Cell-like State Research Articles

Paradoxical cancer cell proliferation after FGFR inhibition through decreased p21 signaling in FGFR1-amplified breast cancer cells

Fibroblast growth factors (FGFs) control various cellular functions through fibroblast growth factor receptor (FGFR) activation, including proliferation, differentiation, migration, and survival. FGFR amplification in ER + breast cancer patients correlate with poor prognosis, and FGFR inhibitors are currently being tested in clinical trials. By comparing three-dimensional spheroid growth of ER + breast cancer cells with and without FGFR1 amplification, our research discovered that FGF2 treatment can paradoxically decrease proliferation in cells with FGFR1 amplification or overexpression. In contrast, FGF2 treatment in cells without FGFR1 amplification promotes classical FGFR proliferative signaling through the MAPK cascade. The growth inhibitory effect of FGF2 in FGFR1 amplified cells aligned with an increase in p21, a cell cycle inhibitor that hinders the G1 to S phase transition in the cell cycle. Additionally, FGF2 addition in FGFR1 amplified cells activated JAK-STAT signaling and promoted a stem cell-like state. FGF2-induced paradoxical effects were reversed by inhibiting p21 or the JAK-STAT pathway and with pan-FGFR inhibitors. Analysis of patient ER + breast tumor transcriptomes from the TCGA and METABRIC datasets demonstrated a strong positive association between expression of FGF2 and stemness signatures, which was further enhanced in tumors with high FGFR1 expression. Overall, our findings reveal a divergence in FGFR signaling, transitioning from a proliferative to stemness state driven by activation of JAK-STAT signaling and modulation of p21 levels. Activation of these divergent signaling pathways in FGFR amplified cancer cells and paradoxical growth effects highlight a challenge in the use of FGFR inhibitors in cancer treatment.

Abstract 7555: Selective inhibition of the CoREST complex as a novel therapeutic target of atypical teratoid rhabdoid tumors

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are deadly infantile brain tumors, primarily arising from a single recurring biallelic loss-of-function mutation in SMARCB1 which is a member of the SWI-SNF chromatin remodeling complex. The partially functional SWI-SNF complex (without SMARCB1) is able to inhibit EZH2 at most locations of the genome except at gene promoter regions where EZH2 co-localizes with the REST complex. This results in hypermethylation and subsequent repression of genes coding for neuronal differentiation and tumor suppressors. CoREST is an important transcriptional repressor and consists of LSD1 and HDAC1/2 bound together by the scaffolding protein RCOR. Increased activity of EZH2 and the CoREST complex may help AT/RT cells maintain a stem cell-like state, driving its aggressive growth and therapy resistance. We hypothesize that functional inhibition of the CoREST complex by targeting LSD1 and HDAC1/2 might help restore the epigenetic balance lost in AT/RT cells.We selectively inhibited the CoREST complex in AT/RT cells (BT37, CHLA05 and CHLA06) by using a bi-functional LSD1 and HDAC1/2 inhibitor, Corin, which has previously been shown to be effective against melanoma and diffuse midline glioma. Administration of corin to AT/RT cells significantly increased H3K4me1, H3K9Ac and H3K27Ac histone marks (p < 0.001, Western blotting). Corin inhibited AT/RT cell growth and proliferation (MUSE Viacount p<0.05, BrdU immunofluorescence compared to control), and increased apoptosis (cPARP Western blotting, CC3 immunofluorescence, MUSE Annexin V assay, p<0.05 compared to control). Corin appears to be specific in targeting AT/RT as it did not affect brain organoids derived from iPSCs (cPARP Western blotting). Corin induced neuronal differentiation in AT/RT as seen by the increased expression of Beta-3-tubulin, MAP2 and Synaptophysin (Immunofluorescence). Enrichment analysis of differentially expressed genes after whole mRNA sequencing of AT/RT cells treated with corin for 24 h revealed upregulation of pathways related to neuronal differentiation and axonal development and downregulation of pathways related to proliferation and cell cycle (pFDR < 0.05, FC>2). In mice bearing orthotopic CHLA06 tumors, intratumoral delivery of a single dose of corin for 24 h increased H3K27me3 and H3K27ac, demonstrating that corin engaged its target in vivo, reduced growth and induced apoptosis (Ki67, cPARP). Survival studies show a trend toward single agent activity against the very aggressive CHLA06 orthotopic xenograft (increased in median survival from 14 to 35 days compared to control). These studies demonstrate that targeting the CoREST complex disrupts AT/RT epigenetic abnormalities, reversing their stem cell-like state, slowing tumor cell growth, and inducing apoptosis, that may translate into the clinical setting to help improve AT/RT’s dismal survival. Citation Format: Anupa Geethadevi, Marianne Collard, Tyler Findlay, Yiming Deng, Robert Fisher, Nikhil Vaidya, Charles Eberhart, Philip A. Cole, Rhoda Alani, Jeffrey Rubens, Eric Raabe. Selective inhibition of the CoREST complex as a novel therapeutic target of atypical teratoid rhabdoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7555.

