Acquisition of Drug Resistance in Basal Cell Nevus Syndrome Tumors through Basal to Squamous Cell Carcinoma Transition.

Although basal cell carcinomas arise from ectopic Hedgehog pathway activation and can be treated with pathway inhibitors, sporadic basal cell carcinomas display high resistance rates, whereas tumors arising in patients with Gorlin syndrome with germline Patched (PTCH1) alterations are uniformly suppressed by inhibitor therapy. In rare cases, patients with Gorlin syndrome on long-term inhibitor therapy will develop individual resistant tumor clones that rapidly progress, but the basis of this resistance remains unstudied. In this study, we report a case of an SMO inhibitor-resistant tumor arising in a patient with Gorlin syndrome on suppressive SMO inhibitor for nearly a decade. Using a combination of multiomics and spatial transcriptomics, we define the tumor populations at the cellular and tissue level to conclude that Gorlin tumors can develop resistance to SMO inhibitors through the previously described basal to squamous cell carcinoma transition. Intriguingly, through spatial whole-exome genomic analysis, we nominate PCYT2, ETNK1, and the phosphatidylethanolamine biosynthetic pathway as genetic suppressors of basal to squamous cell carcinoma transition resistance. These observations provide a general framework for studying tumor evolution and provide important clinical insight into mechanisms of resistance to SMO inhibitors for not only Gorlin syndrome but also sporadic basal cell carcinomas.

Epigenetic regulation of p63 blocks squamous-to-neuroendocrine transdifferentiation in esophageal development and malignancy.

While cell fate determination and maintenance are important in establishing and preserving tissue identity and function during development, aberrant cell fate transition leads to cancer cell heterogeneity and resistance to treatment. Here, we report an unexpected role for the transcription factor p63 (Trp63/TP63) in the fate choice of squamous versus neuroendocrine lineage in esophageal development and malignancy. Deletion of p63 results in extensive neuroendocrine differentiation in the developing mouse esophagus and esophageal progenitors derived from human embryonic stem cells. In human esophageal neuroendocrine carcinoma (eNEC) cells, p63 is transcriptionally silenced by EZH2-mediated H3K27 trimethylation (H3K27me3). Upregulation of the major p63 isoform ΔNp63α, through either ectopic expression or EZH2 inhibition, promotes squamous transdifferentiation of eNEC cells. Together these findings uncover p63 as a rheostat in coordinating the transition between squamous and neuroendocrine cell fates during esophageal development and tumor progression.

Depression and incident cardiovascular disease among patients with chronic kidney disease.

Background: Depression is associated with an increased risk of cardiovascular disease (CVD) and is prevalent among patients with chronic kidney disease (CKD). We aimed to identify the association of depression with incident CVD. Methods: We studied patients with CKD stages 2-4 enrolled in the Chronic Renal Insufficiency Cohort (CRIC) and excluded participants with preexisting CVD. The Cox proportional hazard model was used to examine the association between baseline depression [Beck's Depression Inventory (BDI) score ≥11] and incidence of CVD (cerebrovascular accident, myocardial infarction, heart failure, or peripheral artery disease). Models were adjusted for age, sex, race, estimated glomerular filtration rate (eGFR), urine albumin-creatinine ratio (UACR), systolic and diastolic blood pressure, and 10-year estimated CVD risk. Results: Among 2585 CRIC study participants, 640 (25%) patients had depression at study baseline. Compared to patients without depression, patients with depression were more likely to be women (56% vs. 46%), non-White (68% vs. 53%), with household income <$20,000 (53% vs. 26%), without a high school degree (31% vs. 15%), uninsured (13% vs. 7%), with lower eGFR (42 vs. 46 ml/min/1.73 m (Palmer et al., 2013 Jul)22), and with higher UACR (90 vs. 33 mg/g). In multivariate analyses, depression was associated with a 29% increased risk of developing CVD (adjusted hazard ratio 1.29, 95% confidence interval 1.03-1.62, p = 0.03). BDI (as a continuous variable) was associated with CVD (adjusted hazard ratio 1.017, 95% confidence interval 1.004-1.030, p = 0.012). Conclusions: Among patients with CKD stages 2-4 enrolled in CRIC without preexisting CVD, depression was associated with a 29% increased risk of incident CVD.

