Mapping the Human Kinome in Response to DNA Damage

bioRxiv preprint doi: https://doi.org/10.1101/385344; this version posted August 6, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Mapping the human kinome in response to DNA damage

2 3

4 Michel Owusu1, Peter Bannauer1, Athanasios Mourikis2, Alistair Jones2, Joana Ferreira da 5 Silva1, Michael Caldera1, Marc Wiedner1, Charles-Hugues Lardeau1,3, Jörg Menche1, Stefan 6 Kubicek1,3, Francesca Ciccarelli2 and Joanna I. Loizou1,4

8 1CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 9 Lazarettgasse 14, AKH BT 25.3, 1090 Vienna, Austria 10 2The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK 11 3Christian Doppler Laboratory for Chemical Epigenetics and Antiinfectives, CeMM Research 12 Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria 13 4Corresponding author 14 15 Running title: Response of kinome to DNA damage 16 Key words: Kinome, kinase, DNA damage, chemotherapeutics, carmustine, temozolomide, 17 synthetic lethality. 18 19 Lead contact: Joanna I. Loizou 20 Email: [email protected] 21 bioRxiv preprint doi: https://doi.org/10.1101/385344; this version posted August 6, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

22 Summary 23 We provide a catalog for the effects of the human kinome on cell survival in response to 24 DNA damaging agents, selected to cover all major DNA repair pathways. By treating 313 25 kinase-deficient cell lines with ten diverse DNA damaging agents, including seven commonly 26 used chemotherapeutics, we were able to identify kinase specific vulnerabilities and 27 resistances. In order to identify novel synthetic lethal interactions, we investigate the cellular 28 response to carmustine for 25 cell lines, by establishing a phenotypic FACS assay designed 29 to mechanistically investigate and validate gene-drug interactions. We show apoptosis, cell 30 cycle, DNA damage and proliferation after alkylation or crosslink-induced damage for 31 selected cell lines and rescue the cellular sensitivity of DYRK4, EPHB6, MARK3, PNCK as a 32 proof of principle for our study. Our data suggest that some cancers with inactivated DYRK4, 33 EPHB6, MARK3 or PNCK gene could be particularly vulnerable to treatment by alkylating 34 chemotherapeutic agents carmustine or temozolomide. 35 36 Introduction (<200 words) 37 The DNA damage response (DDR) is elicited by a complex and far-reaching network of 38 proteins that are commonly deregulated in human pathologies, including cancer1. Besides 39 surgery, the most common treatment for cancer patients is radio- or chemotherapy. To date, 40 some of the commonly used chemotherapeutic compounds are DNA damaging agents2. 41 DNA damaging agents can cause cell death by targeting either DNA directly or proteins 42 implicated in DNA repair and replication, cell cycle regulators or signal transducers. Protein 43 kinases are an important group of signal transducers and are often deregulated in human 44 cancer3, making them particularly interesting to study within the signaling context of the DNA 45 damage response. Moreover, kinases represent an important group of drug targets4, due to 46 their enzymatic function, and therefore results from loss-of-function studies with kinases are 47 more likely to be translated into a therapeutic setting. Kinases have broad functions following 48 DNA damage. For instance, the ATM superfamily which includes ATM, ATR and DNA-PKcs 49 (encoded by the gene PRKDC) is involved in sensing or amplifying initial signals of DNA 50 lesions5,6. CHK1 and CHK2 kinases, regulate cell cycle progression in response to DNA 51 damage, providing time for DNA repair7,8. Other kinases such as ABL1 are involved in 52 transducing or fine tuning signals resulting from DNA damage, which can ultimately lead to 53 survival, senescence or cell death9. Though some kinases have been studied in depth, the 54 role of many is still not known10. Here, we used CRISPR-Cas9 to individually delete 55 expressed and non-essential kinases in human HAP1 cells. Next, we performed a drug 56 screen using DNA damaging agents, selected to cover all major DNA repair pathways, to 57 map drug specific sensitivities and resistances. We validated selected drug-gene 58 interactions in response to alkylation-induced damage and assessed the contribution of

