Doxycycline-inducible Promoter Research Articles

Co-regulatory Proteins Integrate Metabolic Signals to Regulate ESRRG Transcriptional Transactivation in Stomach Parietal Cell Differentiation

Acid-producing stomach parietal cells (PC) are metabolically active and energy demanding cells. Aberrant PC function and census leads to complications ranging from peptic ulcers to stomach adenocarcinoma. Effcient energy expenditure and catabolic metabolism are required for normal PC differentiation from progenitor cells. Here, our data show that the master metabolic transcriptional factor, estrogen-related receptor gamma ( ESRRG), governs murine and human PC differentiation. We are further investigating how known metabolically controlled co-regulatory proteins — Sirtuin 1 (SIRT1), Nuclear Receptor Subfamily 0 Group B Member 2 (NR0B2), and CREB/ATF BZIP Transcription Factor (CREBZF, or SMILE) — integrate stimuli to enable ESRRG transcriptional activation during PC emergence from precursors. Specifically, our lab has developed an acute and reversible PC ablation model to monitor PC recovery in which Simian diphtheria toxin receptor (DTR) expression under the Atp4b promoter targets PC-specific ablation by diphtheria toxin (DT). Additionally, we employ high-dose tamoxifen (HD TAM), which causes a more general gastric injury (independent of tamoxifen effects on estrogen) from PC death along with reactive spasmolytic polypeptide expressing metaplasia (SPEM) of remaining cells. Immunofluorescence, western blotting, and qRT-PCR revealed SIRT1 is upregulated before emergence of early PC clones post-DT and decreases later, suggesting positive regulation of early PC lineage-commitment from stem cells. During the same early time frame, factors NR0B2 and CREBZF decreased, suggesting contrasting roles. Administration of Resv to mice accelerated PC recovery after injury and expanded PCs even at homeostasis. ChIP and ongoing ChIP-seq and Cut&Run assays show ESRRG binds cis-regulatory elements of the metabolic genes and the co-regulatory proteins to promote PC lineage commitment in both mice and in a human gastric cell line in which stable ESRRG-GFP is expressed under a doxycycline-inducible promoter. In other ongoing work, we are generating mice with PC-specific Sirt1/ Nr0b2 ablation to further dissect PC regeneration dynamics and conducting gastric organoid experiments with NR0B2/SIRT1 pharmacological modulators to monitor PC differentiation effects. These organoid samples will be further subjected to epigenetic analysis (Cut&Run, ATACseq) to study chromatin landscape changes. We conclude that cellular metabolic integration by co-regulators enables ESRRG-mediated transactivation during PC differentiation, and SIRT1 promotes early PC lineage-commitment that might offer a promising pro-PC differentiation therapeutic strategy. This work is supported by funding from the NIH through the National Institute of Diabetes and Digestive and Kidney Diseases and the National Cancer Institute. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Elevated α-synuclein levels inhibit mitophagic flux

The pathogenic effect of SNCA gene multiplications indicates that elevation of wild-type α-synuclein levels is sufficient to cause Parkinson’s disease (PD). Mitochondria have been proposed to be a major target of α-synuclein-induced damage. PINK1/parkin/DJ-1-mediated mitophagy is a defense strategy that allows cells to selectively eliminate severely damaged mitochondria. Here, we quantified mitophagic flux and non-mitochondrial autophagic flux in three models of increased α-synuclein expression: 1/Drosophila melanogaster that transgenically express human wild-type and mutant α-synuclein in flight muscle; 2/human skin fibroblasts transfected with α-synuclein or β-synuclein; and 3/human induced pluripotent stem cell (iPSC)-derived neurons carrying an extra copy of wild-type SNCA under control of a doxycycline-inducible promoter, allowing titratable α-synuclein upregulation. In each model, elevated α-synuclein levels potently suppressed mitophagic flux, while non-mitochondrial autophagy was preserved. In human neurons, a twofold increase in wild-type α-synuclein was already sufficient to induce this effect. PINK1 and parkin activation and mitochondrial translocation of DJ-1 after mitochondrial depolarization were not affected by α-synuclein upregulation. Overexpression of the actin-severing protein cofilin or treatment with CK666, an inhibitor of the actin-related protein 2/3 (Arp2/3) complex, rescued mitophagy in neurons with increased α-synuclein, suggesting that excessive actin network stabilization mediated the mitophagy defect. In conclusion, elevated α-synuclein levels inhibit mitophagic flux. Disruption of actin dynamics may play a key role in this effect.

Generation of three isogenic gene-edited Huntington’s disease human embryonic stem cell lines with DOX-inducible NGN2 expression cassette in the AAVS1 safe locus

Neurogenin 2 (NGN2), a neuronal transcription factor, can expedite differentiation of stem cells into mature glutamatergic neurons. We have utilized an allelic series of previously published and characterized isogenic Huntington's disease (IsoHD) human embryonic stem cell lines (Ooi et al., 2019), carrying different CAG repeat lengths in the first exon of the huntingtin gene. These IsoHDs were modified using CRISPR/Cas9 to insert NGN2 under the TET-ON doxycycline inducible promoter. The resulting IsoHD-NGN2 cell lines retained pluripotency in the absence of doxycycline (DOX), and via addition of DOX to the culturing media differentiation to neurons was achieved within 14 days.

