Achaete-scute Complex Homolog Research Articles

ASCL1 restrains ERK1/2 to promote survival of a subset of neuroendocrine lung cancers.

The transcription factor achaete-scute complex homolog 1 (ASCL1) is a lineage oncogene that is central in growth and survival of the majority of small cell lung cancers (SCLC) and neuroendocrine non-small cell lung cancers (NSCLC-NE) that express it. Targeting ASCL1, or its downstream pathways, remains a challenge. SCLCs and NSCLC-NE that express ASCL1 exhibit relatively low ERK1/2 activity, in dramatic contrast to NSCLCs in which the ERK pathway has a major role in pathogenesis. ERK1/2 inhibition in ASCL1-expressing lung tumor cells revealed down-regulation of ERK1/2 pathway suppressors SPRY4, SPRED1, DUSP6, and the transcription factor ETV5, which regulates DUSP6. CHIP-seq demonstrated that these genes are bound by ASCL1. Availability of a pharmacological inhibitor directed mechanistic studies towards DUSP6, an ERK1/2-selective phosphatase, in a subset of ASCL1-high NE lung tumors. Inhibition of DUSP6 increased active ERK1/2, which accumulated in the nucleus. Pharmacologic and genetic inhibition of DUSP6 reduced proliferation and survival of these cancers. Resistance developed in DUSP6 KO cells, indicating a bypass mechanism. Although targeting ASCL1 remains a challenge, our findings suggest that expression of ASCL1, DUSP6 and low phospho-ERK1/2 identify neuroendocrine lung cancers for which DUSP6 may be a therapeutic target.

Generation of ASCL1-mCherry knock-in reporter in human embryonic stem cell line, WAe001-A-2E, using CRISPR/Cas9-based gene targeting

Achaete-Scute Complex Homolog 1 (ASCL1) is a key regulator in the development and function of the nervous system, particularly in the process of neuronal and neuroendocrine cell differentiation. By employing the CRISPR/Cas9 system, we successfully established an ASCL1-mCherry knock-in human embryonic stem cell (hESC) line by inserting a P2A-mCherry fragment at the ASCL1 locus. The mCherry reporter effectively demonstrated the expression level of endogenous ASCL1 during the process of inducing pulmonary neuroendocrine cells (PNECs) from hESC. This reporter cell line holds significant value as a research tool for investigating the process of lung neuroendocrine cell differentiation, conducting drug screening, and exploring the underlying mechanisms of lung diseases associated with PNECs dysfunction.

Molecular pathology of small cell lung cancer: Overview from studies on neuroendocrine differentiation regulated by ASCL1 and Notch signaling.

Pulmonary neuroendocrine (NE) cells are rare airway epithelial cells. The balance between Achaete-scute complex homolog 1 (ASCL1) and hairy and enhancer of split 1, one of the target molecules of the Notch signaling pathway, is crucial for NE differentiation. Small cell lung cancer (SCLC) is a highly aggressive lung tumor, characterized by rapid cell proliferation, a high metastatic potential, and the acquisition of resistance to treatment. The subtypes of SCLC are defined by the expression status of NE cell-lineage transcription factors, such as ASCL1, which roles are supported by SRY-box 2, insulinoma-associated protein 1, NK2 homeobox 1, and wingless-related integration site signaling. This network reinforces NE differentiation and may induce the characteristic morphology and chemosensitivity of SCLC. Notch signaling mediates cell-fate decisions, resulting in an NE to non-NE fate switch. The suppression of NE differentiation may change the histological type of SCLC to a non-SCLC morphology. In SCLC with NE differentiation, Notch signaling is typically inactive and genetically or epigenetically regulated. However, Notch signaling may be activated after chemotherapy, and, in concert with Yes-associated protein signaling and RE1-silencing transcription factor, suppresses NE differentiation, producing intratumor heterogeneity and chemoresistance. Accumulated information on the molecular mechanisms of SCLC will contribute to further advances in the control of this recalcitrant cancer.

A role of Achaete-scute complex homolog 2 in T follicular regulatory cell development

T follicular regulatory (Tfr) cells, a subset of CD4+ Foxp3+ regulatory T (Treg) cells, locate to the lymphoid follicle and germinal center (GC) and regulate antibody responses. Tfr cells express the functional molecules of follicular helper T (Tfh) cells, including CXCR5 and Bcl6. CD25− mature Tfr cells differentiate from CD25+ Treg cells through CD25+ immature Tfr cells. Others and we have shown that Achaete-scute complex homolog 2 (Ascl2) plays a role in Tfh cell development; however, the role of Ascl2 in the development of Tfr cells remains unclear. Here, we found that Ascl2 was highly and preferentially expressed in CD25+ Tfr cells and CD25− Tfr cells, and that the differentiation from CD25+ Tfr cells to CD25− Tfr cells was impaired by the absence of Ascl2. Furthermore, the forced Ascl2 expression in Treg cells downregulated CD25 expression and suppressed IL-2-induced phosphorylation of STAT5, which is known to suppress CD25− Tfr cell development. Finally, we found that the downregulation of CD25 by Ascl2 in Treg cells is independent of Bach2, which also regulates CD25 downregulation in CD25+ Tfr cells. These results suggest that Ascl2 plays a vital role in developing Tfr cells, possibly by downregulating CD25 expression in a Bach2-independent mechanism.

