1. Introduction
Neuroendocrine neoplasias (NENs) are a clinically heterogeneous group of cancers, characterised by the presence of somatostatin receptors (SSTRs) on the tumour surface [
[1]Tumour biology and histopathology of neuroendocrine tumours.
]. The SSTR family comprises of five widely distributed G-protein coupled receptors that mediate intracellular signalling pathways with roles in cell proliferation, cell differentiation and angiogenesis [
[2]- Volante M.
- Bozzalla-Cassione F.
- Papotti M.
Somatostatin receptors and their interest in diagnostic pathology.
]. The expression of the SSTRs on NENs can be exploited for therapeutic benefits with somatostatin analogues (SSAs) and peptide receptor radionuclide therapy (PRRT). Indeed, the long acting SSA preparations octreotide and lanreotide are considered standard of care for the treatment of Grade 1 and 2 NENs within the current European Neuroendocrine Tumour Society Consensus Guidelines, with proven anti-proliferative effects in phase 3 clinical trials [
3- Pavel M.
- O'Toole D.
- Costa F.
- et al.
ENETS Consensus guidelines update for the management of distant metastatic disease of intestinal, pancreatic, bronchial neuroendocrine neoplasms (NEN) and NEN of unknown primary site.
,
4- Rinke A.
- Müller H.H.
- Schade-Brittinger C.
- et al.
Placebo-controlled, double-blind, prospective, randomized study on the effect of octreotide LAR in the control of tumor growth in patients with metastatic neuroendocrine midgut tumors: a report from the PROMID study group.
,
5- Caplin M.E.
- Pavel M.
- Ruszniewski P.
Lanreotide in metastatic enteropancreatic neuroendocrine tumors.
]. The NETTER-1 trial has arguably provided the biggest clinical impact in the treatment of SSTR2-expressing NENs [
[6]177Lu-Dotatate for midgut neuroendocrine tumors.
]. By comparing ‘double dose’ SSA treatment with PRRT in midgut NET patients progressing on standard dose SSA treatment, the progression-free survival over the first 30 months of the trial for SSA treatment was reported at 8.4 months, with the progression-free survival for
177Lu-DOTATATE not having been reached. This translated to a 79% risk reduction of progression or death for patients treated with PRRT over SSA.
Selection for treatment with PRRT is based on the presence of SSTR2 as illustrated by positive receptor imaging; most frequently using [
68Ga]-labelled SSAs ([
68Ga]-DOTA-PET) [
[7]- Sanli Y.
- Garg I.
- Kandathil A.
- et al.
Neuroendocrine tumor diagnosis and management: (68)Ga-dotatate PET/CT.
]. The only validated predictor of response to PRRT is positive SSTR2 imaging to confirm presence of target, in a binary manner. However, the NETTER1 trial reports an objective response rate for PRRT of 18%, suggesting that a significant number of patients who theoretically should respond do not, and the reasons for this remain unclear [
[6]177Lu-Dotatate for midgut neuroendocrine tumors.
].
Previous studies have demonstrated that the expression of the SSTR2 receptor is controlled by two epigenetic modifications of a novel SSTR2 upstream promoter: cytosine DNA methylation of key CpG islands and histone acetylation [
[8]- Torrisani J.
- Hanoun N.
- Laurell H.
- et al.
Identification of an upstream promoter of the human somatostatin receptor, hSSTR2, which is controlled by epigenetic modifications.
]. CpG islands are found most commonly within the regulatory regions of genes: their promoter and 5’ coding regions, where methylation induces transcriptional silencing [
[8]- Torrisani J.
- Hanoun N.
- Laurell H.
- et al.
Identification of an upstream promoter of the human somatostatin receptor, hSSTR2, which is controlled by epigenetic modifications.
]. This putative upstream promoter area for SSTR2 is conserved across species and is responsible for between 40 and 60% of total SSTR2 production across multiple cell lines representing different cancer types. Methylation of this promoter was demonstrated to be reversible
in vitro with the first-generation DNA methyltransferase inhibiting agent decitabine [
[8]- Torrisani J.
- Hanoun N.
- Laurell H.
- et al.
Identification of an upstream promoter of the human somatostatin receptor, hSSTR2, which is controlled by epigenetic modifications.
