Obligate Partner Research Articles

Alternative splicing is an FXRα loss-of-function mechanism and impacts energy metabolism in hepatocarcinoma cells

Farnesoid X receptor α (FXRα, NR1H4) is a bile acid-activated nuclear receptor that regulates the expression of glycolytic and lipogenic target genes by interacting with the 9-cis-retinoic acid receptor α (RXRα, NR2B1). Along with cofactors, the FXRα proteins reported thus far in humans and rodents have been observed to regulate both isoform (α1-4)- and tissue-specific gene expression profiles to integrate energy balance and metabolism. Here, we studied the biological functions of an FXRα naturally occurring spliced exon 5 isoform (FXRαse5) lacking the second zinc-binding module of the DNA binding domain (DBD). We demonstrate FXRαse5 expression in all FXRα-expressing human and mouse tissues and cells, and that it is unable to bind to its response element or activate FXRα dependent transcription. In parallel, this spliced variant displays differential interaction capacities with its obligate heterodimer partner RXRα that may account for silencing of this permissive dimer for signal transduction. Finally, deletion of exon 5 by gene edition in HepG2 cells leads to FXRα loss-of-function, increased expression of LRH1 metabolic sensor and CD36 fatty acid transporter in conjunction with changes in glucose and triglycerides homeostasis. Together, these findings highlight a novel mechanism by which alternative splicing may regulate FXRα gene function to fine-tune adaptive and/or metabolic responses. This finding deepens our understanding on the role of splicing events in hindering FXRα activity to regulate specific transcriptional programs and their contribution in modifying energy metabolism in normal tissues and metabolic diseases.

Surprising Features of Nuclear Receptor Interaction Networks Revealed by Live Cell Single Molecule Imaging.

Type 2 Nuclear Receptors (T2NRs) require heterodimerization with a common partner, the Retinoid X Receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and over-expression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged retinoid X receptor (RXR) and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.

Cytochrome b5 diversity in green lineages preceded the evolution of syringyl lignin biosynthesis.

Lignin production marked a milestone in vascular plant evolution, and the emergence of syringyl (S) lignin is lineage specific. S-lignin biosynthesis in angiosperms, mediated by ferulate 5-hydroxylase (F5H, CYP84A1), has been considered a recent evolutionary event. F5H uniquely requires the cytochrome b5 protein CB5D as an obligatory redox partner for catalysis. However, it remains unclear how CB5D functionality originated and whether it coevolved with F5H. We reveal here the ancient evolution of CB5D-type function supporting F5H-catalyzed S-lignin biosynthesis. CB5D emerged in charophyte algae, the closest relatives of land plants, and is conserved and proliferated in embryophytes, especially in angiosperms, suggesting functional diversification of the CB5 family before terrestrialization. A sequence motif containing acidic amino residues in Helix 5 of the CB5 heme-binding domain contributes to the retention of CB5D function in land plants but not in algae. Notably, CB5s in the S-lignin-producing lycophyte Selaginella lack these residues, resulting in no CB5D-type function. An independently evolved S-lignin biosynthetic F5H (CYP788A1) in Selaginella relies on NADPH-dependent cytochrome P450 reductase as sole redox partner, distinct from angiosperms. These results suggest that angiosperm F5Hs coopted the ancient CB5D, forming a modern cytochrome P450 monooxygenase system for aromatic ring meta-hydroxylation, enabling the reemergence of S-lignin biosynthesis in angiosperms.

Mouse models to explore the biological and organismic role of DNA polymerase beta.

Gene knock-out (KO) mouse models for DNA polymerase beta (Polβ) revealed that loss of Polβ leads to neonatal lethality, highlighting the critical organismic role for this DNA polymerase. While biochemical analysis and gene KO cell lines have confirmed its biochemical role in base excision repair and in TET-mediated demethylation, more long-lived mouse models continue to be developed to further define its organismic role. The Polb-KO mouse was the first of the Cre-mediated tissue-specific KO mouse models. This technology was exploited to investigate roles for Polβ in V(D)J recombination (variable-diversity-joining rearrangement), DNA demethylation, gene complementation, SPO11-induced DNA double-strand break repair, germ cell genome stability, as well as neuronal differentiation, susceptibility to genotoxin-induced DNA damage, and cancer onset. The revolution in knock-in (KI) mouse models was made possible by CRISPR/cas9-mediated gene editing directly in C57BL/6 zygotes. This technology has helped identify phenotypes associated with germline or somatic mutants of Polβ. Such KI mouse models have helped uncover the importance of key Polβ active site residues or specific Polβ enzyme activities, such as the PolbY265C mouse that develops lupus symptoms. More recently, we have used this KI technology to mutate the Polb gene with two codon changes, yielding the PolbL301R/V303R mouse. In this KI mouse model, the expressed Polβ protein cannot bind to its obligate heterodimer partner, Xrcc1. Although the expressed mutant Polβ protein is proteolytically unstable and defective in recruitment to sites of DNA damage, the homozygous PolbL301R/V303R mouse is viable and fertile, yet small in stature. We expect that this and additional targeted mouse models under development are poised to reveal new biological and organismic roles for Polβ.

