PNAS Research Articles

Discovering the Intriguing Properties of Extrasynaptic γ-Aminobutyric Acid Type A Receptors.

Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by α5 subunit-containing γ-aminobutyric acid type A receptors. By Caraiscos VB, Elliott EM, You-Ten KE, Cheng VY, Belelli D, Newell JG, Jackson MF, Lambert JJ, Rosahl TW, Wafford KA, MacDonald JF, Orser BA. Proc Natl Acad Sci U S A 2004; 101:3662-7. Reprinted with permission. In this Classic Paper Revisited, the author recounts the scientific journey leading to a report published in the Proceedings of the National Academy of Sciences (PNAS) and shares several personal stories from her formative years and "research truths" that she has learned along the way. Briefly, the principal inhibitory neurotransmitter in the brain, γ-aminobutyric acid (GABA), was conventionally thought to regulate cognitive processes by activating synaptic GABA type A (GABAA) receptors and generating transient inhibitory synaptic currents. However, the author's laboratory team discovered a novel nonsynaptic form of tonic inhibition in hippocampal pyramidal neurons, mediated by extrasynaptic GABAA receptors that are pharmacologically distinct from synaptic GABAA receptors. This tonic current is highly sensitive to most general anesthetics, including sevoflurane and propofol, and likely contributes to the memory-blocking properties of these drugs. Before the publication in PNAS, the subunit composition of GABAA receptors that generate the tonic current was unknown. The team's research showed that GABAA receptors containing the α5 subunit (α5GABAARs) generated the tonic inhibitory current in hippocampal neurons. α5GABAARs are highly sensitive to GABA, desensitize slowly, and are thus well suited for detecting low, persistent, ambient concentrations of GABA in the extracellular space. Interest in α5GABAARs has surged since the PNAS report, driven by their pivotal roles in cognitive processes and their potential as therapeutic targets for treating various neurologic disorders.

Abstract 6340: Novel conditionally active tetravalent B7-H3 x CD3 T-cell engagers targeting solid tumors

Abstract B7-H3, also known as CD276, is an immune-checkpoint molecule and a type 1 transmembrane protein that exerts a variety of biological functions. In cancer biology, the overexpression of B7-H3 is correlated with tumor cell invasion and metastasis, decreased T-cell infiltration and function, poor prognosis, and resistance to therapy. As B7-H3 is expressed in multiple solid tumors and plays an important role in modulating anti-tumor immune response as well as promoting cancer cell formation, it became an important target for the development of antibody-based immunotherapy. Many forms of bispecific antibodies targeting B7-H3 have shown antitumor response in preclinical studies. However, bispecific T-cell engagers (TCE) targeting B7H3 and CD3 have been correlated with cytokine release syndrome and hepatic adverse events, a common toxicity observed using TCEs which has limited the development of this immunotherapeutic in clinic. We leveraged BioAtla’s Conditionally Active Biologic (CAB) platform (1) to develop a DualCAB T-cell engager targeting B7-H3 and CD3. This DualCAB-TCE molecule, BA3142, was engineered to bind with high affinity to both recombinant B7-H3 and CD3 receptor/antigens under conditions that mimic the acidic tumor microenvironment, but with low affinity in alkaline physiological conditions. In vitro and in vivo efficacy and toxicity data for the clone BA3142 will be presented. Our data demonstrated that BA3142 promotes cytotoxicity of B7-H3-expressing cancer cells in vitro and induces significant tumor regression in vivo. In Non-Human Primates BA3142 bispecific antibody was tolerated up to 25 mg/kg with minimal cytokine release and no clinical and histopathological signs of toxicities. The CAB technology offers the opportunity to expand the number of targets suitable for drug development in a new class of DualCAB TCEs with superior safety margin and potency in the clinic. (1) Chang HW, Frey G, Liu H, Xing C, Steinman L, Boyle WJ, and Short, J.M. Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches. Proc Natl Acad Sci U S A 2021;118. Citation Format: Ana Paula Cugnetti, PhD, Haizhen Liu, Patricia McNeeley, Kathryn Woodard, Cathy Chang, Gerhard Frey, William J. Boyle, Jay M. Short. Novel conditionally active tetravalent B7-H3 x CD3 T-cell engagers targeting solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6340.

Abstract 261: Akt1 and Cyclin D1 govern polarity of self-renewing divisions in breast cancer stem cells

Abstract Background. Stem-like cells contribute to the development and progression of human cancers. Genes that control symmetric cell divisions have an evolutionarily conserved role in the regulation of cell polarity and in tumorigenesis. Two cells generated by asymmetric divisions have reduced proliferative potential compared with symmetric divisions. The cell survival oncoprotein Akt (Protein Kinase B) is encoded by three isoforms that are altered in human cancers. The Akt isoform-specific impact on breast cancer stem cell function and polarity is controversial, with Akt1 either reducing (1) or promoting (2) cell polarity and migration in tissue culture. Deletion of Akt1 reduced mammary tumor progression and metastasis in transgenic mice (2,3). Methods. Analysis of Akt1, cyclin D1 and Notch signaling was conducted from public databases, and human as well as murine breast cancer samples. Cells derived from Akt1 gene deletion MMTV-ErbB2 breast oncomice (MMTV-ErbB2/Akt1+/+ and MMTV-ErbB2/Akt1−/−) and from the human MCF-7 breast cancer cell line with regulated expression of cyclin D1 and p53 were analyzed for the symmetry of cell division, stem cell function, and signaling pathways. Results. In breast tumors derived from MMTV-ErbB2 transgenics, associated with reduced metastasis, Akt1 genetic deletion reduced the frequency of symmetric cell divisions and the production of mammospheres. Akt1 deletion increased p53 and reduced cyclin D1. Pharmacological reactivation of p53 with Nutlin correlated with the reinduction of asymmetric cellular divisions and reduction in mammospheres in murine and human BCa cells. Akt1 deletion abrogated Nutlin-mediated induction of asymmetric cell divisions. Cyclin D1 induced symmetrical cell divisions, mammosphere formation and reduced p53 and Numb. Conclusions. Akt1 promoted symmetrical cell division at least in part via cyclin D1 suppression of Numb, and induction of γ secretase activity and thereby Notch signaling. Reference. (1) Irie HY, Pearline RV, Grueneberg D, Hsia M, Ravichandran P, Kothari N, et al. Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial-mesenchymal transition. J Cell Biol 2005;171:1023-34. (2) Ju X, Katiyar S, Wang C, Liu M, Jiao X, Li S, et al. Akt1 governs breast cancer progression in vivo. Proc Natl Acad Sci U S A 2007;104:7438-43. (3) Chen X, Ariss MM, Ramakrishnan G, Nogueira V, Blaha C, Putzbach W, et al. Cell-Autonomous versus Systemic Akt Isoform Deletions Uncovered New Roles for Akt1 and Akt2 in Breast Cancer. Mol Cell 2020;80:87-101 e5 Citation Format: Xuanmao Jiao, Xin Sun, Zhiping Li, Xiaoming Ju, Danni Li, Ritika Harish, Gordon Robertson, Anthony W. Ashton, Richard G. Pestell. Akt1 and Cyclin D1 govern polarity of self-renewing divisions in breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 261.

