bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license.

1 Gain-of-function genetic screen of the kinome reveals BRSK2 as an inhibitor of the NRF2 2 transcription factor 3 Tigist Y Tamir1, Brittany M Bowman2, Megan J Agajanian1, Dennis Goldfarb5, 6, Travis P 4 Schrank2, Trent Stohrer2, 3, Andrew E Hale4, Priscila F Siesser2, Seth J Weir2, Ryan M Murphy1, 5 Kyle M LaPak5, Bernard E Weissman2,7, Nathaniel J Moorman2, 4, M. Ben Major1, 2, 5, 8* 6 1Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 7 2Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 8 3Department of Computer Science, University of North Carolina at Chapel Hill, Chapel Hill, NC 9 4Department of Microbiology and Immunology, University of North Carolina at Chapel Hill 10 School of Medicine, Chapel Hill, NC 11 5Department of Cell Biology and Physiology, Washington University in St. Louis, St. Louis, MO 12 6Institute for Informatics, School of Medicine, Washington University in St. Louis, St. Louis, MO 13 7Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, 14 Chapel Hill, NC 15 8Department of Otolaryngology, School of Medicine, Washington University in St. Louis, St. 16 Louis, MO 17 18 *Correspondence to: M. Ben Major, PhD, Washington University in St. Louis, 660 Euclid Ave. 19 Avenue Box 8228, St. Louis, MO 63110. Email: [email protected] 20 21 Summary Statement: BRSK2 suppresses NRF2 signaling by inhibiting protein synthesis 22 through mTOR downregulation. 23 24 Key Words: NRF2, oxidative stress response, phosphoproteomics, functional genomics, 25 kinase, BRSK2, BRSK1, AMPK, mTOR. 26 bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license.

27 Abstract 28 NFE2L2/NRF2 is a transcription factor and master regulator of cellular antioxidant response. 29 Aberrantly high NRF2-dependent transcription is recurrent in human cancer, and conversely 30 NRF2 protein levels as well as activity is diminished with age and in neurodegenerative disorders. 31 Though NRF2 activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here we 32 used a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 33 signaling. We found that the understudied protein kinase Brain Specific Kinase 2 (BRSK2) and 34 the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in 35 an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that 36 BRSK2 drives AMPK activation and suppresses mTOR signaling. As a result, BRSK2 kinase 37 activation suppressed ribosome-RNA complexes, global protein synthesis, and NRF2 protein 38 levels. Collectively, our data establish the catalytically active BRSK2 kinase as a negative 39 regulator of NRF2 via the AMPK/mTOR signaling. This signaling axis may prove useful for 40 therapeutically targeting NRF2 in human diseases. 41 bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

42 Introduction 43 The transcription factor nuclear factor erythroid 2-related factor 2 (NFE2L2, hereafter referred to 44 as NRF2) is central to the cellular response to oxidative and electrophilic stress (Itoh et al., 2010; 45 Suzuki and Yamamoto, 2015). When active, NRF2 provides strong cytoprotective functions by 46 upregulating expression of: 1) xenobiotic metabolism enzymes, 2) phase II detoxification 47 enzymes, 3) drug efflux pumps, and 4) the thioredoxin and glutathione antioxidant systems (Itoh 48 et al., 2010; Kensler et al., 2007). Under homeostatic conditions, the E3 ubiquitin ligase adaptor 49 kelch like ECH-associated protein 1 (KEAP1) binds ETGE and DLG motifs within NRF2 to 50 promote NRF2 ubiquitylation and proteasomal degradation (Cullinan et al., 2004; Itoh et al., 1999; 51 Tong et al., 2006). Electrophilic attack of reactive cysteines within KEAP1 during oxidative stress 52 suppresses KEAP1-dependent ubiquitylation/degradation of NRF2, resulting in NRF2 53 stabilization, nuclear translocation, and transcriptional activation of genes containing Antioxidant 54 Response Elements (AREs)(Baird et al., 2013; Ichikawa et al., 2009; Kensler et al., 2007; Suzuki 55 et al., 2019). Though KEAP1/CUL3 are prominent NRF2 regulators in most tissues and disease 56 models, data describing additional mechanisms are still emerging. 57 As a key regulator of metabolism and redox-balance, it is not surprising that alterations in NRF2 58 contribute to numerous human diseases. NRF2 expression and transcriptional activity declines 59 with age and is decreased in several neurodegenerative diseases (Cuadrado et al., 2018; Tsakiri 60 et al., 2019; Zhang et al., 2015). Conversely, NRF2 transcriptional activity is constitutively active 61 in many cancers, including those of the lung, head/neck, kidney, liver, and bladder cancer 62 (Menegon et al., 2016; Rojo de la Vega et al., 2018). Multiple mechanisms promote NRF2 63 activation in cancer: activating mutations in NRF2, inactivating mutations in KEAP1, NRF2 copy 64 number amplifications, the over-expression of KEAP1 binding proteins which competitively 65 displace NRF2, and various post-translational modifications to KEAP1 and NRF2(Cloer et al., 66 2019; Rojo de la Vega et al., 2018). High NRF2 activity promotes tumor growth, metastasis, and 67 chemo-/radiation-resistance (Homma et al., 2009; Tao et al., 2014). Patients with mutations in 68 KEAP1 or NRF2 leading to NRF2 stabilization have poor survival and are comparatively resistant 69 to many commonly used cytotoxic cancer therapies(Homma et al., 2009; Satoh et al., 2013; Tao 70 et al., 2014). While there are numerous small molecule activators of NRF2 signaling, there are no 71 approved NRF2 inhibitors (Cuadrado et al., 2018). 72 Here, we performed a gain-of-function (GOF) screen of human kinases to identify new regulators 73 of NRF2-dependent transcription. We focused on kinases given their exceptional druggability and 74 because a rigorous, comprehensive annotation of the kinome for NRF2 activity is lacking. Our 75 previous phosphoproteomic analysis of KEAP1 and NRF2 shows both proteins are 76 phosphorylated at multiple sites. The majority of these phosphorylation events are not linked to 77 specific kinases and are of unknown functional importance (Tamir et al., 2016). That said, recent 78 studies have revealed a few kinases that influence NRF2 protein stability, subcellular localization 79 and transcriptional activity. NRF2 is directly phosphorylated by GSK3β, resulting in NRF2 80 ubiquitylation by βTrCP and subsequent proteasomal degradation (Chowdhry et al., 2012; 81 Cuadrado, 2015). PKC and AMPK mediated phosphorylation of NRF2, at S40 and S550, 82 respectively, leads to increased NRF2 stability and signaling (Huang, 2002; Joo et al., 2016). 83 NRF2 is reported to be a substrate of several MAPKs (e.g. JNK, p38, ERK1/2, ASK1, and TAK1), 84 however the functional relevance of MAPK-directed phosphorylation is uncertain (Naidu et al., 85 2009; Shen et al., 2004; Sun et al., 2009). Activation of the PERK kinase during the unfolded 86 protein response (UPR) leads to increased NRF2 nuclear accumulation and cell survival (Cullinan 87 et al., 2003; Del Vecchio et al., 2014). The casein kinase 2 and TAK1 kinases phosphorylate 88 NRF2 to induce NRF2 nuclear localization (Apopa et al., 2008; Shen et al., 2004). There is also 89 evidence that activation of the PI3K/AKT/PKB pathway and PIM kinase signaling induces NRF2- 90 activity and cellular protection (Lim et al., 2008; Nakaso et al., 2003; Warfel et al., 2016). bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

91 Phosphorylation of KEAP1 or its interacting partners also induces NRF2 stability and signaling. 92 For example, a recent study reports that phosphorylation of KEAP1 by MST1/2 on 93 T51/S53/S55/S80 reduces NRF2 ubiquitylation (Wang et al., 2019). Additionally, proteins 94 containing ETGE-like motifs, such as p62/SQSTM1, bind to KEAP1 and stabilize NRF2 upon 95 phosphorylation by upstream kinases (e.g. mTORC1, TAK1) (Hashimoto et al., 2016; Ichimura et 96 al., 2013; Lau et al., 2010). Overall, these findings suggest a wide array of kinase-directed 97 signaling inputs for NRF2. 98 99 Our GOF kinome screen revealed that Brain Specific Kinase 2 and 1 (BRSK2/1, also known as 100 SAD-A and SAD-B, respectively) as negative regulators of NRF2. The BRSKs are understudied 101 members of the AMPK-related family of kinases(Bright et al., 2008). Both BRSK2/1 contain an N- 102 terminal kinase domain, followed by a Ubiquitin associated domain (UBA), a Proline rich region 103 (PRR), and a kinase associated domain (KA1) with an auto-inhibitory sequence (AIS) at the C- 104 terminus (Wang et al., 2018; Wu et al., 2015). These kinases are known to function downstream 105 of Liver Kinase B1 (LKB1) signaling, but are also activated by PAK1, CAMKII, and PKA (Bright et 106 al., 2008; Lizcano et al., 2004; Nie et al., 2012). In various model organisms and mammals, 107 BRSK2 and BRSK1 are expressed the most in the brain. BRSK2 is also expressed in pancreas; 108 BRSK1 is expressed in gonads as well as endocrine tissues (Uhlen et al., 2015; Uhlen et al., 109 2010). In C. elegans, the BRSK2/1 homologue SAD-1 is required for neuron polarization and 110 synaptic vesicle transport (Kim et al., 2010; Morrison et al., 2018). In mouse neurons, Brsk2/1 111 promote formation of vesicles, neurite differentiation, and synapse formation (Kishi, 2005; Lilley 112 et al., 2014). In pancreatic islets, BRSK2 promotes insulin secretion in response to glucose 113 stimulation (Chen et al., 2012; Nie et al., 2018; Nie et al., 2012). To date BRSK2/1 have been 114 implicated in positive regulation of cell cycle progression, ER associated protein degradation 115 (ERAD), neuronal polarity, autophagy, and insulin signaling (Li et al., 2012; Muller et al., 2010; 116 Wang et al., 2012; Wang et al., 2013). Specifically, BRSK2 kinase activity is induced by starvation 117 to inhibit mTOR and promote autophagy, similarly to AMPK, and new evidence suggests BRSK2 118 leads to activation of PI3K/AKT signaling (Bakula et al., 2017; Chen et al., 2012; Saiyin, 2017). 119 While BRSK2 is not yet directly linked to NRF2 signaling, several studies show that BRSK2 is 120 protective during ER stress (Wang et al., 2012; Wang et al., 2013). Here we identified BRSK2 as 121 a novel repressor of NRF2 signaling, and mechanistically establish that BRSK2 activates AMPK, 122 suppresses mTOR and decreases protein translation. 123 124 Results 125 Kinome gain of function screen identifies regulators of NRF2 activity 126 To identify kinase regulators of NRF2 signaling, we employed a gain-of-function arrayed screen 127 where 385 kinases and kinase associated proteins were over-expressed in HEK293T cells. NRF2 128 transcriptional activity was quantified with the hQR41-Firefly luciferase reporter normalized to 129 constitutively expressed Renilla luciferase. The hQR41 reporter contains a NRF2-responsive 130 fragment of the human NQO1 promoter, which is a consensus NRF2 target gene across species 131 and tissue types. Over-expression of the positive controls NRF2 and DPP3 activated hQR41- 132 luciferase, whereas KEAP1 expression suppressed the reporter activity (Hast et al., 2013). 133 Kinases that activated hQR41 include MAP3K8, MOS, MAP3K7/TAK1, and MAP2K6; while 134 HIPK4, BRD3, and BRSK2 were identified as repressors of NRF2-mediated transcription (Fig. 135 1A). Hits from this primary screen were validated in focused reporter assays across multiple cell 136 lines, including HEK293Ts, MEFs and H2228 lung adenocarcinoma cells (Fig. 1B-E). Several of 137 the validated kinases are confirmed in recent reports. MAP3K7/TAK1 activates NRF2 via 138 phosphorylation of p62/SQSTM to drive degradation of KEAP1 (Hashimoto et al., 2016). Though 139 BRD3 has not been directly tested, other members of the BRD family of proteins negatively bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

