IdentiﬁcationofaSmallMoleculeThatOvercomes HdmX-Mediated Suppression of … · key, cellular negative regulators, such as Hdm2 and HdmX. Derivatives of the Hdm2 antagonist Nutlin-3

Small Molecule Therapeutics

Identification of a Small Molecule ThatOvercomesHdmX-Mediated Suppression of p53Goutam Karan1, Huaiyu Wang2, Amit Chakrabarti1, Sukanya Karan1, Zhigang Liu3,Zhiqiang Xia1, Mahesh Gundluru1, Stephen Moreton1, Yogen Saunthararajah4,Mark W. Jackson3, Mukesh K. Agarwal1,5, and David N.Wald1,3,5

Abstract

Inactivation of the p53 tumor suppressor by mutation oroverexpression of negative regulators occurs frequently incancer. As p53 plays a key role in regulating proliferationor apoptosis in response to DNA-damaging chemotherapies,strategies aimed at reactivating p53 are increasingly beingsought. Strategies to reactivate wild-type p53 include the useof small molecules capable of releasing wild-type p53 fromkey, cellular negative regulators, such as Hdm2 and HdmX.Derivatives of the Hdm2 antagonist Nutlin-3 are in clinicaltrials. However, Nutlin-3 specifically disrupts Hdm2-p53,leaving tumors harboring high levels of HdmX resistant to

Nutlin-3 treatment. Here, we identify CTX1, a novel smallmolecule that overcomes HdmX-mediated p53 repression.CTX1 binds directly to HdmX to prevent p53–HdmX complexformation, resulting in the rapid induction of p53 in a DNAdamage–independent manner. Treatment of a panel of cancercells with CTX1 induced apoptosis or suppressed proliferationand, importantly, CTX1 demonstrates promising activity asa single agent in a mouse model of circulating primaryhuman leukemia. CTX1 is a small molecule HdmX inhibitorthat demonstrates promise as a cancer therapeutic candidate.Mol Cancer Ther; 15(4); 574–82. �2016 AACR.

IntroductionDNA-damaging chemotherapy is the first-line therapy for

most types of cancer. Unfortunately, side effects from thisapproach are significant and often dose limiting, as DNAdamage leads to toxicity in normal cells. As one of the mainpathways responsible for the anticancer activity of DNA-dam-aging chemotherapy involves the activation of p53, strategiesto activate p53 and subsequently p53-dependent cancer celldeath without DNA damage are highly valuable. The stabili-zation and subsequent activation of p53 protein is well knownto lead to either cell-cycle arrest and/or apoptosis (1–5).Although p53 is mutated in approximately 50% of all humancancers, wild-type p53 is extremely common in some malig-nancies, such as acute myeloid leukemia (AML) and melano-ma (6–9). Malignancies in which p53 is often wild-type areparticularly attractive targets for p53 activation strategies.

Although p53 is wild-type in certain cancers, it is generallyaccepted that it is functionally inactivated. In these cases, theinactivation of p53 often occurs through one or both of itsnegative regulators, Hdm2 and HdmX.

Hdm2 and HdmX are highly homologous proteins thatdirectly bind p53 and impair p53 functional activity. Hdm2exhibits E3 ubiquitin ligase activity and leads to p53 ubiqui-tination and subsequent proteasomal degradation. This activityhelps to maintain low basal levels of p53. Hdm2, similar toHdmX, can inhibit p53 transcriptional activity by interactingwith the N-terminal domain of p53. In normal cells, a definedbalance in the relative levels of Hdm2 and HdmX, eitherindividually or cooperatively, regulate p53 functions, keepingtarget gene transcription at levels that do not cause apoptosis orcell-cycle arrest (10, 11). However, in cancer, overexpression ofone or both of these proteins can lead to impaired p53 functionand cancer development and/or progression. It is important tonote that HdmX and Hdm2 regulate p53-independent antitu-mor pathways as well leading to activity of Hdm2/HdmXinhibitors in p53-deficient cells as well (12–14).

To induce p53 in a nongenotoxic manner, investigators havefocused for a number of years on the development of potentsmall molecule inhibitors of the Hdm2/p53 interaction (15). Forexample, the small molecule nutlin-3 has been identified as aninhibitor of Hdm2 and p53 (16). Nutlin compounds mimic thecritical amino acids within the p53 alpha-helix that bind withinthe hydrophobic pocket of Hdm2, where p53 normally resides,and show p53-specific antitumor properties (17). Studies sur-rounding nutlin-3 have proven successful against malignanciessuch as B-cell chronic lymphocytic leukemia, multiple myeloma,and AML (18–20). Currently, there are several clinical trials inprogress to evaluate nutlin-3 derivatives as anticancer agents. Ofnote, in addition to strategies targeting wild-type p53, severalcomplementary approaches have been developed for cases inwhich p53 is mutant or deficient (21–23).

1Invenio Therapeutics, Lexington, Kentucky. 2Department of Hema-tology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an,China. 3Department of Pathology, Case Western Reserve Universityand University Hospitals Case Medical Center, Cleveland, Ohio.4Department of Translational Hematology & Oncology Research, Cle-veland Clinic Foundation, Cleveland, Ohio. 5MirX Pharmaceuticals,Cleveland, Ohio.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

G. Karan and H. Wang contributed equally to this article.

Corresponding Authors: David N. Wald, Case Western Reserve University andUniversity Hospitals Case Medical Center, 2103 Cornell Road,WRB3-530, Cleve-land, OH 44106. Phone: 216-368-5668; Fax: 216-368-0495; E-mail:[email protected]; and Mukesh K. Agarwal, Invenio Therapeutics, 111000 CedarAvenue, Cleveland, OH 44106. E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0467

�2016 American Association for Cancer Research.

