USP7 inhibitors suppress tumour neoangiogenesis and promote synergy with immune checkpoint inhibitors by downregulating fibroblast VEGF.

BACKGROUND: Understanding how to modulate the microenvironment of tumors that are resistant to immune checkpoint inhibitors represents a major challenge in oncology.Here we investigate the ability of USP7 inhibitors to reprogram the tumor microenvironment (TME) by inhibiting secretion of vascular endothelial growth factor (VEGF) from fibroblasts. METHODS: To understand the role played by USP7 in the TME, we systematically evaluated the effects of potent, selective USP7 inhibitors on co-cultures comprising components of the TME, using human primary cells. We also evaluated the effects of USP7 inhibition on tumor growth inhibition in syngeneic models when dosed in combination with immune checkpoint inhibitors (ICIs). RESULTS: Abrogation of VEGF secretion from fibroblasts in response to USP7 inhibition resulted in inhibition of tumor neoangiogenesis and increased tumor recruitment of CD8-positive T-lymphocytes, leading to significantly improved sensitivity to immune checkpoint inhibitors. In syngeneic models, treatment with USP7 inhibitors led to striking tumor responses resulting in significantly improved survival. CONCLUSIONS: USP7-mediated reprograming of the TME is not linked to its previously characterized role in modulating MDM2 but does require p53 and UHRF1 in addition to the well-characterized VEGF transcription factor, HIF-1α. This represents a function of USP7 that is unique to fibroblasts, and which is not observed in cancer cells or other components of the TME. Given the potential for USP7 inhibitors to transform "immune desert" tumors into "immune responsive" tumors, this paves the way for a novel therapeutic strategy combining USP7 inhibitors with immune checkpoint inhibitors (ICIs).

Successful Proof-of-Concept for Topical Delivery of Novel Peptide ALM201 with Potential Usefulness for Treating Neovascular Eye Disorders.

Purpose: To evaluate the therapeutic benefit of a novel peptide, ALM201, in ocular pathologic vascularization. Design: Experimental study in mouse, rat, and rabbit animal models. Participants: Ten-week-old Lister Hooded male rats, 8-week-old Brown Norway male rats, 9-day-old C57BL/6J mice, and 12-month-old New Zealand male rabbits. Methods: Corneal vascularization was scored for vessel density and vessel distance to suture in a rat corneal suture model. Ocular penetration and biodistribution were evaluated by matrix-assisted laser desorption/ionization mass spectrometry imaging after topical ALM201 application to rabbit eyes. A mouse choroidal sprouting assay, with aflibercept as positive control, was used to evaluate choroidal neovascularization (CNV) in the posterior segment tissue. Efficacy of topical ALM201 was assessed using a rat laser CNV model of neovascular age-related macular degeneration. Main Outcome Measures: Clinical scoring and histologic analysis of vascularized corneas, sprouting area, lesion size, and vessel leakiness in posterior segments. Results: Assessment of ALM201 treatment in the rat corneal suture model showed a significant decrease in vessel density (P = 0.0065) and vessel distance to suture (P = 0.021) compared with vehicle control (phosphate-buffered saline [PBS]). Infiltration of inflammatory cells into the corneal stroma also was reduced significantly compared with PBS (724.5 ± 122 cells/mm2 vs. 1837 ± 195.9 cells/mm2, respectively; P = 0.0029). Biodistribution in rabbit eyes confirmed ALM201 bioavailability in anterior and posterior ocular segments 1 hour after topical instillation. ALM201 treatment significantly suppressed choroid vessel sprouting when compared with PBS treatment (44.5 ± 14.31 pixels vs. 120.9 ± 33.37 pixels, respectively; P = 0.04) and was not inferior to aflibercept (65.63 ± 11.86 pixels; P = 0.7459). Furthermore, topical ALM201 significantly improved vessel leakiness (leakage scores: 2.1 ± 0.7 vs. 2.9 ± 0.1; P = 0.0274) and lesion size (144,729 ± 33,239 µm3 vs. 187,923 ± 28,575 µm3; P = 0.03) in the rat laser CNV model when compared with topical PBS vehicle. Conclusions: ALM201 is a promising novel molecule with anti-inflammatory and antivascularization activity and is a strong candidate to meet the clinical need of a new, topically delivered therapeutic agent for treating inflammation and pathologic vascularization in the anterior and posterior segments of the eye.

