Phosphoproteomic characterization of primary AML samples and relevance for response towards FLT3-inhibitors

Kinase hyperactivity is a common driver of acute myeloid leukemia (AML) and serves as a therapeutic target. The most frequent genetic aberration leading to hyperactive kinase signaling and poor prognosis is internal tandem duplication (ITD) of the FMS-tyrosine-like Kinase 3-gene (FLT3). FLT3-ITD induces ligand independent activation of FLT3 and downstream pathways leading to proliferation, decreased apoptosis and partial differentiation block. Combined with chemotherapy, FLT3-Tyrosine Kinase Inhibitor (FLT3-TKI) midostaurin improves overall survival (OS) of newly diagnosed FLT3-mutated AML patients, whereas single agent gilteritinib proved superior to chemotherapy in relapsed/refractory FLT3-mutated AML patients . As the primary targets of currently approved FLT3-TKIs are tyrosine (Y) kinases, we hypothesized that direct evaluation of tyrosine phosphorylation status could reveal pY phosphorylation profiles associated with FLT3-TKI response. Therefore, we used label-free pTyr-based phosphoproteomics in 35 primary AML samples (18 FLT3-WT, 17 FLT3-ITD), to identify differentially phosphorylated proteins underlying response to the FLT3-TKIs gilteritinib and midostaurin. We identified a total of 3024 unique phosphosites (median 1299 per sample, range 286 – 1612, pS:pT:pY 11.9%:9.5%:78.6%). Due to low number of identified phosphosites, we excluded two samples from further analyses. On the remaining 33 samples, we additionally performed IMAC global phosphoproteomics and on 17 samples protein expression analysis.

激酶活性异常升高是急性髓系白血病（acute myeloid leukemia, AML）的常见驱动因素，同时也是重要的治疗靶点。最常见的导致激酶信号通路异常激活且预后不良的遗传变异，是FMS样酪氨酸激酶3基因（FMS-tyrosine-like Kinase 3-gene, FLT3）的内部串联重复（internal tandem duplication, ITD）。FLT3-ITD可诱导FLT3及其下游通路发生配体非依赖性激活，进而引发细胞增殖、凋亡抑制及部分分化阻滞。将FLT3酪氨酸激酶抑制剂（FLT3-Tyrosine Kinase Inhibitor, FLT3-TKI）米哚妥林与化疗联合使用，可改善初诊FLT3突变型AML患者的总生存期（overall survival, OS）；而单药吉瑞替尼在复发/难治性FLT3突变型AML患者中的疗效已被证实优于化疗。由于目前获批的FLT3-TKI主要靶向酪氨酸（Y）激酶，我们推测直接评估酪氨酸磷酸化状态，可揭示与FLT3-TKI应答相关的pY磷酸化谱。为此，我们对35例原发性AML样本（18例FLT3野生型、17例FLT3-ITD型）采用无标记pTyr磷酸化蛋白质组学技术，以筛选与FLT3-TKI吉瑞替尼和米哚妥林应答相关的差异磷酸化蛋白。本研究共鉴定到3024个独特磷酸化位点（中位数为1299个/样本，范围286~1612个，pS（丝氨酸磷酸化位点）:pT（苏氨酸磷酸化位点）:pY（酪氨酸磷酸化位点）占比为11.9%:9.5%:78.6%）。由于部分样本鉴定得到的磷酸化位点数量较少，我们将2例样本排除在后续分析之外。针对剩余33例样本，我们额外开展了金属亲和色谱（IMAC）全局磷酸化蛋白质组学检测，并对其中17例样本进行了蛋白质表达分析。