Biology subjects

Murray, H. C.

Publications and source records attributed to Murray, H. C..

16 recordsLinked to original sources

bioRxiv2026

(E,E)-bisantrene suppresses MYC expression and displays anti-leukemic activity in acute myeloid leukemia

Background: Acute myeloid leukemia (AML) is genetically diverse with a high unmet clinical need for improved treatment options. Dysregulation of the transcription factor MYC plays a central role in AML progression and therapeutic resistance. (E,E)-bisantrene was recently found to inhibit MYC transcription and downstream activity via G-quadruplex DNA stabilization. This study aimed to evaluate the mechanism of action and preclinical activity of (E,E)-bisantrene in AML. Methods: The in vitro and in vivo activity of (E,E)-bisantrene was determined in a variety of AML models (cell lines, xenograft mouse models, and ex vivo human AML mononuclear cells). Transcriptomic, proteomic and phosphoproteomic analyses were performed after treatment with (E,E)-bisantrene. Analyses of -omics data to identify enriched pathways and upstream regulators were performed. Results: (E,E)-bisantrene demonstrated potent anti-proliferative activity across a panel of AML cell lines, inducing apoptosis and reducing S phase proportions. (E,E)-bisantrene significantly prolonged survival in cell- and patient-derived xenograft models of AML. Mechanistically, RNA-seq and proteomic analysis of MOLM13 and MV4-11 cells treated with (E,E)-bisantrene showed significant reductions in the activity of MYC and E2F, together with the cell cycle regulators CDK1/2/4/5. Transcript and protein levels of MYC were reduced in a dose- and time-dependent manner. TP53 and inflammation-associated transcript signatures were also observed. Conclusion: Anti-proliferative activity of (E,E)-bisantrene in preclinical AML models was associated with a downregulation of MYC, CDK1/2/4/5 and E2F. This study supports the ongoing clinical evaluation of (E,E)-bisantrene in AML where MYC is a clinically relevant driver of disease aggressiveness and therapy resistance.

cancer biology↗

bioRxiv2026

Systematic screening identifies elevated neurovascular BCL-XL in Parkinson's disease.

BackgroundVascular cells are emerging as active players in Parkinsons disease (PD), yet their molecular contribution to -Synuclein (-Syn) pathology remains undefined. Here, we show that human brain pericytes respond in a distinct manner to unique -Syn strains, with systematic validation identifying BCL-XL as a potential regulator of the neurovascular unit in PD. MethodsPrimary human brain-derived pericytes were exposed to five recombinant -Syn strains (Fibrils, Fibrils-65, Fibrils-91, Fibrils-110, and Ribbons). Transcriptomic profiling identified differentially expressed genes (DEGs), which were validated at the protein level using multiplex immunocytochemistry and in situ labelling of post-mortem middle temporal gyrus (MTG) tissue microarrays from PD (n = 24) and neurologically normal (n = 24) cases. Results-Syn strain exposure produced 300 DEGs with limited overlap between strains. BCL-XL and CSNK1D were upregulated in -Syn-treated pericytes. In post-mortem PD tissue BCL-XL showed marked pericyte-specific elevation in the MTG and increased pericytic and microglial expression in the substantia nigra. ConclusionBCL-XL emerges as a potential regulator of pericyte and microglial resilience in PD, linking acute -Syn strain-specific responses in pericytes to broader neurovascular alterations. Its upregulation likely represents a generalised compensatory response to chronic -Syn-associated stress beyond individual strain effects, identifying BCL-XL as a possible therapeutic target within the neurovascular unit.

neuroscience↗

bioRxiv2025

Bisantrene potentiates tyrosine kinase inhibitor activity in clear cell renal cell carcinoma

Clear Cell Renal Cell Carcinoma (ccRCC) is the most prevalent kidney cancer and often develops resistance to standard therapies. This study aimed to assess if bisantrene, a multi-mechanistic agent with broad anticancer activity, can enhance the activity of standard of care ccRCC treatments. A panel of ccRCC cell lines were treated with bisantrene alone and in combination with common ccRCC drugs. Bisantrene showed moderate activity as a single agent, but strongly synergized with several ccRCC treatments, especially the tyrosine kinase inhibitors (TKIs) lenvatinib, pazopanib and cabozantinib. Cellular signaling pathways assessed by immunoblotting revealed the TKIs inhibit MET as well as downstream AKT and ERK signaling pathways as single agents. Combination of these TKIs with bisantrene was able to induce sustained downstream AKT inhibition and negate the rebound effect seen with TKI resistance. Overall, bisantrene shows promise as a new therapeutic agent for ccRCC in combination with TKIs.

