Critical realism has a reputation for requiring substantial intellectual commitment and time to gain understanding, and being difficult to operationalize as an empirical research methodology. Our paper employs the theoretical framework of 'threshold concepts' to explore these issues. We illustrate the five qualities of 'threshold concepts' using existing literature by scholars describing their journey of engagement with critical realism. We then apply the framework from three viewpoints. First, we offer the perspective of a budding critical realist scholar who transforms their understanding through reflexivity. Second, we consider the role of critical realist communities of practice, peer-to-peer and social learning. Finally, we look from the standpoint of a more experienced critical realist academic advisor or supervisor working with a student. The paper concludes by asking: what kind of learning and teaching frameworks of engagement can the critical realist community build to better support apprentice critical realists?

List of differentially expressed proteins annotated by cluster with pathway analysis in BLM cells

Background Targeted BRAF and MEK therapy (TT) and immune-checkpoint inhibitors (ICI: anti-CTLA4/anti-PD1), have revolutionised treatment for cutaneous melanoma, however both have their own limitations. To increase the frequency of durable disease control while avoiding the high-grade toxicities associated with the combination of ICI and TT treatments, clinical strategies have shifted toward optimising sequencing of these therapies. Current standard of care favours initiating treatment with ICI, followed by TT upon progression. Notably, emerging trial data suggests that switching from TT to ICI while patients are still responding can achieve outcomes comparable to ICI to TT given sequentially, underscoring the need to investigate how treatment timing and sequencing influences therapeutic efficacy and tumour immune dynamics. Method We evaluated sequencing strategies in a mouse melanoma model involving upfront combination of TT and ICI (TT+ICI) and a short term first-line therapy of either TT followed by ICI (TT→ICI) or ICI followed by TT (ICI→TT). Results No significant differences in survival were observed across treatment strategies; however, both TT+ICI and ICI→TT schedules were associated with delayed tumour growth. Analysis of the tumour immune microenvironment at the time of treatment switch revealed a more immunosuppressive landscape in response to first-line TT, marked by an increase in regulatory T cells (Treg) compared to ICI. In contrast, administering TT after ICI led to an enriched CD8+ T cell pool with a progenitor-exhausted, stem-like phenotype. Conclusion These findings suggest that ICI as a first-line therapy effectively primes the tumour immune microenvironment, enhancing the immune stimulating effects of second-line TT. This priming limits the accumulation of suppressive immune cells, ultimately leading to improved progression-free survival.

Background Resistance to BRAF and MEK inhibitors (BRAFi/MEKi) in metastatic melanoma frequently results in cross-resistance to immune checkpoint inhibitors (ICI), limiting effective treatment options. However, a subset of BRAFi/MEKi-resistant patients remains responsive to second-line ICI, suggesting heterogeneous underlying resistance mechanisms. This study aimed to explore the tumor immune microenvironment in BRAFi/MEKi-resistant melanoma to uncover factors influencing sensitivity to second-line ICI therapy.Method To investigate mechanisms underlying resistance and responsiveness to second-line ICIs, BRAFi/MEKi-resistant melanoma mouse models were used. Flow cytometry was employed to analyze immune cell populations within the tumor microenvironment, focusing on changes in CD8+T effector cells and other key immune subsets. RNA sequencing was performed to profile transcriptomic changes in resistant tumors, providing insights into the signaling pathways associated with resistance. Clinical samples from BRAFi/MEKi-resistant patients were further evaluated for correlations between immune profiles and key signaling pathways to support findings from the preclinical models.Results Using BRAFi/MEKi-resistant melanoma mouse models, we observed distinct alterations in the tumor-immune microenvironment. Tumors exhibiting resistance showed a significant increase in CD8+T effector cells following BRAFi/MEKi treatment, suggesting an immune-stimulatory response. Mechanistic analysis identified the activation of the EGFR-STAT signaling pathway as a key driver of intrinsic resistance in these models. Notably, these tumors retained sensitivity to second-line ICI therapy, contrasting with NRAS-driven BRAFi/MEKi-resistant tumors, which demonstrated cross-resistance to ICIs. Supporting these findings, clinical samples from BRAFi/MEKi-resistant patients revealed a correlation between elevated EGFR activation and higher immune scores, indicating potential sensitivity to ICI therapy in this subset of patients.Conclusion EGFR overexpression emerges as a potential predictive biomarker for responsiveness to second-line ICIs in BRAFi/MEKi-resistant melanoma. These findings underscore the need for stratified therapeutic approaches and highlight EGFR as a target for improving outcomes in ICI therapy.

