COTI-2, A Novel Thiosemicarbazone Derivative, Exhibits Antitumor … · Translational Cancer...

Translational Cancer Mechanisms and Therapy

COTI-2, A Novel Thiosemicarbazone Derivative,Exhibits Antitumor Activity in HNSCC throughp53-dependent and -independent MechanismsAntje Lindemann1, Ameeta A. Patel1, Natalie L. Silver2, Lin Tang3, Zhiyi Liu1, Li Wang4,Noriaki Tanaka5, Xiayu Rao6, Hideaki Takahashi1, Nakachi K. Maduka1, Mei Zhao1,Tseng-Cheng Chen7,WeiWei Liu8, Meng Gao1, Jing Wang6, Steven J. Frank4,Walter N. Hittelman9, Gordon B. Mills10, Jeffrey N. Myers1, and Abdullah A. Osman1

Abstract

Purpose: TP53 mutations are highly prevalent in head andneck squamous cell carcinoma (HNSCC) and associated withincreased resistance to conventional treatment primarily con-sisting of chemotherapy and radiation. Restoration of wild-type p53 function in TP53-mutant cancer cells represents anattractive therapeutic approach and has been explored inrecent years. In this study, the efficacy of a putative p53reactivator called COTI-2 was evaluated in HNSCC cell lineswith different TP53 status.

Experimental Design: Clonogenic survival assays and anorthotopic mouse model of oral cancer were used to examinein vitro and in vivo sensitivity of HNSCC cell lines with eitherwild-type, null, or mutant TP53 to COTI-2 alone, and incombination with cisplatin and/or radiation. Western blot-ting, cell cycle, live-cell imaging, RNA sequencing, reverse-phase protein array, chromatin immunoprecipitation, and

apoptosis analyses were performed to dissect molecularmechanisms.

Results: COTI-2 decreased clonogenic survival of HNSCCcells and potentiated response to cisplatin and/or radiationin vitro and in vivo irrespective of TP53 status. Mechanistically,COTI-2 normalized wild-type p53 target gene expression andrestoredDNA-binding properties to the p53-mutant protein inHNSCC. In addition, COTI-2 induced DNA damage andreplication stress responses leading to apoptosis and/or senes-cence. Furthermore, COTI-2 lead to activation of AMPK andinhibition of the mTOR pathways in vitro in HNSCC cells.

Conclusions: COTI-2 inhibits tumor growth in vitro andin vivo in HNSCC likely through p53-dependent and p53-independent mechanisms. Combination of COTI-2 with cis-platin or radiation may be highly relevant in treating patientswith HNSCC harboring TP53 mutations.

IntroductionThe overall 5-year survival rate for patients with advanced

head and neck squamous cell carcinoma (HNSCC) remains inthe 25%–40% range due to resistance to standard therapy,primarily consisting of platinum-based chemoradiotherapy (1).Recent studies have revealed that TP53 is the most commonsomatically mutated gene in HNSCC, with mutations detectedin 65%–85% of non-human papillomavirus (HPV) associatedHNSCC (2–4). The p53 protein, which is encoded by theTP53 gene, plays a central role in coordinating the response tocell stressors such as DNA damage, hypoxia, and oncogenicstress, largely through sequence-specific transcriptional regula-tion of multiple important target genes leading to G1 arrest,senescence, and induction of apoptosis. While most mutationsin TP53 lead to a decrease in DNA-binding activity to p53-responsive elements, some p53-mutant proteins also have adominant negative effect as they can bind and inhibit thefunction of remaining wild-type (wt) p53 protein (5). More-over, some mutant p53s display oncogenic properties, termed"gain-of-function (GOF)," which are independent of wtp53functions (5). Accordingly, GOF p53-mutant proteins canenhance cell transformation, increase tumor formation inmice, and confer cellular resistance to chemotherapy andradiation (5, 6).

1Department of Head and Neck Surgery, The University of Texas MD AndersonCancer Center, Houston, Texas. 2Department of Otolaryngology, Division ofHead and Neck Oncologic Surgery, University of Florida College of Medicine,Gainesville, Florida. 3Department of Cellular and Molecular Medicine, The Uni-versity of Arizona Health Sciences, College of Medicine, Tucson, Arizona.4Department of Radiation Oncology, The University of Texas MD AndersonCancer Center, Houston, Texas. 5Department of Dentistry and Oral Surgery,Osaka Police Hospital, Osaka, Japan. 6Department of Bioinformatics andComputational Biology, The University of Texas MD Anderson Cancer Center,Houston, Texas. 7Department of Otolaryngology, National Taiwan UniversityHospital, Taipei, Taiwan. 8Department of Oral Surgery, School of Stomatology,Jilin University, Changchun, Jilin, China. 9Department of Experimental Thera-peutics, The University of Texas MDAnderson Cancer, Houston, Texas. 10OregonHealth & Science University, Knight Cancer Institute, Portland, Oregon.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

A. Lindemann, A.A. Patel, J.N. Myers, and A.A. Osman contributed equally to thisarticle.

Corresponding Authors: Jeffrey N. Myers and Abdullah A. Osman, The Univer-sity of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston,TX 77030. Phone: 713-745-2667; Fax: 713-794-4662; E-mail:[email protected]; and [email protected]

Clin Cancer Res 2019;25:5650–62

doi: 10.1158/1078-0432.CCR-19-0096

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Because mutation of TP53 in HNSCC is associated with resis-tance to standard treatment, there is a major need for noveltherapeutic approaches to overcome this resistance. Becausewild-type p53 can induce apoptosis and/or senescence whenexpressed in tumor cells, reactivation of wild-type function inmutant p53 is an attractive therapeutic strategy. A variety ofcompounds that can restore wild-type (wt) p53 protein functionhave been identified (7, 8). One approach exploits the require-ment of WT p53 binding to the metal ion Zn2þ to fold correct-ly (9, 10). The thiosemicarbazonemetal ion chelator NSC319726(ZMC1), was found to restore wt p53 function in a subset of, butnot all, mutant p53 protein–expressing tumor cell lines (10).Unfortunately, ZMC1demonstrated toxicity in dogmodels, likelydue to its high affinity for Zn2þ, precluding further clinicaldevelopment. COTI-2 is a novel related third-generation thiose-micarbazone that has been proposed to modulate p53 proteinfunction (11, 12). Upon addition, COTI-2 binds to themisfoldedmutant forms of the p53 protein, thereby inducing conforma-tional change thatnormalizesp53and restores its activity (11,12).COTI-2 has demonstrated activity against multiple human ovar-ian and breast cancer cell lines in vitro and in vivo (11, 12).However, its efficacy in HNSCC has not been investigated andthe specific molecular mechanisms of its action are largelyunknown. This exploration will provide important conceptualinformation as COTI-2 is currently being investigated in a phase Iclinical trial in advanced or recurrent gynecologic and head andneck malignancies (13).

In this study, we showed that COTI-2 decreased cell survivalof both GOF-mutant p53 and wild-type HNSCC cells andsynergized with cisplatin (CDDP) and radiation in vitro.Furthermore, COTI-2 potentiated response to cisplatin andradiation in vivo in an orthotopic mouse model of oral cancer.Notably, the reduction in cell survival was associated withDNA damage and replication stress responses leading to apo-ptosis and/or senescence. Using RNA sequencing coupled withChIP, COTI-2 lead to normalization of wild-type p53 targetgene expression and restoration of DNA-binding properties toa GOF p53-mutant protein in HNSCC. Furthermore, pharma-coproteomic profiling revealed that COTI-2 resulted in acti-vation of AMPK and inhibition of the oncogenic mTOR path-ways in HNSCC cells independent of p53 status. Our data

suggest that combination of COTI-2 with cisplatin or radiationmay be novel therapy for treatment of HNSCC harboring TP53mutations.

