Summary Advanced basal cell carcinomas (BCCs) frequently acquire resistance to Smoothened (SMO) inhibitors through unknown mechanisms. Here, we identify SMO mutations in 50% (22/44) of resistant BCCs and show that these mutations maintain Hedgehog signaling in the presence of SMO inhibitors. Alterations include four ligand binding pocket mutations defining sites of inhibitor binding and four variants confering constitutive activity and inhibitor resistance, illuminating pivotal residues that ensure receptor autoinhibition. In the presence of a SMO inhibitor, tumor cells containing either class of SMO mutants effectively outcompete cells containing the wild type SMO. Finally, we show that both classes of SMO variants respond to aPKC-ι/λ or GLI2 inhibitors that operate downstream of SMO, setting the stage for the clinical use of GLI antagonists.

Basal cell carcinoma (BCC) growth requires high levels of Hedgehog (Hh) signaling through the transcription factor Gli1. While inhibitors of membrane protein Smoothened (Smo) effectively suppress Hh signaling, early tumor resistance illustrates the need for additional downstream targets for therapy1–6. Here we identify atypical Protein Kinase C iota/lambda (aPKC) as a novel Gli regulator. aPKC and its polarity signaling partners7 colocalize at the centrosome and form a complex with Missing-in-Metastasis (MIM), a scaffolding protein that potentiates Hh signaling8,9. Genetic or pharmacological loss of aPKC function blocks Hh signaling and proliferation of BCC cells. aPKC is a Hh target gene that forms a positive feedback loop with Gli and exhibits elevated levels in BCCs. Genome-wide transcriptional profiling shows that aPKC and Smo control the expression of similar genes in tumor cells. aPKC functions downstream of Smo to phosphorylate and activate Gli1, resulting in maximal DNA binding and transcriptional activation. Activated aPKC is upregulated in Smo-inhibitor resistant tumors and targeting aPKC suppresses signaling and growth of resistant BCC cell lines. These results demonstrate aPKC is critical for Hh-dependent processes and implicates aPKC as a new, tumor-selective therapeutic target for the treatment of Smo-inhibitor resistant cancers.

Hedgehog signaling drives oncogenesis in several cancers and strategies targeting this pathway have been developed, most notably through inhibition of Smoothened. However, resistance to Smoothened inhibitors occurs via genetic changes of Smoothened or other downstream Hedgehog components. Here, we overcome these resistance mechanisms by modulating GLI transcription via inhibition of BET bromodomain proteins. We show the BET bromodomain protein, BRD4, regulates GLI transcription downstream of SMO and SUFU and chromatin immunoprecipitation studies reveal BRD4 directly occupies GLI1 and GLI2 promoters, with a substantial decrease in engagement of these sites upon treatment with JQ1, a small molecule inhibitor targeting BRD4. Globally, genes associated with medulloblastoma-specific GLI1 binding sites are downregulated in response to JQ1 treatment, supporting direct regulation of GLI activity by BRD4. Notably, patient- and GEMM-derived Hedgehog-driven tumors (basal cell carcinoma, medulloblastoma and atypical teratoid/rhabdoid tumor) respond to JQ1 even when harboring genetic lesions rendering them resistant to Smoothened antagonists.

Summary Background Asymmetric cell divisions generate daughter cells with distinct fates by polarizing fate determinants into separate cortical domains. Atypical protein kinase C (aPKC) is an evolutionarily conserved regulator of cell polarity. In Drosophila neuroblasts, apically restricted aPKC is required for segregation of neuronal differentiation factors such as Numb and Miranda to the basal cortical domain. While Numb is polarized by direct aPKC phosphorylation, Miranda asymmetry is thought to occur via a complicated cascade of repressive interactions (aPKC –| Lgl –| Myosin II –| Miranda). Results Here we provide biochemical, cellular, and genetic data to show that aPKC directly phosphorylates Miranda to exclude it from the cortex and Lgl antagonizes this activity. Miranda is phosphorylated by aPKC at several sites in its cortical localization domain and phosphorylation is necessary and sufficient for cortical displacement suggesting that the repressive cascade model is incorrect. In investigating key results that led to this model, we found that Y-27632, a Rho Kinase inhibitor used to implicate Myosin II, efficiently inhibits aPKC. Lgl3A, a non-phosphorylatable Lgl variant used to implicate Lgl in this process, inhibits the formation of apical aPKC crescents in neuroblasts. Furthermore, Lgl directly inhibits aPKC kinase activity. Conclusions Miranda polarization during neuroblast asymmetric cell division occurs by displacement from the apical cortex by direct aPKC phosphorylation. Lgl does not mediate Miranda cortical displacement but instead promotes Par-6/aPKC asymmetry by directly inhibiting aPKC. The role of Myosin II in this process, if any, is unknown.

How stem cells give rise to epidermis is unclear despite the crucial role the epidermis plays in barrier and appendage formation. Here we use single cell-RNA sequencing to interrogate basal stem cell heterogeneity of human interfollicular epidermis and find four spatially distinct stem cell populations at the top and bottom of rete ridges and transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling suggests that basal cell populations serve as crucial signaling hubs to maintain epidermal communication. Combining pseudotime, RNA velocity, and cellular entropy analyses point to a hierarchical differentiation lineage supporting multi-stem cell interfollicular epidermal homeostasis models and suggest that transitional basal stem cells are stable states essential for proper stratification. Finally, alterations in differentially expressed transitional basal stem cell genes result in severe thinning of human skin equivalents, validating their essential role in epidermal homeostasis and reinforcing the critical nature of basal stem cell heterogeneity.