Threonine 2609 phosphorylation of the DNA-dependent ......2012/08/23 · 100-permeabilized cells...

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Threonine 2609 phosphorylation of the DNA-dependent Protein Kinase is a critical prerequisite for

epidermal growth factor receptor mediated radiation resistance

Prashanthi Javvadi*, Haruhiko Makino*, Amit K. Das*, Yu-Fen Lin*, David J. Chen*†, Benjamin P. Chen*†,

and Chaitanya S. Nirodi*†.

*Departments of Radiation Oncology and †Simmons Comprehensive Cancer Center, University of Texas

Southwestern Medical Center, Dallas, Texas, USA.

Running Title: Radiation induced EGFR-DNA-PKcs interactions Conflict of Interest: NONE. The authors of this manuscript have no conflicts of interest. Correspondence Chaitanya S. Nirodi, Ph.D. University of Texas Southwestern Medical Center Department of Radiation Oncology Division of Molecular Radiation Biology 2201, Inwood Road, NC 7.208 Mail code 9187 Dallas, TX 75390 Telephone: 214-648-7318 Fax: 214-648-5595 E-mail: [email protected]

on July 11, 2021. © 2012 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 23, 2012; DOI: 10.1158/1541-7786.MCR-12-0482-T

http://mcr.aacrjournals.org/

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Abbreviations DNA-PK: DNA-dependent protein kinase

DNA-PKcs: DNA-dependent protein kinase, catalytic subunit

DSB: double strand breaks

EGFR: epidermal growth factor receptor

Gy: Gray, unit of ionizing radiation equivalent to 1 joule per kilogram

HBEC: human bronchial epithelial cell

IR: ionizing radiation

NHEJ: non-homologous end joining

NSCLC: non-small cell lung carcinoma

PLA: proximity ligation assay

SF: surviving fraction




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Abstract

The epidermal growth factor receptor (EGFR) contributes to tumor radioresistance, in part, through

interactions with the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs), a key enzyme in the

non homologous end joining DNA repair pathway. We previously demonstrated that EGFR-DNA-PKcs

interactions are significantly compromised in the context of activating mutations in EGFR in non small cell

lung carcinoma (NSCLC) and human bronchial epithelial cells. Here, we investigate the reciprocal

relationship between phosphorylation status of DNA-PKcs and EGFR-mediated radiation response. The

data reveal that both the kinase activity of DNA-PKcs and radiation-induced phosphorylation of DNA-

PKcs by the Ataxia Telangiectasia Mutated (ATM) kinase are critical prerequisites for EGFR-mediated

radioresponse. Alanine substitutions at 7 key serine/threonine residues in DNA-PKcs or inhibition of DNA-

PKcs by NU7441 completely abrogated EGFR-mediated radioresponse and blocked EGFR binding. ATM-

deficiency or ATM inhibition with KU55933 produced a similar effect. Importantly, alanine substitution at

an ATM-dependent DNA-PKcs phosphorylation site, T2609, was sufficient to block binding or

radioresponse of EGFR. However, mutation of a DNA-PKcs auto-phosphorylation site, S2056 had no such

effect indicating that DNA-PKcs auto-phosphorylation is not necessary for EGFR-mediated radioresponse.

Our data reveal that in both NSCLCs and HBECs, activating mutations in EGFR specifically abolished the

DNA-PKcs phosphorylation at T2609, but not S2056. Our study underscores the critical importance of a

reciprocal relationship between DNA-PKcs phosphorylation and EGFR mediated radiation response and

elucidates mechanisms underlying mutant EGFR associated radiosensitivity in NSCLCs.




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Introduction

The epidermal growth factor receptor (EGFR), a 170 kDa receptor tyrosine kinase, is an important

determinant of tumor resistance to ionizing radiation (IR) in a number of cancers including non-small cell

lung cancer (NSCLC). In addition to cell proliferation, (1-3) and apoptosis inhibition (4, 5), EGFR has a

direct role in the repair of IR-induced double strand breaks (DSB) (6) and (reviewed in (7, 8)).

Evidence from a number of laboratories shows that, in response to IR, EGFR is rapidly internalized and

translocates to the nucleus (9-11). Moreover, nuclear EGFR has been shown to interact with the catalytic

and regulatory subunits of the DNA-dependent protein kinase (DNA-PK) in an IR-dependent manner (6,

9). The precise domains in EGFR and DNA-PK that are involved in this interaction are not known.

DNA-PK plays an important role in the non homologous end joining DNA repair pathway (NHEJ). It is

composed of the regulatory DNA binding heterodimer, Ku70/80, and a catalytic subunit, DNA-PKcs.

