Genome expansion by a CRISPR trimmer-integrase.

CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity1. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1-Cas2 integrase is necessary but not sufficient2-5. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation6,7, but many CRISPR-Cas systems lack Cas48. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1-Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited9,10 exonucleases for faithful acquisition of new CRISPR immune sequences.

Dotatate PET/CT and 225Ac-Dotatate Therapy for Somatostatin Receptor-expressing Metastatic Breast Cancer.

Background Somatostatin receptors, and specifically somatostatin receptor type 2 (SSTR2), have primarily been associated with neuroendocrine tumors and have revolutionized the imaging and therapy of patients with these tumors. SSTR2 is expressed on other tumors at lower prevalence. Purpose To evaluate the potential of SSTR2-targeted imaging and therapy in patients with breast cancer. Materials and Methods In a preclinical experiment, SSTR2 expression was assessed in tissue microarrays of breast cancer samples using H-score analysis. H-scores higher than 50 (0-300 scale) were considered positive. Then, a prospective phase 2 clinical trial of SSTR2-targeted tetraazacyclododecane tetraacetic acid octreotate (Dotatate) PET/CT was performed in participants with biopsy-proven estrogen receptor (ER)-positive breast cancer from January to August 2023. A positive Dotatate PET/CT scan was defined as tumors with a Krenning score of 3 (avidity greater than liver) or 4 (avidity greater than spleen). The proportion of positive scans and the 95% CI were calculated. One participant with metastatic ER-positive breast cancer and a Krenning 4 Dotatate PET/CT result underwent treatment with SSTR2-targeted actinium 225 (225Ac) Dotatate. Results Preclinical microarrays demonstrated that 63 of 123 ER-positive breast cancer tissue samples (51% [95% CI: 42, 60]) but only 22 of 121 ER-negative breast cancer tissue samples (18% [95% CI: 12, 26]) were enriched for SSTR2 (P < .001). Thirty female participants (mean age, 66 years ± 15) with metastatic ER-positive breast cancer were accrued to the phase 2 SSTR2-targeted imaging trial and underwent Dotatate PET/CT. Dotatate PET/CT demonstrated that nine of 30 participants (30% [95% CI: 15, 49]) had tumors with Krenning scores of 3 or 4, indicating strong SSTR2 expression. SSTR2-targeted therapy with alpha-emitting 225Ac-Dotatate resulted in a near complete response in a heavily pretreated participant with metastatic ER-positive breast cancer and a Krenning 4 Dotatate PET result. Conclusion Molecular imaging targeting SSTR2 and radioligand therapy with SSTR2-targeted 225Ac-Dotatate enables a new therapeutic option for patients with metastatic breast cancer. Clinical trial registration no. NCT05880394 © RSNA, 2024 See also the editorial by Lin and Choyke in this issue.

Fibroblast growth factor receptors in cancer: genetic alterations, diagnostics, therapeutic targets and mechanisms of resistance.

Fibroblast growth factor receptors (FGFRs) are aberrantly activated through single-nucleotide variants, gene fusions and copy number amplifications in 5-10% of all human cancers, although this frequency increases to 10-30% in urothelial carcinoma and intrahepatic cholangiocarcinoma. We begin this review by highlighting the diversity of FGFR genomic alterations identified in human cancers and the current challenges associated with the development of clinical-grade molecular diagnostic tests to accurately detect these alterations in the tissue and blood of patients. The past decade has seen significant advancements in the development of FGFR-targeted therapies, which include selective, non-selective and covalent small-molecule inhibitors, as well as monoclonal antibodies against the receptors. We describe the expanding landscape of anti-FGFR therapies that are being assessed in early phase and randomised controlled clinical trials, such as erdafitinib and pemigatinib, which are approved by the Food and Drug Administration for the treatment of FGFR3-mutated urothelial carcinoma and FGFR2-fusion cholangiocarcinoma, respectively. However, despite initial sensitivity to FGFR inhibition, acquired drug resistance leading to cancer progression develops in most patients. This phenomenon underscores the need to clearly delineate tumour-intrinsic and tumour-extrinsic mechanisms of resistance to facilitate the development of second-generation FGFR inhibitors and novel treatment strategies beyond progression on targeted therapy.

Persistent Sputtering Yield Reduction in Plasma-Infused Foams.