Study on the Biological Function of Abemecilib in Inhibiting the Proliferation, Invasion and Migration of Small Cell Lung Cancer with High c-Myc Expression

Small cell lung cancer (SCLC) with high c-Myc expression is prone to relapse and metastasis, leading to extremely low survival rate. Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor Abemaciclib plays a key role in the treatment of tumors, but the effects and mechanisms on SCLC remain unclear. This study was to analyze the effect and molecular mechanism of Abemaciclib in inhibiting proliferation, migration and invasion of SCLC with high c-Myc expression, with a view to expanding a new direction for reducing the recurrence and metastasis. Proteins interacting with CDK4/6 were predicted using the STRING database. The expressions of CDK4/6 and c-Myc in 31 cases of SCLC cancer tissues and paired adjacent normal tissues were analyzed by immunohistochemistry. The effects of Abemaciclib on the proliferation, invasion and migration of SCLC were detected by CCK-8, colony formation assay, Transwell and migration assay. Western blot was used to detect the expressions of CDK4/6 and related transcription factors. Flow cytometry was used to analyze the effects of Abemaciclib on the cell cycle and checkpoint of SCLC. The expression of CDK4/6 was associated with c-Myc by STRING protein interaction network. c-Myc can directly modalize achaete-scute complex homolog 1 (ASCL1), neuronal differentiation 1 (NEUROD1) and Yes-associated protein 1 (YAP1). Moreover, CDK4 and c-Myc regulate the expression of programmed cell death ligand 1 (PD-L1). Immunohistochemistry showed that the expressions of CDK4/6 and c-Myc in cancer tissues were higher than those in adjacent tissues(P<0.0001). CCK-8, colony formation assay, Transwell and migration assay verified that Abemaciclib could effectively inhibit the proliferation, invasion and migration of SBC-2 and H446OE(P<0.0001). Western blot analysis further showed that Abemaciclib not only inhibited CDK4 (P<0.05) and CDK6 (P<0.05), but also affected c-Myc (P<0.05), ASCL1 (P<0.05), NEUROD1 (P<0.05) and YAP1 (P<0.05), which are related to SCLC invasion and metastasis. Flow cytometry showed that Abemaciclib not only inhibited the cell cycle progression of SCLC cells (P<0.0001), but also significantly increased PD-L1 expression on SBC-2 (P<0.01) and H446OE (P<0.001). Abemaciclib significantly inhibits the proliferation, invasion, migration and cell cycle progression of SCLC by inhibiting the expressions of CDK4/6, c-Myc, ASCL1, YAP1 and NEUROD1. Abemaciclib can also increase the expression of PD-L1 in SCLC.

Abstract CT570: KISIMA-01: A first-in-human trial of the heterologous prime-boost vaccine ATP128/VSV-GP128 with ezabenlimab (BI 754091) in patients with stage IV colorectal cancer

