Cancer-Associated Loss-of-Function Mutations...

Tumor and Stem Cell Biology

Cancer-Associated Loss-of-Function Mutations ImplicateDAPK3 as a Tumor-Suppressing Kinase

John Brognard1,2, You-Wei Zhang2, Lorena A. Puto2, and Tony Hunter2

AbstractCancer kinome sequencing studies have identified several protein kinases predicted to possess driver (i.e.,

causal) mutations. Using bioinformatic applications, we have pinpointed DAPK3 (ZIPK) as a novel cancer-associated kinase with functional mutations. Evaluation of nonsynonymous point mutations, discovered inDAPK3 in various tumors (T112M, D161N, and P216S), reveals that all three mutations decrease or abolish kinaseactivity. Furthermore, phenotypic assays indicate that the three mutations observed in cancer abrogate thefunction of the kinase to regulate both the cell cycle and cell survival. Coexpression of wild-type (WT) and cancermutant kinases shows that the cancer mutants dominantly inhibit the function of the WT kinase. Reconstitutionof a non–small cell lung cancer cell line that harbors an endogenous mutation in DAPK3 (P216S) with WTDAPK3resulted in decreased cellular aggregation and increased sensitivity to chemotherapy. Our results suggest thatDAPK3 is a tumor suppressor in which loss-of-function mutations promote increased cell survival, proliferation,cellular aggregation, and increased resistance to chemotherapy. Cancer Res; 71(8); 3152–61. �2011 AACR.

Introduction

Sequencing technologies are evolving at a rapid rate result-ing in decreasing costs associated with sequencing a singlegenome. Estimates predict that the cost of sequencing a singlegenome will drop significantly and exome sequencing costsare already estimated at $5,000, making it feasible that cancerpatients may soon be able to have both their normal andcancer genomes sequenced (1). Comparison of these genomeswill provide a snapshot of the genetic aberrations thatoccurred during the evolution of a cancer (2, 3). Althoughthe sequencing of a patient's normal and cancer genome isbecoming feasible, our understanding of the many geneticaberrations that contribute to various cancer phenotypes lagsfar behind the development and application of this technol-ogy. If this information is to become clinically relevant wemust understand and catalog the functional consequences ofthe observed genetic changes. Understanding both the func-tional consequences of genetic changes and the signaling

networks they impact will be essential if this information isto be used as a guide for therapeutic treatment (4).

In an effort to identify protein kinases with candidate cancerdriver mutations, we used bioinformatic tools to analyzekinases with somatic mutations reported in a cancer kinomesequencing study performed by the Sanger Centre that eval-uated 518 kinases in 210 cancers (5–8).We identified DAPK3 asa strong candidate to possess functional cancer-associatedmutations. DAPK3 (also called ZIPK) is a member of the DAPK(death-associated protein kinase) family (9), and in addition tothe N-terminal catalytic domain, this kinase has a leucinezipper domain and 2 putative NLS (10). DAPK3 is proapoptotic(11) and is proposed to be a tumor suppressor, suggesting thatmutations inDAPK3 could result in loss of function. Consistentwith this notion, the gene for DAPK1, which has 83% homologywith DAPK3 in the kinase domain (9), is frequently methylatedin cancers, and DAPK1 is considered a tumor suppressingkinase (12–14). All 3 observed cancer mutations in DAPK3 areat residues conserved in the other 2 DAPK family members,DAPK1 and DAPK2 (Fig. 1A). D161 is in the DFG motif, and itsmutation to Asn would be expected to greatly reduce catalyticactivity; on the basis of the DAPK3 catalytic domain structure(15), T112 is in a surface accessible loop at the start of the C-lobe, and P216 lies in an interhelix loop largely buried in the C-lobe (Fig. 1B). The DAPK3 mutations are heterozygous andeach cancer patient retains a wild-type (WT) allele. TheDAPK3gene is mutated at a frequency of 3.2% in the panel of lung,ovarian, and colon cancers examined (the frequency of muta-tions was 1.4% in all cancers examined; 8). The T112M andD161N mutations were identified in the primary tumors of acolon cancer and ovarian cancer patient, respectively, and noDAPK3 mutations were identified in normal cells from thesame patients. The P216Smutationwas identified in the H1770non–small cell lung cancer (NSCLC) cell line and no DAPK3

Authors' Affiliations: 1Signalling Networks in Cancer Group, CancerResearch UK, Paterson Institute for Cancer Research, The University ofManchester, Manchester, United Kingdom; and 2Molecular and CellularBiology Laboratory, The Salk Institute, La Jolla, California

Current address for Y-W. Zhang: Department of Pharmacology, CaseWestern University, 2109 Adelbert Road, Cleveland, OH 44106.