]. However, decitabine, and its deoxy derivative azacitidine, has limited use in the management of solid tumours due to rapid deamination by cytidine deaminase, limiting tumour drug exposure together with significant dose-limiting myelosuppression [
9- Fenaux P.
- Mufti G.J.
- Hellstrom-Lindberg E.
- et al.
Azacitidine prolongs overall survival and reduces infections and hospitalizations in patients with WHO-defined acute myeloid leukaemia compared with conventional care regimens: an update.
,
10- Kaminskas E.
- Farrell A.T.
- Wang Y.C.
- et al.
FDA drug approval summary: azacitidine (5-azacytidine, Vidaza) for injectable suspension.
,
11- Momparler R.L.
- Goodman J.
In vitro cytotoxic and biochemical effects of 5-aza-2'-deoxycytidine.
].
The second-generation DNA methyltransferase inhibitor guadecitabine (SGI-110) couples deoxyguanosine to decitabine, thus resistant to cytidine deaminase with a clinically meaningful longer half-life [
[12]- Rogstad D.K.
- Herring J.L.
- Theruvathu J.A.
- et al.
Chemical decomposition of 5-aza-2'-deoxycytidine (Decitabine): kinetic analyses and identification of products by NMR, HPLC, and mass spectrometry.
]. The described toxicities of myelosuppression and fatigue, and the need for care in renal failure remain similar to those of the first-generation drugs; however, the reduced dosing frequency of guadecitabine may mean that toxicities are encountered less frequently in clinical practice [
[13]- Yoo C.B.
- Jeong S.
- Egger G.
- et al.
Delivery of 5-aza-2'-deoxycytidine to cells using oligodeoxynucleotides.
,
[14]- Derissen E.J.
- Beijnen J.H.
- Schellens J.H.
Concise drug review: azacitidine and decitabine.
]. Guadecitabine is currently under evaluation as combination therapy in multiple clinical trials across several different solid tumour types [
15- Oza A.M.
- Matulonis U.A.
- Alvarez Secord A.
- et al.
A randomized phase II trial of epigenetic priming with guadecitabine and carboplatin in platinum-resistant, recurrent ovarian cancer.
,
16- Matei D.
- Ghamande S.
- Roman L.
- et al.
A phase I clinical trial of guadecitabine and carboplatin in platinum-resistant, recurrent ovarian cancer: clinical, pharmacokinetic, and pharmacodynamic analyses.
,
17- Di Giacomo A.M.
- Covre A.
- Finotello F.
- et al.
Guadecitabine plus ipilimumab in unresectable melanoma: the NIBIT-M4 clinical trial.
,
18- Lee V.
- Wang J.
- Zahurak M.
- et al.
A phase I trial of a guadecitabine (SGI-110) and irinotecan in metastatic colorectal cancer patients previously exposed to irinotecan.
].
We hypothesised that treatment with a robust DNA methyltransferase inhibitor would increase SSTR2 expression, as visualised by PET imaging. We demonstrate that promoter methylation in SSTR2 can be reversed using guadecitabine resulting in increased uptake of the SSTR2-directed radioligand
18F-FET-βAG-TOCA ([
18F]-FETO) [
[19]- Dubash S.R.
- Keat N.
- Mapelli P.
- et al.
Clinical translation of a click-labeled 18F-octreotate radioligand for imaging neuroendocrine tumors.
] both
in vitro and
in vivo, demonstrating for the first time that PET imaging can be used to image epigenetic regulation in NET
. We further assessed the safety of combination guadecitabine, and PRRT was assessed
in vivo and the prevalence of methylation in NENs in clinical samples with a view to moving forward with clinical studies
.2. Materials and methods
2.1 Cell line characteristics and cell culture
All cell lines were obtained from in-house stock and were authenticated (short tandem repeat profiling, Public Health England); CM cells could not be authenticated as no cell line data were available. Each line was expanded into 10 tubes (1.5 × 106/mL/tube) and frozen immediately to provide passage calibrated stock for subsequent experiments. QGP1 and CM cells were cultured in Roswell Park Memorial Institute 1640 media. BON-1 cell line was cultured in Dulbecco's modified Eagle's medium. All were supplemented with 10% foetal bovine serum, 2% penicillin-streptomycin (5000 U/mL) and 1% L-glutamine. Cells were seeded at 150,000 cells/mL and grown as a semi-confluent monolayer in a 5% CO2 humidified incubator at 37 °C. Cells were kept in culture in Falcon flasks for a maximum of 10 passages, before being replaced. Cells were confirmed as mycoplasma free every 4 weeks.
