Wild-type Overexpression Research Articles

Piezo activity levels need to be tightly regulated to maintain normal morphology and function in pericardial nephrocytes.

Due to their position on glomerular capillaries, podocytes are continuously counteracting biomechanical filtration forces. Most therapeutic interventions known to generally slow or prevent the progression of chronic kidney disease appear to lower these biomechanical forces on podocytes, highlighting the critical need to better understand podocyte mechano-signalling pathways. Here we investigated whether the mechanotransducer Piezo is involved in a mechanosensation pathway in Drosophila nephrocytes, the podocyte homologue in the fly. Loss of function analysis in Piezo depleted nephrocytes reveal a severe morphological and functional phenotype. Further, pharmacological activation of endogenous Piezo with Yoda1 causes a significant increase of intracellular Ca++ upon exposure to a mechanical stimulus in nephrocytes, as well as filtration disturbances. Elevated Piezo expression levels also result in a severe nephrocyte phenotype. Interestingly, expression of Piezo which lacks mechanosensitive channel activity, does not result in a severe nephrocyte phenotype, suggesting the observed changes in Piezo wildtype overexpressing cells are caused by the mechanosensitive channel activity. Moreover, blocking Piezo activity using the tarantula toxin GsMTx4 reverses the phenotypes observed in nephrocytes overexpressing Piezo. Taken together, here we provide evidence that Piezo activity levels need to be tightly regulated to maintain normal pericardial nephrocyte morphology and function.

Unique tau- and synuclein-dependent metabolic reprogramming in neurons distinct from normal aging.

Neuronal cells are highly specialized cells and have a specific metabolic profile to support their function. It has been demonstrated that the metabolic profiles of different cells/tissues undergo significant reprogramming with advancing age, which has often been considered a contributing factor towards aging-related diseases including Alzheimer's (AD) and Parkinson's (PD) diseases. However, it is unclear if the metabolic changes associated with normal aging predispose neurons to disease conditions or a distinct set of metabolic alterations happen in neurons in AD or PD which might contribute to disease pathologies. To decipher the changes in neuronal metabolism with age, in AD, or in PD, we performed high-throughput steady-state metabolite profiling on heads in wildtype Drosophila and in Drosophila models relevant to AD and PD. Intriguingly, we found that the spectrum of affected metabolic pathways is dramatically different between normal aging, Tau, or Synuclein overexpressing neurons. Genetic targeting of the purine and glutamate metabolism pathways, which were dysregulated in both old age and disease conditions partially rescued the neurodegenerative phenotype associated with the overexpression of wildtype and mutant tau. Our findings support a "two-hit model" to explain the pathological manifestations associated with AD where both aging- and Tau/Synuclein- driven metabolic reprogramming events cooperate with each other, and targeting both could be a potent therapeutic strategy.

In vitro study of ATP1A3 p.Ala275Pro mutant causing alternating hemiplegia of childhood and rapid-onset dystonia-parkinsonism.

We previously reported that ATP1A3 c.823G>C (p.Ala275Pro) mutant causes varying phenotypes of alternative hemiplegia of childhood and rapid-onset dystonia-parkinsonism in the same family. This study aims to investigate the function of ATP1A3 c.823G>C (p.Ala275Pro) mutant at the cellular and zebrafish models. ATP1A3 wild-type and mutant Hela cell lines were constructed, and ATP1A3 mRNA expression, ATP1A3 protein expression and localization, and Na+-K+-ATPase activity in each group of cells were detected. Additionally, we also constructed zebrafish models with ATP1A3 wild-type overexpression (WT) and p.Ala275Pro mutant overexpression (MUT). Subsequently, we detected the mRNA expression of dopamine signaling pathway-associated genes, Parkinson's disease-associated genes, and apoptosisassociated genes in each group of zebrafish, and observed the growth, development, and movement behavior of zebrafish. Cells carrying the p.Ala275Pro mutation exhibited lower levels of ATP1A3 mRNA, reduced ATP1A3 protein expression, and decreased Na+-K+-ATPase activity compared to wild-type cells. Immunofluorescence analysis revealed that ATP1A3 was primarily localized in the cytoplasm, but there was no significant difference in ATP1A3 protein localization before and after the mutation. In the zebrafish model, both WT and MUT groups showed lower brain and body length, dopamine neuron fluorescence intensity, escape ability, swimming distance, and average swimming speed compared to the control group. Moreover, overexpression of both wild-type and mutant ATP1A3 led to abnormal mRNA expression of genes associated with the dopamine signaling pathway and Parkinson's disease in zebrafish, and significantly upregulated transcription levels of bad and caspase-3 in the apoptosis signaling pathway, while reducing the transcriptional level of bcl-2 and the bcl-2/bax ratio. This study reveals that the p.Ala275Pro mutant decreases ATP1A3 protein expression and Na+/K+-ATPase activity. Abnormal expression of either wild-type or mutant ATP1A3 genes impairs growth, development, and movement behavior in zebrafish.

