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Spatial transcriptomic technologies and spatially annotated single-cell RNA sequencing datasets provide unprecedented opportunities to dissect cell–cell communication (CCC). However, incorporation of the spatial information and complex biochemical processes required in the reconstruction of CCC remains a major challenge. Here, we present COMMOT (COMMunication analysis by Optimal Transport) to infer CCC in spatial transcriptomics, which accounts for the competition between different ligand and receptor species as well as spatial distances between cells. A collective optimal transport method is developed to handle complex molecular interactions and spatial constraints. Furthermore, we introduce downstream analysis tools to infer spatial signaling directionality and genes regulated by signaling using machine learning models. We apply COMMOT to simulation data and eight spatial datasets acquired with five different technologies to show its effectiveness and robustness in identifying spatial CCC in data with varying spatial resolutions and gene coverages. Finally, COMMOT identifies new CCCs during skin morphogenesis in a case study of human epidermal development.

Figure S1. Growth and survival of SMB cells depends on Shh signaling. Figure S2. SMB cell lines are a faithful model to study resistance to Smo inhibitors. Table S1. List of candidate genes with mutations used in the study. Figure S3. RAS signaling confers resistance to Smo inhibitor LDE225. Figure S4. FGF/RAS signaling abrogates dependence on Shh signaling. Figure S5. FGF/RAS-driven resistance to Smo inhibition is reversible and can be treated with MEK inhibitors. Figure S6. MEK inhibitors have therapeutic potential for HRAS SMB cells. Figure S7. Morphology of SMB(HRAS) cells differs from SMB parental cells. Figure S8. Immunohistochemistry of SFRP1, pERK and FGF in primary and metastatic samples from human Shh-subtype medulloblastoma patients. Table S2. Clinical diagnosis for human patient samples shown in Figure 5.

Several methods for generating human skin equivalent (HSE) organoid cultures are regularly used to study skin biology and test pharmaceuticals, however few studies have thoroughly characterized these systems. To fill this gap, we used single cell-RNA sequencing to compare the cellular states of in vitro HSEs generated from distinct culture methods, HSEs xenografted onto mice, and in vivo epidermis. By combining differential gene expression, pseudotime analyses, splicing kinetics, and spatial localization, we reconstructed HSE keratinocyte differentiation trajectories that recapitulated known in vivo epidermal differentiation pathways and show that HSEs contain many of the major in vivo cellular states. However, HSEs also develop several unique keratinocyte states, an expanded basal stem cell program, and disrupted terminal differentiation. In addition, cell-cell communication modeling showed the presence of EMT-associated signaling pathways not normally active in homeostatic skin and we show that EGF supplementation influences the EMT signature. Lastly, xenografted HSEs at early timepoints post-transplantation significantly rescued many of the observed in vitro deficits, while undergoing a hypoxic response that drove an alternative differentiation lineage. This study highlights the strengths and limitations of organoid cultures and identifies areas for potential innovation.

How basal cell carcinoma (BCC) interacts with its tumor microenvironment to promote growth is unclear. We use singe-cell RNA sequencing to define the human BCC ecosystem and discriminate between normal and malignant epithelial cells. We identify spatial biomarkers of tumors and their surrounding stroma that reinforce the heterogeneity of each tissue type. Combining pseudotime, RNA velocity–PAGA, cellular entropy, and regulon analysis in stromal cells reveals a cancer-specific rewiring of fibroblasts, where STAT1, TGF-β, and inflammatory signals induce a noncanonical WNT5A program that maintains the stromal inflammatory state. Cell-cell communication modeling suggests that tumors respond to the sudden burst of fibroblast-specific inflammatory signaling pathways by producing heat shock proteins, whose expression we validated in situ. Last, dose-dependent treatment with an HSP70 inhibitor suppresses in vitro vismodegib-resistant BCC cell growth, Hedgehog signaling, and in vivo tumor growth in a BCC mouse model, validating HSP70’s essential role in tumor growth and reinforcing the critical nature of tumor microenvironment cross-talk in BCC progression.

