Altered redox biology challenges all cells, with compensatory responses often determining a cell’s fate. When 15 lipoxygenase 1 (15LO1), a lipid-peroxidizing enzyme abundant in asthmatic human airway epithelial cells (HAECs), binds phosphatidylethanolamine-binding protein 1 (PEBP1), hydroperoxy-phospholipids, which drive ferroptotic cell death, are generated. Peroxidases, including glutathione peroxidase 4 (GPX4), metabolize hydroperoxy-phospholipids to hydroxy derivatives to prevent ferroptotic death, but consume reduced glutathione (GSH). The cystine transporter SLC7A11 critically restores/maintains intracellular GSH. We hypothesized that high 15LO1, PEBP1, and GPX4 activity drives abnormal asthmatic redox biology, evidenced by lower bronchoalveolar lavage (BAL) fluid and intraepithelial cell GSH:oxidized GSH (GSSG) ratios, to enhance type 2 (T2) inflammatory responses. GSH, GSSG (enzymatic assays), 15LO1, GPX4, SLC7A11, and T2 biomarkers (Western blot and RNA-Seq) were measured in asthmatic and healthy control (HC) cells and fluids, with siRNA knockdown as appropriate. GSSG was higher and GSH:GSSG lower in asthmatic compared with HC BAL fluid, while intracellular GSH was lower in asthma. In vitro, a T2 cytokine (IL-13) induced 15LO1 generation of hydroperoxy-phospholipids, which lowered intracellular GSH and increased extracellular GSSG. Lowering GSH further by inhibiting SLC7A11 enhanced T2 inflammatory protein expression and ferroptosis. Ex vivo, redox imbalances corresponded to 15LO1 and SLC7A11 expression, T2 biomarkers, and worsened clinical outcomes. Thus, 15LO1 pathway–induced redox biology perturbations worsen T2 inflammation and asthma control, supporting 15LO1 as a therapeutic target.

Pancreatic ductal adenocarcinoma (PDAC) has been classified into classical and basal-like transcriptional subtypes by bulk RNA measurements. However, recent work has uncovered greater complexity to transcriptional subtypes than was initially appreciated using bulk RNA expression profiling. To provide a deeper understanding of PDAC subtypes, we developed a multiplex immunofluorescence (mIF) pipeline that quantifies protein expression of six PDAC subtype markers (CLDN18.2, TFF1, GATA6, KRT17, KRT5, and S100A2) and permits spatially resolved, single-cell interrogation of pancreatic tumors from resection specimens and core needle biopsies. Both primary and metastatic tumors displayed striking intra-tumoral subtype heterogeneity that was associated with patient outcomes, existed at the scale of individual glands, and was significantly reduced in patient-derived organoid cultures. Tumor cells co-expressing classical and basal markers were present in >90% of tumors, existed on a basal-classical polarization continuum, and were enriched in tumors containing a greater admixture of basal and classical cell populations. Cell-cell neighbor analyses within tumor glands further suggested that co-expressor cells may represent an intermediate state between expression subtype poles. The extensive intra-tumoral heterogeneity identified through this clinically applicable mIF pipeline may inform prognosis and treatment selection for patients with PDAC.

The action potential (AP) waveform controls the opening of voltage-gated calcium channels and contributes to the driving force for calcium ion flux that triggers neurotransmission at presynaptic nerve terminals. Although the frog neuromuscular junction (NMJ) has long been a model synapse for the study of neurotransmission, its presynaptic AP waveform has never been directly studied, and thus the AP waveform shape and propagation through this long presynaptic nerve terminal are unknown. Using a fast voltage-sensitive dye, we have imaged the AP waveform from the presynaptic terminal of male and female frog NMJs and shown that the AP is very brief in duration and actively propagated along the entire length of the terminal. Furthermore, based on measured AP waveforms at different regions along the length of the nerve terminal, we show that the terminal is divided into three distinct electrical regions: A beginning region immediately after the last node of Ranvier where the AP is broadest, a middle region with a relatively consistent AP duration, and an end region near the tip of nerve terminal branches where the AP is briefer. We hypothesize that these measured changes in the AP waveform along the length of the motor nerve terminal may explain the proximal-distal gradient in transmitter release previously reported at the frog NMJ.

3,4-Diaminopyridine (3,4-DAP) increases transmitter release from neuromuscular junctions (NMJs), and low doses of 3,4-DAP (estimated to reach ∼1 μM in serum) are the Food and Drug Administration (FDA)-approved treatment for neuromuscular weakness caused by Lambert–Eaton myasthenic syndrome. Canonically, 3,4-DAP is thought to block voltage-gated potassium (Kv) channels, resulting in prolongation of the presynaptic action potential (AP). However, recent reports have shown that low millimolar concentrations of 3,4-DAP have an off-target agonist effect on the Cav1 subtype (“L-type”) of voltage-gated calcium (Cav) channels and have speculated that this agonist effect might contribute to 3,4-DAP effects on transmitter release at the NMJ. To address 3,4-DAP’s mechanism(s) of action, we first used the patch-clamp electrophysiology to characterize the concentration-dependent block of 3,4-DAP on the predominant presynaptic Kv channel subtypes found at the mammalian NMJ (Kv3.3 and Kv3.4). We identified a previously unreported high-affinity (1–10 μM) partial antagonist effect of 3,4-DAP in addition to the well-known low-affinity (0.1–1 mM) antagonist activity. We also showed that 1.5-μM DAP had no effects on Cav1.2 or Cav2.1 current. Next, we used voltage imaging to show that 1.5- or 100-μM 3,4-DAP broadened the AP waveform in a dose-dependent manner, independent of Cav1 calcium channels. Finally, we demonstrated that 1.5- or 100-μM 3,4-DAP augmented transmitter release in a dose-dependent manner and this effect was also independent of Cav1 channels. From these results, we conclude that low micromolar concentrations of 3,4-DAP act solely on Kv channels to mediate AP broadening and enhance transmitter release at the NMJ.

