Figure 1.
Immunophenotyping analysis of the original lymphoma and the derived cell line LM1.
A: Determination (from top to bottom) of ALK, CD138, CD30 and Ig kappa chain expression and distribution by immunohistochemistry in the original tumor (left column), LM1 cell line grown in vitro (middle column) and LM1 xenografted tumor in NOD-SCID mice (right column). B: The expression of CD30 (left column) and ALK (right column) were determined by flow cytometry in the LM1 cell line before and after engraftment in the NOD-SCID mouse model. The ALCL cell line SU-DHL1 was used as positive control for CD30 and ALK expression while the BL cell line DG75 served as negative control.
Figure 2.
Genetic characterization of the LM1 cell line.
A: The presence of the CLTC-ALK translocation in the patient's lymph node (LN), bone marrow infiltrating tumor cells (BM) and the established cell line (LM1) was determined with RT-PCR. The ALCL cell line SU-DHL1 was used as positive control for the NPM-ALK translocation. The BL cell line DG75 was used as negative control for both translocations involving ALK. ABL was used as loading control. B: Sequencing analysis indicated the presence of the CLTC-ALK fusion transcript in the LM1 cell line. C: Western blot for ALK showing exclusive cytoplasmic localization in the LM1 cell line corresponding to the expected molecular weight of the ALK-CLCT fusion protein. SU-DHL1 showed nuclear and cytosolic localization of Alk1 secondary to the NPM-ALK translocation. DG75 was used as negative control. D: SNP profiling of the LM1 cell line. Gains and losses are indicated in blue and red, respectively. Due to the nearly tetraploid chromosome status of this cell line the single loss of one copy in 2q22.1-qtel not detected by the software was determined manually (light red).
Figure 3.
In vitro effect of the ALK inhibitor TAE-684.
A: The cell lines SU-DHL1 (ALCL), Karpas299 (ALCL) and LM1 (DLBCL) harboring ALK translocations and the cell line Karpas422 (as negative control) were exposed in six replicates to 8 concentrations (from 1 to 400 nM) of the ALK inhibitor TAE-684 or vehicle control (DMSO) for 48 h and analyzed for viability. Dose-response curves were plotted. The X-axis shows the dose of TAE-684 in nM. The Y-axis shows the effect of TAE-684 as compared to vehicle control on cell viability. The experiment was done in triplicates. The goodness of fit for the experimental data to the median-effect equation (linear correlation coefficient) obtained from the logarithmic form of this equation was equal or higher than 0.90 for each curve. The concentration of TAE-684 that inhibits 50% the growth of cell lines compared to control treated cells (GI50) is shown in parentheses. B: Cell proliferation determined by EdU incorporation in DMSO- and TAE-684-treated cells (X-axis, concentration in nM). Results are expressed as percentage to control after normalization to viable cells. C: Apoptosis in LM1 cells measured by Annexin-V (and 7-AAD for nuclear staining) after incubation with DMSO or TAE-684 10 nM for 24 h (one representative of triplicate experiments is shown). D: Caspase-3 and -7 activity in LM1 cells determined after 24 h of exposure to DMSO- or TAE-684- at several concentration (X-axis). Results are expressed in relative fluorescent units (Y-axis) normalized to cell number.
Figure 4.
TAE-684 inhibits signalling downstream of CLTC-ALK in LM1 cells.
A: Phosphoprotein abundance in LM1 cells treated with DMSO (D) or TAE-684 10 nM for 4 h (T). The heat map represent the relative abundance normalized by the positive control in the proteome array (see also Supp. Fig. 2). The particular protein residue phosphorylated is shown as superscript. B: Western blot for phospho-ALK, ALK, phospho-ERK1, ERK1, phospho-RPS6, RPS6, phospho-STAT3 and STAT3 in cytosol lysates of LM1 cells treated with DMSO or TAE-684 10 nM for 4 h. C: STAT 3 and STAT5 DNA binding activity (Y-axis, arbitrary units, AU) in Karpas422 and LM1 cells treated with DMSO (representing the baseline activity) and after 4 h of exposure to 10 nM of TAE-684. Experiments represent duplicates with SEM. D: Transcript abundance for FOSL2, JUNB, CDC25A, CCND1, CCND2, CCND3, BCL2 and MYC in LM1 cells treated with DMSO (control) or TAE-684 10 nM for 12 h. Data was normalized to RPL13A levels. Data is presented as fold decrease compared to control (DMSO treated cells). E: Signaling pathways modified in LM1 cells by treatment with TAE-684. Phosphoproteins (transcripts are represented bellow the double dotted line) with significant decreases by TAE-684 are shown in green boxes, with no changes in yellow boxes and those not determined in white boxes.
Figure 5.
Effect of the ALK inhibitor TAE-684 on the growth of LM1 and Karpas422 xenografts.
A: Tumor growth plot in LM1 xenografted mice treated with vehicle (blue circles) or TAE-684 at 10 mg/kg/day 5 days per week for 2 weeks (red circles). The mice were followed without treatment until the tumors reached 1500 mm3 when the mice were killed (beginning of week 5). The treated mice were followed without further treatment for a total of 16 weeks when they were also killed. The treated mice had no macro or microscopic evidence of tumor relapse. The Y-axis indicates tumor volume (in mm3) and X-axis days of treatment. B: The same experiment as in panel A but using Karpas422 mice. In this case all the mice were killed when the tumors reached 1500 mm3 (at the end of week 5). C: Adjusted body weight at week 5 in the Karpas422 and LM1 mice for the control and TAE-684-treated animals. The error bars represent the SEM.