Proteomics profiling identifies induction of caveolin-1 in chronic lymphocytic leukemia cells by bone marrow stromal cells

Abstract Contact between chronic lymphocytic leukemia (CLL) cells and accessory stromal cells in tissue microenvironments is considered to play a major role in regulating CLL cell survival and disease progression. Stromal cells of various origins and species, and variable stromal-CLL cell ratios have been used in the past to study CLL-stromal cell interactions and to assess cell-adhesion mediated drug resistance (CAM-DR). Because of the heterogeneity of the currently used in vitro systems to study CLL-MSC interactions, and the importance of these co-culture systems for development and testing of novel agents, we tested a panel of murine and human MSC lines for their capacities to support CLL cell survival and CAM-DR, using various CLL-MSC ratios and fludarabine (F-ara-A) to induce CLL cell apoptosis. We tested four murine, non-transformed MSC lines derived from bone marrow: M210B4, KUM4, ST-2 and KUSA-H1. Also, we tested three human transformed cell lines: Stroma-NKtert, derived from bone marrow and immortalized by human telomerase reverse transcriptase (hTERT), UE6E7-T2 derived from bone marrow and transformed with human papilloma viruses (HPV) E6, E7 and hTERT, and UCB408E6E7Tert33 derived from umbilical cord blood and transformed with hTERT and HPV E6, E7. CLL cells were isolated from peripheral blood of untreated patients and each cell line was tested with at least three different patients according to the following protocol: viability of CLL was tested after 24, 48 and 72 hours by flow cytometry after staining with DiOC6 and propidium iodide. The following conditions were assayed on each of the MSC lines: CLL cells in suspension culture, CLL cells in suspension culture with 10 mM F-ara-A, CLL cells in co-culture with MSC, and CLL cells in co-culture with MSC and with 10 mM F-ara-A. Firstly, we performed titration experiments in order to identify the most appropriate ratio between stromal and CLL cells, using CLL-MSC ratios of 5:1, 10:1, 20:1, 50:1 and 100:1. We found a decline in MSC-derived CLL cell protection at the highest ratio of 100:1, suggesting that ratios of 50:1 or lower provide optimal conditions for in vitro assays. Results shown in Table 1 were assayed using a 20:1 ratio and represented relative viabilities when compared to untreated controls (mean±SEM). Regarding the protective effect of different MSC, we found that all MSC lines demonstrated remarkable protection of CLL cells from spontaneous and F-ara-A-induced apoptosis. We also found that stromal cells that had round shape morphology and easily formed confluent monolayer (M210B4, KUSA-H1, Stroma-NKTert) showed more prolonged protective effect in comparison to cell lines with more spindle shaped morphology (ST-2, KUM4, UE6E7-T2). The failure of UE6E7-T2 and UCB408E6E7Tert33 to demonstrate long-term protection of CLL cells could be related to their own sensitivity to F-ara-A. In this comparative study we demonstrated that both murine and human MSC provide substantial and comparable levels of protection from spontaneous and drug-induced apoptosis. CLL:MSC ratios of 50:1 or lower can be considered ideal for co-culture experiments. Further experiments have to be done to determine the levels of MSC-derived protection in a larger series of CLL samples and in different laboratories for validation. Collectively, in these co-culture assays we can study CLL-MSC interactions and CLL drugs under more standardized conditions that may allow us to evaluate the efficacy of new treatments that target the CLL microenvironment. Time points 24 hours 48 hours 72 hours +Flu + MSC + MSC +Flu +Flu + MSC + MSC +Flu +Flu +MSC + MSC +Flu M210B4 85.2±2.4 117.2±5.0 110.5±4.9 30.8±12.6 138.1±9.5 113.0±2.2 5.2±3.1 138.1±5.1 120.4±3.4 ST-2 93.6±3.0 99.9±2.6 103.1±0.5 51.6±9.4 111.9±2.6 89.8±8.7 13.9±6.3 112.6±5.7 87.0±16.4 KUM-4 93.6±3.0 106.4±1.8 104.2±1.9 51.6±9.4 112.4±2.6 100.8±2.8 13.9±6.3 111.8±6.7 88.5±11.4 KUSA-H1 79.4±7.4 125.1±3.7 118.2±2.0 33.9±10.9 136.0±3.6 107.2±7.0 11.3±6.1 133.6±5.4 84.9±7.6 Stroma-NKTert 79.3±7.0 118.6±7.0 111.0±7.0 30.5±9.5 130.7±9.5 115.6±8.0 7.1±4.3 133.0±11.5 122.7±9.0 UE6E7-T2 79.3±7.0 113.4±3.9 109.3±3.0 30.5±9.5 118.4±4.8 85.0±7.1 7.1±4.3 119.2±6.9 51.0±10.1 UCB408 E6E7Tert33 81.5±7.2 120.2±5.4 111.8±2.7 36.7±9.4 123.7±6.3 86.7±7.7 8.5±6.7 119.7±6.1 50.8±13.0 Table 1. Flu: fludarabine (10mM/ml), MSC: marrow stromal cells