Abstract 744: Novel conditionally active biologic (CAB) tetravalent T-cell engagers targeting solid tumors

Abstract The use of T-cell engagers (TCEs) has great therapeutic potential in oncology. This potential, however, is diminished due to severe toxicity (cytokine release effects, as well as on-target off-tumor toxicities). We leveraged BioAtla’s Conditionally Active Biologic (CAB) technology (1) to develop bispecific antibodies which have no or very little binding to CD3 and to the tumor target antigen (TAA) in healthy tissue (normal physiological conditions), yet have strong binding in diseased tissues (i.e. tumor microenvironment, TME) derived from glycolytic tumor metabolism, which supports tumor growth (Warburg effect). The conditional activity in the TME is achieved by optimizing the sequences of the binding domains and does not require masking and subsequent activation of the binding domain as used in pro-drugs. We have developed dual-CAB T-cell engagers targeting serval well-established tumor associated antigens. Data demonstrating superior potency with reduced off-target binding through in vitro and in vivo characterization of three new DualCAB TCEs, targeting PSMA, Trop2, and Tissue Factor (TF) will be presented.(1) Chang HW, Frey G, Liu H, Xing C, Steinman L, Boyle WJ and Short, JM Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches. Proc Natl Acad Sci U S A 2021;118. Citation Format: Ana Paula Cugnetti, Haizhen Liu, Patricia McNeeley, Charles Xing, Kathryn Woodard, Hwai Chang, Gerhard Frey, William J. Boyle, Jay M. Short. Novel conditionally active biologic (CAB) tetravalent T-cell engagers targeting solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 744.

Abstract 1634: Cyclin D1 induces Akt1 activity in RB deficient prostate cancer

Abstract Background: Prostate cancer (PCa), the second leading cause of death in American men, is a genetically heterogeneous disease. The aberrant activation of AKT is one of the most frequent alterations in human cancers, and an elevated level of phosphorylated AKT at Ser473 site (pSer473) is associated with metastatic cancers (1). In prostate cancer (PCa), PI3K/AKT activation induces tumorigenesis (2), androgen receptor (AR)-mediated signaling, enzalutamide resistance and stemness, synergizing with other pathways including the notch intracellular domain (NICD). The cyclin D1 gene (CCND1), which is overexpressed in PCa, conveys canonical function by encoding the regulatory subunit of a holoenzyme that phosphorylates the retinoblastoma gene (RB) product, and non-canonical functions through phosphorylating other substrates and transcriptional activities. Cyclin D1 expression augments growth of some castrate-resistant PCa (CRPC) and clinical trials are targeting the cyclin D1 related CDK kinase activity in pRB+ PCa. Several Rb-independent mechanisms of action have been described (3). In recent studies the effect of CDK inhibitors was similar in patients with Rb+ vs. Rb− PCa (4). Whether Rb expression is required for the antiproliferative effects of CKD4/6 inhibition in PCa remains to be established. Methods: The kinetics of Akt1 induction by mitogens was assessed using the phosphorylation of the dichromic fluorescent (DCF) dye substrate LS456. We used cyclin D1 shRNA to show endogenous cyclin D1 augmented AKT1 signaling in response to mitogens. Results: Cyclin D1 reintroduction into cyclin D1−/− cells augmented Akt1 phosphorylation, requiring the cyclin D1 K112 residue. CDK inhibitors reduced Akt activity in RB+ and RB− human prostate cancer cell lines. Prostate-specific epithelial cell deletion of cyclin D1 (probasin-CRE-cyclin D1fl/fl) reduced prostate epithelial cell Akt1 activity assessed by immunohistochemical (IHC) staining. pAktSer473 was reduced in cell nuclei and the cytoplasm of both epithelial cells and stromal fibroblasts without a significant change in the abundance of Akt1. The downstream targets of Akt signaling (pTSC2Ser939, and pFKHRSer319) and ARSer213P were reduced in both epithelial cells and stromal fibroblasts. Genetic deletion of both epithelial cell and stromal cyclin D1 further reduced Akt1 activity, prostate epithelial cell proliferation, and markers of cancer stem cells. Proteomic analysis of cyclin D1-expressing stroma identified heterotypic chemokine signals that augment PCa AKT activity. Conclusions: Cyclin D1 induces Akt1 activity in both RB+ and RB− PCa cells which may contribute to tumor progression and therapy resistance. Reference: (1) Chen YL, Law PY, Loh HH. Curr Med Chem Anticancer Agents 2005;5:575-892. (2) Stoyanova T, et al. Proc Natl Acad Sci U S A 2013;110:20111-63. (3) Chen K, et al. Cell Rep 2020;32:1081514. (4) de Kouchkovsky I, et al. Clin Cancer Res 2022;28:1531-9 Citation Format: Xuanmao Jiao, Anthony W. Ashton, Zhiping Li, Ritika Harish, Danni Li, Gordon Robertson, Duanwen Shen, Xiaoming Ju, Ke Chen, Wei Zhang, Samuel Archilefu, Kenneth A. Iczkowski, Hallgeir Rui, Beatrice Knudsen, Paul Timpson, Max Nobis, Richard G. Pestell. Cyclin D1 induces Akt1 activity in RB deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1634.

Abstract 743: Using a novel NextGen linker system to generate a conditionally active biologic (CAB) anti-nectin4-ADC demonstrates improved efficacy in pancreatic PDX cancer models and improved tolerability and toxicity profile in non-human primates

Abstract Antibody-drug conjugates (ADCs) are a promising treatment for various forms of cancer. The treatment efficacy is often limited by on-target/off-tumor toxicity caused by the antigen expression in healthy tissue and by unwanted payload release outside the tumor caused by cleavage of the linker via circulating proteases. Our conditionally active biologics (CAB) technology addresses the on-target/off-tumor toxicity and assists in reducing the off-target/off-tumor toxicity by reducing binding to the target under normal physiological conditions (1). To eliminate the remaining off-target/off-tumor toxicity, we developed a novel linker with superior serum stability, solubility, and tumor-specific payload release. Conjugation can be accomplished using regular IgG backbones without the need for sequence modification. Here we report the in vitro and in vivo characterization of a NextGen linker system to generate a CAB anti-Nectin4-ADC that combines the advantages of CAB antibody pH selectivity with the new linker technology. In vivo efficacy data demonstrated complete tumor regression in several cell line derived xenograft models as well as superior efficacy to an enfortumab vedotin analogue in a patient derived pancreatic cancer model. PK and tox data from a toxicology study in non-human primates will be presented. In addition, data demonstrating the influence of linker technology on specific cancer models will also be presented. In conclusion, the NextGen Nectin4 CAB ADC represents a potentially more effective treatment with increased safety in the clinic. (1) Chang HW, Frey G, Liu H, Xing C, Steinman L, Boyle WJ,and Short JM Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches. Proc Natl Acad Sci U S A 2021;118 Citation Format: Jing Wang, Jian Chen, Gerhard Frey, Haizhen Liu, Charles Xing, Kathryn Woodard, Hwai Chang, William J. Boyle, Jay M. Short. Using a novel NextGen linker system to generate a conditionally active biologic (CAB) anti-nectin4-ADC demonstrates improved efficacy in pancreatic PDX cancer models and improved tolerability and toxicity profile in non-human primates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 743.