Abstract 2111: Impact of HER2 Expression on trastuzumab deruxtecan(T-DXd) efficacy

Abstract Background: T-DXd is an FDA-approved anti-HER2 Antibody Drug Conjugate (ADC), which has shown clinical efficacy in breast cancers with either overexpression or low-level expression of HER2. The benefit of T-DXd across the spectrum of HER2 expression has called into question the relevance of HER2 expression level as relevant parameter for T-DXd sensitivity. We sought to experimentally address the relevance of HER2 expression level upon the degree of T-DXd response. Methods: A series of isogenic models was generated that expressed different levels of surface HER2 using a doxycycline-inducible promoter. These were analyzed for impact of differential expression level upon T-DXd internalization and drug response. Human breast cancer samples were analyzed for HER2 expression levels and association with clinical response to T-DXd under an IRB approved protocol. Results: Induction of different levels of HER2 at specified doxycycline concentrations was verified in multiple models using immunoblotting and FACS. These different levels appeared to correspond to clinical breast cancer immunohistochemistry (IHC) scoring levels to 0, 1+, 2+, and 3+. The models were analyzed for T-DXd response and showed increasing levels of HER2 led to increase lysosomal uptake of T-DXd (p<0.0001). Moreover, elevation of HER2 levels led to lower concentrations of T-DXd needed to suppress tumor growth. Analyses of the effects of increasing levels of HER2 upon xenograft response to T-DXd are ongoing. Finally, retrospective analyses of a cohort of 50 breast cancer patients treated with T-DXd revealed that higher levels of HER2 corresponded to longer time on treatment (time to progression). Conclusion: We observe a consistent relationship between elevated expression of HER2 and breast cancer response to T-DXd. The findings highlight the relevance of HER2 expression level as a biomarker of T-DXd response and support the potential value of therapeutic strategies that can induce HER2 surface expression levels to enhance response. Citation Format: Wanyi Chen, Joshua Drago, Avantika Gupta, Bo Liu, Sarat Chandarlapaty. Impact of HER2 Expression on trastuzumab deruxtecan(T-DXd) efficacy [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 2111.

Postnatal Expression of Kitl Affects Pigmentation of the Epidermis

KITL signaling is important for melanocyte development in mammals; however, its function in the melanocyte stem cells in adult skin is not well-understood. In this study, we have generated genetically modified mice that express a Kitl transgene under the control of a doxycycline-inducible promoter to investigate the impact of its overexpression in embryo, young postnatal, and adult skin with intact hair follicles. We report that overexpression of KITL influences the proliferation and differentiation of melanocytes as well as the self-renewal capacity of resident melanocyte stem cells within the follicular niche. Notably, activation of Kit-KITL signaling induced the migration of melanocytes from hair follicles to the epidermis. In addition, we demonstrate that a single pulse of Kitl transgene expression in postnatal mice results in long-lasting effects on melanocyte stem cells and their differentiated progeny as pigmented skin cells that persist through adulthood. Our findings indicate that regulation of KITL signaling in melanocyte lineage is crucial for melanocyte stem cell homeostasis and melanocyte cell differentiation in postnatal and adult mice.

Promoting the Differentiation of Neural Progenitor Cells into Oligodendrocytes through the Induction of Olig2 Expression: A Transcriptomic Study Using RNA-seq Analysis.

Oligodendrocytes are the myelinating cells of the central nervous system that facilitate efficient signal transduction. The loss of these cells and the associated myelin sheath can lead to profound functional deficits. Moreover, oligodendrocytes also play key roles in mediating glial-neuronal interactions, which further speaks to their importance in health and disease. Neural progenitor cells (NPCs) are a promising source of cells for the treatment of oligodendrocyte-related neurological diseases due to their ability to differentiate into a variety of cell types, including oligodendrocytes. However, the efficiency of oligodendrocyte differentiation is often low. In this study, we induced the expression of the Olig2 transcription factor in tripotent NPCs using a doxycycline-inducible promoter, such that the extent of oligodendrocyte differentiation could be carefully regulated. We characterized the differentiation profile and the transcriptome of these inducible oligodendrogenic NPCs (ioNPCs) using a combination of qRT-PCR, immunocytochemistry and RNA sequencing with gene ontology (GO) and gene set enrichment analysis (GSEA). Our results show that the ioNPCs differentiated into a significantly greater proportion of oligodendrocytes than the NPCs. The induction of Olig2 expression was also associated with the upregulation of genes involved in oligodendrocyte development and function, as well as the downregulation of genes involved in other cell lineages. The GO and GSEA analyses further corroborated the oligodendrocyte specification of the ioNPCs.

