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. 2023 Nov 8;20(12):6140–6150. doi: 10.1021/acs.molpharmaceut.3c00496

Cyanine Dye Conjugation Enhances Crizotinib Localization to Intracranial Tumors, Attenuating NF-κB-Inducing Kinase Activity and Glioma Progression

Kathryn M Pflug †, Dong W Lee †, Ashutosh Tripathi †, Vytas A Bankaitis †, Kevin Burgess ‡, Raquel Sitcheran †,*
PMCID: PMC10698717  PMID: 37939020

Abstract

graphic file with name mp3c00496_0006.jpg

Glioblastoma (GBM) is a highly aggressive form of brain cancer with a poor prognosis and limited treatment options. The ALK and c-MET inhibitor Crizotinib has demonstrated preclinical therapeutic potential for newly diagnosed GBM, although its efficacy is limited by poor penetration of the blood brain barrier. Here, we identify Crizotinib as a novel inhibitor of nuclear factor-κB (NF-κB)-inducing kinase, which is a key regulator of GBM growth and proliferation. We further show that the conjugation of Crizotinib to a heptamethine cyanine dye, or a near-infrared dye (IR-Crizotinib), attenuated glioma cell proliferation and survival in vitro to a greater extent than unconjugated Crizotinib. Moreover, we observed increased IR-Crizotinib localization to orthotopic mouse xenograft GBM tumors, which resulted in impaired tumor growth in vivo. Overall, IR-Crizotinib exhibited improved intracranial chemotherapeutic delivery and tumor localization with concurrent inhibition of NIK and noncanonical NF-κB signaling, thereby reducing glioma growth in vitro, as well as in vivo, and increasing survival in a preclinical rodent model.

Keywords: GBM, kinase inhibitor, Crizotinib, NF-κB-inducing kinase, cyanine dye, HMCD

Introduction

Glioblastoma multiforme (GBM) is a rare, malignant form of brain cancer that is characterized as a grade four glioma, or the most aggressive form of gliomas.1 GBM cells are highly invasive and resistant to treatment, leading to high rates of recurrence and poor patient outcomes. Median survival of GBM patients is only 12.6 months with a 5 year survival rate of about 4–5%.2,3 A major impediment to pharmacological approaches for treating GBM is drug accessibility to the brain. Given the need to improve chemotherapeutics, not only for extremely aggressive cancers like GBM, but also for other cancers, recent studies have interrogated the therapeutic potential of near-infrared fluorescence (NIRF), heptamethine cyanine dyes (HMCDs) as they have been shown to accumulate in solid tumors and preferentially localize to tumor sites over healthy tissues.4−6 It has been demonstrated that the preferential uptake of HMCDs by tumor cells involves covalent bonds with albumin and transport by organic anion-transporting polypeptides (OATPs). Notably, OATPs, which regulate cellular uptake of salts, bile acids, hormones, toxins, and drugs,7−11 exhibit increased expression in many cancer cells.12 Furthermore, the fluorescent properties of HMCDs allow visualization of tumor cells, thereby facilitating resection before surgery.13,14 The conjugation of HMCDs to chemotherapeutics is being explored as a means to improve drug delivery, tumor targeting, and efficacy of traditional cancer treatments, especially with therapy-resistant cancers. In particular, the ability of HMCDs to cross the blood brain barrier holds promise for the treatment of GBM.7,15

Similar to other malignancies, GBMs exhibit aberrantly elevated expression and/or activity of the nuclear factor-κB (NF-κB) pathway, which promotes several aspects of tumor growth. We have previously demonstrated that NF-κB-inducing kinase (NIK) is a key regulator of GBM invasiveness and metabolism.16,17 NIK is best known for its role in inducing noncanonical NF-κB signaling and regulation of immunity and inflammation through activation of RelB/p52 transcription factors.18−20 We have also established NF-κB-independent roles for NIK in cancer cell progression through regulation of mitochondrial dynamics and metabolic adaptation to glucose deprivation.21,22 While the use of small-molecule NIK inhibitors is being developed for several diseases, the application of NIK inhibitors to treat GBM has not been explored. Here, we report that Crizotinib, an FDA-approved tyrosine kinase inhibitor that targets anaplastic lymphoma kinase (ALK), c-MET (mesenchymal-epithelial transition), ROS (ROS1 proto-oncogene, receptor tyrosine kinase), and other tyrosine growth receptors, is also an inhibitor of NIK. Additionally, we investigate whether Crizotinib conjugation to HMCDs improves its therapeutic potential for GBM in orthotopic GBM mouse xenografts.