The Grammar of Persuasion: A Meta-Analytic Review Disconfirming the Role of Nouns as Linguistic Cues of Subsequent Behavior

Recent discoveries suggest that nouns, as a word class, exhibit a greater capacity to model real-life behavior. In the studies by [Proc Natl Acad Sci USA 108, 12653–12656 (2011)] participants prompted with a noun label rather than a semantically corresponding verb ( be a voter vs. vote) were more likely to vote. However, many attempts to replicate the original results have shown unsuccessful [Proc Natl Acad Sci USA 113, 7112–7117 (2016)]. To shed light on the effectiveness of subtle linguistic manipulations, we conducted a meta-analysis of 28 effect sizes ( N = 13,376), being an exact or conceptual replications of Bryan et al. Although the average effect was significant ( g = 0.12), the concurrent diagnostics indicated a low trust in the replicability or stability of the effect. Based on that, we cannot conclude that the greater effectiveness of nouns as cues has been reliably demonstrated. We discuss theoretical prospects involving the verb-action link as well as the self-verification motives.

Generalized Partitioned Local Depth

In this paper, we provide a generalization of the concept of cohesion as introduced recently by Berenhaut et al. (Proc Natl Acad Sci 119:2003634119, 2022). The formulation presented builds on the technique of partitioned local depth by distilling two key probabilistic concepts: local relevance and support division. Earlier results are extended within the new context, and examples of applications to revealing communities in data with uncertainty are included. The work sheds light on the foundations of partitioned local depth, and extends the original ideas to enable probabilistic consideration of uncertain, variable and potentially conflicting information.

A Countable-Type Branching Process Model for the Tug-of-War Cancer Cell Dynamics.

We consider a time-continuous Markov branching process of proliferating cells with a countable collection of types. Among-type transitions are inspired by the Tug-of-War process introduced by McFarland et al. (Proc Natl Acad Sci 111(42):15138-15143, 2014) as a mathematical model for competition of advantageous driver mutations and deleterious passenger mutations in cancer cells. We introduce a version of the model in which a driver mutation pushes the type of the cell L-units up, while a passenger mutation pulls it 1-unit down. The distribution of time to divisions depends on the type (fitness) of cell, which is an integer. The extinction probability given any initial cell type is strictly less than 1, which allows us to investigate the transition between types (type transition) in an infinitely long cell lineage of cells. The analysis leads to the result that under driver dominance, the type transition process escapes to infinity, while under passenger dominance, it leads to a limit distribution. Implications in cancer cell dynamics and population genetics are discussed.

Highly Durable Electrocatalyst with Low-Loading Pt-Co Dispersed over Graphene Nanofiber for Oxygen Reduction Reaction Under PEMFC Condition

Proton exchange membrane fuel cell (PEMFC) has been demonstrated as a highly efficient energy conversion technology. The sluggish oxygen reduction reaction (ORR) at the fuel cell cathode requires 3 to 5 times more catalysts than the hydrogen oxidation reaction at the anode (1). Currently, platinum is still the catalyst material of choice. However, the heavy use of scarce Pt in the electrodes represents a major cost barrier to the commercialization of PEMFCs. Tremendous efforts have been dedicated to reduce or remove Pt usage through the development of Pt-transition metal catalysts, as highlighted recently by dealloyed PtNi (2), Mo-doped PtNi(3), and ordered PtCo (4); or through the development of transition metals-nitrogen-carbon based PGM-free catalysts (5, 6).In the fuel cell, catalysts should be highly dispersed over the electrode surface to be easily accessible by the reactants, especially at the high fuel cell current density where a large influx of O2 must be converted. For the Pt alloy catalyst of large crystallites, there won’t be enough crystallites available to spread over the electrode surface to encounter the incoming O2 if the total loading of platinum has to be maintained ultralow. Furthermore, unprotected Pt nano-alloys may lose their nanostructure and crystallinity from the dissolution and agglomeration during the ORR process. The PGM-free catalysts, on the other hand, have high catalytic site density with uniform distribution when prepared by homogenous precursors such as metal-organic framework or porous organic polymer, rendering them highly efficient in interacting with O2 flux. Their key drawback, however, is the poor stability operated under PEMFC condition.In this presentation, we will describe a method of preparing highly active yet stable electrocatalysts containing ultralow Pt content using polymer fiber containing cobalt zeolitic imidazolate framework as precursor. The new catalyst contains Pt-Co nanoparticels situated over the PGM-free catalystically active support (7,8). The ORR activities of the new catalyst was first tested in O2 saturated acidic electrolyte by rotating ring disk electrode (RRDE) before fabricated into the membrane electrode assembly (MEA) and evaluated under fuel cell operating condition. The synergistic catalysis between PtCo NPs and the PGM-free active substrate led to an unprecedented oRR performance in both RRDE and fuel cell. For example, the fuel cell test demonstrated a mess activity of 2.43 A/mgPt for the new catalysts and > 80% retention of the initial mass activity after 60000 continuous voltage cycles from 0.6 – 1.0 V.The characterizations of the fresh and the post-electrochemical test samples were investigated by x-ray photoelectron spectroscopy (XPS), x-ray diffraction (XRD), x-ray absorption near-edge structure (XANES), extended x-ray absorption fine structure (EXAFS), transmission electron microscopy (TEM), Raman spectroscopy and BET surface analysis. We also performed DFT calculation on the ORR thermodynamic barriers over both Pt-Co NPs and PGM-free site as the descriptors for reaction pathway. The results reveal that the synergistic interaction between Pt-Co and PGM active substrate contribute significantly to both activity and durability enhancements.Acknowledgments:This work was supported by Argonne National Laboratory through Maria Goeppert Mayer Fellowship, US; Overseas Outstanding Youth Fund project, shanghai Jiao Tong university, Shanghai, China, State Key Laboratory of Metal Matrix Composites, shanghai Jiao Tong university, Shanghai, China. The works performed at Argonne National Laboratory’s Center for Nanoscale Materials and Advanced Photo Source, and Hydrogen research center, shanghai Jiao Tong university.Reference: H. A. Gasteiger, N. M. Markovic, Just a Dream-or Future Reality? Science 324, 48-49 (2009).B. Han et al., Record activity and stability of dealloyed bimetallic catalysts for proton exchange membrane fuel cells. Energy & Environmental Science 8, 258-266 (2015).X. Huang et al., High-performance transition metal–doped Pt3Ni octahedra for oxygen reduction reaction. Science 348, 1230-1234 (2015).D. Wang et al., Structurally ordered intermetallic platinum-cobalt core-shell nanoparticles with enhanced activity and stability as oxygen reduction electrocatalysts. Nat Mater 12, 81-87 (2013).L. Chong et al., Investigation of Oxygen Reduction Activity of Catalysts Derived from Co and Co/Zn Methyl-Imidazolate Frameworks in Proton Exchange Membrane Fuel Cells. ChemElectroChem 3, 1541-1545 (2016).J. Shui, C. Chen, L. Grabstanowicz, D. Zhao, D. J. Liu, Highly efficient nonprecious metal catalyst prepared with metal-organic framework in a continuous carbon nanofibrous network. Proc Natl Acad Sci U S A 112, 10629-10634 (2015).L. Chong et al., Ultralow-loading platinum-cobalt fuel cell catalysts derived from imidazolate frameworks. Science, (2018).L. Chong et al., Highly durable fuel cell electrocatalyst with low-loading Pt-Co nanoparticles dispersed over single-atom Pt-Co-N-graphene nanofiber. Chem Catalysis 3, 100541.