Level of constitutively expressed BMAL1 affects the robustness of circadian oscillations

The circadian rhythm is a biological oscillation of physiological activities with a period of approximately 24 h, that is driven by a cell-autonomous oscillator called the circadian clock. The current model of the mammalian circadian clock is based on a transcriptional-translational negative feedback loop in which the protein products of clock genes accumulate in a circadian manner and repress their own transcription. However, several studies have revealed that constitutively expressed clock genes can maintain circadian oscillations. To understand the underlying mechanism, we expressed Bmal1 in Bmal1-disrupted cells using a doxycycline-inducible promoter and monitored Bmal1 and Per2 promoter activity using luciferase reporters. Although the levels of BMAL1 and other clock proteins, REV-ERBα and CLOCK, showed no obvious rhythmicity, robust circadian oscillation in Bmal1 and Per2 promoter activities with the correct phase relationship was observed, which proceeded in a doxycycline-concentration-dependent manner. We applied transient response analysis to the Bmal1 promoter activity in the presence of various doxycycline concentrations. Based on the obtained transfer functions, we suggest that, at least in our experimental system, BMAL1 is not directly involved in the oscillatory process, but modulates the oscillation robustness by regulating basal clock gene promoter activity.

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells.

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

Adenovirus Co-Opts Neutrophilic Inflammation to Enhance Transduction of Epithelial Cells.

Human adenoviruses (HAdV) cause a variety of infections in human hosts, from self-limited upper respiratory tract infections in otherwise healthy people to fulminant pneumonia and death in immunocompromised patients. Many HAdV enter polarized epithelial cells by using the primary receptor, the Coxsackievirus and adenovirus receptor (CAR). Recently published data demonstrate that a potent neutrophil (PMN) chemoattractant, interleukin-8 (IL-8), stimulates airway epithelial cells to increase expression of the apical isoform of CAR (CAREx8), which results in increased epithelial HAdV type 5 (HAdV5) infection. However, the mechanism for PMN-enhanced epithelial HAdV5 transduction remains unclear. In this manuscript, the molecular mechanisms behind PMN mediated enhancement of epithelial HAdV5 transduction are characterized using an MDCK cell line that stably expresses human CAREx8 under a doxycycline inducible promoter (MDCK-CAREx8 cells). Contrary to our hypothesis, PMN exposure does not enhance HAdV5 entry by increasing CAREx8 expression nor through activation of non-specific epithelial endocytic pathways. Instead, PMN serine proteases are responsible for PMN-mediated enhancement of HAdV5 transduction in MDCK-CAREx8 cells. This is evidenced by reduced transduction upon inhibition of PMN serine proteases and increased transduction upon exposure to exogenous human neutrophil elastase (HNE). Furthermore, HNE exposure activates epithelial autophagic flux, which, even when triggered through other mechanisms, results in a similar enhancement of epithelial HAdV5 transduction. Inhibition of F-actin with cytochalasin D partially attenuates PMN mediated enhancement of HAdV transduction. Taken together, these findings suggest that HAdV5 can leverage innate immune responses to establish infections.

Inhibition of PINK1-Dependent Mitochondrial Quality Control Pathways Induces Senescence of Acute Myeloid Leukemia Stem Cells

Current treatments for acute myeloid leukemia (AML) are often ineffective in eliminating leukemic stem cells (LSCs), which perpetuate the disease. Novel therapies that target LSCs have the potential to improve clinical outcomes. An important step towards achieving this goal is identifying the fundamental processes that regulate cell fate decisions in LSCs. Perturbation of these processes may impair LSC activity and form the basis of novel therapies.Here, we investigated the role of mitochondrial quality control in the regulation of LSCs by inhibiting PTEN-induced kinase 1 (PINK1). PINK1 is a serine/threonine kinase that serves as a critical sensor of mitochondrial damage and is at the apex of multiple quality control pathways that maintain mitochondrial homeostasis. Damage to mitochondria results in activation of PINK1 on the outer mitochondrial membrane, which in turn triggers mitochondrial fission and mitophagy.We first determined if the overall quality of the mitochondrial pool differs between LSCs and their more differentiated progenies (henceforth termed “non-LSCs”) in two human AML cultures known as OCI-AML-8227 and OCI-AML-21. In both cultures, LSCs assayed by xenotransplantation are found only in the CD34 +CD38 -fraction and not in the CD34 +CD38 + and CD34 - fractions. We monitored the turnover rate of the mitochondrial pool in the different fractions by expressing MitoTimer, a mitochondria-targeted fluorescent protein that matures from green to red fluorescence over 48 hours. A high ratio of green to red fluorescence is indicative of active mitochondrial protein turnover and high overall mitochondrial quality. In both cell lines, the fluorescence ratio was highest in the CD34 +CD38 - fraction compared with the other fractions, suggesting that LSCs maintain a higher quality pool of mitochondria than non-LSCs.Based on the above findings, we hypothesized that PINK1-dependent mitochondrial quality control mechanisms are involved in the regulation of LSC fate. To test this hypothesis, we silenced the expression of PINK1 in OCI-AML-8227 cells using lentiviral vectors expressing validated shRNAs under a doxycycline-inducible promoter. Depletion of PINK1 shifted the MitoTimer fluorescence from green to red, reduced oxygen consumption rate, and disrupted mitochondrial ultrastructure in all cell fractions, consistent with a reduction in mitochondrial quality. Unexpectedly, PINK1 downregulation resulted in a block in differentiation and cell cycle arrest at G1/G0 phase in CD34 +CD38 - cells. Re-expression of PINK1 was unable to reverse the cell cycle arrest, suggestive of a state of cellular senescence. Indeed, we observed other hallmarks of senescence including an increase in p21 WAF1 and p16 INK4a expression and SA-β-gal activity. To determine the mechanism by which PINK1 depletion causes senescence, we performed RNA sequencing analysis of sorted CD34 +CD38 - cells expressing PINK1 shRNAs. This analysis revealed a marked decrease in the expression of MYC target genes, with TERT (Telomerase Reverse Transcriptase) being the most downregulated gene. Consistent with the role of TERT in telomere maintenance and telomere shortening as a trigger of senescence, we found that PINK1 knockdown decreased telomere length in CD34 +CD38 - cells, and overexpression of TERT effectively rescued the senescence phenotype. These findings collectively indicate that inhibition of PINK1-dependent mitochondrial quality control pathways induces senescence of LSCs through telomere shortening.To determine whether these changes translated to a reduction in functional LSC activity, we performed colony forming unit assays and serial xenotransplantation assays in immunodeficient NSG mice. Depletion of PINK1reduced the colony forming capacity and engraftment potential of 3 primary AML samples in primary and secondary recipients. Importantly, PINK1 depletion had minimal impact on the colony forming capacity and engraftment potential of normal CD34 + hematopoietic stem and progenitor cells (HSPCs) derived from cord blood, suggestive of a therapeutic window in vivo.In summary, our results demonstrate that mitochondrial quality control pathways regulate cell fate decision in LSCs. Inhibition of PINK1 activity impairs LSC activity by inducing senescence, while sparing normal HSPCs. Our findings provide the basis for exploring PINK1 as a therapeutic target against LSCs in AML. DisclosuresChan: AbbVie: Research Funding; BMS: Research Funding.