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Erythroid Differentiation Regulator 1 (ERDR1) From Nurse-Like Cells Promotes the Survival of Chronic Lymphocytic Leukemia Cells

Blood ◽

10.1182/blood.v116.21.1372.1372 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1372-1372

Author(s):

Hendrik W. Van Deventer ◽

Robert Mango ◽

Jonathan Serody

Keyword(s):

Bone Marrow ◽

Chronic Lymphocytic Leukemia ◽

Stromal Cells ◽

Stromal Cell ◽

Conflicts Of Interest ◽

Erythroid Differentiation ◽

Mesenchymal Cells ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Wild Type

Abstract Abstract 1372 Background: Chemotherapy resistance in chronic lymphocytic leukemia (CLL) can be mediated by anti-apoptotic signals produced by stromal or nurse-like cells. Developing strategies to overcome this resistance is hindered by the lack of suitable “stromal” targets responsible for these signals. We have discovered that erythroid differentiation regulator 1 (ERDR1) may be a candidate target for such a strategy. In this study, we show Erdr1 is generated by several stromal cell types including bone marrow stromal cells, fibrocytes, and nurse-like cells. Furthermore, inhibition of stroma-generated Erdr1 results in increased apoptosis of co-cultured CLL cells. Methods/Results: We initially identified Erdr1 on an Affymetrix array that compared the gene expression of wild type and CCR5-/- mice with pulmonary metastasis. The increased expression of Erdr1 in the wild type mice was particularly pronounced in the pulmonary mesenchymal cells. Therefore, these cells were transfected with one of two shRNAs (shRNA #9 or shRNA#11) and the survival of these cells was compared with mesenchymal cells transfected with a non-targeted control vector. After 15 days in culture, the control cells expanded normally; however, no significant expansion was seen in either the shRNA#9 or shRNA#11 transfected cells. These differences in cellular expansion were associated with differences in apoptosis. 21.4+1.6% of the Erdr1 knockdown cells were annexin V+ compared to 11.2+1.9% of the non-targeted control (p<0.03). Using GFP as a marker for transfection, we were also able to show that knockdown of Erdr1 increased the apoptosis of surrounding non-transfected mesenchymal cells. Thus, Erdr1 is a critical protein for the survival of stromal cells. Further analysis of the mesenchymal cell subpopulations revealed the greatest expression of Erdr1 in the CD45+, thy1.1+/− fibrocytes. When compared to CD45- fibroblasts, the fibrocytes expressed CCR5 and increased Erdr1 expression by 14.2+/−2.9 fold when treated with the CCR5 ligand CCL4. Given the similarities between fibrocytes and nurse-like cells, we went on to measure the effect of Erdr1 inhibition on CLL cells. In these experiments, stable Erdr1 knockdown and control clones were selected after the transfection of the bone marrow stromal cell line M2-10B4. These clones were then co-cultured with primary CLL cells. At 96 hours, leukemia cells co-cultured with the control lines had expanded by 1.33 + 0.9 compared to 0.74 + 0.22 fold in the knock-down lines (p<0.03). As before, the lack of cellular expansion was associated with an increase in apoptosis. To further show the relevance of these findings to CLL, we demonstrated that human fibrocytes and nurse-like cells expressed mRNA and protein for ERDR1 in all patient samples tested. Implications for the treatment of human disease: Our data demonstrate that ERDR1 is a critically important protein for the survival of nurse-like cells. These data suggest that targeting ERDR1 or the upstream pathway through CCR5 might be a novel approach for the treatment of CLL. Disclosures: No relevant conflicts of interest to declare.

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