CDK13 in Triple-Negative Breast Cancer

Article

Therapeutic Targeting of CDK12/CDK13 in TripleNegative Breast Cancer Graphical Abstract

Authors Victor Quereda, Simon Bayle, Francesca Vena, Sylvia M. Frydman, Andrii Monastyrskyi, William R. Roush, Derek R. Duckett

Correspondence [email protected]

In Brief Quereda et al. develop a selective dual CDK12/CDK13 inhibitor that reduces the expression of core DNA damage response genes by increasing intronic polyadenylation site cleavage, resulting in DNA damage repair deficiency and conferring sensitivity to DNA-damaging agents and PARP inhibitors.

Highlights d

SR-4835, a potent dual inhibitor of CDK12/CDK13, provokes TNBC cell death

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CDK12/CDK13 inhibition/loss promotes cleavage at intronic polyadenylation sites

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CDK12 inhibition causes a BRCAness phenotype by blocking homologous recombination

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SR-4835 acts in synergy with DNA-damaging chemotherapy and PARP inhibitors

Quereda et al., 2019, Cancer Cell 36, 1–14 November 11, 2019 ª 2019 Elsevier Inc. https://doi.org/10.1016/j.ccell.2019.09.004

Please cite this article in press as: Quereda et al., Therapeutic Targeting of CDK12/CDK13 in Triple-Negative Breast Cancer, Cancer Cell (2019), https://doi.org/10.1016/j.ccell.2019.09.004

Cancer Cell

Article Therapeutic Targeting of CDK12/CDK13 in Triple-Negative Breast Cancer Victor Quereda,1 Simon Bayle,1 Francesca Vena,1 Sylvia M. Frydman,1 Andrii Monastyrskyi,1 William R. Roush,2 and Derek R. Duckett1,3,* 1Department

of Drug Discovery, Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA of Chemistry, The Scripps Research Institute, Jupiter, FL 33458, USA 3Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.ccell.2019.09.004 2Department

SUMMARY

Epigenetic regulation enables tumors to respond to changing environments during tumor progression and metastases and facilitates treatment resistance. Targeting chromatin modifiers or catalytic effectors of transcription is an emerging anti-cancer strategy. The cyclin-dependent kinases (CDKs) 12 and 13 phosphorylate the C-terminal domain of RNA polymerase II, regulating transcription and co-transcriptional processes. Here we report the development of SR-4835, a highly selective dual inhibitor of CDK12 and CDK13, which disables triple-negative breast cancer (TNBC) cells. Mechanistically, inhibition or loss of CDK12/CDK13 triggers intronic polyadenylation site cleavage that suppresses the expression of core DNA damage response proteins. This provokes a ‘‘BRCAness’’ phenotype that results in deficiencies in DNA damage repair, promoting synergy with DNA-damaging chemotherapy and PARP inhibitors.

INTRODUCTION Breast cancer is the most frequently diagnosed cancer of women worldwide (Ferlay et al., 2015; Siegel et al., 2017). Currently, treatment selection for breast cancer is based on the expression status of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 receptor (HER2/ERBB2), where targeted treatments blocking receptor function have improved overall survival (McDonald et al., 2016). The subset of breast cancers characterized by the absence of ER, PR, and HER2 overexpression, so-called triple-negative breast cancers (TNBCs), accounts for approximately 20% of all breast cancers and fail to respond to these targeted treatments. Surgery and chemotherapy are the mainstay treatments for most TNBC patients. Although early stage TNBC responds well to treatment, advanced disease has a higher probability of relapse and worse overall survival compared with other breast cancer subtypes (Carey et al., 2010; Ovcaricek et al., 2011).

Cyclin-dependent kinases (CDKs) are serine/threonine kinases the activity of which depends on the interaction with a cyclin regulatory subunit (Malumbres, 2014). The CDK family of enzymes orchestrate the regulation of key eukaryotic cellular processes including the cell cycle and transcription (Malumbres and Barbacid, 2009). CDKs regulate transcription via phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNA Pol II). In humans, the CTD comprises a 52-repeat unit with the consensus sequence YSPTSPS, where differential phosphorylation of the tyrosine, serine, and threonine residues facilitates temporal control of the different stages of transcription (Harlen and Churchman, 2017; Heidemann et al., 2013). In complex with the general transcription factor TFIIH, CDK7 phosphorylates the CTD of RNA Pol II at Ser5 upon recruitment to the promoter during transcription initiation (Murakami et al., 2015). Release from transcriptional pausing, productive transcription, and co-transcriptional processes requires phosphorylation of Ser2 of the heptad repeat by CDK9, CDK12, and CDK13

Significance Triple-negative breast cancer (TNBC) is an aggressive cancer, and in advanced disease rapidly progresses following relapse. Development of targeted therapies to improve TNBC outcome has been challenging, and chemotherapy remains the mainstay treatment. A subset of TNBCs with mutations in pathway components that direct homologous recombination (HR) such as BRCA1 are highly sensitive to PARP inhibitors and platinum agents. Developing strategies to exploit DNA damage response (DDR) vulnerabilities in TNBC is thus highly warranted. We developed a dual CDK12/CDK13 inhibitor that suppresses the expression of several DDR genes, provoking lethal accumulation of chemotherapy-induced DNA damage and cancer cell death. Using orthotopic patient-derived xenografts, we show that CDK12 is an exploitable vulnerability, even in HR-competent TNBC tumors. Cancer Cell 36, 1–14, November 11, 2019 ª 2019 Elsevier Inc. 1

Please cite this article in press as: Quereda et al., Therapeutic Targeting of CDK12/CDK13 in Triple-Negative Breast Cancer, Cancer Cell (2019), https://doi.org/10.1016/j.ccell.2019.09.004

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Figure 1. SR-4835 Is a Potent, Selective Dual CDK12/CDK13 Inhibitor Having Activity Triple-Negative Breast Cancer Cells (A) Structure of SR-4835. (B) Docking pose of SR-4835 (green sticks) in the active site of CDK12 (PDB: 6B3E; surface representation). Key hydrogen bond interactions with protein residues (blue sticks) are shown in yellow dash. (C) Kinome profiling of SR-4835 at 10 mM, against over 450 kinases. Size of the circles reflects degree of inhibition. The kinome tree was produced using the KinMap interface (Eid et al., 2017). (D) SR-4835 kinase affinity (Kd) or kinase inhibition (half maximal inhibitory concentration [IC50]), as tested by DiscoverX (Kd) or Reaction Biology Corp. (IC50). (E) CDK12 in vitro kinase assay, as tested by ADP-glo. Results are presented as mean values ± SEM of three technical replicates. (F) In-Cell western blot of MDA-MB-231 cells treated with increasing concentrations of SR-4835 for 4 h and stained with pSer2 RNA Pol II CTD antibody (green) and total RNA Pol II CTD (red). Representation of merge images are shown at the top, and the percentage of the ratio of pSer2 CTD signal versus total CTD signal relative to DMSO control is shown at the bottom. The experiment was performed in biological triplicates. Results are presented as mean values ± SEM. (legend continued on next page)

2 Cancer Cell 36, 1–14, November 11, 2019

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(Bartkowiak and Greenleaf, 2011). The precise roles of these kinases in transcription elongation and co-transcriptional mRNA processing are only beginning to be understood. Recent studies have shown that CDK12 is associated with the expression of a select set of DNA damage response (DDR) genes (Bajrami et al., 2014; Blazek et al., 2011; Johnson et al., 2016; Joshi et al., 2014; Liang et al., 2015; Wu et al., 2018) and that CDK12 promotes an increase in the rate of elongation reducing the likelihood of cleavage at internal polyadenylation sites and increasing the probability of cleavage at the 30 polyadenylation site. Notably, several DNA damage repair genes, including those of the ‘‘BRCAness’’ phenotype (Lord and Ashworth, 2016), have multiple intronic polyadenylation sites and thus are particularly sensitive to CDK12 inhibition (Dubbury et al., 2018). The DDR is an evolutionary conserved mechanism that detects DNA damage and triggers a complex response that decides cell fate, by promoting cell-cycle arrest and DNA repair, or cell death in cases where DNA lesions persist (Roos et al., 2016). The anti-cancer activity of many chemotherapy drugs rely on DNA replication fork collapse and the induction of DNA double-strand breaks, and tumors with mutations in DDR proteins are particularly sensitive to DNA-damaging chemotherapy (Bouwman and Jonkers, 2012; Cheung-Ong et al., 2013). Synthetic lethal approaches have revealed specific anti-cancer targets in tumors defective in DDR pathways, where, for example, BRCA-deficient tumor cells are highly sensitive to poly(ADPribose) polymerase (PARP) inhibition (Farmer et al., 2005; Fong et al., 2009). Accordingly, strategies to exploit DDR-response pathways as therapeutic approaches are of high priority. CDK12 controls the transcription of a cast of DDR genes, and unbiased screening approaches demonstrate that silencing CDK12 is synthetically lethal in combination with PARP inhibition (Bajrami et al., 2014). To address whether CDK12 is an exploitable vulnerability pharmacologically, we developed and tested if small-molecule inhibitors of CDK12 enhanced the anti-breast cancer activity of PARP inhibitors or other DNA-damaging chemotherapeutics. RESULTS SR-4835 Is a Selective, Potent Inhibitor of CDK12 and CDK13 The most selective inhibitor of CDK12/13 reported to date is THZ531, which covalently targets a remote cysteine residue outside of the kinase ATP binding pocket (Zhang et al., 2016). Structure-guided optimization of selected hits from an in-house library of kinase inhibitors led to the identification of N9 heteroaromatic purines. Analogs in this series, including SR-4835 (Figure 1A), have high nanomolar affinity against CDK12 and CDK13. Molecular modeling studies using the X-ray structure of CDK12 predict the binding mode of SR-4835 to be ATP

