Biology subjects

Mangadu, R.

Publications and source records attributed to Mangadu, R..

3 recordsLinked to original sources

bioRxiv2023

A Light sheet fluorescence microscopy and machine learning-based approach to investigate drug and biomarker distribution in whole organs and tumors.

Tissue clearing and Light sheet fluorescence microscopy (LSFM) provide spatial information at a subcellular resolution in intact organs and tumors which is a significant advance over tools that limit imaging to a few representative tissue sections. The spatial distribution of drugs, targets, and biomarkers can help inform relationships between exposure at the site of action, efficacy, and safety during drug discovery. We demonstrate the use of LSFM to investigate distribution of an oncolytic virus (OV) and vasculature in xenograft tumors, as well as brain A{beta} pathology in an Alzheimers disease (AD) mouse model. Machine learning-based image analysis tools developed to segment vasculature in tumors showed that random forest and deep learning methods provided superior segmentation accuracy vs intensity-based thresholding. Sub-cellular resolution enabled detection of punctate and diffuse intracellular OV distribution profiles. LSFM investigation in the brain in a TgCRND8 AD mouse model at 6.5 months of age enabled evaluation of A{beta} plaque density in different brain regions. The utility of LSFM data to support quantitative systems pharmacology (QSP) and physiology-based pharmacokinetics (PBPK) modeling to inform drug development are also discussed. In summary, we showcase how LSFM can expand our understanding of macromolecular drug and biomarker distribution to advance drug discovery and development.

cancer biology↗

bioRxiv2022

Targeted degradation of PCNA outperforms stoichiometric inhibition to result in programed cell death

Targeted protein degradation has emerged as a powerful technology - both as a biological tool and for broadening the therapeutic proteome. As tools to probe this approach on historically intractable targets, we have previously advanced biodegraders -- targeted degradation fusion constructs composed of mini-proteins/peptides linked to modified E3 ligase receptors. Herein, we gain deeper insights into the utility and potential of biodegraders, through a detailed study on Con1-SPOP, a biodegrader which rapidly degrades the potential cancer target, proliferating cell nuclear antigen (PCNA). In a variety of settings, the active biodegrader (Con1-SPOP) proved pharmacologically superior to its stoichiometric (non-degrading) inhibitor equivalent (Con1-SPOPmut). Specifically, in addition to more potent anti-proliferative effects in both 2D cell culture and 3D spheroids, PCNA degradation uniquely induced DNA damage, cell apoptosis and necrosis. Global proteomic profiling of a stable cell-line expressing Con1-SPOP under doxycycline (Dox) induction revealed that impaired mitotic division and mitochondria dysfunction is a direct consequence of PCNA degradation, effects not seen with the stoichiometric inhibitor protein. To evaluate the therapeutic potential of biodegraders, we showed that Dox-induced Con1-SPOP achieved complete tumor-growth inhibition in a xenograft model. To explore application of biodegraders as a novel therapeutic modality, modified mRNA encoding Con1-SPOP was synthesized and encapsulated into lipid nanoparticles (LNPs). The approach successfully delivered mRNA in vitro to deplete endogenous PCNA within hours of application and with nanomolar potency. Overall, our results demonstrate the utility of biodegraders as biological tools and highlight target-degradation as a more efficacious approach versus stoichiometric inhibition. Finally, once in vivo delivery and expression are optimized, biodegraders may be leveraged as an exciting therapeutic modality.

cell biology↗

bioRxiv2020

STUB1 is an intracellular checkpoint for interferon gamma sensing

Immune checkpoint blockade (ICB) leads to durable and complete tumour regression in some patients but in others gives temporary, partial or no response. Accordingly, significant efforts are underway to identify tumour-intrinsic mechanisms underlying ICB resistance. Results from a published CRISPR screen in a mouse model suggested that targeting STUB1, an E3 ligase involved in protein homeostasis, may overcome ICB resistance but the molecular basis of this effect remains unclear. Herein, we report an under-appreciated role of STUB1 to dampen the interferon gamma (IFN{gamma}) response. Genetic deletion of STUB1 increased IFNGR1 abundance on the cell surface and thus enhanced the downstream IFN{gamma} response as showed by multiple approaches including Western blotting, flow cytometry, qPCR, phospho-STAT1 assay, immunopeptidomics, proteomics, and gene expression profiling. Human prostate and breast cancer cells with STUB1 deletion were also susceptible to cytokine-induced growth inhibition. Furthermore, blockade of STUB1 protein function recapitulated the STUB1-null phenotypes. Despite these encouraging in vitro data and positive implications from clinical datasets, we did not observe in vivo benefits of inactivating Stub1 in mouse syngeneic tumour models - with or without combination with anti-PD-1 therapy. However, our findings elucidate STUB1 as a barrier to IFN{gamma} sensing, prompting further investigations to assess if broader inactivation of human STUB1 in both tumors and immune cells could overcome ICB resistance.

cancer biology↗