Combination of menin and kinase inhibitors as a novel treatment for NUP98-rearranged leukemia

Chromosomal translocations involving the nucleoporin 98 (NUP98) gene at chromosome 11p15 are found in pediatric and adult Acute Myeloid Leukemia (AML) patients, representing one of the most common genetic alterations in pediatric AMLs found in ~10% of these patients. Previous studies demonstrated that NUP98 fusion proteins interact with MLL1 (Mixed Lineage Leukemia 1 also known as KMT2A) protein complex, and colocalize with MLL1 on Hoxa/b cluster genes, supporting MLL1 as a molecular dependency in the NUP98-r leukemia. Importantly, recent work has validated an important role of the scaffold protein menin, which directly interacts with the N-terminus of MLL1, in the NUP98-r leukemia. Here, we explored the effect of combining our menin inhibitor MI-3454 with CDK6 or FLT3 kinase inhibitors in the NUP98-r leukemia models as a possible way to enhance the anti-leukemic effect. Overall design: This project explored the effects of combining MENIN inhibition with kinase inhibitors on the growth and gene expression of acute myeloid leukemia cells. Primary AML patient samples with NUP98 translocations were obtained from 1) adult bone marrow harboring a NUP98-HOXA9 mutation or 2) pediatric bone marrow harboring NUP98-NSD1 mutation. The adult AML primary sample was treated with MI3454 (MENIN inhibitor), Gilteritinib (FLT3 kinase inhibitor), Palbociclib (CDK4/6 kinase inhibitor), or combinations of MI3454 with each of the kinase inhibitors (in triplicate for each treatment group). The pediatric primary AML sample was amplified in vivo by transplantation to mice, then cells were harvested and treated with MI3454, Palbociclib, or a combination of the two (in triplicate for each treatment group). Differential gene expression for each single agent treatment or combination compared to vehicle controls were then calculated and compared across treatments and patient samples.

位于11号染色体短臂1区5带的核孔蛋白98（nucleoporin 98, NUP98）基因发生染色体易位，可见于儿童及成人急性髓系白血病（Acute Myeloid Leukemia, AML）患者中；该易位是儿童AML最常见的遗传学改变之一，约占此类患者的10%。既往研究显示，NUP98融合蛋白可与混合谱系白血病蛋白1（Mixed Lineage Leukemia 1, 又名KMT2A，即MLL1）复合物相互作用，并与MLL1共同定位于Hoxa/b簇基因上，证实MLL1是NUP98重排白血病的分子依赖靶点。尤为重要的是，近期研究证实了支架蛋白menin（scaffold protein menin）可直接结合MLL1的N端，在NUP98重排白血病中发挥关键作用。本研究探索了将我们开发的menin抑制剂MI-3454与CDK6或FLT3激酶抑制剂联合使用，在NUP98重排白血病模型中增强抗白血病效应的可行性。
总体实验设计：本项目探究了抑制MENIN与激酶抑制剂联合使用，对急性髓系白血病细胞生长及基因表达的影响。本研究纳入的携带NUP98易位的原发性AML患者样本来自两类：1）携带NUP98-HOXA9融合突变的成人骨髓样本；2）携带NUP98-NSD1融合突变的儿童骨髓样本。对成人原发性AML样本分别施加如下处理：MI3454（MENIN抑制剂）、吉瑞替尼（Gilteritinib，FLT3激酶抑制剂）、帕博西尼（Palbociclib，CDK4/6激酶抑制剂），以及MI3454与各激酶抑制剂的联合给药组，每组设置3个生物学重复。儿童原发性AML样本通过小鼠体内移植进行扩增，随后收集细胞并施加如下处理：MI3454、帕博西尼，以及二者联合给药组，每组设置3个生物学重复。最后计算各单药处理组或联合给药组与溶剂对照组之间的差异基因表达水平，并在不同处理组及患者样本间进行比较分析。