140 regulate NRF2-mediated transcription (Hussong et al., 2014; Michaeloudes et al., 2014). 141 Similarly, the HIPK4-related protein HIPK2 is a direct transcriptional target of NRF2 and a positive 142 regulator of NRF2 cytoprotection (Torrente et al., 2017). Because of its novelty within the NRF2 143 pathway, we sought to better understand BRSK2 and its role in NRF2 signaling. 144 BRSK2 and BRSK1 repress NRF2 signaling. 145 Redox regulation of cysteines within KEAP1 controls KEAP1-catalyzed NRF2 ubiquitylation and 146 degradation. Through reactivity with cysteine 151 in KEAP1, the triterpenoid of oleanolic acid (OA) 147 derivative CDDO-methyl (CDDO-me) potently suppresses NRF2 degradation (Suzuki et al., 148 2019). We first tested whether BRSK2 over-expression suppressed CDDO-me driven NRF2 149 activation, as quantified by hQR41-luciferase expression in HEK293T cells. We compared murine 150 Brsk2 and two human BRSK2 splice variants, where BRSK2-Isoform4 is missing 20 amino acids 151 compared to BRSK2-Isoform3. KEAP1 and Musculoapoeneuorotic Factor G (MAFG) served as 152 positive controls for NRF2 inhibitors. Compared to the negative controls (hcRED and 153 Glucuronidase Beta (pGUS)), all BRSK2 variants suppressed NRF2 transcriptional activity under 154 both vehicle and CDDO-me treated conditions (Fig. 2A). Similarly, murine Brsk2 blocked NRF2- 155 transcriptional activation. To confirm BRSK2 as an inhibitor of endogenous NRF2, we quantified 156 endogenous NRF2 target gene expression by qRT-PCR. Following over-expression of controls 157 or BRSK2 variants in HEK293T cells, we measured changes in NRF2 target genes HMOX1, 158 GCLM, and SLC7A11 normalized to GAPDH. BRSK2 variants downregulated NRF2 targets 159 similar to MAFG in both vehicle and CDDO-me treated conditions (Fig. 2B-D). Since key 160 regulation of NRF2 is post-translational, we tested whether BRSK2 over-expression impacted 161 steady state NRF2 protein levels. Compared to controls, BRSK2 over-expression decreased 162 NRF2 protein levels in HEK293T cells (Fig. 2E, compare lanes 5–7 with 1 and 2). To further 163 explore a role for KEAP1 in mediating BRSK2-suppression of NRF2, we tested if BRSK2 over- 164 expression could block a constitutively active mutant of NRF2 that does not bind KEAP1 (NRF2 165 ∆ETGE). BRSK2 repressed both wild type NRF2 and NRF2 ∆ETGE (Fig. 2F). 166 BRSK1 is a paralog to BRSK2, sharing ~68% amino acid sequence similarity and having similar 167 signaling functions. Leveraging the hQR41 reporter assay in HEK293T cells, we over-expressed 168 human or mouse BRSK1, BRSK2 or both. Like BRSK2, BRSK1 expression suppressed NRF2- 169 dependent transcriptional activation (Fig. 2G). We also performed the hQR41 reporter assay in 170 Keap1 -/- MEFs, which express high levels of NRF2 (Fig. 2H). Again, both BRSK1 and BRSK2 171 inhibited NRF2-driven transcription, suggesting that the mechanism of suppression was 172 independent of KEAP1-mediated ubiquitylation. Finally, we evaluated the impact of BRSK1 and 173 BRSK2 on a panel of pathway specific transcriptional reporters in HEK293T cells (Fig. 2I). BRSK1 174 and BRSK2 suppressed NRF2 and retinoic acid receptor (RAR) and activated the Activator 175 Protein-1 (AP1), Activating Transcription Factor 3 (ATF3), and Transforming Growth Factor β 176 (TGFβ) reporters. Neither BRSK family member regulated the WNT/β-catenin reporter, NFκB 177 reporter or STAT reporter. As such, BRSK1 and BRSK2 have redundant and conserved functions 178 in NRF2 suppression, and do not impact all signaling pathways equally. 179 Finally, we evaluated the effect of BRSK2 silencing on NRF2 protein expression and NRF2-driven 180 transcription. HEK293T cells were engineered to express dead KRAB-dCas9 nuclease before 181 stable introduction of 4 scrambled control sgRNAs or 5 independent BRSK2-specific sgRNAs. 182 W.blot analysis of the resulting cell lines confirmed efficient sgRNA-mediated CRISPRi silencing 183 of BRSK2 protein and no effect on NRF2 protein levels (Fig. S1A). NRF2 hQR41 reporter assays 184 in these cells did not show a BRSK2-silencing phenotype on NRF2 activity. Since BRSK2 RNA 185 levels are highest in brain and pancreas, we evaluated BRSK2 expression in pancreatic cancer 186 cell lines (Fig. S1C). Two cell lines, PANC1 and MIA PaCa-2, expressed the most BRSK2, of 187 which we used MIA PaCa-2 for CRISPRi silencing. Like HEK293T cells, MIA PaCa-2 cells bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

188 deficient for BRSK2 expressed comparable levels of NRF2 as control cells (Fig. S1D). These data 189 suggest that under homeostatic conditions, endogenous levels of BRSK2 does not control NRF2 190 activity. Further experiments in BRSK2 null background as opposed to silenced background are 191 needed. 192 BRSK2 kinase function is required to suppress NRF2 signaling. 193 We next determined whether BRSK2-mediated inhibition of NRF2 required BRSK2 kinase activity. 194 We mutated K48 and D141 in the kinase domain of BRSK2; these residues are required for ATP 195 binding and substrate phosphorylation (Lizcano et al., 2004). We also mutated T174, which is 196 phosphorylated by LKB1 to activate members of the AMPK kinase family (Fig. 3A) (Lizcano et al., 197 2004). Compared to controls, over-expression of wild type BRSK2 decreased NRF2-dependent 198 hQR41 luciferase expression whereas kinase dead variants had no significant affect (Fig. 3B). 199 The kinase dependency of BRSK2 was further confirmed using qRT-PCR of endogenous NRF2 200 target gene HMOX1, which was repressed by wild type BRSK2, but not by the kinase dead 201 mutants (Fig. 3C). qRT-PCR for the NRF2 transcript showed that expression of neither wild type 202 nor kinase dead BRSK2 mutants affected NRF2 mRNA levels (Fig. 3D). Finally, we evaluated 203 NRF2 protein levels in HEK293T cells over-expressing BRSK2 or the kinase dead mutants. As 204 expected, NRF2 protein levels were decreased by over-expression of wild type, but not kinase 205 dead BRSK2 (Fig. 3E). Similarly, kinase dead mutants of BRSK1 did not impact NRF2 signaling 206 (Fig. 3F). 207 Beyond kinase activity, structure-function studies of BRSK2 and the AMPK-like family of kinases 208 have revealed numerous features of functional importance (Bright et al., 2009; Wu et al., 2015). 209 We created a series of point mutations and truncations to determine which domains within BRSK2 210 are important for NRF2 suppression. Binding of the regulatory UBA and AIS region of the KA1 211 domain to the catalytic fold of the kinase domain is thought to hold BRSK2 in an inactive state 212 until phosphorylated by upstream regulators, bound by interaction partners or recruited to the 213 plasma membrane (Wu et al., 2015). As such, mutations in residues of the UBA domain and loss 214 of the AIS or KA1 domain results in an active BRSK2 kinase. We created and expressed mutant 215 proteins in HEK293T cells followed by hQR41 reporter quantitation and NRF2 W.blot analysis 216 (Fig. 3G-J). The L309D and ∆YFLLL mutations within the UBA domain did not suppress the 217 hQR41 reporter and NRF2 or its target genes in W.blot analysis (Fig.3G and 3H, respectively). 218 Based on previous reports on the role of the BRSK2 UBA domain, it is likely that loss of YFLLL 219 motif increases auto-inhibition by the AIS/KA1 region (Wu et al., 2015). Mutations of UBA residues 220 that contact the kinase domain, M332K and Y334F, repressed NRF2 activity. We next deleted 221 the kinase domain (∆N), C-terminus (∆C), PRR (∆PRR), AIS (∆AIS), or KA1 (∆KA1) domain and 222 evaluated their effect on NRF2-mediated transcription via reporter assays (Fig. 3I). Compared to 223 controls, BRSK2 ∆KA1 and ∆AIS inhibited NRF2 activity to similar or more than BRSK2 WT, while 224 loss of either kinase, C-terminal, or PRR regions abolished regulation of NRF2 signaling (Fig. 3J). 225 BRSK2 does not repress NRF2 via the ubiquitin proteasome system (UPS). 226 NRF2 is rapidly stabilized by electrophilic compounds that react with cysteines in KEAP1. NRF2 227 protein levels also accumulate within minutes of chemical inactivation of the ubiquitin proteasome 228 system (UPS). We tested whether BRSK2 expression would suppress NRF2 stabilized by KEAP1 229 reactive electrophiles or inhibitors of the UPS. First, we treated HEK293T cells with vehicle, 230 Sulforaphane, or tBHQ for 6 hours after 24 hours of BRSK2 expression. Like CDDO-me, these 231 compounds modify cysteine residues on KEAP1 to stabilize NRF2 (Suzuki et al., 2019). Cells 232 over-expressing BRSK2 significantly downregulated NRF2 protein levels compared to the 233 corresponding control (Fig. 4A, quantification below). Second, we tested the proteasomal 234 inhibitors MG132 and Bortezomib as well as the CUL3 neddylation inhibitor MLN4924. NRF2 235 protein levels were decreased by BRSK2 in all treatment groups compared to the control, bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

236 although to varying degrees (Fig. 4B, quantification below). Lastly, we asked whether BRSK2- 237 mediated downregulation of NRF2 involved the autophagy pathway. To evaluate this, we treated 238 BRSK2 overexpressing HEK293T cells with vehicle or Bafilomycin A (BafA1) for 12 hours. 239 Compared to vehicle, BafA1 treatment did not significantly affect NRF2 in either control or BRSK2 240 over-expressing conditions (Fig. 4C, quantified below). LC3B conversion confirmed efficacy of 241 BafA1 treatment. These data suggest that BRSK2-mediated downregulation of NRF2 is not via 242 the UPS or autophagy. 243 RNAseq and phosphoproteomic characterization BRSK2 and BRSK1 expression. 244 Unbiased comprehensive screening and molecular annotation has improved significantly over the 245 past decade, with newly empowered informatics that distill the resulting large datasets into 246 pathways and biological processes. To better understand BRSK2/1 function in cells, and to 247 possibly reveal how BRSK2 suppresses NRF2, we performed RNAseq and global quantitative 248 phosphoproteomic analysis on HEK293T cells expressing BRSK2 and BRSK1 as compared to 249 mock transfected or hcRED expression. Hierarchical clustering analysis revealed genes altered 250 by BRSK2/1 over-expression, where genes that passed FDR (FDR < 5%) and fold change (FC ≥ 251 2) cutoff are highlighted (Fig. 5A and B). Compared to control, we observed 723 and 879 252 differentially expressed genes that pass FDR < 5% for BRSK2 and BRSK1, respectively. Based 253 on fold change, differential gene expression due to BRSK2 expression was more robust than 254 BRSK1. Gene set enrichment analysis (GSEA) using the Hallmark and Oncogenic gene sets in 255 MSigDB revealed statistically significantly altered signaling pathways (Table S5 – S8). Genes 256 associated with mTOR signaling were robustly downregulated in BRSK2/1 expressing cells (Fig. 257 5C). GSEA for several NRF2 gene signatures revealed downregulation by BRSK2 (Fig. S2, Table 258 S9). Close examination by pathway analysis of the downregulated genes revealed enrichment for 259 those involved in pyruvate, glutathione, and amino acid metabolism. Interestingly, several of the 260 genes are known players in ferroptosis, a non-canonical and iron dependent cell death pathway 261 (Table S9). 262 Independently of the RNAseq analyses, we performed tandem mass tags (TMT)-based 263 quantitative phosphoproteomics on BRSK2/1 expressing cells where we used hcRED as control. 264 Biological triplicate samples were analyzed, revealing ~10,000 phosphosites in ~8,400 phospho- 265 peptides. Following the RNAseq trend, BRSK2 impacted phospho-proteome more robustly than 266 did BRSK1. Compared to control, at FDR < 5%, BRSK2 over-expression induced 307 differentially 267 phosphorylated peptides compared to 189 observed in BRSK1 over-expression (Table S10). We 268 leveraged PTMSigDB and enrichment analysis to map the observed phospho-peptide changes 269 to annotated signaling pathways (Fig. 5D and E) (Krug et al., 2019). BRSK2/1 positively regulated 270 AMPK and AKT signaling, while negatively impacting the mTOR pathway. BRSK2 also 271 suppressed signaling through the CDK1, CDK2, and CDC7 pathways (Table S12). 272 To confirm activation of AMPK signaling, BRSK2 was expressed in HEK293T cells before W.blot 273 analysis for phosphorylation of AMPK substrates (LxRxx(pS/pT)). We expressed either wild type 274 (WT), kinase active (∆KA1 or ∆AIS), or kinase dead (K48A or D141N) BRSK2 for 24 hours. Over- 275 expression of wild type and kinase active BRSK2 (lane 2, 5, & 6) upregulated pS/T AMPK motif 276 compared to control and kinase dead BRSK2 (lane 1, 3, & 4) (Fig 6A). We then measured 277 changes in mTOR substrate phosphorylation by evaluating p-S6K T389, p-4EBP1 T37/46, and p- 278 4EBP1 S65 in cells expressing hcRED control, BRSK2, kinase dead BRSK2 (K48A), or BRSK1. 279 BRSK2/1 expression decreased phosphorylation of both S6K and 4EBP1 (Fig. 6B). Total protein 280 levels of 4EBP1 increased following BRSK2/1 expression. Together, these data suggest that 281 BRSK2/1 downregulates mTOR signaling while upregulating AMPK substrate phosphorylation. 282 Finally, because BRSK2/1 over-expression suppressed mTOR, we quantified the rate of protein 283 translation after BRSK2/1 expression (Fig. 6C). HEK293T cells expressing BRSK2/1 or control bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