Abstract 2006: Deciphering the interplay of β-catenin/YAP signaling and alveolar lineage plasticity during targeted therapy in non-small cell lung cancer

Abstract Molecularly targeted therapies have significantly enhanced clinical outcomes by directly addressing oncogenic driver mutations in genes such as EGFR. However, despite the success of treatments targeting these mutations, long-term patient survival remains limited. This study investigates the molecular mechanisms underlying drug-tolerance in response to targeted therapies in non-small cell lung cancer (NSCLC). Using single-cell RNA sequencing of oncogene-driven human NSCLCs at the residual disease (RD) state during active targeted therapy, our investigation reveals an increase of an alveolar type I/II (AT1/2) mixed lineage program in surviving cancer cells, resembling an alveolar cell-like state. We observed increased WNT3A/4 ligands, FZD2 receptors, β-catenin signaling, and AT1/2 signatures at the RD stage. Consistent with clinical results, AT1/2 signatures and target genes of β-catenin and YAP are increased in drug-tolerant persister (DTP) cells in preclinical models. Additionally, we discovered that β-catenin and YAP form a stable nuclear protein complex in DTP cells. Disrupting β-catenin/YAP protein expression and stability in DTP cells by knocking down FZD2 or interrupting the destruction complex of β-catenin/YAP leads to re-sensitization to the EGFR inhibitor Osimertinib, suggesting a potential therapeutic approach. Furthermore, this therapeutic strategy decreased AT1/2 gene signatures. Our results highlight an interdependence between β-catenin and YAP, with both proteins playing crucial roles in sustaining DTP cells and promoting the alveolar cell like lineage plasticity. These findings shed light on the complex interplay between β-catenin/YAP signaling and alveolar signatures in the residual cancer state, offering mechanistic insights and potential therapeutic strategies to address drug tolerance and improve clinical outcomes. Citation Format: Yu-Ting Chou, Wei Wu, Daniel Kerr, Macey Slota, Trever Bivona. Deciphering the interplay of β-catenin/YAP signaling and alveolar lineage plasticity during targeted therapy in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2006.

ARID1A orchestrates SWI/SNF-mediated sequential binding of transcription factors with ARID1A loss driving pre-memory B cell fate and lymphomagenesis

ARID1A, a subunit of the canonical BAF nucleosome remodeling complex, is commonly mutated in lymphomas. We show that ARID1A orchestrates B cell fate during the germinal center (GC) response, facilitating cooperative and sequential binding of PU.1 and NF-kB at crucial genes for cytokine and CD40 signaling. The absence of ARID1A tilts GC cell fate toward immature IgM+CD80-PD-L2- memory B cells, known for their potential to re-enter new GCs. When combined with BCL2 oncogene, ARID1A haploinsufficiency hastens the progression of aggressive follicular lymphomas (FLs) in mice. Patients with FL with ARID1A-inactivating mutations preferentially display an immature memory B cell-like state with increased transformation risk to aggressive disease. These observations offer mechanistic understanding into the emergence of both indolent and aggressive ARID1A-mutant lymphomas through the formation of immature memory-like clonal precursors. Lastly, we demonstrate that ARID1A mutation induces synthetic lethality to SMARCA2/4 inhibition, paving the way for potential precision therapy for high-risk patients.

An arms-race against resistance: leukemic stem cells and lineage plasticity.