Aluminum microfoams are found to exhibit persistent sputtering yield reductions of 40%-80% compared to a flat aluminum surface under 100 to 300 eV argon plasma bombardment. An analytical model reveals a strong dependency of the yield on the foam geometry and plasma sheath. For foam pore sizes near or larger than the sheath thickness, the plasma infuses the foam and transitions the plasma-surface interactions from superficial to volumetric phenomena. By defining a plasma infusion parameter, the sputtering behavior of foams is shown to be separated into the plasma-facing and plasma-infused regimes. While plasma infusion leads to a larger effective sputtering area, geometric recapture of ejected particles facilitates an overall reduction in yield. For a given level of plasma infusion, the reductions in normalized yield are more pronounced at lower ion energies since angular sputtering effects enable more effective geometric recapture of sputterants.

Infigratinib in upper tract urothelial carcinoma versus urothelial carcinoma of the bladder and its association with comprehensive genomic profiling and/or cell-free DNA results.

BACKGROUND: Infigratinib (BGJ398) is a potent and selective fibroblast grown factor receptor 1 to 3 (FGFR1-3) inhibitor with significant activity in patients with advanced or metastatic urothelial carcinoma bearing FGFR3 alterations. Given the distinct biologic characteristics of upper tract urothelial carcinoma (UTUC) and urothelial carcinoma of the bladder (UCB), the authors examined whether infigratinib had varying activity in these settings. METHODS: Eligible patients had metastatic urothelial carcinoma with activating FGFR3 mutations and/or fusions. Comprehensive genomic profiling was performed on formalin-fixed, paraffin-embedded tissues. Blood was collected for cell-free DNA analysis using a 600-gene panel. Patients received infigratinib at a dose of 125 mg orally daily (3 weeks on/1 week off) until disease progression or intolerable toxicity occurred. The overall response rate (ORR; partial response [PR] plus complete response [CR]) and disease control rate (DCR; CR plus PR plus stable disease [SD]) were characterized. RESULTS: A total of 67 patients were enrolled; the majority (70.1%) had received ≥2 prior antineoplastic therapies. In 8 patients with UTUC, 1 CR and 3 PRs were observed (ORR, 50%); the remaining patients achieved a best response of SD (DCR, 100%). In patients with UCB, 13 PRs were observed (ORR, 22%), and 22 patients had a best response of SD (DCR, 59.3%). Notable differences in genomic alterations between patients with UTUC and those with UCB included higher frequencies of FGFR3-TACC3 fusions (12.5% vs 6.8%) and FGFR3 R248C mutations (50% vs 11.9%), and a lower frequency of FGFR3 S249C mutations (37.5% vs 59.3%). CONCLUSIONS: Differences in the cumulative genomic profile were observed between patients with UTUC and those with UCB in the current FGFR3-restricted experience, underscoring the distinct biology of these diseases. These results support a planned phase 3 adjuvant study predominantly performed in this population.

Cysteine as a Monothiol Reducing Agent to Prevent Copper-Mediated Oxidation of Interferon Beta During PEGylation by CuAAC.

Bioconjugation by copper-catalyzed azide-alkyne cycloaddition (CuAAC) provides a powerful means to produce site-specifically modified proteins. However, the use of a copper catalyst brings about the possible generation of reactive oxygen species that could cause degradation of vulnerable amino acid residues. We investigated whether PEGylation by CuAAC caused any modifications to the therapeutic protein interferon beta-1b, which was produced via global amino acid substitution with azidohomo-alanine at the N-terminus and contains no methionine residues. Using previously reported reaction conditions, LC-MS peptide mapping detected +32 Da and +48 Da oxidation modifications of tryptic peptides 28-33 (LEYCLK) and 137-147 (EYSHCAWTIVR) in the protein post-PEGylation. The oxidative degradation increased with reaction time, whereas reducing the copper concentration slowed the PEGylation rate as well as the oxidation rate. Replacing dithiothreitol (DTT) with any of five different monothiol reducing agents in anaerobic conditions allowed efficient PEGylation in 2-4 h and abrogated oxidative degradation. Free cysteine provided reproducible reaction results as a reducing agent in this system and has been successfully applied to other protein conjugations. Monothiol reducing agents, such as cysteine, may be useful tools as protective reducing agents for CuAAC in some bioconjugation systems.