Impact of Reinforced Polymer Electrolyte Membrane Scratch on Fuel Cell Durability Using 4D X-Ray Computed Tomography Technique

PEMFCs, or Polymer Electrolyte Membrane Fuel Cells, can be used in both stationary power and transportation applications and are promising for use in heavy duty electric vehicles due to their high energy density, ability to be refueled quickly, and extended range, which are key challenges for battery-powered vehicles [1]. Despite these advantages, there are still obstacles preventing the widespread commercialization of PEMFCs, one of which is production cost [2]. To reduce manufacturing costs, it is imperative to ensure that quality control rejects and scraps are minimized. Moreover, enhancing component integration defect detection would ensure the durability of the fuel cell and enhance its operational lifetime. However, robust quality control requires a thorough understanding of potential non-uniformities and their effects on fuel cell performance and operational degradation.Non-uniformities of the membrane electrolyte assembly (MEA) can result in durability issues by damaging the membrane and making it more vulnerable to mechanical and chemical stress during the operation [3]. Several studies have been done on cation contamination and membrane pinholes and their effect on fuel cell performance and durability [4,5] but little attention has been paid to other types of non-uniformities such as membrane scratches and foreign particles, which may also conceivably affect durability [6]. By implementing 4D in-situ X-ray computed tomography (XCT) [7,8], the current work aims to track the evolution of scratches that appear on a reinforced membrane surface during fabrication and its specific objective is to determine the impacts of membrane scratches on membrane durability in fuel cells.The study was conducted by using small-scale fuel cells with GORE-SELECT® membranes that were intentionally scratched with a razor blade, subjecting them to combined chemomechanical membrane accelerated stress test (AST), and monitoring the membrane degradation and evolution of the scratches using 4D-XCT throughout the AST. During the fabrication step, the depth of scratches was regulated by a weak bond between the reinforcement layer and the perfluorosulfonic acid (PFSA) layer, i.e., the scratches did not tear through the reinforcement. Furthermore, the scratches did not adversely impact the initial cell performance. However, during AST operation the evolution of the scratched region was affected by several factors, including the friction between the layers of the membrane electrode assembly, the width of the scratch (Figure 1), the presence of membrane debris, and the extent of membrane strain. Additionally, it was observed that changes in the quality of catalyst layer transfer onto the scratched region can alter the degradation pattern and potentially create a self-mitigating mechanism. These findings may be used to develop more optimized quality control measures for membrane scratches in fuel cell production. Acknowledgements This research was supported by the Natural Sciences and Engineering Research Council of Canada, Canada Foundation for Innovation, British Columbia Knowledge Development Fund, Western Economic Diversification Canada, Ballard Power Systems, and W.L. Gore & Associates. This research was undertaken, in part, thanks to funding from the Canada Research Chairs program. Keywords: fuel cell; membrane durability; X-ray computed tomography; membrane scratch; manufacturing References [1] Cullen DA, Neyerlin KC, Ahluwalia RK, Mukundan R, More KL, Borup RL, et al. New roads and challenges for fuel cells in heavy-duty transportation. Nat Energy 2021;6:462–74. https://doi.org/10.1038/s41560-021-00775-z.[2] Whiston MM, Azevedo IL, Litster S, Whitefoot KS, Samaras C, Whitacre JF. Expert assessments of the cost and expected future performance of proton exchange membrane fuel cells for vehicles. Proc Natl Acad Sci U S A 2019;116:4899–904.[3] De Bruijn FA, Dam VAT, Janssen GJM. Review: Durability and degradation issues of PEM fuel cell components. Fuel Cells 2008;8:3–22.[4] Tavassoli A, Lim C, Kolodziej J, Lauritzen M, Knights S, Wang GG, et al. Effect of catalyst layer defects on local membrane degradation in polymer electrolyte fuel cells. J Power Sources 2016;322:17–25.[5] Lü W, Liu Z, Wang C, Mao Z, Zhang M. The effects of pinholes on proton exchange membrane fuel cell performance. Int J Energy Res 2011;35:24–30.[6] Kundu S, Fowler MW, Simon LC, Grot S. Morphological features (defects) in fuel cell membrane electrode assemblies. J Power Sources 2006;157:650–6.[7] Ramani D, Singh Y, White RT, Wegener M, Orfino FP, Dutta M, et al. 4D in situ visualization of mechanical degradation evolution in reinforced fuel cell membranes. Int J Hydrogen Energy 2020;45:10089–103.[8] White RT, Eberhardt SH, Singh Y, Haddow T, Dutta M, Orfino FP, et al. Four-dimensional joint visualization of electrode degradation and liquid water distribution inside operating polymer electrolyte fuel cells. Sci Rep 2019;9:1–12. Figure 1

Experimental and Theoretical Investigations of Shunt Currents between Alkaline Water Electrolyzers

A pivotal component of national climate strategies is the transition of the energy sector from fossil fuels to green energy. In this context, electrochemical processes will play a significant role in the future, requiring electrochemical systems with high efficiency and maximum system life cycles.1,2 For industrial electrochemical systems capital and operating costs are often reduced by a system design with a shared and circulating electrolyte supply providing ionically conductive cell-to-cell pathways. Under such conditions, parasitic ion migration occurs between adjacent cells, known as shunt currents.3,4 Shunt currents can have severe implications, such as decrease in faraday efficiency, material corrosion or interferences with instrumentation 3,5,6.To ensure high efficiency and maximum system life cycle, shunt current estimation is of high importance for the design of electrochemical multi-cell systems. A widely used approach in the literature is the theoretical, model-based approach using equivalent circuit models for shunt current determination7–9. Only a few publications demonstrate experimental determination methods 10,11. Furthermore, most shunt current studies in the field of electrochemistry focus on redox flow batteries.This work shows an innovative experimental approach for direct shunt current measurements between two alkaline water electrolysis cells with shared and circulating electrolyte feed under various conditions. The flow field design enables the insertion of reference electrodes into the flow cells for direct measurement of the potential differences between adjacent electrodes. Combined with the experimental data of the ionic tube resistances, the cell-to-cell shunt currents were accurately determined. Furthermore, an equivalent circuit model was created, fed with experimental data and validated with measured results. After successful validation the model was extended to electrolysis systems with more than two cells.Experimental data and simulations are in good agreement. The conducted experiments show the impact of temperature, cell voltage and tube manifold geometry on shunt current formation between alkaline water electrolyzers. Simulations performed are carried out to calculate shunt currents as a function of these parameters in large multi-cell systems. Furthermore, efficiency losses and corrosion processes as a result of shunt currents are estimated based on the results of this work.Literature Baños R, Manzano-Agugliaro F, Montoya FG, Gil C, Alcayde A, Gómez J. Optimization methods applied to renewable and sustainable energy: A review. Renew Sustain Energy Rev. 2011;15(4):1753-1766. doi:10.1016/j.rser.2010.12.008Yan Z, Hitt JL, Turner JA, Mallouk TE. Renewable electricity storage using electrolysis. Proc Natl Acad Sci U S A. 2020;117(23):12558-12563. doi:10.1073/pnas.1821686116Delgado NM, Monteiro R, Cruz J, Bentien A, Mendes A. Shunt currents in vanadium redox flow batteries – a parametric and optimization study. Electrochim Acta. 2022;403:139667. doi:10.1016/j.electacta.2021.139667Kaminski EA, Savinell RF. A Technique for Calculating Shunt Leakage and Cell Currents in Bipolar Stacks Having Divided or Undivided Cells. J Electrochem Soc. 1983;130(5):1103-1107. doi:10.1149/1.2119891Yin C, Guo S, Fang H, Liu J, Li Y, Tang H. Numerical and experimental studies of stack shunt current for vanadium redox flow battery. Appl Energy. 2015;151:237-248. doi:10.1016/j.apenergy.2015.04.080Pletcher D, Walsh FC. Industrial Electrochemistry. Springer Science & Business Media; 2012.Schaeffer JA, Chen L Der, Seaba JP. Shunt current calculation of fuel cell stack using Simulink®. J Power Sources. 2008;182(2):599-602. doi:10.1016/j.jpowsour.2008.04.014Wandschneider FT, Röhm S, Fischer P, Pinkwart K, Tübke J, Nirschl H. A multi-stack simulation of shunt currents in vanadium redox flow batteries. J Power Sources. 2014;261:64-74. doi:10.1016/j.jpowsour.2014.03.054Ye Q, Hu J, Cheng P, Ma Z. Design trade-offs among shunt current, pumping loss and compactness in the piping system of a multi-stack vanadium flow battery. J Power Sources. 2015;296:352-364. doi:10.1016/j.jpowsour.2015.06.138Rous̆ar I, Cezner V. Experimental Determination and Calculation of Parasitic Currents in Bipolar Electrolyzers with Application to Chlorate Electrolyzer. J Electrochem Soc. 1974;121(5):648. doi:10.1149/1.2401878Fink H, Remy M. Shunt currents in vanadium flow batteries: Measurement, modelling and implications for efficiency. J Power Sources. 2015;284:547-553. doi:10.1016/j.jpowsour.2015.03.057