2.2 Guadecitabine
Astex Pharmaceuticals (Cambridge, UK) kindly donated guadecitabine. A 16 mM working stock was prepared by dissolving guadecitabine in the provided diluent (Astex Pharmaceuticals) and stored at 4 °C. Cell lines were treated with 0, 5 or 10μM of guadecitabine, which was replenished, along with fresh media, every 24 h in order to counter against drug inactivation by hydrolysis. Cells were harvested at 72 h and were washed, spun down in to a pellet and stored at −80 °C.
2.3 Tumour tissue
Human tumour tissue from 65 patients was retrieved from the Imperial College Healthcare Tissue Bank (ICHTB). Demographics are shown in
Table 1. ICHTB is approved by Wales REC3 to release human material for research (17/WA/0161), and the samples for this project were issued from subcollection reference number R14014.
Table 1Characteristics of study population.
2.4 DNA extraction
Prior to DNA extraction, optimal tumour slices were selected and deparaffinised. DNA was extracted from human tissue using the QIAamp DNA FFPE Tissue Kit (Qiagen) according to in-house protocols extrapolated from manufacturer's instructions. DNA was extracted from cell lines using the DNeasy Blood and Tissue Kit (Qiagen), as per manufacturer's instructions. The quality of the extraction was assessed by Nanodrop ND-1000 spectrophotometer (Thermo Scientific), and the amount of DNA extracted was determined using Qubit 2.0 fluorometer (Invitrogen).
2.5 Pyrosequencing of tissue and cell lines
Bisulphite modification of DNA was performed using the EZ DNA Methylation-Lightning™ Kit (Zymo Research Corp), according to manufacturer's instructions. Bisulfite-converted DNA was amplified by methylation-specific PCR (MSP) using SSTR2-specific primers designed and optimised in-house. The resulting amplicon (120 base pairs) included the SSTR2 Transcriptional Start Site and CpG sites of interest as previously described [
[20]Torrisani J, Hanoun N, Laurell H, et al: Identification of an upstream promoter of the human somatostatin receptor, hSSTR2, which is controlled by epigenetic modifications. [academic.oup.com].
]. Pyrosequencing was conducted using Pyromark Q96 (Qiagen), according to manufacturer's instructions. Cell lines and human samples were compared with
in vitro methylated standards at 25, 50, 75 and 100% to monitor the efficiency of the pyrosequencing reaction, and linear regression analysis applied to generate a standard curve with a correlation coefficient to correct for any assay bias. Human genomic DNA pooled from healthy female and male individuals was used as reference (Promega, G1512 and G1471).
2.6 Cytotoxicity assay
Cells were seeded in a 96-well plate and grown in media with various conditioned media for 72 h. After treatment, 10% trichloroacetic acid was added to the media and the plate incubated at 4 °C for 1 h to fix cells. Plates were washed thoroughly in water and left to dry overnight. Cells were stained in 0.4% SRB dye in 1% acetic acid. Unbound SRB was removed by washing with 1% acetic acid and left overnight; 10 mM Tris was added and optical density measured at 564 nm (Tecan infinite M200). Each unknown optical density value was standardised against the vehicle control.
2.7 qRT-PCR
Total RNA was isolated from cell pellets using the RNeasy Mini Kit (Qiagen), measured by Nanodrop ND-1000 spectrophotometer (Thermo Scientific) and reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems). Real-time quantitative PCR was conducted using ‘TaqMan Fast Advanced Master Mix’ (Applied Biosystems) and probes Hs00265624_s1 for SSTR2, Hs02786624_g1 for GAPDH from TaqMan gene expression assays (Applied Biosystems) in a 7900HT Fast Real-Time PCR System (Applied Biosystems). Data were analysed using comparative C
t method as previously described with GAPDH (Fwd.; Rev.) as an internal control [
[21]- Schmittgen T.D.
- Livak K.J.
