Lineage-specific intolerance to oncogenic drivers restricts histological transformation.

Lung adenocarcinoma (LUAD) and small cell lung cancer (SCLC) are thought to originate from different epithelial cell types in the lung. Intriguingly, LUAD can histologically transform into SCLC after treatment with targeted therapies. In this study, we designed models to follow the conversion of LUAD to SCLC and found that the barrier to histological transformation converges on tolerance to Myc, which we implicate as a lineage-specific driver of the pulmonary neuroendocrine cell.

Unlocking New Prenylation Modes: Azaindoles as a New Substrate Class for Indole Prenyltransferases.

Aza-substitution, the replacement of aromatic CH groups with nitrogen atoms, is an established medicinal chemistry strategy for increasing solubility, but current methods of accessing functionalized azaindoles are limited. In this work, indole-alkylating aromatic prenyltransferases (PTs) were explored as a strategy to directly functionalize azaindole-substituted analogs of natural products. For this, a series of aza-l-tryptophans (Aza-Trp) featuring -substitution of every aromatic CH position of the indole ring and their corresponding cyclic Aza-l-Trp-l-proline dipeptides (Aza-CyWP), were synthesized as substrate mimetics for the indole-alkylating PTs FgaPT2, CdpNPT, and FtmPT1.

LKB1 suppresses growth and promotes the internalization of EGFR through the PIKFYVE lipid kinase.

The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1- mediated growth suppression we developed a spheroid-based cell culture assay to study LKB1- dependent growth.

FORMATION OF MALIGNANT, METASTATIC SMALL CELL LUNG CANCERS THROUGH OVERPRODUCTION OF cMYC PROTEIN IN TP53 AND RB1 DEPLETED PULMONARY NEUROENDOCRINE CELLS DERIVED FROM HUMAN EMBRYONIC STEM CELLS.

We recently described our initial efforts to develop a model for small cell lung cancer (SCLC) derived from human embryonic stem cells (hESCs) that were differentiated to form pulmonary neuroendocrine cells (PNECs), a putative cell of origin for neuroendocrine-positive SCLC. Although reduced expression of the tumor suppressor genes and allowed the induced PNECs to form subcutaneous growths in immune-deficient mice, the tumors did not display the aggressive characteristics of SCLC seen in human patients. Here we report that the additional, doxycycline-regulated expression of a transgene encoding wild-type or mutant cMYC protein promotes rapid growth, invasion, and metastasis of these hESC-derived cells after injection into the renal capsule.

Generation of precision preclinical cancer models using regulated in vivo base editing.

Although single-nucleotide variants (SNVs) make up the majority of cancer-associated genetic changes and have been comprehensively catalogued, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a mouse system for temporal and regulatable in vivo base editing. The inducible base editing (iBE) mouse carries a single expression-optimized cytosine base editor transgene under the control of a tetracycline response element and enables robust, doxycycline-dependent expression across a broad range of tissues in vivo.

LKB1-Dependent Regulation of TPI1 Creates a Divergent Metabolic Liability between Human and Mouse Lung Adenocarcinoma.

Unlabelled: KRAS is the most frequently mutated oncogene in human lung adenocarcinomas (hLUAD), and activating mutations frequently co-occur with loss-of-function mutations in TP53 or STK11/LKB1. However, mutation of all three genes is rarely observed in hLUAD, even though engineered comutation is highly aggressive in mouse lung adenocarcinoma (mLUAD). Here, we provide a mechanistic explanation for this difference by uncovering an evolutionary divergence in the regulation of triosephosphate isomerase (TPI1).

Transposon Mutagenesis Reveals RBMS3 Silencing as a Promoter of Malignant Progression of BRAFV600E-Driven Lung Tumorigenesis.

