Biology subjects

Geng, H.

Publications and source records attributed to Geng, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Rethinking Syndecan-1 as a Marker of Trauma-Induced Endothelial Injury

Syndecan-1 (SDC1), a transmembrane proteoglycan and component of the glycocalyx, is widely considered the key circulating biomarker of endothelial dysfunction in trauma and critical illness. However, the tissue distribution of SDC1 and the cellular origin of plasma SDC1 in health and disease are incompletely understood. This series of experiments integrates immunostaining, spatial transcriptomics (GeoMx Digital Spatial Profiler (DSP) and Xenium), and single-cell atlases to map SDC1, as well as the other three members of the syndecan family, syndecan-2 (SDC2), syndecan-3 (SDC3), and syndecan-4 (SDC4), across mouse and human tissues, including the lung, gastrointestinal tract, and brain. Across modalities, SDC1 primarily localizes to epithelial compartments, as demonstrated by colocalization with epithelial markers and enrichment in epithelial cell types in the lung and gut. Spatial profiling further identifies SDC1 and SDC4 enrichment in airway epithelial niches, SDC3 in endothelial-rich regions, and SDC2 distributed more broadly across compartments. Mouse and human single-cell atlases confirm epithelial predominance of SDC1. These findings define the cellular and spatial landscape of syndecan expression in healthy tissues and support SDC1 as an epithelial-enriched proteoglycan. These results also challenge the specificity of circulating SDC1 as a biomarker of endotheliopathy and raise the possibility that epithelial injury contributes substantially to circulating SDC1 after trauma and critical illness.

cell biology↗

bioRxiv2026

Integrative Genomic, Single-Cell, and Functional Profiling of the CD48-CD244 Axis and NK-Cell Dysfunction in Multiple Myeloma

Multiple myeloma (MM) orchestrates immune evasion by subverting natural killer (NK) cell function. CD48, one of the most abundant NK-ligands on MM cells, paradoxically enhances NK-cell activation yet is associated with high-risk cytogenetics and poor patient survival. We integrated multi-omics (bulk and single-cell RNA-seq, ATAC-seq), genome-wide CRISPR-KO/a screens, and machine learning to dissect CD48 regulation and function. In human MM and V{kappa}*MYC mice scRNA-seq datasets, NK cells exhibit stepwise increases in inflammatory and exhaustion signatures and loss of cytotoxic potential as disease progresses. In vitro co-culture assays show CD48 overexpression on MM enhances initial NK-cell cytotoxicity and cytokine secretion, whereas chronic exposure leads to ex vivo NK dysfunction. In vivo, CD48-overexpressing V{kappa}*MYC tumors progress more slowly and extend host survival, while NK-cell depletion accelerates disease. These findings support a context-dependent role for CD48, potentiating acute NK responses while coexisting with chronic NK exhaustion, and suggest strategies to modulate CD48 for therapeutic benefit.

cancer biology↗

bioRxiv2025

Isoform-Dependent Loss- and Gain-of-Function of the Gαs K53N Variant in Human Disease

The K53N mutation in Gs has been identified in patients with Albrights Hereditary Osteodystrophy (AHO), pseudohypoparathyroidism type 1A (PHP1a), and dilated cardiomyopathy; however, its molecular mechanism remains unclear. Here, we characterize the molecular, cellular, and physiological consequences of the K53N mutation in both long and short isoform of Gs. Biochemical analyses reveal that K53N disrupts nucleotide exchange and GTP hydrolysis, rendering both the short (Gs-S) and long (Gs-L) isoforms unresponsive to activation by G protein-coupled receptors (GPCRs) or cholera toxin. Both isoforms display a loss-of-function phenotype, failing to trigger cAMP production in response to {beta}2-adrenergic, parathyroid hormone, or vasopressin receptor stimulation. Notably, only the long isoform (Gs-L K53N) displays constitutive, receptor-independent cAMP generation. The mutation also reduces protein stability, weakens G{beta}{gamma} subunit interaction, and reduces plasma membrane localization. In neonatal rat ventricular cardiomyocytes, K53N impairs cAMP signaling and exerts dominant-negative effects on isoproterenol-induced responses. Strikingly, only Gs-L K53N abolishes isoproterenol-stimulated calcium release, directly implicating this isoform in the pathogenesis of cardiomyopathy. Collectively, these findings identify K53N as a unique Gs mutation that confers both loss- and gain-of-function properties in an isoform-specific manner, providing mechanistic insight into its complex pathogenicity in endocrine and cardiac tissues.