Analyzing real-time PCR data by the comparative C(T) method.
]. All samples were assayed in triplicate, with appropriate non-template controls.
2.8 Western blot analysis
Protein samples were prepared by resuspending cell pellets in ice-cold PBS and washing three times. Cell lysis was achieved using ice-cold RIPA buffer (Thermo Scientific). The lysates were sonicated, and protein concentration was determined using the Pierce Detergent Compatible Bradford Assay Kit (Thermo Scientific) and 30μg of total protein was assayed per sample. Tissue samples were homogenised in RIPA buffer containing protease and phosphatase inhibitors (all Sigma–Aldrich) using a Precellys 24 homogeniser with CK14 beads. Homogenates were cleared of debris by centrifugation at 5000×g at 4 °C for 5 min. Supernatants were recovered and 30 μg protein assayed. Skimmed dried milk (1% w/v) diluted in TBST solution was used for blocking of non-specific binding sites for 1 h, at room temperature. Membranes were incubated at 4 °C overnight with the anti-SSTR2 primary antibody, SSTR2 (Santa Cruz) (1:1000). After three washings with TBST, the membranes were incubated with a HRP-conjugated secondary antibody. Membranes were incubated with ECL substrate solution (GE Healthcare) according to manufacturer's instructions. β-tubulin and β-actin were used as loading controls.
2.9 Uptake studies
BON-1 cells were grown in six-well plates, seeded at 150,000 cells/well. Guadecitabine was added to the wells at two dilutions (5 and 10 M) on Day 2, cells replaced with fresh drug containing media every 24 h [18F]-FETO uptake was conducted 72 h after initiating treatment, by adding the radiotracer in fresh drug-containing medium; each well contained 0.74 MBq of [18F]-FETO in a volume of 1 ml. Cells were incubated at 37 °C and 5% CO2 for 1 h and then were washed three times with 1×PBS and lysed on ice for 15 min using 1 ml RIPA buffer/well. Cell lysates were transferred to respective radioactivity counting tubes. Radioactivity in each sample was counted using an auto gamma counter (Perkin Elmer, London, UK). The amount of protein in each gamma-counted sample was quantified using the Pierce™ BCA protein assay method. Decay-corrected counts were normalised to protein concentration and expressed as percentage incubated dose per milligramme of cellular protein (%ID/mg protein) in each sample.
2.10 PET imaging following guadecitabine treatment and [18F]FETO uptake
All animal experiments were done by licensed investigators in accordance with the UK Home Office Guidance on the Operation of the Animal (Scientific Procedures) Act 1986 (HMSO, London, UK, 1990) and within guidelines set out by the UK National Cancer Research Institute Committee on Welfare of Animals in Cancer Research [
[22]- Workman P.
- Aboagye E.O.
- Balkwill F.
- et al.
Guidelines for the welfare and use of animals in cancer research.
].
The
in vivo models were set up in female athymic nude mice aged 6–8 weeks (Harlan, Bicester, UK Ltd). Xenografts were established under 2.5% isoflurane anaesthesia by subcutaneous injection of either BON-1 or QGP-1 cells on the back of the neck (4 × 10 [
[6]177Lu-Dotatate for midgut neuroendocrine tumors.
] cells in 100 μL of 50% PBS and 50% Matrigel (Corning, Amsterdam, The Netherlands). Tumour dimensions were measured by calliper and volumes calculated using the ellipsoid formula for estimating tumour mass: volume (mm
3) = (π/6) x a x b x c, where a, b and c represent three orthogonal axes of the tumour. When tumours reached 50–70 mm
3 (8 weeks post-induction), mice were randomised into vehicle and guadecitabine treatment groups. Guadecitabine was prepared fresh before administration, according to manufacturer's instructions. Guadecitabine-treated mice were injected with 2 mg/kg of the drug subcutaneously twice weekly for 2 weeks; control groups were injected with vehicle (1:50 diluent:PBS) (
Supplementary Fig. 1) [
[23]- Fang F.
- Munck J.
- Tang J.
- et al.
The novel, small-molecule DNA methylation inhibitor SGI-110 as an ovarian cancer chemosensitizer.
].