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59 apoptosis, DNA damage, cell cycle arrest and proliferation leading to cellular sensitivities or 60 resistances by designing and utilizing a phenotypic assay. 61 62 Results 63 We used CRISPR-Cas9 to target 313 expressed and non-essential kinases in human HAP1 64 cells and produce clonal knock-out cell lines11 (Supp. Table 1). The kinase genes targeted 65 cover all groups, according to the standard classification scheme of kinases12, hence 66 ensuring coverage (Figure 1a). To examine the response of the non-essential human 67 kinome to a broad range of DNA damaging agents, we first designed and optimized our 68 approach using DNA repair deficient cell lines, where we were able to recover known gene- 69 drug interactions (Figure 1b & Supp. Figure 1). Based on this approach, we selected 10 70 compounds that (1) induce different types of DNA damage and thus utilize distinct DNA 71 repair pathways and (2) are frequently used as chemotherapeutics (Figure 1c). Next, we 72 exposed the 313 kinase-deficient cell lines to these compounds at four concentrations and 73 assessed cellular survival after three days (Figure 1b). 74 75 Based on literature, most cell lines showing strong sensitivity or resistance to the 76 compounds were anticipated (Figure 1d, Supp. Table 1). For instance, PRKDC depleted 77 cells showed the strongest sensitivity to DNA double-strand break inducing agents, 78 etoposide and doxorubicin, whereas ABL1 depleted cells showed resistance those agents9. 79 80 A clustering of the cell lines by their sensitivity to the 10 compounds revealed 3 clusters, 81 characterized by high sensitivity to carmustine (Cluster 1), hydroxyurea (Cluster 2) and DNA 82 double-strand break inducing agents, such as etoposide and doxorubicin (Cluster 3) (Figure 83 1e, Supp. Table 1). As expected, we found that compounds with similar modes of action 84 were closer in the clustering, as illustrated by topoisomerase II inhibitors (doxorubicin and 85 etoposide), topoisomerase I inhibitors (topotecan and camptothecin) and agents that 86 induced replicative stress (aphidicolin and cytarabine). Due to their clustering with DNA 87 double-strand break inducing agents, our data support the notion that DNA double-strand 88 breaks, following replication fork stalling and collapse, are one of the primary sources of 89 cellular death after treatment with lethal concentrations of aphidicolin, cytarabine or 90 topoisomerase I inhibitors13 (Figure 1e). Interestingly, Cluster 1 was significantly enriched 91 for genes associated with increased chromatin accessibility, compared to clusters 2 and 3 92 (Figure 1f). Alkylating agents, carmustine and temozolomide (TMZ), have been reported to 93 have a global effect on nuclear organization and chromatin structure, inducing chromatin 94 condensation and gene silencing14. We therefore reasoned that in the absence of a kinase in 95 Cluster 1, cellular death upon carmustine treatment may be due to an alkylation induced

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96 synthetic lethality. Differential gene ontology (GO) term enrichment analysis (Figure 1g, 97 Supp. Table 2) confirmed that Cluster 1 was uniquely enriched for terms previously 98 associated to cellular response to alkylating or crosslinking agents: upregulation of vascular 99 endothelial growth factor receptors15, induction of oxidative stress16 (cellular response to 100 hydrogen peroxide, positive regulation of cytochrome-c oxidase activity), which in turn leads 101 to actin cytoskeleton reorganization17 (Figure 1h). 102 103 Since kinases are highly associated with cancer18, and moreover being enzymes they are 104 potentially amenable to chemical inhibition, we focused on carmustine dependent synthetic 105 lethal interactions. Moreover, we found that cell lines lacking MARK3, PRKACA, CSNK1G1, 106 PNCK, DYRK4 or EPHB6 in combination with carmustine, showed the strongest unreported 107 synthetic lethal interaction in the screen (Figure 1d). To validate and further dissect the 108 mechanism of cellular sensitivity to the drug, we measured DNA damage, apoptosis, cell 109 cycle phases and proliferation in those cell lines in a FACS-based phenotypic assay (Figure 110 2a) with the markers γH2AX, TUNEL, DAPI and EdU, respectively. As controls, we included 111 cell lines lacking proteins previously linked to the signaling of DNA damage or DNA repair 112 (PRKDC, ABL119, PDK220, PIM221 and TNK222), cell cycle (CDK10, CLK1 and CDKL1), cell 113 death (GRK623,24 , GSK3B25, MAST126, STK1027 and STK328) and a gene with a strong 114 general resistance to DNA damaging agents as revealed in our study (TSSK3) (Figure 1h). 115 Carmustine is a chemotherapeutic agent used for the treatment of several types of cancers, 116 particularly those relating to the nervous system, such as glioblastoma29,30. It is a bifunctional 117 alkylating agent that produces DNA mono-alkylation adducts as well as DNA intra- and 118 interstrand crosslinks (ICLs)31,32. Almost all of the lesions (90 - 95%) produced by 119 bifunctional alkylating agents are mono alkylation adducts33, such as N7-methylguanine or 120 O6-methylguanine. However, the less abundant (ca. 5%) DNA crosslinks, particularly ICLs, 121 form the most deleterious lesions33 and can interfere with replication or transcription and 122 trigger apoptosis and cell cycle arrest32. In order to confirm that the predominant cause of 123 cellular toxicity to carmustine was due to the effects of alkylation, we used the 124 monofunctional alkylating agent temozolomide (TMZ), which does not produce crosslinks 125 and is often used as a superior replacement therapy to carmustine34, and the crosslinking 126 agent oxaliplatin (Supp. Figure 1b). We chose concentrations of the compounds that 127 moderately affect wild-type cells and as expected, both compounds induced apoptosis, DNA 128 damage, G2/M cell cycle arrest and a reduction of proliferating cells in a dose- and time- 129 dependent manner35,36 (Figure 2b-c). Hierarchical clustering of the cell lines according to 130 apoptosis confirmed that the cellular sensitivity to carmustine was predominantly due to 131 alkylation induced synthetic lethality: the most sensitive survival interactions after carmustine 132 (Figure 1h) showed the highest apoptosis after TMZ in the phenotypic assay (Figure 2b).

