3D genomic analysis reveals novel enhancer-hijacking caused by complex structural alterations that drive oncogene overexpression.

Cancer genomes are composed of many complex structural alterations on chromosomes and extrachromosomal DNA (ecDNA), making it difficult to identify non-coding enhancer regions that are hijacked to activate oncogene expression. Here, we describe a 3D genomics-based analysis called HAPI (Highly Active Promoter Interactions) to characterize enhancer hijacking. HAPI analysis of HiChIP data from 34 cancer cell lines identified enhancer hijacking events that activate both known and potentially novel oncogenes such as MYC, CCND1, ETV1, CRKL, and ID4. Furthermore, we found enhancer hijacking among multiple oncogenes from different chromosomes, often including MYC, on the same complex amplicons such as ecDNA. We characterized a MYC-ERBB2 chimeric ecDNA, in which ERBB2 heavily hijacks MYC's enhancers. Notably, CRISPRi of the MYC promoter led to increased interaction of ERBB2 with MYC enhancers and elevated ERBB2 expression. Our HAPI analysis tool provides a robust strategy to detect enhancer hijacking and reveals novel insights into oncogene activation.

EstroGene2.0: A multi-omic database of response to estrogens, ER-modulators, and resistance to endocrine therapies in breast cancer.

Endocrine therapies targeting the estrogen receptor (ER/ESR1) are the cornerstone to treat ER-positive breast cancers patients, but resistance often limits their effectiveness. Understanding the molecular mechanisms is thus key to optimize the existing drugs and to develop new ER-modulators. Notable progress has been made although the fragmented way data is reported has reduced their potential impact. Here, we introduce EstroGene2.0, an expanded database of its precursor 1.0 version. EstroGene2.0 focusses on response and resistance to endocrine therapies in breast cancer models. Incorporating multi-omic profiling of 361 experiments from 212 studies across 28 cell lines, a user-friendly browser offers comprehensive data visualization and metadata mining capabilities (https://estrogeneii.web.app/). Taking advantage of the harmonized data collection, our follow-up meta-analysis revealed substantial diversity in response to different classes of ER-modulators including SERMs, SERDs, SERCA and LDD/PROTAC. Notably, endocrine resistant models exhibit a spectrum of transcriptomic alterations including a contra-directional shift in ER and interferon signaling, which is recapitulated clinically. Furthermore, dissecting multiple ESR1-mutant cell models revealed the different clinical relevance of genome-edited versus ectopic overexpression model engineering and identified high-confidence mutant-ER targets, such as NPY1R. These examples demonstrate how EstroGene2.0 helps investigate breast cancer's response to endocrine therapies and explore resistance mechanisms.

Estrogen-induced chromatin looping changes identify a subset of functional regulatory elements.

Transcriptional enhancers can regulate individual or multiple genes through long-range three-dimensional (3D) genome interactions, and these interactions are commonly altered in cancer. Yet, the functional relationship between changes in 3D interactions associated with regulatory regions and differential gene expression appears context-dependent. In this study, we used HiChiP to capture changes in 3D genome interactions between active regulatory regions of endometrial cancer cells in response to estrogen treatment and uncovered significant differential long-range interactions that are strongly enriched for estrogen receptor α (ER) bound sites (ERBS). The ERBS anchoring differential loops with either a gene's promoter or distal regions were correlated with larger transcriptional responses to estrogen compared to ERBS not involved in differential interactions. To functionally test this observation, CRISPR-based Enhancer-i was used to deactivate specific ERBS, which revealed a wide range of effects on the transcriptional response to estrogen. However, these effects are only subtly and not significantly stronger for ERBS in differential loops. In addition, we observed an enrichment of 3D interactions between the promoters of estrogen up-regulated genes and found that looped promoters can work together cooperatively. Overall, our work suggests that changes in 3D genome structure upon estrogen treatment identify some functionally important regulatory regions; however, these changes aren't required for a transcriptional response to E2 in endometrial cancer cells.

Estrogen Receptor Alpha Mutations, Truncations, Heterodimers, and Therapies.