For imaging, mice were anesthetized with 2.5% isoflurane and placed in a thermostatically controlled rig in a dedicated small animal Genisys PET scanner (SOFIE Biosciences, Culver City, USA) [
[24]- Witney T.H.
- Pisaneschi F.
- Alam I.S.
- et al.
Preclinical evaluation of 3-18F-fluoro-2,2-dimethylpropionic acid as an imaging agent for tumor detection.
]. Following injection of 0.925 MBq of [
18F]FETO via lateral tail vein cannula, PET scans were acquired in a list-mode format over either 0–60 min after injection to give decay-corrected values of radioactivity accumulation in tissues. The collected data were reconstructed with a three-dimensional maximum likelihood estimation method (3D ML-EM). Volumes of interest for tumours were defined using Siemens Inveon Research Workplace software (Siemens Molecular Imaging Inc., Knoxville, USA) and the count densities (MBq/mL) averaged for the time points corresponding to 3–20 min (where equilibrium was observed). Tissue radioactivity values were normalised to average whole-body radioactivity.
2.11 Combination therapy of guadecitabine and [177Lu]-DOTATATE in vivo
The
in vivo models were set up in female athymic nude mice aged 6–8 weeks (Harlan, Bicester, UK Ltd). Xenografts were established under 2.5% isoflurane anaesthesia by subcutaneous injection of BON-1 and QGP-1 cell lines (5 × 10 [
[6]177Lu-Dotatate for midgut neuroendocrine tumors.
] cells in 100 μL of 50% PBS and 50% Matrigel (Corning, Amsterdam, The Netherlands) on the upper flank of the mice. Tumour dimensions were measured by calliper as described above. When tumours reached 200 mm
3, mice were randomised into vehicle and guadecitabine treatment groups. Guadecitabine-treated mice were injected with 2 mg/kg of the drug intravenously daily for 5 days; control groups were injected with normal saline [
[23]- Fang F.
- Munck J.
- Tang J.
- et al.
The novel, small-molecule DNA methylation inhibitor SGI-110 as an ovarian cancer chemosensitizer.
]. On Day 7, mice were then randomised to receive either saline or 7.5MBq of [
177Lu]-DOTATATE (six mice in each treatment group) (
Supplementary Fig. 1). Tumour growth was assessed by calliper measurements daily. End-point was set to a tumour size of 1000 mm
3 or weight loss of more than 10% compared with day of treatment start. Upon reaching end-point, animals were sacrificed and the tumour was collected and snap frozen in liquid nitrogen for further analysis.
3. Histopathology
Formalin-fixed, paraffin-embedded specimens and matching haematoxylin and eosin (H&E) slides were retrieved from the local pathology archive. Five μm thick sections were de-paraffinized in xylene and rehydrated in graded alcohols. Optimal heat-mediated antigen retrieval conditions were applied according to manufacturer's recommendations in relation to the primary antibody, using a water bath heated to 100 °C. Slides were then incubated in citrate buffer at pH 6.0 for 20 min. Before immunostaining, slides were cooled at room temperature, and endogenous peroxidase activity was suppressed by incubation with CAS-Block (Invitrogen, Camarillo, California, USA) for 5 min. The primary antibody against SSTR2A (UMB1, Abcam, Cambridge, UK) was used at a 1:250 dilution overnight. Slides were washed with buffered TRIS solution and blocked with Novolink polymer (Leica, Milton Keynes, UK) for 30 min and subsequently developed with diaminobenzidine and Mayer's Haematoxylin counterstaining. Appropriately, selected tissue sections were used according to the manufacturer's instruction as external positive control during each reaction. Negative control reactions were performed omitting the primary antibodies from the dilution buffer. This resulted in a complete absence of staining in all cases. A trained histopathologist (FM) blinded to the clinical data scored all the cases. Tissue samples were scored manually using the immunohistochemical score (IHS) [
[25]- Pinato D.J.
- Tan T.M.
- Toussi S.T.
- et al.
An expression signature of the angiogenic response in gastrointestinal neuroendocrine tumours: correlation with tumour phenotype and survival outcomes.
]. Briefly, each sample can be assigned an IHS ranging between 0 and 300, based on the multiplication of the percentage of cells showing immunohistochemical expression (0–100) by the intensity of the signal (graded 1–3).
