Stabilization of Mutant BRCA1 Protein Confers PARP Inhibitor and Platinum Resistance

Stabilization of mutant BRCA1 protein confers PARP inhibitor and platinum resistance

Neil Johnsona,b,1,2, Shawn F. Johnsona, Wei Yaoa, Yu-Chen Lia, Young-Eun Choic, Andrea J. Bernhardyd, Yifan Wangd, Marzia Capellettia, Kristopher A. Sarosieka, Lisa A. Moreauc,e, Dipanjan Chowdhuryc, Anneka Wickramanayakef, Maria I. Harrellf, Joyce F. Liua,b, Alan D. D’Andreac,e, Alexander Mirong, Elizabeth M. Swisherf, and Geoffrey I. Shapiroa,b,2

Departments of aMedical Oncology, cRadiation Oncology, and gCancer Biology, Dana–Farber Cancer Institute and Harvard Medical School, Boston, MA 02215; bDepartment of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02215; dDevelopmental Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA 19111; eDepartment of Pediatrics, Children’s Hospital and Harvard Medical School, Boston, MA 02215; and fDepartments of Obstetrics and Gynecology and Medicine, University of Washington, Seattle, WA 98195

Edited by Stephen J. Elledge, Harvard Medical School, Boston, MA, and approved August 26, 2013 (received for review March 18, 2013)

Breast Cancer Type 1 Susceptibility Protein (BRCA1)-deficient cells inhibitor-resistant BRCA1- and 53BP1-deficient tumors and have compromised DNA repair and are sensitive to poly(ADP-ribose) derived cell lines, RAD51 γ-irradiation–induced foci were detec- polymerase (PARP) inhibitors. Despite initial responses, the devel- ted, although at a lower level than in BRCA1- and 53BP1-proficient opment of resistance limits clinical efficacy. Mutations in the BRCA C- cells (9). Previous studies demonstrated that RAD51 foci were terminal (BRCT) domain of BRCA1 frequently create protein products partially reduced in BRCA1- or partner and localizer of BRCA2 unable to fold that are subject to protease-mediated degradation. (PALB2)-deficient cells reconstituted with BRCA1 or PALB2 Here, we show HSP90-mediated stabilization of a BRCT domain constructs carrying mutations that disrupt the BRCA1–PALB2 in- mutant BRCA1 protein under PARP inhibitor selection pressure. The teraction (12, 13), suggesting that BRCA1 may enlist PALB2, which stabilized mutant BRCA1 protein interacted with PALB2-BRCA2- in turn organizes the recruitment of BRCA2 and RAD51. RAD51, was essential for RAD51 focus formation, and conferred To date, the described mechanisms of PARP inhibitor resistance occur in only a fraction of the BRCA1 mutant patient PARP inhibitor as well as cisplatin resistance. Treatment of resistant Brca1 cells with the HSP90 inhibitor 17-dimethylaminoethylamino-17- population or in PARP inhibitor-resistant -mutated mouse mammary tumors (8, 10). Here, we used a human breast cancer demethoxygeldanamycin reduced mutant BRCA1 protein levels BRCA1 and restored their sensitivity to PARP inhibition. Resistant cells also cell line that contains a BRCT domain mutation to identify additional mechanisms of acquired PARP inhibitor re- acquired a TP53BP1 mutation that facilitated DNA end resection in sistance, and demonstrate that stabilization of the mutant BRCA1 the absence of a BRCA1 protein capable of binding CtIP. Finally, protein is critical for the restoration of RAD51 focus formation. concomitant increased mutant BRCA1 and decreased 53BP1 protein expression occur in clinical samples of BRCA1-mutated recurrent Results ovarian carcinomas that have developed resistance to platinum. MDA-MB-436 Clones Are Resistant to PARP Inhibitors and Cisplatin. These results provide evidence for a two-event mechanism by which To study PARP inhibitor resistance, we cultured the triple- BRCA1-mutant tumors acquire anticancer therapy resistance. Significance homologous recombination | cancer therapy