Corresponding Author: John Brognard, Paterson Institute for CancerResearch, Wilmslow Road, Withington, Manchester M20 4BX, UnitedKingdom. Phone: 44-161-446-3039; fax: 44-161-446-3109; E-mail:[email protected]; or Tony Hunter, Salk Institute for BiologicalStudies, Molecular and Cell Biology Laboratory, 10010 N. Torrey PinesRoad, La Jolla, CA 92037-1099. Phone: 858-453-4100; Fax: 858-457-4765; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-3543

�2011 American Association for Cancer Research.

CancerResearch

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mutations were observed in the pair-matched normal cells(NCI-BL1770–available through ATCC).Our results indicate that all 3 mutations in DAPK3 decrease

or abolish the function of the kinase and render it unable toregulate cell cycle progression and cell survival. Furthermore,we observe that the cancer mutants can suppress the functionof the WT kinase, suggesting that these mutants can act in adominant-negative fashion. Reconstitution of the DAPK3pathway in a cancer cell line harboring an endogenous muta-tion in DAPK3 results in loss of cellular aggregation andincreased sensitivity to chemotherapy.

Materials and Methods

MaterialsDAPK3-specific SMARTpool siRNA was purchased from

Dharmacon and targeted the following sequences in DAPK3:50-ccacgcgtctgaaggagta-30 (si-1); 50-gatcccaagcggagaatga-30

(si-2); 50-ggacgtggaggaccattat-30 (si-3); and 50-gaacgtgcgtggt-gaggac-30 (si-4). Single DAPK3 siRNA targeted the followingsequences: 50-acgacatcttcgagaacaa-30 (siDAPK3-1), 50-cagc-caagttcatcaagaa-30 (siDAPK3-2), and 50-acatcatgctggtggacaa-30 (siDAPK3-3). The following phospho-specific antibodies

Figure 1. Mutational analysis and biochemical characterization of DAPK3 cancer variants. A, sequence alignment of DAPK family members highlightingconservation ofmutated residues in all 3 family members. Yellow boxes indicate amino acidsmutated in cancer. B, crystal structure of DAPK3 indicating wherethe mutations occurred. C, CanPredict analysis to show how this enhances the selection of putative cancer-associated kinases, the kinase RIPK1, which ispredicted to have a driver mutation is not predicted to have a cancer-related mutation and therefore is not a good candidate for further investigation. Incontrast, evaluation of somatic mutations of DAPK3 (also predicted to possess a driver mutation; 99%) reveal that all 3 somatic mutations are predicted to becancer related. D, H157 and 293T cells were transfected with vector or indicated FLAG-tagged DAPK3 constructs for 48 hours under high-serum conditionsprior to lysis. Immunoblots of lysates from H157 NSCLC or 293T cells were probed with antibodies to phospho-MLC2 (P-MLC2), MLC2, ZIPK, or anti-FLAG. E,H157 cells were transfected with vector or FLAG-tagged DAPK3 constructs as indicated under high-serum conditions (10% FBS DMEM); thereafter, FLAG-DAPK3 was immunoprecipitated and incubated with MLC2 generated from bl-21 bacterial cells. MLC2 phosphorylation was detected by using phospho-specific MLC2 (Thr18/Ser19) antibodies.

Loss-of-Function Cancer Mutations in DAPK3

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were purchased from Cell Signaling: P-MLC2 (Thr18/Ser19)and P-histone H3 (Thr11). DAPK3 (ZIPK) antibody waspurchased from ProSci Incorporated. MLC2 antibody waspurchased from Santa Cruz (sc-28329) and anti-FLAG mono-clonal antibody was purchased from Sigma (F3165). Propi-dium iodide and etoposide were both purchased from Sigma.Cell lines were originally purchased from ATCC, and wereauthenticated by the ATCC cell biology program and were notpassaged for more than 6 months before bringing new cellsout of freeze or purchasing a new cell aliquot from the ATCC.The H157 NSCLC cell line was a gift from Dr. Phillip Dennis,National Cancer Institute, and was established at the NCI/Navy Medical oncology.