Acquisition of drug resistance in basal cell nevus syndrome tumors through basal to squamous cell carcinoma transition.

While basal cell carcinomas (BCCs) arise from ectopic hedgehog pathway activation and can be treated with pathway inhibitors, sporadic BCCs display high resistance rates while tumors arising in Gorlin syndrome patients with germline Patched ( PTCH1 ) mutations are uniformly suppressed by inhibitor therapy. In rare cases, Gorlin syndrome patients on long-term inhibitor therapy will develop individual resistant tumor clones that rapidly progress, but the basis of this resistance remains unstudied. Here we report a case of an SMO i -resistant tumor arising in a Gorlin patient on suppressive SMO i for nearly a decade. Using a combination of multi-omics and spatial transcriptomics, we define the tumor populations at the cellular and tissue level to conclude that Gorlin tumors can develop resistance to SMO i through the previously described basal to squamous cell carcinoma transition (BST). Intriguingly, through spatial whole exome genomic analysis, we nominate PCYT2, ETNK1, and the phosphatidylethanolamine biosynthetic pathway as novel genetic suppressors of BST resistance. These observations provide a general framework for studying tumor evolution and provide important clinical insight into mechanisms of resistance to SMO i for not only Gorlin syndrome but sporadic BCCs as well.

Skin basal cell carcinomas assemble a pro-tumorigenic spatially organized and self-propagating Trem2+ myeloid niche.

Cancer immunotherapies have revolutionized treatment but have shown limited success as single-agent therapies highlighting the need to understand the origin, assembly, and dynamics of heterogeneous tumor immune niches. Here, we use single-cell and imaging-based spatial analysis to elucidate three microenvironmental neighborhoods surrounding the heterogeneous basal cell carcinoma tumor epithelia. Within the highly proliferative neighborhood, we find that TREM2+ skin cancer-associated macrophages (SCAMs) support the proliferation of a distinct tumor epithelial population through an immunosuppression-independent manner via oncostatin-M/JAK-STAT3 signaling. SCAMs represent a unique tumor-specific TREM2+ population defined by VCAM1 surface expression that is not found in normal homeostatic skin or during wound healing. Furthermore, SCAMs actively proliferate and self-propagate through multiple serial tumor passages, indicating long-term potential. The tumor rapidly drives SCAM differentiation, with intratumoral injections sufficient to instruct naive bone marrow-derived monocytes to polarize within days. This work provides mechanistic insights into direct tumor-immune niche dynamics independent of immunosuppression, providing the basis for potential combination tumor therapies.

A scalable, GMP-compatible, autologous organotypic cell therapy for Dystrophic Epidermolysis Bullosa.

Background: Gene editing in induced pluripotent stem (iPS) cells has been hailed to enable new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies. Methods: We developed Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a new generation GMP-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7). DEBCT uses a combined high-efficiency reprogramming and CRISPR-based genetic correction single step to generate genome scar-free, COL7A1 corrected clonal iPS cells from primary patient fibroblasts. Validated iPS cells are converted into epidermal, dermal and melanocyte progenitors with a novel 2D organoid differentiation protocol, followed by CD49f enrichment and expansion to minimize maturation heterogeneity. iSC product characterization by single cell transcriptomics was followed by mouse xenografting for disease correcting activity at 1 month and toxicology analysis at 1-6 months. Culture-acquired mutations, potential CRISPR-off targets, and cancer-driver variants were evaluated by targeted and whole genome sequencing. Findings: iPS cell-derived iSC grafts were reproducibly generated from four recessive DEB patients with different pathogenic mutations. Organotypic iSC grafts onto immune-compromised mice developed into stable stratified skin with functional C7 restoration. Single cell transcriptomic characterization of iSCs revealed prominent holoclone stem cell signatures in keratinocytes and the recently described Gibbin-dependent signature in dermal fibroblasts. The latter correlated with enhanced graftability. Multiple orthogonal sequencing and subsequent computational approaches identified random and non-oncogenic mutations introduced by the manufacturing process. Toxicology revealed no detectable tumors after 3-6 months in DEBCT-treated mice. Interpretation: DEBCT successfully overcomes previous roadblocks and represents a robust, scalable, and safe cGMP manufacturing platform for production of a CRISPR-corrected autologous organotypic skin graft to heal DEB patient wounds.

