Synergistic Targeting of DNA-PK and KIT Signaling Pathways in KIT Mutant Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the most common and aggressive form of acute leukemia, with a 5-year survival rate of just 24%. Over a third of all AML patients harbor activating mutations in kinases, such as the receptor tyrosine kinases FLT3 (receptor-type tyrosine-protein kinase FLT3) and KIT (mast/stem cell growth factor receptor kit). FLT3 and KIT mutations are associated with poor clinical outcomes and lower remission rates in response to standard-of-care chemotherapy. We have recently identified that the core kinase of the non-homologous end joining DNA repair pathway, DNA-PK (DNA-dependent protein kinase), is activated downstream of FLT3; and targeting DNA-PK sensitized FLT3-mutant AML cells to standard-of-care therapies. Herein, we investigated DNA-PK as a possible therapeutic vulnerability in KIT mutant AML, using isogenic FDC-P1 mouse myeloid progenitor cell lines transduced with oncogenic mutant KIT (V560G and D816V) or vector control. Targeted quantitative phosphoproteomic profiling identified phosphorylation of DNA-PK in the T2599/T2605/S2608/S2610 cluster in KIT mutant cells, indicative of DNA-PK activation. Accordingly, proliferation assays revealed that KIT mutant FDC-P1 cells were more sensitive to the DNA-PK inhibitors M3814 or NU7441, compared with empty vector controls. DNA-PK inhibition combined with inhibition of KIT signaling using the kinase inhibitors dasatinib or ibrutinib, or the protein phosphatase 2A activators FTY720 or AAL(S), led to synergistic cell death. Global phosphoproteomic analysis of KIT-D816V cells revealed that dasatinib and M3814 single-agent treatments inhibited extracellular signal-regulated kinase and AKT (RAC-alpha serine/threonine-protein kinase)/MTOR (serine/threonine-protein kinase mTOR) activity, with greater inhibition of both pathways when used in combination. Combined dasatinib and M3814 treatment also synergistically inhibited phosphorylation of the transcriptional regulators MYC and MYB. This study provides insight into the oncogenic pathways regulated by DNA-PK beyond its canonical role in DNA repair and demonstrates that DNA-PK is a promising therapeutic target for KIT mutant cancers.

Pharmaco-phosphoproteomic analysis of cancer-associated KIT mutations D816V and V560G.

The CD117 mast/stem cell growth factor receptor tyrosine kinase (KIT) is critical for haematopoiesis, melanogenesis and stem cell maintenance. KIT is commonly activated by mutation in cancers including acute myeloid leukaemia, melanoma and gastrointestinal stromal tumours (GISTs). The kinase and the juxtamembrane domains of KIT are mutation hotspots; with the kinase domain mutation D816V common in leukaemia and the juxtamembrane domain mutation V560G common in GISTs. Given the importance of mutant KIT signalling in cancer, we have conducted a proteomic and phosphoproteomic analysis of myeloid progenitor cells expressing D816V- and V560G-KIT mutants, using an FDCP1 isogenic cell line model. Proteomic analysis revealed increased abundance of proteases and growth signalling proteins in KIT-mutant cells compared to empty vector (EV) controls. Pathway analysis identified increased oxidative phosphorylation in D816V- and V560G-mutant KIT cells, which was targetable using the inhibitor IACS010759. Dysregulation of RNA metabolism and cytoskeleton/adhesion pathways was identified in both the proteome and phosphoproteome of KIT-mutant cells. Phosphoproteome analysis further revealed active kinases such as EGFR, ERK and PKC, which were targetable using pharmacological inhibitors. This study provides a pharmaco-phosphoproteomic profile of D816V- and V560G-mutant KIT cells, which reveals novel therapeutic strategies that may be applicable to a range of cancers.

Management of patients with diffuse intrinsic pontine glioma in Australia and New Zealand: Australian and New Zealand Children's Haematology/Oncology Group position statement.