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Although there was initial excitement in targeting the Hdm2/p53 interaction as a therapeutic strategy, this initial excitementover selective Hdm2 inhibitors has been dampened by observa-tions that HdmX, which also inhibits p53 activity, is overex-pressed in a relatively large percentage of cancers. In these rela-tively frequent cases, the HdmX/p53 complex can prevent theoptimal induction of p53-dependent antitumor activity byHdm2inhibitors such as nutlin-3 (24). To identify novel small mole-cules that can overcome HdmX-mediated p53 suppression, weperformed a compound library screen. Our screen led to theidentification of the small molecule, CTX1, which exhibits poten-tial as a cancer therapeutic that can bind HdmX, induce p53, andkill cancer cells without the necessity of DNA damage. Impor-tantly, CTX1 exhibits potent activity even as a single agent anddemonstrated superior efficacy to nutlin-3 in amouse AMLmodelsystem.

Materials and MethodsChemicals

CTX1 was obtained from the Development Therapeutics Pro-gram at the National Cancer Institute. CTX-1 biotin was synthe-sized in our laboratory. Doxorubicin, Trypan blue, and nutlin-3were obtained from Sigma. Recombinant GST-FXR, GST-p53, andGST-HdmX were purchased from Abnova.

Cell linesAll cell lines were obtained from ATCC except OCI-AML3,

which were obtained from DSMZ. Upon receiving the cell lines,frozen stocks were prepared within one to two passages and newstocks were thawed frequently to keep the original condition. Thecell lines were passaged for less than 6 months after receipt orresuscitation. They were also routinely authenticated based ongrowth rate, morphology, and viability and were frequentlyconfirmed to be mycoplasma free. The generation of the MCF7shp53 cells and IMR90 cells were previously described and thevalidation of the stable knockdown of p53 and overexpressionof HdmX and Hdm2 in these cells (24). Cells were culturedin RPMI1640 media (Invitrogen) supplemented with 10% FBS(Invitrogen), penicillin G (100 mg/mL), and streptomycin(100 mg/mL). The OCI-AML3 HDMX–overexpressing cells weregenerated by stable transfection of plvx-mcherry-HA-HDMX andisolation of m-cherry–expressing cells by flow sorting (BDFACSAria).

Cell-cycle analysisCells were treated as described and fixed at �20�C in 90%

methanol. Cells were washed in PBS, treated with RNase A (finalconcentration 0.5 mg/mL; Sigma) and stained with propidiumiodide (Sigma; 50 mg/mL). The cells were kept at room temper-ature for 60 minutes and analyzed by flow cytometry on a BD XLcytometer. The cell cycle was modeled using the Modfit software(Verity House).

Western blot analysisWestern blot analysis was performed with p21, p53, p-ser15

p53, p-H2AX, PARP (Santa Cruz Biotechnology), HdmX (BethylLaboratories), and b-actin antibodies (Sigma). Cells were treatedas indicated and washed in PBS. Cells were centrifuged and lysedwith a Triton-containing lysis buffer. Protein lysates (50 mg perlane) were electrophoresed on SDS-polyacrylamide gels and thentransferred to polyvinylidene difluoride membranes (Millipore)

using a wet transfer apparatus (Bio-Rad). The membranes wereblocked, incubated with the indicated primary antibodies at4�C overnight, and then with the appropriate HRP-conjugatedsecondary antibody. Protein bands were visualized by autora-diography after incubation with enhanced chemiluminescencereagent (Millipore).

Biotin immunoprecipitationBriefly, binding/wash buffer (Tris-buffered saline containing

0.1% Tween-20 detergent) containing 2 mmol/L CTX1-biotinwas incubated with 150 ng of either recombinant HdmX and/orp53 for 30 minutes. The samples were desalted (to removeunincorporated biotin) using Zeba Desalt Spin columns(Thermo Scientific). The sample was mixed with prewashedStreptavidin Magnetic Beads (Pierce) and incubated at roomtemperature for 1 hour with mixing. The beads were washed,the bound antigen was eluted with sample buffer, and Westernblot analysis was performed.

P53 protein immunoprecipitationp53antibodywas coupled tomagnetic beadswithborate buffer

using the Direct Magnetic IP/Co-IP Kit (Thermo Scientific),according to the manufacturer's instructions. Next, CTX1,Nutlin-3, 9-aminoacridine, or DMSO (5 mmol/L final concentra-tion) were added to individual tubes containing 100 ng ofrecombinant HdmX. The mixture was incubated at 4�C for90 minutes under mixing. One hundred nanograms of recombi-nant p53was added to each tube and it was incubated for another2 hours at 4�C. The antibody-coupled beads were then added for2 hours at room temperature on a mixer. The beads were washedtwice with IP lysis buffer and the bound protein was eluted withsample buffer and assessed by Western blot analysis.

Cell-based immunoprecipitaitonsExponentially growing OCI cells cultures were treated with

3 mmol/L CTX1, 8 mmol/L Nutlin, and or 15 mmol/L RO-5963for 4.5 hours. Whole-cell extracts were generated using modifiedRIPA lysis buffer 25 mmol/L Tris (pH 8.0), 100 mmol/L NaCl,0.5mmol/L EDTA, 0.50%NP-40, and cOmplete ProteaseMixtureTablet (Roche). Protein extracts (�750 mg) were precleared andimmune precipitationwas performed using the directmagnetic IPKit (Pierce/Thermo Scientific), as per themanufacturer's protocol.For the immunoprecipitation, mouse monoclonal anti-p53(Santa Cruz Biotechnology) and rabbit polyclonal anti-HDMX(Bethyl Laboratories) were used. Immune complexes were thencollected, and proteins were eluted and subjected to Westernblotting with the indicated antibodies.

Spectrophotometric analysisUV-Vis spectral scanning or wave scan measurement methods

were adopted (Genesys 10S UV-VIS Spectrophotometer; ThermoScientific) in this study. Briefly either CTX1 or 9AA compoundsweremixed individually with recombinant HdmXprotein in a 2:1molar ratio in normal saline. Samples were scanned with orwithout passing through a desalting column (Thermo Scientific)to ensure bound protein–compound complex were separatedfrom unbound compound. The absorbance of various sampleswasmeasuredover a specifiedwavelength rangeof 200 to700nm.

