With various concentrations BAY 73-4506 Regorafenib of the JAK2 inhibitor TG101348. The equal exposure of both shRNA transduced and non transduced cells to the JAK2 inhibitor allowed us to compare the effects of JAK2 inhibition in the two populations and observe a cooperative effect of JAK2 inhibition and JMJD2C knockdown. Knockdown of JMJD2C alone was toxic for the K1106 PMBL line and the UH 01 HL line, but treatment with the JAK2 inhibitor increased the loss of shJMJD2C transduced cells in a dose dependent manner. By contrast, expression of a control shRNA did not alter the sensitivity of lymphoma cells to TG101348. In these experiments in which JAK2 and JMJD2C were simultaneously inhibited, the effect of JMJD2C knockdown was still restricted to cell cycle blockade while JAK2 inhibition primarily induced apoptosis.
Hence, the cooperative toxicity of JAK2 and JMJD2C inactivation stems from their dual inhibition of two primary oncogenic processes, proliferation CP-466722 and survival. Of particular interest were the HL lines L540 and KM H2, in which JMJD2C knockdown alone was not toxic but did sensitize the cells to the JAK2 inhibitor. This result suggests that JAK2 signaling and JMJD2C may affect the same regulatory pathway in these cells in a partially redundant fashion. The L428 HL line was not affected by combined inhibition of both of these factors, indicating either that there is further functional redundancy in this cell line for other amplicon genes or that other survival pathways that are active in this line play a dominant role, such as NF kB.
The functional cooperation of the JAK2 inhibitor with JMJD2C knockdown was not observed in the control GCB DLBCL line SUDHL4. To confirm the cooperation between JAK2 and JMJD2C, we examined the effect of shRNAmediated knockdown of these two genes, either alone or in combination. Cells were first transduced with a vector expressing a JAK2 shRNA or with an empty vector control and selected for stable retroviral integration. These two cell populations were then transduced with vectors expressing GFP together with a JMJD2C shRNA or a control shRNA. We monitored the fraction of GFP cells over time following shJMJD2C induction and compared the stable pools expressing the JAK2 shRNA or the control shRNA. As single agents, the JAK2 and JMJD2C shRNAs were toxic for K1106 PMBL and L1236 HL cells but not for control GCB DLBCL cells, as expected.
The toxicity of JMJD2C knockdown was increased with dual knockdown of JAK2, confirming the functional cooperation between these two factors. Also of note, combined knockdown of JAK2 and JMJD2C was toxic to L540 HL cells despite the fact that these cells were not sensitive to knockdown of either gene alone, again suggesting that JAK2 and JMJD2C may redundantly regulate the same pathway in these cells. Activation of the MYC transcriptional network by JAK2 and JMJD2C To investigate the molecular mechanisms of JAK2/JMJD2C cooperation, we profiled gene expression in K1106 PMBL cells following knockdown of these two genes. We identified a set of genes that were downregulated both by JAK2 inhibition and by JMJD2C inhibition, and compared this gene list to a database of previously characterized gene expression signatures. We observed a striking overlap between the genes downmodulated by these treatment