Materials and Methods

Animal Work

All animal experiments were done in accordance with the animal use protocol (2018-0432) with approved IACUC guidelines.

Molecular Docking and Visualization

The PDB ID: 4G3D crystal structure of the catalytic domain of human NIK was used from the RCSB Protein Data Bank. Crizotinib and IR-786-conjugated Crizotinib compound structures were obtained from PubChem, Crizotinib CID: 11626560 and IR-Crizotinib CID: 155521147, and drawn using ChemDraw. Docking was predicted using Autodock Vina23 through PyRX with an exhaustiveness of 16–32, and results were visualized using Discovery Studio.

Compound Synthesis

An IR-786 dye, Crizotinib, and IR-Crizotinib were all synthesized and purchased from TOCRIS and were validated by mass spectrometry and NMR (Supp. Figure 1C). MWs of Crizotinib and IR-Crizotinib were 450.34 and 1024.87 g/mol, respectively. For the conjugated form, Crizotinib was bound to the meso-chloride of the IR-786 dye (see Figure 2B), as previously described.24

Figure 2.

Figure 2

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Docking of Crizotinib and IR-786 Crizotinib to the catalytic domain of NIK. (A) 2D structure of Crizotinib. (B) 2D structure of Crizotinib conjugated to the IR-786 dye. (C, D) Predicted docking by Autodock Vina of (C) Crizotinib or (D) IR-Crizotinib in the back pocket of the catalytic (kinase) domain of NIK (PDB: 4G3D). (E, F) Predicted docking by Autodock Vina of (E) Crizotinib or (F) IR-Crizotinib in the open cleft of the catalytic (kinase) domain of NIK (PDB: 4G3D).

Cell Culture

The BT25 cell line was obtained from human glioma patients as previously described.25 BT25 cells were maintained as spheroids in a neural stem cell (NSC) medium containing DMEM/F-12, 1× B-27 supplement minus Vitamin A, 1× Glutamax, 25 ng/mL EGF, 25 ng/mL bFGF, and 1× Pen/Strep (Life Technologies). U87 MG human glioma cells were grown in the DMEM medium with 10% FBS. 1× Glutamax, and 1× Pen/Strep.

Lentivirus Production

Lentiviral plasmids (24 μg) and 72 μg of polyethylenimine (Sigma-Aldrich) were used to transfect 293T cells. After 3 days of transfection, the viral supernatant was concentrated 20-fold to 500 μL using a Lenti-X concentrator (Clontech, Mountain View, CA), and 100 μL of the concentrated virus was used to infect cells. Stably transduced cells were selected for 72 h in the medium containing 0.6 μg/mL puromycin or 6 μg/mL blasticidin (Thermo Fisher Scientific). BT25 and U87 cells were transduced by a lentivirus packaged from pCD516-Luc. pCD516B-Luc was constructed by inserting the firefly’s luciferase coding sequence into the pCDH-CMV-MCS-EF1a-RFP+Puro (SBI, Palo Alto, CA).

Immunoblot

Cells were lysed in RIPA lysis buffer with a protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific). Equal amounts of protein were mixed with NuPage 4× LDS sample buffer (Thermo Fisher Scientific) containing a reducing agent and denatured at 100 °C for 7 min. Proteins were separated on 8–12% SDS-PAGE and transferred to nitrocellulose membranes (Amersham). Membranes were blocked for 1 h with 5% nonfat dry milk in 0.1% Tween-20/TBS (TBST) and incubated with primary antibodies diluted in blocking buffer at 4 °C overnight. After washing in TBST, membranes were incubated with a goat antirabbit IRDye800CW (LI-COR Biosciences), goat antimouse IRDye680 (LI-COR Biosciences), or goat antirabbit HRP conjugate (Thermo Scientific Scientific) diluted in blocking buffer for 1 h at room temperature. Blots were washed with TBST and developed using a chemiluminescent HRP substrate (EMD Millipore) for the detection of HRP or an Odyssey Infrared Imaging system (LI-COR Biosciences) for the detection of IRDye fluorescent dyes.

