Metabolic rewiring by oncogenic BRAF V600E links ketogenesis pathway to BRAF-MEK1 signaling

描述(申请人提供)：“代谢重编程”和“代谢重连”已被用来描述癌细胞中的代谢变化，其中生物能量、合成代谢生物合成和适当的氧化还原状态被协调以促进细胞增殖和肿瘤生长。我们认为“代谢重编程”代表了癌细胞的“软件”变化，描述了通常由生长因子引起的、被致癌信号“劫持”的增殖细胞中的代谢变化，而“代谢重连”代表了癌细胞的“硬件”变化，描述了由于不同的致癌突变的“新功能”而新“伪造”的、但在正常细胞中没有的代谢变化。尽管越来越多的证据表明，不同的人类癌症可能具有共同的代谢特性，如Warburg效应，但目前尚不清楚不同癌症类型中不同的癌基因突变，包括癌基因和肿瘤抑制基因(TSG)，是否需要不同的代谢特性来促进肿瘤的发展，从而具体地对癌细胞的新陈代谢进行重新连接和重新编程。我们通过鉴定致癌基因BRAF V600E突变体在人黑色素瘤细胞中所需的独特的“代谢易损性”来解决这个问题，这是其他癌基因如NRAS Q61R/K所不需要的。我们发现HMG-CoA裂解酶(HMGCL)是产生酮体的关键酶，是BRAF V600E的“合成致命”伙伴。HMGCL在表达BRAF V600E的人原发黑色素瘤和患者组织样本中的毛细胞白血病细胞中表达上调。抑制HMGCL特异性地抑制表达BRAF V600E的人黑色素瘤细胞的增殖和肿瘤生长潜力。在机制上，HMGCL控制其产物乙酰乙酸酯的细胞内水平，以促进MEK-ERK信号的激活，其中乙酰乙酸酯特异性地增强BRAF V600E而不是BRAF野生型与MEK的结合。我们还发现，BRAF V600E可能以不依赖于MEK1-ERK的方式磷酸化并激活转录因子Oct-1上调HMGCL。此外，高脂饮食导致血清乙酰乙酸酯水平增加，但选择性地促进了表达BRAF V600E的小鼠黑色素瘤细胞的肿瘤形成。因此，我们假设致癌的BRAF V600E通过OCT-1重新连接代谢和细胞信号网络和信号，以促进HMGCL-乙酰乙酸轴，选择性地增强BRAF V600E依赖的肿瘤的发展。具体目的有三：(1)确定生酮饮食(高脂肪和低碳水化合物)是否会加重各种BRAF V600E阳性黑色素瘤或毛细胞白血病小鼠的疾病负担；(2)验证HMGCL-乙酰乙酸轴作为治疗BRAF V600E阳性人类恶性肿瘤的替代治疗靶点；(3)阐明乙酰乙酸酯特异性促进BRAF V600E与MEK1结合的分子机制，以及包括V600E突变株在内的BRAF活性形式激活Oct-1。

DESCRIPTION (provided by applicant): "Metabolic reprogramming" and "metabolic rewiring" have been used to describe the metabolic alterations in cancer cells where bioenergetics, anabolic biosynthesis and appropriate redox status are coordinated to promote cell proliferation and tumor growth. We believe that "metabolic reprogramming" represents "software" changes in cancer cells and describes metabolic alterations normally induced by growth factors in proliferative cells that are "hijacked" by oncogenic signals, whereas "metabolic rewiring" represents "hardware" changes in cancer cells and describes metabolic alterations that are newly "forged" due to "neo-function" of distinct oncogenic mutants, but not found in normal cells. Although increasing evidence emerges and suggests that different human cancers may share common metabolic properties, such as the Warburg effect, it is not clear whether distinct oncogene mutations, including oncogenes as well as tumor suppressor genes (TSGs), in different cancer types may require different metabolic properties for tumor development, and thus specifically "rewire" and "reprogram" cancer cell metabolism. We approached to this question by identifying unique "metabolic vulnerability" required by oncogenic BRAF V600E mutant in human melanoma cells, which are not required by other oncogenes such as NRas Q61R/K. We found that HMG-CoA lyase (HMGCL), a key enzyme in ketogenesis producing ketone bodies, is a "synthetic lethal" partner of BRAF V600E. HMGCL expression is upregulated in BRAF V600E-expressing human primary melanoma and hairy cell leukaemia cells in tissue samples from patients. Suppression of HMGCL specifically attenuates proliferation and tumor growth potential of human melanoma cells expressing BRAF V600E. Mechanistically, HMGCL controls the intracellular levels of its product, acetoacetate, to promote activation of MEK-ERK signaling, where acetoacetate specifically enhances binding of BRAF V600E but not BRAF wild type to MEK. We also found that BRAF V600E may phosphorylate and activate a transcription factor Oct-1 to upregulate HMGCL in a MEK1-ERK independent manner. Moreover, a high-fat diet resulted in increased serum levels of acetoacetate but selectively promoted tumor formation of BRAF V600E-expressing melanoma cells in mice. Thus, we hypothesize that oncogenic BRAF V600E "rewires" metabolic and cell signaling networks and signals through Oct-1 to promote the HMGCL-acetoacetate axis that selectively enhances BRAF V600E-dependent tumor development. Three Specific Aims: (1) To determine whether a ketogenic diet (high-fat and low carbohydrate) would worsen the disease burden in diverse BRAF V600E-positive mouse models with melanoma or hairy cell leukemia; (2) To validate the HMGCL-acetoacetate axis as an alternative therapeutic target in treatment of BRAF V600E-positive human malignancies; (3) To elucidate the molecular mechanisms by which acetoacetate specifically promotes BRAF V600E-MEK1 binding, and active forms of BRAF including V600E mutant activate Oct-1.