Novel Acylguanidine Derivatives Targeting Smoothened Induce Antiproliferative and Pro-Apoptotic Effects in Chronic Myeloid Leukemia Cells

The most relevant therapeutic approaches to treat CML rely on the administration of tyrosine kinase inhibitors (TKIs) like Imatinib, which are able to counteract the activity of Bcr-Abl protein increasing patient’s life expectancy and survival. Unfortunately, there are some issues TKIs are not able to address; first of all TKIs are not so effective in increasing survival of patients in blast crisis, second they are not able to eradicate leukemic stem cells (LSC) which represent the major cause of disease relapse, and third patients often develop resistance to TKIs due to mutations in the drug binding site. For all these reasons it’s of primary interest to find alternative strategies to treat CML. Literature shows that Hedgehog signaling pathway is involved in LSC maintenance, and pharmacological inhibition of Smoothened (SMO), one of the key molecules of the pathway, has been demonstrated to reduce Bcr-Abl positive bone marrow cells and LSC. Consequently, targeting SMO could be a promising way to develop a new treatment strategy for CML overcoming the limitations of current therapies. In our work we have tested some compounds able to inhibit SMO, and among them MRT92 appears to be a very potent SMO antagonist. We found that almost all our compounds were able to reduce Gli1 protein levels in K-562 and in KU-812 CML cell lines. Furthermore, they were also able to increase Gli1 and SMO RNA levels, and to reduce cell proliferation and induce apoptosis/autophagy in both the tested cell lines. Finally, we demonstrated that our compounds were able to modulate the expression of some miRNAs related to Hedgehog pathway such as miR-324-5p and miR-326. Being Hedgehog pathway deeply implicated in the mechanisms of CML we may conclude that it could be a good therapeutic target for CML and our compounds seem to be promising antagonists of such pathway.

治疗慢性髓系白血病（Chronic Myeloid Leukemia, CML）最常用的临床手段为施用酪氨酸激酶抑制剂（tyrosine kinase inhibitors, TKIs），例如伊马替尼，该类药物可通过拮抗Bcr-Abl蛋白的活性以延长患者的生存预期与总生存期。然而，TKIs仍存在诸多无法解决的局限：其一，对于处于急变期的患者，TKIs对生存率的提升效果有限；其二，TKIs无法根除白血病干细胞（leukemic stem cells, LSC）——而后者正是疾病复发的核心诱因；其三，患者常因药物结合位点发生突变而对TKIs产生耐药性。鉴于上述问题，开发针对CML的替代治疗策略具有首要研究价值。现有文献表明，刺猬信号通路（Hedgehog signaling pathway）参与维持LSC的存活，而抑制该通路的关键分子平滑蛋白（Smoothened, SMO）已被证实可减少Bcr-Abl阳性骨髓细胞与LSC的数量。因此，靶向SMO有望成为克服现有疗法局限、开发CML新型治疗方案的极具前景的方向。本研究测试了多款可抑制SMO的化合物，其中MRT92展现出极强的SMO拮抗活性。实验结果显示，绝大多数受试化合物均可降低K-562与KU-812两种CML细胞系中的Gli1蛋白水平。此外，这些化合物还可上调Gli1与SMO的RNA表达水平，同时抑制两种细胞系的增殖并诱导其发生细胞凋亡/自噬。进一步研究发现，受试化合物能够调控与刺猬信号通路相关的部分microRNA的表达，包括miR-324-5p与miR-326。鉴于刺猬信号通路在CML发病机制中的关键作用，我们可以认为该通路是CML的优质治疗靶点，而本研究中的化合物有望成为该通路的高效拮抗剂。