Proteogenomic characterization of small cell lung cancer identifies biological insights and subtype-specific therapeutic strategies.

We performed comprehensive proteogenomic characterization of small cell lung cancer (SCLC) using paired tumors and adjacent lung tissues from 112 treatment-naive patients who underwent surgical resection. Integrated multi-omics analysis illustrated cancer biology downstream of genetic aberrations and highlighted oncogenic roles of FAT1 mutation, RB1 deletion, and chromosome 5q loss. Two prognostic biomarkers, HMGB3 and CASP10, were identified. Overexpression of HMGB3 promoted SCLC cell migration via transcriptional regulation of cell junction-related genes. Immune landscape characterization revealed an association between ZFHX3 mutation and high immune infiltration and underscored a potential immunosuppressive role of elevated DNA damage response activity via inhibition of the cGAS-STING pathway. Multi-omics clustering identified four subtypes with subtype-specific therapeutic vulnerabilities. Cell line and patient-derived xenograft-based drug tests validated the specific therapeutic responses predicted by multi-omics subtyping. This study provides a valuable resource as well as insights to better understand SCLC biology and improve clinical practice.

Haploinsufficiency of ZFHX3, encoding a key player in neuronal development, causes syndromic intellectual disability.

Neurodevelopmental disorders (NDDs) result from impaired development and functioning of the brain. Here, we identify loss-of-function (LoF) variation in ZFHX3 as a cause for syndromic intellectual disability (ID). ZFHX3 is a zinc-finger homeodomain transcription factor involved in various biological processes, including cell differentiation and tumorigenesis. We describe 42 individuals with protein-truncating variants (PTVs) or (partial) deletions of ZFHX3, exhibiting variable intellectual disability and autism spectrum disorder, recurrent facial features, relative short stature, brachydactyly, and, rarely, cleft palate. ZFHX3 LoF associates with a specific methylation profile in whole blood extracted DNA. Nuclear abundance of ZFHX3 increases during human brain development and neuronal differentiation. ZFHX3 was found to interact with the chromatin remodeling BRG1/Brm-associated factor complex and the cleavage and polyadenylation complex, suggesting a function in chromatin remodeling and mRNA processing. Furthermore, ChIP-seq for ZFHX3 revealed that it predominantly binds promoters of genes involved in nervous system development. We conclude that loss-of-function variants in ZFHX3 are a cause of syndromic ID associating with a specific DNA methylation profile.

Exonic trinucleotide repeat expansions in ZFHX3 cause spinocerebellar ataxia type 4: A poly-glycine disease.

Autosomal-dominant ataxia with sensory and autonomic neuropathy is a highly specific combined phenotype that we described in two Swedish kindreds in 2014; its genetic cause had remained unknown. Here, we report the discovery of exonic GGC trinucleotide repeat expansions, encoding poly-glycine, in zinc finger homeobox 3 (ZFHX3) in these families. The expansions were identified in whole-genome datasets within genomic segments that all affected family members shared. Non-expanded alleles carried one or more interruptions within the repeat. We also found ZFHX3 repeat expansions in three additional families, all from the region of Skåne in southern Sweden. Individuals with expanded repeats developed balance and gait disturbances at 15 to 60 years of age and had sensory neuropathy and slow saccades. Anticipation was observed in all families and correlated with different repeat lengths determined through long-read sequencing in two family members. The most severely affected individuals had marked autonomic dysfunction, with severe orthostatism as the most disabling clinical feature. Neuropathology revealed p62-positive intracytoplasmic and intranuclear inclusions in neurons of the central and enteric nervous system, as well as alpha-synuclein positivity. ZFHX3 is located within the 16q22 locus, to which spinocerebellar ataxia type 4 (SCA4) repeatedly had been mapped; the clinical phenotype in our families corresponded well with the unique phenotype described in SCA4, and the original SCA4 kindred originated from Sweden. ZFHX3 has known functions in neuronal development and differentiation n both the central and peripheral nervous system. Our findings demonstrate that SCA4 is caused by repeat expansions in ZFHX3.