Genetically Encoded Azide Containing Amino Acid in Mammalian Cells Enables Site-Specific Antibody-Drug Conjugates Using Click Cycloaddition Chemistry.

Antibody-drug conjugates (ADC) have emerged as potent antitumor drugs that provide increased efficacy, specificity, and tolerability over chemotherapy for the treatment of cancer. ADCs generated by targeting cysteines and lysines on the antibody have shown efficacy, but these products are heterogeneous, and instability may limit their dosing. Here, a novel technology is described that enables site-specific conjugation of toxins to antibodies using chemistry to produce homogeneous, potent, and highly stable conjugates. We have developed a cell-based mammalian expression system capable of site-specific integration of a non-natural amino acid containing an azide moiety. The azide group enables click cycloaddition chemistry that generates a stable heterocyclic triazole linkage. Antibodies to Her2/neu were expressed to contain N6-((2-azidoethoxy)carbonyl)-l-lysine at four different positions. Each site allowed over 95% conjugation efficacy with the toxins auristatin F or a pyrrolobenzodiazepine (PBD) dimer to generate ADCs with a drug to antibody ratio of >1.9. The ADCs were potent and specific in in vitro cytotoxicity assays. An anti Her2/neu conjugate demonstrated stability in vivo and a PBD containing ADC showed potent efficacy in a mouse tumor xenograph model. This technology was extended to generate fully functional ADCs with four toxins per antibody. The high stability of the azide-alkyne linkage, combined with the site-specific nature of the expression system, provides a means for the generation of ADCs with optimized pharmacokinetic, biological, and biophysical properties.

SwinCross: Cross-modal Swin transformer for head-and-neck tumor segmentation in PET/CT images.

BACKGROUND: Radiotherapy (RT) combined with cetuximab is the standard treatment for patients with inoperable head and neck cancers. Segmentation of head and neck (H&N) tumors is a prerequisite for radiotherapy planning but a time-consuming process. In recent years, deep convolutional neural networks (DCNN) have become the de facto standard for automated image segmentation. However, due to the expensive computational cost associated with enlarging the field of view in DCNNs, their ability to model long-range dependency is still limited, and this can result in sub-optimal segmentation performance for objects with background context spanning over long distances. On the other hand, Transformer models have demonstrated excellent capabilities in capturing such long-range information in several semantic segmentation tasks performed on medical images. PURPOSE: Despite the impressive representation capacity of vision transformer models, current vision transformer-based segmentation models still suffer from inconsistent and incorrect dense predictions when fed with multi-modal input data. We suspect that the power of their self-attention mechanism may be limited in extracting the complementary information that exists in multi-modal data. To this end, we propose a novel segmentation model, debuted, Cross-modal Swin Transformer (SwinCross), with cross-modal attention (CMA) module to incorporate cross-modal feature extraction at multiple resolutions. METHODS: We propose a novel architecture for cross-modal 3D semantic segmentation with two main components: (1) a cross-modal 3D Swin Transformer for integrating information from multiple modalities (PET and CT), and (2) a cross-modal shifted window attention block for learning complementary information from the modalities. To evaluate the efficacy of our approach, we conducted experiments and ablation studies on the HECKTOR 2021 challenge dataset. We compared our method against nnU-Net (the backbone of the top-5 methods in HECKTOR 2021) and other state-of-the-art transformer-based models, including UNETR and Swin UNETR. The experiments employed a five-fold cross-validation setup using PET and CT images. RESULTS: Empirical evidence demonstrates that our proposed method consistently outperforms the comparative techniques. This success can be attributed to the CMA module's capacity to enhance inter-modality feature representations between PET and CT during head-and-neck tumor segmentation. Notably, SwinCross consistently surpasses Swin UNETR across all five folds, showcasing its proficiency in learning multi-modal feature representations at varying resolutions through the cross-modal attention modules. CONCLUSIONS: We introduced a cross-modal Swin Transformer for automating the delineation of head and neck tumors in PET and CT images. Our model incorporates a cross-modality attention module, enabling the exchange of features between modalities at multiple resolutions. The experimental results establish the superiority of our method in capturing improved inter-modality correlations between PET and CT for head-and-neck tumor segmentation. Furthermore, the proposed methodology holds applicability to other semantic segmentation tasks involving different imaging modalities like SPECT/CT or PET/MRI. Code:https://github.com/yli192/SwinCross_CrossModalSwinTransformer_for_Medical_Image_Segmentation.

