Article IKKa Kinase Regulates the DNA Damage Response and Drives Chemo-resistance in Cancer Graphical Abstract Authors Carlota Colomer, Pol Margalef, Alberto Villanueva, ..., Anna Bigas, Simon J. Boulton, Lluı́s Espinosa Vemurafenib Correspondence BRAF AZ628 BRAF TAK1 TAK1 Damage Damage abigas@imim.es (A.B.), p38 MAPK p38 MAPK simon.boulton@crick.ac.uk (S.J.B.), lespinosa@imim.es (L.E.) IKKα(p45) IKKα(p45) Cytoplasm Cytoplasm In Brief IKKα(p45) IKKα(p45) P P Colomer et al. discover that IKKa kinase P P ATM ATM contributes to the chemo- and radio- DSB P DSB P H2A.X KAP1 H H2A.X KAP1 H 53BP1 53 5 3 53BP1 5P 3 resistance of cancer cells by facilitating P RIF1 RIF1 Nucleus Nucleus ATM activation and DNA repair. BRAF DNA damage DNA damage repair and survival repair and survival inhibitors prevent damage-induced IKKa activation, leading to the attenuation of Therapy resistant Therapy sensitive ATM signaling and DNA repair. IKKa depletion or BRAF inhibitors combined with 5-FU and irinotecan synergistically enhance the killing of patient-derived xenograft tumors. Highlights d IKKa kinase is activated by BRAF-TAK1-p38-MAPK in response to DNA damage d Loss of IKKa or BRAF attenuates ATM signaling and compromises DNA repair d Loss of IKKa or BRAF in combination with DNA damage potentiates tumor eradication d Combination treatment of patient-derived tumors prolongs survival in mice Colomer et al., 2019, Molecular Cell 75, 669–682 August 22, 2019 ª 2019 The Author(s). Published by Elsevier Inc. https://doi.org/10.1016/j.molcel.2019.05.036 Molecular Cell Article IKKa Kinase Regulates the DNA Damage Response and Drives Chemo-resistance in Cancer Carlota Colomer,1 Pol Margalef,1,2 Alberto Villanueva,3 Anna Vert,1 Irene Pecharroman,1 Laura Solé,1 Mónica González-Farré,4 Josune Alonso,1 Clara Montagut,5 Maria Martinez-Iniesta,3 Joan Bertran,6 Eva Borràs,7,8 Mar Iglesias,4 Eduard Sabidó,7,8 Anna Bigas,1,* Simon J. Boulton,2,* and Lluı́s Espinosa1,9,* 1Cancer Research Program, Institut Mar d’Investigacions Mèdiques, CIBERONC, Hospital del Mar, Doctor Aiguader 88, Barcelona 08003, Spain 2DSB Repair Metabolism Laboratory, The Francis Crick Institute, London NW1 1AT, UK 3Translational Research Laboratory, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), Institut Català d’Oncologia, Hospitalet, Barcelona 08907, Spain 4Department of Pathology, Institut Mar d’Investigacions Mèdiques, CIBERONC, Universitat Autònoma de Barcelona, Barcelona 08003, Spain 5Department of Oncology, Institut Mar d’Investigacions Mèdiques, Universitat Pompeu Fabra, CIBERONC, Barcelona 08003, Spain 6Faculty of Science and Technology, Bioinformatics and Medical Statistics Group, University of Vic-Central University of Catalonia, Vic 08500, Spain 7Proteomics Unit, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona 08003, Spain 8Proteomics Unit, Universitat Pompeu Fabra, Barcelona 08003, Spain 9Lead Contact *Correspondence: abigas@imim.es (A.B.), simon.boulton@crick.ac.uk (S.J.B.), lespinosa@imim.es (L.E.) https://doi.org/10.1016/j.molcel.2019.05.036 SUMMARY cell-cycle stage, ATM coordinates the recruitment of 53BP1 or BRCA1 to damaged DNA, which determines DSB repair pathway Phosphorylated IKKa(p45) is a nuclear active form of choice and whether the lesion is repaired by non-homologous end the IKKa kinase that is induced by the MAP kinases joining (NHEJ) or homologous recombination (HR). In G1, 53BP1, BRAF and TAK1 and promotes tumor growth inde- RIF1, and the Sheldin co-factors oppose DSB resection, thereby pendent of canonical NF-kB signaling. Insights into favoring NHEJ and opposing HR (Panier and Boulton, 2014). the sources of IKKa(p45) activation and its down- Conversely, in S-G2, when an intact sister chromatid is available as a template following the completion of S phase, BRCA1 antag- stream substrates in the nucleus remain to be onizes 53BP1 and co-factors, thereby promoting a switch in DSB defined. Here, we discover that IKKa(p45) is rapidly repair pathway to favor HR (Bothmer et al., 2010, 2011; Chapman activated by DNA damage independent of ATM- et al., 2012a, 2012b; Escribano-Dı́az et al., 2013; Zimmermann ATR, but dependent on BRAF-TAK1-p38-MAPK, et al., 2013). In non-proliferating cells (cells in G0-G1 phases), and is required for robust ATM activation and effi- classical NHEJ is the preferred option for DSB repair since there cient DNA repair. Abolishing BRAF or IKKa activity is no homology donor for HR. From yeast to mammals, multiple attenuates ATM, Chk1, MDC1, Kap1, and 53BP1 genotoxic agents such as UV, ionizing radiation (IR), reactive oxy- phosphorylation, compromises 53BP1 and RIF1 co- gen species, and 5-fluorouracil (5-FU) also induce the protein ki- recruitment to sites of DNA lesions, and inhibits nase p38, which plays an essential role in the regulation of cellular 53BP1-dependent fusion of dysfunctional telomeres. checkpoints (Alao and Sunnerhagen, 2008; Bulavin et al., 2001; Furthermore, IKKa or BRAF inhibition synergistically Preta et al., 2010; Rouse et al., 1994). Recently, it was demon- strated that p38a modulates the ATR pathway through direct enhances the therapeutic potential of 5-FU and irino- phosphorylation of CtIP, which promotes therapy resistance in tecan to eradicate chemotherapy-resistant metasta- cancer cells (Canovas et al., 2018). tic human tumors in vivo. Our results implicate BRAF Contemporary treatments for most solid cancers involve sur- and IKKa kinases in the DDR and reveal a combina- gery, radiotherapy, and combinations of chemotherapies, such tion strategy for cancer treatment. as 5-FU, oxaliplatin, and irinotecan (Iri), which eradicate tumors by inducing DSBs in highly proliferative cells (reviewed in Brenner et al., 2014). However, it is well established that tumors also INTRODUCTION contain low proliferating and quiescent cells that are therapy resis- tant and contribute to tumor relapse and metastasis (Batlle and The DNA damage response (DDR) maintains genome stability by Clevers, 2017). Beyond non-specific DNA-damaging agents, anti- coordinating the cell cycle, DNA repair, and apoptosis in response bodies targeting epidermal growth factor receptor (EGFR) consti- to DNA lesions. Following DNA double-strand breaks (DSBs), the tute a second therapeutic option for cancer treatment, with muta- DDR is activated by ATM-dependent phosphorylation of tions in KRAS and BRAF (two essential elements of the EGFR numerous targets, including the effector kinase Chk2 and the his- signaling pathway) that are predictive of treatment failure (Amado tone H2A.X (called gH2A.X when phosphorylated). Dependent on et al., 2008; Di Nicolantonio et al., 2008) and poor prognosis Molecular Cell 75, 669–682, August 22, 2019 ª 2019 The Author(s). Published by Elsevier Inc. 669 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). (Richman et al., 2009). Based on the structural characteristics of IKKa, we sought to identify its kinase substrates through quanti- mutated RAS, the possibility of developing small molecules that tative phospho-proteomic analysis of control and IKKa-knock- revert its activation remains a significant challenge (Vetter and down HT29 cells. Unexpectedly, this approach identified several Wittinghofer, 2001). Thus, there is a growing interest in developing DDR pathway components, including 53BP1 and KAP1 BRAF inhibitors and other inhibitors of the mitogen-activated pro- (also known as TRIM28 or transcription intermediary factor 1b tein kinase (MAPK) pathway that are being used for treating [TIF-1b]), as proteins whose phosphorylation depends on IKKa BRAF-mutated metastatic melanoma (Hu-Lieskovan et al., (see experimental strategy in Figure S1A and the results in 2015; Ribas et al., 2014). However, recent BRAF inhibitor trials Table S1). This observation raised the possibility that IKKa may in cancer patients carrying BRAF-mutated tumors have produced function at some level in the DDR. largely negative results (Hong et al., 2016). To examine this possibility, we exposed HT29 cells to UV light The nuclear factor kB (NF-kB) signaling pathway regulates and performed western blot analysis at different time points. UV innate and acquired immunity and is essential for most physiolog- treatment rapidly induced the phosphorylation of nuclear ical processes but also for cancer progression (reviewed in Zhang IKKa(p45) on Ser180 (referred to as p-IKKa(p45)), which pre- et al., 2017). Multiple extracellular stimuli, including the inflamma- ceded the phosphorylation of canonical DDR proteins, including tory cytokines tumor necrosis factor a (TNF-a) and interleukin 1b Chk1, KAP1, and H2A.X (Figure 1A). Different UV doses compa- (IL-1b), induce NF-kB through a series of signaling events that rably increased p-IKKa(p45) levels in the nucleus (Figure S1B), lead to the phosphorylation and activation of a kinase complex and this occurred in a range of different cancer cells independent that consists of IKKa, IKKb, and IKKg/NEMO. The IKKa subunit of the mutational status of KRAS or BRAF (Figures 1B and 1C). is dispensable for NF-kB activation, but it has been found to exert To determine whether the induction of nuclear p-IKKa(p45) ex- multiple pro-tumorigenic