t(8;9)(p22;p24)/PCM1-JAK2 Activates SOCS2 and SOCS3 via STAT5
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.