Unlabelled: Mutationally activated BRAF is detected in approximately 7% of human lung adenocarcinomas, with BRAFT1799A serving as a predictive biomarker for treatment of patients with FDA-approved inhibitors of BRAFV600E oncoprotein signaling. In genetically engineered mouse (GEM) models, expression of BRAFV600E in the lung epithelium initiates growth of benign lung tumors that, without additional genetic alterations, rarely progress to malignant lung adenocarcinoma. To identify genes that cooperate with BRAFV600E for malignant progression, we used Sleeping Beauty-mediated transposon mutagenesis, which dramatically accelerated the emergence of lethal lung cancers.

Indole C6 Functionalization of Tryprostatin B Using Prenyltransferase CdpNPT.

Tryprostatin A and B are prenylated, tryptophan-containing, diketopiperazine natural products, displaying cytotoxic activity through different mechanisms of action. The presence of the 6-methoxy substituent on the indole moiety of tryprostatin A was shown to be essential for the dual inhibition of topoisomerase II and tubulin polymerization. However, the inability to perform late-stage modification of the indole ring has limited the structure-activity relationship studies of this class of natural products.

Chemoenzymatic synthesis of daptomycin analogs active against daptomycin-resistant strains.

Daptomycin is a last resort antibiotic for the treatment of infections caused by many Gram-positive bacterial strains, including vancomycin-resistant Enterococcus (VRE) and methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA). However, the emergence of daptomycin-resistant strains of S. aureus and Enterococcus in recent years has renewed interest in synthesizing daptomycin analogs to overcome resistance mechanisms.

Role of mTOR As an Essential Kinase in SCLC.

Objectives: SCLC represents 15% of all lung cancer diagnoses in the United States and has a particularly poor prognosis. We hypothesized that kinases regulating SCLC survival pathways represent therapeutically targetable vulnerabilities whose inhibition may improve SCLC outcome.

Methods: A short-hairpin RNA (shRNA) library targeting all human kinases was introduced in seven chemonaive patient-derived xenografts (PDX) and the cells were cultured in vitro and in vivo.

Acceptor substrate determines donor specificity of an aromatic prenyltransferase: expanding the biocatalytic potential of NphB.

Aromatic prenyltransferases are known for their extensive promiscuity toward aromatic acceptor substrates and their ability to form various carbon-carbon and carbon-heteroatom bonds. Of particular interest among the prenyltransferases is NphB, whose ability to geranylate cannabinoid precursors has been utilized in several in vivo and in vitro systems. It has therefore been established that prenyltransferases can be utilized as biocatalysts for the generation of useful compounds.

FgaPT2, a biocatalytic tool for alkyl-diversification of indole natural products.

Aromatic prenyltransferases from natural product biosynthetic pathways display relaxed specificity for their aromatic substrates. While a growing body of evidence suggests aromatic prenyltransferases to be more tolerant towards their alkyl-donor substrates, most studies aimed at probing their donor-substrate specificity are limited to only a small set of alkyl pyrophosphate donors, restricting their broader utility as biocatalysts for synthetic applications. Here, we assess the donor substrate specificity of an l-tryptophan C4-prenyltransferase, also known as C4-dimethylallyltryptophan synthase, FgaPT2 from , using an array of 34 synthetic unnatural alkyl-pyrophosphate analogues, and demonstrate FgaPT2 can catalyze the transfer of 25 of the 34 non-native alkyl groups from their corresponding synthetic alkyl-pyrophosphate analogues at N1, C3, C4 and C5 position of tryptophan in a normal and reverse manner.

Structural and Functional Consequences of the Weak Binding of Chlorin e6 to Bovine Rhodopsin.

The chlorophyll-derivative chlorin e6 (Ce6) identified in the retinas of deep-sea ocean fish is proposed to play a functional role in red bioluminescence detection. Fluorescence and H NMR spectroscopy studies with the bovine dim-light photoreceptor, rhodopsin, indicate that Ce6 weakly binds to it with μm affinity. Absorbance spectra prove that red light sensitivity enhancement is not brought about by a shift in the absorbance maximum of rhodopsin.

Anthrax toxin receptor 1 is the cellular receptor for Seneca Valley virus.

