Abstract NG06: Autocrine LIF signaling: A novel therapeutic target to eliminate inflammatory and immunosuppressive cancer stem cells in LKB1 mutant lung adenocarcinoma

Abstract As tumors proliferate from a single cell of origin, they develop adaptations to circumvent stressors faced in the tumor microenvironment (TME). These adaptations include acquisition of mutations that enable tumors to suppress immune surveillance. Liver Kinase B1 (LKB1), encoded by the gene STK11, is mutated in approximately 20% of lung adenocarcinoma (LUAD) cases. While immune checkpoint blockade (ICB) is the standard of care therapy for advanced LUAD, LKB1 mutant LUAD is associated with poor response rates to ICB. The mechanism in which LKB1 regulates the TME and ICB responses remains unknown and understanding this mechanism can lead to the development of novel immune based therapies that can target LKB1 mutant LUAD. To investigate how LKB1 mutations in cancer cells alter the surrounding TME we utilized a genetically engineered mouse model (GEMM) combined with in vivo CRISPR/Cas9 editing where a lentivirus expressing a sgRNA targeting Lkb1 (sgLkb1) or non-targeting control (sgNeo) and Cre recombinase, are intratracheally administered to KrasLSL-G12D/+ Tp53fl/fl Rosa26LSL-Cas9-P2A-GFP/LSL-Cas9-P2A-GFP mice to initiate autochthonous KrasG12D/+ Tp53-/- (KP) lung tumors with and without deletion of Lkb1. Eleven weeks after tumor initiation, immune cells were isolated from the lungs and immune profiling using flow cytometry and scRNA-seq was performed. Flow cytometry analysis of tumor bearing lungs revealed that both CD4 and CD8 T cell function was heavily impaired specifically in the setting of Lkb1 mutant tumors as demonstrated by decreased expression of IFNγ and TNFα. Further analysis of immune populations through scRNA-seq identified two subsets of myeloid cells enriched in Lkb1 mutant tumors: SiglecFHi neutrophils and Arg1+ interstitial macrophages, each expressing immunosuppressive gene signatures. To delineate the mechanism in which Lkb1 mutant lung tumors remodel the TME with immunosuppressive myeloid cells, we performed RNA-seq on Lkb1 mutant and wildtype tumors sorted from our GEMM. This revealed that Lkb1 mutant tumors upregulate a range of cytokines involved in myeloid cell recruitment and polarization including Il6, Ccl2, and Csf3. One notable gene upregulated in Lkb1 mutants was the cytokine Leukemia inhibitory factor (Lif). Elevation of these cytokines, including LIF, was verified at the protein level through multiplex cytokine analysis of bronchoalveolar lavage (BAL) fluid. Furthermore, analysis of TCGA dataset confirmed that LKB1 mutant LUAD have increased expression of LIF. Using KP LUAD cell lines we mapped out that LKB1 transcriptionally regulates Lif through the SIK/CRTC2 pathway. Since LIF signals through its receptor (LIFR) primarily through STAT3, we measured the expression of pSTAT3, the active form of STAT3. Remarkably, there was increased intra-tumoral pSTAT3 in murine and human tumor samples with LKB1 mutations. Because of the increased intra-tumoral pSTAT3 expression, we hypothesized that Lkb1 mutant tumor derived LIF was signaling on tumors in an autocrine loop. To investigate the presence of autocrine LIF signaling, we utilized a paired guide approach to generate Lkb1 mutant tumors with and without deletion of either Lif (sgLif) or Lifr (sgLifr). Both deletion of Lif and Lifr in tumor cells led to a decrease in intra-tumoral pSTAT3 and reduction in tumor burden compared to Lkb1 mutant tumor controls verifying that autocrine LIF signaling was active and driving tumorigenesis. Reflective of decreased inflammation, we found that BAL fluid levels of IL6, CSF3, and CCL2 were lower with disruption of LIF signaling. To examine the immune infiltrate in this model, we employed the platform Expanded Cellular Indexing of Transcriptomes and Epitopes by sequencing (ExCITE-seq) which enables analysis of gene expression, surface protein expression, and TCR sequencing at a single cell resolution. This analysis revealed that disruption of autocrine LIF signaling in tumors led to decreased infiltration by SiglecFHi neutrophils