Skin in the game: a review of single-cell and spatial transcriptomics in dermatological research.

Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) are two emerging research technologies that uniquely characterize gene expression microenvironments on a cellular or subcellular level. The skin, a clinically accessible tissue composed of diverse, essential cell populations, serves as an ideal target for these high-resolution investigative approaches. Using these tools, researchers are assembling a compendium of data and discoveries in healthy skin as well as a range of dermatologic pathophysiologies, including atopic dermatitis, psoriasis, and cutaneous malignancies. The ongoing advancement of single-cell approaches, coupled with anticipated decreases in cost with increased adoption, will reshape dermatologic research, profoundly influencing disease characterization, prognosis, and ultimately clinical practice.

A Pre-Leukemic DNA Methylation Signature in Healthy Individuals at Higher Risk for Developing Myeloid Malignancy.

PURPOSE: DNA methylation alterations are widespread in acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS), some of which appear to have evolved independently of somatic mutations in epigenetic regulators. Although the presence of somatic mutations in peripheral blood can predict the risk of development of AML and MDS, its accuracy remains unsatisfactory. EXPERIMENTAL DESIGN: We performed global DNA methylation profiling in a case control study nested within the Singapore Chinese Health Study to evaluate whether DNA methylation alterations were associated with AML/MDS development. Targeted deep sequencing and methylated DNA immunoprecipitation sequencing (MeDIP-seq) were performed on peripheral blood collected a median of 9.9 years before diagnosis of AML or MDS, together with age-matched still-healthy individuals as controls. RESULTS: Sixty-six individuals who developed AML or MDS displayed significant DNA methylation changes in the peripheral blood compared with 167 age- and gender-matched controls who did not develop AML/MDS during the follow-up period. Alterations in methylation in the differentially methylation regions were associated with increased odds of developing AML/MDS. CONCLUSIONS: The epigenetic changes may be acquired independently and before somatic mutations that are relevant for AML/MDS development. The association between methylation changes and the risk of pre-AML/MDS in these individuals was considerably stronger than somatic mutations, suggesting that methylation changes could be used as biomarkers for pre-AML/MDS screening.

Aberrant Upregulation of RUNX3 Activates Developmental Genes to Drive Metastasis in Gastric Cancer.

Gastric cancer metastasis is a major cause of mortality worldwide. Inhibition of RUNX3 in gastric cancer cell lines reduced migration, invasion, and anchorage-independent growth in vitro. Following splenic inoculation, CRISPR-mediated RUNX3-knockout HGC-27 cells show suppression of xenograft growth and liver metastasis. We interrogated the potential of RUNX3 as a metastasis driver in gastric cancer by profiling its target genes. Transcriptomic analysis revealed strong involvement of RUNX3 in the regulation of multiple developmental pathways, consistent with the notion that Runt domain transcription factor (RUNX) family genes are master regulators of development. RUNX3 promoted "cell migration" and "extracellular matrix" programs, which are necessary for metastasis. Of note, we found pro-metastatic genes WNT5A, CD44, and VIM among the top differentially expressed genes in RUNX3 knockout versus control cells. Chromatin immunoprecipitation sequencing and HiChIP analyses revealed that RUNX3 bound to the enhancers and promoters of these genes, suggesting that they are under direct transcriptional control by RUNX3. We show that RUNX3 promoted metastasis in part through its upregulation of WNT5A to promote migration, invasion, and anchorage-independent growth in various malignancies. Our study therefore reveals the RUNX3-WNT5A axis as a key targetable mechanism for gastric cancer metastasis. SIGNIFICANCE: Subversion of RUNX3 developmental gene targets to metastasis program indicates the oncogenic nature of inappropriate RUNX3 regulation in gastric cancer.

Third-Generation Single-Molecule Sequencing for Preimplantation Genetic Testing of Aneuploidy and Segmental Imbalances.

