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Week 5 In the Books!

Hello Dear Abstract Watchers!

 

Today I did manage to almost do an abstract a day, and then was flu-ish on Friday so finished 'em up today. This week, we got to read about

• how women & men metabolize sugar differently (which affects pathology),
• a new molecular mechanism of glioblastoma,
• a negative regulator of human T cell effector function & its potential in immunotherapy,
• the role of mRNA secondary structure in ribosome scanning and chosing the right ATG,
• a previously unknown mechanism that recycles the outer segments of photoreceptors which shed light on a disease-causing gene,
• a pioneer factor essential for reprogramming to totipotency, and
• the cellular mechanism of skin stem cell differentiation. 

 

 

Lots of good stuff here but I particularly loved Dr. Pleasantine Mill's preprint, using developmental biology to uncover a disease mechanism. 

 

Now I have to pick next week's abstracts! Please email or DM me with any suggestions! I'm not devoted to any particular theme because it's all interesting and because it's more fun to see themes emerge than to contrive them. 

 

Have a great week!

Ang

 

Abstract #28 Chronic intake of high dietary sucrose induces sexually dimorphic metabolic adaptations in mouse liver and adipose tissue from Dr. Joan Han's lab. Open access, preprint posted about 6 months earlier.

Abstract on Nature Communications: Almost all effective treatments for non-alcoholic fatty liver disease (NAFLD) involve reduction of adiposity, which suggests the metabolic axis between liver and adipose tissue is essential to NAFLD development. Since excessive dietary sugar intake may be an initiating factor for NAFLD, we have characterized the metabolic effects of liquid sucrose intake at concentrations relevant to typical human consumption in mice. We report that sucrose intake induces sexually dimorphic effects in liver, adipose tissue, and the microbiome; differences concordant with steatosis severity. We show that when steatosis is decoupled from impairments in insulin responsiveness, sex is a moderating factor that influences sucrose-driven lipid storage and the contribution of de novo fatty acid synthesis to the overall hepatic triglyceride pool. Our findings provide physiologic insight into how sex influences the regulation of adipose-liver crosstalk and highlight the importance of extrahepatic metabolism in the pathogenesis of diet-induced steatosis and NAFLD.

 

Abstract on biorxiv: identical

 

The authors found that hepatic triglyceride accumulation following sucrose intake was primarily driven by the re-esterification of fatty acids derived from adipocyte lipolysis in male mice, and by a combination of re-esterification and hepatic de novo fatty acid synthesis in female mice. In male mice, adipocyte lipolysis is necessary for the development of hepatic steatosis following chronic liquid sucrose intake, whereas in female mice, impairment of adipocyte lipolysis attenuates but does not completely prevent hepatic triglyceride accumulation. Compared to males, females showed a dramatic upregulation of enzymes involved in de novo fatty acid synthesis (Acetyl CoA) in liver in response to sugar. Also, under control & sucrose conditions, compared to males, female mice had 2-5x increased concentrations of all measured unconjugated and conjugated primary bile acids, two unconjugated secondary bile acids and two conjugated secondary bile acids. Sucrose intake led to significant changes in the microbiome in females but not males, which would be predicted to reduce amino acid metabolism and increase fructose and mannose metabolism as well as glycolysis and gluconeogenesis pathways in females following chronic sucrose intake. 

 

As they mention, "In silico modeling had suggested that NAFLD develops through distinct metabolic processes in males and females, and epidemiological data strongly support an increased prevalence of NAFLD in men, yet worsened NAFLD severity in women. High fructose, or high fat, high fructose diets, also lead to worse steatosis in female vs male rates. Women increase de novo fatty acid synthesis in response to acute fructose ingestion whereas men do not."

Male mice were able to convert excess dietary sucrose into fatty acids that were readily stored as triglyceride in the adipose tissue, whereas female mice were unable to expand their adipose tissue as effectively as males, leading to additional manufacturing (and storage) of new fatty acids in the liver. 

