Activating mutations in KRAS occur in approximately 30% of lung adenocarcinomas. Despite advances in RAS-targeted therapies, intrinsic resistance limits their long-term efficacy. Here, we identify elevated levels of wild-type KRAS (WT-KRAS) protein as a key driver of intrinsic resistance in KRAS-mutant lung tumors. KRAS accumulation results from impaired LZTR1-mediated degradation, triggered either by LZTR1 loss or pharmacological RAS inhibition. Stabilized WT-KRAS activates the mTOR/HIF1α pathway by promoting lysosomal recruitment of the SLC3A2/SLC7A5 amino acid transporter complex, reprogramming lysosomal amino acid sensing. Shallow deletions of LZTR1, present in up to 40% of KRAS-mutant lung adenocarcinomas, are associated with increased mTOR activity and may contribute to therapeutic resistance to RAS inhibitors. Co-inhibition of mTOR or the SLC3A2/SLC7A5 complex using dactolisib or JPH203 restores sensitivity to KRAS inhibitors in vitro and in vivo. These findings support combinatorial targeting of mTOR signaling or amino acid transport to overcome intrinsic resistance in KRAS-mutant lung cancer.

Almost 30% of lung adenocarcinomas are driven by activating KRAS mutations. The heterogeneous clinical behavior observed in these cancers could be due to the imbalance of wild-type and oncogenic KRAS alleles. However, the role of RAS dysregulation at the protein level needs to be further explored. A genome-wide global protein stability screen identified the CUL3 ubiquitin ligase adaptor LZTR1, as a major proteostatic regulator of wild-type but not mutant KRAS. In KRAS -mutant lung adenocarcinoma, shallow deletion of LZTR1 is observed in up to 50% of patients and is associated with hypoxic signatures and poorer progression-free disease survival. In a Kras -mutant lung cancer mouse model, heterozygous loss of Lztr1 promoted tumor growth, led to peritumoral vascular remodeling, and limited the response to the KRAS-G12D inhibitor MRTX1133. The vascular alteration in LZTR1 -depleted lung cancer was mediated by increased wild-type KRAS protein dosage, which promoted mTOR pathway activation and a subsequent increase in VEGFA secretion. The inhibition of the PI3K/mTOR pathway using dactolisib normalized tumor vasculature, improved drug delivery, and overcame resistance to KRAS-G12D inhibitors. In summary, the dysregulation of RAS proteostasis contributes to lung tumorigenesis, and targeting wild-type KRAS signaling is crucial to overcome intrinsic resistance to inhibitors of mutant KRAS.Significance Our study highlights the impact of RAS proteostasis on lung cancer development and progression. Vascular normalization achieved by suppressing wild-type KRAS signaling improved the delivery and response to MRTX1133 in KRAS -mutant lung cancer with shallow LZTR1 deletion.### Competing Interest StatementThe authors have declared no competing interest.

The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not entirely understood. The most prevalent post-translational modification within the GTPase core domain of NRAS and KRAS is ubiquitination at lysine 128 (K128), which is significantly decreased in cancer samples compared to normal tissue. Here, we found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAPs), NF1 and RASA1, thus increasing RAS binding to GAP proteins and promoting GAP-mediated GTP hydrolysis. Stimulation of cultured cancer cells with growth factors or cytokines transiently induces K128 ubiquitination and restricts the extent of wild-type RAS activation in a GAP-dependent manner. In KRAS mutant cells, K128 ubiquitination limits tumor growth by restricting RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Reduction of K128 ubiquitination activates both wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven pancreatic tumorigenesis.

Blood flow produces shear stress exerted on the endothelial layer of the vessels. Spatial characterization of the endothelial proteome is required to uncover the mechanisms of endothelial activation by shear stress, as blood flow varies in the vasculature. An integrative ubiquitinome and proteome analysis of shear-stressed endothelial cells demonstrated that the non-degradative ubiquitination of several GTPases is regulated by mechano-signaling. Spatial analysis reveals increased ubiquitination of the small GTPase RAP1 in the descending aorta, a region exposed to laminar shear stress. The ubiquitin ligase WWP2 is identified as a novel regulator of RAP1 ubiquitination during shear stress response. Non-degradative ubiquitination fine-tunes the function of GTPases by modifying their interacting network. Specifically, WWP2-mediated RAP1 ubiquitination at lysine 31 switches the balance from the RAP1/ Talin 1 (TLN1) toward RAP1/ Afadin (AFDN) or RAP1/ RAS Interacting Protein 1 (RASIP1) complex formation, which is essential to suppress shear stress-induced reactive oxygen species (ROS) production and maintain endothelial barrier integrity. Increased ROS production in endothelial cells in the descending aorta of endothelial-specific Wwp2-knockout mice leads to increased levels of oxidized lipids and inflammation. These results highlight the importance of the spatially regulated non-degradative ubiquitination of GTPases in endothelial mechano-activation.