competitive, with SR-4835 interacting via hydrogen bonding with the hinge region of the kinase (Tyr-815, Met-816, and Asp-819, Figure 1B). SR-4835 was highly selective toward CDK12 and CDK13 when tested in a panel of over 450 kinases at 10 mM (Figure 1C; Table S1), with comparatively weak affinity for six kinases compared with CDK12 and CDK13. SR-4835 when tested against full-length active LRRK2 (at 10 mM) inhibited less than 20% activity. The other five kinases having weak affinity (CDK4, CDK6, CDK9, GSK3A, and GSK3B) for SR-4835 compared with CDK12 and CDK13 were further analyzed in concentration response curves, and their half maximal inhibitory concentration values were an order of magnitude higher than those for CDK13 or CDK12, highlighting the highly selective nature of SR-4835 for inhibiting CDK12/13 (Figures 1D and 1E). CDK12 and CDK13 are required for productive transcription and phosphorylate Ser2 within the YSPTSPS heptad repeats present in the CTD of RNA Pol II. In-Cell western assays revealed that SR-4835 blocks Ser2 phosphorylation with a half maximal effective concentration (EC50) of 100 nM, consistent with in-cell targeting of CDK12/13 (Figure 1F). In addition, we tested if SR-4835 had affinity for binding to BRD4, because BRD4 inhibition has been linked with reduced phosphorylation of the CTD region at Ser2 (Devaiah et al., 2012). SR-4835 had no affinity to BRD4 at any of the concentrations tested (Table S2), nor did it inhibit PARP activity (Table S3). In cell proliferation studies, TNBC cell lines were found to be highly sensitive to SR-4835, with median EC50 values in the low nanomolar range (Figure 1G). This sensitivity was also manifest in long-term growth assays, in which SR-4835 completely blocked clonogenic growth and survival of MDA-MB-231 cells (Figure 1H). SR-4835 has slightly increased potency over THZ531 on MDA-MB-231 cells in short-term proliferation assays with minimal effects on primary fetal human colon cells (Figure 1I). CDK12/13 Inhibition Suppresses Expression of DDR Proteins Genetic studies have revealed that CDK12 controls transcription of genes encoding a subset of DDR proteins, and that this is due to CDK12-dependent control of cleavage at intronic polyadenylation sites (Dubbury et al., 2018; Krajewska et al., 2019). We therefore assessed if blocking CDK12/13 kinase activity alters expression of DDR genes. SR-4835 treatment of MDA-MB-231 cells reduced the expression of a cast of DDR genes as early as 6 h after treatment. Notably, the expression of many other key cancer-related genes was not affected after SR-4835, suggesting a bias toward DDR genes as reported previously (Blazek et al., 2011; Liang et al., 2015) (Figure 2A). Furthermore, SR-4835 treatment provoked DNA damage and triggered apoptosis, as monitored by g-H2AX and PARP cleavage, respectively (Figure 2B). This was confirmed using confocal immunofluorescence microscopy of MDA-MB-231 cells, in which SR-4835 treatment induced a marked increase in g-H2AX foci formation at 24 h after

(G) Anti-proliferative potency of SR-4835 in the indicated TNBC lines after treatment for 72 h. Data are plotted as the percentage of luminescence relative to DMSO controls. The experiment was performed in biological triplicate. Results are presented as mean values ± SEM. (H) Clonogenic growth of MDA-MB-231 cells in the presence of SR-4835 or vehicle. Data are presented as mean values ± SD of triplicate points. ***p < 0.001 by t test. (I) Cell proliferation studies of THZ531 or SR-4835 in MDA-MB-231 cells or in the primary human fetal colon (FHC) cell line after treatment for 72 h at the indicated concentrations. Data are presented as mean values ± SD of triplicate points. See also Tables S1, S2, and S3.

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Figure 2. SR-4835 Downregulates DNA Damage Repair Proteins, and Provokes DNA Damage and Apoptosis in TNBC (A) Analysis of indicated gene expression by qPCR at 6 h with the indicated concentrations of SR-4835 or vehicle in MDA-MB-231 cells. Data are presented as mean values ± SD of triplicate points. *p < 0.05, **p < 0.01, ***p < 0.001 by t test. (B) Analysis of the indicated proteins at selected time points and concentrations of SR-4835 by immunoblot in MDA-MB-231 cells. GAPDH used as loading control. (C) Representative images of MDA-MB-231 cells treated with the indicated concentrations of SR-4835 or vehicle for the indicated times and then assessed for BRCA1 (green) and g-H2AX (red) foci by confocal immunofluorescence microscopy. Nuclei were stained with Hoechst (blue). Scale bar represents 10 mm. (D) Quantification of experiment presented in (C) using IN Cell Analyzer software. + g-H2AX represents cells with more than ten g-H2AX foci detected. + g-H2AX pan-nuclear are cells in which less than ten foci could be detected, but the intensity of the signal surpasses a threshold selected for each experiment based on (legend continued on next page)

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CRISPR Editing of CDK12 and CDK13 Mimics Effects of SR-4835 in TNBC Cells To test if genetic inactivation of CDK12 or CDK13 recapitulates the phenotype observed pharmacologically, lentiviral CRISPR-Cas9 vectors were generated that express single guide RNAs (sgRNA) directed against CDK12 and CDK13. As anticipated from knockout studies (Juan et al., 2016), no CDK12 knockout or CDK12/CDK13 double knockout clones were obtained, suggesting that CDK12 loss is lethal (data not shown). Thus, we analyzed phenotypes manifest in response to CDK12 or CDK13 deletion after short-term infection using the CRISPR sgRNA most efficient for each gene. Notably, while downregulation of CDK12 and CDK13 reduced colony formation of MDAMB-231 cells, their combined silencing augmented the killing effect versus knockdown of either single gene (Figure 3A). Inhibition of Ser2 phosphorylation within the CTD heptad repeat was only observed in the double knockdown (Figure 3B). This may be due to residual CDK12 or CDK13 activity, but perhaps is

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treatment. This increase in g-H2AX foci in SR-4835-treated cells occurred without changes in cell-cycle profile, and these foci lacked the presence of BRCA1, which differed significantly from vehicle-treated cells (Figures 2C and 2D and data not shown). Neutral comet assays confirmed the generation of DNA damage upon treatment with SR-4835 (Figures 2E and 2F). Interestingly, improved recovery was observed for human fetal colon (FHC) cells after treatment by SR-4835, in keeping with the reduced toxicity of CDK12/13 inhibition in these primary cells (Figure 2G). Thus, inhibition of Ser2 phosphorylation on the CTD of RNA Pol II by SR-4835 treatment compromises the

qualitative observations. + g-H2AX/BRCA1 are cells with more than ten g-H2AX foci and more than ten BRCA1 foci. The experiment was performed in biological triplicates. Results are presented as mean values of a representative experiment ± SD of 8 random fields of view where at least 100 cells were counted in each. *p < 0.05, ***p < 0.001 by t test. (E) Representative images from comet assay of MDA-MB-231 cells after treatment with vehicle or SR-4835 (30 or 90 nM) for 6 or 24 h stained with propidium iodide. Scale bar represents 20 mm. (F) Tail moments obtained from comet assay of MDA-MB-231 cells after treatment with vehicle or SR-4835 (30 or 90nM) for 6 or 24 h. Boxplots represent interquartile ranges, horizontal bars denote the median, whiskers indicate 10th to 90th percentile and points are outliers. For each condition, 50 cells were analyzed. *p < 0.05, ***p < 0.001 by t test. (G) Tail moments obtained from comet assay of primary FHC cells after treatment with vehicle for 24 h and 90 nM SR-4835 for 6 or 24 h. Boxplots represent interquartile ranges, horizontal bars denote the median, whiskers indicate 10th to 90th percentile and points are outliers. For each condition, 50 cells were analyzed. **p < 0.01, ***p < 0.001 by t test.

Cancer Cell 36, 1–14, November 11, 2019 5

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Figure 4. CDK12/CDK13-Dependent Transcriptome in MDA-MB-231 Cells (A–D) Ingenuity Pathway Analysis of the top ten significantly regulated pathways of SR-4835-treated versus vehicle-treated MDA-MB-231 cells (A), CRISPR/Cas9 sgCDK12 versus control (B), CRISPR/Cas9 sgCDK13 versus control (C), and CRISPR/Cas9 sgCDK12 and sgCDK13 versus control (D). Pathways that are upregulated are presented in orange and those that are downregulated are in blue. (E) Comparative ingenuity Pathway Analysis of indicated conditions heatmap. (F) Heatmap of significantly regulated genes (i.e., those in the DNA damage response of cells, repair of DNA, and formation of g-H2AX) in SR-4835-treated MDAMB-231 cells versus vehicle-treated cells, compared with the status of these genes in CRISPR/Cas9 sgCDK12 versus control, CRISPR/Cas9 sgCDK13 versus control, and CRISPR/Cas9 sgCDK12 and sgCDK13 versus control conditions. (G) log2 fold change for all expressed genes, or for the ‘‘BRCAness’’ dataset of MDA-MB-231 cells treated with SR-4835 versus vehicle or having silenced CDK12 and/or CDK13. **p < 0.01, ***p < 0.001 by t test. (legend continued on next page)

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indicative of additional roles of CDK12 and CDK13 beyond those manifested by transcriptional regulation, as indicated by Ser2 phosphorylation. Furthermore, dual knockdown of CDK12 and CDK13 led to more profound suppression of some DDR mRNAs and proteins, specifically RAD51, ATR, and SMARCC (Figures 3B, 3C, and S1), while others were more CDK12 dependent (i.e., BRCA1, ATM, FANCI, and FANCD2), suggesting differential control of DDR by CDK12 and CDK13. Interestingly, whereas CDK12 knockdown triggered DNA damage and apoptosis, as indicated by increased levels of g-H2AX and cleaved-PARP (Figure 3B), CDK13 knockdown induced apoptosis without triggering a DNA damage signal; thus, CDK12 and CDK13 inhibition act cooperatively to induce cancer cell death. To assess the global effects of CDK12/CDK13 knockdown or inhibition on gene expression, RNA sequencing (RNA-seq) analyses were performed on cells treated with either SR-4835 or vehicle or following knockdown of CDK12 and/or CDK13 (Table S4). Ingenuity Pathway Analysis indicated that CDK12/CDK13 inhibition led to significant suppression of genes involved in DNA repair, DNA recombination, and cell-cycle checkpoint control, while significantly upregulated genes included those involved in S and G2-M progression and apoptosis. Furthermore, there was a high coincidence of differentially expressed pathways affected by pharmacologic and genetic silencing of CDK12 and CDK13 (Figures 4A–4E). For example, the ATM pathway was suppressed by both SR-4835 and dual CRISPR CDK12/CDK13 knockdown (Figures 4A–4E and S2A). Notably, only three pathways were significantly regulated by CDK13 knockdown (Figure 4C), whereas there was significant overlap of pathways affected by SR-4835 treatment and silencing of CDK12, or of CDK12 plus CDK13 (Figure 4E). Analyzing genes included in the ingenuity pathway analysis functions: ‘‘DNA damage response of cells, repair of DNA and formation of g-H2AX’’ (Figure 4F; Table S5), revealed that almost 200 genes significantly regulated by SR-4835 (p < 0.05) were also altered in CDK12/CDK13 dual knockdown cells. Furthermore, the number of genes regulated by CDK12 knockdown, while smaller, were altered in the same direction as those affected by SR-4835 treatment. Both SR-4835 and dual CDK12/CDK13 knockdown regulated the expression of genes involved in DDR. Synthetic lethality in response to PARP inhibition has revealed a gene list, coined the BRCAness signature (Lord and Ashworth, 2016). Importantly, expression of BRCAness signature genes was downregulated in the cells treated with SR-4835 (Figure 4G). Indeed, 13 BRCAness genes were profoundly suppressed when compared with global changes provoked by SR-4835 treatment (Figure 4H; Table S6). Recently, it has been shown that genes having intronic polyadenylation (poly(A)) sites are especially sensitive to CDK12 inhibition (Dubbury et al., 2018). Notably, SR-4835 treatment or dual CDK12/CDK13 knockdown led to global suppression of