Ku70/80 heterodimer binds broken DNA ends of DSBs. Two molecules of DNA-PKcs are then recruited to

the DNA break (12). In response to radiation, DNA-PKcs is rapidly activated and phosphorylated at several

serine and threonine residues which are organized into distinct clusters (13). These clusters include the

2609 (or ABCDE) cluster (14, 15), the 2056 (or PQR) cluster (16) and a C’ terminal site (17, 18). IR-

induced serine 2056 (S2056) auto-phosphorylation (19) is mediated by DNA-PKcs itself, but the

phosphorylation of many residues in the 2609 and 2056 clusters, particularly threonine 2609 (T2609), is

mediated by the Ataxia Telangiectasia Mutated kinase (ATM) (20). The auto-phosphorylation of the 2609

cluster promotes end-processing (13), notably through the activity of the Artemis endonuclease (21), while

the auto-phosphorylation at the 2056 cluster inhibits end processing and promotes ligation of DNA ends

(16). Both DNA-PKcs S2056 auto-phosphorylation and ATM-mediated T2609 phosphorylation appear to

be essential for DNA-PKcs-mediated DSB repair and radioresistance (20). Whether the phosphorylation

status of DNA-PKcs affects EGFR-mediated radiation response is not known.




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Somatic activating mutations in EGFR have been clinically linked to dramatic responses in NSCLC

patients to the EGFR inhibitors, gefitinib and erlotinib (22-25). We previously demonstrated that NSCLCs

harboring either an in-frame deletion (ΔE746-E750) in the 19th exon or an leucine to arginine substitution

(L858R) in the 21st exon of the EGFR tyrosine kinase domain exhibit dramatic sensitivity to IR (26).

Moreover, ectopic expression of L858R or ΔE746-E750 EGFR in different NSCLC cells lines or HBEC

cells significantly reduced cellular radioresistance in a dominant negative manner (26). We showed that

mutant EGFR associated radiosensitivity manifests as pronounced delays in repair of IR induced DSBs,

inhibition of IR-induced nuclear translocation and absence of IR-induced EGFR-DNA-PKcs interactions

(27). However, how mutant EGFR expression affects DNA-PKcs activity and function is not fully

understood.

Here, we ectopically express wild type, L858R and ΔE746-E750 forms of EGFR and evaluate their relative

contributions to clonogenic survival in the genetic background of various site-specific, phospho-ablating

mutations in DNA-PKcs. We interrogate key DNA-PKcs residues for their ability to modulate IR-induced

interactions between EGFR and DNA-PKcs and support EGFR-mediated radiation response. Our search

reveals that IR-induced phosphorylation of T2609 in DNA-PKcs is a critical requirement for this

interaction. Surprisingly, T2609 phosphorylation is also under the influence of EGFR. Our data support a

model in which EGFR modulates DNA-PKcs function through stabilization of T2609 phosphorylation.

Materials and Methods

Cell culture

The NSCLC cell lines, NCI-A549, NCI-H820, NCI-HCC827 and NCI-H1975 were from the American

Type Culture Collection (ATCC). The immortalized Human Bronchial Epithelial (HBEC) cell line was

originally obtained from John D. Minna (UT Southwestern Medical center) (28). All cell lines were




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maintained as previously described (27). CHO cell lines,V3-7A, V3-WT DNA-PKCS, V3-S2056A and

V3-T2609A, and fibroblast cell lines, 1BR3, and AT5, were a generous gift from Dr. David J. Chen and

were maintained as previously described (20, 29). The wild-type EGFR, L858R, and ΔE746-E750 forms of

EGFR were tagged with V5 epitope in lentiviral vectors through recombinational cloning using the

Gateway system (Invitrogen/Gibco-BRL, Carlsbad, CA). Immortalized HBEC or CHO cell lines, were

genetically modified by lentivirus infection of V5-tagged EGFR forms or an unrelated LacZ construct and

maintained as previously described (28, 30).

Clonogenic cell survival assay

Clonogenic survival was measured as described before (26, 27). Where inhibitors were used, cells received

a 2 h pretreatment with vehicle, 10 μM NU7441, or KU55933 prior to irradiation and were plated at

various densities 8 h later (delayed plating). Mean SF was plotted as a function of radiation dose from ≥3

independent experiments, each performed in triplicate samples per dose. Curves were fitted to the linear

quadratic equation.

Co-immunoprecipitation (Co-IP) and Western blot (WB) assays

EGFR was immunoprecipitated using an anti-EGFR antibody (Clone R19/48, Biosource,44-796G) and

DNA-PKcs and EGFR were detected by Western blot (WB) using anti-DNA-PKcs, and EGFR antibodies

as described previously (27). Phosphorylated ATM was detected by WB assay using p-ATM antibody (p-

S1981, 200-301-400, Rockland Inc,) and blots were stripped and re-probed with ATM antibody (5C2,

GeneTex).

