Biology subjects

Kuehn, M.

Publications and source records attributed to Kuehn, M..

9 recordsLinked to original sources

bioRxiv2026

Menin-Inhibition Sensitizes Acute Myeloid Leukemia to CLEC12A-Directed CAR Cell Therapy

Menin inhibitors targeting the Menin-KMT2A chromatin complex have emerged as highly selective therapies for KMT2A-rearranged (KMT2A-r) and NPM1-mutated (NPM1mut) acute myeloid leukemia (AML), with recent regulatory approval and increasing interest in combination strategies. In contrast, CAR cell therapies have not yet been successfully established for AML. Here, we show that menin-inhibition primes KMT2A-r and NPM1mut AML for CAR-based targeting by inducing robust and uniform expression of the myeloid antigen CLEC12A (CLL-1). Menin inhibitors did not impair T or NK cell viability, phenotype, or effector function. We engineered second-generation CLEC12A-directed CAR T cells that efficiently eliminated CLEC12A-positive AML. Across in vitro systems and xenograft models, the combination therapy consistently outperformed either monotherapy, resulting in profound disease control and significantly prolonged survival, with evidence of near-complete leukemia eradication in vivo. These findings support epigenetic priming with menin inhibitors to enhance CLEC12A-directed CAR cell-therapy in these AML subtypes. SignificanceMenin inhibitors, now approved for AML treatment, induce the immune target CLEC12A in NPM1mut and KMT2A-r AML subtypes and sensitize AML cells to CLEC12A-directed CAR T cells without compromising immune function. As CLEC12A-CARs are already in clinical testing, this combination is immediately actionable for clinical investigation.

cancer biology↗

bioRxiv2023

Fast myosin binding protein C knockout in skeletal muscle alters length-dependent activation and myofilament structure.

In striated muscle, some sarcomere proteins regulate crossbridge cycling by varying the propensity of myosin heads to interact with actin. Myosin-binding protein C (MyBP-C) is bound to the myosin thick filament and is predicted to interact and stabilize myosin heads in a docked position against the thick filament and limit crossbridge formation, the so-called OFF state. Via an unknown mechanism, MyBP-C is thought to release heads into the so-called ON state, where they are more likely to form crossbridges. To study this proposed mechanism, we used the C2-/- mouse line to knock down fast-isoform MyBP-C completely and total MyBP-C by [~]24%, and conducted mechanical functional studies in parallel with small-angle X-ray diffraction to evaluate the myofilament structure. We report that C2-/- fibers presented deficits in force production and reduced calcium sensitivity. Structurally, passive C2-/- fibers presented altered SL-independent and SL-dependent regulation of myosin head ON/OFF states, with a shift of myosin heads towards the ON state. Unexpectedly, at shorter sarcomere lengths, the thin filament was axially extended in C2-/- vs. non-transgenic controls, which we postulate is due to increased low-level crossbridge formation arising from relatively more ON myosins in the passive muscle that elongates the thin filament. The downstream effect of increasing crossbridge formation in a passive muscle on contraction performance is not known. Such widespread structural changes to sarcomere proteins provide testable mechanisms to explain the etiology of debilitating MyBP-C-associated diseases.

physiology↗

bioRxiv2023

Myosin-binding protein C forms C-links and stabilizes OFF states of myosin

Contraction force in muscle is produced by the interaction of myosin motors in the thick filaments and actin in the thin filaments and is fine-tuned by other proteins such as myosin-binding protein C (MyBP-C). One form of control is through the regulation of myosin heads between an ON and OFF state in passive sarcomeres, which leads to their ability or inability to interact with the thin filaments during contraction, respectively. MyBP-C is a flexible and long protein that is tightly bound to the thick filament at its C-terminal end but may be loosely bound at its middle- and N-terminal end (MyBP-CC1C7). Under considerable debate is whether the MyBP-CC1C7 domains directly regulate myosin head ON/OFF states, and/or link thin filaments ("C-links"). Here, we used a combination of mechanics and small-angle X-ray diffraction to study the immediate and selective removal of the MyBP-CC1C7 domains of fast MyBP-C in permeabilized skeletal muscle. After cleavage, the thin filaments were significantly shorter, a result consistent with direct interactions of MyBP-C with thin filaments thus confirming C-links. Ca2+ sensitivity was reduced at shorter sarcomere lengths, and crossbridge kinetics were increased across sarcomere lengths at submaximal activation levels, demonstrating a role in crossbridge kinetics. Structural signatures of the thick filaments suggest that cleavage also shifted myosin heads towards the ON state - a marker that typically indicates increased Ca2+ sensitivity but that may account for increased crossbridge kinetics at submaximal Ca2+ and/or a change in the force transmission pathway. Taken together, we conclude that MyBP-CC1C7 domains play an important role in contractile performance which helps explain why mutations in these domains often lead to debilitating diseases.