Advanced basal cell carcinomas (BCCs) are driven by the Hedgehog (HH) pathway and often possess inherent resistance to SMO inhibitors. Identifying and targeting pathways that bypass SMO could provide alternative treatments. Here, we use a combination of RNA-sequencing analysis of advanced BCC tumor-normal pairs and immunostaining of human and mouse BCC samples to identify an MTOR signature in BCC. Pharmacological inhibition of MTOR activity in BCC cells significantly reduces cell proliferation without affecting HH signaling. Similarly, treatment ofPtch1fl/fl;Gli1-CreERT2mice with everolimus reduces tumor growth and aPKC activity, suggesting that MTOR promotes tumor growth by activating aPKC and demonstrating that suppressing MTOR could be a promising target for BCC patients.

Tissues and organs are composed of many diverse cell types, making cell-specific gene expression profiling a major challenge. Herein we report that endogenous enzymes, unique to a cell of interest, can be utilized to enable cell-specific metabolic labeling of RNA. We demonstrate that appropriately designed "caged" nucleosides can be rendered active by serving as a substrate for cancer-cell specific enzymes to enable RNA metabolic labeling, only in cancer cells. We envision that the ease and high stringency of our approach will enable expression analysis of tumor cells in complex environments.

How basal cell carcinoma (BCC) interacts with its tumor microenvironment to promote growth is unclear. Here we use singe-cell RNA sequencing to define the human BCC ecosystem and discriminate between normal and malignant epithelial cells. We identify spatial biomarkers of both tumors and their surrounding stroma that reinforce the heterogeneity of each tissue type. Combining pseudotime, RNA velocity, cellular entropy, and regulon analysis in stromal cells reveal a cancer-specific rewiring of fibroblasts where STAT1, TGF-β, and inflammatory signals induce a non-canonical WNT5A program that maintains the stromal inflammatory state. Cell-cell communication modeling suggests that tumors respond to the sudden burst of fibroblast-specific inflammatory signaling pathways by producing heat shock proteins, which we validated in situ. Finally, dose-dependent treatment with an HSP70 inhibitor suppresses in vitro BCC cell growth and Hedgehog signaling and in vivo tumor growth in a BCC mouse model, validating HSP70’s essential role in tumor growth and reinforcing the critical nature of tumor microenvironment crosstalk in BCC progression.

Psoriasis is a common inflammatory skin disease characterized by aberrant inflammation and epidermal hyperplasia. Molecular mechanisms that regulate psoriasis-like skin inflammation remain to be fully understood. Here, we show that the expression of Ovol1 (encoding ovo-like 1 transcription factor) is upregulated in psoriatic skin, and its deletion results in aggravated psoriasis-like skin symptoms following stimulation with imiquimod. Using bulk and single-cell RNA sequencing, we identify molecular changes in the epidermal, fibroblast, and immune cells of Ovol1-deficient skin that reflect an altered course of epidermal differentiation and enhanced inflammatory responses. Furthermore, we provide evidence for excessive full-length IL-1α signaling in the microenvironment of imiquimod-treated Ovol1-deficient skin that functionally contributes to immune cell infiltration and epidermal hyperplasia. Collectively, our study uncovers a protective role for OVOL1 in curtailing psoriasis-like inflammation and the associated skin pathology.

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 carcinoma (BCC) cells with increased nuclear myocardin-related transcription factor (nMRTF) levels are resistant to Smoothened inhibitors, however how MRTF activity is regulated is still elusive. Here, the authors identify a LYPD3+/TACSTD2+/LY6D+ BCC subpopulation of resistant BCC cells where AP-1 and TGFß drive nMRTF activation and amplify noncanonical Gli1 activity

The advent of immune checkpoint therapy for metastatic skin cancer has greatly improved patient survival. However, most skin cancer patients are refractory to checkpoint therapy, and furthermore the differential intra-immune signaling between cancers driving the response to checkpoint therapy remains uncharacterized. When comparing the immune transcriptome in the tumor microenvironment of melanoma and basal cell carcinoma (BCC), we found that the presence of memory B cells and macrophages negatively correlate when stratifying patients by response, with memory B cells more present in responders. Inhibitory immune signaling is reduced in melanoma responders and is increased in BCC responders. We further explored the relationships between macrophages, B cells and response to checkpoint therapy by developing a stochastic differential equation model which qualitatively agrees with the data analysis. Our model predicts BCC to be more refractory to checkpoint therapy than melanoma. We show differences in tumor progression and regression that could serve as a diagnostic and predict the optimal ratio of macrophages and memory B cells for successful treatment.