BACKGROUND: Current strategies for preimplantation genetic testing for aneuploidy or structural rearrangements (PGT-A/SR) rely mainly on next-generation sequencing (NGS) and microarray platforms, which are robust but require expensive instrumentation. We explored the suitability of third-generation single-molecule sequencing as a PGT-A/SR screening platform for both aneuploidy and segmental imbalance. METHODS: Single-cell and multicell replicates from aneuploid or segmentally unbalanced cell lines (n = 208) were SurePlex-amplified, randomized, and subjected to (a) Nanopore-based single-molecule sequencing (Oxford Nanopore Technologies) and (b) NGS using a leading commercial PGT-A solution (Illumina VeriSeq PGS). Archival SurePlex-amplified trophectoderm biopsy samples (n = 96) previously analyzed using the commercial kit were blinded and reanalyzed using Nanopore. RESULTS: Nanopore-based PGT-A identified the specific aberration in 95.45% (84/88) and 97.78% (88/90) of single-/multicells with an aneuploidy or segmental imbalance (10-30.5 Mb), respectively. Comparison against the commercial kit's results revealed concordances of 98.86% (87/88) and 98.89% (89/90) for the aneuploid and segmentally unbalanced (10-30.5 Mb aberration) samples, respectively. Detection sensitivity for smaller segmental imbalances (5-5.8 Mb aberration, n = 30) decreased markedly on both platforms. Nanopore-based PGT-A reanalysis of trophectoderm biopsy samples was 97.92% (94/96) concordant with the commercial kit results. CONCLUSION: Up to 24 SurePlex-amplified single-cell, multicell, or trophectoderm samples could be sequenced in a single MinION flow-cell for subsequent preimplantation genetic testing for aneuploidy or structural rearrangements (PGT-A/SR) analysis, with results obtainable in ≤3 days and at per-sample costs that are competitive with commercial offerings. Nanopore's third-generation single-molecule sequencing represents a viable alternative to current commercial NGS-based PGT-A solutions for aneuploidy and segmental imbalance (≥10 Mb) screening of single-/multicell or trophectoderm biopsy samples.

Core-binding factor fusion downregulation of ADAR2 RNA editing contributes to AML leukemogenesis.

Adenosine-to-inosine RNA editing, which is catalyzed by adenosine deaminases acting on RNA (ADAR) family of enzymes, ADAR1 and ADAR2, has been shown to contribute to multiple cancers. However, other than the chronic myeloid leukemia blast crisis, relatively little is known about its role in other types of hematological malignancies. Here, we found that ADAR2, but not ADAR1 and ADAR3, was specifically downregulated in the core-binding factor (CBF) acute myeloid leukemia (AML) with t(8;21) or inv(16) translocations. In t(8;21) AML, RUNX1-driven transcription of ADAR2 was repressed by the RUNX1-ETO additional exon 9a fusion protein in a dominant-negative manner. Further functional studies confirmed that ADAR2 could suppress leukemogenesis specifically in t(8;21) and inv16 AML cells dependent on its RNA editing capability. Expression of 2 exemplary ADAR2-regulated RNA editing targets coatomer subunit α and component of oligomeric Golgi complex 3 inhibits the clonogenic growth of human t(8;21) AML cells. Our findings support a hitherto, unappreciated mechanism leading to ADAR2 dysregulation in CBF AML and highlight the functional relevance of loss of ADAR2-mediated RNA editing to CBF AML.

FAM3C in circulating tumor-derived extracellular vesicles promotes non-small cell lung cancer growth in secondary sites.