genes having intragenic poly(A) sites, including BRCAness genes, many of which have several intra-poly(A) sites (Figure 4I; Tables S6 and S7). A direct effect on intra-poly(A) was confirmed through qPCR analysis targeting regions before or after the intrapoly(A) sites on selected BRCAness genes and GABPB1 and CSTF2 genes for which their intra-poly(A) sites were previously characterized (Tian et al., 2007) (Figure S2B). As anticipated, due to the presence of multiple intra-poly(A) sites in some genes (i.e., ATM), expression of mRNA distal regions was greatly downregulated (Figure S2C). We conclude that targeting CDK12 induces a BRCAness phenotype in TNBC. CDK12/CDK13 Inhibition Synergizes with DNADamaging Agents or PARP Inhibition to Trigger TNBC Cell Death We hypothesize that downregulation of DDR proteins by CDK12/ CDK13 inhibition would hypersensitize TNBC cells to DNA crosslinkers (cisplatin), topoisomerase I inhibitors (irinotecan), DNA replication targeting agents (doxorubicin), and PARP inhibitors (olaparib). Using the Chou and Talalay (1984) method, doseresponse assays established potent synergy between SR-4835 and cisplatin, olaparib, doxorubicin, and irinotecan in MDAMB-231, HS578T, and MDA-MB-468 cells (Figures 5A, S3, and S4A–S4C), but not between SR-4835 and cisplatin in primary FHC cells (Figures S4D). Consistent with these findings, levels of g-H2AX and apoptosis of TNBC cells were augmented by these combination treatments in the tumor cells (Figures 5B, S5A, and S5B). As anticipated from expression studies, ATM and RAD51 protein levels were suppressed following treatment with SR-4835 (Figures 5C, S5A, and S5B). Interestingly, while the DNA damage checkpoint was triggered by cisplatin or irinotecan treatment, as indicated by phosphorylation of Ser15/Ser9 residues of p53 (Blackford and Jackson, 2017), SR-4835 co-treatment markedly impaired p53 phosphorylation (Figures 5C and S5B). These data are consistent with the notion that downregulation of the DNA damage checkpoint by CDK12 inhibition augments the killing effect of DNA-damaging agents. To further validate this hypothesis, cells treated with the single agents or the combinations were analyzed by confocal microscopy and by neutral comet assay. In the absence of treatment, DNA damage was low (as quantified by the number of g-H2AX foci per cell, Figures 5D, and S6 or tail moment Figures 5E and S6). Nevertheless, in the few DNA lesions that were detected, BRCA1 foci colocalized with g-H2AX (Figure S6), consistent with the notion that the DNA repair machinery had been recruited to sites of damage. Cisplatin-treated cells showed marked increases in DNA damage, with some cells having pan-nuclear g-H2AX staining, and in cells having g-H2AX foci there were co-localizing BRCA1 foci. This, however, did not occur in SR4835/cisplatin-treated cells, which underwent similar levels of

(H) log2 fold change for all expressed genes having at least one predicted intragenic polyadenylation (IPA) site. Red squares indicate CDK12-sensitive BRCAness genes. (I) log2 fold change in expressed genes that did not changed significantly (n.s.) or that changed significantly containing at least one predicted IPA site. Red squares indicate CDK12-sensitive BRCAness genes that harbor IPA sites. Boxplots in (G–I) represent median with 25th and 75th quartiles; whiskers show 1.5 3 interquartile range. 12 + 13, lentivirus expressing CDK12 and CDK13 sgRNAs. See also Figure S2 and Tables S4, S5, S6, and S7.

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8 Cancer Cell 36, 1–14, November 11, 2019

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DNA damage and rapid apoptosis (Figures 5D and S6). Accordingly, the ‘‘comets’’ observed in the combination treatment were larger than those in the individual treatments (Figures 5E and S6). CDK12/CDK13 Inhibition Impairs TNBC Tumor Growth and Cooperates with DNA-Damaging Therapeutics To test the anti-tumor activity of SR-4835 we used an orthotopic, patient-derived xenograft (PDX) model (PDX4013) derived from a TNBC patient who had limited response to treatment with dasatinib and docetaxel (Zhang et al., 2013). Based on pharmacokinetic analysis, we determined that SR-4835 is orally bioavailable (Figure S7A). Once tumors reached 100 mm3, animals were randomized into four groups and the cohorts were administered vehicle, SR-4835, cisplatin, or the combination of SR-4835 and cisplatin. There was a marked decrease in tumor growth in mice treated with SR-4835 or cisplatin compared with vehicletreated mice (Figures 6A and 6B). Notably, the combination treatment provoked rapid tumor regression with none of the animals in this treatment group reaching the defined endpoint over the course of the experiment. Endpoint studies determined that expression of DDR genes (both mRNA and protein) were reduced in tumors treated with SR-4835 or the combination, whereas protein levels of g-H2AX were elevated in all conditions compared with vehicle-treated mice (Figures 6C–6E). The dual treatment increase in g-H2AX was confirmed by immunohistochemistry (IHC) analysis (Figures 6F and 6G). In all drug-treated groups there were reductions in proliferation (KI67+ cells) and an increase in apoptosis (cleaved caspase-3; Figures 6F and 6G). Importantly, SR-4835 treatment alone did not provoke weight loss and, as anticipated, the body weight loss in combination treatments was recovered after removal of the DNA-damaging agent (Figure S7B). Moreover, no significant changes in blood cell counts were observed in endpoint analysis (Figure S7C), and histological analyses of mouse tissues showed no deleterious effects of SR-4835 treatment (data not shown). Collectively, these data support our thesis that SR-4835 is well tolerated with no obvious gross toxicity issues. The efficacy of SR-4835 alone and in combination with irinotecan was also tested using a second pre-clinical PDX model of TNBC (PDX3887) established from a primary BRCA1 mutant tumor from a patient who had limited response to 5-fluorouracil

(Zhang et al., 2013). Treatment with SR-4835 significantly impaired tumor growth and irinotecan was potent at reducing the tumor growth, where 20% of the mice showed complete tumor regression (2 out of 10; Figures 7A and 7B). Notably the combination of SR-4835 and irinotecan was even more striking where 50% of this cohort lacked detectable disease (Figures 7A and 7B). In accord with results observed in the PDX4013 model, SR-4835 treatment reduced the mRNA and protein levels of DDR genes (Figures 7C–7E). Moreover, DNA damage and cell death induction were most prominent in combination-treated animals observed by western blot or IHC endpoint analysis (Figures 7D–7G). Collectively, these data support SR-4835 as a promising therapeutic candidate for TNBC, especially when combined with DNA-damaging agents. DISCUSSION Although patients with TNBC respond to current chemotherapies, once relapse occurs disease progression is invariably rapid. Promising clinical trials with platinum salts and the approval of PARP inhibitors in BRCA-deficient metastatic breast cancers (Kilburn, 2008; Litton et al., 2018; Robson et al., 2017) have established the potential of exploiting DNA repair deficiencies as targeted treatment in this subset of breast cancers. Indeed, great efforts are now underway to identify the full extent of the BRCAness phenotype across multiple malignancies. Accordingly, identifying new targets and modulators of homologous recombination (HR) to enhance the efficacy and broaden the utility of PARP inhibitors and platinum salts is a priority in the therapeutics arena. Notably, CDK12 controls the expression of core DDR genes, and silencing CDK12 is synthetic lethal with PARP inhibitors (Johnson et al., 2016; Kwiatkowski et al., 2014). Thus, developing a small-molecule inhibitor of CDK12 as an anticancer therapeutic is highly desired. Here we present SR-4835, an orally bioavailable inhibitor of CDK12/13 that has excellent isoform selectivity and few offtarget interactions when tested across a panel of 460 kinases. Importantly, SR-4835 has potent cell-based and in vivo antiTNBC activity and augments the anti-cancer activity of cisplatin, irinotecan, and olaparib, which are standard-of-care therapeutics for TNBC. Moreover, SR-4835 is well tolerated in mice after long-term dosing. Mechanistically, we demonstrate that

Figure 5. SR-4835 Synergizes with DNA-Damaging Chemotherapeutics and Provokes TNBC Cell Death by Downregulating DNA Repair Proteins (A) Anti-proliferative potency of the indicated drugs alone or combined with SR-4835 in MDA-MB-231 cells after treatment for 72 h. Average of Chou-Talalay slope values are indicated (combination index [CI]). Data are plotted as the percentage of luminescence relative to DMSO controls. The experiment was performed in biological triplicates. Results are presented as mean values ± SEM. (B) Apoptosis assay of the indicated drugs alone or combined with SR-4835 in MDA-MB-231 cells after treatment for 48 h. The experiment was performed in biological triplicates. Results are presented as mean values ± SEM. (C) Immunoblot analysis of the indicated proteins at select time points in MDA-MB-231 cells treated with cisplatin, SR-4835, or the combination. GAPDH used as loading control. (D) Quantification of MDA-MB-231 cells treated with cisplatin, SR-4835, or the combination for the indicated times using IN Cell Analyzer software. + g-H2AX are cells with more than ten g-H2AX foci detected. + g-H2AX pan-nuclear are cells in which less than ten foci could be detected, but the intensity of the signal surpasses a threshold selected for each experiment based on qualitative observations. + g-H2AX/BRCA1 are cells with more than ten g-H2AX foci and more than ten BRCA1 foci detected in the cell. The experiment was performed in biological triplicate. Results are presented as mean values ± SD of 8 random fields of view where at least 100 cells were counted in each. **p < 0.01, ***p < 0.001 by t test. (E) Tail moments from comet assay obtained from MDA-MB-231 cells after treatment with the indicated drugs for 6 or 24 h. Boxplots represent interquartile ranges, horizontal bars denote the median, whiskers indicate 10th to 90th percentile and points are outliers. For each condition, 50 cells were analyzed. *p < 0.05, **p < 0.01, ***p < 0.001 by t test. See also Figures S3–S6.

