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Immune checkpoint blockade has emerged as a potent new tool in the war on cancer. However, only a subset of cancer patients benefit from this therapeutic modality, sparking a search for combination therapies to increase the fraction of responding patients. We argue here that inhibition of protein phosphatase 2A (PP2A) is a promising approach to increase responses to immune checkpoint blockade and other therapies that rely on the presence of tumor-reactive T cells. Inhibition of PP2A increases neoantigen expression on tumor cells, activates the cGAS/STING pathway, suppresses regulatory T cells, and increases cytotoxic T cell activation. In preclinical models, inhibition of PP2A synergizes with immune checkpoint blockade and emerging evidence indicates that patients who have tumors with mutations in PP2A respond better to immune checkpoint blockade. Therefore, inhibition of PP2A activity may be an effective way to sensitize cancer cells to immune checkpoint blockade and cell-based therapies using tumor-reactive T cells.

Sustained delivery of protein therapeutics remains a largely unsolved problem across anatomic locations. Miniaturized devices that can provide sustained delivery of protein formulations have the potential to address this challenge via minimally invasive administration. In particular, methodologies that can optimize protein formulation independent of device manufacture have the greatest potential to provide a platform suitable for wide applications. The techniques developed here demonstrate the fabrication of tubular devices for sustained release of protein therapeutics. Utilizing a dip-casting process, fine-scale tubes can be reliably produced with wall thickness down to 30 μm. Techniques were developed that enabled effective loading of either solid or liquid formulations, while maintaining a cylindrical form-factor compatible with placement in a 22-gauge needle. Further, highly compacted protein pellets that approach the expected density of the raw materials were produced with a diameter (∼300 μm) suitable for miniaturized devices. Release from a solid-loaded device was capable of sustaining release of a model protein in excess of 400 days. Given significant interest in ocular applications, intravitreal injection was demonstrated in a rabbit model with these devices. In addition, to simulate repeated injections in ocular applications, serial intravitreal injection of two devices in a rabbit model demonstrated acceptable ocular safety without significant intraocular inflammation from clinical exam and histology.

BRAFV600E mutation confers a poor prognosis in metastatic colorectal cancer (CRC) despite combinatorial targeted therapies based on the latest understanding of signaling circuitry. To identify parallel resistance mechanisms induced by BRAF-MEK-EGFR co-targeting, we used a high-throughput kinase activity mapping platform. Here we show that SRC kinases are systematically activated in BRAFV600E CRC following targeted inhibition of BRAF ± EGFR and that coordinated targeting of SRC with BRAF ± EGFR increases treatment efficacy in vitro and in vivo. SRC drives resistance to BRAF ± EGFR targeted therapy independently of ERK signaling by inducing transcriptional reprogramming through β-catenin (CTNNB1). The EGFR-independent compensatory activation of SRC kinases is mediated by an autocrine prostaglandin E2 loop that can be blocked with cyclooxygenase-2 (COX2) inhibitors. Co-targeting of COX2 with BRAF + EGFR promotes durable suppression of tumor growth in patient-derived tumor xenograft models. COX2 inhibition represents a drug-repurposing strategy to overcome therapeutic resistance in BRAFV600E CRC.

As a result of ongoing regional disparities, the United Network for Organ Sharing (UNOS) implemented policy in May 2019 limiting exception points for waitlisted patients with hepatocellular carcinoma (HCC) to median Model for End-Stage Liver Disease at transplant in the area surrounding a transplant center minus 3 points (MMAT-3). The impact of this policy change remains unknown. We included adult patients with HCC (n = 4567) and without HCC (n = 19,773) in the UNOS database added to the waiting list before this policy change (May 7, 2017-May 18, 2019) and after (May 19, 2019-March 7, 2020). Cumulative incidence analysis estimated the probability of dropout within 1 year of listing decreased from 12.9% before the policy to 11.1% after the policy in candidates without HCC and from 14% to 10.7% in candidates with HCC. Incidence rates of liver transplantation (LT) and waitlist dropout varied significantly before the policy in patients with HCC and without HCC but nearly equalized in the postpolicy era. These effects were observed in both shorter and longer wait regions. With policy change being modeled as a time-dependent covariate, competing risk regression analyses estimated a decreased risk of dropout after policy change in the non-HCC group (cause-specific hazard ratio, 0.91; P = 0.02) after adjusting for demographic variables. These results suggest that the MMAT-3 policy has successfully reduced disparities in access to LT including across UNOS wait regions, although certain patients with HCC are now disadvantaged.