Materials and MethodsTissue culture, reagents, and generation of stable cell lines

The HNSCC cell line PCI13 lacking endogenous p53 wasobtained from the laboratory of Dr. Jennifer Grandis (Universityof Pittsburgh, Pittsburgh, Pennsylvania) in August 2008 andengineered to stably express constructs containing either wild-type p53 or high-risk EA (Evolutionary Action) score mutant p53(C238F and G245D, R175H, R273H, R282W) as described pre-viously (6). Additional HNSCC cell lines used in this study wereobtained from an established cell repository in the laboratory ofDr. Jeffrey N. Myers (University of Texas MD Anderson CancerCenter,Houston, TX) under approved institutional protocols. Thecell lines were maintained in DMEM supplemented with 10%FBS, L-glutamine, sodium pyruvate, nonessential amino acids,and a vitamin solution, and incubated at 37�C in 5% CO2 and95%air. The identity of all cell lines was authenticated using shorttandem repeat (STR) testing within 6 months of cell use. COTI-2drug was supplied by Critical Outcome Technologies Inc (cur-rently known as Cotinga Pharmaceuticals). For in vitro studies,COTI-2 was prepared as a 1.0 mmol/L stock solution in DMSOand stored at �20�C.

Clonogenic survival assayFor the clonogenic survival studies,HNSCCcellswere seeded in

6-well plates at predetermined densities, concurrently exposed todifferent fixed-ratio combinations of COTI-2 (dose range, 0.01–40nmol/L) and cisplatin (dose range, 0.1–2mmol/L) for 24 hoursand clonogenic cell survival was determined as described previ-ously (14). For radiosensitivity assays, cells were treated withdifferent doses ofCOTI-2, as indicated, then followedby exposureto either 2, 4, or 6 Gray (Gy) radiation and the surviving fraction(SF2) values were determined.

Analysis of combined drug effectsDrug synergy between COTI-2 and cisplatin was assessed by

combination-index and conservative isobologram analyses,which were generated according to the median-effect methodof Chou and Talalay (15) using CalcuSyn software (Biosoft).See the Supplementary Materials and Methods section fordetails.

Western blot analysisCells grown on 10-cm plates were treated with physiologically

relevant doses ofCOTI-2 (1.0mmol/L), CDDP(1.5mmol/L) eitheralone or in combination for 16or 48hours. For radiosensitizationstudies, cells were radiated with 4 Gray. Whole-cell lysates wereprepared and Western blot analyses were conducted with indi-cated antibodies as described previously (14). Densitometricquantifications were performed with ImageJ (v1.50i). Antibodiesused forWestern blotting are described in SupplementaryMateri-als and Methods section.

Cell-cycle analysis and Annexin V-FITC/PI stainingCells were seeded in 60-mm dishes, treated the next day with

COTI-2 (1 mmol/L), CDDP (1.5 mmol/L) either alone or incombination and then harvested at 12, 24, 48, or 72 hours. The

Translational Relevance

TP53mutations are highly prevalent in HPV-negative headand neck cancer and are often associated with aggressivedisease and resistance to curative therapy with cisplatin andradiation. The majority of these TP53 mutations gain onco-genic properties which are independent of wild-type p53functions. In this study, we have demonstrated that COTI-2,a novel p53 reactivator, has the ability to restore mutant p53function, possesses single-agent activity, and further potenti-ates responses to cisplatin and/or radiation in vitro and in vivoin preclinical models of HNSCC with different TP53 status.Our data provided a preclinical foundation for testing thesafety and efficacy of COTI-2 in an ongoing phase I study ofpatients with refractory gynecologic and head and neckmalignancies.

COTI-2 Synergizes with Cisplatin and Radiation in HNSCC

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cell-cycle analysis was performed as described previously (14).Annexin V-FITC/PI staining was used to detect apoptotic celldeath using the BD Biosciences apoptotic detection kit accordingto the manufacturer's instructions.

Live-cell imaging and EdU labelingHNSCC cell lines (PCI13-pBabe, PCI13-G245D) were stably

transfectedwith histoneH2B-RFP lentiviral vector (Addgene) andsortedbyflowcytometry to enrich for highly expressing cells. Cellswere treated with drugs as indicated and live video imaging, EdUlabeling, andDNA content measured by laser scanning cytometryanalyses, were all carried out as described previously (16).

RNA-Seq profilingHNSCC cell lines (PCI13) stably expressing either wild-type

p53 or high-risk mutp53 (G245D) were treated with COTI-2(1.0 mmol/L) and subjected to RNA sequencing analysisas described in the Supplementary Materials and Methodssection.

qRT-PCR analysesPCI13 cells expressing pBabe (null TP53), wild-type TP53, or

TP53-mutant (G245D) constructs were treated with COTI-2(1.0 mmol/L) for 24 or 48 hours before isolation of total RNAusing RNeasy Mini Kit (QIAGEN). Detailed description of theqRT-PCR procedures is included in the Supplementary Materialsand Methods.

Chromatin immunoprecipitationPCI13 cells expressing pBabe, wild-type TP53, or high-risk

mutant TP53 (G245D) were treated with DMSO (controls) orCOTI-2 as indicated for 24 hours and cross-linked using para-formaldehyde. The DNA lysates were then immunoprecipitatedwith anti-p53 (DO-1X) antibody and the recovered ChIP DNAwas subjected to PCR analysis. The experimental procedureswere described in details in the Supplementary Materials andMethods.

3D cell culture and in vivo TUNEL assayA "3D cell culture" was established as described in previous

publication (17, 18). Briefly, a total of 3,000 cells (PCI13-G245D)embedded in 150mL of collagen gel (n ¼ 3/group) were thenlayered on top of the solidified collagen gel. The "3D tumors"were treated with a calculated corresponding maximum physio-logic (given to mice) dose of COTI-2 (2 mmol/L) for 1 day or5 days and effect of the drug on apoptosis was determined usingthe in vivo TUNEL assay as described in Supplementary Materialsand Methods.

In vivo orthotopic mouse model of oral tongue cancerAll animal experimentation was approved by the Institution-

al Animal Care and Use Committee (IACUC) of the Universityof Texas MD Anderson Cancer Center (Houston, TX). Ourorthotopic nude mouse tongue model has been describedpreviously (14, 16, 17). PCI13 cells (3 � 104) containing eitherwild-type p53 or high-risk mutant p53 (G245D), and HN31were injected into the tongues of male athymic nude micebecause oral cancer is mostly prevalent and represented in malepatients. Mice were randomized into different groups 8–10 daysafter injection. Treatment with COTI-2 (75 mg/kg), cisplatin, orin combination was initiated when tumors were about 1–3

mm3 in size. Treatment with COTI-2, radiation, or in combi-nation was initiated when tumors reached about 10–20mm3 insize. Details of the treatment protocol and tumor growthinhibition measurement are described in the SupplementaryMaterials and Methods.

Statistical analysisThe Student t and one-way ANOVA tests were utilized to

analyze in vitro data. For mouse studies, a two-way ANOVA testwas used to compare tumor volumes between control andtreatment groups. Survival following drug treatment and radi-ation was analyzed by the Kaplan–Meier method and com-pared with log-rank test. All data were expressed as mean � SE,and P values of 0.05 or less were considered to indicatestatistical significance.

