Mark D. Borromeo

ASCL1 is a lineage oncogene providing therapeutic targets for high-grade neuroendocrine lung cancers

Significance New advances in the treatment of aggressive neuroendocrine lung cancers are needed to improve survival in patients with this class of tumors. The current treatment approach, which has remained unchanged for the past 30 years, involves combination chemotherapy and radiation. To uncover novel drug targets, we identified the transcriptome of achaete-scute homolog 1 (ASCL1), a transcription factor that is both necessary for the proper development of neuroendocrine cells and essential for the growth and survival of neuroendocrine lung cancers. Analysis of downstream targets of ASCL1 has revealed unique molecular vulnerabilities that can be exploited for future therapeutic use. Aggressive neuroendocrine lung cancers, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), represent an understudied tumor subset that accounts for approximately 40,000 new lung cancer cases per year in the United States. No targeted therapy exists for these tumors. We determined that achaete-scute homolog 1 (ASCL1), a transcription factor required for proper development of pulmonary neuroendocrine cells, is essential for the survival of a majority of lung cancers (both SCLC and NSCLC) with neuroendocrine features. By combining whole-genome microarray expression analysis performed on lung cancer cell lines with ChIP-Seq data designed to identify conserved transcriptional targets of ASCL1, we discovered an ASCL1 target 72-gene expression signature that (i) identifies neuroendocrine differentiation in NSCLC cell lines, (ii) is predictive of poor prognosis in resected NSCLC specimens from three datasets, and (iii) represents novel “druggable” targets. Among these druggable targets is B-cell CLL/lymphoma 2, which when pharmacologically inhibited stops ASCL1-dependent tumor growth in vitro and in vivo and represents a proof-of-principle ASCL1 downstream target gene. Analysis of downstream targets of ASCL1 represents an important advance in the development of targeted therapy for the neuroendocrine class of lung cancers, providing a significant step forward in the understanding and therapeutic targeting of the molecular vulnerabilities of neuroendocrine lung cancer.

Ascl1 and Neurog2 form novel complexes and regulate Delta-like3 (Dll3) expression in the neural tube.

Delta-like 3 (Dll3) is a Delta family member expressed broadly in the developing nervous system as neural progenitor cells initiate differentiation. A proximal promoter sequence for Dll3 is conserved across multiple species and is sufficient to direct GFP expression in a Dll3-like pattern in the neural tube of transgenic mice. This promoter contains multiple E-boxes, the consensus binding site for bHLH factors. Dll3 expression and the activity of the Dll3-promoter in the dorsal neural tube depends on the basic helix-loop-helix (bHLH) transcription factors Ascl1 (Mash1) and Neurog2 (Ngn2). Mutations in each E-box identified in the Dll3-promoter allowed distinct enhancer or repressor properties to be assigned to each site individually or in combination. In addition, each E-box has distinct characteristics relative to binding of bHLH factors Ascl1, Neurog1, and Neurog2. Surprisingly, novel Ascl1 containing DNA binding complexes are identified that interact with specific E-box sites within the Dll3-promoter in vitro. These complexes include Ascl1/Ascl1 homodimers and Ascl1/Neurog2 heterodimers, complexes that in some cases require additional undefined factors for efficient DNA binding. Thus, a complex interplay of E-box binding proteins spatially and temporally regulate Dll3 levels during neural tube development.

A transcription factor network specifying inhibitory versus excitatory neurons in the dorsal spinal cord

The proper balance of excitatory and inhibitory neurons is crucial for normal processing of somatosensory information in the dorsal spinal cord. Two neural basic helix-loop-helix transcription factors (TFs), Ascl1 and Ptf1a, have contrasting functions in specifying these neurons. To understand how Ascl1 and Ptf1a function in this process, we identified their direct transcriptional targets genome-wide in the embryonic mouse neural tube using ChIP-Seq and RNA-Seq. We show that Ascl1 and Ptf1a directly regulate distinct homeodomain TFs that specify excitatory or inhibitory neuronal fates. In addition, Ascl1 directly regulates genes with roles in several steps of the neurogenic program, including Notch signaling, neuronal differentiation, axon guidance and synapse formation. By contrast, Ptf1a directly regulates genes encoding components of the neurotransmitter machinery in inhibitory neurons, and other later aspects of neural development distinct from those regulated by Ascl1. Moreover, Ptf1a represses the excitatory neuronal fate by directly repressing several targets of Ascl1. Ascl1 and Ptf1a bind sequences primarily enriched for a specific E-Box motif (CAGCTG) and for secondary motifs used by Sox, Rfx, Pou and homeodomain factors. Ptf1a also binds sequences uniquely enriched in the CAGATG E-box and in the binding motif for its co-factor Rbpj, providing two factors that influence the specificity of Ptf1a binding. The direct transcriptional targets identified for Ascl1 and Ptf1a provide a molecular understanding of how these DNA-binding proteins function in neuronal development, particularly as key regulators of homeodomain TFs required for neuronal subtype specification.

