Combined Targeting of STAT3 and STAT5: A Novel Approach to Overcome Drug Resistance in Ph⁺ Cml

Chronic myeloid leukemia (CML) is a stem cell disease characterized by BCR-ABL1. Most patients in chronic phase (CP) CML achieve long-lasting molecular responses when treated with BCR-ABL1 tyrosine kinase inhibitors (TKI). However, resistance against TKI occurs in a subset of patients. Several molecular mechanisms, including BCR-ABL1 mutations, contribute to TKI resistance. For imatinib-resistant patients, second- and third generation TKI, including nilotinib, dasatinib, bosutinib, and ponatinib, are available. Using these drugs, it is now possible to cover most of the known BCR-ABL1 mutations, including the multi-resistant mutation T315I. Ponatinib, a third generation TKI, induces growth-inhibitory effects in drug-resistant patients even if T315I is expressed. However, not all mutant forms of BCR-ABL1 are responsive to ponatinib. Moreover, it has been described that multiple secondary mutations in BCR-ABL1, especially T315I-involving compound mutations, confer resistance against ponatinib. Furthermore, resistance against TKI may develop independent of BCR-ABL1 mutations. Therefore, drug combinations covering a broad range of targets, are currently under investigation with the aim to overcome drug resistance in advanced CML. Bardoxolone methyl (CDDO-Me) is an oleanane triterpenoid that has been described to induce ROS generation and to suppress a number of survival-related molecules, including AKT, mTOR, and STAT3. The aim of the current project was to evaluate the anti-leukemic effects of CDDO-Me in TKI-resistant CML cells. As assessed by ³H-thymidine uptake experiments, CDDO-Me was found to inhibit growth of various CML cell lines, including K562, an imatinib resistant sub-clone of K562, KU812, and imatinib-resistant KCL22 cells (IC₅₀: 0.1-0.5 µM). These effects were accompanied by induction of apoptosis as assessed by staining for AnnexinV and propidium iodide. Furthermore, CDDO-Me was found to block the growth of Ba/F3 cells harboring the BCR-ABL1 mutations T315I, E255K, G250E, H396P, or F359V as well as Ba/F3 cells expressing TKI-resistant compound mutations, such as T315I/E255V, T315I/F311L, T315I/F359V, or T315I/G250E (IC₅₀: 0.1-0.25 µM). The anti-proliferative effects of CDDO-Me were also confirmed in primary CML cells isolated from 13 patients with chronic phase (CP) CML (4 TKI-resistant patients, 3 with BCR-ABL1 mutations), one in blast phase (BP), and one suffering from ponatinib-resistant Ph+ ALL harboring BCR-ABL1^T315I/E255K. IC₅₀ values were comparable between samples isolated from freshly diagnosed patients (IC₅₀: 0.1-0.5 µM) and samples isolated from heavily pre-treated patients, (IC₅₀: 0.1-0.5 µM) suggesting that BCR-ABL1 mutations do not influence responses to this drug. In consecutive experiments, CDDO-Me was found to produce synergistic growth-inhibitory effects when combined with second- or third-generation BCR-ABL1 TKI. The combination ´CDDO-Me+ponatinib´ was found to be effective in Ba/F3 cells expressing various BCR-ABL1 mutations, including T315I-involving compound mutations. We also found that the combination ´CDDO-Me+TKI´ leads to simultaneous dephosphorylation of STAT3 and STAT5. To clarify whether this drug action contributes to the synergistic drug-interactions observed, we performed experiments with shRNA directed against STAT3 or STAT5 and the specific STAT5-inhibitor AC-3-019. Knockdown of STAT3 was found to produce synergistic effects with TKI and with AC-3-019 in K562 and KCL22 cells, whereas STAT5-knockdown sensitized CML cells against CDDO-Me, pointing to a new effective concept of dual STAT3+STAT5 inhibition. However, CDDO-Me was also found to increase expression of heme-oxygenase-1 (HO-1), a heat-shock-protein known to trigger drug resistance and cell survival in CML cells. We therefore combined CDDO-Me with the HO-1 inhibitor SMA-ZnPP, which also resulted in synergistic growth-inhibitory effects in human CML cells and BCR-ABL1⁺Ba/F3 cells. Moreover, SMA-ZnPP was found to sensitize KU812 cells and Ba/F3 cells expressing BCR-ABL1^T315I/F311L against the combination ´CDDO-Me+TKI´. Together, combined targeting of STAT3, STAT5, and HO-1 overcomes multiple forms of TKI resistance in highly resistant CML clones expressing BCR-ABL1^T315I or T315I-containing compound mutations. Whether such drug combinations are effective in vivo in TKI-resistant patients remains to be elucidated.