Abstract Background: Most microsatellite stable/mismatch repair proficient (MSS/MMRp) stage IV colorectal cancers (CRC) do not respond to PD-1 inhibition. ATP128, based on the KISIMA platform, is a single chimeric fusion protein, composed of 3 elements essential to generate potent antitumoral cellular immunity: a proprietary cell-penetrating peptide (CPP) for antigen delivery, a proprietary Toll-like receptor (TLR)-peptide agonist with self-adjuvant properties and a modulable multi-antigenic domain (Mad), where the Mad for CRC includes 3 antigens: carcinoembryonic antigen (CEA), Survivin, Achaete-scute complex homolog 2 (ASCL2). VSV-GP128 is a recombinant vesicular stomatitis virus (VSV) carrying the glycoprotein (GP) of a non-neurotropic strain of lymphocytic choriomeningitis virus (LCMV) instead of the native VSV-GP. The GP of the LCMV abrogates neurotoxicity. As viral vector, VSV-GP128 expresses a Mad with identical sequence to ATP128 integrated in a linear, negative-sense, single-stranded RNA genome. Preclinical data have shown that priming with KISIMA vaccine followed by VSV-GP boost induced a large pool of polyfunctional and persistent antigen-specific cytotoxic T cells in the periphery as well as within the tumor in several tumor models. Combining heterologous vaccination with a PD-1 inhibitor further improved its therapeutic efficacy (Das et al., Nature Communication 2021). Methods: KISIMA-01 (NCT04046445) is an open-label, multi-center Phase 1b umbrella trial to investigate the safety, tolerability and immunogenicity of the heterologous prime-boost vaccine AT128/VSV-GP128 in combination with the anti-PD1 ezabenlimab in patients with stage IV MSS CRC. Two different patient cohorts are investigated: 1) maintenance setting (during a chemotherapy free period) after a minimum of 4 months of 1st line standard chemotherapy with clinical benefit (defined as PR or SD) (n=30); 2) resectable liver-limited disease (n=15). ATP128 is given subcutaneously on day 1 (prime); VSV-GP128 is administered intravenously on day 15 as the heterologous boost. ATP128 is again administered on day 29 (injection 3) and then every 4 weeks for the last 3 immunizations. Ezabenlimab is administered every 3 weeks starting with the first ATP128 administration. Blood and tissue samples are collected before, during and after ATP128/VSV-GP128 treatment to monitor the induction of a tumor associated antigen-specific immune response and immune-related changes. Immunogenicity will be analyzed in the peripheral blood using ELISpot, and in the tumor microenvironment by immunohistochemical assessment of tumor-infiltrating lymphocytes (TILs). Clearance of ctDNA is analyzed as an exploratory endpoint. The heterologous prime-boost cohorts (ATP128/VSV-GP128) of the KISIMA-01 trial are recruiting in the US, Switzerland and Belgium. Citation Format: Heinz-Josef Lenz, Thibaud Koessler, Paul Oberstein, Eric Van Cutsem, Sunnie Kim, Ralph Fritsch, Hans Prenen, Michael Morse, Delphine Gani, Madiha Derouazi, Thomas Bogenrieder, Scott Kopetz. KISIMA-01: A first-in-human trial of the heterologous prime-boost vaccine ATP128/VSV-GP128 with ezabenlimab (BI 754091) in patients with stage IV colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT570.

Abstract CT507: ATP128 vaccine with ezabenlimab promotes antigen-specific immune responses in stage IV colorectal cancer in the KISIMA-01 phase 1b trial

Abstract Background: KISIMA࣪ is a vaccine platform based on a single chimeric fusion protein, containing a proprietary cell-penetrating peptide (CPP) for antigen delivery, a proprietary Toll-like receptor (TLR)-peptide agonist with self-adjuvant properties and a modulable multi-antigenic domain (Mad). ATP128 vaccine targets 3 antigens: carcinoembryonic antigen (CEA), Survivin and Achaete-scute complex homolog 2 (ASCL2); it is used in combination with a PD-1 inhibitor in the treatment of MSS/MMR proficient stage IV colorectal cancer (CRC) patients, after first line of standard of care therapy or as perioperative administration in patients with resectable liver metastases. Method: KISIMA-01 (NCT04046445) is an open-label, multi-center Phase 1b trial to investigate the safety, tolerability and immunogenicity of AT128 alone or in combination with the anti-PD-1 antibody ezabenlimab in patients with stage IV CRC. ATP128 is given SC q2w for the first 3 immunizations (prime) and q4w for the last 3 immunizations (boost). Ezabenlimab is administered q3w starting with the first ATP128 administration. Blood and tissue samples are collected before, during and after ATP128 treatment to monitor the induction of a tumor associated antigen-specific immune response (ELISpot) and immune-related changes in the peripheral blood and in the tumor microenvironment by immunohistochemistry (IHC) and flow cytometry. Results: In more than 50% of evaluated patients treated with ATP128 alone or with ezabenlimab, a cellular immune response against at least one out of three antigens was observed as determined by IFN-γ ELISpot analyses of patient PBMCs after the 3rd vaccination. Analysis of liver metastases by IHC indicated that evaluated patients were positive for all 3 antigens in ATP128. Furthermore, for the patients with paired biopsies, a significant increase in CD8 T cells infiltration into the tumor parenchyma was observed after 3 vaccine administrations, along with a significantly higher proportion of CD45RO expressing memory cells within the CD4 population as compared to baseline. Tumor infiltrating lymphocytes (TILs) flow cytometry analysis comparing untreated (historical controls) and ATP128/ezabenlimab-treated patients showed a similar quantity of the different infiltrated subsets but an improved quality of infiltrated T cells, indicated by an increase (more than 2-fold) in proportion of central memory T cells and an impressive decrease of the proportion of cells positive for exhaustion markers expression in KISIMA-01 patients. Conclusions: Analyses indicate induction of ATP128-specific immune response in the peripheral circulation and increased infiltration of TILs into liver metastases with an improved quality of T cells. Citation Format: Heinz-Josef Lenz, Hanz Prenen, Eric Van Cutsem, Thibaud Kössler, Jean-François Mayol, Francesca Trapani, Matthieu Tihy, Laura Rubbia-Brandt, Christian Toso, Thomas Bogenrieder, Elodie Belnoue, Madiha Derouazi, Scott Kopetz. ATP128 vaccine with ezabenlimab promotes antigen-specific immune responses in stage IV colorectal cancer in the KISIMA-01 phase 1b trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT507.

