Enhanced Delivery of Aurora Kinase A Inhibitor Alisertib via Tumor-Targeting Immunoliposome Nanocomplex for Improved Treatment of Cancers Including Atypical Teratoid/Rhabdoid Tumor

Abstract

Purpose

This study explores a nanoparticle-based delivery system for alisertib, a selective aurora kinase A (AURKA) inhibitor. This nanocomplex is designed to enhance tumor-specific delivery. AURKA is overexpressed in various cancers, including atypical teratoid/rhabdoid tumors (ATRTs) and is associated with poor clinical outcomes. Alisertib has demonstrated promising efficacy in multiple preclinical studies, which supported its advancement into clinical trials across various cancer types. However, alisertib has not met key efficacy endpoints primarily due to its poor biodistribution to tumors and inadequate penetration of biological barriers, including the blood-brain barrier (BBB). Our study aims to address these issues.

Methods

To address the limitations of alisertib, we developed scL-ALI, a nanocomplex formulation designed to improve BBB penetration and to enhance tumor uptake of alisertib, to achieve higher effective intratumoral doses. Our new formulation of alisertib comprises a cationic liposome encapsulating the kinase inhibitor. This formulation incorporates an anti-transferrin receptor antibody fragment to enable efficient delivery into the central nervous system and facilitate tumor targeting. Here, we assessed the therapeutic efficacy of nano-formulated scL-ALI versus conventional alisertib utilizing patient-derived tumor cells and xenograft tumor models of various cancer types, including ATRT. Additionally, we investigated the potential synergistic effects of combining scL-ALI with radiation therapy, a standard treatment modality for brain tumors.

Results

Compared to conventional alisertib, scL-ALI more effectively inhibited AURKA activity and enhanced tumor cell killing of multiple cancer cell lines. In mouse models of ATRT, glioblastoma, and lung cancer, scL-ALI significantly improved tumor growth inhibition relative to conventional unencapsulared alisertib. Furthermore, when combined with radiation therapy, scL-ALI produced further improved antitumor effects, leading to extended survival in ATRT-bearing mice.

Conclusion

These findings highlight the superiority of scL-ALI in overcoming delivery barriers and enhancing therapeutic efficacy of alisertib, while possibly minimizing undesirable side effects in normal tissues. The scL-ALI nanocomplex shows enhanced therapeutic potential for treating AURKA-driven malignancies, particularly in combination with radiation therapy.

Introduction

Aurora kinase A (AURKA), a serine/threonine kinase, plays a crucial role in regulating mitotic cell division.¹ Aberrant expression of AURKA has been linked to mitotic defects that drive uncontrolled cancer cell proliferation and survival, and is frequently associated with poor prognosis across a range of human cancers.^2,3 Beyond mitosis, AURKA influences several non-mitotic pathways, contributing to enhanced cell invasion, metastasis, and resistance to chemotherapy and radiation therapy.⁴

A particularly promising application of AURKA inhibition is in the treatment of atypical teratoid/rhabdoid tumor (ATRT), a rare and highly malignant pediatric brain cancer. Despite intensive multimodal approaches, conventional treatment options have shown limited efficacy, resulting in poor patient outcomes. ATRT is characterized by the loss or mutation of SMARCB1 tumor suppressor gene that encodes a core subunit of the SWI/SNF chromatin remodeling complex. This complex regulates gene expression by modifying chromatin structure.⁵ SMARCB1 protein binds regulatory regions of the AURKA gene and directly represses its transcription.⁶ In ATRT, loss of SMARCB1 abrogates its transcriptional repression over several cell cycle and mitosis regulators, including AURKA.⁶ Compared with normal tissues or SMARCB1-intact controls, rhabdoid tumors and ATRTs with SMARCB1 loss display consistent overexpression and hyperactivation of AURKA demonstrated by immunohistochemistry and gene expression profiling^7,8 The absence of SMARCB1 function that leads to AURKA overexpression compromises the mitotic checkpoint and drives ATRT proliferation. Re-expression of SMARCB1 in SMARCB1-deficient rhabdoid tumor cells leads to a marked decrease in AURKA mRNA and protein levels, indicating that restoring SMARCB1 is sufficient to down-modulate AURKA expression.⁹ These molecular features position AURKA as an attractive therapeutic target in ATRT.^10,11

Alisertib (MLN8237) is a selective small-molecule inhibitor of AURKA that disrupts spindle assembly and chromosome segregation during mitosis. By arresting cancer cells at the G2/M phase, alisertib inhibits proliferation and promotes apoptosis.¹² This antitumor activity of alisertib has been demonstrated in multiple preclinical models, and it is currently being evaluated in clinical trials as both monotherapy and in combination with other therapeutic agents.^11,13–15 However, alisertib’s clinical translation has faced significant hurdles and the agent failed to achieve its primary efficacy endpoints in Phase II clinical trials for ATRT.¹⁶ Similarly, a recent Phase III trial in relapsed or refractory peripheral T-cell lymphoma was terminated early due to lack of clinical benefit.¹⁷ These setbacks underscore the need to optimize the current alisertib-based treatment strategies to improve its clinical utility.¹⁸ Despite these setbacks, alisertib has been granted orphan drug designation by the FDA for extensive-stage small cell lung cancer, reflecting its therapeutic potential.

The lack of tumor targeting in conventional alisertib regimens contributes to limited cellular uptake in tumor tissues and to off-target effects in normal tissues resulting in potential adverse events in patients. The challenges associated with alisertib are not unique and highlight a broader need for improved drug delivery strategies that enhance selective tumor uptake while minimizing systemic exposures. With the goal being an improved therapeutic index for alisertib, we developed new formulation based on a specialized nanoparticle-based delivery system involving a tumor-targeting immunoliposome nanocomplex (designated “scL” for single-chain Liposome). The current study involves head-to-head comparisons of the immunoliposome formulation encapsulating alisertib, referred to as “scL-ALI” with conventional, unencapsulated alisertib. The selection of the scL platform is supported by previous success and its ability to overcome physiological barriers. In previous work, we successfully encapsulated the poorly water-soluble drug temozolomide into this system (referred to as “scL-TMZ”), demonstrating enhanced antitumor efficacy and reduced nonspecific toxicity in an intracranial mouse model of glioblastoma.¹⁹ We have also previously shown that systemically administered scL nanocomplexes can traverse the blood-brain barrier (BBB) and effectively deliver encapsulated payloads to intracranial tumors.²⁰ In the current study, we have adapted the scL platform to carry alisertib, with the goal of achieving receptor-mediated transcytosis across the BBB and enhanced uptake by brain tumor cells.