Rationale: Metastasis is a complex process with a molecular underpinning that remains unclear. We hypothesize that cargo proteins conducted by extracellular vesicles (EVs) released from tumors may confer growth and metastasis potential on recipient cells. Here, we report that a cytokine-like secreted protein, FAM3C, contributes to late-stage lung tumor progression. Methods: EV protein profiling was conducted with an unbiased proteomic mass spectrometry analysis on non-small cell lung cancer (NSCLC) and normal lung fibroblast cell lines. Expression of FAM3C was confirmed in a panel of NSCLC cell lines, and correlated to the invasive and metastatic potentials. Functional phenotype of endogenous FAM3C and tumor-derived EVs (TDEs) were further investigated using various biological approaches in RNA and protein levels. Metastasis potential of TDEs secreted by FAM3C-overexpressing carcinoma cells was validated in mouse models. Results: Transcriptomic meta-analysis of pan-cancer datasets confirmed the overexpression of FAM3C - a gene encoding for interleukin-like EMT inducer (ILEI) - in NSCLC tumors, with strong association with poor patient prognosis and cancer metastasis. Aberrant expression of FAM3C in lung carcinoma cells enhances cellular transformation and promotes distant lung tumor colonization. In addition, higher FAM3C concentrations were detected in EVs extracted from plasma samples of NSCLC patients compared to those of healthy subjects. More importantly, we defined a hitherto-unknown mode of microenvironmental crosstalk involving FAM3C in EVs, whereby the delivery and uptake of FAM3C via TDEs enhances oncogenic signaling - in recipient cells that phenocopies the cell-endogenous overexpression of FAM3C. The oncogenicity transduced by FAM3C is executed via a novel interaction with the Ras-related protein RalA, triggering the downstream activation of the Src/Stat3 signaling cascade. Conclusions: Our study describes a novel mechanism for FAM3C-driven carcinogenesis and shed light on EV FAM3C as a driver for metastatic lung tumors that could be exploited for cancer therapeutics.

Targeting RNA Exonuclease XRN1 Potentiates Efficacy of Cancer Immunotherapy.

Despite the remarkable clinical responses achieved with immune checkpoint blockade therapy, the response rate is relatively low and only a subset of patients can benefit from the treatment. Aberrant RNA accumulation can mediate IFN signaling and stimulate an immune response, suggesting that targeting RNA decay machinery might sensitize tumor cells to immunotherapy. With this in mind, we identified an RNA exoribonuclease, XRN1, as a potential therapeutic target to suppress RNA decay and stimulate antitumor immunity. Silencing of XRN1 suppressed tumor growth in syngeneic immunocompetent mice and potentiated immunotherapy efficacy, while silencing of XRN1 alone did not affect tumor growth in immunodeficient mice. Mechanistically, XRN1 depletion activated IFN signaling and the viral defense pathway; both pathways play determinant roles in regulating immune evasion. Aberrant RNA-sensing signaling proteins (RIG-I/MAVS) mediated the expression of IFN genes, as depletion of each of them blunted the elevation of antiviral/IFN signaling in XRN1-silenced cells. Analysis of pan-cancer CRISPR-screening data indicated that IFN signaling triggered by XRN1 silencing is a common phenomenon, suggesting that the effect of XRN1 silencing may be extended to multiple types of cancers. Overall, XRN1 depletion triggers aberrant RNA-mediated IFN signaling, highlighting the importance of the aberrant RNA-sensing pathway in regulating immune responses. These findings provide the molecular rationale for developing XRN1 inhibitors and exploring their potential clinical application in combination with cancer immunotherapy. SIGNIFICANCE: Targeting XRN1 activates an intracellular innate immune response mediated by RNA-sensing signaling and potentiates cancer immunotherapy efficacy, suggesting inhibition of RNA decay machinery as a novel strategy for cancer treatment.

Oncofetal protein IGF2BP1 regulates IQGAP3 expression to maintain stem cell potential in cancer.