Annual breast cancer (BCa) deaths have declined since its apex in 1989 concomitant with widespread adoption of hormone therapies that target estrogen receptor alpha (ERα), the prominent nuclear receptor expressed in ∼80% of BCa. However, up to ∼50% of patients who are ER+ with high-risk disease experience post endocrine therapy relapse and metastasis to distant organs. The vast majority of BCa mortality occurs in this setting, highlighting the inadequacy of current therapies. Genomic abnormalities to ESR1, the gene encoding ERα, emerge under prolonged selective pressure to enable endocrine therapy resistance. These genetic lesions include focal gene amplifications, hotspot missense mutations in the ligand binding domain, truncations, fusions, and complex interactions with other nuclear receptors. Tumor cells utilize aberrant ERα activity to proliferate, spread, and evade therapy in BCa as well as other cancers. Cutting edge studies on ERα structural and transcriptional relationships are being harnessed to produce new therapies that have shown benefits in patients with ESR1 hotspot mutations. In this review we discuss the history of ERα, current research unlocking unknown aspects of ERα signaling including the structural basis for receptor antagonism, and future directions of ESR1 investigation. In addition, we discuss the development of endocrine therapies from their inception to present day and survey new avenues of drug development to improve pharmaceutical profiles, targeting, and efficacy.

Cis-regulatory control of transcriptional timing and noise in response to estrogen.

Cis-regulatory elements control transcription levels, temporal dynamics, and cell-cell variation or transcriptional noise. However, the combination of regulatory features that control these different attributes is not fully understood. Here, we used single-cell RNA-seq during an estrogen treatment time course and machine learning to identify predictors of expression timing and noise. We found that genes with multiple active enhancers exhibit faster temporal responses. We verified this finding by showing that manipulation of enhancer activity changes the temporal response of estrogen target genes. Analysis of transcriptional noise uncovered a relationship between promoter and enhancer activity, with active promoters associated with low noise and active enhancers linked to high noise. Finally, we observed that co-expression across single cells is an emergent property associated with chromatin looping, timing, and noise. Overall, our results indicate a fundamental tradeoff between a gene's ability to quickly respond to incoming signals and maintain low variation across cells.

3D genomic analysis reveals novel enhancer-hijacking caused by complex structural alterations that drive oncogene overexpression.

Cancer genomes are composed of many complex structural alterations on chromosomes and extrachromosomal DNA (ecDNA), making it difficult to identify non-coding enhancer regions that are hijacked to activate oncogene expression. Here, we describe a 3D genomics-based analysis called HAPI (Highly Active Promoter Interactions) to characterize enhancer hijacking. HAPI analysis of HiChIP data from 34 cancer cell lines identified enhancer hijacking events that activate both known and potentially novel oncogenes such as MYC, CCND1 , ETV1 , CRKL , and ID4 . Furthermore, we found enhancer hijacking among multiple oncogenes from different chromosomes, often including MYC , on the same complex amplicons such as ecDNA. We characterized a MYC - ERBB2 chimeric ecDNA, in which ERBB2 heavily hijacks MYC 's enhancers. Notably, CRISPRi of the MYC promoter led to increased interaction of ERBB2 with MYC enhancers and elevated ERBB2 expression. Our HAPI analysis tool provides a robust strategy to detect enhancer hijacking and reveals novel insights into oncogene activation.

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97.

The t(X,17) chromosomal translocation, generating the ASPSCR1::TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCCs), frustrating efforts to identify therapeutic targets for these rare cancers. Here, proteomic analysis identifies VCP/p97, an AAA+ ATPase with known segregase function, as strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1::TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1::TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributes with ASPSCR1::TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrate the oncogenic transcriptional signature of ASPSCR1::TFE3, by facilitating assembly of higher-order chromatin conformation structures demonstrated by HiChIP. Finally, ASPSCR1::TFE3 and VCP demonstrate co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97.

The t(X,17) chromosomal translocation, generating the ASPSCR1-TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCC), frustrating efforts to identify therapeutic targets for these rare cancers. Proteomic analysis showed that VCP/p97, an AAA+ ATPase with known segregase function, was strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1-TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1-TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributed with ASPSCR1-TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrated the oncogenic transcriptional signature of ASPSCR1-TFE3, by facilitating assembly of higher-order chromatin conformation structures as demonstrated by HiChIP. Finally, ASPSCR1-TFE3 and VCP demonstrated co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.

Global expression analysis of endometrial cancer cells in response to progesterone identifies new therapeutic targets.