The traversing of the BBB and tumor-targeting by scL nanocomplexes arises from an anti-transferrin receptor (TfR) single-chain antibody fragment (TfRscFv) located on their surface. This targeting moiety specifically binds to TfR, which is overexpressed on cancer cells, enabling receptor-mediated internalization of the nanocomplex.²¹ In addition, TfR is involved in receptor-mediated transcytosis, facilitating the transport across brain capillary endothelial cells that comprise the BBB.²² Leveraging this physiological pathway, scL nanocomplexes are capable of dual targeting ie, they are designed to bind to cerebral endothelial cells to cross the BBB and subsequently to deliver their therapeutic payloads to tumor cells within the brain parenchyma.

The primary objective of the current study is to overcome the physicochemical and pharmacological limitations of alisertib. Specifically, the study aims to: 1) enhance drug delivery by utilizing scL-ALI to increase tumor-specific accumulation of alisertib thereby achieving higher effective intratumoral drug concentrations; and 2) improve efficacy for more potent and selective AURKA inhibition within tumor cells.

In our comparative studies, we assessed the therapeutic efficacy of scL-ALI head-to-head versus conventional unencapsulated alisertib (referred to as “free ALI”) in preclinical models of ATRT and other cancers. Additionally, the study investigates the potential synergistic effects of combining scL-ALI with radiation therapy to determine if this combination can further improve therapeutic outcomes. Radiation therapy is a core, standard modality in the management of many primary and metastatic brain tumors.²³ In non-small cell lung cancer, the alisertib combined with radiation reduced tumor cell survival in vitro and enhanced tumor growth delay in xenografts compared with either modality alone, consistent with AURKA blockade being a radiosensitizer.²⁴ In other solid tumor models, ionizing radiation plus AURKA inhibition either via siRNA or a small‑molecule inhibitor increased apoptosis and loss of clonogenic survival.^25,26

Materials and Methods

Patient Data Analysis

AURKA expression across multiple human cancer types was analyzed using publicly available RNA-seq datasets from anonymized patients. This study uses publicly available, de-identified gene expression data and therefore is not considered human subjects research and is exempt from IRB review under US federal regulations—specifically, the Common Rule (45 CFR Part 46). Expression profiles in tumor versus corresponding normal tissues were assessed via the TCGA database accessed through the University of Alabama at Birmingham Cancer Data Analysis (UALCAN) portal.²⁷ To evaluate the prognostic significance of AURKA, Kaplan–Meier survival analyses were performed using the ICGC/TCGA Pan-Cancer datasets available through the cBioPortal for Cancer Genomics.²⁸ For ATRT-specific analysis, AURKA expression levels in tumor and normal brain tissues were examined using the Henriques_2013 dataset accessed through the GlioVis data portal. Additional analyses of glioblastoma patients were conducted using the CGGA and TCGA_GBMLGG datasets from the GlioVis to assess associations between AURKA expression, tumor stage, and patient survival. For lung cancer, the Okayama_2012 lung adenocarcinoma dataset was accessed via the Lung Cancer Explorer (LCE)²⁹ to evaluate the relationship between AURKA expression and patient survival outcomes.

Nanocomplex Preparation

Alisertib was encapsulated into cationic liposomes using the ethanol injection method as previously described.¹⁹ Alisertib (#S1133, Selleck Chemicals, Houston, TX, USA) was diluted in DMSO to 1 mM. DOTAP (1,2-dioleoyl-3-trimethylammonium propane; #890890E, Avanti Polar Lipids, Alabaster, AL, USA) and DOPE (dioleoyl phosphatidylethanolamine; #850725E, Avanti Polar Lipids) were prepared in ethanol at 0.25 mg/mL and pre-warmed to 65°C. The DOPE and DOTAP solutions were then combined at a 1:1 molar ratio, and the alisertib solution was added to the lipid mixture with continuous stirring. The alisertib-lipid mixture was injected into pre-warmed sterile, endotoxin-free water (65°C) under stirring. After cooling to room temperature, the final volume was adjusted with sterile endotoxin-free water. The TfR–targeted liposomal formulation (scL-ALI) was generated by mixing the alisertib-lipid solution with the TfRscFv solution, enabling non-covalent association of targeting moiety with the alisertib-encapsulated liposome. Hydrodynamic diameter and surface charge (zeta potential) were measured using a Zetasizer Nano ZS (Malvern Panalytical, Malvern, UK) across at least five independent batches. To calculate efficiency of encapsulation, unencapsulated free alisertib was separated from the alisertib-encapsulated immunoliposome (scL-ALI) by filtration through Vivaspin 500 (5kDa MWCO, Cytiva, Marlborough, MA, USA) according to the manufacturer’s instruction. Unencapsulated alisertib in the flow-through was quantified using NanoDrop UV-Vis spectrophotometer (Thermo Scientific, Rockford, IL, USA) at a wavelength of 314 nm.³⁰ The encapsulation efficiency of alisertib in the scL complex was 73.10 ± 1.40%. For all subsequent experiments, unencapsulated alisertib was not removed from the final scL-ALI formulation. For in vitro studies, the scL-ALI was diluted in serum-free medium immediately before use. For in vivo administration, nanocomplexes were formulated in 5% dextrose.

Cell Lines

The human ATRT cell lines BT-12 and CHLA-06 were obtained from the Children’s Oncology Group (Monrovia, CA, USA) and the American Type Culture Collection (ATCC, Manassas, VA, USA), respectively. The mouse lung carcinoma cell line LL2 and the mouse glioblastoma cell line GL261 were acquired from ATCC and the NCI Division of Cancer Treatment and Diagnosis Tumor Repository (Frederick, MD, USA), respectively. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂ in the following media: BT-12 cells in RPMI 1640 (Cytiva) supplemented with 15% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA); CHLA-06 cells in DMEM/F12 (Corning Cellgro, Manassas, VA, USA) supplemented with 20 ng/mL human FGF, 20 ng/mL human EGF, and B27 supplement (all from Thermo Fisher, Waltham, MA, USA); LL2 and GL261 cells in DMEM (Mediatech, Manassas, VA, USA) supplemented with 10% FBS. All cell lines were confirmed to be mycoplasma-free prior to experimentation.