functions. Several studies have demon- tends beyond UV to other DNA-damaging agents, we also exam- strated that IKKa enhances the metastatic activity of prostate tu- ined p-IKKa(p45) in response to IR and the topoisomerase II in- mors (Luo et al., 2007) and squamous cell carcinomas (Toll et al., hibitor, etoposide. p-IKKa(p45) was rapidly induced in response 2015) by regulating the Maspin gene. Epithelial IKKa is required for to all of the tested DNA-damaging agents, although the largest the initiation and progression of intestinal adenomas (Colomer effect was produced by UV treatment (Figure 1D). Since p-IK- et al., 2018) and lung adenocarcinomas (Vreka et al., 2018) in Ka(p45) is the nuclear form of activated IKKa, we asked whether mice. Recently, we identified a nuclear active form of the IKKa ki- it is also recruited to sites of DNA damage. Immunofluorescence nase, IKKa(p45), which is localized in the nuclear compartment of analysis revealed that p-IKKa(p45) co-localized with 53BP1 in cancer cells. IKKa(p45) induces the phosphorylation of histone H3 laser-induced stripes (Figure 1E). These results reveal that p-IK- and nuclear co-repressors, which is dependent on its interaction Ka(p45) is rapidly induced by and accumulates at sites of DNA with non-activated full-length IKKa and promotes tumor growth damage, which raised the possibility that p-IKK (p45) may independent of canonical NF-kB signaling (Margalef et al., contribute in some way to the regulation of the DDR pathway. 2012). The phosphorylation and activation of IKKa(p45) require the MAP kinases BRAF and TAK1 and take place in proximity to p38-MAPK Activates IKKa in Response to DNA Damage the endosomal compartment (Margalef et al., 2012, 2015). The ki- The activation of ATM or ATR is an early event in the cellular nase p38a has also been linked to TAK1 (MAP3K7) and IKK acti- response to DNA damage, both kinases being essential for initi- vation in several situations, including the DDR (Chen et al., 2015; ating the DDR signaling cascade in response to different DNA Hindi et al., 2018; Yang et al., 2011). In fact, exposure to chemo- lesions. To determine whether ATM or ATR contributes to the in- therapeutic agents leads to the activation of canonical NF-kB, duction of nuclear p-IKKa(p45) in response to DNA damage, we downstream of ATM and NEMO (Wu et al., 2006). treated UV-irradiated cells with selective ATM or ATR inhibitors Here, we make the unexpected discovery that the activity of and blotted for IKKa(p45) Ser180 phosphorylation. While ATM BRAF and IKKa kinases is important for a proper DDR and for effi- and ATR inhibitors effectively abolished the phosphorylation of cient DNA repair. In response to DNA damage, IKKa(p45) is rapidly their downstream effector kinases Chk2 (pT68) and Chk1 activated by phosphorylation on Ser180, it translocates to the nu- (pS345; Figure 2A), this had no impact on the damage-induced cleus, and it co-localizes with 53BP1 at sites of damage. BRAF in- Ser180 phosphorylation of IKKa(p45). We also considered the hibition or loss of IKKa(p45) attenuates ATM activation and down- possibility that the DNA-dependent protein kinase, catalytic stream checkpoint signaling, reduces the phosphorylation of key subunit (DNA-PKcs) may act redundantly to phosphorylate DDR factors, and compromises DNA repair, including 53BP1- IKKa(p45) in response to damage. However, the combined inhi- dependent end joining. Finally, we show that BRAF inhibition or IK- bition of ATM and DNA-PKc also had no effect on the induction Ka(p45) depletion synergizes with DNA-damaging chemothera- of p-IKKa(p45) following damage (Figure S2A). These data, peutic agents to induce tumor eradication in mice, thus revealing coupled with the fact that p-IKKa(p45) induction precedes a potential therapeutic strategy for cancer treatment. many other early damage markers, including gH2A.X and Chk1 (Figure 1A), suggested that IKKa(p45) phosphorylation either RESULTS occurs upstream of or parallel to ATM-ATR activation. Since IKKa is known to be activated by BRAF, TAK1, and IKKa(p45) Is Rapidly Activated in Response to DNA p38-MAPK, we explored whether these kinases may also promote Damage p-IKKa(p45) activation following UV treatment. Selective inhibitors IKKa(p45) is a largely uncharacterized kinase with no known sub- of TAK1, BRAF, or p38-MAPK (but not MEKi) effectively abolished strates. In an attempt to identify processes that are regulated by IKKa(p45) Ser180 phosphorylation in response to UV (Figure 2B). 670 Molecular Cell 75, 669–682, August 22, 2019 A B Time Post-UV (min.) 0 20 LIM 9 (B K) LIM 9 (B K) ) ) Time post-UV 0 5 15 30 60 180 Wi 80 (K Wi 80 (K ( ( DL R (B) HC 1 (K) (B) HC 1 (K) 1 ) 1 ) SW 215 SW 215 HT 116 HT 116 (min.) 85 kD DR 4 4 T D T D 2 D 2 p-IKK S180 DL 45 kD 85 kD 85 kD p-IKK S180 IKKα 45 kD 45 kD 85 kD p-Chk1 S345 IKKα Chk1 45 kD p-KAP1 S824 p-ERK KAP1 ERK γH2A.X γH2A.X Histone H3 Histone H2A Tubulin Tubulin D C HT29 WiDr Post-treatment γIR UV Etoposide γIR UV Etoposide Time Post-UV (min.) 0 30 (min.) 0 30 60 30 60 60 180 0 30 60 30 60 60 180 85 kD P1 1 P1 1 RW EL13 RW EL13 T2 7 T2 7 MC 9 p-IKK S180 29 F F M M 2 4 4 MC HT HT SK SK 45 kD 85 kD p-IKK S180 85 kD 45 kD IKKα 85 kD IKKα 45 kD 45 kD γH2A.X γH2A.X Tubulin Tubulin E p-IKK S180 53BP1 Merged 10 μm Figure 1. Nuclear IKKa Is Activated by Different DNA-Damaging Agents (A) Western blot (WB) analysis of HT29 exposed to UV light (130 mJ) and collected at the indicated time points. (B) WB analysis of various CRC cells left untreated or collected 20 min after UV (130 mJ) exposure. K and B indicate the presence of mutations in KRAS (K) or BRAF (B) in the different cell lines. (C) WB analysis of different colorectal (HT29), breast (MCF7), bladder (T24), melanoma (SKMEL 131), and pancreatic (RWP1) cancer cell lines collected 30 min after UV exposure (130 mJ). (D) WB analysis of HT29 and WiDr cells treated with the indicated DNA-damaging agents and collected at different time points. (E) Immunofluorescence analysis of HT29 cells to determine the co-localization of 53BP1 and p-IKK in laser-induced stripes. See also Figure S1 and Table S1. Of these kinases, only p38-MAPK and IKKa(p45) were observed to TAK1 and BRAF are required to prime p38-MAPK for subsequent be significantly induced in response to UV treatment (Figure 2B). damage-induced activation (Figure 2B). Nevertheless, TAK1 and BRAF inhibitors prevented the activation To add further support to these findings and to exclude poten- of p38-MAPK in response to UV, indicating that the basal activity of tial off-target effects of the p38-MAPK inhibitors, we examined Molecular Cell 75, 669–682, August 22, 2019 671 A C Time Post-UV p38α MEFs Time Post-UV (min.) 0 20 40 60 (min) - 30 ATMi - + - - + - - + - - + - - - + - - + - - + - - + KO KO T T ATRi W W 85 kD p-IKK S180 45 kD p-IKK s180 85 kD IKKα 45 kD IKKα p-Chk1 S345 p38α-MAPK p-Chk2 T68 Tubulin Tubulin B NO UV UV + 30’ E DLD1 HCT116 HT29 Bafilomycin - - - - - + - - - - - + MEKi - - - - + - - - - - + - UV - - + + - - + + - - + + BRAFi - + - + - + - + - + - + TAK1i - - - + - - - - - + - - BRAFi - - + - - - - - + - - - 85 kD p38i - + - - - - - + - - - - p-IKK S180 85 kD 45 kD p-IKK S180 45 kD 85 kD IKKα 85 kD 45 kD IKKα γH2A.X 45 kD Tubulin p-p38 MAPK p38 MAPK F p-RAF S445 Time Post-UV (min) 0 15 30 BRAF C N Chr C N Chr C N Chr 85/87 kDa p-TAK1 S412 p-IKK S180 TAK1 45 kDa p-ERK 85 kDa p-HSP27 IKKα 45 kDa p-SMAD2/3 p-p38 ERK Tubulin p38 D p-TAK1 Time Post-UV (min) 0 5 15 30 60 180 TAK1 p38i - + - + - + - + - + - + p-RAF p-IKK S180 45 kD BRAF p-p38α-MAPK NEMO p-HSP27 85 kD IKKβ IKKα 45 kD Tubulin p38α-MAPK Lamin B Tubulin (legend on next page) 672 Molecular Cell 75, 669–682, August 22, 2019 IKKa(p45) phosphorylation in UV-treated p38a-MAPK knockout rescued by lentiviral transduction of mCherry-IKKa in the IKKa- cells (Adams et al., 2000). In agreement with our inhibitor exper- deficient cells (Figure 3C). iments, p38a-MAPK knockout cells failed to induce IKKa(p45) Since BRAF is required for IKKa(p45) activation following DNA phosphorylation in response to UV (Figure 2C). p38a-MAPK inhi- damage, we assessed the impact of BRAF inhibitors on ATM bition also prevented IKKa(p45) Ser180 phosphorylation at mul- activation. In agreement with our previous data, inhibition of tiple time points following UV treatment (Figure 2D). Moreover, BRAF significantly reduced ATM activation, as measured by BRAF inhibition was found to abrogate p-IKKa(p45) induction in- the induction of ATM Ser1981 auto-phosphorylation, and also dependent of BRAF mutational status (Figure 2E), irrespective of compromised downstream phosphorylation of DDR markers in DNA-damaging stimulus (Figure S2B), and at a range of different UV- (Figure 3D) and IR-treated (Figure S3E) HT29 cells. Similar time points (Figure S2C). Comparable effects on p-IKKa(p45) in- results were also observed in breast, pancreatic, and melanoma duction were also observed using two different BRAF inhibitors, cancer cell lines (Figure S3F). vemurafenib and sorafenib (Figures S2D and S2E), which are Since the MRE11, RAD50, and NBS1 (MRN) complex is used in clinical practice. These results establish that IKKa(p45) responsible