N6-methyladenosine demethylase fat mass and obesity-associated protein suppresses hyperglycemia-induced endothelial cell injury by inhibiting reactive oxygen species formation via autophagy promotion

IntroductionHyperglycemia-induced endothelial cell injury is one of the main causes of diabetic vasculopathy. Fat mass and obesity-associated protein (FTO) was the first RNA N6-methyladenosine (m6A) demethylase identified; it participates in the pathogenesis of diabetes. However, the role of FTO in hyperglycemia-induced vascular endothelial cell injury remains unclear. Materials and methodsThe effects of FTO on cellular m6A, autophagy, oxidative stress, proliferation, and cytotoxicity were explored in human umbilical vein endothelial cells (HUVECs) treated with high glucose (33.3 mmol/mL) after overexpression or pharmacological inhibition of FTO. MeRIP-qPCR and RNA stability assays were used to explore the molecular mechanisms by which FTO regulates autophagy. ResultsHigh glucose treatment increased m6A levels and reduced FTO protein expression in HUVECs. Wild-type overexpression of FTO markedly inhibited reactive oxygen species generation by promoting autophagy, increasing endothelial cell proliferation, and decreasing the cytotoxicity of high glucose concentrations. The pharmacological inhibition of FTO showed the opposite results. Mechanistically, we identified Unc-51-like kinase 1 (ULK1), a gene responsible for autophagosome formation, as a downstream target of FTO-mediated m6A modification. FTO overexpression demethylated ULK1 mRNA and inhibited its degradation in an m6A-YTHDF2-dependent manner, leading to autophagy activation. ConclusionsOur study demonstrates the functional importance of FTO-mediated m6A modification in alleviating endothelial cell injury under high glucose conditions and indicates that FTO may be a novel therapeutic target for diabetic vascular complications.

Variant-specific pathophysiological mechanisms of AFF3 differently influence transcriptome profiles

BackgroundWe previously described the KINSSHIP syndrome, an autosomal dominant disorder associated with intellectual disability (ID), mesomelic dysplasia and horseshoe kidney, caused by de novo variants in the degron of AFF3. Mouse knock-ins and overexpression in zebrafish provided evidence for a dominant-negative mode of action, wherein an increased level of AFF3 resulted in pathological effects.MethodsEvolutionary constraints suggest that other modes-of-inheritance could be at play. We challenged this hypothesis by screening ID cohorts for individuals with predicted-to-be damaging variants in AFF3. We used both animal and cellular models to assess the deleteriousness of the identified variants.ResultsWe identified an individual with a KINSSHIP-like phenotype carrying a de novo partial duplication of AFF3 further strengthening the hypothesis that an increased level of AFF3 is pathological. We also detected seventeen individuals displaying a milder syndrome with either heterozygous Loss-of-Function (LoF) or biallelic missense variants in AFF3. Consistent with semi-dominance, we discovered three patients with homozygous LoF and one compound heterozygote for a LoF and a missense variant, who presented more severe phenotypes than their heterozygous parents. Matching zebrafish knockdowns exhibit neurological defects that could be rescued by expressing human AFF3 mRNA, confirming their association with the ablation of aff3. Conversely, some of the human AFF3 mRNAs carrying missense variants identified in affected individuals did not rescue these phenotypes. Overexpression of mutated AFF3 mRNAs in zebrafish embryos produced a significant increase of abnormal larvae compared to wild-type overexpression further demonstrating deleteriousness.To further assess the effect of AFF3 variation, we profiled the transcriptome of fibroblasts from affected individuals and engineered isogenic cells harboring + / + , KINSSHIP/KINSSHIP, LoF/ + , LoF/LoF or KINSSHIP/LoF AFF3 genotypes. The expression of more than a third of the AFF3 bound loci is modified in either the KINSSHIP/KINSSHIP or the LoF/LoF lines. While the same pathways are affected, only about one third of the differentially expressed genes are common to the homozygote datasets, indicating that AFF3 LoF and KINSSHIP variants largely modulate transcriptomes differently, e.g. the DNA repair pathway displayed opposite modulation.ConclusionsOur results and the high pleiotropy shown by variation at this locus suggest that minute changes in AFF3 function are deleterious.

Golgi-associated retrograde protein (GARP) complex-dependent endosomes to trans Golgi network retrograde trafficking is controlled by Rab4b