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., The mechanisms regulating stem cells to give rise to human interfollicular epidermis are unclear. Here, the authors use single cell RNA sequencing to identify heterogeneity within the human neonatal interfollicular epidermis and distinct spatial positioning of at least four basal stem cell populations.

Primary cilia loss is a common feature of advanced cancers. While primary cilia are necessary to initiate Hedgehog (HH)-driven cancers, how HH pathway activity is maintained in advanced cancers devoid of primary cilia is unclear. Here, we find that HH-driven basal cell carcinoma (BCC) accumulate mutations in the Alström and Usher syndrome genes in advanced and SMO inhibitorresistant tumors. Loss of Alström and Usher syndrome gene expression, which are common underlying causes of deafness and blindness, suppresses ciliogenesis and HH signaling. Atypical protein kinase C iota/lambda (aPKC) is a GLI1 kinase with higher expression in advanced BCCs and we show that a constitutively active isoform drives HH pathway activity and mutually antagonizes primary cilia. Overexpression of the constitutively active aPKC variant can maintain HH pathway activity in the absence of primary cilia and can drive resistance to the SMO antagonist vismodegib regardless of cilia status. Finally, superficial BCCs display less primary cilia and higher aPKC expression, which is inversely correlated in nodular BCC subtypes. Our results suggest aPKC may serve as a biomarker for SMO inhibitor sensitivity and a target for clinical application.

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.

Actin polymerization is important to generate primary cilia. Drummond et al. show that upstream actin regulators are necessary for this process by controlling aPKC and Src kinase activity to promote Hedgehog signaling and restrict primary cilia., Primary cilia are polarized organelles that allow detection of extracellular signals such as Hedgehog (Hh). How the cytoskeleton supporting the cilium generates and maintains a structure that finely tunes cellular response remains unclear. Here, we find that regulation of actin polymerization controls primary cilia and Hh signaling. Disrupting actin polymerization, or knockdown of N-WASp/Arp3, increases ciliation frequency, axoneme length, and Hh signaling. Cdc42, a potent actin regulator, recruits both atypical protein pinase C iota/lambda (aPKC) and Missing-in-Metastasis (MIM) to the basal body to maintain actin polymerization and restrict axoneme length. Transcriptome analysis implicates the Src pathway as a major aPKC effector. aPKC promotes whereas MIM antagonizes Src activity to maintain proper levels of primary cilia, actin polymerization, and Hh signaling. Hh pathway activation requires Smoothened-, Gli-, and Gli1-specific activation by aPKC. Surprisingly, longer axonemes can amplify Hh signaling, except when aPKC is disrupted, reinforcing the importance of the Cdc42–aPKC–Gli axis in actin-dependent regulation of primary cilia signaling.

Basosquamous carcinoma (BSC) is a rare form of skin cancer with both basaloid and squamous morphology. Chiang et al. (2019) genetically define BSCs and demonstrate that BSCs likely originate as basal cell carcinomas that partially squamatize through accumulation of ARID1A mutations and RAS/MAPK pathway activation.

Stringent chemical methods to profile RNA expression within discrete cellular populations remains a key challenge in biology. To address this issue, we developed a chemical-genetic strategy for metabolic labeling of RNA. Cell-specific labeling of RNA can be profiled and imaged using bioorthogonal chemistry. We anticipate that this platform will provide the community with a much-needed chemical toolset for cell-type specific profiling of cell-specific transcriptomes derived from complex biological systems.

Sporadic and basal cell nevus syndrome basal cell carcinomas show differential response rates to Smoothened inhibitors. Chiang et al. demonstrate notable decreases in UV-induced mutagenesis, total mutation load, genomic instability, and drug-resistant mutations among basal cell nevus syndrome basal cell carcinomas using whole exome sequencing, which may explain the differences in drug response rates.

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.

How stem cells give rise to human interfollicular 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 at least four spatially distinct stem cell populations that decorate the top and bottom of rete ridge architecture and hold transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling through co-variance of cognate ligand-receptor pairs indicate that the basal cell populations distinctly serve as critical signaling hubs that 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 the “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.

