Medical student motivation in specialised contexts.

PURPOSE: During their clinical years, medical students rotate in diverse environments, each with unique factors motivating or demotivating learning. Student motivation to learn in specialised clinical settings has not been robustly described. One framework to understand motivation to learn is self-determination theory (SDT), which posits that intrinsic motivation requires fulfilment of three innate psychological needs: competence, relatedness and autonomy. Referencing SDT, the authors aimed to understand factors influencing student motivation to learn in the specialised context of internal medicine (IM) subspecialty consult services, with the goal of optimising teaching and learning during these experiences. METHOD: Focus groups were conducted with 12 fourth-year medical students who had completed at least one inpatient IM subspecialty consult elective at the University of California, San Francisco, in 2020-2021. Students discussed factors that promoted and hindered their learning and motivation. The authors performed abductive thematic analysis using SDT as a sensitising framework. RESULTS: Three themes were identified and provided insight into how student motivation to learn can be supported: teaching at the appropriate level; integration into the team and workflow; and self-directed learning and career exploration. These themes were overlaid onto the needs of SDT, demonstrating that, in specialised clinical contexts, fulfilment of the needs is interconnected. CONCLUSION: This study provided insight into how students' innate needs can be satisfied in the learning environment of IM subspecialty consult electives, thereby promoting students' intrinsic motivation. Based on insights from the study, the authors offer recommendations for how educators can optimise student motivation to learn.

Epimorphic regeneration in the mammalian tympanic membrane.

Adult mammals are generally believed to have limited ability to regenerate complex tissues and instead, repair wounds by forming scars. In humans and across mammalian species, the tympanic membrane (TM) rapidly repairs perforations without intervention. Using mouse models, we demonstrate that the TM repairs itself through a process that bears many hallmarks of epimorphic regeneration rather than typical wound healing. Following injury, the TM forms a wound epidermis characterized by EGFR ligand expression and signaling. After the expansion of the wound epidermis that emerges from known stem cell regions of the TM, a multi-lineage blastema-like cellular mass is recruited. After two weeks, the tissue architecture of the TM is largely restored, but with disorganized collagen. In the months that follow, the organized and patterned collagen framework of the TM is restored resulting in scar-free repair. Finally, we demonstrate that deletion of Egfr in the epidermis results in failure to expand the wound epidermis, recruit the blastema-like cells, and regenerate normal TM structure. This work establishes the TM as a model of mammalian complex tissue regeneration.

Why do we not have more drugs approved for MDS? A critical viewpoint on novel drug development in MDS.

Approval of new agents to treat higher risk (HR) myelodysplastic syndrome (MDS) has stalled since the approval of DNA methyltransferase inhibitors (DNMTi). In addition, the options for patients with lower risk (LR) MDS who have high transfusion needs and do not harbor ring sideroblasts or 5q- syndrome are limited. Here, we review the current treatment landscape in MDS and identify areas of unmet need, such as treatment after failure of erythropoiesis-stimulating agents or DNMTis, TP53-mutated disease, and MDS with potentially targetable mutations. We discuss how our understanding of MDS pathogenesis can inform therapy development, including treating HR-MDS similarly to AML and pursuing therapies to address splicing factor mutations and dysregulated inflammation. We then bring a critical lens to current methodology of MDS studies and propose solutions to improve the efficiency and yield of these clinical trials, including using the most meaningful response metrics and expanding enrollment.

A Hierarchy of Proliferative and Migratory Keratinocytes Maintains the Tympanic Membrane.

The tympanic membrane (TM) is critical for hearing and requires continuous clearing of cellular debris, but little is known about homeostatic mechanisms in the TM epidermis. Using single-cell RNA sequencing, lineage tracing, whole-organ explant, and live-cell imaging, we show that homeostatic TM epidermis is distinct from other epidermal sites and has discrete proliferative zones with a three-dimensional hierarchy of multiple keratinocyte populations. TM stem cells reside in a discrete location of the superior TM and generate long-lived clones and committed progenitors (CPs). CP clones exhibit lateral migration, and their proliferative capacity is supported by Pdgfra+ fibroblasts, generating migratory but non-proliferative progeny. Single-cell sequencing of the human TM revealed similar cell types and transcriptional programming. Thus, during homeostasis, TM keratinocytes transit through a proliferative CP state and exhibit directional lateral migration. This work forms a foundation for understanding TM disorders and modeling keratinocyte biology.

Cellular Dynamics in Early Healing of Mouse Tympanic Membranes.

AIM: To better elucidate the cellular dynamics by which perforations in the tympanic membrane (TM) are healed. BACKGROUND: Under normal conditions, epidermal cells are born and then migrate radially outward from the malleus in the TM. It is unknown what the relative contribution of newly proliferated cells from different lineages is in the healing of TM perforations. METHODS: Thirty-six female mice were used in this study. Ethynyl deoxyuridine, a thymidine analogue that labels newly proliferated cells, was injected intraperitoneally into each mouse and then subsequently supplied in the drinking water. Acute perforations were performed on the right TM and the left TM served as the control and remained intact. The animals were sacrificed at six time points between 2 hours and 6 days. We stained for proliferative, epithelial, mesenchymal markers, and ethynyl deoxyuridine and analyzed the distribution of cells. RESULTS: In control TMs, newly proliferated cells were detected around the malleus handle and then migrated radially outward. Perforated TMs had a significantly higher number of newly proliferated cells throughout the tympanic membrane with a marked proliferative response of epithelial, mesenchymal, and mucosal cells in the region of the malleus and perforation. The majority of cells in the healed perforation were newly proliferated. In the anterior TM opposite the perforation, an increased turnover of keratinocytes was noted, but not mesenchymal cells. CONCLUSIONS: Perforation of the TM alters the cellular dynamics throughout the entire TM, rather than simply adjacent to the perforation. This argues for long distance signaling occurring in the perforated TM.