 

Abstract #29 PDGF signaling inhibits mitophagy in glioblastoma stem cells through N6-methyladenosine from Dr. Jeremy Rich's lab. Not open access, no preprint available. Abstract in Dev Cell: Dysregulated growth factor receptor pathways, RNA modifications, and metabolism each promote tumor heterogeneity. Here, we demonstrate that platelet-derived growth factor (PDGF) signaling induces N6-methyladenosine (m6A) accumulation in glioblastoma (GBM) stem cells to regulate mitophagy. PDGF ligands stimulate early growth response 1 (EGR1) transcription to induce methyltransferase-like 3 (METTL3) to promote GBM stem cell proliferation and self-renewal. Targeting the PDGF-METTL3 axis inhibits mitophagy by regulating m6A modification of optineurin. Forced optineurin expression phenocopies PDGF inhibition, and optineurin levels portend longer survival of GBM patients; these results suggest a tumor-suppressive role for optineurin. Pharmacologic targeting of METTL3 augments anti-tumor efficacy of PDGF receptor and mitophagy inhibitors in vitro and in vivo. Collectively, we define PDGF signaling as an upstream regulator of oncogenic m6A regulation, driving tumor metabolism to promote cancer stem cell maintenance, highlighting PDGF-METTL3-OPTN signaling as a GBM therapeutic target.

 

PDGF signaling promotes GBM protression - this is a mechanism. It induces METTL3, which leads to m6A modification of optineurin, triggering its degradation. Optineurin is an autophagy receptor that is important for clearing damaged mitochondria during mitophagy. Restoring optineurin levels recapitulates PDGF inhibition suggesting that this is the main mechanism, and it's relevant to patients: higher optineurin = longer survival. Pharmacological inhibition of METTL3 combined with PDGFr inhibitors showed additive effects.

 

Abstract #30 Internal checkpoint regulates T cell neoantigen reactivity and susceptibility to PD1 blockade from Dr. Nicholas Restifo's lab. Open access, and posted on biorxiv 1 month earlier. I had never noticed that the format of the abstracts is different on Med than on other Cell Press journals. Perhaps not surprisingly more like clinical journals:

Context and significance The inconsistent success of therapies based on tumor-infiltrating lymphocytes (TIL) recognizing “neoantigens” (tumor-specific targets) has been the focus of intense study. Authors from the National Cancer Institute and University of Minnesota describe a key role for the cytokine-induced SH2 protein (CISH) in regulating human T cell function and reactivity to neoantigens. To explore the functional and mechanistic basis of these findings, the authors developed an efficient, clinical-grade process to eliminate CISH in primary human TIL. CISH deficiency increased TIL function and neoantigen recognition. In a preclinical in vivo model, CISH knockout also increased tumor vulnerability to checkpoint inhibition (a type of cancer immunotherapy). This work served as the basis for a first-in-human clinical trial of CISH knockout TIL in patients with metastatic gastrointestinal cancers (why gastrointestinal?**).

 

Summary

Background Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit T cell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key (presumably negative?) regulator of T cell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human T cell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade.

Methods Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood T cells and TIL. Tumor-specific T cells with disrupted Cish function were adoptively transferred into tumor-bearing mice (what kind of tumors? orthotopic?*) and evaluated for efficacy with or without checkpoint blockade.

Findings CISH expression was associated with T cell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing T cell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade in vivo.

Conclusions CISH negatively regulates human T cell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy.

Funding This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

* Combination of CISH deletion and PD1 blockade significantly enhances adoptive immunotherapy (adoptive cell transfer of melanoma-specific T cells knocked out for Cish into B16-melanoma-bearing mice, with or without administration of antibodies blocking PD1 blockade. Modified B16- mhgp100 (H-2Db), a mouse melanoma cell line, was transduced as previously described to express glycoprotein 100 (gp100) mutated to express human amino acid residues at positions 25–27 (EGS to KVP); this line was used as the tumor model. - I guess this must be subcutaneous & therefore orthotopic injection?)