The genetically heterogeneous spinocerebellar ataxias (SCAs) are caused by Purkinje neuron dysfunction and degeneration, but their underlying pathological mechanisms remain elusive. The Src family of nonreceptor tyrosine kinases (SFK) are essential for nervous system homeostasis and are increasingly implicated in degenerative disease. Here we reveal that the SFK suppressor Missing-in-metastasis (MTSS1) is an ataxia locus that links multiple SCAs. MTSS1 loss results in increased SFK activity, reduced Purkinje neuron arborization, and low basal firing rates, followed by cell death. Surprisingly, mouse models for SCA1, SCA2, and SCA5 show elevated SFK activity, with SCA1 and SCA2 displaying dramatically reduced MTSS1 protein levels through reduced gene expression and protein translation, respectively. Treatment of each SCA model with a clinically approved Src inhibitor corrects Purkinje neuron basal firing and delays ataxia progression in MTSS1 mutants. Our results identify a common SCA therapeutic target and demonstrate a key role for MTSS1/SFK in Purkinje neuron survival and ataxia progression.

The genetically heterogeneous Spinocerebellar ataxias (SCAs) are caused by Purkinje neuron dysfunction and degeneration, but their underlying pathological mechanisms remain elusive. The Src family of non-receptor tyrosine kinases (SFK) are essential for nervous system homeostasis and are increasingly implicated in degenerative disease. Here we reveal that the SFK suppressor Missing-in-Metastasis (MTSS1) is a novel ataxia locus that links multiple SCAs. MTSS1 loss results in increased SFK activity, reduced Purkinje neuron arborization, and low basal firing rates, followed by cell death. Surprisingly, mouse models for SCA1, SCA2, and SCA5 show elevated SFK activity, with SCA1 and SCA2 displaying dramatically reduced MTSS1 protein levels through reduced gene expression and protein translation, respectively. Treatment of each SCA model with a clinically-approved Src inhibitor corrects Purkinje basal firing, and delays ataxia progression in MTSS1 mutants. Our results identify a common SCA therapeutic target and demonstrate a key role for MTSS1/SFK in Purkinje neuron survival and ataxia progression.Significance StatementThe Src family of non-receptor tyrosine kinases (SFK) are essential for nervous system function, and may contribute to neurodegeneration. Spinocerebellar ataxias (SCAs) are neurodegenerative diseases where Purkinje neurons fire irregularly and degenerate leading to motor problems. We show that the SFK suppressor Missing-in-Metastasis (MTSS1) is a novel ataxia gene that links multiple SCAs. MTSS1 loss results in increased SFK activity, degenerating Purkinje neurons with low basal firing rates, and cell death. Surprisingly, mouse models for three different SCAs show elevated SFK activity, with SCA1 and SCA2 models displaying dramatically reduced MTSS1 protein levels. Treatment of each SCA model with SFK inhibitor corrects Purkinje basal firing, and delays ataxia progression in MTSS1 mutants. Our results identify a common link among disparate neurodegenerative diseases.

SummaryAdvanced basal cell carcinomas (BCCs) frequently acquire resistance to Smoothened (SMO) inhibitors through unknown mechanisms. Here we identify SMO mutations in 50% (22 of 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 conferring 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.

Advanced basal cell carcinomas (BCCs) circumvent Smoothened (SMO) inhibition by activating GLI transcription factors to sustain the high levels of Hedgehog (HH) signaling required for their survival. Unfortunately, there is a lack of efficacious therapies. We performed a gene expression-based drug repositioning screen in silico and identified the FDA-approved histone deacetylase (HDAC) inhibitor, vorinostat, as a top therapeutic candidate. We show that vorinostat only inhibits proliferation of BCC cells in vitro and BCC allografts in vivo at high dose, limiting its usefulness as a monotherapy. We leveraged this in silico approach to identify drug combinations that increase the therapeutic window of vorinostat and identified atypical PKC Ɩ/ʎ (aPKC) as a HDAC costimulator of HH signaling. We found that aPKC promotes GLI1-HDAC1 association in vitro, linking two positive feedback loops. Combination targeting of HDAC1 and aPKC robustly inhibited GLI1, lowering drug doses needed in vitro, in vivo, and ex vivo in patient-derived BCC explants. We identified a bioavailable and selective small-molecule aPKC inhibitor, bringing the pharmacological blockade of aPKC and HDAC1 into the realm of clinical possibility. Our findings provide a compelling rationale and candidate drugs for combined targeting of HDAC1 and aPKC in HH-dependent cancers.

