Biology subjects

Koide, S.

Publications and source records attributed to Koide, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Exitron-type splicing of intrinsically disordered regions recurs across jawed vertebrates despite sequence divergence: donor-proximal cis control in TARDBP

Intrinsically disordered regions (IDRs) mediate molecular interactions but can also promote aberrant self-association. Using donors embedded within IDR-coding sequences, exitron-type splicing can remove these sequences, giving them both protein-coding and splice-regulatory roles. Yet IDR amino-acid sequences often diverge rapidly, raising the question of whether and how their coding sequences support IDR-removing splicing across species. Using RNA-seq data from jawed vertebrates, we identified events predicted to remove most of an IDR while preserving annotated structured regions. Across species, donor-flanking nucleotide identity exceeded expectations from synonymous alternatives encoding the same amino-acid sequence. TARDBP, which encodes TDP-43, provided a model for examining how coding sequences both encode IDRs and regulate exitron-type splicing. Sequence-divergent vertebrate TARDBP terminal regions supported this splicing in human cells. In teleosts, TARDBP ohnologs (tardbpa and tardbpb) encoded compositionally similar disordered tails but differed markedly in IDR-removing exitron-type splicing. Reciprocal donor-proximal substitutions in zebrafish minigenes redirected donor choice and altered the balance between IDR-retaining and IDR-removing transcripts. In corresponding reporters, these substitutions altered protein-product balance and cytoplasmic puncta formation. These findings show that IDR-removing exitron-type splicing can recur despite sequence divergence and that donor-proximal TARDBP sequences both encode IDR segments and regulate IDR inclusion, linking RNA processing to protein behavior.

molecular biology↗

bioRxiv2026

A Novel Metric for Quantifying the Sustainability of Phage-Mediated Bacterial Suppression

The global spread of antimicrobial-resistant (AMR) bacteria represents one of the greatest threats to modern medicine, making the development of novel therapeutic strategies increasingly urgent. Phage therapy, which exploits bacteriophages (phages, viruses that specifically infect and kill bacteria) has regained attention as a therapeutic approach for multidrug-resistant infections. One critical determinant of treatment outcome is the capacity of phages to sustain bacterial growth suppression; however, no metric has previously existed to directly quantify the duration of effective lytic activity. Here, we propose the Sustainability Index (SusI), a novel metric that quantifies both the duration and extent of phage-mediated bacterial growth suppression, which is restricted to the primary lysis period from lysis initiation to resistance emergence. Evaluation of individual phages and two-phage cocktails against both laboratory and clinical isolates of Escherichia coli demonstrated that SusI provides information independent of the Virulence Index, which primarily reflects bactericidal activity during the initial phase of infection, and serves as a complementary metric to the Suppression Index, which may incorporate behavior beyond primary lysis. Cocktails composed of phages targeting different receptors specificities consistently exhibited higher SusI values, consistent with the notion that multifaceted selective pressure delays resistance emergence. Furthermore, in a mouse model of systemic infection established by intraperitoneal administration, cocktails with higher SusI values demonstrated superior therapeutic efficacy. These results confirm SusI as a practical metric for rational phage cocktail design. As phage therapy advances toward clinical implementation, standardized quantitative metrics such as SusI are expected to facilitate evidence-based selection of therapeutic phages across diverse pathogens and infection conditions. ImportanceThe global spread of antimicrobial-resistant bacteria is making bacterial infections increasingly difficult to treat. Phage therapy, which uses bacteriophages (viruses that specifically infect bacteria), has re-emerged as a therapeutic alternative; however, reliable methods to determine in advance which phages will be therapeutically effective remain limited. Current evaluation metrics are well-suited for quantifying how rapidly phages kill bacteria but were not designed to directly measure how long lytic activity is sustained before resistant bacteria emerge. Here, we developed the Sustainability Index (SusI), a novel metric that specifically quantifies the duration of effective bacterial growth suppression. Evaluation of multiple phages and their combinations (cocktails) against both laboratory and clinical bacterial isolates demonstrated that SusI can distinguish phage combinations that existing metrics fail to differentiate. Moreover, in a mouse model of lethal bacterial infection, higher SusI values correlated with improved therapeutic outcomes. SusI has potential as a practical tool for selecting phages with greater likelihood of therapeutic success.