Western Blot Analysis

For in vitro study, BT-12 cells were treated with scL-ALI at 100, 200, or 500 nM. Forty-eight hours after scL-ALI treatment, cells were harvested for Western blot analysis. For in vivo experiments, athymic mice bearing subcutaneous BT-12 tumors were treated with free ALI or scL-ALI (2.4 mg/kg). Twenty-four hours after treatment, tumors were harvested for Western blot analyses. Samples were lysed using radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors. Total protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instruction. Equal amounts of total cellular protein (20 μg per sample) were resolved on NuPAGE 4–12% Bis-Tris Midi gels (Thermo Fisher) and transferred to nitrocellulose membranes. Membranes were incubated with a rabbit monoclonal antibody against phospho-AURKA (Thr288) (#3079S, 1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), followed by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (#7074S, 1:10,000 dilution; Cell Signaling Technology). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for protein loading, detected using a rabbit polyclonal antibody (#2275-PC-100, 1:1000 dilution; Novus Bio, Centennial, CO, USA). Bands were visualized using Radiance Q chemiluminescent substrate (Azure Biosystems, Dublin, CA, USA) and the Amersham Imager 600 (GE HealthCare, Chicago, IL, USA). Densitometric analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cell Viability Assay

Human ATRT cell lines (BT-12 and CHLA-06), mouse glioblastoma cells (GL261), and mouse lung carcinoma cells (LL2) were seeded at a density of 3×10³ cells/well in 96-well plates. After 24 hours, cells were treated with serial dilutions (1–1000 nM) of scL-ALI, free ALI, empty nanocomplex (ie, scL, without payload), or vehicle control (DMSO), each in triplicate. Seventy-two hours post-treatment, cell viability was assessed using sodium 3′-[1-(phenylamino-carbonyl)-3, 4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonate (XTT) assay (Sigma-Aldrich) according to the manufacturer’s instructions. Half-maximal inhibitory concentration (IC₅₀) values (ie, the drug concentration resulting in 50% reduction in cell number) were determined by nonlinear regression analysis of the dose-response curves using SigmaPlot software (Systat Software, San Jose, CA, USA).

Flow Cytometry

Apoptosis was assessed using sub-G1 DNA content analysis and detection of apoptotic markers. For cell cycle analysis, harvested cells were washed with cold PBS and fixed in ice-cold 75% ethanol at least 30 minutes at 4°C. After fixation, cells were washed twice with PBS and stained with 25 μg/mL propidium iodide in the presence of RNase A (100 μg/mL) for 30 minutes at room temperature. DNA content was analyzed to identify the sub-G1 population using an LSRFortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). To further evaluate apoptosis, cells were fixed and permeabilized with Perm/Wash buffer (#421002, BioLegend, San Diego, CA, USA) for 30 min according to the manufacturer’s instructions followed by incubation with rabbit monoclonal antibodies against cleaved PARP (cPARP; #5625S, 1:200 dilution; Cell Signaling Technology) and cleaved caspase-3 (cCASP3; #9579S, 1:200 dilution; Cell Signaling Technology). Stained cells were analyzed on a FACSAria flow cytometer (BD Biosciences). Flow cytometry data were processed and analyzed using FCS Express 7 software (De Novo Software, Pasadena, CA, USA).

Clonogenic Survival Assay

BT-12 cells were seeded at 2×10⁵ cells/well in 6-well plates and treated with either scL-ALI or free ALI (200 nM) for 24 hours. Following treatment, cells were trypsinized, counted, and re-plated at a density of 200 cells/well in 6-well plates. Cells were further incubated for 13 days to allow colony formation, with the culture medium replaced every 3–4 days. Colonies were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and stained with Giemsa solution (Sigma-Aldrich) for visualization. Colony images were captured using a high-resolution digital scanner.

Quantitative Real-Time PCR

Total RNA was extracted from tumor tissues using the RNeasy Plus Mini Kit (QIAGEN, Germantown, MD, USA), and cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturers’ instructions. Quantitative real-time PCR (qRT-PCR) was performed using TaqMan Fast Advanced Master Mix (Thermo Fisher) on a StepOnePlus RT-PCR system (Life Technologies, Carlsbad, CA, USA). TaqMan gene expression assays (Thermo Fisher) and PrimePCR Probe Assays (Bio-Rad) were used to quantify the expression of the human genes associated to apoptosis, cell cycle and transcriptional regulation, immune regulation as summarized in Supplementary Table S1 and Supplementary Figure S1. Relative gene expression was calculated using the ΔΔCt method with GAPDH as the endogenous control. A heatmap of relative gene expression changes was generated using Morpheus (https://software.broadinstitute.org/morpheus/).

Animal Studies

All animal experiments were reviewed and approved by the Georgetown University Institutional Animal Care and Use Committee (IACUC) under protocol #2016-1157, and were performed in accordance with the Guide for the Care and Use of Laboratory Animals as required by the Office of Laboratory Animal Welfare (OLAW) and in accordance with PHS guidelines for the humane treatment of animals. All animal experiments were performed in the AAALAC-accredited, USDA-registered animal facility of Georgetown University.

For intracranial tumor experiments, six-week-old female athymic nude mice (Hsd: Athymic Nude-Foxn1nu, Envigo, Indianapolis, IN, USA) were inoculated stereotactically with BT-12 cells (0.25×10⁶/mouse) into the right hemisphere of the brain (2.0 mm posterior to lambda, 1.5 mm lateral to midline, and 3.0 mm depth from the skull). Following tumor establishment, mice received intraperitoneal injections of scL-ALI or free ALI (1.2 mg/kg, based on alisertib content) three times per week for seven weeks (Figure 1A). In prior in vivo studies, scL without a payload did not impact tumor growth. For this reason, we did not perform additional scL-only testing in the animal models reported here. In select experiments, localized cranial radiation (5 Gy) was administered 24 hours after the first scL-ALI injection, followed by two additional 5 Gy doses on the specified days for a total radiation dose of 15 Gy. Body weights were monitored regularly to assess potential systemic toxicity. Intracranial tumor response was evaluated by measuring tumor volumes over time using magnetic resonance imaging (MRI). Animals were imaged on a 7T Bruker horizontal spectrometer/imager (Bruker Biospin MRI GmbH, Karlsruhe, Germany) without contrast agents. Brain tumors were delineated from normal tissue based on inherent contrast differences, and regions of interest (ROIs) were manually traced using ImageJ software. Tumor volumes were calculated by multiplying the traced area by the MRI slice thickness. Survival was monitored until mice became moribund, at which point they were humanely euthanized.

Figure 1.

Treatment schedules for animals bearing (A) BT-12, (B) GL261, and (C) LL2 tumors.

For the subcutaneous ATRT model, six-week-old female athymic nude mice were inoculated in the flank with BT-12 cells (2×10⁶/mouse). Upon tumor establishment, mice received an intraperitoneal injection of scL-ALI or free ALI (2.4 mg/kg, based on alisertib content). Tumors were harvested at 24- and 48-hours post-treatment for gene expression analyses.