A first-in-human Phase I dose-escalation trial of the novel therapeutic peptide, ALM201, demonstrates a favourable safety profile in unselected patients with ovarian cancer and other advanced solid tumours.

BACKGROUND: We aimed to assess the safety, tolerability and pharmacokinetics of a novel anti-angiogenic peptide. METHODS: We used an open-label, multicentre, dose-escalation Phase I trial design in patients with solid tumours. ALM201 was administered subcutaneously once daily for 5 days every week in unselected patients with solid tumours. RESULTS: Twenty (8 male, 12 female) patients with various solid tumours were treated (18 evaluable for toxicity) over eight planned dose levels (10-300 mg). ALM201 was well-tolerated at all dose levels without CTCAE grade 4 toxicities. Adverse events were predominantly grades 1-2, most commonly, localised injection-site reactions (44.4%), vomiting (11%), fatigue (16.7%), arthralgia (5.6%) and headache (11%). Thrombosis occurred in two patients at the 100 mg and 10 mg dose levels. The MTD was not reached, and a recommended Phase II dose (RP2D) based on feasibility was declared. Plasma exposure increased with dose (less than dose-proportional at the two highest dose levels). No peptide accumulation was evident. The median treatment duration was 11.1 (range 3-18) weeks. Four of 18 evaluable patients (22%) had stable disease. CONCLUSIONS: Doses up to 300 mg of ALM201 subcutaneously are feasible and well-tolerated. Further investigation of this agent in selected tumour types/settings would benefit from patient-selection biomarkers.

A Three-Dimensional In Vitro Coculture Model to Quantify Breast Epithelial Cell Adhesion to Endothelial Cells.

Three-dimensional (3D) in vitro culture models better recapitulate the tissue microenvironment, and therefore may provide a better platform to evaluate therapeutic effects on adhesive cell-cell interactions. The objective of this study was to determine if AD-01, a peptide derivative of FK506-binding protein like that is reported to bind to the adhesion receptor CD44, would induce a greater reduction in breast epithelial spheroid adhesion to endothelial tube-like networks in our 3D coculture model system compared to two-dimensional (2D) culture. MCF10A, MCF10A-NeuN, MDA-MB-231, and MCF7 breast epithelial cells were pretreated with AD-01 either as single cells or as spheroids. Breast epithelial cell adhesion to 2D tissue culture substrates was first measured, followed by spheroid formation (breast cell-cell adhesion) and spheroid adhesion to Matrigel or endothelial networks. Finally, CD44 expression was quantified in breast epithelial cells in 2D and 3D culture. Our results show that AD-01 had the largest effect on spheroid formation, specifically in breast cancer cell lines. AD-01 also inhibited breast cancer spheroid adhesion to and migration along endothelial networks. The different breast epithelial cell lines expressed more CD44 when cultured as 3D spheroids, but this did not universally translate into higher protein levels. This study shows that 3D coculture models can enable unique insights into cell adhesion, migration, and cell-cell interactions, thereby enhancing understanding of basic biological mechanisms. Furthermore, such 3D coculture systems may also represent a more relevant testing platform for understanding the mechanism-of-action of new therapeutic agents. Impact Statement Cell adhesion is inherently different in two dimensional (2D) compared to three dimensional (3D) culture; yet, most adhesion assays in academia and industry are still conducted in 2D because few simple, yet effective, adhesion models exist in 3D. Recently we developed a 3D in vitro coculture model to examine breast epithelial spheroid interactions with endothelial tubes. We now show that this 3D coculture model can effectively be used to interrogate and quantify drug-induced differences in breast epithelial cell adhesion that are unique to 3D cocultures. This 3D coculture adhesion model can furthermore be modified for use with other cell types to better predict drug effects on cell-vasculature adhesion.