microbiology↗

bioRxiv2026

DNA barcoding-based paired daughter cell analysis reveals division preferences of hematopoietic stem cells

Hematopoietic stem cells(HSCs) maintain their pools by stem-stem division and produce mature blood cells through stem-progenitor or progenitor-progenitor division. A paired daughter cell(PDC) assay combined with single cell transplantation is a powerful method to compare the lineage outputs of two HSC daughter cells. However, single-cell transplantation precludes large-scale analysis of daughter-cell pairs, as only one cell can be transplanted per recipient. Here, we developed a DNA barcoding-based PDC assay to overcome this limitation, enabling simultaneous analysis of 476 daughter pairs from individual HSC divisions and revealing that daughter-cell fates are coordinated and that HSC division patterns are biased toward stem-stem and progenitor-progenitor divisions rather than stem-progenitor divisions. These findings indicate that HSC fate outcomes are directed toward symmetric division outcomes. Integration of single-cell RNA sequencing with DNA barcoding revealed a continuum of HSC states--from balanced HSCs to myeloid-biased HSCs and ultimately to a low-output HSC subset--in which progressively reduced production of mature blood cells relative to stem cell expansion, a proxy for stem-stem division bias, exhibits distinct activities of transcription factors and signaling pathways. Overall, our analysis uncovers characteristic patterns of HSC division and links stem maintenance with distinct molecular features.

cell biology↗

bioRxiv2026

Structural basis for direct NGF/TrkA blockade by an analgesic antibody

The NGF/TrkA signaling axis is a central mediator of inflammatory and chronic pain, where injury-induced NGF binds and activates TrkA on nociceptive neurons to drive peripheral sensitization and persistent pain states. Despite its therapeutic promise, targeting this pathway is limited by adverse effects of systemic NGF sequestration such as rapidly progressive osteoarthritis and poor isoform selectivity of Trk kinase inhibitors leading to off-target neurological effects. Targeting the TrkA extracellular domain (TrkAECD) offers a pathway to achieve high isoform selectivity while avoiding these complications. However, the precise structural basis for selective TrkA neutralization remains poorly understood. Monoclonal antibody (mAb) 42F5-15 inhibits TrkA-mediated signaling and increases pain threshold. Here, we report the high-resolution (2.60 [A]) cryo-EM structure of the TrkAECD in complex with the Fab region of the TrkA-neutralizing mAb 42F5-15. Structural analysis reveals that the antibody epitope overlaps the NGF-binding interface, consistent with orthosteric inhibition and distinct from previously proposed allosteric mechanisms. The epitope includes residues conserved in TrkA but divergent in TrkB and TrkC, providing a structural basis for receptor isoform selectivity. Furthermore, we demonstrate in vivo that the mAb 42F5-15 potently mitigates mechanical allodynia and nociceptive sensitization. These findings establish a structural framework for the development of selective extracellular TrkA-targeted therapies for safer, non-opioid chronic pain management.

neuroscience↗

bioRxiv2026

Promiscuous RNA binding by WDR5 remodels the KMT2A (MLL1) histone methyltransferase complex to an inactive state

Chromatin-modifying complexes are critical in gene regulation, yet their proposed interactions with RNA remain poorly understood. Here, we show that WDR5, an essential subunit of the MLL1 (KMT2A) histone methyltransferase complex, binds RNA with high affinity but without sequence specificity. Using a stringent approach, we demonstrate that WDR5 directly engages a diverse pool of RNAs in cells, predominantly as a function of RNA abundance rather than specific motifs. Equilibrium binding assays further show that RNA length, rather than sequence, dictates WDR5 affinity. We map multiple RNA-binding regions on WDR5, some of which overlap with surfaces used in MLL1 complex subunit interfaces. Strikingly, we find that RNA binding disrupts the MLL1 complex by competitively displacing WDR5 from these critical protein-protein interactions, leading to a marked inhibition of MLL1 catalytic activity. This regulatory mechanism provides a layer of control over histone methylation, potentially integrating transcriptional activity with chromatin state.