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B Vehicle SR-4835 (20 mg/kg) Cisplatin (6 mg/kg) Combo

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Figure 6. SR-4835/Cisplatin Combination Provokes Tumor Regression in an Orthotopic TNBC PDX Model (A) Tumor volume measurements of BCM-4013 PDX treated with SR-4835 (orally 20 mg/kg 5 days on, 2 days off [5:2] schedule), cisplatin (intraperitoneally [IP] 6 mg/kg once a week), combination, or vehicle (30% hp-BCD in water orally 5:2 schedule and saline IP once a week). Data are plotted as mean values ± SD (n = 8 per arm). *p < 0.05, ***p < 0.001 by t test. (B) Kaplan-Meyer curves for experiment described in (A) presenting percentage of animals with tumors smaller than 1,000 mm3 at the indicated day. (legend continued on next page)

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inhibition of CDK12 and CDK13 provokes anti-cancer activity through cellular stress responses that include a marked modulation of the DDR, and that SR-4835 cooperates with DNAdamaging chemotherapeutics. In tumor cells, this is manifest in a marked reduction of DNA repair foci, in which CDK12/CDK13 inhibition or loss compromises the expression of key DNA damage repair proteins by provoking intragenic poly(A) usage, leading to the rapid accumulation of DNA lesions and apoptosis. Notably, these effects were also manifest in orthotopic PDX models of therapy-resistant TNBC, where combining SR-4835 with cisplatin or irinotecan increases DNA damage, induces apoptosis, and provokes tumor regression. CDK12 and CDK13 inhibition have been shown to perturb the expression of a limited set of genes and both kinases have been shown to phosphorylate the Ser2 residue of the heptad repeat within the CTD of RNA Pol II with proposed roles in transcription and co-transcriptional processes (Greifenberg et al., 2016; Liang et al., 2015). Interestingly, while our genetic silencing studies minimally altered Ser2 phosphorylation levels, Rad51 and ATM were downregulated, suggesting that CDK12 may exert effects on downstream target genes via mechanisms independent of Ser2 phosphorylation. Importantly, recent studies of Sharp and colleagues have shown that CDK12 enhances the rate of transcription elongation and suppresses cleavage at internal polyadenylation sites, and that this is required to permit the production of key HR repair proteins (Dubbury et al., 2018). Notably, several DNA damage repair genes have multiple intronic polyadenylation sites and thus are sensitive to CDK12 silencing (Dubbury et al., 2018). Here, by comparing RNA-seq derived from TNBC cells treated with SR4835 to those infected with CRISPR vectors targeting CDK12 and CDK13, we identified 13 BRCAness-related genes that are CDK12 dependent, which overlaps with the DDR gene set in mouse embryonic stem cells (Dubbury et al., 2018). Notably, cancers with functional inactivating mutations in CDK12, such as ovarian and prostate cancers, have defective DNA damage repair pathways that result in genomic instability. Deep sequencing studies of patient tumors have demonstrated that cancers with CDK12 loss-of-function mutations result in a unique genomic structural variant that involves large focal tandem duplications that differ significantly from the short-span BRCA1 mutant cancers (Menghi et al., 2018; Popova et al., 2016; Vanderstichele et al., 2017; Viswanathan et al., 2018). Interestingly, Wu et al. (2018) demonstrate that the altered structural phenotype induced by CDK12 mutations produces fusioninduced neoantigens that are reporters for immune checkpoint inhibitor sensitivity (Wu et al., 2018). Further studies are thus warranted to assess whether SR-4835 would also act to augment immune checkpoint inhibitor sensitivity.

Interestingly, in TNBC cells silencing CDK13 provokes cell death without affecting DDR genes. Like CDK12, CDK13 phosphorylates the Ser2 position of the heptad repeat in the CTD of RNA Pol II (Greifenberg et al., 2016), yet our studies have established that it is required for the proper expression of a distinct class of genes in TNBC cells. Precisely how CDK13 controls the expression of these genes, and which of these are necessary for TNBC survival, are important for future investigations. Notably, both CDK12 and CDK13 control the transcription of additional pathways that have known roles in tumorigenesis, including the Wntb-catenin, insulin growth factor-1, and eukaryotic initiation factor 2 pathways (Hart et al., 2012; Nusse and Clevers, 2017; Tao et al., 2007). Accordingly, using SR-4835 as a molecular probe of these or other gene sets may enable the selection of target genes that are synthetic lethal in a cancer type-specific manner. Moreover, elucidating the full spectrum of roles that CDK12 and CDK13 signaling plays in tumorigenesis is required to best position the use of optimized SR-4835 analogs in the oncology clinic. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

d

KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODELS AND SUBJECT DETAILS B Cell Lines B Tumor Models and TNBC PDX B SR-4835 and Cisplatin Efficacy Studies B SR-4835 and Irinotecan Efficacy Studies METHOD DETAILS B SR-4835 Synthesis B In Vitro Biochemical Kinase Profiling of SR-4835 B In-Cell Western Analyses B ADP-Glo CDK12 Assay B Cell Proliferation Assay B Clonogenic Assays B Western Blot Analyses B qRT-PCR Analyses B Immunofluorescence B Comet Assay B CRISPR/cas9 Editing B Apoptosis Assay B RNA-seq Analyses B Gene Expression Analyses B Chou-Talalay Combination Index Analysis B Mouse Pharmacokinetic Studies B Immunohistochemistry

(C) qRT-PCR analyses of the expression of the indicated genes in tumors from mice treated with the indicated drug or vehicle (n = 2 mice for vehicle and combination, and n = 3 for SR-4835 and cisplatin) done in triplicates. Data are presented as mean values ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by t test. (D) Immunoblot analysis of the indicated proteins from mice treated with the indicated drug or vehicle at the end of the experiment. GAPDH used as loading control. (E) Quantification of experiment presented in (C) normalized against GAPDH. Data are presented as mean values ± SD. *p < 0.05, ***p < 0.001 by t test. (F) Representative immunohistochemistry images of the indicated proteins in tumors from mice treated with the indicated drug or vehicle. CC3, cleaved caspase-3. Scale bar represents 40 mm. (G) Quantification of immunohistochemistry images. Results are presented as mean values of a representative experiment ± SD of three random fields of view of three different mice. *p < 0.05, **p < 0.01, ***p < 0.001 by t test. See also Figure S7.

Cancer Cell 36, 1–14, November 11, 2019 11

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12 Cancer Cell 36, 1–14, November 11, 2019

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d

QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND CODE AVAILABILITY

SUPPLEMENTAL INFORMATION

Blazek, D., Kohoutek, J., Bartholomeeusen, K., Johansen, E., Hulinkova, P., Luo, Z., Cimermancic, P., Ule, J., and Peterlin, B.M. (2011). The cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes. Genes Dev. 25, 2158–2172.

Supplemental Information can be found online at https://doi.org/10.1016/j. ccell.2019.09.004.

Bouwman, P., and Jonkers, J. (2012). The effects of deregulated DNA damage signalling on cancer chemotherapy response and resistance. Nat. Rev. Cancer 12, 587–598.

d

ACKNOWLEDGMENTS We sincerely thank Dr. Jenny C Chang (Methodists Cancer Center, Houston, TX) for the patient-derived xenograft models BCM-4013 and BCM-3887. We also thank Wayne Grant for technical assistance and Tina Van Meter for her help processing pathological samples. We are grateful to the Genomics and Bioinformatics cores at Scripps Florida for their help processing the RNAseq samples. We also thank Drs. John Cleveland and Patsy McDonald for input and editing of the manuscript. This work was supported in part by the Rendina Family Foundation (to D.R.D.) and by funds from the Moffitt Cancer Center and Research Institute. Support for V.Q. was provided by a postdoctoral fellowship from the FCBTR/ABC2 Brain Tumor Grants Program. AUTHOR CONTRIBUTIONS V.Q. was the primary author of the manuscript and designed and executed experiments; collected, analyzed, and interpreted data; and wrote the manuscript. S.B., F.V., and S.M.F. designed and executed experiments, collected data, and reviewed the manuscript, with contributions from A.M. that were critical to this work. D.R.D. oversaw the study, contributed to experimental design, interpreted the data, and co-wrote the manuscript with substantial contribution from W.R.R. DECLARATION OF INTERESTS

Carey, L., Winer, E., Viale, G., Cameron, D., and Gianni, L. (2010). Triple-negative breast cancer: disease entity or title of convenience? Nat. Rev. Clin. Oncol. 7, 683–692. Cheung-Ong, K., Giaever, G., and Nislow, C. (2013). DNA-damaging agents in cancer chemotherapy: serendipity and chemical biology. Chem. Biol. 20, 648–659. Chou, T.C., and Talalay, P. (1984). Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 22, 27–55. Devaiah, B.N., Lewis, B.A., Cherman, N., Hewitt, M.C., Albrecht, B.K., Robey, P.G., Ozato, K., Sims, R.J., 3rd, and Singer, D.S. (2012). BRD4 is an atypical kinase that phosphorylates serine2 of the RNA polymerase II carboxy-terminal domain. Proc. Natl. Acad. Sci. U S A 109, 6927–6932. Dubbury, S.J., Boutz, P.L., and Sharp, P.A. (2018). CDK12 regulates DNA repair genes by suppressing intronic polyadenylation. Nature 564, 141–145. Eid, S., Turk, S., Volkamer, A., Rippmann, F., and Fulle, S. (2017). KinMap: a web-based tool for interactive navigation through human kinome data. BMC Bioinformatics 18, 16. Farmer, H., McCabe, N., Lord, C.J., Tutt, A.N., Johnson, D.A., Richardson, T.B., Santarosa, M., Dillon, K.J., Hickson, I., Knights, C., et al. (2005). Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434, 917–921.