Cloning and expression3X-FLAG full-length DAPK3 cDNA, T180A, and D273 were

provided by Dr. Timothy Haystead (Department of Pharma-cology and Cancer Biology, Duke University Medical Center,Durham, NC). HA-DAPK3 was provided by Dr. Kevan Shokat(Department of Cellular and Molecular Pharmacology, Uni-versity of California, San Francisco, CA). The cancer mutantDAPK3 variants were generated by converting the nucleotideat position 335 from a C to a T (T112M), nucleotide at position481 from a G to an A (D161N), and nucleotide at position 646from a C to a T (P216S) by using QuikChange site-directedmutagenesis kit (Stratagene). These variants were identical tothe somatic variants observed in cancer patients or in theNSCLC cell line H1770 (P216S). Glutathione S-transferase(GST)-tagged myosin light chain for bacterial expression, usedfor in vitro DAPK3 kinase assays, was a gift from Dr. Ruey-HwaChen (Institute of Biological Chemistry, Academia Sinica,Taipei, Taiwan).

Cell transfections and immunoblottingThe H1770 NSCLC cell line was maintained in RPMI 1640

(Cellgro), and all other cell lines were maintained inDulbecco's modified Eagle's medium (DMEM; Cellgro); bothmedia were supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were maintained at 37�C in 5% CO2.Transient transfections of all cell types were carried out byusing Effectene reagents (Qiagen). Lipofectamine 2000 (Invi-trogen) was used to transfect siRNAs into all cells. Transienttransfections and siRNA experiments were performed aspreviously described (16), except for H1770 cells. H1770 cellswere pipetted repeatedly to break up cell aggregates andtransfected immediately as previously described (16). Trans-fection efficiencies for 293T and H157 cell lines averagedbetween 70% and 90% for each experiment; efficiencies forall other cell lines averaged between 50% and 80%. Forimmunoblotting, transfected cells were lysed in buffer 1 atroom temperature [50 mmol/L Na2HPO4 (pH 7.5), 1 mmol/Lsodium pyrophosphate, 20 mmol/L NaF, 2 mmol/L EDTA, 2mmol/L EGTA, 1% SDS, 1 mmol/L DTT, 200 mmol/L benza-midine, 40 mg/mL leupeptin, and 1 mmol/L phenylmethylsul-fonyl fluoride (PMSF)] and sonicated for 4 seconds. Lysatescontaining equal amounts of protein were analyzed on SDS-PAGE gels, and individual blots were probed by using eachantibody.

Kinase assays and coimmunoprecipitationsGST-MLC2 was expressed in BL21 bacterial cells and

kinase assays were performed by immunoprecipitatingFLAG-DAPK3 from cells and incubating with GST-MLC2as a substrate. The activity of full-length FLAG-DAPK3 andother indicated variants was assessed by expressing andimmunoprecipitating FLAG-DAPK3 from H157 cell lysates.Cells were lysed in buffer 2 [20 mmol/L HEPES (pH 7.4), 1%Triton X-100, 1 mmol/L DTT, 200 mmol/L benzamidine,40 mg/mL leupeptin, 1 mmol/L PMSF], sonicated for 3seconds, and precleared. Detergent soluble lysates (buffer2) were incubated overnight at 4�C with FLAG antibody andUltraLink Protein A/G beads (Pierce). Beads were thenwashed 3 times with buffer 1 and incubated in kinase buffer[25 mmol/L Tris (pH 7.5), 5 mmol/L b-glycerophosphate,2 mmol/L DTT, 0.1 mmol/L Na3VO4, 10 mmol/L MgCl2], and200 mmol/L ATP with purified GST-MLC2, 1 mg per reactionin 50 mL kinase reaction buffer. Kinase reactions were per-formed at 30�C for 25 minutes, terminated by the addition ofsample buffer and phosphorylation was detected by usingthe P-MLC2 antibody (Thr18/Ser19).

Proliferation and apoptosis assaysFor apoptotic assays, cells were switched to low-serum

media conditions (0.1% FBS) and transfected with theindicated DAPK3 constructs for 48 hours. Floating cellswere collected and adherent cells were harvested by trypsi-nization and then centrifuged at 1,000 � g for 5 minutes.Cells were fixed in ice-cold 70% methanol, added drop wise,and then incubated at 20�C for 30 minutes. Cells werecentrifuged and incubated with propidium iodide (25 mg/mL) supplemented with RNase A (30 mg/mL) for 30 minutesat room temperature. Quantification of sub-2N DNA inapoptosis assays was determined by flow cytometry analysisby using a Becton Dickinson FACSort and by manual gatingby using CellQuest software. Gating was performed onblinded samples. For all cell lines, apoptotic assays wereperformed on whole cell population. To determine G1/Sratios, cells were transfected with FLAG-DAPK3 constructsand incubated under high-serum conditions (10% FBS) for48 hours. For bromodeoxyuridine (BrdUrd) incorporationassays, cells were maintained in high growth media (10%FBS) and transfected DAPK3 constructs, and incubated for48 hours prior to performing assays following the man-ufacturer's protocol (Calbiochem, QIA58). For DAPK3siRNA-depleted cells, BrdUrd incorporation assays wereperformed on cells incubated under low-serum conditions(0.1% FBS) and transfected with 100 nmol/L SMARTpoolsiRNA for 48 hours.