molecular biology↗

bioRxiv2025

Export-biased, 3'UTR-preserving TDP-43 model links nuclear loss to cytoplasmic aggregation in ALS/FTLD

Nuclear depletion and cytoplasmic mislocalization of TDP-43 are central pathological features of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 protein levels are normally maintained by autoregulation through its native 3' untranslated region (3' UTR). It remains untested whether this feedback is still protective once the perturbation is chronic, and whether the resulting rise in TARDBP transcripts restores the functional nuclear pool or is instead diverted into non-functional species. To address this, we engineered full-length human TDP-43 carrying an N-terminal nuclear export signal (NES) while leaving the native 3' UTR autoregulatory module intact. Expression was titrated so that whole-cell RIPA-soluble exogenous TDP-43 remained [≤]30% of endogenous levels. In HEK293T cells, export bias drove TDP-43 into the cytoplasm and produced detergent-insoluble species. In differentiated SH-SY5Y cells, nuclear splicing defects and autoregulatory changes scaled with export-biased load, and detergent-insoluble accumulation was already present within the same low-load range. Human iPSC-derived neurons showed a comparable cytoplasmic shift, discrete TDP-43-positive foci, and TDP-43-dependent splicing defects. When endogenous TDP-43 was selectively depleted under transgene induction, 3' UTR-coupled autorepression was weakened and transgene-derived TARDBP transcripts rose; however, the additional exogenous TDP-43 did not expand the soluble, splice-competent pool but partitioned into insoluble fractions. Splicing defects in differentiated SH-SY5Y cells persisted even when soluble NES-TDP-43 reached endogenous-equivalent levels. We therefore propose that under native 3' UTR control, sustained cytoplasmic bias uncouples compensatory TARDBP upregulation from recovery of the functional nuclear pool; the extra output is diverted into insoluble, fragmented species rather than restoring nuclear function.

molecular biology↗

bioRxiv2025

Engineering Antibody-Drug Conjugates targeting an Adhesion GPCR, CD97

Adhesion G protein-coupled receptors (aGPCRs) are key cell-adhesion molecules involved in many cellular functions and contribute to human diseases, including cancer. aGPCRs are characterized by large extracellular regions that could serve as readily accessible antigens. However, the potential of aGPCRs as targets for biologic therapeutics has not been extensively explored. CD97, also known as ADGRE5, is an aGPCR that is upregulated in various cancer types, including acute myeloid leukemia (AML) and glioblastoma (GBM), and their respective cancer stem cells. Here, we developed antibody-drug conjugates (ADCs) targeting CD97 and assessed their efficacy against AML and GBM cells. We generated a panel of synthetic human antibodies targeting distinct epitopes of CD97, from which we identified an antibody that was efficiently internalized. This antibody binds to all isoforms of human CD97 but not to its close homolog, EMR2. Structure determination by single-particle cryo-electron microscopy revealed that this antibody targets the CD97 GPCR autoproteolysis-inducing (GAIN) domain, whose presence is conserved in aGPCRs, through an unconventional binding mode where it extensively utilizes the light chain framework for antigen recognition. Screening of conjugation methods and payloads resulted in a stable ADC that effectively killed AML and GBM cell lines, as well as patient-derived GBM stem cells, with minimal cytotoxicity against peripheral blood mononuclear cells from healthy donors. Our study demonstrates the therapeutic potential of targeting CD97, as well as the aGPCR GAIN domain in general, and broadens our mechanistic understanding of antibody-antigen interactions.

biochemistry↗

bioRxiv2025

Niche-targeted therapy via YAP/TAZ activation enhances hematopoietic regeneration