GRHL2 and AP2a coordinate early surface ectoderm lineage commitment during development.

Ectodermal dysplasias including skin abnormalities and cleft lip/palate result from improper surface ectoderm (SE) patterning. However, the connection between SE gene regulatory networks and disease remains poorly understood. Here, we dissect human SE differentiation with multiomics and establish GRHL2 as a key mediator of early SE commitment, which acts by skewing cell fate away from the neural lineage. GRHL2 and master SE regulator AP2a balance early cell fate output, with GRHL2 facilitating AP2a binding to SE loci. In turn, AP2a restricts GRHL2 DNA binding away from de novo chromatin contacts. Integration of these regulatory sites with ectodermal dysplasia-associated genomic variants annotated within the Biomedical Data Commons identifies 55 loci previously implicated in craniofacial disorders. These include ABCA4/ARHGAP29 and NOG regulatory regions where disease-linked variants directly affect GRHL2/AP2a binding and gene transcription. These studies elucidate the logic underlying SE commitment and deepen our understanding of human oligogenic disease pathogenesis.

LY6D marks pre-existing resistant basosquamous tumor subpopulations.

Improved response to canonical therapies requires a mechanistic understanding of dynamic tumor heterogeneity by identifying discrete cellular populations with enhanced cellular plasticity. We have previously demonstrated distinct resistance mechanisms in skin basal cell carcinomas, but a comprehensive understanding of the cellular states and markers associated with these populations remains poorly understood. Here we identify a pre-existing resistant cellular population in naive basal cell carcinoma tumors marked by the surface marker LY6D. LY6D+ tumor cells are spatially localized and possess basal cell carcinoma and squamous cell carcinoma-like features. Using computational tools, organoids, and spatial tools, we show that LY6D+ basosquamous cells represent a persister population lying on a central node along the skin lineage-associated spectrum of epithelial states with local environmental and applied therapies determining the kinetics of accumulation. Surprisingly, LY6D+ basosquamous populations exist in many epithelial tumors, such as pancreatic adenocarcinomas, which have poor outcomes. Overall, our results identify the resistant LY6D+ basosquamous population as an important clinical target and suggest strategies for future therapeutic approaches to target them.

The HHIP-AS1 lncRNA promotes tumorigenicity through stabilization of dynein complex 1 in human SHH-driven tumors.

Most lncRNAs display species-specific expression patterns suggesting that animal models of cancer may only incompletely recapitulate the regulatory crosstalk between lncRNAs and oncogenic pathways in humans. Among these pathways, Sonic Hedgehog (SHH) signaling is aberrantly activated in several human cancer entities. We unravel that aberrant expression of the primate-specific lncRNA HedgeHog Interacting Protein-AntiSense 1 (HHIP-AS1) is a hallmark of SHH-driven tumors including medulloblastoma and atypical teratoid/rhabdoid tumors. HHIP-AS1 is actively transcribed from a bidirectional promoter shared with SHH regulator HHIP. Knockdown of HHIP-AS1 induces mitotic spindle deregulation impairing tumorigenicity in vitro and in vivo. Mechanistically, HHIP-AS1 binds directly to the mRNA of cytoplasmic dynein 1 intermediate chain 2 (DYNC1I2) and attenuates its degradation by hsa-miR-425-5p. We uncover that neither HHIP-AS1 nor the corresponding regulatory element in DYNC1I2 are evolutionary conserved in mice. Taken together, we discover an lncRNA-mediated mechanism that enables the pro-mitotic effects of SHH pathway activation in human tumors.