Supplementary Material and Methods

The RAS pathway is the most frequently activated signaling node in human cancer. Nevertheless, the mechanisms leading to the hyperactivation of wild-type RAS in human pathologies have not been fully elucidated. The GTPase activity of RAS proteins is tightly controlled through a series of post-transcriptional mechanisms, which are commonly distorted in the context of cancer. Here, we explored the functional role of KRAS and NRAS ubiquitination at lysine 128 (K128), which is the most abundant post-translational modification of the RAS proto-oncogenes. Our data indicate that RAS ubiquitination at K128 promotes GTP hydrolysis and impedes downstream signaling via a bimodal mechanism. Ubiquitination at K128 not only destabilizes the active RAS clusters at the plasma membrane but also facilitates the interaction of RAS with RAS GTPase-activating proteins. Understanding the complexities of RAS biology may be translated into novel therapeutic approaches to defeating RAS-driven cancers. Citation Format: Wout Magits, Mikhail Steklov, Benoit Lechat, Ruth Nussinov, Anna Sablina. A bimodal mechanism of RAS inactivation by monoubiquitination [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR04.

Dear Editor, Schwannomas are peripheral nervous system tumors that cause chronic pain, numbness, and potentially life-threatening organ dysfunction. Surgery is a conventional treatment for schwannoma patients. However, those who undergo surgery may experience neurological deficits. Moreover, the management of schwannomas can be problematic due to their unpredictable growth. Thus, there is a pressing need for nonsurgical treatments to manage both tumor growth and pain control. Germline mutations of leucine zipper like post-translational regulator 1 (LZTR1), a 22q tumor suppressor gene, account for approximately 30% of all non-NF2-related schwannomatosis cases [1]. LZTR1 loss is associated with mitogen-activated protein kinases (MAPK)1 activation [2]. Given that standard clinical trials are exceedingly difficult for schwannoma patients, mouse models offer the possibility of bespoke therapeutic regimens. Only a few animal models of schwannomatosis exist in mice, as only a model combining Lztr1 and cyclin-dependent kinase inhibitor 2A (Cdkn2A) mutations in the nervous system using a Glial fibrillary acidic protein-driven Cre recombinase was reported [3], highlighting a need for models that faithfully recapitulate LZTR1-mutated schwannoma, using proper Schwann cell-specific promoters. To model germline Lztr1 loss of function, we induced Lztr1 knockout in Schwann cell precursors using a Cre recombinase (cre) driven by the myelin protein zero (P0) (Mpz) promoter. We observed a decrease in Lztr1 expression in oligodendrocyte marker O4 (OLIG4)-positive Schwann cells from P0 cre Lztr1flox allele (fl);fl mice, confirming the specific deletion of Lztr1 in Schwann cells (Supplementary Figure S1A-B). However, we did not detect any phenotypic changes in the Mpz-specific Lztr1 knockout mice (Figure 1A), which could be explained by the fact that germline LZTR1 mutations in patients are frequently accompanied by a somatic 22q chromosomal region loss, containing LZTR1 and the other 22q tumor suppressors, such as Neurofibromatosis type 2 (NF2) and SWI/SNF related, matrix associated, actin dependent regulator of chromatin subfamily B member 1 (SMARCB1) [4]. NF2 encodes a moesin-ezrin-radixin-like tumor suppressor (MERLIN), which negatively regulates the Hippo pathway through large tumor suppressor kinases, LATS1 and LATS2. Either Schwann-cell specific Nf2 or a double Lats1/2 knockout leads to schwannomatogenesis in mice [5, 6], whereas MAPK signaling has been proposed as a modifier of schwannoma formation [5]. The role of LZTR1 in peripheral nerve tumor progression. (A) T2-weighted MRI imaging of 30-week-old mice. The yellow boxes indicate the vestibulocochlear nerve. H&E staining was performed on the isolated vestibulocochlear nerve of the same mice. Scale bar: 1 mm and 100 μm. Immunostaining for S100, F4/80, phosphorylated MEK1/2 (p-MEK1/2), and Ki-67 of the vestibulocochlear nerves. Scale bar: 100μm and 30 μm, respectively. (B) Quantification of the vestibulocochlear nerve area based on the T2-weighted MRI images. n = 6 per group. (C) Maximum intensity projection PET-CT images of 100-week-old mice after [18F]-FDG injection. The tumor volume in the abdominal area was quantified using PET-CT volumes. n = 8 per group. (D) Heatmap of proteins differentially expressed in human primary Schwann cells expressing shGFP or shLZTR1. n = 3 per group. (E) ARCHSF4 and IPA upstream analyses of the differentially expressed proteins. (F) Analysis of OLIG4-positive cells isolated from the sciatic nerves by immunoblotting. Values are means of phosphorylated relative to total STAT1 levels ± SEM. n = 4 per group. (G) Analysis of HEI-193 cells expressing shGFP or shLZTR1 treated with fludarabine (0.01 mmol/L, 48 hours) by immunoblotting. Values are means of phosphorylated (p) relative to total STAT1 levels. n = 4 per group. (H) Quantification of mRNA expression in OLIG4-positive cells isolated from the sciatic nerve. n = 5 per group. (I) Quantification of mRNA expression in HEI-193 cells treated with fludarabine (0.01 mmol/L, 48 hours). n = 4 per group. (J) Secretome analysis of OLIG4-positive cells isolated from the sciatic nerves. n = 5 per group. (K) Secretome analysis of HEI-193 cells expressing shGFP or shLZTR1. n = 5 per group. (L) PET-CT images of 100-week-old mice injected with [18F]-FDG after treatment with fludarabine (100 mg/kg, 14 days every 2 days). SUV-scaled tracer uptake quantification in the abdomen. n = 4 per group. (M, N) Immunohistochemical analysis for Ki67 and F4/80 expression in the uterus schwannomas after treatment with fludarabine (100 mg/kg, 14 days every 2 days). Scale bar: 100 μm. Data are shown as mean ± SEM. B, C, J, K and L, P values: Wilcoxon Mann-Whitney test. F, G and H, P values: Mann Whitney test. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001. Abbreviations: [18F]-FDG, 18F-fluorodeoxyglucose; +, wild type; ARCHSF4, All RNA-seq and ChIP-seq sample and signature search; F4/80 (EMR1), EGF-like module-containing mucin-like hormone receptor-like 1; Fl, flox allele; GFP, Green Fluorescent Protein; HEI-193, House Ear Institute 193; IPA, Ingenuity Pathway Analysis; Ki67, marker of proliferation Ki67; LZTR1, leucine zipper like post-translational regulator; MEK1/ 2, mitogen-activated protein kinase 1/ 2; MRI, magnetic resonance imaging; ns, not significant; PET-CT, Positron Emission Tomography and Computed Tomography; OLIG4, oligodendrocyte marker O4; SEM, standard error of the mean; sh, short hairpin; STAT1, signal transducer and activator of transcription 1; SUV, standardized uptake value. To model LZTR1-mutant schwannoma development in humans, we generated a Schwann cell-specific triple Lztr1, Lats1, and Lats2 knockout mice. Although in the P0 cre Lztr1fl;fl; Lats1/2fl;+ mice, the loss of Lztr1 and Lats1/2 is not sequential, our model partially recapitulates the LZTR1-mutant schwannoma occurring in patients. We observed sporadic unilateral sciatic, nasofacial, and peripheral nerve schwannomas in P0 cre Lztr1+;+ Lats1/2fl;+ and P0 cre Lztr1fl;fl Lats1/2fl;+ mice (Supplementary Figure S1C-E). Hence, our model partially recapitulates schwannoma development, which also appears sporadically and displays unpredictable clinical behavior in patients. To unbiasedly detect Schwann cell-derived tumors, we performed magnetic resonance imaging on 30-week-old mice. Vestibulocochlear nerve enlargement was detected in the P0 cre Lztr1fl;fl Lats1/2fl;+ when compared to the P0 cre Lztr1fl;fl Lats1/2+;+ and P0 cre Lztr1+;+ Lats1/2fl;+ mice (Figure 1A-B). We also detected tumor masses in the abdomen of 100-week-old mice using a positron emission tomography-computed tomography (PET-CT) scan with 18F-fluorodeoxyglucose ([18F]-FDG) (Figure 1C). Male P0 cre Lztr1fl;fl Lats1/2fl;+ mice developed seminal vesicle Schwann-cell-derived tumors (Supplementary Figure S1F) that mimic rare prostate-type schwannomas [7]. Female P0 cre Lztr1fl;fl Lats1/2fl;+ mice presented cervical Schwann-cell-derived tumors (Supplementary Figure S1G) that approximate rare uterus-derived schwannomas [8]. Volumetric analysis of PET-CT scans with [18F]-FDG revealed that loss of Lztr1 was associated with higher metabolic activity and increased tumor burden (Figure 1C). Tumor origin was confirmed by S100 and SRY-box transcription factor 2 (SOX2) immunostainings (Figure 1A, Supplemental Figures S1H-I). Concordant to previous reports [2], immunohistochemical analysis of isolated vestibulocochlear nerves revealed that Lztr1 loss led to increased phosphorylation of mitogen-activated protein kinase MEK1/2 in both wild-type and Lats1/2-knockout mice (Figure 1A). Similarly, we observed an activation of extracellular signal-regulated kinases 1/2 (ERK1/2) upon LZTR1 depletion in NF2-mutated schwannoma cell line HEI-193 (Supplementary Figure S2A). Moreover, immunohistochemical analysis of vestibulocochlear nerves isolated from the triple knockout mice also revealed increased cell proliferation and higher levels of macrophage infiltration as detected by Ki67 and F4/80 immunostainings, respectively (Figure 1A, Supplementary Figure S2B). This indicates that concurrent loss of Lztr1 and Lats1/2 could promote schwannoma development intrinsically as well as by altering the tumor microenvironment, which is commonly observed during schwannoma progression in patients [9]. To investigate LZTR1-mediated molecular alterations promoting schwannomatosis, we performed proteomics analysis of human Schwann cells expressing either shGFP or shLZTR1 (Figure 1D). The upstream regulator analyses of the differentially expressed proteins (Supplementary Table S1) showed the activation of the signal transducer and activator of the transcription 1 (STAT1) pathway triggered by LZTR1 depletion (Figure 1E), suggesting the contribution of the STAT1 pathway to LZTR1-mediated disease pathogenesis. STAT1 function is regulated through phosphorylation on Y701 by Janus kinases (JAK) and S727 by MAPK kinases [10]. We found that the Lats1/2 co-deletion induced phosphorylation of STAT1 on Y701 but not on S727 (Figure 1F, Supplementary Figure S2C). In contrast, LZTR1 depletion in Lats1/2 knockout mouse Schwann cells or NF2-mutant HEI-193 cells increased the phosphorylation of STAT1 on S727 (Figure 1F-G, Supplementary Figure S2D), suggesting that LZTR1-mediated increase in the MAPK activity modulates STAT1 function by inducing its phosphorylation on S727. LATS1/2-regulated phosphorylation of Y701 led to increased expression of well-known STAT1 transcriptional targets B-cell lymphoma-extra-large (Bcl-xL) and cyclin D1 (Ccnd1) (Figure 1H). On the other hand, phosphorylation at both sites in the triple knockout cells induced transcription of additional STAT1 targets, such as pro-immunogenic secreted factors, C-C motif chemokine ligand (CCL2) and interleukin 6 (IL6)/ interleukin 8 (IL8) (Figure 1H-I). Concordantly, the cytokine array analysis revealed an increased secretion of multiple cytokines by the P0 cre Lztr1fl;fl Lats1/2fl;+ mouse-isolated Schwann cells (Figure 1J). Similarly, LZTR1 depletion in the HEI-193 cells increased secretion of IL6, IL8, and CCL2 (Figure 1K). The STAT1 inhibitor fludarabine suppressed STAT1 phosphorylation at both sites and decreased CCL2 and IL8 expression (Figure 1G, I). Inhibition of the STAT signaling by either fludarabine or nifuroxazide also abolished the increased secretion of IL6 and IL8 induced by LZTR1 depletion (Supplementary Figure S2E). These results indicate that depletion of the LATS1/2 kinases triggers STAT1-induced expression of cell proliferation and survival regulators, whereas concurrent LZTR1 loss results in a STAT1-dependent increase in cytokine production. We next assessed the therapeutic effect of STAT1 inhibition for schwannoma treatment. We found that in vivo treatment with fludarabine led to a significant decrease of [18F]-FDG uptake by sexual tissue-derived nerve sheath tumors (Figure 1L). Immunohistochemistry analysis confirmed that STAT1 inhibition led to decreased tumor cell proliferation and lower macrophage infiltration within the tumor environment as detected by Ki67 and F4/80 immunostainings, respectively (Figure 1M-N, Supplementary Figure S2F). These results indicate that STAT1 activation contributes to schwannomatosis via both tumor cell-intrinsic and extrinsic mechanisms. The synergistic effect of the Hippo and MAPK pathway activities up-regulates a broader range of STAT1 target genes, resulting in both pro-proliferative and pro-inflammatory phenotypes (Supplementary Figure S2G). In summary, we demonstrated that LZTR1-mediated activation of the MAPK cascade leads to the phosphorylation of STAT1 at S727, whereas LATS1/2 loss promotes the phosphorylation of STAT1 at Y701. Anti-STAT1 therapy decreased inflammation and tumor cell proliferation in schwannomas. These results identified anti-STAT1 therapy as a potential strategy for schwannomas with the loss of LZTR1 function. Tonci Ivanisevic and Raj N Sewduth performed the in vivo and in vitro experiments; Mikhail Steklov performed the sample preparation for Mass Spectrometry; Benoit Lechat performed the genotyping and cloning; Christopher Cawthorne performed the PET-CT under the supervision of Christophe Deroose and Koen Van Laere; Christophe Deroose and Koen Van Laere provided the tracer for PET-CT; Tonci Ivanisevic performed the CT-scan under the supervision of Greetje Vande Velde; Willy Gsell performed the MRI under the supervision Uwe Himmelreich; the data analysis was performed by Tonci Ivanisevic, Raj N Sewduth under the supervision of Anna A Sablina, Christopher Cawthorne, Greetje Vande Velde and Uwe Himmelreich; Raj N Sewduth, Anna A Sablina wrote the paper and designed the figures. We thank Dr. Jeremie Vitte and Prof. Marco Giovannini (University of California, Los Angeles) for providing comprehensive training for Tonci Ivanisevic and gifting the HEI-193 cells. We thank Prof. Georg Halder (VIB-KU Leuven Center for Cancer Biology) for gifting transgenic Lats1fl/+; Lats2fl/+ mice. We also thank Jens Wouters (Nuclear Medicine and Molecular Imaging, Department of Imaging and Pathology, KU Leuven) for the help with the MRI acquisition. All authors declare no conflicts of interest. This research was supported by Ub-RASdisease (AS, H2020 ERC, Grant agreement ID: 772649) and the Young Investigator Award (TI, CTF, award ID: 2022-01-004). All procedures involving animals were performed following the guidelines of the KU Leuven institutional animal care and use committee and approved in the project application 179/2020, titled ‘Role of the ubiquitin system in nerve sheath cancer’. Not applicable. All the data are available upon request from the corresponding author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Exosomal transfers represent an important mode of intercellular communication. Syntenin is a small scaffold protein that, when binding ALIX, can direct endocytosed syndecans and syndecan cargo to budding endosomal membranes, supporting the formation of intraluminal vesicles that compose the source of a major class of exosomes. Syntenin, however, can also support the recycling of these same components to the cell surface. Here, by studying mice and cells with syntenin-knock out, we identify syntenin as part of dedicated machinery that integrates both the production and the uptake of secreted vesicles, supporting viral/exosomal exchanges. This study significantly extends the emerging role of heparan sulfate proteoglycans and syntenin as key components for macromolecular cargo internalization into cells.