Spinocerebellar ataxia type 4 is caused by a GGC expansion in the ZFHX3 gene and is associated with prominent dysautonomia and motor neuron signs.

BACKGROUND: Spinocerebellar ataxia 4 (SCA4), characterized in 1996, features adult-onset ataxia, polyneuropathy, and linkage to chromosome 16q22.1; its underlying mutation has remained elusive. OBJECTIVE: To explore the radiological and neuropathological abnormalities in the entire neuroaxis in SCA4 and search for its mutation. METHODS: Three Swedish families with undiagnosed ataxia went through clinical, neurophysiological, and neuroimaging tests, including PET studies and genetic investigations. In four cases, neuropathological assessments of the neuroaxis were performed. Genetic testing included short read whole genome sequencing, short tandem repeat analysis with ExpansionHunter de novo, and long read sequencing. RESULTS: Novel features for SCA4 include dysautonomia, motor neuron affection, and abnormal eye movements. We found evidence of anticipation; neuroimaging demonstrated atrophy in the cerebellum, brainstem, and spinal cord. [18F]FDG-PET demonstrated brain hypometabolism and [11C]Flumazenil-PET reduced binding in several brain lobes, insula, thalamus, hypothalamus, and cerebellum. Moderate to severe loss of Purkinje cells in the cerebellum and of motor neurons in the anterior horns of the spinal cord along with pronounced degeneration of posterior tracts was also found. Intranuclear, mainly neuronal, inclusions positive for p62 and ubiquitin were sparse but widespread in the CNS. This finding prompted assessment for nucleotide expansions. A polyglycine stretch encoding GGC expansions in the last exon of the zink finger homeobox 3 gene was identified segregating with disease and not found in 1000 controls. CONCLUSIONS: SCA4 is a neurodegenerative disease caused by a novel GGC expansion in the coding region of ZFHX3, and its spectrum is expanded to include dysautonomia and neuromuscular manifestations.

Evaluation of Potential Roles of Zinc Finger Homeobox 3 (Zfhx3) Expressed in Chondrocytes and Osteoblasts on Skeletal Growth in Mice.

Bone formation is tightly modulated by genetically encoded molecular proteins that interact to regulate cellular differentiation and secretion of bony matrix. Many transcription factors are known to coordinate these events by controlling gene transcription within networks. However, not all factors involved are known. Here, we identified a novel function for Zinc Finger Homeobox 3 (Zfhx3), a gene encoding a transcription factor, as a regulator of bone metabolism. We knocked out Zfhx3 conditionally in mice in either chondrocytes or osteoblasts and characterized their bones by micro-CT in 12-week-old mice. We observed a negative effect in linear bone growth in both knockout mice but reduced bone mass only in mice with Zfhx3 deleted in osteoblasts. Loss of Zfhx3 expression in osteoblasts affected trabecular bone mass in femurs and vertebrae in both sexes but influenced cortical bone volume fraction only in females. Moreover, transcriptional analysis of femoral bones in osteoblast Zfhx3 conditional knockout mice revealed a reduced expression of osteoblast genes, and histological evaluation of trabecular bones suggests that Zfhx3 causes changes in bone formation and not resorption. The loss of Zfhx3 causes reductions in trabecular bone area and osteoid volume, but no changes in the expression of osteoclast differentiation markers or number of TRAP stained osteoclasts. These studies introduce Zfhx3 as a relevant factor toward understanding gene regulatory networks that control bone formation and development of peak bone mass.

A missense mutation in zinc finger homeobox-3 (ZFHX3) impedes growth and alters metabolism and hypothalamic gene expression in mice.