 

** In the discussion: The increase in T cell functional avidity after CISH KO presents unique safety concerns that must be considered. For instance, autoimmune uveitis and vitiligo can occur in melanoma patients who respond after TIL ACT because of the expression of tumor antigens on retinal pigment epithelium and normal melanocytes. Our previous work in a mouse melanoma model indicated that CISH can exacerbate such toxicities. Thus, in the setting of melanoma where off-tumor toxicities are common, the risk of exacerbating such toxicities must be weighed in the context of prognosis and the availability of alternative therapies. Conversely, epithelial malignancies, such as those of the gastrointestinal tract, are appealing from a clinical standpoint because of a comparatively lower mutational load and overall poor immunogenicity. ACT may maximally benefit from increased functional avidity in this setting, while the lower overall TCR diversity in TIL from such tumors may reduce the probability of off-tumor toxicity, particularly when neoantigen selection is used...To this end, these preclinical data are the foundation for a recently initiated human clinical trial entitled “A Study of Metastatic Gastrointestinal Cancers Treated with Tumor Infiltrating Lymphocytes in Which the Gene Encoding the Intracellular Immune Checkpoint CISH Is Inhibited Using CRISPR Genetic Engineering” (ClinicalTrials.gov Identifier NCT04426669). Results from this trial will hopefully shed light on the feasibility, safety, and efficacy of novel checkpoint inhibition using neoantigen-selected, CRISPR genetically engineered CISH KO T cell therapy for solid tumors."

 

The Abstract on biorxiv was more like a traditional abstract (and it had a few nice extra details but nothing about the clinical trial): While neoantigen-specific tumor infiltrating lymphocytes (TIL) can be derived from in antigen-expressing tumors, their adoptive transfer fails to consistently elicit durable tumor regression. There has been much focus on the role of activation/exhaustion markers such as PD1, CD39 and TOX in TIL senescence. We found these markers were inversely expressed to Cytokine-Induced SH2 protein (CISH), a negative regulator of TCR signaling and tumor immunity in mice. To evaluate the physiological role of CISH in human TIL we developed a high-efficiency CRIPSR-based method to knock out CISH in fully mature TIL. CISH KO resulted in increased T cell receptor (TCR) avidity, tumor cytolysis and neoantigen recognition. (Ang added: TP53 mutations are frequent in many cancers and can be presented as neoantigens; however, only a subset of patients harbor T cells that elicit a functional immune response.) CISH expression in the tumor resections correlated with TIL inactivity against p53 hotspot mutations and CISH KO in TIL unmasked reactivity against these universal neoantigens. While CISH KO resulted in T cell hyperactivation and expansion it did not alter maturation, perhaps by preferential PLCγ-1 and not AKT inhibition. Lastly, CISH KO in T cells increased PD1 expression and the adoptive transfer of Cish KO T cells synergistically combines with PD1 antibody blockade resulting in durable tumor regression and survival in a preclinical animal model. These data offer new insights into the regulation of neoantigen recognition, expression of activation/exhaustion markers, and functional/maturation signals in tumor-specific T cells.

 

Abstract #31 Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation from Dr. Joseph Puglisi's lab. Not open access but availabe on biorxiv about 11 months ago. I picked this one because I loved Dr. Puglisi's work from my grad student days, and because I remember sitting at the back of a standing room only talk at an RNA society conference in Santa Cruz and he was standing in the aisle vocally annoyed with the speaker for not presenting unpublished data. 

Summary: How the eukaryotic 43S preinitiation complex scans along the 5′ untranslated region (5′ UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A. Once engaged, 43S scanning occurs rapidly and directionally at ∼100 nucleotides per second, independent of multiple cycles of ATP hydrolysis by RNA helicases post ribosomal loading. Scanning ribosomes can proceed through RNA secondary structures, but 5′ UTR hairpin sequences near start codons drive scanning ribosomes at start codons backward in the 5′ direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5′ UTR structures and upstream near-cognate start codons.

 

The biorxiv summary - almost a year earlier - is identical. That being said, neither of these abstracts tell me the punchline :( so I have to go in and look at the paper (except for the backward movement & rescanning). 

 

