Synthetic Lethal Interactions for Kinase Deficiencies

Author Manuscript Published OnlineFirst on August 6, 2019; DOI: 10.1158/0008-5472.CAN-19-1364 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Synthetic lethal interactions for kinases to DNA damage Robinson-Garcia et al, 2019

1 Synthetic lethal interactions for kinase deficiencies

2 to DNA damage chemotherapeutics

3 4 5 Lydia Robinson-Garcia1,2, Joana Ferreira da Silva1,2 and Joanna I. Loizou1 6 7 1CeMM Research Centre for Molecular Medicine of the Austrian Academy of Sciences, 8 Lazarettgasse 14, AKH BT 25.3, 1090 Vienna, Austria 9 2Equal contribution 10 11 12 13 14 15 16 17 18 Running title: Synthetic lethal interactions for kinases to DNA damage 19 20 Keywords: Kinases, DNA damage, chemotherapeutics, MARK3, alkylating lesions. 21 22 Financial support: JFdaS is funded by a DOC Fellowship from the Austrian Academy of 23 Sciences (OAW25035). The Loizou lab is funded by two grants from the Austrian Science 24 Fund (FWF; P29555 and P29763). CeMM is funded by the Austrian Academy of Sciences. 25 26 Corresponding author: Joanna I. Loizou, CeMM Research Centre for Molecular Medicine 27 of the Austrian Academy of Sciences, Lazarettgasse 14, AKH BT 25.3, 1090 Vienna, 28 Austria. 29 Phone +43-1/40160-70 058, Fax +43-1/40160-970 000, Email [email protected] 30 31 Conflict of interest statement: The authors declare no conflict of interest. 32 33 Word count: 2,852 (excluding abstract and references) 34 Number of figures: 2 35 Number of tables: 0 36

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Synthetic lethal interactions for kinases to DNA damage chemotherapeutics Robinson-Garcia et al, 2019

37 Abstract 38 39 Kinases are signaling enzymes that regulate diverse cellular processes. As such, they are 40 frequently mutated in cancer and therefore represent important targets for drug discovery. 41 However, until recently, systematic approaches to identify vulnerabilities and resistances of 42 kinases to DNA damaging chemotherapeutics have not been possible, partially due to the 43 lack of appropriate technologies. With the advent of CRISPR-Cas9, a comprehensive study 44 has investigated the cellular survival of more than 300 kinase-deficient isogenic cell lines to 45 a diverse panel of DNA damaging agents, enriched for chemotherapeutics. Here, we discuss 46 how this approach has allowed for the rational development of combination therapies that 47 are aimed at utilizing synthetic lethal interactions between kinase deficiencies and DNA 48 damaging agents that are used as chemotherapeutics.

50 Main Text

51 Kinases are enzymes that catalyze the addition of a phosphate from adenosine triphosphate 52 (ATP) onto a substrate, a modification that is reversible via the enzymatic activity of 53 phosphatases. Protein phosphorylation is one of the main post-translational modifications 54 and can rapidly change either the activity, localization or interaction network of the target 55 protein (1). As such, kinases regulate diverse fundamental cellular processes including 56 cellular differentiation, cell cycle progression, apoptosis and DNA repair, hence being 57 implicated in several of the hallmarks of cancer. The human kinome is estimated to include 58 some 518 kinases and, of these, 120 -157 are suggested to function as drivers of cellular 59 transformation (2). Mutations within these kinases can be either gain- or loss-of-function and 60 can promote tumor initiation or progression, leading to a range of cancer types (3). For 61 example, the gene PIK3CA can harbor mutations that lead to the up-regulation of the AKT- 62 mTOR pathway and promote cell growth and proliferation (2). In contrast, ATM loss-of- 63 function mutations dysregulate the signaling of DNA damage and promote genomic 64 instability (4,5).