BackgroundThe trafficking of cargoes from endosomes to the trans-Golgi network requires numerous sequential and coordinated steps. Cargoes are sorted into endosomal-derived carriers that are transported, tethered, and fused to the trans-Golgi network. The tethering step requires several complexes, including the Golgi-associated retrograde protein complex, whose localization at the trans-Golgi network is determined by the activity of small GTPases of the Arl and Rab family. However, how the Golgi-associated retrograde protein complex recognizes the endosome-derived carriers that will fuse with the trans-Golgi network is still unknown.MethodsWe studied the retrograde trafficking to the trans-Golgi network by using fluorescent cargoes in cells overexpressing Rab4b or after Rab4b knocked-down by small interfering RNA in combination with the downregulation of subunits of the Golgi-associated retrograde protein complex. We used immunofluorescence and image processing (Super Resolution Radial Fluctuation and 3D reconstruction) as well as biochemical approaches to characterize the consequences of these interventions on cargo carriers trafficking.ResultsWe reported that the VPS52 subunit of the Golgi-associated retrograde protein complex is an effector of Rab4b. We found that overexpression of wild type or active Rab4b increased early endosomal to trans-Golgi network retrograde trafficking of the cation-independent mannose-6-phosphate receptor in a Golgi-associated retrograde protein complex-dependent manner. Conversely, overexpression of an inactive Rab4b or Rab4b knockdown attenuated this trafficking. In the absence of Rab4b, the internalized cation-independent mannose 6 phosphate receptor did not have access to VPS52-labeled structures that look like endosomal subdomains and/or endosome-derived carriers, and whose subcellular distribution is Rab4b-independent. Consequently, the cation-independent mannose-6-phosphate receptor was blocked in early endosomes and no longer had access to the trans-Golgi network.ConclusionOur results support that Rab4b, by controlling the sorting of the cation-independent mannose-6-phosphate receptor towards VPS52 microdomains, confers a directional specificity for cargo carriers en route to the trans-Golgi network. Given the importance of the endocytic recycling in cell homeostasis, disruption of the Rab4b/Golgi-associated retrograde protein complex-dependent step could have serious consequences in pathologies.

Real-time resolution studies of the regulation of lactate production by hexokinases binding to mitochondria in single cells.

During hypoxia accumulation of lactate may be a key factor in acidosis-induced tissue damage. Binding of hexokinase (HK) to the outer membrane of mitochondria may have a protective effect under these conditions. We have investigated the regulation of lactate metabolism by hexokinases (HKs), using HEK293 cells in which the endogenous hexokinases have been knocked down to enable overexpression of wild type and mutant HKs. To assess the real-time changes in intracellular lactate levels the cells were also transfected with a lactate specific FRET probe. In the HKI/HKII double knockdown HEK cells, addition of extracellular pyruvate caused a large and sustained decrease in lactate. Upon inhibition of the mitochondrial electron transfer chain by NaCN this effect was reversed as a rapid increase in lactate developed which was followed by a slow and sustained increase in the continued presence of the inhibitor. Incubation of the HKI/HKII double knockdown HEK cells with the inhibitor of the malic enzyme, ME1*, blocked the delayed accumulation of lactate evoked by NaCN. With replacement by overexpression of HKI or HKII the accumulation of intracellular lactate evoked by NaCN was prevented. Blockage of the pentose phosphate pathway with the inhibitor 6-aminonicotinamide (6-AN) abolished the protective effect of HK expression, with NaCN causing again a sustained increase in lactate. The effect of HK was dependent on HK's catalytic activity and interaction with the mitochondrial outer membrane (MOM). Based on these data we propose that transformation of glucose into G6P by HK activates the pentose phosphate pathway which increases the production of NADPH, which then blocks the activity of the malic enzyme to transform malate into pyruvate and lactate.

Mechanism of Hirudin-Mediated Inhibition of Proliferation in Ovarian Cancer Cells.

To investigate the inhibitory effect of hirudin on the cell proliferation of human ovarian cancer A2780 cells by preventing thrombin and its underlying molecular mechanism. Cell Counting Kit-8 (CCK-8) method was used to detect the effect of different concentrations of hirudin and thrombin on the cell proliferation of A2780 cells. PAR-1 wild-type overexpression plasmid was constructed utilizing enzyme digestion identification, and it was transferred to A2780 cells. Sequencing and Western blot were used to detect the changes in PAR-1 protein expression. Western blot detection of PKCα protein phosphorylation in A2780 cells was performed. We also implemented quantitative PCR to detect the mRNA expression levels of epithelial-mesenchymal transition (EMT)-related genes, CDH2, Snail, and Vimentin, in A2780 cells. 1μg/ml hirudin treatment maximally inhibited the promotion of A2780 cell proliferation by thrombin. Hirudin inhibited the binding of thrombin to the N-terminus of PAR-1, hindered PKCα protein phosphorylation in A2780 cells, and downregulated the mRNA expression levels of CDH2, Snail, and Vimentin. In conclusion, hirudin inhibits the cell proliferation of ovarian cancer A2780 cells, and the underlying mechanism may be through downregulating the transcription level of EMT genes, CDH2, Snail, and Vimentin. This study indicates that hirudin may have a therapeutic potential as an anti-cancer agent for ovarian cancer.