ResultsCOTI-2 shows single-agent activity and synergy with cisplatinand radiation in vitro in HNSCC cells expressing GOF-mutantp53

To determine the relative degree of sensitivity of HNSCC cellsto COTI-2 as a single agent, isogenic HNSCC PCI13 cells eitherlacking p53 expression (pBabe null), or expressing wild-typep53, or p53 mutations (C238F, G245D, R175H, R273H,R282W) were treated with COTI-2 for 24 hours and thenassessed for clonogenic survival. As shown in Fig. 1A and B,the PCI13 isogenic cells displayed exquisite sensitivity to COTI-2 as a single agent (IC50; 1.4–13.2 nmol/L). Representativeimages of clonogenic survival assays with corresponding IC50for COTI-2 as a single agent are shown in SupplementaryFig. S1A–S1C. The results suggest that COTI-2 appears to targetHNSCC regardless of TP53 status. In addition, MTT viabilityassays demonstrated that various HNSCC cell lines with nat-urally occurring p53 mutations and HPV positivity (wtp53)were sensitive to COTI-2 as monotherapy (SupplementaryFig. S1D). HNSCC cells expressing mutant p53, but notwild-type p53, are relatively resistant to cisplatin and radiationin vitro and in vivo (3, 14, 19). We next examined whether COTI-2 was synergistic with CDDP treatment in isogenic PCI13 celllines, using the combination index (CI) method of Chou andTalalay (15). HNSCC cells expressing mutant p53 were resistantto cisplatin, and addition of COTI-2 significantly decreasedtheir survival in vitro, compared with each drug alone (Fig. 1C,E, and G). Conservative isobologram plots of effective doses(ED50; 50% inhibition), ED75 (75% inhibition), and ED90(90% inhibition) show the combination index (CI) at eachinhibitory concentration is less than 1.0, indicating strongsynergism in all HNSCC cells tested (Fig. 1D, F, and H). Strongsynergy between COTI-2 and cisplatin was also demonstratedin additional HNSCC cell lines with naturally occurring mutantp53 (UMSCC10A) and HPVþ (UMSCC47; SupplementaryFig. S2A–S2D). To evaluate with COTI-2 radiosensitizesHNSCC cell lines, PCI13-Wtp53 and PCI13-G245D cells weretreated with radiation and COTI-2 and subjected to clonogenicsurvival assays as indicated. The degree of radiosensitizationwas quantified from the survival curves by comparing survivingfractions at the radiation dose of 2 Gy (SF2). The SF2 isparticularly relevant because 2 Gy is the typical dose given ona daily basis in clinical radiotherapy for HNSCC. Comparedwith control and each treatment alone, COTI-2 markedly

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Figure 1.

COTI-2 shows single agent activity and strong synergy with cisplatin and radiation in vitro in HNSCC cells expressing GOFmutant p53. A and B, Clonogenicsurvival curves for isogenic HNSCC PCI13 cells lacking p53 (pBabe null), wtp53, or various GOFmutp53 (C238F, G245D, R175H, R273H, R282W) treated with arange of COTI-2 concentrations (0.01–40 nmol/L) for 24 hours to determine the IC50. C, E, and G, Representative images of clonogenic survival assays forcombined treatment with COTI-2 and cisplatin (CDDP). D, F, and H, Assessment of the degree of synergy between COTI-2 and CDDP in PCI13-pBabe (p53 null),PCI13-G245D, and PCI13-R273H cells, using the Chou and Talalay method. CDDP and COTI-2 were used at constant ratios (50:1, respectively). The CI values forcombination of effective drug doses (ED) that result in growth inhibition of 50% (ED50; fraction affected; fa¼ 0.5), 75% (ED75; fa¼ 0.75), and 90% (ED90;fa¼ 0.90) were generated from the conservative isobolograms. The ED50 (red X), ED75 (green crosses) and ED90 (blue circles) graphed against fractionalconcentrations of CDDP and COTI-2 on the y and x-axis, respectively are indicated. The diagonal line is the line of additivity. The conservative isobologram plotsshow CI values less than 1.0 falling below diagonal line, demonstrating that COTI-2 and CDDP acts synergistically to inhibit in vitro growth of HNSCC cells. Alltreatments were performed in triplicate and each experiment was repeated at least three times.






decreased the SF2 fractions when combined with radiationin vitro in HNSCC cells in a dose-dependent manner (Supple-mentary Fig. S3). Taken together, these data clearly demon-strated that COTI-2 synergized with cisplatin and/or radiationand inhibited in vitro tumor cell growth in HNSCC independentof the TP53 status.

COTI-2 increases the levels of DNA damage response andreplication stress markers with subsequent induction ofapoptosis and cellular senescence in HNSCC cells

Chemotherapy and radiation elicitDNAdamage responses andcan confer anticancer synergy in tumor cells. Therefore, phos-phorylation levels of the DNA damage and replicative stressindicators, gH2AX (S139), CHK1 (S345), and CDK1 (Y15) wereexamined by Western blotting. Increased phosphorylation ofgH2AX, CHK1, and increased activity of CDK1 (manifested bydecreased level of phosphorylation at tyrosine 15) were observedfollowing COTI-2 treatment and in combination with cisplatinand/or radiation (Fig. 2A and B). Modification of these proteinsoccurred in a time-dependent manner, indicating induction of

DNA damage and replication stress responses during cell cycle inthese cells. Blots were quantified and results were shown inSupplementary Fig. S4A and S4B. The p21 protein levels wereinduced in all cells tested in a time-dependent manner (Fig. 2Aand B; Supplementary Fig. S4A and S4B), suggesting that COTI-2activates p53-dependent transcription similar to that regulated bywild-type p53. To determine whether COTI-2 inhibits growth ofHNSCC cells via apoptosis, the apoptotic marker, PARP cleavagewas examined at 16 and 48 hours of treatment by Westernblotting. Cleavage of PARP, caspase-3, and to less extent cas-pase-9 was observed only in cell lines with mutant (G245D) ornull (PCI13-pBabe) p53 when exposed to COTI-2 alone or incombination with cisplatin and radiation (Fig. 2A and B; Sup-plementary Fig. S5A–S5C). Induction of apoptosis in these cellsfollowing treatment with cisplatin and COTI-2 was further con-firmed with AnnexinV/PI–positive staining (Fig. 2C and D).Treatment of the wild-type p53 cells with COTI-2 showed verylow levels of AnnexinV/PI–positive staining (SupplementaryFig. S6A and S6B). The p53 can cause growth arrest throughsenescence and can be assessed by expression of the acidic

Figure 2.

COTI-2 increases the levels of DNAdamage response and replicationstress markers with subsequentinduction of apoptosis in defectiveTP53 HNSCC cells. PCI13-pBabe(null TP53) and PCI13-G245D(mutant p53) cells were treatedwith either COTI-2, cisplatin,radiation (XRT) alone, or incombination for 16 and 48 hoursand subjected to immunoblotanalysis using antibodies asindicated. A and B, Proteinexpression levels of p53, p21,phospho-gH2AX (S139), phospho-Chk1 (S345), and total Chk1. Themitotic entry marker histone H3 S10phosphorylation, CDK1 (CDC2) tyr15phosphorylation, and total proteinlevels were also analyzed. Thepresence of PARP-1 cleavage asmarker of apoptosis is shown.b-Actin served as a loading control.C and D, AnnexinV/PI–positivestaining confirming induction ofapoptosis in PCI13-pBabe andPCI13-G245D HNSCC cells followingtreatment with COTI-2 and cisplatin.E and F, Unsynchronized PCI13-pBabe and PCI13-G245D cells weretreated with COTI-2, CDDP alone, orin combination for various timepoints as indicated and subjected tocell-cycle analysis. NT, no treatmentcontrol. Percentage of cell-cycledistribution determined by flowcytometry is presented as bargraphs. An increase in S-phase andsub-G1 fractions indicative ofreplicative stress and cumulativeapoptosis respectively wereobserved over time. Data shown arerepresentative of two independentexperiments.