INTRODUCTION: The main mission of the Australian and New Zealand Children's Haematology and Oncology Group (ANZCHOG) is to develop and facilitate local access to the world's leading evidence-based clinical trials for all paediatric cancers, including brain tumours, as soon as practically possible. Diffuse intrinsic pontine gliomas (DIPGs) - a subset of a larger group of tumours now termed diffuse midline glioma, H3K27-altered (DMG) - are paediatric brain cancers with less than 10% survival at two years. In the absence of any proven curative therapies, significant recent advancements have been made in pre-clinical and clinical research, leading many to seek integration of novel therapies early into standard practice. Despite these innovative therapeutic approaches, DIPG remains an incurable disease for which novel surgical, imaging, diagnostic, radiation and systemic therapy approaches are needed. MAIN RECOMMENDATIONS: All patients with DIPG should be discussed in multidisciplinary neuro-oncology meetings (including pathologists, neuroradiologists, radiation oncologists, neurosurgeons, medical oncologists) at diagnosis and at relapse or progression. Radiation therapy to the involved field remains the local and international standard of care treatment. Proton therapy does not yield a superior survival outcome compared with photon therapy and patients should undergo radiation therapy with the available modality (photon or proton) at their treatment centre. Patients may receive concurrent chemotherapy or radiation-sensitising agents as part of a clinical trial. Biopsy should be offered to facilitate consideration of experimental therapies and eligibility for clinical trial participation. After radiation therapy, each patient should be managed individually with either observation or considered for enrolment on a clinical trial, if eligible, after full discussion with the family. Re-irradiation can be considered for progressive disease. CHANGES IN MANAGEMENT AS A RESULT OF THE GUIDELINE: Every child diagnosed with DIPG should be offered enrolment on a clinical trial where available. Access to investigational drugs without biological rationale outside the clinical trial setting is not supported. In case of potentially actionable target identification with molecular profiling and absence of a suitable clinical trial, rational targeted therapies can be considered through compassionate access programs.

Proteomic Dissection of the Impact of Environmental Exposures on Mouse Seminal Vesicle Function.

Seminal vesicles are an integral part of the male reproductive accessory gland system. They produce a complex array of secretions containing bioactive constituents that support gamete function and promote reproductive success, with emerging evidence suggesting these secretions are influenced by our environment. Despite their significance, the biology of seminal vesicles remains poorly defined. Here, we complete the first proteomic assessment of mouse seminal vesicles and assess the impact of the reproductive toxicant acrylamide. Mice were administered acrylamide (25 mg/kg bw/day) or control daily for five consecutive days prior to collecting seminal vesicle tissue. A total of 5013 proteins were identified in the seminal vesicle proteome with bioinformatic analyses identifying cell proliferation, protein synthesis, cellular death, and survival pathways as prominent biological processes. Secreted proteins were among the most abundant, and several proteins are linked with seminal vesicle phenotypes. Analysis of the effect of acrylamide on the seminal vesicle proteome revealed 311 differentially regulated (FC ± 1.5, p ≤ 0.05, 205 up-regulated, 106 downregulated) proteins, orthogonally validated via immunoblotting and immunohistochemistry. Pathways that initiate protein synthesis to promote cellular survival were prominent among the dysregulated pathways, and rapamycin-insensitive companion of mTOR (RICTOR, p = 6.69E-07) was a top-ranked upstream driver. Oxidative stress was implicated as contributing to protein changes, with acrylamide causing an increase in 8-OHdG in seminal vesicle epithelial cells (fivefold increase, p = 0.016) and the surrounding smooth muscle layer (twofold increase, p = 0.043). Additionally, acrylamide treatment caused a reduction in seminal vesicle secretion weight (36% reduction, p = 0.009) and total protein content (25% reduction, p = 0.017). Together these findings support the interpretation that toxicant exposure influences male accessory gland physiology and highlights the need to consider the response of all male reproductive tract tissues when interpreting the impact of environmental stressors on male reproductive function.

Proteome and secretome analysis of pancreatic cancer cells.

Pancreatic cancer is a lethal malignancy and no screening biomarker or targeted therapy is currently available. Here, we performed a shotgun proteomic label-free quantification (LFQ) to define protein changes in the cellular proteome and secretome of four pancreatic cancer cell lines (PANC1, Paca44, Paca2, and BXPC3) versus normal human pancreatic ductal epithelial cells (HPDE). In the cellular proteome and secretome, 149 and 43 proteins were dysregulated in the most cancer cell lines, respectively. Using Ingenuity Pathway Analysis (IPA), the most dysregulated signaling pathways in pancreatic cancer cells included the activation of epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), extracellular regulated kinase (ERK), and the deactivation of type-I interferon (IFN) pathways, which could promote cancer cell progression and decrease antitumor immunity. Parallel reaction monitoring (PRM) mass spectrometry was used to confirm the changes of seven regulated proteins quantified by LFQ: EGFR, growth/differentiation factor 15 (GDF15), protein-glutamine gamma-glutamyltransferase 2 (TGM2), leukemia inhibitory factor (LIF), interferon-induced GTP-binding protein Mx1 (MX1), signal transducer and activator of transcription 1 (STAT1), and serpin B5 (SERPINB5). Together, this proteomic analysis highlights protein changes associated with pancreatic cancer cells that should be further investigated as potential biomarkers or therapeutic targets.