Transcription Factor ASCL1 Acts as a Novel Potential Therapeutic Target for the Treatment of the Cushing's Disease.

The pathogenesis of Cushing's disease (CD) is still not adequately understood despite the identification of somatic driver mutations in USP8, BRAF, and USP48. In this multiomics study, we combined RNA sequencing (RNA-seq) with Sanger sequencing to depict transcriptional dysregulation under different gene mutation backgrounds. Furthermore, we evaluated the potential of achaete-scute complex homolog 1 (ASCL1), a pioneer transcription factor, as a novel therapeutic target for treatment of CD and its possible downstream pathway. RNA-seq was adopted to investigate the gene expression profile of CD, and Sanger sequencing was adopted to detect gene mutations. Bioinformatics analysis was used to depict transcriptional dysregulation under different gene mutation backgrounds. The function of ASCL1 in hormone secretion, cell proliferation, and apoptosis were studied in vitro. The effectiveness of an ASCL1 inhibitor was evaluated in primary CD cells, and the clinical relevance of ASCL1 was examined in 68 patients with CD. RNA-seq in AtT-20 cells on Ascl1 knockdown combined with published chromatin immunoprecipitation sequencing data and dual luciferase assays were used to explore downstream pathways. ASCL1 was exclusively overexpressed in USP8-mutant and wild-type tumors. Ascl1 promoted adrenocorticotrophin hormone overproduction and tumorigenesis and directly regulated Pomc in AtT-20 cells. An ASCL1 inhibitor presented promising efficacy in both AtT-20 and primary CD cells. ASCL1 overexpression was associated with a larger tumor volume and higher adrenocorticotrophin secretion in patients with CD. Our findings help to clarify the pathogenesis of CD and suggest that ASCL1 is a potential therapeutic target the treatment of CD. The pathogenesis of Cushing's disease (CD) is still not adequately understood despite the identification of somatic driver mutations in USP8, BRAF, and USP48. Moreover, few effective medical therapies are currently available for the treatment of CD. Here, using a multiomics approach, we first report the aberrant overexpression of the transcription factor gene ASCL1 in USP8-mutant and wild-type tumors of CD. Ascl1 promoted adrenocorticotrophin hormone overproduction and tumorigenesis and directly regulated Pomc in mouse AtT-20 cells. Notably, an ASCL1 inhibitor presented promising efficacy in both AtT-20 and primary CD cells. Importantly, ASCL1 overexpression was associated with a larger tumor volume and higher adrenocorticotrophin secretion in patients with CD. Thus, our findings improve understanding of CD pathogenesis and suggest that ASCL1 is a potential therapeutic target the treatment of CD.

SO-11 KISIMA-01 trial: Safety, tolerability and immunogenicity of ATP128 with or without ezabenlimab (BI 754091) in patients with stage IV colorectal cancer – preliminary results from a phase 1b study

Microsatellite stable/mismatch repair proficient (MSS/MMRp) stage IV colorectal cancer (CRC) remains an area of high unmet medical need, with the vast majority not responding to anti-PD1 checkpoint inhibitors (CPI). It is believed that a cancer vaccine approach could turn such “cold” tumors into “hot” tumors. ATP128 is a single chimeric fusion protein composed of three elements essential to generate potent antitumoral cellular immunity: a proprietary cell-penetrating peptide (CPP) for antigen delivery, a proprietary Toll-like receptor (TLR)-peptide agonist with self-adjuvant properties and a modulable multi-antigenic domain (Mad), where the Mad for CRC includes3 antigens: carcinoembryonic antigen (CEA), Survivin, Achaete-scute complex homolog 2 (ASCL2).

ASCL1 promotes tumor progression through cell-autonomous signaling and immune modulation in a subset of lung adenocarcinoma.

The master regulator of neuroendocrine differentiation, achaete-scute complex homolog 1 (ASCL1) defines a subgroup of lung adenocarcinoma. However, the mechanistic role of ASCL1 in lung tumorigenesis and its relation to the immune microenvironment is principally unknown. Here, the immune landscape of ASCL1-positive lung adenocarcinomas was characterized by immunohistochemistry. Furthermore, ASCL1 was transduced in mouse lung adenocarcinoma cell lines and comparative RNA-sequencing and secretome analyses were performed. The effects of ASCL1 on tumorigenesis were explored in an orthotopic syngeneic transplantation model. ASCL1-positive lung adenocarcinomas revealed lower infiltration of CD8+, CD4+, CD20+, and FOXP3+ lymphocytes and CD163+ macrophages indicating an immune desert phenotype. Ectopic ASCL1 upregulated cyclin transcript levels, stimulated cell proliferation, and enhanced tumor growth in mice. ASCL1 suppressed secretion of chemokines, including CCL20, CXCL2, CXCL10, and CXCL16, indicating effects on immune cell trafficking. In accordance with lower lymphocytes infiltration, ASCL1-positive lung adenocarcinomas demonstrated lower abundance of CXCR3-and CCR6-expressing cells. In conclusion, ASCL1 mediates its tumor-promoting effect not only through cell-autonomous signaling but also by modulating chemokine production and immune responses. These findings suggest that ASCL1-positive tumors represent a clinically relevant lung cancer entity.