Acute myeloid leukemia (AML) therapy is undergoing rapid development, but primary and acquired resistance to therapy complicates the prospect of a durable cure. Recent functional and single-cell multi-omics approaches have greatly expanded our knowledge of the diversity of lineage trajectories in AML settings. AML cells range from undifferentiated stem-like cells to more differentiated myeloid or megakaryocyte/erythroid cells. Current clinically relevant drugs predominantly target the myeloid progenitor lineage, while monocyte- or stem cell-like states can evade current AML treatment and may be targeted in the future with lineage-specific inhibitors. The extent of aberrant lineage plasticity upon therapeutic pressure in AML cells in conjunction with hijacking of normal differentiation pathways is still a poorly understood topic. Insights into the mechanisms of lineage plasticity of AML stem cells could identify both therapy-specific and cross-drug resistance pathways and reveal novel strategies to overcome them.

Temporal change of DNA methylation subclasses between matched newly diagnosed and recurrent glioblastoma

The longitudinal transition of phenotypes is pivotal in glioblastoma treatment resistance and DNA methylation emerged as an important tool for classifying glioblastoma phenotypes. We aimed to characterize DNA methylation subclass heterogeneity during progression and assess its clinical impact. Matched tissues from 47 glioblastoma patients were subjected to DNA methylation profiling, including CpG-site alterations, tissue and serum deconvolution, mass spectrometry, and immunoassay. Effects of clinical characteristics on temporal changes and outcomes were studied. Among 47 patients, 8 (17.0%) had non-matching classifications at recurrence. In the remaining 39 cases, 28.2% showed dominant DNA methylation subclass transitions, with 72.7% being a mesenchymal subclass. In general, glioblastomas with a subclass transition showed upregulated metabolic processes. Newly diagnosed glioblastomas with mesenchymal transition displayed increased stem cell-like states and decreased immune components at diagnosis and exhibited elevated immune signatures and cytokine levels in serum. In contrast, tissue of recurrent glioblastomas with mesenchymal transition showed increased immune components but decreased stem cell-like states. Survival analyses revealed comparable outcomes for patients with and without subclass transitions. This study demonstrates a temporal heterogeneity of DNA methylation subclasses in 28.2% of glioblastomas, not impacting patient survival. Changes in cell state composition associated with subclass transition may be crucial for recurrent glioblastoma targeted therapies.

Single-nucleus expression characterization of non-enhancing region of recurrent high-grade glioma.

Non-enhancing (NE) infiltrating tumor cells beyond the contrast-enhancing (CE) bulk of tumor are potential propagators of recurrence after gross total resection of high-grade glioma. We leveraged single-nucleus RNA sequencing on 15 specimens from recurrent high-grade gliomas (n = 5) to compare prospectively identified biopsy specimens acquired from CE and NE regions. Additionally, 24 CE and 22 NE biopsies had immunohistochemical staining to validate RNA findings. Tumor cells in NE regions are enriched in neural progenitor cell-like cellular states, while CE regions are enriched in mesenchymal-like states. NE glioma cells have similar proportions of proliferative and putative glioma stem cells relative to CE regions, without significant differences in % Ki-67 staining. Tumor cells in NE regions exhibit upregulation of genes previously associated with lower grade gliomas. Our findings in recurrent GBM paralleled some of the findings in a re-analysis of a dataset from primary GBM. Cell-, gene-, and pathway-level analyses of the tumor microenvironment in the NE region reveal relative downregulation of tumor-mediated neovascularization and cell-mediated immune response, but increased glioma-to-nonpathological cell interactions. This comprehensive analysis illustrates differing tumor and nontumor landscapes of CE and NE regions in high-grade gliomas, highlighting the NE region as an area harboring likely initiators of recurrence in a pro-tumor microenvironment and identifying possible targets for future design of NE-specific adjuvant therapy. These findings also support the aggressive approach to resection of tumor-bearing NE regions.

The mitochondrial fission protein DRP1 influences memory CD8+ T cell formation and function.