Proximity ligation assay for protein-protein interactions




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Stable complexes of EGFR and DNA-PKcs or PP2A and DNA-PKcs were detected using the Duolink®

proximity ligation assay (PLA) kit according to the manufacturer’s instructions (Olink Bioscience,

Uppsala, Sweden). NSCLC or HBEC cells expressing wild type, L858R and ΔE746-E750 mutant EGFR

were exposed to 4 Gy IR. For inhibition experiments, cells were pretreated for 2 h prior to IR with 10 μM

of NU7441 or KU55933. At various time points, cells were fixed for immunofluorescence staining as

described before (27) and simultaneously incubated with mouse DNA-PKcs antibody (Clone 25-4, Lab

Vision, dilution 1:150) and rabbit anti-EGFR antibody (Santa Cruz, sc-03, 1:1000) or rabbit anti-PP2A

antibody (Clone 81G5, Cell Signaling, S2041, 1:100 dilution). Cells were incubated with complementary

oligonucleotide-conjugated anti-rabbit and anti-mouse secondary antibodies followed by ligation and

rolling circle amplification in the presence of a Texas Red conjugated nucleotide. The fluorescent

amplicons manifest as red fluorescent dots, with each dot representing a specific and stable interaction

between the two interacting proteins. Cells were co-stained with 4',6-Diamidino-2-phenylindole (DAPI)

and images were acquired using Zeiss Axiophot fluorescence microscope with a 40x objective. After

correcting for illumination, integrated fluorescence intensity of foci in 600-800 nuclei per experiment was

measured using the Cell profiler image analysis software (31). Mean integrated fluorescence intensity per

nucleus and standard error of means from ≥3 independent experiments was used to quantify EGFR-DNA-

PKcs or PP2A-DNA-PKcs binding at various time points following IR.

Quantification of phosphorylated forms of DNA-PKcs by immunocytochemical staining

DNA-PKcs phosphorylation in response to 4 Gy IR was measured by staining formalin fixed, Triton-X-

100-permeabilized cells with antibodies against pT2609 or S2056 (20) which were detected by Alexa-488-

conjugated secondary antibodies. Images were acquired using a 40x objective of the Zeiss Axiophot

fluorescence microscope. After correcting for illumination, integrated fluorescence intensity of phospho

DNA-PKcs per nucleus in 450-600 nuclei per experiment was measured using the Cell profiler image




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analysis software (31). Mean integrated fluorescence intensity per nucleus and standard error of means

from ≥2 independent experiments were reported.

Results

Mutations in the 2609 and 2056 clusters of DNA-PKcs abrogate EGFR-mediated radiation response

To examine the effect of DNA-PKcs phosphorylation on wild type or mutant EGFR-mediated radiation

responses, we stably expressed the wild type, L858R, or ΔE746-E750 forms of EGFR in three different

DNA-PKcs backgrounds: DNA-PKcs-deficient V3 CHO cells, V3 cells stably expressing wild type human

DNA-PKcs (V3-WT) or V3 cells stably expressing a catalytically active, but DSB-repair defective, mutant

form of human DNA-PKcs (V3-7A) in which 7 alanine replacements in the 2609 and 2056 clusters

(Figure 1A and B). Consistent with previous observations (27), in V3-WT DNA-PKCS cells, ectopic

expression of wild type EGFR significantly increased clonogenic survival, whereas expression of the

L858R and ΔE746-E750 activating mutant forms of EGFR had a pronounced dominant-negative

radiosensitizing effect relative to untransfected cells (Figure 1C, middle). This contrasting pattern of

radiation responses associated with wild type and mutant EGFR expression was completely abrogated in

V3 cells (Figure 1C, left) or V3-7A cells (Figure 1C, right). The data indicate EGFR-mediated radiation

response is not only DNA-PKcs-dependent but also requires phosphorylation of residues in the 2056 and

2609 clusters of DNA-PKcs.

ATM deficiency abrogates EGFR-mediated radiation responses

Previous reports have shown that IR-induced phosphorylation of many of the residues in the 2056 and 2609

cluster of DNA-PKcs is ATM-dependent (20). To test whether ATM-deficiency has any effect on EGFR

mediated effects on cellular radiosensitivity, we over-expressed wild type, L858R, or ΔE746-E750 forms of

EGFR in ATM-proficient, 1BR3, or ATM-deficient, AT5, backgrounds (Figure 2A). The data in Figure 2B




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show that both wild type EGFR-mediated radioprotection and mutant EGFR-mediated radiosensitization

are intact in 1BR31 cells. In AT5 cells, on the other hand, expression of either wild type or mutant EGFR

did not significantly affect clonogenic survival relative to mock- or LacZ-transfected cells. The data

indicate that IR-induced ATM-driven DNA-PKcs phosphorylation is an essential requirement for EGFR-

mediated radiation response.