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senescence-associated b-galactosidase (SA-b-gal) activity. The iso-genic PCI13-WTp53 and naturally occurring wild-type p53(HN30) cell lines were treated as indicated, and tested for SA-b-gal positivity. A substantial fraction of cells showed increasedlevels of SA-b-gal activity following COTI-2 treatment (Supple-mentary Fig. S6C). Cells treated with cisplatin served as positivecontrols. Quantification of senescence staining is shown in Sup-plementary Fig. S6D and S6E. These results indicate that HNSCCcells expressing wild-type p53 are sensitive to COTI-2 treatmentthrough induction of cellular senescence rather than apoptosis atthe concentrations of drug evaluated.

To better understand the longer term effects of drug treatment,PCI13-pBabe and G245D cells were treated with cisplatin andCOTI-2, and examined at 12–72 hours for cell-cycle kinetics.Treatment with COTI-2 arrested the cells at G1 and G2–M cell-cycle phases in a time-dependent manner (12–48 hours); how-ever, at 72 hours, cell number in these phases gradually declinedbecause proportion of cells went into apoptosis as revealed byincreased sub-G1 fractions (Fig. 2E and F). Moreover, cisplatinarrested the cells at the G2–M phase within 48 hours and uponaddition of COTI-2, increased fraction of cells was observed in theS-phase, suggesting slow rate of DNA synthesis associated withinduction of replication stress (Fig. 2E and F). While cells with nop53 (pBabe) showed increased sub-G1 fraction only at 72 hours(Fig. 2E), a significant increase in the sub-G1 fraction of cellsindicative of apoptosis was seen in PCI13-G245D cells after48 hours of combination treatment and remained elevated at72 hours (Fig. 2E). Interestingly, cells with wild-type p53 werearrested at the G1 cell-cycle phase upon treatment with COTI-2 orin combination with cisplatin (Supplementary Fig. S7). Further-more, these cells showed insignificant sub-G1 fraction of cells,consistent with the low levels of the PARP-1 cleavage and Annex-inV/PI staining observed previously with the drug treatment(Supplementary Fig. S6A and S6B).

Multiple cell-cycle perturbations leading to significantapoptosis in HNSCC cells treated with combination of COTI-2and cisplatin

To better understand the dynamics of cell-cycle perturbationsand cell death response to COTI-2 treatment alone and in com-bination with cisplatin, live-cell imaging studies were carried outon histone H2B-RFP-tagged HNSCC cells. The results of thesestudies are depicted in Fig. 3A as event charts for the cell linesPCI13-pBabe (p53 null) and PCI13-G245D (mutp53), whereeach graphed line represents the history of a cell over time andthe color of the line represents whether the cell was in interphase(gray) or in mitosis (blue). A red asterisk marks the time ofapoptosis. Over the 50-hour time period of these studies, bothuntreated cell lines were able to complete nearly three completecell cycles (i.e., three waves of mitoses). While most cells treatedwith cisplatin were able to reach the first mitosis, albeit increas-ingly delayed with duration of treatment, subsequent cell cycleswere slowed as demonstrated by longer time intervals from thefirst to the second mitosis and fewer transitions through subse-quent cell cycles. On the other hand, only two-thirds of COTI-2–treated cells were able to reach the first mitosis with similarkinetics as untreated cells (Supplementary Fig. S8A), suggestinga block in the early phase of the cell cycle.Moreover, theywere notable to attain a second mitosis and a high fraction of cellsunderwent apoptosis at variable times after the first mitosis.Interestingly, COTI-2–treated cells showed increasing cyto-

plasmic vacuolization after 12hours that persisted until apoptosis(Fig. 3B; Supplementary Fig. S9; Supplementary Videos S1–S4).Combined treatment with cisplatin and COTI-2 resulted in fewercells reaching the first mitosis and slightly earlier onset and extentof apoptosis. Cells expressing wild-type p53 showed very littleapoptosis upon treatment with COTI-2 alone that increased withaddition of cisplatin (Supplementary Fig. S10), but seemedmuchlower than that observed in cells with pBabe or G245D, respec-tively. Because COTI-2 treatment was found to block cells fromreaching a second mitosis, it was of interest to determine whereand howCOTI-2 treatment delayed cell-cycle progression. PCI13-pBabe and PCI13-G245D cells were therefore treated with COTI-2, pulsedwith EdU for 1 hour prior to cell harvest after 12, 18, and24 hours of treatment, and analyzed for cell-cycle position andEdU uptake levels (i.e., rate of DNA synthesis). As shownin Fig. 3C and Supplementary Fig. S8B, by 12 hours of COTI-2treatment, there was an increase in the fraction of cells in S-phasebut with decreased EdU uptake per cell, suggesting a generalizedslowing ofDNA synthesis. By 18hours ofCOTI-2 treatment, fewercells were transitioning from G1 to S-phase, and DNA synthesiswas increasingly slowed, resulting in an increased fraction of cellsin late S-phase. By 24 hours of COTI-2 treatment, the rate of DNAsynthesis was even lower but the relative fraction of cells in S-phase dropped, suggesting that cells were dying in late S/G2 phaseand were not able to reach the second mitosis.

COTI-2 restores the DNA-binding properties andtranscriptional activity of a subset of p53-mutant proteins inHNSCC cells

To further assess the cellular phenotype associated with theactivity of COTI-2 monotherapy in HNSCC cells expressing wild-type or mutp53, cells were treated for 48 hours with the drug anddifferentially expressed genes (DEGs) were examined by RNAsequencing (RNA-seq). A linear regression model [false discoveryrate (FDR) cutoff ¼ 0.05] and filtering criteria (i.e., DEG with anPadj value of < 0.01, genes with 50 average reads among thecomparison groups, exclusion of noncoding RNAs) were used.Clustering via principal component analysis (PCA) revealed thatDEGs among treatment groups are different between cell types.Approximately, 1,772 and 2,764 genes (up- and downregulated)out of the total 26,119 DEGs that are significantly differentbetween COTI-2 and untreated groups with P < 0.01 were iden-tified in PCI13-wtp53 and PCI13-G245D cells, respectively (Sup-plementary Table S1). Because our in vitro Western blotting datasuggested that COTI-2–induced p21 (CDKN1A), a known p53target gene inHNSCC cells tested (Fig. 2), we focused our analysison comparing the p53-induced gene expression levels in PCI13cells expressing wild-type and mutant TP53 following COTI-2treatment within 48 hours as an indication of normalization ofp53 transcriptional activity. A list of high confidence p53 targetgenes has been described in the literature (20). Results demon-strated that COTI-2 treatment produced a p53 target expressionsignature remarkably different from untreated controls (Fig. 4Aand B; Supplementary Tables S2–S4), suggesting that this agentrestored normal DNA-binding activity to the p53 G245D mutantsimilar to wild-type p53 and resulted in a transcriptionally activeprotein. The results further suggest thatCOTI-2 inhibits cell growththrough a p53-dependent mechanism. The cluster heatmap repre-sents significant differentially expressed p53 target genes (82 and94genes forwtp53andmutp53, respectively) as indicatedwith thecolor scale (Fig. 4A and B).mRNA expression levels of selected p53






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Figure 3.

