ALCL is a highly malignant form of Non-Hodgkin’s lymphoma and frequently associated with a chromosomal translocation generating the oncogenic fusion protein NPM-ALK. We take advantage of a NPM-ALK transgenic mouse model for ALCL lymphomagenesis driven by the human CD4 promoter (Chiarle et al. 2003). These mice develop high malignant T-cell lymphomas. Human ALCLs were recently shown to constitutively overexpress the AP-1 proteins c-Jun and JunB. The role of c-Jun and JunB in T-cell lymphomas has not been fully understood. Interestingly, a tumor suppressive role of JunB was recently demonstrated in mice lacking JunB in the myeloid lineage which develop a CML-like disease. In this project we use mice carrying floxed alleles of JunB and/or c-Jun and the CD4-Cre mice which specifically delete these genes in T-cells. We conditionally delete c-Jun and JunB in NPM-ALK induced lymphomas to study the requirement of AP-1 in lymphoma formation. In this context we analyze latency, proliferation and the apoptotic index of transformed cells. These experiments define the function of AP-1 targets as potential therapeutic target in T-cell lymphomas before and/or after lymphoma formation.
Mice carrying the NPM-ALK fusion gene under the human CD4 promoter (Chiarle et al. 2003) is the backbone of our experiments. We conditionally delete JunB and cJun in T-cells taking advantage of a CD4 Cre mouse deleter strain.
These TFs directly upregulate platelet derived growth factor receptor B (PDGFRB) expression in the lymphoma cells and promote tumor progression and dissemination in a murine NPM-ALK lymphomagenesis model. Therapeutic inhibition of PDGFRB with the kinase inhibitor imatinib markedly prolonged the life of NPM-ALK transgenic mice. Moreover, imatinib treatment in a late-stage patient with refractory NPM-ALK-positive PDGFR-expressing ALCL resulted in rapid, complete and sustained remission. In a large cohort of fully annotated ALK+ ALCL patients of the European Intergroup for Childhood NHL (EICNHL) consortium PDGFRB expression in ALK+ but not ALK- ALCL correlated with poor prognosis suggesting an ALK dependent function of PDGFRB. The focus of our future work is to analyze PDGFRB function in both the tumor cells as well as in the microenvironment of ALCL.
To dissect the role of PDGFRB signalling in NPM-ALK driven T cell lymphomas, we have crossed mice carrying the CD4-promotor driven human NPM-ALK to PDGFRB floxed mice. In a second interbreeding step these mice have been crossed to mice with CD4 promoter driven Cre recombinase expression to yield specific deletion of PDGFRB in T cells (furthermore assigned as CD4-NPM-ALK-CD4ΔPDGFRB). As PDGFRB is absent in normal T-cells no phenotypic effects are expected towards normal T-cell development.
Kaplan Meier survival of CD4-NPM-ALK mice (black, n=21), CD4-NPM-ALK mice with PDGFRB deletion (red, n=21, CD4-NPM-ALK-CD4ΔPDGFRB) and wild-type litter-mates (green, n=3). PDGFRB deletion specifically in tumor cells led to significantly prolonged survival as tested by log-rank statistics (p=0.0023). All of the wild-type litter-mate control mice were alive at the end of the observation period.
We have developed a novel cell culture system to functionally address tumor-stroma interactions of lymphomas in vitro. Mouse thymic stromal cells (THSC) were isolated and co-cultured with mouse lymphoma cell lines. The lymphoma cells attached to the stromal fibroblasts and partially grow beneath the stromal fibroblast layer. This co-culture set-up Therefore, physiologically mimics a stromal niche for the lymphoma cells. Interestingly, imatinib treatment of THSC resulted in decreased CXCL12 (SDF-1), PDGFB and fibronectin expression as well as impaired AKT phosphorylation.
ALCL Tumor-stroma interaction in vitro. (a) Effect of imatinib on stromal cells in vitro. Stromal cells from ALCL mice were cultured in vitro. Western blot analysis of NPM-ALK lymphoma associated stromal cells revealed that increasing concentrations of imatinib resulted in a decrease in SDF-1, PDGFB as well as fibronectin (FN) and pAKT. (0) control; (1) 1 µM; (5) 5 µM imatinib (b) NPM-ALK lymphoma cells were co-cultured with thymic stromal cells and treated with imatinib as indicated, which decreased cell density (upper panels) due to increased apoptotic rates as measured by PI-staining in a dose dependent fashion. This effect could be largely rescued by treatment with SDF-1. (c) Tumor cells from CD4-NPM-ALK cJUNΔ/ΔJUNBΔ/Δ lymphomas were isolated and analyzed for deletion of cJUN and JUNB by PCR. Cells from CD4-NPM-ALK cJUNf/+JUNBf/+ tumors are shown as controls and cultivated on (d) THSCs which are positive for cJUN and JUNB. (e) Co-cultivation of CD4-NPM-ALK cJUNΔ/ΔJUNBΔ/Δ with THSCs or bone marrow stromal cells (BMSC). Tumor cells cultivated in co-culture with BMSCs (red) died within 8 days as assessed by cell number. In comparison, lymphoma cells cultivated with THSCs were viable and showed slow proliferation (blue). Upon removal of stromal cells these cells died rapidly (green). (f) Cell proliferation/viability as measured by the XTT method in CD4-NPM-ALK cJUNΔ/ΔJUNBΔ/Δ lymphoma cells in the absence (blue) or presence (red) of THSCs.
Expression of PDGFRB and correlation to survival data in mouse and human NPM-ALK positive lymphomas. (A) Kaplan Meier survival curve of CD4-NPM-ALKCD4ΔPDGFRB mice (green, n=26) shows a highly significant survival advantage (p≤0.0001; log-rank statistics) compared to CD4-NPM-ALK littermates (red, n=29). All of the wild-type littermate control mice were alive at the end of the observation period (black, n=3). (B) CD4-NPM-ALKCD4ΔPDGFRB tumor extracts show significant reduction of PDGFRB protein levels by Western blot. (C) CD4-NPM-ALKCD4ΔPDGFRB mice show significantly reduced tumor/body weight ratio. (D) Diagram representing the strategy employed to knockout or overexpress PDFlorian GrebienFRB in tumor and stromal cell lines established from primary mouse tumors. (E) Western blot for PDGFRB, ALK and GAPDH to show successful CRISPR mediated knockout, overexpression and controls in tumor cell lines. (F) Proliferation curves with cell lines characterized in (E). (G) Tissue culture medium change observed in cell lines for proliferation curves. (H) Diagram showing the strategy for breeding of FSP Cre and CD4 Cre mice in the NPM-ALK system with a double fluorescent reporter mouse strain. This strategy enables us to efficiently separate tumor cells from stromal cells by a dual color discrimination.
This research is supported through funding from the European Union's Horizon 2020 Marie Sklodowska-Curie Innovative Training Netweorks (ITN-ETN) under grant agreement No.: 675712 towards project Alkatras