Boston Biomedical (United States)

Abstract Traditional pooled genetic dependency screens, such as DepMap, have proven powerful for identifying essential genes in cancer cell lines. However, these pooled formats are prone to the bystander effect, where secreted factors from wild-type cells support the survival of knockout (KO) cells within the same culture, potentially masking dependencies on specific secreted proteins. To address this limitation, we performed a well-by-well CRISPR screen targeting genes encoding secreted factors in the pancreatic ductal adenocarcinoma (PDAC) cell line HPAFII. This arrayed format eliminates cross-feeding and enables precise assessment of cell-intrinsic dependencies on targeted proteins and could thus expose hidden vulnerabilities. CellTiter-Glo (CTG) viability assays were used to assess gene lethality following target gene KOs. Furthermore, RNA sequencing verified effective disruption of target genes, establishing the specificity and reliability of the screening workflow. We observed strong growth inhibition in EFNA1 (Ephrin-A1) KO samples compared to lipofectamine-treated controls. However, this inhibitory effect was missed and underrepresented in the conventional pooled format. EFNA1 is a ligand for EPH receptors, involved in cell-cell communication, migration, and proliferation signaling. Differential gene expression analysis comparing EFNA1 KO to lipofectamine-treated controls revealed enriched pathways of oxidative phosphorylation, E2F targets, MYC targets, and DNA repair in EFNA1 KO samples. This result supports our observation of lethality in EFNA1 KO samples. It suggests that EFNA1 contributes to cell-cell communication and is critical for HPAFII cell survival. Its KO triggers stress pathways, including E2F, MYC, OXPHOS, and DNA repair, and acts as a suppressor of excessive proliferative programs. In conclusion, we identified EFNA1 as a critical non-cell-autonomous essential gene through an arrayed CRISPR screen, with its loss showing lethality to HPAFII cells exclusively in isolated settings, likely as a result of disrupted cell-cell interactions. Defining such dependencies may facilitate the discovery of directly druggable and more tumor-selective therapeutic strategies. Citation Format: Xingyi Shi, David Ruddy, Millicent Gabriel, Michelle Piquet, Kris Muskiewicz, Joel Wagner, Joshua Korn, Cory Johannessen. Arrayed CRISPR Screening Reveals EFNA1 as a Non-Cell-Autonomous Essential Gene in Pancreatic Cancer Cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C035.

Patients with fibrotic interstitial lung diseases (ILDs) such as Idiopathic Pulmonary Fibrosis (IPF) and Connective Tissue Disease-associated ILD (CTD-ILD) experience progressive lung function decline, with few biomarkers predicting transplant-free survival. We aimed to identify soluble factors in bronchoalveolar lavage (BAL) associated with poor outcomes in these patients. High-throughput aptamer-based proteomics (SomaLogic) was used to measure 7,596 somamers (6,398 proteins) in BAL samples from IPF (n=115), CTD-ILD (n=128), and controls (n=20). Proteins associated with transplant-free survival were identified using Cox regression. Associations with baseline forced vital capacity (FVC) and diffusion capacity (DLCO) were assessed using generalized linear models. All models were adjusted for age, sex, and multiple comparisons (Benjamini–Hochberg) assuming significance with with adjusted p values ≤0.05. In the combined IPF and CTD-ILD cohort (n=243), 20 BAL proteins were significantly associated with poor survival. SMURF1 showed the strongest association (aHR=49.50, adj. p=0.035). Kaplan-Meier analysis confirmed shorter survival in IPF patients with high SMURF1 levels (Q4 median: 1,045 days vs. Q1: 2,079 days; p=0.0026). Seven proteins were linked to lower baseline FVC, and 266 to lower baseline DLCO. Only PRSS2, GDF15, and TNC were significantly associated with all three metrics, transplant-free survival, FVC and DLCO. These data suggest BAL protein signatures in both patients with IPF and CTD-ILD to be predictive of adverse outcomes with SMURF1 being of particular interest for further investigation.

"What's in a name? That which we call a rose / By any other name would smell as sweet" (Juliet, from Romeo and Juliet by William Shakespeare). Shakespeare's implication is that a name is nothing but a word and it therefore represents a convention with no intrinsic meaning. Whilst this may be relevant to romantic literature, disease names do have real meanings, and consequences, in medicine. Hence, there must be a very good rational for changing the name of a disease that has a centuries-old historical context. A working group of representatives from national and international endocrinology and endocrine pediatric societies now proposes changing the name of "diabetes insipidus" to "Arginine Vasopressin Deficiency (AVP-D)" for central etiologies, and "Arginine Vasopressin Resistance (AVP-R)" for nephrogenic etiologies This editorial provides both the historical context and the rational for this proposed name change.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Combining live imaging with the ability to retrieve individual cells of interest remains a technical challenge. Combining imaging with precise cell retrieval is of particular interest when studying highly dynamic or transient, asynchronous, or heterogeneous cell biological and developmental processes. Here, we present a method to encapsulate live cells in a 3D hydrogel matrix, via hydrogel bead compartmentalisation. Using a small-scale screen, we optimised matrix conditions for the culture and multilineage differentiation of mouse embryonic stem cells. Moreover, we designed a custom microfluidic platform that is compatible with live imaging. With this platform we can long-term culture and subsequently extract individual cells-in-beads by media flow only, obviating the need for enzymatic cell removal from the platform. Specific beads may be extracted from the platform in isolation, without disrupting the adjacent beads. We show that we can differentiate mouse embryonic stem cells, monitor reporter expression by live imaging, and retrieve individual beads for functional assays, correlating reporter expression with functional response. Overall, we present a highly flexible 3D cell encapsulation and microfluidic platform that enables both monitoring of cellular dynamics and retrieval for molecular and functional assays.

Abstract Translation products encoded by non canonical or novel open reading frame (ORF) genomic regions are generally considered too small to play any significant biological role, and dismissed as inconsequential. In this study, we show that mutations mapping to novel ORFs have significantly higher pathogenicity scores than mutations in protein-coding regions. Importantly, novel ORFs can translate into protein-like structures with putative independent biological functions that can be of relevance in disease states, including cancer. We thus provide strong evidence to support the systematic study of novel ORFs to gain new insights into normal biological and disease processes.One Sentence SummaryNon coding regions may encode protein-like products that are important to understand diseases.

Most determinants of health originate from the “contexts” in which we live, which has remained outside the confines of the U.S. healthcare system. This issue has left providers unprepared to operate with an ample understanding of the challenges patients may face beyond their purview. The recent shift to value-based care and increasing prevalence of Electronic Health Record (EHR) systems provide opportunities to incorporate upstream contextual factors into care. We discuss that incorporating context into care is hindered by a chicken-and-egg dilemma – ie, lack of evidence on the utility of contextual data at the point of care, where contextual data are missing due to the lack of an informatics infrastructure. We argue that if we build the informatics infrastructure today, EHRs can give the tomorrow’s clinicians the tools and the data they need to transform the U.S. healthcare from episodic and reactive to preventive and proactive. We also discuss system design considerations to improve efficacy of the suggested informatics infrastructure, which include systematically prioritizing contextual data domains, developing interoperability standards, and ensuring that integration of contextual data does not disrupt clinicians’ workflow.