Design, synthesis, and activity evaluation of 2-iminobenzimidazoles as c-Myc inhibitors for treating multiple myeloma

Multiple myeloma (MM) is a plasma cell malignancy that remains incurable and poses a significant threat to global public health. The multifunctional transcription factor c-Myc plays a crucial role in various cellular processes and is closely associated with MM progression. As part of the basic-helix-loop-helix-leucine zipper (bHLHZip) family, c-Myc forms heterodimers with its obligate partner Max, binds to the Enhancer-box (E-box) of DNA, and ultimately co-regulates gene expression. Therefore, impeding the capacity for heterodimerization to bind to DNA represents a favored strategy in thwarting c-Myc transcription. In this study, we first synthesized a series of novel 2-iminobenzimidazole derivatives and further estimated their potential anti-MM activity. Notably, among all the derivatives, 5b and 5d demonstrated remarkable inhibitory activity against RPMI-8226 and U266 cells, with IC50 values of 0.85 μM and 0.97 μM for compound 5b, and 0.96 μM and 0.89 μM for compound 5d. Western blot and dual-luciferase reporter assays demonstrated that compounds 5b and 5d effectively suppressed both c-Myc protein expression and transcriptional activity of the c-Myc promoter in RPMI-8226 and U266 cells. Furthermore, these compounds induced apoptosis and G1 cell cycle arrest in the aforementioned MM cells. Molecular docking studies revealed that 5b and 5d exhibited strong binding affinity to the interface between c-Myc/Max and E-box of DNA. Taken together, our findings suggest that further investigations are warranted for potential therapeutic applications of 5b and 5d for c-Myc-related diseases.

Intrinsically Disordered Regions in the Transcription Factor MYC:MAX Modulate DNA Binding via Intramolecular Interactions.

The basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor (TF) MYC is in large part an intrinsically disordered oncoprotein. In complex with its obligate heterodimerization partner MAX, MYC preferentially binds E-Box DNA sequences (CANNTG). At promoters containing these sequence motifs, MYC controls fundamental cellular processes such as cell cycle progression, metabolism, and apoptosis. A vast network of proteins in turn regulates MYC function via intermolecular interactions. In this work, we establish another layer of MYC regulation by intramolecular interactions. We used nuclear magnetic resonance (NMR) spectroscopy to identify and map multiple binding sites for the C-terminal MYC:MAX DNA-binding domain (DBD) on the intrinsically disordered regions (IDRs) in the MYC N-terminus. We find that these binding events in trans are driven by electrostatic attraction, that they have distinct affinities, and that they are competitive with DNA binding. Thereby, we observe the strongest effects for the N-terminal MYC box 0 (Mb0), a conserved motif involved in MYC transactivation and target gene induction. We prepared recombinant full-length MYC:MAX complex and demonstrate that the interactions identified in this work are also relevant in cis, i.e., as intramolecular interactions. These findings are supported by surface plasmon resonance (SPR) experiments, which revealed that intramolecular IDR:DBD interactions in MYC decelerate the association of MYC:MAX complexes to DNA. Our work offers new insights into how bHLH-LZ TFs are regulated by intramolecular interactions, which open up new possibilities for drug discovery.

Abstract B006: Targeting BARD1 in human pancreatic ductal adenocarcinoma: How to expand the target population of vulnerable tumors?