MSphere of Influence: The complex world of bacterial biogeography.

Drew Bridges works in the field of bacterial signal transduction and studies the formation and disassembly of bacterial biofilms. In this mSphere of Influence article, he reflects on how the paper "Biogeography of a human oral microbiome at the micron scale" by Mark Welch et al. (J. L. Mark Welch, B. J. Rossetti, C. W. Rieken, F. E. Dewhirst, et al., Proc Natl Acad Sci U S A 113:E791-E800, 2016, https://doi.org/10.1073/pnas.1522149113) inspired him to change his research trajectory.

Iron Restricted Erythropoiesis Under Hepcidin Mimetic Treatment (PN23114) Improved Disease Parameters in a Mouse Model for Sickle Cell Disease

Sickle Cell Disease (SCD) is an inherited hemolytic disorder that results from a single point mutation in the hemoglobin-beta (HBB) gene that encodes for the beta-globin subunit of hemoglobin, causing the production of abnormal hemoglobin S (HbS). HbS polymerizes under low oxygen conditions, causing sickling effects on red blood cells (RBCs). Sickling of RBCs initiates vaso-occlusion, downstream inflammatory responses, and hemolysis (Zhang D, et al., Blood. 2016;127:801-9). Previous studies have shown that polymerization under low oxygen conditions is extremely sensitive to HbS concentrations within the RBCs (Hofrichter J, et al, Proc Natl Acad Sci USA. 1974;71:4864-8; Sunshine HR, et al. Nature. 1978;275:238-40). Controlling iron delivery for erythropoiesis provides a mechanism to potentially down-modulate the HbS concentration, thereby reducing the potential for HbS polymerization and sickling of RBCs (Parrow NL, et al., Blood. 2021;137:1553-5). The potent hepcidin mimetic peptide PN23114 targets the iron exporter membrane protein ferroportin to trigger its degradation, thus preventing iron export from cells. We hypothesize that the resulting pharmacodynamic effect of limiting iron availability for erythropoiesis should reduce HbS concentrations within the RBCs that may prevent sickling and hemolysis. Townes mice (genetically altered to contain the human SCD HbSS homozygous mutation) are a well-established experimental model for human SCD (Li-Chen W, et al., Blood. 2006;108:1183-8). They exhibit severe hemolysis as indicated by elevated bilirubin and lactate dehydrogenase (LDH) levels, and a shortened RBC half-life compared to wild type (WT) mice. These mice also develop vaso-occlusive events and elevated inflammatory responses (for example, increases in peripheral blood white blood cell [WBC] and neutrophil counts). To test whether iron restriction mediated by a hepcidin mimetic may alter outcomes in this model, PN23114 was administered subcutaneously to Townes HbSS mice. Mice were treated over 4-weeks (3 times weekly) with vehicle or PN23114 at 1 or 2.5 mg/kg (vehicle treated WT mice served as controls). At the end of 4 weeks, total bilirubin was elevated in the HbSS-vehicle group compared to WT-vehicle (Figure 1A). Total bilirubin was dose-dependently reduced by PN23114 treatment compared to the HbSS-vehicle group (Figure 1A). LDH was also elevated in the HbSS-vehicle group and was dose-dependently reduced with PN23114 treatment (557±105 units/L in WT-vehicle, 809±145 units/L in HbSS-vehicle, 611±341 units/L in HbSS-PN23114-1mg/kg and 372±71 units/L in HbSS-PN23114-2.5mg/kg groups). Peripheral blood WBC counts were dose-dependently reduced by PN23114 treatment compared to HbSS-vehicle (Figure 1B), with concomitant decreases in peripheral blood neutrophil counts. Reduction in hemoglobin values was observed with PN23114 treatment in HbSS (5.9±0.9 g/dL in HbSS-1mg/kg group and 4.4±0.4 g/dL in HbSS-2.5 mg/kg group) compared to the HbSS-vehicle group (7.1±0.3 g/dL). These results suggest that pharmacologic iron restriction with PN23114, a hepcidin mimetic peptide, led to a reduction in hemolysis and inflammatory markers in a SCD model. Therefore, a hepcidin mimetic therapeutic could potentially be a novel approach to prevent hemolysis, inflammation and downstream clinical complications associated with human SCD.

Transgenic Mouse Model for Translational Immunotherapy Studies on Multiple Myeloma