Divisional Memory Drives Hematopoietic Stem Cell Functional Diversity

Hematopoietic stem cells are endowed with high regenerative potential but their actual self-renewal capacity is limited. Studies using the H2B-retention labeling system show HSC functional decline at each round of division (Qiu, Stem Cell Reports 2014). We have shown that mitochondria drive HSC functional decline with division history after transplantation (Cell Stem Cell 2020). Here we examined the link between mitochondrial metabolism, in vivo division at steady state, and HSC functions using the GFP label-Histone 2B (GFP-H2B) mouse model driven by a doxycycline-inducible promoter. Five months after doxycycline removal, mitochondrial membrane potential (MMP) was examined using TMRE in HSC with varying GFP intensity. HSC were separated into an H2B-labeled retention population and an H2B-labeled population. Interestingly, within the H2B-labeled retention population, HSC could be further subdivided into GFP high, medium, and low. MMP increased in a stepwise fashion with GFP dilution in HSC. We noted the presence of 2 TMRE peaks within each GFP high and medium populations leading to 5 populations: GFP-high;MMP-low (G1), GFP-high;MMP-high (G2), GFP-medium;MMP-low (G3), GFP-medium;MMP-high (G4), GFP-low;MMP-high (G5). We examined the repopulation activity of each population in a serial competitive transplant assay. G1 and G2 maintained higher peripheral blood chimerism up to 24 weeks post-transplant than G3 and G4. G5 did not engraft at all. However, only G1 reconstituted high frequency of HSC in primary recipients. In secondary recipients, G1, G2, G3 but not G4 gave rise to positive engraftment. Interestingly, G1 and G2 grafts showed myeloid/lymphoid balanced engraftment whereas the G3 graft was myeloid-bias, suggesting that myeloid skewing can be acquired upon HSC division. We further examined lineage fate maps of bone marrow cells derived from G1 or G3 population in vivo, using single cell RNA sequencing, 10X genomics. Surprisingly, G3-derived bone marrow cells displayed a distinct myeloid cell trajectory from G1-derived bone marrow cells, in which G3 gave rise to increased immature neutrophils but fewer myeloid precursors. Remarkably, each lineage population derived from G3 donor cells had different gene expression signatures than those derived from G1 donor cells. Therefore, HSC that have divided in vivo in the same bone marrow microenvironment are intrinsically and molecularly different such that not only do they exhibit lineage potential differences but they also produce progeny that are transcriptionally different. These findings imply that cellular division rewires HSC and that this rewiring is passed down to their fully differentiated progeny. When G1 and G3 single HSC were cultured in-vitro, G1 had a slower entry into cell-cycle which has been associated with increased stemness. Additionally, when single HSC from G1 and G3 were assessed for their multipotency in a lineage differentiation assay, G1 HSC had a higher propensity to produce all four myeloid lineages (megakaryocytes, neutrophils, macrophages, and erythroid), further supporting increased stemness in G1 compared to G3 HSC. Finally, HSC from G1, G2, G3 and G4 populations carried mitochondria that were morphologically different, and express distinct levels of Sca-1, CD34 and EPCR, with Sca-1 high, CD34-, EPCR+ cells more enriched in G1. In summary, this study suggests that HSC transition into distinct metabolic and functional states with division history that may contribute to HSC diversity and functional heterogeneity. It also suggests the existence of a cell-autonomous mechanism that confers HSC divisional memory to actively drive HSC functional heterogeneity and aging. DisclosuresNo relevant conflicts of interest to declare.

