Novel Targeted Therapy for AML

描述(申请人提供)：开发治疗急性髓系白血病(AML)的新的、有针对性的和更安全的疗法显然是一个尚未得到满足的需求。约30%的AML携带核磷蛋白1(NPM1)杂合性突变，导致突变型NPM1(MtNPM1)胞质错位。野生型NPM1(WtNPM1)是一种核仁磷酸蛋白，其功能是蛋白质和核酸的分子伴侣(4)。NPM1的N端保守的寡聚区是其多功能的关键。在AML中，细胞质错位的mtNPM1的存在会减弱核仁wtNPM1的水平。作为AML的创建者突变，mtNPM1是髓系恶性肿瘤的单倍体缺陷肿瘤抑制基因，似乎也赋予了AML表型功能的获得。尽管mtNPM1的存在与AML相对较好的预后有关，但mtNPM1经常与突变的Flt-3-ITD(内部串联复制)共表达，突变的Flt-3-ITD代表了一种侵袭性和相对治疗难治的AML亚型。最近，我们报道了下调NPM1的水平和/或功能可以通过mtNPM1诱导AML细胞的生长停滞、分化和凋亡，并使AML细胞对全反式维甲酸(ATRA)诱导的分化敏感。因此，有必要在靶向下调NPM1的水平和/或功能的同时，结合分化诱导剂或Flt3-TKI，全面评估抗AML的疗效及其机制。基于此，本研究的目的是填补我们对NPM1基因敲除诱导分化和凋亡机制的认识的空白，并增加携带mtNPM1的AML细胞对分化诱导治疗和Flt3-TKI的敏感性。提出的在小鼠模型上的体外和体内研究将为进一步开发以NPM1拮抗剂为基础的AML治疗的新组合的临床前支持数据。这些研究将检验一个统一的假设，即下调NPM1水平/功能可以克服分化停滞，并使表达mtNPM1的AML细胞对分化诱导剂敏感，以及使共同表达Flt3-ITD和mtNPM1的AML细胞对Flt3-TKI敏感。该提案的具体目的是：目的1：确定下调NPM1(mtNPM1与wtNPM1+mtNPM1)水平或功能对培养的和患者来源的原代表达mtNPM1的AML细胞的生长、分化和存活的影响。目的：研究下调NPM1水平或功能对全反式维甲酸(ATRA)或组蛋白脱乙酰酶(HDAC)抑制剂等分化诱导剂对培养和原代AML细胞体外和体内活性的增敏作用。目的：检测NPM1和Flt3-ITD联合靶向在体外和原代共表达mtNPM1和Flt3-ITD的AML细胞中的抗AML活性。总体而言，这些目标将使NPM1成为“可用药”的靶标，并为进行高通量筛选和开发针对NPM1的新化合物以治疗表达mtNPM1的急性髓系白血病铺平道路。

DESCRIPTION (provided by applicant): There is a clear unmet need to develop novel, targeted and safer therapies for acute myeloid leukemia (AML). Approximately 30% of AML carry a heterozygous mutation in the nucleophosmin 1 (NPM1), which results in the cytoplasmic dislocation of the mutant NPM1 (mtNPM1). Wild-type NPM1 (wtNPM1) is a nucleolar phosphoprotein, which functions as a molecular chaperone for proteins and nucleic acids (4). The N-terminal conserved oligomerization domain of NPM1 is critical for its multiple functions. In AML, presence of the cytoplasmic, dislocated mtNPM1 attenuates the levels of the nucleolar wtNPM1. As a founder mutation in AML, mtNPM1 is a haploinsufficient tumor suppressor of myeloid malignancies and appears to also confer a gain of function for the AML phenotype. Although its presence is associated with a relatively better prognosis in AML, mtNPM1 is often co-expressed with mutant FLT-3-ITD (internal tandem duplication), which represents an aggressive and relatively treatment-refractory sub-type of AML. Recently, we reported that knockdown of the levels and/or function of NPM1 induces growth arrest, differentiation and apoptosis of AML cells with mtNPM1, as well as sensitizes the AML cells to differentiation induced by all-trans retinoic acid (ATRA). Therefore, there is a strong rationale and need for fully evaluating the anti-AML efficacy and its mechanism following the targeted knockdown of the level and/or function of NPM1 along with a differentiation inducing agent or a FLT3-TKI. Based on this, the objectives of this proposal are to fill the gap in our knowledge about the mechanism by which knockdown of NPM1 induces differentiation and apoptosis and increases sensitivity of AML cells with mtNPM1 to differentiation-inducing therapy and FLT3-TKI. Proposed in vitro and in vivo studies in mouse models will create the supportive pre-clinical data for further clinical development of the novel combinations of NPM1 antagonist-based therapy of AML. These studies will test the unifying hypothesis that knockdown of NPM1 levels/function overcomes differentiation arrest and sensitizes AML cells that express mtNPM1 to differentiation-inducing agents, as well as sensitizes AML cells that co-express FLT3-ITD and mtNPM1 to FLT3-TKI. The specific aims of the proposal are: AIM 1: To determine the in vitro and in vivo effects of the knockdown of NPM1 (mtNPM1 versus wtNPM1 plus mtNPM1) levels or function on cell growth, differentiation and survival of cultured and patient-derived primary AML cells expressing mtNPM1. AIM 2: To determine the sensitizing effect of knockdown of NPM1 levels or function on the in vitro and in vivo activity of differentiation- inducing agents, e.g., all-trans retinoic acid (ATRA) or a histone deacetylase (HDAC) inhibitor, against cultured and primary AML cells. AIM 3: To determine the in vitro and in vivo anti-AML activity of combined targeting of NPM1 and FLT3-ITD in cultured and primary AML cells that co-express mtNPM1 and FLT3-ITD. Overall, these aims will establish NPM1 as a 'druggable' target and pave the way for performing high throughput screening and development of novel compounds that target NPM1 for the therapy of AML expressing mtNPM1.