Biology subjects

Nixon, B.

Publications and source records attributed to Nixon, B..

18 recordsLinked to original sources

bioRxiv2026

Evasion of Hepatic ILC1 Immunosurveillance Drives Breast Cancer Liver Metastasis

Metastasis, the dissemination of cancer cells to distant organs, is an inefficient process1, yet remains the leading cause of cancer-related mortality2. The cellular barriers and evasion mechanisms governing organ-specific susceptibility remain incompletely understood3-5, hindering the development of effective anti-metastatic therapies6. Here, we show that liver-resident type 1 innate lymphoid cells (ILC1s) constitute an essential immunosurveillance barrier against breast cancer liver metastasis that can be bypassed through either cancer cell intrinsic or host-dependent mechanisms. In a multi-organ metastatic breast cancer patient cohort, liver metastasis occurred later and was associated with poorer survival than lung metastasis, with elevated body mass index emerging as a clinical risk factor. This metabolic vulnerability was recapitulated in mice, where a high-fat diet induced hepatosteatosis and selectively enhanced breast cancer colonization of the liver. Mechanistically, liver and lung colonization were restrained by ILC1s and circulating natural killer (NK) cells, respectively. Following cancer cell challenge, NK cells expanded and activated, employing lytic granule pathways and the death receptor DR5 for clearance, whereas activated ILC1s selectively required high DR5 expression on cancer cells, supported by AP-2a (TFAP2A) mediated transcriptional activation, for effective immunosurveillance. Heterozygous copy number deletion at TNFRSF10B (encoding DR5) in human tumours reduced DR5 expression and predisposed to liver metastasis. Hepatosteatosis further compromised this barrier by reducing IL-15 expression in macrophages, thereby depleting hepatic ILC1s effects reversible with glucagon-like peptide-1 (GLP-1) receptor agonists. Thus, hepatic ILC1s form a critical organ-specific barrier to breast cancer metastasis, vulnerable to both cancer cell-intrinsic DR5 downregulation and host metabolic dysfunction.

immunology↗

bioRxiv2026

Mitochondrial and metabolic plasticity distinguish stem cells from progenitors in the male germline

Metabolism and mitochondrial remodelling are increasingly recognised as key regulators of stem cell identity, governing the balance between self-renewal and differentiation. Here we compared mitochondrial and metabolic properties of stem and progenitor cells from the male germline using transmission electron microscopy, quantitative proteomics and extracellular flux analysis. While the stem cell-to-progenitor transition was accompanied by increased mitochondrial abundance and cristae density, our analyses unveiled a remarkable and unexpected capacity for spermatogonial stem cells (SSCs) to engage oxidative phosphorylation. When oxygen availability was unrestricted, SSCs displayed markedly greater maximal and spare respiratory capacity than progenitors, despite comparable glycolytic flux. SSCs were also enriched for glutathione-dependent antioxidant enzymes and maintained lower basal mitochondrial superoxide levels than progenitors. Collectively, these findings challenge the view that SSCs are predominantly glycolytic and instead identify metabolic plasticity, marked by latent oxidative capacity and enhanced redox control, as a hallmark of the stem cell state.

cell biology↗

bioRxiv2026

A proteomic and phosphoproteomic comparison of mouse spermatogonial stem cells and progenitor spermatogonia