BRCA1 Poly(ADP-ribose) polymerase (PARP) inhibitors have pro- he breast cancer 1, early onset ( ) gene is commonly duced responses in homologous recombination (HR) repair- Tmutated in hereditary breast and ovarian cancers. The BRCA1 deficient cancers, such as those with a mutated breast cancer 1, protein has multiple domains that mediate protein interactions; early onset (BRCA1) gene. We have delineated a two-event BRCA1 gene mutations may produce truncated proteins that lose mechanism of acquired resistance by using a BRCA1 BRCA C- the ability to interact with associated proteins. Additionally, terminal (BRCT) domain-mutated breast cancer cell line, in- mutations in the BRCA C-terminal (BRCT) domain of BRCA1 volving heat shock protein (HSP)90-mediated stabilization of create protein folding defects that result in protease-mediated the mutant protein coupled with tumor protein p53 binding degradation (1–3). protein 1 (TP53BP1) gene mutation, which together restore Cells that contain dysfunctional BRCA1 proteins are hyper- DNA end resection and RAD51 filament formation, critical steps sensitive to DNA damaging agents (4). In particular, BRCA1- in HR. Similar events may occur in primary BRCA1-mutated fi de cient cell lines are exquisitely sensitive to poly(ADP-ribose) ovarian cancers as cells develop resistance to platinum. The MEDICAL SCIENCES polymerase (PARP) inhibition (5). Despite initial responses of data demonstrate that, even though BRCA1 BRCT domain BRCA1 -mutant cancers to PARP inhibitor treatment (6), acquired mutant proteins cannot promote DNA end resection, they re- resistance universally develops. Resistance may result from sec- tain partial function and can contribute to RAD51 loading and ondary mutations in the BRCA1 gene that restore the reading Brca1 HR. Finally, HSP90 inhibition may prove useful for resensitizing frame and produce a functional BRCA1 protein (7, 8). In - resistant BRCA1-mutant cancer cells to drug treatment. mutated mouse mammary tumors, activation of p-glycoprotein or loss of p53 binding protein 1 (53BP1) expression resulting from Author contributions: N.J., E.M.S., and G.I.S. designed research; N.J., S.F.J., W.Y., Y.-C.L., Y.-E.C., truncating TP53BP1 mutations confers PARP inhibitor resistance A.J.B., Y.W., M.C., K.A.S., L.A.M., A.W., M.H., J.F.L., and A.M. performed research; N.J., D.C., (9). Loss of 53BP1 in BRCA1-deficient cells provides the C- A.D.D., A.M., E.M.S., and G.I.S. analyzed data; and N.J. and G.I.S. wrote the paper. terminal binding protein interacting protein (CtIP) with un- The authors declare no conflict of interest. restricted access to DNA breaks, facilitating DNA end resection, This article is a PNAS Direct Submission. – an early step in homologous recombination (HR) (9 11). 1Present address: Developmental Therapeutics Program, Fox Chase Cancer Center, Following BRCA1-CtIP–mediated activation of DNA end Philadelphia, PA 19111. resection, eventual BRCA2-mediated assembly of the RAD51 2To whom correspondence may be addressed. E-mail: [email protected] or recombinase in nucleoprotein filaments is a critical step in HR. A [email protected]. role for BRCA1 in RAD51 loading and the mechanisms by which This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. it participates have not been fully clarified. Of note, in PARP 1073/pnas.1305170110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1305170110 PNAS | October 15, 2013 | vol. 110 | no. 42 | 17041–17046 Downloaded by guest on October 2, 2021 decrease in the number of aberrant chromosome structures after treatment with rucaparib compared with the parental cell line, with 10- to 20-fold (P < 0.0001) and 7- to 15-fold (P < 0.0001) feweraberrationsandradialsper cell, respectively (Fig. 1B). To rule out drug efﬂux as a mechanism of PARP inhibitor resistance, we measured the ability of rucaparib to inhibit the PARP enzyme by assessing cellular poly(ADP-ribose) (PAR) levels by Western blot in the absence of activated DNA. Ruca- parib reduced the levels of PAR to a similar degree in MDA- MB-436 parental cells and in all the resistant clones except for RR-1 (Fig. S1A). Of note, clones RR-5 and RR-6 had reduced basal PAR levels. To assess if the lack of PARP inhibition in RR- 1 cells accounted for drug resistance, we used siRNA to deplete PARP-1 and PARP-2 levels. PAR levels were reduced after siRNA treatment (Fig. S1B); however, the colony forming po- tential of RR-1 cells was not signiﬁcantly impacted (Fig. S1C).