Supplementary methods and Tables 1, 2, 3, 4, and 6

Abstract Melanoma, a highly metastatic skin cancer, exhibits variations in prognosis and response to therapy based on the site of metastasis. Despite the success of immunotherapy and targeted therapies in melanoma, over half of metastatic melanoma patients will experience disease progression due to therapy resistance. The heterogeneity and plasticity of melanoma cells contribute to metastatic dissemination and therapy resistance. Our aim is to determine whether distinct clones and/or their transcriptional cell states can predict the formation of tumors in various organs and assess how these clones change over time. To identify melanoma clones across different metastatic sites, NOD scid gamma (NSG) mice and C57BL/6 mice were injected subcutaneously, intravenously or intracranially with the same pool of cells of barcoded luciferase expressing YUMMER1.7 murine melanoma cells. Transduction conditions ensured that one DNA barcode integrated into cell genomes at one barcode per cell, serving as a lineage tag. Bioluminescence imaging was performed once a week to monitor tumor growth of mice injected intravenously and intracranially. Subcutaneous tumors were measured by calliper. Mice were euthanized at different time points; day 8, 15, 22, 29 post-implantation and at ethical endpoints. Tumors were harvested and DNA sequencing was performed to identify barcodes expressed by the tumor cells. All mice developed tumors, with 100% penetrance in NSG mice. However, in C57BL/6 mice, 10% of mice intravenously injected and 27% of subcutaneously implanted mice showed complete lesion regression, suggesting that the immune system may be responsible for tumor regression. Analysis of barcodes allowed us to assess the heterogeneity of melanoma tumors at different metastatic sites and their evolution over time. Lineage tracing and clonal heterogeneity will be evaluated using the state of art technology, SPLINTR (Single-cell Profiling and LINeage Tracing), enabling us to match a cells evolution with changes in transcriptional states. Barcode analysis performed before implanting the cells and, at different timepoints in subcutaneous and lung tumors showed that dominant subclones at the baseline were also dominant in subcutaneous and lung tumors in immunocompromised mice. In contrast, dominant subclones in immunocompetent mice was those present in lower proportion at baseline. Additionally, greater variability of subclones was observed especially in lung and brain tumors, across immunocompetent mice, likely as a mechanism of resistance, enabling these tumors to overcome immunoediting. Understanding the variability of clonality between different metastatic sites and over time will improve our comprehension of the role of different subclones in organ- specific metastasis and their transcriptional cell states. Citation Format: Veronica L. Aedo Lopez, Reem Saleh, Benjamin Blyth, Xin Du, Dane Vassiliadis, Katie Fennell, Davide Moi, Roberta Mazzieri, Riccardo Dolcetti, Karen E. Sheppard, Grant A. McArthur. Intra- and inter-tumoral heterogeneity of melanoma across different metastatic sites [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B001.

An assessment for learning approach is foundational for student learning. The necessity to shift teaching and learning online as a response to COVID-19 has propelled digital assessment into the mainstream within higher education institutions. User experience is a common indicator of effectiveness of technologically enhanced initiatives; however, measuring the pedagogical impact of digital assessment initiatives has not been straightforward. This paper presents a pedagogical evaluation, incorporating staff and student perspectives, of a digital assessment platform (DAP) at a large Australian metropolitan university. Data were collected using surveys, interviews, focus groups and case studies and incorporated findings from student evaluations of courses and a student- staff partnership project. Results highlight strong pedagogical reasons for continuing the digital assessment initiative, while also identifying constraints and opportunities for improvement. The insights generated through this evaluation emphasise the importance of considering technical, pedagogical and contextual factors and the interplay between these factors. In a quality assurance climate, where the demand for this type of institution-wide evaluation is increasing, this study contributes an approach that addresses some of the complexities and challenges of evaluating digital assessment initiatives in higher education. Future research is needed to understand more fully the relationships between pedagogical and contextual factors when undertaking an evaluation. Implications for practice or policy: center dot The DAP enabled the (re)design of authentic, inclusive, engaging and secure assessment tasks . center dot Educator support is critical in learning the system and creating assessments with high pedagogical merit. center dot Student familiarisation opportunities with using the DAP are beneficial. center dot The DAP's longevity within the institution is crucial considering the significant effort required of educators and students. center dot Contextual influences blur understanding of pedagogical factors.

Abstract Aberrant cell-cycle progression is characteristic of melanoma, and CDK4/6 inhibitors, such as palbociclib, are currently being tested for efficacy in this disease. Despite the promising nature of CDK4/6 inhibitors, their use as single agents in melanoma has shown limited clinical benefit. Herein, we discovered that treatment of tumor cells with palbociclib induces the phosphorylation of the mRNA translation initiation factor eIF4E. When phosphorylated, eIF4E specifically engenders the translation of mRNAs that code for proteins involved in cell survival. We hypothesized that cancer cells treated with palbociclib use upregulated phosphorylated eIF4E (phospho-eIF4E) to escape the antitumor benefits of this drug. Indeed, we found that pharmacologic or genetic disruption of MNK1/2 activity, the only known kinases for eIF4E, enhanced the ability of palbociclib to decrease clonogenic outgrowth. Moreover, a quantitative proteomics analysis of melanoma cells treated with combined MNK1/2 and CDK4/6 inhibitors showed downregulation of proteins with critical roles in cell-cycle progression and mitosis, including AURKB, TPX2, and survivin. We also observed that palbociclib-resistant breast cancer cells have higher basal levels of phospho-eIF4E, and that treatment with MNK1/2 inhibitors sensitized these palbociclib-resistant cells to CDK4/6 inhibition. In vivo we demonstrate that the combination of MNK1/2 and CDK4/6 inhibition significantly increases the overall survival of mice compared with either monotherapy. Overall, our data support MNK1/2 inhibitors as promising drugs to potentiate the antineoplastic effects of palbociclib and overcome therapy-resistant disease.