pharmacology and toxicology↗

bioRxiv2025

The antioxidant networks act as a firewall in maintenance of genomic integrity

Reactive oxygen species (ROS) threatens genomic integrity by both destruction of DNA metabolic enzymes and oxidative DNA damage. We find a negative correlation between oxidative DNA damage and activity of antioxidant enzymes. An antioxidant network formed by interactions between the antioxidant enzymes and the proteins involved in maintenance of genomic integrity was indentified in the nucleus by multi-proteomic investigations. The protein-protein interactions in the antioxidant network are up-regulated under oxidative stress, as proved by quantitative proximity ligation assays in situ within single cells. The antioxidant network catalyzes the ROS dismutation cascade before ROS attacks DNA-processing enzymes, thereby preventing DNA and DNA metabolic enzymes from oxidative damage and dysfunction. Therefore, the antioxidant network plays a firewall role in maintenance of genomic integrity.

biochemistry↗

bioRxiv2025

Selective Sensitivity of Ph-like B-ALL to BRG1 Inhibition Reveals a Novel Targeted Therapy Strategy

Despite therapeutic advances, high-risk subtypes of B-cell acute lymphoblastic leukemia (B-ALL) such as Philadelphia chromosome-like (Ph-like) and KMT2A-rearranged (KMT2A-R) remain a formidable clinical challenge. BRG1 (gene name SMARCA4), the ATPase subunit of the SWI/SNF chromatin-remodeling complex, has been extensively studied in solid tumors, where inactivating mutations are linked to aggressive disease and poor prognosis. Although BRG1 is known to be essential for early B cell development, its role in B-ALL remains poorly understood. Therefore, we investigated the therapeutic relevance of BRG1 in high-risk B-ALL. Meta-analysis of gene expression data revealed that BRG1-inactivating mutations are exceedingly rare (0.35%) in B-ALL, suggesting that intact BRG1 function may be critical for leukemogenesis. Subtype-specific analyses revealed that elevated BRG1 expression is associated with significantly shorter overall survival in children with Ph-like B-ALL, while the opposite trend was observed in KMT2A-R B-ALL. We confirmed higher BRG1 expressions in Ph-like compared to KMT2A-R B-ALL via gene expression analysis, RT-PCR, and Western blotting. The pharmacologic inhibition of BRG1 using two selective inhibitors, BRM014 and FHD-286, revealed marked sensitivity in Ph-like B-ALL cell lines, whereas KMT2A-R B-ALL was resistant. Mechanistically, we found that BRG1 inhibition results in cell cycle arrest via downregulation of cell cycle regulators (CCND3, CDK4, CDK6, E2F1, and MYC) and upregulation of the cell cycle inhibitor CDKN1B (p27). Importantly, treatment with FHD-286 significantly prolonged the survival of NSG mice engrafted with Ph-like B-ALL cells. Taken together, these findings establish BRG1 as a critical, subtype-specific dependency in Ph-like B-ALL and demonstrate that its pharmacologic inhibition effectively suppresses leukemic cell proliferation through induction of cell cycle arrest. The pronounced in vitro sensitivity and improved in vivo survival upon BRG1 inhibition provide compelling preclinical evidence for its therapeutic targeting. These results support the advancement of BRG1-directed strategies as a viable treatment approach for patients with Ph-like B-ALL, with the potential to improve outcomes in this high-risk population. HighlightsO_LIHigher levels of BRG1 correlate to poor clinical outcomes in Ph-like but KMT2A-R B-ALL C_LIO_LIInhibition of BRG1 induces cell cycle arrest in Ph-like cells in vitro and extends the survival of mice in pre-clinical in vivo studies C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/661805v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1b0a6cforg.highwire.dtl.DTLVardef@977257org.highwire.dtl.DTLVardef@d3bf7forg.highwire.dtl.DTLVardef@1c12d52_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Affinity-matured CD72-targeting Nanobody CAR T-cells Enhance Elimination of Antigen-Low B-cell Malignancies