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133 134 DNA damage as measured by γH2AX can either be a cause or consequence of apoptosis37. 135 For a gene to be involved in the signaling or repair of DNA damage, we would expect to see 136 higher levels of γH2AX preceding or coinciding with higher levels of apoptosis. For instance, 137 DYRK4-deficient cells showed a peak of apoptosis 24 hours after treatment followed by a 138 peak of γH2AX at 48 hours of treatment (Figure 2b). Hence, the γH2AX signal at the 48 hour 139 time point may therefore be a consequence of apoptosis in DYRK4-deficient cells. This may 140 also be the case for PNCK and PKN2 (Figure 2b). In contrast, PRKDC-deficient cells, which 141 have a deficiency in DNA repair, showed the maximum levels of γH2AX and apoptosis early, 142 at 24 hours after treatment, followed by a slow but coinciding recovery of γH2AX and 143 apoptosis at 48 hours after treatment (Figure 2b). A similar, albeit weaker phenotype can be 144 observed in cells deficient for CSNK1G1, EPHB6, MARK3, PRKACA (Figure 2b). 145 Interestingly, we also observed a strong and persistent G2/M arrest in cells deficient for 146 CSNK1G1 or EPHB6 (Figure 2b). Although the sensitivity of these kinase-deficient cells to 147 alkylating agents revealed in our assay is as of yet unreported, it is in line with what is known 148 about their function: CSNK1G1 has previously been reported to regulate the kinase CHK1, 149 which is a cell cycle regulator following DNA damage38 whereas EPHB6 has been linked to 150 the regulation of NPAT, a DNA damage signaling and cell cycle regulator39. 151 152 After confirming selected cellular survival phenotypes in our phenotypic screen, we next 153 sought to validate gene-drug interactions in knockout cell lines by reconstitution of the wild- 154 type genes. We selected the understudied kinases, DYRK4, EPHB6, PNCK and MARK3, as 155 well as control kinases with resistance phenotypes, ABL1 and TSSK3 (Figure 1h, Supp. 156 Figure 2a). After expression of HA-tagged inducible proteins (Figure 2d) in the deficient cell 157 lines, we assessed whether cellular survival to DNA damage was reverted. Indeed, the 158 sensitivity and resistance phenotypes could be corrected by recombinant expression of the 159 relevant proteins hence establishing a coherent genotype-phenotype relationship (Figure 160 2e, Supp. Figure 2c-e). ABL1 and TSSK3 deficient cells, which showed resistance to 161 doxorubicin or hydroxyurea in the survival screen, became significantly sensitive after 162 reconstitution (Supp. Figure 2c-e), whereas DYRK4, EPHB6, PNCK and MARK3 which 163 showed sensitivities in survival after alkylation-induced damage, became significantly more 164 resistant after reconstitution of the respective wild-type genes (Figure 2e). 165 166 Discussion 167 Unperturbed signaling of DNA damage is essential in guarding the genome against cancer40. 168 At the same time targeting the DNA damage response has proven to be a successful

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169 strategy in cancer therapy. In this study, we have shown the response of 313 cell lines, 170 lacking kinases involved in different cellular signaling pathways, against 10 diverse DNA 171 damaging agents, including 7 commonly used chemotherapeutics. In doing so, we have 172 identified unreported synthetic lethal and resistance gene-drug interactions. Moreover, we 173 reveal that a sensitivity to carmustine may be important for a cluster of genes associated 174 with chromatin accessibility. For selected cell lines, we further probe the synthetic lethality 175 with carmustine by designing a phenotypic assay to investigate and validate gene-drug 176 interactions in a broad manner. We show apoptosis, cell cycle, DNA damage and 177 proliferation after alkylation or crosslink-induced damage for those cell lines. Moreover, we 178 rescue the survival phenotype of DYRK4, EPHB6, MARK3, PNCK as a proof of principle for 179 our study in reconstitution experiments. Our data suggests that some cancers with 180 inactivated DYRK4, EPHB6, MARK3 or PNCK could be particularly vulnerable to alkylating 181 agents. For example, EPHB6 is found to be downregulated in diverse metastatic cancers, 182 including lung41, breast42 and brain43 cancers. Treatment of EPHB6 deficient cancers with 183 the chemotherapeutic agents carmustine or TMZ may therefore represent a promising 184 therapeutic strategy. 185 186 Experimental Procedures (<100 words) 187 Experimental procedures can be found in the Supplemental Information. 188 189 Author Contributions 190 M.O., P.B., MW and C.-H.L. carried out the experimental work. M.O., P.B., A.M., A.J., M.C. 191 and J.F.daS. performed data analysis. Writing was by M.O. with J.I.L. The project was 192 conceptualized by M.O. and J.I.L. Project administration was by S.K., J.M., F.C. and J.I.L. 193 Funding acquisition was by S.K., F.C., J.M. and J.I.L. All authors reviewed and commented 194 on the manuscript. 195 196 Acknowledgements 197 Research in the Kubicek lab is supported by the Austrian Federal Ministry of Science, 198 Research and Economy, the National Foundation for Research, Technology, and 199 Development. M.O., P.B. and J.F.daS. were supported by FWF grant awarded to J.L. 200 (29555 and 29763). 201 202 203 References 204 205 1. Jackson, S. P. & Bartek, J. The DNA-damage response in human biology and

