Development of novel anti-leukemia agents targeting the menin-MLL interaction

描述(由申请人提供)：影响原癌基因MLL(混合血统白血病)的染色体易位发生在侵袭性人类急性白血病中，无论是儿童还是成人。MLL与60多个配对基因中的一个融合，产生MLL融合癌蛋白，上调正常造血所需的HOX基因的表达，最终导致白血病的发生。携带MLL基因融合的患者患有非常侵袭性的白血病，对现有治疗方法的反应很差，仅有~35%的五年存活率，强调迫切需要新的治疗方法。所有致癌的MLL融合蛋白都有一个保留的MLL的N端片段，该片段与MEN1(多发性内分泌肿瘤1)基因编码的蛋白Menin相互作用。重要的是，薄荷素与MLL融合蛋白的相互作用对MLL融合的白血病活性至关重要。因此，在MLL白血病中，薄荷素是一种重要的致癌辅助因子，是治疗干预的一个有价值的分子靶点。我们假设，直接靶向Menin和MLL融合蛋白与小分子之间的相互作用将逆转MLL融合的致癌潜力，并将抑制白血病的发展。通过应用两个独立的高通量筛选，我们鉴定了20多个小分子，这些小分子与薄荷素特异结合，并抑制薄荷素-MLL相互作用。这些化合物属于几种不同的化学类型，代表了第一批针对这种相互作用的小分子抑制剂。应用生化、生物物理和细胞分析相结合的方法，我们能够评估它们的效力、特异性和作用机制。最有效的化合物竞争性地取代来自薄荷素的MLL衍生肽，其IC50值在低微摩尔范围内，最有效的MI-1和MI-2代表两种不同的支架。这些化合物选择性地抑制MLL易位白血病细胞的生长，并诱导MLL白血病细胞的凋亡和分化。我们已经测试了MI-1和MI-2的类似物，以建立初步的构效关系，并提高它们的效力。本实验室合成的最有效的MI-1类似物对脑膜素-MLL相互作用的抑制IC50=430 nM。该化合物选择性地抑制细胞生长，诱导MLL白血病细胞的凋亡和分化，显著下调Hoxa9和Meis1基因的表达，并抑制MLL融合癌蛋白的转化，显示出高度特异性的作用方式。在这个项目中，我们建议通过结合药物化学、核磁共振、生化和生物物理方法来进一步优化这些先导化合物。化合物将在白血病细胞和正常骨髓细胞上进行活性测试，以验证其作用机制，并排除可能导致毒性问题的化合物。对于选定的化合物，还将进行MLL白血病小鼠模型的体内疗效研究。如果成功，我们的研究将产生化合物，可能为治疗伴有MLL易位的白血病提供一种新的治疗方法。 公共卫生相关性：我们建议开发小分子来逆转急性白血病中MLL融合蛋白的致癌功能。最终，这些化合物可以用作高度特异的药物，用于治疗侵袭性白血病患者，这些患者对目前的治疗方法反应不佳。

DESCRIPTION (provided by applicant): Chromosomal translocations that affect the proto-oncogene MLL (Mixed Lineage Leukemia) occur in aggressive human acute leukemias, both in children and adults. Fusion of MLL to one of more than 60 partner genes results in generation of the MLL fusion oncoprotein which upregulates expression of Hox genes required for normal hematopoiesis, and ultimately leads to the development of leukemia. Patients harboring fusion of the MLL gene suffer from very aggressive leukemias and respond poorly to available therapies, with only ~35% five-year survival rate, emphasizing that novel therapeutic treatments are urgently needed. All oncogenic MLL fusion proteins have a preserved N-terminal fragment of MLL that interacts with menin, a protein encoded by MEN1 (Multiple Endocrine Neoplasia 1) gene. Importantly, the interaction of menin with the MLL fusion proteins is critical to the leukemogenic activity of the MLL fusions. Therefore, menin functions as an essential oncogenic cofactor in MLL leukemias, and represents a valuable molecular target for therapeutic intervention. We hypothesize that direct targeting of the interaction between menin and MLL fusion proteins with small molecules will reverse the oncogenic potential of MLL fusions and will inhibit the development of leukemia. By applying two independent high throughput screens, we identified over 20 small molecules which specifically bind to menin and inhibit the menin-MLL interaction. These compounds, which belong to several distinct chemotypes, represent the first small molecule inhibitors targeting this interaction. Applying a combination of biochemical, biophysical and cellular assays we were able to evaluate their potency, specificity, and mechanism of action. The most potent compounds competitively displace the MLL-derived peptide from menin with the IC50 values at low micromolar range, with the most potent MI-1 and MI-2 representing two different scaffolds. These compounds selectively inhibit the growth of leukemia cells with MLL translocations, and induce apoptosis and differentiation of MLL leukemia cells. We have tested analogues of MI-1 and MI-2 to establish an initial structure-activity relationship and improve their potency. The most potent analogue of MI-1 synthesized in our laboratory has IC50 = 430 nM for inhibition of the menin-MLL interaction. This compound selectively inhibits the cell growth, induces apoptosis and differentiation of MLL leukemia cells, substantially downregulates expression of Hoxa9 and Meis1 genes, and inhibits transformation by MLL fusion oncoprotein, demonstrating a highly specific mode of action. In this project we propose to further optimize these lead compounds by combining medicinal chemistry, NMR, biochemical and biophysical methods. Compounds will be tested for their activity in leukemia cells and on normal bone marrow cells to validate their mechanism of action and exclude compounds which might cause toxicity issues. In vivo efficacy studies in mouse models of MLL leukemia will also be pursued for selected compounds. If successful, our studies will result in compounds which may provide a novel therapeutic approach for the treatment of leukemias with MLL translocations. PUBLIC HEALTH RELEVANCE: We are proposing to develop small molecules that may reverse the oncogenic function of MLL fusion proteins in acute leukemias. Ultimately, such compounds could be used as highly specific drugs for treatment of patients with aggressive forms of leukemia, which poorly respond to current therapies.