BackgroundChimeric antigen receptor (CAR) T-cell therapies are highly efficacious for several different hematologic cancers. However, for most CAR T targets it is observed that low surface antigen density on tumors can significantly reduce therapeutic efficacy. Here, we explore this dynamic in the context of CD72, a surface antigen we recently found as a promising target for refractory B-cell cancers, but for which CD72 low antigen density can lead to therapeutic resistance in preclinical models. MethodsPrimary samples were accessed via institutional review board-approved protocols. Affinity-matured and humanized nanobody clones were previously described in Temple et al.1 CAR T-cells were generated via lentiviral transduction. In vitro cytotoxicity assays were performed using luciferase-labeled cell lines. In vivo studies were performed using cell line- or patient-derived xenografts implanted in NOD scid gamma (NSG) mice. ResultsWe first confirmed ubiquitous CD72 expression across a range of primary B-cell non-Hodgkin lymphomas. We further found that after resistance to CD19-directed therapies, across both B-cell acute lymphoblastic leukemia (B-ALL) models and primary tumor samples, surface CD72 expression was largely preserved while CD22 expression was significantly diminished. Affinity maturation of a nanobody targeting CD72, when incorporated into chimeric antigen receptor (CAR) T-cells, led to more effective elimination in vitro of isogenic models of CD72 low-expressing tumors. These results suggested that nanobody-based CAR T-cells (nanoCARs) may exhibit a similar relationship between binder affinity, antigen expression, and efficacy as previously demonstrated only for scFv-based CAR T-cells. Surprisingly, however, this significantly improved in vitro efficacy only translated to modest in vivo survival benefit. As a parallel strategy to enhance CAR T function, we found that the small molecule bryostatin could also significantly increase CD72 surface antigen density on B-cell malignancy models. Structural modeling and biochemical analysis identified critical residues improving CD72 antigen recognition of our lead affinity-matured nanobody. ConclusionsTogether, these findings support affinity-matured CD72 nanoCARs as a potential immunotherapy product for CD19-refractory B-cell cancers. Our results also suggest that for B-ALL in particular, CD72 may be a preferable second-line immunotherapy target over CD22. What is already known on this topicPrevious work using single chain variable fragment (scFv) based CAR Ts has suggested that improving affinity for target antigen could potentially help mitigate tumor resistance mediated by antigen downregulation, or baseline low antigen density. However, it is unknown whether this same dynamic holds for CAR T-cells that utilize different antigen recognition elements, such as nanobodies. What this study addsHere we show that affinity maturation of nanobody-based CAR T-cells (nanoCARs) targeting CD72 can improve their in vitro efficacy versus CD72-low tumors; however, in vivo efficacy differences are more modest. Furthermore, we show that for refractory B-cell malignancies, surface CD72 appears preserved after CD19 resistance even in situations where CD22 is strongly downregulated. How this study might affect research, practice or policyCD72 warrants further investigation as a preferred immunotherapy target in the context of CD19-refractory B-cell cancers, though nanobody affinity maturation is not a universal solution to the challenge of low tumor surface antigen density.

immunology↗

bioRxiv2024

Enhancer heterogeneity in acute lymphoblastic leukemia drives differential gene expression between patients

Genetic alterations alone cannot account for the diverse phenotypes of cancer cells. Even cancers with the same driver mutation show significant transcriptional heterogeneity and varied responses to therapy. However, the mechanisms underpinning this heterogeneity remain under-explored. Here, we find that novel enhancer usage is a common feature in acute lymphoblastic leukemia (ALL). In particular, KMT2A::AFF1 ALL, an aggressive leukemia with a poor prognosis and a low mutational burden, exhibits substantial transcriptional heterogeneity between individuals. Using single cell multiome analysis and extensive chromatin profiling, we reveal that much transcriptional heterogeneity in KMT2A::AFF1 ALL is driven by novel enhancer usage. Using high resolution Micro-Capture-C in primary patient samples, we also identify patient-specific enhancer activity at key oncogenes such as MEIS1 and RUNX2, driving high levels of expression of both oncogenes in a patient-specific manner. Overall, our data show that enhancer heterogeneity is highly prevalent in KMT2A::AFF1 ALL and may also be a mechanism that drives transcriptional heterogeneity in cancer more generally. Key PointsO_LILeukemia patients with the same driver mutations often display gene expression differences C_LIO_LIUsing chromatin profiling and high resolution 3C methods we show that enhancer heterogeneity drives gene expression differences C_LI

cancer biology↗

bioRxiv2024

Understanding the development of enzalutamide resistance based on a functional single-cell approach