Statistical and cell aggregation analysisStatistical comparison of values was performed by the

Student t test comparing empty vector controls or untreatedcells to indicated DAPK3 constructs. Quantification of cellularaggregation was determined by Image J software and spheroidsize was correlated to the number of pixels in an area of agiven spheroid. Eight cellular aggregates were included in eachmeasurement.

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Results

To determine whether mutations identified in DAPK3 arelikely to alter the protein function, we used the CanPredict(http://www.cgl.ucsf.edu/Research/genentech/canpredict/)web application to determine whether the mutations are likelyto be cancer-associated mutations (6). This web applicationemploys an algorithm based on 3 measurements (SIFT, LogR.E-vlaue, and GOSS values) to determine whether a givenmutation is a probable cancer mutation (6). To show howthis analysis could enhance the selection of potential cancer-associated kinases, evaluation of mutations in DAPK3 byCanPredict reveals that all 3 somatic mutations are predictedto be cancer-related, and therefore we considered this kinaseto be an excellent candidate for further investigation (Fig. 1C;at least one mutation in DAPK3 is also predicted to be a drivermutation based on biostatistical analysis; ref. 8). These resultswere verified by PMUT (5), and all 3 DAPK3 mutations werepredicted to be driver mutations (pathologic). To show thatmany of the mutations analyzed by CanPredict are suggestedto be passenger mutations, we have included the analysis ofRIPK1 (Fig. 1C).We generated the 3 observed DAPK3 somatic mutations—

T112M (colon cancer), D161N (ovarian cancer), and P216S(NSCLC)—in full-length DAPK3 cDNA by using site-directedmutagenesis. FLAG-tagged versions were expressed in theH157 NSCLC cell line, which was selected for initial studiesbecause it is amenable to transfection and is sensitive toapoptotic stimuli, and 293T cells. To assess kinase activity,we monitored phosphorylation of MLC2, a reported DAPK3substrate (17), at T18/S19 by blotting with anti-pT18/pS19antibodies. Expression of WT DAPK3 increased MLC2 phos-phorylation (Fig. 1D), whereas expression of D161N, T112M,and P216S did not. These data suggest that all 3 somaticmutations inactivate DAPK3, because overexpression of theDAPK3 cancer mutants did not increase MLC2 phosphoryla-tion. As a positive control we expressed the constitutivelyactive D273 DAPK3 mutant (ZIPKD273), which is a C-terminaltruncation variant that consists of only the kinase domain andwas previously reported to have increased kinase activity (18).As a negative control we expressed T180A DAPK3 (mutated atthe key activation loop autophosphorylation site), which wasreported to have decreased kinase activity (18). We comple-mented these studies by carrying out in vitro kinase assayswith DAPK3 immunoprecipitated from H157 cells by usingrecombinant GST-MLC2 purified from bacteria cells as asubstrate (Fig. 1E). Consistent with the in vivo results,D161N, P216S, and T180A DAPK3 had very low kinase activitycompared with WT DAPK3; T112M had significantlydecreased activity. Combined, these data indicate that DAPK3mutations identified in cancer patients significantly suppressthe activity of the kinase.To determine whether the DAPK3 cancer mutations alter

the function of the WT kinase, we transiently expressed themin H157 cells (transfection efficiency is 70%–90%, based ongating green fluorescent protein–positive cells, and overex-pression of WT DAPK3 is approximately 3-fold based onImage J densitometry) and monitored cell survival and cell

cycle progression of the whole cell population. Overexpressionof WT DAPK3 under high-serum (10% FBS) media conditionscaused cells to accumulate in G1, resulting in an increase inthe G1/S ratio (Fig. 2A). This phenotype was more pronouncedwith DAPK3 D273, but all 3 cancer variants of DAPK3, as wellas T180A were ineffective at inhibiting cell cycle progression.To determine whether this inhibition of cell cycle progressiontranslated into a decrease in cellular proliferation we over-expressed DAPK3 constructs in H157 cells and monitoredBrdUrd incorporation. Consistent with the cell cycle analysis,overexpression of WT DAPK3 and the constitutively activeD273 DAPK3 significantly decreased BrdUrd incorporation(Fig. 2B), whereas the DAPK3 T180A mutant and all 3 cancermutants did not (Fig. 2B). These data are consistent with amodel in which loss of functional growth inhibitory DAPK3may increase cell proliferation, a hallmark of tumorigenesis.

