Loss of ribonuclease DIS3 hampers genome integrity in myeloma by disrupting DNA:RNA hybrid metabolism.

The ribonuclease DIS3 is one of the most frequently mutated genes in the hematological cancer multiple myeloma, yet the basis of its tumor suppressor function in this disease remains unclear. Herein, exploiting the TCGA dataset, we found that DIS3 plays a prominent role in the DNA damage response. DIS3 inactivation causes genomic instability by increasing mutational load, and a pervasive accumulation of DNA:RNA hybrids that induces genomic DNA double-strand breaks (DSBs). DNA:RNA hybrid accumulation also prevents binding of the homologous recombination (HR) machinery to double-strand breaks, hampering DSB repair. DIS3-inactivated cells become sensitive to PARP inhibitors, suggestive of a defect in homologous recombination repair. Accordingly, multiple myeloma patient cells mutated for DIS3 harbor an increased mutational burden and a pervasive overexpression of pro-inflammatory interferon, correlating with the accumulation of DNA:RNA hybrids. We propose DIS3 loss in myeloma to be a driving force for tumorigenesis via DNA:RNA hybrid-dependent enhanced genome instability and increased mutational rate. At the same time, DIS3 loss represents a liability that might be therapeutically exploited in patients whose cancer cells harbor DIS3 mutations.

Myeloma and DNA damage.

ABSTRACT: DNA-damaging agents have represented the first effective treatment for the blood cancer multiple myeloma, and after 65 years since their introduction to the clinic, they remain one of the mainstay therapies for this disease. Myeloma is a cancer of plasma cells. Despite exceedingly slow proliferation, myeloma cells present extended genomic rearrangements and intense genomic instability, starting at the premalignant stage of the disease. Where does such DNA damage stem from? A reliable model argues that the powerful oncogenes activated in myeloma as well the phenotypic peculiarities of cancer plasma cells, including the dependency on the proteasome for survival and the constant presence of oxidative stress, all converge on modulating DNA damage and repair. Beleaguered by these contraposing forces, myeloma cells survive in a precarious balance, in which the robust engagement of DNA repair mechanisms to guarantee cell survival is continuously challenged by rampant genomic instability, essential for cancer cells to withstand hostile selective pressures. Shattering this delicate equilibrium has been the goal of the extensive use of DNA-damaging agents since their introduction in the clinic, now enriched by novel approaches that leverage upon synthetic lethality paradigms. Exploiting the impairment of homologous recombination caused by myeloma genetic lesions or treatments, it is now possible to design therapeutic combinations that could target myeloma cells more effectively. Furthermore, DNA-damaging agents, as demonstrated in solid tumors, may sensitize cells to immune therapies. In all, targeting DNA damage and repair remains as central as ever in myeloma, even for the foreseeable future.

KDM6A Regulates Immune Response Genes in Multiple Myeloma.

The histone H3K27 demethylase KDM6A is a tumor suppressor in multiple cancers, including multiple myeloma (MM). We created isogenic MM cells disrupted for KDM6A and tagged the endogenous protein to facilitate genome wide studies. KDM6A binds genes associated with immune recognition and cytokine signaling. Most importantly, KDM6A binds and activates NLRC5 and CIITA encoding regulators of Major Histocompatibility Complex (MHC) genes. Patient data indicate that NLRC5 and CIITA, are downregulated in MM with low KDM6A expression. Chromatin analysis shows that KDM6A binds poised and active enhancers and KDM6A loss led to decreased H3K27ac at enhancers, increased H3K27me3 levels in body of genes bound by KDM6A and decreased gene expression. Reestablishing histone acetylation with an HDAC3 inhibitor leads to upregulation of MHC expression, offering a strategy to restore immunogenicity of KDM6A deficient tumors. Loss of Kdm6a in murine RAS-transformed fibroblasts led to increased growth in vivo associated with decreased T cell infiltration.

Scalable integration of multiomic single-cell data using generative adversarial networks.

