IB-DNQ and Rucaparib dual treatment alters cell cycle regulation and DNA repair in triple negative breast cancer cells.

Background: Triple negative breast cancer (TNBC), characterized by the lack of three canonical receptors, is unresponsive to commonly used hormonal therapies. One potential TNBC-specific therapeutic target is NQO1, as it is highly expressed in many TNBC patients and lowly expressed in non-cancer tissues. DNA damage induced by NQO1 bioactivatable drugs in combination with Rucaparib-mediated inhibition of PARP1-dependent DNA repair synergistically induces cell death. Methods: To gain a better understanding of the mechanisms behind this synergistic effect, we used global proteomics, phosphoproteomics, and thermal proteome profiling to analyze changes in protein abundance, phosphorylation and protein thermal stability. Results: Very few protein abundance changes resulted from single or dual agent treatment; however, protein phosphorylation and thermal stability were impacted. Histone H2AX was among several proteins identified to have increased phosphorylation when cells were treated with the combination of IB-DNQ and Rucaparib, validating that the drugs induced persistent DNA damage. Thermal proteome profiling revealed destabilization of H2AX following combination treatment, potentially a result of the increase in phosphorylation. Kinase substrate enrichment analysis predicted altered activity for kinases involved in DNA repair and cell cycle following dual agent treatment. Further biophysical analysis of these two processes revealed alterations in SWI/SNF complex association and tubulin / p53 interactions. Conclusions: Our findings that the drugs target DNA repair and cell cycle regulation, canonical cancer treatment targets, in a way that is dependent on increased expression of a protein selectively found to be upregulated in cancers without impacting protein abundance illustrate that multi-omics methodologies are important to gain a deeper understanding of the mechanisms behind treatment induced cancer cell death.

Identification of type 2 diabetes- and obesity-associated human ß-cells using deep transfer learning.

Diabetes affects >10% of adults worldwide and is caused by impaired production or response to insulin, resulting in chronic hyperglycemia. Pancreatic islet ß-cells are the sole source of endogenous insulin and our understanding of ß-cell dysfunction and death in type 2 diabetes (T2D) is incomplete. Single-cell RNA-seq data supports heterogeneity as an important factor in ß-cell function and survival. However, it is difficult to identify which ß-cell phenotypes are critical for T2D etiology and progression. Our goal was to prioritize specific disease-related ß-cell subpopulations to better understand T2D pathogenesis and identify relevant genes for targeted therapeutics. To address this, we applied a deep transfer learning tool, DEGAS, which maps disease associations onto single-cell RNA-seq data from bulk expression data. Independent runs of DEGAS using T2D or obesity status identified distinct ß-cell subpopulations. A singular cluster of T2D-associated ß-cells was identified; however, ß-cells with high obese-DEGAS scores contained two subpopulations derived largely from either non-diabetic or T2D donors. The obesity-associated non-diabetic cells were enriched for translation and unfolded protein response genes compared to T2D cells. We selected DLK1 for validation by immunostaining in human pancreas sections from healthy and T2D donors. DLK1 was heterogeneously expressed among ß-cells and appeared depleted from T2D islets. In conclusion, DEGAS has the potential to advance our holistic understanding of the ß-cell transcriptomic phenotypes, including features that distinguish ß-cells in obese non-diabetic or lean T2D states. Future work will expand this approach to additional human islet omics datasets to reveal the complex multicellular interactions driving T2D.

Small Molecule-mediated Insulin Hypersecretion Induces Transient ER Stress Response and Loss of Beta Cell Function.

Pancreatic islet beta cells require a fine-tuned endoplasmic reticulum (ER) stress response for normal function; abnormal ER stress contributes to diabetes pathogenesis. Here, we identified a small molecule, SW016789, with time-dependent effects on beta cell ER stress and function. Acute treatment with SW016789 potentiated nutrient-induced calcium influx and insulin secretion, while chronic exposure to SW016789 transiently induced ER stress and shut down secretory function in a reversible manner. Distinct from the effects of thapsigargin, SW016789 did not affect beta cell viability or apoptosis, potentially due to a rapid induction of adaptive genes, weak signaling through the eIF2α kinase PERK, and lack of oxidative stress gene Txnip induction. We determined that SW016789 acted upstream of voltage-dependent calcium channels (VDCCs) and potentiated nutrient- but not KCl-stimulated calcium influx. Measurements of metabolomics, oxygen consumption rate, and G protein-coupled receptor signaling did not explain the potentiating effects of SW016789. In chemical cotreatment experiments, we discovered synergy between SW016789 and activators of protein kinase C and VDCCs, suggesting involvement of these pathways in the mechanism of action. Finally, chronically elevated calcium influx was required for the inhibitory impact of SW016789, as blockade of VDCCs protected human islets and MIN6 beta cells from hypersecretion-induced dysfunction. We conclude that beta cells undergoing this type of pharmacological hypersecretion have the capacity to suppress their function to mitigate ER stress and avoid apoptosis. These results have the potential to uncover beta cell ER stress mitigation factors and add support to beta cell rest strategies to preserve function.