2018 Synaptic vesicle 2 receptors as a novel targets for
neuroendocrine cancer therapy
Authors: Jaskula-Sztul Renata , Whitt Jason D. , Ou Jianfa , Liu Margaret X. , Aburjania Zviadi , Chen Herbert 
Published: November 2018

OBJECTIVES/SPECIFIC AIMS: (1) To delineate the function of the
heavy-chain receptor binding domain (HCR), a portion of botulinum neurotoxin
type A (BoNT/A) and synaptic vesicle 2 (SV2) signaling pathway, which
provide a novel multipurpose biologic with potential clinical applications in
tumor detection/imaging, inhibition of tumor progression, and
reduction of bioactive hormone secretion in metastatic neuroendocrine (NE)
cancers. (2) To evaluate the expression pattern of SV2 receptors in NE cancer
patient-derived tissues for prediction of patient response for recombinant HCR
(rHCR) treatment. (3) To assess the in vivo efficacy and toxicity of rHCR in a
NE cancer liver metastasis mouse models and in the NE patient-derived 3D
MicroTumor system. (4) To collect preclinical data to design and conduct a
clinical trial with NE cancer patients, a major goal toward translating our
discoveries into much needed therapies. METHODS/STUDY POPULATION:
Recombinant botulinum heavy chain (rHCR) was produced using an IPTG-inducible
expression vector in E. coli BL21. The rHCR was His-Tag
purified and stored in PBS buffer before usage. Cytotoxicity: H727, TT, and MZ
cells were plated at a density of 5000 cells/well in 96-well plates
and incubated under standard conditions overnight. The next day, cells were
treated with 10, 100, or 500 nmol/L of rHCR and incubated for 72
hours. Following incubation, cell viability was assessed by ATP quantification
using the CellTiter-Glo (Promega) assay. Fresh NE tumors were dissociated and
injected into polydimethylsiloxane bioreactors in a matrigel and collagen
suspension for 3D culture experiments. The viability of 3D cultures incubated
with various doses of rHCR was assessed by measuring the uptake of the
near-infrared dye IR-783 using an IVIS imaging system. Western blot: H727, TT,
and MZ cells were seeded in 6-well plates at a density of 3×105
cells/well for 24 hours followed by treatment with 100
nmol/L for 72 hours. Total cellular proteins were isolated and
analyzed to assess the level of SV2A expression and the effect of rHCR on the
expression levels of NET marker proteins. Immunohistochemistry: Deparaffinized
tissue culture slides were incubated with SV2A primary antibody in 1%
BSA and incubated overnight at 4°C. Slides were rinsed twice with TBS
containing 0.025% Triton, followed by 0.3%
H2O2 for 15 minutes. Slides were then incubated with
HRP-conjugated secondary antibody for 1 hour at room temperature. Detection of
protein-protein interaction: Precleared cell lysate was incubated with
glutathione-agarose beads in the presence of 10 μg of GST-tagged rHCR
for 2 h at 4°C with end-over end mixing. Samples were then
centrifuged at 10,000 g for 2 minutes and the supernatant was analyzed by
SDS-PAGE. Preclinical models: To allow rHCR testing on NET patient derived cells
in a very novel 3D surrogates, sterile collected fresh NET tissues will be
obtained from the UAB Tissue Procurement, dissociated into a single cell
suspension and injected into a polydimethylsiloxane bioreactors containing
extracellular matrix composed of bovine collagen and matrigel. Such 3D cell
culture will be maintained in bioreactors with constant supply of media through
the microchannels and treated with rHCR at concentrations ranging from 10 nM to
1 mM. Following histologic confirmation of growth and morphology of NET
patient-derived 3D surrogates we will test the anticancer activity of rHCR in
this system using the standard cytotoxicity assays as well as we will validate
the NET hormone expression using immunohistochemistry assay. To create an animal
model of NE cancer progression, we will perform intrasplenic injection of NET
cell lines. In approximately 4 weeks, the animals should develop NE liver
metastases based upon our previous experience. rHCR-iFPs accumulation in the
tumor mass: rHCR-iFPs will be injected to the tumor bearing mice after 4 weeks
of cells implantation in 1 week interval for total of 4 treatments at the
concentrations of 0.125, 1.25, and 12.5 mg/kg.
RESULTS/ANTICIPATED RESULTS: Based on the preliminary data, we expect
to detect rHCR-iFPs in NE cancer xenografts and in patient derived 3D explants.
Our preliminary data revealed that treating NETcells with rHCR significantly
reduced NE peptide expression in 3 days. Thus, we expect to see the decrease of
NE tumor markers even if the fluorescent detection method is not sensitive
enough to monitor the signal. The reduction of the NET markers can be used as an
indicator of the rHCR-iFPs uptake by the tumor mass. If HCR exhibit high binding
affinity to SV2 receptors in NET models but moderate anticancer efficacy, we
plan to use rHCR peptide to conjugate with the nanocarrier for targeted drug
delivery. In this case rHCR peptide can be used as a ligand that specifically
binds to NE cancer cells and delivers anticancer drug. In 3D NET patient derived
explants we expect significant reduction of NET markers and hormones (serotonin
and calcitonin) in NE cancer cells upon long-term rHCR-iFPs treatment. In
addition, we will perform multiplex protein quantification assay using Luminex
to assess the various hormones, cytokines, and growth factors to be repurposed
into a diagnostic and detection reagent, or a drug delivery ligand for targeted
therapies. DISCUSSION/SIGNIFICANCE OF IMPACT: NE cancers are highly
metastatic: NE cancers such as carcinoid, islet cell tumors, and medullary
thyroid cancer frequently metastasize to the liver. They are the second most
prevalent GI malignancy. Ninety percent of patients with pancreatic carcinoid
tumors and 50% of patients with islet cell tumors develop isolated
hepatic metastases. Patients with untreated, isolated NE liver metastases have
<30% 5-year survival. Thus, there is a critical need for
new therapies for NE cancers. Surgery is the only curative therapy available for
patients with NE cancers but most cannot be cured: surgical removal is the most
effective treatment for NE cancers; however, a very high percentage of patients
present with metastatic disease. While surgical resection can be potentially
curative, many patients are not candidates for operative intervention due to
widespread metastases or the degree of hepatic involvement by the NE cancers.
Moreover, other forms of therapy including chemoembolization, radioembolization,
cryoablation, and chemotherapy have had limited efficacy. We hope that our in
vitro data on rHCR toxicity and specificity to NET, validated in the
pre-clinical models will allow the first in-human application of this technology
in clinical studies.