Most metastatic prostate cancers (PCa) initially depend on androgen for survival and proliferation. Thus, anti-androgen or castration therapies are the mainstay treatment. Although effective at first, androgen-dependent PCa (ADPC) universally develops therapy resistance, thereby evolving to the incurable disease, called castration resistant PCa (CRPC). Currently, mechanisms underlying the emergence of CRPC from ADPC are largely unclear. We used single-cell RNA-sequencing (scRNA-Seq) to determine how a therapy-naive ADPC cell line - LNCaP responds to the anti-androgen drug, enzalutamide. We found that most cells expressed the drug-target androgen receptor (AR+), while a small subpopulation ([~]12%) expressed low or no AR (ARlow/-). Gene set enrichment analysis (GSEA) revealed that AR+ and ARlow/- cells were enriched with significantly different gene expressions and signaling pathways. Unexpectedly, ARlow/- cells displayed robust transcriptional response, including upregulations of genes and pathways involved in clinical CRPC. Next, we isolate ARlow/- and AR+ cells from the LNCaP cell line, and functionally confirmed the enzalutamide resistant phenotype of ARlow/- cells in vitro and in xenograft models in vivo. Finally, to explore a therapeutic option for ARlow/- cells, we found that ARlow/- cells expressed low levels of NAD+ biosynthesis genes, notably NAPRT, indicating a possible vulnerability to inhibitors blocking NAD+ synthesis. Indeed, treating ARlow/- cells with NAD+ synthesis inhibitors, FK866 and OT-82, significantly inhibited the survival and proliferation of ARlow/- cells, thus suggesting a possible novel therapeutic option for ADT and enzalutamide resistant PCa. SUMMARYSingle-cell RNA-Sequencing reveals heterogeneities of tumor cell populations. In most cases, however, the functional significance of the observed heterogeneity is not tested. In this study, we first identified a possible therapy-resistant prostate cancer cell subpopulation with scRNA-Seq, then confirmed the resistant phenotype with single cell and colony - based cloning and functional testing. In addition, we also identified a therapeutic vulnerability of the resistant cells.

cancer biology↗

bioRxiv2024

Targeting high-risk multiple myeloma genotypes with optimized anti-CD70 CAR-T cells

Despite the success of BCMA-targeting CAR-Ts in multiple myeloma, patients with high-risk cytogenetic features still relapse most quickly and are in urgent need of additional therapeutic options. Here, we identify CD70, widely recognized as a favorable immunotherapy target in other cancers, as a specifically upregulated cell surface antigen in high risk myeloma tumors. We use a structure-guided design to define a CD27-based anti-CD70 CAR-T design that outperforms all tested scFv-based CARs, leading to >80-fold improved CAR-T expansion in vivo. Epigenetic analysis via machine learning predicts key transcription factors and transcriptional networks driving CD70 upregulation in high risk myeloma. Dual-targeting CAR-Ts against either CD70 or BCMA demonstrate a potential strategy to avoid antigen escape-mediated resistance. Together, these findings support the promise of targeting CD70 with optimized CAR-Ts in myeloma as well as future clinical translation of this approach. One sentence summaryStructure-optimized CD27-based CAR-T cells targeting CD70 are a promising therapeutic option for high-risk multiple myeloma patients who are most likely to relapse on current BCMA-targeting cellular therapies.

cancer biology↗

bioRxiv2023

Short-term evolution of antibiotic responses in highly dynamic environments favors loss of regulation

To adjust to sudden shifts in conditions, microbes possess regulated genetic mechanisms that sense environmental challenges and induce the appropriate responses. The initial evolution of microbes in new environments is thought to be driven by regulatory mutations, but it is not clear how this evolution is affected by how quickly conditions change (i.e. dynamics). Here, we perform experimental evolution on continuous cultures of tetracycline resistant E. coli in different dynamical regimens of drug administration. We find that cultures evolved under gradually increasing drug concentrations acquire fine-tuning mutations adapting an alternative efflux pump to tetracycline. However, cultures that are instead periodically exposed to large drug doses evolve transposon insertions resulting in loss of regulation of the main mechanism of tetracycline resistance. A mathematical model shows that sudden drug exposures overwhelm regulated responses, which cannot induce resistance fast enough. These results help explain the frequent loss of regulation of resistance in clinical pathogens.