MOTIVATION: Single-cell profiling has become a common practice to investigate the complexity of tissues, organs, and organisms. Recent technological advances are expanding our capabilities to profile various molecular layers beyond the transcriptome such as, but not limited to, the genome, the epigenome, and the proteome. Depending on the experimental procedure, these data can be obtained from separate assays or the very same cells. Yet, integration of more than two assays is currently not supported by the majority of the computational frameworks avaiable. RESULTS: We here propose a Multi-Omic data integration framework based on Wasserstein Generative Adversarial Networks suitable for the analysis of paired or unpaired data with a high number of modalities (>2). At the core of our strategy is a single network trained on all modalities together, limiting the computational burden when many molecular layers are evaluated. AVAILABILITY AND IMPLEMENTATION: Source code of our framework is available at https://github.com/vgiansanti/MOWGAN.

The endothelin-1-driven tumor-stroma feed-forward loops in high-grade serous ovarian cancer.

The high-grade serous ovarian cancer (HG-SOC) tumor microenvironment (TME) is constellated by cellular elements and a network of soluble constituents that contribute to tumor progression. In the multitude of the secreted molecules, the endothelin-1 (ET-1) has emerged to be implicated in the tumor/TME interplay; however, the molecular mechanisms induced by the ET-1-driven feed-forward loops (FFL) and associated with the HG-SOC metastatic potential need to be further investigated. The tracking of the patient-derived (PD) HG-SOC cell transcriptome by RNA-seq identified the vascular endothelial growth factor (VEGF) gene and its associated signature among those mostly up-regulated by ET-1 and down-modulated by the dual ET-1R antagonist macitentan. Within the ligand-receptor pairs concurrently expressed in PD-HG-SOC cells, endothelial cells and activated fibroblasts, we discovered two intertwined FFL, the ET-1/ET-1R and VEGF/VEGF receptors, concurrently activated by ET-1 and shutting-down by macitentan, or by the anti-VEGF antibody bevacizumab. In parallel, we observed that ET-1 fine-tuned the tumoral and stromal secretome toward a pro-invasive pattern. Into the fray of the HG-SOC/TME double and triple co-cultures, the secretion of ET-1 and VEGF, that share a common co-regulation, was inhibited upon the administration of macitentan. Functionally, macitentan, mimicking the effect of bevacizumab, interfered with the HG-SOC/TME FFL-driven communication that fuels the HG-SOC invasive behavior. The identification of ET-1 and VEGF FFL as tumor and TME actionable vulnerabilities, reveals how ET-1R blockade, targeting the HG-SOC cells and the TME simultaneously, may represent an effective therapeutic option for HG-SOC patients.

Antibody Drug Conjugates for Cancer Therapy: From Metallodrugs to Nature-Inspired Payloads.

This review highlights significant advancements in antibody-drug conjugates (ADCs) equipped with metal-based and nature-inspired payloads, focusing on synthetic strategies for antibody conjugation. Traditional methods such us maleimide and succinimide conjugation and classical condensation reactions are prevalent for metallodrugs and natural compounds. However, emerging non-conventional strategies such as photoconjugation are gaining traction due to their milder conditions and, in an aspect which minimizes side reactions, selective formation of ADC. The review also summarizes the therapeutic and diagnostic properties of these ADCs, highlighting their enhanced selectivity and reduced side effects in cancer treatment compared to non-conjugated payloads. ADCs combine the specificity of monoclonal antibodies with the cytotoxicity of chemotherapy drugs, offering a targeted approach to the elimination of cancer cells while sparing healthy tissues. This targeted mechanism has demonstrated impressive clinical efficacy in various malignancies. Key future advancements include improved linker technology for enhanced stability and controlled release of cytotoxic agents, incorporation of novel, more potent, cytotoxic agents, and the identification of new cancer-specific antigens through genomic and proteomic technologies. ADCs are also expected to play a crucial role in combination therapies with immune checkpoint inhibitors, CAR-T cells, and small molecule inhibitors, leading to more durable and potentially curative outcomes. Ongoing research and clinical trials are expanding their capabilities, paving the way for more effective, safer, and personalized treatments, positioning ADCs as a cornerstone of modern medicine and offering new hope to patients.

Revealing and harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells.