Prdm13 Mediates the Balance of Inhibitory and Excitatory Neurons in Somatosensory Circuits

Generating a balanced network of inhibitory and excitatory neurons during development requires precise transcriptional control. In the dorsal spinal cord, Ptf1a, a basic helix-loop-helix (bHLH) transcription activator, maintains this delicate balance by inducing homeodomain (HD) transcription factors such as Pax2 to specify the inhibitory lineage while suppressing HD factors such as Tlx1/3 that specify the excitatory lineage. We uncover the mechanism by which Ptf1a represses excitatory cell fate in the inhibitory lineage. We identify Prdm13 as a direct target of Ptf1a and reveal that Prdm13 actively represses excitatory cell fate by binding to regulatory sequences near the Tlx1 and Tlx3 genes to silence their expression. Prdm13 acts through multiple mechanisms, including interactions with the bHLH factor Ascl1, to repress Ascl1 activation of Tlx3. Thus, Prdm13 is a key component of a highly coordinated transcriptional network that determines the balance of inhibitory versus excitatory neurons in the dorsal spinal cord.

Comprehensive functional characterization of cancer-testis antigens defines obligate participation in multiple hallmarks of cancer.

Tumours frequently activate genes whose expression is otherwise biased to the testis, collectively known as cancer–testis antigens (CTAs). The extent to which CTA expression represents epiphenomena or confers tumorigenic traits is unknown. In this study, to address this, we implemented a multidimensional functional genomics approach that incorporates 7 different phenotypic assays in 11 distinct disease settings. We identify 26 CTAs that are essential for tumor cell viability and/or are pathological drivers of HIF, WNT or TGFβ signalling. In particular, we discover that Foetal and Adult Testis Expressed 1 (FATE1) is a key survival factor in multiple oncogenic backgrounds. FATE1 prevents the accumulation of the stress-sensing BH3-only protein, BCL-2-Interacting Killer (BIK), thereby permitting viability in the presence of toxic stimuli. Furthermore, ZNF165 promotes TGFβ signalling by directly suppressing the expression of negative feedback regulatory pathways. This action is essential for the survival of triple negative breast cancer cells in vitro and in vivo. Thus, CTAs make significant direct contributions to tumour biology.

Program specificity for ptf1a in pancreas versus neural tube development correlates with distinct collaborating cofactors and chromatin accessibility

ABSTRACT The lineage-specific basic helix-loop-helix transcription factor Ptf1a is a critical driver for development of both the pancreas and nervous system. How one transcription factor controls diverse programs of gene expression is a fundamental question in developmental biology. To uncover molecular strategies for the program-specific functions of Ptf1a, we identified bound genomic regions in vivo during development of both tissues. Most regions bound by Ptf1a are specific to each tissue, lie near genes needed for proper formation of each tissue, and coincide with regions of open chromatin. The specificity of Ptf1a binding is encoded in the DNA surrounding the Ptf1a-bound sites, because these regions are sufficient to direct tissue-restricted reporter expression in transgenic mice. Fox and Sox factors were identified as potential lineage-specific modifiers of Ptf1a binding, since binding motifs for these factors are enriched in Ptf1a-bound regions in pancreas and neural tube, respectively. Of the Fox factors expressed during pancreatic development, Foxa2 plays a major role. Indeed, Ptf1a and Foxa2 colocalize in embryonic pancreatic chromatin and can act synergistically in cell transfection assays. Together, these findings indicate that lineage-specific chromatin landscapes likely constrain the DNA binding of Ptf1a, and they identify Fox and Sox gene families as part of this process.

Prdm12 specifies V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes in Xenopus

V1 interneurons are inhibitory neurons that play an essential role in vertebrate locomotion. The molecular mechanisms underlying their genesis remain, however, largely undefined. Here, we show that the transcription factor Prdm12 is selectively expressed in p1 progenitors of the hindbrain and spinal cord in the frog embryo, and that a similar restricted expression profile is observed in the nerve cord of other vertebrates as well as of the cephalochordate amphioxus. Using frog, chick and mice, we analyzed the regulation of Prdm12 and found that its expression in the caudal neural tube is dependent on retinoic acid and Pax6, and that it is restricted to p1 progenitors, due to the repressive action of Dbx1 and Nkx6-1/2 expressed in the adjacent p0 and p2 domains. Functional studies in the frog, including genome-wide identification of its targets by RNA-seq and ChIP-Seq, reveal that vertebrate Prdm12 proteins act as a general determinant of V1 cell fate, at least in part, by directly repressing Dbx1 and Nkx6 genes. This probably occurs by recruiting the methyltransferase G9a, an activity that is not displayed by the amphioxus Prdm12 protein. Together, these findings indicate that Prdm12 promotes V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes, and suggest that this function might have only been acquired after the split of the vertebrate and cephalochordate lineages. Summary: In vertebrates, V1 interneuron specification requires Prdm12, whose expression depends on Pax6 and retinoic acid and is restricted to the p1 domain by Dbx1 and Nkx6.1/2, themselves repressed by Prdm12.