Gibbin mesodermal regulation patterns epithelial development.

Proper ectodermal patterning during human development requires previously identified transcription factors such as GATA3 and p63, as well as positional signalling from regional mesoderm1-6. However, the mechanism by which ectoderm and mesoderm factors act to stably pattern gene expression and lineage commitment remains unclear. Here we identify the protein Gibbin, encoded by the Xia-Gibbs AT-hook DNA-binding-motif-containing 1 (AHDC1) disease gene7-9, as a key regulator of early epithelial morphogenesis. We find that enhancer- or promoter-bound Gibbin interacts with dozens of sequence-specific zinc-finger transcription factors and methyl-CpG-binding proteins to regulate the expression of mesoderm genes. The loss of Gibbin causes an increase in DNA methylation at GATA3-dependent mesodermal genes, resulting in a loss of signalling between developing dermal and epidermal cell types. Notably, Gibbin-mutant human embryonic stem-cell-derived skin organoids lack dermal maturation, resulting in p63-expressing basal cells that possess defective keratinocyte stratification. In vivo chimeric CRISPR mouse mutants reveal a spectrum of Gibbin-dependent developmental patterning defects affecting craniofacial structure, abdominal wall closure and epidermal stratification that mirror patient phenotypes. Our results indicate that the patterning phenotypes seen in Xia-Gibbs and related syndromes derive from abnormal mesoderm maturation as a result of gene-specific DNA methylation decisions.

A conserved YAP/Notch/REST network controls the neuroendocrine cell fate in the lungs.

The Notch pathway is a conserved cell-cell communication pathway that controls cell fate decisions. Here we sought to determine how Notch pathway activation inhibits the neuroendocrine cell fate in the lungs, an archetypal process for cell fate decisions orchestrated by Notch signaling that has remained poorly understood at the molecular level. Using intratumoral heterogeneity in small-cell lung cancer as a tractable model system, we uncovered a role for the transcriptional regulators REST and YAP as promoters of the neuroendocrine to non-neuroendocrine transition. We further identified the specific neuroendocrine gene programs repressed by REST downstream of Notch in this process. Importantly, we validated the importance of REST and YAP in neuroendocrine to non-neuroendocrine cell fate switches in both developmental and tissue repair processes in the lungs. Altogether, these experiments identify conserved roles for REST and YAP in Notch-driven inhibition of the neuroendocrine cell fate in embryonic lungs, adult lungs, and lung cancer.

Is hypoimmunogenic stem cell therapy safe in times of pandemics?

The manipulation of human leukocyte antigens (HLAs) and immune modulatory factors in "universal" human pluripotent stem cells (PSCs) holds promise for immunological tolerance without HLA matching. This paradigm raises concerns should "universal" grafts become virally infected. Furthermore, immunological manipulation might functionally impair certain progeny, such as hematopoietic stem cells. We discuss the risks and benefits of hypoimmunogenic PSCs, and the need to further advance HLA matching and autologous strategies.

c-FOS drives reversible basal to squamous cell carcinoma transition.

While squamous transdifferentiation within subpopulations of adenocarcinomas represents an important drug resistance problem, its underlying mechanism remains poorly understood. Here, using surface markers of resistant basal cell carcinomas (BCCs) and patient single-cell and bulk transcriptomic data, we uncover the dynamic roadmap of basal to squamous cell carcinoma transition (BST). Experimentally induced BST identifies activator protein 1 (AP-1) family members in regulating tumor plasticity, and we show that c-FOS plays a central role in BST by regulating the accessibility of distinct AP-1 regulatory elements. Remarkably, despite prominent changes in cell morphology and BST marker expression, we show using inducible model systems that c-FOS-mediated BST demonstrates reversibility. Blocking EGFR pathway activation after c-FOS induction partially reverts BST in vitro and prevents BST features in both mouse models and human tumors. Thus, by identifying the molecular basis of BST, our work reveals a therapeutic opportunity targeting plasticity as a mechanism of tumor resistance.