cancer biology↗

bioRxiv2025

The genetic origin of fetal growth restriction and mitochondrial complex I dysregulation

BackgroundMitochondria are organelles required for bioenergetic homeostasis, providing energy in the form of ATP to support cellular function, growth, and proliferation. Mitochondria synthesise ATP using the electron transport chain (ETC) to produce an electrochemical gradient that facilitates the conversion of ADP to ATP by ATP synthase. Mitochondrial function is governed by an intricate bidirectional relationship between the mitochondrial genome encoding 1-2% of proteins and the nuclear genome which encodes 98-99% of mitochondrial proteins. Pregnancy creates a unique environment whereby the mitochondrial genome of the placenta is maternally inherited, while the nuclear genome is comprised of both maternal and paternal contributions. Thus, mitochondrial structure and function are largely dependent on the adaptability of mitochondria to conform to the new mixed nuclear genome. In pregnancy, fetal growth restriction (FGR) is characterised by poor placental development, underlying trophoblast insufficiencies, and is associated with mitochondrial dysfunction within the placenta, although the mechanisms which underpin these changes remain unclear. This study investigated if mitochondrial dysfunction in FGR is programmed by genetic incompatibility arising from single nucleotide polymorphisms (SNPs). Methods and findingsWe performed a targeted meta-analysis of over 100 genome wide association studies within the early growth genetics (EGG) consortium, assessing 289 genes encoding mitochondrial function across the ETC and ATP synthase. This study identified 37 SNPs across 32 genes associated with low birthweight. Sanger sequencing was performed to validate the presence of 10 SNPs of interest located within the nuclear genome in a cohort of fetal growth restricted placentas. Of the 10 SNPs assessed we confirmed the presence of 6 in FGR, and identified an additional previously unidentified SNP, and detected 3 nucleotide deletions across numerous components of the electron transport chain. This analysis identified 5 mutations within genes that encode complex I. Subsequent analysis of the gene and proteins that correspond to complex I mutations was performed using PCR, proteomics, and western blotting. Notably, we identified significant downregulation of NDUFA6 at the gene and protein level in FGR, lower protein levels of NDUFS3 and NDUFS6, and confirmed the largest log2 fold change identified in NDUFS6 by western blot. Subsequent investigation of mitochondrial respiratory capacity identified decreased ATP-linked respiration in FGR using Seahorse XF Analysis. ConclusionOur results identify that there is an inheritable contribution of SNPs within the maternal and paternal nuclear genome in fetal growth restriction. These SNPs alter gene expression and protein abundance of mitochondrial complex I components, impacting structural assembly and subsequent bioenergetic function within the placenta. Collectively these findings suggest that SNPs that affect mitochondrial assembly and function are associated with low birthweight and fetal growth restriction.

developmental biology↗

bioRxiv2025

Placental Iron Utilisation in Fetal Growth Restriction: Alterations in Mitochondrial Heme Synthesis and Iron-Sulfur Cluster Assembly Pathways