Background Genetically engineered mouse models that recapitulate key immunological features of human multiple myeloma may lend themselves to preclinical design and testing of novel immune therapies that target members of the tumor necrosis factor (TNF) receptor superfamily in addition to BCMA. One model of this sort are double-transgenic IL6Myc mice that spontaneously develop myeloma-like plasma cell tumors (PCTs) with full genetic penetrance (100% tumor incidence) and relatively short latency (4-5 months) in presence of an intact innate and adaptive immune system (Rutsch et. al. Blood 2010). IL6Myc mice exhibit serum M-spikes, respond to proteasome inhibition, and develop osteolytic lesions mimicking myeloma bone disease (Duncan K et. al. Blood Cancer J 2013, Sun F et. al. Haematologica 2020). Here, we examined changes in the immune tumor microenvironment during PCT progression in IL6Myc mice. We found that neoplastic plasma cell development was accompanied by accumulation of inhibitory T cells, T cell exhaustion, and elevated serum levels of MIP-3α and MCP-5. A subset of aberrant plasma cells expressed the TNF receptors, TACI and BAFF-R, which, together with their ligands, APRIL and BAFF, may serve as immunotherapeutic targets in myeloma not responding to BCMA-targeted treatment (Wong DP et. al. Nat Commun 2022, Braunstein M et. al. Expert Rev Hematol 2021). Materials and methods Double-transgenic IL6Myc mice were bred by intercrossing homozygous transgenic C.iMyc ΔEμ (Duncan K et. al. Blood Cancer J 2013) and heterozygous transgenic C.IL6 mice (Kovalchuk AL. et. al. Proc Natl Acad Sci USA 2002). Age-matched IL6Myc mice were investigated at an early (5-7 weeks), intermediate (10-12 weeks) and late (14-17 weeks) stage of PCT development using flow cytometry (Cytek Aurora) and serum cytokine levels (Bio-Plex) as main research tools. Results Bone marrow (BM) plasma cells, defined as CD45-gated B220 -CD138 + cells, increased during PCT development, as one might have expected: 10.5 ± 5.8% and 26.8 ± 6.4% ( p< 0.05) in early- and late-stage mice, respectively. The proportion of B220 +CD19 + B cells fell accordingly: 29.6 ± 4.1% vs 5.5 ± 4.9% ( p< 0.0001). Consistent with the notion that the BM microenvironment of PCT-bearing mice is immunosuppressive, there was a 2-fold elevation of TGF-β expressing T cells and a 4-fold elevation of IL-10 expressing T cells in the BM when late-stage mice were compared to early-stage mice. A sizeable fraction of aberrant plasma cells expressed TACI and BAFF-R in addition to BCMA ( Figure 1, left). Spleens of IL6Myc mice undergoing tumor development contained increasing numbers of FoxP3 + regulatory T cells (8.6 ± 2.1% at early stage vs 14.0 ± 4.6% at late stage, p< 0.05) and CD11b +Ly-6G/C + myeloid-derived suppressor cells (21.2 ± 9.2% vs 51.6 ± 10.1%, p< 0.01). Exhausted T cells, detected via expression of PD-1, TIM-3, LAG-3 and TIGIT, increased in sync with PCT progression ( Figure 1, right). Elevated serum levels of inflammatory cytokines, including MIP-3α (95.3 ± 41.9 pg/ml in early-stage mice vs 514 ± 279 pg/ml in late-stage mice, p< 0.01) and MCP-5 (121 ± 43.9 pg/ml vs 284 ± 70.5 pg/ml, p< 0.05), agreed with flow-based tissue data. Conclusion Neoplastic plasma cell development in IL6Myc mice takes place in an immune suppressed tumor microenvironment similar to that promoting human myeloma. IL6Myc-driven PCTs may provide a good experimental model system for novel immunotherapeutic approaches that target TACI and BAFF-R or their ligands, APRIL and BAFF. Previous work to that end holds great promise but has been challenging thus far (Goodman DB et. al. Sci Transl Med 2022, Lee L et. al. J Immunother Cancer 2023).

Phase 1 Study of SC291, a Hypoimmune, Allogeneic CD19-Directed CAR T Cell Therapy for Relapsed/Refractory B-Cell Malignancies (ARDENT) - Initial Clinical Data

ARDENT is a phase 1 study evaluating safety and tolerability of SC291, a hypoimmune (HIP), allogeneic, CD19-directed CAR T cell therapy in subjects with Non-Hodgkin's Lymphoma and Chronic Lymphocytic Leukemia (CLL). SC291 is derived from healthy donor CD4+ and CD8+ T cells that are genetically engineered to disrupt function of CD3 and HLA class I/II, overexpress CD47, and express a CD19-directed CAR. A primary obstacle for allogeneic CAR T cell approaches remains their immune recognition and rejection. Patient immune responses prevent durable allogeneic CAR T clinical responses despite the use of enhanced lymphodepletion (LD) regimens. We therefore developed hypoimmune CAR T cells that avoid immune recognition and have the potential to deliver clinical efficacy while using a standard LD chemotherapy regimen (as defined by approved autologous CAR T products). The HIP engineering approach involves the depletion of HLA class I/II to prevent adaptive immune rejection and the overexpression of CD47 to prevent innate immune rejection, which has previously demonstrated alloimmune protection in preclinical models ( Nat Biotechnol 2019;37(3):252-258; Proc Natl Acad Sci U S A 2021;118(28):e2022091118; J Exp Med. 2021;218(3):e20200839; Nat Biotechnol. 2023; doi: 10.1038/s41587-023-01784-x, and Nat Commun. 2023;14(1):2020). The initial patient, a 74-year-old male with CLL (unmutated IGHV, del(11q), BTK C481S mutation) and 3 prior lines of therapy (FCR, ibrutinib, venetoclax + rituximab), received a LD regimen of cyclophosphamide 500 mg/m 2 and fludarabine 24 mg/m 2 (daily for 3 days) followed by a starting dose of 60 million CAR+ SC291 cells (of which approximately 80% are fully HIP engineered cells). SC291 was well tolerated with no observed CRS or ICANS. The patient developed non-neutropenic fever after LD (prior to SC291 infusion). At the Day 28 visit post-SC291 infusion, the patient had a partial response (per iwCLL 2018 criteria), along with significant (99%) B-cell reduction and improvements in platelet levels. Analysis of cellular kinetics is ongoing. SC291 is a mixture of T cells, in which portions are partially or fully HIP-engineered. As such, we evaluated the initial patient's immune response against SC291 subpopulations using a panel of previously described in vitro assays. Blood samples from the patient were taken pre-treatment (Day -5) and at the Day 13 and Day 28 visits following SC291 treatment and were assessed for immune evasion. The patient's PBMCs were sorted into CD3+ T cells and CD3-CD56+ NK cells and incubated with the following subpopulations sorted from SC291 drug product (DP): (i) fully HIP engineered (CD19 HIP CAR T cells), (ii) HLA I/II negative T cells without CD47 overexpression (DKO), and (iii) HLA I/II positive CAR T cells overexpressing CD47 (WT CD19 CAR T cells). Patient serum from Day -5 and the Day 28 visit were used for donor-specific antibody analyses, complement-dependent cytotoxicity (CDC), and antibody-dependent cellular cytotoxicity (ADCC) against the DP subpopulations. At Day -5, T cell EliSpot assays showed no immune responses against any of the SC291 sorted subpopulations, but by Days 13 and 28, these assays produced high and increasing IFN-g spot frequencies against the WT CD19 CAR T cells. Similar results were shown with T cell cytotoxicity assays (Fig. 1B). In contrast, no T cell activation and no T cell killing was observed with DKO and CD19 HIP CAR T cells at any time point. As expected, patient NK cells vigorously killed DKO T cells at all time points but spared WT CD19 CAR T and CD19 HIP CAR T cells (Fig. 1B). No antibodies against any sorted subpopulation were detected at Day -5, but IgG antibody binding of the WT CD19 CAR T population was observed at Day 28. These antibodies did not bind to DKO or CD19 HIP CAR T cells, and there was no CDC or ADCC response against these subpopulations. Interestingly, with the LD regimen used, we observed a 95% and 99% reduction of CD19+ cells on Days 0 and 28, respectively, while T cells rebounded from an 88% reduction on day 0 to pretreatment levels on Day 28 (Fig. 1A). These data demonstrate the preliminary safety and tolerability of SC291 in the initial treated patient. Immune assays demonstrated that the CD19 HIP CAR T cell subpopulation effectively evades the host adaptive and innate immune responses and could overcome the allogeneic barrier in humans. Additional data from the ARDENT study will be presented at the time of the conference.

