Cabozantinib Exhibits Potent Antitumor Activity in ... · 10 PDX mouse models. In vivo angiogenesis and glucose uptake were assessed using dynamic contrast-enhanced (DCE)-MRI and

Small Molecule Therapeutics

Cabozantinib Exhibits Potent Antitumor Activityin Colorectal Cancer Patient-Derived TumorXenograft Models via Autophagy and SignalingMechanismsAaron J. Scott1, John J. Arcaroli2, Stacey M. Bagby2, Rachel Yahn2, Kendra M. Huber3,Natalie J. Serkova3, Anna Nguyen2, Jihye Kim2, Andrew Thorburn4, Jon Vogel5,Kevin S. Quackenbush2, Anna Capasso2, Anna Schreiber2, Patrick Blatchford2,Peter J. Klauck2, Todd M. Pitts2, S. Gail Eckhardt6, and Wells A. Messersmith2

Abstract

Antiangiogenic therapy used in treatment of metastaticcolorectal cancer (mCRC) inevitably succumbs to treatmentresistance. Upregulation of MET may play an essential roleto acquired anti-VEGF resistance. We previously reportedthat cabozantinib (XL184), an inhibitor of receptor tyrosinekinases (RTK) including MET, AXL, and VEGFR2, had potentantitumor effects in mCRC patient-derived tumor explantmodels. In this study, we examined the mechanisms ofcabozantinib sensitivity, using regorafenib as a control. Thetumor growth inhibition index (TGII) was used to comparetreatment effects of cabozantinib 30 mg/kg daily versusregorafenib 10 mg/kg daily for a maximum of 28 days in10 PDX mouse models. In vivo angiogenesis and glucoseuptake were assessed using dynamic contrast-enhanced(DCE)-MRI and [18F]-FDG-PET imaging, respectively.RNA-Seq, RTK assay, and immunoblotting analysis wereused to evaluate gene pathway regulation in vivo and

in vitro. Analysis of TGII demonstrated significant antitumoreffects with cabozantinib compared with regorafenib(average TGII 3.202 vs. 48.48, respectively; P ¼ 0.007).Cabozantinib significantly reduced vascularity and glucoseuptake compared with baseline. Gene pathway analysisshowed that cabozantinib significantly decreased proteinactivity involved in glycolysis and upregulated proteinsinvolved in autophagy compared with control, whereasregorafenib did not. The combination of two separate anti-autophagy agents, SBI-0206965 and chloroquine, pluscabozantinib increased apoptosis in vitro. Cabozantinibdemonstrated significant antitumor activity, reduction intumor vascularity, increased autophagy, and altered cellmetabolism compared with regorafenib. Our findingssupport further evaluation of cabozantinib and combina-tional approaches targeting autophagy in colorectal cancer.Mol Cancer Ther; 17(10); 2112–22. �2018 AACR.

IntroductionColorectal cancer is the third most common cancer diagnosed

annually in the United States (1). Metastatic colorectal cancer(mCRC) is the second leading cause of cancer-related death withan estimated 50,000 deaths in theUnited States and over 500,000deaths worldwide annually (1). Despite substantial improve-ments in treatment over the past decade, mCRC carries a poorprognosis with a median overall survival of roughly 30 monthswith optimal combination chemotherapy. Although regorafenibhas been approved for refractorymCRC, the benefit of this therapyis modest at best with a median overall survival benefit of 1.4months comparedwith best supportive care (2, 3).Unfortunately,resistance to regorafenib occurs quickly for most individuals.

Expressionof VEGFand its receptors, VEGFR1 andVEGFR2, hasbeen shown to be upregulated inmany tumor types. VEGF ligandbinding and activation of VEGFR2 is likely the major driver oftumor neovascularization (4). Regorafenib, a multikinase inhib-itor targeting VEGFR2, KIT, RET, TIE2, BRAF, and PDGFR, hasreceived FDA approval for treatment of refractory mCRC. Theinhibition of VEGFR2 by regorafenib is thought to be a central

1Division of Hematology and Oncology, Banner University of Arizona CancerCenter, Tucson, Arizona. 2Division of Medical Oncology, University of ColoradoAnschutz Medical Campus and University of Colorado Cancer Center, Aurora,Colorado. 3Department of Anesthesia, University of Colorado Anschutz MedicalCampus and University of Colorado Cancer Center, Aurora, Colorado.4Department of Pharmacology, University of Colorado Anschutz Medical Cam-pus andUniversity of ColoradoCancerCenter, Aurora, Colorado. 5Department ofSurgery, University of Colorado Anschutz Medical Campus and University ofColorado Cancer Center, Aurora, Colorado. 6Division of Medical Oncology, TheUniversity of Texas at Austin, Austin, Texas.

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

A.J. Scott and J.J. Arcaroli contributed equally to this work.

Current address for S. Gail Eckhardt: Dell Medical School, TheUniversity of Texasat Austin, Chair, Department of Oncology, CPRIT Scholar, Professor and Asso-ciate Dean of Cancer Programs, Austin, Texas.