Phospho-heavy-labeled-spiketide FAIMS stepped-CV DDA (pHASED) provides real-time phosphoproteomics data to aid in cancer drug selection.

Global high-throughput phosphoproteomic profiling is increasingly being applied to cancer specimens to identify the oncogenic signaling cascades responsible for promoting disease initiation and disease progression; pathways that are often invisible to genomics analysis. Hence, phosphoproteomic profiling has enormous potential to inform and improve individualized anti-cancer treatment strategies. However, to achieve the adequate phosphoproteomic depth and coverage necessary to identify the activated, and hence, targetable kinases responsible for driving oncogenic signaling pathways, affinity phosphopeptide enrichment techniques are required and often coupled with offline high-pressure liquid chromatographic (HPLC) separation prior to nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS). These complex and time-consuming procedures, limit the utility of phosphoproteomics for the analysis of individual cancer patient specimens in real-time, and restrict phosphoproteomics to specialized laboratories often outside of the clinical setting. To address these limitations, here we have optimized a new protocol, phospho-heavy-labeled-spiketide FAIMS Stepped-CV DDA (pHASED), that employs online phosphoproteome deconvolution using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and internal phosphopeptide standards to provide accurate label-free quantitation (LFQ) data in real-time. Compared with traditional single-shot LFQ phosphoproteomics workflows, pHASED provided increased phosphoproteomic depth and coverage (phosphopeptides = 4617 pHASED, 2789 LFQ), whilst eliminating the variability associated with offline prefractionation. pHASED was optimized using tyrosine kinase inhibitor (sorafenib) resistant isogenic FLT3-mutant acute myeloid leukemia (AML) cell line models. Bioinformatic analysis identified differential activation of the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) pathway, responsible for sensing and repairing DNA damage in sorafenib-resistant AML cell line models, thereby uncovering a potential therapeutic opportunity. Herein, we have optimized a rapid, reproducible, and flexible protocol for the characterization of complex cancer phosphoproteomes in real-time, a step towards the implementation of phosphoproteomics in the clinic to aid in the selection of anti-cancer therapies for patients.

A novel role for milk fat globule-EGF factor 8 protein (MFGE8) in the mediation of mouse sperm-extracellular vesicle interactions.

Spermatozoa transition to functional maturity as they are conveyed through the epididymis, a highly specialized region of the male excurrent duct system. Owing to their transcriptionally and translationally inert state, this transformation into fertilization competent cells is driven by complex mechanisms of intercellular communication with the secretory epithelium that delineates the epididymal tubule. Chief among these mechanisms are the release of extracellular vesicles (EV), which have been implicated in the exchange of varied macromolecular cargo with spermatozoa. Here, we describe the optimization of a tractable cell culture model to study the mechanistic basis of sperm-extracellular vesicle interactions. In tandem with receptor inhibition strategies, our data demonstrate the importance of milk fat globule-EGF factor 8 (MFGE8) protein in mediating the efficient exchange of macromolecular EV cargo with mouse spermatozoa; with the MFGE8 integrin-binding Arg-Gly-Asp (RGD) tripeptide motif identified as being of particular importance. Specifically, complementary strategies involving MFGE8 RGD domain ablation, competitive RGD-peptide inhibition and antibody-masking of alpha V integrin receptors, all significantly inhibited the uptake and redistribution of EV-delivered proteins into immature mouse spermatozoa. These collective data implicate the MFGE8 ligand and its cognate integrin receptor in the mediation of the EV interactions that underpin sperm maturation.

Proteomic analysis of koala (phascolarctos cinereus) spermatozoa and prostatic bodies.