Peptide Binder to Glypican-3 as a Theranostic Agent for Hepatocellular Carcinoma.

Glypican-3 (GPC3) is a membrane-associated glycoprotein that is significantly upregulated in hepatocellular carcinomas (HCC) with minimal to no expression in normal tissues. The differential expression of GPC3 between tumor and normal tissues provides an opportunity for targeted radiopharmaceutical therapy to treat HCC, a leading cause of cancer-related deaths worldwide. Methods: DOTA-RYZ-GPC3 (RAYZ-8009) comprises a novel macrocyclic peptide binder to GPC3, a linker, and a chelator that can be complexed with different radioisotopes. The binding affinity was determined by surface plasma resonance and radioligand binding assays. Target-mediated cellular internalization was radiometrically measured at multiple time points. In vivo biodistribution, monotherapy, and combination treatments with 177Lu or 225Ac were performed on HCC xenografts. Results: RAYZ-8009 showed high binding affinity to GPC3 protein of human, mouse, canine, and cynomolgus monkey origins and no binding to other glypican family members. Potent cellular binding was confirmed in GPC3-positive HepG2 cells and was not affected by isotope switching. RAYZ-8009 achieved efficient internalization on binding to HepG2 cells. Biodistribution study of 177Lu-RAYZ-8009 showed sustained tumor uptake and fast renal clearance, with minimal or no uptake in other normal tissues. Tumor-specific uptake was also demonstrated in orthotopic HCC tumors, with no uptake in surrounding liver tissue. Therapeutically, significant and durable tumor regression and survival benefit were achieved with 177Lu- and 225Ac-labeled RAYZ-8009, as single agents and in combination with lenvatinib, in GPC3-positive HCC xenografts. Conclusion: Preclinical in vitro and in vivo data demonstrate the potential of RAYZ-8009 as a theranostic agent for the treatment of patients with GPC3-positive HCC.

A microneedle vaccine printer for thermostable COVID-19 mRNA vaccines.

Decentralized manufacture of thermostable mRNA vaccines in a microneedle patch (MNP) format could enhance vaccine access in low-resource communities by eliminating the need for a cold chain and trained healthcare personnel. Here we describe an automated process for printing MNP Coronavirus Disease 2019 (COVID-19) mRNA vaccines in a standalone device. The vaccine ink is composed of lipid nanoparticles loaded with mRNA and a dissolvable polymer blend that was optimized for high bioactivity by screening formulations in vitro. We demonstrate that the resulting MNPs are shelf stable for at least 6 months at room temperature when assessed using a model mRNA construct. Vaccine loading efficiency and microneedle dissolution suggest that efficacious, microgram-scale doses of mRNA encapsulated in lipid nanoparticles could be delivered with a single patch. Immunizations in mice using manually produced MNPs with mRNA encoding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain stimulate long-term immune responses similar to those of intramuscular administration.

RYZ101 (Ac-225 DOTATATE) Opportunity beyond Gastroenteropancreatic Neuroendocrine Tumors: Preclinical Efficacy in Small-Cell Lung Cancer.

Overexpression of somatostatin receptors (SSTR), particularly SSTR2, is found in gastroenteropancreatic neuroendocrine tumors (GEP-NET), and subsets of other solid tumors such as small-cell lung cancer (SCLC). SCLC accounts for approximately 13% to 15% of lung cancer and lacks effective therapeutic options. IHC analysis indicates that up to 50% of SCLC tumors are SSTR2-positive, with a substantial subset showing high and homogenous expression. Peptide receptor radionuclide therapy with radiolabeled somatostatin analogue, Lu-177 DOTATATE, has been approved for GEP-NETs. Different strategies aimed at improving outcomes, such as the use of alpha-emitting radioisotopes, are currently being investigated. RYZ101 (Ac-225 DOTATATE) is comprised of the alpha-emitting radioisotope actinium-225, chemical chelator DOTA, and octreotate (TATE), a somatostatin analogue. In the cell-based competitive radioligand binding assay, RAYZ-10001-La (lanthanum surrogate for RYZ101) showed high binding affinity (Ki = 0.057 nmol/L) to human SSTR2 and >600-fold selectivity against other SSTR subtypes. RAYZ-10001-La exhibited efficient internalization to SSTR2-positive cells. In multiple SSTR2-expressing SCLC xenograft models, single-dose intravenous RYZ101 3 µCi (0.111 MBq) or 4 µCi (0.148 MBq) significantly inhibited tumor growth, with deeper responses, including sustained regression, observed in the models with higher SSTR2 levels. The antitumor effect was further enhanced when RYZ101 was combined with carboplatin and etoposide at clinically relevant doses. In summary, RYZ101 is a highly potent, alpha-emitting radiopharmaceutical agent, and preclinical data demonstrate the potential of RYZ101 for the treatment of patients with SSTR-positive cancers.