Exposure-response relationship between CX-5461 levels and rDNA transcription rate in normal PBMCs

PDF file - 155K, Screen control metrics

Abstract Background Cutaneous melanoma is a lethal form of skin cancer with morbidity and mortality rates highest amongst European, North American and Australasian populations. The developments of targeted therapies (TTs) directed at the oncogene BRAF and its downstream mediator MEK, and immune checkpoint inhibitors (ICI), have revolutionized the treatment of metastatic melanoma, improving patient outcomes. However, both TT and ICI have their limitations. Although TTs are associated with high initial response rates, these are typically short‐lived due to resistance. Conversely, although ICIs provide more durable responses, they have lower initial response rates. Due to these distinct yet complementary response profiles, it has been proposed that sequencing ICI with TT could lead to a high frequency of durable responses whilst circumventing the toxicity associated with combined ICI + TT treatment. However, several questions remain unanswered, including the mechanisms underpinning this synergy and the optimal sequencing strategy. The key to determining this is to uncover the biology of each phase of the therapeutic response. Aims and methods In this review, we show that melanoma responds to TT and ICI in three phases: early response, minimal residual disease (MRD) and disease progression. We explore the effects of ICI and TT on melanoma cells and the tumour immune microenvironment, with a particular focus on MRD which is predicted to underpin the development of acquired resistance in the third phase of response. Conclusion In doing so, we provide a new framework which may inform novel therapeutic approaches for melanoma, including optimal sequencing strategies and agents that target MRD, thereby ultimately improving clinical outcomes for patients.

Targeted and immune therapies have transformed outcomes for melanoma patients, nevertheless, significant challenges remain to maximize their clinical benefit. BRAF/MEK targeted therapy (TT) is associated with very high initial response rates that are typically short-lived, while in contrast Immune Checkpoint Inhibitors (ICI) provide more durable responses but have lower initial response rates. Hence, it has been proposed that sequencing ICIs with TT would lead to a higher frequency of durable responses. However, there are many unanswered questions regarding the choice of immunotherapy, the best sequencing strategy and the mechanism leading to this synergy. To address these questions, we have optimized a syngeneic mouse melanoma model, YUMMER1.7-PV1 that is sensitive to both TT and ICIs. Importantly, this model recapitulates the known three therapeutic response phases of targeted therapies: initial response, followed by drug tolerance and then resistance. Investigation of the tumor immune microenvironment (TIME) at these three drug-induced phases, demonstrates that the immune suppressive microenvironment present during the acquired resistant phase is primed at the drug tolerance phase. Utilizing this model, we have explored the time-dependent therapy-induced changes in both tumor cells and the TIME during various TT and ICI sequencing strategies. This information will determine the optimal sequencing strategy of targeted therapy and immunotherapy that will deliver better outcomes for melanoma patients. Citation Format: Riyaben Patel, Anna Trigos, Nicole Haynes, Emily Lelliott, Grant McArthur, Karen E. Sheppard. Sequencing of targeted- and immune-therapy: delineating time dependent changes in both melanoma cells and the immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5941.

All Supplementary Methods and associated references

PDF file - 1.8MB, Supp Fig S1. Variation in DNA copy number and gene expression among primary ovarian tumors and associated abdominal deposits Supp Fig S2. Heatmap of CNC in paired primary - relapse tumors for oncogenes/tumour suppressors and literature derived candidates Supp Fig S3. Box plots of gene expression changes of 19 literature derived candidates associated with resistance in ovarian cancer Supp Fig S4. Copy number genome in line view of primary sensitive tumor - relapse resistant ascites pairs Supp Fig S5. Genome in line view of primary resistant tumor - relapse resistant ascites pairs Supp Fig S6. Genome in line view of primary tumor - primary ascites samples Supp Fig S7. Schematic of reported mutations in LRP1B Supp Fig S8. LRP1B expression in 32 ovarian cancer and normal cell lines Supp Fig S9. LRP1B siRNA and minigene sequence targets Supp Fig S10. siRNA knockdown of LRP1B in vitro using individual siRNA constructs alters drug sensitivity in ovarian cancer cell lines, Kuramochi and JHOS3

XLS file 72K, Table describing the 300 genes whose mRNA expression were most significantly changed by vemurafenib treatment in A375 cells