CCRL2 as a Regulator of Growth and Novel Target in Myelodysplastic Syndrome

项目摘要 高危骨髓增生异常综合征（MDS）是由恶性原始细胞产生的，这些细胞对目前的化疗药物具有抗性。 治疗这些细胞在MDS微环境中表现出生长益处，并且它们的消除具有挑战性 这是因为缺乏将它们与健康原始细胞区分开的靶向标记。我们发现， MDS和骨髓增生性肿瘤（MPN）在其原发性骨髓增生异常综合征（MDS）中具有更差的生存率和更高的疾病负担。 与女性相比，为了阐明这些性别差异，我们评估了已知的 MDS/MPN样品中的雄激素受体（AR）靶基因。我们发现一个最重要的AR调节基因， CCRL 2在MDS患者的原始细胞中过表达。CCRL 2是一种非典型趋化因子受体， 缺乏G蛋白结合结构域，通过内吞作用内化，其C末端区域是其表面所需的 本地化我们发表的结果表明，CCRL 2诱导MDS生长并激活JAK 2，突出了这一点。 受体作为MDS的潜在靶点。CCRL 2敲低抑制生长相关途径， 主要是转铁蛋白受体（TFRC）和E2 F靶点。然而，由于CCRL 2缺乏信号传导结构域， 机制尚不清楚。我们假设CCRL 2在早期内体中与TFRC相互作用并激活它， 而CDK/E2 F受CCRL 2通过JAK 2调节。我们还假设CCRL 2 C端区域是必需的， 因为它的致癌特性。我们发现CCRL 2缺失使MDS细胞对阿扎胞苷敏感， 常用的MDS治疗方法。JAK 2（CCRL 2靶点）的抑制增加阿扎胞苷在CCRL 2野生型中的疗效。 而在CCRL 2敲除细胞中则没有。因此，JAK 2可能介导CCRL 2调节的阿扎胞苷耐药性。 这也表明靶向其他CCRL 2调节的途径可以选择性地有效对抗CCRL 2 - 1。 表达细胞。因此，我们假设抑制CCRL 2调节的途径或抑制剂， CCRL 2水平对表达CCRL 2的细胞具有毒性，并且可以增加阿扎胞苷的功效。在目标1中， 证实CCRL 2诱导TFRC生长相关活性，确定JAK 2在CCRL 2介导的生长中的作用 途径的调节，并确定CCRL 2 C-末端区域参与其生长效应， 进行免疫荧光、蛋白质组学和CCRL 2基因编辑。在目标2中，我们将确认 JAK 2/STAT在CCRL 2介导的阿扎胞苷耐药性中的作用，并发现其他具有选择性疗效的药物 通过进行药物和CRISPR-Cas9敲除筛选来对抗CCRL 2表达细胞。选定的代理人将 与阿扎胞苷在诱导型CCRL 2 MDS异种移植模型中组合。这些研究将为PI提供 有机会获得关键技能和专业知识，以研究MDS生长的分子生物学，重点是 CCRL 2的作用，并发现这种疾病的新的靶向治疗。这些目标将得以实现 通过正式的课程，科学的编程，以及分子基因组学专家的直接指导， 药理学和计算生物学。拟议的职业发展计划和研究目标将提供 一个途径的职业生涯作为一个独立的研究者研究MDS/MPN分子生物学。

Project Summary High-risk myelodysplastic syndrome (MDS) arises from malignant primitive cells that are resistant to current therapies. These cells exhibit a growth benefit in the MDS microenvironment and their elimination is challenging due to the lack of targetable markers distinguishing them from healthy primitive cells. We found that men with MDS and myeloproliferative neoplasms (MPN) have worse survival and higher disease burden in their primitive cells compared to women. To shed light into these sex differences, we evaluated the expression of known androgen receptor (AR) target genes in MDS/MPN samples. We found that one of the top AR-regulated genes, CCRL2, is overexpressed in primitive cells from MDS patients. CCRL2 is an atypical chemokine receptor as it lacks G-protein binding domain, is internalized via endocytosis and its C-terminal region is required for its surface localization. Our published results showed that CCRL2 induces MDS growth and activates JAK2 highlighting this receptor as a potential target in MDS. CCRL2 knockdown suppresses growth-related pathways, most prominently transferrin receptor (TFRC) and E2F targets. However, as CCRL2 lacks signaling domains, the exact mechanism is unclear. We hypothesized that CCRL2 interacts with TFRC in early endosomes and activates it while CDK/E2F is regulated by CCRL2 via JAK2. We also hypothesize that CCRL2 C-terminal region is essential for its oncogenic properties. We found that CCRL2 deletion sensitizes MDS cells to azacitidine, the most commonly used MDS therapy. Inhibition of JAK2, a CCRL2 target increases azacitidine efficacy in CCRL2 wild- type but not in CCRL2 knockdown cells. Thus, JAK2 may mediate the CCRL2-regulated azacitidine resistance. This also suggests that targeting other CCRL2-regulated pathways can be selectively effective against CCRL2- expressing cells. Thus, we hypothesize that inhibition of CCRL2-regulated pathways or agents suppressing CCRL2 levels are toxic against CCRL2-expressing cells and can increase azacitidine efficacy. In Aim 1 we will confirm the induction of TFRC growth-related activity by CCRL2, define JAK2 role in CCRL2-mediated growth pathways regulation and determine the involvement of CCRL2 C-terminal region in its growth effects by performing immunofluorescence, proteomics, and CCRL2 gene editing. In Aim 2 we will confirm the implication of JAK2/STAT in the CCRL2-mediated azacitidine resistance and discover other agents with selective efficacy against CCRL2-expressing cells by performing drug and CRISPR-Cas9 knockout screens. Selected agents will be combined with azacitidine in an inducible-CCRL2 MDS xenograft model. These studies will provide the PI the opportunity to gain critical skills and expertise to study the molecular biology of MDS growth with emphasis in the role of CCRL2 and discover novel targeted therapies for this disease. These objectives will be accomplished through formal coursework, scientific programing, and direct mentorship by experts in molecular genomics, pharmacology and computational biology. The proposed career development plan and research aims will provide a pathway to a career as an independent investigator studying MDS/MPN molecular biology.