microbiology↗

bioRxiv2023

Pharmacologically inducing regenerative cardiac cells by small molecule drugs

Adult mammals, unlike some lower organisms, lack the ability to regenerate damaged hearts through cardiomyocytes (CMs) dedifferentiation into cells with regenerative capacity. Developing conditions to induce such naturally unavailable cells with potential to proliferate and differentiate into CMs, i.e., regenerative cardiac cells (RCCs), in mammals will provide new insights and tools for heart regeneration research. In this study, we demonstrate that a two-compound combination, CHIR99021 and A-485 (2C), effectively induces RCCs from human embryonic stem cell (hESC)-derived TNNT2+ CMs in vitro, as evidenced by lineage tracing experiments. Functional analysis shows that these RCCs express a broad spectrum of cardiogenesis genes and have the potential to differentiate into functional CMs, endothelial cells (ECs), and smooth muscle cells (SMCs). Importantly, similar results were observed in neonatal rat CMs both in vitro and in vivo. Remarkably, administering 2C in adult mouse hearts significantly enhances survival and improves heart function post-myocardial infarction. Mechanistically, CHIR99021 is crucial for the transcriptional and epigenetic activation of genes essential for RCC development, while A-485 primarily suppresses H3K27Ac and particularly H3K9Ac in CMs. Their synergistic effect enhances these modifications on RCC genes, facilitating the transition from CMs to RCCs. Therefore, our findings demonstrate the feasibility and reveal the mechanisms of pharmacological induction of RCCs from endogenous CMs, which could offer a promising regenerative strategy to repair injured hearts.

cell biology↗

bioRxiv2023

Data-driven optimization yielded a highly-efficient CRISPR/Cas9 system for gene editing in Arabidopsis

The CRISPR/Cas9 technology is widely used for plant gene editing. In Arabidopsis, the early CRISPR/Cas9 systems using constitutive promoters to drive Cas9 expression usually have low heritable mutation efficiencies in the T1 generation. Germ cell-specific or cell division-specific promoters, like EC1.2en-EC1.1p fusion, YAO and CDC45, have been used as alternatives to direct Cas9 expression, but the efficiency of getting nonchimeric mutations is still not high. To further improve gene editing efficiency, we used gene co-expression network analysis to identify NUC1 as a candidate promoter for driving Cas9 expression. NUC1 has expression patterns similar to YAO, but expresses at a much higher level. We constructed a CRISPR/Cas9 system pHY07 that uses the NUC1 promoter to drive Cas9 and carries the mCherry protein as a fluorescent marker for selecting transgenic seeds. Using this system to edit the GL2 gene, we obtained apparently nonchimeric mutations in 55% of the T1 transgenic plants. Among the Cas9-free T2 plants 99% harbored mutations that are stably inherited from the previous generation. Therefore, our system exhibited extremely high editing efficiency, and through fluorescent screening of transgenic seeds, it become easy to obtain Cas9-free and stable genetic mutants in the T2 generation.

plant biology↗

bioRxiv2023

Structural insights into abscisic acid exporter AtABCG25

Cellular hormone homeostasis is essential for the precise spatial and temporal signaling responses and plant fitness. Abscisic acid (ABA) plays pivotal roles in orchestrating various developmental and stress responses and confers fitness benefits over ecological and evolutionary timescales in terrestrial plants. Cellular ABA levels is regulated by complex processes including biosynthesis, catabolism, and transport. AtABCG25 is the first identified ABA exporter through genetic screen which affects diverse ABA responses. Resolving the structure basis of ABCG25 in ABA exporting is critical for further manipulating ABA homeostasis and plant fitness. We utilized cryo-electron microscopy to elucidate the structural dynamics of AtABCG25, and successfully characterized different states including apo AtABCG25, ABA-bound AtABCG25 and ATP-bound AtABCG25(E232Q). Notably, AtABCG25 forms a homodimer, featuring a deep, slit-like cavity in the transmembrane domain. The critical residues in the cavity where ABA binds are precisely characterized. Moreover, ATP binding triggers the closure of nucleotide-binding domains and conformational transitions in the transmembrane domains. Collectively, these findings provide valuable insights into the intricate substrate recognition and transport mechanisms of ABA exporter ABCG25, paving the way towards genetical manipulating of ABA homeostasis and plant fitness.