To identify genes that promote facial skin youthfulness (SY), a genome-wide association study on an Ashkenazi Jewish discovery group (n=428) was performed using Affymetrix 6.0 Single-Nucleotide Polymorphism (SNP) Array. After SNP quality controls, 901,470 SNPs remained for analysis. The eigenstrat method showed no stratification. Cases and controls were identified by global facial skin aging severity including intrinsic and extrinsic parameters. Linear regression adjusted for age and gender, with no significant differences in smoking history, body mass index, menopausal status, or personal or family history of centenarians. Six SNPs met the Bonferroni threshold with P allele

The Hedgehog pathway is a potent regulator of cellular growth and plays a central role in the development of many cancers including basal cell carcinoma (BCC). The majority of BCCs arise from mutations in the Patched receptor resulting in constitutive activation of the Hedgehog pathway. Secondary driver mutations promote BCC oncogenesis and occur frequently due to the high mutational burden resulting from sun exposure of the skin. Here, we uncover novel secondary mutations in Suppressor of Fused (SUFU), the major negative regulator of the Hedgehog pathway. SUFU normally binds to a Hedgehog transcriptional activator, GLI1, in order to prevent it from initiating transcription of Hedgehog target genes. We sequenced tumor-normal pairs from patients with early sporadic BCCs. This resulted in the discovery of nine mutations in SUFU, which were functionally investigated to determine whether they help drive BCC formation. Our results show that four of the SUFU mutations inappropriately activate the Hedgehog pathway, suggesting they may act as driver mutations for BCC development. Indeed, all four of the loss of function SUFU variants were found to disrupt its binding to GLI, leading to constitutive pathway activation. Our results from functional characterization of these mutations shed light on SUFU’s role in Hedgehog signaling, tumor progression, and highlight a way in which BCCs can arise.

Patched (Ptch) receptors are critical negative regulators of Hedgehog signaling, where Ptch1 loss causes basal cell carcinoma and Ptch1;Ptch2 loss disrupts skin and hair follicle development. Adolphe et al. use single molecule fluorescent in situ hybridization to show quantitatively that Ptch receptors create a Hedgehog signaling gradient that may specify hair follicle development.

Most cutaneous basal cell carcinomas (BCCs) are successfully treated by surgical resection. However, locally advanced (unresectable) or metastatic BCC (mBCC) have a poor prognosis, with a mean survival ranging from 8 months to 3.6 years.1 Basal cell carcinomas require the hedgehog (HH) pathway for growth. Hedgehog pathway inhibitors, such as vismodegib2 and sonidegib phosphate, target the G-protein–coupled receptor Smoothened (SMO) and are recommended as first-line treatment for advanced BCC or mBCC by the National Comprehensive Cancer Network.3 The emergence of resistance has limited vismodegib's efficacy2,4 and led the search for therapeutics downstream from SMO. Investigators5 found that 50% of SMO inhibitor–resistant BCCs had SMO mutations with disruption of ligand responsiveness or release of autoinhibition. Itraconazole, a widely used oral antifungal agent, is a potent HH pathway antagonist that suppresses BCC carcinogenesis and reduces messenger RNA (mRNA) expression of the GLI family zinc finger 1 (GLI1 [OMIM 165220]) gene in murine BCCs6 and in patients with nonadvanced cutaneous BCCs.7 Arsenic trioxide, which is approved by the US Food and Drug Administration for treatment of acute promyelocytic leukemia, inhibits the HH pathway downstream from SMO by preventing ciliary trafficking and destabilizing GLI2. Arsenic trioxide can bypass acquired mutations in SMO found in resistant tumors.8 Combined treatment with arsenic trioxide and itraconazole (ATO-ITRA) has an additive HH inhibitory effect shown to be effective in murine BCCs.9 To assess the HH inhibitory effect and clinical efficacy of ATO-ITRA in human BCC, we performed an open-label proof-of-concept, phase 2 biomarker trial in patients with mBCC whose tumors were clinically resistant to SMO inhibitors.10 The primary goal of our study was to determine whether administration of intravenous (IV) arsenic trioxide and oral itraconazole in patients with mBCC is associated with a reduction in GLI1 mRNA expression in tumor and/or normal skin biopsy samples. Secondary end points included the evaluation of tumor response, safety profile, and presence of HH pathway mutations in SMO inhibitor–resistant BCCs before treatment.