For the subcutaneous glioblastoma model, six-week-old female C57BL/6NHsd mice (Envigo) were inoculated in the flank with GL261 cells (1×10⁶/mouse). Mice bearing the established syngeneic tumors received intraperitoneal injections of scL-ALI or free ALI (1.2 mg/kg) three times per week for three weeks (Figure 1B). In certain cohorts, a single 5 Gy dose of ionizing radiation was administered 24 hours after the second scL-ALI injection.

For the subcutaneous lung cancer model, six-week-old female C57BL/6NHsd mice were inoculated subcutaneously with LL2 cells (0.5×10⁶/mouse). Mice with the established syngeneic tumors received intravenous injections of scL-ALI or free ALI (0.3 mg/kg) twice weekly for four weeks (Figure 1C). In radiation combination studies, a 5 Gy dose was administered 48 hours after the second injection, followed by a second 5 Gy dose one week later for a total radiation dose of 10 Gy. Throughout the treatment period, body weights were monitored to assess systemic toxicity. Tumor growth was evaluated by semi-weekly caliper measurements, and tumor volumes were calculated using the formula L×W×H (in mm³), where L = length, W = width, and H = height.

Statistical Analysis

All data are presented as means ± standard deviations. Statistical analyses were performed using one-way ANOVA for group comparisons. When comparing three or more groups, Tukey’s post-hoc test was applied to adjust for multiple comparisons. Bonferroni correction was used in cases where pairwise comparisons were conducted across multiple conditions. A two-tailed p-value of <0.05 was considered statistically significant. Survival data were analyzed using the Kaplan–Meier method, and differences between survival curves were evaluated using the Log rank test. All graphs and statistical analyses were conducted using SigmaPlot software.

Results

Increased AURKA Associated with Poor Survival in Human Cancers

To assess AURKA expression across various types of human cancers, RNA-seq data from the TCGA database were analyzed through the UALCAN portal.²⁷ Compared to corresponding normal tissues, AURKA expression was significantly elevated in the majority of cancer types, including brain and lung cancers, with increased levels observed in 21 out of 24 analyzed cancer types (p<0.001; Table 1). To evaluate the prognostic relevance of AURKA expression, Kaplan–Meier survival analyses were conducted using the ICGC/TCGA pan-cancer datasets accessed through the cBioPortal.²⁸ The results demonstrated a strong association between high AURKA expression and poor patient outcomes; patients with elevated AURKA levels (AURKA HIGH) exhibited significantly shorter overall survival compared to those with lower expression (AURKA LOW), with a log-rank p<0.001 (Figure 2A). These observations suggest as might be expected that therapeutic strategies targeting AURKA may be more relevant in tumor types with elevated AURKA expression.

Table 1.

AURKA Expression in Human Cancers

Cancer	Abbreviation	Normal		Primary Tumor		Statistical Significance (p)
		N	Median (TPM)	N	Median (TPM)
Bladder urothelial carcinoma	BLCA	19	1.73	408	4.63	1.62E-12
Breast invasive carcinoma	BRCA	114	1.63	1097	4.14	<1E-12
Cervical squamous cell carcinoma	CESC	3	1.11	305	29.55	1.62E-12
Cholangiocarcinoma	CHOL	9	0.72	36	8.36	5.00E-10
Colon adenocarcinoma	COAD	41	11.83	286	36.81	1.62E-12
Esophageal carcinoma	ESCA	11	2.89	184	30.97	1.62E-12
Glioblastoma multiforme	GBM	5	1.32	156	12.64	<1E-12
Head and Neck squamous cell carcinoma	HNSC	44	5.11	520	23.45	<1E-12
Kidney chromophobe	KICH	25	1.97	67	7.13	2.92E-13
Kidney renal clear cell carcinoma	KIRC	72	2.24	533	3.61	7.66E-10
Kidney renal papillary cell carcinoma	KIRP	32	2.05	290	3.55	<1E-12
Liver hepatocellular carcinoma	LIHC	50	0.84	371	8.61	<1E-12
Lung adenocarcinoma	LUAD	59	0.23	515	13.92	1.62E-12
Lung squamous cell carcinoma	LUSC	52	2.27	503	24.45	<1E-12
Pancreatic adenocarcinoma	PAAD	4	2.83	178	7.11	0.241
Prostate adenocarcinoma	PRAD	52	1.16	497	2.03	4.52E-12
Pheochromocytoma and Paraganglioma	PCPG	3	5.62	179	2.73	0.816
Rectum adenocarcinoma	READ	10	10.42	166	37.68	3.83E-12
Sarcoma	SARC	2	2.46	260	16.51	1.62E-12
Skin cutaneous melanoma	SKCM	1	19.28	104	17.60	N/A
Thyroid Carcinoma	THCA	59	1.94	505	1.58	0.003
Thymoma	THYM	2	17.36	120	14.55	0.806
Stomach adenocarcinoma	STAD	34	5.35	415	29.17	1.62E-12
Uterine corpus endometrial carcinoma	UCEC	35	1.25	546	14.55	1.62E-12

Note: Comparison of AURKA expression across various tumor types and their corresponding normal tissues.

Abbreviation: TPM, transcript per million.

Figure 2.

AURKA expression in human cancers. (A) Kaplan–Meier survival analysis of AURKA expression in cancer patients. (B) AURKA expression in ATRT compared to normal brain tissue. **p<0.001. (C and D) Survival analysis based on AURKA expression in glioma patients from the CGGA and TCGA databases, respectively. (E and F) AURKA expression in glioma stratified by tumor stages from CGGA and TCGA databases, respectively. (G) AURKA expression in lung cancer versus normal lung tissue. (H) Survival analysis of AURKA expression in lung cancer patients.