66 The long-standing precedence of kinases in cancer, as well as other diseases, has identified 67 them as important drug targets. The first kinase inhibitors were discovered in the 1980s and 68 currently, numerous are under development for different purposes. In the USA alone, around 69 10,000 patent applications for kinase inhibitors have been filed since 2001 (2). As of 2018, 70 31 kinase inhibitors were approved by the Food and Drug Association (FDA) for cancer 71 therapy (2). These functioned by blocking the ATP binding domain, a region that is highly 72 conserved, hence making these inhibitors unspecific and of low potency. Strikingly, it was

Synthetic lethal interactions for kinases to DNA damage chemotherapeutics Robinson-Garcia et al, 2019

73 not until 1998 when trastuzumab (Herceptin) became the first example of an approach to 74 block the activity of a kinase, in the clinics. Trastuzumab is a monoclonal antibody that 75 inhibits ERBB2 and is used for the treatment of ErbB2-positive metastatic breast cancer (6). 76 ERBB2 is a receptor tyrosine kinase that belongs to the EGFR family. A variety of ligands 77 bind to these receptors, activating different signaling pathways, such as those regulated by 78 AKT and RAS. Mutations in these receptors cause over-activation of these pathways and 79 hence, promote tumor development (6). Tyrosine kinases have also been targeted using 80 small molecules, an example of which is imatinib (Gleevec) that was approved by the FDA in 81 2001. Imatinib was developed to target dysregulated tyrosine kinase activity, that results 82 from the BCR-ABL fusion oncoprotein. However, the therapeutic benefits of such kinase 83 inhibitors can be short-lived, due to the acquisition of resistance mechanisms. For imatinib 84 this is mainly due to point mutations in the tyrosine kinase domain of BCR-ABL (7).

86 To increase the efficacy of kinase inhibitors, they can be used in combination with 87 chemotherapeutic drugs. An important group of such chemotherapeutic agents includes 88 those that induce DNA damage, hence functioning to stop proliferation or induce cell death 89 (8). Since the 1960s, many chemotherapeutic agents have been developed that react 90 chemically with DNA or block essential DNA-associated functions (9). Capecitabine (a 91 fluoropyrimidine), as well as radiation therapy, have been used in combination with erlotinib, 92 an EGFR small-molecule inhibitor, for the treatment of advanced pancreatic cancer (10). 93 Moreover, the multi-kinase small molecule inhibitor, sunitinib, has been used in combination 94 with radiation for the treatment of a range of cancers, including renal cell carcinoma and 95 prostate cancer (11,12). In addition, kinase inhibitors have been shown to increase the 96 efficacy of drugs that block the catalytic activity of DNA repair enzymes. Representative 97 examples are the inhibitors targeting the receptor tyrosine kinase c-Met (a proto-oncogene; 98 using foretinib and crizotinib) that synergize with inhibitors of PARP (a molecule involved in 99 signaling DNA damage; using ABT-888 and AG014699) and hence this combination therapy 100 may be beneficial for tumors with high c-Met expression (13). 101 102 103 It is noteworthy that kinases play important functions in the repair of DNA damage, or in the 104 regulation of the cell cycle in response to DNA lesions. The DNA damage response is 105 activated by three main kinases that are members of the PIKK family, namely ATM, ATR 106 and DNA-PKcs (14). Inhibitors have been developed to target these kinases and clinical 107 trials are ongoing to test their efficacy either alone, or in combination with other treatments. 108 Amongst others, the DNA-PKcs inhibitor MSC2490484A (study number: NCT02516813) as

Synthetic lethal interactions for kinases to DNA damage chemotherapeutics Robinson-Garcia et al, 2019

