Phosphoproteomic Landscaping Identifies Non-canonical cKIT Signaling in Polycythemia Vera Erythroid Progenitors

Abstract

Although stem cell factor (SCF)/cKIT interaction plays key functions in erythropoiesis, cKIT signaling in human erythroid cells is still poorly defined. To provide new insights into cKIT-mediated erythroid expansion in development and disease, we performed phosphoproteomic profiling of primary erythroid progenitors from adult blood (AB), cord blood (CB), and Polycythemia Vera (PV) at steady-state and upon SCF stimulation. While AB and CB, respectively, activated transient or sustained canonical cKIT-signaling, PV showed a non-canonical signaling including increased mTOR and ERK1 and decreased DEPTOR. Accordingly, screening of FDA-approved compounds showed increased PV sensitivity to JAK, cKIT, and MEK inhibitors. Moreover, differently from AB and CB, in PV the mature 145kDa-cKIT constitutively associated with the tetraspanin CD63 and was not endocytosed upon SCF stimulation, contributing to unrestrained cKIT signaling. These results identify a clinically exploitable variegation of cKIT signaling/metabolism that may contribute to the great erythroid output occurring during development and in PV.

Over-Expression of Centromere Protein U Participates in the Malignant Neoplastic Progression of Breast Cancer

“…This is the first report on the location of CENP-U protein both in the nucleus and cytoplasm of four breast cancer cell lines and normal epithelial cell line. Early studies about the subcellular localization of CENP-U in normal tissue showed that CENP-U is mainly distributed in the nucleus (30)(31)(32), but it remains unclear in cancer cells. In malignant cells, CENP-A or CENP-C would appear non-chromatin ectopic expression (8,37).…”

“…Instead of the CENP-U antibody, phosphate buffered saline (PBS) was used as a negative control. CENP-U is located in both cytoplasm and nucleus (30)(31)(32). The criteria for assessing positive cells were the presence of brown or dark brown yellow particles in the cytoplasm or nucleus.…”

The Glucocorticoid Receptor Polymorphism Landscape in Patients With Diamond Blackfan Anemia Reveals an Association Between Two Clinically Relevant Single Nucleotide Polymorphisms and Time to Diagnosis

“…Healthy controls (adult blood and cord blood samples) used for the CD34+ cell expansion studies were published in Federici et al (2019).…”

“…At day 10, cell numbers and viability were assessed by microscopic evaluation after trypan blue staining (Boston Bioproducts, Ashland, MA, United States), and phenotypic analysis was performed by flow-cytometry using Fluorescein Thiocyanate (FITC)-conjugated CD36 and phycoerythrin (PE)-conjugated CD235a, as described or appropriate isotype controls (all from Becton Dickinson Biosciences, Franklin Lakes, NJ, United States). Fluorescence intensities were measured with FACS ARIA (Becton Dickinson Biosciences;Falchi et al, 2015;Federici et al, 2019). The cells were then subjected to reverse-phase protein array (RPPA) analyses as described (Federici et al, 2019).…”

“…Fluorescence intensities were measured with FACS ARIA (Becton Dickinson Biosciences;Falchi et al, 2015;Federici et al, 2019). The cells were then subjected to reverse-phase protein array (RPPA) analyses as described (Federici et al, 2019). Internal standardization was performed using the software package JMP v6 (SAS Institute, Cary, NC, United States), two-way hierarchical clustering was performed with the Wards method and significance was evaluated by two-group Wilcoxon test (significance cut off p ≤ 0.05).…”