The aims of this study were to investigate the proteome of koala spermatozoa and that of the prostatic bodies with which they interact during ejaculation. For this purpose, spermatozoa and prostatic bodies were fractionated from the semen of four male koalas and analysed by HPLC MS/MS. This strategy identified 744 sperm and 1297 prostatic body proteins, which were subsequently attributed to 482 and 776 unique gene products, respectively. Gene ontology curation of the sperm proteome revealed an abundance of proteins mapping to the canonical sirtuin and 14-3-3 signalling pathways. By contrast, protein ubiquitination and unfolded protein response pathways dominated the equivalent analysis of proteins uniquely identified in prostatic bodies. Koala sperm proteins featured an enrichment of those mapping to the functional categories of cellular compromise/inflammatory response, whilst those of the prostatic body revealed an over-representation of molecular chaperone and stress-related proteins. Cross-species comparisons demonstrated that the koala sperm proteome displays greater conservation with that of eutherians (human; 93%) as opposed to reptile (crocodile; 39%) and avian (rooster; 27%) spermatozoa. Together, this work contributes to our overall understanding of the core sperm proteome and has identified biomarkers that may contribute to the exceptional longevity of koala spermatozoa during ex vivo storage.

Post-testicular sperm maturation in the saltwater crocodile Crocodylus porosus: assessing the temporal acquisition of sperm motility.

Conservation efforts to secure the long-term survival of crocodilian species would benefit from the establishment of a frozen sperm bank in concert with artificial breeding technologies to maintain genetic diversity among captive assurance populations. Working towards this goal, our research has focused on the saltwater crocodile Crocodylus porosus as a tractable model for understanding crocodilian sperm physiology. In extending our systematic characterisation of saltwater crocodile spermatozoa, in this study we examined the development of motility during sperm transport through the excurrent duct system of the male crocodile. The results show that approximately 20% of crocodile testicular spermatozoa are immediately motile but experience a gradient of increasing motility (percentage motile and rate of movement) as they transit the male reproductive tract (epididymis). Moreover, we confirmed that, as in ejaculated crocodile spermatozoa, increased intracellular cAMP levels promoted a significant and sustained enhancement of sperm motility regardless of whether the cells were isolated from the testis or epididymis. Along with the development of artificial reproductive technologies, this research paves the way for the opportunistic recovery, storage and potential utilisation of post-mortem spermatozoa from genetically valuable animals.

Time-resolved proteomic profiling of cigarette smoke-induced experimental chronic obstructive pulmonary disease.

BACKGROUND AND OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is the third leading cause of illness and death worldwide. Current treatments aim to control symptoms with none able to reverse disease or stop its progression. We explored the major molecular changes in COPD pathogenesis. METHODS: We employed quantitative label-based proteomics to map the changes in the lung tissue proteome of cigarette smoke-induced experimental COPD that is induced over 8 weeks and progresses over 12 weeks. RESULTS: Quantification of 7324 proteins enabled the tracking of changes to the proteome. Alterations in protein expression profiles occurred in the induction phase, with 18 and 16 protein changes at 4- and 6-week time points, compared to age-matched controls, respectively. Strikingly, 269 proteins had altered expression after 8 weeks when the hallmark pathological features of human COPD emerge, but this dropped to 27 changes at 12 weeks with disease progression. Differentially expressed proteins were validated using other mouse and human COPD bronchial biopsy samples. Major changes in RNA biosynthesis (heterogeneous nuclear ribonucleoproteins C1/C2 [HNRNPC] and RNA-binding protein Musashi homologue 2 [MSI2]) and modulators of inflammatory responses (S100A1) were notable. Mitochondrial dysfunction and changes in oxidative stress proteins also occurred. CONCLUSION: We provide a detailed proteomic profile, identifying proteins associated with the pathogenesis and disease progression of COPD establishing a platform to develop effective new treatment strategies.

Modification of Crocodile Spermatozoa Refutes the Tenet That Post-testicular Sperm Maturation Is Restricted To Mammals.