Cooperative Somatic Alterations in XPO1 and TNFAIP3 (A20) Promote Immune Modulation and Tissue Infiltration in Primary Mediastinal B Cell Lymphoma

Background: Primary Mediastinal B cell lymphoma (PMBCL) is an aggressive large B cell non-Hodgkin lymphoma typically presenting as a mass in the anterior mediastinum in early adulthood, disproportionately affecting females (2:1). Previous studies have identified constitutive activation of the NF-κB pathway and immune evasion as hallmark features of PMBCL. The master negative regulator of canonical NF-κB, TNFAIP3 (A20), is inactivated in ~25-60% of PMBCL cases via disruptive mutations or deletions. Gains of the 2p16.1-15 locus ( REL, XPO1, BCL11A) are seen in ~40-75% of PMBCL cases. Interestingly, PMBCL cases with 2p16.1-15 gain/amplification display concomitant increases in XPO1 expression but lack correlative increases in REL or BCL11A. In addition, XPO1 mutations are seen in ~15-33% of PMBCL cases, with the most prevalent mutation (E571K) implicated in NF-KB activation. Despite the established occurrence of genetic events impacting XPO1 and A20 in PMBCL, the cooperative potential of these alterations remains unexplored. Consequently, we conducted an extensive review of literature describing the PMBCL genomic landscape and utilized in vivo models to examine the combined potential of XPO1 and A20 alterations to promote lymphomagenesis. Methods: Genomic data was obtained from NGS analyses from a total of 233 PMBCL patients across 6 independent published research studies and analyzed for cooccurrence of A20 & XPO1 alterations (Camus et al. 2022; Chapuy et al. 2019; Lacy et al. 2020; Mareschal et al. 2015; Mottock et al. 2019; Tuveri et al 2022). For in vivo analysis, we developed a murine model of B cell specific overexpression of wildtype (WT) or E571K- XPO1 with CD19.cre driven depletion of A20 (Eµ-XPO1xA20 KO). Whole body histopathologic evaluation of FFPE tissue with H&E staining was performed on 6-month-old, age and sex matched littermate mice. Results: We found co-occurrence of inactivating events in A20 and XPO1 missense mutations impacting 15% of PMBCL evaluated cases, with consistent results seen across all studies (range: 13.8-20%). As expected, A20 mutations/del were more prevalent than XPO1 mutations across all studies (OR 2.46, 95% CI 0.84-7.18, p = 0.02). XPO1 mutated cases trended towards a greater likelihood of having co-occurring A20 mutations (OR 8.51, 95% CI 0.84-86.47, p = 0.08). XPO1 mutations and gain (2p16) were found to be mutually exclusive events with gains co-occurring with A20 mutations/del in ~17% of PMBCL cases evaluated. Analysis of survival of PMBCL patients with cooccurring A20 inactivating alterations and XPO1 mutations is ongoing although preliminarily trend towards reduced mean 2-year survival (p=0.056) compared to all others are observed. Taken together, co-occurring genetic alterations effecting A20 and XPO1 are prevalent in PMBCL the alternative biology stemming from potential cooperative mechanisms may represent an aggressive subgroup of PMBCL. To evaluate concurrent B cell-specific alterations of A20 and XPO1 in vivo we utilized Eµ-XPO1xA20 KO mouse models. Performing histopathology in 6-month-old mice we observed enlarged lymph nodes due to marked increases in lymphocytes within the cortex, paracortex, medulla, and sinuses in mice with loss of functional A20, particularly in Eµ-XPO1xA20 KO mice with overexpression of WT XPO1. Increased lymphocyte populations drove overall enlargement of the node (mandibular, mesenteric, pancreatic, and mediastinal) and distorted normal architecture, featuring small lymphocytes populating sinus regions and larger lymphocytes populating areas resembling follicles. Eµ-XPO1(WT & E571K)xA20 KO mice additionally featured mild mononuclear infiltrates in the lung and salivary glands; all of which were observed with increased severity compared to A20KO, Eµ-XPO1, or non-transgenic littermates. Significance: We identified co-occurring genetic lesions impacting A20 and XPO1 in PMBCL tumors and show that these alterations synergize in vivo leading to nodal/extranodal lymphocyte infiltration. The immunomodulatory effects appear to be driven by the cooperation of XPO1 overexpression, more so than mutation, and A20 inactivation in young mice. Further investigation of these models will allow elucidation of underlying cooperative mechanisms and reveal targetable pathways in PMBCL.

Naturally Occurring Mutations in Human T Cell Lymphomas Enhance Engineered T Cell Therapies