The distinctive milieu of the bone marrow (BM), known as the BM niche, supports hematopoietic stem cells (HSCs) and serves as a foundation for hematopoietic regeneration. Myeloablative stress disrupts not only hematopoietic stem and progenitor cells but also essential BM niche components, including endothelial cells (ECs) and mesenchymal stromal cells (MSCs); disruption of the latter impairs efficient hematopoietic recovery. However, therapeutic strategies targeting niche restoration remain largely underdeveloped. Here, we demonstrate that the Hippo pathway effectors YAP/TAZ are critical for enabling ECs and MSCs to respond to BM injury, and that YAP/TAZ activation accelerates BM niche recovery, thereby promoting hematopoietic regeneration. We found that YAP/TAZ are rapidly activated in both MSCs and ECs following myeloablative stress, maintaining MSC multipotency and orchestrating vascular remodeling. Mechanistically, YAP/TAZ function as transcriptional hubs in MSCs, regulating key transcriptional factors such as Ebf1 and Ebf3. This regulation preserves MSC identity by preventing osteogenic and fibrogenic differentiation while promoting the expression of hematopoietic factors such as Cxcl12 and angiogenic factors. In parallel, YAP/TAZ signaling in ECs facilitates pro-angiogenic responses and drives sinusoidal vessel remodeling after injury. These YAP/TAZ-mediated niche responses are essential for HSC retention and hematopoietic regeneration following diverse myelosuppressive therapies. Notably, pharmacological activation of YAP/TAZ enhances BM niche reorganization and augments hematopoietic regeneration following myeloablative therapies. These findings establish YAP/TAZ as central regulators of BM niche resilience, providing a rationale for niche-targeted therapeutic strategies to enhance hematopoietic regeneration. Key points{blacksquare} YAP/TAZ enable BM niche cells to sense injury and restore their structure and function, thereby promoting hematopoietic regeneration. {blacksquare}Pharmacological activation of YAP/TAZ enhances BM niche resilience and accelerates hematopoietic recovery after myelosuppressive therapies.

cell biology↗

bioRxiv2025

Exploiting macropinocytosis for therapeutic intervention in RAS mutant Multiple Myeloma

Roughly 50% of newly diagnosed multiple myeloma (MM) cases harbor KRAS (25%) or NRAS (24%) mutations with an even greater frequency of these mutations observed at relapse. By and large, mutant RAS-driven MM is more resistant to existing therapies including proteasome inhibitors, immunomodulator drugs (IMiDs), and monoclonal anti-CD38 antibodies. In the present study, we demonstrate that mutant RAS-dependent macropinocytosis (MP) can be leveraged for selective delivery of a monobody-drug conjugate (MDC) to mutant RAS MM cells. This MDC delivery platform consists of monobody, a fragment of human fibronectin (FN), used as the carrier to which monomethyl auristatin E (MMAE) is site-specifically conjugated (FN-MMAE). In comparison to standard of care (SoC) therapeutics, FN-MMAE displays a substantially improved anti-tumor effect in vitro and in vivo when administered alone or in combination with SoC treatments. Furthermore, the in vivo safety profile of FN-MMAE is tolerable affording increased drug dosing compared to clinically used ADCs. This MDC platform offers a way of selectively targeting mutant RAS MM and RRMM to potentially improve patient outcomes. Statement of SignificanceRas mutations are present in approximately 50% of MM patients and are associated with poor prognosis and drug resistance. Herein, we describe a novel protein-drug conjugate designed to target selectively mutant RAS harboring MM cells. Our findings uncover a new therapeutic modality for improving the outcomes for patients with mutant Ras MM.

cancer biology↗

bioRxiv2024

CH-related Mutant ASXL1 Promotes Atherosclerosis in Mice via Dysregulated Innate Immunity