Antibodies

IKKα (CST2682), pIKKα/β (CST2697), NFKB2 (p100/p52) (CST4882), NIK (CST4994), p-p65 (CST3033), p65 (sc-8008), RelB (CST4992), GAPDH (sc-137179), and Lamin A/C (CST4777) were used.

Kinase Assay

A NIK kinase activity assay was performed using Promega’s ADP-GloTM + NIK kinase enzyme system (V4077; Promega, Madison, WI) following the manufacturer’s protocol. NIK (50 ng) was used in the reaction.

MTS Proliferation Assay

BT25-RFP-LUC or U87- RFP-LUC cells were plated 5,000 cells per well among four 96-well plates for a 4 day time course. Compounds were treated at a final concentration of 5 μM at day 0 in 100 μL of media. Each day, 20 μL of MTS (Promega, G3582) was mixed with the media and incubated at 37 °C for 1 h and then absorbance was read at 490 nm.

Sphere Assay

BT25-RFP-LUC or U87-RFP-LUC cells (200 cells/200 μL) were plated in triplicate–quadruplet in a 96-well plate. Cells were grown for 3 days, treated with 5 μM of the various compounds, and then grown for the rest of the week with the inhibitors. Wells were then imaged under brightfield at 4×.

Invasion Assay

Collagen was prepared for a final concentration of 2.0 mg/mL. The collagen mixture (28 μL) was plated in half-well 96-well plates. The plate with collagen was then incubated for 45 min at 37 °C. BT25-RFP-LUC cells were then plated at 50,000 cells/well in complete DMEM F-12, and compounds were added to the media at a final concentration of 5 μM. Two days later, cells were fixed with 3% glutaraldehyde for 30 min and then stained with toluidine blue. Invading cells were counted under a 25× objective.

Xenograft Experiments

All animal experiments were done in accordance with AUP/IACUC guidelines. Seven week old Foxn1 nu females were ordered from Jackson Lab. BT25-RFP-LUC or U87-RFP-LUC (5 × 105) cells were injected intracranially into the right hemisphere of the mice. Tumor growth was monitored by luminescence, with subcutaneous d-luciferin injections, or by fluorescence (RFP; 570–620 nm).

Intravenous Chemotherapeutic Injections

Three to four days post tumor cell injection, intravenous (IV) injections of the drugs and vehicle were administered by retro-orbital. IR-786 and IR-786-conjugated Crizotinib were diluted in 10% DMSO and 2% Tween-80 in PBS. IR-CRIZ/CRIZ were injected by IV once a week at 3 mg/kg from fresh 2 mM stocks. Drug clearance/localization of cyanine dyes and conjugated compounds was monitored by fluorescence imaging (745–840 nm).

Fluorescence/Luminescence In Vivo Imaging

Mice were anesthetized with isoflurane and then imaged for fluorescence or luminescence on an IVIS spectrum (in vivo imaging system; PerkinElmer). For luminescence imaging, mice were subcutaneously injected with d-luciferin (15 mg/mL; PerkinElmer, cat. no. 122796) in microliters at 10× the mouse’s weight. During imaging, mice remained anesthetized by isoflurane. Fluorescence was measured by radiant efficiency, and luminescence was measured by counts. Red fluorescence protein (RFP) from BT25 or U87 cells was captured at an excitation wavelength of 570 nm and an emission wavelength of 620 nm. The near IR dye fluorescence was captured at an excitation wavelength of 745 nm and an emission wavelength of 840 nm. Images were captured, and data were analyzed using Living Image Software (PerkinElmer).

H&E Staining

Brain tissue was fixed in 4% PFA overnight and then dehydrated in 30% sucrose. The tissue was then embedded in OCT and frozen at −80 °C. Tissue was cryo-sectioned at 20 μm and then stained with Mayer’s Hematoxylin and Eosin. Slides were imaged on a Lion Heart imager by Biotek with 4× images stitched together.

Tissue Immunofluorescence

Twenty micron cryosections of brain samples were cleared of excess OCT with water, blocked/permeabilized with 5% goat serum and 0.5% Triton-X in PBS for 1 h at room temperature, washed with PBS, and then primary antibodies were added in 1% BSA and 0.1% Triton-X in PBS overnight at 4 °C. NF-κB2 (CST 3017) was added at 1:200, and cPARP (Biolegend 660993) was added at 1:150. Following PBS washing, fluorescent seconday antibodies were added at 1:1000 in 1% BSA and 0.1% Triton-X in PBS at room temperature for 1 hour. Mounting media with DAPI (Life Technologies) were used in the coverslipping slides.