T cell therapies, including chimeric antigen receptor (CAR) T cells, have revolutionized cancer therapy. However, impressive responses are limited to a subset of patients with hematological cancers and have not been unlocked in patients with solid tumors, which represent 90% of adult cancers. Adoptive T cell therapy in both treatment-resistant hematological malignancies and solid cancers is limited by a complex combination of factors, including poor in vivo persistence, immunosuppressive environmental factors, and T cell exhaustion. New solutions to overcome these limitations are needed to address the significant unmet need for effective cell therapies. To overcome this engineering problem, we turned to spontaneously occurring T cell cancers. Over time, T cells, like other cells in the adult, acquire somatic mutations. When these mutations increase the fitness of the T cell, T cell clones emerge via positive selection. These T cell cancers develop genetic means to overcome similar cell-intrinsic and cell-extrinsic immunosuppressive factors facing anti-tumor T cells. Additionally, mutations from T cell cancers may unlock modifications not available by current screening approaches, because evolution employs any mutation available in nature. Like existing approaches, these mutations can modify gene expression by gene locus duplication or deletion. However, unlike current approaches, gain-of-function point mutations or translocations can be introduced that have unique, outsized effects unachievable by modulation of wild-type gene expression. Here, we have generated a first-of-its-kind library of 71 mutations and 45 wild-type over-expression controls. We performed in vitro and in vivo screening to identify the effects of these mutations on CAR signaling, cytokine production, and accumulation in vivo in tumors. These screens converged on a gene fusion, CARD11-PIK3R3. Mechanistically, CARD11-PIK3R3 enhances CAR-dependent CARD11-BCL10-MALT1 complex signaling including NF-κB/AP-1 transcriptional activity and MALT1 substrate cleavage, in a BCL10 dependent manner. In vivo, CARD11-PIK3R3 transduced T cells exhibit enhanced intratumoral accumulation, effector functions, and therapeutic efficacy. Importantly, CARD11-PIK3R3 expression was sufficient to reduce T cell dose requirements and protect against tumor re-challenge in an immunotherapy refractory syngeneic model in the absence of lymphodepletion. Our results indicate that exploiting naturally occurring mutations represents a promising approach to explore the extremes of T cell biology and discover how solutions derived from evolution of malignant T cells can improve a broad range of T cell therapies. Notably, the gene fusion we identified would be inaccessible through previous T cell screening efforts such as loss of function, CRISPR activation, or wild-type gene over-expression screens. Finally, our results implicate the CARD11-BCL10-MALT1 signalosome as a key regulator of therapeutic T cell function. Altogether, investigation of T cell lymphoma mutations in the context of adoptively transferred T cells has the promise to both improve cellular therapies and to elucidate new T cell biology.

Real-time resolution studies of the regulation of pyruvate-dependent lactate metabolism by hexokinases in single cells.

Lactate is a mitochondrial substrate for many tissues including neuron, muscle, skeletal and cardiac, as well as many cancer cells, however little is known about the processes that regulate its utilization in mitochondria. Based on the close association of Hexokinases (HK) with mitochondria, and the known cardio-protective role of HK in cardiac muscle, we have investigated the regulation of lactate and pyruvate metabolism by hexokinases (HKs), utilizing wild-type HEK293 cells and HEK293 cells in which the endogenous HKI and/or HKII have been knocked down to enable overexpression of wild type and mutant HKs. To assess the real-time changes in intracellular lactate levels the cells were transfected with a lactate specific FRET probe. In the HKI/HKII double knockdown cells, addition of extracellular pyruvate caused a large and sustained decrease in lactate. This decrease was rapidly reversed upon inhibition of the malate aspartate shuttle by aminooxyacetate, or inhibition of mitochondrial oxidative respiration by NaCN. These results suggest that in the absence of HKs, pyruvate-dependent activation of the TCA cycle together with the malate aspartate shuttle facilitates lactate transformation into pyruvate and its utilization by mitochondria. With replacement by overexpression of HKI or HKII the cellular response to pyruvate and NaCN was modified. With either hexokinase present, both the decrease in lactate due to the addition of pyruvate and the increase following addition of NaCN were either transient or suppressed altogether. Blockage of the pentose phosphate pathway with the inhibitor 6-aminonicotinamide (6-AN), abolished the effects of HK replacement. These results suggest that blocking of the malate aspartate shuttle by HK may involve activation of the pentose phosphate pathway and increased NADPH production.

Physiologic and Transcriptomic Effects Triggered by Overexpression of Wild Type and Mutant DNA Topoisomerase I in Streptococcus pneumoniae.

Topoisomerase I (TopoI) in Streptococcus pneumoniae, encoded by topA, is a suitable target for drug development. Seconeolitsine (SCN) is a new antibiotic that specifically blocks this enzyme. We obtained the topARA mutant, which encodes an enzyme less active than the wild type (topAWT) and more resistant to SCN inhibition. Likely due to the essentiality of TopoI, we were unable to replace the topAWT allele by the mutant topARA version. We compared the in vivo activity of TopoIRA and TopoIWT using regulated overexpression strains, whose genes were either under the control of a moderately (PZn) or a highly active promoter (PMal). Overproduction of TopoIRA impaired growth, increased SCN resistance and, in the presence of the gyrase inhibitor novobiocin (NOV), caused lower relaxation than TopoIWT. Differential transcriptomes were observed when the topAWT and topARA expression levels were increased about 5-fold. However, higher increases (10-15 times), produced a similar transcriptome, affecting about 52% of the genome, and correlating with a high DNA relaxation level with most responsive genes locating in topological domains. These results confirmed that TopoI is indeed the target of SCN in S. pneumoniae and show the important role of TopoI in global transcription, supporting its suitability as an antibiotic target.