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the deadliest malignancy of the pancreas, with an overall five-year survival rate of only 12%. Despite therapeutic advances, effective therapies are still limited. It is now known that many of the DNA damage repair (DDR) genes that are mutated in PDAC are crucial for the proper functioning of the homologous recombination repair (HRR) pathway. Loss-of-function alterations in DDR genes such as BRCA1/2 mutations render PDAC cells vulnerable to certain DNA damage agents, for example, poly (ADP-ribose) polymerase inhibitors (PARPi) and platinum-based agents. Since this treatment strategy is currently limited to only 5-10% of PDAC patients, it underscores a dire need to find new therapeutic avenues and expand the PDAC patient population that could benefit from these therapies. We have recently published that BARD1 (BRCA1- Associated- Ring- Domain- 1), an obligate binding partner of BRCA1 in the process of HRR, is upregulated in PDAC cells exposed to PARPi/platinums. PDAC tissue microarray stained for BARD1 also shows higher BARD1 expression in PDAC tissues compared to normal pancreas. Here, we explored the effects of targeting BARD1 on PDAC cells. For this, we created genetic models of BARD1 inhibition and utilized these model systems to study if loss of BARD1 slows PDAC growth, invasion, and chemo-sensitizes PDAC cells to PARPis and DNA damage agents. Doxycycline inducible shRNA and BARD1 CRISPR KO models were created to inhibit gene expression of BARD1 in PDAC cells. Loss of BARD1 protein and mRNA expression was confirmed by western blot and qRT-PCR analyses. Using colony formation assays, Pico green cell survival assays, and Boyden matrigel invasion assays, we found that inhibition of BARD1 resulted in slower growth and decrease in invasion of pancreatic cancer cells in vitro. The expression levels of EMT markers such as zeb-1 and vimentin, were also decreased. These studies were recapitulated in a mouse xenograft subcutaneous model, where BARD1 CRISPR KO (+/- and -/-) and WT PDAC cells were injected subcutaneously on flanks of mice and tumor growth was monitored over time. Kaplan Meier curves were calculated and plotted using Log-rank test. We found that inhibiting BARD1 significantly (P=0.026) delayed PDAC tumor growth in vivo and extended survival (P<0.001). CRISPR KO of BARD1 also enhanced the efficacies of PARP inhibitors like olaparib, rucaparib and veliparib, and lowered their IC50s (fold changes >10). Our work indicates that targeting BARD1 affects key hallmarks of cancer (growth and invasion), such that the window of therapeutic opportunity is extended, which further allows chemo-sensitization of PDAC cells to DNA damaging agents. Our ongoing studies will explore if BARD1 loss is a predictive marker of PDAC sensitivity to other DNA-damaging agents besides PARPi and allow the development of therapeutic strategies to target BARD1 in PDAC. Citation Format: Aditi Jain, Rutuj P. Kusurkar, Eleanor Jenkins, Avinoam Nevler, Wei Jiang, Charles J. Yeo. Targeting BARD1 in human pancreatic ductal adenocarcinoma: How to expand the target population of vulnerable tumors? [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B006.

Scale insect (Hemiptera: Coccomorpha) morphology is transformed under trophobiosis

Abstract Ants (Hymenoptera: Formicidae) have great potential to exert influence over the morphological evolution of their obligate mutualist partners. Obligately myrmecophilic mealybugs are noted for their unusual morphology, and while this is often attributed to their relationship with ants, a quantitative assessment of this link is lacking. We address this need by evaluating morphological change among mealybugs as a function of ant association. This study considers the associates of 2 independent ant clades—Acropyga Roger, 1862 ants associated with root mealybugs from the families Xenococcidae and Rhizoecidae and herdsmen ants from the Dolichoderus cuspidatus (Smith, F., 1857) species-group associated with mealybugs from the tribe Allomyrmococcini (Pseudococcidae)—and compares them to free-living or potentially myrmecophilic species sampled from among the mealybugs and root mealybugs. We use a combination of geometric morphometric and linear datasets to evaluate characteristics of body shape, body size, leg metrics, and ostiole development. Obligate myrmecophily significantly influences both body shape and size. Myrmecophilous mealybugs are smaller than their free-living counterparts and are either pyriform or rotund in shape rather than oval. Ant-associates from Rhizoecidae also have significantly reduced anterior pairs of ostioles compared to free-living species. Ostioles are involved in defense against natural enemies and mutualist ants typically protect their partners, presumably supplanting the need for structures like ostioles among myrmecophilous species. We discuss the influence ants have on the evolution of their associates in the context of domestication and offer avenues for future exploration.