3.1 Statistical analysis
Quantitative data are presented as mean ± SEM, and p < 0.05 was considered significant. Statistical significance for multiple comparisons between control and treated groups was determined by non-parametric one-way ANOVA, while for statistical significance between two groups, Student's t-test was performed. Data analysis was performed by using GraphPad Prism 6.0 or SPSS 23.0 software package (SPSS, Inc., IL, USA).
5. Discussion
PRRT treatment represents the only biomarker driven treatment for NENs, with treatment targeted to SSTR2 expression as determined by [
68Ga]-DOTA imaging [
[6]177Lu-Dotatate for midgut neuroendocrine tumors.
]. Emerging data suggest that qualitative-positive [
68Ga]-DOTA imaging
per se does not accurately predict outcome to PRRT but that semi-quantitative tumour SUV measure is a better predictor of therapeutic outcome, whereby patients with low [
68Ga]-DOTA SUV have poor outcomes compared to those with higher uptake [
[30]- Sharma R.
- Wang W.M.
- Yusuf S.
- et al.
68)Ga-DOTATATE PET/CT parameters predict response to peptide receptor radionuclide therapy in neuroendocrine tumours.
,
[31]- Haug A.R.
- Auernhammer C.J.
- Wangler B.
- et al.
68Ga-DOTATATE PET/CT for the early prediction of response to somatostatin receptor-mediated radionuclide therapy in patients with well-differentiated neuroendocrine tumors.
]. Moreover, some patients will have negative SSTR2-specific imaging making them unsuitable for therapy [
[6]177Lu-Dotatate for midgut neuroendocrine tumors.
]. Transient enhancement of SSTR2 expression in patients with negative [
68Ga]-DOTA imaging may represent a novel method by which the number of patients that receive and respond to PRRT could be increased. We confirm that the SSTR2 gene expression is controlled by methylation-specific gene silencing via an upstream promoter, as previously demonstrated [
[8]- Torrisani J.
- Hanoun N.
- Laurell H.
- et al.
Identification of an upstream promoter of the human somatostatin receptor, hSSTR2, which is controlled by epigenetic modifications.
]. We add to this mechanistically relevant information by revealing that promoter methylation can be transiently reversed by the demethylating agent guadecitabine in a drug concentration related manner, leading to both increased transcription and translation of the SSTR2 gene product. Crucially, we illustrate that the upregulation of SSTR2 is detectable by PET imaging, thus illustrating for the first time that PET imaging can be used to monitor the epigenetic change.
In the largest published patient cohort to date, we report the MI of the SSTR2 upstream promotor in 65 tumour samples, compared to non-NET standardised controls, revealing a statistically significant difference in MI that correlated inversely with SSTR2 expression. Several studies have illustrated that SUV is correlated with SSTR2 tissue expression and is predictive of outcome to PRRT [
30- Sharma R.
- Wang W.M.
- Yusuf S.
- et al.
68)Ga-DOTATATE PET/CT parameters predict response to peptide receptor radionuclide therapy in neuroendocrine tumours.
,
31- Haug A.R.
- Auernhammer C.J.
- Wangler B.
- et al.
68Ga-DOTATATE PET/CT for the early prediction of response to somatostatin receptor-mediated radionuclide therapy in patients with well-differentiated neuroendocrine tumors.
,
32- Kratochwil C.
- Stefanova M.
- Mavriopoulou E.
- et al.
SUV of [68Ga]DOTATOC-PET/CT predicts response probability of PRRT in neuroendocrine tumors.
,
33- Brunner P.
- Jorg A.C.
- Glatz K.
- et al.
The prognostic and predictive value of sstr2-immunohistochemistry and sstr2-targeted imaging in neuroendocrine tumors.
,
34- Kaemmerer D.
- Prasad V.
- Daffner W.
- et al.
Neoadjuvant peptide receptor radionuclide therapy for an inoperable neuroendocrine pancreatic tumor.
]. Taken together with the findings in this study, it can be hypothesised that methylation of SSTR2 does not result in complete epigenetic silencing of the receptor but a reduction in expression which can be enhanced using a DNA hypomethylating agent to improve the efficacy of PRRT.