The PARP Inhibitor AZD2461 Provides Insights into the Role of PARP3 Inhibition for Both Synthetic Lethality and Tolerability with Chemotherapy in Preclinical Models.

The PARP inhibitor AZD2461 was developed as a next-generation agent following olaparib, the first PARP inhibitor approved for cancer therapy. In BRCA1-deficient mouse models, olaparib resistance predominantly involves overexpression of P-glycoprotein, so AZD2461 was developed as a poor substrate for drug transporters. Here we demonstrate the efficacy of this compound against olaparib-resistant tumors that overexpress P-glycoprotein. In addition, AZD2461 was better tolerated in combination with chemotherapy than olaparib in mice, which suggests that AZD2461 could have significant advantages over olaparib in the clinic. However, this superior toxicity profile did not extend to rats. Investigations of this difference revealed a differential PARP3 inhibitory activity for each compound and a higher level of PARP3 expression in bone marrow cells from mice as compared with rats and humans. Our findings have implications for the use of mouse models to assess bone marrow toxicity for DNA-damaging agents and inhibitors of the DNA damage response. Finally, structural modeling of the PARP3-active site with different PARP inhibitors also highlights the potential to develop compounds with different PARP family member specificity profiles for optimal antitumor activity and tolerability. Cancer Res; 76(20); 6084-94. ©2016 AACR.

Clinical development of new drug-radiotherapy combinations.

In countries with the best cancer outcomes, approximately 60% of patients receive radiotherapy as part of their treatment, which is one of the most cost-effective cancer treatments. Notably, around 40% of cancer cures include the use of radiotherapy, either as a single modality or combined with other treatments. Radiotherapy can provide enormous benefit to patients with cancer. In the past decade, significant technical advances, such as image-guided radiotherapy, intensity-modulated radiotherapy, stereotactic radiotherapy, and proton therapy enable higher doses of radiotherapy to be delivered to the tumour with significantly lower doses to normal surrounding tissues. However, apart from the combination of traditional cytotoxic chemotherapy with radiotherapy, little progress has been made in identifying and defining optimal targeted therapy and radiotherapy combinations to improve the efficacy of cancer treatment. The National Cancer Research Institute Clinical and Translational Radiotherapy Research Working Group (CTRad) formed a Joint Working Group with representatives from academia, industry, patient groups and regulatory bodies to address this lack of progress and to publish recommendations for future clinical research. Herein, we highlight the Working Group's consensus recommendations to increase the number of novel drugs being successfully registered in combination with radiotherapy to improve clinical outcomes for patients with cancer.

Tumor growth inhibition by olaparib in BRCA2 germline-mutated patient-derived ovarian cancer tissue xenografts.

PURPOSE: Most patients with ovarian carcinomas succumb to their disease and there is a critical need for improved therapeutic approaches. Carcinomas arising in BRCA mutation carriers display defective DNA double-strand break repair that can be therapeutically exploited by inhibition of PARP-1, a key enzyme in the repair of DNA single-strand breaks, creating synthetic lethality in tumor cells. EXPERIMENTAL DESIGN: To investigate synthetic lethality in vivo, we established a BRCA2 germline-mutated xenograft model that was developed directly from human ovarian cancer tissue, treated with the PARP inhibitor olaparib (AZD2281) alone and in combination with carboplatin. RESULTS: We show that olaparib alone and in combination with carboplatin greatly inhibit growth in BRCA2-mutated ovarian serous carcinoma. This effect was not observed in a serous carcinoma with normal BRCA function, showing a specific antitumor effect of olaparib in mutation carriers. Immunohistochemistry (cleaved caspase-3 and Ki-67 stains) of remnant tissue after olaparib treatment revealed significantly decreased proliferation and increased apoptotic indices in these tumors compared with untreated controls. Furthermore, olaparib-treated tumors showed highly reduced PARP-1 activity that correlated with olaparib levels. CONCLUSIONS: We established a BRCA2-mutated human ovarian cancer xenograft model suitable for experimental drug testing. The demonstrated in vivo efficacy of olaparib extends on the preclinical rationale for further clinical trials targeting ovarian cancer patients with BRCA mutations.