CTCF Is a Haploinsufficient Tumor Suppressor in B-Cell Lymphoma

Alteration of epigenetic regulators is a hallmark of diffuse large B cell lymphoma (DLBCL), the most common type of non-Hodgkin B cell malignancy. It has been recently shown that haploinsufficiency of the cohesin complex ATPase subunit Smc3 cooperates with the oncogene IμBcl6 to induce lymphoma in mice, by a mechanism that involves decreased intra-topological associating domain (TAD) chromosomal interactions and downregulation of tumor suppressor genes. The transcription factor CTCF is an obligate partner of the cohesin complex in defining TADs and regulatory gene loops, acting as the boundaries of TADs and restricting the interaction of promoters and enhancers. Alterations in CTCF have been shown in several cancer types, including lymphomas. In this study, we aim to define the role of CTCF during B-cell lymphoma and lymphomagenesis. We generated germinal center (GC) B cell-specific conditional knockout mice for CTCF by crossing the CTCF floxed mice to the Ighg1-Cre (Cγ1-Cre) allele. In these experiments, we compared haploinsufficient CTCF conditional knockout mice-in which only one allele of CTCF was knocked out-versus wild-type mice expressing the Cγ1-Cre. Eight days after immunization with the T-cell-dependent antigen sheep red blood cells, we excised spleens and surveyed B cell populations by flow cytometry and immunohistochemistry. Remarkably, CTCF haploinsufficient mice contained a similar percentage of follicular GC B cells compared to wild-type mice (Live B220 +CD95 +GL7 + or live B220 +CD95 +CD38 DIM splenocytes). However, CTCF haploinsufficient GCs displayed a bias in the GC subpopulations, namely an increased percentage of light zone centrocytes (LZ: GC + markers CXCR4 loCD86 hi) and decreased dark zone centroblasts (DZ: GC + markers CXCR4 hiCD86 lo). Because biases in GCs were an early indication of malignant transformation in previous studies, we wondered whether CTCF haploinsufficiency would cooperate with lymphoma oncogenes to induce B-cell lymphoma. To address the lymphomagenic potential of CTCF haploinsufficiency, we crossed CTCF conditional knockout mice to the vavP-Bcl2 mice that develop GC-derived follicular-like lymphoma with long latency. We harvested bone marrow blood cell precursors from Cγ1-Cre (“WT”), Cγ1-Cre;CTCF +/- (“CTCF”), vavP-BCL2 +;Cγ1-Cre (“BCL2”), and vavP-BCL2 +;Cγ1-Cre;CTCF +/- (“BCL2/CTCF”) and transplanted them into lethally irradiated C57BL6/J (30 per group). Mice that survived more than two weeks after irradiation and bone marrow transplant were considered stably engrafted and monitored for any signs of disease. Lethargic mice were sacrificed and macroscopically inspected for the presence of tumors. Survival analysis by Kaplan-Meier showed that BCL2/CTCF mice had the shortest survival from all four groups, having a median survival of 230 days, while survival for the BCL2 alone group did not fall below 50% up to day 370. Immunohistochemistry against B220 + was performed in tissue sampling from dying mice to confirm the cause of death. Curiously, CTCF haploinsufficiency accelerated BCL2-induced lymphomagenesis but did not affect the survival of IμBCL6-induced lymphoma. To explore the mechanistic basis of CTCF haploinsufficiency-induced lymphoma in mice, we FACS sorted DZ and LZ GC B-cells from 8-day immunized mice and explored global transcriptomics by RNA-sequencing. While the transition from DZ to LZ involves the regulation of around ~1,500 genes, we failed to detect changes in gene expression between wild-type and CTCF haploinsufficiency cells. ATAC-seq assay on the same cells showed little to no change in chromatin accessibility between cells from CTCF haploinsufficient and wild-type mice. TOBIAS footprinting analysis, which identifies biases in Tn5 transposase activity due to protein binding, showed that CTCF haploinsufficient cells maintained a deep depletion in CTCF binding. Hi-C chromosomal conformation analysis identified an overlapping set of gene regulatory loops that lose interactions and showed decreased signal by gene set enrichment analysis. In this study, we characterized the role of CTCF haploinsufficiency phenotypically and are currently exploring the molecular mechanisms that contribute to the acceleration of the lymphomagenesis induced by the BCL2 oncogene.

HER3: Toward the Prognostic Significance, Therapeutic Potential, Current Challenges, and Future Therapeutics in Different Types of Cancer.

Human epidermal growth factor receptor 3 (HER3) is the only family member of the EGRF/HER family of receptor tyrosine kinases that lacks an active kinase domain (KD), which makes it an obligate binding partner with other receptors for its oncogenic role. When HER3 is activated in a ligand-dependent (NRG1/HRG) or independent manner, it can bind to other receptors (the most potent binding partner is HER2) to regulate many biological functions (growth, survival, nutrient sensing, metabolic regulation, etc.) through the PI3K-AKT-mTOR pathway. HER3 has been found to promote tumorigenesis, tumor growth, and drug resistance in different cancer types, especially breast and non-small cell lung cancer. Given its ubiquitous expression across different solid tumors and role in oncogenesis and drug resistance, there has been a long effort to target HER3. As HER3 cannot be targeted through its KD with small-molecule kinase inhibitors via the conventional method, pharmaceutical companies have used various other approaches, including blocking either the ligand-binding domain or extracellular domain for dimerization with other receptors. The development of treatment options with anti-HER3 monoclonal antibodies, bispecific antibodies, and different combination therapies showed limited clinical efficiency for various reasons. Recent reports showed that the extracellular domain of HER3 is not required for its binding with other receptors, which raises doubt about the efforts and applicability of the development of the HER3-antibodies for treatment. Whereas HER3-directed antibody-drug conjugates showed potentiality for treatment, these drugs are still under clinical trial. The currently understood model for dimerization-induced signaling remains incomplete due to the absence of the crystal structure of HER3 signaling complexes, and many lines of evidence suggest that HER family signaling involves more than the interaction of two members. This review article will significantly expand our knowledge of HER3 signaling and shed light on developing a new generation of drugs that have fewer side effects than the current treatment regimen for these patients.