In this manuscript, we used an Id4-eGfp mouse line to produce the first proteomic and phosphoproteomic database for mouse spermatogonial stem cells (SSCs) and progenitor spermatogonia. Our proteomic analyses identified 8,464 proteins in spermatogonia, superseding the depth of previously published spermatogonia datasets by >2000 proteins. While the comparison of SSCs and progenitors revealed few unique proteins (18 and 3, respectively), 532 proteins exhibited significantly different abundance (FC {+/-} 1.5, p-value [≤] 0.05) between these sub-populations. Interestingly, in overlaying the proteome with transcriptomic data, correlation was poor (R2 = 0.236), re-iterating discordance between transcript and protein abundance in the testis. In our phosphoproteomic analyses, phosphosites were identified in 19.5% of proteins (3,604 total phosphosites). Unique protein phosphorylation was substantially more common than unique protein expression when comparing SSCs and progenitors, with 38 and 191 unique phosphosites identified, respectively, in addition to significant differences in abundance at 60 and 257 phosphosites. In identifying a wave of phosphorylation that accompanies the progenitor transition, we performed predictive analyses to identify three potential master kinases for follow up validation studies (PAK1, BUB1, ABL2). The inhibition of these kinases resulted in a significant reduction in the capacity for progenitor spermatogonia to differentiate, and caused elevated apoptosis and DNA damage. Finally, we explored the testis phenotype of 42 knockout mouse lines for proteins that were either differentially expressed (26) or differentially phosphorylated (15) in our dataset. Of these lines, 21 exhibited a testis phenotype in at least one of two biological replicates observed (severity score [≥] 1, compared to 0.15 for controls), with increased numbers of Sertoli-only tubules being evident in four of these lines. This manuscript provides a comprehensive roadmap to understand the multifaceted layers of regulation over fate decisions in undifferentiated spermatogonia. These data have been provided in an accessible platform via our ShinySpermatogoniaCells app to encourage future progress in the field.

cell biology↗

bioRxiv2026

Decoding human sperm signalling: Phosphoproteomic discovery of kinases governing fertilization competency

Capacitation, the process whereby sperm gain the functional competence to fertilize an egg in the absence of de novo transcription and translation, is orchestrated by a hierarchy of kinases driving the phosphorylation of sperm proteins. While increased phosphorylation, in particular tyrosine phosphorylation, is a revered hallmark of fertilization competency in our species, only a limited repository of phosphorylated substrates and kinases have ever been reported from human sperm. To broaden therapeutic targets for sperm targeted contraceptives and infertility therapies, we adapted a contemporary phosphoproteomic technique termed EasyPhos to generate bespoke methodology for the investigation of human sperm signalling. This approach yielded high depth phosphoproteomes of non-capacitated and capacitated human spermatozoa with in silico investigation of the phosphosites revealing 52 kinases with previously uncharacterized roles in sperm capacitation. Investigating the function of the putative sperm capacitation kinases identified yielded several kinases with novel roles in the regulation of sperm function. Of particular interest, polo like kinase 1 (PLK1) inhibition significantly reduced progressive sperm motility, attenuated capacitation-associated tyrosine phosphorylation and reduced the sperm acrosome reaction, an essential step to achieve fertilization. These findings reveal extensive phosphoproteome remodelling during human sperm capacitation, expanding the landscape of molecular targets for fertility control.

Cell Biology↗

bioRxiv2025

Developing a nuclear transplantation model for Australian amphibian conservation using a common species of Australian Frog (Limnodynastes peronii)

As amphibian populations continue to decline, there is a need to establish procedures to store and recover genetic diversity. Nuclear transplantation (NT) is a potentially useful reproductive technology with applications to conservation in specific circumstances. Although sperm have been successfully cryopreserved and used in in vitro fertilization to produce sexually mature offspring, there are many endangered and even extinct amphibian species that only exist as stored frozen adult or larval tissues. The only way to recover that genetic diversity is through NT. Nuclear transplantation can introduce new genes into captive breeding programs from individuals where no viable gametes from that individual have been cryopreserved. Amphibian NT research was common during the middle to late 20th century; however, it was never conducted for amphibian conservation. Early amphibian NT experiments were conducted for the purpose of determining whether differentiation was a terminal process that involved the loss of reprogramming genes. However, following the success of mammalian NT, amphibian NT research declined, and the field has been at a standstill for decades. This study developed the first nuclear transplantation protocol for a native Australia frog using Limnodynastes peronii, as a model. Specifically, this paper aimed to test: 1) nuclear transplantation with fresh embryonic cells in Limnodynastes peronii and trial the implementation of different egg enucleation methods (pricked eggs only, UV only, and pricked + UV), 2) use of cryopreserved cells in nuclear transplantation and 3) use single nucleotide polymorphisms to determine if the nuclear genomes of nuclear transplant embryos were derived from the injected donor cell nuclei. With both fresh and cryopreserved embryonic cells as donor cell nuclei, development of the nuclear transplant embryos was low compared to the fertilized controls. Only one nuclear transplant embryo derived from a fresh embryonic cell (injected into a UV only enucleated egg) developed into a tadpole. This attained stage 36 of the Gosner Staging System for Anurans (hind limbs) before dying. As an egg activation technique, pricking the eggs resulted in higher cleavage rates and development to blastula than UV only and pricked + UV nuclear transplants (in trials with cryopreserved cells), but none of the nuclear transplants from the pricked only group developed beyond blastula. None of the nuclear transplants derived from cryopreserved embryonic cells developed to tadpoles. The most advanced stage attained from cryopreserved embryonic cells was stage 13 (neurula) from UV only eggs. Single nucleotide polymorphism (SNP) analysis testing for genotype mismatches was used to determine if the genomes of developing nuclear transplant embryos were derived from the injected donor cell nucleus. SNP analysis confirmed that two true clones were generated in this study. This is the first confirmed nuclear transplantation in any Australian native frog.