To our knowledge this was the first time DAPK3 has beenimplicated in regulating cell cycle progression and prolifera-tion. To verify that endogenous DAPK3 regulates the cell cyclewe depleted endogenous DAPK3 from H157 NSCLC cells andU87 glioma cells, which both have WT DAPK3, using SMART-pool siRNA transfection. Immunoblots showed that the levelof endogenous DAPK3 was significantly reduced, correlatingwith a decrease in phosphorylation of downstream substrates,including histone H3 (T11; ref. 19) and MLC2 (Thr18/Ser19;Fig. 2C, left). In parallel, we measured relative BrdUrd incor-poration in cells depleted of DAPK3, and found that thisincreased BrdUrd incorporation in both H157 and U87 cells(Fig. 2C, right), consistent with DAPK3 regulating both cellcycle progression and cell proliferation. To verify that theobserved effect was not due to off-target effects, we depletedDAPK3 in H157 cells by using DAPK3-specific single siRNAoligos. As displayed in Figure 2D, depletion of DAPK3 in theH157 cells correlated with a decrease in MLC2 phosphoryla-tion as well as a decrease in the G1/S ratio indicating the cellsare progressing through the cell cycle at an increased rate(Fig. 2D).

To determine whether DAPK3 regulated cell survival, weoverexpressed DAPK3 constructs under low-serum conditions(0.1% FBS) and monitored apoptosis. Under these conditions,expression of WT DAPK3 caused a greater than 2-fold increasein apoptosis, whereas expression of T112M, D161N, and P216SDAPK3 did not, again suggesting that these mutants are notfunctional (Fig. 2E; a similar increase in apoptosis is observedin these cells when treated with other cytotoxic agents such asetoposide, cis-platinum, or taxol; ref. 20).

All the DAPK3 mutations identified to date in cancerpatients are heterozygous, suggesting that a loss-of-functionmutation in one allele could suppress the function of the WTallele by preventing activation of the WT allele followingdimerization between an inactive DAPK3 cancer mutantand the WT DAPK3 proteins (15, 18). To determine whetherthese cancer mutants did indeed suppress the function of WTkinase, we first evaluated endogenous MLC2 phosphorylationin several cancer cell lines with high levels of phospho-MLC2(T18/S19). Overexpression of WT DAPK3 (2- to 5-fold over-expression compared with endogenous DAPK3 based onImage J densitometry for the 3 cell lines examined) did not






Figure 2. Mutations in DAPK3 result in loss-of-function. A, H157 NSCLC cells were transfected with indicated DAPK3 variants or vector alone, underhigh-serum conditions (10% FBS DMEM) for 48 hours and apoptosis (sub-2N DNA content) and G1/S ratios were determined by propidium iodideincorporation assays and flow cytometry. An increasewas observed in the G1/S ratio in cells transfectedwith FLAG-DAPK3 or FLAG-D273 comparedwith cellstransfected with vector alone, T180A, or cancer variants. *, P < 0.01. B, overexpression of FLAG-DAPK3 or FLAG-D273 for 48 hours under high-serumconditions decreased BrdUrd incorporation in H157 cells, whereas T180A or cancer mutants did not significantly alter BrdUrd incorporation. *, P < 0.05. C,depletion of DAPK3 for 48 hours under high-serum conditions increased BrdUrd incorporation in H157 and U87 cells. Immunoblots were performed in parallelto ensure that DAPK3 was being adequately depleted and we observed a concomitant decrease in MLC2 and histone H3 phosphorylation. *, P < 0.05. D,depletion of DAPK3 with 3 unique siRNAs for 48 hours under high-serum conditions decreased the G1/S ratio in the H157 cells. Immunoblots were performedin parallel to ensure that DAPK3 was being depleted. E, H157 cells were transfected with indicated constructs for 48 hours under low-serum conditions.Histograms show sub-2N DNA; quantitation of sub-2N DNA is indicated in bar graph. *, P < 0.01. A–E, the data in the bar graphs are representative of assaysperformed in triplicate, with error bars indicating SD, and are representative of 3 independent experiments.