A voltammetric study of nitrogenase MoFe-protein using low-potential electron transfer mediators

The molybdenum-iron protein (MoFeP), a component of the enzyme nitrogenase, catalyzes the reduction of an array of small molecules, including N2 to NH3. In microorganisms, during the catalytic cycle, MoFeP receives electrons from the obligate biological redox partner iron protein (FeP) in a process coupled to the hydrolysis of two MgATP per one electron transferred. Despite the favorable redox properties of the cofactors, the requirement of the MgATP hydrolysis significantly decreases the energy efficiency of MoFeP. Therefore, remarkable efforts have been devoted to electrochemically activating MoFeP without FeP and MgATP. Previously, MoFeP was adsorbed on an electrode surface and revealed a slow catalysis with and without electron transfer mediators. However, enzyme adsorption can cause conformational and structural changes in a fragile protein molecule and alter its catalytic activity. In this work, MoFeP was electrochemically studied in solution. Various electron transfer mediators with potentials ranging from −0.3 V to −1 V (vs. NHE) were examined with MoFeP using cyclic voltammetry. No significant catalytic activity of the MoFeP was observed with any of the tested mediators. This indicates that efficient electrochemical activation of MoFeP cannot be achieved exclusively by increasing the driving force between the MoFeP redox cofactors and an electron donor.

Data-driven large-scale genomic analysis reveals an intricate phylogenetic and functional landscape in J-domain proteins

The 70-kD heat shock protein (Hsp70) chaperone system is a central hub of the proteostasis network that helps maintain protein homeostasis in all organisms. The recruitment of Hsp70 to perform different and specific cellular functions is regulated by the J-domain protein (JDP) co-chaperone family carrying the small namesake J-domain, required to interact and drive the ATPase cycle of Hsp70s. Besides the J-domain, prokaryotic and eukaryotic JDPs display a staggering diversity in domain architecture, function, and cellular localization. Very little is known about the overall JDP family, despite their essential role in cellular proteostasis, development, and its link to a broad range of human diseases. In this work, we leverage the exponentially increasing number of JDP gene sequences identified across all kingdoms owing to the advancements in sequencing technology and provide a broad overview of the JDP repertoire. Using an automated classification scheme based on artificial neural networks (ANNs), we demonstrate that the sequences of J-domains carry sufficient discriminatory information to reliably recover the phylogeny, localization, and domain composition of the corresponding full-length JDP. By harnessing the interpretability of the ANNs, we find that many of the discriminatory sequence positions match residues that form the interaction interface between the J-domain and Hsp70. This reveals that key residues within the J-domains have coevolved with their obligatory Hsp70 partners to build chaperone circuits for specific functions in cells.

Targeting Endothelial HIF2α/ARNT Expression for Ischemic Heart Disease Therapy.

Ischemic heart disease (IHD) is a major cause of mortality and morbidity worldwide, with novel therapeutic strategies urgently needed. Endothelial dysfunction is a hallmark of IHD, contributing to its development and progression. Hypoxia-inducible factors (HIFs) are transcription factors activated in response to low oxygen levels, playing crucial roles in various pathophysiological processes related to cardiovascular diseases. Among the HIF isoforms, HIF2α is predominantly expressed in cardiac vascular endothelial cells and has a key role in cardiovascular diseases. HIFβ, also known as ARNT, is the obligate binding partner of HIFα subunits and is necessary for HIFα's transcriptional activity. ARNT itself plays an essential role in the development of the cardiovascular system, regulating angiogenesis, limiting inflammatory cytokine production, and protecting against cardiomyopathy. This review provides an overview of the current understanding of HIF2α and ARNT signaling in endothelial cell function and dysfunction and their involvement in IHD pathogenesis. We highlight their roles in inflammation and maintaining the integrity of the endothelial barrier, as well as their potential as therapeutic targets for IHD.

Neofunctionalization of S-adenosylmethionine decarboxylase into pyruvoyl-dependent L-ornithine and L-arginine decarboxylases is widespread in bacteria and archaea