Statistical Analysis

Statistical analysis was done using GraphPad PRISM software, and specifics on data representation and tests used for analysis can be found in figure legends. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Unpaired Student's t-tests were run as two-tailed. All statistically significant analyses were run based on a 95% confidence interval.

Results

While investigating potential repurposing of FDA-approved small-molecule inhibitors for targeting NIK in GBM cells, we observed that among a panel of compounds, Crizotinib had the greatest effect on impairing phosphorylation of IKKα/β and nuclear translocation of the NIK-dependent NF-κB transcription factors RelB and p52 (Figure S1A). Crizotinib, a potent inhibitor of the receptor tyrosine kinase MET and anaplastic lymphoma kinase (ALK),26,27 has demonstrable antitumor efficacy in mouse xenografts and shows great promise in clinical trials of patients with cancers harboring ALK rearrangements. In GBM patients, Crizotinib has exhibited potential as an adjuvant therapy in addition to radiation and temozolomide in newly diagnosed GBM patients28 but has limited efficacy on its own,29 likely due to its poor BBB permeability. A recent report demonstrated that the conjugation of Crizotinib to the infrared cyanine dye 786 (referred to as IR-Criz) resulted in increased cellular uptake and inhibition of cell viability in vitro in U87 GBM cells.24 Given the current interest in Crizotinib for treatment of GBM patients and high efficacy of IR-Criz in impairing GBM in vitro, we sought to determine whether IR-Crizotinib enhanced impediment of GBM tumor growth in vivo with an orthotopic GBM xenograft model.

Crizotinib and IR-Crizotinib Inhibit NIK Kinase Activity and Noncanonical NF-κB Signaling

To determine whether Crizotinib attenuated IKK phosphorylation and induction of noncanonical NF-κB RelB/p52 nuclear accumulation through the inhibition of NIK kinase activity, we performed in vitro kinase assays with purified recombinant NIK protein. Co-incubation with Crizotinib reduced NIK activity (IC50 = 2.851 μM), similar to the known NIK inhibitor B022 (Figure 1A and Figure S1B), suggesting that it inhibits NIK through direct interaction. The IR-conjugated form of Crizotinib was produced by binding to the meso-chloride of the heptamethine cyanine dye/infrared dye, IR-786 (IR-Crizotinib) as previously described.24 As we observed with Crizotinib, IR-Crizotinib also inhibited NIK kinase activity in vitro (IC50 = 3.381 μM), whereas the IR-786 dye alone had no effect (Figure 1A). Immunoblot analysis of patient-derived glioma cells (BT25) for proteins in the noncanonical NF-κB pathway demonstrated that treatment with Crizotinib or IR-Crizotinib inhibited nuclear translocation of p52, whose proteolytic processing from its p100 precursor in response to tumor necrosis factor-weak-like inducer of apoptosis (TWEAK) treatment is dependent on NIK. Though the IR-786 dye alone did not appear to inhibit the activity of purified recombinant NIK in vitro, some inhibition of p52 nuclear translocation was observed with treatment of the dye alone, possibly due to nonspecific effects of the IR dye in vivo. While both Crizotinib and IR-Crizotinib had minor effects on p65 activity in the canonical NF-κB pathway, observed by p-p65 reduction, we observed a greater impairment of nuclear accumulation of RelB and p52 (Figure 1B). Several studies have shown the endocytosis of HMCDs by cancer cells. Here, we demonstrate that human BT25 glioma cells coincubated with IR-786 or IR-Crizotinib also take up the compounds where both compounds localize within the cytoplasm of the cells (Figure 1C). Overall, these data demonstrate that the IR-786 dye and its conjugated form to Crizotinib are actively imported into patient-derived GBM cells and inhibit NIK activity.

Figure 1.