Abstract Meningiomas are the most common benign brain tumors. Mutations of the E3 ubiquitin ligase TRAF7 occur in 25% of meningiomas and commonly cooccur with mutations in KLF4, yet the functional link between TRAF7 and KLF4 mutations remains unclear. By generating an in vitro meningioma model derived from primary meningeal cells, we elucidated the cooperative interactions that promote meningioma development. By integrating TRAF7-driven ubiquitinome and proteome alterations in meningeal cells and the TRAF7 interactome, we identified TRAF7 as a proteostatic regulator of RAS-related small GTPases. Meningioma-associated TRAF7 mutations disrupted either its catalytic activity or its interaction with RAS GTPases. TRAF7 loss in meningeal cells altered actin dynamics and promoted anchorage-independent growth by inducing CDC42 and RAS signaling. TRAF deficiency–driven activation of the RAS/MAPK pathway promoted KLF4-dependent transcription that led to upregulation of the tumor-suppressive Semaphorin pathway, a negative regulator of small GTPases. KLF4 loss of function disrupted this negative feedback loop and enhanced mutant TRAF7-mediated cell transformation. Overall, this study provides new mechanistic insights into meningioma development, which could lead to novel treatment strategies. Significance: The intricate molecular cross-talk between the ubiquitin ligase TRAF7 and the transcription factor KLF4 provides a first step toward the identification of new therapies for patients with meningioma.