In vivo crystallization of human IgG in the endoplasmic reticulum of engineered Chinese hamster ovary (CHO) cells.

Protein synthesis and secretion are essential to cellular life. Although secretory activities may vary in different cell types, what determines the maximum secretory capacity is inherently difficult to study. Increasing protein synthesis until reaching the limit of secretory capacity is one strategy to address this key issue. Under highly optimized growth conditions, recombinant CHO cells engineered to produce a model human IgG clone started housing rod-shaped crystals in the endoplasmic reticulum (ER) lumen. The intra-ER crystal growth was accompanied by cell enlargement and multinucleation and continued until crystals outgrew cell size to breach membrane integrity. The intra-ER crystals were composed of correctly folded, endoglycosidase H-sensitive IgG. Crystallizing propensity was due to the intrinsic physicochemical properties of the model IgG, and the crystallization was reproduced in vitro by exposing a high concentration of IgG to a near neutral pH. The striking cellular phenotype implicated the efficiency of IgG protein synthesis and oxidative folding exceeded the capacity of ER export machinery. As a result, export-ready IgG accumulated progressively in the ER lumen until a threshold concentration was reached to nucleate crystals. Using an in vivo system that reports accumulation of correctly folded IgG, we showed that the ER-to-Golgi transport steps became rate-limiting in cells with high secretory activity.

In situ microscopy for plasma erosion of complex surfaces.

A novel method for the in situ visualization and profilometry of a plasma-facing surface is demonstrated using a long-distance microscope. The technique provides valuable in situ monitoring of the microscopic temporal and morphological evolution of a material surface subject to plasma-surface interactions, such as ion-induced sputter erosion. Focus variation of image stacks enables height surface profilometry, which allows a depth of field beyond the limits associated with high magnification. As a demonstration of this capability, the erosion of a volumetrically featured aluminum foam is quantified during ion-bombardment in a low-temperature argon plasma where the electron temperature is ∼7 eV and the plasma is biased relative to the target surface such that ions impinge at ∼300 eV. Three-dimensional height maps are reconstructed from the images captured with a long-distance microscope with an x-y resolution of 3 × 3 µm2 and a focus-variation resolution based on the motor step-size of 20 µm. The time-resolved height maps show a total surface recession of 730 µm and significant ligament thinning over the course of 330 min of plasma exposure. This technique can be used for developing plasma-facing components for a wide range of plasma devices for applications such as propulsion, manufacturing, hypersonics, and fusion.

Generation and validation of APOE knockout human iPSC-derived cerebral organoids.

Apolipoprotein E (apoE) is a major lipid carrier in the brain and closely associated with the pathogenesis of Alzheimer's disease (AD). Here, we describe a protocol for efficient knockout of APOE in human induced pluripotent stem cells (iPSCs) using the CRISPR-Cas9 system. We obtain homozygous APOE knockout (APOE-/- ) iPSCs and further validate the deficiency of apoE in iPSC-derived cerebral organoids. APOE-/- cerebral organoids can serve as a useful tool to study apoE functions and apoE-related pathogenic mechanisms in AD. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020).

Immuno-oncological Efficacy of RXDX-106, a Novel TAM (TYRO3, AXL, MER) Family Small-Molecule Kinase Inhibitor.