Figure 1

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Crizotinib and IR-Crizotinib inhibit NIK kinase activity and noncanonical NF-κB signaling. (A) In vitro ADP-glo kinase assay of purified NIK protein with IR-786, Crizotinib, or IR-786-conjugated Crizotinib. (B) Immunoblot of human glioma, brain tumor (BT25) cells nontreated (No) or treated with 10 ng/mL TWEAK, and then with 5 μM IR-786, Crizotinib, IR-Crizotinib, or DMSO. Cytoplasmic and nucleic fractions were probed for NF-κB proteins including noncanonical proteins (p100/p52 and RelB) and canonical proteins (p65 and p-p65) with cytoplasmic and nuclear controls GAPDH and Lamin A, respectively. The asterisk denotes a nonspecific band on the p100/p52 blot. (C) BT25 human glioma cells treated with a 5 μM IR-786 dye or IR-Crizotinib, both of which exhibit a far-red fluorescence (purple), and the nuclei were stained with Hoechst (blue). Pictures captured at 60× with 1.5× zoom.

Docking of Crizotinib and IR-786 Crizotinib to the Catalytic Domain of NIK

The inhibition of the NIK catalytic activity by Crizotinib and IR-Crizotinib in vitro strongly suggested their direct interaction. To determine whether direct interaction was feasible, we modeled potential molecular interactions between NIK and Crizotinib or IR-Crizotinib. Overlay of the crystal structures of the catalytic domain of NIK demonstrated conservation in structure between the N-lobe, C-lobe, and hinge area with the most structure flexibility observed in the activation loop (Figure S2A,B). After conservation of NIK kinase structures was confirmed, the crystal structure, PDB: 4G3D, was used to investigate the docking capabilities of Crizotinib and IR-Crizotinib (Figure 2A,B). Modeling of NIK with Crizotinib and IR-Crizotinib demonstrated that though Crizotinib is a known tyrosine kinase inhibitor, it is able to bind within the kinase domain of NIK in its unconjugated and conjugated form. Modeling predictions demonstrate possible ligand binding both in the back pocket (Figure 2C,D) and in the open cleft region (Figure 2E,F) between the two lobes of the kinase domain of NIK. Both with binding capabilities adjacent to the ATP pocket of NIK. IR-Crizotinib is nearly double the size of Crizotinib alone; nevertheless, it was predicted to have better virtual binding affinity than that of Crizotinib adjacent to the ATP pocket (Table S1). Predicted binding in the back pocket of the kinase domain, adjacent to the ATP pocket, demonstrates common receptor residues for Crizotinib binding of ARG 408, SER 410, PHE 411, GLU 413, VAL 414, ASP 515, ASP 519, LEU 522, CYS 533, ASP 534, and HIS 537. Possible NIK-Crizotinib binding sites consisted of key residues ARG 408, the catalytic base ASP 515, ASP 534 of the DFG, and the gatekeeper methionine 469 (Supp. Figure 2C,D). Predicted IR-Crizotinib binding demonstrated common receptor residues of SER 410, PHE 411, VAL 431, ARG 432, VAL 435, HIS 537, and GLY 558 (Supp. Figure 2E,F). Overall, these data demonstrate not only possible binding for Crizotinib and its dye-conjugated form to the kinase domain of NIK but also with probable binding adjacent to the ATP pocket and to binding of key catalytic residues, hindering ATP binding and NIK activity.

Crizotinib and IR-Crizotinib Impair Glioma Cell Growth and Invasion

Thus far, we have shown that Crizotinib and IR-Crizotinib are able to bind to NIK and inhibit its activity, impeding downstream noncanonical NF-κB activation while being able to accumulate in patient-derived glioma cells. Growth analysis of BT25 cells demonstrated significant impairment by Crizotinib and IR-Crizotinib, while the IR-786 dye alone slowed cell growth by about half compared to the DMSO-treated control (Figure 3A). Compound treatment in U87 glioma cells yielded results similar to those of BT25 cells in inhibition of the noncanonical NF-κB pathway (Figure S3A); however, while IR-Crizotinib completely impeded cell growth, Crizotinib decreased cell growth by about 2-fold and IR-786 treatment did not impair U87 growth (Supp. Figure 3B). Natural sphere formation of nonadherently growing glioma cells was also reduced by compound treatments, more so with Crizotinib and IR-Crizotinib than with the dye alone (Figure 3B and Figure S3C). In addition to limiting glioma growth, Crizotinib and IR-Crizotinib significantly reduced the glioma invasion potential in a collagen matrix. BT25 cells were treated with TWEAK to induce NIK/noncanonical NF-κB-specific invasion followed by compound treatment. While Crizotinib negated the TWEAK-induced invasion, BT25 Crizotinib-treated cells exhibited similar invasiveness to those of nontreated cells, treatment with IR-Crizotinib completely inhibited glioma cell invasion (Figure 3C). In U87 cells, Crizotinib and IR-Crizotinib also impeded TWEAK-induced invasion similar to untreated cells (Figure 3D). Further investigation of the collagen monolayer of the invading cells showed that the reduction in invasion for the IR-Crizotinib-treated cells was due to cell death (Supp. Figure 3D).