We reported earlier that IQGAP3 is an important stem cell factor in rapidly proliferating isthmus stem cells in the stomach and that IQGAP3 expression is robustly induced in terminally differentiated chief cells and de-differentiated cells following tissue damage. The elevated IQGAP3 expression in cancer and its association with metastasis suggest a fundamental role for IQGAP3 in proliferating cancer stem cells. What causes IQGAP3 upregulation in cancer is unclear. Here, we show that IGF2BP1 and IQGAP3 expression levels are highest in the blastocyst, with both decreasing during adulthood. This suggests that IQGAP3, like IGF2BP1, is an early developmental gene that is aberrantly upregulated upon re-expression of IGF2BP1 during carcinogenesis. IGF2BP1 binds and stabilizes m6A-modified IQGAP3 transcripts. Downstream targets of IGF2BP1, namely SRF and FOXM1, also upregulate IQGAP3 expression. These multiple layers of IQGAP3 regulation, which may safeguard against inappropriate stem cell proliferation, present additional drug targets to inhibit IQGAP3-driven malignant growth.

Pan-cancer pervasive upregulation of 3' UTR splicing drives tumourigenesis.

Most mammalian genes generate messenger RNAs with variable untranslated regions (UTRs) that are important post-transcriptional regulators. In cancer, shortening at 3' UTR ends via alternative polyadenylation can activate oncogenes. However, internal 3' UTR splicing remains poorly understood as splicing studies have traditionally focused on protein-coding alterations. Here we systematically map the pan-cancer landscape of 3' UTR splicing and present this in SpUR ( http://www.cbrc.kaust.edu.sa/spur/home/ ). 3' UTR splicing is widespread, upregulated in cancers, correlated with poor prognosis and more prevalent in oncogenes. We show that antisense oligonucleotide-mediated inhibition of 3' UTR splicing efficiently reduces oncogene expression and impedes tumour progression. Notably, CTNNB1 3' UTR splicing is the most consistently dysregulated event across cancers. We validate its upregulation in hepatocellular carcinoma and colon adenocarcinoma, and show that the spliced 3' UTR variant is the predominant contributor to its oncogenic functions. Overall, our study highlights the importance of 3' UTR splicing in cancer and may launch new avenues for RNA-based anti-cancer therapeutics.

Demethylation and Up-Regulation of an Oncogene after Hypomethylating Therapy.

BACKGROUND: Although hypomethylating agents are currently used to treat patients with cancer, whether they can also reactivate and up-regulate oncogenes is not well elucidated. METHODS: We examined the effect of hypomethylating agents on SALL4, a known oncogene that plays an important role in myelodysplastic syndrome and other cancers. Paired bone marrow samples that were obtained from two cohorts of patients with myelodysplastic syndrome before and after treatment with a hypomethylating agent were used to explore the relationships among changes in SALL4 expression, treatment response, and clinical outcome. Leukemic cell lines with low or undetectable SALL4 expression were used to study the relationship between SALL4 methylation and expression. A locus-specific demethylation technology, CRISPR-DNMT1-interacting RNA (CRISPR-DiR), was used to identify the CpG island that is critical for SALL4 expression. RESULTS: SALL4 up-regulation after treatment with hypomethylating agents was observed in 10 of 25 patients (40%) in cohort 1 and in 13 of 43 patients (30%) in cohort 2 and was associated with a worse outcome. Using CRISPR-DiR, we discovered that demethylation of a CpG island within the 5' untranslated region was critical for SALL4 expression. In cell lines and patients, we confirmed that treatment with a hypomethylating agent led to demethylation of the same CpG region and up-regulation of SALL4 expression. CONCLUSIONS: By combining analysis of patient samples with CRISPR-DiR technology, we found that demethylation and up-regulation of an oncogene after treatment with a hypomethylating agent can indeed occur and should be further studied. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).

A Runx1-enhancer Element eR1 Identified Lineage Restricted Mammary Luminal Stem Cells.