DNA-PKcs phosphorylation of 2609 and 2056 cluster is critical for IR-induced binding to EGFR

A key step in EGFR-mediated radiation response requires binding of EGFR and DNA-PKcs which is

abrogated by activating mutations, L858R or ΔE746-E750 in EGFR (27) and supplemental figure, S1C. We

first tested whether phospho-ablating mutations in 7A-DNA-Pkcs affected EGFR binding. Figure 3A shows

that in V3-WT cells, interactions between wild type DNA-PKcs and wild type EGFR occurred as early as 5

minutes following 4 Gy IR, persisted until 90 minutes and diminished shortly thereafter. As expected, WT

DNA-PKcs failed to co-precipitate with L858R or the ΔE746-E750 mutant form of EGFR (27).

Interestingly, the 7A mutant form of DNA-PKcs was undetectable in immune complexes of wild type or

mutant EGFR. The data indicate that alanine substitution in 7 of the 11 phosphorylation sites in DNA-

PKcs completely abolished radiation-induced binding of EGFR. We reasoned that the reported abrogation

of DNA-PKcs phosphorylation in the context of ATM-deficiency (20) should similarly affect EGFR-DNA-

PKcs binding. As expected, DNA-PKcs co-precipitated with wild type EGFR (Figure 3B) in ATM-

proficient 1BR31 fibroblast cells but such interaction was undetectable in ATM-deficient AT5 cells even at

90 minutes following IR. The data indicate that radiation-induced interactions between EGFR and DNA-

PKcs are dependent, in part, on ATM-driven phosphorylation of DNA-PKcs.

Inhibition of DNA-PKcs or ATM abrogates EGFR DNA-PKcs interactions and EGFR-mediated

radioresponse




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We next examined whether EGFR-mediated radioresponses and radiation-induced EGFR-DNA-PKcs

binding are dependent on the kinase activity of DNA-PKcs or ATM. NU7441 (2-N-morpholino-8-

dibenzothiophenyl-chromen-4-one) and KU55933 (2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one) are

highly potent, selective inhibitors of DNA-PKcs and ATM respectively (32, 33). In HBEC cells, a 2 hour

pre-treatment with either NU7441 or KU55933 completely abolished the survival responses associated with

both wild type and mutant EGFR (Figure 4A). Moreover, NU7441 or KU55933 pretreatment completely

eliminated IR-induced associations between DNA-PKcs and EGFR in co-immunoprecipitation assays

(Figure 4B). To quantify the effects of NU7441 or KU55933 on EGFR-DNA-PKcs binding in NSCLCs

and HBEC we used the proximity ligation assay (PLA). PLA relies on in situ detection of protein-protein

interactions through a fluorescent signal which is generated only when two interacting proteins are

physically associated with each other. PLA not only allows the quantitative assessment of protein-protein

interactions but also reveals the sub-cellular location where they predominate. Figure 4C shows that in wild

type EGFR expressing A549 cells, relative to untreated cells, at 1 hour following 4 Gy IR, there was a

significant (12.5-fold) increase in EGFR-DNA-PKcs complexes, occurring predominantly in the nuclear

region of the cells. Both basal and radiation-induced PLA fluorescence was undetectable in the ΔE746-

E750 expressing H820 cells. More importantly, NU7441 or KU55933 treatment completely abrogated both

basal and radiation-induced EGFR-DNA-PKcs complexes in A549 cells and showed no appreciable change

over baseline in H820 cells. In the isogenic settings of HBEC cells (Figure 4C, bottom panel), NU7441 or

KU55933 pre-treatment similarly eliminated the ~200-fold IR-induced increase in EGFR-DNA-PKcs

associations in wild type EGFR expressing HBEC cells. By contrast, cells expressing the ΔE746-E750

EGFR showed no such increase and were not further affected by treatment with either inhibitor. The data

indicate that kinase activities of both, DNA-PKcs and ATM, are critical for the radiation-induced binding

of DNA-PKcs and EGFR in NSCLCs and HBEC.




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DNA-PKcs phosphorylation at T2609 but not S2056 is critical for radiation-induced binding to EGFR

Previous studies demonstrate that radiation-induced phosphorylation of DNA-PKcs at T2609 in the 2609

cluster is ATM-dependent while S2056 is auto-phosphorylated by DNA-PKcs in an ATM-independent

manner (20). Our next objective was to examine whether alanine substitution at either of these residues

would affect EGFR-DNA-PKcs binding and in turn alter EGFR-mediated survival responses. Towards this

end, we stably expressed wild type and mutant EGFR in V3 cells expressing either a S2056A-mutated (V3-

S2056A) or T2609A-mutated (V3-T2609A) forms of DNA-PKcs (Figure 5A). V3-S2056A cells were more

radiosensitive compared to V3-T2609A cells (Figure 5B). However, only V3-S2056A cells exhibited the

characteristic decrease in radiosensitivity associated with wild type EGFR and increase in radiosensitivity

typical of L858R or ΔE746-E750 EGFR. By contrast, survival of V3-T2609A cells was strikingly

unresponsive to wild type or mutant EGFR expression, indicating that T2609 was crucial in supporting

EGFR-mediated survival response to radiation. We compared IR-induced binding of EGFR with S2056A

or T2609A mutant forms of DNA-PKcs. In V3-S2056A cells we observed a robust 40-fold IR-induced

increase in nuclear complexes of EGFR and the S2056A mutant DNA-PKcs (Figure 5C). In V3-T2609A

cells, however, IR-induced complexes between EGFR and the T2609A mutant DNA-PKcs were virtually

undetectable. Co-immunoprecipitiation assay essentially confirmed these findings in Figure 5D. The data in

Figure 5 offer compelling evidence that DNA-PKcs phosphorylation at T2609, but not S2056 is critical for

radiation-induced EGFR-DNA-PKcs binding and EGFR-mediated radioresponses.