Targeting MTHFD2 in acute myeloid leukemia.

Drugs targeting metabolism have formed the backbone of therapy for some cancers. We sought to identify new such targets in acute myeloid leukemia (AML). The one-carbon folate pathway, specifically methylenetetrahydrofolate dehydrogenase-cyclohydrolase 2 (MTHFD2), emerged as a top candidate in our analyses. MTHFD2 is the most differentially expressed metabolic enzyme in cancer versus normal cells. Knockdown of MTHFD2 in AML cells decreased growth, induced differentiation, and impaired colony formation in primary AML blasts. In human xenograft and MLL-AF9 mouse leukemia models, MTHFD2 suppression decreased leukemia burden and prolonged survival. Based upon primary patient AML data and functional genomic screening, we determined that FLT3-ITD is a biomarker of response to MTHFD2 suppression. Mechanistically, MYC regulates the expression of MTHFD2, and MTHFD2 knockdown suppresses the TCA cycle. This study supports the therapeutic targeting of MTHFD2 in AML.

Molecular rationale for the use of PI3K/AKT/mTOR pathway inhibitors in combination with crizotinib in ALK-mutated neuroblastoma.

Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALK(F1174L) mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALK(F1174L)/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALK(F1174L)/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors.

Selective HDAC1/HDAC2 inhibitors induce neuroblastoma differentiation.

While cytotoxic chemotherapy remains the hallmark of cancer treatment, intensive regimens fall short in many malignancies, including high-risk neuroblastoma. One alternative strategy is to therapeutically promote tumor differentiation. We created a gene expression signature to measure neuroblast maturation, adapted it to a high-throughput platform, and screened a diversity oriented synthesis-generated small-molecule library for differentiation inducers. We identified BRD8430, containing a nine-membered lactam, an ortho-amino anilide functionality, and three chiral centers, as a selective class I histone deacetylase (HDAC) inhibitor (HDAC1 > 2 > 3). Further investigation demonstrated that selective HDAC1/HDAC2 inhibition using compounds or RNA interference induced differentiation and decreased viability in neuroblastoma cell lines. Combined treatment with 13-cis retinoic acid augmented these effects and enhanced activation of retinoic acid signaling. Therefore, by applying a chemical genomic screening approach, we identified selective HDAC1/HDAC2 inhibition as a strategy to induce neuroblastoma differentiation.

Targeting MYCN in neuroblastoma by BET bromodomain inhibition.

Bromodomain inhibition comprises a promising therapeutic strategy in cancer, particularly for hematologic malignancies. To date, however, genomic biomarkers to direct clinical translation have been lacking. We conducted a cell-based screen of genetically defined cancer cell lines using a prototypical inhibitor of BET bromodomains. Integration of genetic features with chemosensitivity data revealed a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. We characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent amplification of MYCN. Genome-wide expression analysis showed downregulation of the MYCN transcriptional program accompanied by suppression of MYCN transcription. Functionally, bromodomain-mediated inhibition of MYCN impaired growth and induced apoptosis in neuroblastoma. BRD4 knockdown phenocopied these effects, establishing BET bromodomains as transcriptional regulators of MYCN. BET inhibition conferred a significant survival advantage in 3 in vivo neuroblastoma models, providing a compelling rationale for developing BET bromodomain inhibitors in patients with neuroblastoma.

A general assay for monitoring the activities of protein tyrosine phosphatases in living eukaryotic cells.

Protein tyrosine phosphatases (PTPs) are key signal-transduction regulators and have emerged as potential drug targets for inhibitor design. Here we report a yeast-based assay that provides a general means of assessing the activity and/or inhibition of essentially any classical PTP in living cells. The assay uses the activity of an exogenously expressed PTP to counter the activity of a coexpressed and toxic tyrosine kinase, such that only active PTPs are capable of rescuing growth. PTP activity gives rise to both increased growth and decreased phosphotyrosine levels; cellular PTP activity can therefore be monitored by either yeast-growth curves or anti-phosphotyrosine Western blots. We show that four PTPs (TCPTP, Shp2, PEST, PTPα) are capable of rescuing the effects of v-Src toxicity. Since these PTPs are chosen from four distinct subfamilies, it is likely that biologically and medicinally important PTPs from other subfamilies can similarly function in the cellular PTP assay. Because many small-molecule PTP inhibitors fail to penetrate cell membranes effectively, this cell-based assay has the potential to serve as a useful screening tool for determining the cellular efficacy of candidate inhibitors in a more biologically relevant context than can be provided by an in vitro PTP assay.

The intersection of genetic and chemical genomic screens identifies GSK-3α as a target in human acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Long-term survival of patients with AML has changed little over the past decade, necessitating the identification and validation of new AML targets. Integration of genomic approaches with small-molecule and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. Here, we identified a role for glycogen synthase kinase 3α (GSK-3α) in AML by performing 2 independent small-molecule library screens and an shRNA screen for perturbations that induced a differentiation expression signature in AML cells. GSK-3 is a serine-threonine kinase involved in diverse cellular processes, including differentiation, signal transduction, cell cycle regulation, and proliferation. We demonstrated that specific loss of GSK-3α induced differentiation in AML by multiple measurements, including induction of gene expression signatures, morphological changes, and cell surface markers consistent with myeloid maturation. GSK-3α-specific suppression also led to impaired growth and proliferation in vitro, induction of apoptosis, loss of colony formation in methylcellulose, and anti-AML activity in vivo. Although the role of GSK-3ß has been well studied in cancer development, these studies support a role for GSK-3α in AML.