Pharmacological methods for promoting mitochondrial elongation suggest that effector T cells can be altered to support a memory T cell-like metabolic state. Such mitochondrial elongation approaches may enhance the development of immunological memory. Therefore, we hypothesized that deletion of the mitochondrial fission protein dynamin-related protein 1 (DRP1) would lead to mitochondrial elongation and generate a large memory T cell population, an approach that could be exploited to enhance vaccination protocols. We find that, as expected, while deletion of DRP1 from T cells in dLckCre × Drp1flfl does compromise the magnitude and functionality of primary effector CD8+ T cells, a disproportionately large pool of memory CD8+ T cells does form. In contrast to primary effector CD8+ T cells, DRP1-deficient memory dLckCre × Drp1flfl CD8+ T cells mount a secondary response comparable to control memory T cells with respect to kinetics, magnitude, and effector capabilities. Interestingly, the relative propensity to form memory cells in the absence of DRP1 was associated with neither differentiation toward more memory precursor CD8+ T cells nor decreased cellular death of effector T cells. Instead, the tendency to form memory CD8+ T cells in the absence of DRP1 is associated with decreased T cell receptor expression. Remarkably, in a competitive environment with DRP1-replete CD8+ T cells, the absence of DRP1 from CD8+ T cells compromised the generation of primary, memory, and secondary responses, indicating that approaches targeting DRP1 need to be carefully tailored.

MODL-23. PAIRED GLIOBLASTOMA CELL CULTURES OF THE FLUORESCENT BULK TUMOR AND NON-FLUORESCENT TUMOR MARGIN DISPLAY DIFFERENTIAL PHENOTYPES AND CELL STATES ACROSS PATIENTS

Abstract Glioblastoma is a devastating disease with a median survival of 8 months after diagnosis. Over 90% of patients have a recurrence that is in contact with the resection cavity. To understand the potential relapse-causing cells of the local infiltrative tumor margin, which have rarely been studied, we have established and analyzed paired cell cultures from the fluorescent bulk tumor and non-fluorescent tumor margin. Tumor samples were resected using 5-ALA fluorescence-guided surgery and employing stringent procedures to separate margin samples from bulk tumor cells. All margin samples were resected at least 1cm beyond the fluorescent border in non eloquent areas as determined intraoperatively. Bulk and margin samples from 13 glioblastoma patients were explanted and 7 paired cultures that could be maintained to at least passage 7 were established. Analysis of self-renewal and orthotopic tumor formation of both acutely dissociated cells and established cultures showed that bulk tumor cells in all cases had a higher capacity than their paired margin cells. Conversely, margin cells always displayed a higher invasive capacity than their paired bulk tumor cells. Combined single nucleus RNA- and ATAC-sequencing was performed on three paired cultures. Analysis of snRNA-seq data showed that the margin cell phenotype was connected with transitions towards more differentiated (AC- and MES-like) cell states compared to paired bulk tumor cells that showed a higher rate of cells in progenitor cell-like states (NPC- and OPC-like). snATAC analysis uncovered that the margin cell phenotype was connected with transcription factor (TF) motifs known to be involved in cell invasion and differentiation, while paired bulk tumor cells were enriched for TF motifs involved in neural development and proliferation. We present a unique collection of margin cell cultures that we believe are more relevant models of the relapse-causing glioblastoma cells than those established from the bulk tumor.

CSIG-02. SHH PATHWAY ACTIVATION DRIVES NEURAL CREST CELL SIGNATURE IN A SUBGROUP OF MPNSTS

Abstract INTRODUCTION Neurofibromatosis Type 1 is hereditary tumor predisposition syndrome that results in the development of innumerable nerve sheath tumors that fall within the spectrum of benign, premalignant and malignant lesions. Our previous work has demonstrated that SHH pathway activation drives malignant transformation in a subset of malignant peripheral nerve sheath tumors (MPNSTs). Here, we demonstrate that SHH pathway activation induces a neural crest cell-like state in a subset of MPNSTs. METHODS We performed single nuclear RNA sequencing on 6 tumors (5 MPNSTS and 1 atypical neurofibroma). We performed in vitro experiments to assess the correlation between SHH pathway activation and neural crest signatures. We inhibited SHH pathway with sonidegib, a SMO inhibitor, in MPNST cell lines and performed RT-PCR to assess markers of neural crest cell signatures. In addition, we assessed cellular viability with treatment. RESULTS We observed that cells that express SMO at high levels, with SHH pathway activation, express neural crest cell makers. Single cell trajectory analysis further demonstrates a pseudotemporal continuum from atypical neurofibromas (Schwann cells), MPNSTs (Schwann cell precursor cells) and other MPNSTs (neural crest cells), which supports that these tumors fall along the developmental trajectory of neural crest cell lineage. SHH pathway activity was high in S462TY MPNST cell line and low in STS-26T MPNST cell line. S462TY also demonstrated prominent elevation in expression of neural crest cell markers TWIST1, SOX9, SNAI2, OTX2, PAX4 and PAX6 . We found that treatment with sonidegib treatment attenuates the expression of neural crest cell signatures. Finally, inhibiting SHH pathway activation reduced cellular viability in MPNST cell lines. CONCLUSION SHH pathway activation drives dedifferentiation into a neural crest cell-like state in a subset of MPNSTs. We confirm that inhibiting SHH pathway activity in a subset of MPNSTS prevent growth and malignant progression, providing a rational for future clinical trials.