Neuroligin 2 governs synaptic morphology and function through RACK1-cofilin signaling in Drosophila

Neuroligins are transmembrane cell adhesion proteins well-known for their genetic links to autism spectrum disorders. Neuroligins can function by regulating the actin cytoskeleton, however the factors and mechanisms involved are still largely unknown. Here, using the Drosophila neuromuscular junction as a model, we reveal that F-Actin assembly at the Drosophila NMJ is controlled through Cofilin signaling mediated by an interaction between DNlg2 and RACK1, factors not previously known to work together. The deletion of DNlg2 displays disrupted RACK1-Cofilin signaling pathway with diminished actin cytoskeleton proteo-stasis at the terminal of the NMJ, aberrant NMJ structure, reduced synaptic transmission, and abnormal locomotion at the third-instar larval stage. Overexpression of wildtype and activated Cofilin in muscles are sufficient to rescue the morphological and physiological defects in dnlg2 mutants, while inactivated Cofilin is not. Since the DNlg2 paralog DNlg1 is known to regulate F-actin assembly mainly via a specific interaction with WAVE complex, our present work suggests that the orchestration of F-actin by Neuroligins is a diverse and complex process critical for neural connectivity.

Differential Effects of Overexpression of Wild Type and Kinase-Dead MELK in Fibroblasts and Keratinocytes, Potential Implications for Skin Wound Healing and Cancer.

Maternal embryonic leucine-zipper kinase (MELK) plays a significant role in cell cycle progression, mitosis, cell migration, cell renewal, gene expression, embryogenesis, proliferation, apoptosis, and spliceosome assembly. In addition, MELK is known to be overexpressed in multiple types of cancer and is associated with cancer proliferation. Tumorigenesis shares many similarities with wound healing, in which the rate of cell proliferation is a critical factor. Therefore, this study aimed to determine the involvement of MELK in the regulation of cell division in two cell types involved in this process, namely fibroblasts and keratinocytes. We examined how temporal overexpression of wild-type and kinase-dead MELK kinase variants affect the rate of proliferation, viability, cell cycle, and phosphorylation state of other kinases involved in these processes, such as ERK1/2, AKT1, MAPK9, p38, and p53. We explored if MELK could be used as a therapeutic stimulator of accelerated wound healing via increased proliferation. We observed that aberrant expression of MELK results in abnormal proliferation, altered cell cycle distribution, and decreased viability of the cells, which challenge the utility of MELK in accelerated wound healing. Our results indicate that, at least in healthy cells, any deviation from precisely controlled MELK expression is harmful to fibroblasts and keratinocytes.

Abstract 3482: METTL3-mediated m6A modification controls splicing factor abundance and contributes to CLL progression

Abstract RNA splicing dysregulation is a hallmark of cancers and underlies the onset and progression of diseases. Chronic lymphocytic leukemia (CLL) is one of the most common adult leukemia in western countries. Spliceosome mutations occur in ~20% of patients. However, the mechanism for splicing defects in spliceosome unmutated CLL cases remains elusive. Through an integrative transcriptomic and proteomic analysis of primary CLL samples, we discovered proteins involved in RNA splicing are post-transcriptionally upregulated. Coupled with clinical annotation, we found spliceosome protein abundance is an independent risk factor and associated with poor prognosis. Splice variants found in CLL are highly overlapped with those driven by high spliceosome abundance but not splicing factor mutations, indicating high spliceosome abundance contributes to genetic lesion-independent splicing defects. To identify potential regulators for spliceosome, we proteome-widely analyzed the proteins that highly correlated with splicing factors expression. Analysis of 113 CLL samples has consistently identified METTL3 upregulation with positive correlation with 77.6% of detected splicing factors. METTL3 is an RNA methyltransferase that modifies N6-methyladenosine (m6A) on mRNA and regulates the translation of m6A-installed transcripts. m6A level on mRNA is increased in CLL cells with differential m6A highly enriched on splicing related transcripts. Moreover, high METTL3 expression in CLL is also associated with poor clinical outcomes. These results suggested that METTL3 translationally controls splicing factors through m6A and plays a role in CLL progression. Toward this end, we demonstrated that METTL3 is essential for CLL growth in both in vitro and in vivo studies. Knock out (KO) and pharmaceutical inhibition of METTL3 decreased cell growth and splicing factor expression. Overexpression of wildtype but not catalytic mutant METTL3 restored both defects in METTL3 KO cells, indicating that the regulation of splicing factor is m6A-dependent. To dissect the underlying mechanism, we performed an integrated Ribo-seq, RNA-seq, and MeRIP-seq on CLL cells with or without METTL3. KO of METTL3 decreased overall translation efficiency (TE) with RNA splicing as the most significantly affected pathway. Splicing factors with reduced TE displayed either hypo-m6A at stop codon region or hyper-m6A at CDS regions upon METTL3 KO, as direct or indirect targets of METTL3. Moreover, we found that m6A at stop codon and CDS regions regulates splicing factor translation via ribosome recycling and ribosome pausing, respectively. Taken together, our results uncovered a novel regulatory axis for METTL3 that controls splicing factor translation and contributes to CLL progression. Our study highlights a post-transcriptional layer of m6A modification as a major contributor to genetic lesion-independent splicing defects in CLL. Citation Format: Yiming Wu, Meiling Jin, Mike Fernandez, Kevyn Hart, Aijun Liao, Stacey M. Fernandes, Tinisha McDonald, Zhenhua Chen, Daniel Röth, Lucy Ghoda, Guido Marcucci, Markus Kalkum, Raju K. Pillai, Alexey V. Danilov, Jianjun Chen, Jennifer R. Brown, Steven T. Rosen, Tanya Siddiqi, Lili Wang. METTL3-mediated m6A modification controls splicing factor abundance and contributes to CLL progression [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 3482.