Certain somatic mutations confer a fitness advantage in hematopoietic stem and progenitor cells (HSPCs) over normal HSPCs, resulting in the clonal expansion of mutant blood cells1, otherwise known as clonal haematopoiesis (CH). CH is frequently observed among healthy elderly people and is closely associated with the risk of cardiovascular diseases (CVDs). The most frequently mutated genes of CH include DNMT3A, TET2, and ASXL12. Among them, even though ASXL1 mutations are clinically associated with the highest risk for developing CVDs, little is known whether and how the mutations contribute to CVDs. Here we show accelerated development of atherosclerosis and increased inflammatory monocytes in mice transplanted with the bone marrow cells (BMCs) from the mice expressing mutant ASXL1 (ASXL1-MT) selectively in hematopoietic cells. RNA sequencing analysis of the plaque-macrophages derived from BMCs expressing ASXL1-MT showed more inflammatory signatures than those from control BMCs. Mechanistically, wild-type ASXL1 inhibited innate immune signalling through direct interactions with IRAK1/TRAF6/TAK1 in the cytoplasm, while ASXL1-MT, which only interacted with TAK1, lost this regulatory function, leading to NF-{kappa}B activation. This mechanism is unique and distinct from those of CH with Tet2 or Dnmt3a mutations, where overactivation of the IL-1{beta}/NLRP3 inflammasome plays critical roles3-5. Intriguingly, IRAK1/4 inhibition decreased the number of inflammatory monocytes and attenuated the development of atherosclerosis driven by ASXL1-MT. The present work connects the mutations of an epigenetic factor, ASXL1, with inflammation and CVDs and gives an indication for the prevention of CVDs in CH.

cell biology↗

bioRxiv2024

ARGX-119, a therapeutic agonist antibody targeting MuSK

ARGX-119 is a novel, humanized, agonist monoclonal SIMPLE Antibody specific for muscle-specific kinase (MuSK) that is being developed for treatment of patients with neuromuscular diseases. ARGX-119 is the first monoclonal antibody (mAb) that binds with high affinity to the Frizzled-like domain of human, non-human primate, rat and mouse MuSK, without off-target binding, making it suitable for clinical development. Within the Fc-region, ARGX-119 harbors L234A, L235A mutations to diminish potential immune-activating effector functions. Its mode-of-action is to activate MuSK without interfering with its natural ligand neural Agrin, and cluster acetylcholine receptors (AChRs) in a dose-dependent manner, thereby stabilizing neuromuscular function. In a mouse model for DOK7 congenital myasthenia (CM), ARGX-119 prevented early postnatal lethality and reversed disease relapse by restoring neuromuscular function and reducing muscle weakness and fatigability in a dose-dependent manner. Pharmacokinetic (PK) studies in non-human primates, rats and mice revealed non-linear PK behavior of ARGX-119, indicative of target-mediated-drug disposition (TMDD) and in vivo target engagement. Instability of neuromuscular synapses contributes to symptoms in many neuromuscular diseases for example congenital myasthenia (CM), amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). ARGX-119 is a novel, first-in-class MuSK agonist mAb in clinical development. Based on this proof-of-concept study, it has the potential to alleviate neuromuscular diseases hallmarked by impaired neuromuscular synaptic function. One sentence summaryARGX-119 is a novel first-in-class MuSK agonist monoclonal antibody in clinical development for treatment of neuromuscular diseases.

neuroscience↗

bioRxiv2024

ASO-enhancement of TARDBP exitron splicing mitigates TDP-43 proteinopathies

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration are fatal neurodegenerative diseases characterized by pathological aggregation and nuclear functional loss of TDP-431,2. Current therapies inadequately address this core pathology3,4, necessitating innovative approaches that target aggregation while preserving TDP-43s essential functions. Here we demonstrate that enhancing the splicing of the TARDBP exitron--a cryptic intron encoding the aggregation-prone intrinsically disordered region (IDR) of TDP-435,6-- effectively mitigates TDP-43 pathology. This exitron splicing event, directly regulated by nuclear TDP-437-9, suppresses the expression of IDR-containing TDP-43 isoforms and generates IDR-spliced-out TDP-43 isoforms7,9,10 (which we term "IDRsTDP"). Our findings reveal that IDRsTDP, known to heterodimerize with full-length TDP-4310, inhibits TDP-43 aggregation by suppressing IDR-mediated clustering and enhances TDP-43 clearance via chaperone-mediated autophagy. In disease states, however, impaired nuclear TDP-43 function disrupts exitron splicing, leading to increased levels of IDR-containing TDP-439,11 and reduced levels of IDRsTDP, exacerbating aggregation and nuclear dysfunction6,12-17. By identifying HNRNPA1 and HNRNPC as key repressors of TARDBP exitron splicing, we designed antisense oligonucleotides (ASOs) to block their binding and restore splicing. These ASOs suppressed TDP-43 pathology and neurodegeneration in both neuronal cell models with impaired nuclear transport and a mouse model of proteasome dysfunction-induced TDP-43 proteinopathy. Our strategy, by rescuing the impaired autoregulatory pathway, inhibits the pathological cycle of TDP-43 aggregation and nuclear dysfunction, offering a promising avenue for treating these currently intractable neurodegenerative diseases.