developmental biology↗

bioRxiv2025

Goldilocks conundrum explains cryoinjury in slow-cooled amphibian embryonic cells

Cryopreservation of intact fish and amphibian eggs and embryos is challenging due to the sheer size and yolk content, which prevents proper dehydration and causes lethal intracellular ice formation during cooling. Alternatively, cryopreservation of dissociated embryonic cells allows to biobank diploid genomes. However, amphibian and fish embryos have cells of varying sizes throughout the developing embryo and cell size distribution remains understudied in cryopreservation. This study examined cell size effects during cryopreservation of dissociated embryonic cells from two amphibian species. Most work used Limnodynatses peronii blastula, gastrula, and neurula cells cryopreserved with 10% dimethyl sulfoxide (DMSO) and sucrose at 0%, 1%, or 10%. Increasing sucrose concentration improved post-thaw recovery of membrane intact cells and cell concentrations, with gastrula and neurula cells having better recovery than blastula cells. An interaction amongst cryoprotectant concentration, cell size, and embryonic stage was observed. Higher sucrose improved recovery of larger cells, but reduced recovery of smaller cells. These results supported a "Goldilocks" model of cryoinjury, in which larger cells require more time to dehydrate adequately, while smaller cells are damaged by excessive dehydration and solute effects, implying that optimal cryoprotectant conditions differ by cell size. Based on post-thaw recovery, 10% DMSO + 10% sucrose was optimal and successfully applied to the cryopreservation of neurula cells from the threatened Rawlinsonia littlejohni, marking the first report of embryonic cell cryopreservation in a threatened amphibian. Our results demonstrated the importance of cell size effects on cryoinjury and their consideration in the application of cryopreservation for amphibian conservation. In brief: Since intact amphibian eggs and embryos cannot be cryopreserved, dissociated embryonic cell cryopreservation provides an alternative avenue for biobanking embryonic diploid genomes; however, cell size effects on cryoinjury is poorly understood and the developing amphibian embryo consists of different sized cells. We demonstrated that cell size influences cryopreservation outcomes by cryopreserving embryonic cells from three developmental stages under varying cryoprotectant concentrations.

developmental biology↗

bioRxiv2025

The functional maturation of mouse spermatozoa is underpinned by global remodeling of the cellular phosphoproteome