DNA-PKcs phosphorylation at T2609 but not S2056 is abrogated in mutant EGFR expressing NSCLCs and

HBEC cells

Radiation-induced EGFR-DNA-PKcs interactions are absent in cells expressing the L858R or ΔE746-E750

mutant forms of EGFR (Figure 3A and (27)). We investigated whether this had any effect on the

phosphorylation status of DNA-PKcs. We used an immune-fluorescence microscopy approach to quantify




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the extent and sub-cellular location of DNA-PKcs phosphorylation. We focused on two specific residues.

Phosphorylation of T2609 is ATM-dependent, whereas S2056 is auto-phosphorylated by DNA-PKcs in an

ATM-independent manner (20). Figure 6A shows that, in wild type EGFR expressing HBEC cells, DNA-

PKcs phosphorylation at both, T2609 and S2056 rapidly increased in response to 4 Gy. Cells expressing the

L858R or ΔE746-E750 forms of EGFR, showed a similar IR-induced increase in S2056 phosphorylation.

Surprisingly, IR-induced DNA-PKcs phosphorylation at T2609 was virtually undetectable even at 60

minutes following IR. The data indicate that mutant EGFR expression specifically abrogates radiation-

induced phosphorylation of DNA-PKcs at T2609 but not S2056.

We next verified the relationship between EGFR mutation status and T2609 phosphorylation in NSCLC

cell lines. Exposure of wild type expressing A549 NSCLC to 4 Gy resulted in a ~1000-fold increase in

DNA-PKcs phosphorylation at T2609 (Figure 6C). Both basal and radiation-induced DNA-PKcs

phosphorylation was undetectable in the L858R expressing H1975 cells or the ΔE746-E750 expressing

H820 cells. However, HCC827, which also harbors the ΔE746-E750 mutant, exhibited a ~75-fold increase

in T2609 phosphorylation with radiation, although this was dramatically lower than the ~1000-fold increase

observed in A549 NSCLCs. Moreover, while IR-induced T2609 phosphorylation in A549 cells was

NU7441-insensitive, but KU55933-sensitive, IR-induced T2609 phosphorylation in HCC827 NSCLC cells

was unaffected by either ATM- or DNA-PKcs-inhibition, indicating a mechanism unrelated to ATM or

DNA-PKcs.

Mutant EGFR expression reverses IR-induced decrease in DNA-PKcs-PP2A binding

The data in Figure 6 indicate that activating mutations in EGFR adversely influence radiation-induced

phosphorylation of DNA-PKcs at T2609. We considered two possibilities. First, we examined whether

mutant EGFR directly influences DNA-PKcs p-T2609 though an ATM-dependent mechanism. Results in




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Figure 7A and 7B indicate that in both NSCLCs and HBEC cells, L858R or ΔE746-E750 expression had

no effect on overall ATM levels or ATM phosphorylation at S1981. Second, we examined whether wild

type or mutant EGFR expression had any effect on interactions between DNA-PKcs and the protein

phosphatase, PP2A. There is evidence that radiation-induced DNA-PKcs phosphorylation is regulated by

protein phosphatase PP2A which de-phosphorylates and inactivates DNA-PKcs (34). Data in Figure 7C

and 7D demonstrate that in un-irradiated A549 and wild type EGFR expressing HBEC cells, levels of

PP2A-DNA-PKcs interactions predominate but are significantly reduced (~3-fold) on exposure to 4 Gy IR.

In striking contrast, ΔE746-E750 mutant EGFR expressing H820 and HBEC cells have higher basal levels

of PP2A-DNA-PKcs complexes and exposure to IR resulted in a further (≥2-fold) increase in PP2A-DNA-

PKcs binding. The data provide compelling evidence that activating mutations in EGFR significantly

augment PP2A-DNA-PKcs interactions and suggest a possible mechanism underlying mutant EGFR-

associated abrogation of T2609 phosphorylation.