ATRT, a highly aggressive pediatric brain tumor, was found to exhibit markedly elevated AURKA expression. Analysis of publicly available gene expression datasets³¹ via the GlioVis portal confirmed that AURKA is significantly overexpressed in ATRT compared to normal brain tissues from both fetuses and adults (p<0.001; Figure 2B). These findings highlight the therapeutic potential of inhibiting/downmodulating AURKA in ATRT. Mechanistically, it has been reported that the loss of SMARCB1, a tumor suppressor and hallmark genetic alteration in ATRT, leads to AURKA overexpression.⁶ Furthermore, SMARCB1 restoration considerably downregulated AURKA mRNA in intracranial BT-12 tumors,³² supporting AURKA’s relevance as a therapeutic target. Additionally, analyses of glioma patient data from the CGGA and TCGA databases, also accessed through the GlioVis, demonstrated that higher AURKA expression is associated with worse overall survival (Figure 2C and D), and correlates with more advanced tumor stages (Figure 2E and F). Similarly, microarray analysis of lung adenocarcinoma datasets using the LCE^29,33 revealed elevated AURKA levels in tumor tissues relative to normal lung samples (Figure 2G). Correspondingly, higher AURKA expression was linked to worse survival outcomes in lung adenocarcinoma patients (Figure 2H). Collectively, these results suggest that AURKA may be a key driver of tumor progression across multiple cancer types, including ATRT, glioma, and lung adenocarcinoma, and therefore represents a promising therapeutic target for the treatment of cancers with abnormally upregulated AURKA.

Enhanced AURKA Inhibition by scL-ALI Nanocomplex

The therapeutic potential of cancer drugs including alisertib has been limited by several pharmacokinetic and delivery challenges, including its short half-life in circulation, poor penetration of biological barriers (eg, BBB), and inefficient uptake by tumor cells. To address these limitations, we employed a tumor-targeted delivery system (referred to as “scL”) to encapsulate alisertib, aiming to enhance its bioavailability and cellular uptake. This formulation, termed “scL-ALI”, yielded a nanosized complex with an average diameter of 135.9 ± 22.2 nm (Figure 3A), a negative surface charge (zeta potential) of −7.5 ± 0.8 mV (Figure 3B), and a relatively uniform size distribution, as indicated by a low polydispersity index (PDI) of 0.148 ± 0.032. The encapsulation efficiency of alisertib in the scL complex was 73.10 ± 1.40%.

Figure 3.

Characterization of the scL-ALI nanocomplex. (A) Representative size distribution profile. (B) Representative zeta potential (surface charge) measurement.

We compared the efficacy of the novel scL-ALI nanocomplex to conventional free ALI in inhibiting AURKA activity. Since phosphorylation at threonine 288 (Thr288) activates AURKA by inducing a conformational change that facilitates substrate binding,³⁴ the phosphorylation level of AURKA (P-AURKA) was used as a surrogate marker of kinase activity. In vitro treatment of BT-12 cells with increasing concentrations of scL-ALI resulted in a dose-dependent decrease in P-AURKA levels (Figure 4A and B), indicating effective intracellular delivery and functional inhibition of AURKA by encapsulated alisertib. In a mouse model of subcutaneous BT-12 tumors, scL-ALI treatment led to a significant reduction in P-AURKA (~43%, p<0.05) compared to untreated controls, while free ALI at the same dosage produced no measurable effect (Figure 4C and D). These results highlight the enhanced efficacy of the scL-ALI formulation in delivering alisertib to tumor cells and inhibiting AURKA activity compared to free ALI.

Figure 4.

Inhibition of AURKA phosphorylation by scL-ALI. (A) Western blot and (B) quantification of phosphorylated AURKA (P-AURKA) of BT-12 cells 48 hours after treatment with scL-ALI at 100, 200, or 500 nM in vitro. (C) Western blot and (D) quantification of P-AURKA levels in BT-12 tumors 24 hours after treatment with free ALI or scL-ALI (2.4 mg/kg). *p<0.05, **p<0.001.

Improved Tumor Cell Growth Suppression by scL-ALI

To evaluate the antitumor efficacy of scL-ALI, we treated various tumor cell types, including human pediatric brain cancer cells (BT-12, CHLA-06), murine brain cancer cells (GL261), and murine lung cancer cells (LL2) with either free ALI or scL-ALI at concentrations ranging from 0 to 1000 nM. Across all cell types and both species, scL-ALI induced dose-dependent cytotoxicity (Figure 5A–D) and demonstrated markedly lower IC₅₀ values compared to free ALI (Table 2), indicating enhanced intracellular delivery and potency. Importantly, control treatments with either empty scL nanocomplexes (lacking alisertib) or the DMSO vehicle showed no cytotoxic effects (Figure 5B and D), confirming that the antitumor activity observed was specifically attributable to alisertib delivered via the scL nanocomplex and not to some non-specific effect attributable to the delivery system.

Figure 5.

Enhanced inhibition of tumor cell growth by scL-ALI. XTT assays 72 hours after transfection with scL-ALI, free ALI, DMSO, or empty nanocomplex (scL) in (A) BT-12, (B) CHLA-06, (C) GL261, and (D) LL2 cells. DMSO and scL lacking a payload served as control groups. Statistical significance between free ALI and scL-ALI (*p<0.05, **p<0.001).

Table 2.

Summary of IC₅₀ Values in Figure 5

Cell line	Tumor Type	Treatment	IC50 (nM)
BT-12	Human pediatric brain cancer (ATRT)	Free ALI	838.41
		scL-ALI	260.40
CHLA-06	Human pediatric brain cancer (ATRT)	Free ALI	493.84
		scL-ALI	127.21
		scL	>1000
GL261	Murine brain cancer (GBM)	Free ALI	>1000
		scL-ALI	581.28
LL2	Murine non-small cell lung cancer	Free ALI	340.33
		scL-ALI	119.27
		DMSO	>1000

Increased Tumor Cell Killing by scL-ALI

To elucidate the mechanisms underlying the enhanced antitumor activity of scL-ALI, we examined cell cycle progression and apoptosis in BT-12 cells treated with either scL-ALI or free ALI at 200 nM. Flow cytometry analysis revealed that scL-ALI induced substantial arrest at G2/M Phase 24 hours after treatment, followed by an accumulation of aneuploid cells at 48 hours (Figure 6A and B). In contrast, free ALI treatment at the same dose did not elicit G2/M arrest or aneuploidy. Furthermore, scL-ALI markedly increased the sub-G1 apoptotic population (Figure 6C) and the apoptosis markers cPARP and cCASP3 (Figure 6D), while free ALI had no measurable effects relative to untreated controls. These findings validate the superior pro-apoptotic and cell cycle-disrupting activity of scL-ALI compared to free ALI. A colony formation assay further corroborated these results, showing a significantly greater reduction in colony-forming ability with scL-ALI than with free ALI (86.0% vs 42.8% reduction compared to untreated cells) (p<0.05; Figure 6E). Collectively, these results indicate that enhanced intracellular delivery of alisertib via the scL-ALI nanocomplex improves AURKA inhibition to disrupt cell cycle progression, promote apoptosis, and reduce cell proliferation in ATRT cells.

Figure 6.