Mammary gland homeostasis is maintained by adult tissue stem-progenitor cells residing within the luminal and basal epithelia. Dysregulation of mammary stem cells is a key mechanism for cancer development. However, stem cell characterization is challenging because reporter models using cell-specific promoters do not fully recapitulate the mammary stem cell populations. We previously found that a 270-basepair Runx1 enhancer element, named eR1, marked stem cells in the blood and stomach. Here, we identified eR1 activity in a rare subpopulation of the ERα-negative luminal epithelium in mouse mammary glands. Lineage-tracing using an eR1-CreERT2 mouse model revealed that eR1+ luminal cells generated the entire luminal lineage and milk-secreting alveoli-eR1 therefore specifically marks lineage-restricted luminal stem cells. eR1-targeted-conditional knockout of Runx1 led to the expansion of luminal epithelial cells, accompanied by elevated ERα expression. Our findings demonstrate a definitive role for Runx1 in the regulation of the eR1-positive luminal stem cell proliferation during mammary homeostasis. Our findings identify a mechanistic link for Runx1 in stem cell proliferation and its dysregulation in breast cancer. Runx1 inactivation is therefore likely to be an early hit in the cell-of-origin of ERα+ luminal type breast cancer.

Loss of METTL3 attenuates blastic plasmacytoid dendritic cell neoplasm response to PRMT5 inhibition via IFN signaling.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematologic malignancy with poor clinical outcomes. Dysregulated MYC expression, which is associated with protein arginine methyltransferase 5 (PRMT5) dependency, is a recurrent feature of BPDCN. Although recent studies have reported a PRMT5 gene signature in BPDCN patient samples, the role of PRMT5 in BPDCN remains unexplored. Here, we demonstrate that BPDCN is highly sensitive to PRMT5 inhibition. Consistent with the upregulation of PRMT5 in BPDCN, we show that pharmacological inhibition (GSK3326595) of PRMT5 inhibits the growth of the patient-derived BPDCN cell line CAL-1 in vitro and mitigated tumor progression in our mouse xenograft model. Interestingly, RNA-sequencing (RNA-seq) analysis revealed that PRMT5 inhibition increases intron retention in several key RNA methylation genes, including METTL3, which was accompanied by a dose-dependent decrease in METTL3 expression. Notably, the function of cellular m6A RNA modification of METTL3 was also affected by PRMT5 inhibition in CAL-1 cells. Intriguingly, METTL3 depletion in CAL-1 caused a significant increase in interferon (IFN) signaling, which was further elevated upon PRMT5 inhibition. Importantly, we discovered that this increase in IFN signaling attenuated the sensitivity of METTL3-depleted CAL-1 cells to PRMT5 inhibition. Correspondingly, stimulation of IFN signaling via TLR7 agonists weakened CAL-1 cell sensitivity to PRMT5 inhibition. Overall, our findings implicate PRMT5 as a therapeutic target in BPDCN and provide insight into the involvement of METTL3 and the IFN pathway in regulating the response to PRMT5 inhibition.

Multilayered control of splicing regulatory networks by DAP3 leads to widespread alternative splicing changes in cancer.

The dynamic regulation of alternative splicing requires coordinated participation of multiple RNA binding proteins (RBPs). Aberrant splicing caused by dysregulation of splicing regulatory RBPs is implicated in numerous cancers. Here, we reveal a frequently overexpressed cancer-associated protein, DAP3, as a splicing regulatory RBP in cancer. Mechanistically, DAP3 coordinates splicing regulatory networks, not only via mediating the formation of ribonucleoprotein complexes to induce substrate-specific splicing changes, but also via modulating splicing of numerous splicing factors to cause indirect effect on splicing. A pan-cancer analysis of alternative splicing across 33 TCGA cancer types identified DAP3-modulated mis-splicing events in multiple cancers, and some of which predict poor prognosis. Functional investigation of non-productive splicing of WSB1 provides evidence for establishing a causal relationship between DAP3-modulated mis-splicing and tumorigenesis. Together, our work provides critical mechanistic insights into the splicing regulatory roles of DAP3 in cancer development.

PLK1 inhibition selectively induces apoptosis in ARID1A deficient cells through uncoupling of oxygen consumption from ATP production.

