Investigation of FoxO3 dynamics during erythroblast development in β-thalassemia major

<div><p>The FoxO3 transcription factor is a key regulator of oxidative stress and erythroid maturation during erythropoiesis. In this study, we explored the involvement of FoxO3 in severe β-thalassemia. Using primary CD34⁺ hematopoietic progenitor cells from patients with β-thalassemia major, we successfully developed an <i>in vitro</i> model of ineffective erythropoiesis. Based on this model, FoxO3 activity was quantified in single cells using high throughput imaging flow cytometry. This study revealed a significant reduction of FoxO3 activity during the late stage of erythroblast differentiation in β-thalassemia, in contrast to erythropoiesis in normal cells that maintain persistent activation of FoxO3. In agreement with the decreased FoxO3 activity in β-thalassemia, the expression of FoxO3 target genes was also found to decrease, concurrent with elevated phosphorylation of AKT, most clearly at the late stage of erythroid differentiation. Our findings provide further evidence for the involvement of FoxO3 during terminal erythropoiesis and confirm the modulation of the PI3K/AKT pathway as a potential therapeutic strategy for β-thalassemia.</p></div

<i>In vitro</i> differentiation of CD34⁺ cells towards erythrocytes recapitulates ineffective erythropoiesis seen in β-thalassemia.

<p>(A) Imaging flow cytometry gating strategy for identifying each distinct stage of erythroblast populations. (B) Morphology of each stage of erythroblasts analyzed by imaging flow cytometry (merged fluorescence images of CD36 (blue) and propidium iodide (orange) representing cytoplasm and nucleus areas respectively), compared with the morphology observed in Giemsa-stained cytospin preparations (brightfield images). (C) Immunofluorescence images of erythroid cell surface markers CD36 (blue), CD71 (red) and GlyA (pink) in each stage of erythroblasts captured using an imaging flow cytometer. (D) Percentage of each stage of erythroblasts in normal and β-thalassemia cell populations on day 11 and 14, quantitated with imaging flow cytometry analysis. (E) Morphology of normal and β-thalassemia maturing erythroid cells at different days observed by light microscopy after Giemsa staining. (F) Imaging flow cytometry analysis of ROS levels in normal and β-thalassemia erythroblast populations on day 14 detected by staining cells with CellROX green (left panel), and percentage of CellROX-positive cells (right panel). (G) Percentage of Annexin V positive erythroblasts in normal and β-thalassemia samples on the final day of culture (day 18). The data are shown as means ± SEM. *<i>P</i> < 0.05, **<i>P</i> < 0.01 by Student’s t test.</p

Decreased FoxO3 nuclear localization during late erythroblast maturation in β-thalassemia.

<p>Imaging flow cytometry analysis with FoxO3/nucleus similarity scores of each stage of erythroblasts from normal and β-thalassemia samples on day 11 (A) and day 14 (B). (C) Analysis of similarity scores of OrthoE in normal and β-thalassemia on day 14 with representative brightfield (BF) images, fluorescence images, and similarity scores (white numbers). Summary of % FoxO3 nuclear translocation in each stage of normal and β-thalassemia erythroblasts on day 11 (D) and day 14 (E). The data from normal cells on day 11 presented in Figs 3A and D were based on those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187610#pone.0187610.g002" target="_blank">Fig 2D</a>. Data are expressed as means ± SEM. **<i>P</i> < 0.01 was calculated using the Student’s t test. Normal group, n = 5 independent experiments (from 5 different normal subjects); β-thalassemia group, n = 4 independent experiments (from 4 different patients).</p

Clinical characteristics of patients.

<p>Clinical characteristics of patients.</p

β-Thalassemia cells shows higher levels of phosphorylated AKT and FoxO3 and exhibit lower expression of FoxO3 target genes in late erythroid differentiation.

<p>(A) Expression of pAKT, total AKT, pFoxO3, and total FoxO3 in cultured cells on day 14 from normal and β-thalassemia groups analyzed by immunoblotting. β-Actin used as a protein loading control. Quantification of relative pAKT and pFoxO3 levels (Right panel). The data are presented as pAKT/AKT or pFoxO3/FoxO3 ratios and shown as means ± SEM. *<i>P</i> < 0.05, n = 3 independent experiments. (B) Relative mRNA expression levels of <i>CAT</i>, <i>SOD2</i>, <i>BIM</i>, <i>RIOK3</i>, <i>PINK1</i>, and <i>ULK1</i> in normal and β-thalassemia on day 11 and day 14 evaluated by real-time RT-PCR. <i>GAPDH</i> used as a reference gene. Data are expressed as means ± SEM. *<i>P</i> < 0.05, **<i>P</i> < 0.01 by Student’s t test.</p

DataSheet_1_Combining the SMAC mimetic LCL161 with Gemcitabine plus Cisplatin therapy inhibits and prevents the emergence of multidrug resistance in cholangiocarcinoma.pdf

Cholangiocarcinoma (CCA) is a highly lethal gastrointestinal malignancy that has one of the worst prognoses among solid tumors. The combination of Gemcitabine + Cisplatin (GEM/CIS) remains the standard first-line treatment for advanced stage CCA. However, this drug combination yields only a modest objective response rate, and in cases that initially respond to this treatment, drug resistance commonly rapidly develops. To improve the efficiency of GEM/CIS therapy for CCA, a thorough understanding of the mechanism of GEM/CIS resistance in CCA is required. To that end – in this study, we developed several acquired GEM/CIS-resistant CCA cell lines and we screened those cell lines for acquired vulnerability. The screening process revealed that subset of CCA with GEM/CIS resistance acquired vulnerability to the small-molecule second mitochondrial-derived activator of caspases (SMAC) mimetics LCL161 and Birinapant. The observed acquired vulnerability was found to be associated with upregulation of an inhibitor of apoptosis protein 2 (cIAP2), a known target of SMAC mimetics. LCL161 or cIAP2-shRNA downregulated cIAP2 and restored the sensitivity to GEM/CIS in GEM/CIS-resistant CCA cell lines and in in vivo GEM/CIS-resistant xenograft models. A strong synergic effect was observed when LCL161 was added to GEM/CIS. Interestingly, this synergism was also observed in drug-naïve CCA cell lines, xenografts, and patient-derived organoids. This triplet therapy also prevented the emergence of multidrug-resistant CCA in in vitro and in vivo models. Our findings suggest that activation of cIAP2 allows CCA to escape GEM/CIS, and that suppression of cIAP2 reestablishes the apoptotic profile of CCA, thus restoring its vulnerability to GEM/CIS. The results of this study indicate that combining the SMAC mimetic LCL161 with GEM/CIS inhibits and prevents the emergence of multidrug resistance in CCA.</p