Targeting the RNA modifying enzyme ALKBH5 in Medulloblastoma

摘要 髓母细胞瘤是儿童最常见的恶性脑肿瘤，占全部脑肿瘤的很大比例。 儿童癌症死亡。MB是生长非常迅速的肿瘤，它会扩散到中枢神经系统(CNS) 通过脑脊液，导致软脑膜转移，在脑癌中发生的比例高达66% 病人。尽管在治疗甲基溴方面取得了进展，但高危甲基溴的5年存活率仍然很低，而且很高 复发。此外，那些幸存下来的孩子的生活质量也因高利率而大幅下降 与高辐射暴露和多种药物化疗相关的毒性必须在这样的环境中忍受 很小的时候。因此，确定新的因素并了解以前未定义的机制至关重要 这推动了甲基溴的生长和进展，因此可以开发安全可行的疗法来治疗甲基溴。在……里面 这一建议，我们提供了令人信服的证据，RNA去甲基酶ALKBH5可能在MB中发挥重要作用 通过支持MB癌症(肿瘤起始)干细胞(MB-CSC)的生长和进展，从而充当 新的治疗靶点。我们的初步结果显示，MB细胞高度依赖于ALKBH5的其 生死存亡。使用有或没有ALKBH5耗竭的多个MB细胞系，我们发现ALKBH5的耗竭 抑制MB细胞的短期和长期生长。此外，ALKBH5的耗尽抑制了迁移 MB细胞的数量。重要的是，我们发现ALKBH5沉默抑制了MB干细胞的自我更新/增殖 手机。支持这一点的是，ALKBH5缺失导致癌症干细胞标记物水平显著下降。这些 有重大发现，因为甲基溴干细胞被认为是甲基溴启动、维持、 复发并使MB细胞对辐射具有抵抗力。这些观察结果使我们假设RNA 去甲基酶ALKBH5通过促进癌症干细胞自我更新来支持MB的生长和进展；以及 这些以ALKBH5水平/活性为目标的方法将使MB患者的辐射反应敏感。至 为了验证这些假设，我们提出了以下两个具体目标：在目标#1中，确立ALKBH5作为一种 甲基溴生长和进展的促进剂。在目标2中，确定ALKBH5调节的机制 通过鉴定和鉴定ALKBH5靶基因及其在MB中的M6A状态来研究MB的茎和生长 细胞。这一提议的成功完成将表明RNA表观遗传学是 髓母细胞瘤干细胞活性和自我更新。鉴于ALKBH5基因敲除小鼠被报道是可行的 我们的初步研究表明，MB细胞的存活高度依赖于ALKBH5，这表明ALKBH5可能 作为治疗MB患者的一种新的、安全的治疗靶点。

Abstract Medulloblastoma is the most common malignant childhood brain tumor, which accounts for large number of all childhood cancer deaths. MB are very fast-growing tumors, which spreads to central nervous system (CNS) through cerebrospinal fluid, which leads to leptomeningeal metastases that occurs up to 66% in brain cancer patients. Despite the progress in treating MB, the 5-year survival rate for high-risk MB remains poor with high recurrence. Moreover, the quality of life for those kids who do survive is substantially reduced due to the high toxicity associated with the high radiation exposure and multiple drug chemotherapy they must endure at such an early age. Therefore, it is critical to identify novel factors and understand previously undefined mechanisms that drive MB growth and progression, so that safe and viable therapeutics can be developed for treating MB. In this proposal, we provide compelling evidence that RNA demethylase ALKBH5 may play an important role in MB growth and progression by supporting MB cancer (tumor initiating) stem cells (MB-CSC) and thus serve as a novel therapeutic target. Our preliminary results revealed that MB cells are highly dependent on ALKBH5 for its survival. Using multiple MB cell lines with or without ALKBH5 depletion, we discovered that depletion of ALKBH5 inhibited both short and long-term growth of MB cells. In addition, depletion of ALKBH5 inhibited the migration of MB cells. Importantly, we show that ALKBH5 silencing suppressed the self-renewal/proliferation of MB stem cell. Supporting that, ALKBH5-depletion led to significantly decreased levels of cancer stem cell markers. These are significant findings as MB stem cells are considered to be the major source of MB initiation, maintenance, relapse and render MB cells resistant to radiation. These observations led us to hypothesize that RNA demethylase ALKBH5 supports MB growth and progression by promoting cancer stem cell self-renewal; and those approaches aimed at targeting ALKBH5 level/activity will sensitize radiation response in MB patients. To test these hypotheses, we propose following two Specific Aims: In aim#1, To establish the role of ALKBH5 as a promoter of MB growth and progression. In aim#2, To determine the mechanism by which ALKBH5 regulates MB stemness and growth by identifying and characterizing ALKBH5 target genes and their m6A status in MB cells. Successful completion of this proposal will indicate RNA epigenetics as an important regulator of medulloblastoma stem cell viability and self-renewal. Given that ALKBH5 knockout mice is reported to be viable and our preliminary studies showing high dependency of MB cell survival on ALKBH5 suggest that ALKBH5 may serve as a novel and safe therapeutic target for treating MB patients.