LMP student seminars: 20 January

Each week during term time, MSc and PhD candidates in the Department of Laboratory Medicine and Pathobiology present their research.

Anyone is welcome. No need to register.

Location: Medical Sciences Building, rooms 4171 or 4279, see below.

As part of the core research curriculum, students taking LMP1001/2/3: Graduate Seminars in Laboratory Medicine and Pathobiology will present their projects. Please see abstracts below.

2. Cancer, Development and Aging 

Location: MSB 4171

Dusan Pesic

• Title: Targetable gene dependencies in Ewing sarcoma subtypes
• Supervisor: Dr. Adam Shlien

Kristyna Gorospe

• Title: In vitro and in vivo modelling of drug tolerance and minimal residual disease to discover effective therapeutic combination strategies for EGFR-mutated lung cancer
• Supervisor: Dr. Ming-Sound Tsao and Dr. Kelsie Thu

1. Brain and Neuroscience

Location: MSB 4279

Ann Mansur

• Title: MEK inhibitors in neurovascular care: Towards targeted therapeutics for vascular malformations
• Supervisor: Dr. Ivan Radovanovic

Xavier Rutherford

• Title: Investigating the role of DNA damage in neurodevelopmental disorders using a patient-derived 15q13.3 microdeletion 3D forebrain model
• Supervisor: Dr. Karun Singh

Abstracts

Dusan Pesic: Targetable gene dependencies in Ewing sarcoma subtypes

Ewing sarcoma (EwS) is a bone and soft tissue cancer primarily driven by the EWSR1::ETS fusion protein, most commonly EWS::FLI1. While localized tumours have a five-year survival rate of 70-80% with multimodal treatment, the prognosis for relapsed or metastatic disease remains dismal, with survival rates below 30%. This underscores the urgent need for improved prognostic markers and innovative therapeutic strategies. Using unsupervised hierarchical clustering, we identified three distinct transcriptional subtypes of EwS: EWS::FLI1-high, neuronal-like, and muscle-like. These subtypes represent a spectrum of transcriptional programs, with variability between the EWS::FLI1-high and neuronal-like extremes observed at single-cell resolution and across commonly used pre-clinical EwS models. Subtype classification in clinical cohorts revealed significant survival differences, with neuronal-like tumours showing the poorest prognosis and the highest metastatic potential. In vivo experiments in patient-derived xenografts (PDXs) reinforced these findings, as neuronal-like tumours exhibited significantly greater metastatic potential. Additionally, functional genomics data from CRISPR-screened cell lines identified subtype-specific gene dependencies–the Fanconi anemia pathway emerged as a promising therapeutic target for EWS::FLI1-high tumours, while neuronal-like tumours showed reliance on distinct transcriptional regulators. These findings provide a framework for leveraging subtype-specific vulnerabilities to inform targeted therapeutic approaches. By integrating transcriptomic profiling with functional genomics, this study highlights the biological and clinical heterogeneity of EwS, offering novel insights into its molecular underpinnings and paving the way for personalized treatment strategies tailored to specific transcriptional subtypes.

Kristyna Gorospe: In vitro and in vivo modelling of drug tolerance and minimal residual disease to discover effective therapeutic combination strategies for EGFR-mutated lung cancer

Resistance to EGFR-targeted therapy is a major barrier to improving survival rates for non-small cell lung cancer (NSCLC) patients whose tumors have activating EGFR mutations. Resistance to EGFR tyrosine kinase inhibitors (TKIs) emerges from drug-tolerant persister cells (DTPs) that survive TKI therapy and manifest in patients as minimal residual disease, which inevitably drives fatal tumor recurrences. DTPs are a rare population of cancer cells in a reversible, slowly proliferating state that survives targeted therapy. Upon TKI withdrawal, they exit the DTP state to recommence rapid proliferation and aggressive tumor growth. Since DTPs provide a reservoir of surviving cells from which outright resistance driven by acquired genetic alterations arises, we hypothesize that discovering DTP survival mechanisms will inform vulnerabilities whose inhibition could enhance the efficacy of EGFR TKIs. However, such mechanisms are poorly understood in EGFR-mutant lung adenocarcinoma (LUAD). To address this knowledge gap, this study aims to generate robust in vitro and in vivo LUAD models of Osimertinib (Osi)-induced drug tolerance. Genetic and functional characterization of these models has the potential to identify mechanisms enabling drug tolerance that could be targeted to prevent TKI resistance from developing. To date, the response of 3 EGFR-mutant LUAD models to Osi treatment has been characterized both in vitro and in vivo [PC9, HCC4006, and 1 patient-derived xenograft (PDX)-derived cell line, X137CL]. Osi treatment of these cells increased apoptosis, but also arrested treated cells in G0-G1 phases of the cell cycle, consistent with the slow cycling nature of DTPs. Osi treatment of the xenografts formed by these lines induced strong tumor regressions and tumor replicates relapsed upon drug cessation, indicating these models are appropriate for studying DTPs in vivo. These models will be leveraged for molecular, functional, and pharmacological studies to identify shared and distinct mechanisms driving drug tolerance, in in vitro and in vivo conditions, including comparative single-cell transcriptomic profiling and pathway analysis. Candidate genes and pathways discovered will be functionally validated using CRISPR screens. Our comparative analyses of DTP versus untreated tumors will reveal novel interventions for preventing tumor relapse following TKI therapy in EGFR-mutant lung cancers.