for the recruitment of ATM to sites of DNA damage, is rapidly activated in response to DNA-damaging agents down- we investigated whether IKKa deficiency or BRAF inhibition may stream of TAK1, BRAF, and p38-MAPK. Notably, we did not affect MRN complex stability. Western blot analysis revealed detect evidence of nuclear TAK1, BRAF, or p38-MAPK following that the protein levels of the MRN complex were unaffected UV treatment (Figure 2F), suggesting that IKKa is principally acti- both in IKKa-deficient cells (Figure S3G) and after BRAF inhibi- vated in the cytoplasm, translocates to the nucleus, and accu- tion (Figure S3H). We also investigated the impact of IKKa mulates on chromatin. knockdown in Rad50s/s mutant cells, which exhibit a hypermor- phic signaling phenotype that suppresses ATM deficiency by IKKa and BRAF Facilitate ATM Activation and activating other phosphatidylinositol 3-kinase (PI3K)-like kinases Downstream DDR Signaling (Morales et al., 2005). In contrast to wild-type cells, knocking In light of our findings that IKKa(p45) is rapidly induced by DNA down IKKa in Rad50s/s mutant cells did not prevent KAP1 and damage, we consider the possibility that p-IKKa(p45) may CHK1 phosphorylation after UV exposure, but instead resulted contribute to the DDR. To investigate this possibility further, we in the robust activation of these DDR markers (Figure S3I). These conducted a phospho-proteomic analysis of control and IKKa- data suggest that similar to ATM deficiency, the Rad50s/s allele is knockdown HT29 cells either untreated, as before, or subject capable of suppressing the signaling defects caused by IKKa to UV irradiation for 30 min. We observed that the UV-induced knockdown, potentially through the activation of other PI3K- phosphorylation of several DDR components, including 53BP1, like kinases. MDC1, and KAP1, were compromised in cells lacking IKKa (Fig- ure 3A; Table S2). Western blot analysis of IKKa-knockdown IKKa Interacts with and Directly Phosphorylates ATM in cells exposed to UV at different time points further confirmed Response to DNA Damage that Chk1 phosphorylation on Ser345 and gH2A.X induction is Next, we examined whether IKKa and ATM interact upon DNA significantly attenuated in IKKa-depleted cells (Figure S3A). In damage. Immunoprecipitation experiments with an anti-IK- contrast, knocking down IKKb or NEMO had no measurable ef- Ka(p45) antibody detected an interaction between endogenous fect on Chk1, Ser345, and histone H2A.X phosphorylation upon ATM and both full-length and IKKa(p45), 15 min after UV treat- UV treatment (Figure S3B). ment (Figure 3E). This observation raised the possibility that Since H2A.X, MDC1, 53BP1, and KAP1 are known substrates IKKa(p45) may directly phosphorylate ATM in response to DNA of ATM, we considered the possibility that IKKa(p54) may facili- damage, potentially contributing to ATM activation. Consistent tate ATM activation after UV exposure. Auto-phosphorylation of with this possibility, kinase assays revealed that recombinant ATM at Ser1981, which is an established marker of ATM activa- IKKa is able to phosphorylate a fragment of ATM comprising tion, was significantly reduced in IKKa knockout cells exposed to amino acids 1,911–2,063 in vitro (Figure 3F). The same fragment UV (Figure 3B). IKKa knockout cells were also compromised for was also phosphorylated in lysates from IKKa wild-type (WT) but ATM activation following IR (Figure S3C) or doxorubicin treat- not from IKKa-deficient cells (Figure 3G). Mass spectrometry ment (Figure S3D) and at multiple time points tested. Impaired analysis of the in vitro phosphorylated ATM fragment showed ATM activation in IKKa knockout cells was also associated that S1974, S1987, S2058, and T2059 are direct substrates of with attenuated p-Chk2 (T68), p-KAP1 (S824), and gH2A.X the IKKa kinase (Figure 3H). It should be noted that once acti- levels. Impaired ATM activation following UV exposure was vated, ATM auto-phosphorylates itself on Ser367, Ser1893, Figure 2. Activation of IKKa by DNA Damage Is Dependent on BRAF and p38a-MAPK Activity (A) WB analysis of HT29 pretreated with ATM or ATR inhibitors and then exposed to UV (130 mJ). (B) WB analysis of HT29 cells treated with the indicated inhibitors (p38i = SB203580, 10 mM; BRAFi = AZ628, 10 mM; TAKi = 5Z-7-oxozeaneol, 10 mM; MEKi = trametinib, 10 mM; bafilomycin, 10 nM) 16 h before UV exposure (130 mJ) and collected 30 min after treatment. (C) WB analysis of p38a wild-type (WT) and knockout (KO) MEFs collected 30 min after UV exposure (130 mJ). (D) WB analysis of WT MEFs treated with p38i for 16 h and then exposed to UV, as indicated. (E) WB of different CRC cell lines treated with the BRAF inhibitor AZ628 (16 h, 10 mM) before UV light (130 mJ) exposure and collected 30 min after treatment. (F) WB of cytoplasmic (C), nuclear (N), and chromatin (Chr) extracts from HT29 cells collected at different time points after UV treatment. See also Figure S2. Molecular Cell 75, 669–682, August 22, 2019 673 A D Time post-UV (min) 0 5 15 30 45 60 180 S405T401 S366 S1618 BRAFi - + - + - + - + - + - + - + S418 S1114 S410 S417 p-ATM S1981 RIPK3 CTTN ATR ATM S462 53BP1 CHEK2 p-ATM/ATM 0 0 0.1 0 0.1 0 0.9 0.3 1.1 0.3 3.4 0.9 3.1 0.7 T541 TRIM28 P53 H2AFX IKKA p-ATR S428 S50 ATM SUMO2 p-Chk1 S345 YY1AP1 S711 CDK1 ANXA2 MDC1 Chk1 JUN S453 KRT18 S63 Y24 SNAP23 T455 p-Chk2 T68 S1775 Chk2 S110 S398/S399 p-KAP1 S824 B Time post-UV 0 5 15 30 60 180 KAP1 (min) +/+ +/+ +/+ +/+ +/+ +/+ p-53BP1 S1618 -/- -/- -/- -/- -/- -/- 53BP1 p-ATM S1981 p-IKK S180 45 kD ATM 85 kD p-ATM/ATM 0 0 0 0 0.4 0 0.7 0 2.3 0 1.1 0 IKKα 45 kD p-ATR S428 p-ERK p-Chk1 S345 ERK Chk1 Tubulin p-Chk2 T68 E Input IP: IKKα(p45) IP: IgG Chk2 Crosslink - + + + - + + + - + + + Time post-UV(min) 0 0 5 15 0 0 5 15 0 0 5 15 p-KAP1 S824 ATM KAP1 IKKα 85 kD γH2A.X 45 kD IKKα Tubulin F G IKKα+/+ IKKα-/- C Autoradiograph UV - + - + Time Post-UV 32 (min.) 0 32 P-ATM 30 60 P-IKKα - + 75 kD Cherry-IKKα - + - + 32 Coomassie P-ATM 50 kD p-ATM S1981 37 kD GST-ATM 25 kD ATM IKKα Coomassie GST-ATM 50 kD p-IKK IKKα 100 kDa 37 kD S180 75 kDa GST 25 kD Tubulin 50 kDa Tubulin H Sequence pRS Site Probabilities Area IonScore Exp Value ∆M[ppm] ALVTYDLETAIPS[ST](p)R S(2058): 48.0; T(2059): 48.0 9.799E6 57 4.395E-05 -0.83 RSLAFEEGSQSTTIS(p)SLSEK S(1987): 99.9 8.563E6 69 3.393E-06 -0.18 SMDDQEKRS(p)LAFEEGSQSTTISSLSEK S(1974): 100.0 2.296E6 68 5.703E-06 -0.10 Figure 3. IKKa Downstream of BRAF Is Required for the Phosphorylation of Specific DDR Elements after DNA Damage (A) Network of selected proteins related to DNA damage with phosphosites increasing upon UV irradiation in control (q < 0.15, positive log2 [fold-change]) but not in IKKa-knockdown cells (delta log2 [fold-change] > 0.5). Color fills represent the differential fold-change upon UV treatment between control and IKKa- knockdown cells, and the width of the phosphosite borders represents the significance of the change in control cells upon UV irradiation (bold: q < 0.05, medium: 0.05 < q < 0.1, light: 0.1 < q < 0.15). (B) WB analysis of IKKa WT (+/+) and IKKa KO (/) cells treated with UV light (130 mJ). (C) WB analysis of UV-treated IKKa KO cells () and the same cells transduced with Cherry-IKKa expression vector (+). (legend continued on next page) 674 Molecular Cell 75, 669–682, August 22, 2019 and Ser1981, with the latter commonly used as a marker of ATM BRAF and IKKa affect 53BP1 and RIF1 function, thereby allow- activation in vivo. However, these auto-phosphorylation sites are ing the effective resolution of DNA damage by NHEJ in G1. dispensable for ATM activation following DNA damage based on the fact that knockin mice mutated for these three sites exhibit BRAF and IKKa Inhibition Synergize with DNA Damage- normal downstream DDR signaling (Daniel et al., 2008). Since Based Therapy in Patient-Derived Tumoroids the loss of IKKa impairs robust ATM activation, our data raise Current protocols for cancer treatment rely to a large extent on the possibility that IKKa(p45) phosphorylation of these distinct DNA-damaging agents that selectively kill highly proliferative tu- sites in ATM may facilitate its activation. mor cells but impose a lesser effect on low proliferating cancer cells. Furthermore, PI3K-like kinase inhibitors, including antago- IKKa and BRAF Contributes to DNA Damage Resolution nists of ATM, ATR, and DNA-PKcs, are in clinical development to Since ATM activation is important for coordinating DNA repair, exploit their impact on the DDR. In light of our findings that BRAF we next tested the possible impact of BRAF and IKKa inhibition and IKKa are required for efficient ATM signaling and DNA repair, in promoting lesion resolution. Inhibiting BRAF did not impair we hypothesized that the inhibition of these kinases may syner- the formation of gH2A.X/53BP1 foci at sites of IR-induced gize with DNA-damaging drugs to promote tumor cell killing. To DNA damage, but instead imposed a significant delay in lesion test this possibility, we treated patient-derived colorectal cancer resolution (Figure 4A), consistent with defective or attenuated (CRC) tumoroids (Figure 5A), which have been recently validated DNA repair. Phosphorylation