Fig. 1. MDA-MB-436 clones are resistant to PARP inhibitors and cisplatin. (A) MDA-MB-436 clones RR-1 to RR-6 were signiﬁcantly more resistant to rucaparib than parental cells (red curve). Cells cultured in the absence of drug for 6 mo remained resistant to rucaparib (+6 mo). Cells were also cross- resistant to olaparib and cisplatin, as measured by colony formation assay (n = 3, mean ± SEM of colonies formed relative to DMSO-treated cells). (B) Metaphase spread analyses of chromosome aberrations and radial for- mations after treatment with rucaparib (1 μM) for 24 h (n = 3, mean ± SEM). (Inset) Representative metaphase spreads.

negative breast cancer cell line MDA-MB-436 in the presence of the PARP inhibitor rucaparib. MDA-MB-436 cells contain a BRCA1 5396 + 1G>A mutation in the splice donor site of exon 20 that results in a BRCT domain-truncated protein (14). Drug- resistant clones, labeled rucaparib-resistant (RR) 1 through 6, Fig. 2. Mutant BRCA1 protein is abundant in MDA-MB-436 resistant clones. emerged ∼2 to 4 mo after initial exposure. Clones were highly (A) BRCA1, RAD51, histone H3, and tubulin levels were measured in cyto- resistant to rucaparib, and cross-resistant to olaparib, as well as plasmic (marked as “c”) and nuclear (marked as “n”) extracts from MCF7 cisplatin (Fig. 1A). Concentrations required to reduce colony cells, MDA-MB-436 parental cells and resistant clones RR-1 to RR-6 by formation by 50% (lethal concentration 50, LC ) were 482- to Western blot. (B) MCF7 cells, MDA-MB-436 parental cells and resistant clones 50 RR-1, RR-5, and RR-6 were treated with DMSO (−)or1μM rucaparib (+) 590-fold (P < 0.0001), 254- to 492-fold (P < 0.0001), 150- to 173- P < P = for 24 h, and BRCA1 protein levels were assessed by using BRCA1 N- or C- fold ( 0.0001), and 27- to 59-fold ( 0.0056) greater than terminal–specific antibodies by Western blot. (C) Detection of BRCA1, those for parental cells for rucaparib, rucaparib after a 6-mo RAD51, γ-H2AX, and DAPI by immunofluorescence in MDA-MB-436 parental holiday from rucaparib selection, olaparib, and cisplatin, respec- and resistant cells (n = 3, mean ± SEM percentage of cells containing more tively. Additionally, MDA-MB-436–resistant clones had a marked than five foci). (Inset) Representative cells.

17042 | www.pnas.org/cgi/doi/10.1073/pnas.1305170110 Johnson et al. Downloaded by guest on October 2, 2021 We conclude that, although rucaparib did not inhibit PARP as effectively in RR-1 cells, additional events may have contributed to rucaparib resistance.

Increased Mutant BRCA1 Protein in Resistant Clones. We next measured BRCA1 and RAD51 protein levels by Western blot. MCF7 cells express WT BRCA1 protein and were used as a positive control. Mutant BRCA1 protein was undetectable in MDA-MB- 436 parental cells, but was abundant in resistant clones. RAD51 protein levels were similar in parental cells and resistant clones (Fig. 2A). To determine whether BRCA1 reversion mutation had occurred, we sequenced BRCA1 gene introns and exons. MDA- MB-436–resistant clones retained the original 5396+1G>Amu- tation, and did not harbor any additional mutations in BRCA1 (Fig. S2A). Furthermore, the BRCA1 mRNA sequences of parental cells and resistant clones were identical (Fig. S2 B and C). The BRCA1 protein detected in resistant clones by an N- terminal BRCA1 antibody was C-terminal truncated and consequently not recognized by a C-terminal–specific antibody (Fig. 2B). The BRCA1 5396+1G>A mutation produces two splice variants (14). We used siRNAs specific to each isoform to determine which variant accounted for the reexpressed protein. MDA-MB-436 parental cells stably expressing an exogenous WT BRCA1 protein (MDA-MB-436+WT) were used as a control for nonspecific BRCA1 protein knockdown. We demonstrated that siRNA specifically targeting the exon 20 deletion variant resulted in knockdown of mutant but not WT BRCA1 protein. Therefore, it is likely that the exon 20 deletion variant accounted for the reexpressed protein in resistant clones (Fig. S2 D and E). The mutant BRCA1 protein could be detected in association with chromatin (Fig. S3). As expected, γ-irradiation–induced BRCA1 foci were not detectable in MDA-MB-436 parental cells; in contrast, BRCA1 foci were readily detectable in resistant clones. Similarly, RAD51 foci were not detected in parental cells, despite the abundance of RAD51 protein; however, resistant clones readily formed RAD51 foci following irradiation. Formation of γ-H2AX foci, a marker of DNA damage, was present to the same degree in parental and resistant cells (Fig. 2C).

