The role of regulation and subcellular localization of GTP biosynthesis in melanoma invasion and metastasis

转移性黑色素瘤是最具侵袭性的皮肤癌类型，无法治疗。获取侵入性 表型是从原发性黑色素瘤进展到转移性黑色素瘤的关键事件， 对于黑色素瘤脑转移（MBM）患者的生存率。 黑色素瘤细胞的侵袭依赖于调节生物合成的几种RHO-GTP酶的激活 细胞突起。在RHO-GTP酶中，RAC 1在黑色素瘤进展中发挥最突出的作用。 事实上，RAC 1的P29 S激活突变（主要在黑色素瘤中检测到）是第三常见的 暴露在阳光下的黑色素瘤的突变。此外，RAC 1和其他RH 0-GTP酶作为效应物和/或 MBM中激活的信号转导程序的调节剂，包括MAPK、PI 3 K/AKT或STAT 3通路。 然而，一般黑色素瘤或MBM中调节RHO-GTdR活性的机制尚未得到充分研究。 细胞内GTP的生理变化不被认为调节RHO-GTP酶活性， 细胞中的总GTP水平被认为远高于RHO蛋白的GTP解离常数 （KdRH0·GTP）。在我们的论文中（Bianchi-Smiraglia et al. Nature Methods 2017 & Nature Communications 2021; Wolff 等人，Cell Chemical Biology 2022），我们证明了不仅游离GTP水平与KdRH 0·GTP相当， 存在于细胞中，而且RAC 1激活依赖于局部游离GTP的可用性。此外，RAC 1 与肌苷一磷酸脱氢酶2（IMPDH 2，GTP生物合成中的限速酶）相互作用 并通过这种相互作用与其他GTP代谢酶（GMEs）相互作用。因此，IMPDH 2和GME 与RAC 1一起在入侵细胞的细胞突起中富集。 通过将IMPDH 2从质膜隔离而破坏IMPDH 2-RAC 1相互作用， 不改变总GTP池，但降低细胞突起中的局部GTP水平、RAC 1活性和细胞侵袭。 IMPDH 2和其他GTP代谢酶不含膜定位序列，然而， 与质膜结合。我们还证明了高IMPDH 2水平与低IMPDH 2相关。 黑色素瘤患者的预后。 因此，在第一个具体目标中，我们将建立将IMPDH 2募集到质膜的机制， 细胞突起在第二个具体目标中，我们将确定和表征维持高IMPDH 2的机制 转移性黑色素瘤细胞中的水平。在第三个具体目标中，我们将询问IMPDH 2的抑制作为一种抑制剂。 治疗MBM的新策略。

Metastatic melanoma is the most aggressive type of skin cancer with no treatment. Acquisition of invasive phenotypes is a critical event driving progression from primary to metastatic melanoma which is most detrimental for patient survival in THE case of melanoma brain metastases (MBMs). Invasion of melanoma cells depends on activation of several RHO-GTPases which regulate the biogenesis of cell protrusions. Among RHO-GTPases, RAC1 plays the most prominent role in melanoma progression. Indeed, P29S activating mutation of RAC1 (detected predominately in melanoma) is the 3rd most common mutation in sun-exposed melanomas. In addition, RAC1 and other RHO-GTPases function as effectors and/or regulators of signal transduction programs activated in MBMs, including MAPK, PI3K/AKT or STAT3 pathways. However, mechanisms regulating RHO-GTPase activity in melanoma in general or in MBMs are understudied. Physiological changes of intracellular GTP have not been thought to regulate RHO-GTPase activity because total GTP levels in the cell were considered to be much higher than GTP dissociation constant of RHO proteins (KdRHO•GTP). In our papers (Bianchi-Smiraglia et al. Nature Methods 2017 & Nature Communications 2021; Wolff et al, Cell Chemical Biology 2022), we demonstrated that not only free GTP levels comparable to KdRHO•GTP existed in the cell, but also that RAC1 activation depended on availability of local free GTP. Furthermore, RAC1 interacted with inosine monophosphate dehydrogenase 2 (IMPDH2, a rate limiting enzyme in GTP biosynthesis) and, through this interaction, with other GTP metabolism enzymes (GMEs). Accordingly, IMPDH2 and GMEs were enriched together with RAC1 in cell protrusions of invading cells. Disruption of IMPDH2-RAC1 interaction via sequestration of IMPDH2 away from the plasma membrane did not alter total GTP pools, but decreased local GTP levels in cell protrusions, RAC1 activity, and cell invasion. IMPDH2 and other GTP metabolism enzymes do not contain membrane localization sequences, however, do associate with the plasma membrane. We also demonstrated that high IMPDH2 levels correlate with poor prognosis in melanoma patients. Thus, in the 1st Specific Aim we will establish the mechanisms that recruit IMPDH2 to plasma membrane and cell protrusions. In the 2nd Specific Aim, we will identify and characterize mechanisms maintaining high IMPDH2 levels in metastatic melanoma cells. In the 3rd Specific Aim, we will interrogate the inhibition of IMPDH2 as a novel strategy for treatment of MBMs.