of 53BP1 by ATM is required to re- as models for therapy prescription (Sato et al., 2011; Vlachogian- cruit Rap1 interacting factor 1 (RIF1) to sites of DNA damage nis et al., 2018), with suboptimal doses of 5-FU+irinotecan (Iri), to counteract DSB end resection to promote NHEJ in G1 alone or in combination with the BRAF inhibitors vemurafenib (Chapman et al., 2012a). In agreement with our previous obser- or AZ628. Consistent with our cellular studies, BRAF inhibition vations, co-recruitment of 53BP1 and its cofactor RIF1 was significantly reduced ATM activation and Chk1 phosphorylation attenuated following BRAF inhibition (Figure 4B) or in IKKa in response to damage (Figure 5B), with the combination treat- knockout mouse embryonic fibroblasts (MEFs) (Figure 4C). ment imposing a striking synergistic effect on the eradication IKKa-deficient cells or cells subject to BRAF inhibition also ex- of tumoroids compared with single treatments alone (Figure 5C). hibited a significant increase in the levels of DNA breaks in UV- Comparable synergistic effects with BRAF inhibitors were also treated MEFs as determined by comet assay (Figures 4D and observed when treating tumoroids with increasing doses of S4A). These results suggest that the loss or inhibition of IKKa 5-FU+Iri (Figures 5D and 5E) or g-irradiation (Figure S5A), or us- or BRAF compromises ATM activation and hinders down- ing the DNA-damaging agent doxorubicin (Figure S5B), which is stream DNA repair. commonly used in breast cancer treatment but is primarily inef- To further examine the contribution of IKKa and BRAF to fective against CRC tumors as a single agent. 53BP1-dependent DNA repair, we used conditional Trf2FL/FL The loss of CRC tumoroid viability in the combination treat- MEFs; inactivation of the shelterin subunit telomeric repeat- ments was also associated with the massive accumulation of binding factor 2 (TRF2) leads to telomere deprotection and chro- DNA damage and induction of apoptosis as determined by mosome end-to-end fusions mediated by 53BP1-dependent gH2A.X and cleaved caspase 3 levels, respectively (Figures 5F, NHEJ (Chapman et al., 2013; Celli and de Lange, 2005). 5G, S5C, and S5D). The enhanced accumulation of DNA breaks Following TRF2 inactivation by adenoviral CRE transduction, in tumoroids subject to the combination treatment was further MEFs were left untreated or treated with BRAF or MEK inhibitors confirmed by comet assays (Figures 5H and S5E). Most impor- (which do not prevent damage-induced IKKa(p45) phosphoryla- tant, tumoroids subjected to the partial depletion of IKKa by tion; see Figure 2D) for 72 h and then the cells were processed for CRISPR-Cas9 and treated with 5-FU+Iri displayed reduced metaphase visualization. Using telomere-specific fluorescence ATM activation (Figure 5I) with no significant cell-cycle alter- in situ hybridization (FISH), cells were scored for the presence ations (Figure S5F), and were not further sensitized by BRAF in- of chromosome end-to-end fusions. Consistent with our previ- hibitors (Figure 5J). IKKa-depleted tumoroids do not show any ous findings, IKKa knockdown (Figure 4E) or treatment with reduction in IKKb levels or activity (Figures 5I and S5G). These BRAF inhibitor (Figure 4F) significantly reduced chromosome fu- data suggest that BRAF and IKKa inhibition could be exploited sions after Trf2 deletion. In contrast, MEK inhibition did not affect to improve tumor killing in combination with DNA-damaging chromosome fusions in Trf2 null MEFs (Figure S4B), even though chemotherapy or radiotherapy. this had a comparable effect on cell-cycle position when compared to the BRAF inhibitor (Figure S4C) and IKKa knock- BRAF Inhibition Synergizes with DNA Damage-Based down (Figure S4D). Consistent with impaired ATM function, Therapy in a Patient-Derived Xenograft Model In Vivo BRAF inhibition precluded RIF1 recruitment to the telomeres af- To further explore the therapeutic potential of drug combinations ter Trf2 deletion (Figure S4E). These results further suggest that involving BRAF inhibitors and DNA-damaging agents, we used (D) WB analysis of HT29 pretreated with the BRAF inhibitor AZ638 (16 h, 10 mM) and then exposed to UV and collected at the indicated time points. (E) Immunoprecipitation assay with anti-IKKa(p45) antibody from HT29 cells treated as indicated. (F and G) In vitro kinase assay using glutathione S-transferase (GST) or GST-ATM (amino acids [aa] 1,854–2,063) as substrate and recombinant active IKKa (F) or lysates from IKKa WT (+/+) and IKKa KO (/) cells untreated or treated with UV for 15 min (G). (H) Mass spectrometry analysis of the GST-ATM peptide phosphorylated in vitro with recombinant IKKa. See also Figure S3 and Table S2. Molecular Cell 75, 669–682, August 22, 2019 675 A B C 10 μm 10 μm D E 10 μm 2 μm F 10 μm 2 μm Figure 4. IKKa Downstream of BRAF Functionally Contributes to DNA Damage Resolution (A) Immunofluorescence analysis of 53BP1 and gH2A.X in control or BRAF inhibitor-treated MEFs exposed to IR (2 Gy) and quantification from 3 independent experiments performed. (B and C) Double immunofluorescence analysis of 53BP1 and RIF1 and quantification of the number of co-localizing foci per nucleus in BRAF-treated (2 h, 10 mM) (B) and IKKa wild-type (+/+) or KO (/) MEFs (C). (D) Comet assay of IKKa wild-type (+/+) or KO (/) MEFs 8 and 24 h after DNA damage exposure (UV, 130 mJ). (E and F) Representative images of FISH analysis from control and BRAF-inhibited Trf2-deficient MEFs using a telomeric probe (green) (E) or IKKa knockdown (small interfering IKKa [siIKKa]) cells (F). Quantification of the relative number of metaphases containing fused chromosomes from three independent experiments performed is shown at right. See also Figure S4. 676 Molecular Cell 75, 669–682, August 22, 2019 A D B E C F H G I J Figure 5. BRAF Inhibition Enhances the Effect of DNA Damage-Based Therapy in CRC Patient-Derived Tumoroids (A) Experimental design used for the expansion of primary CRC tumors, generation of patient-derived tumoroids, and drug testing. (B) Western blot analysis of control and BRAF-inhibited IMIM-TD#5, collected at the indicated time points after IR treatment. (C) Quantification of tumoroid viability after treatment with the indicated compounds as a single treatment or in combination. (D and E) Dose-response curves of two different tumoroids, IMIM-TD#6 (D) and IMIM-TD#5 (E), treated as indicated. (legend continued on next page) Molecular Cell 75, 669–682, August 22, 2019 677 two independent patient-derived metastatic tumors (IMIM-X#1 mechanisms. However, insights into the substrates of IKKa and IMIM-X#3) that possess acquired resistance to regimes and the pathways that IKKa regulates were unknown. Using an of DNA damage-based and EGFR antibody-based therapies unbiased proteomic analysis to identify IKKa substrates, we (Montagut et al., 2012). Equivalent volumes of tumor cells were have uncovered an unappreciated role for IKKa and its activating implanted in the cecum of nude mice, and tumor growth was kinase BRAF in the DDR pathway. Loss or inhibition of these monitored by palpation. At the time of tumor detection, animals kinases compromised ATM activation and downstream DDR were randomly ascribed to the treatment groups: control, signaling, impaired DNA repair by 53BBP1-dependent end 5-FU+Iri, vemurafenib, or 5-FU+Iri plus vemurafenib. The combi- joining, and synergized with commonly used chemotherapies nation of 5-FU+Iri plus vemurafenib significantly reduced tumor to induce tumor eradication in vivo. Since these tumors are re- growth in vivo compared with single-agent treatments (Figures fractory to killing by single-agent treatments, our proposed com- 6A and 6B), and this was associated with the presence of exten- bination strategy may have important implications for cancer sive areas of necrosis and fibrosis (Figure 6C). Morphologically, treatment. residual neoplastic cells displayed a severe pleomorphism after A role for IKKa in the DDR is supported by our findings that combination treatment, but not with other conditions tested (Fig- IKKa is rapidly activated by phosphorylation on Ser180 in ure 6C), and was associated with the accumulation of DNA dam- response to a range of DNA-damaging agents and proceeds age in the epithelial tumor component (Figures 6D and 6E). to accumulate on chromatin at sites of DNA damage. Dam- Notably, the combination treatment did not lead to measurable age-induced IKKa activation was found to occur independently toxicity, as indicated by the overall aspect of the animals and of the canonical damage-responsive kinases ATM and ATR, but the absence of anomalous cleaved caspase 3 staining in the is instead dependent on BRAF, TAK1, and p38-MAPK kinases. colonic tissue adjacent to the implanted tumors (Figure S6A). We also showed that IKKa interacts with ATM shortly after the The combination treatment may also affect the invasive nature introduction of DNA damage, which raised the possibility that of these tumors, as we detected only 1 animal with peritoneal IKKa may directly regulate ATM function. Recombinant IKKa implants from 5 animals in this group compared with the con- was found to directly