Phase II Open-Label, Single-Arm Trial to Investigate the Efficacy and Safety of Topical Remetinostat Gel in Patients with Basal Cell Carcinoma.

PURPOSE: The mainstay of treatment for basal cell carcinoma (BCC) is surgical excision, which can result in significant associated morbidity, particularly for patients with recurrent tumors. We previously conducted a drug repositioning screen using molecular data from human BCCs and identified histone deacetylase (HDAC) inhibitors as a potential treatment for BCC. Here we conduct the first proof-of-principle study of a topical pan-HDAC inhibitor, remetinostat, in human BCC. PATIENTS AND METHODS: We conducted a phase II, open-label, single-arm, single-institution trial of a topical HDAC inhibitor. Participants with at least one BCC were recruited. All participants applied 1% remetinostat gel three times daily for 6 weeks, with measurements of tumor diameter conducted at baseline and week 8. Surgical excision of the remaining tumor was conducted at the end of the study and microscopic evaluation was performed. RESULTS: Thirty-three per-protocol tumors from 25 participants were included in the analysis. The overall response rate, defined as the proportion of tumors achieving more than 30% decrease in the longest diameter from baseline to week 8, was 69.7% [90% confidence interval (CI), 54%-82.5%]. On pathologic examination, 54.8% of tumors demonstrated complete resolution. Pharmacodynamic analysis demonstrated similar levels of acetylated histone H3 in skin tissue before and after treatment, however, phosphorylation was increased. No systemic adverse events were reported. CONCLUSIONS: The HDAC inhibitor remetinostat is a well-tolerated and effective topical treatment for reducing BCC disease burden in a clinically significant manner. This provides in-human validation of HDAC inhibitors as a therapy for BCC.

Challenging proximal hypospadias repairs: An evolution of technique for two stage repairs.

INTRODUCTION: Proximal hypospadias repair remains challenging. Our approach to the first stage of two-stage proximal hypospadias repairs has evolved from using Byars' flaps to preputial inlay grafts in anatomically suitable cases and pedicled preputial flaps in more complex repairs. We reviewed our outcomes, hypothesizing that inlay grafts and pedicled preputial flaps were associated with lower complication risks than Byars' flaps. STUDY DESIGN: A single institution, retrospective, cohort study of consecutive two-stage, primary, proximal hypospadias repairs performed from 2007 to 2017 was conducted. Patients with <6 months follow-up and incomplete operative reports were excluded. Risk of complications (fistula, dehiscence, diverticulum, meatal stenosis, stricture) were evaluated following urethroplasty and stratified by first-stage repair technique. As technique refinements have been made since 2012, comparisons between two temporal subgroups (those who underwent repair in 2007-2012 and in 2013-2017) were made. RESULTS: 78 of 127 patients met inclusion criteria. Overall complication rate was 47% (Summary Table). Median follow-up was 25.4 months (range 6.4-128.5 months) after urethroplasty. Pedicled preputial flaps (hazards ratio [HR] 0.30; 95% Confidence Interval [CI] 0.14-0.65) and inlay grafts (HR 0.32; 95% CI 0.11-0.95) were associated with lower complication risks compared to Byars' flaps (Summary Table). Median time to complication was significantly shorter for Byars' flaps (5.7 months) than for inlay grafts (40.6 months) and pedicled preputial flaps (79.2 months) by Kaplan Meier analysis. Temporal subgroup comparisons showed that overall complication rates decreased from 70% to 31% (p = 0.001), but differences in complication rates by first-stage technique were not statistically significant. DISCUSSION: In our cohort, repairs with Byars' flaps had the highest complication rate, which is consistent with our observations that urethras tubularized from Byars' flaps lack appropriate backing and are hypermobile and irregular. To overcome these shortcomings, modifications were made to our approach to two-stage proximal hypospadias repairs with the use of inlay grafts and pedicled preputial flaps quilted to the underlying corporal bodies to optimize the stability of the urethral plate. Our preliminary results are promising. CONCLUSION: Approach to the first stage of two-stage repairs affects outcomes. Pedicled preputial flaps and inlay grafts were associated with lower complication risks than Byars' flaps. Refinement of technique and patient selection may have resulted in fewer complications in the short term. However, long-term follow-up is needed.