While previous studies investigated the use of decitabine, this drug has limited use in the management of solid tumours due to rapid deamination by cytidine deaminase limiting tumour drug exposure, and significant dose-limiting myelosuppression. Guadecitabine is a novel second generation hypomethylating agent, made of the active moiety of decitabine and deoxyguanosine linked by a phosphodiester bond. The phosphodiester bond undergoes gradual cleavage by phosphorylases and other enzymes over an extended period of time, prolonging drug exposure [
[23]- Fang F.
- Munck J.
- Tang J.
- et al.
The novel, small-molecule DNA methylation inhibitor SGI-110 as an ovarian cancer chemosensitizer.
]. Guadecitabine has a half-life of 4 h in humans compared to decitabine that has a half-life of 30 min [
[26]- Issa J.J.
- Roboz G.
- Rizzieri D.
- et al.
Safety and tolerability of guadecitabine (SGI-110) in patients with myelodysplastic syndrome and acute myeloid leukaemia: a multicentre, randomised, dose-escalation phase 1 study.
]. Moreover, guadecitabine is resistant to cytidine deaminase resulting in improved tumour drug exposure. In clinical studies, guadecitabine had a lower C
max compared to decitabine resulting in less toxicity, in particular myelosuppression, a key dose-limiting side-effect that limits the use of decitabine [
[26]- Issa J.J.
- Roboz G.
- Rizzieri D.
- et al.
Safety and tolerability of guadecitabine (SGI-110) in patients with myelodysplastic syndrome and acute myeloid leukaemia: a multicentre, randomised, dose-escalation phase 1 study.
,
35Digging deep into “dirty” drugs - modulation of the methylation machinery.
,
36- Schrump D.S.
- Fischette M.R.
- Nguyen D.M.
- et al.
Phase I study of decitabine-mediated gene expression in patients with cancers involving the lungs, esophagus, or pleura.
,
37- Stewart D.J.
- Issa J.P.
- Kurzrock R.
- et al.
Decitabine effect on tumor global DNA methylation and other parameters in a phase I trial in refractory solid tumors and lymphomas.
]. Consistent with previous findings using decitabine, we illustrated that treatment of NET cell lines with the demethylating agent, guadecitabine, not only resulted in re-expression of SSTR2 but importantly resulted in enhanced uptake of [
18F]-FETO [
[38]- Veenstra M.J.
- van Koetsveld P.M.
- Dogan F.
- et al.
Epidrug-induced upregulation of functional somatostatin type 2 receptors in human pancreatic neuroendocrine tumor cells.
,
[39]- Taelman V.F.
- Radojewski P.
- Marincek N.
- et al.
Upregulation of key molecules for targeted imaging and therapy.
] [
18F]-FETO, a [
18F]labelled octreotate, was developed by our group to obviate the need for an on-site gallium generator – due to the short half-life of [
68Ga] [
[29]- Leyton J.
- Iddon L.
- Perumal M.
- et al.
Targeting somatostatin receptors: preclinical evaluation of novel 18F-fluoroethyltriazole-Tyr3-octreotate analogs for PET.
]. [
18F]-FETO has recently been shown to result in clinical quality images and was therefore taken forward in this study with a view to taking this tracer forward in the clinical setting [
[19]- Dubash S.R.
- Keat N.
- Mapelli P.
- et al.
Clinical translation of a click-labeled 18F-octreotate radioligand for imaging neuroendocrine tumors.
].
We extended our
in vitro findings into a mouse model of NET using BON-1 cells (high methylation and low basal expression SSTR2) and QGP-1 (low methylation and high basal expression SSTR2). At the guadecitabine doses studied, we did not observe any change in tumour size. However, the dose administered was sufficient to significantly increase SSTR2 expression within the tumour as reflected by an increased in [
18F]-FETO uptake, rendering undetectable BON-1 xenografts into clearly detectable tumours. In addition we conducted a
in vivo tolerability study using a single dose of [
177]Lu-DOTATATE in tumour models from the cell lines studied
in vitro. We did not observe any additive toxicity when guadecitabine was combined with [
177Lu]-DOTATATE a key consideration as both drugs are associated with myelotoxicity. Changes in tumour size were not observed with combination therapy. While these observations somehow challenges the downstream consequences of our hypothesis of reversing SSTR2 silencing, we also observed slow tumour growth of the NEN models studied, such that a single dose of [
177Lu]-DOTATATE was unlikely to induce changes in tumour size within the bounds of the study including consideration of animal welfare. Future work should consider either multiple dosing experiments or models with more robust growth, considering differential methylation states. Moreover [
177Lu]-DOTATATE was administered when tumour size was relatively large which have may have impacted on tumour response. [
177Lu] is a short-range particle, tissue penetration 1.4 mm, and tumoural size has a significant impact on the biodistribution of [
177Lu] [
[40]- de Jong M.