Cell-cycle perturbation and subsequent apoptosis in HNSCC cells treated with combination of COTI-2 and cisplatin. Histone-H2B-RFP–expressing PCI13-pBabeand PCI13-G245D cells were treated with the drugs as indicated and followed by live cell imaging.A, Live cell imaging results depicted as event charts, whereeach line represents one cell. The cell's time in interphase is labeled gray, its time in mitosis is labeled blue, and the time of apoptosis is marked by a red symbol.B, Representative still image frames from the live-cell imaging movies illustrating mitotic and interphase events leading to increased cytoplasmic vacuolizationand apoptosis in PCI13-G245D cell lines treated with COTI-2 compared with untreated control. C, Scatter plots showing cell-cycle position and EdU uptake levels(i.e., rate of DNA synthesis) in PCI13-pBabe and PCI13-G245D cells harvested and analyzed after 12-, 18-, or 24-hour time points following treatment with COTI-2as indicated. Decreased EdU uptake and eventual disappearance of S-phase cells at later time point is shown.

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target genes presented in the heatmap, including p21, PUMA,NOXA, MDM2, EphA2, PLK2, and ATF3 in PCI13-Wtp53 andPCI13-G245D cells treated with COTI-2 for 24 hours were vali-datedwith qRT-PCR analysis (Fig. 4C andD). InHNSCCcellswithdefective p53, basalmRNAandprotein expression levels of the keyprosurvival geneMDM2 were increased compared with wild-typep53 cells (Supplementary Fig. S11A–S11D). Increased expressionof TAp63 was also observed in the PCI13-G245D cells followingCOTI-2 treatment (Fig. 4A and B), suggesting that the drug canenhance tumor suppression through induction of this TP53 fam-ily–related gene. We further confirmed the ability of COTI-2 torestoreDNA-bindingproperties to thep53G245D-mutant proteinby ChIP coupledwith qRT-PCR analyses. Results in PCI13-G245Dcells treated with COTI-2 for 24 hours revealed significant resto-ration of site-specific DNA binding of p53 G245D mutant to thepromoters of p21, PUMA, NOXA, MDM2, PLK3, and ATF3

(Fig. 4F). While wtp53 showed significant binding activity toMDM2 andATF3 promoters upon COTI-2 treatment, this bindingactivity was not significant toward p21, PUMA, NOXA, and PLK3promoters in these cells (Fig. 4E), consistent with publisheddata (9). No p53 DNA-binding activity to these promoters wasdetected in the PCI13-pBabe (p53 null) cells following COTI-2treatment (Supplementary Fig. S12). As TAp63a can alsoinduce many of the prototypical p53-inducible target genes(PIG) that can mediate cell-cycle arrest and induction of senes-cence and apoptosis (21), it is conceivable that activation ofthis isoform could mediate COTI-2 antitumor effects observedin in the p53-null HNSCC cells. qRT-PCR analysis revealed thattreatment with COTI-2 for 48 hours increased expression ofthe p53 target genes and TAp63a isoform following COTI-2treatment in HNSCC cells lacking TP53 (PCI13-pBabe; Sup-plementary Fig. S13A and S13B). Furthermore, ChIP analysis

Figure 4.

COTI-2 restores the DNA-bindingproperties and transcriptionalactivity of the p53-mutant protein inHNSCC cells. An RNA-sequencinganalysis of COTI-2 (1 mmol/L, 48hours) and control treated PCI13-wtp53 and PCI13-G245D HNSCCcells comparing gene expressionlevels of 126 of 310 p53-induciblegenes (PIG) was performed. A andB, Cluster heatmap representssignificant differentially expressed82 and 94 p53 target genes asindicated with the color scale (foldchange) for PCI13-wtp53 and PCI13-G245D cells, respectively. Statisticalsignificance was set based on theFDR cutoff of 0.05. C and D, qRT-PCR analyses to validate mRNAexpression levels of selected p53target genes presented in theheatmap, including p21, PUMA,NOXA, MDM2, PLK3, and ATF3 inthese cells treated as indicated.E and F, Chromatinimmunoprecipitation (ChIP) assay.Lysates from PCI13-wtp53 andPCI13-G245D cells treated withCOTI-2 for 24 hours were subjectedto immunoprecipitation using anti-p53 antibody (DO-1) and followedby qRT-PCR of p53 recognitionelements (p53REs) of p21, PUMA,NOXA, MDM2, PLK3, and ATF3.Results of functional DNA-bindingproperties with COTI-2 treatmentwere presented as enrichment foldchange (� , P < 0.05; �� , P < 0.001compared with untreated control,respectively).






using the p63a antibody followed by qRT-PCR analysis showedthat COTI-2 significantly enhanced DNA binding of TAp63 top21 and PUMA promoters consistent with this contention(Supplementary Fig. S13C).

COTI-2 leads to activation of AMPK and inhibition of theoncogenic mTOR pathways in HNSCC cells independent ofp53 status

Our results demonstrated that regardless of p53 status,HNSCC cells were similarly sensitive to COTI-2 in vitro. Thissuggests that a portion of the response to COTI-2 is p53independent and further COTI-2 may have additional targets.Therefore, to identify other signaling pathways mediating theeffects of COTI-2, proteomic profiling using a reverse-phaseprotein array (RPPA) was performed in PCI13-wtp53, PCI13-pBabe (p53 null), and PCI13-G245D cells treated with COTI-2for 8 and 36 hours as indicated. Significant changes in proteinexpression levels were selected on the basis of Padj < 0.05 foreach comparison. The principal component analysis (PCA) forthe normalized protein expression revealed that all sampleswere clustered by cell type, treatment type, and time. Unlikethe 36-hour time point, no significant differences betweentreatments versus control groups were observed at 8-hour timepoint in all cell lines tested. Therefore, we focused our attentionon detecting differences in protein levels in samples from thesecells treated with the drug for 36 hours. A total of 13, 64, and76 of 301 total proteins assessed by RPPA were significantlydifferentially expressed (Padj < 0.05) in wtp53, pBabe, andG245D-expressing cells respectively (Supplementary TablesS5–S7). To focus our investigation, we limited our analysisto protein level changes of > 1.5 fold, with a false discoveryrate of < 1% and P < 0.05 (22). No significant differences inprotein expression levels were observed in the wild-type p53expressing cells among untreated and COTI-2–treated groups(Supplementary Table S8). However, we identified significantdifferences in protein expression levels in 13 of 64 and 18 of76 proteins in cells null for p53 (pBabe) or expressing mutantp53 (G245D), respectively (Supplementary Tables S9 and S10).The hierarchical cluster heatmap representing significantlydifferentially expressed proteins is shown in Fig. 5A and B.Proteins involved in the AMPK and mTOR signaling pathwayswere among the top proteins that were significantly upregu-lated and downregulated, respectively, following COTI-2 treat-ment in both pBabe and G245D cells (Fig. 5A and B). Proteinsinvolved in cell-cycle regulation, apoptosis, gap junction, andcalcium regulation were also downregulated in pBabe andG245D cells (Fig. 5A and B). Differential expression of theseproteins was further confirmed by Western blotting (Fig. 5C–E). These results further suggest that COTI-2 activates apoptosisin HNSCC cells through reduction of the antiapoptotic proteinMCl-1 and induction of proapoptotic protein PDCD4, respec-tively. COTI-2 decreased expression of connexin-43, a trans-membrane protein that regulates gap junction intercellularcommunication (23–26). Therefore, we examined whetherCOTI-2–mediated decreased connexin-43 resulted in inhibi-tion of gap junction–mediated cellular communication usingthe scrape loading and dye transfer technique (23–26). COTI-2significantly decreased the number of cells labeled with the dye,indicating a possible involvement in regulation of cellularcommunication in HNSCC (Supplementary Fig. S14A andS14B). Taken together, the RPPA analysis suggests that COTI-

2 acquires an additional non-p53 mechanism to inhibit tumorgrowth in HNSCC cells.

