Differential expression of genes and gene signatures in RPM and RPMA allografts.

Genotypes of prostate organoids and resulting allograft tumors.

IHC characterization of RPM tumors and histopathology assessment of allografts.

Castration does not alter growth or heterogeneity of RPM and RPMA allograft tumors

Sorting parameters for TdTom + tumor cells used for seeding secondary allograft tumors.

Genotypes of RPMA organoids and differences in metastatic burden.

PCR genotyping gels and unmerged images from immunofluorescence.

ASCL1 is a neural basic helix-loop-helix (bHLH) transcription factor that plays essential roles during neural development, including neural differentiation and neuronal subtype specification. bHLH factors are defined by their motifs, including a basic region interacting with DNA and an HLH domain involved in protein-protein interactions. We previously defined specific regions within the bHLH domain of ASCL1 as important for its specific functions directing neuronal differentiation in the chick neural tube. Here, we build upon these findings to show how specific mutations within the basic region block DNA binding but not heterodimer formation with E-protein partners TCF3 (E12/E47) and TCF12 (HEB) yet have differential abilities to show dominant negative phenotypes. Additionally, truncating domains outside the bHLH define a nuclear localization signal, a requirement for the C-terminal acidic residues, and the non-essentiality of the N-terminal glutamine/alanine repeats. This structure/function analysis identifies functional domains for ASCL1 activity.

Shared genetic developmental programs in which specific transcription factors affect similar cell fate decisions in distinct tissues are common. In the developing dorsal neural tube and cerebellum, PTF1A is essential for specification of GABAergic inhibitory neurons and suppression of alternative glutamatergic excitatory neuronal fates. Previous studies in the mouse dorsal neural tube identified the transcriptional repressor PRDM13 as a transcriptional target of PTF1A that functions to suppress the alternate cell fates to ensure precision in neuronal cell identity. The presence of PRDM13 in PTF1A + cerebellar progenitors suggests a similar role for PRDM13 in cerebellar neuronal specification. Cerebellar agenesis in humans with missense mutations in PRDM13, and perturbations in cerebellar development in Prdm13 mutant mice and zebrafish, confirm PRDM13 requirement in this tissue. Here we add to these findings showing additional mutant alleles in mouse Prdm13 phenocopy the perturbation in cerebellar cell fates seen with the absence of PTF1A, including loss of PAX2+ interneuron and Purkinje cell inhibitory neuronal lineages, increases in TLX3+ excitatory neuronal lineages, increased apoptosis, and reduced cerebellar size. Additional defects are seen in the placement of TBR1+ cerebellar cells. Thus, using Prdm13 mutant mice, we support conclusions that PRDM13 functions to specify balanced numbers of inhibitory and excitatory neuronal progenitors in the developing cerebellum.

ABSTRACTGlioblastomas (GBMs) are highly aggressive, infiltrative, and heterogeneous brain tumors driven by complex driver mutations and glioma stem cells (GSCs). The neurodevelopmental transcription factors ASCL1 and OLIG2 are co-expressed in GBMs, but their role in regulating the heterogeneity and hierarchy of GBM tumor cells is unclear. Here, we show that oncogenic driver mutations lead to dysregulation of ASCL1 and OLIG2, which function redundantly to initiate brain tumor formation in a mouse model of GBM. Subsequently, the dynamic levels and reciprocal binding of ASCL1 and OLIG2 to each other and to downstream target genes then determine the cell types and degree of migration of tumor cells. Single-cell RNA sequencing (scRNA-seq) reveals that a high level of ASCL1 is key in defining GSCs by upregulating a collection of ribosomal protein, mitochondrial, neural stem cell (NSC), and cancer metastasis genes – all essential for sustaining the high proliferation, migration, and therapeutic resistance of GSCs.