Discussion

The data so far indicate that EGFR mediates survival response to radiation through IR-induced interactions

with DNA-PKcs which are abrogated in a dominant negative manner in the context of radiosensitizing

L858R or ΔE746-E750 EGFR mutations. Our study demonstrates for the first time that DNA-PKcs

phosphorylation is an essential prerequisite for EGFR-DNA-PKcs interaction. Alanine substitutions in

DNA-PKcs that prevent its radiation-induced phosphorylation abrogate EGFR-DNA-PKcs binding (Figures

3A, 5C and 5D). Of these, the T2609A and S2056A mutations are particularly relevant. Evidence shows

that p-T2609 is an ATM-dependent phosphorylation event, whereas p-S2056 is an ATM-independent

DNA-PKcs auto-phosphorylation event (20). Our data indicate that p-T2609 is a site of interaction between

DNA-PKcs and EGFR because T2609A, but not S2056A, substitution abrogated EGFR binding (Figures

5C and 5D). Moreover, ATM deficiency (Figure 2) or inhibition (Figure 4), which abrogates T2609 (Figure




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6C), but not S2056 phosphorylation (20), also blocked EGFR-DNA-PKcs binding. DNA-PKcs or ATM

alterations had no effect on IR-induced EGFR nuclear translocation (Supplemental figures, S1A -S1C)

suggesting a direct impact on nuclear EGFR-DNA-PKcs interaction.

Both, DNA-PKcs-7A and T2609A, mutants are catalytically active but do not bind EGFR (Figures 3A and

5D). Moreover, alanine substitution at S2056, a substrate of DNA-PKcs kinase activity, did not affect

EGFR-DNA-PKcs binding (Figures 5C and 5D). However, DNA-PKcs inhibition by NU7441 completely

inhibited EGFR-DNA-PKcs binding (Figure 4B) raising the possibility that other DNA-PKcs auto-

phosphorylation sites could be involved.

Surprisingly, in addition to its pivotal role in EGFR-DNA-PKcs binding, T2609 phosphorylation is also

influenced by EGFR. Our study demonstrates for the first time, that activating mutations in EGFR

specifically inhibit IR-induced phosphorylation of DNA-PKcs at T2609, but not S2056, a pattern that

closely resembles EGFR blockade by anti-EGFR antibody, C225 (9, 35) or ATM inhibition by KU55933

(Figure 6 and (20)). At least with mutant EGFRs, the mechanism is likely ATM-independent, because IR-

induced ATM S1981 phosphorylation (Figures 7A and 7B) was not affected.

Our data reveal an inverse relationship between EGFR-DNA-PKcs binding and DNA-PKcs association

with protein phosphatase, PP2A. Wild-type EGFR expression was associated with dramatic IR-induced

increases in EGFR-DNA-PKcs binding, corresponding reductions in PP2A-DNA-PKcs complexes and

robust IR-induced DNA-PKcs-T2609 phosphorylation. By contrast, in both basal and IR-induced settings,

mutant EGFR expression was associated with absence of EGFR-DNA-PKcs complexes (Figure 3A),

significantly elevated levels of PP2A-DNA-PKcs complexes (Figure 7C and 7D) and abrogation of IR-

induced DNA-PKcs-T2609 phosphorylation (Figure 6). However, PP2A phosphorylation at Y307 (Figure

7E, bottom panel) or EGFR-PP2A binding (Figure 7E, top panel) was unaffected by radiation or EGFR




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mutation status. Thus, EGFR modulates radiation response predominantly through interactions with DNA-

PKcs which likely stabilizes DNA-PKcs phosphorylation.

Mutant EGFR-associated abrogation of T2609 phosphorylation has important implications on DNA-PKcs

function. Studies show that DNA-PKcs phosphorylation at different sites govern either the stability of the

DSB-DNA-PKcs complex required for end processing, or the timely dissociation of DNA-PKcs, which

appears critical for DNA end ligation (14, 16, 37, 38). Uematsu et al observed that wild type and T2609A-

mutated DNA-PKcs had similar kinetics of association or dissociation at DSBs (29). We observed no

change in the dissociation kinetics of wild type DNA-PKcs with L858R or ΔE746-E750 mutant EGFR

expression (data not shown). Two groups have shown that phosphorylation of the 2609 cluster in DNA-

PKcs plays a critical role in regulating the intra-strand endonuclease activity of the Artemis nuclease (21,

39). It is therefore conceivable that mutant EGFR-mediated inhibition of p-T2609 adversely influences

Artemis activity and DNA end-processing.