and Arg1+ interstitial macrophages. We leveraged our ExCITE-seq data set and assessed T cell clonality in each of the tumor genetic conditions and found an expansion of T cell clones specifically in the context of Lif or Lifr deletion. Corresponding with this increased clonality, we detected an increase in T cell expression of IFNγ and TNFα with disruption of LIF signaling. Overall, genetic deletion of LIF signaling in Lkb1 mutant tumors reversed the immunosuppressive myeloid infiltrate and augmented T cell function. To understand the signaling process downstream of LIFR, we extended our ExCITE-seq analysis to tumor cells. Subclustering tumor cells identified unique cancer cell states labeled 1 - 8 exhibiting the significant heterogeneity within Lkb1 mutant tumors. Pathway analysis revealed that cancer cell states 5 and 8 have high expression of an Inflammatory Response pathway and genes associated with stemness. Furthermore, states 5 and 8 lacked expression of Nkx2-1 reflecting a dedifferentiated state compared to state 1 which had high expression of Nkx2-1 and a gene signature associated with alveolar type 2 (AT2) cells. Notably, deletion of Lif and Lifr resulted in the selective elimination of these inflammatory cancer stem cell states. Differential gene analysis identified that cell state 5 highly expressed the gene Sox17. Given that the transcription factor Sox17 was specifically expressed in the inflammatory cancer stem cells, we questioned whether Sox17 was driving the inflammatory and immunosuppressive nature of Lkb1 mutant tumors. Deletion of Sox17 in Lkb1 mutant tumors resulted in a significant decrease in tumor burden. Furthermore, loss of Sox17 also led to a reduction in cytokines such as IL6, CSF3, and CCL2 as well as an increase in CD4 and CD8 T cell function. From a clinical perspective, LKB1 mutant LUAD reflects an aggressive and treatment resistant subtype of lung cancer. To evaluate the therapeutic potential of targeting LIF in Lkb1 mutant tumors, we initiated Lkb1 mutant lung tumors using our KP LUAD GEMM. Eight weeks after tumor initiation we treated tumor bearing mice with an anti-LIF neutralizing antibody for three weeks. Tumor burden was significantly reduced with anti-LIF therapy demonstrating that targeting LIF is effective in impairing tumorigenesis in established tumors. ExCITE-seq analysis confirmed that anti-LIF therapy selectively eliminated the Sox17+ cancer cell state supporting the notion that the LIF is needed for the maintenance of this subpopulation. With the loss of the inflammatory cancer stem cells, the immunosuppressive TME was reversed with anti-LIF therapy as demonstrated by a decrease in SiglecFHi neutrophils and Arg1+ interstitial macrophages along with a reduction of cytokines such as IL6, CSF3, and CCL2. To establish the importance of T cells on the effect of LIF neutralization, we treated mice bearing Lkb1 mutant lung tumors with a combination of anti-LIF antibody, anti-CD4/anti-CD8 antibodies, or the combination of these antibodies. Anti-LIF therapy was ineffective in the absence of T cells, demonstrating that the inhibitory effect of LIF neutralization on tumor growth is T cell mediated. In summary, we established through a comprehensive mechanistic analysis of the TME of lung tumors derived from a KP GEMM, that Lkb1 mutant lung tumors transcriptionally upregulate the cytokine Lif. These tumors harness autocrine LIF signaling to induce tumor plasticity and nurture an inflammatory cancer stem cell subpopulation driven by Sox17. These cancer stem cells produce cytokines that remodel the TME with immunosuppressive SiglecFHi neutrophils and Arg1+ interstitial macrophages that dampen anti-tumor T cell function. Targeting LIF signaling is a novel therapeutic approach to reverse the immunosuppressive nature of LKB1 mutant lung tumors, enhance anti-tumor immune response, and ultimately impair tumor growth. Citation Format: Ray Pillai. Autocrine LIF signaling: A novel therapeutic target to eliminate inflammatory and immunosuppressive cancer stem cells in LKB1 mutant lung adenocarcinoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (8Suppl): Abstract nr NG06.