Abstract Glioblastomas (GBMs) are highly aggressive, infiltrative, and heterogeneous brain tumors driven by complex genetic alterations. The neurodevelopmental transcription factors ASCL1 and OLIG2 are highly co-expressed in GBMs. However, their combinatorial roles in regulating the hierarchy and heterogeneity of GBM cells are unknown. Here, we show that induction of somatic mutations in neural progenitor cells lead to the dysregulation of ASCL1 and OLIG2, which then function redundantly and are required for brain tumor formation in a mouse model of GBM. Subsequently, the binding of ASCL1 and OLIG2 to each other’s loci and to downstream target genes then determine the cell types and degree of migration of tumor cells. Notably, single-cell RNA sequencing (scRNA-seq) reveals that a high level of ASCL1 is key in promoting neural stem cell (NSC)/astrocyte-like tumor cell types, which are highly proliferative, migratory, and are marked by upregulation of ribosomal protein, oxidative phosphorylation, cancer metastasis, and therapeutic resistance genes.

Glioblastomas (GBMs) are highly aggressive, infiltrative, and heterogeneous brain tumors driven by complex genetic alterations. The basic-helix-loop-helix (bHLH) transcription factors ASCL1 and OLIG2 are dynamically co-expressed in GBMs; however, their combinatorial roles in regulating the plasticity and heterogeneity of GBM cells are unclear. Here, we show that induction of somatic mutations in subventricular zone (SVZ) progenitor cells leads to the dysregulation of ASCL1 and OLIG2, which then function redundantly and are required for brain tumor formation in a mouse model of GBM. Subsequently, the binding of ASCL1 and OLIG2 to each other's loci and to downstream target genes then determines the cell types and degree of migration of tumor cells. Single-cell RNA sequencing (scRNA-seq) reveals that a high level of ASCL1 is key in specifying highly migratory neural stem cell (NSC)/astrocyte-like tumor cell types, which are marked by upregulation of ribosomal protein, oxidative phosphorylation, cancer metastasis, and therapeutic resistance genes. ASCL1 and OLIG2 are two basic-helix-loop-helix transcription factors that are highly co-expressed in glioblastoma (GBM). Here the authors find these two transcription factors function redundantly and are required for brain tumor initiation in a mouse model of GBM, while they possess inverse roles in determining tumor cell types and cell migration ability.

Supplementary Table from Inhibition of Karyopherin β1-Mediated Nuclear Import Disrupts Oncogenic Lineage-Defining Transcription Factor Activity in Small Cell Lung Cancer

Most patients with prostate adenocarcinoma (PAd), an androgen receptor (AR) driven cancer, develop resistance to therapies targeting AR. Consequently, a portion of these patients develop neuroendocrine prostate cancer (NEPC), a rapidly progressing cancer with limited therapies and poor survival outcomes. Current research to understand the transition of PAd to NEPC suggests a model of lineage plasticity, where AR-dependent luminal tumors progress towards an AR-independent neuroendocrine lineage. Several groups have shown human NEPC tumors have lost RB1 and TP53, and in experimental models, loss of both genes mediates the transition to a neuroendocrine lineage. Notably, NEPC histology and gene expression resemble another neuroendocrine cancer, small cell lung carcinoma (SCLC), also characterized by loss of RB1 and TP53. In SCLC, transcription factor ASCL1 is required for tumor cell growth in vitro and for SCLC formation in vivo. ASCL1 is also present in NEPC tumors and in NEPC cell line models. In fact, ASCL1 was shown to regulate neuronal stem cell-like lineage programming in NEPC in vitro. We aimed to determine if ASCL1 is required in the transition of PAd to NEPC in vivo and to define its function in NEPC. To model the PAd to NEPC transition, we established genetically engineered mouse models (GEMMs) harboring loss of RB1 and TP53 with MYC overexpression (RPM) by administering adenovirus expressing Cre recombinase directly to the prostate of these GEMMs. These animals display prostate tumors with small cell histology and are heterogeneous for neuroendocrine markers such as ASCL1, NEUROD1, and INSM1. We recently determined that the concomitant loss of ASCL1 in this model (RPMA) does not stop the formation of tumors with small cell histology. Notably, a subset of the cells with small cell histology are NEUROD1+, with a higher proportion of NEUROD1+ cells present compared to the RPM model with intact ASCL1, suggesting the possibility that NEUROD1 in the same or distinct cells is compensating for the absence of ASCL1. In a separate paradigm using these same GEMMs to establish prostate organoids, we show their capacity to generate subcutaneous allograft tumors displaying mixed histology including neuroendocrine small cell features expressing ASCL1, NEUROD1, and INSM1. Strikingly, subcutaneous allografts from prostate organoids lacking ASCL1 exhibit a dramatic reduction in tumor formation efficiency, lack small cell histology including NEUROD1 and INSM1 and other neuroendocrine markers, and often result in the development of cysts rather than tumors. These results demonstrate complex functions for ASCL1 in neuroendocrine lineage programming while suggesting additional functions in supporting prostate tumor formation. Citation Format: Kathia E. Rodarte, Lydia Flores, Vishal Kandagatla, Juan Villarreal, Trisha K. Savage, Su Deng, Ping Mu, Rajal B. Shah, Trudy G. Oliver, Jane E. Johnson. Exploring the role of ASCL1 in neuroendocrine prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR004.