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significantly increase MLC2 phosphorylation, although D273DAPK3 did (Fig. 3A). Consistent with the notion that theDAPK3 cancer mutants may suppress endogenousWTDAPK3function, overexpression of all 3 cancer mutants as well as theT180A DAPK3 suppressed MLC2 phosphorylation. To verifythat mutant endogenous DAPK3 could suppress WT DAPK3,we coexpressed the 3 cancer mutants individually with WTDAPK3 in H157 NSCLC cells and monitored phosphorylationof MLC2 (Fig. 3B). Overexpression of WT DAPK3 increasedMLC2 phosphorylation and this was blocked by coexpressionof each of the cancer mutants when transfected at equal inputDNA level (Fig. 3B). To verify that the cancer mutants actedthrough association with WT DAPK3, we performed pull-down experiments with transiently coexpressed HA-tagged

WT DAPK3 and FLAG-tagged WT DAPK3 or FLAG-taggedDAPK3 cancer mutants. Cancer mutants associated with WTDAPK3 to a level comparable to the WT DAPK3 (FLAG;Fig. 3C). Together these data suggest that the cancer mutantscan act in a dominant manner to suppress the activity of theWT allele.

To determine whether the DAPK3 cancer mutants couldsuppress the function of the WT kinase, we coexpressedT112M, D161N, or P216S DAPK3 with WT DAPK3 in H157NSCLC cells and monitored cell cycle progression. Coexpres-sion of the cancer mutants caused a reduction in the G1/Sratio observed when WT DAPK3 is expressed alone (Fig. 4A).This suggests that the cancer mutants interfere with theability of WT DAPK3 to slow cell cycle progression. Consistent

Figure 3.DAPK3 cancer mutants inhibit the activity of theWT kinase. A, A549, HeLa, and U87 cells were transfected with the indicated constructs for 48 hoursunder high-serum conditions prior to lysis. The phosphorylation state of MLC2 in lysates was detected by immunoblot analysis. B, coexpression ofcancer mutants suppresses the activity of WT DAPK3. H157 cells were cotransfected with the indicated constructs for 48 hours under high-serum conditionsand MLC2 phosphorylation was detected by immunoblot analysis. C, cancer mutants associate with WT DAPK3. H157 cells were cotransfected withthe indicated constructs for 48 hours under high-serum conditions, lysed, immunoprecipitated overnight at 4�C, and association was detected byimmunoblot analysis.






with these results, coexpression of DAPK3 cancer mutantswith WT DAPK3 suppressed apoptosis induced byWT DAPK3(Fig. 4B). This provides further evidence that the cancer-associated mutations in DAPK3 exert a dominant-negativeeffect over WT DAPK3 function.

An important goal of this research is to address the func-tional relevance of mutations in cancer-associated kinases inthe context of the cancer in which they occur. To this end, weused a cell line, H1770, with an endogenous mutation inDAPK3 that occurred in a NSCLC patient, and was presentin only the tumor cells. H1770 cells harbor a P216S mutation inDAPK3, which we have shown is a loss-of-function mutation(this cell line is heterozygous WT/P216S). Sequencing ofDAPK3 RNA verified that H1770 cells carry the P216S endo-genous mutation, and the sequencing chromatograms suggestthat both alleles are expressed at equal levels (not shown).This NSCLC cell line is a suspension cell line that grows aslarge cellular aggregates (Fig. 5A). Depletion of total DAPK3from these cells with SMARTpool siRNA did not decreaseMLC2 or histone H3 phosphorylation (levels of phosphoryla-tion for these proteins were already very low and barelydetectable with the antibodies; Fig. 5C), or alter cell cycledistribution, suggesting that DAPK3 and downstream signal-ing are inactive in this cell line (data not shown). Reconstitu-tion of these cells with WT DAPK3 resulted in decreasedcellular aggregation (Fig. 5A; quantified in Fig. 5B) and anincrease in MLC2 phosphorylation (Fig. 5C). Similar resultswere observed in cells expressing D273 DAPK3 (Fig. 5A and5C), whereas no significant changes were observed in cellsexpressing P216S DAPK3. This suggests that loss of DAPK3may enhance cellular aggregation and cell-to-cell adherence.Because cell-to-cell adhesion can promote chemotherapeuticresistance, we next determined whether reconstitution of

H1770 cells with WT DAPK3 rendered them more sensitiveto chemotherapy. As shown in Figure 5D, cells expressing WTDAPK3 were somewhat more sensitive to etoposide than cellstransfected with empty vector or P216S DAPK3. These dataunderscore the importance of the P216S mutation in this cellline, as reactivation of the DAPK3 pathway decreased cellularaggregation and rendered these cells more sensitive to apop-tosis.