A protein altering variant in the gene encoding zinc finger homeobox-3 (ZFHX3) has recently been associated with lower BMI in a human genome-wide association study. We investigated metabolic parameters in mice harboring a missense mutation in Zfhx3 (Zfhx3Sci/+ ) and looked for altered in situ expression of transcripts that are associated with energy balance in the hypothalamus to understand how ZFHX3 may influence growth and metabolic effects. One-year-old male and female Zfhx3Sci/+ mice weighed less, had shorter body length, lower fat mass, smaller mesenteric fat depots, and lower circulating insulin, leptin, and insulin-like growth factor-1 (IGF1) concentrations than Zfhx3+/+ littermates. In a second cohort of 9-20-week-old males and females, Zfhx3Sci/+ mice ate less than wildtype controls, in proportion to body weight. In a third cohort of female-only Zfhx3Sci/+ and Zfhx3+/+ mice that underwent metabolic phenotyping from 6 to 14 weeks old, Zfhx3Sci/+ mice weighed less and had lower lean mass and energy expenditure, but fat mass did not differ. We detected increased expression of somatostatin and decreased expression of growth hormone-releasing hormone and growth hormone-receptor mRNAs in the arcuate nucleus (ARC). Similarly, ARC expression of orexigenic neuropeptide Y was decreased and ventricular ependymal expression of orphan G protein-coupled receptor Gpr50 was decreased. We demonstrate for the first time an energy balance effect of the Zfhx3Sci mutation, likely by altering expression of key ARC neuropeptides to alter growth, food intake, and energy expenditure.

Association between cyclin-dependent kinase inhibitor 2B antisense RNA 1 and zinc finger homeobox 3 gene polymorphisms and COVID-19 severity.

BACKGROUND: There is no doubt about the cardiovascular complications of coronavirus disease 2019 (COVID-19). Several genetic studies have demonstrated an association between genetic variants in a region on chromosome 9p21 and in a region on chromosome 16q22 with myocardial infarction (MI) and atrial fibrillation (AF) accompanied by cerebral infarction (CI), respectively. OBJECTIVES: MI and CI susceptibility in patients with CDKN2B-AS1 and ZFHX3 polymorphisms, respectively, may have an effect on COVID-19 severity. We aimed to investigate whether there is an association between the cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) rs1333049 and zinc finger homeobox 3 (ZFHX3) rs2106261 single nucleotide polymorphisms (SNPs) and the degree of COVID-19 severity. SUBJECTS AND METHODS: This current work was carried out on 360 subjects. They were classified into three groups: 90 severe COVID-19 cases, 90 moderate COVID-19 cases and 180 age- and gender-matched healthy controls. All subjects underwent genotyping of CDKN2B-AS1 (rs1333049) and ZFHX3 (rs2106261) by real-time PCR. RESULTS: The frequency of G/C in CDKN2B-AS1 (rs1333049) was higher in severe and moderate COVID-19 patients than in controls (71.1% and 53.3% vs. 37.8%). The frequency of the C/C of CDKN2B-AS1 (rs1333049) was higher in moderate COVID-19 patients than in controls (26.7% vs. 13.3%). There were no significant differences regarding genotype frequency and allelic distribution of ZFHX3 (rs2106261) between COVID-19 patients and healthy controls. CONCLUSION: CDKN2B-AS1 (rs1333049) gene polymorphism may play a role in determining the degree of COVID-19 severity. Further studies on its effect on cyclins and cyclin-dependent kinases (CDKs) [not measured in our study] may shed light on new treatment options for COVID-19.

AR imposes different effects on ZFHX3 transcription depending on androgen status in prostate cancer cells.

Both androgen receptor (AR) and the ZFHX3 transcription factor modulate prostate development. While AR drives prostatic carcinogenesis, ZFHX3 is a tumour suppressor whose loss activates the PI3K/AKT signalling in advanced prostate cancer (PCa). However, it is unknown whether ZFHX3 and AR are functionally related in PCa cells and, if so, how. Here, we report that in AR-positive LNCaP and C4-2B PCa cells, androgen upregulates ZFHX3 transcription via androgen-induced AR binding to the androgen-responsive elements (AREs) of the ZFHX3 promoter. Androgen also upregulated ZFHX3 transcription in vivo, as castration dramatically reduced Zfhx3 mRNA and protein levels in mouse prostates, and ZFHX3 mRNA levels correlated with AR activities in human PCa. Interestingly, the binding of AR to one ARE occurred in the absence of androgen, and the binding repressed ZFHX3 transcription as this repressive binding was interrupted by androgen treatment. The enzalutamide antiandrogen prevented androgen from inducing ZFHX3 transcription and caused excess ZFHX3 protein degradation. In human PCa, ZFHX3 was downregulated and the downregulation correlated with worse patient survival. These findings establish a regulatory relationship between AR and ZFHX3, suggest a role of ZFHX3 in AR function and implicate ZFHX3 loss in the antiandrogen therapies of PCa.