OBJECTIVE: Colorectal tumours are often densely infiltrated by immune cells that have a role in surveillance and modulation of tumour progression but are burdened by immunosuppressive signals, which might vary from primary to metastatic stages. Here, we deployed a multidimensional approach to unravel the T-cell functional landscape in primary colorectal cancers (CRC) and liver metastases, and genome editing tools to develop CRC-specific engineered T cells. DESIGN: We paired high-dimensional flow cytometry, RNA sequencing and immunohistochemistry to describe the functional phenotype of T cells from healthy and neoplastic tissue of patients with primary and metastatic CRC and we applied lentiviral vectors (LV) and CRISPR/Cas9 genome editing technologies to develop CRC-specific cellular products. RESULTS: We found that T cells are mainly localised at the front edge and that tumor-infiltrating T cells co-express multiple inhibitory receptors, which largely differ from primary to metastatic sites. Our data highlighted CD39 as the major driver of exhaustion in both primary and metastatic colorectal tumours. We thus simultaneously redirected T-cell specificity employing a novel T-cell receptor targeting HER-2 and disrupted the endogenous TCR genes (TCR editing (TCRED)) and the CD39 encoding gene (ENTPD1), thus generating TCREDENTPD1KOHER-2-redirected lymphocytes. We showed that the absence of CD39 confers to HER-2-specific T cells a functional advantage in eliminating HER-2+ patient-derived organoids in vitro and in vivo. CONCLUSION: HER-2-specific CD39 disrupted engineered T cells are promising advanced medicinal products for primary and metastatic CRC.

Gold(I)-N-Heterocyclic Carbene Synthons in Organometallic Synthesis.

The prominent role of gold-N-heterocyclic carbene (NHC) complexes in numerous research areas such as homogeneous (photo)catalysis, medicinal chemistry and materials science has prompted organometallic chemists to design gold-based synthons that permit access to target complexes through simple synthetic steps under mild conditions. In this review, the main gold-NHC synthons employed in organometallic synthesis are discussed. Mechanistic aspects involved in their synthesis and reactivity as well as applications of gold-NHC synthons as efficient pre-catalysts, antitumor agents and/or photo-emissive materials are presented.

Tumor suppressor PNRC1 blocks rRNA maturation by recruiting the decapping complex to the nucleolus.

Focal deletions occur frequently in the cancer genome. However, the putative tumor-suppressive genes residing within these regions have been difficult to pinpoint. To robustly identify these genes, we implemented a computational approach based on non-negative matrix factorization, NMF, and interrogated the TCGA dataset. This analysis revealed a metagene signature including a small subset of genes showing pervasive hemizygous deletions, reduced expression in cancer patient samples, and nucleolar function. Amid the genes belonging to this signature, we have identified PNRC1, a nuclear receptor coactivator. We found that PNRC1 interacts with the cytoplasmic DCP1α/DCP2 decapping machinery and hauls it inside the nucleolus. PNRC1-dependent nucleolar translocation of the decapping complex is associated with a decrease in the 5'-capped U3 and U8 snoRNA fractions, hampering ribosomal RNA maturation. As a result, PNRC1 ablates the enhanced proliferation triggered by established oncogenes such as RAS and MYC These observations uncover a previously undescribed mechanism of tumor suppression, whereby the cytoplasmic decapping machinery is hauled within nucleoli, tightly regulating ribosomal RNA maturation.

Chest CT in the emergency department for suspected COVID-19 pneumonia.

PURPOSE: In overwhelmed emergency departments (EDs) facing COVID-19 outbreak, a swift diagnosis is imperative. CT role was widely debated for its limited specificity. Here we report the diagnostic role of CT in two EDs in Lombardy, epicenter of Italian outbreak. MATERIAL AND METHODS: Admitting chest CT from 142 consecutive patients with suspected COVID-19 were retrospectively analyzed. CT scans were classified in "highly likely," "likely," and "unlikely" COVID-19 pneumonia according to the presence of typical, indeterminate, and atypical findings, or "negative" in the absence of findings, or "alternative diagnosis" when a different diagnosis was found. Nasopharyngeal swab results, turnaround time, and time to positive results were collected. CT diagnostic performances were assessed considering RT-PCR as reference standard. RESULTS: Most of cases (96/142, 68%) were classified as "highly likely" COVID-19 pneumonia. Ten (7%) and seven (5%) patients were classified as "likely" and "unlikely" COVID-19 pneumonia, respectively. In 21 (15%) patients a differential diagnosis was provided, including typical pneumonia, pulmonary edema, neoplasia, and pulmonary embolism. CT was negative in 8/142 (6%) patients. Mean turnaround time for the first COVID-19 RT-PCR was 30 ± 13 h. CT diagnostic accuracy in respect of the first test swab was 79% and increased to 91.5% after repeated swabs and/or BAL, for 18 false-negative first swab. CT performance was good with 76% specificity, 99% sensitivity, 90% positive predictive value and 97% negative predictive value. CONCLUSION: Chest CT was useful to streamline patients' triage while waiting for RT-PCR in the ED, supporting the clinical suspicion of COVID-19 or providing alternative diagnosis.