Corresponding Author: Aaron J. Scott, Banner University of Arizona CancerCenter, 1501 N. Campbell Avenue, Rm 1916, Tucson, AZ 85724. Phone: 520-891-0957; Fax: 520-626-2224; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-0131

�2018 American Association for Cancer Research.

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mechanism for its activity in treatment of mCRC. In addition toregorafenib, three other antiangiogenic agents have received FDAapproval in combination with chemotherapy to treat mCRC:bevacizumab, an mAb targeting VEGF; ziv-aflibercept, a recom-binant fusion protein decoy receptor of VEGFR1 and 2; andramucirumab, anmAb targeting VEGFR2. These agents add incre-mental benefit in progression-free survival and overall survivalcompared with chemotherapy alone in mCRC (5).

Treatment with antiangiogenics is often short lived due to thedevelopment of acquired resistance. Previously published datahave shown that the MET kinase pathway promotes resistanceto antiangiogenic therapy (6–8). The RTK MET, also known asthe hepatocyte growth factor receptor (HGFR), has been clas-sified as a proto-oncogene that drives tumor cell survival,metastasis, and resistance to anticancer therapies (9). METexpression alone has correlated with worse prognosis, tumorinvasion, and lymph node invasion in colorectal cancer (10). Inaddition, evidence from multiple preclinical studies has dem-onstrated that MET and VEGF receptor pathways may cooperateto promote tumor angiogenesis and provides a mechanism forresistance to selective blockade of either pathways (11–13).Increased MET activation also allows cancer cells to undergoepithelial–mesenchymal transition (EMT), facilitating cellmetastases to less hypoxic conditions and a more favorableenvironment for cell survival (14, 15). Because of this, interestin inhibiting the VEGF and MET axes for treatment in a varietyof malignancies continues to grow.

Cabozantinib (XL184) is an orally bioavailable, small-mole-cule inhibitor of multiple kinases central to cancer cell growth,angiogenesis, and metabolism including VEGFR2/KDR, MET,AXL, RET, TIE2, and KIT. Although the kinase target profile ofregorafenib is similar, it does not inhibit MET. Cabozantinib hasdemonstrated activity in multiple tumor types and has receivedFDA approval for treatment of previously treated metastatic renalcell carcinoma and metastatic medullary thyroid cancer (16, 17).Previously we demonstrated that treatment of colorectal cancerpatient–derived xenografts (PDX)with cabozantinib led topotentinhibition of tumor growth in 80%of treated tumors in colorectalcancer explant models (18). The conclusion was that the dualinhibition of MET and VEGFR2 is central to the antitumor effectsof cabozantinib.

In this study, we aimed to investigate mechanisms of theantitumor effects of cabozantinib in colorectal cancer PDX mod-els, using regorafenib as a comparator. We hypothesized thatantitumor activity of cabozantinib would be superior to regor-afenib in colorectal cancer PDX models due to dual inhibition ofMET and VEGFR2, as well as potentially other metabolic andautophagy mechanisms.

Materials and MethodsColorectal cancer PDX model

Patient-derived tumor tissues were acquired from the Univer-sity of Colorado Hospital (Aurora, CO). All patients consentedtheir tissue to be used in the study in accordance with protocolsapproved by the Colorado Multiple Institutional Review Board.Female athymic nudemice ages 4 to 8weekswere purchased fromEnvigo. All animal experiments for this study were approved bythe Institutional Animal Care and Use Committee. Tumor speci-mensobtained frompathologywerefinelyminced and implantedin mice and passed into subsequent generations (19). For treat-

ment studies involving cabozantinib and regorafenib, tumorswere expanded subcutaneously in the left and right flanks of mice(�10 tumors/group) and randomized into groups receiving vehi-cle, cabozantinib (30mg/kg) or regorafenib (10mg/kg) accordingto published studies (18, 20–22). Treatment started when theaverage tumor volumes reached approximately 200 mm3. Micewere treated with vehicle (no drug), cabozantinib, or regorafenibfor 28 days. Mice were monitored daily for signs of toxicity andtumor size was evaluated twice per week by calipermeasurementsusing the following formula: tumor volume ¼ (length � width2)� 0.52.

MET kinase active HCT116 cell line xenograft modelTheHCT116MET kinase active Y1253D andHCT116 (control)

isogenic colorectal cancer cell lines were obtained from Horizon.Cells were cultured in RPMI media containing 10% FBS, 1%nonessential amino acids, and 1% penicillin/streptomycin. TheMET kinase active and HCT116 isogenic cell lines were harvested,washed with complete media, and resuspended 50% media and50% matrigel. One million cells in a volume of 100 mL wereinjected subcutaneously in both flanks of each mouse (�10tumors/group). Mice were randomized into groups receivingvehicle, cabozantinib (30 mg/kg) or regorafenib (10 mg/kg).Treatment started when the average tumor volumes reachedapproximately 200 mm3. Mice were monitored daily for signsof toxicity and tumor sizewas evaluated twice per week by callipermeasurements using the following formula: tumor volume ¼(length � width2) � 0.52.