Protein Stability Accounts for Increased Mutant BRCA1 Protein. We Fig. 3. HSP90 stabilizes mutant BRCA1. (A) HSP90 was immunoprecipitated next investigated factors that could contribute to changes in from MDA-MB-436 control (GFP) cells, MDA-MB-436+WT cells, and RR clones 1 BRCA1 protein levels in PARP inhibitor-resistant clones. There to 6, and HSP90 and BRCA1 protein levels were analyzed by Western blot (WCE, were no changes in BRCA1 gene copy number (Fig. S4A); ad- whole cell extract). (B) BRCA1 was immunoprecipitated from MDA-MB-436 ditionally, resistant clones demonstrated only a 1.5- to 2.7-fold control (GFP) cells, MDA-MB-436+WT cells, and RR clones 1 to 3, and BRCA1 and (P = 0.0061) increase in BRCA1 mRNA by quantitative RT-PCR HSP90 protein levels were analyzed by Western blot. (C) MDA-MB-436+WT, analyses (Fig. S4B). To determine if increased BRCA1 protein RR-1, RR-5, and RR-6 were treated with 100 nM 17-DMAG for the indicated expression was dependent on transcription or translation, we times, and BRCA1, HSP70, and tubulin protein levels were measured by West- “ ” treated parental and resistant clones with cycloheximide to in- ern blot. (D) RR-1, RR-5, and RR-6 were treated with vehicle (marked as V )or 50 nM 17-DMAG (marked as “D”) in the presence of vehicle (marked as “V”)or hibit protein translation. We could detect a faint BRCA1 band in “ ” = MDA-MB-436 parental cells when we increased protein loading 100 nM rucaparib (marked as R ), and colony formation was assessed (n 3, mean ± SEM of colonies formed relative to vehicle + vehicle-treated cells). and ﬁlm exposure time; however, BRCA1 protein was undetectable at 6 h after cycloheximide treatment. In contrast, BRCA1 protein levels were maintained for as long as 24 h after cyclo- C we investigated the dependency of BRCA1 mutant protein levels heximide treatment in RR clones (Fig. S4 ). These data suggest on HSP90 activity. First, we assessed the association of BRCA1 MEDICAL SCIENCES that the increase in mutant BRCA1 protein in resistant cells was proteins with HSP90 by determining levels of BRCA1 protein likely a result of protein stabilization rather than hyperactivation of in HSP90 immunoprecipitates from MDA-MB-436+WT cells BRCA1 transcription or translation. BRCT domain mutations often result in an inability of the or PARP inhibitor-resistant clones. Mutant and ectopically ex- mutant protein to fold correctly; consequently, the unfolded pressed WT BRCA1 protein from the parental cell line were protein is more susceptible to protease-mediated degradation absent or weakly in complex with HSP90. In contrast, mutant – BRCA1 protein from resistant clones was readily found in (1 3). It is therefore possible that the mutant BRCA1 protein in A MDA-MB-436 parent cells is undetectable as a result of an in- association with HSP90 (Fig. 3 ). Similarly, when we immuno- + ability to correctly fold, with subsequent degradation by the precipitated BRCA1 from MDA-MB-436 WT cells or RR cells, proteasome. Consistent with this hypothesis, MDA-MB-436 pa- HSP90 could only be found in association with the mutant rental cells treated with the proteasome inhibitors MG132 or BRCA1 proteins (Fig. 3B). Next, we treated MDA-MB-436+WT bortezomib had detectable levels of mutant BRCA1 protein, BRCA1 cells, as well as RR cells, with the HSP90 inhibitor suggesting protein was being generated but rapidly degraded due 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17- to folding defects (Fig. S4D). DMAG). WT BRCA1 protein remained at levels comparable to untreated cells at 72 h posttreatment. In contrast, RR cells had HSP90 Stabilizes Mutant BRCA1 Protein. Because HSP90 has been reduced mutant BRCA1 protein levels by 48 h posttreatment. implicated in the folding of cancer-related mutant proteins (15), HSP70 levels increased in response to 17-DMAG, indicating

