Biology subjects

McGlade, C. J.

Publications and source records attributed to McGlade, C. J..

7 recordsLinked to original sources

bioRxiv2026

Targeting CBL ubiquitin ligase activation to downregulate tyrosine kinase signalling

The CBL E3 ubiquitin ligase is a critical regulator of tyrosine kinase (TK) signalling. CBL activity is regulated by a feedback loop in which an active TK phosphorylates CBL, relieving its autoinhibited conformation, and allowing ubiquitination of substrates, including TKs, leading to their degradation. Binding of the Src Like Adapter Protein 2 (SLAP2) to CBL can also activate autoinhibited CBL and promote substrate ubiquitination. Using an engineered CBL mutant in the SLAP2 binding interface, termed RE CBL, that mimics activation by SLAP2 binding, we characterized the cellular functions of this CBL activation mechanism. Comparison of wildtype and RE CBL interactomes using MiniTurboID showed extensive and overlapping interaction networks, with a discrete subset of proteins, including the known substrate, epidermal growth factor receptor (EGFR), as well as endocytic factors such as EPS15, in higher abundance with RE CBL compared to wildtype. Consistent with these observations, RE CBL interacted more readily with EGFR, enhanced EGFR internalization, and attenuated downstream signalling compared to WT. In Cbl null hematopoietic cells, RE CBL expression reduced sensitivity to cytokines IL-3 and GM-CSF, and decreased activation of the Src-family kinase Lyn. Furthermore, we optimized and conducted a small molecule screen to identify a group of structurally related compounds that, like SLAP2 binding, promoted CBL activation in vitro. Together these findings provide proof of concept for targeting CBL activity to downregulate TK signalling.

biochemistry↗

bioRxiv2025

SRRM1 coordinates an alternative splicing program that promotes expression of oncogenic protein isoforms.

The alternative splicing of the adapter protein NUMB is dysregulated in multiple cancer types, regulating its functional divergence towards either tumor suppression or oncogenesis in an isoform-dependant manner. Here we utilized a NUMB exon 9 (E9) splicing reporter in a genome-wide CRISPR screen to identify splicing regulators SRRM1 and SRSF11 that promote NUMB oncogenic splicing in colorectal, lung and breast cancer cell lines. We show that SRRM1 and SRSF11 share common protein interactors, RNA targets and effects on an oncogenic splicing program which favors the expression of pro-tumorigenic isoforms. In addition to NUMB E9, SRRM1 regulates cancer-associated splicing events in genes encoding signaling proteins, transcription factors and actin cytoskeleton regulators, many of which are also developmentally regulated, including CD44, MKNK2, ECT2, DIAPH1, KAT5, TCF7L2, FOXM1 and TBX3. Loss of SRRM1 in colorectal and lung cancer cells reduces cell proliferation, colony formation and invasion capabilities, as well as expression of tumour promoters Cyclin D1, Notum, and PRDX2. Our data indicate that SRRM1 regulation of alternative splicing represents a node to target multiple properties of malignant cells, with broad effects on cellular signaling, proliferation, EMT, apoptosis resistance and stemness.

cancer biology↗

bioRxiv2025

Alternative splicing of NUMB correlates with tumor immune evasion and metabolic adaptation.

Alternative splicing of the NUMB gene at exon 9 generates protein isoforms linked to tumor growth and metastasis, but its impact on the tumor immune microenvironment remains unclear. Using RNA sequencing data from over 5,000 tumors across 16 cancer types, including breast cancer subtypes, we found that tumors with high NUMB exon 9 inclusion exhibit lower expression of immune-related genes, reduced immune cell infiltration, and decreased cytolytic activity, consistent with an immune "cold" phenotype. This pattern was consistent across multiple cancer types and breast cancer subtypes. Additionally, exon 9-high tumors showed evidence of increased oxidative phosphorylation, suggesting metabolic adaptations that support tumor progression. These findings identify NUMB exon 9 inclusion as a potential biomarker of immune evasion and highlight opportunities for patient stratification and targeted therapies based on NUMB exon 9 inclusion levels.

cancer biology↗

bioRxiv2023

Pan tissue analysis of human NUMB alternative splicing.