Figure 3.

Figure 3

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Crizotinib and IR-Crizotinib impair glioma cell growth and invasion. (A) MTS growth assay of BT25 cells treating DMSO, IR-786, Crizotinib, or IR-Crizotinib. Data represented by mean ± SD, one-way ANOVA. (B) Sphere assay of glioma and BT25 cells in culture treated with DMSO, IR-786, Crizotinib, or IR-Crizotinib. (C) Collagen invasion assay of BT25 cells either untreated or treated with TWEAK followed by sequential treatment with DMSO, IR-786, Crizotinib, or IR-Crizotinib. Data represented by mean ± SD, one-way ANOVA. (D) Images of collagen invading BT25 cells under different treatment conditions stained with Trypan blue.

Cyanine Dye and Conjugated Crizotinib Cross the Blood Brain Barrier and Localize to Intracranial Tumors

After establishing the inhibition on growth and invasion of glioma cells in vitro, we next sought to test the efficacy of these compounds in intracranial xenograft models. Both U87 and BT25 human glioma cells expressing RFP and luciferase reporters were injected intracranially into immunocompromised mice followed by IV drug treatment. Fluorescent tracing from the cyanine dye showed the dye alone and IR-Crizotinib was found stably throughout the mouse for several days, in conjunction with intracranial localiazation in vivo (Figure 4A) (Figure S4A). Clearance of the compounds began around day 3 after injection but remained at least 5 days post-injection while maintaining intracranial localization (Figure 4A,B). Post-mortem analysis of the brain and other tissues verified intracranial tumor localization of the compounds. Overlay between the RFP-tumor signal and far-red dye fluorescence is observed in the excised brain tissue, with the compound signal still observable a few days post IV injection. The fluorescent signal from the dye was also seen in other tissues, specifically the liver and kidney, most likely due to drug metabolism and excretion. Moreover, RFP-tumor signal demonstrated smaller intracranial tumors in IR-Crizotinib treated mice than control (Figure 4C and Figure S4B). These in vivo data demonstrated the credibility of HMCDs and HMCD-conjugated therapeutics to be able to cross the blood brain barrier, unlike many other drugs, and localize to tumor cells.

Figure 4.

Figure 4

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Cyanine dye and conjugated Crizotinib cross the blood brain barrier and localize to intracranial tumors. (A) In vivo fluorescence imaging of dye signal localization in intravenous injected mice with either the IR-786 dye alone or IR-786-conjugated Crizotinib in mice with intracranial tumors with U87 or BT25 glioma cells. (B) In vivo fluorescence imaging following clearance of the dye signal of the IR-786 dye or IR-786-conjugated Crizotinib 1 or 5 days after IV injection. (C) Ex vivo fluorescence imaging of isolated tissues (brain, heart, kidneys, liver, and spleen) from mice with BT25 intracranial tumors. Fluorescence shows either the RFP signal from the tumor cells or far-red dye fluorescence from IR-786 or IR-786 conjugated to Crizotinib. Imaging of the IR-786 (dye) injected mouse was 24 h post IV injection, and imaging of the IR-Crizotinib injected mouse was 72 h post IV injection.