S-adenosylmethionine decarboxylase (AdoMetDC/SpeD) is a key polyamine biosynthetic enzyme required for conversion of putrescine to spermidine. Autocatalytic self-processing of the AdoMetDC/SpeD proenzyme generates a pyruvoyl cofactor from an internal serine. Recently, we discovered that diverse bacteriophages encode AdoMetDC/SpeD homologs that lack AdoMetDC activity and instead decarboxylate L-ornithine or L-arginine. We reasoned that neofunctionalized AdoMetDC/SpeD homologs were unlikely to have emerged in bacteriophages and were probably acquired from ancestral bacterial hosts. To test this hypothesis, we sought to identify candidate AdoMetDC/SpeD homologs encoding L-ornithine and L-arginine decarboxylases in bacteria and archaea. We searched for the anomalous presence of AdoMetDC/SpeD homologs in the absence of its obligatory partner enzyme spermidine synthase, or the presence of two AdoMetDC/SpeD homologs encoded in the same genome. Biochemical characterization of candidate neofunctionalized genes confirmed lack of AdoMetDC activity, and functional presence of L-ornithine or L-arginine decarboxylase activity in proteins from phyla Actinomycetota, Armatimonadota, Planctomycetota, Melainabacteria, Perigrinibacteria, Atribacteria, Chloroflexota, Sumerlaeota, Omnitrophota, Lentisphaerota, and Euryarchaeota, the bacterial candidate phyla radiation and DPANN archaea, and the δ-Proteobacteria class. Phylogenetic analysis indicated that L-arginine decarboxylases emerged at least three times from AdoMetDC/SpeD, whereas L-ornithine decarboxylases arose only once, potentially from the AdoMetDC/SpeD-derived L-arginine decarboxylases, revealing unsuspected polyamine metabolic plasticity. Horizontal transfer of the neofunctionalized genes appears to be the more prevalent mode of dissemination. We identified fusion proteins of bona fide AdoMetDC/SpeD with homologous L-ornithine decarboxylases that possess two, unprecedented internal protein-derived pyruvoyl cofactors. These fusion proteins suggest a plausible model for the evolution of the eukaryotic AdoMetDC.

Carm1-arginine methylation of the transcription factor C/EBPα regulates transdifferentiation velocity.

Here, we describe how the speed of C/EBPα-induced B cell to macrophage transdifferentiation (BMT) can be regulated, using both mouse and human models. The identification of a mutant of C/EBPα (C/EBPαR35A) that greatly accelerates BMT helped to illuminate the mechanism. Thus, incoming C/EBPα binds to PU.1, an obligate partner expressed in B cells, leading to the release of PU.1 from B cell enhancers, chromatin closing and silencing of the B cell program. Released PU.1 redistributes to macrophage enhancers newly occupied by C/EBPα, causing chromatin opening and activation of macrophage genes. All these steps are accelerated by C/EBPαR35A, initiated by its increased affinity for PU.1. Wild-type C/EBPα is methylated by Carm1 at arginine 35 and the enzyme's perturbations modulate BMT velocity as predicted from the observations with the mutant. Increasing the proportion of unmethylated C/EBPα in granulocyte/macrophage progenitors by inhibiting Carm1 biases the cell's differentiation toward macrophages, suggesting that cell fate decision velocity and lineage directionality are closely linked processes.

Abstract 6102: Targeting BARD1 slows PDAC growth in vitro and in vivo and sensitizes cells to DNA damaging agents

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent malignant neoplasm of the pancreas, responsible for over 90% of pancreatic cancer cases. Despite recent therapeutic advances, the five-year overall survival rate for PDAC is only 11% and therapies remain limited. It is now known that deficiency in DNA damage repair (DDR) renders PDAC cells vulnerable to certain DNA damage agents, for example, poly (ADP-ribose) polymerase inhibitors (PARPi). Most of the DDR genes mutated in PDAC are crucial for the proper functioning of the homologous recombination repair (HRR) pathway, and loss-of-function alterations in DDR genes sensitize cells to DNA-damaging agents. Since this treatment strategy is currently limited to 5-10% of PDAC cases with known mutations in the DDR genes, it underscores a dire need to understand new therapeutic avenues and expand the PDAC patient population that could benefit from this therapeutic approach. We have recently published that BARD1 (BRCA1- Associated- Ring- Domain- 1), an obligate binding partner of BRCA1, is upregulated in PDAC cells exposed to PARPi/platinums. We found that transient inhibition of BARD1 rendered PDAC cells extremely sensitive to olaparib (PARPi)/oxaliplatin (platinum), and enhanced DNA damage. Based on these results, we created two genetic models of BARD1 inhibition and utilized these model systems to investigate if loss of BARD1 chemo-sensitizes PDAC cells to other PARPis and DNA damage agents. Doxycycline inducible shRNA and BARD1 CRISPR KO models were created to inhibit gene expression of BARD1 in PDAC cells. We first characterized these model systems to confirm the loss of BARD1 protein and mRNA expression by western blot and qRT-PCR analyses. Using colony formation and Pico green cell survival assays, we found that inhibiting BARD1 resulted in slower growth of pancreatic cancer cells in vitro. Moreover, expression of several cell cycle genes from G2-M phase was significantly decreased (p=0.05). These studies were recapitulated in a mouse xenograft subcutaneous model, where BARD1 KO and WT PDAC cells were injected subcutaneously and tumor growth was analyzed over 25-30 days. Kaplan-Meier curves were calculated and plotted using Log-rank test. We found that inhibiting BARD1 significantly (p=0.026) delayed formation of PDAC tumors in vivo. Next, we analyzed the efficacies of various DNA damaging agents when BARD1 was inhibited. Silencing or CRISPR KO of BARD1 enhanced efficacy of mitomycin C, irinotecan, as well as other PARPis, which are currently in clinical trials (rucaparib, veliparib) in vitro and lowered their IC50s. These results indicate that targeting BARD1 has the potential to benefit patients in terms of novel or improved treatments. Our ongoing and future studies will expand on these findings to understand BARD1-mediated pathway and evaluate sensitivity of multiple DNA damage agents by screening a library of > 250 compounds. Citation Format: Sohum Patel, Anoohya Arkala, Avinoam Nevler, Carolyn Coats, Charles J. Yeo, Aditi Jain. Targeting BARD1 slows PDAC growth in vitro and in vivo and sensitizes cells to DNA damaging agents. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6102.