Johnson et al. PNAS | October 15, 2013 | vol. 110 | no. 42 | 17043 Downloaded by guest on October 2, 2021 HSP90 was inhibited to an equal degree in all cell lines (Fig. 3C). mediated depletion of 53BP1 conferred only a slight degree of Furthermore, 17-DMAG treatment of resistant clones restored resistance to PARP inhibitor treatment in MDA-MB-436 cells sensitivity to rucaparib; compared with DMSO/rucaparib, clonal (3.4-fold increase in rucaparib LC50 value vs. scrambled siRNA survival of RR-1, RR-5, and RR-6 in 17-DMAG/rucaparib was treatment; P = 0.1295, unpaired t test; Fig. S7D). In contrast, reduced 4.7-fold (P < 0.0001), 13.1-fold (P = 0.0007), and 4.9- ectopic expression of WT BRCA1 (MDA-MB-436+WT) con- fold (P = 0.0023), respectively (Fig. 3D). Together, these data ferred substantial rucaparib resistance (426-fold increase in ruca- suggest that HSP90 promotes mutant BRCA1 protein folding parib LC50 value compared with GFP control cells; P < 0.0001, and conformational stability in RR cells. Of note, 17-DMAG unpaired t test; Fig. S7E), similar to that seen in our RR clones treatment also sensitized MDA-MB-436+WT cells to rucaparib (Fig. 1A). These data indicate that disruption of 53BP1 function treatment (Fig. S5A), likely mediated through a reduction in alone could not fully account for the resistance acquired by the BRCA2 and RAD51 protein levels (16) (Fig. S5B). MDA-MB-436 clones derived under rucaparib selection pressure. Because 53BP1 deletion was previously shown to provide Reduced 53BP1 Facilitates BRCA1-Independent DNA End Resection. PARP inhibitor resistance in mouse Brca1 mutant cell lines, we We next analyzed the contribution of stabilized mutant BRCA1 further investigated the effect of 53BP1 depletion on additional in RR cells to two critical steps of HR, DNA end resection and human BRCA1 mutated cancer cell lines, including SUM1315 RAD51 loading. First, we investigated the ability of exogenous (185delAG) and HCC1395 (5251C>T). Consistent with the data WT BRCA1 in parental cells and the C-terminal truncated in MDA-MB-436 cells, siRNA mediated-depletion of 53BP1 in mutant BRCA1 protein in resistant clones to interact with SUM1315 and HCC1395 cells conferred a 5.1-fold and 5.7-fold proteins known to complex with BRCA1. Analyses of immuno- increase in rucaparib LC50 value compared with scrambled precipitated exogenous WT BRCA1 protein from MDA-MB-436+ siRNA treatment (P = 0.145 and P = 0.083, unpaired t test), WT cells demonstrated that BARD1, PALB2, BRCA2, RAD51, respectively (Fig. S7 F and G). CtIP, and RAP80 could all be detected in association with WT We hypothesized that the reduction in 53BP1 protein levels in BRCA1. Similarly, BARD1, PALB2, BRCA2, and RAD51 PARP inhibitor-resistant clones enabled CtIP to activate DNA associated with endogenous mutant BRCA1 protein immu- end resection and RPA32 loading in the absence of CtIP– noprecipitated from resistant clones. However, the BRCT do- BRCA1 protein interaction. We demonstrated that a twofold main interacting proteins CtIP and RAP80 were not found to increase (by densitometry) in 53BP1 protein levels in RR-1 cells interact with the C-terminal truncated BRCA1 protein from engineered to express ectopic WT 53BP1 resulted in a 1.5-fold MDA-MB-436 resistant cells (Fig. S6A). (P = 0.005, unpaired t test) and 1.3-fold (P = 0.025, unpaired DNA end resection is dependent on the activities of BRCA1 t test) reduction in the percentage of RPA32 and RAD51 foci- and CtIP proteins (17, 18). We investigated the role of the mu- positive cells compared with control RR-1 cells, respectively tant BRCA1 protein in DNA end resection by measuring the (Fig. S8A). Furthermore, reexpression of 53BP1 increased RR-1 formation of RPA32 foci after γ-irradiation (Fig. S6 B and C). sensitivity to PARP inhibitor treatment with a twofold decrease + MCF7 and MDA-MB-436 WT cells express WT BRCA1 pro- (P = 0.049, unpaired t test) in the LC50 value of rucaparib tein and were used for comparison with mutant BRCA1 pro- compared with control cells (Fig. S8B). teins. Depletion of WT BRCA1 by using three individual siRNAs resulted in a fourfold (P = 0.0001) and 3- to 15-fold (P = RAD51 Focus Formation Is Dependent on Mutant BRCA1. To de- 0.0011) decrease in the formation of RPA32 foci compared with termine why decreased 53BP1 protein levels conferred only scrambled siRNA control-treated MCF7 and MDA-MB-436+ modest PARP inhibitor resistance in MDA-MB-436 cells, we WT cells, respectively. In contrast, depletion of mutant BRCA1 studied RAD51 assembly following DNA damage in these cells. protein from RR clones RR-1 and RR-5 did not affect the Of note, RNF8 and RNF168 have been implicated in RAD51 formation of RPA32 foci. Depletion of CtIP by using three loading during HR in the absence of BRCA1 and 53BP1 (21). individual siRNAs resulted in a three- to fivefold (P < 0.0001), 3- However, levels of these proteins remained unchanged in re- to 15-fold (P = 0.0042), 5- to 21-fold (P < 0.0001), and 13- to sistant clones (Fig. S8C). 25-fold (P < 0.0001) decrease in the formation of RPA32 foci We measured the effect of 53BP1 depletion on RPA32 and compared with scrambled siRNA control-treated MCF7, MDA- RAD51 foci after γ-irradiation treatment in MDA-MB-436 cells MB-436+WT, RR-1, and RR-5 cells, respectively (Fig. S6C). engineered to express GFP control or exogenous WT BRCA1 These data indicate that the truncated C-terminal BRCA1 pro- (Fig. 4A). Depletion of 53BP1 increased RPA32 foci 3.4-fold tein that did not interact with CtIP did not influence DNA end (P < 0.001) and 4.9-fold (P < 0.001) in MDA-MB-436 control (+ resection, and that CtIP activates DNA end resection independently GFP) and MDA-MB-436+WT cells, respectively. Therefore, the of BRCA1 interaction in MDA-MB-436 resistant clones. presence or absence of BRCA1 protein did not affect the in- TP53BP1 mutation and loss of function have been demon- crease in formation of RPA32 foci after 53BP1 depletion. In strated to confer PARP inhibitor resistance (9–11). We se- contrast, depletion of 53BP1 resulted in a 3.3-fold increase (P < quenced TP53BP1 gene introns and exons in parental cells and 0.001) in RAD51 foci in MDA-MB-436+WT cells that contained resistant clones. MDA-MB-436 parental cells contained homo- WT BRCA1 protein; however, RAD51 foci remained completely zygous WT TP53BP1 gene sequences. In contrast, all resistant absent in MDA-MB-436 control cells. Similar to MDA-MB-436+ clones contained a heterozygous 3708 del 11 mutation located WT cells, depletion of 53BP1 in MCF7 cells expressing en- in exon 18 (Fig. S7A). This was a microhomology-mediated de- dogenous WT BRCA1 also resulted in a 2.2-fold (P < 0.005) and letion (Fig. S7B), a mechanism of deletion common to BRCA1/2- 2.4-fold (P < 0.001) increase in RPA32 and RAD51 focus for- mutant cancers (19). The mutation creates a frameshift and an mation, respectively (Fig. S8 D and E). Therefore, in MDA-MB- early stop codon predicted to produce a truncated protein (p. 436 cells that lack BRCA1 protein, reducing 53BP1 protein P1235PfsX37). Consistent with a heterozygous TP53BP1 gene levels enabled CtIP to activate DNA end resection and enhance loss-of-function mutation, PARP inhibitor-resistant clones had RPA32 loading, but did not facilitate efficient RAD51 recruit- lower levels of 53BP1 protein compared with parental cells (Fig. ment. Consequently, 53BP1 depletion did not afford dramatic S7C). 53BP1 protein was reduced four- to sevenfold compared PARP inhibitor resistance in MDA-MB-436 parental cells, in with parental cells (by densitometry), a greater reduction than contrast to the more substantial effects of 53BP1 depletion in expected from loss of one allele, suggesting possible transcrip- other model systems (10, 11). tional silencing of the remaining WT allele. MDA-MB-436 resistant clones readily form RAD51 foci (Fig. To investigate if loss of function of 53BP1 accounted for 2C). We therefore investigated the role of the mutant BRCA1 PARP inhibitor resistance, we depleted 53BP1 from parental protein in restoring RAD51 focus formation. Depletion of BRCA1 MDA-MB-436 cells by using siRNA and measured PARP in- by using three individual siRNAs abolished formation of RAD51 hibitor sensitivity. Consistent with a previous report (20), siRNA- foci in MDA-MB-436 resistant clones, indicating that recruitment