SummaryThe primary cilium is critical for transducing Sonic hedgehog (Shh) signaling, but the mechanisms of its transient assembly are poorly understood. Previously we showed that the actin regulatory protein Missing-in-Metastasis (MIM) regulates Shh signaling, but the nature of MIM's role was unknown. Here we show that MIM is required at the basal body of mesenchymal cells for cilia maintenance, Shh responsiveness, and de novo hair follicle formation. MIM knockdown results in increased Src kinase activity and subsequent hyperphosphorylation of the actin regulator Cortactin. Importantly, inhibition of Src or depletion of Cortactin compensates for the cilia defect in MIM knockdown cells, whereas overexpression of Src or phospho-mimetic Cortactin is sufficient to inhibit ciliogenesis. Our results suggest that MIM promotes ciliogenesis by antagonizing Src-dependent phosphorylation of Cortactin and describe a mechanism linking regulation of the actin cytoskeleton with ciliogenesis and Shh signaling during tissue regeneration.

HHS Public Access Author manuscript Author Manuscript J Invest Dermatol. Author manuscript; available in PMC 2016 February 01. Published in final edited form as: J Invest Dermatol. 2015 August ; 135(8): 2138–2141. doi:10.1038/jid.2015.115. Rolling the genetic dice: neutral and deleterious Smoothened mutations in drug-resistant basal cell carcinoma Scott X. Atwood 1 , Kavita Y. Sarin 1 , Jiang R. Li 1 , Catherine Yao 1 , Nicole M. Urman 1 , Anne Lynn S. Chang 1 , Jean Y. Tang 1 , and Anthony E. Oro 1 1 Program in Epithelial Biology and Department of Dermatology, Stanford University School of Medicine, Stanford, CA, USA Author Manuscript To the editor Author Manuscript Basal cell carcinomas (BCCs) are locally invasive epithelial tumors that are caused by activating mutations in the Hedgehog (HH) pathway, typically through the loss of the receptor Patched1 or by activating the G-protein coupled receptor Smoothened (SMO). Genomic analysis by our group and others have revealed that BCCs are typically diploid and carry a high frequency of non-silent single nucleotide variants (SNVs) compared to other cutaneous and non-cutaneous tumors (Alexandrov et al. 2013; Atwood et al. 2014; Atwood et al. 2015; Jayaraman et al. 2014). Given their high mutational load, how these variants confer selective tumor growth without deleterious effects remains poorly understood. We previously identified and functionally validated nine SMO mutations that drive the majority of drug resistance in BCC through two distinct mechanisms that maintain HH signaling in the presence of drug: induction of constitutive activity or disruption of ligand binding (Atwood et al. 2015). However, SMO mutations with unclear function are frequently found across many HH and non-HH dependent cancers with drug-resistant BCCs bearing the highest rate of recurrent mutations at 66% (Figure 1a). Author Manuscript To determine how these additional SMO mutations promote tumor growth, we identified 28 mutations through our genomic analysis of 44 drug-resistant and 36 sporadic BCC that were either recurrent, found to overlap with the COSMIC database, or were regional-specific (ligand binding pocket or pivot region) and interrogated their ability to promote HH signaling (Figure 1b, c). We expressed wildtype human SMO (SMO-WT) or SMO mutants in Smo-null mouse embryonic fibroblasts (MEFs) to assess the ability of these variants to activate the HH pathway with or without ligand. SMO-W535L is a known constitutively active mutant that was present in many of our tumor samples and significantly increased basal HH activity in the absence of HH ligand as determined by mRNA levels of the HH target gene Gli1 (Figure 2a). No other SMO variant induced constitutive activity, including SMO-WT and the known ligand binding pocket mutant SMO-D473G (Yauch et al. 2009), Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Address Correspondence to: Anthony E. Oro, oro@stanford.edu, 269 Campus Drive, CCSR 2140, Stanford, CA 94305, T: (650) 723-7843 F: (650) 725-8762. Conflict of Interest: AO and AC are investigators in Genentech, Novartis, and Eli Lilly clinical trials. JT is a consultant to Genentech.

