The Aurora A and B kinases are up-regulated in bone marrow-derived chronic lymphocytic leukemia cells and represent potential therapeutic targets

Abstract Abstract 359 Chromosomal translocations involving c-Myc are frequently found in high grade lymphoma and multiple myeloma. In contrast, c-Myc translocations rarely occur in low-grade lymphomas/leukemias like chronic lymphocytic leukemia (CLL), but when present they are associated with rapid disease progression and bad prognosis. Overexpression of c-myc may also be the result of increased transcription by several proto-oncogene transcription factors, including NF-kB. Mice with c-Myc de-regulation at different stages of B cell development develop either aggressive B cells lymphomas or plasma cell neoplasm. So far, no c-Myc mouse model developed low-grade lymphoma/leukemia. iMycCa mice develop an expansion of CD5+ peritoneal B1 cells, as compared with WT littermates mice. These mice have a normal life-span and very rarely develop B cell lymphoma at older age. Interestingly, in iMycCa mice mature B cells, but not plasma cells,could be rescued from apoptosis by administration of B cell-activating factor belonging to the TNF family (BAFF). To our surprise, double transgenic iMycCa/Baff-Tg (Myc/Baff) mice developed a disease resembling human CLL, with dramatically shorter mean survival than parental strains, due to early onset and rapid clonal expansion of a mature CD5+B220low B cell population. Those cells transferred the disease into Baff-Tg (Baff) mice with marked infiltration in lymphoid organs and bone marrow. Gene-expression analyses revealed that among the genes altered in Myc/Baff CD5+B220lowleukemia cells were those with known relevance to human CLL disease, including elevated anti-apoptotic Bcl2 family members. Apart from studies on individual genes, sub-network analysis was performed which showed enrichment of apoptosis-related and stress-induced gene sets in Myc/Baff CD5+CD3- leukemia cells. The NF-kB gene set, a major target downstream of BAFF signaling, was also enriched in Myc/Baff CD5+CD3- leukemia cells. We observed a continuum in levels of c-MYC mRNA in 166 samples using Affymetrix array analyses. Changes in c-Myc protein expression were confirmed by immunoblot analyses and correlated with disease progression. In accordance with the functions of c-Myc as a promoter of cell cycle progression, as well as apoptosis, we found enhanced spontaneous cell death in vitro in CLL cells expressing high levels of c-Myc, which could be abrogated by co culture with BAFF expressing nurse-like cells (NLC) or recombinant BAFF. In addition to its anti-apoptotic role, BAFF treatment of primary human CLL cells led to dramatically enhanced expression of c-Myc through the IKK/NF-kB pathway. Inhibition of the NF-kB pathway significantly reduced viability of both Myc/Baff CD5+CD3- leukemia cells and human CLL cells co-cultured with NLC. Also it significantly lowered CD5+B220low leukemia cell population in blood and spleen, and prevented the infiltration of leukemia cells into lymph nodes and bone marrow of transplanted mice. This study demonstrates a potential pathologic role for c-Myc, in the pathogenesis and progression of CLL. Disclosures: No relevant conflicts of interest to declare.

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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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