Targeting beta1 integrin in JAK2V617F+ stem cells

项目摘要/摘要 目前癌症治疗的主要缺点是不能靶向白血病干细胞，尽管最初 成功地消除了大量白血病细胞。原发性骨髓纤维化(PMF)属于 骨髓增生性肿瘤(MPN)，第四大最常见的血液系统恶性肿瘤。在美国， 约有10万人患有MPN或缓解了MPN。最常见的驱动突变是JAK2V617F。JAK2 抑制剂Ruxolitinib显著缓解结构性症状，但不能延长生存期或逆转疾病， 进展为骨髓纤维化或转化为急性髓系白血病。小鼠模型和患者数据 提示这一缺陷是由于它不能靶向JAK2V617F+干细胞。此应用程序用于 迫切需要开发专门针对JAK2V617F+干细胞的治疗方法，以改善长期 当前治疗的结果。之前关于SERCA K01奖项的工作揭示了表达的去调控 整合素是一种黏附分子，将细胞外环境与肌动蛋白细胞骨架整合在一起 JAK2V617F转基因小鼠和PMF患者的巨核细胞。这项提议的中心假设是 1整合素是JAK2V617F+骨髓间充质干细胞维持的关键调节因子。在我们最近的初步研究中， 我们发现JAK2V617F转基因小鼠的干细胞中1整合素的活性高于野生型(WT)小鼠。 控制。体内用抗1整合素抑制抗体HM1-1治疗能够显著减少 JAK2V617F+干细胞的数量，对WT动物影响不大。然而，重要的是要了解 是什么使JAK2V617F+干细胞特别容易受到1整合素的抑制，以及干细胞是否 减排将长期持续下去。这项具有机械意义的提案的目标1将定义抗1如何 整合素抗体HM1-1通过解决两个假设而减少体内JAK2V617F+干细胞：1)诱导 2)HM1-1诱导 JAK2V617F+干细胞的凋亡和细胞死亡具有翻译意义的目标2将解决这一假设 HM1-1治疗后JAK2V617F+干细胞减少将长期存在，并将导致ALL减少 从JAK2V617F+干细胞衍生的后代。将在JAK2V617F转基因小鼠上进行研究，携带 人类的突变，使用最先进的流式细胞术分析造血干细胞。重要的是，尽管 它在机体发育中的重要作用--成年小鼠造血细胞-1整合素的条件性缺失 不会影响血细胞的产生或干细胞的维持。相比之下，正如我们的数据所表明的那样，JAK2V617F+ 干细胞似乎特别依赖整合素信号，并对其抑制高度敏感，提供了一种 可能的治疗机会窗口。这项提议解决了尚未满足的识别分子的需求 针对JAK2V617F+干细胞的靶点，具有治疗开发的潜力。该提案的目的将是 提供重要的初步数据，这些数据将作为将在 随后的R01拨款申请，以支持申请人的独立研究事业。

PROJECT SUMMARY/ABSTRACT The major shortcoming of current cancer therapies is the inability to target the leukemic stem cells, despite initial success in eliminating the bulk of leukemic cells. Primary myelofibrosis (PMF) belongs to the group of myeloproliferative neoplasms (MPNs), the fourth most prevalent hematological malignancy. In the United States, ~100,000 people are living with or in remission from MPNs. The most frequent driver mutation is JAK2V617F. JAK2 inhibitor ruxolitinib remarkably alleviates constitutive symptoms, but does not prolong survival or reverse disease, which progresses to myelofibrosis or transforms to acute myeloid leukemia. Mouse models and patient data suggest that this shortcoming is due to its inability to target the JAK2V617F+ stem cell. This application addresses the urgent need to develop treatments that specifically target JAK2V617F+ stem cells to improve the long-term outcome of current therapies. Previous work on the SERCA K01 award revealed de-regulated expression of integrins, adhesion molecules that integrate the extracellular environment with the actin cytoskeleton, in megakaryocytes of JAK2V617F transgenic mice and PMF patients. The central hypothesis of this proposal is that 1 integrin is a key mediator of JAK2V617F+ stem cell maintenance in PMF. In our most recent preliminary studies, we found higher activation of 1 integrin in stem cells of JAK2V617F transgenic mice compared to wild-type (WT) controls. Treatment with an anti-1 integrin inhibitory antibody HM1-1 in vivo was able to dramatically reduce the number of JAK2V617F+ stem cells, with negligible effect on WT animals. However, it is critical to understand what makes the JAK2V617F+ stem cells particularly susceptible to 1 integrin inhibition, and whether the stem cell reduction persists long-term. AIM 1 of this proposal, of mechanistic significance, will define how the anti-1 integrin antibody HM1-1 reduces JAK2V617F+ stem cells in vivo, by addressing two hypotheses: 1) that induction of cell cycle leads JAK2V617F+ stem cells to exit quiescence and differentiate, and 2) that HM1-1 induces apoptosis and cell death in JAK2V617F+ stem cells. AIM 2, of translational significance, will address the hypothesis that JAK2V617F+ stem cell reduction upon HM1-1 treatment persists long-term, and will lead to reduction of all progeny derived from JAK2V617F+ stem cells. Studies will be performed on JAK2V617F transgenic mice, carrying the human mutation, using state-of-art flow cytometric analysis of hematopoietic stem cells. Importantly, despite its essential role in organism development, conditional deletion of 1 integrin in hematopoietic cells of adult mice did not affect blood cell production or maintenance of stem cells. In contrast, as our data suggest, JAK2V617F+ stem cells seem to be particularly dependent on integrin signaling and highly sensitive to its inhibition, offering a possible therapeutic opportunity window. This proposal addresses the unmet need of identifying a molecular target specific to JAK2V617F+ stem cells with potential for therapeutic development. The AIMS of the proposal will provide important preliminary data which will serve as a basis for mechanistic studies to be proposed in an ensuing R01 grant application in support of applicant’s independent research career.