The functional maturation of mammalian spermatozoa is driven by modification of their intrinsic proteome as the cells transit the male (epididymal sperm maturation) and female reproductive tracts (capacitation). Here, high-resolution mass spectrometry was used to interrogate the central role that phosphoproteomic changes play in the functional remodeling of mouse spermatozoa. This strategy identified 14,586 site-specific phosphorylation events, including the phosphorylation of 573 proteins and dephosphorylation of 426 during epididymal maturation and additional phosphorylation changes in 211 proteins linked to capacitation. We identified over 300 kinases that putatively govern these events, including three novel kinases (STK33, HIPK4, and PAK1) implicated in acrosomal exocytosis. The functional relevance of these data was confirmed via the use of knockout mouse models, which demonstrated several phosphoproteins as being essential for sperm motility and fertilization capacity. These findings illustrate that large-scale phosphorylation remodeling occurs during sperm maturation with implications extending to novel means of fertility regulation. HIGHLIGHTSO_LISperm phosphoproteome is dramatically remodeled during post-testicular maturation C_LIO_LIIdentification of >14,000 site-specific phosphorylation events providing comprehensive insight into sperm cell signaling events associated with functional maturation C_LIO_LIMajor changes in the sperm phosphoproteome coincide with epididymal maturation whereas capacitation results in more modest changes C_LIO_LIIdentification of 343 novel kinases potentially important for conferring functional maturity to spermatozoa and demonstrated role for STK33, HIPK4 and PAK1 kinases C_LIO_LIKnockout mouse models of 23 genes provided in vivo validation, with loss of these proteins leading to pronounced defects in sperm motility and fertilization capacity C_LIO_LIAll data is available via our interactive ShinySpermPhospho application - https://reproproteomics.shinyapps.io/ShinySpermPhospho/ C_LI

cell biology↗

bioRxiv2025

Towards a Kingdom of Reproductive Life - the Core Sperm Proteome

Reproductive biology is often considered in three siloed research areas; humans, agriculture and wildlife. Yet, each demand solutions for treatment of subfertility, fertility biomarkers, development of assisted reproductive technologies and effective contraception. To efficiently develop solutions applicable to all species, we must improve our understanding of the common biology underpinning reproductive processes. Accordingly, we integrate proteomic data from 29 publicly available datasets (>2 TB of data) to characterize mature sperm proteomes spanning 12 vertebrate species, identifying 13,853 proteins. Although human and mouse have relatively well-annotated sperm proteomes, many non-model species rely heavily on predicted or homology-inferred identifications. Despite variation in proteome size, composition and reproductive strategies, comparative analyses revealed that vertebrates share a fundamental molecular framework essential for sperm function. A core set of 45 species-level and 135 order-level conserved proteins mapped to critical processes, including energy generation, acrosome function, as well as novel signalling pathways (BAG2 and FAT10). Knockout mouse models further validate the significance of these conserved proteins, demonstrating that their disruption impairs sperm motility and fertilization capacity. Moreover, we discovered loss-of-function variants of two additional core sperm proteins in clinical samples, linking them to severe sperm defects. Intriguingly, in-silico analysis reveals function-driven, context-dependent diversity surpassing evolutionary patterns. Collectively, these results highlight the value of integrating publicly available datasets and underscore the need for improved genome/proteome annotation in non-model species in mammals. This work provides a foundation for developing cross-species strategies to enhance fertility treatments, assisted reproductive technologies, and conservation efforts. All data is available via ShinySpermKingdom (https://reproproteomics.shinyapps.io/ShinySpermKingdom/). IN BRIEFSperm function is essential for fertility across humans, agriculture, and wildlife, yet comparative studies remain limited. This study integrates multi-species proteomic data to identify a core sperm proteome, uncovering conserved molecular pathways and validating novel sperm proteins critical for motility and fertilization.

cell biology↗

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

Sub-chronic elevation in ambient temperature drives alterations to the sperm epigenome and accelerates early embryonic development in mice