Competition to achieve paternity has contributed to the development of a multitude of elaborate male reproductive strategies. In one of the most well-studied examples, the spermatozoa of all mammalian species must undergo a series of physiological changes, termed capacitation, in the female reproductive tract before realizing their potential to fertilize an ovum. However, the evolutionary origin and adaptive advantage afforded by capacitation remains obscure. Here, we report the use of comparative and quantitative proteomics to explore the biological significance of capacitation in an ancient reptilian species, the Australian saltwater crocodile (Crocodylus porosus,). Our data reveal that exposure of crocodile spermatozoa to capacitation stimuli elicits a cascade of physiological responses that are analogous to those implicated in the functional activation of their mammalian counterparts. Indeed, among a total of 1119 proteins identified in this study, we detected 126 that were differentially phosphorylated (± 1.2 fold-change) in capacitated versus, noncapacitated crocodile spermatozoa. Notably, this subset of phosphorylated proteins shared substantial evolutionary overlap with those documented in mammalian spermatozoa, and included key elements of signal transduction, metabolic and cellular remodeling pathways. Unlike mammalian sperm, however, we noted a distinct bias for differential phosphorylation of serine (as opposed to tyrosine) residues, with this amino acid featuring as the target for ∼80% of all changes detected in capacitated spermatozoa. Overall, these results indicate that the phenomenon of sperm capacitation is unlikely to be restricted to mammals and provide a framework for understanding the molecular changes in sperm physiology necessary for fertilization.

Proteomic Profiling of Mouse Epididymosomes Reveals their Contributions to Post-testicular Sperm Maturation.

The functional maturation of spermatozoa that is necessary to achieve fertilization occurs as these cells transit through the epididymis, a highly specialized region of the male reproductive tract. A defining feature of this maturation process is that it occurs in the complete absence of nuclear gene transcription or de novo, protein translation in the spermatozoa. Rather, it is driven by sequential interactions between spermatozoa and the complex external milieu in which they are bathed within lumen of the epididymal tubule. A feature of this dynamic microenvironment are epididymosomes, small membrane encapsulated vesicles that are secreted from the epididymal soma. Herein, we report comparative proteomic profiling of epididymosomes isolated from different segments of the mouse epididymis using multiplexed tandem mass tag (TMT) based quantification coupled with high resolution LC-MS/MS. A total of 1640 epididymosome proteins were identified and quantified via this proteomic method. Notably, this analysis revealed pronounced segment-to-segment variation in the encapsulated epididymosome proteome. Thus, 146 proteins were identified as being differentially accumulated between caput and corpus epididymosomes, and a further 344 were differentially accumulated between corpus and cauda epididymosomes (i.e., fold change of ≤ -1.5 or ≥ 1.5; p, < 0.05). Application of gene ontology annotation revealed a substantial portion of the epididymosome proteins mapped to the cellular component of extracellular exosome and to the biological processes of transport, oxidation-reduction, and metabolism. Additional annotation of the subset of epididymosome proteins that have not previously been identified in exosomes revealed enrichment of categories associated with the acquisition of sperm function (e.g., fertilization and binding to the zona pellucida). In tandem with our demonstration that epididymosomes are able to convey protein cargo to the head of maturing spermatozoa, these data emphasize the fundamental importance of epididymosomes as key elements of the epididymal microenvironment responsible for coordinating post-testicular sperm maturation.

Cold Shock Domain Containing E1 (CSDE1) Protein is Overexpressed and Can be Targeted to Inhibit Invasiveness in Pancreatic Cancer Cells.

Pancreatic cancer has a dismal prognosis and to date there are no targeted therapies for this malignancy. Using shotgun proteomics, the mRNA binding protein cold shock domain containing E1 (CSDE1), also called upstream-of-N-Ras, is detected in pancreatic cancer cell lines but not in normal pancreatic epithelial cells. The expression of CSDE1 in pancreatic cancer cells is confirmed by Western blotting and immunohistochemistry of human pancreatic tumors. In vitro functional assays show that siRNA downregulation of CSDE1 or gene knockout using CRISPR-Cas9 significantly reduce the invasiveness of pancreatic cancer cells. Together, this study reveals that CSDE1 is overexpressed in pancreatic cancer and is a potential therapeutic target to inhibit pancreatic cancer cell invasion.

Mechanisms of tethering and cargo transfer during epididymosome-sperm interactions.

