MOLECULAR PATHOGENESIS OF CHROMOSOME 16 INVERSION IN HUMAN LEUKEMIA

核心结合因子(CBF)白血病，即那些影响转录因子基因RUNX1或Cbfb的易位或倒位的白血病，约占成人急性髓系白血病(AML)的24%和儿童急性淋巴细胞白血病的25%。我们部门一直在开发临床相关的转基因小鼠模型来研究人类CBF白血病。我们之前培育了表达人类Cbfb-MYH11融合基因的小鼠，这是AML最常见的原因之一。在过去的几年里，我们一直在培育含有Cbfb-MYH11和其他AML常见突变的转基因小鼠，如KIT的激活突变和Flt3的内部串联复制(Flt3-ITD)，以展示和分析它们在白血病发生过程中的合作。在过去的一年里，我们产生了数据，证明Cbfb-MYH11和突变的KIT基因在小鼠模型中用于白血病发生的合作。KIT突变是CBF AML中最常见的继发突变之一，与预后不良有关。因此，验证KIT突变与Cbfb-MYH11在白血病发生中的协同作用是重要的。在本研究中，我们将Kit D816V/Y突变的野生型(WT)和条件性Cbfb-MYH11敲击型(KI)小鼠骨髓(BM)细胞导入成年健康受体小鼠体内。我们发现，分别表达D816V和D816Y Kit的Ki-BM细胞移植的小鼠中，60%和80%的小鼠在9个月内死于白血病，而对照组小鼠没有死亡。有限稀释移植的结果表明，在KIT诱导的白血病中，白血病起始细胞的频率更高。信号通路分析显示，P44/42MAPK和STAT3在白血病细胞中有强烈的磷酸化，而AKT和STAT5没有。最后，携带KIT D816突变的白血病细胞对激酶抑制剂PKC412敏感。我们的数据为突变KIT和Cbfb-MYH11在白血病发生过程中的合作提供了明确的证据。这些发现发表在最近(赵等人，血液119：1511-21,2012)。 目前治疗CBF白血病的方法与显著的发病率和死亡率有关，5年存活率约为50%。我们假设，RUNX1和Cbfb分别编码的蛋白质RUNX1和Cbf之间的相互作用对CBF白血病至关重要，可以作为药物开发的靶点。我们开发了高通量的筛选方法来量化RUNX1-CBF的相互作用，并筛选了243,398个化合物的文库集合。从筛选到的苯二氮卓类药物Ro5-3335能够直接与RUNX1和CBF相互作用，在报告实验中抑制RUNX1/Cbfb依赖的反式激活，并抑制斑马鱼胚胎中依赖RUNX1的造血。Ro5-3335优先杀伤人CBF白血病细胞系，在转RUNX1-ETO(白血病中的另一个CBF融合基因)的斑马鱼中拯救白血病前期表型，并在小鼠Cbfb-MYH11白血病模型中减轻白血病负担。因此，我们的数据证实了RUNX1-CBF相互作用可以作为白血病治疗的靶点，我们已经确定了一种有希望的先导化合物用于此目的。这些结果最近已经发表(Cunningham等人，Proc Natl Acad Sci USA 109：14592-7,2012年)。 该实验室的一个新方向是开发人类诱导多能干细胞(IPSCs)来模拟人类疾病。IPSCs作为研究疾病的模型和细胞治疗的来源的效用取决于它们基因组的完整性。尽管最近发表了关于ipscs中DNA序列变化的文章，但这种变化对整个基因组的真实范围尚不清楚。我们首次对三个人IPSC株进行了全基因组测序，这三个株系来自一名成人供体的两种细胞类型，通过附体载体。在测序较深的IPSC品系基因组中未检测到该载体序列。我们在每个IPSC系中发现了1058-1808个杂合单核苷酸变异(SNV)，但没有拷贝数变异。这些SNV中有6到12个在每个IPSC系的编码区内，但其中50%是同义变化，其余的不是选择性地丰富与癌症或其他疾病相关的已知基因。因此，我们的数据表明，在IPSC诱导过程中，Episome介导的重编程不是固有的突变。这些发现已在今年早些时候发表(程等人，细胞干细胞，10：337-44,2012)。

Core binding factor (CBF) leukemias, those with translocations or inversions that affect transcription factor genes RUNX1 or CBFB, account for approximately 24% of adult acute myeloid leukemia (AML) and 25% of pediatric acute lymphocytic leukemia. Our section has been developing clinically relevant transgenic mouse models to study human CBF leukemia. We previously generated mice that express the human CBFB-MYH11 fusion gene, which is one of the most common causes of AML. During the last several years we have been generating transgenic mice that harbor CBFB-MYH11 and other common mutations in AML, such as activating mutations of KIT and internal tandem duplication of FLT3 (FLT3-ITD), to demonstrate and analyze their cooperation during leukemogenesis. In the past year we generated data demonstrating cooperation between CBFB-MYH11 and the mutated KIT gene for leukemogenesis in a mouse model. KIT mutations are among the most common secondary mutations in CBF AML and are associated with poor prognosis. It is therefore important to verify that KIT mutations cooperate with CBFB-MYH11 for leukemogenesis. In this study, we transduced wild type (WT) and conditional Cbfb-MYH11 knockin (KI) mouse bone marrow (BM) cells with KIT D816V/Y mutations, and the transduced cells were then transplanted to adult healthy recipient mice. We found that 60% and 80% of mice transplanted