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: 10740923
• 负责人: Thomas E. Smithgall
• 金额: $ 5.37万
• 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/10740923
• 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。急性髓系白血病(AML)是一种治疗有限的破坏性血液病。 选择。虽然不同的基因变化与AML有关，但酪氨酸激酶信号的上调 通路是为靶向治疗提供机会的一个共同特征。这些途径涉及非 在髓系细胞中表达的受体酪氨酸激酶Fes、Syk和三个Src家族激酶(HCK、FGR和 LYN)。本应用的重点是一种独特的小分子激酶抑制剂(TL02-59)，具有显著的抗 急性髓系白血病疗效。这种化合物有效地抑制了来自20个国家的骨髓样本的增殖。 26例急性髓系白血病患者，抑制物敏感性与其表达水平显著相关 髓系Src家族激活FGR、HCK和LYN。未观察到与Flt3表达或突变相关 状态，与四个最敏感的患者样本的Flt3野生型。全动蛋白组靶向图谱和蛋白激酶 分析表明，TL02-59具有较窄的特异性，具有抗髓系SrC-1的微克分子效力。 家族成员FGR在体外和细胞内均有表达。在AML小鼠异种移植模型中，口服TL02- 59连续三周以10 mg/kg的剂量完全清除脾和外周血中的白血病细胞 同时显著减少骨髓植入。在这一应用中，我们建议探索这种机制 TL02-59的作用、获得性耐药倾向和体内疗效的研究 以下具体目标：目标1.确定TL02-59的结构基础 AML相关的Src家族激酶FGR。初步数据表明，TL02-59在体外对FGR有抑制作用 IC50值为30皮摩尔，而其他被测试的AML相关激酶(包括Flt3)在100%的 纳摩尔范围。FGR与TL02-59络合物的X射线结晶学研究及基于PCR的密码子突变 FGR将结合起来探索抑制剂专一性的结构和功能基础。目标2.测试 假设FGR是AML中TL02-59的主要靶点。表达TL02-的AML工程化细胞系 59个抗性的FGR突变体将进行抑制剂敏感性丧失测试。此外，AML细胞群体将被 进化出对TL02-59的获得性抗性，并由下一代探索抗性机制 测序(整个外显子组和RNAseq)。这种不偏不倚的方法可能会发现意料之外的变构 TL02-59对急性髓系白血病细胞的抑制机制及作用靶点。目标3.评估 TL02-59对急性髓系白血病小鼠模型的疗效初步数据显示TL02-59疗效显著 在使用AML细胞系的小鼠异种移植模型中进行短期治疗后。这一目标将用于研究缓蚀剂 急性髓系白血病患者来源的异种移植小鼠对长期存活的影响，包括其清除人类的能力 骨髓中的白血病干细胞及TL02-59主要靶点的相关表达 (FGR)对体内反应。这些体内模型的成功结果将为以下方面提供令人信服的理由 以FGR为靶点的TL02-59或相关类似物用于AML治疗的未来临床试验。

项目成果

In Vitro Evolution Reveals a Single Mutation as Sole Source of Src-Family Kinase C-Helix-out Inhibitor Resistance.

• DOI: 10.1021/acschembio.0c00373
• 发表时间: 2020-08-21
• 期刊: ACS chemical biology
• 影响因子: 4
• 作者: Patel RK;Patel YK;Smithgall TE
• 通讯作者: Smithgall TE

ATP-site inhibitors induce unique conformations of the acute myeloid leukemia-associated Src-family kinase, Fgr.

• DOI: 10.1016/j.str.2022.08.008
• 发表时间: 2022-11-03
• 期刊: STRUCTURE
• 影响因子: 5.7
• 作者: Du, Shoucheng;Alvarado, John J.;Wales, Thomas E.;Moroco, Jamie A.;Engen, John R.;Smithgall, Thomas E.
• 通讯作者: Smithgall, Thomas E.

Discovery of Non-peptide Small Molecule Allosteric Modulators of the Src-family Kinase, Hck.

Src 家族激酶 Hck 的非肽小分子变构调节剂的发现。

• DOI: 10.3389/fchem.2019.00822
• 发表时间: 2019
• 期刊: Frontiers in chemistry
• 影响因子: 5.5
• 作者: Dorman,HeatherR;Close,David;Wingert,BentleyM;Camacho,CarlosJ;Johnston,PaulA;Smithgall,ThomasE
• 通讯作者: Smithgall,ThomasE