phosphorylate ATM on four sites distinct trols (5/5 animals with implants) and the single treated animals from the known ATM auto-phosphorylation sites, including the (5/6 and 3/6 for vemurafenib and 5-FU+Iri, respectively) commonly used activation maker pSer1981. IKKa-deficient cell (Figure S6B). extracts were also compromised for ATM phosphorylation Finally, we examined the long-term therapeutic potential of the in vitro, and the loss of IKKa or the inhibition of BRAF was found vemurafenib and 5-FU+Iri combination treatment of IMIM-X#1 to attenuate ATM activation both in cells and in tumoroids. In tumors in vivo. After a 3-month follow-up, we observed a signif- agreement with the observed impairment in ATM activation, icant impact on the survival of animals treated with 5-FU+Iri in IKKa-deficient cells or inhibition of BRAF also resulted in the combination with vemurafenib when compared with vehicle- diminished phosphorylation of Chk1, Chk2, gH2A.X, MDC1, treated, vemurafenib-only, and 5-FU+Iri-treated animals. All of 53BP1, and Kap1 following the induction of DNA damage. We the mice treated with 5-FU+Iri or vemurafenib only died during propose that IKKa affects the DDR by facilitating ATM activa- the course of the experiment due to IMIM-X#1-derived tumor tion. Since ATM auto-phosphorylation sites are dispensable growth. In contrast, six of seven animals treated with 5-FU+Iri for its activation (Daniel et al., 2008), future studies should plus vemurafenib survived for the duration of the study and assess the importance of the ATM residues phosphorylated were tumor-free at the end of the experiment. There was no ev- by IKKa. idence of tumor growth in the only mouse that succumbed in this DDR signaling also plays an important role in coordinating the treatment group, which appeared to have died from other repair of DNA lesions. The loss or inhibition of IKKa or BRAF was causes. The resulting Kaplan-Meier curves (Figures 6F and 6G) found to delay the resolution of DNA damage, which manifests demonstrate the potent effect of combining BRAF inhibitors as the persistence of H2A.X and 53BP1 foci at late time points with DNA-damaging agents in promoting tumor eradication following the induction of damage. The inhibition of BRAF or and long-term survival in vivo. loss of IKKa also resulted in persistent tail moments after dam- age in comet assays. Attenuated damage signaling in these cells DISCUSSION is the likely cause of the DNA repair defects, as IKKa-deficient cells exhibit impaired recruitment of RIF1 to the sites of DNA IKKa was previously found to influence tumor initiation and can- damage, which is dependent on the phosphorylation of 53BP1 cer progression through NF-kB-dependent and-independent by ATM (Chapman et al., 2013). A role for BRAF and IKKa in (F and G) Representative images of gH2A.X and cleaved caspase 3 staining in a representative tumoroid treated as indicated (F) and quantification of the percentage of positive cells from 20 tumoroids per condition counted (G). (H) Comet assay of IMIM-TD#9, treated as indicated. (I and J) Western blot analysis of control and IKKa-depleted IMIM-TD#9 using CRISPR-Cas9 technology (I) and quantification of cell viability after 72 h of culture with the indicated treatments (J). The statistical analysis in (C) and (I) was performed by unpaired t test, comparing the combination treatments with single 5FU+irinotecan (red) or BRAF/IKK inhibitors (green); ****p < 0.0001, n.s., non-significant. For the statistical analysis in (G), we used two-way ANOVA, and the p values are indicated as **p < 0.01 and ****p < 0.0001. Vem, vemurafenib; 5FU, 5-fluorouracil; Dox, doxorubicin; and Iri, irinotecan. See also Figure S5. 678 Molecular Cell 75, 669–682, August 22, 2019 A B C E D F G Figure 6. BRAF Inhibition Enhances the Effect of DNA Damage-Based Therapy in Orthotopic Xenograft Model from Metastatic CRC (A) Photograph of the tumors recovered at the end of the experiment with the indicated treatments. (B) Quantification of the weight of the tumors in the different groups of treatment from two independent experiments performed using IMIM-X#1 and IMIM-X#3. Two-way ANOVA was used for statistical analysis, and the p values are indicated as *p < 0.05, **p < 0.01, and ****p < 0.0001. (C) H&E staining of representative tumors treated as indicated. Note the various scales shown for the different treated tumors. (D and E) Immunohistochemistry analysis of gH2A.X in IMIM-X#1 tumors obtained at the time of sacrifice (D) and quantification of the percentage of gH2A.X+ tumor cells (E) from a minimum of 10 fields (203) counted per group of treatment. Two-way ANOVA was used for statistical analysis, and the p values are indicated as *p < 0.05, **p < 0.01, and ****p < 0.0001. (F and G) Kaplan-Meier curves for mice bearing human tumor IMIM-X#1, treated as indicated. Overall (F) and cancer-related (G) survival analysis for the indicated treatments. Statistical significance was determined using the Mantel-Cox log-rank test. Vem, vemurafenib; 5FU, 5-fluorouracil; Dox, doxorubicin; and Iri, irinotecan. See also Figure S6. Molecular Cell 75, 669–682, August 22, 2019 679 promoting efficient ATM activation and 53BP1-dependent end B Kinase Assay joining was further supported by experiments in conditional B Mass Spectrometry Analysis Trf2 knockout MEFs. Conditional inactivation of the Shelterin d QUANTIFICATION AND STATISTICAL ANALYSIS subunit TRF2 results in telomere deprotection, which normally d DATA AND CODE AVAILABILITY leads to ‘‘spaghetti chromosomes,’’ formed as a consequence of the fusion of dysfunctional telomeres by 53BP1-dependent SUPPLEMENTAL INFORMATION end joining (Chapman et al., 2013). We found that treating Trf2- deficient MEFs with BRAF inhibitors (but not MEKi) or depletion Supplemental Information can be found online at https://doi.org/10.1016/j. molcel.2019.05.036. of IKKa reduced telomere-telomere fusions. We attribute this ef- fect to the reduction in the co-recruitment of 53BP1 and RIF1 at ACKNOWLEDGMENTS dysfunctional telomeres. These results highlight the importance of BRAF and IKKa signaling for efficient ATM signaling and sub- We want to thank the Bigas and Boulton lab members for constructive discus- sequent repair following DNA damage. sions and suggestions and Kitty van Zwieten, Grazzia Giuffrida, and Marta Finally, we present evidence that clinically approved BRAF in- Garrido for technical support. This work was funded by grants from Instituto hibitors could be repurposed for use in combination with DNA- de Salud Carlos III FEDER (PIE15/00008 and PI16/00437), Generalitat de Cat- damaging chemotherapies to target relevant cancers. This alunya 2017SGR135, and the ‘‘Xarxa de Bancs de Tumors’’ sponsored by Pla conclusion is based on our findings that the BRAF inhibitors ve- Director d’Oncologia de Catalunya (XBTC). C.C. is supported by FPI BES- 2014-068451 and the EMBO Short-Term Fellowship (na7084). P.M. is sup- murafenib or AZ628, when combined with 5-FU+Iri, effectively ported by funding from the European Union’s Horizon 2020 research and inno- eradicate two different metastatic tumors that had acquired ther- vation program under the Marie Sklodowska-Curie grant agreement #702430. apeutic resistance in human patients, although we cannot The Boulton lab is supported by The Francis Crick Institute, which receives its directly ascribe these results to the effect of BRAF inhibition on core funding from Cancer Research UK (FC0010048), the UK Medical IKKa. Until now, the clinical use of BRAF inhibitors has focused Research Council (FC0010048), and the Wellcome Trust (FC0010048). on BRAF mutant (primarily BRAFV600E) tumors. Our findings S.J.B. is funded by European Research Council (ERC) Advanced Investigator Grants (TelMetab) and a Wellcome Trust Senior Investigator Grant. The Centre demonstrating that BRAF inhibition synergizes with DNA- de Regulació Genòmica/Universitat Pompeu Fabra Proteomics Unit is part of damaging drugs independent of BRAF mutation status therefore the ‘‘Plataforma de Recursos Biomoleculares y Bioinformáticos (ProteoRed)’’ expands the potential target population that would benefit from supported by grant PT13/0001 of Instituto de Salud Carlos III from the Spanish this therapeutic approach. In addition, we have shown that com- government and ‘‘Secretaria d’Universitats i Recerca del Departament pounds that are primarily ineffective in CRC but are used in other d’Economia i Coneixement de la Generalitat de Catalunya’’ (2014SGR678). types of cancer (e.g., doxorubicin) should be reconsidered in We acknowledge support from the Spanish Ministry of Economy and Compet- itiveness and ‘‘Centro de Excelencia Severo Ochoa 2013-2017’’ (SEV- combination treatments with BRAF inhibitors. Our results will 2012-0208). prompt further preclinical and clinical studies to understand the broader utility of BRAF inhibitors beyond their current AUTHOR CONTRIBUTIONS applications. A.B., S.J.B., and L.E. conceptualized the study, designed the experiments, and wrote the manuscript. E.B. and E.S. designed the proteomic analysis STAR+METHODS and evaluated the results. C.C., P.M., A. Villanueva, A. Vert, I.P., J.B., and M.M.