Human colorectal cancer cell lines were obtained from ATCC,DSMZ Cell Line Bank, ECACC (Sigma), and the Korean Cell LineBank. The GEO cell line was a generous gift from Dr. FortunatoCiardiello (Cattedra di OncologiaMedica, DipartimentoMedico-Chirurgico di Internistica Clinica e Sperimentale "F Magrassi e ALanzara," SecondaUniversit�a degli Studi di Napoli, Naples, Italy).All cell lines were cultured in RPMI media supplemented with10% FBS, 1% penicillin–streptomycin, and 1% MEM nonessen-tial amino acids and routinely screened for the presence ofMycoplasma (Mycoplasma Detection Kit, Biotool). Cell lines weremaintained at 37�Cwith 5%CO2. Cell lines were validated by theMolecular Biology Service Center in the Barbara Davis Center forDiabetes.

In vivo angiogenesis assessment: DCE-MRINoninvasive measurements of tumor vascularity were per-

formed on two sensitive colorectal cancer PDX models (CRC098and 162) tomeasure treatment effects on angiogenesis. Mice weretreated with cabozantinib and regorafenib and subjected todynamic contrast-enhanced MRI (DCE-MRI; n ¼ 4–8 tumors/group). DCE-MRI was done at baseline (prior to treatment), andon day 7 and day 28 (end of the treatment cycle). Briefly, theanimals were anesthetized with an intraperitoneal injection ofketamine/xylazine (60/10 mg/kg) and a tail vein catheter (filledwith 0.4 mmol/kg of MultiHance, gadobenate dimeglubine) wasplaced. The animal was positioned inside a warmed animalholder and inserted into the Bruker 4.7 Tesla MRI scanner. Allimages were acquired using a 36-mm volume receiver and trans-mitter coin and Bruker ParaVision v4.0 software. First, a fasttripilot was obtained for anatomic localization. Then, a series offast gradient echo (GRE) T1-weighted scanswas obtained for totalacquisition time of 5 minutes. After 30 seconds of image acqui-sition (precontrast), gadolinium (Gd) contrast was injected

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through the tail vein catheter. T1-weighted Gd-enhanced MRIscans were continuously taken for another 4.5 minutes. For eachdynamic frame, the T1-signal voxel intensities were calculated foreach lesion and blood vessel. A compartmental pharmacokineticmodel, using SAAM program (version 2, University of Washing-ton, Seattle, WA) and Bruker Advanced ParaVision v4.0, inwhich correlation of tissue clearance of Gd and transcapillaryexchange between tumor and blood vessels, was constructed onthe basis of T1-signal curves. The volume transfer constants(Ktrans) and the areas under the T1-intensity curves (AUC andIAUC), total and the first 60 seconds of enhancement, werereported as quantitative assessments of tumor perfusion andpermeability of Gd uptake (23).

Measurement of glucose uptake by FDG-PET followingcabozantinib and regorafenib treatment

18-Fluoro-deoxyglucose PET (FDG-PET) studies were con-ducted on two colorectal cancer PDX models (CRC098 and162) to determine whether treatment alters glucose uptake. Theanimals were scanned at baseline (prior to treatment) and onday 7 and day 28 following treatment with cabozantinib orregorafenib. Mice were fasted for 4 hours and injected via tailvein with 150 mCi (5.55 MBq) of 18F-fluorodeoxyglucose (FDG,obtained through PetNet). Animals were then anesthetizedwith 2% to 2.5% isoflurane, placed onto a warming pad inthe Siemens mouse holder and inserted into a Siemens InveonmPET/CT scanner. All scans were performed using SiemensInveon Acquisition Workplace software (IAW v1.5). First, afast CT scan was performed for anatomical localization(270� rotation with 18 rotation steps; 2,048 � 2,048 field ofview; 4 binning with 80 kV and 450 mA; 30 msec exposure time;low-medium magnification; 60 mm effective pixel size; and 6minutes total scan time). Then, the bed was moved into themPET scanner. All PET scans were acquired in a double-sam-pling mode to improve spatial resolution (1.2 mm). PET/CTimage fusion for precise anatomical identification of theregions of interest (ROI) was performed by Siemens InveonWorkstation Research (IWR v3.0) software. Regions of interestwere manually drawn within the tumor lesions and totalradioactivity of the ROI was determined (in kBq/mL). The SUVwas calculated as described by our group and others (24, 25).All PET/CT scans were performed at the UCCC Animal ImagingShared Resources (AISR, N. Serkova).

Gene pathway analysis by RNA-SeqTotal RNA from cabozantinib-treated colorectal cancer

explants at day 3 of treatment was extracted using RNeasy Kit(Qiagen). RNA sequencing (RNS-Seq) was used to determinegene expression in tumors of control and treated mice. Rawexpression values were obtained and normalized by the Affy-metrix Power Tools based on a multiarray average. Multipleprobe sets representing the same gene were collapsed by themaximum value. To assess pathways enriched in the controlversus cabozantinib-treated explants, we used the softwareversion 2.0.13 obtained from the Broad Institute (http://www.broad.mit.edu/gsea). We used the pathways defined bythe Kyoto Encyclopaedia of Genes and Genomes (KEGG) as thegene set (26). Gene set permutations were performed 1,000times for each analysis. We used the nominal P value andnormalized enrichment score obtained from gene set enrich-ment analysis to sort the pathways regulated in the cabozanti-

nib-treated groups. RNA-Seq data have been uploaded in theNCBI GEO repository database (GSE60939).