Progesterone prevents development of endometrial cancers through its receptor (PR) although the molecular mechanisms have yet to be fully characterized. In this study, we performed a global analysis of gene regulation by progesterone using human endometrial cancer cells that expressed PR endogenously or exogenously. We found progesterone strongly inhibits multiple components of the platelet derived growth factor receptor (PDGFR), Janus kinase (JAK), signal transducer and activator of transcription (STAT) pathway through PR. The PDGFR/JAK/STAT pathway signals to control numerous downstream targets including AP-1 transcription factors Fos and Jun. Treatment with inhibitors of the PDGFR/JAK/STAT pathway significantly blocked proliferation in multiple novel patient-derived organoid models of endometrial cancer, and activation of this pathway was found to be a poor prognostic signal for the survival of patients with endometrial cancer from The Cancer Genome Atlas. Our study identifies this pathway as central to the growth-limiting effects of progesterone in endometrial cancer and suggests that inhibitors of PDGFR/JAK/STAT should be considered for future therapeutic interventions.

Allele-Specific Gene Regulation, Phenotypes, and Therapeutic Vulnerabilities in Estrogen Receptor Alpha-Mutant Endometrial Cancer.

Activating estrogen receptor alpha (ER; also known as ESR1) mutations are present in primary endometrial and metastatic breast cancers, promoting estrogen-independent activation of the receptor. Functional characterizations in breast cancer have established unique molecular and phenotypic consequences of the receptor, yet the impact of ER mutations in endometrial cancer has not been fully explored. In this study, we used CRISPR-Cas9 to model the clinically prevalent ER-Y537S mutation and compared results with ER-D538G to discover allele-specific differences between ER mutations in endometrial cancer. We found that constitutive activity of mutant ER resulted in changes in the expression of thousands of genes, stemming from combined alterations to ER binding and chromatin accessibility. The unique gene expression programs resulted in ER-mutant cells developing increased cancer-associated phenotypes, including migration, invasion, anchorage-independent growth, and growth in vivo. To uncover potential treatment strategies, we identified ER-associated proteins via Rapid Immunoprecipitation and Mass Spectrometry of Endogenous Proteins and interrogated two candidates, CDK9 and NCOA3. Inhibition of these regulatory proteins resulted in decreased growth and migration, representing potential novel treatment strategies for ER-mutant endometrial cancer. IMPLICATIONS: This study provides insight into mutant ER activity in endometrial cancer and identifies potential therapies for women with ER-mutant endometrial cancer.

Estrogen receptor alpha mutations regulate gene expression and cell growth in breast cancer through microRNAs.

Estrogen receptor α (ER) mutations occur in up to 30% of metastatic ER-positive breast cancers. Recent data has shown that ER mutations impact the expression of thousands of genes not typically regulated by wildtype ER. While the majority of these altered genes can be explained by constant activity of mutant ER or genomic changes such as altered ER binding and chromatin accessibility, as much as 33% remain unexplained, indicating the potential for post-transcriptional effects. Here, we explored the role of microRNAs in mutant ER-driven gene regulation and identified several microRNAs that are dysregulated in ER mutant cells. These differentially regulated microRNAs target a significant portion of mutant-specific genes involved in key cellular processes. When the activity of microRNAs is altered using mimics or inhibitors, significant changes are observed in gene expression and cellular proliferation related to mutant ER. An in-depth evaluation of miR-301b led us to discover an important role for PRKD3 in the proliferation of ER mutant cells. Our findings show that microRNAs contribute to mutant ER gene regulation and cellular effects in breast cancer cells.

TXNIP loss expands Myc-dependent transcriptional programs by increasing Myc genomic binding.

