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Tumor heterogeneity and lack of knowledge about resistant cell states remain a barrier to targeted cancer therapies. Basal cell carcinomas (BCCs) depend on Hedgehog (Hh)/Gli signaling, but can develop mechanisms of Smoothened (SMO) inhibitor resistance. We previously identified a nuclear myocardin-related transcription factor (nMRTF) resistance pathway that amplifies noncanonical Gli1 activity, but characteristics and drivers of the nMRTF cell state remain unknown. Here, we use single cell RNA-sequencing of patient tumors to identify three prognostic surface markers (LYPD3, TACSTD2, and LY6D) which correlate with nMRTF and resistance to SMO inhibitors. The nMRTF cell state resembles transit-amplifying cells of the hair follicle matrix, with AP-1 and TGFß cooperativity driving nMRTF activation. JNK/AP-1 signaling commissions chromatin accessibility and Smad3 DNA binding leading to a transcriptional program of RhoGEFs that facilitate nMRTF activity. Importantly, small molecule AP-1 inhibitors selectively target LYPD3+/TACSTD2+/LY6D+ nMRTF human BCCs ex vivo, opening an avenue for improving combinatorial therapies.

Basal cell carcinomas (BCCs) rely on Hedgehog (HH) pathway growth signal amplification by the microtubule-based organelle, the primary cilium. Despite naive tumor responsiveness to Smoothened inhibitors (Smoi), resistance in advanced tumors remains common. Although the resistant BCCs usually maintain HH pathway activation, squamous cell carcinomas with Ras/MAPK pathway activation also arise, and the molecular basis of tumor type and pathway selection are still obscure. Here, we identify the primary cilium as a critical determinant controlling tumor pathway switching. Strikingly, Smoothened inhibitor-resistant BCCs have an increased mutational load in ciliome genes, resulting in reduced primary cilia and HH pathway activation compared with naive or Gorlin syndrome patient BCCs. Gene set enrichment analysis of resistant BCCs with a low HH pathway signature showed increased Ras/MAPK pathway activation. Tissue analysis confirmed an inverse relationship between primary cilia presence and Ras/MAPK activation, and primary cilia removal in BCCs potentiated Ras/MAPK pathway activation. Moreover, activating Ras in HH-responsive cell lines conferred resistance to both canonical (vismodegib) and noncanonical (atypical protein kinase C and MRTF inhibitors) HH pathway inhibitors and conferred sensitivity to MAPK inhibitors. Our results provide insights into BCC treatment and identify the primary cilium as an important lineage gatekeeper, preventing HH-to-Ras/MAPK pathway switching.

Hedgehog pathway-dependent cancers can escape Smoothened (SMO) inhibition through mutations in genes encoding canonical hedgehog pathway components; however, around 50% of drug-resistant basal cell carcinomas (BCCs) lack additional variants of these genes. Here we use multidimensional genomics analysis of human and mouse drug-resistant BCCs to identify a noncanonical hedgehog activation pathway driven by the transcription factor serum response factor (SRF). Active SRF along with its coactivator megakaryoblastic leukemia 1 (MKL1) binds DNA near hedgehog target genes and forms a previously unknown protein complex with the hedgehog transcription factor glioma-associated oncogene family zinc finger-1 (GLI1), causing amplification of GLI1 transcriptional activity. We show that cytoskeletal activation through Rho and the formin family member Diaphanous (mDia) is required for SRF-MKL-driven GLI1 activation and for tumor cell viability. Remarkably, nuclear MKL1 staining served as a biomarker in tumors from mice and human subjects to predict tumor responsiveness to MKL inhibitors, highlighting the therapeutic potential of targeting this pathway. Thus, our study illuminates, for the first time, cytoskeletal-activation-driven transcription as a personalized therapeutic target for combatting drug-resistant malignancies.