-I. performed the biochemical assays and the in vitro and in vivo drug Detailed methods are provided in the online version of this paper testing experiments. C.C. prepared the figures. M.G-.F. and M.I. performed and include the following: the clinicopathological characterization of human tumors. C.M. provided clin- ical advice. d KEY RESOURCES TABLE d LEAD CONTACT AND MATERIALS AVAILABILITY DECLARATION OF INTERESTS d EXPERIMENTAL MODEL AND SUBJECT DETAILS The authors declare no competing interests. B Animal Studies B Patient-derived tumoroids Received: April 25, 2018 B Cell lines Revised: March 13, 2019 d METHOD DETAILS Accepted: May 30, 2019 B Tumoroid viability assays Published: July 10, 2019 B Cell lysis and Western Blot (WB) B Cell fractionation REFERENCES B immunoprecipitation Assay Adams, R.H., Porras, A., Alonso, G., Jones, M., Vintersten, K., Panelli, S., B Immunohistochemical Staining (IHC) Valladares, A., Perez, L., Klein, R., and Nebreda, A.R. (2000). Essential role B Immunofluorescence (IF) analysis of p38alpha MAP kinase in placental but not embryonic cardiovascular devel- B Peptide Nucleic Acid (PNA) Fluorescence In Situ Hy- opment. Mol. Cell 6, 109–116. bridization (FISH) and IF-FISH Alao, J.P., and Sunnerhagen, P. (2008). Rad3 and Sty1 function in B Laser microirradiation induced DNA damage Schizosaccharomyces pombe: an integrated response to DNA damage and B Knock down and knock out assays environmental stress? Mol. Microbiol. 68, 246–254. B Cell cycle analysis Amado, R.G., Wolf, M., Peeters, M., Van Cutsem, E., Siena, S., Freeman, D.J., B Comet Assay Juan, T., Sikorski, R., Suggs, S., Radinsky, R., et al. (2008). Wild-type KRAS is 680 Molecular Cell 75, 669–682, August 22, 2019 required for panitumumab efficacy in patients with metastatic colorectal can- Vemurafenib in Combination with Irinotecan and Cetuximab in Patients with cer. J. Clin. Oncol. 26, 1626–1634. Metastatic Colorectal Cancer with BRAFV600E Mutation. Cancer Discov. 6, Batlle, E., and Clevers, H. (2017). Cancer stem cells revisited. Nat. Med. 23, 1352–1365. 1124–1134. Hu-Lieskovan, S., Mok, S., Homet Moreno, B., Tsoi, J., Robert, L., Bothmer, A., Robbiani, D.F., Feldhahn, N., Gazumyan, A., Nussenzweig, A., Goedert, L., Pinheiro, E.M., Koya, R.C., Graeber, T.G., Comin-Anduix, B., and Nussenzweig, M.C. (2010). 53BP1 regulates DNA resection and the choice and Ribas, A. (2015). Improved antitumor activity of immunotherapy with between classical and alternative end joining during class switch recombina- BRAF and MEK inhibitors in BRAF(V600E) melanoma. Sci. Transl. Med. tion. J. Exp. Med. 207, 855–865. 7, 279ra41. Bothmer, A., Robbiani, D.F., Di Virgilio, M., Bunting, S.F., Klein, I.A., Feldhahn, Luo, J.L., Tan, W., Ricono, J.M., Korchynskyi, O., Zhang, M., Gonias, S.L., N., Barlow, J., Chen, H.T., Bosque, D., Callen, E., et al. (2011). Regulation of Cheresh, D.A., and Karin, M. (2007). Nuclear cytokine-activated IKKalpha DNA end joining, resection, and immunoglobulin class switch recombination controls prostate cancer metastasis by repressing Maspin. Nature 446, by 53BP1. Mol. Cell 42, 319–329. 690–694. Brenner, H., Kloor, M., and Pox, C.P. (2014). Colorectal cancer. Lancet 383, Margalef, P., Fernández-Majada, V., Villanueva, A., Garcia-Carbonell, R., 1490–1502. Iglesias, M., López, L., Martı́nez-Iniesta, M., Villà-Freixa, J., Mulero, M.C., Bulavin, D.V., Higashimoto, Y., Popoff, I.J., Gaarde, W.A., Basrur, V., Andreu, M., et al. (2012). A truncated form of IKKa is responsible for specific Potapova, O., Appella, E., and Fornace, A.J., Jr. (2001). Initiation of a G2/M nuclear IKK activity in colorectal cancer. Cell Rep. 2, 840–854. checkpoint after ultraviolet radiation requires p38 kinase. Nature 411, Margalef, P., Colomer, C., Villanueva, A., Montagut, C., Iglesias, M., Bellosillo, 102–107. B., Salazar, R., Martı́nez-Iniesta, M., Bigas, A., and Espinosa, L. (2015). BRAF- Canovas, B., Igea, A., Sartori, A.A., Gomis, R.R., Paull, T.T., Isoda, M., Perez- induced tumorigenesis is IKKa-dependent but NF-kB-independent. Sci. Montoyo, H., Serra, V., Gonzalez-Suarez, E., Stracker, T.H., and Nebreda, A.R. Signal. 8, ra38. (2018). Targeting p38alpha Increases DNA Damage, Chromosome Instability, Montagut, C., Dalmases, A., Bellosillo, B., Crespo, M., Pairet, S., Iglesias, and the Anti-tumoral Response to Taxanes in Breast Cancer Cells. Cancer Cell M., Salido, M., Gallen, M., Marsters, S., Tsai, S.P., et al. (2012). 33, 1094–1110.e8. Identification of a mutation in the extracellular domain of the Epidermal Celli, G.B., and de Lange, T. (2005). DNA processing is not required for ATM- Growth Factor Receptor conferring cetuximab resistance in colorectal can- mediated telomere damage response after TRF2 deletion. Nat. Cell Biol. 7, cer. Nat. Med. 18, 221–223. 712–718. Morales, M., Theunissen, J.W., Kim, C.F., Kitagawa, R., Kastan, M.B., and Chapman, J.R., Sossick, A.J., Boulton, S.J., and Jackson, S.P. (2012a). Petrini, J.H. (2005). The Rad50S allele promotes ATM-dependent BRCA1-associated exclusion of 53BP1 from DNA damage sites underlies tem- DNA damage responses and suppresses ATM deficiency: implications poral control of DNA repair. J. Cell Sci. 125, 3529–3534. for the Mre11 complex as a DNA damage sensor. Genes Dev. 19, Chapman, J.R., Taylor, M.R., and Boulton, S.J. (2012b). Playing the end game: 3043–3054. DNA double-strand break repair pathway choice. Mol. Cell 47, 497–510. Panier, S., and Boulton, S.J. (2014). Double-strand break repair: 53BP1 comes Chapman, J.R., Barral, P., Vannier, J.B., Borel, V., Steger, M., Tomas-Loba, A., into focus. Nat. Rev. Mol. Cell Biol. 15, 7–18. Sartori, A.A., Adams, I.R., Batista, F.D., and Boulton, S.J. (2013). RIF1 is essential for 53BP1-dependent nonhomologous end joining and suppression Preta, G., de Klark, R., Chakraborti, S., and Glas, R. (2010). MAP kinase- of DNA double-strand break resection. Mol. Cell 49, 858–871. signaling controls nuclear translocation of tripeptidyl-peptidase II in response to DNA damage and oxidative stress. Biochem. Biophys. Res. Commun. 399, Chen, I.T., Hsu, P.H., Hsu, W.C., Chen, N.J., and Tseng, P.H. (2015). 324–330. Polyubiquitination of Transforming Growth Factor b-activated Kinase 1 (TAK1) at Lysine 562 Residue Regulates TLR4-mediated JNK and p38 Ribas, A., Gonzalez, R., Pavlick, A., Hamid, O., Gajewski, T.F., Daud, A., MAPK Activation. Sci. Rep. 5, 12300. Flaherty, L., Logan, T., Chmielowski, B., Lewis, K., et al. (2014). Combination of vemurafenib and cobimetinib in patients with advanced Colomer, C., Margalef, P., Gonzalez, J., Vert, A., Bigas, A., and Espinosa, L. BRAF(V600)-mutated melanoma: a phase 1b study. Lancet Oncol. 15, (2018). IKKa is required in the intestinal epithelial cells for tumour stemness. 954–965. Br. J. Cancer 118, 839–846. Daniel, J.A., Pellegrini, M., Lee, J.H., Paull, T.T., Feigenbaum, L., and Richman, S.D., Seymour, M.T., Chambers, P., Elliott, F., Daly, C.L., Meade, Nussenzweig, A. (2008). Multiple autophosphorylation sites are dispensable A.M., Taylor, G., Barrett, J.H., and Quirke, P. (2009). KRAS and BRAF muta- for murine ATM activation in vivo. J. Cell Biol. 183, 777–783. tions in advanced colorectal cancer are associated with poor prognosis but do not preclude benefit from oxaliplatin or irinotecan: results from the MRC Di Nicolantonio, F., Martini, M., Molinari, F., Sartore-Bianchi, A., Arena, S., FOCUS trial. J. Clin. Oncol. 27, 5931–5937. Saletti, P., De Dosso, S., Mazzucchelli, L., Frattini, M., Siena, S., and Bardelli, A. (2008). Wild-type BRAF is required for response to panitumumab Rouse, J., Cohen, P., Trigon, S., Morange, M., Alonso-Llamazares, A., or cetuximab in metastatic colorectal cancer. J. Clin. Oncol. 26, 5705–5712. Zamanillo, D., Hunt, T., and Nebreda, A.R. (1994). A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP ki- Escribano-Dı́az, C., Orthwein, A., Fradet-Turcotte, A., Xing, M., Young, J.T., , J., Cook, M.A., Rosebrock, A.P., Munro, M., Canny, M.D., et al. nase-2 and phosphorylation of the small heat shock proteins. Cell 78, Tkác 1027–1037. (2013). A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice. Mol. Cell 49, 872–883. Sato, T., Stange, D.E., Ferrante, M., Vries, R.G., Van Es, J.H., Van den Brink, Espinosa, L., Inglés-Esteve, J., Aguilera, C., and Bigas, A. (2003). S., Van Houdt, W.J., Pronk, A., Van Gorp, J., Siersema, P.D., and Clevers, Phosphorylation by glycogen synthase kinase-3 beta down-regulates H. (2011). Long-term expansion of epithelial organoids from human colon, ad- Notch activity, a link for Notch and Wnt pathways. J. Biol. Chem. 278, enoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 32227–32235. 1762–1772. Hindi, S.M., Sato, S., Xiong, G., Bohnert, K.R., Gibb, A.A., Gallot, Y.S., Toll, A., Margalef, P., Masferrer, E., Ferrándiz-Pulido, C., Gimeno, J., Pujol, McMillan, J.D., Hill, B.G., Uchida, S., and Kumar, A. (2018). TAK1 regu- R.M., Bigas, A., and Espinosa, L. (2015). Active nuclear IKK correlates with lates skeletal muscle mass and mitochondrial function. JCI Insight metastatic risk in cutaneous squamous cell carcinoma. Arch. Dermatol. Res. 3, 98441. 