284 hcRED were pulsed with 35S-Methionine before lysis and quantitation of nascent polypeptides, 285 where Cyclohexamide (CHX) served as a negative control. Compared to control and kinase dead 286 BRSK2, expression of wild type BRSK2 and BRSK1 downregulated protein translation by 40% 287 and 10%, respectively (Fig. 6C). To further confirm decreased translation, we measured ribosome 288 binding to mRNA in cells expressing BRSK2/1. Cells transfected with the indicated construct were 289 fractionated in a 10% - 50% sucrose gradient followed by absorbance measurement for 290 polyribosome tracing. Compared to control and kinase dead BRSK2, over-expression of wild type 291 BRSK2 decreased heavy polyribosome formation on mRNA (Fig. 6D). 292 293 Discussion 294 The NRF2 transcription factor is central to a growing number of human pathologies. The 295 transcriptional program it governs enables life in the presence of oxygen, electrophiles, and 296 environmental stressors. As such, aberrant activation or suppression of NRF2 contributes to and 297 causes a number of human diseases, including cancer, inflammation, diabetes, and 298 neurodegeneration. Though its relevance and centrality to human health is well-established, we 299 have yet to realize the full potential of NRF2-directed therapeutics. In this study, we focused on 300 kinase regulators of NRF2 because the kinome is exceptionally druggable and the mechanistic 301 impact of NRF2 phosphorylation remains elusive. We discovered that, in an activity-dependent 302 manner, the BRSK2 kinase suppresses NRF2 signaling. We show that BRSK2 induces AMPK 303 activity and inhibits mTOR, resulting in decreased ribosome loading on mRNAs which 304 downregulates protein synthesis. 305 Beyond revealing the BRSK kinases as indirect suppressors of NRF2 translation, this work has 306 several implications. Signaling through the mTOR pathway has been reported to both promote 307 and inhibit NRF2 in a context dependent manner (Aramburu et al., 2014). Our data argue that 308 mTOR suppression by BRSK2, irrespective of cell type or duration, results in NRF2 protein loss. 309 Specifically, BRSK2 over-expression suppresses NRF2 protein at basal and electrophilic induced 310 conditions. Though NRF2 suppression was greatest after 24 hours of BRSK2 expression, through 311 an unknown mechanism, BRSK2 over-expression for greater than 36 hours resulted in cell death 312 (not shown). While we connect BRSK2 to NRF2 through activation of AMPK and suppression of 313 mTOR, it is possible that additional mechanisms contribute to the phenotype. For example, it 314 remains to be determined if BRSK2/1 bind or directly phosphorylate KEAP1 or NRF2. Our 315 immunoprecipitation and mass spectrometry experiments for KEAP1, NRF2, BRSK1 and BRSK2 316 did not reveal a co-complex, but false-negatives are common in pull-down mass spectrometry 317 experiments, particularly for kinase-substrate interactions. 318 The small molecule NRF2 inhibitors halofuginone and brusatol offer further support for a requisite 319 role of protein synthesis as a key point of NRF2 regulation (Harder et al., 2017; Tsuchida et al., 320 2017). Both halofuginone and brusatol are noted as potent NRF2 inhibitors that sensitize cells to 321 chemotherapy. Halofuginone inhibits prolyl-tRNA synthetase while brusatol acts on peptidyl 322 transferase to suppress protein translation and ultimately decrease proteins with short half-life 323 like NRF2, a phenotype observed with over-expression of BRSK2 (Harder et al., 2017; Tsuchida 324 et al., 2017). This also highlights the underlying role of stress signaling in protein synthesis. Under 325 moderate oxidative stress, mTOR regulated 5’ Cap-dependent translation is inhibited, but can be 326 bypassed via Internal Ribosomal Entry Site (IRES)-mediated translation found in the 5’ 327 untranslated region (5’UTR) (Komar and Hatzoglou, 2011). However, under severe stress 328 translation is inhibited via localization of ribosome-RNA complexes to stress granules to protect 329 nescient polypeptides and translation complex from damage. While we have not evaluated 330 changes in NRF2 mRNA levels associated with polysomes, the NRF2 mRNA does contain an 331 IRES-like region that promotes de novo translation under oxidative stress (Lee et al., 2017). bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

332 Conversely, NRF2 loss decreases protein translation rate in pancreatic cancer due to increased 333 oxidation of proteins in the translational machinery as well as decreased 4EBP1 phosphorylation 334 (DeNicola et al., 2012). Since BRSK2/1 decreases mTOR activity, possible mechanism of NRF2 335 depletion may be block of translation initiation/elongation under low oxidative stress where NRF2 336 mRNA is still dependent on 5’Cap-dependent translation. 337 Gain-of-function screening and extensive validation confirm BRSK2 as a suppressor of NRF2, yet 338 it is unclear if BRSK2 loss impacts NRF2 or oxidative stress response signaling. In an acute 339 experiment, we designed and tested a panel of short interfering RNAs against BRSK2, and 340 observed inconsistent results despite greater than 95% BRSK2 silencing (not shown). Long-term 341 suppression of BRSK2 expression with stable CRISPRi technology did not impact NRF2 protein 342 levels. Since kinases are catalytic enzymes, it is likely that our loss-of-function approaches failed 343 to eliminate enough BRSK2 activity to impact NRF2. Alternatively, it is possible that our 344 experiments lacked the necessary context, for example the presence of an upstream activating 345 signal. Further studies are needed in neuronal and pancreatic systems to evaluate BRSK loss 346 and NRF2 signal transduction. 347 BRSK2 and BRSK1 impact cellular signaling pathways beyond NRF2, AMPK and mTOR, as 348 gleaned from the RNAseq and phosphoproteomic experiments. BRSK2 expression activated 349 CHEK1, PKC, and AKT1 among other kinase signaling pathways. In a focused screen of various 350 engineered transcriptional reporters, we show that BRSK1 and BRSK2 induced AP1 and ATF3 351 stress response signaling, as well at TGFβ signaling. It is possible that some of these responses 352 are coupled indirectly to the suppression of mTOR; further work is needed to determine this. 353 These data provide a foundational resource to better interrogate signaling regulated by BRSK2/1. 354 355 Material and Methods 356 Cell Culture 357 All cell lines were obtained from American Type Culture Collection (ATCC) and were used within 358 10 passages after receipt from ATCC to ensure their identities. Cells were cultured in a humidified o 359 incubator at 37 C and 5% CO2. Cells were passaged with 0.05% Trypsin/0.53mM EDTA in 360 Sodium Bicarbonate (Corning, 25-052-CI), and maintained in media supplemented with 10% fetal 361 bovine serum (FBS) as follows: HEK293T, MIA PaCa2– Dulbecco’s Modified Eagle Medium 362 (DMEM) (Corning, 10-013-CV); H2228– RPMI-1640 (Corning, 10-040-CV); and Keap1 +/+ and 363 Keap1 -/- Mouse Embryonic Fibroblasts (MEFs) generated as previously described (Cloer, 2018; 364 Wakabayashi et al., 2003) – DMEM/Ham’s F-12 50/50 mix supplemented with Sodium Pyruvate 365 and Non-essential amino acids (Corning, 10-092-CV). 366 Generation of CRISPRi Cell lines 367 Lentivirus for KRAB-dCas9 was generated by using PsPax2 and PMD2G packaging vectors 368 (Gilbert et al., 2014). HEK293T and MIA PaCa-2 cells were infected with KRAB-dCas9 lentivirus 369 and monoclonal lines were generated via single cell sorting following 10µg/mL Blasticidin (GIBCO, 370 A11139-03) selection for 5 passages. Each monoclonal line was cultured in 5µg/mL Blasticidin 371 following sorting. Single guide RNA (sgRNA) vectors were generated by ligating oligonucleotides 372 into AarI (ThermoFisher Scientific, ER1582) digested VDB783 vector (Table S1). Each sgRNA 373 was lentivirally introduced to the above mentioned cell lines, and cells were cultured for 3 374 passages in 2.5µg/mL Puromycine (Corning, 61-385-RA) and 5µg/mL Blasticidin before further 375 analysis. Stable cell lines were maintained in 1µg/mL Puromycine and 5µg/mL Blasticidin. 376 Plasmids and Reagents bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

377 The human kinome ORF library in pDONOR223 was obtained from Addgene (Cambridge, MA, 378 1000000014) and cloned into a custom pHAGE-CMV-FLAG destination vector using Gateway 379 cloning technology, as previously described (Agajanian et al., 2019). Luciferase reporter plasmids 380 for WNT (BAR), NFκB, AP-1, ATF3, STAT, Retinoic acid (RAR), and TGFβ (SMAD) were cloned 381 into transient expression vectors as previously done (Matthew P. Walker, 2015; Travis L. 382 Biechele, 2008). The NOTCH (CSL) reporter was a gift from Raphael Kopan (Addgene, 41726) 383 (Saxena et al., 2001). 384 All ORFs were cloned into pHAGE-CMV-FLAG via LR clonase (Thermo Fisher, 11791-020): 385 pDONOR223.1 BRSK2 (splice isoform 3, BRSK2_Iso3) and Brsk2 were obtained from Harvard 386 PlasmID repository (HsCD00297097 and MmCD00295042). BRSK2 Kinase dead (K48A, D141N, 387 and T174A) and UBA domain (L309D, M332K/Y334F, and ∆YFLLL) mutants were generated 388 using Q5 Hot Start Site Direct Mutagenesis kit (New England BioLabs, E0552S). BRSK2 domain 389 deletion mutations ∆N (∆ kinase), ∆C (∆ C-terminal), ∆PRR (∆ proline-rich region), ∆KA1 ( ∆ 390 kinase associated domain), and ∆AIS (∆ auto-inhibitory sequence) were generated via Phusion 391 DNA polymerase (New England BioLabs, M0530S), where the PCR product was treated with 392 DpnI (New England BioLabs, R0176S) then purified with Monarch PCR & DNA Purification Kit 393 (New England BioLabs, T1030S) followed by T4 DNA Ligase reaction (New England BioLabs, 394 M0202S). BRSK1 WT and T189A vectors were obtained from MRC PPU Reagents and Services 395 (DU1236 and DU1242) in a pCMV5-HA backbone. Brsk1 was obtained from Origene 396 (MR220008). They were then gateway converted into pDONOR223.1. Cloning primers are listed 397 in Table S1. 398 Kinome gain-of-function screen 399 HEK293T cells were plated in 384-well clear-bottom plates (Corning, 3764), and transfected with 400 a cocktail of 20ng FLAG-Kinase, 4ng hQR41 (Moehlenkamp and Johnson, 1999) (a generous gift 401 from Jeffery Johnson, University of Wisconsin), and 1ng HSV-thymidine kinase promoter driven 402 Renilla luciferase (pRL-TK-Renilla, referred to as Renilla) (Promega, E2241) per well using 403 Lipofectamine 2000 (Life technologies, 11668-019) in OptiMEM (Gibco, 31985-070). Each kinase 404 was transfected in four technical replicates and biological triplicate using the Promega Dual-Glo 405 Luciferase Assay System per the manufacturer’s protocol (Promega, E2940) (Agajanian et al., 406 2019). NRF2 transcriptional activity was determined by measuring levels of Firefly luciferase and 407 was normalized to Renilla luciferase for well-to-well variability. Fold activation of the reporter was 408 determined compared to GFP expressing cells, and false discovery rate (FDR) was calculated 409 following adjustment via Benjamini & Hochberg correction. 410 Western blot 411 All samples were lysed in RIPA (10% glycerol, 50mM Tris-HCL, 100mM NaCl, 2mM EDTA, 0.1% 412 SDS, 1% Nonidet P-40, 0.2% Sodium Deoxycholate) supplemented with protease inhibitor 413 cocktail (ThermoFisher Scientific, 78429), phosphatase inhibitor cocktail (ThermoFisher 414 Scientific, 78426), NEM (Thermo Scientific, 23030), and Benzonase (Sigma, E1014). Lysis was 415 done on ice for 30min and lysates were centrifuged at 4oC for 15min at 21,000xg. Following 416 normalization of protein concentration via BCA (Pierce, 23225), samples were denatured in 417 NuPAGE LDS buffer (Invitrogen, NP0007) with 1mM DTT. Samples were treated with the 418 following small molecules to stabilize NRF2: Sigma-- Bardoxolone Methyl/CDDO-me (S8078), 419 tert-Butal hydroquinone/tBHQ (112941), Sulforaphane (S6317); Calbiochem-- MG132 (474790), 420 MLN4924 (505477001); Selleck Chem-- Bortezomib (PS-341). Antibodies are listed in Table S2. 421 qRT-PCR and RNA sequencing analysis bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

