<i>NPRL2</i> gene therapy induces effective antitumor immunity in <i>KRAS/STK11</i> mutant anti-PD1 resistant metastatic non-small cell lung cancer (NSCLC) in a humanized mouse model.

Abstract

Expression of <i>NPRL2/TUSC4</i>, a tumor-suppressor gene, is reduced in many cancers including NSCLC. Restoration of <i>NPRL2</i> induces DNA damage, apoptosis, and cell-cycle arrest. We investigated <i>NPRL2</i> antitumor immune responses in aPD1^R/<i>KRAS/STK11^mt</i> NSCLC in humanized-mice. Humanized-mice were generated by transplanting fresh human cord blood-derived CD34 stem cells into sub-lethally irradiated NSG mice. Lung-metastases were developed from <i>KRAS/STK11^mt</i>/aPD1^R A549 cells and treated with <i>NPRL2</i> w/wo pembrolizumab. <i>NPRL2</i>-treatment reduced lung metastases significantly, whereas pembrolizumab was ineffective. Antitumor effect was greater in humanized than non-humanized-mice. <i>NPRL2</i> + pembrolizumab was not synergistic in <i>KRAS/STK11^mt</i>/aPD1^R tumors but was synergistic in <i>KRAS^wt</i>/aPD1^S H1299. <i>NPRL2</i> also showed a significant antitumor effect on <i>KRAS^mt</i>/aPD1^R LLC2 syngeneic-tumors. The antitumor effect was correlated with increased infiltration of human cytotoxic-T, HLA-DR⁺DC, CD11c⁺DC, and downregulation of myeloid and regulatory-T cells in TME. Antitumor effect was abolished upon in-vivo depletion of CD8-T, macrophages, and CD4-T cells whereas remained unaffected upon NK-cell depletion. A distinctive protein-expression profile was found after <i>NPRL2</i> treatment. <i>IFNγ, CD8b</i>, and <i>TBX21</i> associated with T-cell functions were significantly increased, whereas <i>FOXP3, TGFB1/B2</i>, and <i>IL-10RA</i> were strongly inhibited by <i>NPRL2</i>. A list of T-cell co-inhibitory molecules was also downregulated. Restoration of <i>NPRL2</i> exhibited significantly slower tumor growth in humanized-mice, which was associated with increased presence of human cytotoxic-T, and DC and decreased percentage of Treg, MDSC, and TAM in TME. <i>NPRL2</i>-stable cells showed a substantial increase in colony-formation inhibition and heightened sensitivity to carboplatin. Stable-expression of <i>NPRL2</i> resulted in the downregulation of MAPK and AKT-mTOR signaling. Taken-together, <i>NPRL2</i> gene-therapy induces antitumor activity on <i>KRAS/STK11^mt</i>/aPD1^R tumors through DC-mediated antigen-presentation and cytotoxic immune-cell activation.