BACKGROUND: The mammalian epididymis is responsible for the provision of a highly specialized environment in which spermatozoa acquire functional maturity and are subsequently stored in preparation for ejaculation. Making important contributions to both processes are epididymosomes, small extracellular vesicles released from the epididymal soma via an apocrine secretory pathway. While considerable effort has been focused on defining the cargo transferred between epididymosomes and spermatozoa, comparatively less is known about the mechanistic basis of these interactions. To investigate this phenomenon, we have utilized an in vitro co-culture system to track the transfer of biotinylated protein cargo between mouse epididymosomes and recipient spermatozoa isolated from the caput epididymis; an epididymal segment that is of critical importance for promoting sperm maturation. RESULTS: Our data indicate that epididymosome-sperm interactions are initiated via tethering of the epididymosome to receptors restricted to the post-acrosomal domain of the sperm head. Thereafter, epididymosomes mediate the transfer of protein cargo to spermatozoa via a process that is dependent on dynamin, a family of mechanoenzymes that direct intercellular vesicle trafficking. Notably, upon co-culture of sperm with epididymosomes, dynamin 1 undergoes a pronounced relocation between the peri- and post-acrosomal domains of the sperm head. This repositioning of dynamin 1 is potentially mediated via its association with membrane rafts and ideally locates the enzyme to facilitate the uptake of epididymosome-borne proteins. Accordingly, disruption of membrane raft integrity or pharmacological inhibition of dynamin both potently suppress the transfer of biotinylated epididymosome proteins to spermatozoa. CONCLUSION: Together, these data provide new mechanistic insight into epididymosome-sperm interactions with potential implications extending to the manipulation of sperm maturation for the purpose of fertility regulation.

Signal Transduction in Diffuse Intrinsic Pontine Glioma.

Diffuse intrinsic pontine glioma (DIPG) is an untreatable, heterogeneous high-grade glioma (HGG) of the brainstem. This highly aggressive cancer affects mostly young children and is uniformly fatal. Genomic studies show that DIPG is driven by somatic mutations to histone H3, either H3.1 or H3.3 variants (HIST1H3B/C and H3F3A), altering the epigenetic landscape of primitive oligodendrocyte or astrocyte precursor cells of the pontine region of the brainstem. Lysine-to-methionine point mutations at amino acid 27 (H3K27M) co-occur with alterations in signaling genes, including the receptor tyrosine kinases (PDGFR/KIT/VEGFR/MET/EGFR), activin A receptor (ACVR1), intracellular kinases (PI3K/AKT/mTOR), cyclin-dependent kinases (CDKs1/4/6), transcriptional regulators (MYCN), and tumor suppressors (PTEN/TP53). This cooperation drives gene expression signatures that inhibit cellular differentiation (ID1/2, Hedgehog) and promotes malignant transformation. Unique to DIPG, is the frequency of co-occurring sets of genomic insults. However, mapping of the oncogenic signaling pathways activated in response to recurring mutations is unresolved. Herein, known oncogenic signal pathways activated in response to recurring somatic mutations and gene amplifications in DIPG are reviewed. Additionally, an important role for high-resolution quantitative proteomics/phosphoproteomics in the characterization of signaling cascades are highlighted. These regulate the cell cycle, epigenetics and anti-apoptotic processes, information critical for the development of improved treatment strategies for DIPG.

Differential cell death decisions in the testis: evidence for an exclusive window of ferroptosis in round spermatids.

Oxidative stress is a major aetiology in many pathologies, including that of male infertility. Recent evidence in somatic cells has linked oxidative stress to the induction of a novel cell death modality termed ferroptosis. However, the induction of this iron-regulated, caspase-independent cell death pathway has never been explored outside of the soma. Ferroptosis is initiated through the inactivation of the lipid repair enzyme glutathione peroxidase 4 (GPX4) and is exacerbated by the activity of arachidonate 15-lipoxygenase (ALOX15), a lipoxygenase enzyme that facilitates lipid degradation. Here, we demonstrate that male germ cells of the mouse exhibit hallmarks of ferroptosis including; a caspase-independent decline in viability following exposure to oxidative stress conditions induced by the electrophile 4-hydroxynonenal or the ferroptosis activators (erastin and RSL3), as well as a reciprocal upregulation of ALOX15 and down regulation of GPX4 protein expression. Moreover, the round spermatid developmental stage may be sensitized to ferroptosis via the action of acyl-CoA synthetase long-chain family member 4 (ACSL4), which modifies membrane lipid composition in a manner favourable to lipid peroxidation. This work provides a clear impetus to explore the contribution of ferroptosis to the demise of germline cells during periods of acute stress in in vivo models.

