IL-7 Receptor Mutations and Steroid Resistance in Pediatric T-Cell Acute Lymphoblastic Leukemia: a Genome Sequencing Study

BACKGROUND: Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood cancer and the leading cause of cancer-related mortality in children. T-cell acute lymphoblastic leukemia (T-ALL) represents about 15% of pediatric ALL cases and is considered a high-risk disease entity. T-ALL is often associated with resistance to treatment modalities, including steroids, which are currently the cornerstone for treating ALL; moreover, the initial steroid response strongly predicts survival and cure. However, the cellular mechanisms underlying steroid resistance in T-ALL patients are poorly understood. In this study, we combined various genomic datasets in order to identify candidate genetic mechanisms that underlie steroid resistance in children undergoing T-ALL treatment. METHODS AND FINDINGS: We performed whole-genome sequencing on paired pre-treatment (diagnostic) and post-treatment (remission) samples from 13 patients. In additions, we performed targeted exome sequencing of pre-treatment samples from 69 additional T-ALL patients. We then integrated mutation data with copy-number data for 151 mutated genes, and this integrated dataset was then correlated with clinical outcome and in vitro drug response. Our analysis revealed that mutations in JAK1 and KRAS, two genes encoding components of the interleukin 7 receptor (IL7R) signaling pathway, are associated with steroid resistance and poor outcome. We then sequenced other genes in this pathway, including IL7Ra, JAK1, JAK3, NF1, NRAS, KRAS, and AKT, in these 69 T-ALL patients and an additional 77 T-ALL patients. We identified mutations in 32% of patients, the majority of whom had a specific T-ALL subtype (ETP-ALL or TLX). Based on the outcome of these patients and their prednisolone responsiveness measured in vitro, we then confirmed that these mutations are associated with both steroid resistance and poor outcome. To functionally explore how these mutations cause steroid resistance and subsequent poor outcome, we expressed wild-type and mutant IL7R signaling molecules in two steroid-sensitive T-ALL cell lines (SUPT1 and P12 Ichikawa cells) using inducible lentiviral expression constructs. We found that expressing mutant IL7Ra, JAK1, or NRAS, wild-type NRAS, or wild-type AKT specifically induced steroid resistance in both lines without affecting sensitivity to vincristine or asparaginase. In contrast, wild-type IL7R, JAK1, and JAK3, as well as mutant JAK3 and mutant AKT, had no effect. We then performed a functional study to examine the mechanisms underlying steroid resistance and found that resistance was not due to a change in the steroid receptor’s ability to activate downstream targets. Rather, steroid resistance was associated with strong activation of MEK-ERK and AKT, downstream components of IL7R signaling pathway, thereby inducing a robust anti-apoptotic response by upregulating MCL1 and BCL-XL expression. Both the MEK-ERK and AKT pathways also inactivate BIM, an essential molecule for steroid-induced death, and inhibit GSK-3B, an important regulator of pro-apoptotic BIM. Importantly, treating our cell line models with IL7R signaling inhibitors restored steroid sensitivity. To show clinical relevance, we treated primary T-ALL cells obtained from 11 patients with steroids either alone or in combination with IL7R signaling inhibitors; we found that including MEK, AKT, mTOR, or a dual PI3K/mTOR inhibitor strongly increased steroid-induced cell death. Therefore, combining these inhibitors with steroid treatment may enhance steroid sensitivity in patients with ALL. CONCLUSIONS: Using an unbiased sequencing approach, we found that specific mutations in IL7R signaling molecules underlie steroid resistance in T-ALL. Therefore, future prospective clinical studies should include MEK, AKT, mTOR or a dual PI3K/mTOR inhibitor in order to restore—or even enhance—steroid sensitivity and improve clinical outcome.