EPCO-41. MICRORNA-MEDIATED DIRECT NEURONAL REPROGRAMMING OF GLIOBLASTOMAS

Abstract Neuron-specific microRNAs miR-9/9* and miR-124 (miR-9/9*-124) are potent neurogenic molecules whose ectopic expression in non-neuronal somatic cells effectively induces direct cell fate conversion into a neuronal fate. Our recent work indicates that the fate transition occurs in distinct steps where miR-9/9*-124 first represses core gene regulatory networks to erase the original non-neuronal cell identity and subsequently activates the neuronal program to confer neuronal fate. Extending these findings, we reason that repression of similar gene regulatory networks by miR-9/9*-124 can also induce fate erasure in cancerous cells using glioblastoma as a paradigm. Glioblastoma multiforme (GBM) is a type of aggressive brain tumor with poor prognosis. Instead of having specific mutation profiles, GBMs present a global genetic network dysregulation which gives rise to a neural stem cell or progenitor cell-like state resembling embryonic neural development. Whereas miR-9/9*-124 plays a key role in inducing mitotic exit and orchestrating neurogenesis during development and neuronal reprogramming, we hypothesize that miR-9/9*-124 will instruct neuronal reprogramming of GBMs. Our results so far demonstrate that miR-9/9*-124 indeed exerts reprogramming activities in patient-derived GBM cell lines. Ectopic expression of miR-9/9*-124 in vitro readily induces GBM cells to exit cell cycle and promotes neuronal identity over time. In GBM intracerebral xenograft mouse model, mice receiving GBM cells overexpressing miR-9/9*-124 were able to survive longer than those with GBMs overexpressing non-specific microRNAs. We are currently carrying out transcriptomic and chromatin accessibility analyses to infer the epigenetic switch as GBM cells are reprogrammed to neurons. Based on these studies, we will further examine whether the GBM cells undergo irreversible fate erasure and are reprogrammed into a stable post-mitotic neuronal state. Our studies will provide insights into the plasticity of GBM fate that can be controlled by microRNAs and the key downstream regulatory nodes underlying fate transition of GBM to permanent post-mitotic state.

Her6 and Prox1a are novel regulators of photoreceptor regeneration in the zebrafish retina

Damage to light-sensing photoreceptors (PRs) occurs in highly prevalent retinal diseases. As humans cannot regenerate new PRs, these diseases often lead to irreversible blindness. Intriguingly, animals, such as the zebrafish, can regenerate PRs efficiently and restore functional vision. Upon injury, mature Müller glia (MG) undergo reprogramming to adopt a stem cell-like state. This process is similar to cellular dedifferentiation, and results in the generation of progenitor cells, which, in turn, proliferate and differentiate to replace lost retinal neurons. In this study, we tested whether factors involved in dedifferentiation of Drosophila CNS are implicated in the regenerative response in the zebrafish retina. We found that hairy-related 6 (her6) negatively regulates of PR production by regulating the rate of cell divisions in the MG-derived progenitors. prospero homeobox 1a (prox1a) is expressed in differentiated PRs and may promote PR differentiation through phase separation. Interestingly, upon Her6 downregulation, Prox1a is precociously upregulated in the PRs, to promote PR differentiation; conversely, loss of Prox1a also induces a downregulation of Her6. Together, we identified two novel candidates of PR regeneration that cross regulate each other; these may be exploited to promote human retinal regeneration and vision recovery.