Seneca Valley virus (SVV) is an oncolytic picornavirus with selective tropism for neuroendocrine cancers. It has shown promise as a cancer therapeutic in preclinical studies and early-phase clinical trials. Here, we have identified anthrax toxin receptor 1 (ANTXR1) as the receptor for SVV using genome-wide loss-of-function screens.

Noninvasive Interrogation of DLL3 Expression in Metastatic Small Cell Lung Cancer.

The Notch ligand DLL3 has emerged as a novel therapeutic target expressed in small cell lung cancer (SCLC) and high-grade neuroendocrine carcinomas. Rovalpituzumab teserine (Rova-T; SC16LD6.5) is a first-in-class DLL3-targeted antibody-drug conjugate with encouraging initial safety and efficacy profiles in SCLC in the clinic.

Chemosensitive Relapse in Small Cell Lung Cancer Proceeds through an EZH2-SLFN11 Axis.

Small cell lung cancer is initially highly responsive to cisplatin and etoposide but in almost every case becomes rapidly chemoresistant, leading to death within 1 year. We modeled acquired chemoresistance in vivo using a series of patient-derived xenografts to generate paired chemosensitive and chemoresistant cancers. Multiple chemoresistant models demonstrated suppression of SLFN11, a factor implicated in DNA-damage repair deficiency.

Macrophage Ontogeny Underlies Differences in Tumor-Specific Education in Brain Malignancies.

Extensive transcriptional and ontogenetic diversity exists among normal tissue-resident macrophages, with unique transcriptional profiles endowing the cells with tissue-specific functions. However, it is unknown whether the origins of different macrophage populations affect their roles in malignancy. Given potential artifacts associated with irradiation-based lineage tracing, it remains unclear if bone-marrow-derived macrophages (BMDMs) are present in tumors of the brain, a tissue with no homeostatic involvement of BMDMs.

Quantitation of Murine Stroma and Selective Purification of the Human Tumor Component of Patient-Derived Xenografts for Genomic Analysis.

Patient-derived xenograft (PDX) mouse models are increasingly used for preclinical therapeutic testing of human cancer. A limitation in molecular and genetic characterization of PDX tumors is the presence of integral murine stroma. This is particularly problematic for genomic sequencing of PDX models.

PARP Inhibitor Activity Correlates with SLFN11 Expression and Demonstrates Synergy with Temozolomide in Small Cell Lung Cancer.

Purpose: PARP inhibitors (PARPi) are a novel class of small molecule therapeutics for small cell lung cancer (SCLC). Identification of predictors of response would advance our understanding, and guide clinical application, of this therapeutic strategy.

Experimental Design: Efficacy of PARP inhibitors olaparib, rucaparib, and veliparib, as well as etoposide and cisplatin in SCLC cell lines, and gene expression correlates, was analyzed using public datasets.

P-selectin is a nanotherapeutic delivery target in the tumor microenvironment.

Disseminated tumors are poorly accessible to nanoscale drug delivery systems because of the vascular barrier, which attenuates extravasation at the tumor site. We investigated P-selectin, a molecule expressed on activated vasculature that facilitates metastasis by arresting tumor cells at the endothelium, for its potential to target metastases by arresting nanomedicines at the tumor endothelium. We found that P-selectin is expressed on cancer cells in many human tumors.

CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer.

Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment.

Anatomic Single-Graft Anterior Cruciate Ligament Reconstruction Restores Rotational Stability: A Robotic Study in Cadaveric Knees.

Purpose: First, we aimed to investigate the ability of a single bone-patellar tendon-bone graft placed in the anatomic center of the femoral and tibial attachment sites to restore normal tibiofemoral compartment translations and tibial rotation. Second, we aimed to investigate what combination of anterior load and internal rotation torque applied during a pivot-shift test produces maximal anterior tibiofemoral subluxations.

Methods: We used a 6-df robotic simulator to test 10 fresh-frozen cadaveric specimens under anterior cruciate ligament (ACL)-intact, ACL-sectioned, and ACL-reconstructed conditions measuring anterior translations of the medial, central, and lateral tibiofemoral compartments and degrees of tibial rotation.