Rationale: Noonan syndrome (NS) is one of the most frequent genetic disorders. Bleeding problems are among the most common, yet poorly defined complications associated with NS. A lack of consensus on the management of bleeding complications in patients with NS indicates an urgent need for new therapeutic approaches. Objective: Bleeding disorders have recently been described in patients with NS harboring mutations of LZTR1 (leucine zipper-like transcription regulator 1), an adaptor for CUL3 (CULLIN3) ubiquitin ligase complex. Here, we assessed the pathobiology of LZTR1-mediated bleeding disorders. Methods and Results: Whole-body and vascular specific knockout of Lztr1 results in perinatal lethality due to cardiovascular dysfunction. Lztr1 deletion in blood vessels of adult mice leads to abnormal vascular leakage. We found that defective adherent and tight junctions in Lztr1 -depleted endothelial cells are caused by dysregulation of vesicular trafficking. LZTR1 affects the dynamics of fusion and fission of recycling endosomes by controlling ubiquitination of the ESCRT-III (endosomal sorting complex required for transport III) component CHMP1B (charged multivesicular protein 1B), whereas NS-associated LZTR1 mutations diminish CHMP1B ubiquitination. LZTR1-mediated dysregulation of CHMP1B ubiquitination triggers endosomal accumulation and subsequent activation of VEGFR2 (vascular endothelial growth factor receptor 2) and decreases blood levels of soluble VEGFR2 in Lztr1 haploinsufficient mice. Inhibition of VEGFR2 activity by cediranib rescues vascular abnormalities observed in Lztr1 knockout mice Conclusions: Lztr1 deletion phenotypically overlaps with bleeding diathesis observed in patients with NS. ELISA screening of soluble VEGFR2 in the blood of LZTR1 -mutated patients with NS may predict both the severity of NS phenotypes and potential responders to anti-VEGF therapy. VEGFR inhibitors could be beneficial for the treatment of bleeding disorders in patients with NS.