109 well as the ATM inhibitor AZD1390 (study number: NCT03423628) are currently being 110 tested in combination with radiotherapy in Phase 1 trials. Similarly, the ATR inhibitors 111 AZD6738, M6620, BAY1895344 and VX-970 are being tested either alone (NCT03718091) 112 or in combination with a plethora of other chemotherapeutic agents, including immune 113 checkpoint inhibition by targeting PDL-1 (study number: NCT02264678), olaparib (study 114 number: NCT03787680), gemcitabine (NCT02595892), PARP inhibition and cisplatin 115 (NCT02723864), as well as topotecan (NCT02487095). Other kinases have been shown to 116 function in the regulation of the cell cycle, an essential process that regulates cellular 117 homeostasis. Proteins like PLK1 or CHK1 and CHK2 are degraded or activated, 118 respectively, to induce checkpoint activation (15,16). The CHK1 inhibitor SRA737 is in 119 Phase 1 trials either alone or in combination with gemcitabine (with or without cisplatin; 120 study numbers: NCT02797964 and NCT02797977). 121

122 Intriguingly, the vast majority of scientific literature on kinases relates to a small number of 123 these proteins (17), suggesting that other kinases may also function to regulate cellular 124 pathways, including those related to DNA repair. Indeed, kinase dependencies have been 125 studied through the use of unbiased and systematic experimental approaches and several 126 kinases have been implicated in the repair of DNA damage (18). Such high-throughput 127 screening approaches have included siRNAs (19–21) or small molecule libraries (22,23) and 128 have unraveled new synthetic lethal interactions in cancer models, suggesting the inhibition 129 of specific kinases, such as FGFR and CHK1, as promising therapeutic targets for 130 osteosarcoma (19) and neuroblastoma (20), respectively. Moreover, a systematic 131 overexpression approach for kinases, and phosphatases, identified the overexpression of 132 tyrosine kinases to induce a MEK-dependent ERK activation that promotes drug-resistance 133 (24). In the context of DNA repair, an siRNA screen revealed that cancers with defects in the 134 DNA repair pathways, Fanconi Anemia and homologous recombination, are hypersensitive 135 to the inhibition of the WEE1 kinase (21). More recently, CRISPR-Cas9 (25) has emerged as 136 a powerful approach to modify genomic loci with higher efficiency and less off-target effects 137 and has been widely used to interrogate gene function at genome scale (26). 138 139 140 To investigate the roles that kinases have in response to DNA damage, we utilized CRISPR- 141 Cas9 to produce an isogenic panel of cell lines deleted for all non-essential and expressed 142 kinases (some 313 in total) in the human near-haploid cell line HAP1 (25). The cell line 143 HAP1 is derived from the chronic myelogenous leukemia cell line KBM7 and it has been 144 extensively used in biomedical research, especially for investigations into gene-gene and

Synthetic lethal interactions for kinases to DNA damage chemotherapeutics Robinson-Garcia et al, 2019

145 gene-drug interactions (27). Even though the HAP1 cell line carries the BCR-ABL fusion, a 146 driver mutation of chronic myeloid leukemia, it does not depend on the translocation for 147 growth, making it a suitable cell model for the identification of gene-drug interactions (25). 148 This was confirmed by revealing the known hypersensitivities of 15 DNA repair defects to an

149 array of 14 DNA damaging agents (25). The collection of kinase knock-out cell lines was 150 designed to comprise kinases that function in different oncogenic pathways, categorized by 151 their roles in cancer (Figure 1A). Included in the collection is the PI3K family that has been 152 implicated in 30-50% of human cancers (28). Similarly, genetic alterations in other kinases 153 such as ALK, JAK, c-KIT, FGFR1 or SRC regulate fundamental molecular mechanisms for 154 tumor cell growth and development (29). Apart from tumor initiation, the collection of kinase 155 deficient cell lines was designed to also include kinases that are important for tumor survival 156 and proliferation. This category includes members of the EGFR family, such as the ERBB 157 family receptor tyrosine kinases, widely implicated in cancer development and progression 158 (6,30). Other examples include those that induce tumor cell survival, such as mTOR and the 159 S6 and MEK families. A further category of kinases implicated in cancer includes those that 160 are overexpressed in tumors and surrounding tissues, and hence of importance in the 161 maintenance of tumors. These include, but are not limited to, the FGFR kinase and CK2 (2). 162 Overall, the 313 kinases that were targeted effectively represent oncogenic kinases involved 163 in multiple hallmarks of cancer, including rapid proliferation, growth, survival and metastasis. 164 Importantly, nine out of the twelve kinases that have been approved by the FDA as 165 therapeutic targets are part of this collection (31), including BRAF, ABL1, CDK4 and CDK6. 166 Additionally, of the 23 kinases currently under evaluation as therapeutic targets in current