ImmunoblottingControl and treated tumor specimens (15 mg/tumor tissue)

were homogenized using a Qiagen tissue lyser. Following 10minutes on ice the lysed tissue was centrifuged at 16,000 � g at4�C for 10 minutes. Protein quantification in each sample wasdetermined using the 660 Protein Assay Kit (Thermo FisherScientific). A total of 50 mg of sample was electrophoresed on aprecast 4% to 12% Bis–Tris gel (Life Technologies). Proteins werethen transferred onto a nitrocellulose membrane using the iBlottransfer system (Life Technologies). Membranes were blocked for1 hour at room temperature using 3% casein. After blocking, themembranes were probed with the following primary antibodies(1:1,000) overnight at 4�C with rocking: phosphorylated andtotal: MET, AKT, hexokinase, pyruvate dehydrogenase, ULK1,ATG3, Beclin, LC3, SQSTM/p62, and actin (Cell Signaling Tech-nologies). Following washing three times with TBST, the mem-branes were incubated for 1 hour at room temperature with anti-rabbit IgG (HþL;DyLight 800Conjugate) secondary antibody at afinal dilution of 1:15,000. Signal images were captured using theOdyssey Infrared Imaging System (Li-Cor).

Receptor tyrosine kinase arrayControl and treated tumor specimens (15 mg/tumor tissue)

were homogenized using a Qiagen tissue lyser and protein wasquantitated using the 660 Protein Assay Kit. After blocking theslides containing 39 antibodies/well (RTK array; Cell SignalingTechnologies) for 15 minutes, 50 mg of protein was added tothe slide and incubated overnight at 4�C with gentle rocking.The slides were then washed and detection antibody was addedto the slide followed by DyLight 680VR–linked Streptavidin.Slide images were captured using the Odyssey Infrared ImagingSystem (Li-Cor) and were quantified using the Odyssey systemsoftware.

AutophagyTreatment effects of cabozantinib, regorafenib, and crizotinib

on autophagy were evaluated on the HCT116 andHT29 cell linesusing the CYTO-ID Autophagy Detection Kit (Enzo -ENZ-51031-K200).HCT116orHT29 cellswere plated in 96black-walled plateat a concentration of 1,000 cells/well. After 24hours, the cellsweretreated with cabozantinib (5 mmol/L), regorafenib (5 mmol/L),and crizotinib (1 mmol/L). Following 24 hours of incubation, thecells were washed twice with 1� assay buffer and then incubatedfor 30 minutes with the CYTO-ID Autophagy Detection reagent.After 30minutes, the cellswerewashed twicewith 1� assay buffer.The effects of treatment were evaluated on the IncuCyte ZOOMlive cell imager using 488 nm-excitable green fluorescentdetection.

Treatment effects on apoptosisNext, we performed in vitro combination studies with the

autophagy inhibitors chloroquine and/or SBI-0206965 (ULK1inhibitor) on the HCT116 and HT29 colorectal cancer cells.Cells were plated in 96 black-well plate at a concentration of1,000 cells/well and incubated overnight to allow cells to adhere.After 24 hours, the media were removed and 100 mL of mediacontaining IncuCyte Kinetic Caspase-3/7 Apoptosis AssayReagent (catalog no 4440) was added to each well. Immediately

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after the cells were treated with cabozantinib (5 mmol/L), SBI-0206965 (5 mmol/L), chloroquine (10 mmol/L), cabozantinib(5 mmol/L), plus SBI-0206965 (5 mmol/L), and cabozantinib(5 mmol/L) plus chloroquine (10 mmol/L). In addition, regora-fenib (5 mmol/L) or crizotinib (1 mmol/L) were used as a singleagent and in combination with SBI-0206965 (5 mmol/L). Treat-ment effects on apoptosis were measured every 2 hours (total of36–48 hours) on the IncuCyte ZOOM live cell imager.

Statistical analysisAn unpaired t test was used to compare the tumor growth

inhibition index (TGII) differences between cabozantinib andregorafenib. The TGII is a standardizedmeasure of tumor growth,which is calculated using the following formula: TGII ¼ (tumorvolume of TX on day 28 – tumor volume of TX on day 0)/(tumorvolume of Con on day 28 – tumor volume of Con on day 0) �100, where TX is the cabozantinib or regorafenib-treated xeno-graft and CX is the control-treated xenograft. The differences wereconsidered significant when the P value was <0.05. All error barsare represented as the SEM. A paired t test was used to examine the

differences in [18F]-FDG-PET uptake and Gd DCE-MRI uptake atbaseline, day 7 and day 28. The differences were consideredsignificant when the P value was < 0.05. A one-way ANOVA wasused to determine whether the means were significantly differentbetween treatments with respect to autophagy and apoptosis. Ifthe overall means were significantly different, we performed apair-wise comparison.P valueswere adjustedusing Tukeymethodfor multiple comparisons. SEM error bars were indicated for eachvalue. All statistical analyses were performed using GraphPadPrism Software.