PurposeCombined MAPK pathway inhibition using dual BRAF and MEK inhibitors has prolonged the duration of clinical response in patients with BRAFV600E-driven tumors compared with either agent alone. However, resistance frequently arises.Experimental designWe generated cell lines resistant to dual BRAF/MEK inhibition and utilized a pharmacologic synthetic lethal approach to identify a novel, adaptive resistance mechanism mediated through the fibroblast growth factor receptor (FGFR) pathway.ResultsIn response to drug treatment, transcriptional upregulation of FGF1 results in autocrine activation of FGFR, which potentiates extracellular signal-regulated kinases (ERK) activation. FGFR inhibition overcomes resistance to dual BRAF/MEK inhibitors in both cell lines and patient-derived xenograft (PDX) models. Abrogation of this bypass mechanism in the first-line setting enhances tumor killing and prevents the emergence of drug-resistant cells. Moreover, clinical data implicate serum FGF1 levels in disease prognosis.ConclusionsTaken together, these results describe a new, adaptive resistance mechanism that is more commonly observed in the context of dual BRAF/MEK blockade as opposed to single-agent treatment and reveal the potential clinical utility of FGFR-targeting agents in combination with BRAF and MEK inhibitors as a promising strategy to forestall resistance in a subset of BRAF-driven cancers.

Pancreatic islet transplantation is a promising treatment for type I diabetes, which is a chronic autoimmune disease in which the host immune cells attack insulin-producing beta cells. The impact of this therapy is limited due to tissue availability and dependence on immunosuppressive drugs that prevent immune rejection of the transplanted cells. These issues can be solved by encapsulating stem cell-derived insulin-producing cells in an immunoprotective device. However, encapsulation exacerbates ischemia, and the lack of vasculature at the implantation site post-transplantation worsens graft survival. Here, an encapsulation device that supplements nutrients to the cells is developed to improve the survival of encapsulated stem cell-derived insulin-producing cells in the poorly vascularized subcutaneous space. An internal compartment in the device is fabricated to provide zero-order release of alanine and glutamine for several weeks. The amino acid reservoir sustains viability of insulin-producing cells in nutrient limiting conditions in vitro. Moreover, the reservoir also increases cell survival by 30% after transplanting the graft in the subcutaneous space.

Topical eye-drop administration and intravitreal injections are the current standard for ocular drug delivery. However, patient adherence to the drug regimen and insufficient administration frequency are well-documented challenges to this field. In this review, we describe recent advances in intraocular implants designed to deliver therapeutics for months to years, to obviate the issues of patient adherence. We highlight recent advances in monolithic ocular implants in the literature, the commercialization pipeline, and approved for the market. We also describe design considerations based on material selection, active pharmaceutical ingredient, and implantation site.

Phosphorylation networks intimately regulate mechanisms of response to therapies. Mapping the phospho-catalytic profile of kinases in cells or tissues remains a challenge. Here, we introduce a practical high-throughput system to measure the enzymatic activity of kinases using biological peptide targets as phospho-sensors to reveal kinase dependencies in tumour biopsies and cell lines. A 228-peptide screen was developed to detect the activity of >60 kinases, including ABLs, AKTs, CDKs and MAPKs. Focusing on BRAFV600E tumours, we found mechanisms of intrinsic resistance to BRAFV600E-targeted therapy in colorectal cancer, including targetable parallel activation of PDPK1 and PRKCA. Furthermore, mapping the phospho-catalytic signatures of melanoma specimens identifies RPS6KB1 and PIM1 as emerging druggable vulnerabilities predictive of poor outcome in BRAFV600E patients. The results show that therapeutic resistance can be caused by the concerted upregulation of interdependent pathways. Our kinase activity-mapping system is a versatile strategy that innovates the exploration of actionable kinases for precision medicine.