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316 visualization of combinatorial gene-gene and gene-drug interaction screens. BMC 317 Bioinformatics 15, 98 (2014). 318 45. Englinger, B. et al. Acquired nintedanib resistance in FGFR1-driven small cell lung 319 cancer: role of endothelin-A receptor-activated ABCB1 expression. Oncotarget 7, 320 (2016). 321 46. Lawrence, M. S. et al. Mutational heterogeneity in cancer and the search for new 322 cancer-associated genes. Nature 499, 214–218 (2013). 323 47. Kuleshov, M. V. et al. Enrichr: a comprehensive gene set enrichment analysis web 324 server 2016 update. Nucleic Acids Res. 44, W90–7 (2016). 325 48. JAIR, P. R. J. A. I. R.1999. Semantic similarity in a taxonomy: An information-based 326 measure and its application to problems of ambiguity in natural language. jair.org 327 328 49. Supek, F., Bošnjak, M., Škunca, N. & Šmuc, T. REVIGO summarizes and visualizes 329 long lists of gene ontology terms. PLoS ONE 6, e21800 (2011). 330 331 332 Figure Titles and Legends 333 Figure 1. Survival of non-essential kinome in response to DNA damage. (a) Kinome 334 tree representing all kinases12. In bold squares are the kinases targeted by CRISPR-Cas9 335 (313) and in light squares are the remaining kinases. (b) Workflow of survival assay. Dose 336 responses were performed with 4 concentrations in 4 replicates. Cells were incubated with 337 compounds for 3 days and survival was performed using a luminescent readout. (c) List of 338 the 10 DNA damaging compounds selected for use in the survival assay. Compounds with 339 similar modes of action share the same color label. (d) Survival response of cell lines to 340 compounds: kinases from the same kinase groups are clustered together in columns. Each 341 vertical line represents a particular knockout cell line with all of its gene-drug interactions. 342 Compounds are depicted as different color-coded bubbles. HypoM (hypomethylating agent, 343 Decitabine), SSB (single-strand break inducing agents), DSB (double-strand break inducing 344 agents), ReS (replication stress inducing agents), HU (replication stress inducing agent 345 hydroxyurea), Alk & Cros (alkylating and crosslinking agent, BNCU), Chkp (Chk1 inhibitor). 346 Z-scores were calculated for the area under the curve (AUC) of 4 concentrations across the 347 mean of 4 replicates, for each cell line. Lines are set at z-scores greater than 1.65 or less 348 than -1.65 (p<0.05). The names of some expected or known interactions are labeled in black 349 font. Names in red font are examples of lethal interactions after carmustine treatment. AGC= 350 protein kinase families A, G and C; CAMK= Calmodulin/Calcium regulated kinases; CK1= 351 Casein kinase 1; CMGC= CDK, MAPK, GSK3, CLK family; RGC= Receptor guanylate 352 cyclases; STE= STE7, STE11 and STE20 homologs; TK= Tyrosine kinases, TKL= Tyrosine 353 kinase like. (e) Clustering of 313 kinase-deficient cell lines in response to diverse DNA 354 damaging agents reveals three distinct clusters (left dendrogram): Cluster 1 is characterized 355 by a sensitivity to carmustine, Cluster 2 by a sensitivity to hydroxyurea and Cluster 3 by a 356 sensitivity to DNA double-strand break inducing agents, notably etoposide and doxorubicin. 357 Compounds with similar modes of action (color labels) are closer in neighborhood (top