Forecasted increases in the prevalence and severity of extreme weather events accompanying changes in climatic behavior pose potential risk to the reproductive capacity of humans and animals of ecological and agricultural significance. While several studies have revealed that heat stress induced by challenges such as testicular insulation can elicit a marked negative effect on the male reproductive system, and particularly the production of spermatozoa, less is known about the immediate impact on male reproductive function following sub-chronic whole-body exposure to elevated ambient temperature. To address this knowledge gap, we exposed unrestrained male mice to heat stress conditions that emulate a heat wave (daily cycle of 8_h at 35{degrees}C followed by 16 h at 25{degrees}C) for a period of seven days. Neither the testes or epididymides of heat exposed male mice exhibited evidence of gross histological change, and similarly, spermatozoa of exposed males retained their functionality and ability to support embryonic development. However, the embryos generated from heat exposed spermatozoa experienced pronounced changes in gene expression linked to acceleration of early embryo development, aberrant blastocyst hatching and increased fetal weight. Such changes were causally associated with an altered sperm small non-coding RNA (sncRNA) profile, such that these developmental phenotypes were recapitulated by microinjection of wild-type embryos sired by control spermatozoa with RNAs extracted from heat exposed spermatozoa. Such data highlight that even a relatively modest excursion in ambient temperature can affect male reproductive function and identify the sperm sncRNA profile as a particular point of vulnerability to this imposed environmental stress. Significance StatementThe fidelity of sperm production underpins successful reproduction yet is highly vulnerable to various forms of environmental challenge, including heat stress. Despite this knowledge, we lack a complete understanding of the immediate impact on male reproduction of whole-body exposure to elevated ambient temperatures such as those encountered during a heatwave. By experimentally emulating heatwave conditions, we demonstrate that the spermatozoa of exposed male mice accumulate changes in their small RNA profile that are causally linked to pronounced changes in embryonic gene expression, accelerated pre-implantation development, aberrant blastocyst hatching, and increased fetal weight. Such data highlight that even a relatively modest alteration in ambient temperature can affect male reproductive function, demonstrating the acute sensitivity of sperm small RNAs to environmental stress.

cell biology↗

bioRxiv2023

Development of peptides for targeting cell ablation agents concurrently to the Sertoli and Leydig cell populations of the testes: an approach to non-surgical sterilization

The surgical sterilization of cats and dogs has been used to prevent their unwanted breeding for decades, but this is an expensive and invasive procedure, and often impractical in wider contexts, for example the control of feral populations. A sterilization agent that could be administered in a single injection would not only eliminate the risks imposed by surgery but also be a much more cost-effective solution to this worldwide problem. In this study, we sought to develop a targeting peptide that would selectively bind to Leydig cells of the testes. Subsequently, after covalently attaching a cell ablation agent, Auristatin, to this peptide we aimed to apply this conjugated product (LH2Auristatin) to adult male mice in vivo, both alone and together with a previously developed Sertoli cell targeting peptide (FSH2Menadione). The application of LH2Auristatin alone resulted in an increase in DNA damage, reduced mean testes weights and mean seminiferous tubule size, along with extensive germ cell apoptosis and a reduction in litter sizes. Together with FSH2Menadione there was also an increase in embryo resorptions. These promising results were observed in around a third of all treated animals. Given this variability we discuss how these reagents might be adjusted in order to increase target cell ablation and improve their efficacy as sterilization agents.

biochemistry↗

bioRxiv2023

CONDITIONALLY MUTANT ANIMAL MODEL FOR INVESTIGATING THE INVASIVE TROPHOBLAST CELL LINEAGE

Placental development involves coordinated expansion and differentiation of trophoblast cell lineages possessing specialized functions. Among the differentiated trophoblast cell lineages are invasive trophoblast cells, which exit the placenta and invade into the uterus where they restructure the uterine parenchyma and facilitate remodeling of uterine spiral arteries. The rat exhibits deep intrauterine trophoblast cell invasion, a feature shared with human placentation, and is also amenable to gene manipulation using genome editing techniques. In this investigation, we generated a conditional rat model targeting the invasive trophoblast cell lineage. Prolactin family 7, subfamily b, member 1 (Prl7b1) is uniquely and abundantly expressed in the rat invasive trophoblast cell lineage. Disruption of Prl7b1 did not adversely affect placental development. We demonstrated that the Prl7b1 locus could be effectively used to drive the expression of Cre recombinase in invasive trophoblast cells. Our rat model represents a new tool for investigating candidate genes contributing to the regulation of invasive trophoblast cells and their contributions to trophoblast-guided uterine spiral artery remodeling.

developmental 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↗

bioRxiv2022

Oocyte and cumulus cell cooperativity and metabolic plasticity under the direction of oocyte paracrine factors