The editor/detective in me noticed that the biorxiv paper has the classic formatting of a Nature or Science paper ;) The Intro gave a good background and a 'what we did' but not a 'what we found' (that of course is up to the author, but I'm a reader who skips to the end in everything...not every reader is like me...). I skipped to the Discussion and the intro was pretty similar to the abstract, and reads quite descriptive in that the data provide a 'mechanistic framework' - but I want to know what is that framework and is it different than what we thought and if so how? There's a paragraph "5′UTR secondary structures play a surprising role in scanning. Here we demonstrate that scanning ribosomes readily melt mRNA hairpin structures up to -52.1 kcal/mol in stability in the 5′UTR upon first encounter to reach the start site rapidly (Fig. 4). However, when the hairpin is located adjacent to the start codon, the ribosome remains at the start site for only a short period (~3 s, fig. S9I); subsequent backwards motion (3′ to 5′) and fluctuations between the upstream position and the start site impair 60S joining. The frequency of such fluctuations plateaued with hairpin stabilities above -12.2 kcal/mol (Fig. 4, H and I). This provides a rationale for the underrepresented (~1%) secondary structures with stability above -12.2 kcal/mol in a 50-nt 280 window in yeast 5′UTRs (13), and explains why secondary structures immediately upstream of the start site (positions -40 to -1) are selected against in yeast (42)" This seemed interesting: "Stable hairpin structures downstream of the CUG codon enhanced initiation at near-cognate codons by two mechanisms."

 

I imagine this paper was a technological breakthrough but conceptually my main take-home was that downstream hairpins can increase the use of near-cognate start codons upstream of an ATG. (I don't know if we already had some evidence of that, but it wouldn't surprise me - but this sheds light on the mechanism (not sure how much we knew there, either...)

 

Abstract #32 Ciliary tip actin dynamics regulate the cadence of photoreceptor disc formation a preprint from Dr. Pleasantine Mill's lab. I chose this one because I know Pleasantine :) and she's a wonderful scientist and advocate for women in science. We were in grad school in the same department, though she was (is :) ) much younger than me. I was in grad school a long time and saw many awesome students at so many different stages.

 

Abstract: As signalling organelles, primary cilia regulate their membrane G protein-coupled receptor (GPCR) content by ectocytosis, a process requiring localised actin dynamics at their tip to alter membrane shape.(1, 2) Mammalian photoreceptor outer segments comprise an expanse of folded membranes (discs) at the tip of highly-specialised connecting cilia (CC), in which photosensitive GPCRs like rhodopsin are concentrated. In an extraordinary feat of biology, outer segment discs are shed and remade daily.(3) Defects in this process, due to genetic mutations, cause retinitis pigmentosa (RP), an untreatable, blinding disease. The mechanism by which photoreceptor cilia generate outer segments is therefore fundamental for vision yet poorly understood. The authors show that the membrane deformation required for outer segment disc genesis is driven by dynamic changes in the actin cytoskeleton in a process akin to ectocytosis. Further, they show RPGR, a leading causal RP gene, regulates activity of actin binding proteins crucial to this process. Disc genesis is compromised in Rpgr mouse models, slowing the actin dynamics required for timely disc formation, leading to aborted membrane shedding as ectosome-like vesicles, photoreceptor death and visual loss. Manipulation of actin dynamics partially rescues the phenotype, suggesting this pathway could be targeted therapeutically. These findings help define how actin-mediated dynamics control outer segment turnover.

 

Very cool finding - studying the mechanism of 'disc' recycling of photoreceptors (specialized neurons found in the retina that convert light into electrical signals that stimulate physiological processes) likely revealed the mechanism of retinitis pigmentosa, which causes blindness. Retinitis pigmentosa is one of the most common inherited diseases of the retina (retinopathies). It is estimated to affect 1 in 3,500 to 1 in 4,000 people in the United States and Europe.

The discs, as their name suggests, look like discs :) . They have GPCRs in them, and GPCRs are known to be recycled by ectocytosis - and here it turns out discs are recycled by a similar mechanism that involves localized actin dynamics. A mutation in RPGR which is thought to cause retinis pigmentosis and defects in disc recycling, regulates the activity of actin binding proteins that are important in the mechanism they discovered here. Manipulating actin dynamics can restore phenotypes of the mutants - so there's therapeutic potential. BEAUTIFUL PAPER!

 

Abstract #33 Zygotic genome activation by the totipotency pioneer factor Nr5a2 from Dr. Kikuë Tachibana's lab. Not open access but preprint available about 6 months earlier. I picked this one because I love Kikuë's work and have had the pleasure to work with her and her group on a number of manuscripts, including this one. I find the process of reprogramming to totipotency after fertilization fascinating, and we know very little about how this is accomplished in mammals. 

