Our results show that bortezomib and paclitaxel combined treatment is able to target the TKIsresistant cell lines with the T315I mutation in Bcr-Abl. Collectively, our findings indicate that the bortezomib in combination with four different mitotic inhibitors, that repress mitosis by different mechanisms are able to shut down Bcr-Abl activity and result in caspase-dependent cell death in TKIs-resistant and -sensitive Bcr-Abl-positive cell lines. A schematic representation of these findings is presented in Figure 7. Our results Screening Libraries demonstrate that regimens of bortezomib combined with mitotic inhibitors are associated with Bcr-Abl and/or P-BcrAbl downregulation. Few other agents have been shown to induce a significant Bcr-Abl downregulation when used in combination with imatinib. Moreover, the pan-CDK PCI-32765 inhibitor flavopiridol, the heat shock protein 90 antagonist 17-AAG and the histone deacetylase inhibitor SAHA were previously revealed to induce apoptosis in combination with bortezomib, an effect associated with Bcr-Abl downregulation. Although the exact mechanism of Bcr-Abl downregulation is still unclear, it seems plausible that the decrease of Bcr-Abl levels and its inactivation contribute, at least in part, to the caspase-mediated cell death induced by these combinations, including the bortezomib/mitotic inhibitors regimens. Our results point out that a bortezomib/paclitaxel combination inhibits STAT3 and STAT5 activation. Bortezomib/BI 2536 combination similarly results in a decrease in P-STAT5 levels in K562 cells. As previously shown, Bcr-Abl phosphorylates and activates STAT3 and STAT5 transcription factors resulting in cellular survival and proliferation. Constitutive activation of STAT5 is known to be critical for the maintenance of chronic myeloid leukemia and STAT3 is also constitutively active in Bcr-Abl-positive embryonic stem cells. Thus, cell death induced by inhibition of Bcr-Abl with imatinib in Bcr-Abl-positive cells 
is at least in part related to the inhibition of STAT signaling. Additionally, it is known that JAKSTAT pathway activation contributes to imatinib and nilotinib resistance in Bcr-Abl-positive progenitors. All these findings suggest that STAT3/STAT5 signaling inhibition plays an important role in bortezomib/paclitaxel- or bortezomib/BI 2536-induced cell death, in Bcr-Abl-positive cells. Several pathways are known to be critical downstream mediators of the Bcr-Abl pro-survival and pro-leukemogenic effects. Bcr-Abl is phosphorylated at multiple phosphorylation sites, resulting in binding/phosphorylation of downstream BcrAbl mediators. Phosphorylation of Tyrosine 177 induces the formation of a Lyn – Gab2 – Bcr-Abl complex, important in BcrAbl-induced tumorigenesis. Lyn tyrosine kinase binding to phosphorylated and active Bcr-Abl leads to Lyn activation by phosphorylation. Lyn further regulates survival and responsiveness of CML cells to inhibition of Bcr-Abl kinase. Interestingly, Lyn kinase can also phosphorylate Bcr-Abl, resulting in a potential feedback mechanism. Additionally, Bcr-Abl phosphorylates CrkL adaptor protein, an event needed for Bcr-Abl-induced leukemia. CrkL can enhance cell migration and Bcr-Abl-mediated leukemogenesis. Thus, Lyn and CrkL are key regulators and downstream mediators of BcrAbl-induced survival and leukemogenesis that can be inhibited by downregulation or inhibition of Bcr-Abl. Our results demonstrate that the combined treatment with bortezomib and paclitaxel is able to inhibit the activity of these important BcrAbl downstream mediators. JNK activation was previously associated with apoptosis induced by bortezomib in Bcr-Abl-positive cells and by bortezomib in combination with the pan-CDK inhibitor Flavopiridol in both Bcr-Abl-positive and negative leukemic cells. In addition, several other studies pointed out the role of JNK activation in cell death of Bcr-Abl-positive or -negative cells. Thus, the activation of JNK seen in our results following bortezomib/paclitaxel treatment in Bcr-Abl-positive cells may contribute to cell death.