Discussion

DAPK3 is a member of the death-associated protein kinasefamily and regulates cellular processes, including apoptosis,adherence, and cell cycle progression/proliferation (21). Sev-eral substrates have been reported for DAPK3, includingMLC2, histone H3, and Par4 (9), and these substrates arelikely to be important for DAPK3-mediated regulation of cellsurvival and cell cycle progression. DAPK3 has been suggestedto be a tumor suppressor (21), and our results strongly impli-cate DAPK3 as a tumor suppressing kinase. In vitro and in vivokinetic assays reveal that mutations in DAPK3 abolish orsignificantly reduce the enzymatic activity of DAPK3. Further-more, we show that the DAPK3 cancer mutants are unable toinhibit cellular processes, including cell proliferation, showingthat these are loss-of-function mutations and suggesting thatacquisition of these mutations by cancer cells will conferselective proliferative or survival advantages.

Previous reports have given us mechanistic insight intoDAPK3 regulation and provide evidence that inactive DAPK3forms a symmetrical homodimer through activation loopstrand exchange between subunits (15, 18). The kinase domainof DAPK3 is critical for the homodimerzation and alone issufficient to interact with full-length DAPK3 (18). We present

Figure 4. DAPK3 cancer mutants inhibit the function of the WT kinase. A and B, coexpression of cancer mutants suppresses the function of WT DAPK3. H157cells were cotransfected with indicated constructs for 48 hours under high-serum (10% FBS; cell cycle analysis) or low-serum (0.1% FBS; apoptosis analysis)conditions. Apoptosis (sub-2N DNA content) and G1/S ratios were determined by propidium iodide incorporation assays and flow cytometry.

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data showing that inactive DAPK3 proteins expressed from amutant allele could act in a dominant-negative fashion bybinding to WT DAPK3 and preventing its activation by T180transphosphorylation (15). The data presented provide amechanism, in which mutations observed in cancer maynot only abolish the activity of the mutant allele, but alsoact to suppress the function of the WT allele.To determine whether DAPK3 loss-of-function mutations

confer an advantage in the context of a tumor where themutation was acquired, we sought to reconstitute the H1770NSCLC cell line with WT DAPK3 and monitor phenotypiceffects. Interestingly, expression of DAPK3 in the H1770 cellline resulted in a decrease in cellular aggregation, consistentwith a loss of cell-to-cell adhesion that is likely to occurfollowing MLC2 phosphorylation. Interestingly, other MLC2kinases are also mutated in this cell line [MYLK2 (A117V) andCDC42BPA (MRCK, E50K)] and the functional consequencesof these mutations have not been determined. If these muta-

tions were loss-of-function mutations, it would suggest adecrease in MLC2 phosphorylation (at both Ser19 andThr18) was a critical step in the evolution of the tumorand potentially explains why increasing MLC2 phosphoryla-tion at T18/S19 would have dramatic phenotypic conse-quences. Additionally, this observation may provide a novelinsight into a pathway that could drive increased proliferationand drug resistance and could be a common pathway utilizedto promote tumorigenesis. Cell-to-cell adhesion has previouslybeen shown to promote drug resistance, and, consistent withthis observation, H1770 cells expressing WT DAPK3, whichpromotes loss of cell-to-cell adhesion, are more sensitive tochemotherapy (22). However, the role MLC2 phosphorylationin tumor formation is likely to be much more complex, andindeed activating mutations in ROCK1 have been describedand increased MLC2 phosphorylation is also likely to play acritical role in tumor progression and increased migration(23). Interestingly, one ROCK1-activating mutation described

Figure 5. LOF mutation in DAPK3 promotes cell–cell adhesion and chemotherapeutic resistance. A, reconstitution of DAPK3 pathway in H1770 cellsthat harbor a P216Smutation in DAPK3 reduces cell–cell adhesion. H1770 cells were transfected with the indicated constructs for 48 hours under high-serumconditions and cells were photographed. White bar, 0.2 mm. B, quantification of cellular aggregates is displayed and was determined by usingImage J software based on the area occupied by a single cellular aggregate. C, cells were then harvested, lysed, and immunoblot analysis was performed.D, H1770 cells expressing WT DAPK3 are more sensitive to etoposide-induced apoptosis. Cells were transfected with WT or cancer mutant P216SDAPK3, maintained in low-serum growth media (0.1% FBS), and treated with vehicle or 50 mmol/L etoposide for 48 hours prior to harvesting cells anddetermining sub-2N DNA content by propidium iodide incorporation assays and flow cytometry. E, model depicting results of loss-of-function mutationof DAPK3 promotes increased adhesion, survival, proliferation, and drug resistance.