 

Abstract: Life begins with a switch in genetic control from the maternal to the embryonic genome during zygotic genome activation (ZGA). Despite its importance, the essential regulators of ZGA remain largely unknown in mammals. Based on de novo motif searches, we identified the orphan nuclear receptor Nr5a2 as a key activator of major ZGA in mouse 2-cell embryos. Nr5a2 is required for progression beyond the 2-cell stage. It binds to its motif within SINE B1/Alu retrotransposable elements found in cis-regulatory regions of ZGA genes. Chemical inhibition suggests that 72% of ZGA genes are regulated by Nr5a2 and potentially other orphan nuclear receptors. Nr5a2 promotes chromatin accessibility during ZGA and binds nucleosomal DNA in vitro. We conclude that Nr5a2 is an essential pioneer factor that regulates ZGA.

 

The preprint abstract had a few more details: Consistent with a role in ZGA, Nr5a2 is required for progression beyond the 2-cell stage. Nr5a2 promotes chromatin accessibility (it opens chromatin at inactive regions) during ZGA and binds to entry/exit sites of nucleosomal DNA in vitro. 

 

To define ZGA genes, they compared transcription profiles of two mouse strains during the oocyte-to-embryo transition (OET). A total of 2508 “extended” ZGA genes were upregulated >4-fold in 2-cell embryos of the two strains. They classified the 985 genes that were common to both as “core” ZGA genes  and found a supermotif that is enriched upstream of ~70% of core ZGA genes (compared to 46% non-ZGA genes). The sequence has 90% similarity to the SINE B1 family of retrotransposons. Within the supermotif were individual motifs for the orphan nuclear receptors Nr5a2 and Esrrb and nuclear hormone receptor “retinoic acid receptor gamma” (Rarg). Rarg is not essential for ZGA (Rarg-/- females are fertile). Oocyte cytoplasm is sufficient to reprogram somatic cell nuclei to totipotency, and they detected Nr5a2 protein in oocytes, zygotes and 2-cell embryos. Using chemical and genetic inhibition they show that Nr5a2 and Esrrb contribute to ZGA. Their data show that Nr5a2 and Esrrb bind to distinct, cell type-specific enhancers in pluripotent versus totipotent cells.

 

Abstract #34 Gradual differentiation uncoupled from cell cycle exit generates heterogeneity in the epidermal stem cell layer from Dr. Valentina Greco's lab. Open access and preprint available about 11 months ago. I chose this one because I work with Valentina quite a bit, she's a fantastic scientist and unbelievably supportive. She is also on the Board of LSEF. I have worked with so many wonderful people in her lab, including Dr. Katie Cockburn, the first author of this paper (who now has a lab of her own). 

 

Abstract: Highly regenerative tissues continuously produce terminally differentiated cells to replace those that are lost. How they orchestrate the complex transition from undifferentiated stem cells towards post-mitotic, molecularly distinct and often spatially segregated differentiated populations is not well understood. In the adult skin epidermis, the stem cell compartment contains molecularly heterogeneous subpopulations whose relationship to the complete trajectory of differentiation remains unknown. Here the authors show that differentiation, from commitment to exit from the stem cell layer, is a multi-day process wherein cells transit through a continuum of transcriptional changes with upregulation of differentiation genes preceding downregulation of typical stemness genes. Differentiation-committed cells remain capable of dividing to produce daughter cells fated to further differentiate, demonstrating that differentiation is uncoupled from cell cycle exit. These cell divisions are not required as part of an obligate transit-amplifying programme but help to buffer the differentiating cell pool during heightened demand. Thus, instead of distinct contributions from multiple progenitors, a continuous gradual differentiation process fuels homeostatic epidermal turnover.

 

If you take a snapshot of the skin - you'll see stem cells at the bottom, along with some 'intermediate cells' and then differentiating cells rising up to the surface (only to be sloughed off!). Those intermediate cells - what do they reflect? Are they progenitors that amplify and expand the pool of differentiating cells? Or are they just lingering on their way our the door? Turns out it's the latter - they may or may not divide, but these intermediate cells neighbor stem cells for a while in the basal layer as they gradually turn off the stem cell program and turn on the differentiation program. "Our results ....argue against the presence of a long-lived and functionally distinct committed epidermal progenitor population. At the same time, we reveal direct evidence of a molecular heterogeneity among the proliferating basal population. This helps to clarify the distinct results obtained by different lineage tracing approachesand explains the strong temporal correlation in lifetimes between sister cells that both go on to delaminate"