Inhibitors of the mitotic kinase PLK1 yield objective responses in a subset of refractory cancers. However, PLK1 overexpression in cancer does not correlate with drug sensitivity, and the clinical development of PLK1 inhibitors has been hampered by the lack of patient selection marker. Using a high-throughput chemical screen, we discovered that cells deficient for the tumor suppressor ARID1A are highly sensitive to PLK1 inhibition. Interestingly this sensitivity was unrelated to canonical functions of PLK1 in mediating G2/M cell cycle transition. Instead, a whole-genome CRISPR screen revealed PLK1 inhibitor sensitivity in ARID1A deficient cells to be dependent on the mitochondrial translation machinery. We find that ARID1A knock-out (KO) cells have an unusual mitochondrial phenotype with aberrant biogenesis, increased oxygen consumption/expression of oxidative phosphorylation genes, but without increased ATP production. Using expansion microscopy and biochemical fractionation, we see that a subset of PLK1 localizes to the mitochondria in interphase cells. Inhibition of PLK1 in ARID1A KO cells further uncouples oxygen consumption from ATP production, with subsequent membrane depolarization and apoptosis. Knockdown of specific subunits of the mitochondrial ribosome reverses PLK1-inhibitor induced apoptosis in ARID1A deficient cells, confirming specificity of the phenotype. Together, these findings highlight a novel interphase role for PLK1 in maintaining mitochondrial fitness under metabolic stress, and a strategy for therapeutic use of PLK1 inhibitors. To translate these findings, we describe a quantitative microscopy assay for assessment of ARID1A protein loss, which could offer a novel patient selection strategy for the clinical development of PLK1 inhibitors in cancer.

ADARs act as potent regulators of circular transcriptome in cancer.

Circular RNAs (circRNAs) are produced by head-to-tail back-splicing which is mainly facilitated by base-pairing of reverse complementary matches (RCMs) in circRNA flanking introns. Adenosine deaminases acting on RNA (ADARs) are known to bind double-stranded RNAs for adenosine to inosine (A-to-I) RNA editing. Here we characterize ADARs as potent regulators of circular transcriptome by identifying over a thousand of circRNAs regulated by ADARs in a bidirectional manner through and beyond their editing function. We find that editing can stabilize or destabilize secondary structures formed between RCMs via correcting A:C mismatches to I(G)-C pairs or creating I(G).U wobble pairs, respectively. We provide experimental evidence that editing also favors the binding of RNA-binding proteins such as PTBP1 to regulate back-splicing. These ADARs-regulated circRNAs which are ubiquitously expressed in multiple types of cancers, demonstrate high functional relevance to cancer. Our findings support a hitherto unappreciated bidirectional regulation of circular transcriptome by ADARs and highlight the complexity of cross-talk in RNA processing and its contributions to tumorigenesis.

Global analysis of RNA-binding proteins identifies a positive feedback loop between LARP1 and MYC that promotes tumorigenesis.

In addition to genomic alterations, aberrant changes in post-transcriptional regulation can modify gene function and drive cancer development. RNA-binding proteins (RBPs) are a large class of post-transcriptional regulators that have been increasingly implicated in carcinogenesis. By integrating multi-omics data, we identify LARP1 as one of the most upregulated RBPs in colorectal cancer (CRC) and demonstrate its oncogenic properties. We perform LARP1:RNA interactome profiling and unveil a previously unexplored role for LARP1 in targeting the 3'UTR of oncogenes in CRC. Notably, we identify the proto-oncogenic transcription factor MYC as a key LARP1-regulated target. Our data show that LARP1 positively modulates MYC expression by associating with its 3'UTR. In addition, antisense oligonucleotide-mediated blocking of the interaction between LARP1 and the MYC 3'UTR reduces MYC expression and in vitro CRC growth. Furthermore, a systematic analysis of LARP1:protein interactions reveals IGF2BP3 and YBX1 as LARP1-interacting proteins that also regulate MYC expression and CRC development. Finally, we demonstrate that MYC reciprocally modulates LARP1 expression by targeting its enhancer. In summary, our data reveal a critical, previously uncharacterized role of LARP1 in promoting CRC tumorigenesis, validate its direct regulation of the proto-oncogene MYC and delineate a model of the positive feedback loop between MYC and LARP1 that promotes CRC growth and development.

p53-NEIL1 co-abnormalities induce genomic instability and promote synthetic lethality with Chk1 inhibition in multiple myeloma having concomitant 17p13(del) and 1q21(gain).