307, 721–729. Hong, D.S., Morris, V.K., El Osta, B., Sorokin, A.V., Janku, F., Fu, S., Overman, Vetter, I.R., and Wittinghofer, A. (2001). The guanine nucleotide-binding switch M.J., Piha-Paul, S., Subbiah, V., Kee, B., et al. (2016). Phase IB Study of in three dimensions. Science 294, 1299–1304. Molecular Cell 75, 669–682, August 22, 2019 681 Vlachogiannis, G., Hedayat, S., Vatsiou, A., Jamin, Y., Fernández-Mateos, J., Yang, Y., Xia, F., Hermance, N., Mabb, A., Simonson, S., Morrissey, S., Khan, K., Lampis, A., Eason, K., Huntingford, I., Burke, R., et al. (2018). Patient- Gandhi, P., Munson, M., Miyamoto, S., and Kelliher, M.A. (2011). A derived organoids model treatment response of metastatic gastrointestinal cytosolic ATM/NEMO/RIP1 complex recruits TAK1 to mediate the cancers. Science 359, 920–926. NF-kappaB and p38 mitogen-activated protein kinase (MAPK)/MAPK- Vreka, M., Lilis, I., Papageorgopoulou, M., Giotopoulou, G.A., Lianou, M., activated protein 2 responses to DNA damage. Mol. Cell. Biol. 31, Giopanou, I., Kanellakis, N.I., Spella, M., Agalioti, T., Armenis, V., et al. 2774–2786. (2018). Ikb kinase ais required for development and progression of KRAS- Zhang, Q., Lenardo, M.J., and Baltimore, D. (2017). 30 Years of NF-kB: A mutant lung adenocarcinoma. Cancer Res. 78, 2939–2951. Blossoming of Relevance to Human Pathobiology. Cell 168, 37–57. Wu, Z.H., Shi, Y., Tibbetts, R.S., and Miyamoto, S. (2006). Molecular linkage Zimmermann, M., Lottersberger, F., Buonomo, S.B., Sfeir, A., and de Lange, T. between the kinase ATM and NF-kappaB signaling in response to genotoxic (2013). 53BP1 regulates DSB repair using Rif1 to control 50 end resection. stimuli. Science 311, 1141–1146. Science 339, 700–704. 682 Molecular Cell 75, 669–682, August 22, 2019 STAR+METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies p-IKK s180 Santa Cruz sc-23470; RRID: AB_2122159 IKKa Merck Millipore OP133; RRID: AB_10681621 IKKa Abcam ab32041; RRID: AB_733070 IKKa(p45) Merck Millipore MABF222; RRID: not available p-Chk1 S345 Cell Signaling #2348; RRID: AB_331212 Chk1 Cell Signaling #2360; RRID: AB_2080320 p-KAP1 S824 Bethyl Laboratories A300-767A; RRID: AB_669740 KAP1 Cell Signaling #4123; RRID: AB_2256670 ɣH2A.X Cell Signaling #2577; RRID: AB_2118010 ɣH2A.X Merck Millipore 05-636; RRID: AB_309864 p-ERK 1/2 Cell Signaling #4370; RRID: AB_2315112 ERK 1/2 Cell Signaling #4696; RRID: AB_390780 Histone H3 Abcam ab1791; RRID: AB_302613 Lamin B Santa Cruz sc-6216; RRID: AB_648156 a-Tubulin Sigma T6074; RRID: AB_477582 IKKb Abcam ab32135; RRID: AB_733071 NEMO Santa Cruz sc-8330; RRID: AB_2124846 Cleaved caspase 3 Cell Signaling #9661; RRID: AB_2341188 p-ATM S1981 Merck Millipore 05-740; RRID: AB_309954 p-53BP1 S1618 Cell Signaling #6209; RRID: AB_11220229 53BP1 Abcam ab21083; RRID: AB_722496 Anti-Rabbit-HRP (2ary) DAKO P0448; RRID: AB_2617138 Anti-Mouse-HRP (2ary) DAKO P0260; RRID: AB_2636929 Anti-Goat-HRP (2ary) DAKO P0449; RRID: AB_2617143 53BP1 Bethyl Laboratories A300-272A; RRID: AB_185520 RIF1 Santa Cruz sc-65191; RRID: AB_2126820 Alexa Fluor 488 donkey anti-rabbit (2ary) Invitrogen A21206; RRID: AB_141708 Alexa Fluor 488 donkey anti-mouse (2ary) Invitrogen A21202; RRID: AB_141607 Alexa Fluor 546 donkey anti-rabbit (2ary) Invitrogen A10040; RRID: AB_2534016 Alexa Fluor 546 donkey anti-goat (2ary) Invitrogen A11056; RRID: AB_142628 ATM Santa Cruz sc-135663; RRID: AB_2062962 Biological Samples PDXIMIM#1-6 Hospital del Mar (Barcelona) N/A Chemicals, Peptides, and Recombinant Proteins DMEM Sigma N/A Advanced DMEM/F12 GIBCO 12634028 AZ628 (BRAFi) Selleckchem S2746 Vemurafenib Selleckchem S1267 Sorafenib Selleckchem S7397 5Z-7-oxozeaneol (TAKi) Selleckchem 499610 Trametinib (MEKi) Selleckchem S2673 5-Fluorouracil (5-FU) Accord 606544.3 Irinotecan Frasenius Kabi 687014.3 Doxorubicin Accord 174247 (Continued on next page) Molecular Cell 75, 669–682.e1–e5, August 22, 2019 e1 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Matrigel Corning 354234 1x B27 supplement GIBCO 17504044 1x N-2 supplement GIBCO 17502048 Y-27632 (ROCK inhibitor) Sigma Y0503 EGF Sigma E9644 Human Noggin PeproTech 120-10C Human R-spondin 1 PeproTech 120-38 Nicotinamide Sigma N3376 A8301 (ALK inhibitor) Sigma SML0788 SB202190 Sigma S70677 Prostaglandin E2 Tocris 2296 Gastrin I human Tocris 3006 Ad-GFP Vector Biolabs 1060 Ad-GFP-Cre Vector Biolabs 1700 PhosSTOP phosphatase inhibitor cocktail Roche PHOSS-RO Complete Mini protease inhibitor cocktail Roche 11-836-170-001 TAMRA-TelG 50 -(TTAGGG)3-30 PNA probe PNA Bio-synthesis F1006 Critical Commercial Assays Envision+ System HRP Labeled Polymer anti-Rabbit DAKO K4003 Envision+ System HRP Labeled Polymer anti-Mouse DAKO K4001 3,30 -diaminobenzidine (DAB) DAKO K3468 ProLong. Diamond with DAPI Thermo Scientific P36971 ECL solution Biological Industries 20-500-120 ECL Prime Western Blotting Detection Reagent GE Healthcare RPN2232 CometAssay Kit Trevigen 4250-050-K DharmaFECT 1 transfection reagent Dharmacon T-2001-03 Deposited Data MS data from shC and shIKKa in UV/non-UV HT29 cells PRIDE EBI-EMBL PXD008932 Raw data from our manuscript is available at N/A N/A http://dx.doi.org/10.17632/k6xkjc2bf7.2 Experimental Models: Cell Lines HT29 ATCC HTB-38D WiDr ATCC CCL-218 HCT116 ATCC CCL-247 LIM1215 ECACC 10092301 DLD1 ATCC CCL-221 SW480 ATCC CCL-228 MCF7 ATCC HTB-22 T24 ATCC HTB-4 SKMEL131 ATCC N/A RWP1 ATCC N/A Mouse Embryonic Fibroblasts p38a KO Gift from A. Nebreda. N/A Mouse Embryonic Fibroblasts Trf2FL/FL Gift from T. de Lange. N/A Experimental Models: Organisms/Strains Athymic nude, nu/nu mice Jackson Laboratories 002019 Oligonucleotides ON-TARGETplus SMART pool siRNA Chuk Dharmacon SO-2621140G ON-TARGETplus Non-targeting Control Pool Dharmacon D-001810-10 (Continued on next page) e2 Molecular Cell 75, 669–682.e1–e5, August 22, 2019 Continued REAGENT or RESOURCE SOURCE IDENTIFIER MISSION shRNA, TRCN0000000508 (human) (shIKKa 1) Sigma N/A MISSION shRNA, TRCN0000199496 (human) (shIKKa 4) Sigma N/A TRC2 pLKO.5-puro Nonmammalian shRNA Control Sigma SHC202 MISSION shRNA, TRCN00001897 (human) (shIKKb) Sigma N/A MISSION shRNA, TRCN000022146 (human) (shNEMO) Sigma N/A Recombinant DNA pMD2.G plasmid Addgene #12259 pCMV-dR8.2 dvpr plasmid (human) Addgene #8455 Software and Algorithms GraphPad Prism 6 Graphpad https://www.graphpad.com/ Volocity 6.3 PerkinElmer http://cellularimaging.perkinelmer.com/ downloads/detail.php?id=14 LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contacts, Lluı́s Espinosa (lespinosa@imim.es) and Simon J. Boulton (simon.boulton@crick.ac.uk). EXPERIMENTAL MODEL AND SUBJECT DETAILS Animal Studies Fragments of human colorectal tumors obtained from MARbiobank with the informed consent of patients and following all recom- mendations of Hospital del Mar’ Ethics Committee, the Spanish regulations, and the Helsinki declaration’s Guide were transplanted and expanded in the cecum of nude mice as orthoxenografts. To perform in vivo drug testing, equivalent pieces of individual tumors were implanted orthotopically in the wall of the cecum of nude mice. When tumors were detectable by palpation (4-5 weeks), animals were randomly ascribed to the different groups of treatment. Vemurafenib (50mg/kg) was administered orally every day, and 5-FU and irinotecan (50mg/kg each) every 4 days intravenously. After 21 days of treatment, mice were euthanized and tumors collected, photographed, measured and processed for immunohistochemistry examination. In a parallel experiment, animals were equally treated and left for survival analysis. In all our procedures, animals were kept under pathogen-free conditions, and animal work was conducted according to the guidelines from the Animal Care Committee at the Generalitat de Catalunya. The Committee for Animal Experimentation at the Institute of Biomedical Research of Bellvitge (Barcelona) approved these studies. Patient-derived tumoroids For tumoroids generation, primary or xenografted tumors were disaggregated in 1mg/mL collagenase II (Sigma) and 20 mg/mL hyaluronidase (Sigma), filtered in 100 mm cell strainer, and seeded in Matrigel (BD Biosciences) as described(Sato et al., 2011). Tumoroids were expanded by serial passaging and kept frozen in liquid Nitrogen for being used in subsequent experiments. Mutations identified in the different tumors were: IMIM#1, EGFR(S464L) and TP53(I254T); IMIM#3, NRAS(Q61K), TP53(R175H), and EGFR(E928K); IMIM#5, KRAS(G12D); IMIM#6, KRAS; and IMIM#9, KRAS, NRAS and BRAF WT. Cell lines CRC cell lines LIM1215 (KRAS and BRAF wild-type), Caco2, DLD1, SW480 and HCT116 (KRAS mutated), WiDr and HT29 (BRAF mutated) were obtained from the ATCC. IKKa wild-type and knock out MEFs were kindly provided by Michael Karin (UCSD, La Jolla). SV40-LT-immortalized TRF2FL/FL were previously described (Celli and de Lange, 2005). All cells were grown in Dulbecco’s modified Eagle’s medium (Invitrogen) plus 10% fetal bovine serum (Biological Industries) and were maintained in a 5% CO2 incubator at 37 C. METHOD DETAILS Tumoroid viability assays 600 single tumoroid cells were plated in 96-well plates in Matrigel. After 4 days in culture, we treated growing tumoroids with 5-FU, Irinotecan, doxorubicin, AZ628, vemurafenib or combinations for 72 hours at the indicated concentrations. Cell