The Significance of IL-3 Receptor beta chain for FLT3-ITD Dependent Oncogeneic Signaling in AML

在AML中，激活FLT3突变与不良预后相关。FLT3酪氨酸激酶抑制剂（TKIs）的临床活性有限。这种耐药性的机制在大多数情况下是无法解释的。在第一个资助期，我们确定了FLT3激酶非依赖性耐药的机制。在体外模型中，我们发现白血病细胞自分泌趋化因子CCL5是一种耐药机制，并在患者样本中进行了验证（Waldeck et al. 2020）。此外，我们证明激活JAK1/2/3突变可以在体外替代FLT3依赖的CSF2RB激活，并且JAK1/2/3突变发生在FLT3抑制剂耐药的AML患者中（Rummelt等）。白血病2020)。我们证明了FLT3和JAK抑制剂的结合可以克服他的耐药性。我们发现CSF2RB直接与FLT3-ITD相互作用，并以flt3依赖的方式磷酸化。CSF2RB敲低抑制细胞生长和STAT5激活，并与FLT3抑制剂协同作用。缺乏csf2rb的骨髓转化潜能较低。在FLT3-ITD阳性的异种移植物中，CSF2RB敲除导致STAT5磷酸化降低并延长存活时间。csf2rb缺失的骨髓减少了flt3 - itd诱导的AML的生长，延长了受体动物的生存期。在本后续研究中，我们旨在阐明FLT-ITD依赖性CSF2RB激活的机制，特别是FLT3-ITD中酪氨酸在CSF3RB结合基序中的作用，以及真正的CSF2RB相互作用伙伴（LYN、SRC、SYK）和内质网应激信号作为FLT3-ITD依赖性CSF2RB激活的可能机制的意义。我们将检验一个与临床高度相关的假设，即已知FLT3-ITD变体的差异转化能力和预后意义是否源于FLT3-ITD中CSF2RB结合基序的差异，从而取决于CSF2RB的结合能力。基于详细阐述的相互作用域，我们将在体外和体内验证优化的REPLACE肽对FLT3-ITD对csf2rb依赖性转化和白血病发生的治疗性阻断。通过排除细胞因子依赖效应的实验模型，我们将在体内研究CSF2RB细胞内在的、FLT3- itd依赖的转化效应，并验证FLT3抑制剂和CSF2RB界面阻断肽模拟物的新型联合疗法的临床应用。

In AML, activating FLT3 mutations are associated with poor prognosis. FLT3 tyrosine kinase inhibitors (TKIs) have limited clinical activity. The mechanism of this resistance is in most cases unexplained. In the first funding period, we identified mechanisms of FLT3 kinase-independent resistance. In an in vitro model, we identified autocrine secretion of the chemokine CCL5 by leukemia cells as a resistance mechanism and verified it in patient samples (Waldeck et al. 2020). Moreover, we demonstrated that activating JAK1/2/3 mutations can substitute FLT3-dependent CSF2RB activation in vitro and that JAK1/2/3 mutations occur in patients with FLT3 inhibitor resistant AML (Rummelt et al. Leukemia 2020). We demonstrated that his resistance can be overcome by a combination of FLT3 and JAK inhibitors. We found that CSF2RB interacts directly with FLT3-ITD and is phosphorylated in a FLT3-dependent manner. CSF2RB knockdown inhibited cell growth and STAT5 activation, and acted synergistically with FLT3 inhibitors. CSF2RB-deficient bone marrow showed lower transformation potential. In FLT3-ITD positive xenografts, CSF2RB knockdown resulted in reduced STAT5 phosphorylation and prolonged survival. CSF2RB-deficient bone marrow reduced growth of FLT3-ITD-induced AML and prolonged survival in recipient animals. In this follow-up proposal, we aim to elucidate the mechanism of FLT-ITD dependent CSF2RB activation, specifically the role of tyrosines within the CSF3RB binding motif of FLT3-ITD and the significance of bona fide CSF2RB interaction partners (LYN, SRC, SYK) and ER stress signaling as possible mechanisms of FLT3-ITD dependent CSF2RB activation. We will test the highly clinically relevant hypothesis whether the differential transformation capacity and prognostic significance of known FLT3-ITD variants are due to differences in the binding motif for CSF2RB in FLT3-ITD and thus due to CSF2RB binding ability. Based on the elaborated interaction domains, we will validate optimized REPLACE peptides for therapeutic blockade of CSF2RB-dependent transformation and leukemogenesis by FLT3-ITD in vitro and in vivo. Using experimental models that exclude cytokine-dependent effects, we will investigate the cell-intrinsic, FLT3-ITD-dependent transforming effect of CSF2RB in vivo and validate novel combination therapies of FLT3 inhibitors and CSF2RB interface blocking peptidomimetics for clinical use.