RET is constitutively activated by novel tandem mutations that alter the active site resulting in multiple endocrine neoplasia type 2B.

Constitutive activation of the RET receptor tyrosine kinase underlies the genesis and progression of multiple endocrine neoplasia type 2 (MEN 2), a dominantly inherited cancer predisposition. Importantly, although kinase activation represents a common theme in neoplasias, not all activating mutations are functionally equivalent. Consistent with this, we ascertained a patient with classical features of MEN 2B, but lacking either of the classical mutations in RET (M918T or A883F). Instead, the patient harbors a novel pair of germ line missense mutations in cis at codons 804 and 805. We evaluated the potential physiochemical effects of these substitutions in silico, predicting both to be moderately deleterious in isolation, but severely deleterious in combination. Consistent with this postulate, we show that the identified tandem mutations (V804M/E805K) are biologically active, transforming cells in culture and that their transforming capacity in combination is distinctly synergistic. Furthermore, the V804M/E805K tandem lesion confers resistance to the small molecule receptor tyrosine kinase inhibitor, PP1, suggesting a mode of action distinct from that known for classical MEN 2B mutations. To address this question, we used homology molecular modeling in silico to model the active site of RET. We predict that RET804 constitutes a critical gatekeeper residue that, when mutated in combination with RET805, induces a conformational change in the hinge region that locks the active site in a position permissive for ATP hydrolysis. Our findings have implications both in the clinic and in the successful development of novel kinase-targeted anticancer drugs.

The Ret(C620R) mutation affects renal and enteric development in a mouse model of Hirschsprung's disease.

In rare families RET tyrosine kinase receptor substitutions located in exon 10 (especially at positions 609, 618, and 620) can concomitantly cause the MEN 2A (multiple endocrine neoplasia type 2A) or FMTC (familial medullary thyroid carcinoma) cancer syndromes, and Hirschsprung's disease (HSCR). No animal model mimicking the co-existence of the MEN 2 pathology and HSCR is available, and the association of these activating mutations with a developmental defect still represents an unresolved problem. The aim of this work was to investigate the significance of the RET(C620R) substitution in the pathogenesis of both gain- and loss-of-function RET-associated diseases. We report the generation of a line of mice carrying the C620R mutation in the Ret gene. Although Ret(C620R) homozygotes display severe defects in kidney organogenesis and enteric nervous system development leading to perinatal lethality. Ret(C620R) heterozygotes recapitulate features characteristic of HSCR including hypoganglionosis of the gastrointestinal tract. Surprisingly, heterozygotes do not show any defects in the thyroid that might be attributable to a gain-of-function mutation. The Ret(C620R) allele is responsible for HSCR and affects the development of kidneys and the enteric nervous system (ENS). These mice represent an interesting model for studying new therapeutic approaches for the treatment of HSCR disease.

Novel SRESPHP peptide mediates specific binding to primary medullary thyroid carcinoma after systemic injection.

The efficient and specific introduction of genes into cancer cells in vivo remains a major challenge for current gene therapy modalities. Peptides possess appropriate properties to serve as tumor-targeting agents. Thus, finding new cancer-selective peptides directing gene transfer to neoplastic cells by reducing transduction of normal cells is a central goal for molecular targeting. We have previously reported identification of a peptide (HTFEPGV) that selectively binds to human medullary thyroid carcinoma (MTC)-derived TT cells in vitro and transplanted tumor xenografts in vivo, using phage display. In the present study, we have performed this approach in primary orthotopically growing murine MTCs of RET-C634R transgenic mice as a clinically relevant model for thyroid cancer by intravenous injection of a complex peptide library. Two rounds of screening on primary tumors yielded multiple copies of a phage that displays a cyclic 7-amino acid peptide, SRESPHP, with a 3000-fold increase in titer between rounds 1 and 2. The selected phage showed highly specific binding to the tumor after systemic administration, whereas binding to other organs such as lung, liver, kidney, and heart was reduced up to 90%. After tail vein injection, homing to the tumor was substantially reduced in the presence of synthetic SRESPHP peptide, indicating that tumor phage interaction strictly depends on the displayed peptide. Immunohistochemical analysis of paraffin sections from mouse tissues revealed direct binding of the SRESPHP peptide to MTC tissue. Moreover, this peptide also mediates binding to human MTC cells in vitro and in vivo, suggesting abundant expression of its cognate receptor in murine and human medullary thyroid carcinoma. Because the SRESPHP peptide is also efficiently internalized into MTC cells, it likely provides the basis for a new selective therapy of medullary thyroid carcinoma.

