others have reported that PRMT5 is essential for normal physiology. This dependence may become dose limiting in a therapeutic setting, warranting the search for combinatorial approaches. Here, we report that PRMT5 depletion or inhibition impairs homologous recombination (HR) DNA repair, leading to DNA-damage accumulation, p53 activation, cell-cycle arrest, and cell death. PRMT5 symmetrically dimethylates histone and non-histone substrates, including several components of the RNA splicing machinery. We find that PRMT5 depletion or inhibition induces aberrant splicing of the multifunctional histone-modifying and DNA-repair factor TIP60/KAT5, which selectively affects its lysine acetyltransferase activity and leads to impaired HR. As HR deficiency sensitizes cells to PARP inhibitors, we demonstrate here that PRMT5 and PARP inhibitors have synergistic effects on acute myeloid leukemia cells.

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Section: Resultsmentioning

confidence: 99%

“…Bliss synergy was calculated using the bioconductor package synergyfinder v1.0.0 using default parameters for calculating bliss independence (He et al, 2018). Excess over bliss was calculated as the difference between the observed response and the expected bliss independence (Iniguez et al, 2018). …”

Section: Methodsmentioning

confidence: 99%

PRMT5 Regulates DNA Repair by Controlling the Alternative Splicing of Histone-Modifying Enzymes

et al. 2018

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“…Furthermore, they are characterized by an increased sensitivity to cisplatin and thus represent potential biomarker for treatment response [29][30][31][32][33]. Although inactivation of CDK12 kinase activity clearly leads to HR defects and sensitivity to PARP inhibitors in cells [21,[34][35][36][37], the discovery of the CDK12 inactivation-specific tandem duplication phenotype indicated a distinct function of CDK12 in maintenance of genome stability. The size and distribution of the tandem duplications suggested that DNA replication stress-mediated defect(s) are a possible driving force for their formation [30,31].…”

Section: Introductionmentioning

confidence: 99%

CDK12 controls G1/S progression by regulating RNAPII processivity at core DNA replication genes

et al. 2019

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CDK12 is a kinase associated with elongating RNA polymerase II (RNAPII) and is frequently mutated in cancer. CDK12 depletion reduces the expression of homologous recombination (HR) DNA repair genes, but comprehensive insight into its target genes and cellular processes is lacking. We use a chemical genetic approach to inhibit analog‐sensitive CDK12, and find that CDK12 kinase activity is required for transcription of core DNA replication genes and thus for G1/S progression. RNA‐seq and ChIP‐seq reveal that CDK12 inhibition triggers an RNAPII processivity defect characterized by a loss of mapped reads from 3′ends of predominantly long, poly(A)‐signal‐rich genes. CDK12 inhibition does not globally reduce levels of RNAPII‐Ser2 phosphorylation. However, individual CDK12‐dependent genes show a shift of P‐Ser2 peaks into the gene body approximately to the positions where RNAPII occupancy and transcription were lost. Thus, CDK12 catalytic activity represents a novel link between regulation of transcription and cell cycle progression. We propose that DNA replication and HR DNA repair defects as a consequence of CDK12 inactivation underlie the genome instability phenotype observed in many cancers.

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“…This response correlates with decreased U1 snRNP levels, with the resultant intronic polyadenylation 60 . Conceivably, the effects we attribute to CDK12 inhibition may not be limited to cells with underlying DNA damage such as NB and possibly Ewing sarcoma 16 , but also in cancers with CDK12 loss-of-function mutations such as ovarian cancer. Indeed, as recently demonstrated in a subset of prostate cancers with CDK12 loss-of-function mutations 14 , the PCPA, as well as intron retention, observed with CDK12 inhibition could facilitate the formation of neoantigens that could be exploited to improve immune therapies or to develop personalized cancer vaccines 61 .…”

