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

在AML中，激活Flt3突变与预后不良相关。Flt3酪氨酸激酶抑制剂(TKI)的临床活性有限。在大多数情况下，这种抗性的机制尚不清楚。在第一个资助阶段，我们确定了Flt3激酶非依赖性耐药的机制。在体外模型中，我们确定白血病细胞自分泌趋化因子CCL5是一种耐药机制，并在患者样本中验证了这一点(Waldeck等人。2020)。此外，我们证明了在体外激活JAK1/2/3突变可以取代依赖于Flt3的CSF2RB的激活，并且JAK1/2/3突变发生在耐Flt3抑制剂的AML患者中(RumMelt等人。白血病2020)。我们证明，他的耐药性可以通过Flt3和JAK抑制剂的组合来克服。我们发现CSF2RB直接与Flt3-ITD相互作用，并以依赖于Flt3的方式被磷酸化。CSF2RB基因敲除抑制细胞生长和STAT5激活，并与Flt3抑制剂协同作用。CSF2RB缺失的骨髓表现出较低的转化潜能。在Flt3-ITD阳性的异种移植物中，CSF2RB基因敲除导致STAT5磷酸化减少，延长了存活期。CSF2RB缺失的骨髓抑制了Flt3-ITD诱导的AML的生长，延长了受体动物的存活时间。在这个后续的方案中，我们旨在阐明依赖Flt-ITD的CSF2RB激活的机制，特别是酪氨酸在Flt3-ITD的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ER应激信号作为依赖Flt3-ITD依赖的CSF2RB激活的可能机制的意义。我们将检验这一高度临床相关的假设，即已知的Flt3-ITD变体的差异转化能力和预后意义是否是由于Flt3-ITD中CSF2RB的结合基序的差异，从而归因于CSF2RB的结合能力。基于详细的相互作用区域，我们将在体内外验证优化的替换多肽用于治疗阻断CSF2RB依赖的转化和Flt3-ITD的白血病发生。利用排除细胞因子依赖效应的实验模型，我们将研究CSF2RB在体内的细胞内固有的、依赖于Flt3-ITD的转化效应，并验证Flt3抑制剂和CSF2RB界面阻断肽类药物的新组合疗法用于临床。

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.