167 clinical trials (31), eleven are included in this collection, such as AKT1/3, BRD4 and ERBB3. 168

169 To uncover combinatorial synthetic lethal and resistant interactions, but also to identify novel 170 kinases in the context of the DNA damage response and repair, these isogenic cell lines 171 were next individually treated with ten different DNA damaging chemotherapeutic agents. 172 These agents were carefully selected to span the engagement of all DNA repair pathways 173 (Figure 1B). The topoisomerase II inhibitors, etoposide (32) and doxorubicin (33), were 174 chosen to induce DNA double-strand breaks, whereas DNA single-strand breaks were 175 induced by the topoisomerase I inhibitors, topotecan (34) and camptothecin (35). Aphidicolin 176 is a polymerase  inhibitor and it was chosen to induce DNA replication stress. Other agents 177 that induce replication stress are hydroxyurea, that contributes to the depletion of the 178 nucleotide pool and cytarabine, a cytidine analogue (36). Decitabine, another cytidine 179 analogue, was chosen to mediate DNA hypomethylation (37). Carmustine was used as a 180 DNA alkylation agent (38) and 7-hydroxystaurosporine as a CHK1/2 inhibitor, for checkpoint

Synthetic lethal interactions for kinases to DNA damage chemotherapeutics Robinson-Garcia et al, 2019

181 abrogation (39). A similar drug panel, covering all classes of chemotherapy consisting of 29 182 drugs, was utilized by Hu and collaborators (40) to map the impact on cellular survival upon 183 depleting (using siRNA) some 625 proteins related to cancer and DNA repair.

184

185 The combination of kinase deficiency, coupled with DNA damage, led to the classification of 186 the kinase-deficient cell lines into three different clusters of sensitivity and confirmed several 187 known interactions of kinases to DNA damaging agents (25). The first cluster was defined by 188 kinases that were hypersensitive to carmustine, a chemotherapeutic drug and a bifunctional 189 alkylating agent that produces DNA mono-alkylating adducts, as well as DNA intra- and 190 interstrand crosslinks. Interestingly, this cluster was significantly enriched for genes 191 associated with increased chromatin accessibility, compared to clusters two and three. 192 Alkylating agents, such as carmustine and temozolomide, have been reported to have a 193 global effect on nuclear organization and chromatin structure, inducing chromatin 194 condensation and gene silencing (41). Hence, it can be reasoned that kinase-deficient cell 195 lines within this cluster are hypersensitive to carmustine due to alkylation-induced synthetic 196 lethality. In support of this hypothesis, this cluster was enriched for gene ontology terms 197 associated with the cellular response to alkylating or crosslinking agents. Such terms 198 included the upregulation of vascular endothelial growth factor receptors (42) and induction 199 of oxidative stress (43) (cellular response to hydrogen peroxide and positive regulation of 200 cytochrome-c oxidase activity), which in turn leads to actin cytoskeleton reorganization (44). 201 Kinases within this cluster included EPHB6, DYRK4 and MARK3. The second cluster was 202 dominated by synthetic lethal interactions with hydroxyurea and it was enriched for terms 203 associated with cell adhesion and apoptosis. The third cluster was enriched for kinases that 204 displayed hypersensitivity to the DNA double-strand break inducing agents, etoposide and 205 doxorubicin. It is worth noting that the approach taken here was limited to one cell line and 206 future directions might include using CRISPR-Cas9 approaches targeting the human kinome 207 across a panel of cell lines that would serve to firstly confirm these findings, but also to 208 identify other potential kinases involved in the resolution of DNA damage.