422 RNA was collected using PureLink RNA Mini Kit (Invitrogen, 12183018A) per manufacturer 423 instruction. The extracted RNA was then reverse transcribed to cDNA using iScript cDNA 424 Synthesis Kit (BioRad, 170-8891), which was then used to perform quantitative RT-PCR (qRT- 425 PCR) using SYBR Green (Applied Biosystems, 4385617) for the specified target genes (Table 426 S3). 427 3µg of RNA was submitted to Novogene Corp. Ltd. (Sacramento, CA) for sequencing using 428 Illumina HiSeq platform where reads were mapped to reference genome Homo sapiens 429 (GRCh37/hg19). Alignments were done using STAR/HTSeq. Differential expression analysis was 430 performed starting with gene level read count quantification provided by Novogene Corp. 431 Preprocessing, normalization, and differential expression analysis were performed according to 432 the analysis pipeline outlined by Law et. al. (Law et al., 2016). Marginally detected genes (<5 total 433 read counts across samples) were filtered out as an initial preprocessing step, yielding a uni- 434 modal distribution of read-counts per million by gene. Data normalization was performed by using 435 the trimmed mean of M-values method as implemented in the calcNormFactors edgeR R-package 436 function(Chen, 2014). Differential expression analysis was subsequently performed using the 437 Limma R-packages functions voom and eBayes (Ritchie et al., 2015). 438 Gene set enrichment analysis (GSEA) was performed based on the above described pre- 439 processed read counts which were converted to counts per million and log2 transformed 440 (logCPM). From 13,000 genes the top 10,000 differentially expressed genes were used for GSEA 441 analysis. Genes were ranked according to signal to noise ratio as defined by the Broad Institute 442 GSEA software using the R-project fgsea package. Test gene sets (Hallmark and Oncogenic 443 gene sets from Molecular Signatures Database (MSigDB, Broad Institute)) were downloaded from 444 the MSIG data bank via the msigdbr R-project package (Liberzon et al., 2015; Subramanian, 445 2005). Raw data and processed datasets are available through GEO (GSE139135). 446 Translation assay and polysome fractionation 447 Rate of protein translation was measured in HEK293T cells expressing controls or BRSK2/1 24 448 hours post transfection using radioactive methionine (35S-Met) labeling, and polysome 449 fractionation was performed as previously described (Graves et al., 2019; Lenarcic et al., 2014). 450 Phosphoproteomics sample processing and data analysis 451 Protein from HEK293Ts expressing control or BRSK2/1 (1.4mg) was precipitated in acetone 452 overnight at -20°C. The sample was pelleted and re-suspended in 7M urea, reduced with 5mM 453 DTT (dithiothreitol) and alkylated with 15mM CAA (chloroacetamide). The sample was adjusted 454 with 50mM ABC (ammonium bicarbonate) such that the urea concentration is 1M or less. A 455 standard tryptic digest was performed overnight at 37°C. Solid Phase Extraction (SPE) was then 456 performed using C18 Prep Sep™ cartridges (Waters, WAT054960), followed by reconstitution in 457 0.5% TFA (trifluoroacetic acid). The SPE cartridge was washed with conditioning solution (90% 458 methanol with 0.1% TFA), then equilibrated with 0.1% TFA. The sample was passed slowly 459 (1drop/sec) through the equilibrated cartridge, then the cartridge was desalted with equilibration 460 solution. The sample was then slowly eluted (1drop/sec) with an elution solution (50% ACN 461 (acetonitrile)) with 0.1% TFA. The sample was then TMT labeled according to kit specifications 462 (ThermoFisher Scientific, 90110), with the exception that labeling was performed for 6hrs instead 463 of 1hr. Following labeling, another SPE was performed, as stated above. 10% of sample was 464 saved for whole proteome input, 90% was phosphopeptide enriched using Titansphere Phos-TiO 465 Kit (GL Sciences, 5010-21312). Before enrichment, samples were reconstituted in 100µL of Buffer 466 B (75% ACN, 1% TFA, 20% lactic acid – solution B in the kit). The tip was conditioned by 467 centrifugation with 100µL of Buffer A (80% ACN, 1% TFA), followed by conditioning with Buffer B 468 (3000xg, 2min). The sample was then loaded onto the tip and centrifuged twice (1000xg, 5min). bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

469 The tip was then washed with 50µL of Buffer B, followed by 2 washes with 50µL of Buffer A 470 (1000xg, 2min). Phosphopeptides were eluted with 100µL of elution 1 (20% ACN, 5% NH4OH) 471 then 100µL of elution 2 (20% ACN, 10% NH4OH) (1000xg, 5min). Following phosphopeptide 472 enrichment, both whole proteome input and phosphopeptides were fractionated utilizing a High 473 pH Reversed-Phase Peptide Fractionation kit (Pierce, 84868), per manufacturers specifications. 474 A final clean-up step was performed using C18 Spin Columns (Pierce, 89870). 475 Mass Spectrometry, Data Filtering, and Bioinformatics

476 Mass spectrometry analysis done as follows: peptides were separated via reverse-phase nano- 477 HPLC using nanoACQUITY UPLC system (Waters Corporation). Peptides were trapped on a 2 478 cm column (Pepmap 100, 3μM particle size, 100 Å pore size; ThermoFisher Scientific, 164946), 479 and separated on a 25cm EASYspray analytical column (75μM ID, 2.0μm C18 particle size, 100 480 Å pore size; ThermoFisher Scientific, ES802) at 45oC. The mobile phases were 0.1% formic acid 481 in water (Buffer A) and 0.1% formic acid in ACN (Buffer B). A 180-minute gradient of 2-30% buffer 482 B was used with a flow rate of 300nl/min. Mass spectral analysis was performed by 483 an Orbitrap Fusion Lumos mass spectrometer (ThermoFisher Scientific). The ion source 484 was operated at 2.4kV and the ion transfer tube was set to 275oC. Full MS scans (350-2000 m/z) 485 were analyzed in the Orbitrap at a resolution of 120,000 and 4e5 AGC target. The MS2 spectra 486 were collected using a 0.7 m/z isolation width and analyzed by the linear ion trap using 1e4 AGC 487 target after HCD fragmentation at 30% collision energy with 50ms maximum injection 488 time. The MS3 scans (100-500 m/z) were acquired in the Orbitrap at a resolution of 50,000 with 489 a 1e5 AGC, 2 m/z MS2 isolation window, and at 105ms maximum injection time after HCD 490 fragmentation with a normalized energy of 65%. Precursor ions were selected in 400-2000 m/z 491 mass range with mass exclusion width of 5 – 18 m/z. Lock mass = 371.10124 m/z (Polysiloxane).

492 MaxQuant (1.6.6.0) search parameters: specific tryptic digestion, up to 2 missed cleavages, a 493 static carbamidomethyl cysteine modification, variable protein N-term acetylation, and variable 494 phospho(STY) as well as methionine oxidation using the human UniProtKB/Swiss-Prot sequence 495 database (Downloaded Feb 1, 2017). MaxQuant data was deposited to PRIDE/Proteome 496 Xchange (PXD015884) (Vizcaino et al., 2014). MaxQuant output files proteinGroups.txt and 497 Phospho(STY) sites.txt were converted using in-house script into GCT format. This GCT file was 498 then rewritten using Morpheus for compatibility with PTM-SEA analysis in R (Krug et al., 2019). 499 500 Acknowledgements 501 We would like to thank Drs. Michael J Emanuele and Thomas R Bonacci for valuable scientific 502 discussion. We thank Drs. Kirsten L Bryant and Channing J Der for providing expertise and 503 resources to explore mTOR signaling as well as autophagy. 504 Funding sources 505 TYT and MJA were funded by HHMI Gilliam Fellowship for Advanced Study and the Initiative for 506 Maximizing Student Diversity Grant (R25-GM055336-16). Additionally, TYT was supported by 507 NIH T32 Pre-doctoral Training Grants in Pharmacology (T32-GM007040- 42). MJA was also 508 funded via NIH/NCI NRSA (F31CA228289) and NCI F99/K00 (1F99CA245724-01). RMM was 509 supported by NIH/NCI NRSA (1F31DE028749-01) and NIH/NIGMS T32 MiBio Training Program 510 (5T32GM119999-03). BMB, TPS, and KML were funded by ITCMS T32 Training Grant 511 (T32CA009156). BMB was also supported by NIH/NCI F32 (1F32CA225040-01). This research 512 was supported by American Cancer Society grant to M. B. Major (RSG-14-1657068-01-TBE), 513 NIH/NCI to M. B. Major and B. E. Weissman (RO1CA216051) and a NIH consortium grant on 514 Illuminating the Druggable Genome (U24- DK116204-01). bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

515 516 Author Contributions 517 This work was designed by MBM and TYT. Experiments were carried out by TYT, BMB, MJA, 518 AEH, PSF, SJW, RMM, and KML. Bioinformatics analysis were performed by DG, TPS, TS, and 519 TYT. Expertise and resources for evaluating protein translation biology were provided by NJM 520 and AEH. Manuscript was written by TYT and MBM. Manuscript was reviewed and edited by BMB 521 and BEW. 522 Competing interests: Authors do not have competing interests to declare. 523 bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