The authors declare that a provisional patent for CDK12 and CDK13 inhibitors and their use in cancer has been filed.

Ferlay, J., Soerjomataram, I., Dikshit, R., Eser, S., Mathers, C., Rebelo, M., Parkin, D.M., Forman, D., and Bray, F. (2015). Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int. J. Cancer 136, E359–E386.

Received: April 4, 2019 Revised: July 29, 2019 Accepted: September 12, 2019 Published: October 24, 2019

Fong, P.C., Boss, D.S., Yap, T.A., Tutt, A., Wu, P., Mergui-Roelvink, M., Mortimer, P., Swaisland, H., Lau, A., O’Connor, M.J., et al. (2009). Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N. Engl. J. Med. 361, 123–134.

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Figure 7. Effect of SR-4835/Irinotecan Combination Provokes Regression of a BRCA1-Deficient PDX Model (A) Tumor volume measurements of BCM-3887 PDX treated with SR-4835 (orally 20 mg/kg 5 days on, 2 days off [5:2] schedule), irinotecan (IP 50 mg/kg single dose), combination, or vehicle (30% hp-BCD in water orally. 5:2 schedule and saline IP single treatment). Data are plotted out as mean values ± SD (n = 10 per arm, except combination, n = 8). *p < 0.05, **p < 0.01 by t test. (B) Kaplan-Meyer curves for the experiment described in (A) presenting percentage of animals with tumors smaller than 1,000 mm3 at the indicated day. (C) qRT-PCR analyses of the expression of the indicated genes in tumors from two mice treated with the indicated drug or vehicle performed in triplicates. Data are presented as mean values ± SD. *p < 0.05, ***p < 0.001 by t test. (D) Immunoblot analysis of the indicated proteins from mice treated with the indicated drug or vehicle at the end of the experiment. CC3, cleaved caspase-3. GAPDH used as loading control. (E) Quantification of experiment presented in (C) normalized against GAPDH. Data are presented as mean values ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by t test. (F) Representative immunohistochemistry images of the indicated proteins in tumors from mice treated with the indicated drug or vehicle for 5 days. CC3. Cleaved caspase-3. Scale bar represents 40 mm. (G) Quantification of immunohistochemistry images. Results are presented as mean values ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by t test. See also Figure S7.

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Joshi, P.M., Sutor, S.L., Huntoon, C.J., and Karnitz, L.M. (2014). Ovarian cancer-associated mutations disable catalytic activity of CDK12, a kinase that promotes homologous recombination repair and resistance to cisplatin and poly(ADP-ribose) polymerase inhibitors. J. Biol. Chem. 289, 9247–9253. Juan, H.C., Lin, Y., Chen, H.R., and Fann, M.J. (2016). Cdk12 is essential for embryonic development and the maintenance of genomic stability. Cell Death Differ. 23, 1038–1048. Kilburn, L.S. (2008). ’Triple negative’ breast cancer: a new area for phase III breast cancer clinical trials. Clin. Oncol. (R Coll. Radiol.) 20, 35–39. Krajewska, M., Dries, R., Grassetti, A.V., Dust, S., Gao, Y., Huang, H., Sharma, B., Day, D.S., Kwiatkowski, N., Pomaville, M., et al. (2019). CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation. Nat Commun 10, 1757. Kwiatkowski, N., Zhang, T., Rahl, P.B., Abraham, B.J., Reddy, J., Ficarro, S.B., Dastur, A., Amzallag, A., Ramaswamy, S., Tesar, B., et al. (2014). Targeting transcription regulation in cancer with a covalent CDK7 inhibitor. Nature 511, 616–620. Lau, H.Y., Tang, J., Casey, P.J., and Wang, M. (2017). Isoprenylcysteine carboxylmethyltransferase is critical for malignant transformation and tumor maintenance by all RAS isoforms. Oncogene 36, 3934. Liang, K., Gao, X., Gilmore, J.M., Florens, L., Washburn, M.P., Smith, E., and Shilatifard, A. (2015). Characterization of human cyclin-dependent kinase 12 (CDK12) and CDK13 complexes in C-terminal domain phosphorylation, gene transcription, and RNA processing. Mol. Cell Biol. 35, 928–938. Litton, J.K., Rugo, H.S., Ettl, J., Hurvitz, S.A., Gonc¸alves, A., Lee, K.-H., Fehrenbacher, L., Yerushalmi, R., Mina, L.A., Martin, M., et al. (2018). Talazoparib in patients with advanced breast cancer and a germline BRCA mutation. N. Engl. J. Med. 379, 753–763. Lord, C.J., and Ashworth, A. (2016). BRCAness revisited. Nat. Rev. Cancer 16, 110–120. Malumbres, M. (2014). Cyclin-dependent kinases. Genome Biol. 15, 122. Malumbres, M., and Barbacid, M. (2009). Cell cycle, CDKs and cancer: a changing paradigm. Nat. Rev. Cancer 9, 153–166. McDonald, E.S., Clark, A.S., Tchou, J., Zhang, P., and Freedman, G.M. (2016). Clinical diagnosis and management of breast cancer. J. Nucl. Med. 57 (Suppl 1 ), 9S–16S. Menghi, F., Barthel, F.P., Yadav, V., Tang, M., Ji, B., Tang, Z., Carter, G.W., Ruan, Y., Scully, R., Verhaak, R.G.W., et al. (2018). The tandem duplicator phenotype is a prevalent genome-wide cancer configuration driven by distinct gene mutations. Cancer Cell 34, 197–210.e195. Monastyrskyi, A., Nilchan, N., Quereda, V., Noguchi, Y., Ruiz, C., Grant, W., Cameron, M., Duckett, D., and Roush, W. (2018). Development of dual casein kinase 1delta/1epsilon (CK1delta/epsilon) inhibitors for treatment of breast cancer. Bioorg. Med. Chem. 26, 590–602. Murakami, K., Tsai, K.L., Kalisman, N., Bushnell, D.A., Asturias, F.J., and Kornberg, R.D. (2015). Structure of an RNA polymerase II preinitiation complex. Proc. Natl. Acad. Sci. U S A 112, 13543–13548.

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Nusse, R., and Clevers, H. (2017). Wnt/b-catenin signaling, disease, and emerging therapeutic modalities. Cell 169, 985–999. Olive, P.L., and Banath, J.P. (2006). The comet assay: a method to measure DNA damage in individual cells. Nat. Protoc. 1, 23–29. Ovcaricek, T., Frkovic, S.G., Matos, E., Mozina, B., and Borstnar, S. (2011). Triple negative breast cancer––prognostic factors and survival. Radiol. Oncol. 45, 46–52. Popova, T., Manie, E., Boeva, V., Battistella, A., Goundiam, O., Smith, N.K., Mueller, C.R., Raynal, V., Mariani, O., Sastre-Garau, X., and Stern, M.H. (2016). Ovarian cancers harboring inactivating mutations in CDK12 display a distinct genomic instability pattern characterized by large tandem duplications. Cancer Res. 76, 1882–1891. Robson, M., Im, S.-A., Senkus, E., Xu, B., Domchek, S.M., Masuda, N., Delaloge, S., Li, W., Tung, N., Armstrong, A., et al. (2017). Olaparib for metastatic breast cancer in patients with a germline BRCA mutation. N. Engl. J. Med. 377, 523–533. Rocca, M.S., Benna, C., Mocellin, S., Rossi, C.R., Msaki, A., Di Nisio, A., Opocher, G., and Foresta, C. (2019). E2F1 germline copy number variations and melanoma susceptibility. J. Transl Med. 17, 181. Roos, W.P., Thomas, A.D., and Kaina, B. (2016). DNA damage and the balance between survival and death in cancer biology. Nat. Rev. Cancer 16, 20–33. Siegel, R.L., Miller, K.D., and Jemal, A. (2017). Cancer statistics, 2017. CA Cancer J. Clin. 67, 7–30. Tao, Y., Pinzi, V., Bourhis, J., and Deutsch, E. (2007). Mechanisms of disease: signaling of the insulin-like growth factor 1 receptor pathway—therapeutic perspectives in cancer. Nat. Clin. Pract. Oncol. 4, 591. Tian, B., Pan, Z., and Lee, J.Y. (2007). Widespread mRNA polyadenylation events in introns indicate dynamic interplay between polyadenylation and splicing. Genome Res. 17, 156–165. Vanderstichele, A., Busschaert, P., Olbrecht, S., Lambrechts, D., and Vergote, I. (2017). Genomic signatures as predictive biomarkers of homologous recombination deficiency in ovarian cancer. Eur. J. Cancer 86, 5–14. Viswanathan, S.R., Ha, G., Hoff, A.M., Wala, J.A., Carrot-Zhang, J., Whelan, C.W., Haradhvala, N.J., Freeman, S.S., Reed, S.C., Rhoades, J., et al. (2018). Structural alterations driving castration-resistant prostate cancer revealed by linked-read genome sequencing. Cell 174, 433–447.e19. Wu, Y.M., Cieslik, M., Lonigro, R.J., Vats, P., Reimers, M.A., Cao, X., Ning, Y., Wang, L., Kunju, L.P., de Sarkar, N., et al. (2018). Inactivation of CDK12 delineates a distinct immunogenic class of advanced prostate cancer. Cell 173, 1770–1782.e14. Zhang, T., Kwiatkowski, N., Olson, C.M., Dixon-Clarke, S.E., Abraham, B.J., Greifenberg, A.K., Ficarro, S.B., Elkins, J.M., Liang, Y., Hannett, N.M., et al. (2016). Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors. Nat. Chem. Biol. 12, 876–884. Zhang, X., Claerhout, S., Prat, A., Dobrolecki, L.E., Petrovic, I., Lai, Q., Landis, M.D., Wiechmann, L., Schiff, R., Giuliano, M., et al. (2013). A renewable tissue resource of phenotypically stable, biologically and ethnically diverse, patientderived human breast cancer xenograft models. Cancer Res. 73, 4885–4897.