Biacore studySurface plasmon resonance (SPR) was performed on a Bia-

core T100 instrument (GE Healthcare) at 25�C using PBSPþ

Identification of a Novel HdmX Inhibitor

www.aacrjournals.org Mol Cancer Ther; 15(4) April 2016 575




(GE Healthcare) and 5% DMSO as running buffer. S seriessensor CM5 sensorchip (GE Healthcare) was used to immobi-lize HdmX. CTX1 with increasing concentrations (156.3 nmol/L, 312.5 nmol/L, 625 nmol/L, 1.25 mmol/L, 2.5 mmol/L, and 5mmol/L 1.56 mmol/L) was injected over the surface at 30 mL/minute for 2 minutes. GnHCl (2 mol/L) was used for regen-eration of the surface. Data analysis and determination ofaffinity constants were made using Biaevaluation3 software(GE Healthcare). The concentration series was fitted using a1:1 kinetics model.

ELISAThe ELISA was done as described previously (25). Briefly,

recombinant p53 was bound to an ELISA plate (96-well format),and recombinant HdmX or Hdm2 were preincubated with theindicated doses of CTX1 and Nutlin-3. The HdmX and Hdm2were added to the wells containing p53 and incubated for 1 hour.After extensive washing, the amount of HdmXorHdm2 bound top53 was quantified using anti-Hdmx or anti-Hdm2.

Library screenApproximately 5,000 MCF7 cells stably overexpressing

HdmX and expressing a p21/ConA-bgal reporter were seededin 96-well plates. The wells were treated with approximately20,000 compounds at 10 mmol/L from libraries from threesources (Enamine, Prestwick, and the National Cancer Insti-tute) for 72 hours. Next, the reported induction was measuredusing the Beta-Glo Reagent (Promega) according to the man-ufacturer's protocol (Promega).

Cell growth and death measurementsCell growth was assessed using the MTT assay (Trevigen)

according to themanufacturer's protocol. Cell death was assessed

using Trypan blue staining by counting at least 200 cells from twoindependent fields and/or using Annexin V staining (Santa CruzBiotechnology), according to manufacturer's suggestions. All celldeath experiment values represent means� SDs from at least twoindependent experiments.

Mouse xenograftSix-week-old female NOD/SCID/IL2Rg�/� mice (Jackson Lab-

oratory) were injected into the tail vein with 5� 106 cells primaryhuman AML cells (n¼ 5 per group). Drug treatment was started 2days after tumor cell injection. Nutlin-3 was given by oral gavage(200mg/kg) and CTX1 was injected intraperitoneally (30mg/kg)5 days aweek for 3weeks. Flow cytometrywas performed onbonemarrow cells isolated from the mouse femur using a human-specific CD45PE antibody (BD Biosciences) using a BD XL cyt-ometer to confirm AML infiltration into the bone marrow. TheCase Western Reserve University Animal Research Committeeapproved all of the animal protocols used in this study.

ResultsScreening for novel HdmX inhibitors

To identify activators of p53 in cells overexpressing HdmX, acell-based screen was employed. In setting up a screening proto-col, we looked for compounds capable of inducing p53-depen-dent transcriptional activity of a p53-dependent reporter greaterthan 4-fold in breast cancer cells in which p53 activity is inhibitedthrough stable overexpression ofHdmX.We screened over 20,000small molecules from a number of compound collections fromPrestwick, NCI, and Enamine. From this screen, 6 compoundsexhibited a reproducible induction of the p53 reporter (Fig. 1A).Next, the p53 dependence of reporter activation by the positivecompounds was assessed using the HdmX-overexpressing report-er cells stably infected with shRNA directed against p53 or a

Figure 1.Identification of novel HdmX inhibitors. A, identification of novel compounds that induce a p21/ConA-b-gal reporter in MCF7 cells. MCF7 cells were treatedfor 24 hours with the indicated compounds (2 mmol/L) and b-galactosidase (b-gal) activity was measured. B, CTX1 and CTX6 exhibit p53-dependentactivation of the reporter construct. MCF7 and MCF7shp53 cells were treated with the indicated compounds and b-gal activity was measured as describedin Fig. 1A. C, CTX1 and CTX6 induce p53 and p21. MCF7 cells were treated with CTX1 (2 mmol/L), CTX6 (3 mmol/L), or nutlin-3 (10 mmol/L) for 6 hoursand a Western blot analysis was performed. D, structure of CTX1 and CTX1-biotin. Error bars, SD.

Karan et al.

Mol Cancer Ther; 15(4) April 2016 Molecular Cancer Therapeutics576




control shRNA (Fig. 1B). Two compounds, CTX1 and CTX6,exhibited strong p53-dependent reporter activation in the pres-ence of high levels of HdmX. As the disruption of the HdmX/p53interaction leads to a rapid induction of p53 protein, we nexttested the ability of CTX1 and CTX6 to induce p53 and itstranscriptional target p21 (Fig. 1C). Both compounds were foundto induce p53 and p21 protein expression. Because of the higheractivity of CTX1 on inducing p53-reported activation, our studieshave focused on this compound (Fig. 1D).

CTX1 rapidly induces p53 in a non–DNA damage-dependentfashion

As the compound library screen was aimed at identifyingcompounds that induce p53 by overcoming HdmX-mediatedsuppression, we predicted that unlike the vast majority of che-motherapeutics, CTX1 can induce p53 independently of DNAdamage. CTX1, unlike doxorubicin (a well-known DNA damageagent) does not induce measurable DNA damage as measured bycommon markers of DNA damage [phosphorylation of g-H2AXor p53 (ser-15)] at doses necessary for p53 induction (Fig. 2A–C).Therefore, CTX1-mediated p53 induction was found to be inde-pendent of genotoxic stress. Of note, CTX1 induces p53 rapidly(ex. 2 hours) supporting its direct role in stabilizing the p53protein as opposed to induction by DNA damage or transcrip-tional mechanisms (Fig. 2A and Supplementary Fig. S1).

