Sykes David B., Haynes Mark K., Waller Anna, Garcia Matthew, Ursu Oleg, Gouveia Kristine E., Sklar Larry, Lewis Timothy A., Dandapani Sivaraman, Munoz Benito, Scadden David T., Palmer Michelle, Schreiber Stuart L.
Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA
University of New Mexico, Albuquerque, NM
Acute Myeloid Leukemia (AML) in adults is a clinically devastating disease with a 5-year survival rate of only 25%. We lack new and effective therapies for AML, and the chemotherapy standard of care remains unchanged in thirty years. One success story has been the discovery of drugs that trigger the differentiation of leukemic blasts in a small subset of patients, approximately 10%, with acute pro-myelocytic leukemia. Differentiation therapy is not available for the remaining 90% of acute myeloid leukemia patients. We generated a novel cell line model of acute myeloid leukemia, based on the potent ability of the homeobox protein HoxA9 to block differentiation in primary murine cultures of immature myeloblasts. These cells were engineered with a built-in reporter of differentiation, permitting a high-throughput flow-cytometry-based phenotypic differentiation screen against more than 330,000 small molecules. 2,500 compounds were retested resulting in twenty-nine confirmed hits. Active compounds and analogs were obtained, validated, and tested in dose against the screening cell line as well as other murine and human cell lines. This identified twelve active compounds from which two distinct scaffolds were chosen for further study based upon chemical tractability and availability. One of the two hits demonstrated stereospecificity, hence we chose to explore SAR about this lead. Of approximately thirty synthesized analogs, we identified four compounds that were more active in three cell lines (THP-1, U937 and ERHOXA9). We have chosen compound ML390 as the probe given its potent activity. Of note, the lead compound and its analog ML390 are both very well-tolerated with limited cytotoxicity at high concentrations when assayed against cultures of normal human primary bone marrow cells. ML390 exerts its potent differentiation effect on multiple leukemia models, though its mechanism of action is currently unknown. Target identification assays as well as experiments for testing the compound are currently underway. Mechanism of action studies will be performed in parallel using a combination of gene expression studies (Library of Integrated Network-Based Cellular Signatures, LINCS), stable isotope labeling by amino acids in cell culture (SILAC), and next-generation sequencing of compound-resistant cell clones. ML390 will be tested in mice harboring a HoxA9-driven acute myeloid leukemia to assess its differentiation effect as well as its effect on leukemic progression and overall survival. The chemotherapy standard of care in acute myeloid leukemia relies on traditional cytotoxic chemotherapy and has not changed in more than thirty years. We need new, and less toxic, therapeutic agents. In the context of our novel cell line model of AML, we hope to have identified a clinically relevant pro-differentiation therapy for acute myeloid leukemia. If successful, we anticipate that ML390 will offer insight into the mechanism of overcoming differentiation arrest, and will translate into a starting point for a much-needed new and potent treatment for patients with acute myeloid leukemia.
成人急性髓系白血病（AML）是一种临床危害极大的疾病，5年生存率仅为25%。我们缺乏针对AML的新型有效疗法，化疗的标准治疗方案在30年里一直未变。一个成功案例是发现了能使一小部分（约10%）急性早幼粒细胞白血病患者的白血病原始细胞发生分化的药物。对于其余90%的急性髓系白血病患者，分化疗法并不适用。我们基于同源盒蛋白HoxA9在未成熟髓母细胞原代小鼠培养物中阻断分化的强大能力，建立了一种新型急性髓系白血病细胞系模型。这些细胞被设计了一个内置的分化报告基因，允许针对超过330,000种小分子进行基于高通量流式细胞术的表型分化筛选。对2500种化合物进行了重新测试，最终确定了29种阳性结果。获得了活性化合物及其类似物，并在针对筛选细胞系以及其他小鼠和人类细胞系的剂量实验中进行了验证和测试。由此确定了12种活性化合物，基于化学可处理性和可得性从中选择了两个不同的骨架进行进一步研究。其中一个阳性结果显示出立体特异性，因此我们选择围绕这个先导物探索构效关系。在大约30种合成类似物中，我们鉴定出4种在三种细胞系（THP - 1、U937和ERHOXA9）中活性更高的化合物。鉴于其强大的活性，我们选择化合物ML390作为探针。值得注意的是，先导化合物及其类似物ML390在针对正常人原代骨髓细胞培养物进行检测时，在高浓度下都具有很好的耐受性，细胞毒性有限。ML39对多种白血病模型都有强大的分化作用，尽管其作用机制目前尚不清楚。目前正在进行靶点鉴定实验以及该化合物的测试实验。将同时使用基因表达研究（基于整合网络的细胞特征库，LINCS）、细胞培养中氨基酸的稳定同位素标记（SILAC）以及化合物抗性细胞克隆的下一代测序等方法进行作用机制研究。ML390将在携带HoxA9驱动的急性髓系白血病的小鼠中进行测试，以评估其分化作用以及对白血病进展和总体生存的影响。急性髓系白血病的标准化疗方案依赖于传统的细胞毒性化疗，30多年来一直没有改变。我们需要新的、毒性更小的治疗药物。在我们新型的AML细胞系模型的背景下，我们希望已经确定了一种与临床相关的急性髓系白血病促分化疗法。如果成功，我们预计ML390将为克服分化停滞的机制提供见解，并将成为急需的针对急性髓系白血病患者的新的有效治疗方法的起点。