Section: Inhibition Of Cdk12 By Thz531 Results In Increased Transcripmentioning

confidence: 99%

“…The selective regulation of these genes by CDK12 is also evident in cancers with loss-of-function CDK12 mutations, such as high-grade serous ovarian carcinoma and metastatic castration-resistant prostate cancer, where a 'BRCAness' phenotype with genomic instability sensitizes cells to DNA cross-linking agents and poly (ADP-ribose) polymerase (PARP) inhibitors [11][12][13][14] . Similarly, suppression of wild-type CDK12 in Ewing sarcoma cells driven by the EWS/FLI fusion oncoprotein using THZ531 15 (a selective inhibitor of CDK12/13) also led to the decreased expression of DDR genes 16 . Hence, CDK12 loss of function, whether spontaneous or induced, appears to preferentially affect genes that have prominent roles in DNA repair and consequently in maintaining the stability of the genome.…”

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CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation

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et al. 2018

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damage response (DDR) and mRNA processing genes. Yet, the mechanism(s) by which it achieves this selectivity remains unclear. Using a highly selective CDK12/13 inhibitor, THZ531, and nascent RNA sequencing, we show that CDK12 inhibition results in gene length-dependent elongation defects, leading to premature cleavage and polyadenylation (PCPA) as well as loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlated with an increased proportion of intronic polyadenylation sites, a feature that was especially prominent among DDR genes. Finally, phosphoproteomic analysis indicated that pre-mRNA processing factors, including those involved in PCPA, are direct phosphotargets of CDKs 12 and 13. These results support a model in which DDR genes are uniquely susceptible to CDK12 inhibition due primarily to their relatively longer lengths and lower ratios of U1 snRNP binding to intronic polyadenylation sites. 5 CDK12 inhibition preferentially affects DDR genes. CDK12 inhibition has been shown to affect the expression of genes involved in the DDR 5,6,10 . To determine whether similar effects are produced by our selective inhibitor in NB cells, we analyzed the gene expression profiles of cells treated with and without THZ531 for 6 hr., a time point at which there were little or no confounding effects due to cell cycle changes ( Supplementary Fig. 1e).Unlike the effects seen with THZ1 17 , predominantly an inhibitor of CDK7 with some activity against CDK12/13 28 , we failed to observe a complete and global transcriptional shutdown in THZ531-treated NB cells; instead, only 57.4% of the transcripts were downregulated (n=10,707), with 0.35% (n=66) upregulated [false discovery rate (FDR) <0.05] (Supplementary Fig. 2a; Supplementary Table 1). Consistent with earlier studies 15,16 , THZ531 led to significant downregulation of both transcription-associated and DDR genes ( Fig. 1c, Supplementary Fig. 2b, c), the latter of which were primarily associated with homologous recombination (HR) repair and are crucial for the maintenance of genome stability, including BRCA1, BARD1 and RAD51 29 (Fig. 1c, Supplementary Fig. 2c, d). To determine whether these effects were due to inhibition of CDK12 or 13, we depleted the expression of each kinase individually in NB cells, and in keeping with prior studies 6,10,30 , observed selective downregulation of DDR genes with CDK12 and not CDK13 knockdown (KD) (Supplementary Fig. 2e). Additionally, the expression of DDR genes was not affected in Kelly E9R THZ531-resistant cells, further implicating the selective role of CDK12 in regulating the DDR (Fig. 1d). Consistent with these observations, THZ531 also led to increased DNA damage with elevated γ-H2AX levels (Fig. 1e) and decreased radiationinduced RAD51 foci, indicating defects in DNA repair (Fig. 1f). Thus, our findings indicate that DDR genes are selectively affected by THZ531 and that such regulation is driven predominantly by CDK12.CDK12/13 inhibition with TH...

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EWS/FLI Confers Tumor Cell Synthetic Lethality to CDK12 Inhibition in Ewing Sarcoma

Cited by 134 publications

References 44 publications

PRMT5 Regulates DNA Repair by Controlling the Alternative Splicing of Histone-Modifying Enzymes

PRMT5 Regulates DNA Repair by Controlling the Alternative Splicing of Histone-Modifying Enzymes

CDK12 controls G1/S progression by regulating RNAPII processivity at core DNA replication genes

CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation

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