Association between intra-operative meatal mismatch and urethrocutaneous fistula development in hypospadias repair.

INTRODUCTION: The Glans-Meatus-Shaft (GMS) Score is a pre-operative phenotypic scoring system used to assess hypospadias severity and risk for post-operative complications. The 'M' component is based on pre-operative meatal location, but meatal location sometimes changes after penile degloving, resulting in 'meatal mismatch.' OBJECTIVE: To identify: 1) the incidence and clinical predictors of meatal mismatch, and 2) the association of meatal mismatch with post-operative urethrocutaneous fistula development. STUDY DESIGN: We performed a retrospective cohort study on patients who underwent primary hypospadias repair at a single center from 2011 to 2018. Meatal mismatch was defined as: upstaging (meatus moving more proximally after degloving), downstaging (moving more distally after degloving), or none. Covariates included: pre-degloving meatal location, chordee severity, penoscrotal anatomy, pre-operative testosterone, and number of stages for repair. To test the association between meatal mismatch and fistula development, we constructed two, nested, multivariable Cox proportional hazards regression models with and without meatal mismatch and compared them with the likelihood ratio test. A sensitivity analysis excluded patients with <6 months of follow-up. RESULTS: Of 485 patients, 99 (20%) exhibited meatal mismatch, including 75 (15%) with upstaging and 24 (5%) patients with downstaging (Figure). Meatal mismatch was significantly associated with penoscrotal webbing, number of stages for repair, and pre-degloving meatal location, with downstaging being associated with more proximal meatal location. Over a median follow-up of 7.3 months (interquartile range 2.0-20.9), fistulae developed in 56 (12%) patients. On multivariable analysis, meatal upstaging was associated with a 3-fold increased risk of fistula development (Hazards Ratio [HR]: 3.04, 95% Confidence Interval [CI]: 1.44-6.45) compared to no mismatch. Meatal downstaging had similar risk of fistula development compared to no mismatch (HR: 0.99, 95% CI: 0.29-3.35). Multi-stage compared to single-stage repair was associated with reduced risk of fistula development (HR: 0.24, 95% CI: 0.09-0.66). The likelihood ratio test favored the model that included meatal mismatch. The sensitivity analysis showed similar findings. DISCUSSION: Our short-term results suggest that meatal mismatch may be an important additional consideration to the GMS score as a tool to assess hypospadias severity, counsel families, and predict outcomes. Longer-term studies are needed to enhance the precision of risk stratification in hypospadias. CONCLUSIONS: Meatal mismatch occurred in 20% of patients undergoing hypospadias repair. Among this cohort, meatal upstaging was associated with a 3-fold increased risk of post-operative urethrocutaneous fistula development.

Urinary Tract Infection After Robot-assisted Laparoscopic Pyeloplasty: Are Urine Cultures and Antibiotics Helpful?