Aberrant Shh signaling promotes tumor growth in diverse cancers. The importance of Shh signaling is particularly evident in medulloblastoma and basal cell carcinoma (BCC), where inhibitors targeting the Shh pathway component Smoothened (Smo) show great therapeutic promise. However, the emergence of drug resistance limits long-term efficacy, and the mechanisms of resistance remain poorly understood. Using new medulloblastoma models, we identify two distinct paradigms of resistance to Smo inhibition. Sufu mutations lead to maintenance of the Shh pathway in the presence of Smo inhibitors. Alternatively activation of the RAS–MAPK pathway circumvents Shh pathway dependency, drives tumor growth, and enhances metastatic behavior. Strikingly, in BCC patients treated with Smo inhibitor, squamous cell cancers with RAS/MAPK activation emerged from the antecedent BCC tumors. Together, these findings reveal a critical role of the RAS–MAPK pathway in drug resistance and tumor evolution of Shh pathway–dependent tumors. Cancer Res; 75(17); 3623–35. ©2015 AACR.

Hedgehog signaling drives oncogenesis in several cancers, and strategies targeting this pathway have been developed, most notably through inhibition of Smoothened (SMO). However, resistance to Smoothened inhibitors occurs by genetic changes of Smoothened or other downstream Hedgehog components. Here we overcome these resistance mechanisms by modulating GLI transcription through inhibition of bromo and extra C-terminal (BET) bromodomain proteins. We show that BRD4 and other BET bromodomain proteins regulate GLI transcription downstream of SMO and suppressor of fused (SUFU), and chromatin immunoprecipitation studies reveal that BRD4 directly occupies GLI1 and GLI2 promoters, with a substantial decrease in engagement of these sites after 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 (genetically engineered mouse model)-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. Altogether, our results reveal BET proteins as critical regulators of Hedgehog pathway transcriptional output and nominate BET bromodomain inhibitors as a strategy for treating Hedgehog-driven tumors with emerged or a priori resistance to Smoothened antagonists. © 2014 Nature America, Inc. All rights reserved.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

080 Mutational landscape of MCPyV-positive and MCPyV-negative Merkel cell carcinomas with implications for immunotherapy T Walradt, V Makarov, R Doumani, N Riaz, K Stafstrom, A Moshiri, L Yelistratova, J Levinsohn, T Chan, P Nghiem, R Lifton and J Choi 1 Dermatology, Northwestern University, Chicago, IL, 2 Dermatology, Yale University, New Haven, CT, 3 Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, 4 Dermatology, University of Washington, Seattle, WA, 5 Genetics, Yale University, New Haven, CT and 6 Biochemistry and Molecular Genetics, Northwestern University, Chicago, IL Merkel cell carcinoma (MCC) is a rare but highly aggressive cutaneous neuroendocrine carcinoma, associated with the merkel cell polyomavirus (MCPyV) in 80% of cases. To define the genetic basis of MCCs, we performed exome sequencing of 49 MCCs. We show that MCPyV-negative MCCs have a high mutation burden (median of 1121 Somatic Single Nucleotide Variants (SSNVs) per-exome with frequent mutations in RB1 and TP53 and additional damaging mutations in genes in the chromatin modification (ASXL1, MLL2, and MLL3), JNK (MAP3K1 and TRAF7), and DNA-damage pathways (ATM, MSH2, and BRCA1). In contrast, MCPyV-positive MCCs harbor few SSNVs (median of 12.5 SSNVs/tumor) with none in the genes listed above. In both subgroups, there are rare cancer-promoting mutations predicted to activate the PI3K pathway (HRAS, KRAS, PIK3CA, PTEN, and TSC1) and to inactivate the NOTCH pathway (NOTCH1 and NOTCH2). Virus-negative MCCs harbor potentially targetable gain-of-function mutations in TP53 at p.R248 and p.P278. Moreover, TP53 mutational status predicts death in early stage MCC (5-year survival in TP53 mutant vs wild-type stage I and II MCCs is 20% vs. 92%, respectively; P1⁄40.0036). Lastly, we identified the tumor neoantigens in MCPyV-negative and MCPyV-positive MCCs. We found that virusnegative MCCs harbor more tumor neoantigens than melanomas or non-small cell lung cancers (median of 173, 65, and 111 neoantigens/sample, respectively), two cancers for which immune checkpoint blockade can produce durable clinical responses. Collectively, these data support the use of immunotherapies for virus-negative MCCs. 081 Resistant basal cell carcinomas require SRF/MRTF to maintain hedgehog pathway activation and tumor growth R Whitson, A Lee, N Urman, J Li, A Mirza, A Brown, C Yao, G Shankar, M Fry, SX Atwood, E Epstein, J Tang and AE Oro 1 Dermatology, Stanford, Stanford, CA, 2 Childrens Hospital Oakland Research Institute, Oakland, CA and 3 UC Irvine, Irvine, CA Basal cell carcinoma (BCC) and other hedgehog-dependent cancers commonly acquire drug resistance to the Smoothened (Smo) inhibitor vismodegib (Atwood et al., 2014; Sekulic et al., 2015). Resistant BCCs maintain activation of the hedgehog pathway suggesting the presence of mutations at or downstream of Smo. Variants in Smo are found in w50% of resistance cases (Atwood et al., 2015b), however, many variants are non-functional (Atwood et al., 2015a) leaving tumor escape mechanisms for a large proportion of patient tumors unexplored. We recently uncovered a novel resistance mechanism using multi-dimensional genomic analysis in human and mouse resistant BCCs to identify a non-canonical hedgehog activation pathway driven by the transcription factor, serum response factor (SRF). The majority of resistant human and mouse BCCs measured acquire the active nuclear form of SRF, which forms a novel protein complex and shares chromosomal occupancy with the hedgehog transcription factor Gli1. SRF is necessary for hedgehog pathway output, and activation by the SRF coactivator myocardin-related transcription factor (MRTF) is sufficient to potentiate hedgehog activation. Remarkably, pharmacological inhibitors targeting MRTF suppress tumor growth in mice harboring resistant BCCs. Our data uncovers a novel non-canonical mechanism for hedgehog transcriptional activation through SRF/MRTF which is activated in a majority of resistant BCCs. Combined with our in vivo data, we highlight SRF/MRTF-driven transcription as a promising therapeutic target to combat resistant BCC growth.