Expression of the TAM (TYRO3, AXL, MER) family of receptor tyrosine kinases (RTK) has been associated with cancer progression, metastasis, and drug resistance. In immune cells, TAM RTKs can dampen inflammation in favor of homeostatic wound-healing responses, thus potentially contributing to the evasion of cancer cells from immune surveillance. Here we characterize the small-molecule RXDX-106 as a selective and potent pan-TAM RTK inhibitor with slow dissociation kinetics and significant antitumor activity in multiple syngeneic tumor models. Expression of AXL and MER on both immune and tumor cells increased during tumor progression. Tumor growth inhibition (TGI) following treatment with RXDX-106 was observed in wild-type mice and was abrogated in immunodeficient mice, suggesting that the antitumor activity of RXDX-106 is, in part, due to the presence of immune cells. RXDX-106-mediated TGI was associated with increased tumor-infiltrating leukocytes, M1-polarized intratumoral macrophages, and activation of natural killer cells. RXDX-106 proportionally increased intratumoral CD8+ T cells and T-cell function as indicated by both IFNγ production and LCK phosphorylation (pY393). RXDX-106 exhibited its effects via direct actions on TAM RTKs expressed on intratumoral macrophages and dendritic cells, leading to indirect activation of other immune cells in the tumor. RXDX-106 also potentiated the effects of an immune checkpoint inhibitor, α-PD-1 Ab, resulting in enhanced antitumor efficacy and survival. Collectively, these results demonstrate the capacity of RXDX-106 to inhibit tumor growth and progression and suggest it may serve as an effective therapy against multiple tumor types. SIGNIFICANCE: The pan-TAM small-molecule kinase inhibitor RXDX-106 activates both innate and adaptive immunity to inhibit tumor growth and progression, indicating its clinical potential to treat a wide variety of cancers.

MerTK inhibition by RXDX-106 in MerTK activated gastric cancer cell lines.

RXDX-106 is a potent and selective type II pseudo-irreversible (slow off-rate) inhibitor of TYRO3, AXL, MER and c-MET. MER tyrosine kinase (MerTK) is expressed in a variety of malignancies, including gastric cancer (GC). The oncogenic potential of MerTK is supported by various lines of evidence. First, we surveyed 10 GC cell lines for MerTK protein overexpression and MerTk phosphorylation. We next evaluated the change of downstream signaling molecules including (p)-ERK and (p)-AKT, following RXDX-106 treatment. We also investigated the effect of RXDX-106 in patient-derived cell lines to mimic the in vivo condition. The prevalence of MerTK protein overexpression was evaluated in 229 cancer tissue specimens. We have found that MerTK inhibitor treatment resulted in considerable inhibition of cell growth and downstream signaling. In addition, MerTK phosphorylation, not total MerTK expression, is likely more predictive of therapeutic success. p-MerTK protein overexpression by IHC was found in 18% (17/87) of GC patients. Lastly, RXDX-106 inhibited cell proliferation in MerTK activated gastric cancer cell line. These findings provide further evidence of oncogenic roles for MerTK in GC, and demonstrate the importance of kinase activity for MerTK tumorigeneicity and validate RXDX-106, a novel MerTK inhibitor, as a potential therapeutic agent for treatment of GC.

Combating autophagy is a strategy to increase cytotoxic effects of novel ALK inhibitor entrectinib in neuroblastoma cells.

Neuroblastoma (NB) is a threatening childhood malignancy. Its prognosis is affected by several morphological, and biological characteristics, including the constitutive expression of ALK tyrosine kinase. In this study we examined the therapeutic potential of a novel ALK inhibitor, entrectinib, in obliterating NB tumor cells. Entrectinib showed the growth-inhibitory effects on NB cells with a 50% inhibitory concentration range of 0.03-5 µM. In the ALK-dependent cells, entrectinib mediated G1-arrest, which was associated with modified expression of multiple cell-cycle regulators. Down-regulation of Ki-67, and attenuated phosphorylation of ERK1/2, and STAT3, correlated with observed antiproliferative capacity of entrectinib. Initial cytostatic activity of entrectinib was followed by concentration-dependent apoptotic cell death, and Caspase-3 activation. However, we delineated a reduced sensitivity of ALK mutated NB cells to entrectinib, and demonstrated strong activation of autophagy in SH-SY5YF1174L NB cell line. Abrogation of autophagy by chloroquine increased significantly the toxicity of entrectinib, as confirmed by enhanced death rate, and PARP protein cleavage in SH-SY5YF1174L cells. In aggregate, our data show that entrectinib inhibits proliferation, and induces G1-arrest, and apoptosis in NB cells. We propose entrectinib for further consideration in treatment of NB, and recommend pharmacological inhibition of autophagy to be explored for a combined therapeutic approach in NB patients that might develop resistance to entrectinib.