Enhanced cell cycle arrest and apoptosis by scL-ALI in BT-12 cells. (A) Cell cycle distribution at 24, 48, and 72 hours after transfection with 200 nM scL-ALI or free ALI. Quantification of (B) hyperploid (>4N) cells and (C) sub-G1 (<2N) apoptotic cells. (D) Apoptosis detection via cPARP and cCASP3 staining in BT-12 cells after transfection with 200 nM scL-ALI or free ALI. (E) Representative images and quantification from colony formation assays performed 14 days after transfection. *p<0.05, **p<0.001.

Gene Modulation by scL-ALI in ATRT Xenografts

To investigate the molecular mechanisms underlying the enhanced antitumor activity of scL-ALI, we analyzed gene expression in BT-12 tumors harvested from athymic mice treated with either free ALI or scL-ALI nanocomplex (2.4 mg/kg). qRT-PCR analyses revealed that both treatments modulated the expression of several genes (Figure 7A), but scL-ALI induced more pronounced and distinct transcriptome changes. Specifically, scL-ALI upregulated multiple pro-apoptotic genes, including BAX, FAS, NOXA1, PUMA, and TRAIL, while downregulating the anti-apoptotic gene BCL2 (Figure 7B). In contrast, free ALI did not notably alter the expression of these genes at the tested dose. Furthermore, only scL-ALI significantly downregulated key genes implicated in ATRT progression, including LIN28B, FOXM1, and PLK1 (p<0.05; Figure 7C). LIN28B, an RNA-binding protein essential for maintaining embryonic stem cell properties, is overexpressed in primitive neuroectodermal tumors and ATRT,³⁵ and its knockdown has been shown to decrease ATRT proliferation and viability.^35,36 Similarly, FOXM1 (a proliferation-associated transcription factor) and PLK1 (a mitotic kinase) are often elevated in ATRT and their inhibition has been shown to produce antitumor effects.^37,38 Notably, scL-ALI also significantly reduced the expression of CCNB1, a cell cycle regulator essential for AURKA-mediated progression of renal cell carcinoma³⁹ (p<0.05; Figure 7D). In terms of tumor-suppressive responses, both free ALI and scL-ALI significantly upregulated CDKN1C, a cyclin-dependent kinase inhibitor that suppresses ATRT growth (p<0.05).⁴⁰ However, only scL-ALI markedly increased the expression of additional tumor suppressor genes, including p53 and p21 (Figure 7E). Moreover, scL-ALI selectively upregulated FBXW7, a p53-dependent E3 ligase that promotes AURKA degradation,^41,42 and GADD45A, a stress-responsive gene that directly inhibits AURKA kinase activity⁴³ (Figure 7F). These molecular changes support the enhanced intracellular accumulation and functional impact of alisertib delivered via the scL nanocomplex. Finally, both free ALI and scL-ALI modulated genes involved in antitumor immune responses. Specifically, both treatments significantly altered the expression of genes involved in antigen presentation (HLA-A, TAP1, TAP2; p<0.05; Figure 7G), NK cell activation (MICA, MICB, ULBP2; p<0.05; Figure 7H), and T cell immune checkpoint regulation (PD-L1; p<0.05; Figure 7I), with no significant differences observed between the two treatment groups. The precise mechanisms by which alisertib influences immune responses remain incompletely understood and needs further study. Collectively, these findings provide mechanistic insight into the superior antitumor efficacy of scL-ALI and highlight key gene expression changes that underpin its enhanced therapeutic activity compared to free ALI.

Figure 7.

Gene expression changes in BT-12 tumors following free ALI or scL-ALI (2.4 mg/kg) treatments. (A) Heatmap showing fold-changes in transcript levels relative to untreated controls 24 and 48 hours after treatment. Expression of genes related to (B) apoptosis, (C) ATRT progression, (D) cell cycle regulation, (E) p53 signaling, (F) AURKA signaling, (G) antigen presentation, (H) NK cell activation, and (I) T cell inhibition. *p<0.05, **p<0.001.

Enhanced Tumor Growth Suppression by scL-ALI in ATRT Xenografts

We evaluated the antitumor efficacy of systemically administered free ALI and scL-ALI in an intracranial ATRT mouse model. Athymic mice bearing MRI-confirmed BT-12 tumors were randomized to receive either free ALI or scL-ALI (1.2 mg/kg), as outlined in Figure 1A. Consistent with in vitro findings, scL-ALI treatment resulted in notable tumor growth inhibition, with mean tumor volumes of 47.5 ± 3.3 mm³ on day 77, compared to 84.4 ± 8.3 mm³ in untreated controls (Figure 8A). In contrast, mice treated with free ALI exhibited only modest tumor growth inhibition (74.4 ± 16.8 mm³ on day 77) (Figure 8B). Quantitative analysis revealed that scL-ALI achieved 56.3% and 45.3% tumor growth inhibition on days 42 and 77, respectively (p<0.05; Figure 8C and D), whereas free ALI achieved only modest inhibition of 15.9% and 12.1% at the same time points. In prior in vivo studies, scL without a payload did not impact tumor growth. For this reason, we did not perform additional scL-only testing in the animal models reported here. These results demonstrate the delivery enhancement across the BBB together with tumor targeting via scL nanocomplex leads to enhanced antitumor efficacy in an intracranial ATRT model.

Figure 8.

Enhanced tumor inhibition by scL-ALI plus radiation treatment in an intracranial ATRT model. Mice bearing intracranial BT-12 tumors were randomized to receive free ALI or scL-ALI (1.2 mg/kg), with or without radiation therapy (n = 5–6/group). (A) Representative MRI images at days 14, 42, and 77 (tumors outlined in yellow). (B) MRI-based measurement of brain tumor volume. Tumor inhibition quantified by percent volume reduction at (C) day 42 and (D) day 77. (E) Relative body weight plotted over time. (F) Kaplan–Meier survival curves. Gray boxes indicate the duration of treatments in (B), (E), and (F). *p<0.05, **p<0.001.