ResultsCabozantinib exhibits significantly greater antitumor effectswhen compared with regorafenib in colorectal cancer PDXmouse models

Cabozantinib demonstrated greater antitumor activity whencompared with regorafenib in 7 of 10 colorectal cancer explantstested (Fig. 1A), with no difference in body weight of the animalsbetween the groups (data not shown). A comparison of the

Figure 1.

Cabozantinib demonstrated significant antitumor effects compared with regorafenib in 7 of 10 of the colorectal cancer explants. A, Five of the explantstreated with cabozantinib showed tumor regression. B, Significant tumor growth inhibition measured by TGII in 10 colorectal cancer explants with cabozantinibcompared with regorafenib. C, Cabozantinib maintained significant TGII in the engineered MET kinase active knock-in mutation Y1253D cell line comparedwith regorafenib in vitro. D and E, percent growth curve of cabozantinib in the parental HCT116 cell line and the engineered MET kinase active cell line. � , P < 0.05;�� , P < 0.007; �� , P < 0.001; statistically significant difference in TGII with cabozantinib compared with regorafenib. #, P < 0.05; statistically significant difference inTGII with cabozantinib compared with control.






combined TGII among the 10 total colorectal cancer explantsrevealed that cabozantinib treatment was significantly better thanregorafenib at tumor growth inhibition (P ¼ 0.007; Fig. 1B).Supplementary Table S1displays thepatient characteristics aswellas gene mutations for each colorectal cancer explant treated onthis study. Next, we determined whether treatment differencesdiffered with a constitutively active MET kinase cell line. Aheterozygous knock-in of MET activating mutation Y1253D wasintroduced in the HCT116 cell line. As displayed in Fig. 1C–E,both compounds demonstrated similar antitumor activity in theHCT116 parental cell line; however, cabozantinib exhibited sig-nificantly improved antitumor effects compared to regorafenib inthe MET kinase active cell line.

Evaluation of cabozantinib and regorafenib on angiogenesisWe next investigated the treatment effects of regorafenib

and cabozantinib on angiogenesis using DCE-MRI. Cabozan-

tinib treatment significantly reduced tumor vascularity inCRC098 and CRC162 after 28 days of treatment comparedwith baseline (Fig. 2; Supplementary Table S2). Evaluation ofVEGFR2 and TIE2 revealed a decrease in activation as early as 4hours, which was sustained for 7 days after cabozantinibtreatment (Fig. 2B–D). Regorafenib treatment of CRC098also exhibited a decrease in vascularity (Supplementary TableS2) as well as in the activation of VEGFR2 and TIE2 (Supple-mentary Fig. S1). In contrast, no changes in vascularity wereobserved in CRC162 after regorafenib treatment (Supplemen-tary Table S2).

Reduction of PI3K/AKT/mTOR signaling pathway followingcabozantinib treatment

RTK activation plays an essential role in facilitating extracel-lular signals to downstream targets including the PI3K signalingpathway. Phosphoprotein levels involved in the PI3K/mTOR

Figure 2.

A, DCE-MRI using Gd contrast revealed significant decrease in vascularity, measured in Ktrans (min�1), at day 28 of cabozantinib compared with baseline. B–D, RTKantibody assay using fluorescent analysis revealed decreased levels of TIE2 and VEGFR2 4 hours after treatment with cabozantinib and were sustainedthrough 7 days of treatment compared with control.

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signaling pathway were significantly reduced after cabozantinibtreatment including PI3K, PDK1, AKT, mTOR, Rictor, Raptor,and S6K1/2 (Fig. 3A). In addition, analysis of RTK activationby an antibody array revealed a decrease in the phosphoryla-tion of MET, RET, and AXL, and a subsequent decrease inAKT and S6 after cabozantinib treatment in CRC098 andCRC162 (Fig. 3B–G). A maximal decrease in activation wasobserved at 8 hours after treatment in CRC098 and 162.Regorafenib did not demonstrate significantly reduced levelsof pMET, pRET, pAXL, AKT, and pS6 in CRC098 or CRC162(Supplementary Fig. S2).

Examination of glucose uptake (18[F] FDG-PET) followingcabozantinib treatment

Upon observation that cabozantinib significantly reduceddownstream effectors in the PI3K/AKT/mTOR signaling path-way, we hypothesized that cabozantinib may be an importantmodulator of tumor cell metabolism. Cabozantinib signifi-cantly reduced glucose uptake measured by [18F]-FDG-PET atdays 7 and 28 compared with baseline in the CRC098 andCRC162 explants (Fig. 4A; Supplementary Table S3). Regor-afenib did not significantly reduce glucose uptake in theCRC098 or 162.