physiology↗

bioRxiv2023

Kremen1 regulates the regenerative capacity of support cells and mechanosensory hair cells in the zebrafish lateral line

Mechanosensory hair cells in the inner ear mediate the sensations of hearing and balance, and in a specialize lateral line sensory system of aquatic vertebrates, the sensation of water movement. In mammals, hair cells lack the ability of regenerate following damage, resulting in sensory deficits. In contrast, non-mammalian vertebrates, such zebrafish, can renew hair cells throughout the life of the animal. Wnt signaling is required for development of inner ear and lateral line hair cells and regulates regeneration. Kremen1 inhibits Wnt signaling and hair cell formation, though its role in regeneration has not been established. We use a zebrafish kremen1 mutant line, to show that when Wnt signaling is overactivated in the lateral line, excessive regeneration occurs in the absence of increased proliferation, due to an increase in support cells. This contrasts with the previously described role of Wnt signaling during hair cell regeneration. This work will allow us to understand the biology of mechanosensory hair cells, and how regeneration might be promoted following damage.

developmental biology↗

bioRxiv2023

The distinctive mechanical and structural signatures of residual force enhancement in myofibers

In muscle, titin proteins connect myofilaments together and are thought to be critical for contraction, especially during residual force enhancement (RFE) when force is elevated after an active stretch. We investigated titins function during contraction using small-angle X-ray diffraction to track structural changes before and after 50% titin cleavage and in the RFE-deficient, mdm titin mutant. We report that the RFE state is structurally distinct from pure isometric contractions, with increased thick filament strain and decreased lattice spacing, most likely caused by elevated titin-based forces. Furthermore, no RFE structural state was detected in mdm muscle. We posit that decreased lattice spacing, increased thick filament stiffness, and increased non-crossbridge forces are the major contributors to RFE. We conclude that titin directly contributes to RFE. One-Sentence SummaryTitin contributes to active force production and residual force enhancement in skeletal muscles.

physiology↗

bioRxiv2020

Protective Plant Immune Responses are Elicited by Bacterial Outer Membrane Vesicles

Bacterial outer membrane vesicles (OMVs) perform a variety of functions in bacterial survival and virulence. In mammalian systems, OMVs activate immune responses and have been exploited as vaccines. However, little work has focused on the role that OMVs play during interactions with plant hosts. Here we report that OMVs from the pathogenic Pseudomonas syringae and the beneficial Pseudomonas fluorescens activate plant immune responses that protect against bacterial and oomycete pathogens. OMVs from these two species display different sensitivity to biochemical stressors, which could indicate differences in OMV cargo packaging. Furthermore, our study shows that OMV-induced protective immune responses are T3SS- and protein-independent, while OMV-mediated seedling growth inhibition largely depends on protein cargo. Importantly, OMV-mediated plant responses are distinct from those triggered by PAMP/MAMPs or effector molecules alone. OMVs provide a unique opportunity to study virulence factors in combination and add a new layer of interaction and complexity to host-microbe interactions.

microbiology↗

bioRxiv2020

Dynamin-related Irgm proteins modulate LPS-induced caspase-4 activation and septic shock

Inflammation associated with gram-negative bacterial infections is often instigated by the bacterial cell wall component lipopolysaccharide (LPS). LPS-induced inflammation and resulting life-threatening sepsis are mediated by the two distinct LPS receptors TLR4 and caspase-4. Whereas the regulation of TLR4 activation by extracellular and phago-endosomal LPS has been studied in great detail, auxiliary host factors that specifically modulate recognition of cytosolic LPS by caspase-4 are largely unknown. This study identifies dynamin-related membrane remodeling proteins belonging to the family of Immunity related GTPases M clade (IRGM) as negative regulators of caspase-4 activation in macrophages. Phagocytes lacking expression of mouse isoform Irgm2 aberrantly activate caspase-4-dependent inflammatory responses when exposed to extracellular LPS, bacterial outer membrane vesicles or gram-negative bacteria. Consequently, Irgm2-deficient mice display increased susceptibility to caspase-4-mediated septic shock in vivo. This Irgm2 phenotype is partly reversed by the simultaneous genetic deletion of the two additional Irgm paralogs Irgm1 and Irgm3, indicating that dysregulated Irgm isoform expression disrupts intracellular LPS processing pathways that limit LPS availability for caspase-4 activation.