plant biology↗

bioRxiv2023

Dynamic phosphatase-recruitment controls B-cell selection and oncogenic signaling

Initiation of B-cell receptor (BCR)1 signaling, and subsequent antigen-encounter in germinal centers2,3 represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL)4 and lymphoma5 also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells6-9, the significance of its expression on B-cells was unclear. Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKC{delta} and SHIP1 and SHP1 phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKC{delta}10-12, SHIP113,14 and SHP114, 15,16, conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early (B-ALL) and late (lymphoma) stages of B-cell development, CD25-loss induced cell death in the former and accelerated proliferation in the latter. Clinical outcome annotations mirrored opposite effects of CD25-deletion: high CD25 expression levels predicted poor clinical outcomes for patients with B-ALL, in contrast to favorable outcomes for lymphoma-patients. Biochemical and interactome studies revealed a critical role of CD25 in BCR-feedback regulation: BCR-signaling induced PKC{delta}-mediated phosphorylation of CD25 on its cytoplasmic tail (S268). Genetic rescue experiments identified CD25-S268 tail-phosphorylation as central structural requirement to recruit SHIP1 and SHP1 phosphatases to curb BCR-signaling. A single point mutation CD25S268A abolished recruitment and activation of SHIP1 and SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function, autonomous BCR-signaling and Ca2+-oscillations induced anergy and negative selection during early B-cell development, as opposed to excessive proliferation and autoantibody production in mature B-cells. These findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and negative selection to prevent autoimmune disease.

immunology↗

bioRxiv2022

Pharmacologic Inhibition of DYRK1A Results in MYC Hyperactivation and ERK Hyperphosphorylation rendering KMT2A-R ALL Cells Sensitive to BCL2 Inhibition

KMT2A-rearranged (KMT2A-R) B cell acute lymphoblastic leukemia (ALL) is a high-risk disease in children and adults that is often chemotherapy resistant. To identify non-cytotoxic approaches to therapy, we performed a domain-specific kinome-wide CRISPR screen in KMT2A-R cell lines and patient derived xenograft samples (PDX) and identified dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) as a potential target. Pharmacologic inhibition of the KMT2A-fusion transcriptional co-regulator Menin released the KMT2A-fusion complex from the DYRK1A promoter thereby lowering DYRK1A expression levels confirming DYRK1A as a direct target of the KMT2A fusion oncogene. Direct pharmacologic inhibition of DYRK1A decreased cell proliferation of KMT2A-R ALL, thereby confirming the requirement of DYRK1A in this ALL subtype. To further understand the biologic function of DYRK1A in KMT2A-R ALL, we leveraged pharmacologic DYRK1A inhibitors in KMT2A-R PDX and cell line models. DYRK1A inhibition consistently led to upregulation of MYC protein levels, and hyperphosphorylation of ERK, which we confirmed via in vivo treatment experiments. Furthermore, DYRK1A inhibition decreased ALL burden in mice. Our results further demonstrate that DYRK1A inhibition induces the proapoptotic factor BIM, but ERK hyperphosphorylation is the driving event that induces cell cycle arrest. In contrast, combined treatment of KMT2A-R ALL cells in vitro and in vivo with DYRK1A inhibitors and the BCL2 inhibitor, venetoclax, synergistically decreases cell survival and reduced the leukemic burden in mice. Taken together these results demonstrate a unique function of DYRK1A specially in KMT2A-R ALL. Synergistic inhibition of DRYK1A and BCL2 may provide a low-toxic approach to treat this high risk ALL subtype.

cancer biology↗

bioRxiv2022

Cell circuits between leukemic cells and mesenchymal stem cells block lymphopoiesis by activating lymphotoxin-beta receptor signaling