524 References 525 Agajanian, M. J., Walker, M. P., Axtman, A. D., Ruela-de-Sousa, R. R., Serafin, D. S., 526 Rabinowitz, A. D., Graham, D. M., Ryan, M. B., Tamir, T., Nakamichi, Y. et al. (2019). WNT Activates the 527 AAK1 Kinase to Promote Clathrin-Mediated Endocytosis of LRP6 and Establish a Negative Feedback 528 Loop. Cell Rep 26, 79-93 e8. 529 Apopa, P. L., He, X. and Ma, Q. (2008). Phosphorylation of Nrf2 in the transcription activation 530 domain by casein kinase 2 (CK2) is critical for the nuclear translocation and transcription activation 531 function of Nrf2 in IMR-32 neuroblastoma cells. Journal of Biochemical and Molecular Toxicology 22, 63- 532 76. 533 Aramburu, J., Ortells, M. C., Tejedor, S., Buxade, M. and Lopez-Rodriguez, C. (2014). 534 Transcriptional regulation of the stress response by mTOR. Sci Signal 7, re2. 535 Baird, L., Llères, D., Swift, S. and Dinkova-Kostova, A. T. (2013). Regulatory flexibility in the 536 Nrf2-mediated stress response is conferred by conformational cycling of the Keap1-Nrf2 protein 537 complex. Proceedings of the National Academy of Sciences 110, 15259-15264. 538 Bakula, D., Muller, A. J., Zuleger, T., Takacs, Z., Franz-Wachtel, M., Thost, A. K., Brigger, D., 539 Tschan, M. P., Frickey, T., Robenek, H. et al. (2017). WIPI3 and WIPI4 beta-propellers are scaffolds for 540 LKB1-AMPK-TSC signalling circuits in the control of autophagy. Nat Commun 8, 15637. 541 Bright, N. J., Carling, D. and Thornton, C. (2008). Investigating the regulation of brain-specific 542 kinases 1 and 2 by phosphorylation. Journal of Biological Chemistry 283, 14946-14954. 543 Bright, N. J., Thornton, C. and Carling, D. (2009). The regulation and function of mammalian 544 AMPK-related kinases. Acta Physiol (Oxf) 196, 15-26. 545 Chen, X. Y., Gu, X. T., Saiyin, H., Wan, B., Zhang, Y. J., Li, J., Wang, Y. L., Gao, R., Wang, Y. F., 546 Dong, W. P. et al. (2012). Brain-selective Kinase 2 (BRSK2) Phosphorylation on PCTAIRE1 Negatively 547 Regulates Glucose-stimulated Insulin Secretion in Pancreatic -Cells. Journal of Biological Chemistry 287, 548 30368-30375. 549 Chen, Y. L., A.T.L.; and Smyth, G.K. (2014). Differential expression analysis of complex RNA-seq 550 experiments using edgeR. In: Statistical Analysis of Next Generation Sequence Data, Somnath Datta and 551 Daniel S Nettleton (eds). Springer. 552 Chowdhry, S., Zhang, Y., McMahon, M., Sutherland, C., Cuadrado, A. and Hayes, J. D. (2012). 553 Nrf2 is controlled by two distinct &beta;-TrCP recognition motifs in its Neh6 domain, one of which 554 can be modulated by GSK-3 activity. Oncogene 32, 3765-3781. 555 Cloer, E. W., Goldfarb, D., Schrank, T. P., Weissman, B. E. and Major, M. B. (2019). NRF2 556 Activation in Cancer: From DNA to Protein. Cancer Res 79, 889-898. 557 Cloer, E. W. S., P. F.; Cousins, E. M.; Goldfarb, D.; Mowrey, D. D.; Harrison, J. S.; Weir, S. J.; 558 Dokholyan, N. V.; Major, M. B. (2018). p62-Dependent Phase Separation of Patient-Derived KEAP1 559 Mutations and NRF2. Molecular and Cellular Biology. 560 Cuadrado, A. (2015). Structural and functional characterization of Nrf2 degradation by glycogen 561 synthase kinase 3/β-TrCP. Free Radical Biology and Medicine 88, 147-157. 562 Cuadrado, A., Manda, G., Hassan, A., Alcaraz, M. J., Barbas, C., Daiber, A., Ghezzi, P., Leon, R., 563 Lopez, M. G., Oliva, B. et al. (2018). Transcription Factor NRF2 as a Therapeutic Target for Chronic 564 Diseases: A Systems Medicine Approach. Pharmacol Rev 70, 348-383. 565 Cullinan, S. B., Gordan, J. D., Jin, J., Harper, J. W. and Diehl, J. A. (2004). The Keap1-BTB Protein 566 Is an Adaptor That Bridges Nrf2 to a Cul3-Based E3 Ligase: Oxidative Stress Sensing by a Cul3-Keap1 567 Ligase. Molecular and Cellular Biology 24, 8477-8486. 568 Cullinan, S. B., Zhang, D., Hannink, M., Arvisais, E., Kaufman, R. J. and Diehl, J. A. (2003). Nrf2 is 569 a direct PERK substrate and effector of PERK-dependent cell survival. Molecular and Cellular Biology 23, 570 7198-7209. bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

571 Del Vecchio, C. A., Feng, Y., Sokol, E. S., Tillman, E. J., Sanduja, S., Reinhardt, F. and Gupta, P. 572 B. (2014). De-Differentiation Confers Multidrug Resistance Via Noncanonical PERK-Nrf2 Signaling. PLoS 573 Biology 12, e1001945. 574 DeNicola, G. M., Karreth, F. A., Humpton, T. J., Gopinathan, A., Wei, C., Frese, K., Mangal, D., 575 Yu, K. H., Yeo, C. J., Calhoun, E. S. et al. (2012). Oncogene-induced Nrf2 transcription promotes ROS 576 detoxification and tumorigenesis. Nature 475, 106-109. 577 Gilbert, L. A., Horlbeck, M. A., Adamson, B., Villalta, J. E., Chen, Y., Whitehead, E. H., 578 Guimaraes, C., Panning, B., Ploegh, H. L., Bassik, M. C. et al. (2014). Genome-Scale CRISPR-Mediated 579 Control of Gene Repression and Activation. Cell 159, 647-61. 580 Graves, P. R., Aponte-Collazo, L. J., Fennell, E. M. J., Graves, A. C., Hale, A. E., Dicheva, N., 581 Herring, L. E., Gilbert, T. S. K., East, M. P., McDonald, I. M. et al. (2019). Mitochondrial Protease ClpP is 582 a Target for the Anticancer Compounds ONC201 and Related Analogues. ACS Chem Biol 14, 1020-1029. 583 Harder, B., Tian, W., La Clair, J. J., Tan, A. C., Ooi, A., Chapman, E. and Zhang, D. D. (2017). 584 Brusatol overcomes chemoresistance through inhibition of protein translation. Mol Carcinog 56, 1493- 585 1500. 586 Hashimoto, K., Simmons, A. N., Kajino-Sakamoto, R., Tsuji, Y. and Ninomiya-Tsuji, J. (2016). 587 TAK1 Regulates the Nrf2 Antioxidant System Through Modulating p62/SQSTM1. Antioxid Redox Signal 588 25, 953-964. 589 Hast, B. E., Goldfarb, D., Mulvaney, K. M., Hast, M. A., Siesser, P. F., Yan, F., Hayes, D. N. and 590 Major, M. B. (2013). Proteomic Analysis of Ubiquitin Ligase KEAP1 Reveals Associated Proteins That 591 Inhibit NRF2 Ubiquitination. Cancer Research 73, 2199-2210. 592 Homma, S., Ishii, Y., Morishima, Y., Yamadori, T., Matsuno, Y., Haraguchi, N., Kikuchi, N., 593 Satoh, H., Sakamoto, T., Hizawa, N. et al. (2009). Nrf2 Enhances Cell Proliferation and Resistance to 594 Anticancer Drugs in Human Lung Cancer. Clinical Cancer Research 15, 3423-3432. 595 Huang, H. C. (2002). Phosphorylation of Nrf2 at Ser-40 by Protein Kinase C Regulates Antioxidant 596 Response Element-mediated Transcription. Journal of Biological Chemistry 277, 42769-42774. 597 Hussong, M., rno, S. T. B. o., Kerick, M., Wunderlich, A., Franz, A., ltmann, H. S. u., 598 Timmermann, B., Lehrach, H., Hirsch-Kauffmann, M. and Schweiger, M. R. (2014). The bromodomain 599 protein BRD4 regulates the KEAP1/NRF2-dependent oxidative stress response. Cell Death and 600 Disease 5, e1195-11. 601 Ichikawa, T., Li, J., Meyer, C. J., Janicki, J. S., Hannink, M. and Cui, T. (2009). Dihydro-CDDO- 602 trifluoroethyl amide (dh404), a novel Nrf2 activator, suppresses oxidative stress in cardiomyocytes. PLoS 603 ONE 4, e8391. 604 Ichimura, Y., Waguri, S., Sou, Y.-s., Kageyama, S., Hasegawa, J., Ishimura, R., Saito, T., Yang, Y., 605 Kouno, T., Fukutomi, T. et al. (2013). Phosphorylation of p62 Activates the Keap1-Nrf2 Pathway during 606 Selective Autophagy. Molecular Cell 51, 618-631. 607 Itoh, K., Mimura, J. and Yamamoto, M. (2010). Discovery of the Negative Regulator of Nrf2, 608 Keap1: A Historical Overview. Antioxidants & Redox Signaling 13, 1665-1678. 609 Itoh, K., Wakabayashi, N., Katoh, Y., Ishii, T., Igarashi, K., Engel, J. D. and Yamamoto, M. 610 (1999). Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding 611 to the amino-terminal Neh2 domain. Genes & Development 13, 76-86. 612 Joo, M. S., Kim, W. D., Lee, K. Y., Kim, J. H., Koo, J. H. and Kim, S. G. (2016). AMPK Facilitates 613 Nuclear Accumulation of Nrf2 by Phosphorylating at Serine 550. Mol Cell Biol 36, 1931-42. 614 Kensler, T. W., Wakabayashi, N. and Biswal, S. (2007). Cell Survival Responses to Environmental 615 Stresses Via the Keap1-Nrf2-ARE Pathway. Annual Review of Pharmacology and Toxicology 47, 89-116. 616 Kim, J. S., Hung, W., Narbonne, P., Roy, R. and Zhen, M. (2010). C. elegans STRADalpha and SAD 617 cooperatively regulate neuronal polarity and synaptic organization. Development 137, 93-102. bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

618 Kishi, M. P., YA; Crump, JG; Sanes, JR. (2005). Mammalian SAD kinases are required for 619 neuronal polarization. Science. 620 Komar, A. A. and Hatzoglou, M. (2011). Cellular IRES-mediated translation: the war of ITAFs in 621 pathophysiological states. Cell Cycle 10, 229-40. 622 Krug, K., Mertins, P., Zhang, B., Hornbeck, P., Raju, R., Ahmad, R., Szucs, M., Mundt, F., 623 Forestier, D., Jane-Valbuena, J. et al. (2019). A Curated Resource for Phosphosite-specific Signature 624 Analysis. Mol Cell Proteomics 18, 576-593. 625 Lau, A., Wang, X.-J., Zhao, F., Villeneuve, N. F., Wu, T., Jiang, T., Sun, Z., White, E. and Zhang, 626 D. D. (2010). A noncanonical mechanism of Nrf2 activation by autophagy deficiency: direct interaction 627 between Keap1 and p62. Molecular and Cellular Biology 30, 3275-3285. 628 Law, C. W., Alhamdoosh, M., Su, S., Dong, X., Tian, L., Smyth, G. K. and Ritchie, M. E. (2016). 629 RNA-seq analysis is easy as 1-2-3 with limma, Glimma and edgeR. F1000Res 5. 630 Lee, S. C., Zhang, J., Strom, J., Yang, D., Dinh, T. N., Kappeler, K. and Chen, Q. M. (2017). G- 631 Quadruplex in the NRF2 mRNA 5' Untranslated Region Regulates De Novo NRF2 Protein Translation 632 under Oxidative Stress. Mol Cell Biol 37. 633 Lenarcic, E. M., Ziehr, B., De Leon, G., Mitchell, D. and Moorman, N. J. (2014). Differential role 634 for host translation factors in host and viral protein synthesis during human cytomegalovirus infection. J 635 Virol 88, 1473-83. 636 Li, R., Wan, B., Zhou, J., Wang, Y., Luo, T., Gu, X., Chen, F. and Yu, L. (2012). APC/CCdh1 Targets 637 Brain-Specific Kinase 2 (BRSK2) for Degradation via the Ubiquitin-Proteasome Pathway. PLoS ONE 7, 638 e45932. 639 Liberzon, A., Birger, C., Thorvaldsdottir, H., Ghandi, M., Mesirov, J. P. and Tamayo, P. (2015). 640 The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst 1, 417-425. 641 Lilley, B. N., Krishnaswamy, A., Wang, Z., Kishi, M., Frank, E. and Sanes, J. R. (2014). SAD 642 kinases control the maturation of nerve terminals in the mammalian peripheral and central nervous 643 systems. Proc Natl Acad Sci U S A 111, 1138-43. 644 Lim, J. H., Kim, K.-M., Kim, S. W., Hwang, O. and Choi, H. J. (2008). Bromocriptine activates 645 NQO1 via Nrf2-PI3K/Akt signaling: Novel cytoprotective mechanism against oxidative damage. 646 Pharmacological Research 57, 325-331. 647 Lizcano, J. M., Goransson, O., Toth, R., Deak, M., Morrice, N. A., Boudeau, J., Hawley, S. A., 648 Udd, L., Makela, T. P., Hardie, D. G. et al. (2004). LKB1 is a master kinase that activates 13 kinases of the 649 AMPK subfamily, including MARK/PAR-1. EMBO J 23, 833-43. 650 Matthew P. Walker, C. M. S., Maria Cederlund, Fang Fang, Christopher Jahn, Alex D. 651 Rabinowitz, Dennis Goldfarb, David M. Graham, Feng Yan, Allison M. Deal, Yuri Fedoriw, Kristy L. 652 Richards, Ian J. Davis, Gilbert Weidinger, Blossom Damania, Michael B. Major. (2015). FOXP1 653 potentiates Wnt/b-catenin signaling in diffuse large B cell lymphoma. Science Signaling. 654 Menegon, S., Columbano, A. and Giordano, S. (2016). The Dual Roles of NRF2 in Cancer. Trends 655 in Molecular Medicine. 656 Michaeloudes, C., Mercado, N., Clarke, C., Bhavsar, P. K., Adcock, I. M., Barnes, P. J. and 657 Chung, K. F. (2014). Bromodomain and Extraterminal Proteins Suppress NF-E2-Related Factor 2- 658 Mediated Antioxidant Gene Expression. The Journal of Immunology 192, 4913-4920. 659 Moehlenkamp, J. D. and Johnson, J. A. (1999). Activation of antioxidant/electrophile-responsive 660 elements in IMR-32 human neuroblastoma cells. Archives of biochemistry and biophysics 363, 98-106. 661 Morrison, L. M., Edwards, S. L., Manning, L., Stec, N., Richmond, J. E. and Miller, K. G. (2018). 662 Sentryn and SAD Kinase Link the Guided Transport and Capture of Dense Core Vesicles in Caenorhabditis 663 elegans. Genetics 210, 925-946. 664 Muller, M., Lutter, D. and Puschel, A. W. (2010). Persistence of the cell-cycle checkpoint kinase 665 Wee1 in SadA- and SadB-deficient neurons disrupts neuronal polarity. J Cell Sci 123, 286-94. bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