Droplet superpropulsion in an energetically constrained insect

Food consumption and waste elimination are vital functions for living systems. Although how feeding impacts animal form and function has been studied for more than a century since Darwin, how its obligate partner, excretion, controls and constrains animal behavior, size, and energetics remains largely unexplored. Here we study millimeter-scale sharpshooter insects (Cicadellidae) that feed exclusively on a plant’s xylem sap, a nutrient-deficit source (95% water). To eliminate their high-volume excreta, these insects exploit droplet superpropulsion, a phenomenon in which an elastic projectile can achieve higher velocity than the underlying actuator through temporal tuning. We combine coupled-oscillator models, computational fluid dynamics, and biophysical experiments to show that these insects temporally tune the frequency of their anal stylus to the Rayleigh frequency of their surface tension-dominated elastic drops as a single-shot resonance mechanism. Our model predicts that for these tiny insects, the superpropulsion of droplets is energetically cheaper than forming jets, enabling them to survive on an extreme energy-constrained xylem-sap diet. The principles and limits of superpropulsion outlined here can inform designs of energy-efficient self-cleaning structures and soft engines to generate ballistic motions.

MBD2a-NuRD Binds to the γ-Globin Gene Promoter and Uniquely Forms a Complex Required for Bcl11a Mediated Silencing of Hb F Expression

A mechanistic understanding of γ-globin silencing is of clinical importance because reversal of silencing in adult erythroid cells results in high level expression Hb F which is therapeutic in sickle cell anemia and beta thalassemia. We describe here studies to elucidate the mechanism by which MBD2a-NuRD is an obligate partner of Bcl11a in silencing of HbF. We have previously shown that depletion of MBD2 or disruption of the MBD2-NuRD chromatin remodeling complex, but not MBD3-NuRD complex results in high levels of Hb F despite a concomitant 2-3 fold increase in expression of Bcl11a, a major silencing transcription factor (Yu etal. Haematologica, 2019). At the same time, Bcl11a can interact with both the MBD3 and MBD2 NuRD complexes. To elucidate the specific role of MBD2-NuRD, Chromatin Immunoprecipitation-qPCR(ChIP-qPCR) assays carried out in both adult erythroid phenotype HUDEP-2 and primary progenitor derived adult erythroid cells showed binding at the γ-globin gene promoter. To clarify the role of MBD2-NuRD in establishing closed chromatin configuration at the γ-globin gene loci, we carried out ATAC seq assays which showed that MBD2 knockout resulted in the same pattern of chromatin opening as reported for Bcl11a knockout in HUDEP-2 cells, consistent with our previous data showing ~40-50% Hb F in MBD2 knockout cells. In order to examine the effect of MBD2-NuRD on nucleosome positioning over the γ-globin gene promoter, NOME-SEQ assays were performed in HUDEP 2 cells which showed a nucleosome positioned at -110 to + 36 relative to the transcription start site (TSS) in MBD +/+ cells but not MBD2 KO HUDEP-2 cells, such that the NFY-A transcriptional activator binding site but not the Bcl11a repressor binding site is occluded. MBD2 exists in two isoforms in erythroid cells, resulting from alternative translation initiation sites. MBD2a contains an arginine rich (GR) domain which is absent in MBD2b and MBD3. Addback of MBD2a but not MBD2b in MBD2 KO HUDEP-2 cells resulted in silencing of γ-globin gene expression. Editing of the endogenous MBD2 gene such that predominantly MBD2b was expressed relieved silencing. To understand how the GR domain contributes to silencing we carried out unbiased proteomic analysis of MBD2a vs MBD2b NuRD complexes, which identified only 3 proteins uniquely associated with MBD2a. One of these, PRMT5, has been implicated in silencing of HbF. Knockdown of PRMT5 resulted in a`from <1% to ~15% γ-globin RNA in parental HUDEP-2 cells and a 4-5 fold increase in primary human progenitor derived erythroid cells, but caused no significant further increase in MBD2 KO HUDEP-2 cells. ChIP qPCR assays showed that PRMT5 and its obligate binding partner MEP50 occupy the γ- promoter in parental HUDEP-2 cells and primary erythroid cells but not in MBD2 KO HUDEP-2 cells. Furthermore, knockdown of PRMT5 resulted in frequent displacement of the nucleosome positioned at -110 to + 36 relative to the TSS. In order to determine the role that the GR domain might have in occupancy of MBD2a-NuRD at the γ-promoter, in vitro binding studies where done, which showed a requirement for both CpG methylation and the GR domain for high affinity binding of the methyl cytosine binding domain of MBD2 to the proximal γ-globin gene promoter sequences. We conclude that MBD2a-NuRD is required for silencing of Hb F due to its ability to localize at the methylated γ-globin gene promoter in conjunction with Bcl11a and recruit PRMT5, thereby stably positioning a nucleosome that blocks binding of the transcriptional activator NFY-A. These results reinforce the concept that targeted disruption of this specific complex has therapeutic potential to increase HbF in sickle cell anemia and β thalassemia.