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358 dendrogram): topoisomerase II inhibitors (doxorubicin, etoposide), topoisomerase I inhibitors 359 (topotecan, camptothecin), replication stress inducing agents by fork staling (cytarabine, 360 aphidicolin). (f) Difference in chromatin accessibility of genes from Cluster 1, defined by 361 sensitivity to carmustine compared to genes from Cluster 2 or 3. Increasing Hi-C values 362 correspond to increasing chromatin accessibility. (g) Gene ontology (GO) term enrichment 363 analysis for Clusters 1 - 3. (h) GO terms enriched for Cluster 1 uniqely. 364 365 Figure 2. Phenotypic and genetic validation of sensitivity to alkylating agents. (a) 366 Workflow of phenotypic screen. Wild-type and 25 CRISPR-Cas9 knockout cell lines were 367 treated with alkylating or crosslinking agents (temozolomide or oxaliplatin, respectively) for 5 368 or 24 hours, after which drug medium was replaced with fresh medium to allow cells to 369 recover from damage. EdU incorporation was performed for 40 minutes before harvest. 370 Cells were fixed and co-stained using the following markers: TUNEL for apoptosis, anti- 371 γH2AX for DNA damage, DAPI for cell cycle and EdU stain for proliferation. Cells were 372 analysed by flow cytometry. For each stain, the six concentrations for each time point were 373 summarized with an area under the curve (AUC) calculation. Bottom: Figure legend and 374 phenotypic plot of wild-type HAP1 cells after temozolomide treatment (right). (b-c) 375 Phenotypic plot for cell lines after alkylation-induced damage by temozolomide (b) or 376 crosslinking induced damage by oxaliplatin (c). The inner most circle of the phenotypic plot 377 shows DNA damage (color gradient of bubbles) and apoptosis (size of bubbles) for 4 378 different time points, t1 - t4, from center to periphery, and indicated cell lines. The next circle 379 (middle) shows cell cycle distribution of cell lines in G1 (green), S (purple), and G2/M 380 (orange) for time points t1 - t4, from center to periphery. The outermost circle shows 381 proliferation changes of cell lines at time point t1 - t4. Cell lines are ordered hierarchically 382 according to apoptosis and names are indicated outside of the phenotypic plot. Cells 383 identified to be sensitive to carmustine (red names) show a high levels of apoptosis to 384 temozolomide (7/10) but not to oxaliplatin (2/10), indicating that the survival response is 385 primarily due to alkylating and not crosslinking lesions. Wild-type HAP1 cells (WT) are 386 highlighted in a box. Figure legend as in (a). (d-e) Rescue of sensitivity to alkylating agents 387 after reconstitution of knockout cell lines with HA-tagged, doxycycline inducible DYRK4, 388 EPHB6, PNCK or MARK3 proteins. (d) Anti-HA immunoblot of the indicated cells lines 389 reconstituted with the relevant cDNA after doxycycline induction. EPHB6, shows a 390 characteristic smear of a fragmented membrane protein by immunoblotting. * indicates non- 391 specific band. Tubulin was used as a loading control. (e) Upon temozolomide (TMZ) 392 treatment, sensitivities of DYRK4, EPHB6, PNCK and MARK3 deficient cells are rescued 393 after expression of exogenous proteins by doxycycline induction. Results are means of 3 394 replicates with standard deviations.

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395 396 Supp. Figure 1. Survival of DNA repair deficient cell lines in response to DNA damage. 397 (a) List of DNA repair deficient cell lines generated by CRISPR-Cas9 in human HAP1 cells 398 (b) and their survival response to DNA damaging compounds are depicted in a circular 399 bubble plot: DNA double-strand break repair deficient ΔPRKDC, ΔXRCC4, ΔXLF and 400 ΔDCLRE1C are sensitive to DNA double-strand break inducing agents, doxorubicin and 401 etoposide. Crosslinking repair deficient ΔFANCA, ΔFANCB, ΔFANCC and ΔFANCG are 402 sensitive to crosslinking agents MMC, cisplatin and oxaliplatin. ΔMGMT and ΔXPC, deficient 403 in the repair of alkylating adducts, are sensitive to the nonfunctional alkylating agent TMZ. 404 Cell lines deficient in the repair of alkylation damage (ΔMGMT and ΔXPC) or crosslink 405 induced lesions (ΔFANCG) are sensitive to the bifunctional alkylating agent carmustine. 406 Each compound is represented by 4 dose points with a mean of 4 replicates per dose point.

407 H2O2, Hydrogen peroxide; MMS, Methyl methanesulfonate; Negative control, DMSO; 408 Positive control, 25X camptothecin (cytotoxic concentration). The color represents the drug- 409 gene interaction, distance from the center indicates score/p-value and bubble size 410 corresponds to the magnitude of the measured effect (over all other perturbations)44. 411 412 Supp. Figure 2. Rescue of resistance phenotypes after reconstitution of knockout cell 413 lines with HA-tagged, doxycycline inducible proteins. (a-b) Validation of resistant 414 survival responses of ΔTSSK3 to hydroxyurea and ΔBL1 to doxorubicin. (c) Anti-HA 415 immunoblot of the indicated cell lines after doxycycline induction of the relevant cDNA. 416 Expressed genes correspond to expected sizes. Wild-type control, WT, shows no affinity to 417 anti-HA. * indicates non-specific band. Tubulin was used as a loading control. (d) Upon 418 hydroxyurea (HU) treatment, resistance of TSSK3 deficient cells is rescued after expression 419 of exogenous protein by doxycycline induction. Results are means of 3 replicates with 420 standard deviations. (e) Setup as in (c) using resistant ALB1 deficient cells after doxorubicin 421 treatment. 422 423 Supplemental Experimental Procedures 424 425 Cell Culture 426 All HAP1 cell lines used in this work were generated using CRISPR-Cas9 gene editing 427 technology in collaboration with Horizon Genomics (Vienna Austria) as single clones. They 428 were grown in Iscove’s Modified Dulbecco’s Medium (IMDM) from GIBCO®, containing L- 429 Glutamine and 25 mM HEPES and supplemented with 10% heat-inactivated fetal bovine 430 serum (FBS) and 1% penicillin-streptomycin (P/S at 100µg/mL) and passaged according to

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431 standard protocol with trypsin and PBS. All cell lines were grown at 37°C in a 3% oxygen

432 and 5% CO2-humidified incubator. 433 434 HEK293T cells used for virus production were expanded in Dulbecco’s Modified Eagle 435 Medium (DMEM), supplemented with 10% FBS and 1% P/S. Cells were grown at 37°C in a

