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

基本信息

批准号：
10388497
负责人：
Thomas E. Smithgall
金额：
$ 8.09万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
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/10388497
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 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 acquired drug resistance acute myeloid leukemia cell analog base cancer cell cell growth cell transformation clinical development cytotoxicity drug action efficacy evaluation 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 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

项目摘要

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。急性髓性白血病 (AML) 是一种破坏性血液癌症，治疗方法有限选项。虽然多种遗传变化与 AML 相关，但酪氨酸激酶信号传导的上调通路是一个共同特征，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 和骨髓细胞中表达的三种 Src 家族激酶（Hck、Fgr 和林）。该应用的重点是一种独特的小分子激酶抑制剂（TL02-59），具有显着的抗- AML疗效。这种化合物有效抑制了来自 20 个人的骨髓样本的增殖。 26 名 AML 患者，抑制剂敏感性与 AML 患者的表达水平之间存在显着相关性骨髓 Src 家族激酶 Fgr、Hck 和 Lyn。未观察到与 Flt3 表达或突变的相关性状态，其中四个对 Flt3 最敏感的患者样本为野生型。全激酶组靶标分析和激酶检测显示 TL02-59 具有较窄的特异性，对骨髓 Src-具有皮摩尔效力。家族成员 Fgr 在体外和细胞中均存在。在 AML 小鼠异种移植模型中，口服 TL02- 59 10 mg/kg 仅三周就完全消除了脾脏和外周血中的白血病细胞同时显着减少骨髓移植。在此应用中，我们建议探索该机制使用 AML 小鼠模型研究 TL02-59 的作用、获得性耐药倾向和体内功效具体目标如下：目标 1. 确定 TL02-59 效力和选择性的结构基础 AML 相关 Src 家族激酶，Fgr。初步数据表明，TL02-59 在体外可抑制 Fgr IC50 值为 30 皮摩尔，而测试的其他 AML 相关激酶（包括 Flt3）在 100 皮摩尔内受到抑制纳摩尔范围。 Fgr 与 TL02-59 复合物的 X 射线晶体学和基于 PCR 的密码子诱变将结合Fgr来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 的主要靶标。表达 TL02- 的工程化 AML 细胞系将测试 59 抗性 Fgr 突变体是否丧失抑制剂敏感性。此外，AML细胞群将随着对 TL02-59 的获得性耐药性的进化，以及下一代探索的耐药机制测序（全外显子组和 RNAseq）。这种公正的方法可能会发现意想不到的变构抑制机制以及 TL02-59 在 AML 细胞中作用的其他靶点。目标 3. 评估 TL02-59 在 AML 小鼠模型中的功效。初步数据显示TL02-59功效显着使用 AML 细胞系对小鼠异种移植模型进行短期治疗后。该目标将研究抑制剂使用 AML 患者来源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来自骨髓的白血病干细胞，并关联 TL02-59 的假定主要靶标的表达 (Fgr) 到体内反应。这些体内模型的成功结果将成为令人信服的理由未来使用 TL02-59 或相关类似物靶向 Fgr 进行 AML 治疗的临床试验。