CTX1 exhibits specificity in targeting HdmXTo determine whether CTX1 is specific for overcoming p53

suppression because of HdmX, Hdm2, or both, we utilized afibroblast cell model system in which p53 was suppressed byHdmX or Hdm2 overexpression. In addition, as HdmX inhi-bition should partially lead to cell death through p53 induc-tion, we also used cells in which p53 expression was knockeddown. This same cell model system was previously shown todefine the specificity of the Hdm2 inhibitor, nutlin-3, for Hdm2(24, 26). CTX1 induced significant p53-dependent cell deathpreferentially in HdmX-expressing cells (Vector or HdmX) as

compared with cells in which p53 is inactivated by Hdm2overexpression or p53 is knocked down using p53 targetingshRNA (Fig. 3A). This study demonstrates CTX1 shows speci-ficity in targeting HdmX over its homolog Hdm2. In addition,this model system further supports the ability of CTX1 toovercome HdmX-mediated p53 suppression. To further con-firm the ability of CTX1 to overcome HdmX-mediated suppres-sion of cell killing, we employed a leukemia cell model system.Consistent with the fibroblast cell model, OCI-AML3 (OCI)cells overexpressing HdmX were found to exhibit a similarsensitivity to CTX1-mediated cell killing as parental cells incontrast to the Hdm2 inhibitor, nutlin-3, that as expecteddemonstrates reduced activity in the presence of high HdmXlevels (Supplementary Fig. S2A and S2B).

CTX1 directly interacts with HdmX and modulatesp53–HdmX binding

To explore how CTX1 leads to p53 induction, we assessedwhether or not CTX1 can directly interact withHdmX and/or p53.To test for direct binding, we synthesized a biotin-conjugatedversion of CTX1 (Fig. 1D). The CTX1-biotin compound exhibitssimilar activity in cell killing to the parent compound (Supple-mentary Fig. S2B). We found that CTX1 can directly interact withGST-HdmX, but not unrelated proteins such as GST-FXR byimmunoprecipitation (Fig. 3B and Supplementary Fig. S3A). Inaddition, GST-HdmX can bind at least weakly with p53. Biotinalone was confirmed not to bind with HdmX demonstrating theCTX1 component of the CTX1-biotin conjugate is responsible forthis interaction (Supplementary Fig. S3A).

In addition to testing the binding of CTX1 and HdmX throughimmunoprecipitations using biotin-tagged CTX1, we also con-firmed the interaction of HdmX with non-tagged CTX1 using twobiophysical based approaches. First, we took advantage of the factthat CTX1, but not HdmX, exhibits a specific absorbance patternthat is detectable by a spectrophotometer. After coincubation ofCTX1 with HdmX (and removal of free CTX1 by size exclusionchromatography), not only does HdmX now exhibit an

Figure 2.CTX1 rapidly induces p53 independent of DNA damage. A and B, MCF7 cells were treated with CTX1 (3 mmol/L) or doxorubicin (1 mmol/L) for the indicated times(hours) and Western blot analysis was performed for markers of DNA damage [p-p53 (Ser15) and p-H2AX]. C, HCT116 cells (left) or OCI cells (right) weretreated with the indicated doses of CTX1 or Doxorubicin for 9 hours and Western blot analysis was performed using the indicated antibodies.






absorbance pattern, but there is also a clear spectral shift of theabsorbance pattern as compared to free CTX1 (Fig 3C). Thisspectral shift is highly suggestive of an HdmX/CTX1 interaction.In addition, size exclusion chromatography performed on CTX1alone as a control demonstrates the absorbance seen with the

HdmX/CTX1 sample is not because of residual non-bound CTX1(postcolumn residual control; Fig. 3C).

Interestingly, CTX1 is an acridine-containing molecule andother acridine-containing compounds such as 9-aminoacridine(9-AA) have previously been shown to rapidly induce p53 in a

Figure 3.CTX1 specifically targets and directly binds to HdmX. A, CTX1 preferentially kills cells transformed by HdmX and not Hdm2 or shp53. IMR90 cells overexpressing theindicated constructs were treated with increasing doses of CTX1 (1, 2.5, and 5 mmol/L) and assessed for cell death at 72 hours by Trypan blue staining.B, CTX1 binds HdmX and to a lesser extent p53. Recombinant p53, HdmX, and/or biotin-CTX1 were incubated in vitro and streptavidin beads were used to pull downthe protein complex. The bound protein was eluted and analyzed byWestern blot analysis with the indicated antibodies. C, spectral studies suggest CTX1 and HdmXdirectly interact. CTX1 and HdmX alone represent the spectral pattern of both agents without purification. The CTX1þHdmX sample and CTX1 postcolumnresidual samples underwent size exclusion chromatography to remove unbound CTX1. D and E, CTX1 disrupts the interaction of recombinant HdmX/p53 but notHdm2/p53 by ELISA and coimmunoprecipitation. The indicated drugs were used as controls. F, CTX1 disrupts the interaction of HdmX/p53 in cells.Immunoprecipitations were performed as indicated using lysate from OCI cells treated with the indicated drugs or a DMSO control. Error bars, SD.

Karan et al.





non–DNA damage-dependent fashion possibly related to itsability to intercalate in DNA (27). Interestingly, we did not detectbinding of 9-AA toHdmX using the same spectral studies suggest-ing a distinct mechanism of action (Supplementary Fig. S3B).

Using another biophysical approach, we also demonstratedHdmX and CTX1 binding using SPR. HdmX demonstrated strongbinding to CTX1 (Kd 450 nmol/L) but not nutlin-3 (Kd 5.1 mmol/L; Supplementary Fig. S3C). Again, 9-AA did not demonstrate anybinding to HdmX using SPR at doses up to 12.5 mmol/L (data notshown).

