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: 9814793
• 负责人: Thomas E. Smithgall
• 金额: $ 43.56万
• 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 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9814793
• 关键词: Acute Myelocytic Leukemia; Adult; Aftercare; Apoptosis; Architecture; Binding; Biological Assay; Bone Marrow; Bone Marrow Stem Cell; Cell Cycle Arrest; Cell Line; Cell Survival; Cells; Chemicals; Clinical; Clinical Trials; Codon Nucleotides; Combined Modality Therapy; Complex; Crystallization; Cytogenetics; Data; Development; Dose; Drug effect disorder; Engineering; Engraftment; Evolution; Family member; Fibroblasts; Future; Hematologic Neoplasms; Hematopoietic Neoplasms; Human; In Vitro; 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; Protein Tyrosine Kinase; Proteins; Reading Frames; Receptor Protein-Tyrosine Kinases; Recombinants; Relapse; Resistance; Rodent; Roentgen Rays; Sampling; Signal Pathway; Signaling Molecule; Signaling Protein; Specificity; Spleen; Structure; Testing; Therapeutic; Up-Regulation; Validation; X-Ray Crystallography; Xenograft Model; Xenograft procedure; acquired drug resistance; acute myeloid leukemia cell; analog; base; cancer cell; cell growth; cell transformation; clinical development; cytotoxicity; exome; in vivo; in vivo Model; inhibitor/antagonist; kinase inhibitor; leukemic stem cell; mouse model; mutant; mutational status; nanomolar; next generation sequencing; novel therapeutic intervention; overexpression; 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。急性骨髓性白血病（AML）是一种毁灭性的血液学癌症，治疗有限 选项。虽然不同的遗传变化与AML有关，但酪氨酸激酶信号的上调 途径是一个常见的特征，为有针对性的治疗提供了机会。这些途径涉及非 受体酪氨酸激酶FES，SYK和三种在髓样细胞中表达的SRC家庭激酶（HCK，FGR和 林恩）。该应用的重点是一种独特的小分子激酶抑制剂（TL02-59），具有明显的抗 AML功效。这种复合有效地抑制了二十个骨髓样品的增殖 二十六名AML患者，抑制剂敏感性与表达水平之间具有显着的相关性 髓样SRC系列激酶FGR，HCK和Lyn。未观察到与FLT3表达或突变的相关性 状态，四个最敏感的患者为FLT3样本野生型。范围内的靶标分析和激酶 测定显示TL02-59的特异性狭窄，对髓样SRC-的皮摩尔效力 家庭成员在体外和细胞中均具有FGR。在AML的小鼠异种移植模型中，口服TL02- 59仅以10 mg/kg的形式出现三个星期 同时显着减少了骨髓植入。在此应用程序中，我们建议探索机制 TL02-59的作用，获得的抗性倾向以及使用AML的小鼠模型与AML的体内功效 以下特定目的：目标1。确定TL02-59的结构基础 AML相关的SRC家庭激酶，FGR。初步数据表明，FGR在体外抑制了TL02-59 IC50值为30 picomolol，而其他与AML相关的激酶（包括FLT3）在100中被抑制 纳摩尔范围。 FGR的X射线晶体学与TL02-59和基于PCR的密码子诱变的X射线晶体学 FGR将合并以探索抑制剂特异性的结构和功能基础。目标2。测试 假设FGR是AML中TL02-59的主要目标。表达TL02-的工程AML细胞系 将测试59种耐药的FGR突变体的抑制剂敏感性。另外，AML细胞群将是 具有对TL02-59的耐药性进化，并通过下一代探索的抗性机制 测序（整个外显子和RNASEQ）。这种公正的方法可能会发现意外的变构 AML细胞中TL02-59作用的抑制机制以及其他目标。目标3。评估 TL02-59在AML小鼠模型中的功效。初步数据显示出显着的TL02-59功效 使用AML细胞系在小鼠异种移植模型中短期治疗后。这个目标将研究抑制剂 使用AML患者衍生的异种移植小鼠对长期生存的影响，包括其清除人类的能力 骨髓的白血病干细胞，并与TL02-59的推定主要靶标相关表达 （FGR）对体内响应。这些体内模型的成功结果将为 未来的TL02-59或针对FGR用于AML治疗的相关类似物的临床试验。