Acute lymphoblastic and myeloblastic leukemias (ALL and AML) have been known to modify the bone marrow microenvironment and disrupt non-malignant hematopoiesis. However, the molecular mechanisms driving these alterations remain poorly defined. Here we show that leukemic cells turn-off lymphopoiesis and erythropoiesis shortly after colonizing the bone marrow. ALL and AML cells express lymphotoxin-1{beta}2 and activate LT{beta}R signaling in mesenchymal stem cells (MSCs), which turns off IL7 production and prevents non-malignant lymphopoiesis. We show that the DNA damage response pathway and CXCR4 signaling promote lymphotoxin-1{beta}2 expression in leukemic cells. Genetic or pharmacologic disruption of LT{beta}R signaling in MSCs restores lymphopoiesis but not erythropoiesis, reduces leukemic cell growth, and significantly extends the survival of transplant recipients. Similarly, CXCR4 blocking also prevents leukemia-induced IL7 downregulation, and inhibits leukemia growth. These studies demonstrate that acute leukemias exploit physiological mechanisms governing hematopoietic output as a strategy for gaining competitive advantage. One Sentence SummaryLeukemias colonize bone marrow niches and disrupt hematopoiesis. However, the cross-talk between leukemia and niche cells remains poorly understood. We show that leukemia activates LT{beta}R in mesenchymal stem cells which suppresses IL7 production and IL7-dependent lymphopoiesis and accelerates leukemia growth.

immunology↗

bioRxiv2022

Development and application of an uncapped mRNA platform

A novel uncapped mRNA platform was developed. Five lipid nanoparticle (LNP)-encapsulated mRNA constructs were made to evaluate several aspects of our platform, including transfection efficiency and durability in vitro and in vivo and the activation of humoral and cellular immunity in several animal models. The constructs were eGFP-mRNA-LNP (for enhanced green fluorescence mRNA), Fluc-mRNA-LNP (for firefly luciferase mRNA), S{delta}T-mRNA-LNP (for Delta strain SARS-CoV-2 spike protein trimer mRNA), gDED-mRNA-LNP (for truncated glycoprotein D mRNA coding ectodomain from herpes simplex virus type 2 (HSV2)) and gDFR-mRNA-LNP (for truncated HSV2 glycoprotein D mRNA coding amino acids 1[~]400). Quantifiable target protein expression was achieved in vitro and in vivo with eGFP-and Fluc-mRNA-LNP. S{delta}T-mRNA-LNP, gDED-mRNA-LNP and gDFR-mRNA-LNP induced both humoral and cellular immune responses comparable to those obtained by previously reported capped mRNA-LNP constructs. Notably, S{delta}T-mRNA-LNP elicited neutralizing antibodies in hamsters against the Omicron and Delta strains. Additionally, gDED-mRNA-LNP and gDFR-mRNA-LNP induced potent neutralizing antibodies in rabbits and mice. The mRNA constructs with uridine triphosphate (UTP) outperformed those with N1-methylpseudouridine triphosphate (N1m{psi}TP) in the induction of antibodies via S{delta}T-mRNA-LNP. Our uncapped, process-simplified, and economical mRNA platform may have broad utility in vaccines and protein replacement drugs.

bioengineering↗

bioRxiv2021

Overcoming microenvironment-mediated chemoprotection through stromal Galectin-3 inhibition in acute lymphoblastic leukemia

Environmentally-mediated drug resistance in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) significantly contributes to relapse. Stromal cells in the bone marrow environment protect leukemia cells by secretion of chemokines as cues for BCP-ALL migration towards, and adhesion to, stroma. Stromal cells and BCP-ALL cells communicate through stromal galectin-3. Here, we investigated the significance of stromal galectin-3 to BCP-ALL cells. We used CRISPR/Cas9 genome editing to ablate galectin-3 in stromal cells and found that galectin-3 is dispensable for steady-state BCP-ALL proliferation and viability. However, efficient leukemia migration and adhesion to stromal cells are significantly dependent on stromal galectin-3. Importantly, loss of stromal galectin-3 production sensitized BCP-ALL cells to conventional chemotherapy. We therefore tested novel carbohydrate-based small molecule compounds (Cpd14 and Cpd17) with high specificity for galectin-3. Consistent with results obtained using galectin-3-knockout stromal cells, treatment of stromal-BCP-ALL co-cultures inhibited BCP-ALL migration and adhesion. Moreover, these compounds induced anti-leukemic responses in BCP-ALL cells including a dose-dependent reduction of viability and proliferation, induction of apoptosis and, importantly, inhibition of drug resistance. Collectively, these findings indicate galectin-3 regulates BCP-ALL cell responses to chemotherapy through the interactions between leukemia cells and the stroma, and show that a combination of galectin-3 inhibition with conventional drugs can sensitize the leukemia cells to chemotherapy.

cancer biology↗