Flotillin-2 regulates epidermal growth factor receptor activation, degradation by Cbl-mediated ubiquitination, and cancer growth

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene (Cbl) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. Here, we used stable isotope labeling with amino acids in cell culture mass spectrometry to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and endosomal trafficking. Furthermore, we determined that FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl dependent, as coknockdown of Cbl and Cbl-b restored EGFR levels. In addition, FLOT2 overexpression decreased EGFR signaling and growth. Overexpression of wildtype (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. Together, these data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation invitro and invivo.

CaMKII T286 phosphorylation has distinct essential functions in three forms of long-term plasticity

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) mediates long-term potentiation or depression (LTP or LTD) after distinct stimuli of hippocampal NMDA-type glutamate receptors (NMDARs). NMDAR-dependent LTD prevails in juvenile mice, but a mechanistically different form of LTD can be readily induced in adults by instead stimulating metabotropic glutamate receptors (mGluRs). However, the role that CaMKII plays in the mGluR-dependent form of LTD is not clear. Here we show that mGluR-dependent LTD also requires CaMKII and its T286 autophosphorylation (pT286), which induces Ca2+-independent autonomous kinase activity. In addition, we compared the role of pT286 among three forms of long-term plasticity (NMDAR-dependent LTP and LTD, and mGluR-dependent LTD) using simultaneous live imaging of endogenous CaMKII together with synaptic marker proteins. We determined that after LTP stimuli, pT286 autophosphorylation accelerated CaMKII movement to excitatory synapses. After NMDAR-LTD stimuli, pT286 was strictly required for any movement to inhibitory synapses. Similar to NMDAR-LTD, we found the mGluR-LTD stimuli did not induce CaMKII movement to excitatory synapses. However, in contrast to NMDAR-LTD, we demonstrate that the mGluR-LTD did not involve CaMKII movement to inhibitory synapses and did not require additional T305/306 autophosphorylation. Thus, despite its prominent role in LTP, we conclude that CaMKII T286 autophosphorylation is also required for both major forms of hippocampal LTD, albeit with differential requirements for the heterosynaptic communication of excitatory signals to inhibitory synapses.

Inhibition of Epidermal Growth Factor Receptor Signaling by Antisense Oligonucleotides as a Novel Approach to Epidermal Growth Factor Receptor Inhibition.

We report a novel method to inhibit epidermal growth factor receptor (EGFR) signaling using custom morpholino antisense oligonucleotides (ASOs) to drive expression of dominant negative mRNA isoforms of EGFR by ASO-induced exon skipping within the transmembrane (16) or tyrosine kinase domains (18 and 21). In vivo ASO formulations induced >95% exon skipping in several models of nonsmall cell lung cancer (NSCLC) and were comparable in efficacy to erlotinib in reducing colony formation, cell viability, and migration in EGFR mutant NSCLC (PC9). However, unlike erlotinib, ASOs maintained their efficacy in both erlotinib-resistant subclones (PC9-GR) and wild-type overexpressing EGFR models (H292), in which erlotinib had no significant effect. The most dramatic ASO-induced phenotype resulted from targeting the EGFR kinase domain directly, which resulted in maximal inhibition of phosphorylation of EGFR, Akt, and Erk in both PC9 and PC9GR cells. Phosphoproteomic mass spectrometry confirmed highly congruent impacts of exon 16-, 18-, and 21-directed ASOs compared with erlotinib on PC9 genome-wide cell signaling. Furthermore, EGFR-directed ASOs had no impact in EGFR-independent NSCLC models, confirming an EGFR-specific therapeutic mechanism. Further exploration of synergy of ASOs with existing tyrosine kinase inhibitors may offer novel clinical models to improve EGFR-targeted therapies for both mutant and wild-type NSCLC patients.