Perturbation of 3D nuclear architecture, epigenomic dysregulation and aging, and cannabinoid synaptopathy reconfigures conceptualization of cannabinoid pathophysiology: part 1-aging and epigenomics.

Much recent attention has been directed toward the spatial organization of the cell nucleus and the manner in which three-dimensional topologically associated domains and transcription factories are epigenetically coordinated to precisely bring enhancers into close proximity with promoters to control gene expression. Twenty lines of evidence robustly implicate cannabinoid exposure with accelerated organismal and cellular aging. Aging has recently been shown to be caused by increased DNA breaks. These breaks rearrange and maldistribute the epigenomic machinery to weaken and reverse cellular differentiation, cause genome-wide DNA demethylation, reduce gene transcription, and lead to the inhibition of developmental pathways, which contribute to the progressive loss of function and chronic immune stimulation that characterize cellular aging. Both cell lineage-defining superenhancers and the superanchors that control them are weakened. Cannabis exposure phenocopies the elements of this process and reproduces DNA and chromatin breakages, reduces the DNA, RNA protein and histone synthesis, interferes with the epigenomic machinery controlling both DNA and histone modifications, induces general DNA hypomethylation, and epigenomically disrupts both the critical boundary elements and the cohesin motors that create chromatin loops. This pattern of widespread interference with developmental programs and relative cellular dedifferentiation (which is pro-oncogenic) is reinforced by cannabinoid impairment of intermediate metabolism (which locks in the stem cell-like hyper-replicative state) and cannabinoid immune stimulation (which perpetuates and increases aging and senescence programs, DNA damage, DNA hypomethylation, genomic instability, and oncogenesis), which together account for the diverse pattern of teratologic and carcinogenic outcomes reported in recent large epidemiologic studies in Europe, the USA, and elsewhere. It also accounts for the prominent aging phenotype observed clinically in long-term cannabis use disorder and the 20 characteristics of aging that it manifests. Increasing daily cannabis use, increasing use in pregnancy, and exponential dose-response effects heighten the epidemiologic and clinical urgency of these findings. Together, these findings indicate that cannabinoid genotoxicity and epigenotoxicity are prominent features of cannabis dependence and strongly indicate coordinated multiomics investigations of cannabinoid genome-epigenome-transcriptome-metabolome, chromatin conformation, and 3D nuclear architecture. Considering the well-established exponential dose-response relationships, the diversity of cannabinoids, and the multigenerational nature of the implications, great caution is warranted in community cannabinoid penetration.

Transient naive reprogramming corrects hiPS cells functionally and epigenetically

Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function1–8. These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory.

Tff2 defines transit-amplifying pancreatic acinar progenitors that lack regenerative potential and are protective against Kras-driven carcinogenesis.

While adult pancreatic stem cells are thought not to exist, it is now appreciated that the acinar compartment harbors progenitors, including tissue-repairing facultative progenitors (FPs). Here, we study a pancreatic acinar population marked by trefoil factor 2 (Tff2) expression. Long-term lineage tracing and single-cell RNA sequencing (scRNA-seq) analysis of Tff2-DTR-CreERT2-targeted cells defines a transit-amplifying progenitor (TAP) population that contributes to normal homeostasis. Following acute and chronic injury, Tff2+ cells, distinct from FPs, undergo depopulation but are eventually replenished. At baseline, oncogenic KrasG12D-targeted Tff2+ cells are resistant to PDAC initiation. However, KrasG12D activation in Tff2+ cells leads to survival and clonal expansion following pancreatitis and a cancer stem/progenitor cell-like state. Selective ablation of Tff2+ cells prior to KrasG12D activation in Mist1+ acinar or Dclk1+ FP cells results in enhanced tumorigenesis, which can be partially rescued by adenoviral Tff2 treatment. Together, Tff2 defines a pancreatic TAP population that protects against Kras-driven carcinogenesis.

Heterogeneity in quiescent Müller glia in the uninjured zebrafish retina drive differential responses following photoreceptor ablation.