Significance of achaete-scute complex homologue 1 (ASCL1) in pulmonary neuroendocrine carcinomas; RNA sequence analyses using small cell lung cancer cells and Ascl1-induced pulmonary neuroendocrine carcinoma cells.

ASCL1 is one of the master transcription factors of small cell lung carcinoma (SCLC). To investigate the significance of ASCL1 in pulmonary neuroendocrine carcinoma, we performed 2 comparative RNA-seq studies between H69 (ASCL1-positive, classical type SCLC) and H69AR (ASCL1-negative, variant type SCLC) and between ASCL1-transfected A549 adenocarcinoma cell lines (A549(ASCL1+) cell lines) and A549(control) cell lines. RNA-seq analyses revealed that 940 genes were significantly different between the H69 and H69AR cell lines, and 728 between the A549(ASCL1+) and A549(control) cell lines. In total, 120 common genes between these analyses were selected as candidate ASCL1-related genes, and included genes with various cellular functions, such as neural development, secretion, growth, and morphology. Their expression degrees in three classical and two variant SCLC cell lines, two A549(ASCL1+) and two A549(control) cell lines were subjected to quantitative PCR analyses. Since the candidate ASCL1-related genes were strongly expressed in the classical SCLC and A549(ASCL1+) cell lines and more weakly expressed in the variant SCLC and A549(control) cell lines, the ASCL1-related 7 molecules INSM1, ISL1, SYT4, KCTD16, SEZ6, MS4A8, and COBL were further selected. These molecules suggested diverse functions for A549(ASCL1+): INSM1 and ISL1 are transcription factors associated with neuroendocrine differentiation, while SYT4, KTCD16, and SEZ6 may be related to neurosecretory functions and MS4A8 and COBL to cell growth and morphology. An immunohistochemistry of these seven molecules was performed on lung carcinoma tissues and the xenotransplanted tumors of A549(ASCL1+), and they were preferentially and positively stained in ASCL1-postive tumor tissues.

Ascl1-induced Wnt11 regulates neuroendocrine differentiation, cell proliferation, and E-cadherin expression in small-cell lung cancer and Wnt11 regulates small-cell lung cancer biology

The involvement of Wnt signaling in human lung cancer remains unclear. This study investigated the role of Wnt11 in neuroendocrine (NE) differentiation, cell proliferation, and epithelial-to-mesenchymal transition (EMT) in human small-cell lung cancer (SCLC). Immunohistochemical staining of resected specimens showed that Wnt11 was expressed at higher levels in SCLCs than in non-SCLCs; 58.8% of SCLC, 5.2% of adenocarcinoma (ADC), and 23.5% of squamous cell carcinoma tissues stained positive for Wnt11. A positive relationship was observed between Achaete-scute complex homolog 1 (Ascl1) and Wnt11 expression in SCLC cell lines, and this was supported by transcriptome data from SCLC tissue. The expression of Wnt11 and some NE markers increased after the transfection of ASCL1 into the A549 ADC cell line. Knockdown of Ascl1 downregulated Wnt11 expression in SCLC cell lines. Ascl1 regulated Wnt11 expression via lysine H3K27 acetylation at the enhancer region of the WNT11 gene. Wnt11 controlled NE differentiation, cell proliferation, and E-cadherin expression under the regulation of Ascl1 in SCLC cell lines. The phosphorylation of AKT and p38 mitogen-activated protein kinase markedly increased after transfection of WNT11 into the SBC3 SCLC cell line, which suggests that Wnt11 promotes cell proliferation in SCLC cell lines. Ascl1 plays an important role in regulating the Wnt signaling pathway and is one of the driver molecules of Wnt11 in human SCLC. Ascl1 and Wnt11 may employ a cooperative mechanism to control the biology of SCLC. The present results indicate the therapeutic potential of targeting the Ascl1–Wnt11 signaling axis and support the clinical utility of Wnt11 as a biological marker in SCLC.

WiNTRLINC1/ASCL2/c-Myc Axis Characteristics of Colon Cancer with Differentiated Histology at Young Onset and Essential for Cell Viability.