The c-Myc protooncogene places a demand on glucose uptake to drive glucose-dependent biosynthetic pathways. To meet this demand, c-Myc protein (Myc henceforth) drives the expression of glucose transporters, glycolytic enzymes, and represses the expression of thioredoxin interacting protein (TXNIP), which is a potent negative regulator of glucose uptake. A Mychigh/TXNIPlow gene signature is clinically significant as it correlates with poor clinical prognosis in triple-negative breast cancer (TNBC) but not in other subtypes of breast cancer, suggesting a functional relationship between Myc and TXNIP. To better understand how TXNIP contributes to the aggressive behavior of TNBC, we generated TXNIP null MDA-MB-231 (231:TKO) cells for our study. We show that TXNIP loss drives a transcriptional program that resembles those driven by Myc and increases global Myc genome occupancy. TXNIP loss allows Myc to invade the promoters and enhancers of target genes that are potentially relevant to cell transformation. Together, these findings suggest that TXNIP is a broad repressor of Myc genomic binding. The increase in Myc genomic binding in the 231:TKO cells expands the Myc-dependent transcriptome we identified in parental MDA-MB-231 cells. This expansion of Myc-dependent transcription following TXNIP loss occurs without an apparent increase in Myc's intrinsic capacity to activate transcription and without increasing Myc levels. Together, our findings suggest that TXNIP loss mimics Myc overexpression, connecting Myc genomic binding and transcriptional programs to the nutrient and progrowth signals that control TXNIP expression.

FOXA1 Reprogramming Dictates Retinoid X Receptor Response in ESR1-Mutant Breast Cancer.

Estrogen receptor alpha (ER/ESR1) mutations occur in 30% to 40% of endocrine resistant ER-positive (ER+) breast cancer. Forkhead box A1 (FOXA1) is a key pioneer factor mediating ER-chromatin interactions and endocrine response in ER+ breast cancer, but its role in ESR1-mutant breast cancer remains unclear. Our previous FOXA1 chromatin immunoprecipitation sequencing (ChIP-seq) identified a large portion of redistributed binding sites in T47D genome-edited Y537S and D538G ESR1-mutant cells. Here, we further integrated FOXA1 genomic binding profile with the isogenic ER cistrome, accessible genome, and transcriptome data of T47D cell model. FOXA1 redistribution was significantly associated with transcriptomic alterations caused by ESR1 mutations. Furthermore, in ESR1-mutant cells, FOXA1-binding sites less frequently overlapped with ER, and differential gene expression was less associated with the canonical FOXA1-ER axis. Motif analysis revealed a unique enrichment of retinoid X receptor (RXR) motifs in FOXA1-binding sites of ESR1-mutant cells. Consistently, ESR1-mutant cells were more sensitive to growth stimulation with the RXR agonist LG268. The mutant-specific response was dependent on two RXR isoforms, RXR-α and RXR-ß, with a stronger dependency on the latter. In addition, T3, the agonist of thyroid receptor (TR) also showed a similar growth-promoting effect in ESR1-mutant cells. Importantly, RXR antagonist HX531 blocked growth of ESR1-mutant cells and a patient-derived xenograft (PDX)-derived organoid with an ESR1 D538G mutation. Collectively, our data support the evidence for a stronger RXR response associated with FOXA1 reprograming in ESR1-mutant cells, suggesting development of therapeutic strategies targeting RXR pathways in breast tumors with ESR1 mutation. IMPLICATIONS: It provides comprehensive characterization of the role of FOXA1 in ESR1-mutant breast cancer and potential therapeutic strategy through blocking RXR activation.

Characterization of HCI-EC-23 a novel estrogen- and progesterone-responsive endometrial cancer cell line.

Most endometrial cancers express the hormone receptor estrogen receptor alpha (ER) and are driven by excess estrogen signaling. However, evaluation of the estrogen response in endometrial cancer cells has been limited by the availability of hormonally responsive in vitro models, with one cell line, Ishikawa, being used in most studies. Here, we describe a novel, adherent endometrioid endometrial cancer (EEC) cell line model, HCI-EC-23. We show that HCI-EC-23 retains ER expression and that ER functionally responds to estrogen induction over a range of passages. We also demonstrate that this cell line retains paradoxical activation of ER by tamoxifen, which is also observed in Ishikawa and is consistent with clinical data. The mutational landscape shows that HCI-EC-23 is mutated at many of the commonly altered genes in EEC, has relatively few copy-number alterations, and is microsatellite instable high (MSI-high). In vitro proliferation of HCI-EC-23 is strongly reduced upon combination estrogen and progesterone treatment. HCI-EC-23 exhibits strong estrogen dependence for tumor growth in vivo and tumor size is reduced by combination estrogen and progesterone treatment. Molecular characterization of estrogen induction in HCI-EC-23 revealed hundreds of estrogen-responsive genes that significantly overlapped with those regulated in Ishikawa. Analysis of ER genome binding identified similar patterns in HCI-EC-23 and Ishikawa, although ER exhibited more bound sites in Ishikawa. This study demonstrates that HCI-EC-23 is an estrogen- and progesterone-responsive cell line model that can be used to study the hormonal aspects of endometrial cancer.