Despite years of effort, sustained delivery of protein therapeutics remains an unmet need due to three primary challenges - dose, duration, and stability. The work presented here provides a design methodology for polycaprolactone reservoir-based thin film devices suitable for long-acting protein delivery to the back of the eye. First, the challenge of formulating highly concentrated protein in a device reservoir was addressed by improving stability with solubility-reducing excipients. Next, predictive correlations between design parameters and device performance were developed to provide a methodology to achieve a target product profile. Prototype devices were designed using this methodology to achieve desired device size, release rate, therapeutic payload, and protein stability, assessed by in vitro studies. Finally, prototype tolerability was established in a non-human primate model. The design methodology presented here is widely applicable to reservoir-based sustained delivery devices for proteins and provides a general device design framework.

ObjectiveInflammation is a key driver of excessive neointimal hyperplasia within vein grafts. Recent work demonstrates that specialized proresolving lipid mediators biosynthesized from omega-3 polyunsaturated fatty acids, such as resolvin D1 (RvD1), actively orchestrate the process of inflammation resolution. We investigated the effects of local perivascular delivery of RvD1 in a rabbit vein graft model.MethodsIpsilateral jugular veins were implanted as carotid interposition grafts through an anastomotic cuff technique in New Zealand white rabbits (3-4 kg; N = 80). RvD1 (1 μg) was delivered to the vein bypass grafts in a perivascular fashion, using either 25% Pluronic F127 gel (Sigma-Aldrich, St. Louis, Mo) or a thin bilayered poly(lactic-co-glycolic acid) (PLGA) film. No treatment (bypass only) and vehicle-loaded Pluronic gels or PLGA films served as controls. Delivery of RvD1 to venous tissue was evaluated 3 days later by liquid chromatography-tandem mass spectrometry. Total leukocyte infiltration, macrophage infiltration, and cell proliferation were evaluated by immunohistochemistry. Elastin and trichrome staining was performed on grafts harvested at 28 days after bypass to evaluate neointimal hyperplasia and vein graft remodeling.ResultsPerivascular treatments did not influence rates of graft thrombosis (23%), major wound complications (4%), or death (3%). Leukocyte (CD45) and macrophage (RAM11) infiltration was significantly reduced in the RvD1 treatment groups vs controls at 3 days (60%-72% reduction; P < .01). Cellular proliferation (Ki67 index) was also significantly lower in RvD1-treated vs control grafts at 3 days (40%-50% reduction; P < .01). Treatment of vein grafts with RvD1-loaded gels reduced neointimal thickness at 28 days by 61% vs bypass only (P < .001) and by 63% vs vehicle gel (P < .001). RvD1-loaded PLGA films reduced neointimal formation at 28 days by 50% vs bypass only (P < .001). RvD1 treatment was also associated with reduced collagen deposition in vein grafts at 28 days.ConclusionsLocal perivascular delivery of RvD1 attenuates vein graft hyperplasia without associated toxicity in a rabbit carotid bypass model. This effect appears to be mediated by both reduced leukocyte recruitment and decreased cell proliferation within the graft. Perivascular PLGA films may also impart protection through biomechanical scaffolding in this venous arterialization model. Our studies provide further support for the potential therapeutic role of specialized proresolving lipid mediators such as D-series resolvins in modulating vascular injury and repair.