Fetal growth restriction (FGR) affects [~]10% of pregnancies worldwide and is often associated with placental insufficiency. Iron is essential for maternal haematopoietic adaptations and placental processes such as mitochondrial iron-sulfur (Fe-S) cluster assembly, heme synthesis, and erythropoiesis. This study aimed to characterise iron transport and downstream utilisation in FGR. Placental tissues from term uncomplicated (n=19) and FGR (n=18) pregnancies were analysed. Maternal iron status was retrospectively assessed from clinical records. Placental mRNA and protein expression of iron-dependent pathways were analysed via RT-qPCR, LC-MS, and western blotting. Placental iron content was assessed histologically, and heme levels were measured by activity assay. FGR pregnancies showed significantly elevated maternal serum ferritin and lower red cell distribution width, although remained within normal clinical values. Placental iron uptake transporters TFRC and DMT1 were significantly upregulated, while the iron exporter to the fetus, ferroportin, was reduced, indicating increased iron retention in the FGR placenta. Despite altered transporter expression, Fe3 iron levels were unchanged, suggesting iron utilisation over storage. Subsequent investigations identified reduced mitochondrial Fe-S synthesis components (FDXR, FDX2, NDUFAB1, HSPA9), and a prioritisation of mitochondrial and cytosolic heme synthesis enzymes in FGR. Protein levels of haemoglobin subunits (HBG1, HBG2, HBB, HBA1) and erythrocyte membrane markers (EPB41, EPB42, SPTA1, SPTB, ANK1) were decreased. These findings reveal a compensatory response in FGR placentae, with increased iron uptake and utilisation favouring heme synthesis over Fe-S cluster formation, possibly to support oxygen handling under poor placental vascularisation and reduced fetal oxygenation, with potential consequences for mitochondrial energy metabolism. Key PointsO_LIIron plays a critical role in placental function, while iron-dependent pathway components are well-characterised, their integrated response and adaptive reprogramming in fetal growth restriction (FGR) remain poorly understood. C_LIO_LIIn FGR, maternal iron status was unchanged, however, placental iron uptake proteins were increased and ferroportin reduced, suggesting that the placenta retains iron. C_LIO_LIFGR placentae showed altered de novo mitochondrial iron-sulfur cluster (Fe-S) formation and a bottleneck in late-stage Fe-S cluster assembly. C_LIO_LIThis shift in Fe-S synthesis prioritises mitochondrial and cytosolic heme synthesis pathways, consistent with increased heme utilisation and breakdown. C_LIO_LIGlobin subunits were lower in protein abundance and impaired placental erythrocyte structure in FGR. C_LIO_LIDysregulation of erythrocyte membrane proteins in FGR placentae suggests altered erythrocyte structure, potentially representing an adaptive response to inadequate vascularisation, attempting to optimise oxygen delivery to the fetus. C_LI

physiology↗

bioRxiv2025

Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington's disease human cerebral cortex

Huntingtons disease (HD) is a hereditary neurodegenerative condition caused by a CAG repeat expansion mutation in the gene encoding the huntingtin (Htt) protein. The accumulation of Htt inclusion bodies is a pathological hallmark of HD and a common target for therapeutic strategies. However, the limited efficacy of treatments targeting the Htt protein highlights the need for a better understanding of the role of Htt inclusion bodies in HD pathogenesis. This study examined the heterogeneity of Htt inclusion body composition by co-labelling with three Htt epitope-specific antibodies to characterize Htt inclusion body immunophenotype. We then characterized the size and sub-cellular location of Htt inclusions with distinct immunophenotypes. Using multiplex immunohistochemistry, we also examined the ubiquitination profile of each immunophenotype. Our findings demonstrate that Htt inclusions have a range of immunophenotypes, with some labelled by only one of the three antibodies and others exhibiting co-labelling by several antibodies, thus demonstrating the heterogeneity in inclusion composition and structure. We outline evidence that inclusion bodies exclusively labelled with the EM48 antibody are small, non-nuclear, and more abundant in HD cases with increased CAG repeat length, higher Vonsattel grade, and earlier age of onset. We also find that Htt inclusion bodies labelled by multiple antibodies are more likely to be ubiquitinated, predominantly by K63-rather than K48-linked ubiquitin, suggesting preferential degradation by autophagy. Lastly, we show that ubiquitinated Htt inclusion bodies are more highly immunoreactive for ubiquilin 2 than p62. Our findings highlight the need for multiple antibodies to capture the full spectrum of Htt pathology in HD and imply that future studies should consider the diversity of inclusion body composition and structure when correlating pathology formation to neurodegeneration, clinical symptoms, or disease severity.

neuroscience↗

bioRxiv2024

Adaptive resistance to FLT3 inhibitors is potentiated by ROS-driven DNA repair signalling

Alterations in the FMS-like tyrosine kinase 3 (FLT3) gene are the most frequent driver mutations in acute myeloid leukaemia (AML), linked to a high risk of relapse in patients with internal tandem duplications (FLT3-ITD). Tyrosine kinase inhibitors (TKIs) targeting the FLT3 protein are approved for clinical use, yet resistance often emerges. This resistance is mainly seen following the acquisition of additional point mutations in the tyrosine kinase domain (TKD), resulting in a double mutant FLT3-ITD/TKD, which sustains cell signalling and survival despite the presence of FLT3 inhibitors. Here, we developed a FLT3-mutant AML model with adaptive resistance to type II TKIs, sorafenib, and quizartinib by in vitro drug selection. Through global multiomic profiling, we identified upregulation of proteins involved in reactive oxygen species (ROS) production, particularly NADPH-oxidases, driving cellular ROS-addiction, with resistant cells relying on ROS for survival, and genome fidelity preserved by ATM-driven DNA repair. Transcriptomic analysis of adult and paediatric AML (pAML) patients identified high ATM expression as a biomarker for shorter median overall survival in both the de novo and relapsed settings. Inhibition of ATM with clinically relevant therapy WSD-0628 effectively killed TKI- and chemotherapy-resistant AML cells in vitro and significantly extended the survival of mice with sorafenib- and quizartinib-resistant FLT3-ITD AML in vivo. We propose a new treatment strategy to improve survival of patients who develop resistance to sorafenib and quizartinib, as well as relapsed and refractory pAML, exploiting resistance mechanisms to precision therapies and cell-intrinsic features of high-risk cases, highlighting a clinically relevant salvage strategy.