Besides assessing for interactions of CTX1 and HdmX, weassessed for the ability of CTX1 to directly impair the interactionof p53 andHdmX that could result in the observed stabilization ofp53 protein. Utilizing an ELISA assay, we found that CTX1disrupted HdmX/p53, but not Hdm2/p53 interactions (Fig.3D). In contrast, the Hdm2 inhibitor, nutlin-3, disrupts theinteraction between p53 and Hdm2, but not p53 and HdmX. Tofurther confirm the ability of CTX1 to disrupt HdmX and p53binding,we performed coimmunoprecipitation assays using bothpurified recombinant HdmX and p53 as well as native protein inOCI cells (Fig. 3E and F). Utilizing these assays, we furtherdemonstrate that CTX1, but not 9-AA or a standard DNA-dam-aging agent, doxorubicin, impairs the binding of HdmX and p53.As a control, we also found that the compound RO-0596, whichhas been reported to impair HdmX/p53 binding, also disruptsbinding in our immunoprecipitations (28). This result suggeststhat CTX10s ability to impair HdmX/p53 binding is specific and isnot simply due to its acridine moiety.

CTX1 impairs cancer cell growthTo explore the effects of CTX1 on cancer cell growth and

survival, we tested the ability of CTX1 to inhibit the growthand/or kill a panel of wild-type p53 cancer cell lines includingHCT116, Hela, A549, MCF7, LNCaP, OCI-AML3 (OCI), andMOLM-13. In addition, mutant p53 cell lines (HT29, DLD1,and K562) and p53-deficient cells (HCT116 p53-/-, A549shp53, and Jurkat) were tested (HdmX and p53 status of celllines are summarized in Table 1). CTX1 was tested alone and atlower doses in combination with the commonly used Hdm2inhibitor, nutlin-3. These studies revealed that low doses ofCTX1 and nutlin-3 led to cooperative killing when combinedwith nutlin-3. In addition, although CTX1 can clearly causedeath of p53-deficient cancer cells, testing of isogenic cells(HCT116 and A549) demonstrated a modest increase in killingof p53-expressing cells. Of note, HdmX can lead to induction ofboth p53-dependent and independent pathways. Also of note,the leukemia cell lines tested demonstrated an increased sen-sitivity to CTX1-mediated cell killing as compared with thesolid tumor cell types (Fig. 4A–C and Supplementary Fig.S4; Table 2). As CTX1 can induce cell killing in p53-mutantcell lines, we also investigated whether it can stabilize mutantp53 levels. As seen in Supplementary Fig. 4C, CTX1 can inducep53 protein levels in the p53-mutant cell line, HT29. Besidescell lines, we have also found that CTX1 exhibits potent activity(LD50 � 1 mmol/L) as a single agent on primary AML patientsamples in a similar fashion to AML cell lines (Table 2).

Cell-cycle analysis of paired p53-expressing and p53-defi-cient cancer cell lines (HCT116 and A549) also demonstratesthat CTX1 mediates growth inhibition partially through a p53-dependent pathway. For example, after 16 hours of treatmentwith CTX1 in HCT116 p53 wild-type cells there is a decrease in

S-phase from 23% to 3%, whereas HCT116 p53-null cellsexhibit a reduction in S-phase from 34% to 28% (Fig. 4B andSupplementary Fig. S4A). Although both HCT116 and A549p53-wild-type cells exhibit a dramatic reduction in S-phase,HCT116 also see a modest accumulation of cells in the G2–Mphase. Interestingly, other agents have also been reported topreferentially accumulate HCT116 cells in G2–M as comparedwith A549 cells suggesting differences in cell-cycle–regulatorypathways (29).

To further characterize the mechanism of cell killing,Annexin-V staining was done to assess for apoptosis in HCT116p53-wild-type and p53-null cells. The combination of lowdoses of CTX1 and nutlin-3 led to a significant enhancementof apoptosis in p53-wild type, but not p53-null cells (Fig. 4Cand Supplementary Fig. S4B).

In agreement with the synergistic and additive induction ofcell death when combining CTX1 and Hdm2 inhibition, we alsoobserved a modest increase in p53 protein induction, the p53target protein Hdm2 and a marker of apoptosis, PARP cleavage,in cells treated with the combination regimen (Fig. 4D and E).

In addition to p53 induction, we also assessed the regulationof Hdm2 and Hdmx in response to CTX1 alone as theseproteins are known to be induced and downregulated bynutlin-3, respectively. We observed that the expression of theseproteins were not markedly impacted by CTX1 alone; however,Hdm2 induction was enhanced by CTX1 (likely due toenhanced p53 induction; Fig 4E).

CTX1 exhibits high in vivo activityAs CTX1 represents one of the few examples of a compound

that can induce p53 and kill cancer cells in a genotoxic-inde-pendent fashion, we performed mouse efficacy studies to beginto explore its clinical potential. We utilized a highly aggressiveAML model system for this study as this is a disease unlike mostmalignancies in which wild-type p53 status is extremely com-mon and new therapeutics are urgently needed. The ability ofCTX1 (30 mg/kg i.p.), nutlin-3 (200 mg/kg orally), or thecombination to impact the growth of primary human AMLcells (wild-type p53) in immunodeficient mice was assessed.This model system closely mimics the human disease as itutilizes a primary patient sample and the leukemic cells circu-late in the mouse and proliferate in the bone marrow. Utilizinga primary human AML sample, CTX1 even as a single agentsignificantly enhanced the survival of mice in this model system(Fig. 5). Of note, this model system is clinically important asthere are no existing therapeutics that are efficacious in this

Table 1. p53 and HdmX status of cell lines used

p53 status HdmX log mRNA expression

OCI Wild-type 9.12Jurkat Null 9.41Hela Wild-type UnknownLnCAP Wild-type 7.54HCT116 Wild-type 7.31Molm-13 Wild-type 7.77K562 Mutant 8.15HT29 Mutant 7.24DLD1 Mutant 6.45MCF7 Wild-type 7.79A549 Wild-type 6.51

NOTE: HdmXmRNA expression values were obtained from the Cancer Cell LineEncyclopedia Project (http://www.broadinstitute.org/ccle/home; ref. 38).






patient population. Although all of the vehicle mice succumbedto disease by 60 days after cell injection, mice treated with CTX1alone or in combination with nutlin-3 had a significantlyincreased survival time (P < 0.0001, log-rank test).