Activation of soluble AC10 induces cytoskeletal reorganisation

The cellular monolayer of the pulmonary endothelium forms a semi-selective barrier intersecting the vascular space, the underlying interstitium, and adjacent airspace. This endothelial barrier maintains tissue homeostasis critically regulating vessel permeability. Endothelial cAMP signals regulate endothelial permeability; however, cAMP signals are highly compartmentalized, such that cAMP signals generated by transmembrane adenylyl cyclase protect the barrier, while cAMP signals generated by soluble AC disrupt the barrier. Indeed, bicarbonate stimulation of soluble AC10 disrupts the endothelial barrier increasing permeability and is required for LPS-induced lung edema. Yet, how AC10 cAMP signals coordinate endothelial barrier regulation is unclear. The endothelial cytoskeleton alters cell shape which in turn regulates endothelial permeability. Thus, we hypothesized that bicarbonate stimulation of AC10 induces cytoskeletal remodeling. PMVECs were engineered to overexpress AC10 fused to a C-terminus hemagglutinin tag (HA). Gene and protein expression were controlled using a two-tier gene expression approach in which the doxycycline inducible promoter couples with a protein destabilizing variant of FKBP12 fused to the AC10-HA construct rendering the protein unstable in the absence of Shield1. In the presence of doxycycline and Shield1, AC10-HA protein began to appear 4-hours after induction and was absent in control cells. In this system, following addition of doxycycline and Shield1 and in the presence of elevated bicarbonate, whole cell cAMP increased over the same time course as protein expression while control cells showed no additional increase above normal bicarbonate stimulation. As cAMP increases above controls, endothelial cells begin to retract inducing inter-endothelial gaps between adjacent cells. This increase in inter-endothelial gaps was consistent with decreased resistance across the endothelial monolayer. To further address cytoskeletal mediated changes during AC10-induced endothelial barrier disruption, we examined actin cytoskeleton dynamics. Significant loss of the cortical actin rim and formation of actin stress fibers progress to complete actin cytoskeletal disruption by 24 hours. Interestingly, over the same course as the restructuring of the actin cytoskeleton, we detected phosphorylation of actin binding proteins, filamin and VASP, at PKA mediated sites. Thus, it appears that AC10 mediated pulmonary endothelial barrier disruption is coupled to actin remodeling.

Inhibition of Human Uracil DNA Glycosylase Sensitizes a Large Fraction of Colorectal Cancer Cells to 5-Fluorodeoxyuridine and Raltitrexed but Not Fluorouracil.

Previous short-hairpin RNA knockdown studies have established that depletion of human uracil DNA glycosylase (hUNG) sensitizes some cell lines to 5-fluorodeoxyuridine (FdU). Here, we selectively inhibit the catalytic activity of hUNG by lentiviral transduction of uracil DNA glycosylase inhibitor protein into a large panel of cancer cell lines under control of a doxycycline-inducible promoter. This induced inhibition strategy better assesses the therapeutic potential of small-molecule targeting of hUNG. In total, 6 of 11 colorectal lines showed 6- to 70-fold increases in FdU potency upon hUNG inhibition ("responsive"). This hUNG-dependent response was not observed with fluorouracil (FU), indicating that FU does not operate through the same DNA repair mechanism as FdU in vitro. Potency of the thymidylate synthase inhibitor raltitrexed (RTX), which elevates deoxyuridine triphosphate levels, was only incrementally enhanced upon hUNG inhibition (<40%), suggesting that responsiveness is associated with incorporation and persistence of FdU in DNA rather than deoxyuridine. The importance of FU/A and FU/G lesions in the toxicity of FdU is supported by the observation that dT supplementation completely rescued the toxic effects of U/A lesions resulting from RTX, but dT only increased the IC50 for FdU, which forms both FU/A and FU/G mismatches. Contrary to previous reports, cellular responsiveness to hUNG inhibition did not correlate with p53 status or thymine DNA glycosylase expression. A model is suggested in which the persistence of FU/A and FU/G base pairs in the absence of hUNG activity elicits an apoptotic DNA damage response in both responsive and nonresponsive colorectal lines. SIGNIFICANCE STATEMENT: The pyrimidine base 5-fluorouracil is a mainstay chemotherapeutic for treatment of advanced colorectal cancer. Here, this study shows that its deoxynucleoside form, 5-fluorodeoxyuridine (FdU), operates by a distinct DNA incorporation mechanism that is strongly potentiated by inhibition of the DNA repair enzyme human uracil DNA glycosylase. The hUNG-dependent mechanism was present in over 50% of colorectal cell lines tested, suggesting that a significant fraction of human cancers may be sensitized to FdU in the presence of a small-molecule hUNG inhibitor.