209

210 Different factors can trigger cellular mechanisms of sensitivity to DNA damaging agents, 211 such as an inability to resolve the damage, cell cycle dysregulation, induction of apoptosis or 212 decreased proliferation. In order to simultaneously assess these different possibilities in 213 selected isogenic cell lines, a fluorescence-activated cell sorting (FACS)-based phenotypic 214 assay was developed. This was performed using a panel of antibodies and dyes: an 215 antibody detecting the histone variant H2AX phosphorylated at serine 139 (H2AX) was

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216 employed as a marker of DNA damage, the DNA binding dye 4′,6-Diamidine-2′-phenylindole 217 dihydrochloride (DAPI) was used to monitor cell cycle phases, the terminal deoxynucleotidyl 218 transferase dUTP nick end labeling staining (also called the TUNEL assay) was used to 219 detect DNA breaks formed during apoptosis and the 5-ethynyl-2’-deoxyuridine (a thymidine 220 analogue) was used to measure proliferation. Twenty-five kinase deficient cell lines from 221 cluster one (sensitive to carmustine) were selected for further validation using this FACS- 222 based phenotypic assay (Figure 2A). Since carmustine can function as both a DNA 223 alkylating and crosslinking agent, these 25 cell lines were treated with the alkylating agent 224 temozolomide and the crosslinking agent oxaliplatin, to determine which type of lesions drive 225 the cellular toxicity. Hierarchical clustering revealed that the hypersensitivity to carmustine 226 was predominantly due to DNA alkylation-induced synthetic lethality and not due to the 227 generation of DNA crosslinks, since the majority of cell lines were hypersensitive to 228 temozolomide but not to oxaliplatin.

229

230 The FACS-based approach also allowed for the identification of four kinases frequently 231 baring loss-of-function mutations in cancer that show cellular sensitivity to temozolomide. 232 These include EPHB6, MARK3, DYRK4 and PNCK (Figure 2B). MARK3 was prioritized for 233 validation, since it was the second most mutated kinase in cancer, after EPHB6 that has 234 been previously classified as a pseudokinase. As an approach to unravel the mechanism by 235 which MARK3 deficient cells are sensitive to temozolomide, proteomic studies were 236 designed to identify changes in protein abundance in MARK3-deficient cells. This led to the 237 observation that MGMT protein levels were significantly reduced in MARK3-deficient cells 238 (Figure 2C). The cytotoxicity of temozolomide is mediated by its addition of methyl groups at 239 N7 and O6 sites on guanines and the O3 sites on adenines in genomic DNA. MGMT is the 240 enzyme that reverses the methylation of the O6 position of guanine, hence allowing for 241 cellular survival, therefore, cells lacking MGMT are hypersensitive to temozolomide. 242 Temozolomide-based therapy is the standard of care for patients with glioblastoma 243 multiforme (GBM). Resistance to this drug is mediated by high MGMT levels and several 244 clinical studies have shown that elevated MGMT protein levels or lack of MGMT promoter 245 methylation (and concomitant loss of promoter silencing) are associated with temozolomide 246 resistance in some GBM tumors (45,46). Hence, the synthetic lethal interaction between 247 MARK3 and MGMT may hold promise for application in the clinics, as a way to revert 248 temozolomide resistance in GBM tumors, through the development of MARK3 inhibitors. 249 Moreover, since MARK3 itself is found to carry loss-of-function mutations in cancer, these 250 findings suggest that such cancers would be hypersensitive to temozolomide and this gene- 251 drug interaction might represent an unexplored avenue for their treatment.