neuroscience↗

bioRxiv2023

Maternal sterol 27-hydroxylase is crucial for securing fetal development

The maternal body helps in providing nutrients and degrading toxic metabolites instead of the fetal body; disruptions in these mechanisms affect normal fetal development. Sterol 27-hydroxylase (Cyp27a1) is involved in the alternative pathway of bile acid synthesis, which is enhanced during pregnancy. However, its role in fetal development remains unclear. Here, we demonstrate that maternal Cyp27a1 activity is essential for progression of normal pregnancy and fetal organ formation. Depletion of maternal Cyp27a1 reduced the pregnancy rate and litter size. Newborn mice died of respiratory distress syndrome resulting from the absence of mature alveolar epithelial cells. These phenotypes were caused by 7-hydroxycholesterol (7-HC) accumulating in Cyp27a1-deficient mice. Mechanistically, 7-HC destabilized the Fau protein, mediating ribosome assembly, the downregulation of which caused poor polysome formation, lower protein synthesis, and impaired lung maturation. Overall, this study revealed an essential mechanism of securing fetal development by degrading a toxic metabolite in the maternal body.

developmental biology↗

bioRxiv2023

The expression profile and tumorigenic mechanisms of CD97 (ADGRE5) in glioblastoma render it a targetable vulnerability

Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Adhesion G protein-coupled receptors (aGPCRs) have attracted interest for their functional role in gliomagenesis and their potential as treatment targets. To identify therapeutically targetable opportunities among aGPCR family members in unbiased fashion, we analyzed expression levels of all aGPCRs in GBM and non-neoplastic brain tissue. Using bulk and single cell transcriptomic and proteomic data, we show that CD97 (ADGRE5), an aGPCR previously implicated in GBM pathogenesis, is the most promising aGPCR target in GBM, by virtue of its abundance in all GBM tumors and its de novo expression profile in GBM compared to normal brain tissue and neural progenitors. CD97 knockdown or knockout significantly reduces the tumor initiation capacity of patient-derived GBM cultures (PDGC) in vitro and in vivo. Transcriptomic and metabolomic data from PDGCs suggest that CD97 promotes glycolytic metabolism. The oncogenic and metabolic effects of CD97 are mediated by the MAPK pathway. Activation of MAPK signaling depends on phosphorylation of the cytosolic C-terminus of CD97 and recruitment of {beta}-arrestin. Using single-cell RNA-sequencing and biochemical assays, we demonstrate that THY1/CD90 is the most likely CD97 ligand in GBM. Lastly, we show that targeting of PDGCs with an anti-CD97 antibody-drug conjugate in vitro selectively kills tumor cells but not human astrocytes or neural stem cells. Our studies identify CD97 as an important regulator of tumor metabolism in GBM, elucidate mechanisms of receptor activation and signaling, and provide strong scientific rationale for developing biologics to target it for therapeutic purposes.

cancer biology↗

bioRxiv2023

Tumor-intrinsic LKB1-LIF signaling axis establishes a myeloid niche to promote immune evasion and tumor growth

Tumor mutations can influence the surrounding microenvironment leading to suppression of anti-tumor immune responses and thereby contributing to tumor progression and failure of cancer therapies. Here we use genetically engineered lung cancer mouse models and patient samples to dissect how LKB1 mutations accelerate tumor growth by reshaping the immune microenvironment. Comprehensive immune profiling of LKB1-mutant vs wildtype tumors revealed dramatic changes in myeloid cells, specifically enrichment of Arg1+ interstitial macrophages and SiglecFHi neutrophils. We discovered a novel mechanism whereby autocrine LIF signaling in Lkb1-mutant tumors drives tumorigenesis by reprogramming myeloid cells in the immune microenvironment. Inhibiting LIF signaling in Lkb1-mutant tumors, via gene targeting or with a neutralizing antibody, resulted in a striking reduction in Arg1+ interstitial macrophages and SiglecFHi neutrophils, expansion of antigen specific T cells, and inhibition of tumor progression. Thus, targeting LIF signaling provides a new therapeutic approach to reverse the immunosuppressive microenvironment of LKB1-mutant tumors.