Design and Synthesis of Pyridone-Containing 3,4-Dihydroisoquinoline-1(2H)-ones as a Novel Class of Enhancer of Zeste Homolog 2 (EZH2) Inhibitors.

A new enhancer of zeste homolog 2 (EZH2) inhibitor series comprising a substituted phenyl ring joined to a dimethylpyridone moiety via an amide linkage has been designed. A preferential amide torsion that improved the binding properties of the compounds was identified for this series via computational analysis. Cyclization of the amide linker resulted in a six-membered lactam analogue, compound 18. This transformation significantly improved the ligand efficiency/potency of the cyclized compound relative to its acyclic analogue. Additional optimization of the lactam-containing EZH2 inhibitors focused on lipophilic efficiency (LipE) improvement, which provided compound 31. Compound 31 displayed improved LipE and on-target potency in both biochemical and cellular readouts relative to compound 18. Inhibitor 31 also displayed robust in vivo antitumor growth activity and dose-dependent de-repression of EZH2 target genes.

AZ17: a new bispecific drug targeting IL-6 and IL-23 with potential clinical use--improves psoriasis in a human xenograft transplantation model.

Targeting more than one molecule in multifactorial diseases involving several disease mediators may provide improved therapeutic efficacy. Psoriasis is a multifactorial disease in which interleukin (IL)-6 and IL-23 are important disease mediators because they facilitate development of Th17 cells; widely accepted to be associated with psoriasis. To meet the need for new therapeutics, we aimed to create a clinically relevant bispecific drug, by combining the inhibitory properties of anti-IL-6 and anti-IL-23 antibodies, exhibiting high affinity, high stability and the ability to be produced in high yield. The bispecific molecule AZ17 was created by combining high affinity binding domains originating from monoclonal antibodies targeting human IL-6 and IL-23. To allow for high and efficient production, AZ17 was assembled by site-specific bioconjugation from two individual single chain fragment variables that were synthesized separately in Escherichia coli. To improve stability and extend pharmacokinetics, a flexible poly-ethylene glycol molecule was used as linker. In preclinical psoriasis models, AZ17 reduced IL-23-induced ear inflammation and improved psoriasis in a xenograft transplantation model where psoriasis skin is transplanted onto immune-deficient mice. The data presented here suggest AZ17 to be a promising drug candidate in psoriasis and other inflammatory diseases associated with Th17 cell development.

Durable Clinical Response to Entrectinib in NTRK1-Rearranged Non-Small Cell Lung Cancer.

INTRODUCTION: Chromosomal rearrangements involving neurotrophic tyrosine kinase 1 (NTRK1) occur in a subset of non-small cell lung cancers (NSCLCs) and other solid tumor malignancies, leading to expression of an oncogenic TrkA fusion protein. Entrectinib (RXDX-101) is an orally available tyrosine kinase inhibitor, including TrkA. We sought to determine the frequency of NTRK1 rearrangements in NSCLC and to assess the clinical activity of entrectinib. METHODS: We screened 1378 cases of NSCLC using anchored multiplex polymerase chain reaction (AMP). A patient with an NTRK1 gene rearrangement was enrolled onto a Phase 1 dose escalation study of entrectinib in adult patients with locally advanced or metastatic tumors (NCT02097810). We assessed safety and response to treatment. RESULTS: We identified NTRK1 gene rearrangements at a frequency of 0.1% in this cohort. A patient with stage IV lung adenocrcinoma with an SQSTM1-NTRK1 fusion transcript expression was treated with entrectinib. Entrectinib was well tolerated, with no grade 3-4 adverse events. Within three weeks of starting on treatment, the patient reported resolution of prior dyspnea and pain. Restaging CT scans demonstrated a RECIST partial response (PR) and complete resolution of all brain metastases. This patient has continued on treatment for over 6 months with an ongoing PR. CONCLUSIONS: Entrectinib demonstrated significant anti-tumor activity in a patient with NSCLC harboring an SQSTM1-NTRK1 gene rearrangement, indicating that entrectinib may be an effective therapy for tumors with NTRK gene rearrangements, including those with central nervous system metastases.