Purpose: We hypothesized that mutations in homologous recombination repair (HRR) genes beyond BRCA1 and BRCA2 improve outcomes for ovarian carcinoma patients treated with platinum therapy and would impact the relative benefit of adding prolonged bevacizumab.Experimental Design: We sequenced DNA from blood and/or neoplasm from 1,195 women enrolled in GOG-0218, a randomized phase III trial in advanced ovarian carcinoma of bevacizumab added to carboplatin and paclitaxel. Defects in HRR were defined as damaging mutations in 16 genes. Proportional hazards models were used to estimate relative hazards for progression-free survival (PFS) and overall survival (OS).Results: Of 1,195 women with ovarian carcinoma, HRR mutations were identified in 307 (25.7%). Adjusted hazards for progression and death compared with those without mutations were lower for women with non-BRCA HRR mutations [HR = 0.73; 95% confidence interval (CI), 0.57-0.94; P = 0.01 for PFS; HR = 0.67; 95% CI, 0.50-0.90; P = 0.007 for OS] and BRCA1 mutations (HR = 0.80; 95% CI, 0.66-0.97; P = 0.02 for PFS; HR = 0.74; 95% CI, 0.59-0.94; P = 0.01 for OS) and were lowest for BRCA2 mutations (HR = 0.52; 95% CI, 0.40-0.67; P < 0.0001 for PFS; HR = 0.36; 95% CI, 0.25-0.53; P < 0.0001 for OS). A test of interaction showed no difference in the effect of bevacizumab on PFS between cases with and without mutations.Conclusions: HRR mutations, including non-BRCA genes, significantly prolong PFS and OS in ovarian carcinoma and should be stratified for in clinical trials. The benefit of adding bevacizumab was not significantly modified by mutation status. Clin Cancer Res; 24(4); 777-83. ©2017 AACR.

BackgroundPatients with BRCA1-like tumors correlate with improved response to DNA double-strand break-inducing therapy. A gene expression-based classifier was developed to distinguish between BRCA1-like and non-BRCA1-like tumors. We hypothesized that these tumors may also be more sensitive to PARP inhibitors than standard treatments.MethodsA diagnostic gene expression signature (BRCA1ness) was developed using a centroid model with 128 triple-negative breast cancer samples from the EU FP7 RATHER project. This BRCA1ness signature was then tested in HER2-negative patients (n = 116) from the I-SPY 2 TRIAL who received an oral PARP inhibitor veliparib in combination with carboplatin (V-C), or standard chemotherapy alone. We assessed the association between BRCA1ness and pathologic complete response in the V-C and control arms alone using Fisher's exact test, and the relative performance between arms (biomarker × treatment interaction, likelihood ratio p

Encapsulation of human embryonic stem-cell-differentiated beta cell clusters (hES-βC) holds great promise for cell replacement therapy for the treatment of diabetics without the need for chronic systemic immune suppression. Here, we demonstrate a nanoporous immunoprotective polymer thin film cell encapsulation device that can exclude immune molecules while allowing exchange of oxygen and nutrients necessary for in vitro and in vivo stem cell viability and function. Biocompatibility studies show the device promotes neovascular formation with limited foreign body response in vivo. The device also successfully prevented teratoma escape into the peritoneal cavity of mice. Long-term animal studies demonstrate evidence of engraftment, viability, and function of cells encapsulated in the device after 6 months. Finally, in vivo study confirms that the device was able to effectively immuno-isolate cells from the host immune system.

Current administration of ranibizumab and other therapeutic macromolecules to the vitreous and retina carries ocular risks, a high patient treatment burden, and compliance barriers that can lead to suboptimal treatment. Here we introduce a device that produces sustained release of ranibizumab in the vitreous cavity over the course of several months. Composed of twin nanoporous polymer thin films surrounding a ranibizumab reservoir, these devices provide release of ranibizumab over 16 weeks in vitro and 12 weeks in vivo, without exhausting the initial drug payload. Following implantation in vivo, devices were well-tolerated and showed no sign of immune response. This platform presents a potential solution to the challenge of delivering protein therapeutics to the vitreous and retina for sustained periods of time.