Inhibition of Critical DNA Dioxygenase Activity in IDH1/2 Mutant Myeloid Neoplasms

Neomorphic mutations in IDH1/2 producing R-2-Hydroxyglutrate (R-2HG), are common in myeloid malignancies and in various solid cancers. A diffuse hypermethylated status is the biological consequence of the R-2HG-mediated inhibition of several α-ketoglutarate (αKG)-dependent enzymes including DNA dioxygenases TET1, TET2 and TET3.1,2 Specifically, the inhibition of TET2, either induced by the interaction with R-2HG or by direct genomic silencing (as in case of TET2 loss of function mutations) is responsible for the block of the DNA cytosine demethylation pathway, inducing changes in expression patterns, (e.g. decreasing expression of tumor suppressor genes) and impairing execution of differentiation programs. Analysis of genomic data from a Cleveland Clinic (CCF) cohort of AML/MDS patients combined in a meta-analytic fashion with BeatAML3 and Tumor Cancer Genome Atlas (TCGA) cohorts (1119 profiled patients) showed that IDH1/2 mutations are mutually exclusive (only 3% [N=4/106] of AML IDH1/2 mutated cases had TET2 mutations, expected to be at a frequency of 18% [N=110/585] in IDH1/2 wild type cases, p=.000125). In this scenario we suggest that the loss of TET2 activity due to mutations prevents the expansion of IDH1/2 mutant myeloid neoplasms (MNs) because of phenotypic redundancies inducing synthetic lethality. With this premise we stipulated that a critical level of DNA dioxygenase activity exists and thus cells with low TET2 activity will not tolerate further inhibition by R-2HG. Here we propose to apply pharmacologic inhibition of TET2 to produce an additive effect on DNA dioxygenases to investigate whether this will result in a synthetic lethality of IDH1/2 mutant cells. Specifically we hypothesize that TET-dioxygenase inhibition may be implemented as a possible therapeutic strategy in neomorphic IDH1/2 mutant MNs. To explore this hypothesis we conducted a series of in vitro experiments in different isogenic cell lines expressing either mutant or wild type IDH1 or IDH2, that were simultaneously mutant, wild type (WT) or knock down (KD) for TET2 (TF1-IDH2R140Q, K562-IDH1R132C both WT for TET2 gene, and K18-IDH1R132CTET2KD and SIGM5-IDH1R132C TET2MT, both with a doxycycline inducible promoter for mutant IDH1). First we found that the doxycycline induction of ectopic IDH1R132C expression led to R-2HG increase (~10,000-fold over the baseline) and induced cell death in TET2-deficient cells (experiments conducted in SIGM5-IDH1R132C cells showing 70% of decrease in cell growth after five days of IDH induction with doxycycline), confirming the cytotoxic effect of cellular R-2HG. We then tested in IDH1/2MT cells sensitivity towards TETi76, a specific TET inhibitor designed on R-2HG scaffold (with more than 200 fold potency compared to R-2HG in cell-free assays of 5-hydroxy-methyl cytosine [5hMC] production).4 This compound showed particular selectivity towards inhibition of DNA dioxygenases when a set of 23 other dioxygenase inhibitors were screened. Most importantly, consistent with our hypothesis, TETi76 preferentially inhibited the proliferation of IDH1/2MT cells either following doxycycline-induction both in TET2WTand TET2 deficient models (K562 TET2WT, K18 TET2kD, SIGM-5 TET2MT cell lines), or in models not carrying the inducible promoter (TF1 TETWT) (Growth inhibition: 20-25% in IDHWT vs 70-80% in IDHMT cell lines after 72h of co-culture with TETi76 treatment for concentrations ranging between 1 and 5 µM. P-value range: 0.04-0.001 in pairwise comparisons with untreated controls). Overall, our findings are consistent with the idea that neomorphic IDH1/2MT phenocopies loss of function TET2MT, through R-2HG, down-modulating pathways fundamental for cell homeostasis, division and differentiation. If a residual TET-activity is needed for the function of IDH1/2MT cells, the complete block of the residual activity appears to inevitably disrupt this phenotype impairing cell growth and proliferation. This is also in agreement with the paucity of TET3 and TET1 mutation in the context of TET2MT carriers. In summary, results shown here represent an important proof of concept that the increased inhibition of DNA dioxygenase activity, instead of being more leukemogenic, can be synthetically lethal. Our observations may have implications with regard to the therapy of IDH1/2 mutated neoplasms including AML and MDS Disclosures Saunthararajah: EpiDestiny: Consultancy, Current equity holder in private company, Patents &amp; Royalties: University of Illinois at Chicago. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Human shelterin protein POT1 prevents severe telomere instability induced by homology-directed DNA repair.

The evolutionarily conserved POT1 protein binds single-stranded G-rich telomeric DNA and has been implicated in contributing to telomeric DNA maintenance and the suppression of DNA damage checkpoint signaling. Here, we explore human POT1 function through genetics and proteomics, discovering that a complete absence of POT1 leads to severe telomere maintenance defects that had not been anticipated from previous depletion studies in human cells. Conditional deletion of POT1 in HEK293E cells gives rise to rapid telomere elongation and length heterogeneity, branched telomeric DNA structures, telomeric R-loops, and telomere fragility. We determine the telomeric proteome upon POT1-loss, implementing an improved telomeric chromatin isolation protocol. We identify a large set of proteins involved in nucleic acid metabolism that engage with telomeres upon POT1-loss. Inactivation of the homology-directed repair machinery suppresses POT1-loss-mediated telomeric DNA defects. Our results unravel as major function of human POT1 the suppression of telomere instability induced by homology-directed repair.