IR-Crizotinib Has Improved Efficacy and Impairs InVivo GBM Growth

Assessment of intracranial xenografts of BT25 cells with weekly chemotherapeutic intervention demonstrated a reduction in tumor growth and improved survival after IR-Crizotinib administration. Tumor growth was tracked by monitoring stable luciferase expression from glioma cells, where we observed heterogeneity in tumor size within the treatment groups, but overall IR-Crizotinib-treated mice maintained smaller, less dense orthotopic tumors (Figure 5A and Figure S5A). Indeed, analysis of changes in glioma size verified a reduction in tumor growth in IR-Crizotinib-treated mice with a significant reduction in tumor size at week 4 (Figure 5B). In U87-treated tumors, IR-Crizotinib and IR-786 had similarly impaired tumor growth by week 5 to that of not being significantly different from mice with no tumors (Figure 5B,C). Chemotherapeutic treatment targeted tumor growth without having deleterious effects systemically as Crizotinib and IR-Crizotinib-treated mice maintained initial weights (Figure 5C and Figure S5D). In fact, the reduction in tumor growth of IR-Crizotinib-treated mice was accompanied by a significant increase in survival (Figure 5D). Similar results were observed in mice with intracranial U87 tumors, where IR-Crizotinib-treated mice maintained smaller, localized tumors (Figure 5E). Coinciding with the reduction in tumor size and growth, analysis of apoptosis among tumor samples by probing for cleaved poly-ADP-ribose polymerases (cPARP)30,31 revealed increasing degrees of apoptosis from Crizotinib to IR-Crizotinib-treated tumor samples. Corresponding evaluation of NF-κB2 (p100/p52), activated by NF-κB-inducing kinase, demonstrated reduced expression at the tumor interior and edge, matching with prior analysis of NIK/NF-κB2 inhibition (Figure 1) by Crizotinib and IR-Crizotinib (Figure 5E). Overall, these data demonstrate with improved tumor targeting therapeutics that there is a heightened level of tumor cell death, impeding overall tumor growth.

Figure 5.

Figure 5

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IR-Crizotinib has improved efficacy and impairs in vivo GBM growth (A) Weekly in vivo luminescence of intracranial GBM tumors of mice treated with weekly IV injections of vehicle, Crizotinib, or IR-786-conjugated Crizotinib. (B) Quantification of weekly average luminescence counts. Data represented as mean ± SEM, unpaired Student's t-test. (C) Average weekly weights of mice in each treatment group. Data represented as mean ± SEM, two-way ANOVA. (D) Survival curve of mice with intracranial tumors treated with vehicle, Crizotinib, or IR-786-conjugated Crizotinib. Data represented as n = 7 for vehicle, n = 5 for Crizotinib, and n = 5 for IR-Crizotinib, log-rank (Mantel–Cox) test, *p = 0.03. (E) Immunofluorescence of the BT25 intracranial tumor sections with DAPI (blue), cleaved PARP (cPARP) (red), and NF-κB2 (green).

Discussion

Improvement of chemotherapeutic intervention is needed not only for highly malignant cancers like Glioblastoma but also for enhancement of tumor targeting overall. Here, we show that an FDA-approved kinase inhibitor, Crizotinib, has the potential to bind and inhibit NIK, resulting in the inhibition of noncanonical NF-κB activation. Furthermore, we demonstrate that the conjugation of Crizotinib to a cyanine dye improves the compound’s efficacy in inhibiting glioma cell growth, adhesion, and invasion. This translates to glioma xenograft models, where not only was IR-Crizotinib able to pass the blood brain barrier and localize to the site of the intracranial tumor, but it also significantly slowed tumor growth. IR-Crizotinib treatment resulted in elevated noncanonical NF-κB pathway inhibition, higher tumor cell death, and overall improved survival of the treated animals.

Crizotinib is well known for inhibiting tyrosine kinases, specifically ALK, c-MET, and ROS1. Crizotinib is a type 1 inhibitor, where inhibition of kinase activity is due to being a competitor of ATP and preferentially binding within or adjacent to the ATP pocket.32 While kinases typically have preferences between tyrosine or serine/threonine phosphorylation, kinases share a similar structure with two distinct lobes and an active site in between.33,34 Owing to the mechanism of type 1 inhibitors, this generally makes them less specific than type 2 inhibitors.35,36 Although Crizotinib is a known tyrosine kinase inhibitor, and due to the conserved structure of kinases and the inhibitory nature of Crizotinib, this does not rule out the ability of this compound to bind to other types of kinases, as we demonstrate in this study. Currently, Crizotinib is approved in treating ALK+ nonsmall cell lung cancers.37 Other studies have investigated the use of these chemotherapeutics in treatment of GBM and have found promising results in the efficacy of Crizotinib against GBM. One study investigated the heterogeneity of Glioblastoma stem cells (GSCs) and found increased expression of ALK, ROS1, and MET in various cell lines, hypothesized to regulate cell stemness. Given the expression of these various proteins, Crizotinib was proposed as a therapeutic to target GSCs.38 Indeed, a later study demonstrated that Crizotinib alone, and in combination with other chemotherapeutics, inhibited neurosphere formation, a phenotype of stemness, in patient-derived GBM cells with high expression of c-MET and EGFR.39 In addition, Crizotinib has also been shown to reduce migration and invasion of GBM cells40 and is undergoing clinical trials for high-grade gliomas.41−43 Given the potential of this kinase inhibitor as a chemotherapeutic to treat GBM, improvement of efficacy is sought by conjugating this drug to a heptamethine cyanine dye to increase penetration of the blood brain barrier and tumor targeting.

