Precision Targeting of Myeloid Src-family Kinases in Acute Myelogenous Leukemia

急性髓系白血病中髓系 Src 家族激酶的精确靶向

• 批准号: 10687861
• 负责人: Thomas E. Smithgall
• 金额: $ 42.69万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-15 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10687861
• 关键词: Acute Myelocytic Leukemia; Adult; Aftercare; Architecture; Binding; Biological Assay; Bone Marrow; Cell Cycle Arrest; Cell Line; Cell Survival; Cells; Chemicals; Clinical; Clinical Trials; Codon Nucleotides; Combined Modality Therapy; Complex; Cytogenetics; Data; Development; Dose; Engineering; Engraftment; Evolution; FLT3 gene; Family member; Fibroblasts; Future; Hematologic Neoplasms; Hematopoietic Neoplasms; Human; In Vitro; Induction of Apoptosis; Lead; Length; Leukemic Cell; Libraries; Maps; Modeling; Mus; Mutagenesis; Mutation; Myelogenous; Myeloid Cells; Oral Administration; Outcome; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphotransferases; Population; Probability; Proliferating; Protein Tyrosine Kinase; Proteins; Reading Frames; Receptor Protein-Tyrosine Kinases; Recombinants; Relapse; Resistance; Risk Reduction; Rodent; Roentgen Rays; Sampling; Signal Pathway; Signaling Molecule; Signaling Protein; Specificity; Spleen; Structure; Testing; Therapeutic; Up-Regulation; Validation; X-Ray Crystallography; Xenograft Model; acquired drug resistance; acute myeloid leukemia cell; analog; cancer cell; cell growth; cell transformation; clinical development; cytotoxicity; drug action; efficacy evaluation; exome; in vivo; in vivo Model; inhibitor; kinase inhibitor; leukemia treatment; leukemic stem cell; mouse model; mutant; mutational status; nanomolar; next generation sequencing; novel therapeutic intervention; overexpression; patient derived xenograft model; peripheral blood; purge; relapse risk; resistance mechanism; resistance mutation; response; small molecule; small molecule inhibitor; src-Family Kinases; targeted treatment; transcriptome sequencing

Abstract. Acute myelogenous leukemia (AML) is a devastating hematologic cancer with limited treatment options. While diverse genetic changes are associated with AML, upregulation of tyrosine kinase signaling pathways is a common feature that offers opportunities for targeted therapy. These pathways involve the non- receptor tyrosine kinases Fes, Syk and the three Src-family kinases expressed in myeloid cells (Hck, Fgr, and Lyn). The focus of this application is a unique small molecule kinase inhibitor (TL02-59) with remarkable anti- AML efficacy. This compound potently suppressed the proliferation of bone marrow samples from twenty of twenty-six AML patients, with a striking correlation between inhibitor sensitivity and expression levels of the myeloid Src family kinases Fgr, Hck, and Lyn. No correlation was observed with Flt3 expression or mutational status, with the four most sensitive patient samples wild-type for Flt3. Kinome-wide target profiling and kinase assays demonstrated a narrow specificity profile for TL02-59, with picomolar potency against the myeloid Src- family member Fgr both in vitro and in cells. In a mouse xenograft model of AML, oral administration of TL02- 59 for just three weeks at 10 mg/kg completely eliminated leukemic cells from the spleen and peripheral blood while significantly reducing bone marrow engraftment. In this application, we propose to explore the mechanism of TL02-59 action, propensity for acquired resistance, and in vivo efficacy using mouse models of AML with the following Specific Aims: Aim 1. Determine the structural basis for TL02-59 potency and selectivity for the AML-associated Src-family kinase, Fgr. Preliminary data show that Fgr is inhibited by TL02-59 in vitro with an IC50 value of 30 picomolar, while other AML-associated kinases tested (including Flt3) are inhibited in the 100 nanomolar range. X-ray crystallography of Fgr in complex with TL02-59 and PCR-based codon mutagenesis of Fgr will be combined to explore the structural and functional basis of inhibitor specificity. Aim 2. Test the hypothesis that Fgr is the primary target for TL02-59 in AML. Engineered AML cell lines expressing TL02- 59-resistant Fgr mutants will be tested for loss of inhibitor sensitivity. In addition, AML cell populations will be evolved with acquired resistance to TL02-59, and resistance mechanisms explored by next-generation sequencing (whole exome and RNAseq). This unbiased approach may uncover unanticipated allosteric mechanisms of inhibition as well as additional targets for TL02-59 action in AML cells. Aim 3. Evaluate the efficacy of TL02-59 in mouse models of AML. Preliminary data demonstrate remarkable TL02-59 efficacy following short-term treatment in a mouse xenograft model using an AML cell line. This Aim will study inhibitor effects on long-term survival using AML patient-derived xenograft mice, including its ability to purge human leukemic stem cells from the bone marrow, and relate expression of the presumptive primary target for TL02-59 (Fgr) to the in vivo response. Successful outcomes with these in vivo models will make a compelling case for future clinical trials with TL02-59 or related analogs targeting Fgr for AML therapy.