Abstract 1390: Inhibition of the catalytic activity of hUNG2 sensitizes multiple cancer cell lines to 5-FdUrd killing

Abstract Previously reported shRNA knockdown studies have established that expression of the DNA repair enzyme human uracil DNA glycosylase (hUNG2) is protective against 5-flourodeoxyuridine (5FdUrd) cell killing in p53 mutated cancer cell lines, but not in cell lines with functional p53. Here we take a different approach to understanding the role of hUNG2 in 5FdUrd chemotherapy by selectively inhibiting the catalytic activity of hUNG2 using the small bacterial inhibitor protein UGI. UGI binds extremely tightly to the DNA binding and catalytic site of hUNG2, effectively mimicking the impact of a small molecule inhibitor and allowing us to explore the potential therapeutic vulnerabilities that loss of hUNG2 activity produces in p53 mutated and wild-type cancer cells. Accordingly, we used lentiviral transduction to introduce UGI, under control of a doxycycline-inducible promoter, into a large panel of cancer cell lines. In vitro hUNG2 activity measurements using cell extracts established that all lines had robust activity before induction of UGI and undetectable activity after induction. We find that some wild-type and mutant p53 cancer lines from different tissues show significant increases in drug sensitivity. For instance, the p53 wild-type MCF7 breast cancer line showed a 10,000-fold increase in sensitivity, while the p53 mutant HT29 colorectal cancer line showed a 50-fold increased sensitivity. Overall, the results do not support the proposal that p53 status itself determines whether a cancer cell will be sensitized to 5FdUrd in the presence of hUNG2 inhibition. Ongoing studies are focused on identifying the cellular factors that contribute to drug sensitivity under conditions of hUNG2 inhibition. Realizing the full therapeutic benefit of 5FdUrd requires identification of the cancer cell phenotypes that are sensitized upon hUNG2 inhibition. Citation Format: Eric S. Christenson, Junru Cui, Anthony Gizzi, Mesfin Meshesha, James T. Stivers. Inhibition of the catalytic activity of hUNG2 sensitizes multiple cancer cell lines to 5-FdUrd killing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1390.

3055 – MITOCHONDRIAL METABOLISM LINKS HSC DIVISIONAL HISTORY AND CELL FATE

Hematopoietic stem cells are endowed with high regenerative potential but their actual self-renewal capacity is limited. Studies using the H2B-retention labeling system show HSC functional decline at each round of division (Qiu, Stem Cell Reports 2014). We showed mitochondria drive HSC functional decline with division history after transplantation (Cell Stem Cell 2020). Here we examined the link between mitochondrial metabolism, in vivo division at steady state and HSC functions using the GFP label-Histone 2B (GFP-H2B) mouse model driven by doxycycline-inducible promoter. Five months after doxycycline removal, mitochondrial membrane potential (MMP) was examined using TMRE in HSCs with different GFP intensity. HSC were subdivided into GFP high, medium and low. Interestingly, MMP increased in a stepwise fashion with GFP dilution in HSC. We noted the presence of 2 TMRE peaks within each GFP high and medium population leading to 5 populations: GFP-high;MMP-low (G1), GFP-high;MMP-high (G2), GFP-medium;MMP-low (G3), GFP-medium;MMP-high (G4), GFP-low;MMP-high (G5) and transplanted with competitors. G1 and G2 maintained higher peripheral blood chimerism up to 24 weeks post-transplant than G3 and G4. G5 did not engraft at all. However, only G1 reconstituted high frequency of HSC in primary recipients. In secondary recipients, G1, G2, G3 but not G4 gave rise to positive engraftment. Interestingly, G1 and G2 grafts were myeloid/lymphoid balanced engraftment whereas G3 graft was myeloid-bias graft. Further, HSC from G1, G2, G3 and G4 groups carry mitochondria that were morphologically different, exhibited distinct metabolomic profiles, proliferated and differentiated into mature lineages at different rate in vitro. These findings suggest that HSC transition into distinct metabolic and functional state with division history that may contribute to HSC cellular memory.

PHGDH supports liver ceramide synthesis and sustains lipid homeostasis

Backgroundd-3-phosphoglycerate dehydrogenase (PHGDH), which encodes the first enzyme in serine biosynthesis, is overexpressed in human cancers and has been proposed as a drug target. However, whether PHGDH is critical for the proliferation or homeostasis of tissues following the postnatal period is unknown.MethodsTo study PHGDH inhibition in adult animals, we developed a knock-in mouse model harboring a PHGDH shRNA under the control of a doxycycline-inducible promoter. With this model, PHGDH depletion can be globally induced in adult animals, while sparing the brain due to poor doxycycline delivery.ResultsWe found that PHGDH depletion is well tolerated, and no overt phenotypes were observed in multiple highly proliferative cell compartments. Further, despite detectable knockdown and impaired serine synthesis, liver and pancreatic functions were normal. Interestingly, diminished PHGDH expression reduced liver serine and ceramide levels without increasing the levels of deoxysphingolipids. Further, liver triacylglycerol profiles were altered, with an accumulation of longer chain, polyunsaturated tails upon PHGDH knockdown.ConclusionsThese results suggest that dietary serine is adequate to support the function of healthy, adult murine tissues, but PHGDH-derived serine supports liver ceramide synthesis and sustains general lipid homeostasis.