A common problem with efficacy of chemotherapeutics in treating glioma is the ability of the compounds to cross the blood brain barrier (BBB).44,45 As with our data shown here, HMCDs have been shown to improve drug delivery with their permeability to the BBB.7,11,46 The increased hypoxic environment of tumors induces elevation of HIF1α and OATPs, which increases uptake of HMCDs.24,47 In the same regard, this mechanism of uptake allows for tumor specificity, as OATPs are more overexpressed on tumor cells, and limits nontumor toxicity. Overall, HMCDs are a viable method to deliver chemotherapeutics specifically to the tumor site without having to increase the permeability of the BBB, though further investigation is needed to elucidate the extent to which compounds remain conjugated to the dyes once at the tumor site.

Similar to our results, other studies have demonstrated that HMCDs alone have some bioactivity. We demonstrate that IR-786 inhibits noncanonical NF-κB activation with TWEAK treatment as well as glioma cell growth and sphere development (see Figures 1 and 2). Additionally, we show that in vivo, IR-786 also reduces U87 tumor growth (see Supp. Figure 5). Other studies have demonstrated similar HMCD bioeffects, with one similarly showing that IR-786 reduced patient-derived glioma growth in vitro with an EC50 of 1.7 μM.24 Cancer cell inhibition was also observed in cancer cell lines with IR-780, IR-783, and MHI-148 dyes in a dose-dependent manner.48−50

Temozolomide (TMZ) is the most standard treatment for GBM. As a lipophilic, alkylating agent, this compound has the ability to cross the blood brain barrier and cause DNA damage within proliferating cells. Though this enables some tumor specificity, as tumor cells are highly proliferative, this does not completely alleviate nontumor toxicity. Furthermore, unfortunately, many patients are either initially resistant to TMZ treatment or develop resistance later followed by a recurrence of the tumor.51−53 The use of HMCDs can improve tumor specificity while granting them the ability to be conjugated with a number of compounds for drug delivery. In addition to our data, Crizotinib has demonstrated efficacy in treating GBM either alone or in combinational therapies, including with TMZ, and underwent phase I clinical trials for GBM treatment.24,38−41 Overall, there is a need to improve upon standard practices with GBM treatment, and application of HMCDs and kinase inhibitors has many benefits as we and others have demonstrated.

Acknowledgments

We thank Dr. Lisa Perez, Director for Advanced Computing Enablement, at the Texas A&M High Performance Research Computing for guidance and assistance with protein–ligand docking analysis with Autodock Vina. BioRender software was used for the creation of the TOC abstract graphic.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.3c00496.

  • Supplemental data consists of NMR analysis of IR-786-conjugated Crizotinib, overlay of human crystal structures of NIK and corresponding sequence alignments, predicted docking structures and 2D map of binding residues between NIK and Crizotinib or IR-Crizotinib, and in vitro/in vivo data obtained with U87 glioma cells. Table S1 is of binding affinity and coordinates from Autodock Vina binding predictions of Crizotinib or IR-Crizotinib with human NIK (PDB: 4G3D) at the back pocket or open cleft region of the kinase domain (PDF)

Author Contributions

Conceptualization: K.M.P., K.B., and R.S.; data acquisition: K.M.P. and D.W.L.; writing—original draft preparation, K.M.P.; writing—review and editing, K.M.P., A.T., V.A.B., K.B., and R.S.; visualization: K.M.P., A.T., and R.S.; funding acquisition: R.S., K.B., and V.A.B.; supervision: R.S. All authors have read and agreed to the publication of the manuscript.

This work was funded by NIH-1R01NS082554, RP180875, and RP160842 to R.S. and NIH R35 GM131804 and award BE-0017 from the Robert A. Welch Foundation to V.A.B.

The authors declare no competing financial interest.

Supplementary Material

mp3c00496_si_001.pdf (1.1MB, pdf)

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Supplementary Materials

mp3c00496_si_001.pdf (1.1MB, pdf)

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