cancer biology↗

bioRxiv2023

In vivo metabolomics identifies CD38 as an emergent vulnerability in LKB1-mutant lung cancer

LKB1/STK11 is a serine/threonine kinase that plays a major role in controlling cell metabolism, resulting in potential therapeutic vulnerabilities in LKB1-mutant cancers. Here, we identify the NAD+ degrading ectoenzyme, CD38, as a new target in LKB1-mutant NSCLC. Metabolic profiling of genetically engineered mouse models (GEMMs) revealed that LKB1 mutant lung cancers have a striking increase in ADP-ribose, a breakdown product of the critical redox co-factor, NAD+. Surprisingly, compared with other genetic subsets, murine and human LKB1-mutant NSCLC show marked overexpression of the NAD+-catabolizing ectoenzyme, CD38 on the surface of tumor cells. Loss of LKB1 or inactivation of Salt-Inducible Kinases (SIKs)--key downstream effectors of LKB1-- induces CD38 transcription induction via a CREB binding site in the CD38 promoter. Treatment with the FDA-approved anti-CD38 antibody, daratumumab, inhibited growth of LKB1-mutant NSCLC xenografts. Together, these results reveal CD38 as a promising therapeutic target in patients with LKB1 mutant lung cancer. SIGNIFICANCELoss-of-function mutations in the LKB1 tumor suppressor of lung adenocarcinoma patients and are associated with resistance to current treatments. Our study identified CD38 as a potential therapeutic target that is highly overexpressed in this specific subtype of cancer, associated with a shift in NAD homeostasis.

cancer biology↗

bioRxiv2023

Context-Dependent Modification of PFKFB3 in Hematopoietic Stem Cells Promotes Anaerobic Glycolysis and Ensures Stress Hematopoiesis

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of adenosine triphosphate (ATP) levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells. Key PointsO_LICombined isotope tracing, mathematical modeling, and single cell ATP analysis enable high-resolution evaluation of blood cell metabolism. C_LIO_LIUnder stress, HSCs quickly accelerate glycolysis to meet ATP demands and maintain hematopoiesis via context-dependent PFKFB3 activation. C_LI

cell biology↗

bioRxiv2022

Polycomb repressive complex 1.1 coordinates homeostatic and emergency myelopoiesis

Polycomb repressive complex (PRC) 1 regulates stem cell fate by mediating mono-ubiquitination of histone H2A at lysine 119. While canonical PRC1 is critical for hematopoietic stem and progenitor cell (HSPC) maintenance, the role of non-canonical PRC1 in hematopoiesis remains elusive. PRC1.1, a non-canonical PRC1, consists of PCGF1, RING1B, KDM2B, and BCOR. We recently showed that PRC1.1 insufficiency induced by the loss of PCGF1 or BCOR causes myeloid-biased hematopoiesis and promotes transformation of hematopoietic cells in mice. Here we show that PRC1.1 serves as an epigenetic switch that coordinates homeostatic and emergency hematopoiesis. PRC1.1 maintains balanced output of steady-state hematopoiesis by restricting C/EBP-dependent precocious myeloid differentiation of HSPCs and the HOXA9- and {beta}-catenin-driven self-renewing network in myeloid progenitors. Upon regeneration, PRC1.1 is transiently inhibited to facilitate formation of granulocyte-macrophage progenitor (GMP) clusters, thereby promoting emergency myelopoiesis. Moreover, constitutive inactivation of PRC1.1 results in unchecked expansion of GMPs and eventual transformation. Collectively, our results define PRC1.1 as a novel critical regulator of emergency myelopoiesis, dysregulation of which leads to myeloid transformation.

immunology↗