Chemotherapy resistance in chronic lymphocytic leukemia (CLL) is in part mediated by anti-apoptotic signals produced by bone marrow stromal cells. Identifying these signals is the first step to overcoming this resistance. ERDR1 was initially described as an inducer of hemoglobinization. We now present evidence that it inhibits apoptosis of CLL cells.
Previously, we showed that wild type (WT) but not CCR5−/− mesenchymal cells increase pulmonary melanoma metastasis in CCR5−/− mice. This observation led us to compare gene expression in the lungs of these mice. Using an Affymetrix expression array, we found Erdr1 was differentially expressed in the wild type mice. To show that the increase in metastasis was mediated by Erdr1, we transferred WT pulmonary mesenchymal cells transfected with Erdr1 shRNA or non-targeted control shRNA. CCR5−/− mice receiving Erdr1 knockdown mesenchymal cells had a 27.3% to 37% decrease in metastasis compared to animals receiving control transfected cells (p<0.01).
One explanation for the decrease in metastasis would be a failure of the Erdr1 knockdown cells to survive in the lung. Since the knockdown and control vectors express EGFP, we were able to compare the quantity of transfected cells surviving in the lung by applying an EGFP ELISA to lung homogenates. These experiments showed no difference in the number of surviving mesenchymal cells. These results suggested Erdr1 was acting as a pro-survival factor for the melanoma cells.
Since ERDR1 expression is the highest in the bone marrow, we compared the survival of CLL cells co-cultured with control and Erdr1 knockdown cells. In these experiments, stable Erdr1 knockdown and control clones were selected after the transfection of the bone marrow stromal cell line M2-10B4. Peripheral blood samples were then collected from 10 untreated CLL patients and co-cultured with these stromal cell lines. After 72 and 96 hours, total cell counts and apoptosis were measured using Annexin V/PI. At both time points, the cell counts were higher when the CLL cells were co-cultured with control cell lines compared to Erdr1 knockdown lines (OR 1.88 ± 0.27, 2.52 ± 0.66 respectively). The increase in total cell number was associated with a decrease in the percentage of apoptotic cells (OR 0.69 ± 0.18, 0.58 ± 0.12 respectively).
Since Erdr1 was differentially expressed in WT compared to CCR5−/− mice, we considered the regulation of this gene by chemokine agonists. In these experiments, 100 ng/ml of CCL4 was added to WT and CCR5−/− PMCs and mRNA was harvested at 12, 24, and 48 hours. Using real-time PCR, we found that Erdr1 expression was increased compared to baseline in the WT mesenchymal cells by 1.33 fold ± 0.06 (p < 0.05) after 24 hours. By 48 hours, expression had increased by 3.36 fold ± 0.14 (p < 0.001). As expected, CCL4 did not increase expression of Erdr1 in CCR5−/− mesenchymal cells.
Since Erdr1 is associated with hemoglobinization, we also investigated the effect of hypoxia on Erdr1 expression. In these experiments, deferoxamine was added to the stromal cell line M2-10B4 at varying concentrations. Significant fold increases in Erdr1 expression were seen after 48 hours of 1.43 ± 0.10 (50nM), 1.96 ± 0.08, (100nM), and 2.44 ± 0.01 (200nM).
Other investigators have shown that stromal cells such as nurse-like cells and CLL cells can interact to induce CCL4 and promote CLL cell survival. Our work suggests a novel pathway by which this may take place.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.