Please cite this article in press as: Quereda et al., Therapeutic Targeting of CDK12/CDK13 in Triple-Negative Breast Cancer, Cancer Cell (2019), https://doi.org/10.1016/j.ccell.2019.09.004

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Phospho-Ser2 CTD

Cell Signaling

Cat#13499S; RRID: AB_2235415

Phospho-Ser5 CTD

Cell Signaling

Cat#13523S; RRID: AB_2798246

Phospho-Ser7 CTD

Abcam

Cat#ab126537

Total CTD

Abcam

Cat#ab817; RRID: AB_306327

ATM

Cell Signaling

Cat#2873S; RRID: AB_2062659

RAD51

Santa Cruz

Cat#sc-8349; RRID: AB_2253533

g-H2AX (Western Blot – IHQ)

Cell Signaling

Cat#9718S; RRID: AB_2118009

g-H2AX (IF)

Millipore

Cat#05-636; RRID: AB_2755003

cPARP

Cell Signaling

Cat#9541S; RRID: AB_331426

Cleaved Caspase 3 (CC3)

Cell Signaling

Cat#9661S; RRID: AB_2341188

BRCA1

Cell Signaling

Cat#9010S; RRID: AB_2228244

Phospho-Ser15 p53

Cell Signaling

Cat#9286S; RRID: AB_331741

Phospho-Ser9 p53

Cell Signaling

Cat#9288S; RRID: AB_331470

Total p53

Santa Cruz

Cat#sc-126; RRID: AB_628082

KI67

Cell Signaling

Cat#9129S; RRID: AB_2687446

Antibodies

IRDye  680RD Goat anti-Mouse IgG (H+L)

Li-Cor

Cat#926-68070; RRID: AB_10956588

CDK12

Novus

Cat#NB100-87011; RRID: AB_1199396

CDK13

Millipore

Cat#ABE1860

IRDye  800CW Goat anti-Rabbit IgG (H+L)

Li-Cor

Cat#926-32211; RRID: AB_621843

Alexa-Fluor 488 Goat anti-Rabbit IgG (H+L)

Life Technologies

Cat#A-11008; RRID: AB_143165

Alexa-Fluor 594 Donkey anti-Mouse IgG (H+L)

Life Technologies

Cat#A-21203; RRID: AB_2535789

GAPDH

Millipore

Cat#CB1001-500UG; RRID: AB_2107426

Life Technologies

C737303

Baylor College of Medicine (Zhang et al., 2013)

N/A

DMSO

Sigma-Aldrich

Cat#D2650

Odyssey Blocking Buffer

Li-Cor

Cat#927-40000

Paraformaldehide (PFA)

Electron Microscopy Sciences

Cat#15710

Cdk12 recombinant protein

Proquinase

Cat#1483-1484-1

Bacterial and Virus Strains Stbl3 Chemically Competent E. coli Biological Samples Patient-derived xenografts (PDX) Chemical, Peptides, and Recombinant Proteins

CTDpS7 peptide

LifeTein

N/A

DMEM

Life Technologies

Cat#11965118

Fetal Bovine Serum

Life Technologies

Cat#16140071

Insulin

Life Technologies

Cat#12585014

Cisplatin

Biotang Inc

Cat#RYG01

Irinotecan

LC Laboratories

Cat#I-4122

Doxorubicin

LC Laboratories

Cat#D-4000

Low-gelling-temperature agarose

Sigma-Aldrich

Cat#A0701

Proteinase K

Millipore

Cat# 706634

Propidium Iodide

Sigma-Aldrich

Cat#P4864

Olaparib

LC Laboratories

Cat#O-9201-50MG

Hydroxypropyl-b-Cyclodextrin (hp-BCD)

Sigma-Aldrich

Cat#389145-25G (Continued on next page)

Cancer Cell 36, 1–14.e1–e7, November 11, 2019 e1

Please cite this article in press as: Quereda et al., Therapeutic Targeting of CDK12/CDK13 in Triple-Negative Breast Cancer, Cancer Cell (2019), https://doi.org/10.1016/j.ccell.2019.09.004

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

Crystal Violet

Alfa Aesar

Cat#22866

qScript cDNA SupeMix

QuantaBio

Cat#95048-100

Power SYBR Green PCR Master Mix

Life Technologies

Cat#4367659

Bovine Serum Albumin (BSA)

Sigma-Aldrich

Cat#A3803-50G

Hoechst

Thermo Scientific

Cat#62249

Lipofectamine3000

Life Technologies

Cat#L3000015

SR-4835

This paper

N/A

RNeasy Plus Mini Kit

Qiagen

Cat#74136

ADP-Glo Kinase Assay

Promega

Cat#V9101

Critical Commercial Assays

CellTiter-Glo Luminescent Cell Viability Assay

Promega

Cat#G7571

Caspase-Glo 3/7

Promega

Cat#G8091

BioProject

PRJNA530774, https://www.ncbi. nlm.nih.gov/bioproject/528296

Human: MDA-MB-231

ATCC

Cat#HTB-26; RRID: CVCL_0062

Human: MDA-MB-436

ATCC

Cat#HTB-130; RRID: CVCL_0623

Human: HS578T

ATCC

Cat#HTB-126; RRID: CVCL_0332

Human: MDA-MB-468

ATCC

Cat#HTB-132; RRID: CVCL_0419

Human: FHC

ATCC

Cat#CRL-1831; RRID: CVCL_3688

Charles River

Cat#250; RRID: IMSR_CRL:250

ATM_F: GCTGACAATCATCACCAAGT

This paper

N/A

ATM_R: GGTTCTCAGCACTATGGGACA

This paper

N/A

ATR_F: CGCTGAACTGTACGTGGAAA

This paper

N/A

ATR_R: CAATTAGTGCCTGGTGAACATC

This paper

N/A

BRCA1_F: CTGCTCAGGGCTATCCTCTCA

This paper

N/A

BRCA1_R: GCTTCTAGTTCAGCCATTTCCTG

This paper

N/A

CD44_F: CGGACACCATGGACAAGTTT

This paper

N/A

CD44_R: GAAAGCCTTGCAGAGGTCAG

This paper

N/A

CDK12_F: CCAATCTGGAACTGGCTCAG

This paper

N/A

Deposited Data Bioproject – Gene Expression Data Experimental Models: Cell Lines

Experimental Models: Organisms/Strains Mouse: Fox Chase SCID Beige Female Oligonucleotides

CDK12_R: CAAGTGCTGCAGAAGGAATG

This paper

N/A

CDK13_F: GGTGTTTGAATATATGGACC

This paper

N/A

CDK13_R: CAAGTCCAAAGTCTGCAAGTT

This paper

N/A

CCNK_F: CTCCCAAAGAAGAGAACAAAGCA

This paper

N/A

CCNK_R: AGGCAACGGTGGATGAGTG

This paper

N/A

E2F1_F: CATCAGTACCTGGCCGAGAG

(Rocca et al., 2019)

N/A

E2F1_R: CCCGGGGATTTCACACCTTT

(Rocca et al., 2019)

N/A

FANCD2_F: CCCAGAACTGATCAACTCTCCT

This paper

N/A

FANCD2_R: CCATCATCACACGGAAGAAA

This paper

N/A

FANCI_F: CACCACACTTACAGCCCTTG

This paper

N/A

FANCI_R: ATTCCTCCGGAGCTCTGAC

This paper

N/A

GAPDH_F: TCACCAGGGCTGCTTTTAAC

This paper

N/A

GAPDH_R: ATCTCGCTCCTGGAAGATGG

This paper

N/A

KRAS_F: CCTGCTGTGTCGAGAATATCCA

(Nakayama et al., 2017)

N/A

KRAS_R: TTGACGATACAGCTAATTCAGAATCA

(Nakayama et al., 2017)

N/A

NRAS_F: GCGAAGGCTTCCTCTGTGTA

(Lau et al., 2017)

N/A (Continued on next page)

e2 Cancer Cell 36, 1–14.e1–e7, November 11, 2019

Please cite this article in press as: Quereda et al., Therapeutic Targeting of CDK12/CDK13 in Triple-Negative Breast Cancer, Cancer Cell (2019), https://doi.org/10.1016/j.ccell.2019.09.004

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

NRAS_R: CTTGTTTCCCACTAGCACCA

(Lau et al., 2017)

N/A

RAD51_F: GCTGATGAGTTTGGTGTAGCAG

This paper

N/A

RAD51_R: GGAAGACAGGGAGAGTCGTAGA

This paper

N/A

SMARCC_F: GAGGGATAAGCAGGTTCTTCTG

This paper

N/A

SMARCC_R: GGGATCCACGTGTCGTAACT

This paper

N/A

TP53_F: CCCAAGCAATGGATGATTTGA

This paper

N/A

TP53_R: GCATTCTGGGAGCTTCATCT

This paper

N/A

VEGFA_F: CTTGCCTTGCTGCTCTACC

(Niki et al., 2000)

N/A

VEGFA_R: CACACAGGATGGCTTGAAG

(Niki et al., 2000)