cancer biology↗

bioRxiv2024

Upregulated cholesterol biosynthesis facilitates the survival of methylation-retaining AML cells following decitabine treatment.

DNA hypomethylating agents (HMAs) are used to treat acute myeloid leukemia (AML) and myelodysplasia patients who are unsuitable for intensive chemotherapy. However, low response rates and therapy-resistant relapse remain significant challenges. To improve outcomes, we must understand how AML cells survive HMA treatment and continue to proliferate following therapy. We combine single-cell multiomics with parallel colony-forming assays to link HMA-induced heterogeneity with functional consequences in AML cellss. Azacytidine (AZA) and decitabine (DAC) induced global epigenetic heterogeneity, associated with upregulation of inflammatory responses and cell death pathways in a subset of hypomethylated cells. Some cells maintained high DNA methylation levels during treatment, and these methylation-retaining cells had increased self-renewal capacity following DAC treatment in two FLT3-ITD AML cell lines. Transcriptional profiling of colonies formed after HMA treatment revealed many genes with altered expression in both methylation-retaining and hypomethylated cells, with increased expression of cholesterol-related genes observed in all cell lines. Inhibition of the cholesterol biosynthesis pathway by rosuvastatin enhanced HMA effects on colony formation in vitro and extended survival in two in vivo models of AML. Our study demonstrates that HMA-induced epigenetic heterogeneity has implications for AML cell growth and identifies statins as a candidate co-treatment strategy to improve HMA efficacy.

cancer biology↗

bioRxiv2023

PI3K/mTOR is a therapeutically targetable genetic dependency in diffuse intrinsic pontine glioma

Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma - DIPG), are uniformly fatal brain tumors that lack effective pharmacological treatment. Analysis of pooled CRISPR-Cas9 loss-of-function gene deletion screen datasets, identified PIK3CA and MTOR as targetable molecular dependencies across DIPG patient derived models, highlighting the therapeutic potential of the blood-brain barrier penetrant PI3K/Akt/mTOR inhibitor paxalisib. At the human equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic feedback resulting in increased blood glucose and insulin levels, commensurate with DIPG patients in Phase 1b clinical trials who experienced hyperglycemia/hyperinsulinemia. To exploit genetic dependences, but maintain compliance and benefit, we optimized a paxalisib treatment regimen that employed reduced dosing more frequently, in combination with the anti-hyperglycemic drug, metformin. Combining optimized dosing with metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptor in vivo, a common mechanism of PI3K-inhibitor resistance, extending the survival of DIPG xenograft models. RNA sequencing and phosphoproteomic profiling of DIPG models treated with paxalisib identified increased calcium-activated PKC signaling. Using the brain penetrant PKC inhibitor, enzastaurin in combination with paxalisib, we synergistically extended the survival of orthotopic xenograft models, benefits further promoted by metformin; thus, identifying a clinically relevant DIPG combinatorial approach. Brief SummaryDiffuse intrinsic pontine glioma is a lethal childhood brain tumor. Here we identify PIK3CA as a genetic dependency targeted by the brain penetrant pan-PI3K-inhibitor paxalisib.

cancer biology↗

bioRxiv2023

Microglial CD68 and L-ferritin upregulation in response to phosphorylated-TDP-43 pathology in the amyotrophic lateral sclerosis brain