Design and in vitro characterization of RXR variants as tools to investigate the biological role of endogenous rexinoids

Retinoid X receptors (RXRα, β, and γ) are essential members of the nuclear receptor (NR) superfamily of ligand-dependent transcriptional regulators that bind DNA response elements and control the expression of large gene networks. As obligate heterodimerization partners of many NRs, RXRs are involved in a variety of pathophysiological processes. However, despite this central role in NR signaling, there is still no consensus regarding the precise biological functions of RXRs and the putative role of the endogenous ligands (rexinoids) previously proposed for these receptors. Based on available crystal structures, we introduced a series of amino acid substitutions into the ligand-binding pocket of all three RXR subtypes in order to alter their binding properties. Subsequent characterization using a battery of cell-based and in vitro assays led to the identification of a double mutation abolishing the binding of any ligand while keeping the other receptor functions intact and a triple mutation that selectively impairs interaction with natural rexinoids but not with some synthetic ligands. We also report crystal structures that help understand the specific ligand-binding capabilities of both variants. These RXR variants, either fully disabled for ligand binding or retaining the property of being activated by synthetic compounds, represent unique tools that could be used in future studies to probe the presence of active endogenous rexinoids in tissues/organs and to investigate their role in vivo. Last, we provide data suggesting a possible involvement of fatty acids in the weak interaction of RXRs with corepressors.

Structural Basis for the Allosteric Inhibition of Hypoxia-Inducible Factor 2 by Belzutifan

Hypoxia-inducible factor (HIF)-2α and its obligate heterodimerization partner aryl hydrocarbon receptor nuclear translocator (ARNT), are both members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factor family. Previous studies have identified HIF-2α as a key oncogenic driver in clear cell renal cell carcinoma (ccRCC), rendering it a promising drug target for this type of kidney cancer. Belzutifan is the first HIF-2α inhibitor approved for treating ccRCC and other cancers associated with the von Hippel-Lindau (VHL) disease. However, the detailed inhibitory mechanism of belzutifan at molecular level is still unclear. Here we obtained the crystal structure of HIF-2α-ARNT heterodimer in complex with belzutifan at 2.75 Å resolution. The complex structure shows that belzutifan binds into the PAS-B pocket of HIF-2α, and it destabilizes the dimerization of HIF-2α and ARNT through allosteric effects mainly mediated by the key residue M252 of HIF-2α near the dimer interface. We further explored the inhibitory effects of belzutifan using biochemical and functional assays. The time-resolved fluorescence energy transfer (TR-FRET)-based binding assay showed that belzutifan disrupts the dimerization of HIF-2α and ARNT with a Ki value of 20 nM. The luciferase reporter assay indicated that belzutifan can efficiently inhibit the transcriptional activity of HIF-2α with an IC50 value of 17 nM. Besides, the real-time PCR assay illustrated that belzutifan can reduce the expression of HIF-2α downstream genes in 786-O kidney cancer cells in a dose-dependent manner. Our work reveals the molecular mechanism by which belzutifan allosterically inhibits HIF-2α and provides valuable information for the subsequent drug development targeting HIF-2α. Significance Statement The bHLH-PAS family of transcription factors are an emerging group of small-molecule drug targets. Belzutifan, originally developed by Peloton Therapeutics, is the first FDA-approved drug directly binding to a bHLH-PAS protein, the hypoxia-inducible factor (HIF)-2α. Based on the protein-drug complex structure, biochemical binding assays, and functional profiling of downstream gene expression, this study reveals the regulatory mechanism of how belzutifan allosterically destabilizes HIF-2α's heterodimerization with its obligate partner protein, thus reducing their transcriptional activity that links to tumor progression.