Nutlin-3a efficacy in sarcoma predicted by transcriptomic and epigenetic profiling

Nutlin-3a is a small-molecule antagonist of p53/MDM2 that is being explored as a treatment for sarcoma. In this study, we examined the molecular mechanisms underlying the sensitivity of sarcomas to Nutlin-3a. In an ex vivo tissue explant system, we found that TP53 pathway alterations (TP53 status, MDM2/MDM4 genomic amplification/mRNA overexpression, MDM2 SNP309, and TP53 SNP72) did not confer apoptotic or cytostatic responses in sarcoma tissue biopsies (n = 24). Unexpectedly, MDM2 status did not predict Nutlin-3a sensitivity. RNA sequencing revealed that the global transcriptomic profiles of these sarcomas provided a more robust prediction of apoptotic responses to Nutlin-3a. Expression profiling revealed a subset of TP53 target genes that were transactivated specifically in sarcomas that were highly sensitive to Nutlin-3a. Of these target genes, the GADD45A promoter region was shown to be hypermethylated in 82% of wild-type TP53 sarcomas that did not respond to Nutlin-3a, thereby providing mechanistic insight into the innate ability of sarcomas to resist apoptotic death following Nutlin-3a treatment. Collectively, our findings argue that the existing benchmark biomarker for MDM2 antagonist efficacy (MDM2 amplification) should not be used to predict outcome but rather global gene expression profiles and epigenetic status of sarcomas dictate their sensitivity to p53/MDM2 antagonists.Kathleen I. Pishas, Susan J. Neuhaus, Mark T. Clayer, Andreas W. Schreiber, David M. Lawrence, Michelle Perugini, Robert J. Whitfield, Gelareh Farshid, Jim Manavis, Steve Chryssidis, Bronwen J. Mayo, Rebecca C. Haycox, Kristen Ho, Michael P. Brown, Richard J. D'Andrea, Andreas Evdokiou, David M. Thomas, Jayesh Desai, David F. Callen and Paul M. Neilse

Dissecting signalling contributions of the alpha and beta subunits of the GM-CSF receptor

Normal tissue homeostasis and appropriate responses to injury and infection are dependent on cellular communication mediated by cell surface receptors that respond to extrinsic stimuli. The GM-CSF receptor was the major focus of this project. This receptor shares a common signalling subunit, β [subscript c], with the IL-3 and IL-5 receptors. The unique GM-CSF receptor α-subunit ( GMRα ) confers ligand binding specificity to the complex and is essential for GM-CSF receptor signalling, although the full complement of signalling events mediated by GMRα remains elusive. Through cloning of candidate interacting proteins, expression and co-immunoprecipitation studies, we have confirmed interactions for two proteins previously reported to interact with the GMRα, p85 and IKKβ. Additionally, we identified the Src family kinase, Lyn, as a novel direct interacting partner of GMRα and provide insights into possible roles of this kinase in initiating signalling from the GM-CSF receptor. In addition to GMRα associated events we aimed to further characterise the role of the common β [subscript c] subunit in GM-CSF mediated signalling. We utilised two classes of consitutively active β [subscript c] mutants ( extracellular or transmembrane ) which transform the bi-potential myeloid FDB1 cell line to either factor-independent growth and survival, or granulocyte-macrophage differentiation, respectively. Here we report a comprehensive biochemical analysis of signalling by these two classes of mutants in this cell line. The two activated GMR mutants displayed distinct and non-overlapping signalling capacity. In particular, expression of a mutant with a substitution in the transmembrane domain ( V449E ) selectively activated JAK / STAT5 and MAPK pathways resulting in a high level of sensitivity to JAK and MEK inhibitors. In contrast, expression of a mutant with a 37 amino acid duplication in its extracellular domain ( FI Δ ) selectively activates the PI3K / AKT and IKKβ / NFkB pathways. Cells responding to this mutant display a relative high level of sensitivity to two independent PI3K inhibitors and relative resistance to inhibition of MEK and JAK2. The non-overlapping nature of signalling by these two activated mutants suggests that there are alternative modes of receptor activation that differentially dependent on JAK2 and that act synergistically in the mature liganded cytokine receptor complex. Further detailed analysis of these mutants will facilitate the dissection of the signalling pathways involved in the GM-CSF response that mediate proliferation, survival and differentiation.Thesis (Ph.D.)--University of Adelaide, School of Medicine, 2007

#ESHREjc report: on the road to preconception and personalized counselling with machine learning models