ATR addiction in multiple myeloma: synthetic lethal approaches exploiting established therapies.

Therapeutic strategies designed to tinker with cancer cell DNA damage response have led to the widespread use of PARP inhibitors for BRCA1/2-mutated cancers. In the haematological cancer multiple myeloma, we sought to identify analogous synthetic lethality mechanisms that could be leveraged upon established cancer treatments. The combination of ATR inhibition using the compound VX-970 with a drug eliciting interstrand cross-links, melphalan, was tested in in vitro, ex vivo, and most notably in vivo models. Cell proliferation, induction of apoptosis, tumor growth and animal survival were assessed. The combination of ATM inhibition with a drug triggering double strand breaks, doxorucibin, was also probed. We found that ATR inhibition is strongly synergistic with melphalan, even in resistant cells. The combination was dramatically effective in targeting myeloma primary patient cells and cell lines reducing cell proliferation and inducing apoptosis. The combination therapy significantly reduced tumor burden and prolonged survival in animal models. Conversely, ATM inhibition only marginally impacted on myeloma cell survival, even in combination with doxorucibin at high doses. These results indicate that myeloma cells extensively rely on ATR, but not on ATM, for DNA repair. Our findings posit that adding an ATR inhibitor such as VX-970 to established therapeutic regimens may provide a remarkably broad benefit to myeloma patients.

Contrasting roles of histone 3 lysine 27 demethylases in acute lymphoblastic leukaemia.

T-cell acute lymphoblastic leukaemia (T-ALL) is a haematological malignancy with a dismal overall prognosis, including a relapse rate of up to 25%, mainly because of the lack of non-cytotoxic targeted therapy options. Drugs that target the function of key epigenetic factors have been approved in the context of haematopoietic disorders, and mutations that affect chromatin modulators in a variety of leukaemias have recently been identified; however, 'epigenetic' drugs are not currently used for T-ALL treatment. Recently, we described that the polycomb repressive complex 2 (PRC2) has a tumour-suppressor role in T-ALL. Here we delineated the role of the histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX in T-ALL. We show that JMJD3 is essential for the initiation and maintenance of T-ALL, as it controls important oncogenic gene targets by modulating H3K27 methylation. By contrast, we found that UTX functions as a tumour suppressor and is frequently genetically inactivated in T-ALL. Moreover, we demonstrated that the small molecule inhibitor GSKJ4 (ref. 5) affects T-ALL growth, by targeting JMJD3 activity. These findings show that two proteins with a similar enzymatic function can have opposing roles in the context of the same disease, paving the way for treating haematopoietic malignancies with a new category of epigenetic inhibitors.

Che-1-induced inhibition of mTOR pathway enables stress-induced autophagy.

Mammalian target of rapamycin (mTOR) is a key protein kinase that regulates cell growth, metabolism, and autophagy to maintain cellular homeostasis. Its activity is inhibited by adverse conditions, including nutrient limitation, hypoxia, and DNA damage. In this study, we demonstrate that Che-1, a RNA polymerase II-binding protein activated by the DNA damage response, inhibits mTOR activity in response to stress conditions. We found that, under stress, Che-1 induces the expression of two important mTOR inhibitors, Redd1 and Deptor, and that this activity is required for sustaining stress-induced autophagy. Strikingly, Che-1 expression correlates with the progression of multiple myeloma and is required for cell growth and survival, a malignancy characterized by high autophagy response.

Pancreatic cancers require autophagy for tumor growth.