Misexpression of Ptf1a in Cortical Pyramidal Cells In Vivo Promotes an Inhibitory Peptidergic Identity

The intracellular transcriptional milieu wields considerable influence over the induction of neuronal identity. The transcription factor Ptf1a has been proposed to act as an identity “switch” between developmentally related precursors in the spinal cord (Glasgow et al., 2005; Huang et al., 2008), retina (Fujitani et al., 2006; Dullin et al., 2007; Nakhai et al., 2007; Lelièvre et al., 2011), and cerebellum (Hoshino et al., 2005; Pascual et al., 2007; Yamada et al., 2014), where it promotes an inhibitory over an excitatory neuronal identity. In this study, we investigate the potency of Ptf1a to cell autonomously confer a specific neuronal identity outside of its endogenous environment, using mouse in utero electroporation and a conditional genetic strategy to misexpress Ptf1a exclusively in developing cortical pyramidal cells. Transcriptome profiling of Ptf1a-misexpressing cells using RNA-seq reveals that Ptf1a significantly alters pyramidal cell gene expression, upregulating numerous Ptf1a-dependent inhibitory interneuron markers and ultimately generating a gene expression profile that resembles the transcriptomes of both Ptf1a-expressing spinal interneurons and endogenous cortical interneurons. Using RNA-seq and in situ hybridization analyses, we also show that Ptf1a induces expression of the peptidergic neurotransmitter nociceptin, while minimally affecting the expression of genes linked to other neurotransmitter systems. Moreover, Ptf1a alters neuronal morphology, inducing the radial redistribution and branching of neurites in cortical pyramidal cells. Thus Ptf1a is sufficient, even in a dramatically different neuronal precursor, to cell autonomously promote characteristics of an inhibitory peptidergic identity, providing the first example of a single transcription factor that can direct an inhibitory peptidergic fate.

Repression by PRDM13 is critical for generating precision in neuronal identity

The mechanisms that activate some genes while silencing others are critical to ensure precision in lineage specification as multipotent progenitors become restricted in cell fate. During neurodevelopment, these mechanisms are required to generate the diversity of neuronal subtypes found in the nervous system. Here we report interactions between basic helix-loop-helix (bHLH) transcriptional activators and the transcriptional repressor PRDM13 that are critical for specifying dorsal spinal cord neurons. PRDM13 inhibits gene expression programs for excitatory neuronal lineages in the dorsal neural tube. Strikingly, PRDM13 also ensures a battery of ventral neural tube specification genes such as Olig1, Olig2 and Prdm12 are excluded dorsally. PRDM13 does this via recruitment to chromatin by multiple neural bHLH factors to restrict gene expression in specific neuronal lineages. Together these findings highlight the function of PRDM13 in repressing the activity of bHLH transcriptional activators that together are required to achieve precise neuronal specification during mouse development.

The Epithelial Sodium Channel (αENaC) Is a Downstream Therapeutic Target of ASCL1 in Pulmonary Neuroendocrine Tumors

Small cell lung cancer (SCLC) is an aggressive neuroendocrine carcinoma, designated as a recalcitrant cancer by the National Cancer Institute, in urgent need of new rational therapeutic targets. Previous studies have determined that the basic helix-loop-helix transcription factor achaete-scute homolog 1 (ASCL1) is essential for the survival and progression of a fraction of pulmonary neuroendocrine cancer cells, which include both SCLC and a subset of non-SCLC. Previously, to understand how ASCL1 initiates tumorigenesis in pulmonary neuroendocrine cancer and identify the transcriptional targets of ASCL1, whole-genome RNA-sequencing analysis combined with chromatin immunoprecipitation-sequencing was performed with a series of lung cancer cell lines. From this analysis, we discovered that the gene SCNN1A, which encodes the alpha subunit of the epithelial sodium channel (αENaC), is highly correlated with ASCL1 expression in SCLC. The product of the SCNN1A gene ENaC can be pharmacologically inhibited with amiloride, a drug that has been used clinically for close to 50 years. Amiloride inhibited growth of ASCL1-dependent SCLC more strongly than ASCL1-independent SCLC in vitro and slowed growth of ASCL1-driven SCLC in xenografts. We conclude that SCNN1A/αENaC is a direct transcriptional target of the neuroendocrine lung cancer lineage oncogene ASCL1 that can be pharmacologically targeted with antitumor effects.