Importantly, CTX1 exhibited significant anticancer activityalone and in combination with nutlin-3. There was a significantsurvival advantage of CTX1 treatment as compared with nutlin-3 in this mouse model using a standard dosing regimen fornutlin-3 while further studies are necessary to identify optimalCTX1 dosing. This work suggests that CTX1 may have potentialeven as a single agent. In addition to efficacy, the mouse studiesalso demonstrate the potential safety of CTX1. The mice treatedwith CTX1 alone or in combination with nutlin-3 gainedweight in a similar fashion to the vehicle-treated mice and didnot show any obvious signs of toxicity. This study shows thepotential of CTX1 as a leukemia therapeutic and that HdmXinhibitors alone may be a promising therapeutic strategy.Overall, we have identified a novel HdmX inhibitor that is apromising potent anticancer agent with apparent low toxicitythat is worthy of further development.

Discussionp53 is a major regulator of cancer cell growth and drug

resistance. Although many cancer cells exhibit p53 mutationsto inactivate p53, another mechanism to regulate p53 functionis through the upregulation of negative regulators such asHdmX and Hdm2. By targeting these negative regulators ofp53, it is possible to directly activate p53 without the necessityof DNA damage. Our objective was to identify a small moleculeinhibitor that could overcome HdmX-mediated suppression ofp53. Because HdmX is overexpressed in a large number ofcancers and Hdm2 inhibitors such as nutlin-3 do not showsignificant efficacy on cancer cells that overexpress HdmX, thedevelopment of small molecules targeting HdmX is important.Targeting HdmX is also known to have anticancer propertiesthat are independent of p53. Another potential advantage ofHdmX inhibition is that, it has been predicted to be safer thanHdm2 inhibition as the lack of the mouse homolog, Mdm2 butnot Mdmx, leads to significant p53-dependent toxicities innormal adult tissues (30).

Figure 4.CTX1 induces apoptosis and growth arrest of cancer cells. A and C, the combination of CTX1 and nutlin-3 leads to significant apoptosis. The indicated cell lines weretreated with CTX1 3 mmol/L (MCF7), 4 mmol/L (A549), 2 mmol/L (HCT116), 4 mmol/L (LNCaP), 0.75 mmol/L (OCI); nutlin-3 (5 mmol/L), or a combination for72 hours and cell deathwas assessed by Trypan blue exclusion. B, CTX1 preferentially impairs the growth of cells expressingwild-type p53. HCT116 p53þ/þ or p53-nullcells were treated with CTX1 (2 mmol/L) and cell-cycle analysis was performed using PI staining at 16 hours. D–E, CTX1 and nutlin-3 cooperate to induce p53,Hdm2, and PARP cleavage. OCI cells were treated for 24 hours (D) and 2 hours (E) with the indicated compounds and Western blot analysis was performed.Error bars, SD.

Karan et al.





Although there are numerous studies demonstrating the po-tential for small molecules capable of inhibiting Hdm2/p53interactions, there are extremely few reported small moleculesthat have been shown to be capable of inhibiting HdmX/p53 andnone that have demonstrated both cell and animal efficacy. It isnot clear that the compounds reported previously as Hdmx/p53inhibitors are suitable for clinical development (reviewed inref. 28). SJ-172550 is a compound recently described as anHdmX/p53 inhibitor, but the authors concluded that the com-pound was not suitable for further development as it formscysteine adducts with Hdm2 and HdmX (31, 32). Another smallmolecule,WK298, was reported to disruptHdmX/p53 interactionsbut does not exhibit any cellular activity (33). Finally, RO-5963 isa dual Hdm2 and HdmX inhibitor, although it exhibits weakeractivity against the Hdm2/p53 interaction in cells (15, 28). Thismolecule exhibits a unique mechanism of action distinct fromCTX1 as it has been reported to impair HdmX/p53 binding bystabilizing HdmX/Hdm2 heterodimers; however, the in vivo activ-ity of this agent has not been described. Besides small moleculeinhibitors, a stapled p53 helix and peptide inhibitors have alsobeen reported (25, 34). Therefore, the identification of CTX1 that

demonstrates both in vitro and mouse in vivo anticancer efficacy isimportant for the potential clinical targeting of the HdmX-medi-ated p53 suppression in patients. Besides direct inhibitors ofHdmx/p53, other investigators have taken alternative and poten-tially complementary approaches to induce p53 in a nongenotoxicmanner. For example, NSC207895 is a compound that modulatesHdmX transcription and other groups have developed E3 ubiqui-tin ligase inhibitors (28, 35, 36).

The identification of CTX1 as an HdmX/p53 inhibitor wasunexpected as CTX1 contains an acridine ring structure which isfound in many other well-known compounds tested as anti-cancer agents that can induce DNA damage. Interestingly,however, there are also several acridine containing compoundsthat like CTX1 can induce p53 in a non–DNA damage-depen-dent fashion. For example, quinacrine and 9-aminoacridine (9-AA) have been shown to exhibit this property and their anti-cancer activities have been attributed to a combination of p53induction and NF-kB inhibition (27, 37). Although CTX1shares some structural similarities with 9-AA, the mechanismsof p53 induction do not appear to completely overlap as 9-AAwas not found to be capable of disrupting HdmX/p53 inter-actions or to interact with HdmX.

Although CTX1 can disrupt HdmX/p53 interactions, inducep53, and causep53-dependent cell death, it clearly also can inducecell death through additional pathways. These p53-independentactivities of CTX1 fit well with the fact that HdmX (as well asHdm2) are known to exhibit many p53-independent antitumorpathways (12–14). It will be interesting to see if some of thesep53-independent pathways overlap with those reported for othernon–DNA-damaging acridine agents such as 9-AA. In addition,these p53-independent pathways suggest CTX1 may have utilityfor p53-deficient tumors as well.

