Abstract 169: Tumor heterogeneity and differential pathway activation in melanoma: insights from spatial profiling of AXL and NGFR upregulated regions

Abstract Tumor heterogeneity remains an obstacle in the treatment of melanoma, with unique tumor microenvironments contributing to therapy resistance and differential tumor growth. Dedifferentiated melanoma states have been associated with resistance to targeted and immunotherapy. AXL and NGFR are dedifferentiation markers linked to these states; however, the mechanisms by which they interact or result in distinct tumor programs remain unclear. Both AXL and NGFR have been linked to therapy resistance. AXL has been associated with cell survival, proliferation and immune suppression and NGFR with tumor progression, invasion, metastasis and therapy resistance. Understanding these interactions is critical for identifying therapeutic strategies and improving patient outcomes. In a melanoma patient with longitudinal tumor samples, we used highly multiplexed cyclic immunofluorescence (CyCIF) and matched H&E to study the tumor and immune states associated with progression. Spatial transcriptomic profiling was performed using GeoMX on selected regions of interest to further study tumor states characterized by different morphology and proteomic differentiation markers. In the primary and in-transit metastasis, we observed intratumoral heterogeneity, with histologically distinct tumor nodules. CyCIF of the in-transit sample showed upregulation of AXL in one nodule and NGFR in the other. The NGFR nodule was characterized by densely packed tumor cells, while the AXL nodule featured an inner cleft region encased by dense peripheral tumor cells. In the NGFR nodule, differential expression analysis (DEA) revealed upregulation of EPAS1, HIF1A, ERBB3 and VEGFA suggesting hypoxic stress, cell signaling, active angiogenesis and cell migration. Transcription factor analysis (TFA) showed upregulation of HDGF, TGFB1I1, HOXA7, IRF6 and SRSF2, TFs associated with proliferation, differentiation and increased translational activity. Downregulated HIP2K and PAWR suggest apoptosis inhibition, further facilitating tumor survival and expansion. In the AXL nodule, DEA revealed increased CDKN1A, indicating stress response and altered cell cycle regulation. Gene set enrichment analysis (GSEA) highlighted WNT and Hedgehog signaling, hormonal responses (e.g., estrogen signaling), and metabolic alterations (bile acid metabolism, myogenesis). EMT upregulation suggested a mesenchymal-like phenotype associated with resistance. TFA showed elevated KLF8 and TGFB1I1 driving EMT and metastasis, while SRSF2, HSF4, CXXC1, and NR1H2 suggested stress resilience, apoptosis regulation, and ER stress mitigation. Distinct signaling in AXL and NGFR regions suggests NGFR drives metabolic activity and proliferation, while AXL promotes resilience via stress and hormonal signaling. These findings offer insights into therapies targeting melanoma heterogeneity and resistance. Citation Format: Mark Woodnorth, Giuseppe Tarantino, Anne Zaremba, Tuulia Vallius, Florian Rambow, Lisa Zimmer, Antje Sucker, Elisabeth Livingstone, Eva Hadaschik, Roxanne Pelletier, Yingxiao Shi, Mariana Lopez, Samira Makhzami, Christine Lian, George Murphy, Genevieve Boland, Peter Sorger, Dirk Schadendorf, David Liu. Tumor heterogeneity and differential pathway activation in melanoma: insights from spatial profiling of AXL and NGFR upregulated regions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 169.