Sensitizing Leukemia Stem Cells to NF-kB Inhibition by Blocking TNF Signaling

描述(由申请人提供)：白血病干细胞(LSCs)是白血病起始、进展和复发所需的白血病细胞(LCs)的关键亚群。彻底消除LSCs是目前白血病治疗的终极目标。2001年，人们发现，在人类急性髓系白血病样本中使用针对核因子-βB(一种许多生存基因的转录调节因子)的抑制剂可导致干细胞显著死亡，同时保留健康的造血干细胞/祖细胞(HSC/Ps)。1不幸的是，由于皮肤、肝脏以及神经和免疫系统的严重炎症相关副作用的发展，在治疗水平上使用核因子-βB抑制剂的临床应用受到限制。这些副作用是在测试小鼠2-4的过程中发现的。因此，我们需要新的治疗方法来减少副作用，要么以更低的剂量使用更好的核因子-βB抑制剂，要么通过开发新的疗法来预防由核因子-βB抑制剂引起的炎症。肿瘤坏死因子α是一种由造血细胞释放的主要炎症信号，与核因子？B调节治疗有关。5、6虽然肿瘤坏死因子的炎症信号会导致正常HSC/Ps的细胞死亡和生长停滞，但其在白血病发生中的作用尚不清楚。7、8明确的是，控制炎症对于白血病的成功治疗至关重要。 我们的初步研究表明，LCS通过激活部分依赖于核因子-βB的信号网络，利用肿瘤坏死因子介导的炎症作为生存和增殖信号。通过敲除这两个受体(Tnfr1a-/-1b-/-)，我们可以使LCS对核因子-βB抑制剂治疗的敏感性提高100倍。因此，我们假设同时抑制肿瘤坏死因子受体信号和核因子-βB将提供双重益处：临床医生可以在使用较低剂量的核因子-βB抑制剂的同时阻断肿瘤坏死因子依赖的炎症。我们在这项研究中的主要目标是确定新的白血病特异性治疗策略，这些策略可以 增强对核因子？B的抑制作用。我们的近期目标是确定阻断肿瘤坏死因子信号是否增强了核因子-B在消除白血病细胞和白血病干细胞方面的抑制作用。在我们的研究中，我们将比较LCS和骨髓(BM)HSC/Ps的治疗方法，并将利用我们建立的小鼠遗传白血病模型。我们将利用药理学方法改变信号通路的活性，以最大限度地杀死LC，同时保持正常的HSC/Ps。我们将通过Annexin V/PI染色检测对细胞死亡的整体影响，通过CFU实验检测祖细胞的克隆形成能力，通过体内移植检测正常HSC/Ps的造血重建能力和LSCs的白血病形成能力。我们研究的第一个目的是探讨阻断肿瘤坏死因子与核因子？B抑制剂联合治疗的效果。我们的第二个目标是探索小分子抑制肿瘤坏死因子受体下游靶点结合核因子-B抑制剂的治疗。这项研究的预期结果将大大有助于加深对白血病的理解，也可能为未来的治疗指明新的策略。

DESCRIPTION (provided by applicant): Leukemia stem cells (LSCs) are the key subset of leukemic cells (LCs) required for leukemia initiation, progression and relapse. Complete elimination of LSCs is currently the ultimate goal of leukemia therapy. In 2001, it was found that using an inhibitor against NF-?B (a transcriptional regulator of many survival genes) resulted in significant death of LSCs in human acute myeloid leukemia samples while preserving healthy hematopoietic stem cells /progenitors (HSC/Ps).1 Unfortunately, the clinical use of NF-?B inhibitors at therapeutic levels is limited by the development of severe inflammation-related side-effects in the skin, the liver, and elements of the nervous, and immune systems. These side effects were uncovered during testing in mice.2-4. Therefore, we need new therapies that reduce side-effects either by using a better NF-?B inhibitor at a lower dose or by developing novel therapies which prevent the inflammation caused by NF-?B inhibitor. Tumor Necrosis Factor alpha (TNF) is a major inflammatory signal released by hematopoietic cells and is implicated in NF-?B modulation therapies.5, 6 While TNF's inflammatory signals causes cell death and growth arrest in normal HSC/Ps, its role in leukemia development is not yet clear.7, 8 What is clear is that managing inflammation is critical for the successful treatment of leukemia.9, 10 Our preliminary studies show that LCs utilize TNF-mediated inflammation as both a survival and proliferation signal by activating a signaling network that depends in part on NF-?B. By knocking out both receptors of TNF (Tnfr1a-/-1b-/-), we can sensitize LCs 100-fold to NF-?B inhibitor treatment when compared to normal HSC/Ps. Therefore, we hypothesize that inhibiting both TNF receptor signaling and NF-?B would provide a double benefit: clinicians could use NF-?B inhibitors at a lower dose while simultaneously blocking TNF-dependent inflammation. Our broad goal in this study is to identify novel leukemia-specific therapeutic strategies that can augment NF-?B inhibition. Our immediate goal is to determine whether blocking TNF signaling augments NF- ?B inhibition in eliminating both leukemic cells and leukemic stem cells. In our study, we will compare treatments in LCs and bone marrow (BM) HSC/Ps, and will take advantage of the murine genetic leukemia models we have generated. We will utilize pharmacological approaches to alter the activity of signaling pathways in order to maximally kill LCs while preserving normal HSC/Ps. We will measure global effects on cell death by Annexin V/PI staining, colonigenic capacity of progenitor cells by CFU assay, and hematopoietic reconstitutive capacity of normal HSC/Ps and leukemogenic capacity of LSCs by in vivo transplantation. The first aim of our study explores the effects of blocking TNF in conjunction with NF-?B inhibitor treatment. Our second aim explores small molecule inhibition of TNF receptor downstream targets in combination with NF-?B inhibitor treatment. The expected results of this study will significantly contribute to furthering the understanding of leukemia, an may also point the way toward novel strategies for future therapies.