The Role of Reactive Oxygen Species in Acute Myeloid Leukaemia.

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with a poor overall survival. Reactive oxygen species (ROS) have been shown to be elevated in a wide range of cancers including AML. Whilst previously thought to be mere by-products of cellular metabolism, it is now clear that ROS modulate the function of signalling proteins through oxidation of critical cysteine residues. In this way, ROS have been shown to regulate normal haematopoiesis as well as promote leukaemogenesis in AML. In addition, ROS promote genomic instability by damaging DNA, which promotes chemotherapy resistance. The source of ROS in AML appears to be derived from members of the "NOX family" of NADPH oxidases. Most studies link NOX-derived ROS to activating mutations in the Fms-like tyrosine kinase 3 (FLT3) and Ras-related C3 botulinum toxin substrate (Ras). Targeting ROS through either ROS induction or ROS inhibition provides a novel therapeutic target in AML. In this review, we summarise the role of ROS in normal haematopoiesis and in AML. We also explore the current treatments that modulate ROS levels in AML and discuss emerging drug targets based on pre-clinical work.

Pharmacological inhibition of arachidonate 15-lipoxygenase protects human spermatozoa against oxidative stress.

One of the leading causes of male infertility is defective sperm function, a pathology that commonly arises from oxidative stress in the germline. Lipid peroxidation events in the sperm plasma membrane result in the generation of cytotoxic aldehydes such as 4-hydroxynonenal (4HNE), which accentuate the production of reactive oxygen species (ROS) and cause cellular damage. One of the key enzymes involved in the metabolism of polyunsaturated fatty acids to 4HNE in somatic cells is arachidonate 15-lipoxygenase (ALOX15). Although ALOX15 has yet to be characterized in human spermatozoa, our previous studies have revealed a strong link between ALOX15 activity and the levels of oxidative stress and 4HNE in mouse germ cell models. In view of these data, we sought to assess the function of ALOX15 in mature human spermatozoa and determine whether the pharmacological inhibition of this enzyme could influence the level of oxidative stress experienced by these cells. By driving oxidative stress in vitro with exogenous H2O2, our data reveal that 6,11-dihydro[1]benzothiopyrano[4,3-b]indole (PD146176; a selective ALOX15 inhibitor) was able to significantly reduce several deleterious, oxidative insults in spermatozoa. Indeed, PD146176 attenuated the production of ROS, as well as membrane lipid peroxidation and 4HNE production in human spermatozoa. Accordingly, ALOX15 inhibition also protected the functional competence of these cells to acrosome react and bind homologous human zonae pellucidae. Together, these results implicate ALOX15 in the propagation of oxidative stress cascades within human spermatozoa and offer insight into potential therapeutic avenues to address male in fertility that arises from oxidative stress.

Mouse quiescin sulfhydryl oxidases exhibit distinct epididymal luminal distribution with segment-specific sperm surface associations.

Sulfhydryl oxidation is part of the sperm maturation process essential for the acquisition of sperm fertilization competency and its structural stabilization; however, the specific sulfhydryl oxidases that fulfill these roles have yet to be identified. In this study, we investigate the potential involvement of one atypical thiol oxidase family called quiescin Q6/sulfhydryl oxidase (QSOX) using the mouse epididymis as our model system. With multidisciplinary approaches, we show that QSOX isoform 1 and 2 exhibit complementary distribution throughout the epididymal duct, but that each variant possesses distinct subcellular localization within the epididymal principal cells. While QSOX2 was exclusively present in the Golgi apparatus of the caput and corpus epididymis, QSOX1c, the most profusely express QSOX1 variant, was abundantly present in the cauda luminal fluids. Moreover, immunohistochemistry studies together with proteomic identification in isolated epididymosomes provided evidence substantiating the release of QSOX2, but not QSOX1c, via an apocrine secretory pathway. Furthermore, we demonstrate for the first time, distinct association of QSOX1c and QSOX2 with the sperm acrosome and implantation fossa, during different stages of their epididymal maturation. In conclusion, our study provides the first comprehensive comparisons between QSOX1 and QSOX2 in the mouse epididymis, revealing their distinct epididymal distribution, cellular localization, mechanisms of secretion and sperm membrane association. Together, these data suggest that QSOX1 and QSOX2 have discrete biological functions in male germ cell development.