Extract Introduction Artificial intelligence (AI) is a tool thought to revolutionize the field of reproductive medicine in the years to come. Specifically, machine learning (ML), which is a subset of AI methods used to detect patterns and make predictions based on large datasets, has been used in ART to predict implantation outcome, embryo transfer strategies or adverse outcomes for IVF treatments (for a review see, Wang et al., 2019). However, ML methods have not yet been widely adopted in medically assisted reproduction (MAR). This slow uptake could be due to a lack of in-depth interdisciplinary communication between ML experts and clinicians/embryologists, as well as some uncertainty on how ML models could be generalized for different populations. The February edition of the ESHRE Journal Club discussed a paper from Yland et al. (2022) where the authors compared different ML algorithms to predict the chance of pregnancy in couples actively trying to conceive (without undergoing MAR). By using data from the Pregnancy Study Online (PRESTO), a web-based prospective cohort study of 4133 couples in North America (Wise et al., 2015), the authors analysed 163 potential variables and used supervised ML classification algorithms to identify the strength of each variable in predicting pregnancy outcomes of couples across the fertility spectrum (infertile: <12 menstrual cycles; subfertile: within 6 menstrual cycles; and fecundability: the average probability of pregnancy per menstrual cycle). The authors were able to predict pregnancy with a discrimination as high as 71.2% and identify the variables that most consistently predicted conception. These variables included lifestyle and reproductive characteristics such as age, BMI, history of infertility, daily use of vitamins or folic acid, intercourse timing, diet quality and reduced stress. ESHRE Journal Club discussion focused on promoting interdisciplinarity among ML and reproductive medicine specialists. It hosted 50 participants on Twitter together with experts Michelle Perugini, Vajira Thambawita and authors Jennifer Yland, Yannis Paschalidis and Lauren Wise. The discussion resulted in 902 tweets and around 1 million impressions over a 24-h perio

Molecular basis of cytokine receptor activation

Cytokines are secreted soluble peptides that precisely regulate multiple cellular functions. Amongst these the GM-CSF/IL-3/IL-5 family of cytokines controls whether hematopoietic cells will survive or apoptose, proliferate, differentiate, migrate, or perform effector functions such as phagocytosis or reactive oxygen species release. Their potent and pleiotropic activities are mediated through binding to high affinity membrane receptors at surprisingly low numbers per cell. Receptor binding triggers a cascade of intracellular signaling events, including reversible phosphorylation of receptor subunits and associated signaling molecules, leading to multiple biological responses, with the prevention of apoptosis or “cell survival” being a key cellular function that underpins all others. Many chronic inflammatory diseases and a number of haematological malignancies are driven by deregulated GM-CSF, IL-3, or IL-5 cytokine receptor signaling, highlighting their importance in disease. A major step in understanding how these cytokine receptors function is to elucidate their three dimensional structure and to relate this to the many signaling pathways emanating from their receptors. We have recently solved the structure of the human GM-CSF receptor complexed to GM-CSF which revealed distinct forms of receptor assembly: a hexamer that comprises two molecules each of GM-CSF, GM-CSF receptor alpha chain and GM-CSF receptor beta chain; and an unexpected dodecamer in which two hexameric complexes associate through a novel site 4. This latter form is necessary to bring JAK2 molecules sufficiently close together to enable full receptor activation. In this review we focus on the most recent insights in cytokine receptor signaling, and in receptor assembly. The stage is now set to link distinct forms of cytokine receptor assembled structures to specific forms of cytokine receptor signaling and function. Armed with this knowledge it may be possible to map distinct cytokine receptor signaling pathways from the cell surface to the cell nucleus which may themselves become new therapeutic targets.

Signalling by the βc family of cytokines

The GM-CSF, IL-3 and IL-5 family of cytokines, also known as the βc family due to their receptors sharing the signalling subunit βc, regulates multiple biological processes such as native and adaptive immunity, inflammation, normal and malignant hemopoieis, and autoimmunity. Australian scientists played a major role in the discovery and biological characterisation of the βc cytokines and their recent work is revealing unique features of cytokine receptor assembly and signalling. Furthermore, specific antibodies have been generated to modulate their function. Characterisation of the structural and dynamic requirements for the activation of the βc receptor family and the molecular definition of downstream signalling pathways are providing new insights into cytokine receptor signalling as well as new therapeutic opportunities.

The GM-CSF receptor utilizes beta-catenin and Tcf4 to specify macrophage lineage differentiation

Granulocyte-macrophage colony stimulating factor (GM-CSF) promotes the growth, survival, differentiation and activation of normal myeloid cells and is essential for fully functional macrophage differentiation in vivo. To better understand the mechanisms by which growth factors control the balance between proliferation and self-renewal versus growth-suppression and differentiation we have used the bi-potent FDB1 myeloid cell line, which proliferates in IL-3 and differentiates to granulocytes and macrophages in response to GM-CSF. This provides a manipulable model in which to dissect the switch between growth and differentiation. We show that, in the context of signaling from an activating mutant of the GM-CSF receptor β subunit, a single intracellular tyrosine residue (Y577) mediates the granulocyte fate decision. Loss of granulocyte differentiation in a Y577F second-site mutant is accompanied by enhanced macrophage differentiation and accumulation of β-catenin together with activation of Tcf4 and other Wnt target genes. These include the known macrophage lineage inducer, Egr1. We show that forced expression of Tcf4 or a stabilised β-catenin mutant is sufficient to promote macrophage differentiation in response to GM-CSF and that GM-CSF can regulate β-catenin stability, most likely via GSK3β. Consistent with this pathway being active in primary cells we show that inhibition of GSK3β activity promotes the formation of macrophage colonies at the expense of granulocyte colonies in response to GM-CSF. This study therefore identifies a novel pathway through which growth factor receptor signaling can interact with transcriptional regulators to influence lineage choice during myeloid differentiation.Anna L. Brown, Diana G. Salerno, Teresa Sadras, Grant A. Engler, Chung H. Kok, Christopher R. Wilkinson, Saumya E. Samaraweera, Timothy J. Sadlon, Michelle Perugini, Ian D. Lewis, Thomas J. Gonda, Richard J. D'Andre