Mammalian oocytes develop and mature in a mutually dependent relationship with surrounding cumulus cells. The oocyte actively regulates cumulus cell differentiation and function by secreting soluble paracrine oocyte-secreted factors (OSFs). We characterized the molecular mechanisms by which two model OSFs, cumulin and BMP15, regulate oocyte maturation and cumulus-oocyte cooperativity. Exposure to these OSFs during maturation altered the proteomic and multispectral autofluorescence profiles of both the oocyte and cumulus cells. In oocytes, cumulin significantly upregulated proteins involved in nuclear function. In cumulus cells, both OSFs elicited marked upregulation of a variety of metabolic processes (mostly anabolic), including lipid, nucleotide, and carbohydrate metabolism, while mitochondrial metabolic processes were downregulated. The mitochondrial changes were validated by functional assays confirming altered mitochondrial morphology, respiration, and content, whilst maintaining ATP homeostasis. Collectively, these data demonstrate that OSFs remodel cumulus cell metabolism during oocyte maturation in preparation for ensuing fertilization and embryonic development. HIGHLIGHTSO_LIDuring oocyte maturation, oocyte-secreted factors promote cell cooperativity between the oocyte and cumulus cells by altering the molecular composition of both cell types. C_LIO_LIOocyte-secreted factors downregulate protein catabolic processes, and upregulate DNA binding, translation, and ribosome assembly in oocytes. C_LIO_LIOocyte-secreted factors alter mitochondrial number, morphology, and function in cumulus cells. C_LIO_LIOocyte-secreted factors further enhance metabolic plasticity in cumulus cells by upregulating anabolic pathways for macromolecules and small molecule organics. C_LIO_LIThe oocyte, via oocyte-secreted factors, instructs cumulus cells to increase metabolic workload on its behalf, thereby subduing oocyte metabolism. C_LI

developmental 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↗

bioRxiv2020

IRF8 Governs Tumor-Associated Macrophage Control of T Cell Exhaustion

Tumor progression is associated with overstimulation of cytotoxic T lymphocytes (CTLs), resulting in a dysfunctional state of exhaustion. How T cell exhaustion is elicited in the tumor remains poorly understood. Here we show that tumor-associated macrophages (TAMs) present cancer cell antigen and induce CTL exhaustion through a gene expression program dependent on the transcription factor interferon regulatory factor-8 (IRF8). In a transgenic model of murine breast cancer, CTL priming was supported by IRF8-dependent dendritic cells; yet, CTL exhaustion required TAM expression of IRF8, and its ablation suppressed tumor growth. An analysis of the highly immune-infiltrated human renal cell carcinoma tumors revealed abundant TAMs that expressed IRF8 and were enriched for an IRF8 gene expression signature. The IRF8 signature co-segregated with T cell exhaustion markers and was negatively associated with long-term patient survival. Thus, CTL exhaustion is promoted by TAMs via IRF8, and this crosstalk may be disrupted in TAM-targeted therapies.

immunology↗

bioRxiv2020

TGFβ Suppresses Type 2 Immunity to Cancer

The immune system employs two distinct defense strategies against infections: pathogen elimination typified by type 1 immunity, and pathogen containment exemplified by type 2 immunity in association with tissue repair. Akin to infectious diseases, cancer progresses with cancer cell acquisition of microorganism-like behavior propagating at the expense of the host. While immunological mechanisms of cancer cell elimination are well defined, whether immune-mediated cancer cell containment can be induced is poorly understood. Here we show that ablation of transforming growth factor-{beta} receptor II (TGF{beta}RII) in CD4+ T cells promotes tumor tissue healing and halts cancer progression. Notably, the restorative response is dependent on the T helper 2 cytokine IL-4 fortifying vasculature organization that spares only proximal layers of cancer cells from hypoxia, nutrient starvation and death. Thus, type 2 immunity represents an effective cancer defense mechanism, and TGF{beta} signaling in helper T cells may be targeted for novel cancer immunotherapy.

immunology↗