Microglia, the innate immune cells of the brain, are activated by damage or disease. In mouse models of amyotrophic lateral sclerosis (ALS), microglia shift from neurotrophic to neurotoxic states with disease progression. It remains unclear how human microglia change relative to the TAR DNA-binding protein 43 (TDP-43) aggregation that occurs in 97% of ALS cases. Here we examine spatial relationships between microglial activation and TDP-43 pathology in brain tissue from people with ALS and from a TDP-43-driven ALS mouse model. Post-mortem human brain tissue from the Neurological Foundation Human Brain Bank was obtained from 10 control and 10 ALS cases in parallel with brain tissue from a bigenic NFFH-tTA/tetO-hTDP-43{Delta}NLS (rNLS) mouse model of ALS at disease onset, early disease, and late disease stages. The spatiotemporal relationship between microglial activation and ALS pathology was determined by investigating microglial functional marker expression in brain regions with low and high TDP-43 burden at end-stage human disease: hippocampus and motor cortex, respectively. Sections were immunohistochemically labelled with a two-round multiplexed antibody panel against; microglial functional markers (L-ferritin, HLA-DR, CD74, CD68, and Iba1), a neuronal marker (NeuN), an astrocyte marker (GFAP), and pathological phosphorylated TDP-43 (pTDP-43). Single-cell levels of microglial functional markers were quantified using custom analysis pipelines and mapped to anatomical regions and ALS pathology. We identified a significant increase in microglial Iba1 and CD68 expression in the human ALS motor cortex, with microglial CD68 being significantly correlated with pTDP-43 pathology load. We also identified two subpopulations of microglia enriched in the ALS motor cortex that were defined by high L-ferritin expression. A similar pattern of microglial changes was observed in the rNLS mouse, with an increase first in CD68 and then in L-ferritin expression, with both occurring only after pTDP-43 inclusions were detectable. Our data strongly suggest that microglia are phagocytic at early-stage ALS but transition to a dysfunctional state at end-stage disease, and that these functional states are driven by pTDP-43 aggregation. Overall, these findings enhance our understanding of microglial phenotypes and function in ALS.

neuroscience↗

bioRxiv2023

Distribution of ubiquilin 2 and TDP-43 aggregates throughout the CNS in UBQLN2 p.T487I-linked amyotrophic lateral sclerosis and frontotemporal dementia

Mutations in the UBQLN2 gene cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The neuropathology of such UBQLN2-linked cases of ALS/FTD is characterised by aggregates of the ubiquilin 2 protein in addition to aggregates of the transactive response DNA-binding protein of 43 kDa (TDP-43). ALS and FTD without UBQLN2 mutations are also characterised by TDP-43 aggregates, that may or may not colocalise with wildtype ubiquilin 2. Despite this, the relative contributions of TDP-43 and ubiquilin 2 to disease pathogenesis remain largely under-characterised, as does their relative deposition as aggregates across the central nervous system (CNS). Here we conducted multiplex immunohistochemistry of three UBQLN2 p.T487I-linked ALS/FTD cases, three non-UBQLN2-linked (sporadic) ALS cases, and eight non-neurodegenerative disease controls, covering 40 CNS regions. We then quantified ubiquilin 2 aggregates, TDP-43 aggregates, and aggregates containing both proteins in regions of interest to determine how UBQLN2-linked and non-UBQLN2-linked proteinopathy differ. We find that ubiquilin 2 aggregates that are negative for TDP-43 are predominantly small and punctate, and are abundant in the hippocampal formation, spinal cord, all tested regions of neocortex, medulla, and substantia nigra in UBQLN2-linked ALS/FTD but not sporadic ALS. Curiously, the striatum harboured small punctate ubiquilin 2 aggregates in all cases examined, while large diffuse striatal ubiquilin 2 aggregates were specific to UBQLN2-linked ALS/FTD. Overall, ubiquilin 2 is mainly deposited in clinically unaffected regions throughout the CNS such that symptomology in UBQLN2-linked cases maps best to the aggregation of TDP-43.

neuroscience↗

bioRxiv2022

Characterization of adult human skeletal cells in different tissues reveals a CD90+CD34+ periosteal stem cell population