Clinical Characteristics, Genetic Profile and Prognosis of Adult Acute Myeloid Leukemia with NUP98 Rearrangement

Introduction: Nucleoporin 98 ( NUP98) rearrangement has been proposed in 2022 as one type of acute myeloid leukemia (AML) with defining genetic abnormalities. It occurred more often in pediatric leukemia with poor outcome. However, in adult AML with NUP98 rearrangement, patients may show different clinical characteristics, genetic profile and outcome, as we reported here. Method: We retrospectively collected and analyzed the data of adult AML patients with NUP98 rearrangement who were diagnosed at Shanghai Institute of Hematology from May 2019 to December 2022, including clinical characteristics, laboratory examination, genetic abnormalities, treatment strategies and outcome. All patients underwent next generation sequencing and RNA sequencing. Result: A total of 21 AML patients with NUP98 rearrangement were included in this study, counting for 2.3% of all AML patients (930 cases). The median age was 40 (19-64) years old. The median white blood cell and platelet count was 31.1 (1.5-313.6) and 48 (15-140) ×10 9/L, respectively. Interestingly, 11 (52.4%) patients suffered from severe or fatal hemorrhage irrelative with platelet count or coagulative function, including 4 cerebral bleeding (3 died), 1 urgent uterine bleeding, 1 gastro-intestinal bleeding, 1 retina bleeding and 4 massive bleeding of subcutaneous soft tissue or mucous membranes. The FAB types involved M2, M4 and M5. Eight patients carried normal karyotype, 6 were found with t(7;11)(p15;p15), all of whom had NUP98::HOXA9 fusion gene, 2 with +8 and 1 complex karyotype. According to ELN 2022 risk classification, 14 (66.7%) were at intermediate risk and 6 (28.6%) at adverse risk, 1 unclassified. Five fusion partners with NUP98 were detected: NUP98::NSD1 (11 cases), NUP98::HOXA9 (7 cases), NUP98::HOXA11, NUP98::HMGB3 and NUP98::TNRC18 (each with 1 case). Of which NUP98::TNRC18 was first reported by our center (see Cheng WY, et al. Proc Natl Acad Sci U S A. 2022;119(49):e2211429119). NUP98 rearrangement also co-existed with several mutations. FLT3-ITD (12 cases, of which 10 were co-existed with NUP98::NSD1) and WT1 (7 cases) were most frequently occurred. Patients with relapse/refractory disease had more additional mutations than de novo status (Figure 1). Nineteen patients were treated with standard dose of “7+3” induction therapy, and 2 with hypomethylating agents (HMA) plus venetoclax (VEN). Complete remission (CR) was achieved by 8 (38.1%) patients after first induction, 2 (9.5%) early deaths were both due to cerebral hemorrhage. Salvage induction varied from intensive chemotherapy (IC, including “7+3” and CLAG±M) to low-intensity therapy (LIT, including priming chemotherapy±HMA and HMA+VEN). FLT3 inhibitors (FLT3i) were added to 5 patients carrying FLT3-ITD, and they all achieved CR. A total of 15 (71.4%) patients achieved CR after 1-2 courses of induction (8 mainly benefited from IC, 7 mainly benefited from LIT±FLT3i), 1 survived from allogeneic hematopoietic stem cell transplantation (allo-HSCT) without reaching remission before, and 3 died from refractory disease within 6 months. Three patients relapsed after CR, 2 died without allo-HSCT, and the other one survived after CR2 and sequential allo-HSCT. A total of 14 patients underwent allo-HSCT after CR, 7 were measurable residual disease (MRD) negative and 7 were positive by flow cytometry before HSCT, none of them relapsed till the last follow-up. With a median follow-up time of 19.2 months, the median overall survival (OS) and event-free survival (EFS) were not reached, estimated 2-year OS and EFS rate were 66.7% and 57.1%, respectively. For patients achieving CR or not, the median OS were not reached vs 3.2 months (95% CI 0-8.361, P<0.001, Figure 2). Ten patients were diagnosed with NUP98::NSD1 co-occurring with FLT3-ITD (once considered as unfavorable prognosis), ending up with a CR rate of 70%, and a 2-year OS and EFS rate of 80% and 60%, respectively, making no significant difference compared to others ( P=0.236 and 0.725, respectively). Conclusion: Adult AML with NUP98 rearrangement may combine with a set of partner fusion genes and mutations, with NUP98::NSD1 and FLT3-ITD as the most common combination. Both IC an LIT can induce CR, and FLT3i will benefit those with FLT3 mutations, also prevention of fatal bleeding should be paid attention to. After achieving CR with whatever method and whatever MRD status, allo-HSCT is the key to long-term survival.

Targeted Protein Degradation for c-MYC Overcomes Therapy Resistance in T-Cell Acute Lymphoblastic Leukemias

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by diffuse infiltration of the bone marrow by immature lymphoblasts of thymic origin expressing T-cell surface markers. Early T cell precursor (ETP)-ALL is a distinct subtype of T-ALL characterized by a lack of CD1a and CD8; low expression of CD5 and the presence of myeloid or stem cell markers, and recent studies have shown resistance to conventional chemotherapies and poor outcome in ETP-ALL compared to thymic or mature T-ALL. A recent study revealed distinct gene expression profiles in adult compared to pediatric-adolescent ETP-ALL, representing higher expression levels of BCL2 and CD34 (Dai et al. Proc Natl Acad Sci 2022), however, detailed proteomic profiling in ETA-ALL has yet to be determined. Amplification of overexpression of MYC oncogene or stabilization of c-MYC protein occurs up to 70% in human cancers. Multifaceted activities of MYC include rapid proliferation of malignant cells supported by increased mitochondrial and ribosome biogenesis, dysregulated protein translation, and altered metabolism. NOTCH1-MYC signaling axis activation is one of the hallmarks that drives T-ALL leukemogenesis (Weng et al, Science 2004; Sanchez-Martin and Ferrando, Blood 2017). We have reported that targeting of BRD4 proteins induces suppression of leukemia initiating cells (LICs) in T-ALL by inhibiting the NOTCH1-MYC-CD44 axis, providing rationale to target MYC in therapy-resistant T-ALL (Piya et al. Leukemia 2022). We recently reported targeted protein degradation of c-MYC utilizing GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC exhibited promising anti-leukemia efficacy in acute myeloid leukemias (Nishida et al. ASH 2022). Here we employ GT19715 to investigate efficacy of targeting protein degradation of c-MYC in T-ALL. GT19715 induced dose-dependent apoptosis and cytoreduction in T-ALL cell lines with IC50 values below 10 nM except HPB-ALL cells. We found substantial decrease of c-MYC protein levels in sensitive but not in resistant cells (HPB-ALL). GT19715 induced apoptosis and ³ 90% cytoreduction at nanomolar concentrations in primary, therapy-resistant T-ALL. GT19715 also enhanced cell death induced by dexamethasone. In a xenograft model of CCRF-CEM cells carrying NOTCH1, PTEN, FBXW7, KRAS and TP53 mutations, GT19715 (3 mg/kg, three IP injections per week, two weeks on and one week off) reduced > 99% circulating human CD45+ leukemia cells compared to vehicle on day 16 after engraftment ( Fig. A), suggesting promising anti-leukemia efficacy in T-ALL in vivo. ETP-ALL cells from an adult patient exhibited distinct clustering compared to those from an adolescent patient as determined by single-cell mass cytometry (CyTOF) (N = 2). Adult ETP-ALL cells were characterized by increased protein levels of BCL2, CD34, CD44 and p-S6 compared to adolescent ETP-ALL cells, suggesting more stem-like properties. On the other hand, adolescent ETP-ALL cells showed higher CD7, CD33 and CD38 levels compared to the adult sample ( Fig. B). Interestingly, GT19715 treatment predominantly reduced p-S6 high clusters in adult ETP-ALL cells, potentially suggesting c-MYC degradation-induced suppression of the AKT-mTOR pathway. Conclusion: Targeted protein degradation of c-MYC induces promising anti-leukemia efficacy in T-ALL cells in vitro and in vivo. Further mechanistic and in vivo studies are ongoing.