WiNTRLINC1 is a long non-coding RNA (lncRNA) that positively regulates the Wnt pathway via achaete-scute complex homolog 2 (ASCL2) in colorectal cancer. ASCL2 was recently reported to play a critical role in chemoresistance, however clinical relevance of the WiNTRLINC1/ASCL2 axis remains obscure in colon cancer. WiNTRLINC1/ASCL2 expression was investigated at messenger RNA (mRNA) level in 40 primary colon cancer tissues and the corresponding normal mucosa tissues, together with Wnt-related genes (c-Myc/PRL-3) and other lncRNAs (H19, HOTAIR, and MALAT1). Knock-down experiments of WiNTRLINC1 clarified its role in their expression and chemoresistance. Real-time quantitative reverse transcriptase-polymerase chain reaction confirmed definite overexpression of WiNTRLINC1 mRNA in primary colon cancer compared with the corresponding normal colon mucosa tissues (p = 0.0005), such as ASCL2, c-Myc, and PRL-3 (p < 0.0001). The four gene expression signatures were tightly associated in the center of the ASCL2 gene (r = 0.72, p < 0.0001) in clinical samples. WiNTRLINC1 was not significantly associated with prognostic factors in colon cancer and other lncRNAs, while the WiNTRLINC1/ASCL2/c-Myc signatures were unique to young-onset colon cancer with differentiated histology. On the other hand, undifferentiated histology was significantly associated with H19 expression. Knockdown of the WiNTRLINC1 gene reduced the expression of ASCL2/c-Myc, but rather augmented PRL-3 at mRNA level, and robustly affected cell viability in colon cancer cell lines. The enhanced WiNTRLINC1/ASCL2/c-Myc axis involved in Wnt pathway activation is a common pathway essential for differentiated colon tumorigenesis, especially with young onset, and may be essential for a viable phenotype of colon cancer.

Ascl2 facilitates IL-10 production in Th17 cells to restrain their pathogenicity in inflammatory bowel disease

Inflammatory bowel disease (IBD) has been well-documented as a chronic gastrointestinal autoimmune disease, but its etiology remains to be elusive. Ascl2 (achaete-scute complex homologue 2), identified as a homologue of the Drosophila achaete-scute gene, has been shown to play an essential for the pathogenesis of autoimmune diseases and cancers. However, whether it is associated with the pathogenesis of IBD remains unclear. Here, we demonstrated that Ascl2 was greatly down-regulated in human IBD and experimental colitis. Interestingly, CD4+ T cell expression of Ascl2 was regulated by intestinal microbiota. Moreover, we revealed that Ascl2 inhibited the differentiation of Th17 cells and restrained their pathogenicity through facilitating IL-10 production. We further showed that Blimp-1 might be involved in the Ascl2-inducing IL-10 expression in CD4+ T cells under Th17 differentiating condition. Notably, lentivirus-mediated overexpression of Ascl2 remarkably alleviated the severity of 2,4,6-trinitrobenzenesulfonic acid solution (TNBS)-induced colitis in mice, with decreased level of colonic IL-17A. Our findings demonstrated an unappreciated mechanism whereby Ascl2 negatively modulates pathogenic Th17 cell differentiation via promoting IL-10 production, and alleviates intestinal inflammation. Thus, Ascl2 may serve as a novel therapeutic target of IBD.

Expression of mammalian ASH1 and ASH4 in Drosophila reveals opposing functional roles in neurogenesis

To investigate whether the members of the mammalian Achaete-Scute Complex homologue (ASH) gene family have evolved functional differences, we used the patterning of bristles as a phenotypic marker. Drosophila uses a single genetic locus - the Achaete-Scute Complex - to demarcate the regions of the body where bristles can form. We found 4–5 Achaete-Scute Complex homologue genes (ASH) in the mammalian genome, which are homologous with scute in Drosophila. Although ASH2 and ASH3 have gained new functions during evolution, the function of ASH4 and its evolutionary changes are still unclear. In this study, we overexpressed mouse and human ASH1 and ASH4 in the Drosophila notum respectively. The results show that both the protein sequence and cis-regulatory elements of mammalian ASH1 have conserved an ancient proneural function during evolution. However, mouse ASH4 has lost proneural function partly due to truncation of a C-terminal amino acid domain. Interestingly, instead of a similar loss of proneural function, we found human ASH4 can actually inhibit Drosophila bristle development, implying that human ASH4 may be a potential factor relating to skin development in human being. Our results demonstrate gene duplication of the ASH family may have led to a novel function during evolution.

An integrative transcriptome analysis reveals a functional role for thyroid transcription factor-1 in small cell lung cancer.