Comprehensive evaluation and efficient classification of BRCA1 RING domain missense substitutions.

BRCA1 is a high-risk susceptibility gene for breast and ovarian cancer. Pathogenic protein-truncating variants are scattered across the open reading frame, but all known missense substitutions that are pathogenic because of missense dysfunction are located in either the amino-terminal RING domain or the carboxy-terminal BRCT domain. Heterodimerization of the BRCA1 and BARD1 RING domains is a molecularly defined obligate activity. Hence, we tested every BRCA1 RING domain missense substitution that can be created by a single nucleotide change for heterodimerization with BARD1 in a mammalian two-hybrid assay. Downstream of the laboratory assay, we addressed three additional challenges: assay calibration, validation thereof, and integration of the calibrated results with other available data, such as computational evidence and patient/population observational data to achieve clinically applicable classification. Overall, we found that 15%-20% of BRCA1 RING domain missense substitutions are pathogenic. Using a Bayesian point system for data integration and variant classification, we achieved clinical classification of 89% of observed missense substitutions. Moreover, among missense substitutions not present in the human observational data used here, we find an additional 45 with concordant computational and functional assay evidence in favor of pathogenicity plus 223 with concordant evidence in favor of benignity; these are particularly likely to be classified as likely pathogenic and likely benign, respectively, once human observational data become available.

ESR1 mutant breast cancers show elevated basal cytokeratins and immune activation.

Estrogen receptor alpha (ER/ESR1) is frequently mutated in endocrine resistant ER-positive (ER+) breast cancer and linked to ligand-independent growth and metastasis. Despite the distinct clinical features of ESR1 mutations, their role in intrinsic subtype switching remains largely unknown. Here we find that ESR1 mutant cells and clinical samples show a significant enrichment of basal subtype markers, and six basal cytokeratins (BCKs) are the most enriched genes. Induction of BCKs is independent of ER binding and instead associated with chromatin reprogramming centered around a progesterone receptor-orchestrated insulated neighborhood. BCK-high ER+ primary breast tumors exhibit a number of enriched immune pathways, shared with ESR1 mutant tumors. S100A8 and S100A9 are among the most induced immune mediators and involve in tumor-stroma paracrine crosstalk inferred by single-cell RNA-seq from metastatic tumors. Collectively, these observations demonstrate that ESR1 mutant tumors gain basal features associated with increased immune activation, encouraging additional studies of immune therapeutic vulnerabilities.

A human breast cancer-derived xenograft and organoid platform for drug discovery and precision oncology.

Models that recapitulate the complexity of human tumors are urgently needed to develop more effective cancer therapies. We report a bank of human patient-derived xenografts (PDXs) and matched organoid cultures from tumors that represent the greatest unmet need: endocrine-resistant, treatment-refractory and metastatic breast cancers. We leverage matched PDXs and PDX-derived organoids (PDxO) for drug screening that is feasible and cost-effective with in vivo validation. Moreover, we demonstrate the feasibility of using these models for precision oncology in real time with clinical care in a case of triple-negative breast cancer (TNBC) with early metastatic recurrence. Our results uncovered a Food and Drug Administration (FDA)-approved drug with high efficacy against the models. Treatment with this therapy resulted in a complete response for the individual and a progression-free survival (PFS) period more than three times longer than their previous therapies. This work provides valuable methods and resources for functional precision medicine and drug development for human breast cancer.

Hotspot ESR1 Mutations Are Multimodal and Contextual Modulators of Breast Cancer Metastasis.