Loss of neurons in the neural retina is a leading cause of vision loss. While humans do not possess the capacity for retinal regeneration, zebrafish can achieve this through activation of resident Müller glia. Remarkably, despite the presence of Müller glia in humans and other mammalian vertebrates, these cells lack an intrinsic ability to contribute to regeneration. Upon activation, zebrafish Müller glia can adopt a stem cell-like state, undergo proliferation and generate new neurons. However, the underlying molecular mechanisms of this activation subsequent retinal regeneration remains unclear. To address this, we performed single-cell RNA sequencing (scRNA-seq) and report remarkable heterogeneity in gene expression within quiescent Müller glia across distinct dorsal, central and ventral retina pools of such cells. Next, we utilized a genetically driven, chemically inducible nitroreductase approach to study Müller glia activation following selective ablation of three distinct photoreceptor subtypes: long wavelength sensitive cones, short wavelength sensitive cones, and rods. There, our data revealed that a region-specific bias in activation of Müller glia exists in the zebrafish retina, and this is independent of the distribution of the ablated cell type across retinal regions. Notably, gene ontology analysis revealed that injury-responsive dorsal and central Müller glia express genes related to dorsal/ventral pattern formation, growth factor activity, and regulation of developmental process. Through scRNA-seq analysis, we identify a shared genetic program underlying initial Müller glia activation and cell cycle entry, followed by differences that drive the fate of regenerating neurons. We observed an initial expression of AP-1 and injury-responsive transcription factors, followed by genes involved in Notch signaling, ribosome biogenesis and gliogenesis, and finally expression of cell cycle, chromatin remodeling and microtubule-associated genes. Taken together, our findings document the regional specificity of gene expression within quiescent Müller glia and demonstrate unique Müller glia activation and regeneration features following neural ablation. These findings will improve our understanding of the molecular pathways relevant to neural regeneration in the retina.

In this Issue.

In this Issue.

ATRT-04. TARGETING THE COREST COMPLEX HELPS REPROGRAM AT/RT’S ABNORMAL EPIGENETIC LANDSCAPE TOWARD A DIFFERENTIATION PHENOTYPE

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are deadly infantile brain tumors. Their aggressive phenotype results from a single recurring biallelic loss-of-function mutation in genes encoding components of the SWI/SNF chromatin-remodeling complex - mainly SMARCB1 and SMARCA4 in remaining tumors. Residual SWI/SNF activity continues to inhibit EZH2 methyltransferase except at promoter regions colocalized to REST, which associates with neuronal differentiation and tumor suppressor genes. At these sites, EZH2 increases repressive H3K27me3 marks, helping maintain AT/RT in a stem cell-like state, and driving its aggressive growth and therapy resistance. We study the consequences of disrupting SWI/SNF inhibition at sites of EZH2/REST co-localization to target AT/RT precisely. The repressive action of REST is carried out mainly by the CoREST complex, consisting of LSD1, HDAC1/2 and RCOR. Lentiviral knockdown of RCOR2 reversed AT/RT’s stem cell-like state and induced differentiation along the glial and neuronal lineage. A decrease in the H3K27Me3 further confirmed the absence of COREST complex assembly at these sites. We used a bifunctional LSD1 and HDAC1/2 inhibitor, Corin, to pharmacologically target the CoREST complex in AT/RT cells. Corin also induced neuronal differentiation in AT/RT with increased expression of neuronal differentiation markers and decreased expression of stem cell markers. Further, Corin reduced AT/RT cell viability and proliferation (p<0.05) and increased apoptosis (p<0.05). In mice bearing orthotopic CHLA06 tumors, convection-enhanced delivery of a single dose of Corin increased H3K27ac, demonstrating that Corin engaged its target in vivo and induced apoptosis (western blot, cPARP). These studies demonstrate that targeting the CoREST complex disrupts AT/RT epigenetic abnormalities, reversing their stem cell-like state, slowing tumor cell growth, and inducing apoptosis. Targeting the CoREST complex with Corin is a promising new strategy that may translate into the clinical setting to help improve AT/RT’s dismal survival.