Ann Mansur: MEK inhibitors in neurovascular care: Towards targeted therapeutics for vascular malformations

Background: Arteriovenous malformations (AVMs) are abnormal tangles of vessels that cause significant morbidity and mortality in young individuals. Sporadic AVMs harbour somatic mutations in the KRAS-MEK-ERK pathway governing cell proliferation, motility and angiogenesis. MEK inhibitors like Trametinib are oral KRAS pathway inhibitors currently approved in oncological care.

Rationale: Given their common pathophysiology, early case reports repurposed Trametinib for pediatric AVMs with clinical promise. Assessments are now required on their clinical safety and efficacy in adult populations with AVMs, alongside investigations on their molecular/cellular mechanisms of action.

Aims: We aim to: (1) explore the clinical efficacy of Trametinib in adult patients with extra- cranial AVMs and those of the central nervous system; and (2) explore the molecular and cellular effects of MEK inhibition in human-derived tissues using in-vitro co-culture models and single-cell analyses.

Methods: Towards our first aim, we prospectively followed patients with palliative AVMs for over 12 months while on Trametinib therapy. We documented changes in symptomatology, radiological response and safety outcomes. Towards our second aim, we isolated single-cell populations from resected AVMs treated with Trametinib in order to discern the effects of the drug on gene expression. Lastly, we isolated cells from resected brain AVMs and magnetically sorted for endothelial (EC) and non-endothelial (SMC) fractions. We then applied Trametinib in vitro to these cell populations in order to dissect the effects of the drug on each cell type’s morphology, protein expression, and cellular functions/interactions.

Results: Trametinib therapy afforded significant improvements in symptomatology for all patients with extra-cranial palliative AVMs. It improved upon AVM natural history and angioarchitecture in some, even bridging a patient to conventional therapy, without serious adverse events. Endothelial and supporting cells were successfully cultured and sorted from 5 brain AVMs. KRAS mutant ECs prime their supporting cells to enhance sprouting phenotype, which is attenuated by Trametinib. Naturally, the drug decreases pERK activation in both cell types, but is met with compensatory activation of parallel signaling cascades. Lastly, MEK inhibitor therapy decreases proliferation without apoptosis in both cell types. Patients taking Trametinib for palliative AVMs of the brain and spinal cord had stabilization of their AVM morphology without evidence of regression. Results from single-cell analyses are pending. Conclusion: MEK inhibition may play a role in AVM care as an adjunct therapy. It can be considered for symptomatic relief and potentially as a bridge to conventional therapy for AVMs of the body. Its clinical utility for AVMs of the brain is more restricted to stabilization. Further analyses are required to determine the drug’s effect on gene expression and its potential for combinatorial therapy approaches as part of a precision-medicine paradigm.

Xavier Rutherford: Investigating the role of DNA damage in neurodevelopmental disorders using a patient-derived 15q13.3 microdeletion 3D forebrain model

The 15q13.3 microdeletion (15q13.3del) arises from a recurrent copy number variation on human chromosome 15 and is strongly associated with neurodevelopmental disorders (NDDs), including autism spectrum disorder, epilepsy, and schizophrenia. Within the deletion locus resides several protein-coding genes, including FAN1, which encodes a nuclease critical for DNA repair. Cortical circuit dysfunction is a hallmark of NDD’s, yet the early disruptions in human cortical development driving these defects are poorly understood. Here, using 15q13.3del patient-derived forebrain assembloids, our multi-omics analyses show inhibitory neuron-specific synaptic deficits and increased DNA damage expression. By virally labelling excitatory and inhibitory neurons, I have found a population-wide decrease in inhibitory neurons. Further, I have used inhibitory-specific calcium imaging to validate that these effects translate functionally, where we see decreased inhibitory activity in our circuit model. Collectively, it can be speculated that inhibitory neurons are particularly vulnerable to impaired DNA repair during neurodevelopment, and that this disruption may drive the cortical circuit abnormalities associated with NDDs. My project aims to explore the specific role of FAN1 and DNA repair in a human NDD context. This may help identify cell type-specific mechanisms driving neural circuit dysfunction which contributes to the intellectual disability, neuropsychiatric, and epileptic phenotypes associated with the 15q13.3del.

Contact

No need to register.

Contact lmp.grad@utoronto.ca with any questions