Abstract P6-10-08: AKT1 alterations following exposure to endocrine-based therapy in patients with hormone-receptor positive (HR+) metastatic breast cancer (MBC): Clinical and functional implications

Abstract Background: The AKT1 oncogene represents a central node in the well-characterized PI3K/AKT/mTOR signal transduction pathway involved in the regulation of cellular proliferation as well as therapeutic resistance. With the emergence of multiple targeted therapies for HR+ MBC, we sought to explore the prevalence of AKT1 alterations, the spectrum of co-occurring mutations, and any potential impact on response to endocrine-based therapy, including CDK 4/6 and PIK3CA inhibitors. Methods: Presence or absence of AKT1 mutation was interrogated via cell-free DNA (cfDNA) analysis (Guardant assay) among patients with HR+/HER2- MBC receiving treatment at the Massachusetts General Hospital who underwent routine clinical cfDNA testing. Genomic sequencing results were then compared to detailed clinical annotation based upon retrospective chart review. In the laboratory, HR+/HER2- T47D breast cancer cells were transfected with AKT1, under the control of a doxycycline-inducible promoter, to induce AKT1 overexpression. Sensitivity to escalating doses of fulvestrant and palbociclib were interrogated via cell-titer-glo viability assay in vitro. Results: Among 272 patients with HR+/HER2- MBC, we identified 13 patients with AKT1 mutation (4.7%). 12/13 (92%) patients had a canonical AKT1E17K alteration, one patient (8%) had an AKT1 E49K alteration. AKT1 mutations occurred concurrently with alterations in ESR1 (31%), PIK3CA (23%), TP53 (23%), ERBB2 (15%), and RB1 (8%). All patients had been exposed to some form of anti-estrogen prior to sequencing, either via (neo)adjuvant therapy and/or in the metastatic setting; 5/13 (38%) had exposure to a CDK4/6 inhibitor prior to cfDNA sequencing. Following the identification of an AKT1 alteration, examples of rapid clinical progression on fulvestrant, CDK4/6i, and/or everolimus were identified, however the presence of an AKT1 alteration was not sufficient to predict pan-resistance to these agents, as counter-examples of clinical benefit were also identified. In an index patient with an AKT1 mutation and prior progression on letrozole and palbociclib, subsequent treatment with fulvestrant and abemaciclib did not yield clinical benefit, highlighting the potential role of AKT1 in mediating clinical resistance to endocrine therapy and CDK4/6i. In the pre-clinical model, HR+/HER2- breast cancer cells demonstrated resistance to both fulvestrant and palbociclib following upregulation of exogenous AKT1 expression. Updated clinical data with additional patients and response to regimens incorporating an anti-estrogen, a CDK4/6 inhibitor, an mTOR inhibitor, and/or a PIK3CA inhibitor will be presented at the meeting. Conclusions: Activating AKT1 mutations can be identified via targeted sequencing of cfDNA in patients with HR+/HER2- MBC, and may play an important role in mediating resistance to anti-estrogens, CDK4/6 inhibitors, and other emerging targeted therapies. Heterogeneity in response to endocrine-based therapy following the identification of an AKT1 alteration may be related to tumor-extrinsic factors, the AKT1 allelic fraction, or cooperativity between multiple resistance drivers. Further research is needed to confirm these findings and guide optimal therapeutic sequencing strategies for patients with HR+/HER2- AKT1 mutant MBC. Citation Format: Seth Wander, Pingping Mao, Maxwell Lloyd, Kailey Kowalski, Gabriela N. Johnson, Giuliana Malvarosa, Beverly Moy, Leif W. Ellisen, John Iafrate, Nikhil Wagle, Aditya Bardia. AKT1 alterations following exposure to endocrine-based therapy in patients with hormone-receptor positive (HR+) metastatic breast cancer (MBC): Clinical and functional implications [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-08.

A human somatic cell culture system for modelling gene silencing by transcriptional interference

Transcriptional interference and transcription through regulatory elements (transcriptional read-through) are implicated in gene silencing and the establishment of DNA methylation. Transcriptional read-through is needed to seed DNA methylation at imprinted genes in the germ line and can lead to aberrant gene silencing by DNA methylation in human disease. To enable the study of parameters and factors influencing transcriptional interference and transcriptional read-through at human promoters, we established a somatic cell culture system. At two promoters of imprinted genes (UBE3A and SNRPN) and two promoters shown to be silenced by aberrant transcriptional read-through in human disease (MSH2 and HBA2) we tested, if transcriptional read-through is sufficient for gene repression and the acquisition of DNA methylation. Induction of transcriptional read-through from the doxycycline-inducible CMV promoter resulted in consistent repression of all downstream promoters, independent of promoter type and orientation. Repression was dependent on ongoing transcription, since withdrawal of induction resulted in reactivation. DNA methylation was not acquired at any of the promoters. Overexpression of DNMT3A and DNMT3L, factors needed for DNA methylation establishment in oocytes, was still not sufficient for the induction of DNA methylation. This indicates that induction of DNA methylation has more complex requirements than transcriptional read-through and the presence of de novo DNA methyltransferases.