Dysregulated splicing is a common event in cancer even in the absence of mutations in the core splicing machinery. The aberrant long non-coding transcriptome constitutes an uncharacterized level of regulation of post-transcriptional events in cancer. Here, we found that the stress-induced long non-coding RNA (lncRNA), LINC02657 or LASTR (lncRNA associated with SART3 regulation of splicing), is upregulated in hypoxic breast cancer and is essential for the growth of LASTR-positive triple-negative breast tumors. LASTR is upregulated in several types of epithelial cancers due to the activation of the stress-induced JNK/c-JUN pathway. Using a mass-spectrometry based approach, we identified the RNA-splicing factor SART3 as a LASTR-interacting partner. We found that LASTR promotes splicing efficiency by controlling SART3 association with the U4 and U6 small nuclear ribonucleoproteins (snRNP) during spliceosome recycling. Intron retention induced by LASTR depletion downregulates expression of essential genes, ultimately decreasing the fitness of cancer cells.

Mild traumatic brain injury (mTBI) can lead to diffuse neurophysical damage as well as cognitive and affective alterations. The nature and extent of behavioral changes after mTBI are still poorly understood and how strong an impact force has to be to cause long-term behavioral changes is not yet known. Here, we examined spatial learning acquisition, retention and reversal in a Morris water maze, and assessed search strategies during task performance after a single, mild, closed-skull traumatic impact referred to as "minimal" TBI. Additionally, we investigated changes in conditioned learning in a contextual fear-conditioning paradigm. Results show transient deficits in spatial memory retention, which, although limited, are indicative of deficits in long-term memory reconsolidation. Interestingly, minimal TBI causes animals to relapse to less effective search strategies, affecting performance after a retention pause. Apart from cognitive deficits, results yielded a sub-acute, transient increase in freezing response after fear conditioning, with no increase in baseline behavior, an indication of a stronger affective reaction to aversive stimuli after minimal TBI or greater susceptibility to stress. Furthermore, western blot analysis showed a short-term increase in hippocampal GFAP expression, most likely indicating astrogliosis, which is typically related to injuries of the central nervous system. Our findings provide evidence that even a very mild impact to the skull can have detectable consequences on the molecular, cognitive and affective-like level. However, these effects seemed to be very transient and reversible.