666 Naidu, S., Vijayan, V., Santoso, S., Kietzmann, T. and Immenschuh, S. (2009). Inhibition and 667 genetic deficiency of p38 MAPK up-regulates heme oxygenase-1 gene expression via Nrf2. Journal of 668 immunology (Baltimore, Md. : 1950) 182, 7048-7057. 669 Nakaso, K., Yano, H., Fukuhara, Y., Takeshima, T., Wada-Isoe, K. and Nakashima, K. (2003). 670 PI3K is a key molecule in the Nrf2-mediated regulation of antioxidative proteins by hemin in human 671 neuroblastoma cells. FEBS Letters 546, 181-184. 672 Nie, J., Sun, C., Chang, Z., Musi, N. and Shi, Y. (2018). SAD-A Promotes Glucose-Stimulated 673 Insulin Secretion Through Phosphorylation and Inhibition of GDIalpha in Male Islet beta Cells. 674 Endocrinology 159, 3036-3047. 675 Nie, J., Sun, C., Faruque, O., Ye, G., Li, J., Liang, Q., Chang, Z., Yang, W., Han, X. and Shi, Y. 676 (2012). Synapses of amphids defective (SAD-A) kinase promotes glucose-stimulated insulin secretion 677 through activation of p21-activated kinase (PAK1) in pancreatic beta-Cells. J Biol Chem 287, 26435-44. 678 Ritchie, M. E., Phipson, B., Wu, D., Hu, Y., Law, C. W., Shi, W. and Smyth, G. K. (2015). limma 679 powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 680 43, e47. 681 Rojo de la Vega, M., Chapman, E. and Zhang, D. D. (2018). NRF2 and the Hallmarks of Cancer. 682 Cancer Cell 34, 21-43. 683 Saiyin, H. N., N; Han, X; Fang, Y; Wu, Y; Lou, W; Yang, X. (2017). BRSK2 induced by nutrient 684 deprivation promotes Akt activity in pancreatic cancer via downregulation of mTOR activity. Oncotarget. 685 Satoh, H., Moriguchi, T., Takai, J., Ebina, M. and Yamamoto, M. (2013). Nrf2 Prevents Initiation 686 but Accelerates Progression through the Kras Signaling Pathway during Lung Carcinogenesis. Cancer 687 Research 73, 4158-4168. 688 Saxena, M. T., Schroeter, E. H., Mumm, J. S. and Kopan, R. (2001). Murine notch homologs (N1- 689 4) undergo presenilin-dependent proteolysis. J Biol Chem 276, 40268-73. 690 Shen, G., Hebbar, V., Nair, S., Xu, C., Li, W., Lin, W., Keum, Y.-S., Han, J., Gallo, M. A. and Kong, 691 A.-N. T. (2004). Regulation of Nrf2 transactivation domain activity. The differential effects of mitogen- 692 activated protein kinase cascades and synergistic stimulatory effect of Raf and CREB-binding protein. 693 The Journal of biological chemistry 279, 23052-23060. 694 Subramanian, A. T., Pablo; Mootha, Vamsi K.; Mukherjee, Sayan; Ebert, Benjamin L.; Gillette, 695 Michael A.; Paulovich, Amanda; Pomeroy, Scott L.; Golub, Todd R.; Lander, Eric S.; Mesirov, Jill P. 696 (2005). Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide 697 expression profiles. Proceedings of the National Academy of Sciences. 698 Sun, Z., Huang, Z. and Zhang, D. D. (2009). Phosphorylation of Nrf2 at multiple sites by MAP 699 kinases has a limited contribution in modulating the Nrf2-dependent antioxidant response. PLoS ONE 4, 700 e6588. 701 Suzuki, T., Muramatsu, A., Saito, R., Iso, T., Shibata, T., Kuwata, K., Kawaguchi, S. I., Iwawaki, 702 T., Adachi, S., Suda, H. et al. (2019). Molecular Mechanism of Cellular Oxidative Stress Sensing by Keap1. 703 Cell Rep 28, 746-758 e4. 704 Suzuki, T. and Yamamoto, M. (2015). Molecular basis of the Keap1–Nrf2 system. Free Radical 705 Biology and Medicine 88, 93-100. 706 Tamir, T. Y., Mulvaney, K. M. and Major, M. B. (2016). Dissecting the Keap1/Nrf2 pathway 707 through proteomics. Current Opinion in Toxicology 1, 118-124. 708 Tao, S., Wang, S., Moghaddam, S. J., Ooi, A., Chapman, E., Wong, P. K. and Zhang, D. D. (2014). 709 Oncogenic KRAS Confers Chemoresistance by Upregulating NRF2. Cancer Research 74, 7430-7441. 710 Tong, K. I., Katoh, Y., Kusunoki, H., Itoh, K., Tanaka, T. and Yamamoto, M. (2006). Keap1 711 Recruits Neh2 through Binding to ETGE and DLG Motifs: Characterization of the Two-Site Molecular 712 Recognition Model. Molecular and Cellular Biology 26, 2887-2900. bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

713 Torrente, L., Sanchez, C., Moreno, R., Chowdhry, S., Cabello, P., Isono, K., Koseki, H., Honda, T., 714 Hayes, J. D., Dinkova-Kostova, A. T. et al. (2017). Crosstalk between NRF2 and HIPK2 shapes 715 cytoprotective responses. Oncogene 36, 6204-6212. 716 Travis L. Biechele, R. T. M. (2008). Assaying b-Catenin/TCF Transcription with b-Catenin/TCF 717 Transcription-Based Reporter Constructs. In Methods in Molecular Biology, (ed. E. Vincan). 718 Tsakiri, E. N., Gumeni, S., Iliaki, K. K., Benaki, D., Vougas, K., Sykiotis, G. P., Gorgoulis, V. G., 719 Mikros, E., Scorrano, L. and Trougakos, I. P. (2019). Hyperactivation of Nrf2 increases stress tolerance at 720 the cost of aging acceleration due to metabolic deregulation. Aging Cell 18, e12845. 721 Tsuchida, K., Tsujita, T., Hayashi, M., Ojima, A., Keleku-Lukwete, N., Katsuoka, F., Otsuki, A., 722 Kikuchi, H., Oshima, Y., Suzuki, M. et al. (2017). Halofuginone enhances the chemo-sensitivity of cancer 723 cells by suppressing NRF2 accumulation. Free Radic Biol Med 103, 236-247. 724 Uhlen, M., Fagerberg, L., Hallstrom, B. M., Lindskog, C., Oksvold, P., Mardinoglu, A., 725 Sivertsson, A., Kampf, C., Sjostedt, E., Asplund, A. et al. (2015). Proteomics. Tissue-based map of the 726 human proteome. Science 347, 1260419. 727 Uhlen, M., Oksvold, P., Fagerberg, L., Lundberg, E., Jonasson, K., Forsberg, M., Zwahlen, M., 728 Kampf, C., Wester, K., Hober, S. et al. (2010). Towards a knowledge-based Human Protein Atlas. Nat 729 Biotechnol 28, 1248-50. 730 Vizcaino, J. A., Deutsch, E. W., Wang, R., Csordas, A., Reisinger, F., Rios, D., Dianes, J. A., Sun, 731 Z., Farrah, T., Bandeira, N. et al. (2014). ProteomeXchange provides globally coordinated proteomics 732 data submission and dissemination. Nat Biotechnol 32, 223-6. 733 Wakabayashi, N., Itoh, K., Wakabayashi, J., Motohashi, H., Noda, S., Takahashi, S., Imakado, 734 S., Kotsuji, T., Otsuka, F., Roop, D. R. et al. (2003). Keap1-null mutation leads to postnatal lethality due 735 to constitutive Nrf2 activation. Nat Genet 35, 238-45. 736 Wang, P., Geng, J., Gao, J., Zhao, H., Li, J., Shi, Y., Yang, B., Xiao, C., Linghu, Y., Sun, X. et al. 737 (2019). Macrophage achieves self-protection against oxidative stress-induced ageing through the Mst- 738 Nrf2 axis. Nat Commun 10, 755. 739 Wang, Y., Wan, B., Li, D., Zhou, J., Li, R., Bai, M., Chen, F. and Yu, L. (2012). BRSK2 is regulated 740 by ER stress in protein level and involved in ER stress-induced apoptosis. Biochemical and Biophysical 741 Research Communications 423, 813-818. 742 Wang, Y., Wan, B., Zhou, J., Li, R. and Yu, L. (2013). BRSK2 is a valosin-containing protein (VCP)- 743 interacting protein that affects VCP functioning in endoplasmic reticulum-associated degradation. 744 Biotechnology Letters 35, 1983-1989. 745 Wang, Y. L., Wang, J., Chen, X., Wang, Z. X. and Wu, J. W. (2018). Crystal structure of the kinase 746 and UBA domains of SNRK reveals a distinct UBA binding mode in the AMPK family. Biochem Biophys Res 747 Commun 495, 1-6. 748 Warfel, N. A., Sainz, A. G., Song, J. H. and Kraft, A. S. (2016). PIM Kinase Inhibitors Kill Hypoxic 749 Tumor Cells by Reducing Nrf2 Signaling and Increasing Reactive Oxygen Species. Mol Cancer Ther 15, 750 1637-47. 751 Wu, J. X., Cheng, Y. S., Wang, J., Chen, L., Ding, M. and Wu, J. W. (2015). Structural insight into 752 the mechanism of synergistic autoinhibition of SAD kinases. Nat Commun 6, 8953. 753 Zhang, H., Davies, K. J. A. and Forman, H. J. (2015). Oxidative stress response and Nrf2 signaling 754 in aging. Free Radical Biology and Medicine 88, 314-336. 755 bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