COTI-2 displays single-agent activity and enhances response tocisplatin and radiation in vivo in an orthotopic mousemodel oforal tongue cancer

3D cell culture systems have been established as reliablemodels formonitoring cell growth, proliferation, and drug testingdue to their evident advantages in providingmore physiologicallyrelevant phenotypes similar to that in vivo (17, 18). Therefore, thePCI13-G245D andHN31 cells were grown in 3D collagen cultureand treated with COTI-2 as indicated previously. A significanteffect on cell growth in 3D culture at day 5was observed followingtreatment with COTI-2 (Fig. 6A). In addition, TUNEL-positivestaining was significantly increased in the 3D culture organoidstreated with COTI-2, indicative of apoptosis (Fig. 6B and C),consistent with 2D in vitro data. Moreover, organoids estab-lished from the 3D culture of these mutant p53 cell lines andtreated with COTI-2 for 5 days exhibited increased positive tonegative ratios of nuclear immunostaining of p21 comparedwith untreated controls (Fig. 6D and E), consistent with the 2Din vitro data. The H&E staining of the 3D organoids is shown inSupplementary Fig. S15I.

On the basis of the in vitro results, the impact of COTI-2 eitheralone or in combination with cisplatin and radiation was eval-uated in an orthotopic nude mouse model implanted withisogenic PCI13-Wtp53 and PCI13-G245D cells in the tongue.COTI-2 (75mg/kg) alone had significant effects on tumor growthin vivo at this tested optimal dose, independent of p53 status(Fig. 6F and G). While cisplatin treatment with physiologic dose(4 mg/kg) had little effect on tumor growth, addition of COTI-2enhanced the response to cisplatin and significantly reduced oraltumor growth (P ¼ 0.0358 and P ¼ 0.0006) in mice bearingPCI13-Wtp53 and PCI13-G245D cells, respectively (Fig. 6F andG). The combination of COTI-2 and cisplatin showed a trendtoward increase in tumor growth inhibition compared withCOTI-2 alone as a result of its strong single-agent activity(Fig. 6F and G). No survival benefits were observed with combi-nation of cisplatin and COTI-2 at the dose tested (SupplementaryFig. S15A and S15B). We next determined the effect of COTI-2 onthe response to radiation in vivo. A fractionated radiation (2 Gy)was delivered to the tumor-bearing tongue of mice expressingPCI13-G245D and HN31-mutant p53 cells, following treatmentwith COTI-2. Compared with untreated controls, significanttumor growth delay was observed with a combination ofCOTI-2 and radiation in mice bearing each cell lines (Fig. 6Hand I). The combination of COTI-2 and radiation showed littletumor growth delay and survival benefits compared with radia-tion or COTI-2 alone (Fig. 6Hand I; Supplementary Fig. S15C andS15D) in the models tested. Taken together, our results suggestthat COTI-2 potentiates in vivo sensitivity of HNSCC-mutant p53cells to cisplatin and radiation. During the study, mice in thetreatment arms showed no more than 10%–20% body weightloss (Supplementary Fig. S15E–S15H).

DiscussionSeveral attempts have been made to design molecules that

target mutant p53 in cancer cells (27). However, with the excep-tion of PRIMA-1Met, all p53 reactivators showed unacceptabletoxicities in mice precluding further clinical development (27).

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COTI-2 is a new smallmolecule that has been proposed to restoreawild-type–likep53function inmutantTP53cancer cells (11,12).In this study, we show for the first time that COTI-2 is active as asingle agent against a variety of head and neck cancer cellsirrespective of TP53 status, consistent with recent reports (11, 12).COTI-2 significantly potentiates sensitivity to cisplatin and/orradiation in vitro and in vivo in HNSCC tumor cells that areinherently resistant to these treatments. The antitumor and apo-ptotic effects of COTI-2 are also evident in 3D organotypiccultures of HNSCC. While COTI-2 induces apoptosis in HNSCCcells lacking TP53 or with mutant TP53, in wild-type TP53 cells,COTI-2 induces senescence rather than apoptosis. This could bedue to the enhanced effect of COTI-2 on p21, a known inducer ofsenescence in these cells (19, 28). Surprisingly, apoptosis was notapparent even though many apoptotic genes were upregulated inwild-type TP53 cells. It is likely that these proapoptotic geneswerenot induced with COTI-2 at the protein levels to cause significantapoptotic death in these cells. While it is not clear how COTI-2

potentiates the combination effect of cisplatin and/or radiation inwild-type TP53 cells, it is possible that this drug exerts antitumoractivity through nonspecific cytotoxic effects. Taken together, thedifferent mechanisms of action in wild-type versus TP53-mutantcells suggests that this agent could have potential therapeutic rolesin different settings.

COTI-2 belongs to the thiosemicarbazone family that can actas metal ion zinc [Zn2þ] chelators to induce correct p53 proteinfolding and restore wild-type structure and function of mutantTP53 (10–12). In our study, COTI-2 showed no effects onintracellular zinc [Zn2þ] levels in HNSCC-mutant TP53 cells(Supplementary Fig. S16). These data suggest that COTI-2either has low binding affinity for zinc or its action is inde-pendent of zinc chelation in HNSCC tumor cells. A recentstudy has shown that the thiosemicarbazone agent, ZMC1(NSC319726), reactivates mutant TP53 (R175H) in cancer cellsthrough modulation of zinc ions and redox changes (9). Redoxchanges often lead to generation of reactive oxygen species that

Figure 5.

COTI-2 leads to activation of AMPKand inhibition of the oncogenicmTOR pathways in HNSCC cellsindependent of p53 status. PCI13-pBabe and PCI13-G245D HNSCCcell lines treated with COTI-2 for36 hours were subjected to RPPAanalysis as indicated. A and B,Hierarchical cluster heatmapanalyses of proteins and phospho-proteins from RPPA significantlydifferent (protein level changes of> 1.5 fold, with an FDR� 1% andP < 0.05) between COTI-2treatment and control in these celllines (performed in triplicate). Redand green color scales indicateincreased or decreased proteins orphosphoproteins levels,respectively. C–E,Western blotanalyses confirming differentialexpression of most of theseproteins, including APMK andmTOR following the drugtreatment.






play a pivotal role in triggering cellular senescence (28). There-fore, the senescence seen in wild-type TP53-expressing HNSCCcells following COTI-2 treatment could be mediated throughROS production. In addition, our in vitro experiments guidedby live-cell imaging demonstrate that COTI-2 not only arrestedcells at the G1 and G2–M cell-cycle phases but also causedaccumulation of fraction of cells in early S-phase as result ofprogressively slowed rates of DNA synthesis. These data furthersuggest that COTI-2–mediated cell death may involves stress-induced signals in HNSCC. Furthermore, addition of cisplatinresulted in significant replication stress and apoptotic death,

perhaps in response to stalled and collapsed replication forks.Interestingly, COTI-2–treated cells showed increasing cyto-plasmic vacuolization that persisted until apoptosis. Whetherthese vacuoles represent induction of autophagy or reflectaltered metabolism remains to be determined.

One significant finding of our study is that COTI-2 restores site-specific DNA-binding properties of p53-mutant protein to thepromoters of several p53 target genes determined by ChIP.Moreover, COTI-2 produced a distinct p53 target gene signaturethat is remarkably different from untreated controls, confirmingthat the structural changes of the PCI13-G245D–mutant TP53

Figure 6.