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We have exploited whole brain microscopy to map the progressive deposition of hyperphosphorylated tau in intact, cleared mouse brain. We found that the three-dimensional spreading pattern of hyperphosphorylated tau in the brain of an aging Tau.P301L mouse model did not resemble that observed in AD patients. Injection of synthetic or patient-derived tau fibrils in the CA1 region resulted in a more faithful spreading pattern. Atlas-guided volumetric analysis showed a connectome-dependent spreading from the injection site and also revealed hyperphosphorylated tau deposits beyond the direct anatomical connections. In fibril-injected brains, we also detected a persistent subpopulation of rod-like and swollen microglia. Furthermore, we showed that the hyperphosphorylated tau load could be reduced by intracranial co-administration of, and to a lesser extent, by repeated systemic dosing with an antibody targeting the microtubule-binding domain of tau. Thus, the combination of targeted seeding and in toto staging of tau pathology allowed assessing regional vulnerability in a comprehensive manner, and holds potential as a preclinical drug validation tool.

The low-density lipoprotein receptor-related protein-1 (LRP1) has a dual role in the metabolism of the amyloid precursor protein (APP). In cellular models, LRP1 enhances amyloid-β (Aβ) generation via APP internalization and thus its amyloidogenic processing. However, conditional knock-out studies in mice define LRP1 as an important mediator for the clearance of extracellular Aβ from brain via cellular degradation or transcytosis across the blood-brain barrier (BBB). In order to analyze the net effect of LRP1 on production and clearance of Aβ in vivo, we crossed mice with impaired LRP1 function with a mouse model of Alzheimer's disease (AD). Analysis of Aβ metabolism showed that, despite reduced Aβ clearance due to LRP1 inactivation in vivo, less Aβ was found in cerebrospinal fluid (CSF) and brain interstitial fluid (ISF). Further analysis of APP metabolism revealed that impairment of LRP1 in vivo shifted APP processing from the Aβ-generating amyloidogenic cleavage by beta-secretase to the non-amyloidogenic processing by alpha-secretase as shown by a decrease in extracellular Aβ and an increase of soluble APP-α (sAPP-α). This shift in APP processing resulted in overall lower Aβ levels and a reduction in plaque burden. Here, we present for the first time clear in vivo evidence that global impairment of LRP1's endocytosis function favors non-amyloidogenic processing of APP due to its reduced internalization and subsequently, reduced amyloidogenic processing. By inactivation of LRP1, the inhibitory effect on Aβ generation overrules the simultaneous impaired Aβ clearance, resulting in less extracellular Aβ and reduced plaque deposition in a mouse model of AD.

Alzheimer's disease is the most common neurodegenerative disease, and many patients also present with vascular dysfunction. In this study, we aimed to assess cerebral blood flow (CBF) and cerebrovascular response (CVR) as early, pre-symptomatic (3 months of age), imaging markers in a bigenic model of Alzheimer's disease (APP.V717IxTau.P301L, biAT) and in the monogenic parental strains. We further developed our previously published combination of pulsed arterial spin labeling perfusion MRI and hypo-ventilation paradigm, which allows weaning of the mice from the ventilator. Furthermore, the commonly used isoflurane anesthesia induces vasodilation and is thereby inherently a vascular challenge. We therefore assessed perfusion differences in the mouse models under free-breathing isoflurane conditions. We report (i) that we can determine CBF and hypoventilation-based CVR under ketamine/midazolam anesthesia and wean mice from the ventilator, making it a valuable tool for assessment of CBF and CVR in mice, (ii) that biAT mice exhibit lower cortical CBF than wild-type mice at age 3 months, (iii) that CVR was increased in both biAT and APP.V717I mice but not in Tau.P301L mice, identifying the APP genotype as a strong influencer of brain CVR and (iv) that perfusion differences at baseline are masked by the widely used isoflurane anesthesia.