Macroautophagy (autophagy) is a regulated catabolic pathway to degrade cellular organelles and macromolecules. The role of autophagy in cancer is complex and may differ depending on tumor type or context. Here we show that pancreatic cancers have a distinct dependence on autophagy. Pancreatic cancer primary tumors and cell lines show elevated autophagy under basal conditions. Genetic or pharmacologic inhibition of autophagy leads to increased reactive oxygen species, elevated DNA damage, and a metabolic defect leading to decreased mitochondrial oxidative phosphorylation. Together, these ultimately result in significant growth suppression of pancreatic cancer cells in vitro. Most importantly, inhibition of autophagy by genetic means or chloroquine treatment leads to robust tumor regression and prolonged survival in pancreatic cancer xenografts and genetic mouse models. These results suggest that, unlike in other cancers where autophagy inhibition may synergize with chemotherapy or targeted agents by preventing the up-regulation of autophagy as a reactive survival mechanism, autophagy is actually required for tumorigenic growth of pancreatic cancers de novo, and drugs that inactivate this process may have a unique clinical utility in treating pancreatic cancers and other malignancies with a similar dependence on autophagy. As chloroquine and its derivatives are potent inhibitors of autophagy and have been used safely in human patients for decades for a variety of purposes, these results are immediately translatable to the treatment of pancreatic cancer patients, and provide a much needed, novel vantage point of attack.

HIF-1α regulates the interaction of chronic lymphocytic leukemia cells with the tumor microenvironment.

Hypoxia-inducible transcription factors (HIFs) regulate a wide array of adaptive responses to hypoxia and are often activated in solid tumors and hematologic malignancies due to intratumoral hypoxia and emerging new layers of regulation. We found that in chronic lymphocytic leukemia (CLL), HIF-1α is a novel regulator of the interaction of CLL cells with protective leukemia microenvironments and, in turn, is regulated by this interaction in a positive feedback loop that promotes leukemia survival and propagation. Through unbiased microarray analysis, we found that in CLL cells, HIF-1α regulates the expression of important chemokine receptors and cell adhesion molecules that control the interaction of leukemic cells with bone marrow and spleen microenvironments. Inactivation of HIF-1α impairs chemotaxis and cell adhesion to stroma, reduces bone marrow and spleen colonization in xenograft and allograft CLL mouse models, and prolongs survival in mice. Of interest, we found that in CLL cells, HIF-1α is transcriptionally regulated after coculture with stromal cells. Furthermore, HIF-1α messenger RNA levels vary significantly within CLL patients and correlate with the expression of HIF-1α target genes, including CXCR4, thus further emphasizing the relevance of HIF-1α expression to CLL pathogenesis.

Highly similar genomic landscapes in monoclonal B-cell lymphocytosis and ultra-stable chronic lymphocytic leukemia with low frequency of driver mutations.

Despite the recent discovery of recurrent driver mutations in chronic lymphocytic leukemia, the genetic factors involved in disease onset remain largely unknown. To address this issue, we performed whole-genome sequencing in 11 individuals with monoclonal B- cell lymphocytosis, both of the low-count and high-count subtypes, and 5 patients with ultra-stable chronic lymphocytic leukemia (>10 years without progression from initial diagnosis). All three entities were indistinguishable at the genomic level exhibiting low genomic complexity and similar types of somatic mutations. Exonic mutations were not frequently identified in putative chronic lymphocytic leukemia driver genes in all settings, including low-count monoclonal B-cell lymphocytosis. To corroborate these findings, we also performed deep sequencing in 11 known frequently mutated genes in an extended cohort of 28 monoclonal B-cell lymphocytosis/chronic lymphocytic leukemia cases. Interestingly, shared mutations were detected between clonal B cells and paired polymorphonuclear cells, strengthening the notion that at least a fraction of somatic mutations may occur before disease onset, likely at the hematopoietic stem cell level. Finally, we identified previously unreported non-coding variants targeting pathways relevant to B-cell and chronic lymphocytic leukemia development, likely associated with the acquisition of the characteristic neoplastic phenotype typical of both monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia.

Modeling multiple myeloma-bone marrow interactions and response to drugs in a 3D surrogate microenvironment.