COTI-2 displays single-agent activity and enhances response to cisplatin and radiation in vivo in an orthotopic mouse model of oral tongue cancer. A, PCI13-G245D and HN31 cells grown in 3D collagen culture and monitored for survival following treatment with equivalent physiologic dose of COTI-2 (2 mmol/L) asindicated for 1 and 5 days. B, TUNEL-positive staining determined at day 5 in the 3D culture organoids made from PCI13-G245D and HN31 cells followingtreatment with COTI-2. C,Quantification of the positive TUNEL staining shown in B.D, Positive nuclear immunostaining of p21 (brown) with addition of COTI-2evaluated at day 5 compared with untreated controls in organoids made from these p53-mutant HNSCC cells. E,Quantification of the positive staining of p21shown in D. F and G, Tumor growth curves in orthotopic mouse model oral tongues bearing HNSCC (PCI13-wtp53 and PCI13-G245D) cells following treatmentwith COTI-2 (75 mg/kg), cisplatin (4 mg/kg) either alone or in combination as described in methods. H and I, Tumor growth curves in orthotopic mouse modeloral tongues bearing these p53-mutant cells following treatment with COTI-2 (75 mg/kg), fractionated radiation dose (2 Gy), and in combination as described inMaterials and Methods. Each treatment group contains 6–12 mice.

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resulted in a transcriptionally active proteinmediating cell killing.These results are in agreement with study demonstrated thatZMC1 resulted in restoration of p53 transcriptional function inTOV112D cell lines (9). In addition, our data suggest that theeffect of COTI-2 in HNSCC lacking TP53 is mediated, at least inpart, through activation of p53 target genes likely induced by theTAp63 gene family. Interestingly, COTI-2 increases expression ofMDM2, a p53-interacting protein that represses p53 transcrip-tional activity. Inhibition of MDM2with drugs such as Nutlin-3Aor AMG-232 may increase p53 function and enhance cell killingin HNSCC tumors with mutant p53 following treatment withCOTI-2.

Our proteomic profiling studies revealed that COTI-2 treat-ment affects other targets that are independent of p53 in HNSCC.Thus, COTI-2 treatment results in activation of the tumor sup-pressor, AMPK, and inactivates the oncogene mTOR either direct-ly or indirectly, possibly culminating in greater cell killing inHNSCC, consistent with published data (11). Activation ofAMPKa is mainly initiated allosterically and by posttranslationalmodification through phosphorylation of its threonine residue172(T172)by at least threekinases, namely liver kinaseB1 (LKB1),calcium-/calmodulin-dependent kinase-kinase 2 (CaMKK2), andTGFb-activated kinase 1 (TAK1; ref. 29). In addition, when energyis depleted, high levels of AMP and ADP bind to the AMPKg-subunit and activates AMPK (29). Our data showed thatCOTI-2 treatment resulted in phosphorylation and activation ofthe CaMKK2 kinase isoforms at threonine 286 (T286). Therefore,it is highly likely that COTI-2 activates AMPK through calcium-mediated activation of CaMKK2. It is also possible that inHNSCCcells, COTI-2 activates AMPK through modulation of energymetabolism involving ATP levels. One of the major downstreamsignaling pathways regulated by nutrient deprivation and AMPKis themTOR pathway (30). AMPKdirectly phosphorylates at leasttwo proteins, mTORC1 andmTORC2, to induce rapid inhibitionof mTOR activity (30). Thus, COTI-2–mediated activation ofAMPK observed in our study may be responsible for inhibitingoncogenic mTOR in HNSCC cells. It is possible that AMPKactivation and inhibition of mTOR pathways with COTI-2 leadsto energy depletion and starvation resulting in the cell-cyclechanges and cell death observed in HNSCC. Our study alsoprovided evidence that COTI-2 either directly or indirectlydecreased cellular communication perhaps through downregula-tion of connexin-43.

In summary, we demonstrate that COTI-2 is highly effectivein preclinical models of mutant TP53-bearing tumor cells thatare inherently resistant to cisplatin and radiation. Furthermore,this drug appears to exert its action in a novel manner through acombination of effects including reactivation of a subset ofmutant p53 and induction of DNA damage and replicationstress responses leading to apoptotic cell death. Furthermore,unlike other thiosemicarbazones, our data indicate that COTI-2is not a traditional zinc metallochaperone, and likely acts toincrease apoptosis through both p53-dependent and p53-inde-pendent mechanisms in HNSCC. In our study, few mice died inthe combination of COTI-2 and cisplatin, suggesting possibletoxicity with the dose tested. However, early results from an

ongoing clinical trial conduced at our institution show thatCOTI-2 and in combination with cisplatin is safe and welltolerated in patients with solid tumors including HNSCC(privileged communications, Dr. Shannon Westin, MD Ander-son Cancer Center, Houston TX). Further studies to identifyoptimal and effective combination dose of COTI-2 with cis-platin and/or radiation in vivo in HNSCC with different TP53mutational status are warranted.

Disclosure of Potential Conflicts of InterestG.B. Mills has ownership interests (including patents) at Myriad Genetics,

NanoString, Catena Pharmaceuticals, ImmunoMet, SignalChem, Spindletop,and Tarveda, is a consultant/advisory boardmember for AstraZeneca, Chrysalis,ImmunoMet, Ionis, Mills Institute for Personalized Care Center (MIPCC),Nuevolution, PDX Pharma, SignalChem Lifescience, Symphogen, and Tarveda,and reports receiving commercial research support from AstraZeneca, Ionis,ImmunoMet, Karus Therapeutics, NanoString, Pfizer, and Takeda/MillenniumPharmaceuticals. J.N. Myers reports receiving commercial research grants fromCOTINGA Pharmaceuticals. No potential conflicts of interest were disclosed bythe other authors.

Authors' ContributionsConception and design:A. Lindemann, A.A. Patel, L.Wang, N.L. Silver,M.Gao,W.N. Hittelman, G.B. Mills, J.N. Myers, A.A. OsmanDevelopment of methodology: A. Lindemann, Z. Liu, L. Wang, N. Tanaka,H. Takahashi, N.K. Maduka, W.W. Liu, M. Gao, W.N. Hittelman, J.N. Myers,A.A. OsmanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Lindemann, A.A. Patel, L. Tang, Z. Liu, L. Wang,N.L. Silver,N. Tanaka,H. Takahashi,N.K.Maduka,M. Zhao, T.-C.Chen,M.Gao,S.J. Frank, W.N. Hittelman, A.A. OsmanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Lindemann, Z. Liu, L. Wang, N. Tanaka, X. Rao,H. Takahashi, N.K. Maduka, M. Gao, J. Wang, S.J. Frank, W.N. Hittelman,G.B. Mills, J.N. Myers, A.A. OsmanWriting, review, and/or revision of the manuscript: A. Lindemann, A.A. Patel,L. Wang, H. Takahashi, S.J. Frank, W.N. Hittelman, G.B. Mills, J.N. Myers,A.A. OsmanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Lindemann, A.A. Patel, N.L. Silver, W.W. Liu,W.N. Hittelman, J.N. Myers, A.A. OsmanStudy supervision: A. Lindemann, L. Wang, J.N. Myers, A.A. OsmanOther (performing animal experiments): L. WangOther (acquisition of in vitro data [Western blotting data]): T.-C. Chen

AcknowledgmentsThis work was supported by grants from the NIH/NIDCR R01DE024601 (to

A.A. Osman and J.N. Myers), Cotinga Pharmaceuticals (to A.A. Osman and J.N.Myers), and The University of Texas MD Anderson Cancer Center-OropharynxCancer Program generously supported by Mr. and Mrs. Charles W. Stiefel(awarded to A.A. Osman and J.N. Myers). This work used the services of MDAnderson's Flow Cytometry and Cellular Imaging Core, Bioinformatics SharedResource, and the Reverse Phase Protein Array (RPPA) Core, which are sup-ported by the NIH through MD Anderson's Cancer Center Support Grant(P30CA016672).