436 3% oxygen and 5% CO2 incubator. 437 438 Knockout Confirmation by Sanger Sequencing 439 HAP1 kinase deficient knockout cell lines were validated for gene editing leading to a frame- 440 shift mutation in the respective genes. We designed forward and reverse primers for each 441 gene and purchased oligonucleotides from Sigma-Aldrich. For genomic DNA extraction, 442 cells were treated with trypsin and washed twice with PBS, then resuspended in 100µL 443 Direct PCR-Cell lysis solution with 2µL Proteinase K (20mg/mL). Wells were sealed and 444 heated for 2.5 hours at 56°C, then 45 minutes at 80°C to inactivate Proteinase K, followed 445 by PCR amplification. PCR amplification conditions: heat lid 110°C; 94°C 2 minutes; loop 446 35x (94°C 30 seconds; 337 55°C 30 seconds; 68°C 1 minute) 68°C 7 minutes. Then the 447 PCR product was purified using Rapid PCR Cleanup Enzyme Set from BioLabs Inc., diluted

448 1:2 with double distilled water (ddH2O) and sequenced by Microsynth AG. Results were 449 aligned to respective genes using Basic Local Alignment Search Tool, BLAST provided by 450 NCBI. 451 452 High-throughput Drug Screen 453 Indicated volumes and concentrations of compounds (Supp. Table 3) per well were 454 transferred into 384-well plates (Corning 3712) from DMSO stock plates using acoustic 455 transfer (Labcyte Echo 520). Wild-type or knockout HAP1 cells (at an amount of 1,000 cells) 456 were seeded in 50 µl media into the compound-containing plates. Three days later cell 457 viability was determined using Cell Titer-Glo (Promega). Compounds were used at 4 dose 458 points with 4 replicates. For data analysis, the percentage of control was calculated and the 459 signal of the DMSO treated sample was used to set values to 100% survival, while the 25X 460 camptothecin (cytotoxic concentrations) signal was used to set the values to 0%. Survival 461 circos plot with DNA repair deficient cells was created using TOPS44 for analysis and basic 462 visualization. For kinome surival to DNA damage, areas under the curve (AUC) were 463 calculated as a cumulative measure of compound potency by taking the sum of the mean of 464 subsequent concentration points (as applied previously45). 465 466 467

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468 FACS Screen 469 Cell Culture: HAP1 cells were cultured as described above for 7 days until 80-90% 470 confluence. Every HAP1 cell line was seeded into 4 Costar® 6-well cell culture plates, each 471 plate at a density (cell number/well) according to the time-points of harvest: 160,000 for 5- 472 hours treatment, 80,000 for 24 hours treatment, 20,000 for 24 hours post treatment and 473 10,000 for 48 hours post treatment. 474 475 Drug treatment: The day after seeding, cells were treated with 6 concentrations of the 476 respective compound. The highest concentration- temozolomide, 250 µM; oxaliplatin, 780 477 nM- was chosen to moderately affect wild-type cells (10 - 30 % cell death). The compounds 478 were serial diluted 1:2 from the highest to lowest dose. For the untreated control we used 479 DMSO at a concentration corresponding to the lowest compound dilution. After 24 hours 480 treatment, media from the remaining time points was aspirated and replaced with 2 mL of 481 fresh (drug-free) IMDM medium. 40 minutes before each harvest 5-ethynyl-2′-deoxyuridine 482 (EdU) at a concentration of 10 µM was added to each well. 483 484 Cell harvest: Cells were washed with 400µL phosphate buffer saline (PBS) and detached 485 with 500µL trypsin, collected with 1 ml of medium, transferred into 96-deep-well (2ml) plates 486 and centrifuged at 2,000 rpm for 6 min. The supernatant was carefully discarded, cell pellets 487 were washed with PBS and re-suspended in 100µL fixing solution, containing 4% para- 488 formaldehyde (PFA) and 0.1% Triton X, transferred into V-bottom shaped 96-well plates, 489 incubated at 4°C and then stained. 490 491 FACS staining: 96-well plates, containing fixed cells, were centrifuged at 1,200 rpm for 6 min 492 then washed with 50µL PBS. Pellets were re-suspended in TUNEL staining solution (In Situ 493 Cell Death Detection Kit, TMR red, Sigma Aldrich) containing anti-phospho-H2A.X (Ser139) 494 i.e. γH2AX, clone JBW301 (1:500 dilution, Sigma Aldrich) and incubated for one hour in the 495 dark at 37°C. Then the pellets were washed three times with PBS (with centrifugations at 496 1,200 rpm for 6 min) and re-suspended with Click-iT EdU Alexa Fluor 488 Flow Cytometry 497 Assay Kit staining solution (Thermo Fisher Scientific) containing secondary antibody (1:500 498 dilution, Alexa Fluor® 647 conjugate, goat anti-Mouse IgG (H+L), Thermo Fisher Scientific) 499 for detection of γH2AX and incubated for one hour in the dark at room temperature (RT). 500 Subsequently pellets were washed three times with PBS and re-suspended in DAPI (Sigma 501 Aldrich) solution (1:1,000 dilution) and kept dark. Samples were measured using the BD 502 LSRFortessa cell analyzer machine and data was analyzed using FlowJo v10.3. 503