Although the in vitro activity of CTX1 is strongly enhanced byconcurrent Hdm2 inhibition using an agent such as nutlin-3,CTX1 alone is a promising lead anticancer agent. The potential ofCTX1 as a single agent can be seen from the efficacy of CTX1 in acirculating AML mouse model system. In these studies, CTX1alone showed significant efficacy that was higher than nutlin-3using a standard nutlin-3 dosing regimen. Of note, the standardAML therapeutic cytarabine also does not demonstrate efficacy inthis aggressive disease model. CTX1 further was well tolerated inmice anddidnot showanyovert evidenceof toxicities.Overall, weidentified anovel potent smallmolecule inhibitor, CTX1,which iscapable of binding Hdmx, overcoming HdmX-mediated p53suppression in a nongenotoxic manner and inducing cancer celldeath particularly in combination with anHdm2 inhibitor. CTX1exhibits anticancer both in vitro and in vivo and, therefore, haspotential to be developed into a novel targeted therapeutic.

Disclosure of Potential Conflicts of InterestM.W. Jackson is a consultant/advisory board member for Invenio Ther-

apeutics. M.K. Agarwal reports receiving a commercial research grant fromInvenio Therapeutics and has ownership interest in MirX-Invenio and hassubmitted a provisional patent on CTX1. D.N. Wald reports receiving acommercial research grant from Invenio Therapeutics and has ownershipinterest in Invenio Therapeutics and has submitted a provisional patent onCTX1. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: G. Karan, H. Wang, M. Gundluru, M.W. Jackson,M.K. Agarwal, D.N. WaldDevelopment of methodology: G. Karan, H. Wang, A. Chakrabarti, S. Karan,Z. Xia, M. Gundluru, S. Moreton, M.W. Jackson, M.K. Agarwal, D.N. Wald

Table 2. LD50 of CTX1 on cancer cell lines

LD50, mmol/L

OCI 0.97Jurkat 1Hela 8.3LnCAP 2.9HCT116 2.81HCT116 p53�/� 5.65Molm-13 1.11K562 1.01HT29 4.05DLD1 7.4MCF7 4.29A549 5.32A549 shp53 7.7AML, PT 1 1.25AML, PT 2 0.75AML, PT 3 1.26

Figure 5.CTX1 demonstrates significant anticancer activity in vivo. NSGmice (n¼ 5 pergroup) were injected with primary human AML cells by the tail vein(5� 106 cells in 100 mL of media) andmice were treated with CTX1 (30mg/kgi.p.) or Nutlin-3 (200 mg/kg orally) and assessed for survival. Log-rank test,P < 0.0001.






Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Karan, H. Wang, A. Chakrabarti, S. Karan, Z. Liu,Z. Xia, S. Moreton, M.W. Jackson, M.K. Agarwal, D.N. WaldAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Karan, H. Wang, S. Karan, Z. Liu, M.W. Jackson,M.K. Agarwal, D.N. WaldWriting, review, and/or revision of the manuscript: G. Karan, Z. Xia,M. Gundluru, Y. Saunthararajah, M.K. Agarwal, D.N. WaldAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):H. Wang, S. Moreton, M.K. Agarwal, D.N. WaldStudy supervision: S. Karan, M.K. Agarwal, D.N. Wald

Grant SupportThis work was supported by the NIH award R43CA139791 (to D.N. Wald

and M.K. Agarwal).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 5, 2015; revised February 1, 2016; accepted February 2, 2016;published OnlineFirst February 16, 2016.

References1. Dumont P, Leu JI, Della Pietra AC III, George DL,MurphyM. The codon 72

polymorphic variants of p53 have markedly different apoptotic potential.Nat Genet 2003;33:357–65.

2. Leu JI, Dumont P, Hafey M, Murphy ME, George DL. Mitochondrial p53activates Bak and causes disruption of a Bak-Mcl1 complex. Nat Cell Biol2004;6:443–50.

3. Mihara M, Erster S, Zaika A, Petrenko O, Chittenden T, Pancoska P, et al.p53 has a direct apoptogenic role at the mitochondria. Mol Cell2003;11:577–90.

4. Chipuk JE, Bouchier-Hayes L, Kuwana T, Newmeyer DD, GreenDR. PUMAcouples the nuclear and cytoplasmic proapoptotic function of p53. Science2005;309:1732–5.

5. Levine AJ.p53, the cellular gatekeeper for growth and division. Cell1997;88:323–31.

6. Volkenandt M, Schlegel U, Nanus DM, Albino AP. Mutational analysis ofthe human p53 gene in malignant melanoma. Pigment Cell Res 1991;4:35–40.

7. Lubbe J, Reichel M, Burg G, Kleihues P. Absence of p53 gene mutations incutaneous melanoma. J Invest Dermatol 1994;102:819–21.

8. Boyapati A, Kanbe E, Zhang DE. p53 alterations in myeloid leukemia. ActaHaematol 2004;111:100–6.

9. Saha MN, Qiu L, Chang H. Targeting p53 by small molecules in hemato-logical malignancies. J Hematol Oncol 2013;6:23.

10. JacksonMW,Berberich SJ. Constitutivemdmx expressionduring cell growth,differentiation, and DNA damage. DNA Cell Biol 1999;18:693–700.

11. Wu X, Bayle JH, Olson D, Levine AJ. The p53-mdm-2 autoregulatoryfeedback loop. Genes Dev 1993;7:1126–32.

12. Zhang Z, Zhang R. p53-independent activities of MDM2 and their rele-vance to cancer therapy. Curr Cancer Drug Targets 2005;5:9–20.

13. de Lange J, Teunisse AF, Vries MV, Lodder K, Lam S, Luyten GP, et al. Highlevels of Hdmx promote cell growth in a subset of uveal melanomas. Am JCancer Res 2012;2:492–507.

14. Li Q, LozanoG.Molecular pathways: targetingMdm2 andMdm4 in cancertherapy. Clin Cancer Res 2013;19:34–41.

15. Wade M, Li YC, Wahl GM. MDM2, MDMX and p53 in oncogenesis andcancer therapy. Nat Rev Cancer 2013;13:83–96.

16. Vassilev LT.Small-molecule antagonists of p53-MDM2 binding: researchtools and potential therapeutics. Cell Cycle 2004;3:419–21.