Small cell lung cancer (SCLC) is a neuroendocrine tumour that exhibits rapid growth and metastatic spread. Although SCLC represents a prototypically undifferentiated cancer type, thyroid transcription factor-1 (TTF-1, gene symbol NKX2-1), a master regulator for pulmonary epithelial cell differentiation and lung morphogenesis, is strongly upregulated in this aggressive cancer type. The aim of this study was to evaluate a functional role for TTF-1 in SCLC. We demonstrated that achaete-scute complex homolog 1 (ASCL1), an essential transcription factor for neuroendocrine differentiation, positively regulated TTF-1 in SCLC cell lines. Subsequently, we described genes and microRNAs (miRNAs) that were possibly controlled by TTF-1 and identified nuclear factor IB (NFIB), a recently characterised driver of SCLC progression, as a transcriptional target of TTF-1. Our findings shine light on a regulatory axis in SCLC consisting of ASCL1/TTF-1/NFIB that potentially contributes to the tumourigenesis of SCLC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract LB-199: Role of ASCL1 in neuroendocrine prostate cancer progression

Abstract INTRODUCTION: Metastatic prostate cancer is a lethal disease. Many patients develop a form of the disease resistant to Androgen Deprivation Therapy (ADT) called castration resistant prostate cancer (CRPC), and androgen refractory tumors emerge. A subset of these involve gaining small cell and neuroendocrine (NE) features, resulting in neuroendocrine prostate cancer (NEPC). The identification of key drivers and the mechanism of emergence is critical, as NEPC is associated with poor outcomes. Of interest is the neuron lineage differentiation gene achaete-scute complex homologue 1 (ASCL1). Mouse models of small cell lung cancer (SCLC) show ASCL1 as a key promoter of tumor growth in vivo. While there is overlap between SCLC and NEPC (inactivation of RB1 and TP53, ASCL1 overexpression (OE)), it's unclear if ASCL1 contributes to transdifferentiation of prostate epithelium into NEPC. In addition to genetic drivers, defined soluble factors can promote reprogramming of fibroblasts into neurons. We thus hypothesize that ASCL1 in combination with soluble factors may promote transdifferentiation of prostate epithelium to NEPC. METHODS: CRISPR technology is used to knockout (KO) RB1 and TP53 in LNCaP cells. Lentiviral vector is used to OE for ASCL1 or RFP in LNCaP or LNCap p53/RB1 KO cells. Gene expression for AR, AR target genes, neuronal, and NE genes is measured via qRT-PCR and Western. Regular FBS or neuronal media containing either FGF2/BDNF/NTF3 or all soluble factors is used for cell culture. RESULTS: RNAseq data from metastatic AR+ CRPC, AR- NEPC, and SCLC tumors and cell lines shows OE of ASCL1 in AR- NEPC and SCLC. LNCaP ASCL1 OE cells show no change in cellular growth in FBS, CSS, or NM + supplements (BDNF, FGF2, NT3) compared to control. However, LNCaP p53/RB1 KO ASCL1 OE cells + 10μM Enzalutamide (ENZ) proliferated similar to the cells without any drug treatment when cultured in NM + sup (96h, p=0.999). Both LNCaP ± ASCL1 and LNCaP p53/RB1 KO ± ASCL1 cells grown in NM + sup show significantly decreased AR and AR target gene expression (~10 fold, p&amp;lt; 0.05). FGF8 (~5 fold), SOX2 (&amp;gt;300 fold), and SYP (&amp;gt;10 fold) gene expression increased significantly (p&amp;lt;0.05) in LNCaP ± ASCL1 grown in NM + sup. LNCaP p53/RB1 KO ± ASCL1 OE in NM + sup show ~3-6 fold increase in SOX2 and ~30-50 fold increase in FGF9 compared to cells cultured in FBS. CONCLUSION: Loss of TP53 and RB1 are not sufficient to induce transdifferentiation to a NEPC state, but ASCL1 OE induced the expression of SOX2 and SYP that may trigger the process. Microenvironment effects operating through paracrine factors induced the expression of a NE gene program and reduced AR expression. Future studies will investigate the role of ASCL1 in promoting resistance to ENZ in prostate adenocarcinoma cells as well as its role in in vivo tumor growth. ASCL1, NEUROD1, and MYTL1 that are OE in NEPC and SCLC in combination with individual soluble factors will be used to delineate the mechanism required for the transdifferentiation process. Citation Format: Seema Sinha, Michael D. Nyquist, Alexandra Corella, Ilsa Coleman, Peter S. Nelson. Role of ASCL1 in neuroendocrine prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-199.

PO-461 A transcriptomic and molecular approach to uncovering achaete-scute complex homolog 2 (ASCL2) as a potential novel driver gene in breast cancer