The costs of publicationof this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 10, 2019; revised April 25, 2019; accepted July 8, 2019;published first July 15, 2019.

References1. Adelstein DJ, Li Y, Adams GL, Wagner H Jr, Kish JA, Ensley JF, et al. An

intergroup phase III comparison of standard radiation therapy and twoschedules of concurrent chemoradiotherapy in patients with unresectablesquamous cell head and neck cancer. J Clin Oncol 2003;21:92–8.






2. Agrawal N, Frederick MJ, Pickering CR, Bettegowda C, Chang K, Li RJ, et al.Exome sequencing of head and neck squamous cell carcinoma revealsinactivating mutations in NOTCH1. Science 2011;333:1154–7.

3. Pickering CR, Zhang J, Yoo SY, Bengtsson L, Moorthy S, Neskey DM, et al.Integrative genomic characterization of oral squamous cell carcinomaidentifies frequent somatic drivers. Cancer Discov 2013;3:770–81.

4. Cancer Genome Atlas Network. Comprehensive genomic characterizationof head and neck squamous cell carcinomas. Nature 2015;517:576–82.

5. Rivlin N, Koifman G, Rotter V. p53 orchestrates between normal differ-entiation and cancer. Semin Cancer Biol 2015;32:10–7.

6. Osman AA, Neskey DM, Katsonis P, Patel AA, Ward AM, Hsu TK, et al.Evolutionary action score of TP53 coding variants is predictive ofplatinum response in head and neck cancer patients. Cancer Res2015;75:1205–15.

7. Bykov VJ, Issaeva N, Shilov A, Hultcrantz M, Pugacheva E, Chumakov P,et al. Restoration of the tumor suppressor function tomutant p53 by a low-molecular-weight compound. Nat Med 2002;8:282–8.

8. Bykov VJ, Issaeva N, Zache N, Shilov A, Hultcrantz M, Bergman J, et al.Reactivation of mutant p53 and induction of apoptosis in human tumorcells by maleimide analogs. J Biol Chem 2005;280:30384–91.

9. Yu X, Vazquez A, Levine AJ, Carpizo DR. Allele-specific p53 mutantreactivation. Cancer Cell 2012;21:614–25.

10. Yu X, Blanden AR, Narayanan S, Jayakumar L, Lubin D, Augeri D, et al.Small molecule restoration of wildtype structure and function of mutantp53 using a novel zinc-metallochaperone based mechanism. Oncotarget2014;5:8879–92.

11. Salim KY, Maleki Vareki S, Danter WR, Koropatnick J. COTI-2, a novelsmall molecule that is active against multiple human cancer cell linesin vitro and in vivo. Oncotarget 2016;7:41363–79.

12. Maleki VS, Salim KY, Danter WR, Koropatnick J. Novel anti-cancer drugCOTI-2 synergizes with therapeutic agents and does not induce resistanceor exhibit cross-resistance in human cancer cell lines. PLoS One 2018;13:e0191766.

13. Westin SN, Nieves-Neira W, Lynam C, Salim KY, Silva AD, Ho RT, et al.Safety and early efficacy signals for COTI-2, an orally available smallmolecule targeting p53, in a phase I trial of recurrent gynecologic cancer[abstract]. In: Proceedings of the AmericanAssociation for Cancer ResearchAnnual Meeting 2018; 2018 Apr 14–18; Chicago, IL. Philadelphia (PA):AACR; 2018. Abstract nr CT033.

14. Osman AA,MonroeMM,Ortega AlvesMV, Patel AA, Katsonis P, FitzgeraldAL, et al.Wee-1 kinase inhibition overcomes cisplatin resistance associatedwith high-risk TP53 mutations in head and neck cancer through mitoticarrest followed by senescence. Mol Cancer Ther 2015;14:608–19.

15. Chou TC. Theoretical basis, experimental design, and computerized sim-ulation of synergism and antagonism in drug combination studies.Pharmacol Rev 2006;58:621–81.

16. Tanaka N, Patel AA, Silver NL, Lindemann A, Tang L, Jaksik R, et al.Synergistic antitumor activity of HDAC inhibitor vorinostat combined

withWEE1 inhibitor AZD1775 in head and neck squamous cell carcinomaharboring high-risk TP53 mutation. Clin Cancer Res 2017;23:6541–54.

17. Tanaka N, Zhao M, Tang L, Patel AA, Xi Q, Van HT, et al. Gain-of-functionmutant p53 promotes the oncogenic potential of head andneck squamouscell carcinoma cells by targeting the transcription factors FOXO3a andFOXM1. Oncogene 2018;37:1279–92.

18. Tanaka N, Osman AA, Takahashi Y, Lindemann A, Patel AA, ZhaoM, et al.Head and neck cancer organoids established by modification of the CTOSmethod canbe used topredict in vivo drug sensitivity.OralOncol 2018;87:49–57.

19. Skinner HD, Sandulache VC, Ow TJ, Meyn RE, Yordy JS, Beadle BM, et al.TP53 disruptive mutations lead to head and neck cancer treatment failurethrough inhibitionof radiation-induced senescence.ClinCancer Res 2012;18:290–300.

20. ChangGS, Chen XA, Park B, RheeHS, Li P, Han KH, et al. A comprehensiveand high resolution genome-wide response of p53 to stress. Cell Rep 2014;8:514–27.

21. DohnM, Zhang S, Chen X. p63alpha and DeltaNp63alpha can induce cellcycle arrest and apoptosis and differentially regulate p53 target genes.Oncogene 2001;20:3193–205.

22. Byers LA, Wang J, Nilsson MB, Fujimoto J, Saintigny P, Yordy J, et al.Proteomic profiling identifies dysregulated pathways in small cell lungcancer and novel therapeutic targets including PARP1. Cancer Discov2012;2:798–811.

23. Ribeiro-Rodrigues TM, Martins-Marques T, Morel S, Kwak BR, Girao H.Role of connexin43 indifferent formsof intercellular communication–gapjunctions, extracellular vesicles and tunnelling nanotubes. J Cell Sci 2017;130:3619–30.

24. El-Fouly MH, Trosko JE, Chang C-C. Scrape-loading and dye transfer, arapid and simple technique to study gap junctional intracellular commu-nication. Expt Cell Res 1987;168:422–30.

25. Babica P, Sovadinova I, Upham BL. Scrape loading/dye transfer assay.Methods Mol Biol 2016;1437:133–44.

26. Azzam EI, De Toledo SM, Little JB. Direct evidence for the participation ofgap junctionmediated intercellular communication in the transmission ofdamage signals from a-particle irradiated to nonirradiated cells. PNAS2001;98:473–8.

27. Lindemann A, Takahashi H, Patel AA, Osman AA, Myers JN. Targeting theDNAdamage response inOSCCwith TP53mutations. J Dent Res 2018;97:635–44.

28. Fitzgerald AL, Osman AA, Xie TX, Patel A, Skinner H, Sandulache V, et al.Reactive oxygen species and p21Waf1/Cip1 are both essential for p52-mediated senescence of head and neck cancer cells. Cell Death Dis 2015;6:e1678.

29. Jeon S-M. Regulation and function of AMPK in physiology and diseases.Exp Mol Med 2016;48:e245.

30. Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism, anddisease. Cell 2017;168:960–76.


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2019;25:5650-5662. Published OnlineFirst July 15, 2019.Clin Cancer Res Antje Lindemann, Ameeta A. Patel, Natalie L. Silver, et al. MechanismsActivity in HNSCC through p53-dependent and -independent COTI-2, A Novel Thiosemicarbazone Derivative, Exhibits Antitumor

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10.1158/1078-0432.CCR-19-0096doi:

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