17. Vassilev LT, VuBT,Graves B, CarvajalD, Podlaski F, Filipovic Z, et al. In vivoactivation of the p53 pathway by small-molecule antagonists of MDM2.Science 2004;303:844–8.

18. Coll-Mulet L, Iglesias-Serret D, Santidrian AF, Cosialls AM, de Frias M,Castano E, et al. MDM2 antagonists activate p53 and synergize withgenotoxic drugs in B-cell chronic lymphocytic leukemia cells. Blood2006;107:4109–14.

19. Kojima K, Konopleva M, Samudio IJ, Shikami M, Cabreira-Hansen M,McQueen T, et al. MDM2 antagonists induce p53-dependent apoptosisin AML: implications for leukemia therapy. Blood 2005;106:3150–9.

20. Secchiero P, Barbarotto E, Tiribelli M, Zerbinati C, di Iasio MG, Gonelli A,et al. Functional integrity of the p53-mediated apoptotic pathway inducedby the nongenotoxic agent nutlin-3 in B-cell chronic lymphocytic leukemia(B-CLL). Blood 2006;107:4122–9.

21. Rana S, Gupta K, Gomez J, Matsuyama S, Chakrabarti A, Agarwal ML, et al.Securinine induces p73-dependent apoptosis preferentially in p53-defi-cient colon cancer cells. FASEB J 2010;24:2126–34.

22. Issaeva N, Bozko P, Enge M, Protopopova M, Verhoef LG,Masucci M, et al. Small molecule RITA binds to p53, blocksp53-HDM-2 interaction and activates p53 function in tumors. NatMed 2004;10:1321–8.

23. Mandinova A, Lee SW. The p53 pathway as a target in cancer therapeutics:obstacles and promise. Sci Trans Med 2011;3:64rv1.

24. Patton JT, Mayo LD, Singhi AD, Gudkov AV, Stark GR, Jackson MW. Levelsof HdmX expression dictate the sensitivity of normal and transformed cellsto Nutlin-3. Cancer Res 2006;66:3169–76.

25. Noguchi T, Oishi S, Honda K, Kondoh Y, Saito T, Kubo T, et al. Affinity-based screening of MDM2/MDMX-p53 interaction inhibitors by chemicalarray: identification of novel peptidic inhibitors. Bioorg Med Chem Lett2013;23:3802–5.

26. Hu B, Gilkes DM, Farooqi B, Sebti SM, Chen J. MDMX overexpressionprevents p53 activation by the MDM2 inhibitor Nutlin. J Biol Chem2006;281:33030–5.

27. Gurova KV, Hill JE, Guo C, Prokvolit A, Burdelya LG, Samoylova E, et al.Small molecules that reactivate p53 in renal cell carcinoma reveal a NF-kappaB-dependent mechanism of p53 suppression in tumors. Proc NatlAcad Sci U S A 2005;102:17448–53.

28. Graves B, Thompson T, Xia M, Janson C, Lukacs C, Deo D, et al.Activation of the p53 pathway by small-molecule-induced MDM2and MDMX dimerization. Proc Natl Acad Sci U S A 2012;109:11788–93.

29. Hoeferlin LA, Oleinik NV, Krupenko NI, Krupenko SA. Activation of p21-dependentG1/G2 arrest in the absence ofDNAdamage as an antiapoptoticresponse to metabolic stress. Genes Cancer 2011;2:889–99.

30. Ringshausen I,O'Shea CC, Finch AJ, Swigart LB, EvanGI.Mdm2 is criticallyand continuously required to suppress lethal p53 activity invivo. CancerCell 2006;10:501–14.

31. Bista M, Smithson D, Pecak A, Salinas G, Pustelny K, Min J, et al. On themechanism of action of SJ-172550 in inhibiting the interaction of MDM4and p53. PLoS One 2012;7:e37518.

32. Reed D, Shen Y, Shelat AA, Arnold LA, Ferreira AM, Zhu F, et al. Identi-fication and characterization of the first small molecule inhibitor ofMDMX. J Biol Chem 2010;285:10786–96.

33. Popowicz GM, Czarna A, Wolf S, Wang K, Wang W, Domling A, et al.Structures of lowmolecularweight inhibitors bound toMDMXandMDM2reveal new approaches for p53-MDMX/MDM2 antagonist drug discovery.Cell Cycle 2010;9:1104–11.

34. Bernal F, Wade M, Godes M, Davis TN, Whitehead DG, Kung AL, et al. Astapled p53 helix overcomes HDMX-mediated suppression of p53. CancerCell 2010;18:411–22.

35. Wang H, Ma X, Ren S, Buolamwini JK, Yan C. A small-moleculeinhibitor of MDMX activates p53 and induces apoptosis. Mol CancerTher 2011;10:69–79.

36. Herman AG, Hayano M, Poyurovsky MV, Shimada K, Skouta R, Prives C,et al. Discovery of Mdm2-MdmX E3 ligase inhibitors using a cell-basedubiquitination assay. Cancer Discov 2011;1:312–25.

37. Wang W, Ho WC, Dicker DT, MacKinnon C, Winkler JD, Marmorstein R,et al. Acridine derivatives activate p53 and induce tumor cell death throughBax. Cancer Biol Ther 2005;4:893–8.

38. Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S,et al. The Cancer Cell Line Encyclopedia enables predictive modelling ofanticancer drug sensitivity. Nature 2012;483:603–7.


Karan et al.




2016;15:574-582. Published OnlineFirst February 16, 2016.Mol Cancer Ther Goutam Karan, Huaiyu Wang, Amit Chakrabarti, et al. Suppression of p53Identification of a Small Molecule That Overcomes HdmX-Mediated

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IdentiﬁcationofaSmallMoleculeThatOvercomes HdmX-Mediated Suppression of … · key, cellular negative regulators, such as Hdm2 and HdmX. Derivatives of the Hdm2 antagonist Nutlin-3