- Breeman W.A.
- Valkema R.
- et al.
Combination radionuclide therapy using 177Lu- and 90Y-labeled somatostatin analogs.
]. Further optimisation of regimen is required to show a reduction in tumour size.
Taken together, this work suggests SSTR2 epigenetic silencing can be reversed, enabling future optimisation of therapeutic options in patients with negative [
68Ga]-DOTA-PET scans or marginal expression of SSTR2, not suitable for PRRT, using [
18F]-FETO- or other SSA-PET imaging procedure as a measure of epigenetic change. As such, we postulate that producing an increased density of functional SSTR2 will (i) augment response in patients with low [
68Ga]-DOTA uptake and (ii) transiently upregulate SSTR2 expression in patients with negative functional imaging such that they can receive PRRT. While this concept has not been assessed clinically, Taelman and colleagues illustrated enhanced cell death with [
177Lu]-DOTATATE following treatment with epigenetic modifiers using a number of other cell lines [
[39]- Taelman V.F.
- Radojewski P.
- Marincek N.
- et al.
Upregulation of key molecules for targeted imaging and therapy.
]. The authors also illustrated that inhibitors of histone deactylation (HDAC) upregulated SSTR expression albeit to a lesser degree. It would be of interest to investigate novel HDAC agents in future combination studies using different
in vivo models to allow maximal re-expression of SSTR2 prior to clinical translational. Any proposed clinical study would have to have clear safety end-points particularly given the potential for additive myelotoxicity and renal toxicity with [
177Lu]-DOTATATE and demethylating agents. While we did not observe additive toxicity
in vivo, only a single dose of [
177Lu]-DOTATATE was administered and repeat dosing experiments should be undertaken
in vivo with relevant safety end-points evaluated prior to clinical translation.
While we have illustrated SSTR2 expression can be modulated using demethylating agents in NENs, this is not limited to this tumour type. Previous work illustrates that SSTR is present in a number of different tumour types [
[41]- Taelman V.F.
- Radojewski P.
- Marincek N.
- et al.
Upregulation of key molecules for targeted imaging and therapy.
], and there is growing interest in pursuing PRRT for these tumour types. The role of methylation remains to be elucidated but the combination of a demethylation agent and PRRT could potentially be extended to other cancer types.
Authors contribution
Joanne S. Evans: Data curation; Formal analysis; Investigation; Methodology; Project administration; Supervision; Validation; Visualization; Roles/Writing – original draft.
Jamie Beaumont: Data curation; Formal analysis; Investigation; Methodology; Roles/Writing – original draft.
Marta Braga: Data curation; Formal analysis; Investigation; Methodology; Roles/Writing – original draft.
Nahal Masrour: Data curation; Formal analysis; Investigation; Methodology; Roles/Writing – original draft
Francesco Mauri: Data curation; Formal analysis; Investigation; Methodology; Roles/Writing – original draft.
Alice Beckley: Data curation; Formal analysis; Investigation; Methodology; Roles/Writing – original draft.
Shamus Butt: Data curation; Formal analysis; Investigation; Methodology; Roles/Writing – original draft.
Christina Simoglou Karali: Data curation; Formal analysis; Investigation; Methodology; Roles/Writing – original draft.
Chris Cawthorne: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Roles/Writing – original draft
Stephen Archibald: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Roles/Writing – original draft
Eric O. Aboagye: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Resources; Software; Writing – review & editing.
Rohini Sharma: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Resources; Software; Supervision; Validation; Visualization; Roles/Writing – original draft; Writing – review & editing.
Article info
Publication history
Published online: October 05, 2022
Accepted:
September 8,
2022
Received:
August 1,
2022
Copyright
Crown Copyright © 2022 Published by Elsevier Ltd.