N/A

ATM pre-intra-polyAdenilation/ex2/ex3_F: GAACATGATAGAGC TACAGAACG

This paper

N/A

ATM pre-intra-polyAdenilation/ex2_R: GCATCCCAATTCAAATA TTTTC

This paper

N/A

ATM post-intra-polyAdenilation ex3_R: GCTTGTGTTGAGGCTG ATAC

This paper

N/A

ATM post-intra-polyAdenilation ex15_F: GATCGCTGTCTTCTGG GATT

This paper

N/A

ATM post-intra-polyAdenilation ex15_R: CCTGCACATTGCATTA GAGA

This paper

N/A

ATM post-intra-polyAdenilation/ex21_F: TGGAGAAGAGTACCC CTTGC

This paper

N/A

ATM post-intra-polyAdenilation/ex21_R: GCAATTTACTAGGGC CATTC

This paper

N/A

ATR pre-intra-polyAdenilation_F: CAGTTGTACAGAAGCCAAG ACAA

This paper

N/A

ATR pre-intra-polyAdenilation_R: TCATGGCTTCCACTCACATT

This paper

N/A

ATR post-intra-polyAdenilation_F: GGATGCCACTGCTTGTTATG

This paper

N/A

ATR post-intra-polyAdenilation_R: AGCATGCACTCCATTCACC

This paper

N/A

BLM pre-intra-polyAdenilation_F: GGGAAGTTTGGATCCTGGTT

This paper

N/A

BLM pre-intra-polyAdenilation_R: TGTTCTGGCTGAGTGACGTT

This paper

N/A

BLM post-intra-polyAdenilation_F: CTGAAACATGAGCGTTTCCA

This paper

N/A

BLM post-intra-polyAdenilation_R: CTGAGTCAGTCTTATCACC TGTC

This paper

N/A

BRCA2 pre-intra-polyAdenilation_F: CCGCTGTACCAATCTCC TGT

This paper

N/A

BRCA2 pre-intra-polyAdenilation_R: GGACAGGAAACATCATCT GCT

This paper

N/A

BRCA2 post-intra-polyAdenilation_F: GCCGTACACTGCTCAAA TCA

This paper

N/A

BRCA2 post-intra-polyAdenilation_R: TCCTTTTGGCCATACAAA GTG

This paper

N/A

GABPB1_F: GGTCAGCCCATCATTGTGAC

(Tian et al., 2007)

N/A

GABPB1 pre-intra-polyAdenilation_R: CAATATTTATTTATTGAG GGCTTGC

(Tian et al., 2007)

N/A

GABPB1 post-intra-polyAdenilation_R: GCTTCATCCAGCTGTT TCTG

(Tian et al., 2007)

N/A

CSTF3_F: GAGGCCATGTCAGGAGAC

(Tian et al., 2007)

N/A

CSTF3 pre-intra-polyAdenilation_R: GCAACTCCAAAATGCAA CAA

(Tian et al., 2007)

N/A

CSTF3 post-intra-polyAdenilation_R: CATAAATCAATGTGCAAA ACC

(Tian et al., 2007)

N/A (Continued on next page)

Cancer Cell 36, 1–14.e1–e7, November 11, 2019 e3

Please cite this article in press as: Quereda et al., Therapeutic Targeting of CDK12/CDK13 in Triple-Negative Breast Cancer, Cancer Cell (2019), https://doi.org/10.1016/j.ccell.2019.09.004

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

sgRNA targeting sequence CDK12_1: TGGCTCAGCTAGAACTGATC

Genescript

Cat#SC1678

sgRNA targeting sequence CDK12_6: CAAATCAAACTAGCAGATTT

Genescript

Cat#SC1678

sgRNA targeting sequence CDK13_1: ATATATGGACCATGATCTGA

Genescript

Cat#SC1805

sgRNA targeting sequence CDK13_3: ACCTATATTTCAAGCAAATC

Genescript

Cat#SC1805

Oligonucleotides

Recombinant DNA MISSION packaging system

Sigma-Aldrich

Cat#SHP001-1.7ML

pLenti CRISPR v2

Genescript

N/A

pLenti CRISPR v2 gRNA-CDK12_1

Genescript

Cat#SC1678

pLenti CRISPR v2 gRNA-CDK12_6

Genescript

Cat#SC1678

pLenti CRISPR v2 gRNA-CDK13_1

Genescript

Cat#SC1805

pLenti CRISPR v2 gRNA-CDK13_3

Genescript

Cat#SC1805

Software and Algortihms PRISM

GraphPad Software

Version 7

Kinome tree

(Eid et al., 2017)

http://www.kinhub.org/kinmap/

Compusyn

ComboSyn, Inc.

V1.0

ImageJ

NIH

https://imagej.nih.gov/ij/

ImageStudio Lite

Li-Cor

V4.0

IN Cell Developer Toolbox

GE Healthcare

V1.6

LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact Derek Duckett ([email protected]). EXPERIMENTAL MODELS AND SUBJECT DETAILS Cell Lines MDA-MB-231 and MDA-MB-468 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) in presence of penicillin/streptomycin. MDA-MB-436 and HS678T cell lines were cultured in DMEM supplemented with 10 mg/ml insulin and 10% FBS in presence of penicillin/streptomycin. FHCs were cultured in DMEM:F12 Medium supplemented with extra 10mM HEPES, 10 ng/ml cholera toxin, 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 100 ng/ml hydrocortisone, 20 ng/ml human recombinant EGF and 10% FBS in presence of penicillin/streptomycin. Tumor Models and TNBC PDX All animal studies were approved by the Moffitt Cancer Center and Scripps Florida Institutional Animal Care and Use Committee. The patient derived xenograft models BCM-4013 and BCM-3887 were a kind gift from Dr. Jenny C Chang (Methodists Cancer Center, Houston, TX) and establishment of these PDX models were performed as described (Zhang et al., 2013). Briefly, fresh xenograft tumor fragments (1 mm3) were transplanted into the cleared mammary fat pad of recipient SCID Beige mice (Charles River Laboratories). Tumors were measured twice a week by caliper and tumor volume was calculated using the formula; Tumor volume = width 3 width 3 length 3 0.52. When tumors measured 100-150 mm3, mice were divided into groups for treatment. SR-4835 and Cisplatin Efficacy Studies Mice were divided into four groups: vehicle control, SR-4835, cisplatin, and SR-4835 and cisplatin in combination. SR-4835 was administered PO 5 days per week at 20 mg/kg prepared in 10/90 DMSO/30% Hydroxypropyl-b-Cyclodextrin (hp-BCD) in water; cisplatin was administered IP once a week at 6 mg/kg for a total of two doses prepared in saline.

e4 Cancer Cell 36, 1–14.e1–e7, November 11, 2019

Please cite this article in press as: Quereda et al., Therapeutic Targeting of CDK12/CDK13 in Triple-Negative Breast Cancer, Cancer Cell (2019), https://doi.org/10.1016/j.ccell.2019.09.004

SR-4835 and Irinotecan Efficacy Studies Mice were divided into four groups: vehicle control, SR-4835, irinotecan, and SR-4835 and irinotecan in combination. SR-4835 was administered PO 5 days per week at 20 mg/kg prepared in 10/90 DMSO/30% Hydroxypropyl-b-Cyclodextrin (hp-BCD) in water; irinotecan was administered IP as a single treatment at 50 mg/kg prepared in saline. METHOD DETAILS SR-4835 Synthesis SR-4835 was prepared according to the reported procedures (Monastyrskyi et al., 2018). 2,6-dichloro-9-(1-methyl-1H-pyrazol-4-yl)9H-purine (6g, 22.30 mmol), and (5,6-dichloro-1H-benzo[d]imidazol-2-yl)methanamine, 2HCl (6.77 g, 23.41 mmol) were charged into 250 ml round bottom flask followed by addition of 2-propanol (89 ml) at room temperature. DIPEA (19.47 ml, 111 mmol) was then added to the suspension and vial was heated in a conventional oil bath at 90 C for 60 min. The reaction mixture was allowed to reach room temperature, and then the precipitate was filtered and washed with Et2O (LCMS m/z 449.7 (M + 1)). 2-chloro-N-((5,6-dichloro1H-benzo[d]imidazol-2-yl)methyl)-9-(1-methyl-1H-pyrazol-4-yl)-9H-purin-6-amine (8g, 17.83 mmol) was then added to a 250 ml round bottom flask followed by addition of morpholine (15.53 ml, 178 mmol). The mixture was then stirred at 130 C for 30 min. The reaction mixture was concentrated under reduced pressure and the crude product was purified by flash chromatography (DCM:MeOH, 10:1, gradient). Fractions containing product were combined, and the organic phase was washed twice with NH4Cl (to remove residual morpholine), and finally once with brine. The organic layer was dried over Na2SO4 and the solvent was evaporated under reduced pressure to yield SR-4835 as a white solid (74% yield over two steps). LCMS m/z 500.7 (M + 1). 1H NMR (400 MHz, DMSO-d6) d 8.31 (s, 1H), 8.21 (s, 2H), 7.99 (s, 1H), 7.71 (s, 2H), 4.82 (s, 2H), 3.91 (s, 3H), 3.58 – 3.45 (m, 8H). 13C NMR (101 MHz, DMSO-d6) d 158.68, 156.53, 154.03, 150.17, 137.49, 136.35, 130.75, 124.18, 122.88, 118.63, 115.89, 113.46, 65.99, 44.56, 39.16, 38.54. In Vitro Biochemical Kinase Profiling of SR-4835 A panel of 465 kinases was tested by ThermoFisher Scientific  using Z0 -LYTE, Adapta and LanthaScreen technologies. Individual CRCs against PARP1, BRD4, CDK1-Cyclin A, CDK2-Cyclin A2 and CDK2-Cyclin E were obtained from Reaction Biology Corp. Kds for CDK12, CDK13, CDK4-cyclin D1, CDK6, CDK9, GSK3A and GSK3B were obtained from DiscoverX. In-Cell Western Analyses MDA-MB-231 cells were seeded in 384-wells clear bottom black plates (7,000 cells/well). The day after, cells were treated with selected compounds for 4 hr in decreasing concentrations, after which the cells were fixed with 4% PFA in PBS for 30 min at room temperature. After washing with PBS, the cells were permeabilized with 0.3% Tx100 in PBS for 15 min at room temperature. Cells were washed with PBS and incubated with blocking buffer for 2 hr at room temperature. Cells were then incubated with primary antibodies against anti-pSer2 RNA Pol II CTD (Cell Signaling) or total RNA Pol II overnight at 4 C. After washing with 0.1% Tween-20 in PBS, the cells were incubated with appropriate secondary antibodies for 1hr at room temperature. After final washes with 0.1% Tween-20 in PBS, the top of the wells was covered with a black seal and the cells were visualized by Li-Cor Odyssey. ADP-Glo CDK12 Assay The reaction was performed following the protocol from Proquinase using CDK12 as an enzyme and a peptide containing three repeats of the CTDpS7 as substrate. Cell Proliferation Assay Cell proliferation was measured 72 hr after compound treatment using CellTiter-Glo (Promega) according to the manufacturer’s instructions. EC50 values were determined by nonlinear regression and a four-parameter algorithm (GraphPad Prism 7). Clonogenic Assays 500 cells per well were plated in six-well dishes in triplicate. After overnight incubation, SR-4835 or vehicle (DMSO) was added to the medium for 72 hr, and cells were allowed to grow for 7 to 10 days, during which medium was changed every 2 to 3 days. Colonies were fixed in 4% PFA in PBS, stained with 0.5% crystal violet in 25% methanol for 20 min at room temperature, and then were destained with water. Pictures of the wells were taken and the stain was extracted by adding 10% acetic acid to each well. The absorbance was then measured at 590 nm and triplicate wells stained without cells were used as a reference. Western Blot Analyses SDS–polyacrylamide gel electrophoresis was performed using NuPAGE 4 to 12% bis-tris gels (Invitrogen), and proteins were transferred to nitrocellulose membranes by semidry transfer using Trans-Blot transfer medium (Bio-Rad). Membranes were blocked in Odyssey blocking buffer (LI-COR Biosciences) and incubated overnight at 4 C with primary antibodies. After repeated washes with TBS-T [20 mM Tris (pH 7.6), 140 mM NaCl, and 0.1% Tween 20], the blots were incubated with the appropriate IRDye-conjugated secondary antibody (LI-COR Biosciences) and imaged using the LI-COR Odyssey. Bands were quantified using the Odyssey software (LI-COR Biosciences). Cancer Cell 36, 1–14.e1–e7, November 11, 2019 e5