DIPG-49. A SYSTEMS BIOLOGY APPROACH TO DEFINING AND TARGETING CELL STATE-SPECIFIC MASTER REGULATOR DEPENDENCIES IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse Midline Glioma (DMG) are fatal pediatric brain tumors. We leveraged network-based methodologies to dissect the heterogeneity of DMG tumors and to discover Master Regulator (MR) proteins representing pharmacologically accessible, mechanistic determinants of molecularly distinct cell states. We produced a DMG regulatory network from 122 publicly available RNAseq profiles with ARACNe, and inferred sample-specific MR protein activity with VIPER. A CRISPR/Cas9 KO screen across 3 DMG patient cell lines identified a set of 73/77 essential genes that were enriched in the MR signature of 80% of patient samples (GSEA p=0.000034). FOXM1 emerged as an essential MR, significantly activated across virtually all patients. We then generated RNAseq profiles following perturbation with ~300 oncology drugs in 2 DMG cell lines most representative of patient MR signatures, and used this to identify drugs that invert patient MR activity profiles using the NYS/CA Dept.of Health approved OncoTreat algorithm. OncoTreat predicted sensitivity to HDAC, MEK, CDK, PI3K, and proteosome inhibitors in subsets of patients. 80%of OncoTreat-predicted drugs (p<10-5) from 3 DMG patient tumor biopsies showed in vitro sensitivity in cultured tumor cells from the respective patients, with overall 68% accuracy among 223 drugs evaluated by both OncoTreat and in vitro (Fisher’s Exact Test p=0.0449). Further analysis of DMG intra-tumor heterogeneity via protein activity inference from published scRNAseq profiles identified 6 tumor clusters with unique MR signatures representing distinct cellular states. Targetable MRs and OncoTreat-predicted drugs were distinct between these states. Bulk RNAseq analysis recapitulated predictions seen in the more prevalent Oligodendrocyte progenitor cell-like states, but failed to capture MR and drug predictions for the Astrocyte-like states. Ongoing validations of cell state-specific drug predictions in vivo in subcutaneous patient-derived xenograft and orthotopic syngeneic DMG models have already shown tumor volume and subpopulation differences (e.g. Trametinib-treated). This provides a platform to nominate much-needed novel drugs to treat DMG.

DIPG-19. BAF COMPLEX PERTURBATION AS A NOVEL THERAPEUTIC OPPORTUNITY IN H3K27M PEDIATRIC GLIOMA

Abstract Epigenetic dysregulation resulting in stalled development plays a crucial role in pediatric cancer tumorigenesis. Diffuse midline gliomas (DMG) are universally fatal pediatric brain cancers refractory to standard of care treatment modalities. These malignancies are driven by heterozygous mutations in genes encoding histone 3 (H3K27M) which create an aberrant epigenetic landscape that keeps glioma cells in an undifferentiated stem-like state. Consequently, targeting epigenetic regulators to restore the epigenome and force glioma cells to exit this stem-like cell state represents a promising new therapeutic strategy for H3K27M-DMG. To interrogate for epigenetic dependencies, we performed a CRISPR/Cas9 inactivation screen in patient-derived H3K27M-DMG neurospheres using an epigenetically focused sgRNA library and identified several core components of the mammalian BAF (SWI/SNF) chromatin remodeling complex as genetic vulnerabilities. Validation assays revealed that knockout of the BAF catalytic subunit BRG1 results in decreased glioma cell proliferation and tumor growth in orthotopic mouse models. Mechanistically, genome wide localization and DNA accessibility studies combined with regulatory network analysis demonstrated that BRG1 controls the transcription factor and enhancer landscapes that maintain H3K27M-DMG cells in a cycling, oligodendrocyte precursor cell-like state. Single cell transcriptome analysis in vitro and immunofluorescence studies in vivo confirmed that genetic perturbation of this chromatin remodeler promotes progression of differentiation along the astrocytic lineage. Similarly, pharmacological suppression of BRG1 activity, using both catalytic inhibitors as well as recently developed degraders, opposes tumor cell proliferation, stimulates cell state transition, and improves overall survival of patient-derived xenograft models. Interestingly, these effects seem to be restricted to H3K27M mutant glioma, as H3 wildtype glioma cells were less sensitive to BRG1 inhibition both in vitro and in vivo. In summary, we demonstrate that the BAF complex contributes to the maintenance of glioma cells in a proliferative stem-like state and that its therapeutic inhibition has translational potential for children bearing H3K27M-DMG.