We investigated whether scL-ALI could potentiate the therapeutic effects of radiation therapy, an established treatment for pediatric brain tumors associated with improved survival. Athymic mice bearing intracranial BT-12 xenografts were treated with whole-brain radiation (5 Gy/week for 3 weeks), either alone or in combination with free ALI or scL-ALI (Figure 1A). Radiation therapy alone reduced tumor growth, with mean tumor volumes of 47.1 ± 13.3 mm³ on day 77, comparable to those observed with scL-ALI monotherapy (Figure 8B). Notably, combining radiation with scL-ALI produced an additive antitumor efficacy, considerably reducing tumor volume to 17.8 ± 8.0 mm³ on day 77. In contrast, the combination of radiation and free ALI provided only minimal benefit over radiation alone, yielding tumor volumes of 37.1 ± 5.8 mm³ (Figure 8B). Quantitatively, radiation plus scL-ALI resulted in 90.5% and 80.6% tumor growth inhibition on days 42 and 77, respectively (p<0.05; Figure 8C and D), whereas the radiation plus free ALI group showed only more modest enhancement (69.0% and 57.9%) over radiation alone (64.0% and 45.8% on days 42 and 77, respectively). These findings demonstrate that scL-ALI not only outperforms free ALI as a monotherapy but also enhances the efficacy of radiation therapy when used in a combination treatment regimen. Consistent with tumor growth inhibition data, mice receiving the combination of scL-ALI and radiation maintained the most stable body weight throughout treatment, whereas earlier weight loss was observed in the other treatment groups (Figure 8E).

Despite the observed antitumor effects, neither scL-ALI nor free ALI monotherapy significantly extended survival in tumor-bearing mice compared to untreated controls (Figure 8F). All untreated mice succumbed to disease by day 84, with a median survival of 73 days. Mice treated with scL-ALI or free ALI alone had similar outcomes, with median survival times of 70 and 73 days, respectively, and no animals surviving beyond day 88. In contrast, combining scL-ALI with radiation therapy significantly improved survival (p<0.05), with a maximum survival of 139 days and a median survival of 91 days. Mice receiving radiation alone or in combination with free ALI had shorter median survival times of 81 and 77 days, respectively. Collectively, these findings demonstrate that scL-ALI significantly enhances the therapeutic benefit of radiation therapy in this intracranial ATRT model. It is important to note that these results are based on the treatment regimen described in Figure 1A; further optimization of dosing and scheduling could potentially yield even greater tumor suppression and survival benefit.

Improved Antitumor Efficacy of scL-ALI in a Glioblastoma Model

To evaluate whether the antitumor efficacy of the scL-ALI nanocomplex extends beyond pediatric brain tumors, we tested its activity in a murine glioblastoma model. C57BL/6 mice bearing subcutaneous syngeneic GL261 tumors were randomized to receive treatments as outlined in Figure 1B. Consistent with results from the BT-12 tumor model, scL-ALI notably suppressed tumor growth compared to untreated controls (618 ± 198 mm³ vs 883 ± 285 mm³ on day 39; Figure 9A). In contrast, free ALI treatment showed no inhibitory effect on tumor growth (882 ± 240 mm³ on day 39). Radiation therapy alone substantially inhibited GL261 tumor growth (237 ± 177 mm³ on day 39), and its efficacy was further enhanced by combination with scL-ALI, leading to complete tumor regression by day 30. Conversely, the addition of free ALI to radiation provided only a modest improvement over radiation alone (183 ± 120 mm³ vs 237 ± 177 mm³; Figure 9B). Importantly, body weights of scL-ALI–treated mice remained comparable to untreated controls, suggesting low systemic toxicity (Figure 9C). These findings suggest that scL-ALI possesses preliminary antitumor potential for brain tumors, particularly when used in combination with radiation therapy, a well-established treatment modality with demonstrated survival benefits for patients with brain tumors.

Figure 9.

Enhanced tumor inhibition by scL-ALI plus radiation in a mouse syngeneic model of glioblastoma. C57BL/6 mice with subcutaneous GL261 tumors were randomized to receive free ALI or scL-ALI (1.2 mg/kg), with or without radiation therapy (n = 6–16/group). (A) Tumor volume. (B) Tumor inhibition quantified by percent volume reduction at day 39. (C) Body weight. Gray boxes indicate the duration of treatments. *p<0.05, **p<0.001.

Improved Antitumor Efficacy of scL-ALI in a Lung Cancer Model

To determine whether the antitumor efficacy of the scL-ALI nanocomplex extends to other cancer types, we examined its effects in a murine syngeneic lung cancer model. Notably, elevated AURKA expression in lung tumors is associated with worse patient survival (Figure 2H). To test this, C57BL/6 mice bearing subcutaneous LL2 tumors were randomized to receive systemically administered free ALI or scL-ALI (0.3 mg/kg), with or without radiation therapy, as outlined in Figure 1C. Consistent with findings in the ATRT and glioblastoma models, scL-ALI treatment notably suppressed tumor growth compared to untreated controls (1989 ± 484 mm³ vs 3534 ± 492 mm³ on day 31; Figure 10A). Free ALI, by contrast, showed only modest tumor inhibition (2826 ± 90 mm³). Radiation therapy alone was effective in reducing tumor burden (1222 ± 18 mm³ on day 31), and this effect was further enhanced when combined with scL-ALI (616 ± 146 mm³). However, combining radiation with free ALI did not substantially improve outcomes over radiation alone (1043 ± 254 mm³; Figure 10B). Importantly, scL-ALI treatment did not induce any drastic body weight loss, suggesting low systemic toxicity (Figure 10C). These results indicate that scL-ALI enhances intracellular delivery and antitumor activity of alisertib across diverse tumor models, including lung cancer. Furthermore, its combination with radiation therapy yields superior therapeutic benefit in our mouse models, underscoring the potential of scL-ALI as a versatile therapeutic option for AURKA-driven malignancies.

Figure 10.

Enhanced tumor inhibition by scL-ALI plus radiation in a mouse syngeneic model of lung cancer. C57BL/6 mice with subcutaneous LL2 tumors were randomized to receive free ALI or scL-ALI (0.3 mg/kg), with or without radiation therapy (n = 3–4/group). (A) Tumor volume. (B) Tumor inhibition quantified by percent volume reduction at day 31. (C) Body weight. Gray boxes indicate the duration of treatments. *p<0.05, **p<0.001.

Discussion

Brain cancer remains one of the leading causes of cancer-related deaths in young children, with ATRT representing one of the most aggressive and lethal forms of CNS tumors. ATRT predominantly affects children under the age of three, with a median age at diagnosis ranging from 16 to 30 months.⁴⁴ Despite current treatment approaches, including surgical resection, intensive chemotherapy, and radiation therapy, ATRT progression is often rapid and difficult to control. Furthermore, the nonspecific cytotoxicity of conventional therapies poses a significant risk of long-term neurological damage in the developing brains of pediatric patients,⁴⁵ further limiting treatment options. Tragically, the majority of children diagnosed with ATRT have a median survival of less than one year, highlighting the urgent need for more effective therapeutic strategies.

