No abstract

Phosphoinositide 3-kinase (PI3K) family members are involved in diverse cellular fates including cell growth, proliferation, and survival. While many molecular details are known about the Class I and III PI3Ks, less is known about the Class II PI3Ks. To explore the function of all eight PI3K isoforms in autophagy, we knock down each gene individually and measure autophagy. We find a significant decrease in autophagy following siRNA-mediated PIK3C2A (encoding the Class 2 PI3K, PI3K-C2α) knockdown. This defective autophagy is rescued by exogenous PI3K-C2α, but not kinase-dead PI3K-C2α. Using confocal microscopy, we probe for markers of endocytosis and autophagy, revealing that PI3K-C2α colocalizes with markers of endocytosis. Though endocytic uptake is intact, as demonstrated by transferrin labeling, PIK3C2A knockdown results in vesicle accumulation at the recycling endosome. We isolate distinct membrane sources and observe that PI3K-C2α interacts with markers of endocytosis and autophagy, notably ATG9. Knockdown of either PIK3C2A or ATG9A/B, but not PI3KC3, results in an accumulation of transferrin-positive clathrin coated vesicles and RAB11-positive vesicles at the recycling endosome. Taken together, these results support a role for PI3K-C2α in the proper maturation of endosomes, and suggest that PI3K-C2α may be a critical node connecting the endocytic and autophagic pathways.

Purpose: There is a need for biomarkers of drug efficacy for targeted therapies in triple-negative breast cancer (TNBC). As a step toward this, we identify multi-omic molecular determinants of anti-TNBC efficacy in cell lines for a panel of oncology drugs. Methods:Using 23 TNBC cell lines, drug sensitivity scores (DSS 3 ) were determined using a panel of investigational drugs and drugs approved for other indications. Molecular readouts were generated for each cell line using RNA sequencing, RNA targeted panels, DNA sequencing, and functional proteomics. DSS 3 values were correlated with molecular readouts using an FDRcorrected significance cutoff of p* < 0.05 and yielded molecular determinant panels that predict anti-TNBC efficacy.Results: Six molecular determinant panels were obtained from 12 drugs we prioritized based on their efficacy. Determinant panels were largely devoid of DNA mutations of the targeted pathway. Molecular determinants were obtained by correlating DSS 3 with molecular readouts. We found that co-inhibiting molecular correlate pathways leads to robust synergy across many cell lines. Conclusions:These findings demonstrate an integrated method to identify biomarkers of drug efficacy in TNBC, where DNA predictions correlate poorly with drug response. Our work outlines a framework for the identification of novel molecular determinants and optimal companion drugs for combination therapy based on these correlates.Terms of use and reuse: academic research for non-commercial purposes, see here for full terms. https://www.springer.com/aamterms-v1

While many resources exist for the drug screening of bladder cancer cell lines in 2D culture, it is widely recognized that screening in 3D culture is more representative of in vivo response. Importantly, signaling changes between 2D and 3D culture can result in changes to drug response. To address the need for 3D drug screening of bladder cancer cell lines, we screened 17 bladder cancer cell lines using a library of 652 investigational small-molecules and 3 clinically relevant drug combinations in 3D cell culture. Our goal was to identify compounds and classes of compounds with efficacy in bladder cancer. Utilizing established genomic and transcriptomic data for these bladder cancer cell lines, we correlated the genomic molecular parameters with drug response, to identify potentially novel groups of tumors that are vulnerable to specific drugs or classes of drugs. Importantly, we demonstrate that MEK inhibitors are a promising targeted therapy for the basal subtype of bladder cancer, and our data indicate that drug screening of 3D cultures provides an important resource for hypothesis generation.

e14003 Background: Brain metastases (BrMet) remain a clinically challenge. There is an increase interest in evaluating the efficiacy of systemic therapy for BrMet. Patient-derived organoids (PDO) and xenografts (PDX) are thought to capture the tumor heterogeneity and molecular alterations of the source tumor, and may be used as ‘avatars’ for therapeutic response assessment of the source patient. PDO has an advantage over PDX with a shorter establishment time, and thus, may allow a real-time drug sensitivity testing. The objective of this study was to determine the feasibility of establishing PDO from resected breast cancer BrMet and to evaluate the drug sensitivities in a time period amenable to eventual implemention into clinical utiliity. Methods: Under an IRB-approved protocol, resected BrMet tissues were prospectively collected at the time of clinically indicated neurosurgical procedure as part of a banking program. Tumors were directly cultured as 3-D organoids. DNA and RNA were collected. Drug testing panels included standard clinical care drugs or drug combinations as well as those selected based on the molecular profiling. When clinically conducted next-generation sequencing testing reports were available, we also tested drugs (or class of drugs) that were suggested as a potential therapeutic consideration in these reports. When possible, Nanostring PanCancer Panel evaluation was conducted at the time of PDO establishment to guide the selection of drugs. Results: To date, 11 breast cancer BrMet samples have been collected. 8/11 were successfully established as ‘direct from operating room’ PDO and underwent drug panel testing. The reasons for 3 cases not drug tested include: no viable tumor (confirmed by clinical pathology report as necrosis only) and primary brain tumor (not metastasis). Of the 8 PDO, the clinical subtypes were 5HER2+, 2HR-HER2-, and 1HR+HER2- cases.The median time from collection to the drug testing results availability was 11 days (range: 7-19 days). The median number of drugs or drug combinations tested was 23 (range: 7-32). The drug testing revealed various patterns of sensitivities to chemotherapies and targeted therapies. Nanostring PanCancer testing of PDO identified potential targetable pathways for which PDO demonstrated sensitivity when the drugs were matched to the associated molecular pathway. Conclusions: While the expected number of cases has been much lower due to COVID-19 pandemic-related change in clinical practice patterns and research operations, we have successfully demonstrated that the real-time establishment of BrMet PDO is feasible and may be used as a platform for further investigations. Real-time PDO could be potentially employed to predict drug sensitives for prioritizing drug therapy options in a clinically meaningful time-frame. PDO platform may be used to investigating biomarkers and mechanisms of response and resistance to therapy.