背景：儿童急性淋巴细胞白血病（Acute Lymphoblastic Leukemia, ALL）是儿童最常见的恶性肿瘤，也是儿童癌症相关死亡的首要原因。T细胞急性淋巴细胞白血病（T-cell Acute Lymphoblastic Leukemia, T-ALL）约占儿童ALL病例的15%，被归类为高危疾病亚型。T-ALL常对多种治疗手段产生耐药性，其中糖皮质激素是当前ALL治疗的基石，而患者初始糖皮质激素应答情况可强烈预测生存与治愈结局。然而，T-ALL患者糖皮质激素耐药的细胞分子机制目前仍未明确。本研究整合多组基因组数据集，旨在明确接受T-ALL治疗的儿童患者中糖皮质激素耐药的潜在遗传机制。

方法与结果：本研究对13例患者的配对治疗前（诊断时）与治疗后（缓解时）样本进行了全基因组测序。此外，我们对另外69例T-ALL患者的治疗前样本开展了靶向外显子测序。随后，我们将151个突变基因的突变数据与拷贝数数据进行整合，并将该整合数据集与临床结局及体外药物应答进行关联分析。分析结果显示，编码白细胞介素7受体（Interleukin 7 Receptor, IL7R）信号通路组分的JAK1与KRAS基因突变，与糖皮质激素耐药及不良临床结局显著相关。随后，我们对上述69例T-ALL患者及额外77例T-ALL患者的该通路中的其他基因（包括IL7Rα、JAK1、JAK3、NF1、NRAS、KRAS及AKT）进行了测序，在32%的患者中检测到突变，其中大多数患者携带特定的T-ALL亚型（早期T细胞前体急性淋巴细胞白血病（Early T-cell Precursor Acute Lymphoblastic Leukemia, ETP-ALL）或TLX阳性亚型）。基于这些患者的临床结局及体外检测的泼尼松龙应答情况，我们进一步证实上述突变与糖皮质激素耐药及不良预后密切相关。
为了功能层面探究这些突变如何导致糖皮质激素耐药及后续不良结局，我们采用诱导型慢病毒表达载体，在两株糖皮质激素敏感的T-ALL细胞系（SUPT1与P12 Ichikawa细胞）中过表达野生型及突变型IL7R信号通路分子。结果发现，在两株细胞系中过表达突变型IL7Rα、JAK1或NRAS，野生型NRAS或野生型AKT，可特异性诱导糖皮质激素耐药，且不影响细胞对长春新碱或天冬酰胺酶的敏感性。与之相反，过表达野生型IL7R、JAK1及JAK3，以及突变型JAK3与突变型AKT，则未产生上述效应。
随后我们开展功能实验以解析糖皮质激素耐药的分子机制，结果显示耐药并非源于糖皮质激素受体激活下游靶标的能力改变。相反，糖皮质激素耐药与IL7R信号通路下游的MEK-ERK及AKT通路的过度激活密切相关，该激活通过上调MCL1与BCL-XL的表达诱导强烈的抗凋亡应答。同时，MEK-ERK与AKT通路还可使BIM（糖皮质激素诱导细胞死亡的关键分子）失活，并抑制促凋亡BIM的重要调控因子糖原合成酶激酶3β（GSK-3β）。值得注意的是，使用IL7R信号通路抑制剂处理细胞系模型，可恢复其对糖皮质激素的敏感性。
为验证该发现的临床相关性，我们用糖皮质激素单药或联合IL7R信号通路抑制剂处理11例患者来源的原代T-ALL细胞，结果发现联合MEK、AKT、哺乳动物雷帕霉素靶蛋白（mTOR）或双靶点磷脂酰肌醇3-激酶/哺乳动物雷帕霉素靶蛋白（PI3K/mTOR）抑制剂，可显著增强糖皮质激素诱导的细胞死亡。因此，将此类抑制剂与糖皮质激素联合使用，或可增强ALL患者对糖皮质激素的敏感性。

结论：本研究通过无偏倚测序策略发现，IL7R信号通路分子的特定突变是T-ALL患者糖皮质激素耐药的潜在分子基础。因此，未来的前瞻性临床研究应纳入MEK、AKT、mTOR或双靶点PI3K/mTOR抑制剂，以恢复甚至增强患者对糖皮质激素的敏感性，进而改善临床结局。