PIK3CA signaling and pancreatic cancer

该提案的目标是探索如何靶向PIK 3CA信号传导以治疗胰腺导管癌。 腺癌（PDAC）。这种致命的癌症对目前的治疗方案具有高度耐药性，包括 检查点抑制剂的免疫疗法。超过90%的PDAC在KRAS中存在致癌突变， PIK 3CA是KRAS的直接效应子。我们的小组首次报道了胰腺中PIK 3CA的基因切除， 完全保护小鼠免受致癌KRAS诱导的肿瘤形成，随后报道， PIK 3CA通过保护胰腺肿瘤免受免疫系统的影响，在维持胰腺肿瘤方面发挥着关键作用。 KrasG 12 D; Trp 53 R172 H; Pdx 1-Cre（KPC）胰腺肿瘤细胞在免疫活性细胞中的原位植入 小鼠引起100%致死率，而植入Pik 3ca-/- KPC（PIK 3CA-KO）KPC细胞的小鼠表现出 肿瘤消退，100%动物存活。全基因组基因缺失筛选，以寻找分子， 可以逆转免疫系统识别的PCCB对PIK 3CA-KO细胞的消除，PCCB催化PIK 3CA-KO细胞的增殖。 丙酰-CoA羧化以产生甲基丙二酰-CoA。PCCB-无效的PIK 3CA-KO KPC细胞是 当植入小鼠体内时，这些肿瘤不会被宿主免疫系统清除。两 将检验以下假设：1）PIK 3CA的致癌性KRAS激活调节PCCB调节的代谢 有利于胰腺癌逃避免疫消除的环境;和2）Alpelisib抑制胰腺癌的免疫消除。 PIK 3CA增强抗PD 1加抗PCSK 9疗法对胰腺肿瘤的活性。要求1 研究KRAS激活PIK 3CA调节PCCB调节的代谢的机制 有利于胰腺癌免疫逃避的环境，包括免疫检查点的上调 和T细胞衰竭。肿瘤浸润性T细胞将被分离并通过单细胞RNA和T细胞免疫印迹分析。 受体（TCR）V（D）J测序、流式细胞术和IHC。T细胞转移实验将在 进行以确定先前暴露于PCCB-无效PIK 3CA-KO肿瘤的T细胞是否将消除PIK 3CA。 移植到SCID小鼠中的KO肿瘤。PCCB-无效PIK 3CA-KO相对于PIK 3CA-KO的细胞代谢和免疫学特征 将测量PIK 3CA-KO和亲本KPC细胞系，以更好地了解代谢改变 PIK 3CA和PCCB控制以及这些变化如何导致免疫抑制。目标2是一个 一项测试alpelisib（一种PIK 3CA抑制剂）与中和药物联合治疗的疗效的治疗性研究 使用原位植入的KPC治疗胰腺癌的针对PD-1和PCSK 9的抗体 小鼠模型还将分离肿瘤浸润性T细胞并通过单细胞测序进行分析。在 总之，本提案将研究PIK 3CA信号传导如何调节代谢和免疫 胰腺癌的症状Alpelisib、抗PD 1抗体和抗PCSK 9抗体均为FDA批准的用于治疗癌症的药物。 临床应用。成功完成我们的研究可能会导致这些获批药物的临床试验， 胰腺癌的治疗

The goal of this proposal is to explore how to target PIK3CA signaling for treatment of pancreatic ductal adenocarcinoma (PDAC). This deadly cancer is highly resistant to current treatment regimens, including immunotherapy with checkpoint inhibitors. More than 90% of PDAC have oncogenic mutations in KRAS, and PIK3CA is a direct effector of KRAS. Our group first reported that genetic ablation of PIK3CA in the pancreas completely protected mice against oncogenic KRAS-induced tumor formation, and subsequently reported that PIK3CA plays a critical role in sustaining pancreatic tumors by shielding them from the immune system. Orthotopic implantation of KrasG12D;Trp53R172H;Pdx1-Cre (KPC) pancreatic tumor cells in immunocompetent mice caused 100% lethality, whereas mice implanted with Pik3ca-/- KPC (PIK3CA-KO) KPC cells exhibited tumor regression with 100% animal survival. A genome-wide gene deletion screen to search for molecules that can reverse the elimination of PIK3CA-KO cells by the immune system identified PCCB, which catalyzes the carboxylation of propionyl-CoA to produce methylmalonyl-CoA. PCCB-null PIK3CA-KO KPC cells were generated, and when implanted in mice, these tumors were not cleared by the host immune system. Two hypotheses will be tested: 1) Oncogenic KRAS activation of PIK3CA modulates a PCCB-regulated metabolic environment that favors evasion of pancreatic cancer from immune elimination; and 2) Alpelisib inhibition of PIK3CA enhances the activity of anti-PD1 plus anti-PCSK9 therapy against pancreatic tumors. Aim 1 investigates mechanisms by which KRAS activation of PIK3CA modulates a PCCB-regulated metabolic environment that favors immune evasion of pancreatic cancer, including upregulation of immune checkpoints and T cell exhaustion. Tumor infiltrating T cells will be isolated and analyzed by single-cell RNA and T cell receptor (TCR) V(D)J sequencing, flow cytometry and IHC. Adoptive T cell transfer experiments will be performed to determine if T cells previously exposed to PCCB-null PIK3CA-KO tumors will eliminate PIK3CA- KO tumors implanted in SCID mice. Cell metabolism and immunological profile of PCCB-null PIK3CA-KO vs. PIK3CA-KO and parental KPC cell lines will be measured to better understand the metabolic alterations controlled by PIK3CA and PCCB and how these changes may lead to immune suppression. Aim 2 is a therapeutic study that tests the efficacy of alpelisib, a PIK3CA inhibitor, in combination with neutralizing antibodies against PD-1 and PCSK9 for treatment of pancreatic cancer using the orthotopically implanted KPC mouse model. Tumor infiltrating T cells will also be isolated and analyzed by single-cell sequencing. In summary, this proposal will investigate how PIK3CA signaling regulates the metabolic and immunological profile of pancreatic cancer. Alpelisib, anti-PD1 antibodies and anti-PCSK9 antibodies are all FDA-approved for clinical use. Successful completion of our studies may lead to clinical trials with these approved drugs for treatment of pancreatic cancer.