Recurrent cytogenetic abnormalities are the main hallmark of multiple myeloma (MM) and patients having 2 or more high-risk prognostic events are associated with extremely poor outcome. 17p13(del) and 1q21(gain) are critical and independent high-risk cytogenetic markers, however, the biological significance underlying the poor outcome in MM patients having co-occurrence of both these chromosomal aberrations has never been interrogated. Herein, we identified that patients harbouring concomitant 17p13(del) with 1q21(gain) demonstrated the worst prognosis as compared to patients with single- (either 17p13(del) or 1q21(gain)) and with no chromosomal events (WT for both chromosomal loci); and they are highly enriched for genomic instability (GI) signature. We discovered that the GI feature in the patients with concomitant 17p13(del)-1q21(gain) was recapitulating the biological properties of myeloma cells with co-existing p53-deficiency and NEIL1 mRNA-hyper-editing (associated with chromosome 17p and 1q, respectively) that have inherent DNA damage response (DDR) and persistent activation of Chk1 pathway. Importantly, this became a vulnerable point for therapeutic targeting whereby the cells with this co-abnormalities demonstrated hyper-sensitivity to siRNA- and pharmacological-mediated-Chk1 inhibition, as observed at both the in vitro and in vivo levels. Mechanistically, this was attributable to the synthetic lethal relationship between p53-NEIL1-Chk1 abnormalities. The Chk1 inhibitor (AZD7762) tested showed good synergism with standard-of-care myeloma drugs, velcade and melphalan, thus further reinforcing the translational potential of this therapeutic approach. In summary, combination of NEIL1-p53 abnormalities with an ensuing Chk1 activation could serve as an Achilles heel and predispose MM cells with co-existing 1q21(gain) and 17p13(del) to therapeutic vulnerability for Chk1 inhibition.

A Point Mutation R122C in RUNX3 Promotes the Expansion of Isthmus Stem Cells and Inhibits Their Differentiation in the Stomach.

BACKGROUND & AIMS: RUNX transcription factors play pivotal roles in embryonic development and neoplasia. We previously identified the single missense mutation R122C in RUNX3 from human gastric cancer. However, how RUNX3R122C mutation disrupts stem cell homeostasis and promotes gastric carcinogenesis remained unclear. METHODS: To understand the oncogenic nature of this mutation in vivo, we generated the RUNX3R122C knock-in mice. Stomach tissues were harvested, followed by histologic and immunofluorescence staining, organoid culture, flow cytometry to isolate gastric corpus isthmus and nonisthmus epithelial cells, and RNA extraction for transcriptomic analysis. RESULTS: The corpus tissue of RUNX3R122C/R122C homozygous mice showed a precancerous phenotype such as spasmolytic polypeptide-expressing metaplasia. We observed mucous neck cell hyperplasia; massive reduction of pit, parietal, and chief cell populations; as well as a dramatic increase in the number of rapidly proliferating isthmus stem/progenitor cells in the corpus of RUNX3R122C/R122C mice. Transcriptomic analyses of the isolated epithelial cells showed that the cell-cycle-related MYC target gene signature was enriched in the corpus epithelial cells of RUNX3R122C/R122C mice compared with the wild-type corpus. Mechanistically, RUNX3R122C mutant protein disrupted the regulation of the restriction point where cells decide to enter either a proliferative or quiescent state, thereby driving stem cell expansion and limiting the ability of cells to terminally differentiate. CONCLUSIONS: RUNX3R122C missense mutation is associated with the continuous cycling of isthmus stem/progenitor cells, maturation arrest, and development of a precancerous state. This work highlights the importance of RUNX3 in the prevention of metaplasia and gastric cancer.