Figure 1

A B HEK293T HIPK4 NRF2 dETGE BRD3 MAP3K8 NRF2 MAP3K8 PRKACA KEAP1 DPP3 NRF2 RIPK2 MOS NRTK2 BRSK2 TRIM27 TYRO3

value) MAP3K7 FASTK MAP2K6 MOS EXOSC10 MAP3K7 MAP2K6 TRIM27 −log10(P BRSK2 EXOSC10 BRD3 HIPK4 MAFG KEAP1 PGUS

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 hcRED −2 0 2 4 -5 -3 -1 1 3 5 7 Fold Activation of NRF2 Reporter (Log2) Fold activation of NRF2 reporter (Log2)

C MEFs D H2228 (Lung) E

NRF2 dETGE NRF2 dETGE HEK293T NRF2 NRF2 FASTK FASTK TRIM27 MAP3K8 MAP3K8 MAP3K7 RIPK2 RIPK2 MOS MOS MOS MAP2K6 NRTK2 NRTK2 HIPK4 MAP3K7 MAP3K7 BRSK2 TYRO3 TYRO3 MEFs BRD3 H2228 PRKACA PRKACA EXOSC10 BRSK2 BRSK2 MAP3K8 EXOSC10 EXOSC10 BRD3 BRD3 HIPK4 HIPK4 MAFG MAFG KEAP1 KEAP1 PGUS PGUS hcRED hcRED -4 -2 0 2 4 6 -4 -2 0 2 4 6 Fold activation of NRF2 reporter (Log2) Fold activation of NRF2 reporter (Log2)

Figure 1. A gain-of-function screen identifies kinases that regulate NRF2-dependent transcription. (A). Volcano plot representation of kinase over-expression screen of NRF2-driven hQR41-firefly luciferase reporter in HEK293T cells. The experiment was performed in biological triplicate and 6 technical replicates. NRF2 transcriptional activity was determined by measuring levels of hQR41-firefly luciferase normalized to TK-Renilla luciferase. Data are plotted relative to GFP over-expression control. Significance was measured with Student T-test including correction for multiple comparison using Benjamini & Hochberg method. Colored circles indicate candidate kinases that passed false discovery rate of less than 10%. NRF2, KEAP1 and DPP3 serve as positive controls. (B-D). hQR41 reporter validation study for the indicated kinase in HEK293T cells, MEFs and H2228 cells, respectively. (E) Venn diagram of validated kinase hits in HEK293T, H2228 and MEFs. bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

Figure 2

A 1.0 E G 0.20 DMSO DMSO hcREDpGUSMAFGKEAP1BRSK2_4BRSK2_3mBrsk2 CDDO-me CDDO-me 0.8 130 kDa - 0.15 100 kDa - NRF2 ill a

ill a 0.6 0.10

250 kDa - 41 /Ren

41 /Ren 0.4 130 kDa - 100 kDa - hQR 0.05 ***

hQR ** ** 75 kDa - BRSK2 0.2 *** 55 kDa - 0.00

0.0 35 kDa -

25 kDa - PGUS MAFG hcRED KEAP1 BRSK2 BRSK1 BRSK2 BRSK1 + mBrsk2 mBrsk1 +mBrsk2 mBrsk1 hcRED PGUS MAFG KEAP1 BRSK2 BRSK2 Iso4 Iso3 mBrsk2 0.3 H B 0.3 DMSO CDDO-me 250 kDa - 0.25 Keap1 +/+ 130 kDa - 100 kDa - Keap1 -/- 0.2 ill a 0.2 * 75 kDa - 0.15 ** ** 55 kDa -

0.1 41 /Ren

HMOX1/GAPDH FLAG 0.05 35 kDa - 25 kDa - hQR 0.1 0 hcRED PGUS MAFG BRSK2 BRSK2 mBrsk2 ** Iso4 Iso3 C 0.35 130 kDa - VCL 0.3 DMSO CDDO-me 1 2 3 4 5 6 7 0.0 0.25 hcRED PGUS MAFG KEAP1 mBrsk2 mBrsk1 0.2 ** * ** 0.15 F I 2.5 4 0.1 hcRED GCLM/GAPDH GFP **** 0.05 NRF2 WT BRSK2 3 **** 0 NRF2 ΔETGE 2.0 BRSK1 hcRED PGUS MAFG BRSK2 BRSK2 mBrsk2 ****

Iso4 Iso3 ill a 2 D **** 0.09 1.5 0.08 DMSO CDDO-me 1 0.07 41 /Ren *** 0.06 **** * 1.0 **** 0.05 ** hQR 0.04 * 0.06 0.03 0.5 0.02 0.04 SLC7A11/GAPDH 0.01 0.02 **

0 0.00 (over hcRED) activation Fold Reporter hcRED PGUS MAFG BRSK2 BRSK2 mBrsk2 0.0 hcRED PGUS MAFG KEAP1 BRSK2 Iso4 Iso3 B AP1 RAR NRF2 WNT NFK STAT ATF3 TGFß NOTCH Figure 2. BRSK2 and BRSK1 inhibit NRF2-mediated transcription. (A) hQR41-luciferase assay in HEK293T cells following expression of control (hcRED, PGUS), positive control (MAFG, KEAP1) or BRSK2 splice variants (BRSK2iso4, BRISK2 iso3) or mouse Brsk2. Cell were treated with vehicle control or CDDO-me (100nM, 12hrs). (B-D) BRSK2 over-expression inhibits endogenous NRF2 target genes (HMOX1, GCLM, and SLC7A11) induction in HEK293T cells (representative of N = 3). (E) Western blot of HEK293T cells 24h after transfection with the indicated expression plasmid. Cells were treated with CDDO-me 12h before lysis. (F) hQR41- luciferase reporter assay in HEK293T cells expressing the indicated plasmid combination for 24h. (G) hQR41-luciferase reporter assay in HEK293T cells expressing the indicated plasmid. Cells were treated with CDDO-me 12h before lysis. (H) hQR41 reporter assay in wild type MEFs and KEAP1 -/- MEFs expressing the indicated plasmids. (I) HEK293T cells were transiently transfected with the indicated firefly transcriptional reporter, Renilla luciferase, hcRED, BRSK2 or BRSK1. Two-tailed Student’s t-test was performed comparing BRSK2 or BRSK1 to hcRED control, and statistical significance was assigned as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

Figure 3

M332 A K48 D141 T174 L309 YFLLL (334 - 338) AIS hBRSK2 Kinase UBA PRR KA1 AA 1- 668 WTK48AD141NT174A E hcREDMAFGBRSK2 ns B C D * NRF2 0.4 DMSO CDDO-me 0.3 DMSO CDDO-me 0.4 DMSO CDDO-me 0.35 SLC7A11 0.25 0.3 ns 0.3

ill a HMOX1 0.2 0.25 Ren 0.2 0.15 0.2 41 / 0.15

0.1 NRF2/GAPDH

HMOX1/GAPDH Flag hQR 0.1 0.1 ** 0.05 0.05

0.0 0 0

WT WT WT K48A MAFG K48A T174A MAFG K48A T174A VCL hcRED PGUS MAFG D141N T174A hcRED D141N hcRED D141N BRSK2 BRSK2 BRSK2 1 2 3 4 5 6 F G H I 0.20 ∆KA1 DMSO CDDO-me DMSO CDDO-me AA 488 - 586 WT K48A∆YFLLLL309DM332K/ ∆AIS hcRED BRSK2 Y334F 0.4 AA 488 - 533 ill a ill a 0.15 ns ∆PRR ns NRF2 AA 340 - 480 ns ∆N AA 22 - 258 HMOX1 41 /Ren ns ∆C 0.10 AA 269 - 586 * hQR hQR41 /Ren 0.2 BLRVB J 0.05 *** *** DMSO CDDO-me * SLC7A11 1.0 ** 0.8 ns ns ns 0.00 0.0 GPX4 0.6 ill a

WT WT WT 0.3 K48A K48A YFLLLM332K *** hcRED PGUS MAFG T174A T189A hcREDPGUS MAFG L309DΔ Y334F 41 /Ren BRSK2 BRSK1 BRSK2 0.2

Flag hQR **

0.1 **

0.0

VCL WT ΔN ΔC ΔKA1 ΔAIS 1 2 3 4 5 6 7 hcRED PGUSMAFG ΔPRR BRSK2

Figure 3. BRSK1 and BRSK2 kinase activity is required for suppression of NRF2- dependent transcription. (A) Cartoon schematic of BRSK2 protein showing residues and domains important for kinase activity. (B) hQR41 reporter assay in HEK293T cells expressing the indicated construct; cells were treated with vehicle or CDDO-me for 12h prior to luciferase quantitation. (C,D). Quantitative RT-PCR for HMOX1 and NRF2 in HEK293T cells following over-expression of the indicated genes (representative of N = 3). (E) Western blot analysis of HEK293T cells expressing the indicated plasmids. Cells were treated with CDDO-me for 4h before lysis. ns = nonspecific. (F) hQR41 luciferase assay in HEK293T cells expressing the indicated proteins. (G) Western blot analysis of HEK293T cells expressing the indicated plasmids. Cells were treated with CDDO-me for 4h before lysis. (H) Cartoon schematic of BRSK2 deletions. (I,J) hQR41 reporter assay in HEK293T cells expressing the indicated construct; cells were treated with vehicle or CDDO-me for 12h prior to luciferase quantitation. Two-tailed Student’s t-test was performed comparing corresponding hcRED control with treatment groups and statistical significance was assigned: * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not statistically significant. bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

Figure 4

A B C Veh. BafA1 CDDO-me Veh. MG132 Bort. MLN4924CDDO-me hcRED Veh. Sulf. tBHQ hcRED BRSK2 hcRED BRSK2 ns* BRSK2 ns* ns* NRF2 ns* NRF2 NRF2 ns* LC3B

Flag Flag Flag

GAPDH GAPDH GAPDH 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6

150 150 150 hcRED BRSK2 hcRED BRSK2 hcRED BRSK2

100 100 100 * ** /G APDH ** ** * *** *** 50 % NR F2/GAPDH % N RF2

% NR F2/GAPDH 50 50 *** ***

0 0 0 Vehicle Sulf. tBHQ Vehicle MG132 MLN4924 Bort. CDDO-me Vehicle BafA1 CDDO-me

Figure 4. BRSK2-mediated NRF2 downregulation is independent of the NRF2 ubiquitylation and degradation. (A) HEK293T cells were transfected with control hcRED or BRSK2 for 24h before treatment with vehicle (veh) or Sulforaphane (Sulf., 2µM) or tert- Butylhydroquinone (tBHQ, 50µM) for 4h. Quantitation of biological triplicate experiments is shown in the lower panel, normalized to GAPDH. (B, C) HEK293T cells were transfected with control hcRED or BRSK2 for 24h before treatment with MG132 [10µM], Bortezomab (Bort., [40nM]), MLN4924 [2.5µM], Bafilomycin A1 (BafA1, [200nM]). All treatments were for 4h, except for BafA1 which was 12h. Two-tailed Student’s t-test was performed comparing hcRED with BRSK2, and statistical significance was assigned as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license. Figure 5