in the study by Lochhead and colleagues, P1193S, which lies inthe PH domain, was shown to be an activating mutation (23),and this mutation is also present in the H1770 cell line. Theprevious report did not examine MLC2 phosphorylation inH1770 cells, and our data suggest that overall MLC2 phos-phorylation in these cells is barely detectable. It could be thatthis mutation promotes an increase in phosphorylation ofanother ROCK1-specific substrate or a small pool of MLC2.Comparing the various ROCK1 mutations evaluated in thisstudy indicates that the P1193S did not appear to increaseMLC2 phosphorylation significantly above WT ROCK1 andmuch less than the other 2 activating mutations. Additionally,P1193S ROCK1 appeared to have a unique cellular localization,and this could be because of a structural change in the PHdomain induced by the mutation. Different consequences ofMLC2 phosphorylation at S19 and combined phosphorylationat T18/S19 may contribute to the complex results.

Consistent with our general hypothesis, recent revelationshave shown that decreased MLC2 phosphorylation may pro-mote tumorigenesis (24). Specifically, loss-of-function muta-tions in LKB1 should lead to decreased NUAK1 activation,resulting in decreased inhibition of the MLC2 phosphatase,MYPT1, due to decreased 14-3-3 binding (24). MYPT1 wouldthen be free to dephosphorylate MLC2 and maintain it in adephosphorylated state (24). In agreement with this hypoth-esis, MCF-10A cells depleted of LKB1 grow as abnormally largeacini with multilayering of acinar cells (25). Loss-of-functionmutations in DAPK3 would be another mechanism to pro-mote decreased MLC2 phosphorylation. Interestingly, theH157 NSCLC cell line has a loss-of-function mutation inLKB1, and this cell line is very responsive to increases inMLC2 phosphorylation, likely because phosphorylation of thesubstrate is normally suppressed by increased MYPT1 activity(26). Thus, loss-of-function mutations in either LKB1 orDAPK3 could lead to suppression of MLC2 phosphorylation,promote cell-to-cell adhesion and lead to increased drugresistance (Fig. 5D). Drugs that could promote MLC2 phos-phorylation (or possibly diphosphorylation at T18/S19) couldbe valuable in the clinic in combination with other chemother-apeutics, particularly in patients with DAPK3 or LKB1 muta-

tions, or decreased DAPK3 expression, such as in gastriccancer, where a recent study showed that 111 of 162 gastriccarcinomas displayed decreased expression of DAPK3, whichwas associated with increased metastasis and invasion (27).

SummaryCancer genomic studies that identify somatic mutations

acquired in cancer cells will provide a roadmap for theidentification and characterization of new oncogenes andtumor suppressors (28). These screens uncover mutationsin known tumor suppressing kinases, such as LKB1 andATM, verifying the ability of these screens to identify muta-tionally inactivated tumor suppressors (8). Functional analysisof the consequences of cancer-associated mutations in signal-ing proteins, such as protein kinases, will lead to a broaderunderstanding of signaling networks that contribute totumorigenesis and define new targets that regulate thesecrucial networks. Combining the genomic screens with evol-ving and improving bioinformatics screens will provide aplatform for basic researchers to identify new critical regula-tors of tumorigenesis that have a lower frequency of muta-tions, yet are still essential for tumorigenesis. The resultspresented illustrate the potential of this approach and suggestthat DAPK3 is a tumor suppressor with loss-of-functionmutations in cancer patients.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This work was supported by Cancer Research UK (J. Brognard), AmericanCancer Society Postdoctoral Grant (#116653; J. Brognard), NCI Training Grant(#T32 CA009523; J. Brognard), US Public Health Service Grants CA14195 andCA82683 from the NCI (T. Hunter), and a sanofi-aventis Discovery grant (T.Hunter). T. Hunter is a Frank and Else SchillingAmericanCancer Society Professor.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 29, 2010; revised January 24, 2011; accepted February 15,2011; published online April 12, 2011.

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2011;71:3152-3161. Published OnlineFirst April 12, 2011.Cancer Res John Brognard, You-Wei Zhang, Lorena A. Puto, et al.

Suppressing Kinase−DAPK3 as a Tumor Cancer-Associated Loss-of-Function Mutations Implicate

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