No abstract

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune-mediated neuromuscular disease thought to be caused by autoantibodies against P/Q-type voltage-gated calcium channels (VGCCs), which attack and reduce the number of VGCCs within transmitter release sites (active zones; AZs) at the neuromuscular junction (NMJ), resulting in neuromuscular weakness. However, LEMS patients also have antibodies to other neuronal proteins, and about 15% of LEMS patients are seronegative for antibodies against VGCCs. We hypothesized that a reduction in the number of P/Q-type VGCCs alone is not sufficient to explain LEMS effects on transmitter release. Here, we used a computational model to study a variety of LEMS-mediated effects on AZ organization and transmitter release constrained by electron microscopic, pharmacological, immunohistochemical, voltage imaging, and electrophysiological observations. We show that models of healthy AZs can be modified to predict the transmitter release and short-term facilitation characteristics of LEMS, and that in addition to a decrease in number of AZ VGCCs, disruption in the organization of AZ proteins, a reduction in AZ number, a reduction in the amount of synaptotagmin, and the compensatory expression of L-type channels outside the remaining AZs are important contributors to LEMS-mediated effects on transmitter release. Furthermore, our models predict that antibody-mediated removal of synaptotagmin in combination with disruption in AZ organization alone could mimic LEMS effects without removal of VGCCs (a seronegative model). Overall, our results suggest that LEMS pathophysiology may be caused by a collection of pathological alterations to AZs at the NMJ, rather than by a simple loss of VGCCs.

<p>Supplementary Figures</p>

Targeted therapies for molecularly-defined subtypes have led to immense clinical benefit for many cancer types but have generally not been successful for pancreatic cancer. Given that the mainstay of treatment remains multi-agent chemotherapy with FOLFIRINOX or gemcitabine/nab-paclitaxel, there remains an urgent need to identify novel actionable vulnerabilities for subsets of PDAC patients. Toward this end, we conducted an integrative, genome-scale examination of genetic dependencies and cell surface targets for PDAC by leveraging CRISPR and RNAi screening data from The Cancer Dependency Map Project, genomic data of bulk patient tumors from The Cancer Genome Atlas, and custom single-nucleus RNA-seq of a 43-patient cohort comprised of untreated and treated specimens. Our results re-affirm the prominence of Ras/MAPK signaling and a synthetically-lethal interaction between VPS4A/B, but also reveal recurrent susceptibilities to genes within the fatty acid metabolism, vesicular transport and exocytosis, and nucleobase synthesis pathways that otherwise have minor to moderate depleting effects on the majority of cell lines. Aberrations in frequent tumor suppressor genes and chromosomal arm-level variations appear to modify the strength of dependencies, including that of KRAS, CCND1, and GPX4, and may serve as predictive biomarkers of response. In addition, we leveraged mRNA profiling of bulk primary tumors as well as metastatic organoid models to conduct a genome-wide search for cell surface targets that are highly-expressed in tumors while lowly or not expressed in other toxicity-prone, non-malignant tissues. These putative drug targets do not need to be cancer dependencies and can be compatible with antibody-based therapeutic strategies that leverage alternative modes of cellular toxicity. Our approach identifies MSLN, NECTIN4, TROP2, and other antigens which have previously been shown to be largely tumor-specific but also reveals the expression of multiple putative targets within the CEACAM, claudin, and tetraspanin families. Finally, molecular subtyping efforts over the past decade have yielded classical and basal-like as consensus subtypes with variations therein, but genetic dependencies and cell surface expression patterns unique to either are insufficiently understood. We identified CLDN18, CEACAM5, and CEACAM6 as cell surface antigens for the classical subtype and MSLN, AQP5, and SLC6A14 for basal-like. Dependency on TLK2 and CCND1 is associated with the basal-like and classical subtype, respectively. Taken together, our integrative genomic approach may provide a precision medicine blueprint for stratifying and targeting pancreatic cancer. Citation Format: Jimmy A. Guo, Daniel Zhao, Scott P. Ginebaugh, Steven Wang, Ananya D. Jambhale, Patrick Z. Yu, Westley W. Wu, Peter Chen, Maryann Zhao, Kristen E. Lowder, Kevin S. Kapner, Hannah I. Hoffman, Stephanie W. Cheng, Daniel Y. Kim, Rebecca Boiarsky, Francois Aguet, Brenton Paolella, John M. Krill-Burger, James M. McFarland, Tobiloba Oni, Tyler Jacks, Aviv Regev, Gad Getz, William L. Hwang, Harshabad Singh, Andrew J. Aguirre. Integrative genomic characterization of therapeutic targets for pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-006.