The Role of over-Expressed β <em>Globin</em> in Driving Relapsed B - Cell Acute Lymphoblastic Leukemia (B-ALL)

Background: The prognosis for patients with relapsed acute lymphoblastic leukemia (ALL) remains suboptimal despite intensification of chemotherapy, immunotherapy and stem cell transplant. We and others have identified genetic and epigenetic alterations enriched at relapse that drive drug resistance and relapse. In our recent work mapping the epigenetic landscape of B-ALL, we discovered novel super-enhancers (SEs), or regions with increased H3K27 acetylation, enriched at relapse in a majority of patients, implicating a role in clonal evolution. One of these SEs was upstream of the β globin locus and lead to aberrant expression of hemoglobin beta subunit ( HBB) mRNA. HBB upregulation at relapse was also found in a large meta-analysis of gene expression data from pediatric diagnosis/relapse pairs. Of note, b globin is normally found in erythrocytes that interacts with other subunits to form functional hemoglobin. We hypothesized that HBB upregulation in blasts plays a role in relapse and aimed to determine whether it leads to drug tolerance, an early recovery phenotype and/or increased clonal potential. Methods: B-lineage REH, RS4;11 and 697 ALL cell lines were transduced with lentiviral vectors overexpressing either wild-type (WT) HBB or an empty vector (EV) control. Real time PCR was performed to confirm RNA expression and Western Blot analysis was performed to detect protein expression. We generated additional cell lines with a mutation in the translation start codon and another leading to a premature stop codon to unequivocally interrupt protein expression. The generated cell lines were then plated and evaluated for differences in apoptosis via Annexin V assay and proliferation, in response to chemotherapeutic agents commonly used in the treatment of ALL. Clonogenic growth in vitro was also assessed using MethoCult TM media. To test the possibility that HBB acted in generating a persistor cell phenotype, cell lines were placed in mercaptopurine and prednisone for 10-14 days at two different concentrations and counted throughout drug exposure and for an additional 10-14 days following removal of drug. Results: We did not observe significant differences in chemosensitivity to various chemotherapies used in the treatment of B-ALL. However, we did observe less cells undergoing apoptosis in HBB expressing cells compared to control following prolonged treatment with 6-mercaptopurine, suggesting an increase in a small population of drug tolerant cells. However, this did not result in a difference in recovery time following removal of the drug. We also demonstrated that β globin overexpression led to increased clonal growth in methylcellulose compared to control (Fig. 1A, REH) and this observation was replicated in a second cell line (Fig. 1B, RS4;11). While we confirmed mRNA expression, we were not able to confirm protein expression by Western blot or by immunoprecipitation. We therefore hypothesized that b globin mRNA, rather than protein, mediates the observed increase in clonal growth. Interestingly, we observed the same phenotype of increased clonal growth (Fig. 1) in mutated cell lines, suggesting a catalytic role for β globin RNA in expanding a leukemia stem cell population. Conclusions: The increased clonal potential upon overexpression of HBB could be indicative of the ability to reconstitute a tumor from a small subpopulation following treatment. Additionally, our data suggests that protein is not required for increased clonal growth leading us to hypothesize that catalytic RNA is responsible. We are also currently analyzing RNAseq data from the cell lines and will compare differential gene expression between the empty vector and HBB expressing cells to discover downstream pathways impacted by HBB. We will be testing this hypothesis further by performing limiting dilution analysis in an immunocompromised mouse model (with David Teachey, Children's Hospital of Philadelphia).

背景：尽管强化了化疗、免疫疗法和干细胞移植，但复发急性淋巴细胞白血病（ALL）患者的预后仍然欠佳。我们及其他研究人员已经确定了在复发时富集的基因和表观遗传改变，这些改变驱动了耐药性和复发。在我们近期绘制B - ALL表观遗传图谱的工作中，我们发现了新的超级增强子（SEs），即H3K27乙酰化增加的区域，在大多数患者复发时富集，这暗示其在克隆演变中起作用。其中一个超级增强子位于β珠蛋白基因座的上游，并导致血红蛋白β亚基（HBB）mRNA的异常表达。在对儿童诊断/复发对的基因表达数据进行的大型荟萃分析中，也发现了复发时HBB的上调。值得注意的是，β珠蛋白通常存在于红细胞中，它与其他亚基相互作用形成有功能的血红蛋白。 我们假设原始细胞中HBB的上调在复发中起作用，并旨在确定它是否导致药物耐受性、早期恢复表型和/或克隆潜能增加。 方法：用慢病毒载体转导B系REH、RS4;11和697 ALL细胞系，使其过表达野生型（WT）HBB或空载体（EV）作为对照。进行实时定量PCR以确认RNA表达，并进行蛋白质印迹分析以检测蛋白质表达。我们构建了在翻译起始密码子处有突变以及另一个导致提前终止密码子的其他细胞系，以明确中断蛋白质表达。然后将构建的细胞系接种，通过膜联蛋白V检测评估细胞凋亡差异，并评估其对治疗ALL常用化疗药物的增殖反应。还使用MethoCult TM培养基评估体外克隆生长。 为了测试HBB在产生持续细胞表型中的可能性，将细胞系置于两种不同浓度的巯嘌呤和泼尼松中10 - 14天，在整个药物暴露期间以及停药后另外10 - 14天进行计数。 结果：我们没有观察到对用于治疗B - ALL的各种化疗药物的化疗敏感性有显著差异。然而，我们确实观察到在长时间使用6 - 巯嘌呤治疗后，与对照相比，表达HBB的细胞中发生凋亡的细胞较少，这表明一小部分耐药细胞增加。然而，这并没有导致停药后恢复时间的差异。我们还证明，与对照相比，β珠蛋白过表达导致在甲基纤维素中的克隆生长增加（图1A，REH），并且这一观察结果在第二个细胞系中得到重复（图1B，RS4;11）。 虽然我们确认了mRNA表达，但我们无法通过蛋白质印迹或免疫沉淀确认蛋白质表达。因此我们假设是β珠蛋白mRNA，而不是蛋白质，介导了观察到的克隆生长增加。有趣的是，我们在突变细胞系中观察到了相同的克隆生长增加的表型（图1），这表明β珠蛋白RNA在扩增白血病干细胞群体中起催化作用。 结论：HBB过表达时克隆潜能增加可能表明在治疗后从一个小的亚群重建肿瘤的能力。此外，我们的数据表明蛋白质对于克隆生长增加不是必需的，这使我们假设催化性RNA是其原因。我们目前也在分析来自细胞系的RNA测序数据，并将比较空载体和表达HBB的细胞之间的差异基因表达，以发现受HBB影响的下游途径。我们将通过在免疫缺陷小鼠模型中进行有限稀释分析（与费城儿童医院的David Teachey合作）进一步验证这一假设。