Abstract Small cell lung cancer (SCLC) and other neuroendocrine (NE) lung tumors account for 25% of all lung cancers, they are the most aggressive type and have the poorest prognosis. SCLC does not present driver oncogenes involved in the regulation of kinase signaling, which makes these tumors hard to treat. The few available treatments yield responses early, but relapse occurs and they become resistant. Due to the poor efficacy of therapies, research in SCLC had been moved to the background, with basically no progress over the past decades. Mutations in the RAS-ERK pathway, elevating ERK activity and enhancing tumorigenesis, are common in other types of cancer, including NSCLC. Nevertheless, they are rare in SCLC. Indeed, previous studies demonstrated that ERK activation interferes with growth of SCLC. However, the mechanism by which ERK hinders tumor progression has not been defined yet. One of the most important players in SCLC and other NE tumors is the transcription factor ASCL1, essential for their survival. It has been reported that ASCL1 increases DUSP6 transcription with the consequent ERK activity suppression. Its downregulation affects cell cycle progression and survival. Conversely, inhibition of ERK activation by MEK inhibitors increases ASCL1 mRNA levels. Previous studies in our laboratory suggested that DUSP6 has a major role in a subset of SCLC and NSCLC-NE cell lines. Inhibition of DUSP6 enhances ERK signaling and significantly decreases survival of NE lung tumor cells. We have considered DUSP6 a good candidate to try to understand the mechanism by which ERK interferes with survival. However, hurdles in studying DUSP6 in SCLC include off-target effects of inhibitors and difficulty of gene-edited cell lines. To date, we have been able to generate a DUSP6 KO NSCLC-NE cell line which shows a clear drop in proliferation. Also, the overexpression of ERK or a constitutively active MEK1 mutant in a few SCLC, NE-NSCLC and MEK inhibitor-insensitive squamous lung cancer cell lines render a decrease in survival. DUSP6 is a cytoplasmic anchor for ERK and may reduce the ability of ERK to suppress ASCL1 transcription. For this reason, we are working on studying the effect on survival of ERK localization in the nucleus or the cytoplasm. With our ongoing experiments we are defining the conditions to examine transcriptional impact of ERK on SCLC. Discovering molecular events antagonized by the ERK pathway in SCLC will reveal novel targets controlling the inhibitory mechanisms. One of the SCLC major questions of translational relevance is what are the mechanisms upholding the development of chemoresistance, where the ERK pathway seems to be involved. Hence, the definition of the underlying mechanisms of tumorigenesis and therapy resistance will uncover new potential antitumor targets that currently escape our notice. A better understanding of the molecular biology of SCLC will let us find new therapeutic strategies to overcome the impasse in chemotherapy. Citation Format: Ana Martin-Vega, Svetlana Eartnest, John D. Minna, Jane E. Johnson, Melanie H. Cobb. Mechanisms of ERK action in MEK inhibitor-insensitive lung cancers [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 LB030.