17044 | www.pnas.org/cgi/doi/10.1073/pnas.1305170110 Johnson et al. Downloaded by guest on October 2, 2021 Fig. 4. Mutant BRCA1 protein promotes RAD51 focus formation. (A) MDA-MB-436 control (GFP) and MDA-MB-436+WT BRCA1 cells (WT) were treated with scrambled (Sc) or 53BP1 siRNA and fixed 6 h after γ-irradiation. RPA32 and RAD51 foci were detected by immunofluorescence. (Left) Western blot dem- onstrating 53BP1 knockdown and images of representative DAPI-stained cells. (Right) Quantification of RPA32 and RAD51 foci (n = 3, mean ± SEM percentage of cells containing more than five foci). (B) MCF7 and MDA-MB-436 resistant clones RR-1, RR-5, and RR-6 were left untreated (−) or treated with scrambled (Sc) control or three individual BRCA1 siRNAs. After 72 h, cells were fixed 6 h after γ-irradiation treatment. (Left) BRCA1 and RAD51 protein knockdown measured by Western blot and images of representative RR-1 cells after detection of BRCA1, RAD51, γ-H2AX and DAPI by immunofluorescence. (Right) Quantification of foci positive cells (n = 3, mean ± SEM percentage of cells containing more than five foci). (C) MDA-MB-436 parental cells or resistant clones were treated with scrambled (Sc) or three individual BRCA1 siRNAs, exposed to increasing concentrations of rucaparib for 72 h and replated for colony formation. Colony formation was calculated as in Fig. 1A (n = 3, mean ± SEM of colonies formed relative to DMSO-treated cells).

