Multiplexed kinase interactome profiling quantifies cellular network activity and plasticity.

Dynamic changes in protein-protein interaction (PPI) networks underlie all physiological cellular functions and drive devastating human diseases. Profiling PPI networks can, therefore, provide critical insight into disease mechanisms and identify new drug targets. Kinases are regulatory nodes in many PPI networks; yet, facile methods to systematically study kinase interactome dynamics are lacking. We describe kinobead competition and correlation analysis (kiCCA), a quantitative mass spectrometry-based chemoproteomic method for rapid and highly multiplexed profiling of endogenous kinase interactomes. Using kiCCA, we identified 1,154 PPIs of 238 kinases across 18 diverse cancer lines, quantifying context-dependent kinase interactome changes linked to cancer type, plasticity, and signaling states, thereby assembling an extensive knowledgebase for cell signaling research. We discovered drug target candidates, including an endocytic adapter-associated kinase (AAK1) complex that promotes cancer cell epithelial-mesenchymal plasticity and drug resistance. Our data demonstrate the importance of kinase interactome dynamics for cellular signaling in health and disease.

CryoGrid-PIXUL-RNA: high throughput RNA isolation platform for tissue transcript analysis.

BACKGROUND: Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in tissue multiomics analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose 96-well microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream multiomics analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such biospecimens remain, in comparison, inefficient. RESULTS: To mitigate this tissue interrogation bottleneck, we have developed a low-cost user-friendly system, CryoGrid, to catalog, cryostore and sample tissue fragments. TRIzol is widely used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. Columns are also commonly used to extract RNA but they involve many steps, are prone to human errors, and are also expensive. Both TRIzol and column protocols use test tubes. We developed a microplate PIXUL-based TRIzol-free and column-free RNA isolation protocol that uses a buffer containing proteinase K (PK buffer). We have integrated the CryoGrid system with PIXUL-based PK buffer, TRIzol, and PureLink column methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. CryoGrid-PIXUL, when integrated with either PK buffer, TRIzol or PureLink column RNA isolation protocols, yielded similar transcript profiles in frozen organs (brain, heart, kidney and liver) from a mouse model of sepsis. CONCLUSIONS: RNA isolation using the CryoGrid-PIXUL system combined with the 96-well microplate PK buffer method offers an inexpensive user-friendly high throughput workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.

Effect of high nucleated cell concentration on product viability and hematopoietic recovery in autologous transplantation.

BACKGROUND: Administering lower total product volumes with high nucleated cell (NC) concentrations may have the potential benefit of decreasing volume- and dimethyl sulfoxide (DMSO)-related patient complications, while maximizing the laboratory's freezer storage capacity. Our study is a retrospective investigation of the effect of HPC(A) products with cell concentrations greater than 3 × 108 NC/mL on clinical and product outcomes in patients undergoing autologous peripheral blood stem cell (PBSC) transplantation. STUDY DESIGN AND METHODS: A total of 113 consecutive patients with hematological malignancies who underwent autologous PBSC transplantation were included in this retrospective analysis. The primary outcomes were days to initial absolute neutrophil count (ANC) recovery and initial platelet recovery. The secondary outcomes included the storage duration, segment thaw viability, and dose of viable CD34+ cells/kg administered. RESULTS: Of 92 patients and 176 apheresis procedures, 81 patients received HPC(A) products with high NC concentration (4.1 × 108 NC/mL), and 11 patients received low NC concentration products (2.4 × 108 NC/mL). There were no observed differences in clinical outcomes with respect to ANC recovery (14 vs. 14 vs. 12 days) and platelet recovery (16 vs. 16 vs. 15 days) when very high NC (5.2 × 108 NC/mL) and high NC (4.1 × 108 NC/mL) groups were compared to the low NC group (2.4 × 108 NC/mL). CONCLUSION: Our retrospective investigation provides further supporting evidence that HPC(A) products with cell concentration greater than 3 × 108 NC/mL did not show detrimental effects on the clinical outcomes in patients undergoing autologous PBSC transplantation.