Next, we performed a comprehensive pathway analysis aidedby the KEGG database using RNA-Seq post-cabozantinib treat-ment in the most sensitive explants (CRC020, 102, and 162).Significant decreases in pathways controlling glycolysis and theTCA cycle were observed (Supplementary Table S4). In addition,Western blot analysis demonstrated downregulation of pyruvatedehydrogenase on days 7 and 28 following cabozantinib treat-ment in the CRC098 and CRC162 explants compared withcontrol (Fig. 4B). In contrast, no decrease in pyruvate dehydro-genase was observed after regorafenib treatment in CRC098 and162 explants (Fig. 4B).

Cabozantinib induces autophagy that may provide amechanism for tumor cell survival

Both glucose deprivation and PI3K/mTOR pathway inhibitionhave been shown to trigger autophagy (27). As shown above, wedetermined that cabozantinib significantly decreased glucoseuptake as well as the PI3K/mTOR signaling pathway. Therefore,we investigated autophagy as an alternate metabolic pathway forcell survival in vitro following cabozantinib treatment. Increasedprotein expression of ATG3, LC3, and beclin-1 occurred as early as7 days after cabozantinib treatment (Fig. 5A). In addition, eval-uation of CRC020 and CRC040 explants (both sensitive) showed

Figure 3.

Significant decrease in levels of activation of components in the PI3K/AKT/mTOR axis was observed using RTK antibody assay after cabozantinib treatment. A,Illustration of the enzymes with significant reduction in activation with cabozantinib are shown in red. B–F, Significant reduction of phosphorylated MET, RET,AXL, AKT, and S6 was observed with cabozantinib treatment after 7 days compared with control. G, RTK antibody fluorescent analysis showing decreasedlevels of the enzymes following 7 days of cabozantinib treatment in CRC098 and CRC162 explants.






a decrease in SQSTM1/p62with cabozantinib treatment ondays 7and 28 (Fig. 5B). No increase in ATG3, LC3A/B, and beclin1 inCRC098 and CRC162 were observed after regorafenib treatment,apart from a modest increase in LC3A/B in the CRC162 explantwhen considered with the actin loading control (SupplementaryFig. S3).

Given that we observed a significant increase in autophagyfollowing cabozantinib treatment, we examined whether com-bining an autophagy inhibitor with cabozantinib would enhancetumor cell death. First, the treatment effects on autophagy wereevaluated in vitro using an autophagy kit (CYTO-ID AutophagyDetection Kit) that monitors autophagic activity at the cellularlevel (28, 29). Twenty-four hours of treatment with cabozantinibsignificantly induced autophagy when compared with controland regorafenib treatment in theHCT116andHT29 cell lines (Fig.5C and D; Supplementary Fig. S4). Next, we tested the combi-nation of cabozantinib with the autophagy inhibitors SBI-0206965, an ULK1 inhibitor, and chloroquine. Using a caspase3/7 fluorescent reagent and IncuCyte live cell imager, we observeda striking increase in apoptosis in the HCT116 cells treated withSBI-0206965 plus cabozantinib and chloroquine plus cabozan-tinib over 48 hours (Fig. 5E; ref. 29). In contrast, no combina-tional effect was seen in the HCT116 cell line when treated withregorafenib (Fig. 5F). Furthermore, although an increase in autop-hagy was observed using crizotinib, a potent MET inhibitor, acombinational effect was not seen with crizitonib plus SBI-020695 in the HCT116 and HT29 colorectal cancer cell lines(Supplementary Fig. S4).

DiscussionWe previously demonstrated that cabozantinib exerts potent

antitumor effects in colorectal PDX models; 7 of 10 colorectalcancer explants showed significantly superior antitumor effects,and 5 of 9 cabozantinib sensitive colorectal cancer explantsdisplayed tumor regression (18). In this study, using regora-fenib (FDA approved for resistant advanced colorectal cancer)as a comparator, we showed that cabozantinib is superior ininhibiting tumor growth in colorectal cancer PDX modelsand mechanistic studies showed unique pharmacodynamiceffects of cabozantinib in this model. Only 1 of 10 colorectalcancer explants, CRC036, showed a trend toward improvedantitumor effect with regorafenib. Dosages used in the PDXmodels were chosen to approximate plasma drug exposureswithin the pharmacodynamic range achievable in humans(20, 30–34).

Preclinical studies have demonstrated the role of MET infacilitating anti-VEGF treatment resistance (6, 7, 35). As withVEGF activation, MET signaling has been shown to be a potentinducer of endothelial angiogenesis (36–38). Although METand the VEGF receptor family are not known to interact with orphosphorylate one another, these kinases synergistically acti-vate similar downstream intermediates including FAK, MAPK,and AKT (12). Data from preclinical studies have also shownthat MET activation promotes angiogenesis through othercommon signaling intermediates such as SRC homology 2domain-containing proteins (SHC). In vivo and in vitro studieshave demonstrated reduction in angiogenesis leading to apo-ptosis with MET inhibition (18). In this study, cabozantinibshowed a significant decrease in tumor vascularity comparedwith regorafenib as measured by DCE-MRI, a technique thathas been shown to correlate with tumor vascularity and angio-genesis in preclinical and clinical studies (39–41). Althoughcabozantinib and regorafenib both have antiangiogenic prop-erties via VEGFR2 inhibition, dual MET and VEGFR2 inhibitionmay explain the observed differences in treatment effectsin tumor vascularity. In addition, a near immediate and sus-tained decrease in protein expression of angiogenic mediatorsVEGFR2 and TIE2 was also observed to a larger degree withcabozantinib.