The transcription factor ZFHX3 is crucial for the angiogenic function of hypoxia-inducible factor 1α in liver cancer cells.

Angiogenesis is a hallmark of tumorigenesis, and hepatocellular carcinoma (HCC) is hypervascular and therefore very dependent on angiogenesis for tumor development and progression. Findings from previous studies suggest that in HCC cells, hypoxia-induced factor 1α (HIF1A) and zinc finger homeobox 3 (ZFHX3) transcription factors functionally interact in the regulation of genes in HCC cells. Here, we report that hypoxia increases the transcription of the ZFHX3 gene and enhances the binding of HIF1A to the ZFHX3 promoter in the HCC cell lines HepG2 and Huh-7. Moreover, ZFHX3, in turn, physically associated with and was functionally indispensable for HIF1A to exert its angiogenic activity, as indicated by in vitro migration and tube formation assays of human umbilical vein endothelial cells (HUVECs) and microvessel formation in xenograft tumors of HCC cells. Mechanistically, ZFHX3 was required for HIF1A to transcriptionally activate the vascular endothelial growth factor A (VEGFA) gene by binding to its promoter. Functionally, down-regulation of ZFHX3 in HCC cells slowed their tumor growth, and addition of VEGFA to conditioned medium from ZFHX3-silenced HCC cells partially rescued the inhibitory effect of this medium on HUVEC tube formation. In human HCC, ZFHX3 expression was up-regulated, and this up-regulation correlated with both HIF1A up-regulation and worse patient survival, confirming a functional association between ZFHX3 and HIF1A in human HCC. We conclude that ZFHX3 is an angiogenic transcription factor that is integral to the HIF1A/VEGFA signaling axis in HCC cells.

SUMOylation of the transcription factor ZFHX3 at Lys-2806 requires SAE1, UBC9, and PIAS2 and enhances its stability and function in cell proliferation.

SUMOylation is a posttranslational modification (PTM) at a lysine residue and is crucial for the proper functions of many proteins, particularly of transcription factors, in various biological processes. Zinc finger homeobox 3 (ZFHX3), also known as AT motif-binding factor 1 (ATBF1), is a large transcription factor that is active in multiple pathological processes, including atrial fibrillation and carcinogenesis, and in circadian regulation and development. We have previously demonstrated that ZFHX3 is SUMOylated at three or more lysine residues. Here, we investigated which enzymes regulate ZFHX3 SUMOylation and whether SUMOylation modulates ZFHX3 stability and function. We found that SUMO1, SUMO2, and SUMO3 each are conjugated to ZFHX3. Multiple lysine residues in ZFHX3 were SUMOylated, but Lys-2806 was the major SUMOylation site, and we also found that it is highly conserved among ZFHX3 orthologs from different animal species. Using molecular analyses, we identified the enzymes that mediate ZFHX3 SUMOylation; these included SUMO1-activating enzyme subunit 1 (SAE1), an E1-activating enzyme; SUMO-conjugating enzyme UBC9 (UBC9), an E2-conjugating enzyme; and protein inhibitor of activated STAT2 (PIAS2), an E3 ligase. Multiple analyses established that both SUMO-specific peptidase 1 (SENP1) and SENP2 deSUMOylate ZFHX3. SUMOylation at Lys-2806 enhanced ZFHX3 stability by interfering with its ubiquitination and proteasomal degradation. Functionally, Lys-2806 SUMOylation enabled ZFHX3-mediated cell proliferation and xenograft tumor growth of the MDA-MB-231 breast cancer cell line. These findings reveal the enzymes involved in, and the functional consequences of, ZFHX3 SUMOylation, insights that may help shed light on ZFHX3's roles in various cellular and pathophysiological processes.