of RAD51 following DNA damage was dependent on mutant 149101 had an increase in BRCA1 staining combined with a re- BRCA1 protein. siRNA-mediated depletion of BRCA1 had no duction in 53BP1 staining, and was resistant to platinum (Fig. 5). γ effect on the formation of -H2AX foci or RAD51 protein levels Discussion MEDICAL SCIENCES (Fig. 4B). Additionally, siRNA-mediated BRCA1 depletion dramatically resensitized resistant clones, but not already-sensitive Mutations in the BRCT domains of BRCA1 often prevent parental cells, to PARP inhibitor treatment. Following expres- proper protein folding, and misfolded proteins are subject to – sion of BRCA1 siRNA, rucaparib LC50 values were reduced 1- to protease-mediated degradation (1 3). In the present study, we 3.6-fold (P = 0.3), 132- to 175-fold (P < 0.0001), 69- to 153-fold show that, under PARP inhibitor selection pressure, HSP90 (P < 0.0001), and 33- to 115-fold (P < 0.0001) compared with interacts with and stabilizes mutant BRCA1 proteins. The sta- scrambled siRNA treatment in MDA-MB-436 parental, RR-1, bilized C-terminal truncated protein is semifunctional, as it is RR-5, and RR-6 cells, respectively (Fig. 4C). unable to interact with CtIP, but retains the protein domains necessary to mediate interactions with PALB2-BRCA2-RAD51 Increased Mutant BRCA1 Protein in Human Cancers. We assessed (12, 22). Importantly, the mutant BRCA1 protein is capable of a panel of carboplatin-treated recurrent BRCA1 mutant ovarian promoting RAD51 loading onto DNA following DNA damage. carcinomas for platinum sensitivity, secondary BRCA1 reversion Because the BRCT domain-deﬁcient BRCA1 protein is in- mutations, and increased BRCA1 and decreased 53BP1 staining capable of interacting with CtIP, cells require further genetic (Table S1). Increased BRCA1 protein expression in the absence adaptations to survive selection pressure. In our model, PARP of BRCA1 reversionmutationoccurredintwooffourtumors inhibitor-resistant MDA-MB-436 cells had reduced 53BP1 protein carrying BRCT domain mutations (5382insC to 5622C>T). Similar levels as a result of a heterozygous loss-of-function mutation, an to RR MDA-MB-436 cells, the recurrent carcinoma from patient event that provided CtIP with unrestricted access to DNA ends