抽象的。急性髓细胞白血病（AML）是一种严重的血液系统恶性肿瘤，治疗方法有限 选项.虽然多种遗传变化与AML相关，但酪氨酸激酶信号转导的上调 通路是一个共同的特征，为靶向治疗提供了机会。这些途径涉及非- 受体酪氨酸激酶Fes、Syk和在髓样细胞中表达的三种Src家族激酶（Hck、Fgr和 林恩）。本申请的重点是一种独特的小分子激酶抑制剂（TL 02 -59），具有显著的抗肿瘤活性。 AML疗效。该化合物有效地抑制了来自20名受试者的骨髓样品的增殖。 26例AML患者，抑制剂敏感性和表达水平之间存在显著相关性。 髓样Src家族激酶Fgr、Hck和林恩。未观察到Flt 3表达或突变与 状态，其中四个最敏感的患者样品为Flt 3野生型。全激酶组靶标分析和激酶 分析显示了对TL 02 -59的窄特异性谱，具有皮摩尔的针对髓样Src的效力。 家族成员Fgr在体外和细胞中。在AML的小鼠异种移植物模型中，口服施用TL 02 - 2000。 59在10 mg/kg剂量下持续三周，完全消除了脾脏和外周血中的白血病细胞 同时显著减少骨髓移植。在本申请中，我们建议探索该机制 TL 02 -59的作用、获得性耐药倾向和体内功效，使用具有TL 02 -59的AML小鼠模型， 具体目标：目标1。确定TL 02 -59效力的结构基础和对 AML相关Src家族激酶，Fgr。初步数据显示，TL 02 -59在体外抑制Fgr， IC 50值为30皮摩尔，而其他AML相关激酶（包括Flt 3）在100 纳摩尔范围。与TL 02 -59复合的Fgr的X射线晶体学和基于PCR的TL 02 - 59的密码子突变 Fgr将结合起来探索抑制剂特异性的结构和功能基础。目标二。测试 假设Fgr是TL 02 -59在AML中的主要靶点。表达TL 02的工程化AML细胞系 59-将测试抗性Fgr突变体抑制剂敏感性的丧失。此外，AML细胞群将被 随着对TL 02 -59的获得性耐药性的发展，下一代研究人员探索了耐药机制， 测序（全外显子组和RNAseq）。这种无偏见的方法可能会发现意想不到的变构 抑制机制以及AML细胞中TL 02 -59作用的其他靶标。目标3.评价 图10示出了TL 02 -59在AML小鼠模型中的功效。初步数据显示TL 02 -59具有显著疗效 在使用AML细胞系的小鼠异种移植物模型中短期治疗后。本课题将研究抑制剂 使用AML患者来源的异种移植小鼠对长期存活的影响，包括其清除人骨髓瘤细胞的能力。 来自骨髓的白血病干细胞，以及TL 02 -59的假定主要靶点的相关表达 (Fgr)to the in vivo体内response响应.这些体内模型的成功结果将成为一个令人信服的案例， 使用靶向Fgr的TL 02 -59或相关类似物进行AML治疗的未来临床试验。