A BRSK1 B BRSK2 D BRSK1 E BRSK2

PERT−P100−DIA_BMS−345541 PERT−P100−PRM_DMSO KINASE−PSP_Akt2/AKT2 PERT−PSP_IONIZING_RADIATION C1orf95 IGSF10 PERT−P100−PRM_VORINOSTAT PATH−WP_Cell_Cycle CTC−479C5.12 PERT−P100−DIA_DEXAMETHASONE PERT−P100−PRM_BMS−345541 PERT−P100−DIA_STAUROSPORINE PERT−P100−PRM_LENALIDOMIDE PERT−PSP_UV PERT−P100−PRM_VORINOSTAT AC007040.11 * KINASE−PSP_Chk1/CHEK1 PERT−PSP_ADRIAMYCIN PERT−P100−DIA_CC−401 PERT−P100−DIA_SEMAGACESTAT PATH−NP_TSLP_PATHWAY KINASE−PSP_PKCD/PRKCD KINASE−PSP_PRKD1 KINASE−PSP_ATM * PERT−P100−PRM_NILOTINIB PERT−P100−DIA_BMS−345541 * PERT−P100−DIA_CHIR99021 PERT−P100−DIA_CHIR99021 PERT−PSP_ADRIAMYCIN * KINASE−PSP_Akt2/AKT2 RENBP PERT−P100−DIA_LAMOTRIGINE PERT−P100−DIA_CC−401 C19orf38 PERT−P100−PRM_LENALIDOMIDE KINASE−PSP_PKACA/PRKACA C2orf48 PERT−P100−PRM_DACTOLISIB PERT−PSP_ANTI_CD3 SH2D3C PERT−P100−PRM_DMSO KINASE−PSP_JNK1/MAPK8 PERT−PSP_SII_ANGIOTENSIN_2 KINASE−PSP_RSK2/RPS6KA3 DHRS2 KINASE−PSP_PLK1 * PERT−PSP_SII_ANGIOTENSIN_2 LOXL4 HIST1H1E PERT−P100−DIA_SEMAGACESTAT * * PERT−PSP_UV NHLH1 PERT−P100−PRM_BMS−345541 * * KINASE−PSP_MAPKAPK2 ALPK3 PERT−P100−DIA_VORINOSTAT * * KINASE−PSP_PRKD1 RELB PERT−P100−DIA_CPI−169 * * PATH−NP_TSLP_PATHWAY CHST8 PERT−P100−PRM_STAUROSPORINE * PERT−P100−DIA_STAUROSPORINE RP11−363E7.4 UNC5A KINASE−PSP_AurB/AURKB * KINASE−PSP_ATR ZC3H12A RP11−61K9.3 PATH−WP_Cell_Cycle PERT−PSP_EGF ADGRG2 DUSP8 KB−1460A1.5 KINASE−PSP_ATM * KINASE−PSP_p90RSK/RPS6KA1 RP11−254F7.2 LINC01125 PERT−P100−DIA_C646_(CHEMBL1797936) * PATH−NP_EGFR1_PATHWAY AMOTL2 PERT−P100−DIA_GELDANAMYCIN KINASE−PSP_Akt1/AKT1 C19orf73 C5 FAAH ZC3H12A KINASE−PSP_ATR * * KINASE−PSP_AMPKA1/PRKAA1 C19orf38 DUSP8 PERT−P100−DIA_DMSO * * * KINASE−PSP_Chk1/CHEK1 EBF4 PERT−PSP_HYDROXYUREA * * * KINASE−PSP_PKCA/PRKCA LINC01125 PIM1 HSPA6 PERT−P100−PRM_VORINOSTAT_HMS * * * KINASE−PSP_CDK1 SOCS3 PERT−PSP_IONIZING_RADIATION * * * KINASE−PSP_CDK2 ALPK3 ARC KINASE−PSP_Src/SRC * * KINASE−PSP_mTOR/MTOR EEF1A1P13 PERT−PSP_ANTI_CD3 * * * PERT−P100−PRM_PS−1145 BAALC−AS1 KINASE−PSP_SGK1 PATH−NP_GLP1_PATHWAY PERT−P100−DIA_LY−294002 DISEASE−PSP_Alzheimer PTK2B PERT−P100−PRM_IPI145 KINASE−PSP_PAK1 EFEMP2 KINASE−PSP_P38A/MAPK14 PERT−PSP_SERUM HIST1H1E DUSP1 KINASE−PSP_PKCD/PRKCD KINASE−PSP_SGK1 CDNF PERT−P100−PRM_PAZOPANIB PATH−NP_PROLACTIN_PATHWAY BRSK1 WNT9A KINASE−PSP_AMPKA1/PRKAA1 PERT−P100−DIA_LAMOTRIGINE TXNIP BRSK2 * * KINASE−PSP_MAPKAPK2 KINASE−PSP_P38A/MAPK14 C5 RNF165 * KINASE−PSP_Akt1/AKT1 PERT−P100−DIA_C646_(CHEMBL1797936) Control (n=4) BRSK1 (n=3) Control (n=4) BRSK2 (n=3) * * KINASE−PSP_p90RSK/RPS6KA1 KINASE−PSP_P38D/MAPK13 * KINASE−PSP_JNK1/MAPK8 * PERT−PSP_INSULIN KINASE−PSP_PKCA/PRKCA KINASE−PSP_GSK3B PERT−P100−PRM_PS−1145 KINASE−PSP_DYRK1A Abs(Log FC) > 1 PATH−NP_EGFR1_PATHWAY KINASE−PSP_ERK2/MAPK1 Relative 2 KINASE−PSP_RSK2/RPS6KA3 * KINASE−PSP_ERK1/MAPK3 −1.5 −1 −0.5 0 0.5 1 1.5 * PATH−NP_PROLACTIN_PATHWAY * KINASE−PSP_CDK5 Expression MTOR Signature PATH−NP_GLP1_PATHWAY * KINASE−PSP_CK2A1/CSNK2A1 KINASE−PSP_DYRK1A * * PERT−P100−DIA_DEXAMETHASONE (Log2) MYC Signature KINASE−PSP_PKACA/PRKACA PERT−P100−DIA_CPI−169 * KINASE−PSP_GSK3B PERT−P100−PRM_DACTOLISIB E2F Targets * PERT−PSP_EGF PERT−P100−DIA_LY−294002 TNFA Signature * PERT−PSP_SERUM PERT−P100−PRM_NILOTINIB * KINASE−PSP_PAK1 PERT−P100−DIA_VORINOSTAT * DISEASE−PSP_Alzheimer PERT−P100−DIA_GELDANAMYCIN * PERT−P100−PRM_LOSMAPIMOD KINASE−PSP_AurB/AURKB KINASE−PSP_CDC7 KINASE−PSP_Src/SRC * KINASE−PSP_CDK7 PERT−PSP_HYDROXYUREA * KINASE−PSP_CK2A1/CSNK2A1 PERT−P100−PRM_VORINOSTAT_HMS * PERT−P100−DIA_MS−275 PERT−PSP_PHORBOL_ESTER KINASE−PSP_P38D/MAPK13 KINASE−PSP_PLK1 C MTORC1 Signaling (BRSK1) MTORC1 Signaling (BRSK2) PERT−P100−DIA_TRAMETINIB PERT−P100−PRM_PAZOPANIB ● ● ● ● ● ● ● ● ● ● ● ● 0.1 ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● KINASE−PSP_HIPK2 PERT−P100−PRM_STAUROSPORINE ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● 0.0 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● PERT−P100−DIA_UNC−0321 PERT−P100−DIA_UNC−0321 ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● 0.0 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● PERT−P100−DIA_MS−275 ● ● ● ● KINASE−PSP_CDK1 ● ● ● ● ● ● ● ●● ● −0.1 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● PERT−P100−DIA_DMSO ● ● ● ● ● KINASE−PSP_CDK2 ● −0.1 ● ● * * ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● PERT−P100−PRM_IPI145 ● ● ●● KINASE−PSP_mTOR/MTOR ● * ● * ●● ●● ● −0.2 ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● −0.2 ● ● ● ● ● ● KINASE−PSP_DNAPK/PRKDC KINASE−PSP_CDC7 ● ● * * ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● PERT−PSP_PHORBOL_ESTER KINASE−PSP_CDK7 ● ● ● ● ● ● ●● * * * * ● ● ●● ● ● ● ● ● ● −0.3 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● −0.3 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● PERT−PSP_NOCODAZOLE PERT−P100−PRM_LOSMAPIMOD ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● * ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● −0.4 −0.4 ● PERT−PSP_INSULIN * KINASE−PSP_HIPK2 0 2500 5000 7500 10000 Enrichment Score KINASE−PSP_ERK2/MAPK1 KINASE−PSP_DNAPK/PRKDC Enrichment Score 0 2500 5000 7500 10000 * * 8 * KINASE−PSP_CDK5 PERT−P100−DIA_TRAMETINIB 8 4 4 0 * KINASE−PSP_ERK1/MAPK3 PERT−PSP_NOCODAZOLE 0 S2N −4 S2N −4 −8 * * −8 0 2500 5000 7500 10000 13 2 1 2 3 0 2500 5000 7500 10000 Replicate Replicate rank rank −4 −2 Log2(NES) −4 −2 Log2(NES) 0 2 4 0 2 4

Figure 5. Genomics and proteomics reveals activation of AMPK and suppression of MTOR following BRSK1/2 expression. (A, B) Hierarchical clustering of RNA sequencing analysis of HEK293T cells transfected with mock, hcRED control or BRSK2 or BRSK1 for 24h. (C) Differentially expressed genes with FDR < 5% were analyzed by Gene Set Enrichment Analysis (GSEA) using the Hallmark and Oncogenic gene sets from MSigDB. Enrichment Score (ES) plots represent genes commonly associated with MTORC1 signaling were decreased by BRSK2/1 overexpression based on GSEA. (D, E) Quantitative TMT phosphoproteomic analysis of HEK293T cells expressing hcRED, BRSK1, or BRKS2 for 24h. TMT ratios from biological triplicate samples were analyzed for enrichment of phosphosites associated with known signaling pathways using the PTMSigDB pipeline (* FDR < 10%). bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license. Figure 6

A B C 200

ns 150 Δ KA1 Δ AIS WT K48A D141N MW hcRED BRSK2 WT BRSK2 K48A BRSK1 WT hcRED BRSK2 (kDa) pS6K 250- T389 100 * 130- ** S6K 100- 50 % Protein Synthesis 75- pS/T AMPK p4EBP1 T37/46 55- 0 p4EBP1 S65 35- CHX hcRED BRSK2WT BRSK2 BRSK1 4EBP1 K48A

D hcRED BRSK2 WT 0.95 BRSK2 K48A BRSK1 FLAG FLAG

0.75 Absorbance 0.55 GAPDH GAPDH

1 2 3 4 5 6 1 2 3 4 0.35 10% 50% Sedimentation

Figure 6. BRSK2 suppresses protein translation. (A) Western blot analysis AMPK consensus phosphorylation sites in HEK293T cells transfected with hcRED, BRSK2, or the indicated BRSK2 mutants (representative of N = 3). (B) Western blot analysis of BRSK1 and BRSK2 expression in HEK293T cells for MTOR targets S6K (T389) and 4EBP1 (T37/46 and S65) which increases 4EBP1 stability (representative of N = 3). (C) Global protein translation assay performed using 35S-Metionine labeling in cells expressing hcRed (control), BRSK2, BRSK2 K48A, or BRSK1. Two-tailed Student’s t-test was performed comparing hcRED to other conditions, and statistical significance was assigned as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. (D) Polyribosome fraction of HEK293T cells overexpressing hcRED control, BRSK2, BRSK2 K48A, or BRSK1 performed on 50% - 10% sucrose gradient. Experiments represent biological triplicates, unless indicated otherwise. bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

Supplemental Figure S1

A B 1.0 sgNT sgBRSK2 A A B B C C 0.8

sgRNA p < 0.0034 Control A Control B BRSK2 A BRSK2 B BRSK2 C BRSK2 D BRSK2 E Control C Control D ill a 0.6

NRF2 Ren 41 / BRSK2 0.4 hQR

GAPDH 0.2

0.0 GFP BRSK2 C D PDAC cell lines MIA PaCa-2 DMSO CDDO-me sgNT sgB2 sgNT sgB2 PANC1MIA BXPC3PaCa2ASPC1CAPAN1CAPAN2CFPAC1HPAFIISW1990 ABABC ABABC ◄ BRSK2

DE

NRF2 BRSK2 ◄

LE KEAP1

GAPDH GAPDH 1 2 3 4 5 6 7 8 9 10

Supplemental Figure S1. BRSK2 CRISPRi silencing does not alter NRF2 protein levels. (A) HEK293T cells stably expressing KRAB-dCas9 and 4 control or 5 BRSK2 sgRNAs. BRSK2 knockdown does not alter NRF2 protein levels. (B) hQR41 reporter assay in CRISPRi silenced cells with GFP or BRSK2 over-expression shows that BRSK2 knockdown does not affect NRF2 activity. (C) BRSK2 expression levels in pancreatic ductal adenocarcinoma (PDAC) cell lines. (D) CRISPRi silencing of BRSK2 in MIA PaCa2 cells treated with DMSO or CDDO-me for 4hrs. Neither NRF2 nor KEAP1 levels were altered by loss of BRSK2 protein. bioRxiv preprint doi: https://doi.org/10.1101/832279; this version posted November 5, 2019. 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-ND 4.0 International license.

Supplemental Figure S2 NRF2 Signaling

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Supplemental Figure S2. BRSK2 overexpression decreased NRF2 target gene signature.