Dependence of basal cell carcinomas and medulloblastomas on the Hedgehog pathway provides an opportunity for targeted or “personalized” therapy. The recent effectiveness and FDA approval of the first Smoothened inhibitors validates this class of agents, but has revealed drug-resistant tumor variants that bypass Smoothened inhibition. Here, we summarize the effectiveness of Hedgehog pathway inhibitors and highlight promising areas for the development of next generation drug antagonists for Hedgehog-dependent cancers.

Growth of basal cell carcinomas (BCCs) requires high levels of hedgehog (HH) signalling through the transcription factor GLI. Although inhibitors of membrane protein smoothened (SMO) effectively suppress HH signalling, early tumour resistance illustrates the need for additional downstream targets for therapy. Here we identify atypical protein kinase C ι/λ (aPKC-ι/λ) as a novel GLI regulator in mammals. aPKC-ι/λ and its polarity signalling partners co-localize at the centrosome and form a complex with missing-in-metastasis (MIM), a scaffolding protein that potentiates HH signalling. Genetic or pharmacological loss of aPKC-ι/λ function blocks HH signalling and proliferation of BCC cells. Prkci is a HH target gene that forms a positive feedback loop with GLI and exists at increased levels in BCCs. Genome-wide transcriptional profiling shows that aPKC-ι/λ and SMO control the expression of similar genes in tumour cells. aPKC-ι/λ functions downstream of SMO to phosphorylate and activate GLI1, resulting in maximal DNA binding and transcriptional activation. Activated aPKC-ι/λ is upregulated in SMO-inhibitor-resistant tumours and targeting aPKC-ι/λ suppresses signalling and growth of resistant BCC cell lines. These results demonstrate that aPKC-ι/λ is critical for HH-dependent processes and implicates aPKC-ι/λ as a new, tumour-selective therapeutic target for the treatment of SMO-inhibitor-resistant cancers.

Atypical protein kinase C (aPKC) controls cell polarity by modulating substrate cortical localization. Aberrant aPKC activity disrupts polarity, yet the mechanisms that control aPKC remain poorly understood. We used a reconstituted system with purified components and a cultured cell cortical displacement assay to investigate aPKC regulation. We find that aPKC is autoinhibited by two domains within its NH(2)-terminal regulatory half, a pseudosubstrate motif that occupies the kinase active site, and a C1 domain that assists in this process. The Par complex member Par-6, previously thought to inhibit aPKC, is a potent activator of aPKC in our assays. Par-6 and aPKC interact via PB1 domain heterodimerization, and this interaction activates aPKC by displacing the pseudosubstrate, although full activity requires the Par-6 CRIB-PDZ domains. We propose that, along with its previously described roles in controlling aPKC localization, Par-6 allosterically activates aPKC to allow for high spatial and temporal control of substrate phosphorylation and polarization.