with KI BM cells expressing D816V or D816Y KIT, respectively, died from leukemia within 9 months, while no control mice did. Results from limiting dilution transplantations indicate higher frequencies of leukemia initiating cells in KIT-induced leukemia. Signaling pathway analysis revealed that p44/42 MAPK and Stat3, but not AKT and Stat5, were strongly phosphorylated in the leukemia cells. Finally, leukemia cells carrying KIT D816 mutations were sensitive to the kinase inhibitor PKC412. Our data provide clear evidence for cooperation between mutated KIT and CBFB-MYH11 during leukemogenesis. These findings were published recently (Zhao et al., Blood 119:1511-21, 2012). Current treatments for CBF leukemias are associated with significant morbidity and mortality, with a 5-year survival rate of approximately 50%. We hypothesize that the interaction between RUNX1 and CBFβ, the proteins encoded by RUNX1 and CBFB respectively, is critical for CBF leukemia and can be targeted for drug development. We developed high-throughput screen methods to quantify the RUNX1-CBFβ interaction and screened a library collection of 243,398 compounds. Ro5-3335, a benzodiazepine identified from the screen, was able to interact with RUNX1 and CBFβ directly, repress RUNX1/CBFB-dependent transactivation in reporter assays, and repress runx1-dependent hematopoiesis in zebrafish embryos. Ro5-3335 preferentially killed human CBF leukemia cell lines, rescued pre-leukemic phenotype in a RUNX1-ETO (another CBF fusion gene in leukemia) transgenic zebrafish, and reduced leukemia burden in a mouse CBFB-MYH11 leukemia model. Our data thus confirmed that RUNX1-CBFβ interaction can be targeted for leukemia treatment and we have identified a promising lead compound for this purpose. These results have been published recently (Cunningham et al., Proc Natl Acad Sci USA 109:14592-7, 2012). A new direction of the lab is the development of human induced pluripotent stem cells (iPSCs) to model human disease. The utility of iPSCs as models to study diseases and as sources for cell therapy depends on the integrity of their genomes. Despite recent publications of DNA sequence variations in the iPSCs, the true scope of such changes for the entire genome is not clear. We conducted the first whole-genome sequencing of three human iPSC lines, which were derived from two cell types of an adult donor by episomal vectors. The vector sequence was not detected in the genomes of the deeply sequenced iPSC lines. We identified 1058-1808 heterozygous single nucleotide variants (SNVs), but no copy number variants, in each iPSC line. Six to twelve of these SNVs were within coding regions in each iPSC line, but 50% of them are synonymous changes and the remaining are not selectively enriched for known genes associated with cancers or other diseases. Our data thus suggest that episome-mediated reprogramming is not inherently mutagenic during iPSC induction. These findings have been published earlier this year (Cheng et al., Cell Stem Cell, 10:337-44, 2012).