Mutations and clinical significance of calcium voltage-gated channel subunit alpha 1E (CACNA1E) in non-small cell lung cancer

CACNA1E is a gene encoding the ion-conducting α1 subunit of R-type voltage-dependent calcium channels, whose roles in tumorigenesis remain to be determined. We previously showed that CACNA1E was significantly mutated in patients with non-small cell lung cancer (NSCLC) who were long-term exposed to household air pollution, with a mutation rate of 19% (15 of 79 cases). Here we showed that CACNA1E was also mutated in 207 (12.8%) of the 1616 patients with NSCLC in The Cancer Genome Atlas (TCGA) datasets. At mRNA and protein levels, CACNA1E was elevated in tumor tissues compared to counterpart non-tumoral lung tissues in NSCLCs of the public datasets and our settings, and its expression level was inversely associated with clinical outcome of the patients. Overexpression of wild type (WT) or A275S or R249G mutant CACNA1E transcripts promoted NSCLC cell proliferation with activation of epidermal growth factor receptor (EGFR) signaling pathway, whereas knockdown of this gene exerted inhibitory effects on NSCLC cells in vitro and in vivo. CACNA1E increased current density and Ca2+ entrance, whereas calcium channel blockers inhibited NSCLC cell proliferation. These data indicate that CACNA1E is required for NSCLC cell proliferation, and blockade of this oncoprotein may have therapeutic potentials for this deadly disease.

HEXIM1 Is Essential for the Establishment of Appropriate Patterns of Gene Expression and Chromatin Architecture during Terminal Erythroid Maturation

Terminal erythroid maturation is associated with dramatic changes in gene expression in the setting of a cell that is undergoing rapid division and nuclear condensation. Disruption of this process is associated with inherited anemias and myelodysplastic syndromes. Recent work from our laboratory revealed that terminal erythroid maturation is associated with a dramatic decline in the level of total and elongation competent RNA polymerase II (Pol II), and that control of pol II activity is a critical step in the regulation of gene expression during terminal erythroid maturation. We further demonstrated that HEXIM1, which is highly expressed in early erythroid cells compared to most other cell types (biogps.org; bloodspot.eu), is essential for erythropoiesis (Murphy Blood 2021). The goal of our current study is to understand the mechanisms by which HEXIM1 regulates erythroid gene expression.HEXIM1 can impact gene expression though multiple mechanisms, most notably by associating with pTEFb, which is required for release of “paused” pol II into active transcription (reviewed in Michels, Transcription, 2018). HEXIM1 can inhibit transcription through sequestration of pTEFb in the 7SK ribonuclear complex, rendering it incapable of facilitating pause release. Alternatively, it can activate transcription by delivering pTEFb to target loci (McNamara Genome Data 2016). In erythroid cells, disruption of HEXIM1 impaired the expression of many erythroid specific genes, such as GYPA and many of the heme synthesis enzymes, while overexpression (OE) of HEXIM1 promoted their expression (Murphy, Blood, 2021). We therefore hypothesized that in maturing erythroblasts, HEXIM1 targets pTEFb to erythroid specific genes, promoting the establishment of appropriate patterns of gene expression and facilitating terminal erythroid maturation. To address this hypothesis, we generated novel HUDEP2 lines that OE HEXIM1 with a tyrosine to alanine mutation (Y271A) that prevents phosphorylation of HEXIM1 and subsequent release of pTEFb (Mbonye Proteomics 2015). Biotinylated 7SK pulldown confirmed that the Y271A mutation maintains the ability to bind the 7SK complex in erythroid cell extracts and RNA immunoprecipitation confirmed that the Y271A mutation increases the affinity of HEXIM1 for the 7SK complex in HUDEP2 cells.The Y271A mutation has significant functional consequences in erythroid cells. OE of wild type (WT) HEXIM1 in HUDEP2 cells resulted in enhanced proliferation in both expansion and maturation conditions, which was accompanied by increased cell and nuclear size, and a dramatic increase in the level of CD235a. Similar to our previously published HEXIM1 mutant with tyrosine to phenylalanine mutations at residues 271 and 274, the Y271A HEXIM1 mutation abrogated the enhanced proliferation seen with HEXIM1 OE in both expansion and maturation conditions. The Y271A mutation also rescued the larger cell and nuclear area associated with HEXIM1 OE, as well as the dramatic increase in the level of CD235a. Conversely, disruption of HEXIM1 via genome editing resulted in poor expansion and viability of HUDEP2 cells, which was rescued by expression of WT but not Y271A mutated HEXIM1, highlighting the importance of HEXIM1-pTEFb interactions for erythroid proliferation and survival. Further, OE of WT HEXIM1, but not the Y271A mutant, promoted erythroid gene expression while facilitating repression of genes that are normally silenced during terminal maturation, such as RPS19. In cells expressing WT HEXIM1 these gene expression changes were accompanied by increases in the global levels of ser2 and ser5 phosphorylated Pol II, as well as genome wide changes in their distribution. In contrast, the Y271A mutant decreased the global level of ser2 and ser5 pol II, consistent with its reduced ability to release pTEFb at target genes. Intriguingly, levels of H3K79me2, a histone mark reflective of active transcription through gene bodies, were decreased with OE of both WT and Y271A mutant HEXIM1, suggesting that the ability of HEXIM1 to promote transcriptional activation or repression is context dependent. Together, these data demonstrate a critical role for HEXIM1 and its interaction with pTEFb and the 7SK complex in the establishment of appropriate patterns of gene expression and chromatin architecture in maturing erythroblasts. DisclosuresNo relevant conflicts of interest to declare.