Figure 1.
A: Depicts the domain structure of JAK2 (amino acid numbering based on Chen et al. [2]. Arrows show breakpoints reported in MAC cells and t(8;9) patients. B: Diagrammatic history of t(8;9) observed in the donor patient and derived cell lines at various times throughout the course of disease. t(8;9) was accompanied by various chromosome 6 rearrangements. Analyses were based on classical methods only. C: G-banding, and D: SKY analyses of MAC-1 and MAC-2B cells which were the most similar to MAC-2A (not depicted). Arrows show t(8;9). E: Shows FISH using BAC clones flanking and straddling PCM1 (left) and JAK2 (right): Note breaks (split signals) within RP11-484L21 (PCM1) and RP11-39K24 (JAK2). F: Shows gPCR for PCM1-JAK2 unique to MAC-1/2A/2B, and G: sequencing of the genomic PCR product of t(8;9) breakpoints in PCM1 and JAK2. Abbreviation: NTC, no template control.
Table 1.
Oligonucleotides.
Figure 2.
A: Shows RT-PCR analysis with minor (1) and major (2) splice variants which were unique to MAC-1/2A cells; similar data for MAC-2B are not shown. Abbreviation: NTC, no template control. B: Fusion sequence of major splice variant and primers used. C: Heatmap of top 30 genes differentially expressed in t(8;9) cells, together with a weakly expressed gene PALMD used as negative control in knockdown experiments. Top upregulated genes are listed separately in Table S3A. D: Heatmap of bottom 20 genes differentially expressed in MAC-1/2A/2B. Top downregulated genes are listed separately in Table S3B. To measure the expression profile of the MAC cell lines, we compared the clustered expression of MAC-1/2A/2B, MAC-2A as one group and calculated differential expression versus non-t(8;9) CTCL and T-ALL cell lines. Salient genes combined maximized differential expression and minimized statistical variance. For creation of heat maps we used MeV-Multi-experiment viewer (http://www.tm4.org/mev/node/33).
Figure 3.
Targets of PCM1-JAK2 signalling.
A: Shows inhibition (RQ-PCR) of the 15/20 top differentially upregulated genes by PCM1-JAK2 knockdown of MAC-2A cells relative to cells transfected with a control vector. Significantly downregulated genes are shown red, upregulated green, and unaffected black. PALMD which was moderately expressed on the array (Fig. 2C) served as a negative control. Data show means of three determinations. Note peak inhibition of SOCS2 followed by SLC26A4 and SOCS3. B: Expression of genes conspicuously downregulated in t(8;9) cell lines was validated by RQ-PCR. Contrast inconspicuous expression of GATA3 in indolent phase MAC-1 cells with silencing in aggressive phase MAC-2A/2B cells (left figure). Right figure shows elevated expression of GATA3 in MAC-1 (4.7×) and MAC-2A (3.7×) after PCM1-JAK2 knockdown, indicating negative regulation by PCM1-JAK2. C: Shows proliferation responses of t(8;9) and control cell lines to treatments with a selective JAK2 inhibitor (TG101348), methotrexate (amethopterin), and SAHA (vorinostat). Note lowest IC50 values (inset) of MAC-1/2A/2B to TG101348, while drugs currently used in therapy flatline (methotrexate) or yield nondescript results (SAHA). Data show means of two or more experiments performed in triplicate with standard errors (omitted from TG101348 for clarity). D: Shows pharmacological inhibition (RQ-PCR) by TG101348 (1 µM 72 h) of top 7 PCM1-JAK2 knockdown targets (red) and 4 additional genes which yielded inconsistent results in knockdown experiments (black). Note peak inhibition of SOCS2 and SOCS3 confirming knockdown data. Figure also shows upregulation of GATA3 (green) consistent with sh-RNA data. E: Relative SOCS2/SOCS3 expression (RQ-PCR) in a chronic eosinophilic leukemia (CEL) patient with PCM1-JAK2, with expression in MAC-2A set to unity. Upper figure shows massive upregulation of SOCS3 (but not SOCS2) in patient peripheral blood lymphocytes (PBC) and bone marrow (BM), while control AML (including EOL-1 established from a patient with an acute eosiniphilic leukemia lacking PCM1-JAK2 rearrangement), CTCL, and T-ALL cells expressed neither gene. Lower figure shows confirmation of PCM1-JAK2 expression in CEL patient PBC by RT-PCR. ETV6 served as control.
Table 2.
Top PCM1-JAK2 transcriptional target genes.
Figure 4.
A: Western blot analysis of STAT3/pSTAT3 and STAT5/pSTAT5 in MAC-2B cells subjected to JAK2 inhibition by exposure to ascending (moderate) concentrations of TG101348 over 72 h. Note dose dependent loss of STAT3 and pSTAT5 only. GAPDH served as loading control. B: Shows expression of SOCS2 and SOCS3 in MAC-1/2A/2B cells exposed to two concentrations of STAT5 inhibitor pimozide for 72 h with vehicle controls set to unity. Note dose dependent losses of SOCS3 expression, followed by SOCS2. Abbreviations: anaplastic large cell lymphoma (ALCL); acute myeloid leukemia (AML); B-lymphoblastoid cell line (B-LCL); cutaneous T-cell lymphoma (CTCL); peripheral T-cell lymphoma (PTCL); T-cell acute lymphoblastic leukemia (T-ALL).