immunology↗

bioRxiv2019

Pseudomonas aeruginosa leucine aminopeptidase influences early biofilm composition and structure via vesicle-associated anti-biofilm activity

Pseudomonas aeruginosa, known as one of the leading causes of disease in cystic fibrosis (CF) patients, secretes a variety of proteases. These enzymes contribute significantly to P. aeruginosa pathogenesis and biofilm formation in the chronic colonization of CF patient lungs, as well as playing a role in infections of the cornea, burn wounds and chronic wounds. We previously characterized a secreted P. aeruginosa peptidase, PaAP, that is highly expressed in chronic CF isolates. This leucine aminopeptidase is highly expressed during infection and in biofilms, and it associates with bacterial outer membrane vesicles (OMVs), structures known to contribute to virulence mechanisms in a variety of Gram-negative species and one of the major components of the biofilm matrix. We hypothesized that PaAP may play a role in P. aeruginosa biofilm formation. Using a lung epithelial cell/bacterial biofilm coculture model, we show that PaAP deletion in a clinical P. aeruginosa background alters biofilm microcolony composition to increase cellular density, while decreasing matrix polysaccharide content, and that OMVs from PaAP expressing strains but not PaAP alone or in combination with PaAP deletion strain-derived OMVs could complement this phenotype. We additionally found that OMVs from PaAP expressing strains could cause protease-mediated biofilm detachment, leading to changes in matrix and colony composition. Finally, we showed that the OMVs could also mediate the detachment of biofilms formed by both non-self P. aeruginosa strains and Klebsiella pneumoniae, another respiratory pathogen. Our findings represent novel roles for OMVs and the aminopeptidase in the modulation of P. aeruginosa biofilm architecture.\n\nImportanceBiofilm formation by the bacterial pathogen P. aeruginosa is known to contribute to drug- resistance in nosocomial infections and chronic lung infections of cystic fibrosis patients. In order to treat these infections more successfully, the mechanisms of bacterial biofilm development must be elucidated. While both bacterially-secreted aminopeptidase and outer membrane vesicles have been shown to be abundant in P. aeruginosa biofilm matrices, the contributions of each of these factors to the steps in biofilm generation have not been well studied. This work provides new insight as to how these bacterial components mediate the formation of a robust, drug-resistant extracellular matrix and implicates outer membrane vesicles as active components of biofilm architecture, expanding our overall understanding of P. aeruginosa biofilm biology.

microbiology↗

bioRxiv2019

Vesicle-associated Pseudomonas aeruginosa leucine aminopeptidase modulates bacterial biofilms grown on host cellular substrates

Pseudomonas aeruginosa, known as one of the leading causes of morbidity and mortality in cystic fibrosis (CF) patients, secretes a variety of virulence-associated proteases. These enzymes have been shown to contribute significantly to P. aeruginosa pathogenesis and biofilm formation in the chronic colonization of CF patient lungs, as well as playing a role in infections of the cornea, burn wounds and chronic wounds. Our lab has previously characterized a secreted P. aeruginosa peptidase, PaAP, that is highly expressed in chronic CF isolates. This leucine aminopeptidase is not only secreted solubly, it also associates with bacterial outer membrane vesicles (OMVs), structures known for their contribution to virulence mechanisms in a variety of Gram-negative species and one of the major components of the biofilm matrix. With this in mind, we hypothesized that PaAP may play a role in P. aeruginosa biofilm formation. Using a lung epithelial cell/bacterial biofilm coculture model, we show that PaAP deletion in a clinical P. aeruginosa background leads to increased early biofilm formation. We additionally found that only native vesicle-bound PaAP, as opposed to its soluble forms, could reconstitute the original PaAP-mediated inhibition phenotype, and that the PaAP-containing vesicles could disperse preformed biofilm microcolonies of Klebsiella pneumoniae, another lung pathogen. These data provide the basis for future work into the mechanism behind PaAP-OMV mediated bacterial microcolony dispersal and the application of these findings to clinical anti-biofilm research.

microbiology↗