DNA methylation is a key regulator of gene expression and a clinical therapeutic predictor. We examined global DNA methylation beyond the generally used promoter areas in human small cell lung cancer (SCLC) and find that gene body methylation is a robust positive predictor of gene expression. Combining promoter and gene body methylation better predicts gene expression than promoter methylation alone including genes involved in the neuroendocrine classification of SCLC and the expression of therapeutically relevant genes including MGMT, SLFN11, and DLL3. Importantly, for super-enhancer (SE) covered genes such as NEUROD1 or MYC, using H3K27ac and NEUROD1, ASCL1, and POU2F3 ChIP-seq data, we show that genic methylation is inversely proportional to expression, thus providing a new approach to identify potential SE regulated genes involved in SCLC pathogenesis. To advance SCLC transitional research, these data are integrated into our web portal ( https://discover.nci.nih.gov/ SclcCellMinerCDB/) for open and easy access to basic and clinical investigators.

Abstract Genomic studies support the classification of small cell lung cancer (SCLC) into subtypes based on the expression of lineage-defining transcription factors ASCL1 and NEUROD1, which together are expressed in ∼86% of SCLC. ASCL1 and NEUROD1 activate SCLC oncogene expression, drive distinct transcriptional programs, and maintain the in vitro growth and oncogenic properties of ASCL1 or NEUROD1-expressing SCLC. ASCL1 is also required for tumor formation in SCLC mouse models. A strategy to inhibit the activity of these oncogenic drivers may therefore provide both a targeted therapy for the predominant SCLC subtypes and a tool to investigate the underlying lineage plasticity of established SCLC tumors. However, there are no known agents that inhibit ASCL1 or NEUROD1 function. In this study, we identify a novel strategy to pharmacologically target ASCL1 and NEUROD1 activity in SCLC by exploiting the nuclear localization required for the function of these transcription factors. Karyopherin β1 (KPNB1) was identified as a nuclear import receptor for both ASCL1 and NEUROD1 in SCLC, and inhibition of KPNB1 led to impaired ASCL1 and NEUROD1 nuclear accumulation and transcriptional activity. Pharmacologic targeting of KPNB1 preferentially disrupted the growth of ASCL1+ and NEUROD1+ SCLC cells in vitro and suppressed ASCL1+ tumor growth in vivo, an effect mediated by a combination of impaired ASCL1 downstream target expression, cell-cycle activity, and proteostasis. These findings broaden the support for targeting nuclear transport as an anticancer therapeutic strategy and have implications for targeting lineage-transcription factors in tumors beyond SCLC. Significance: The identification of KPNB1 as a nuclear import receptor for lineage-defining transcription factors in SCLC reveals a viable therapeutic strategy for cancer treatment.

Natural killer (NK) cells serve as an important role in suppressing tumor growth by directly attacking malignant cells. These cells recognize the tumor cells through moleclar patterns associated with oncogenic transformation. NKG2D ligands (NKG2DLs) are one of these markers present on tumor cells which can activate NK cells and T cells by interacting with NKG2D receptors on these lymphocytes. Majority of tumors express NKG2DLs. Interestingly, tumors carrying neuroendocrine features such as small cell lung cancer (SCLC) and neuroblastoma express very low level of ligands for NK cell activating receptor NKG2D (MICA, MICB, ULBP1, ULBP2, ULBP3), which may help them escape from innate immune surveillance. Further characterization of SCLC immune environment showed reduced antigen presentation, reduced infiltration of total immune cells specifically NK cells, as compared to NSCLC, a more immunogenic tumor. Transcriptional inducers of NKG2DLs did not induce NKG2DL in SCLCs due to hypo-acetylation of MICA/B region. Histone deacetylase (HDAC) inhibitors were able to induce NKG2DL on tumor cell surface. Restoring NKG2DL in preclinical SCLC and neuroblastoma models either by ectopic expression or HDAC inhibitors stimulated direct NK cell killing in co-culture assays. NKG2DL stimulation also caused potentiation of antibody directed therapies such as CD3-EpCAM in neuroendocrine tumors. In vivo studies showed suppressed tumor growth and metastasis in an NK and CD8 T cell dependent manner when NKG2DLs are stimulated. We conclude that: restoring innate immune visibility can target neuroendocrine tumors to both innate and adaptive immune cytotoxicity.