Constitutively active estrogen receptor α (ER/ESR1) mutations have been identified in approximately one-third of ER+ metastatic breast cancers. Although these mutations are known as mediators of endocrine resistance, their potential role in promoting metastatic disease has not yet been mechanistically addressed. In this study, we show the presence of ESR1 mutations exclusively in distant but not local recurrences in five independent breast cancer cohorts. In concordance with transcriptomic profiling of ESR1-mutant tumors, genome-edited ESR1 Y537S and D538G-mutant cell models exhibited a reprogrammed cell adhesive gene network via alterations in desmosome/gap junction genes and the TIMP3/MMP axis, which functionally conferred enhanced cell-cell contacts while decreasing cell-extracellular matrix adhesion. In vivo studies showed ESR1-mutant cells were associated with larger multicellular circulating tumor cell (CTC) clusters with increased compactness compared with ESR1 wild-type CTCs. These preclinical findings translated to clinical observations, where CTC clusters were enriched in patients with ESR1-mutated metastatic breast cancer. Conversely, context-dependent migratory phenotypes revealed cotargeting of Wnt and ER as a vulnerability in a D538G cell model. Mechanistically, mutant ESR1 exhibited noncanonical regulation of several metastatic pathways, including secondary transcriptional regulation and de novo FOXA1-driven chromatin remodeling. Collectively, these data provide evidence for ESR1 mutation-modulated metastasis and suggest future therapeutic strategies for targeting ESR1-mutant breast cancer. SIGNIFICANCE: Context- and allele-dependent transcriptome and cistrome reprogramming in mutant ESR1 cell models elicit diverse metastatic phenotypes related to cell adhesion and migration, which can be pharmacologically targeted in metastatic breast cancer.

MS4A3 promotes differentiation in chronic myeloid leukemia by enhancing common ß-chain cytokine receptor endocytosis.

The chronic phase of chronic myeloid leukemia (CP-CML) is characterized by the excessive production of maturating myeloid cells. As CML stem/progenitor cells (LSPCs) are poised to cycle and differentiate, LSPCs must balance conservation and differentiation to avoid exhaustion, similar to normal hematopoiesis under stress. Since BCR-ABL1 tyrosine kinase inhibitors (TKIs) eliminate differentiating cells but spare BCR-ABL1-independent LSPCs, understanding the mechanisms that regulate LSPC differentiation may inform strategies to eliminate LSPCs. Upon performing a meta-analysis of published CML transcriptomes, we discovered that low expression of the MS4A3 transmembrane protein is a universal characteristic of LSPC quiescence, BCR-ABL1 independence, and transformation to blast phase (BP). Several mechanisms are involved in suppressing MS4A3, including aberrant methylation and a MECOM-C/EBPε axis. Contrary to previous reports, we find that MS4A3 does not function as a G1/S phase inhibitor but promotes endocytosis of common ß-chain (ßc) cytokine receptors upon GM-CSF/IL-3 stimulation, enhancing downstream signaling and cellular differentiation. This suggests that LSPCs downregulate MS4A3 to evade ßc cytokine-induced differentiation and maintain a more primitive, TKI-insensitive state. Accordingly, knockdown (KD) or deletion of MS4A3/Ms4a3 promotes TKI resistance and survival of CML cells ex vivo and enhances leukemogenesis in vivo, while targeted delivery of exogenous MS4A3 protein promotes differentiation. These data support a model in which MS4A3 governs response to differentiating myeloid cytokines, providing a unifying mechanism for the differentiation block characteristic of CML quiescence and BP-CML. Promoting MS4A3 reexpression or delivery of ectopic MS4A3 may help eliminate LSPCs in vivo.

A predominant enhancer co-amplified with the SOX2 oncogene is necessary and sufficient for its expression in squamous cancer.

Amplification and overexpression of the SOX2 oncogene represent a hallmark of squamous cancers originating from diverse tissue types. Here, we find that squamous cancers selectively amplify a 3' noncoding region together with SOX2, which harbors squamous cancer-specific chromatin accessible regions. We identify a single enhancer e1 that predominantly drives SOX2 expression. Repression of e1 in SOX2-high cells causes collapse of the surrounding enhancers, remarkable reduction in SOX2 expression, and a global transcriptional change reminiscent of SOX2 knockout. The e1 enhancer is driven by a combination of transcription factors including SOX2 itself and the AP-1 complex, which facilitates recruitment of the co-activator BRD4. CRISPR-mediated activation of e1 in SOX2-low cells is sufficient to rebuild the e1-SOX2 loop and activate SOX2 expression. Our study shows that squamous cancers selectively amplify a predominant enhancer to drive SOX2 overexpression, uncovering functional links among enhancer activation, chromatin looping, and lineage-specific copy number amplifications of oncogenes.