IntroductionBreast cancer is highly heterogeneous and despite ongoing advancements in diagnostics and targeted therapeutics, incidence and mortality continues to rise. Thus, there is a need for greater characterisation of driver genes to understand their mechanistic role in breast carcinogenesis and their respective signalling pathways.With the advent of high-throughput technologies in transcriptomics, it is now possible to examine thousands of genes in parallel, generating an unprecedented amount of information. However, the task of synthesising the vast amount of ‘OMICs’ data produced and extracting the clinically significant results is a prominent challenge. The aim of this study was to use in silico and in vitro methods to identify and functionally investigate a novel genetic driver gene that plays a key role in breast carcinogenesis.Material and methodsGene expression profiles were obtained from public databases (Array Express and Gene Expression Omnibus) and subjected to an extreme variation analysis. Pathway analysis tools were used to identify a novel potential candidate gene for further investigation. Gene expression was assessed in vitro by quantitative real-time RT-PCR (qRT-PCR) to ensure concordance with transcriptomic data. siRNA knockdown was carried out in breast cancer cells, and sufficient knockdown was validated using qRT-PCR. Subsequently, to assess the effect of gene knockdown, assays were used to measure proliferation, wound-healing and apoptosis.Results and discussionsWe have established a basic, novel and user-friendly pipeline for in silico pathway analysis of transcriptomic data. This has allowed for selection and in vitro validation of a candidate driver gene, Achaete-scute complex homolog 2 (ASCL2), seen to be elevated in breast cancer. This gene is a transcription factor and regulator of stem cell identity, as well as a known Wnt-target gene. Functional work has suggested that ASCL2 may be involved in the migration of breast cancer cells, as upon siRNA knockdown of ASCL2, cellular migration was inhibited compared to negative controls. Additional analysis has revealed that ASCL2 knockdown alters the expression of various Wnt-associated genes, such as Survivin (BIRC5) and CD44.ConclusionThis work suggests that ASCL2, functioning within the Wnt signalling pathway, may be a potential novel driver of breast tumourigenesis. Investigation using both in vitro methods and data mining, will continue to further elucidate the functional role of ASCL2 in breast cancer tumourigenesis.

Unconventional function of an Achaete-Scute homolog as a terminal selector of nociceptive neuron identity.

Proneural genes are among the most early-acting genes in nervous system development, instructing blast cells to commit to a neuronal fate. Drosophila Atonal and Achaete-Scute complex (AS-C) genes, as well as their vertebrate orthologs, are basic helix-loop-helix (bHLH) transcription factors with such proneural activity. We show here that a C. elegans AS-C homolog, hlh-4, functions in a fundamentally different manner. In the embryonic, larval, and adult nervous systems, hlh-4 is expressed exclusively in a single nociceptive neuron class, ADL, and its expression in ADL is maintained via transcriptional autoregulation throughout the life of the animal. However, in hlh-4 null mutants, the ADL neuron is generated and still appears neuronal in overall morphology and expression of panneuronal and pansensory features. Rather than acting as a proneural gene, we find that hlh-4 is required for the ADL neuron to function properly, to adopt its correct morphology, to express its unusually large repertoire of olfactory receptor–encoding genes, and to express other known features of terminal ADL identity, including neurotransmitter phenotype, neuropeptides, ion channels, and electrical synapse proteins. hlh-4 is sufficient to induce ADL identity features upon ectopic expression in other neuron types. The expression of ADL terminal identity features is directly controlled by HLH-4 via a phylogenetically conserved E-box motif, which, through bioinformatic analysis, we find to constitute a predictive feature of ADL-expressed terminal identity markers. The lineage that produces the ADL neuron was previously shown to require the conventional, transient proneural activity of another AS-C homolog, hlh-14, demonstrating sequential activities of distinct AS-C-type bHLH genes in neuronal specification. Taken together, we have defined here an unconventional function of an AS-C-type bHLH gene as a terminal selector of neuronal identity and we speculate that such function could be reflective of an ancestral function of an “ur-” bHLH gene.

Evidence for newly generated interneurons in the basolateral amygdala of adult mice

New neurons are continually generated from the resident populations of precursor cells in selective niches of the adult mammalian brain such as the hippocampal dentate gyrus and the olfactory bulb. However, whether such cells are present in the adult amygdala, and their neurogenic capacity, is not known. Using the neurosphere assay, we demonstrate that a small number of precursor cells, the majority of which express Achaete-scute complex homolog 1 (Ascl1), are present in the basolateral amygdala (BLA) of the adult mouse. Using neuron-specific Thy1-YFP transgenic mice, we show that YFP+ cells in BLA-derived neurospheres have a neuronal morphology, co-express the neuronal marker βIII-tubulin, and generate action potentials, confirming their neuronal phenotype. In vivo, we demonstrate the presence of newly generated BrdU-labeled cells in the adult BLA, and show that a proportion of these cells co-express the immature neuronal marker doublecortin (DCX). Furthermore, we reveal that a significant proportion of GFP+ neurons (~23%) in the BLA are newly generated (BrdU+) in DCX-GFP mice, and using whole-cell recordings in acute slices we demonstrate that the GFP+ cells display electrophysiological properties that are characteristic of interneurons. Using retrovirus-GFP labeling as well as the Ascl1CreERT2 mouse line, we further confirm that the precursor cells within the BLA give rise to mature and functional interneurons that persist in the BLA for at least 8 weeks after their birth. Contextual fear conditioning has no effect on the number of neurospheres or BrdU-labeled cells in the BLA, but produces an increase in hippocampal cell proliferation. These results demonstrate that neurogenic precursor cells are present in the adult BLA, and generate functional interneurons, but also show that their activity is not regulated by an amygdala-dependent learning paradigm.