FRI144 Using Single Cell Technology To Predict The Cell Of Origin Of Aldosterone-producing Adrenal Adenomas

Abstract Disclosure: K. Laycock: None. C. Cabrera: None. E. Wozniak: None. C. Mein: None. E. Azizan: None. X. Wu: None. G. Argentesi: None. E. Goodchild: None. W. Drake: None. M.J. Brown: None. Background: Aldosterone-producing adrenal adenomas (APAs) are the commonest surgically curable cause of hypertension, but individual prediction of postoperative outcome is challenging. Emerging data suggest that outcomes are influenced by APA genotype; identifying the cell of origin of APAs could facilitate a greater understanding of why certain genotypes are more/less likely to be associated with residual/recurrent autonomous aldosterone production(1, 2). Aims: 1. To develop an adult adrenal single cell atlas. 2. To identify the likely cell of origin for APAs with different mutations. Methodology: Bulk RNA sequencing from 67 APAs discovered known or novel somatic genotypes in 55, and replicated or discovered distinctive transcriptomes for each genotype. Single nuclei RNA sequencing was performed on 13 adrenals adjacent to the genotyped APAs. After aggregation of all 13 datasets, cluster analysis was undertaken to identify cell types and some of the transitions between these. Similarities were sought between the genotype-enriched transcriptomes of APAs, and the transcriptomes of individual cell clusters in adjacent adrenal. Results: A single nuclei atlas of 56,442 nuclei was developed. Clusters included each of the 3 cortical zones, aldosterone-producing micronodules, capsular cells, adrenal medulla, macrophages, T cells, stromal cells, endothelial cells and neurones. Several zona glomerulosa selective transcripts in previous laser-capture studies(3, 4) clustered separately from CYP11B2-positive cells (e.g. LGR5, GABBR2) or in distinct non-steroidogenic clusters (e.g. KIAA1210, RSPO3, PTGDS).A sub-cluster of stromal cells, positive for KIAA1210, RSPO3, CDH13 and MFAP5 (each p<10-254 cf other clusters) is likely to represent the distinctive KIAA1210+ capsular/interstitial cells on adrenal immunohistochemistry (www.proteinatlas.org). Bulk RNA sequencing of a rare CLCN2-mutant APA showed 13-30-fold higher expression of these and multiple other genes localising to the single nuclei stromal-cell cluster. Conclusion: Single nuclei sequencing documents the heterogeneity of adrenal cells, and potential for multiple different cell-types to develop into APAs. Ongoing analysis is underway to replicate the CLCN2-mutant transcriptome and identify cells-of-origins for tumours with commoner mutations. 1.Wu X et al. 11C metomidate PET-CT versus AVS for diagnosing surgically curable PA: a prospective within-patient trial. Nat Med. In press 2.Zhou J et al. Somatic mutations of GNA11 and GNAQ in CTNNB1-mutant APAs presenting in puberty, pregnancy or menopause. Nat Genet. 2021 3.Nishimoto K et al. Aldosterone-stimulating somatic gene mutations are common in normal adrenal glands. Proc Natl Acad Sci USA. 2015 4.Shaikh LH et al. LGR5 Activates Noncanonical Wnt Signaling and Inhibits Aldosterone Production in the Human Adrenal. J Clin Endocrinol Metab. 2015 Presentation: Friday, June 16, 2023

THU650 Prevalence Of Hyperandrogenemia, PCOS And Obesity In Mothers Of Daughters With PCOS

Abstract Disclosure: J. Cai: None. K. Brewer: None. R. Sisk: None. R.S. Legro: None. A.E. Dunaif: None. Prenatal exposure to androgens produces phenocopies of PCOS in animal models. In mice, there is evidence for transgenerational prenatal androgen effects. Human studies considered to support the hypothesis that intrauterine actions of androgens play a major role in PCOS pathogenesis come from (1) health registries where PCOS in mothers and daughters was diagnosed by ICD codes and (2) retrospective assessment of mother’s PCOS status in cohorts of affected women. Maternal PCOS status has not been directly assessed. As part of our genetic analyses of PCOS, we have prospectively recruited 678 mothers and 540 fathers of 678 women fulfilling NIH criteria for PCOS with confirmed hyperandrogenism and ovulatory dysfunction (OD). All subjects had fasting blood samples for reproductive hormones measured in central labs. Mothers completed a validated questionnaire for reproductive history. We tested the hypothesis that maternal PCOS accounted for PCOS in her daughter. We also investigated the contribution of maternal compared to paternal obesity to daughters’ obesity. We limited the analysis to mothers age 50 yrs and younger, with FSH <40 mIU/mL; 108 fathers were available from these families. Continuous data are reported as median and data range. Age (yrs): daughters 23 (13-24), mothers 46 (34-50), fathers 48 (37-69). Daughters had significantly higher BMI (kg/m2) (35.3, 18.5-60.0) than mothers (28.9, 18.7-54.1) and fathers (29.6, 18.4-55.5) (both P <0 .0001), while BMI did not differ in mothers and fathers. Hyperandrogenemia (HA), defined as total testosterone >58 ng/dL, biologically available testosterone >15 ng/dL or DHEAS >2683ng/mL, was present in 14.4% (20/139) of mothers, of these, 2.2% (3/139) fulfilled NIH criteria for PCOS, HA+OD. The prevalence of obesity, BMI ≥ 30.0 kg/m2, (66.2% daughter, 44.8% mother, and 45.7% fathers) was increased in daughters compared to both mothers (p= 0.0004) and fathers (p< 0.0001). Mothers with HA had higher BMI than those without it (P= 0.011). The odds ratio for obesity in daughters was 5.16 (1.97, 13.48) with maternal obesity and 2.29 (0.95, 5.52) with paternal obesity. Only ∼14% of reproductive-age mothers had HA, which we have shown in an underlying reproductive phenotype in PCOS families (Legro. Proc Natl Acad Sci USA, 1998); ∼ 2% of mothers fulfilled NIH criteria for PCOS. Although the PCOS reproductive phenotype ameliorates with age, it is clear that many affected daughters did not have a mother with hormonal features of PCOS. These findings do not support the hypothesis that prenatal androgen exposure is a final common path to PCOS in humans. The prevalence of obesity was higher in daughters than in parents and in HA mothers compared mothers without HA suggesting that obesity was associated with features of PCOS. Maternal obesity conferred greater risk for obesity in daughters than paternal obesity. Presentation: Thursday, June 15, 2023