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504 FACS Analysis 505 For analysis, dead cells were discarded using forward and side scatter and next single cell 506 populations were gated using DAPI width and DAPI area following the Abcam PI staining 507 protocol. Gates for γH2AX and TUNEL were set for all concentrations according to the 508 untreated (DMSO) control. For all time points, to take all drug concentrations into account, 509 an area-under-the-curve (AUC) of all six dose points was calculated and compared to 510 untreated controls. This data is visualized in the phenotypic FACS plot. The cell cycle 511 phases were determined by gating G1- and G2-phase as well as S-phase of diploid 512 populations using a DAPI against EdU plot. Proliferating cells were determined by setting a 513 threshold for cells with positive EdU incorporation and EdU positive signals were plotted 514 against untreated control cells. 515 516 Reconstitution of Knockout Cell Lines 517 For reconstitution of the respective wild-type genes in knockout cell lines, we used Gateway- 518 cloning compatible vector backbones containing the gene of interest and a spectinomycin 519 resistance cassette, from Addgene. These plasmids of the kind “pDONR223-XXX” were a 520 gift from William Hahn & David Root and are published in Nature: Johannessen et al (2010 521 Nov 24.). Bacterial DH5α were grown on agar plates containing spectinomycin, from which a 522 single clone was picked and cultured in LB-media containing spectinomycin overnight at 523 37°C. Plasmids were purified using Quiagen MidiPrep Kit and the LR-reaction was 524 performed according to the Gateway Technology protocol provided by Invitrogen. We 525 transferred the cDNA of the gene of interest into the doxycycline inducible pLIX_402 entry 526 vector for mammalian expression and lentivirus production containing an AmpR cassette. 527 The entry clones were transformed into Mg2+/Ca2+ competent DH5α strains, amplified in an 528 overnight culture in LB-media containing ampicillin and plasmids were extracted using 529 Qiagen MidiPrep Kit. Lentivirus particles were produced using following plasmids: CMV- 530 GFP, VSV-G and dR8.91. Virus was harvested for two days in the mornings and evenings. 531 Knockout cells were infected with virus particles containing the respective gene, selected for 532 2 days with puromycin at 2 µg/mL and cells were propagated for another 2 days. 533 534 Dose Responses 535 Dose response curves for temozolomide, oxaliplatin, hydroxyurea and doxorubicin were 536 generated by seeding cells in 96-well plates (1,000 cells/well). The next day, compounds 537 were added at the indicated concentrations. Cells harboring the reconstituted gene of 538 interest were additionally treated with doxycycline at 1µg/mL every day. Three days after

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539 drug treatment, cell viability was measured using the CellTiter-Glo assay protocol 540 (Promega). 541 542 Immunoblotting and Antibodies 543 Cells were harvested and then lysed with RIPA lysis buffer (NEB) supplemented with 544 protease and phosphatase inhibitors from Sigma. Western blots were performed according 545 to standard protocols. Protein samples were separated using NuPAGE™ 4-12% gradient 546 Bis-Tris Protein Gels from Invitrogen and MOPS running buffer at 120 V for 2 hours running 547 time. The separated proteins were then transferred onto nitrocellulose membranes. To 548 prevent unspecific protein binding, membranes were treated with blocking solution (5% milk 549 in TBST) for 1 hour and primary antibodies were added at 1:1,000 to the blocking solution 550 and incubated overnight at 4°C. The next day, membranes were washed 3x with TBST and 551 incubated with secondary antibodies at 1:5,000 in 5% milk/TBST solution. Then membranes 552 were treated with immunoblotting developer solution (GSE) for 1 minute and imaged in the 553 dark. The following antibodies were used: primary; Rabbit Anti-HA tag antibody - ChIP 554 Grade (ab9110, Abcam), secondary; Goat Anti-Rabbit IgG, HRP-linked Antibody (#7074, 555 Cell Signaling). 556 557 Clustering and HiC analysis 558 Cells were clustered with agglomerative clustering using the R package 559 ConsensusClusterPlus. Cells and compounds were randomly sub-sampled for 10,000 times 560 and clustered using complete linkage to increase the robustness of clustering. The distance 561 was calculated using the Pearson correlation. Hi-C values for all genes in each cluster were 562 retrieved from the covariate matrix of MutSigCV v1.2.0146 and the corresponding 563 distributions were compared with Wilcoxon rank-sum test. 564 565 Gene ontology term enrichment analysis 566 Gene ontology (GO) term enrichments were calculated using Enricher, a comprehensive tool 567 for gene set enrichment analysis47. P-values were calculated using a Fisher's exact test and 568 corrected for multiple hypotheses using a cut-off of p-value < 0.05. In order to filter for 569 redundant and unspecific GO terms, we first removed all GO terms that are annotated to 570 more than 70 genes and further summarized terms based on their Resnik semantic 571 similarity48 using the tool ReviGO49. 572

