Abstract 1420: Mechanism of NPRL2 gene therapy induced antitumor immunity in KRAS/STK11mt aPD1 resistant metastatic NSCLC
Authors: Meraz Ismail M. , Majidi Mourad , Song Renduo , Meng Feng , Gao Lihui , Wang Qi , Wang Jing , Shpall Elizabeth , Roth Jack A. 
Published: March 2024

NPRL2/TUSC4 is a tumor suppressor gene whose expression is reduced in many cancers including NSCLC. Restoration of NPRL2 induces cell cycle arrest and apoptosis. We investigated the antitumor immune responses to NPRL2 gene therapy in aPD1 resistant KRAS/STK11mt NSCLC in a humanized mouse model. Humanized mice were generated by transplanting human cord blood-derived CD34 stem cells into NSG mice. Mice harboring &gt; 25% human CD45 cells were considered humanized. Lung metastases were developed in humanized mice by injecting KRAS/STK11mt/aPD1R A549 cells, which were treated with i.v. injection of NPRL2 nanoparticles (DOTAP-NPRL2) with or without pembrolizumab (aPD1). NPRL2 treatment reduced lung metastases, whereas pembrolizumab was ineffective. The antitumor effect was greater in humanized than non-humanized mice suggesting the role of immune cells. The antitumor effect was associated with increased infiltration of human cytotoxic immune cells and decreased numbers of Treg in tumors. NPRL2+ pembrolizumab was not synergistic in this resistant model but was synergistic in KRASwt/aPD1S H1299 tumors. Cytotoxic immune cells in tumors were associated with the antitumor effect. Consistent with A549, NPRL2 showed a significantly strong antitumor effect on other KRASmt/aPD1R syngeneic LLC2 tumors whereas aPD1 was not effective. The antitumor effect of NPRL2 was correlated with HLA-DR+ DC, CD11c DC, TILs and NK cells in TME. The antitumor effect of NPRL2 was abolished upon in-vivo depletion of CD4, CD8 T, and MΦ in the LLC2 tumor model. However, no effect was found on in-vivo depletion of NK cells. Nanostring analysis on lung metastasis resulted in a distinct pattern of human gene expression by NPRL2. T cell functional genes, including IFNγ, CD8b, CD7, TNFSF18, ITGA1, GATA3 and TBX21 were significantly increased. Conversely, the negative regulatory genes, including FOXP3, TGFB1, TGFB2, and IL-10RA were inhibited. NPRL2 also downregulated T cell co-inhibitory molecules, including CTLA4, ICOS, LAG3, PDCD1, CD274, IDO1, PDCD1LG2, CD47, and KLRB1. Stably expressing NPRL2 clones were established, and tumors in humanized mice with these clones exhibited significantly slower growth compared to controls. TME analysis showed an upregulation of human CD45, CD3, CD8 T, HLA-DR+ DC and a downregulation of Tregs, CD3+PD1+T, MDSC, and CD163+ TAM in tumors expressing NPRL2. The stable cells showed a substantial increase in both colony formation inhibition and apoptosis. Stable clones showed heightened sensitivity to carboplatin in colony formation, apoptosis, and PARP cleavage assays. Stable expression of NPRL2 resulted in the downregulation of both AKT-mTOR and MAPK pathways by inhibition of pAKT, pmTOR, pPRAS40, p4E-BP1, pS6, and pERK1/2. Taken together, NPRL2 gene therapy induces antitumor activity on KRAS/STK11mt/aPD1R tumors through DC-mediated antigen presentation and cytotoxic immune cell activation

Citation Format: Ismail M. Meraz, Mourad Majidi, Renduo Song, Feng Meng, Lihui Gao, Qi Wang, Jing Wang, Elizabeth Shpall, Jack A. Roth. Mechanism of NPRL2 gene therapy induced antitumor immunity in KRAS/STK11mt aPD1 resistant metastatic NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1420.