Our study proffers important insights on EGFR’s contribution to cellular radiosensitivity. In V3-WT DNA-

PKcs cell, effects of L858R or ΔE746-E750 mutant EGFR expression (Figures 1C, middle) on

radiosensitivity were not as dramatic compared to DNA-PKcs ablation (Figure 1C, left) or NU7441

inhibition (Figure 4A, middle). This is logical because DNA-PKcs has multiple roles in DSB repair and

cellular radioresistance, only some of which may be EGFR-dependent. Nagasawa et al recently

demonstrated that, relative to wild type DNA-PKcs, single T2609A or S2056A mutations exhibit modest

radiosensitivity, whereas a T2609A/S2056A double mutation has a synergistic effect on radiosensitivity

(36). The synergism of the T2609A/S2056A double mutation suggests that p-T2609 and p-S2056 govern

distinct, non-overlapping functions of DNA-PKcs. Our study reveals that at least one of these non-

overlapping DNA-PKcs functions, pT2609, is modulated by EGFR. Wild type or mutant EGFR effects on




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radiosensitivity were evident in the S2056A, but not T2609A, genetic background. Moreover, L858R or

ΔE746-E750 expression had a strikingly similar radiosensitizing effect on V3-S2056A cells (Figure 5B,

left) as the T2609A/S2056A double mutation (36).

Our study underscores a modulatory role for EGFR in DSB repair and radiation resistance. In its simplest

form, our model suggests that initial IR-induced, ATM-dependent DNA-PKcs phosphorylation at T2609 is

a prerequisite for the binding of nuclear EGFR to DNA-PKcs. This association adversely affects PP2A-

DNA-PKcs interactions and stabilizes DNA-PKcs-T2609 phosphorylation, which is critical for Artemis-

mediated DSB end possessing. Activating mutations in EGFR prevent EGFR-DNA-PKcs interaction, favor

DNA-PKcs-PP2A association and compromise p-T2609 stability which adversely affects DSB processing.

Our study has important implications on how radiotherapy in combination with EGFR blockade may

benefit NSCLC patients, especially those that harbor radioresistant tumors with wild type EGFR.




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Acknowledgements This study was funded by NIH/NCI grant CA129364 (CN) and the Simmons Comprehensive Cancer Center Bridge funds (CN).




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Figure legends

Figure 1 Mutations in the 2609 and 2056 cluster of DNA-PKcs abrogate EGFR-mediated radiation

response (A) Schematic illustration of 7A-mutated DNA-PKcs with alanine substitutions in the 2056 and

2609 clusters (B) Western blot: Relative expression of DNA-PKcs (top panel) or V5 epitope-tagged EGFR

(bottom panel) in V3, V3-WT and V3-7A CHO cells. (C) Clonogenic survival in V3 (left), V3-

WT(middle) and V3-7A (right) CHO cells mock-transfected or stably expressing LacZ, (closed circle)

wild type EGFR (square), L858R (triangle up) ΔE746-E750 (triangle down). Symbols: Mean surviving

fraction (SF) and error bars: standard deviation (SD) from 3 independent experiments.

Figure 2 ATM deficiency abrogates EGFR-mediated radiation responses (A) Western blot: relative levels

of V5-tagged EGFR, DNA-PKcs and ATM in ATM-proficient 1BR3 and AT5 fibroblast cells. (B)

Clonogenic survival in 1BR3 (left) and AT5 (right) cells mock transfected (open circle) or stably

expressing LacZ, (closed circle) wild type EGFR (square), L858R (triangle up) ΔE746-E750 (triangle

down). Symbols: Mean SF and error bars: SD from 3 independent experiments.

Figure 3 DNA-PKcs phosphorylation of 2609 and 2056 cluster is critical for IR-induced binding to EGFR.

Co-IP/WB assay: EGFR and DNA-PKcs in complexes with EGFR immunoprecipitated from (A) V3-WT

and V3-7A transfectants of wild type L858R and ΔE7460-E750 EGFR or (B) 1BR3 and AT5 transfectants

of wild type EGFR at indicated time-points following 4 Gy IR.

Figure 4 Inhibition of DNA-PKcs or ATM abrogates EGFR DNA-PKcs interactions and EGFR-mediated

radioresponse (A) Clonogenic survival in HBEC-3KT cells, mock transfected (dashed gray line), stably

expressing LacZ, (green), wild type EGFR (black), L858R (red) or ΔE746-E750 (blue) following a 2 h

pre-treatment with either 10 μM NU74421 or 10 μM KU55933. Mean SF (symbols) and SD (error bars).




24

(B) Co-IP/WB assay: WB of EGFR and DNA-PKcs in complexes with EGFR immunoprecipitated, pre-

treated for 2 h with either 10 μM NU74421 or 10 μM KU55933 at indicated times following 4 Gy IR (C)

Proximity ligation assay (PLA): NSCLCs or HBEC-3KT cells expressing wild type or ΔE746-E750 forms

of EGFR following a 2 h pretreatment with vehicle, 10 μM NU7441 or 10 μM KU55933 were mock

irradiated or exposed to 4 Gy IR and fixed at 60 minutes. Left panel: representative images of nuclei (blue)

showing EGFR-DNA-PKcs complexes (intense red dots). Original images in Figure S2. Right panel: Mean

integrated fluorescence intensity (columns) and SEM (error bars) from 2 independent experiments.