Alternative splicing (AS) of pre-mRNA generates multiple protein isoforms from a single gene. It is developmentally regulated and mis-regulation is associated with many diseases. Previous studies that examined AS of the cell fate determinant NUMB demonstrated that it undergoes a regulated switch in the inclusion of protein coding exons 3 and 9 during rodent development and the differentiation of human cell lines. Here we extend this work by comparing exon 9 and exon 3 inclusion levels of the human NUMB gene across different normal human tissues by analysis of RNA sequencing data deposited in the Genotype-Tissue Expression (GTEx) and the Vertebrate Alternative Splicing and Transcription (VastDB) databases. Our results support earlier studies and reveal specific Numb isoform expression patterns in previously unexamined tissue and cell types, suggesting that Numb isoform expression is regulated in the normal development of a broad range of tissue types throughout the body.

molecular biology↗

bioRxiv2021

Isoform-specific functions of Numb in breast cancer progression, metastasis and proteome remodeling.

Deregulated alternative splicing of the endocytic adaptor NUMB resulting in high expression of Exon9in (exon 9-containing) isoforms has been reported in several cancer types. However, the role of Numb isoform expression in tumor progression and the underlying mechanisms remain elusive. Here, we report greater exon 9 inclusion in multiple cancer types including all subtypes of breast cancer, and correlation of higher exon 9 inclusion in patients with worse prognosis. Deletion of Exon9in in breast cancer cells leads to reduced cell growth and a significant decrease of lung metastasis in orthotopic xenograft experiments. Quantitative mass spectrometry revealed downregulation of proteins involved in EMT and ECM organization and remodeling of the endocytic protein network in cells lacking the Exon9in Numb isoforms. Exon 9 deletion also results in reduced surface levels of ITG{beta}5, and downstream signaling to ERK and SRC, consistent with enhance lysosomal targeting mediated by the remaining Exon9sk (exon 9 skipping) Numb isoforms. SIGNIFICANCEExpression of NUMB Exon9in protein isoforms correlate with worse progression free survival, particularly in breast cancer. Our findings also reveal that Exon9in isoforms promote breast cancer progression by relieving Numb mediated down regulation of integrins and implicate Numb alternative splicing as a progression factor in multiple cancer types.

cancer biology↗

bioRxiv2020

Sleuthing biochemical evidence to elucidate unassigned electron density in a CBL/SLAP2 crystal complex

The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL E3 ubiquitin ligase to downregulate antigen, cytokine, and tyrosine kinase receptor signaling. In contrast to phospho-tyrosine dependent binding of CBL substrates through its tyrosine kinase binding domain (TKBD), CBL TKBD associates with the C-terminal tail of SLAP2 in a phospho-independent manner. To understand the distinct nature of this interaction, a purification protocol for SLAP2 in complex with CBL TKBD was established and the complex crystallized. However, determination of the complex crystal structure was hindered by apparent SLAP2 degradation during the crystallization process, such that only CBL TKBD residues could be modeled initially. Close examination of the CBL TKBD structure revealed a unique dimer interface that included two short segments of electron density of unknown origin. To elucidate which residues of SLAP2 to model in this unassigned density, a co-expression system was generated to test SLAP2 deletion mutants and define the minimal SLAP2 binding region. As well, SLAP2 degradation products were analyzed by mass spectrometry. Model building and map generation features of the Phenix software package were employed, leading to successful modeling of the C-terminal tail of SLAP2 in the unassigned electron density segments.

biochemistry↗

bioRxiv2020

Structural basis for phosphorylation independent activation of c-CBLby Src-like adaptor protein 2.

CBL is a RING type E3 ubiquitin ligase that functions as a negative regulator of tyrosine kinase signaling and loss of CBL E3 function is implicated in several forms of leukemia. The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL and are required for CBL-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling. Despite the established role of SLAP/SLAP2 in regulating CBL activity, the nature of the interaction and the mechanisms involved are not known. To understand the molecular basis of the interaction between SLAP/SLAP2 and CBL, we solved the crystal structure of CBL tyrosine kinase binding domain (TKBD) in complex with SLAP2. The carboxy-terminal region of SLAP2 adopts an -helical structure which binds in a cleft between the 4H, EF-hand, and SH2 domains of the TKBD. This SLAP2 binding site is remote from the canonical TKBD phospho-tyrosine peptide binding site but overlaps with a region important for stabilizing CBL in its autoinhibited conformation. In addition, binding of SLAP2 to CBL in vitro activates the ubiquitin ligase function of autoinhibited CBL. Disruption of the CBL/SLAP2 interface through mutagenesis demonstrated a role for this protein-protein interaction in regulation of CBL E3 ligase activity in cells. Our results reveal that SLAP2 binding to a regulatory cleft of the TKBD provides an alternative mechanism for activation of CBL ubiquitin ligase function.

molecular biology↗