Skeletal stem and progenitor cells are critical for bone homeostasis and healing, but their identity and diversity in humans are not well understood. In this study, we compared stromal populations in matched tissues from the femoral head and neck of 21 human participants using spectral flow cytometry of freshly isolated cells. High-level analysis indicated significant differences in marker distribution between periosteum, articular cartilage, endosteum and bone marrow stromal populations, and identified populations that were highly enriched or unique to specific tissues. Periosteum-enriched markers included CD90 and CD34. Articular cartilage, which has very poor regenerative potential, showed enrichment of multiple markers, including the PDPN+CD73+CD164+ population previously reported to represent human skeletal stem cells. We further characterized periosteal populations by combining CD90 with other strongly expressed markers. CD90+CD34+ cells sorted directly from periosteum showed significant colony-forming unit fibroblasts (CFU-F) enrichment, rapid expansion, and consistent multi-lineage differentiation of clonal populations. In situ, CD90+CD34+ cells include a perivascular population in the outer layer of the periosteum and non-perivascular cells closer to the bone surface. In conclusion, our study indicates considerable diversity in the stromal cell populations in different tissue compartments within the adult human skeleton, and suggests that periosteal stem cells reside within the CD90+CD34+ population.

cell biology↗

bioRxiv2022

High-Throughput Global Phosphoproteomic Profiling Using Phospho Heavy-Labeled-Spiketide FAIMS Stepped-CV DDA (pHASED)

Global high-throughput profiling of oncogenic signaling pathways by phosphoproteomics is increasingly being applied to cancer specimens. Such quantitative unbiased phosphoproteomic profiling of cancer cells identifies oncogenic signaling cascades that drive disease initiation and progression; pathways that are often invisible to genomics sequencing strategies. Therefore, phosphoproteomic profiling has immense potential for informing individualized anti-cancer treatments. However, complicated and extensive sample preparation protocols, coupled with intricate chromatographic separation techniques that are necessary to achieve adequate phosphoproteomic depth, limits the clinical utility of these techniques. Traditionally, phosphoproteomics is performed using isobaric tagged based quantitation coupled with TiO2 enrichment and offline prefractionation prior to nLC-MS/MS. However, the use of isobaric tags and offline HPLC limits the applicability of phosphoproteomics for the analysis of individual patient samples in real-time. To address these limitations, here we have optimized a new protocol, phospho-Heavy-labeled-spiketide FAIMS Stepped-CV DDA (pHASED). pHASED maintained phosphoproteomic coverage yet decreased sample preparation time and complexity by eliminating the variability associated with offline prefractionation. pHASED employed online phosphoproteome deconvolution using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and internal phosphopeptide standards to provide accurate label-free quantitation data. Compared with our traditional tandem mass tag (TMT) phosphoproteomics workflow and optimized using isogenic FLT3-mutant acute myeloid leukemia (AML) cell line models (n=18/workflow), pHASED halved total sample preparation, and running time (TMT=10 days, pHASED=5 days) and doubled the depth of phosphoproteomic coverage in real-time (phosphopeptides = 7,694 pHASED, 3,861 TMT). pHASED coupled with bioinformatic analysis predicted differential activation of the DNA damage and repair ATM signaling pathway in sorafenib-resistant AML cell line models, uncovering a potential therapeutic opportunity that was validated using cytotoxicity assays. Herein, we optimized a rapid, reproducible, and flexible protocol for the characterization of complex cancer phosphoproteomes in real-time, highlighting the potential for phosphoproteomics to aid in the improvement of clinical treatment strategies.

biochemistry↗

bioRxiv2022

Blockade of redox second messengers inhibits JAK/STAT and MEK/ERK signaling sensitizing FLT3-mutant acute myeloid leukemia to targeted therapies

FLT3-mutations are diagnosed in 25-30% of patients with acute myeloid leukemia (AML) and are associated with a poor prognosis. AML is associated with the overproduction of reactive oxygen species (ROS), which drives genomic instability through the oxidation of DNA bases, promoting clonal evolution, treatment resistance and poor outcomes. ROS are also important second messengers, triggering cysteine oxidation in redox sensitive signaling proteins, however, the specific pathways influenced by ROS in AML remain enigmatic. Here we have surveyed the posttranslational architecture of primary AML patient samples and assessed oncogenic second messenger signaling. Signaling proteins responsible for growth and proliferation were differentially oxidized and phosphorylated between patient subtypes either harboring recuring mutation in FLT3 compared to patients expressing the wildtype-FLT3 receptor, particularly those mapping to the Src family kinases (SFKs). Patients harboring FLT3-mutations also showed increased oxidative posttranslational modifications in the GTPase Rac activated-NADPH oxidase-2 (NOX2) complex to drive autocratic ROS production. Pharmacological and molecular inhibition of NOX2 was cytotoxic specifically to FLT3-mutant AMLs, and reduced phosphorylation of the critical hematopoietic transcription factor STAT5 and MAPK/ERK to synergistically increase sensitivity to FLT3-inhibitors. NOX2 inhibition also reduced phosphorylation and cysteine oxidation of FLT3 in patient derived xenograft mouse models in vivo, highlighting an important link between oxidative stress and oncogenic signaling. Together, these data raise the promising possibility of targeting NOX2 in combination with FLT3-inhibitors to improve treatment of FLT3-mutant AML. One Sentence SummaryFLT3-precision therapies have entered the clinic for AML however, their durability is limited. Here we identify the Rac-NOX2 complex as the major driver of redox second messenger signaling in FLT3-mutant AML. Molecular and pharmacological inhibition of NOX2 decreased FLT3, STAT5 and MEK/ERK signaling to delay leukemia progression, and synergistically combined with FLT3 inhibitors.