Figure 5 DNA-PKcs phosphorylation at T2609 is critical for EGFR-mediated radioresponse (A) Western

blot analysis showing relative levels of DNA-PKcs (left) and wild type, L858R and ΔE746-E750 EGFR in

V3-S2056A and V3-T2609A CHO cells. (B) Clonogenic survival in V3-S2056A and V3-T2609A CHO

cells either mock transfected (open circle), or stably expressing LacZ, (closed circle), wild type EGFR

(square), L858R (triangle up) or ΔE746-E750 (triangle down). Symbols: mean SF and error bars SD. (C)

PLA to detect EGFR-DNA-PKcs complexes in V3-WT DNA-PKCS, V3-S2056A and V3-T2609A CHO

cells expressing wild type EGFR which were either mock-irradiated or exposed to 4 Gy IR. Left panel:

representative images of nuclei (blue) and EGFR-DNA-PKcs complexes (red PLA fluorescence dots) after

processing for PLA Original images in Figure S3. Right Panel: Fold increase in fluorescence (columns)

relative to untreated cells at 1 h hour following IR, and SEM (error bars) from two independent

experiments each with ≥350 nuclei per sample. (D) Co-IP/WB assay: WB analysis of EGFR and DNA-

PKcs in complexes with EGFR was immunoprecipitated from V3-S2056A and V3-T2609A transfectants of

wild type L858R and ΔE7460-E750 EGFR at indicated time-points following 4 Gy IR.

Figure 6 Activating mutations in EGFR specifically block IR-induced DNA-PKcs phosphorylation at

T2609. (A) Immuno-fluorescence detection of DNA-PKcs phosphorylation in HBEC cells stably




25

expressing wild type, L858R or ΔE746-E750 forms of EGFR at various time-points following 4 Gy IR.

Top panel: Representative images with anti-p-T2609 and anti-p-S2056 DNA-PKcs antibodies showing

nuclei (blue) and phosphorylated DNA-PKcs (green). Original images in Figure S4. Bottom panel:

Fluorescence intensity from p-T2609 and p-S2056 signals as a function of time. Columns: mean

fluorescence intensity relative to 100% fluorescence at 60 minutes. Error bars: SEM from 3 experiments,

each with 300-450 nuclei per sample (B) Representative images of p-T2609 immunofluorescence (green)

in nuclei (blue) in indicated NSCLCs (left panel). Original images in Figure S5. Fluorescence intensity

levels (right panel) in indicated NSCLCs (C) p-T2609 immunofluorescence intensity levels (right panel) in

A549 and HCC827 cells, from images (left panel) captured 1 h post-irradiation following a 2 h

pretreatment with 10 μM NU7441 or 10 μM KU55933. Original images in Figure S6. For (B) and (C),

columns: mean p-T2609 fluorescence per nucleus and error bars: SEM from 2 independent experiments,

each with 450-600 nuclei per sample.

Figure 7 Mutant EGFR expression reverses IR-induced decrease in DNA-PKcs-PP2A binding. WB

analysis with antibodies against phospho-serine 1981 ATM or total ATM in (A) NSCLC or (B) HBEC

cells expressing wild type, L858R or ΔE746-E750 forms of EGFR at time-points following 4 Gy IR. (C)

PLA to detect PP2A-DNA-PKcs interactions in NSCLCs and HBEC cells expressing WT or ΔE746-E750

EGFR fixed 1 h following 0 Gy or 4 Gy IR. Left panel: representative images of nuclei (blue) and PP2A-

DNA-PKcs complexes (red: PLA fluorescence). Original images in Figure S7. Right Panel: Mean

integrated fluorescence intensity per nucleus (columns) and SEM from 4 independent experiments, each

with 500-750 nuclei per sample. Single asterisk (*): statistically significant decrease in EGFR-PP2A

interactions determined by Student’s t-test between 0 Gy and 4 Gy samples of WT EGFR expressing

NSCLCs or HBEC (p < 0.01) Double asterisk (**): statistically significant increase in EGFR-PP2A

interactions between 0 Gy and 4 Gy samples of ΔE746-E750 EGFR expressing NSCLCs or HBEC (p <




26

0.01). (E) Co-IP/WB assay: Top panel: EGFR and of PP2A (subunits a and c) in complexes with EGFR

immunoprecipitated from A549 and H820 cell lines. Bottom panel: WB assay to detect levels of total PP2A

and Y307 phosphorylated PP2A in NSCLCs and HBEC cells.




Figure 1

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Figure 2

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Figure 3

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Figure 4

(A) Vehicle NU7441 KU55933( )

(B) (C)

(D)




(B)

(A)

(C)

(D)

Figure 5




Figure 6

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(B)

(C)




Figure 7

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Published OnlineFirst August 23, 2012.Mol Cancer Res Prashanthi Javvadi, Haruhiko Makino, Amit K Das, et al. receptor mediated radiation resistanceKinase is a critical prerequisite for epidermal growth factor Threonine 2609 phosphorylation of the DNA-dependent Protein

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