Johnson et al. PNAS | October 15, 2013 | vol. 110 | no. 42 | 17045 Downloaded by guest on October 2, 2021 irrespective of the primary BRCA1 mutation, this event may be obligatory in BRCT domain-mutated cells so that end-resection can occur in the absence of a BRCA1 BRCT domain–CtIP interaction. Finally, our data suggest that HSP90 inhibition may reverse PARP inhibitor resistance and may be a rational strategy particularly germane in BRCA1 BRCT domain mutant cancers. Materials and Methods Cell Culture. MDA-MB-436 cells were cultured in the presence of gradually increasing concentrations of rucaparib until resistant clones emerged that grew in 1 μM drug. Colonies were labeled RR 1 through 6. Cells were cultured in the absence of rucaparib for at least 1 mo before they were used for experiments. Cells were routinely analyzed 6 h after 10 Gy γ-irradiation treatment.

Genomic Manipulations and Immunoprecipitation. Lentiviral generation and infections and siRNA transfections were carried out according to standard protocols. Protein knockdown or reexpression was routinely assessed 72 h after transfection or 96 h after infection. BRCA1 or HSP90 complexes were recovered from 2 mg of nuclear extract by using the Pierce Classic IP Kit (Thermo Scientiﬁc) according to the manufacturer’s instructions.

Preparation of DNA. Genomic DNA was isolated from cells using the DNeasy tissue kit (Qiagen). BRCA1 gene sequencing was carried out as previously Fig. 5. Increased mutant BRCA1 protein in a platinum-resistant ovarian car- described (7). SNP chip array was carried out using the human SNP 6.0 array ’ cinoma. BRCA1 and 53BP1 protein levels measured by immunohistochemistry according to the manufacturer s instructions (Affymetrix). Total RNA was ﬁ from patient 149101. Representative stains of biopsies taken from the plati- isolated from cell lines by using TRIzol (Invitrogen) and puri ed by using an num-sensitive primary ovarian tumor and the recurrent resistant tumor. RNeasy cleanup kit (Qiagen).

Statistics. Mean and SE values were compared by using unpaired t tests. and facilitated BRCA1-independent DNA end resection (10, 11). For multiple comparisons we used one-way ANOVA. P < 0.05 was con- Consistent with other studies, 53BP1 depletion alone contributed sidered statistically significant. to PARP inhibitor resistance (10, 11), but conferred only a slight degree of resistance in this BRCA1 mutant human cancer cell line Further Details. Further details are provided in SI Materials and Methods. model (20). Although 53BP1 depletion hyperactivated DNA end resection and RPA32 loading, without stabilization and increased ACKNOWLEDGMENTS. Rucaparib and olaparib were supplied by Clovis and AstraZeneca, respectively. This work was supported by US National Institutes expression of the mutant BRCA1 protein, RAD51 assembly could of Health Grants R01 CA090687 (to G.I.S.), P50 CA089393 [Dana-Farber/ not occur following DNA damage. Harvard Cancer Center (DF/HCC) Specialized Program of Research Excellence The present study shows inherent partial function of a BRCT (SPORE) in Breast Cancer (to G.I.S.)], P50 CA090578 [DF/HCC SPORE in Lung domain-mutated BRCA1 protein that can contribute to HR. Cancer (to G.I.S.)], P50 CA83636 [Pacific Ovarian Cancer Research Consortium Other studies have demonstrated functionality of N-terminal SPORE in Ovarian Cancer (to E.M.S.)], P30 CA006927 [Fox Chase Cancer missense mutations, and knock-in Brca1-deficient mouse models Center Developmental New Investigator Funds (to N.J.)], and R01 CA142698 (to D.C.); Susan G. Komen Investigator Initiated Research Grant 12223953 (to expressing these mutants responded poorly to platinum drugs, G.I.S.); Susan G. Komen Career Catalyst Award CCR12226280 (to N.J.); the mitomycin C, or PARP inhibition (23, 24). Additionally, although Wendy Feuer Ovarian Cancer Research Fund (to E.M.S.); and American a reduction in 53BP1 expression may facilitate DNA end resection Cancer Society Research Scholar Grant RSG-12-079-01 (to D.C.).

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