Enhanced antitumor effects of cabozantinib were also dis-played in the constitutively activated MET kinase cell line.Physiologic activation of MET through HGF ligand bindingelicits a complex network of downstream signaling that affectscell-cycle regulation, cell survival, and cell migration and adhe-sion mainly through the PI3K/AKT/mTOR, Src, and RAS/MEKpathways (42, 43). Aberrant MET activation after exposureto anti-VEGF treatment leads to acquired resistance and inducesan upregulation in crosstalk pathways responsible for cellsurvival, angiogenesis, and EMT (44–46). In addition to reduc-ing VEGFR2 and TIE2 levels and tumor vascularization, path-way analysis showed that many components involved inthe PI3K/AKT/mTOR pathway were significantly decreasedwith cabozantinib treatment, including PI3K, PDK1, AKT,mTOR, Rictor, Raptor, and S6K1/2. This observed decrease inboth tumor vascularity and activation of MET, RET, and AXLsuggests that inhibition of multiple crosstalk pathwaysmay abrogate acquired resistance observed with other anti-VEGF therapies.

The PI3K/AKT/mTOR pathway regulates cellular metabolismthrough multiple downstream targets including hypoxia

Figure 4.

A, Cabozantinib significantly reduced glucose uptake measured by[18F] FDG-PET on day 7 and day 28 of treatment compared with baseline. B,Western blot analysis revealed a decrease in protein expression of pyruvatedehydrogenase after cabozantinib treatment compared with regorafeniband control.


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inducible factor-1 (HIF-1), which is involved in glucose metab-olism, and ULK-1, which is involved in autophagy (47, 48).Inhibition of the PI3K/AKT/mTOR pathway leads to disruptionof glucose uptake. This was confirmed with cabozantinib treat-ment, where both a significant decrease in PI3K/AKT/mTORmediators and glucose uptake measured by [18F]-FDG-PETwas observed. In contrast, a change in glucose uptake was notsignificantly altered after regorafenib treatment. Further investi-gation demonstrated a significant decrease in pyruvate metabo-lism and gene analysis of the TCA cycle with cabozantinibtreatment compared with baseline, which was also not observedafter regorafenib treatment. These alterations suggest that inhibi-tion of the PI3K/AKT/mTOR pathway leads to a decrease inglucose metabolism and glycolytic inhibition with cabozantinibtreatment.

Glucose deprivation and inhibition of PI3K/AKT/mTOR path-way have previously been shown to induce autophagy in preclin-ical studies (27, 48, 49). Protein expression of key enzymes in

autophagy including ATG3, LC3, and beclin1 increased duringcabozantinib treatment compared with baseline. These changeswere not observed after regorafenib treatment. Under normalconditions, basal levels of autophagy are usually low and arerestricted to maintaining important cellular functions. However,during times of nutrient deprivation and hypoxic conditions,autophagy is upregulated to promotemetabolic homeostasis andsurvival of cells (50–53). Pharmacologic inhibition of the PI3K/AKT/mTOR pathway enhances autophagy by inhibiting mTOR-mediated activation of the ULK1 complex, leading to subsequentlysosomal degradation of cellular components for ATP energy(Fig. 6; refs. 54, 55). Together with glycolytic inhibition andupregulation of components involved in autophagy, an increasein autophagy occurred during cabozantinib treatment in theHCT116 and HT29 colorectal cancer cell lines. Using an autop-hagy detection kit, significant induction of autophagy wasobserved in vitro after 24 hours of cabozantinib compared withregorafenib and control. These results further support the concept

Figure 5.

A, Autophagy markers ATG3, LC3A/B, and beclin-1 increased after cabozantinib treatment in the CRC098 and CRC162 explant models. B, SQSTM1/p62 levelsalso increased after cabozantinib treatment in the CRC020 and CRC040 explant models. C–F, Autophagy levels measured by CYTO-ID AutophagyDetection Kit demonstrated a significant increase in autophagy after 24 hours of cabozantinib treatment compared with regorafenib and control. G, Significantincrease in levels of apoptosis measured by caspase-3/7 fluorescent assay after treatment with combination cabozantinib plus SBI-0206965, an ULK1inhibitor, and cabozantinib plus chloroquine compared with control or single-agent treatment. H, Significant increase in levels of caspase-3/7 were not seenafter combination treatment with regorafenib plus SBI-0206965 compared with control or single-agent treatment. ��, Statistically significant increase influorescence after cabozantinib treatment compared with control. ###, Statistically significant increase in fluorescence after cabozantinib treatmentcompared with regorafenib.






that multikinase inhibition with cabozantinib, specifically thePI3K/AKT/mTOR pathway throughMET inhibition, elicits a tran-sition from glycosis-dependent to autophagy-dependent metab-olism as a means for continued cell survival.