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252

253 Taken together, kinases represent an important family of enzymes, holding great potential 254 as therapeutic targets for the treatment of cancer. Hence, investigations that systematically 255 unravel interactions between kinases and chemotherapeutic agents are of tremendous value 256 to the scientific community and ultimately to the clinics. Over the coming years, the 257 outcomes of trails consisting of targeting kinases along with the administration of DNA 258 damaging chemotherapeutics will be known and may lead to new treatment regimes. 259 Another exciting development is the combination of kinase inhibitors with immune 260 checkpoint inhibition. In line with this, several clinical trials are currently investigating the 261 combination of VEGF inhibition along with immune checkpoint inhibitors. The findings from 262 these and related studies open the possibility for new and rational combination therapies 263 that share a remarkable potential to unravel important clinical therapeutic benefit for cancer 264 patients.

265

266 Acknowledgements

267 We thank Drs Bensimon (CeMM, Austria), Nagy (CeMM, Austria) and Owusu (IRB 268 Barcelona, Spain) as well as members of the Loizou lab for critically reading and 269 commenting on this review. We also thank Michael Caldera (CeMM, Austria) for curating the 270 kinome plot. We apologize to all authors whose original research was not cited due to space 271 limitations. JFdaS is funded by a DOC Fellowship (OAW25035). The Loizou lab is funded by 272 two grants from the Austrian Science Fund awarded to JIL (FWF; P29555 and P29763). 273 CeMM is funded by the Austrian Academy of Sciences. 274

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401

402 Figure Legends

403 Figure 1: A. Isogenic cell lines each deficient for one out of 313 kinases were generated 404 using CRISPR-Cas9, in the near-haploid human cell line HAP1. Colors represent different 405 kinase families B. Next, gene-drug interactions were identified across a panel of ten diverse 406 DNA damage-inducing agents, including seven chemotherapeutic agents (highlighted in 407 green). 408

409 Figure 2: A. Synthetic lethal interactions to carmustine were prioritized and further 410 investigated for 25 kinase deficient cell lines using a fluorescence-activated cell sorting 411 (FACS)-based phenotypic assay, to measure cell cycle, proliferation, apoptosis and DNA 412 damage. Since carmustine is both an alkylating and a crosslinking agent, these experiments 413 were performed using oxaliplatin and temozolomide, to decipher which of the two properties 414 of carmustine resulted in the synthetic lethality. B. This approach led to the prioritization of 415 four novel DNA damage kinases that are frequently mutated in cancer (indicated in the pie 416 chart). C. Investigations into the role of MARK3 revealed that cells lacking MARK3 had

Synthetic lethal interactions for kinases to DNA damage chemotherapeutics Robinson-Garcia et al, 2019

417 reduced levels of MGMT protein, suggesting this as the mechanism leading to the sensitive 418 to temozolomide. 419

A B Drugs Damage Decitabine Hypomethylation Doxorubicin Etoposide Double-strand breaks AGC Topotecan CK1 Single-strand breaks CMGC Camptothecin Other STE Carmustine Alkylation and crosslinks TK Atypical Aphidicolin TKL Cytarabine Replication stress Other CAMK Hydroxyurea RGC 7-hydroxystaurosporin Cell cycle checkpoint abrogation Chemotherapeutic drugs

ABValidation of twenty-ﬁve Clinical relevance in cancer selected cell lines

Oxaliplatin Temozolomide O H O N2 O MARK3 EPHB6 N N Pt N 0.31 0.42 N O N O N H2 NH O 2

FACS-based assays 0.18 0.24 PNCK DYRK4

Frequency of loss-of-function mutations

Cell cycle arrest Apoptosis DNA damage Proliferation

C Temozolomide

O CH3 N N N N N Cell death NH CH CH3 O 2 3

Glioblastoma MARK3 ? Pi O MGMT CH3

NN N N N Resistance NH CH CH3 O 2 3 resistance MARK3 Glioblastoma ? Pi O MGMT CH3 N N N N N Cell death NH CH CH3 O 2 3 Glioblastoma + loss of MARK3

Synthetic lethal interactions for kinase deficiencies to DNA damage chemotherapeutics

Lydia Robinson-Garcia, Joana Ferreira da Silva and Joanna I Loizou

Cancer Res Published OnlineFirst August 6, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-1364

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.

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