Alisertib, a potent small-molecule inhibitor of AURKA, represents a promising therapeutic agent currently under investigation. An ongoing phase II clinical trial evaluating alisertib as monotherapy for recurrent ATRT (NCT02114229) has demonstrated acceptable tolerability; however, an early report indicated that the study did not meet its predetermined efficacy endpoint.¹⁶

A major challenge in treating brain cancer is the presence of the BBB, a highly selective diffusion barrier that protects the CNS from pathogens, toxins, and most circulating molecules in blood. The BBB restricts the entry of nearly 100% of large-molecule neurotherapeutics and over 98% of small-molecule drugs, presenting a formidable obstacle to effective CNS drug delivery.⁴⁶ This limitation is particularly relevant for alisertib, as demonstrated in a recent biodistribution study in mice, where brain (ie, the critical site for brain tumor treatment) exposure to intravenously administered alisertib was extremely poor.⁴⁷ In contrast, high drug accumulation was observed in the bone marrow, a site associated with myelosuppression-related toxicity, underscoring the potential for systemic adverse effects.⁴⁷ Further studies have highlighted additional limitations of systemic alisertib delivery. In a subcutaneous patient-derived xenograft (PDX) model of diffuse midline glioma (DMG), a common pediatric brain cancer, oral alisertib significantly reduced tumor burden compared to controls.⁴⁸ However, this effect was not replicated in intracranial PDX models, where the drug failed to impact tumor growth or improve survival presumably reflecting poor access to the CNS. Similarly, in a genetically engineered mouse model (GEMM) of brainstem high-grade glioma, alisertib treatment did not reduce tumor burden or prolong survival.⁴⁸ Collectively, these findings illustrate the substantial difficulty in achieving therapeutically relevant concentrations of alisertib in brain tumors through conventional systemic administrations. These underscore the need for delivery strategies that can enhance BBB penetration and improve tumor-specific accumulation to maximize therapeutic efficacy while minimizing off-target toxicity.

scL-ALI is engineered to address these limitations by crossing the BBB via TfR-mediated transcytosis and subsequently entering tumor cells through TfR-mediated endocytosis. Our previous studies have consistently shown that this scL-based targeted delivery platform markedly improves drug delivery efficiency and tumor specificity compared to non-targeted approaches.^21,49 Owing to its dual targeting capabilities (ie, binding to both BBB endothelial cells and tumor cells), systemic administration of scL-ALI demonstrated the enhanced antitumor efficacy of alisertib in vivo. In animal models, scL-ALI treatment led to substantial tumor growth inhibition and increased cancer cell apoptosis. This enhanced efficacy is likely due to the nanocomplex’s ability to actively enter tumor cells via TfRs and deliver its therapeutic payload intracellularly, resulting in more effective AURKA inhibition and superior tumor suppression compared to conventional alisertib. A limitation of this study is the lack of direct measurements of in vivo biodistribution, BBB crossing efficiency, and drug release kinetics for the scL-ALI nanocomplex, and further validation will be required to assess scL-ALI using quantitative biodistribution studies.

The antitumor activity of alisertib appears to be mediated through modulation of multiple pathways, including those directly affecting tumor cell behavior—such as induction of apoptosis, regulation of the cell cycle, and inhibition of cancer progression—as well as pathways that influence the immune microenvironment. Notably, both scL-ALI and free ALI treatments were found to upregulate the expression of natural killer (NK) cell activation ligands, genes involved in antigen presentation, and T cell inhibitory molecules. Although the precise mechanisms by which AURKA influences immune responses remain incompletely understood, emerging evidence suggests a role for AURKA in immune regulation. For instance, a recent study reported that alisertib attenuates immunosuppression mediated by myeloid-derived suppressor cells, thereby reducing breast cancer progression.⁵⁰ Conversely, AURKA inhibition has also been associated with increased PD-L1 expression and suppression of T cell activity in preclinical models of breast and colorectal cancers.^51,52 These findings underscore the complexity of AURKA’s role in tumor immunity and suggest that combining alisertib with immune checkpoint inhibitors, such as PD-1/PD-L1 blockers, may offer enhanced therapeutic benefit.

AURKA overexpression has also been implicated in resistance to conventional cancer therapies, including both radiation and chemotherapy.^53,54 Although radiation therapy remains a mainstay in the treatment of brain tumors, its use in young children is often limited due to the risk of long-term adverse neurocognitive effects. As a result, radiation therapy is typically delayed, dose-reduced, or avoided in pediatric patients to minimize harm to the developing brain. Combining radiation therapy with novel agents like scL-ALI offers a promising strategy to enhance therapeutic efficacy while potentially reducing the radiation dose required and associated toxicities. In our study, co-treatment with scL-ALI significantly augmented the therapeutic effects of radiation in an ATRT model, resulting in enhanced tumor growth inhibition and a marked extension in survival compared to radiation therapy alone. These findings suggest that scL-ALI may sensitize tumor cells to radiation, thereby improving treatment outcomes. However, further investigation is warranted to elucidate the underlying mechanisms and to determine the optimal dosing and scheduling parameters for combining scL-ALI with radiation therapy in future clinical applications.

Given that AURKA overexpression is characteristic of various malignancies, including glioblastoma and lung cancer, and AURKA appears to play a central role in their pathogeneses, the therapeutic potential of scL-ALI likely extends beyond pediatric brain tumors. In our study, scL-ALI demonstrated activity in selected models (brain and lung cancer), suggesting its preliminary potential as a tumor-targeted nanomedicine pending further evaluation.

Conclusion

This study demonstrates that scL-ALI effectively addresses the poor tumor delivery that has apparently limited alisertib’s clinical success. scL-ALI achieved stronger AURKA inhibition, enhanced tumor cell killing, and improved tumor growth suppression across ATRT, glioblastoma, and lung cancer models. Its combination with radiation therapy further increased antitumor efficacy and extended survival in ATRT-bearing mice. Overall, these results highlight scL-ALI as a promising novel nanomedicine that overcomes key delivery barriers and enhances the therapeutic potential of alisertib for CNS and other AURKA-driven malignancies.

Disclosure

E.H.C. is a named inventor on multiple patents related to the described technology, which are owned by Georgetown University and licensed to SynerGene Therapeutics, Inc. for commercial development. E.H.C. holds equity in SynerGene Therapeutics, Inc. A.R. serves as a paid scientific consultant for the company. S.S.K. is a full-time employee of SynerGene Therapeutics, Inc. M.M. was a graduate student supported by a research agreement between Georgetown University and SynerGene Therapeutics, Inc. J.B.H. is the salaried President and CEO of SynerGene Therapeutics, Inc. and holds stock in the company. The authors report no other conflicts of interest in this work.