Chronic lymphocytic leukemia cells in a lymph node microenvironment depict molecular signature associated with an aggressive disease

Abstract

Chronic lymphocytic leukemia (CLL) cells survive longer in vivo than in vitro, suggesting that the tissue microenvironment provides prosurvival signals to tumor cells. Primary and secondary lymphoid tissues are involved in the pathogenesis of CLL, and the role of these tissue microenvironments has not been explored completely. To elucidate host-tumor interactions, we performed gene expression profiling (GEP) of purified CLL cells from peripheral blood (PB; n = 20), bone marrow (BM; n = 18), and lymph node (LN; n = 15) and validated key pathway genes by real-time polymerase chain reaction, immunohistochemistry and/or TCL1 trans-genic mice. Gene signatures representing several pathways critical for survival and activation of B cells were altered in CLL cells from different tissue compartments. Molecules associated with the B-cell receptor (BCR), B cell-activating factor/a proliferation-inducing ligand (BAFF/APRIL), nuclear factor (NF)-κB pathway and immune suppression signature were enriched in LN-CLL, suggesting LNs as the primary site for tumor growth. Immune suppression genes may help LN-CLL cells to modulate antigen-presenting and T-cell behavior to suppress antitumor activity. PB CLL cells overexpressed chemokine receptors, and their cognate ligands were enriched in LN and BM, suggesting that a chemokine gradient instructs B cells to migrate toward LN or BM. Of several chemokine ligands, the expression of CCL3 was associated with poor prognostic factors. The BM gene signature was enriched with antiapoptotic, cytoskeleton and adhesion molecules. Interestingly, PB cells from lymphadenopathy patients shared GEP with LN cells. In Eμ-TCL1 transgenic mice (the mouse model of the disease), a high percentage of leukemic cells from the lymphoid compartment express key BCR and NF-κB molecules. Together, our findings demonstrate that the lymphoid microenvironment promotes survival, proliferation and progression of CLL cells via chronic activation of BCR, BAFF/APRIL and NF-κB activation while suppressing the immune response.

Figure 1

Survival, proliferation and upregulation of the NF-κB–associated genes in cocultured CLL cells in vitro with human- or mouse-derived stromal cells. Analyses of cell survival, proliferation and change in expression of key genes in primary CLL cells cocultured with mouse BM-derived OMA-AD and human endothelial-derived HMEC stromal cells at 72 h. The results were analyzed using a Student t test (p < 0.05). (A) Measurement of the proliferation of CLL cells (n = 6) cocultured on stroma using Ki-67 staining. (B) Measurement of the survival of CLL cells (n > 7) cocultured on stroma using annexin V staining. (C) Validation of the upregulation of key genes in CLL cells (n = 6) cocultured on stroma using real-time PCR.

Figure 2

Unsupervised and supervised clustering of gene expression profiles of PB-CLL (n = 20), BM-CLL (n = 18) and LN-CLL (n = 15) cells in vivo from patients. (A) Unsupervised hierarchical clustering of genes obtained from CLL enriched lymphoid compartments. (B) Supervised clustering of genes of CLL cells isolated from different lymphoid compartments (p < 0.01, FDR < 0.08).

Figure 3

Mean expression of genes associated with seven major signaling pathways and validation of expression of key genes using real-time PCR. (A) Supervised cluster analyses of differentially expressed genes (p < 0.05) associated with seven major signaling pathways. The mean expressions of these significant genes are shown among PB, BM and LN cells. (B) Confirmation of the differentially expressed genes among PB-CLL, BM-CLL and LN-CLL using real-time PCR. Expression of the selected genes from each major pathway was studied by using real-time PCR. Significant differences in expression levels between PB-CLL, BM-CLL and LN-CLL were determined by using the Student t test (p values when comparing with LN cells: *p < 0.05, **p < 0.01, ^#p < 0.005, ^##p < 0.001; p values for comparison with PB is denoted with *p and comparison with BM is denoted as *b). First row: BCR signaling: SYK, BTK, ZAP70; second row: BAFF/APRIL signaling: BAFF, BCMA, TRAF2; third row: MAPK signaling: MAP2K6, CAMLG, STAT1; fourth row: PI3K/Akt pathway: PDK1, IGFBP6, AKT1; fifth row: NF-κB pathway: NF-κBIB, FCER2, CCND2; sixth row: chemokine ligands/receptors: CXCR4, CCL21, CCR7; seventh row: tolerogenic signature: CAV1, MCM3, IDH; microarray analyses had shown all genes were differentially expressed among PB-CLL (n = 20), BM-CLL (n = 18) and LN-CLL (n = 15).

Figure 4

Confirmation of expression of pSyk and p-P65 in lymphoid tissue of a TCL-1 transgenic mouse model and human patients using immunohistochemistry. Panel I: A representative immunohistochemistry of LN tissue from TCL-1 transgenic mouse. Lymphoid tissue were collected at wk 37 from the mouse and stained for B220 (A), CD5 (B), p-P65 (C), pSYK (D) and control antibody (E). Average of percent positive B220/CD5⁺ cells for pSYK and p-P65 from the transgenic mice (n = 3) is shown (F). Panel II: A representative immunohistochemistry of LN from CLL patient. LN tissue from CLL patient is stained with CD5 (A), CD19 (B), p-P65 (C), p-SYK (D) and control antibody (E). Average of percent positive CD19/CD5⁺ CLL cells for p-SYK and p-P65 from patient LNs (n = 3) is shown (F).

Figure 5

Correlation of differential gene expression with a prognostic indicator such as BLA and validation of expression of key molecules such as pSyk and CCL3 expression. Gene expression profiles of PB-CLL samples were grouped on the basis of the presence and absence of BLA. (A) Gene expression signature of PB-CLL cells from BLA patients. PB-CLL samples were grouped based on a known prognostic indicator, presence or absence of BLA, and gene expression profiles of these PB-CLL samples were correlated by using significant analysis of microarray analyses. Comparison of expression profiles between PB-CLL with BLA (n = 9) and without BLA (n = 11) are shown. (B) Comparison of the expression of p-SYK in CLL samples from patients with BLA (n = 4) and those without BLA (n = 5). (C) Expression of CCL3 transcripts in BM-CLL with poor chromosomal abnormalities (ChAb) (n = 6) versus good ChAb (n = 11) and LN-CLL with poor ChAb (n = 11) versus good ChAb (n = 3). (D) Relationship between expression of CCL3 transcripts and time to treatment among the additional 40 PB-CLL samples using the Kaplan-Meier log-rank test.

Figure 6

A hypothetical model of molecular determinants of CLL cell survival, proliferation and migration in the PB, BM and LN microenvironments. Peripheral blood: CLL cells use different NF-κB– and MAPK-associated genes for survival and proliferation. PB-CLL cells express chemokine receptors, which stimulate PB-CLL migration to the LN or BM. Lymph nodes: CLL cells attract stromal cells to form a suitable microenvironment. Here, CLL cells undergo chronic activation via the BCR, BAFF/APRIL, NF-κB, MAPK and PI3K/Akt pathways, which leads to survival and proliferation. In addition, because of expression of a tolerogenic signature in LN-CLL, the immune cells are tolerogenic and unable to identify or kill CLL cells in LNs; Bone marrow: CLL cells attract stromal cells to form a suitable microenvironment. Here, CLL cells use different NF-κB and MAPK pathway genes for survival and proliferation.