Role of MEN2A-derived RET in maintenance and proliferation of medullary thyroid carcinoma.

BACKGROUND: Dominant-activating mutations in the RET protooncogene, a receptor tyrosine kinase, have been identified as a cause of medullary thyroid carcinoma. Such oncogenic RET mutations induce its ligand-independent constitutive trans-autophosphorylation. We investigated the role of endogenous oncogenic RET autophosphorylation in maintaining the neoplastic phenotype in medullary thyroid carcinoma cells and orthotopic medullary thyroid carcinomas in RET transgenic mice. METHODS: We constructed adenoviral vectors expressing a dominant-negative truncated form of RET, termed RET(DeltaTK), and analyzed its effect on cell viability, apoptosis, and proliferation of TT medullary thyroid carcinoma cells. We investigated the effect of RET(DeltaTK) on downsteam signaling by assessing alterations in phosphorylation or in gene expression. The effect of RET(DeltaTK) in primary medullary thyroid carcinomas in transgenic mice was assessed by monitoring tumor growth. All statistical tests were two-sided. RESULTS: Cell viability was reduced. Phosphorylation of Akt and extracellular signal-regulated kinase (ERK), components of downstream signal transduction pathways, was abolished, and cell cycle progression was reduced. Expression of cell cycle regulator cyclin D1 was decreased, and expression of cell cyle regulators p21(CIP1/WAF1) and p27(KIP1) was increased. Apoptosis was stimulated and concurrently the expression of BCL-2 was decreased. All in vitro experiments compared TT cells expressing RET(DeltaTK) with untreated control cells or control vector-treated cells. Furthermore, 2 weeks after injecting adenovirus-carrying RET(DeltaTK) into thyroid glands of transgenic mice with orthotopic medullary thyroid carcinoma, tumors were statistically significantly smaller than their initial size in mice treated with RET(DeltaTK) (43.6%, 95% confidence interval [CI] = 30.7% to 56.5%; P =.010; two-sided unpaired Student's t test), whereas tumors in mice treated with a control vector were larger than their initial size (139.8%, 95% CI = 120.3% to 159.3%; P<.001). CONCLUSION: Selective disruption of oncogenic RET signaling in medullary thyroid carcinoma in vitro and in vivo is associated with loss of the neoplastic phenotype of medullary thyroid carcinoma and should be investigated further as the basis for new therapeutic approaches for this disease.

Modulation of medullary thyroid carcinoma penetrance suggests the presence of modifier genes in a RET transgenic mouse model.

We described previously a thyroid phenotype for transgenic mice expressing an activated Ret oncogene driven from a human calcitonin promoter. Medullary thyroid carcinomas (MTC), a tumor of the thyroid parafollicular C cells, occur in this transgenic line with a pathology analogous to that seen in patients with multiple endocrine neoplasia type 2 (MEN2). When the transgene was introgressed onto four different genetic backgrounds, between 0 and 98% of transgenic progeny developed thyroid tumors by 10 months of age, indicating that tumor penetrance could be modulated by genetic background. Furthermore, tumors on the CBA/ca and C57BL/6J backgrounds were significantly larger than those arising in BALB/c transgenic mice. These results are relevant to human MEN2 disease, because this model system may be used to study genes modifying thyroid tumor penetrance in the dominantly inherited human cancer syndrome.