viability was determined using the CellTiter-Glo 3D Cell Viability Assay (Promega) following manufacturer’s instructions in an Orion II multiplate luminometer (Berthold detection systems). Data were calculated as mean ± standard deviation from 3 independent experiments conducted in triplicates. Molecular Cell 75, 669–682.e1–e5, August 22, 2019 e3 Cell lysis and Western Blot (WB) Cells were lysed 20 min at 4 C in 300 mL of PBS plus 0.5% Triton X-100, 1 mM EDTA, 100 mM NA-orthovanadate, 0.25 mM phenyl- methylsulfonyl fluoride, and complete protease inhibitor cocktail (Roche). Lysates were analyzed by western blotting using standard SDS–polyacrylamide gel electrophoresis (SDS-PAGE) techniques. In brief, protein samples were boiled in Laemmli buffer, run in polyacrylamide gels, and transferred onto polyvinylidene difluoride membranes. The membranes were incubated overnight at 4 C with the appropriate primary antibodies. After being washed, the membranes were incubated with specific secondary horseradish peroxidase–linked antibodies from Dako and visualized using the enhanced chemiluminescence reagent from Amersham. Cell fractionation For cytoplasm/nuclear/chromatin separations, cells were lysed in 10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, and 0.05% NP-40 (pH 7.9) for 10 min on ice and centrifuged at 3,000 rpm. Supernatants were recovered as the cytoplasmic fraction, and the pellets were lysed in 5 mM HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 26% glycerol and sonicated for 5 min three times to recover the soluble nuclear fractions. The remaining pellet included the chromatin fraction. Lysates were run in SDS-PAGE and transferred onto Immobilon-P transfer membranes (Millipore) for western blot analysis. immunoprecipitation Assay For precipitation assay, control and UV-treated cells were crosslinked with dithiobis succinimidyl propionate (DSP, Pierce) 10 min at room temperature and then disrupted in RIPA buffer plus protease inhibitor cocktail (Roche). Cells were then centrifuged at 13,000 rpm for 15 min, and supernatants were incubated with 5 mg of anti-IKKa(p45) antibody. Precipitates were captured with 35 mL of protein A-Sepharose, extensively washed, and analyzed by WB. In most of the experiments, we used the Clean-Blot IP Detection Kit as secondary antibody. Immunohistochemical Staining (IHC) Tissues were fixed in 4% formaldehyde overnight at room temperature and embedded in paraffin. 4 mm paraffin embedded sections were first deparaffinized in xylene. IHC was performed following standard techniques with EDTA- or citrate-based antigen retrieval and developed with the Envision+ System HRP Labeled Polymer anti-Rabbit or anti-Mouse and 3,30 -diaminobenzidine (DAB) from DAKO. Images were obtained with an Olympus BX61 microscope. Immunofluorescence (IF) analysis For cell lines and tumoroid immune-fluorescence, cells were directly fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 (Pierce), washed and incubated overnight with the corresponding primary antibodies. Secondary antibodies were the Alexa FluorTM from Invitrogen. ProLongTM Diamond Antifade Mountant plus DAPI was used as mounting medium. Images were taken in an SP5 upright confocal microscope (Leica). Peptide Nucleic Acid (PNA) Fluorescence In Situ Hybridization (FISH) and IF-FISH Cells were treated with 0.2 mg/ml of colcemid for 90 minutes to arrest cells in metaphase. Trypsinized cells were incubated in 75 mM KCL, fixed with methanol:acetic acid (3:1), and spread on glass slides. To preserve chromosome architecture the slides were rehy- drated in PBS for 5 minutes, fixed in 4% formaldehyde for 5 minutes, treated with 1 mg/ml of pepsin for 10 minutes at 37 C, and fixed in 4% formaldehyde for 5 minutes. Next, slides were dehydrated in 70%, 85%, and 100% (v/v) ethanol for 15 minutes each and then air-dried. Metaphase chromosome spreads were hybridized with telomeric TAMRA-TelG 50 -(TTAGGG)3-30 PNA probe (Bio-synthe- sis) and slides were mounted using ProLong Gold antifade with DAPI (Life Technologies). Chromosome images and telomere signals were captured using Zeiss Axio Imager M1 microscope equipped with an ORCA-ER camera (Hamamatsu) controlled by Volocity 6.3 software (Improvision). For IF-FISH, cells grown on #1.5 glass coverslips were fixed for 5 minutes in cold methanol. Cells were washed twice for 5 min in PBS, incubated for 30 min in blocking solution (1 mg/ml BSA, 3% goat serum, 0.1% Triton X-100, 1 mM EDTA in PBS), and then incubated overnight with primary antibody against 53BP1 and secondary antibody anti-rabbit Alexa Fluor 488 secondary antibody for 1 hr and 30 min, in blocking solution with 5 min washes in PBS in-between. After dehydration of the cells, FISH experiments were performed as described above. Slides were mounted with ProLong Gold antifade containing DAPI and images were acquired with an Olympus FLV1000 inverted microscope equipped with a 63X oil objective. Laser microirradiation induced DNA damage HT29 cells were seeded on 35 mm glass bottom dish (Ibidi, 81158) and pre-sensitized for 48h with 10 mM BrdU. Cells were transferred to Olympus FV1000 confocal LSM with heated stage. Laser microirradition was performed in a stripe shape with a 405 nm laser focused through 40x objective (400mW at objective, 50 scans). 1 hour after DNA damage induction, cells were fixed and processed for IF. Knock down and knock out assays Lentiviral particles including shRNAs against IKK subunits were obtained from Sigma (table). The TRC2 pLKO.5-puro non-mamma- lian shRNA plasmid (SHC202) was used as negative control. siRNAs employed were ON-TARGET plus siRNA SMARTpool purchased e4 Molecular Cell 75, 669–682.e1–e5, August 22, 2019 from GE Dharmacon. RNA interference (RNAi) transfections were performed using Dharmafect Trasfection Reagent (Dharmacon) in a forward transfection mode using manufacturer’s guidelines. Deletion of floxed alleles in Trf2FL/FL cells was carried out with either Ad-GFP or Ad-GFP-Cre adenovirus (Vector Biolabs, ref. 1060 and 1700) and checked by WB. Cell cycle analysis Cell cycle was determined by flow cytometry using the standard Propidium Iodide staining-based protocol in the LSR-Fortessa analyzer (BD Biosciences). Comet Assay Comet assays were performed using CometAssay Trevigen Kit (4250-050-K) following manufacturer’s instructions. Pictures were taken using a Nikon Eclipse Ni-E epifluorescence microscope and tail moment was calculated using the OPENCOMET plugin for Fiji. Kinase Assay In vitro kinase assays were performed as previously described (Espinosa et al., 2003). In brief, 5 mg of GST or GST-ATM (Aa1911-2063) were incubated with 200 ng of recombinant human IKKa (ab102103) or 10 mg of cell lysates from WT or IKKa KO cells at 30 C for 30 minutes in the presence of ATPgP32. Reactions were stopped by adding loading buffer, run in a polyacrylamide gel and developed in autoradiograph film. Mass Spectrometry Analysis Cell lysates obtained in the different experimental conditions were processed and digested with trypsin and endoproteinase LysC with a ratio enzyme:sample of 1:10 for both enzymes (w:w). Samples were then subjected to phospho-peptide enrichment using titanium dioxide (TiO2) beads, and phospho-enriched samples were analyzed by LC-MS/MS. To identify IKKa-dependent phospho- peptides, samples were injected with a 120-minute chromatographic gradient in an Orbitrap Velos Pro with a data-dependent acqui- sition method using CID fragmentation for the top 20 most intense precursor ions and multistage activation. In the UV-activation experiment, samples were acquired with a 90-minute gradient in an Orbitrap Fusion Lumos with a data-dependent acquisition method using top speed, HCD fragmentation and ion-trap detection. In both cases, the resulting data were analyzed with the Pro- teome Discoverer software v1.4, using the search algorithm Mascot (v2.5) against a Human protein database (Uniprot, v2015) with oxidation (Met), and phosphorylation (Ser, Thr, Tyr) as variable modifications. Carbamidomethylation (Cys) was set as fixed modifi- cation and a mass tolerance of 7 ppm (MS1) and 0.5 Da (MS2) were used. Only peptides with a false discovery rate below 5% were considered for quantitative analysis. Peptides relative abundance was estimated with the area under the curve of extracted ion chromatograms. Protein network was generated using cytoscape software (https://cytoscape.org). QUANTIFICATION AND STATISTICAL ANALYSIS Statistical parameters, including number of events quantified, standard deviation, and statistical significance are reported in the figures and in the figure legends. Statistical analysis has been performed using GraphPad Prism6 software (GraphPad) and p < 0.05 is considered significant. Two-sided Student’s t test was used to compare differences between two groups and Two- Way ANOVA test was used to compare differences among multiple groups. Each experiment has been repeated at least twice. DATA AND CODE AVAILABILITY MS data are available at PRIDE EBI-EMBL database with identifier PRIDE: PXD008932. Row data from our manuscript is available at https://doi.org/10.17632/k6xkjc2bf7.1 Molecular Cell 75, 669–682.e1–e5, August 22, 2019 e5