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Kinase Group AGC Atypical CAMK CK1 CMGC Other STE TK TKL 3.5 CDK10 ACVR2B RPS6KB2 TSSK3 CDKL1 MAST1 PIM2 2.5 ABL1 PKN1 TNK2 STK10 PDK2 Kinase Groups 1.5 AGC (39) Atypical (22) CAMK (42) 0.5 CK1 (6) CMGC (40) 0 Other (49) Z-score -0.5 RGC (1) STE (32) TK (51) -1.5 TKL (31) GRK6 PKN2 GSK3B CRISPR-Cas9 kinase knock-out remaining kinases CLK1 MAP4K5 transparency: PRKACA PNCK cell lines used in this study -2.5 PRKDC MARK3 CSNK1G1 DYRK4 STK3 EPHB6 b Cell line 384-well plates HAP1 K.O. cell lines Color by HypoM SSB DSB ReS HU Alk & Cros Chkp DNA damaging agents damage 4 doses 4 replicates 3 days Measurement of survival and analysis c f g

Chromatin accessibility Enriched GO Terms Shared Between Clusters 0.003 0.42 0.04 Cluster 1 Cluster 2

44 107 106 Hi-C

0 86 106 19 e drug sensitivity + - -50 Cluster 1 Cluster 3 Cluster 2 293 -3 +3 z-score

Cluster 3 h

Unique Cluster 1 GO-Terms

Cluster 1 (85) Vascular Endothelial Growth Factor Receptor 14

Number of GO-Terms Summarized

Cluster 2 (89) 11

positive regulation of cytochrome-c oxidase activity

Response To Hydrogen Peroxide 9 Actin Cytoskeleton Reorganization -log(pValue) CRISPR-Cas9 kinase knockout cell lines (313) Regulation of Endothelial Cell Proliferation 7 positive regulation of mitotic cell cycle Regulation of Mitotic Checkpoint Regulation Of Transcription

Cluster 3 (139) DNA Damage Checkpoint 5 petide crosslinking

1 2 3 4 Z-Score UCN1 etoposide topotecan decitabine cytarabine aphidicolin carmustine doxorubicin hydroxyurea camptothecin Figure 1 DNA damaging agents bioRxiv preprint doi: https://doi.org/10.1101/385344; this version posted August 6, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made a available under aCC-BY-NC-ND 4.0 International license. Workflow of phenotypic assay

cell line: wild type and 25 CRISPR-Cas9 kinase knockout cell lines

treatment: DNA alkylation (temozolomide) or DNA crosslinks (oxaliplatin)

time point: untreated 5h 24h 24h treatment and 24h treatment and (t0) treatment (t1) treatment (t2) 24h recovery (t3) 48h recovery (t4)

marker: TUNEL γH2AX DAPI EdU

readout: apoptosis DNA damage cell cycle proliferation

Figure legend WT + temozolomide DNA- Apoptosis damage Cell Cycle Proliferation 48h recovery (t4) high G1 S G2 t0 t2t1 t4t3 24h recovery (t3) t4 t3 100% 24h treatment (t2) t2 50% 5h treatment (t1) t1 low t0 0% untreated (t0)

MAP4K5 PIM2 KN2 PRKDC b 2 c P G 6KB WT 1G1 RK PS K 6 R SN C PK W 3 N L1 T TK 1 K S D C P T R S K 1 S A T K 2 S 3 C A IM A P M R

C P

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d e TMZ (5 μM) 2 106 - doxycycline + doxycycline *** ** **** *** ΔDYRK4 ΔEPHB6 ΔPNCK ΔMARK3 doxycycline - + - + - + - + 1 106 anti-HA

anti-tubulin 6 0.5 10 cell viability (a.u.)

WT PNCK DYRK4 EPHB6 Δ MARK3 Δ Δ Δ

Figure 2 bioRxiv preprint doi: https://doi.org/10.1101/385344; this version posted August 6, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made a b available under aCC-BY-NC-ND 4.0 International license. ntrol tive co Knock-out cell lines ega cisp n latin

ca Fanconi Anemia in rm ic u ub s ΔFANCA (FA) r tin xo e o d ΔFANCB (FB)

PK c a ΔFANCC (FC) e m d XL p i XR t s XR FG o ΔFANCG (FG) o XL FB FG t p PK XR XC h o XR FA e t XR FG MG c e i n PK DC XL Double-strand-break WT PK

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Figure S1 bioRxiv preprint doi: https://doi.org/10.1101/385344; this version posted August 6, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made a available under aCC-BY-NC-NDb 4.0 International license.

150 150 WT WT ΔTSSK3 ΔABL1

100 100

50 50 Cell viabilit y (%) Cell viabilit y (%)

0 0 100 101 102 103 104 10-1 100 101 102 103 hydroxyurea (μM) doxorubicin (nM)

c d HU (125 μM)

- doxycycline + doxycycline 2 106 ** ΔABL1 ΔTSSK3 doxycycline - + - + 1 106 anti-HA * anti-tubulin 0.5 106 cell viability (a.u.)

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doxorubicin (16 nM) e - doxycycline + doxycycline *** 25 105

20 105

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Figure S2