OBJECTIVE: To evaluate how variations in peri-operative urine culture (UCx) and antibiotic prophylaxis utilization following robot assisted laparoscopic pyeloplasty (RALP) affect post-RALP urinary tract infection (UTI) rates in children, then use data to generate a standardized care pathway. METHODS: Patients undergoing RALP at a single institution from January 2014 to October 2018 were retrospectively reviewed. Patients with vesicoureteral reflux, neurogenic bladder, intermittent catheterization, <=2 months follow-up after stent removal, or age >=18 years were excluded. UCx use, UCx results, and pre- and post-RALP antibiotic use were recorded. The primary outcome was symptomatic UTI, tracked until 60 days after stent removal. UTI was defined as presence of fever or urinary symptoms, a positive UCx with >=10,000 colony forming units of one uropathogen, and a positive urinalysis. RESULTS: A total of 152 patients were included (72% male [73% circumcised], 61% white, and 23% Hispanic). One underwent a re-operative pyeloplasty, yielding 153 encounters. Eight patients (5.2%; 95% CI 1.7-8.7%) developed post-RALP UTI. Uncircumcised status and use of pre-operative prophylactic antibiotics were associated with post-RALP UTI (P = .03 and P < .01, respectively). Use of post-RALP antibiotics, whether prophylactic or therapeutic, was not associated with lower UTI rates (P = .92). Positive pre-RALP UCx and positive intra-operative stent removal UCx were associated with higher UTI rates (P = .03 and P < .01, respectively). CONCLUSION: UTI occurred in 5.2% of our cohort of >150 patients. As post-RALP antibiotic use was not associated with lower UTI rates, prophylactic antibiotics may be reserved for patients with risk factors. A standardized care pathway could safely reduce unnecessary utilization of UA/UCx and antibiotics.

AP-1 and TGFß cooperativity drives non-canonical Hedgehog signaling in resistant basal cell carcinoma.

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.

Rapid Ex-Vivo Ciliogenesis and Dose-Dependent Effect of Notch Inhibition on Ciliogenesis of Respiratory Epithelia.

Background: Cilia are actin based cellular protrusions conserved from algae to complex multicellular organisms like Homo sapiens. Respiratory motile cilia line epithelial cells of the tracheobronchial tree, beat in a synchronous, metachronal wave, moving inhaled pollutants and pathogens cephalad. Their role in both congenital disorders like primary ciliary dyskinesia (PCD) to acquired disorders like chronic obstructive pulmonary disease (COPD) continues to evolve. In this current body of work we outline a protocol optimized to reciliate human nasal epithelial cells and mouse tracheal cells in vitro. Using this protocol, we knocked down known cilia genes, as well as use a small molecule inhibitor of Notch, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl Ester (DAPT), to assess the effect of these on ciliogenesis in order to show the validity of our protocol. Methods: Tracheas were harvested from wild-type, adult C57B6 mice, pronase digested and sloughed off epithelial cells grown to confluence in stationary culture on rat-tail collagen coated wells. Upon reaching confluence, collagen was digested and cells placed suspension culture protocol to reciliate the cells. Using this suspension culture protocol, we employed siRNA gene knockdown to assay gene functions required for airway ciliogenesis. Knock down of Dynein axonemal heavy chain 5 (Dnah5), a ciliary structural protein, was confirmed using immunostaining. Mouse tracheal cells were treated in suspension with varying doses of DAPT, an inhibitor of Notch, with the purpose of evaluating its effect and dose response on ciliogenesis. The optimum dose was then used on reciliating human nasal epithelial cells. Results: siRNA knockdown of Foxj1 prevented ciliation, consistent with its role as a master regulator of motile cilia. Knockdown of Dnai1 and Dnah5 resulted in immotile cilia, and Cand1 knockdown, a centrosome protein known to regulate centrosome amplification, inhibited airway ciliogenesis. Dnah5 knockdown was confirmed with significantly decreased immunostaining of cilia for this protein. Inhibiting Notch signaling by inhibiting gamma secretase with DAPT enhanced the percentage of ciliation, and resulted in longer cilia that beat with higher frequency in both mouse and human airway epithelia. Conclusions: Modifying existing reciliation protocols to suit both human nasal epithelial and mouse tracheal tissue, we have shown that knockdown of known cilia-related genes have the expected effects. Additionally, we have demonstrated the optimal dosage for significantly improving reciliation of airway epithelia using DAPT. Given that cilia length and function are significantly compromised in COPD, these findings open up interesting avenues for further exploration.