These findings are consistent with previously published datademonstrating autophagy as a mechanism for acquired resis-tance to certain anticancer therapies (56). Striking in vitroeffects of combination treatment using antiautophagy agentsSBI-0206965, an ULK1 inhibitor, or chloroquine plus cabo-zantinib revealed increased apoptotic marker caspase 3/7 fluo-rescent assay. Regorafenib in combination with these antiau-tophagy agents did not reveal an increase in caspase 3/7activity. Although crizotinib, a potent MET inhibitor, producedsimilar MET suppression compared with cabozantinib, therewas no increase in autophagy when SBI-020695 was added tocrizotinib. These findings suggest that there are multiple cross-talk pathways that allow ongoing glycolysis in the presence ofMET inhibition. However, multiple kinase inhibition as seenwith cabozantinib interrupts crosstalk as an escape mechanismfor ongoing glycolysis and leads to induction of autophagy.Combining an antiautophagy agent with cabozantinib con-firmed this idea as both combinations with SBI-020695 andchloroquine augmented apoptotic response compared with

baseline and single-agent treatment in the HCT116 and HT29cell lines.

Our results show that cabozantinib has superior antitumoreffects compared with regorafenib in vitro and in vivo usingcolorectal cancer explant models. Dual inhibition of MET andVEGFR2 act synergistically to potentiate antiangiogenic responseby inhibiting multiple cross-talk pathways including PI3K/AKT/mTOR. The inhibition of multiple kinase pathways produces achange in metabolism from glycolysis to autophagy, serving as amethod for acquired resistance and cell survival during cabozan-tinib treatment. Combination of cabozantinib plus an antiauto-phagy agent increased apoptosis in the HCT116 and HT29 colo-rectal cancer cell lines. These findings warrant further investiga-tion of cabozantinib in colorectal cancer and combinationapproaches targeting autophagy. A clinical trial is currently underdevelopment to study the efficacy of cabozantinib in patientswithrefractory mCRC.

Disclosure of Potential Conflicts of InterestJ.J. Arcaroli is Medical Science Liaison at Bayer. T.M. Pitts reports receiving a

commercial research grant from Exelixis. W.A. Messersmith reports receiving acommercial research grant from Exelixis. No potential conflicts of interest weredisclosed.

Figure 6.

In this illustration, potentialmechanisms of tumor cell survival viacross-talk between components of theMET pathway, glycolysis, andautophagy are depicted. Basal levelsof autophagy are typically low throughmTOR inhibition of ULK1, a keyenzyme in upregulation of autophagy,and glycolysis predominates as themain mechanism for ATP generation.Aberrant MET activation leads toupregulation of downstreameffectors, including components of thePI3K/AKT/mTOR pathway, andglycolytic metabolism is maintained.Through blockade of MET andsubsequent indirect activation of ULK1through the absence of negativefeedback with mTOR inhibition,upregulation in autophagy occurs tomaintain cellular homeostasis. Asimplified canonical pathway ofautophagy (steps 1–4) is shown.


Scott et al.




Authors' ContributionsConception and design: A.J. Scott, J.J. Arcaroli, T.M. Pitts, W.A. MessersmithDevelopment of methodology: A.J. Scott, N.J. Serkova, T.M. Pitts,W.A. MessersmithAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.J. Scott, J.J. Arcaroli, S.M. Bagby, R. Yahn,K.M. Huber, N.J. Serkova, J. Vogel, K.S. Quackenbush, A. Capasso, A. Schreiber,P.J. Klauck, T.M. Pitts, W.A. MessersmithAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):A.J. Scott, J.J. Arcaroli, N.J. Serkova, A. Nguyen, J. Kim,P. Blatchford, T.M. Pitts, W.A. MessersmithWriting, review, and/or revisionof themanuscript:A.J. Scott, J. Kim, T.M. Pitts,S.G. Eckhardt, W.A. MessersmithAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.J. Scott, J.J. ArcaroliStudy supervision: W.A. Messersmith, J.J. ArcaroliOther (technical advice and experimental design): A. Thorburn

AcknowledgmentsThis work was supported by the NIH (1R01CA152303-01; to W.A. Messer-

smith) and the University of Colorado Cancer Center Shared Resource(P30CA046934; to W.A. Messersmith). Support was also received by Exelixis(to T.M. Pitts). We also thank the patients who consented to allow their tumortissue to be used in these experiments.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 10, 2017; revisedDecember 1, 2017; accepted July 9, 2018;published first July 19, 2018.

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Scott et al.




2018;17:2112-2122. Published OnlineFirst July 19, 2018.Mol Cancer Ther Aaron J. Scott, John J. Arcaroli, Stacey M. Bagby, et al. and Signaling MechanismsCancer Patient-Derived Tumor Xenograft Models via Autophagy Cabozantinib Exhibits Potent Antitumor Activity in Colorectal

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