Page 131 - Haematologica-April 2018

Global mRNA changes in stroma-bound lymphoma cells
enriched gene sets from GSEA are presented in Online Supplementary Tables S6-S9.
Most functional gene clusters can be attributed to a small number of cellular themes, including B-cell activa- tion and immune cell signaling (c2, c4, c7, c10, c17), apop- tosis and anti-apoptosis (c1, c5), cell adhesion and migra- tion (c6, c8, c15, c16), and early mitosis (c19, c22, c23, c24). The first three themes contain up-regulated genes in adherent cells while the fourth cluster contains down-reg- ulated genes. Genes in the B-cell activation-related clusters included B-cell receptor components, co-stimulatory sur- face molecules and soluble factors (e.g. CD79A, CD86, CD180, ICOSLG, PDCD1 and TGFB1). The apoptosis- related clusters contained anti-apoptotic BCL2-family genes (e.g. BCL2A1, BCL2L1 and MCL1) while adhesion- and migration-related clusters included ACTB, ACTG1 and TUBA4A. The mitosis-related genes are primarily involved in early mitosis steps such as mitotic onset (e.g. AURKA, CCNB2 and PLK1), spindle establishment (e.g. BUB1, CENPA, CENPE), and kinetochore formation (e.g. KIF20A, KIF4A, KIF5A). These themes are each compati- ble with survival strategies resulting from adherence dependent changes in immune cell characteristics, apopto- sis pathways and cell cycle status. Other clusters (c9, c11, c13, c20, c21) represent more general functional character- istics, which could in principle contribute to one or more of the process-related themes. Finally, there are 4 clusters (c3, c12, c14, c18) for which fewer than 10% of member genes are differentially regulated even though they do form part of the GSEA leading edge. These clusters may be important but they contain only 11 significantly differ- entially regulated genes. For example, the c12, containing proteasome subunits, is of interest given the use of the proteasome inhibitor, bortezomib, for treatment of refrac- tory MCL.
Apart from the clustered genes there are several leading- edge genes that are not easily clustered because they occur in many functional classes (Groups i–iv, Figure 4A). Examples include NFKB1, NFKB2, ICAM1, CCL3 and CCL4 and the groups, as well as other results are detailed in the Online Supplementary Appendix. Secreted CCL3/4 levels increased in co-cultures and upon MCL-cell simula- tion with anti-IgM (Online Supplementary Figure S1).
Altogether 199 of the 1050 genes with significantly altered transcript levels (19%) were functionally classified by GSEA. Network analysis connected an additional 495 adhesion regulated genes to the 24 GSEA clusters, thus functionally connecting approximately 65% of the 1050 differentially expressed genes. Network analysis did not identify any gene networks not connected to GSEA clus- ters, strongly suggesting that we have identified the main processes and pathways defined by differential expression of genes in adherent cells.
Overlap between adhesion-regulated MCL cell genes and microenvironment-regulated genes in MCL and CLL patients
Analysis of publicly available data from two independ- ent studies of CLL and MCL30,31 identified 2090 and 4136 differentially expressed genes, respectively, between cells from lymph node and blood (FDR q-value≤0.05). Comparison of the 1050 adhesion-associated genes observed in the in vitro co-culture system with differential- ly expressed genes from the CLL and MCL datasets showed significant overlaps of 228 genes (22%,
P=2.7x10-38) and 348 genes (34%, P=8.2x10-38), respectively (Figure 4A). In all, 116 genes were differentially expressed in all three datasets (Figure 4B). Forty-eight (41%) of these genes are included in the GSEA leading edge used to define cell-adhesion-related processes and 32 (28%) of these are members of one of the 24 functional clusters (Table 2), with 29 being in clusters that primarily define the four functional themes discussed previously. Thus the 116 genes that overlap between the three studies are rep- resentative of the gene clusters defining adhesion-associ- ated gene expression. For 65 of the genes, the direction of regulation (up or down) was the same in each of the three datasets, assuming similarity between adherent cells in vitro and lymph node cells in vivo. Similarities are also indi- cated by fold change correlations for genes that are differ- entially regulated in both the in vitro co-culture system and the MCL and CLL datasets (Figure 4C and D). Although there are differences between in vivo and in vitro studies, the co-culture system faithfully reproduces a significant subset of differential gene regulation events observed in MCL and CLL patients.
Thirteen of the 65 genes with conserved direction of regulation (CCL3, CCL4, DUSP4, ETV5, ICAM1, IL15RA, IL21R, IL4I1, MFSD2A, NFKB1, NFKBIE, SEMA7A, TMEM2) had a fold-change value of 2 or more in the pres- ent study (Figure 4E). CCL3, CCL4 and NFKBIE are mem- bers of previously identified NF-κB and BCR signatures30 and are likely to represent a broader activation of NF-κB and BCR pathways (Online Supplementary Figure S2). Of the 51 genes with a different regulation direction in one of the three datasets, 43 showed a discrepancy in the in vitro data in relation to the patient studies; 38 of these genes were related to proliferation, early mitosis or mitotic spin- dle formation.
Discussion
Mantle cell lymphoma is a B-cell lymphoma that is dif- ficult to cure and patients experience frequent relapses, often resulting from minimal residual disease. As described above, adherence of lymphoma cells to stromal cells within microenvironmental niches is thought to be essential for their proliferation, survival and drug resist- ance, as well as for their immunomodulatory ability to recruit other cell types to the microenvironment.8-11,17,18
Here, we developed a co-culture model system to sys- tematically dissect differences in gene expression that occur in MCL cells and stromal cells that adhere to each other, using genome-wide RNA sequencing. A total of 1050 adherence-specific genes in MCL cells represent 24 functionally defined gene clusters, many of which can be attributed to four main functional themes. These corre- spond well with the important biological and pathological characteristics that functionally define lymphoma cells in microenvironments. The identified functions may be acquired characteristics specific to cancer cells or charac- teristics associated with normal B cells. The 1050 differen- tially regulated genes in adherent MCL cells significantly overlap with genes that are differentially expressed in lymph node, compared to blood, in MCL and CLL patients. The most differentially regulated genes included B-cell receptor signature genes that were not seen among highly regulated genes in a previous study,32 perhaps either due to the different study designs used or because differ-
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