cancer biology↗

bioRxiv2022

Hippocampal protein aggregation signatures fully distinguish pathogenic and wildtype UBQLN2 in amyotrophic lateral sclerosis

Mutations in the UBQLN2 gene cause X-linked dominant amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD) characterised by ubiquilin 2 aggregates in neurons of the motor cortex, hippocampus, and spinal cord. However, ubiquilin 2 neuropathology is also seen in sporadic and familial ALS or FTD cases not caused by UBQLN2 mutations, particularly C9orf72-linked cases. This makes the mechanistic role of ubiquilin 2 mutations and the value of ubiquilin 2 pathology for predicting genotype unclear. Here we examine a cohort of 41 genotypically diverse ALS cases with or without FTD, including five cases with UBQLN2 variants (resulting in p.S222G, p.P497H, p.P506S, and two cases with p.T487I). Using multiplexed (5-label) fluorescent immunohistochemistry, we mapped the co-localisation of ubiquilin 2 with phosphorylated TDP-43 (pTDP-43), dipeptide repeat aggregates, and p62, in the hippocampus of controls (n=5), or ALS with or without FTD in sporadic (n=20), unknown familial (n=3), SOD1-linked (n=1), FUS-linked (n=1), C9orf72-linked (n=5), and UBQLN2-linked (n=5) cases. We differentiate between i) ubiquilin 2 aggregation together with pTDP-43 or dipeptide repeat proteins, and ii) ubiquilin 2 self-aggregation promoted by UBQLN2 gene mutations that cause ALS/FTD. Overall, we describe a hippocampal protein aggregation signature that fully distinguishes mutant from wildtype ubiquilin 2 in ALS with or without FTD, whereby mutant ubiquilin 2 is more prone than wildtype to aggregate independently of driving factors. This neuropathological signature can be used to assess the pathogenicity of UBQLN2 gene variants and to understand the mechanisms of UBQLN2-linked disease.

neuroscience↗

bioRxiv2019

Blood-spinal cord barrier leakage is not caused by neurodegeneration in amyotrophic lateral sclerosis

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving progressive degeneration of upper and lower motor neurons. Both lower motor neuron loss and the deposition of phosphorylated TDP-43 inclusions display regional patterning along the spinal cord. The blood-spinal cord barrier (BSCB) ordinarily restricts entry into the spinal cord parenchyma of blood components that are neurotoxic, but in ALS there is evidence for barrier breakdown. Here we sought to examine whether BSCB breakdown, motor neuron loss, and TDP-43 proteinopathy display the same regional patterning across and along the spinal cord.\n\nMethodsWe measured cerebrospinal fluid (CSF) hemoglobin in living ALS patients (n=87 controls, n=236 ALS) as a potential biomarker of BSCB and blood-brain barrier leakage. We then immunostained cervical, thoracic, and lumbar post mortem spinal cord tissue (n=5 controls, n=13 ALS) and employed semi-automated imaging and analysis to quantify and map lower motor neuron loss and phosphorylated TDP-43 inclusion load against hemoglobin leakage.\n\nResultsMotor neuron loss and TDP-43 proteinopathy were seen at all three levels of the ALS spinal cord, with most abundant TDP-43 deposition in the ventral grey (lamina IX) of the cervical and lumbar cord. In contrast, hemoglobin leakage was observed along the ALS spinal cord axis but was most severe in the dorsal grey and white matter in the thoracic spinal cord.\n\nConclusionsOur data show that leakage of the blood-spinal cord barrier occurs during life but at end-stage its distribution is independent from the major motor neuron pathology and is unlikely to be a major contributor to pathogenesis in ALS.

neuroscience↗