Please cite this article in press as: Quereda et al., Therapeutic Targeting of CDK12/CDK13 in Triple-Negative Breast Cancer, Cancer Cell (2019), https://doi.org/10.1016/j.ccell.2019.09.004

qRT-PCR Analyses Total RNA was obtained with the RNeasy Plus Mini Kit (Qiagen), and 1 mg of RNA was reverse transcribed with qScriptTM cDNA SuperMix (QuantaBio) or SuperScript III First-Strand Synthesis System (ThermoFisher Scientific) primed with oligo(dT) for specific intra-polyadenylation studies. qRT-PCR was performed with the Power SYBR Green PCR Master Mix (Life Technologies) on the Applied Biosystems 7900HT Fast Real-Time PCR System. Intron-spanning gene specific primer pairs were designed using the Primer3 algorithm, and relative expression values for each gene of interest were obtained by normalizing to GAPDH mRNA expression using the DDCt method. Immunofluorescence Cells were fixed with 3% PFA-2% sucrose in PBS for 10 min at room temperature, permeabilized with 0.5% Tx-100 in PBS for 5 min on ice, and blocked in 3%BSA in PBS for 15 min at room temperature. Cells were then incubated in primary antibodies against BRCA1 (Cell Signaling, Cat#9010S), and g-H2AX (S139, Millipore, clone JBW301). Alexa-Fluor conjugated secondary antibodies were used (Life Technologies). Cell nuclei were stained using Hoescht reagent (Thermo Scientific, Cat#62249). Images were acquired using INCELL6000 and eight fields of view per well were analyzed using INCELL Developer software to automatically count BRCA1 or g-H2AX foci per nucleus. At least 100 cells were counted per field of view. A cell was classified as g-H2AX or BRCA1 positive only if >10 foci per nucleus were identified. Comet Assay Neutral comet assays were performed as described by Olive et al. (Olive and Banath, 2006). Briefly, 1.2 ml of 1% low-gelling-temperature agarose was added to 8000 cells in 0.4 ml of PBS and pipetted onto Trevigen comet slides (Cat#4250-004-03). Once gel had solidified, slides were incubated in N1 lysis solution (2% sarkosyl, 0.5 M Na2-EDTA, 0.5 mg/ml Proteinase K, pH 8) overnight at 37 C. After three washes with N2 buffer (90 mM Boric Acid, 2 mM Na2-EDTA, 90 mM Tris-HCl pH 8.5), electrophoresis was performed for 20 min at 25 V in N2 buffer. After washes, slides were stained with 2.5 mg/ml propidium iodide. 50 cells from each condition were evaluated with the HCSA Comet analysis system from pictures obtained with a microscope Olympus bx51. CRISPR/cas9 Editing Lentivirus was generated by transfecting 293T cells with the appropriate lentiCRISPRv2 sgRNA vector and the packaging plasmids MISSION, using Lipofectamine3000 per the manufacturer’s instructions (Promega). 24 hr after transfection, the medium was changed and media containing virus was collected at 48 and 72 hr post-transfection and filtered through a 0.20 mM filter. MDAMB-231 cells were transduced in 6-well plates. 48 hr post-infection, cells were selected in puromycin–containing media. 5 days following selection, cells were harvested for protein and RNA analyses. Colony formation assays were performed for an extra two days. Apoptosis Assay Apoptosis was measured 48 hr after compound treatment using Caspase-Glo 3/7 (Promega) according to the manufacturer’s instructions. Curves were determined by nonlinear regression and a four-parameter algorithm (GraphPad Prism 7). RNA-seq Analyses Sequencing libraries were prepared using standard methods and sequenced on an Illumina NextSeq 500 in the Genomics Core at Scripps Research, Florida. Reads were trimmed using trimmomatic (v0.35) using the command ‘‘trimmomatic-0.35.jar PE -phred33 -trimlog TrimmomaticLog.out R1.fastq.gz R2.fastq.gz R1.pair.trim.fastq.gz R1.unpair.trim.fastq.gz R2.pair.trim.fastq.gz R2.unpair. trim.fastq.gz ILLUMINACLIP:Adapter.RNA_strandLT.fa:2:30:10 LEADING:5 TRAILING:5 SLIDINGWINDOW:4:15 MINLEN:36’’. The contents of the Adapter.RNA_strandLT.fa file were as follows: >PrefixPE/1 TACACTCTTTCCCTACACGACGCTCTTCCGATCT >PrefixPE/2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT >PE1 TACACTCTTTCCCTACACGACGCTCTTCCGATCT >PE2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT >PE1_rc AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA >PE2_rc AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC Trimmed reads were mapped to the human genome (human-ENSEMBL-grch38.r91 : H.sapiens-ENSEMBL-GRCh38.r91 : downloaded February 16, 2018), using star (v2.5.2a) with the following command: ‘‘STAR –genomeDir Overhang79 –readFilesCommand zcat –readFilesIn R1.pair.trim.fastq.gz R2.pair.trim.fastq.gz –outFileNamePrefix star’’. Gene counts were obtained using python

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Please cite this article in press as: Quereda et al., Therapeutic Targeting of CDK12/CDK13 in Triple-Negative Breast Cancer, Cancer Cell (2019), https://doi.org/10.1016/j.ccell.2019.09.004

(v2.7.11) and htseq (v0.9.1) with the command: ‘‘htseq-count –mode=union –stranded=reverse –idattr=gene_id starAligned.out.sam Annotation.gtf’’. Genes were tested for differential gene expression using DESeq2 (R v3.4.3, deseq2 v1.18.1). Gene Expression Analyses The list of significantly (padj < 0.01) regulated genes for each comparison were uploaded for Ingenuity pathway analysis and all mapped genes were analyzed using Core Analysis to identify statistically significant canonical pathways via right-tailed Fisher’s exact test. Ingenuity pathway databases also estimate regulatory direction for a subset of the canonical pathways and an activation z value is calculated to identify the direction and statistical significance of the regulation for each of the pathways. Only this particular subset of canonical pathways was considered and relevant pathways were chosen for further consideration. Additionally, using Comparison Analysis tool, the individual datasets (padj < 0.01) were compared and a heatmap that displayed activation z score per pathway in each dataset was created and filtered by selecting cancer relevant pathways. Chou-Talalay Combination Index Analysis To assess combination index, different volume combinations (1:1, 1:4 or 4:1) of the initial stock concentration of each drug (cisplatin, olaparib and irinotecan equal to 400 mM, doxorubicin 10 mM and for SR-4835 either 4 mM or 10 mM) was used to generate 10-point 1:3 dilution concentration response curves. Loewe additivity is a dose-effect model, which states that additivity occurs in a two-drug combination if the sum of the ratios of the dose vs. the median-effect for each individual drug is 1. In this model, combination index (CI) scores estimate the interaction between the two drugs. If CI < 1, the drugs have a synergistic effect and if CI > 1, the drugs have an antagonistic effect. CI = 1 means the drugs have an additive effect. Chou and Talalay (Chou and Talalay, 1984) showed that Loewe equations are valid for enzyme inhibitors with similar mechanisms of action – either competitive or non-competitive toward the substrate. The combination index (CI) coefficients were computed based on the Chou-Talalay Median Effect model as implemented in CalcuSyn v2.11 (http://www.biosoft.com/w/calcusyn.htm). The degree of interaction between drugs was estimated according to the classification presented by Chou-Talalay (Chou and Talalay, 1984). Mouse Pharmacokinetic Studies Pharmacokinetic analyses were determined in male C57Bl-6 mice (n = 3). Compounds were dosed as indicated in the text via intravenous or oral gavage. Plasma was collected into EDTA containing tubes which were kept on ice until plasma collected by standard centrifugation techniques. Time points for determination of pharmacokinetic parameters were 5 min, 15 min, 30 min, 1 hr, 2 hr, 4 hr, 6 hr, and 8 hr. Plasma concentrations were determined via LC-MS/MS using a nine point standard curve between 2 and 2000 ng/ml prepared in mouse plasma. Samples above the curve were diluted with blank mouse plasma. Pharmacokinetic analysis was performed with WinNonLin, Pharsight inc. using a noncompartimental model. Immunohistochemistry Paraffin sections (3 mm) were mounted on Plus slides and dried in a 60 C oven. The slides were placed on a Leica BondMax Immunostainer. Antibodies were optimized with a predetermined staining protocol: g-H2AX (Phospho-Histone H2A.X (Ser139), 1:4000; Rabbit mAb KI67, 1:800; and cleaved Caspase-3 (Asp175), 1:1000. Slides were dehydrated and cover-slipped with Cytoseal 60 (Richard-Allan Scientific) mounting medium. QUANTIFICATION AND STATISTICAL ANALYSIS Statistical analyses and graphical presentations were performed using Prism 7.0 (GraphPad). Power analyses were used to determine number of mice in in vivo experiments. Statistical assays performed are specified in figure legends. DATA AND CODE AVAILABILITY RNA seq data deposited in the NCBI BioProject database: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA530774.

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