Multiple myeloma develops primarily inside the bone marrow microenvironment, that confers pro-survival signals and drug resistance. 3D cultures that reproduce multiple myeloma-bone marrow interactions are needed to fully investigate multiple myeloma pathogenesis and response to drugs. To this purpose, we exploited the 3D Rotary Cell Culture System bioreactor technology for myeloma-bone marrow co-cultures in gelatin scaffolds. The model was validated with myeloma cell lines that, as assessed by histochemical and electron-microscopic analyses, engaged contacts with stromal cells and endothelial cells. Consistently, pro-survival signaling and also cell adhesion-mediated drug resistance were significantly higher in 3D than in 2D parallel co-cultures. The contribution of the VLA-4/VCAM1 pathway to resistance to bortezomib was modeled by the use of VCAM1 transfectants. Soluble factor-mediated drug resistance could be also demonstrated in both 2D and 3D co-cultures. The system was then successfully applied to co-cultures of primary myeloma cells-primary myeloma bone marrow stromal cells from patients and endothelial cells, allowing the development of functional myeloma-stroma interactions and MM cell long-term survival. Significantly, genomic analysis performed in a high-risk myeloma patient demonstrated that culture in bioreactor paralleled the expansion of the clone that ultimately dominated in vivo Finally, the impact of bortezomib on myeloma cells and on specialized functions of the microenvironment could be evaluated. Our findings indicate that 3D dynamic culture of reconstructed human multiple myeloma microenvironments in bioreactor may represent a useful platform for drug testing and for studying tumor-stroma molecular interactions.

Structural basis for PHDVC5HCHNSD1-C2HRNizp1 interaction: implications for Sotos syndrome.

Sotos syndrome is an overgrowth syndrome caused by mutations within the functional domains ofNSD1 gene coding for NSD1, a multidomain protein regulating chromatin structure and gene expression. In particular, PHDVC5HCHNSD1 tandem domain, composed by a classical (PHDV) and an atypical (C5HCH) plant homeo-domain (PHD) finger, is target of several pathological missense-mutations. PHDVC5HCHNSD1 is also crucial for NSD1-dependent transcriptional regulation and interacts with the C2HR domain of transcriptional repressor Nizp1 (C2HRNizp1)in vitro To get molecular insights into the mechanisms dictating the patho-physiological relevance of the PHD finger tandem domain, we solved its solution structure and provided a structural rationale for the effects of seven Sotos syndrome point-mutations. To investigate PHDVC5HCHNSD1 role as structural platform for multiple interactions, we characterized its binding to histone H3 peptides and to C2HRNizp1 by ITC and NMR. We observed only very weak electrostatic interactions with histone H3 N-terminal tails, conversely we proved specific binding to C2HRNizp1 We solved C2HRNizp1 solution structure and generated a 3D model of the complex, corroborated by site-directed mutagenesis. We suggest a mechanistic scenario where NSD1 interactions with cofactors such as Nizp1 are impaired by PHDVC5HCHNSD1 pathological mutations, thus impacting on the repression of growth-promoting genes, leading to overgrowth conditions.

p63 Sustains self-renewal of mammary cancer stem cells through regulation of Sonic Hedgehog signaling.

The predominant p63 isoform, ΔNp63, is a master regulator of normal epithelial stem cell (SC) maintenance. However, in vivo evidence of the regulation of cancer stem cell (CSC) properties by p63 is still limited. Here, we exploit the transgenic MMTV-ErbB2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2) mouse model of carcinogenesis to dissect the role of p63 in the regulation of mammary CSC self-renewal and breast tumorigenesis. ErbB2 tumor cells enriched for SC-like properties display increased levels of ΔNp63 expression compared with normal mammary progenitors. Down-regulation of p63 in ErbB2 mammospheres markedly restricts self-renewal and expansion of CSCs, and this action is fully independent of p53. Furthermore, transplantation of ErbB2 progenitors expressing shRNAs against p63 into the mammary fat pads of syngeneic mice delays tumor growth in vivo. p63 knockdown in ErbB2 progenitors diminishes the expression of genes encoding components of the Sonic Hedgehog (Hh) signaling pathway, a driver of mammary SC self-renewal. Remarkably, p63 regulates the expression of Sonic Hedgehog (Shh), GLI family zinc finger 2 (Gli2), and Patched1 (Ptch1) genes by directly binding to their gene regulatory regions, and eventually contributes to pathway activation. Collectively, these studies highlight the importance of p63 in maintaining the self-renewal potential of mammary CSCs via a positive modulation of the Hh signaling pathway.