Abstract
Intracellular phase separation is emerging as a universal principle for organizing biochemical reactions in time and space. It remains incompletely resolved how biological function is ...encoded in these assemblies and whether this depends on their material state. The conserved intrinsically disordered protein PopZ forms condensates at the poles of the bacterium
Caulobacter crescentus
, which in turn orchestrate cell-cycle regulating signaling cascades. Here we show that the material properties of these condensates are determined by a balance between attractive and repulsive forces mediated by a helical oligomerization domain and an expanded disordered region, respectively. A series of PopZ mutants disrupting this balance results in condensates that span the material properties spectrum, from liquid to solid. A narrow range of condensate material properties supports proper cell division, linking emergent properties to organismal fitness. We use these insights to repurpose PopZ as a modular platform for generating tunable synthetic condensates in human cells.
Human exportin-1 (XPO1) is the key nuclear-cytoplasmic transport protein that exports different cargo proteins out of the nucleus. Inducing nuclear accumulation of these proteins by inhibiting XPO1 ...causes cancer cell death. First clinical validation of pharmacological inhibition of XPO1 was obtained with the Selective Inhibitor of Nuclear Export (SINE) compound selinexor (KPT-330) demonstrating activity in phase-II/IIb clinical trials when dosed 1 to 3 times weekly. The second-generation SINE compound KPT-8602 shows improved tolerability and can be dosed daily. Here, we investigate and validate the drug-target interaction of KPT-8602 and explore its activity against acute lymphoblastic leukemia (ALL).
We examined the effect of KPT-8602 on XPO1 function and XPO1-cargo as well as on a panel of leukemia cell lines. Mutant XPO1 leukemia cells were designed to validate KPT-8602's drug-target interaction.
, anti-ALL activity was measured in a mouse ALL model and patient-derived ALL xenograft models.
KPT-8602 induced caspase-dependent apoptosis in a panel of leukemic cell lines
Using CRISPR/Cas9 genome editing, we demonstrated the specificity of KPT-8602 for cysteine 528 in the cargo-binding groove of XPO1 and validated the drug target interaction.
, KPT-8602 showed potent anti-leukemia activity in a mouse ALL model as well as in patient-derived T- and B-ALL xenograft models without affecting normal hematopoiesis.
KPT-8602 is highly specific for XPO1 inhibition and demonstrates potent anti-leukemic activity supporting clinical application of the second-generation SINE compound for the treatment of ALL.
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Unraveling the mechanism of action and molecular target of small molecules remains a major challenge in drug discovery. While many cancer drugs target genetic vulnerabilities, loss-of-function ...screens fail to identify essential genes in drug mechanism of action. Here, we report CRISPRres, a CRISPR-Cas-based genetic screening approach to rapidly derive and identify drug resistance mutations in essential genes. It exploits the local genetic variation created by CRISPR-Cas-induced non-homologous end-joining (NHEJ) repair to generate a wide variety of functional in-frame mutations. Using large sgRNA tiling libraries and known drug-target pairs, we validate it as a target identification approach. We apply CRISPRres to the anticancer agent KPT-9274 and identify nicotinamide phosphoribosyltransferase (NAMPT) as its main target. These results present a powerful and simple genetic approach to create many protein variants that, in combination with positive selection, can be applied to reveal the cellular target of small-molecule inhibitors.
BackgroundDespite the success of PD1 blockade in various cancers, overcoming resistance to cancer immunotherapy remains challenging. Targeting CD8+ T cell-associated alternative immune-checkpoints is ...anticipated to overcome this issue. However, such immune-checkpoints are also expressed on myeloid cells, but their therapeutic and clinical impact remains enigmatic. Thus, the aim of this study was to reveal underappreciated TAMs-ontology enriching immune-inhibitory receptors, to design biomarker-driven immunotherapy.MethodsWe used reverse translational methodologies starting from human tumour multi-omics bioinformatics to inform pre-clinical experimental research, culminating into human multi-omics prognostic/predictive validationResultsWe identified a unique niche of tumour-associated macrophages (TAMs), preferentially co-expressing the TIM3 and VISTA immune-checkpoints, that dominated the human and mouse tumours resistant to PD(L)1 blockade. Subcutaneous epithelial-origin tumours and orthotopic melanoma in mouse showed that TIM3+VISTA+TAMs were sustained by IL4/IL13-enriching tumours with low (neo)antigenic and non-immunogenic milieu. TIM3/VISTA were instrumental in sustaining a hyper-efferocytotic and anti-inflammatory TAM phenotype, and blunting type I interferon (IFN) sensing, thereby fuelling immune subversion. This was established with cancer cells succumbing to immunogenic cell death (ICD). Herein, while dying cancer cells triggered autocrine type I IFN production, yet they also exposed extracellular HMGB1 and surface VISTA as ligands to engage TIM3 and VISTA on TAMs respectively, to suppress paracrine IFN responses. Consequently, TIM3/VISTA blockade preferentially synergized with paclitaxel, an ICD inducing chemotherapy in vivo, to replace the anti-inflammatory TIM3+VISTA+TAMs with pro-inflammatory TAMs-driven cytotoxicity, thus blunting the immuno-resistant tumours. In vivo macrophage-specific genetic knockout of TIM3/VISTA confirmed this synergism, while immune/genetic ablation of type I IFN sensing, macrophages (but not CD8+T cells), or cancer cell associated HMGB1/VISTA disrupted it. Finally, TIM3+VISTA+TAM signature exhibited pan-cancer negative prognostic impact and predicted resistance to immunotherapy in patients.ConclusionsWe discovered that as-yet-uncharacterized TIM3+VISTA+TAMs, enriched by human and mouse non-immunogenic tumours, mediate chemo-immunotherapy resistance. Thus, targeting TIM3+VISTA+TAMs is a novel and conserved strategy to overcome low neo-antigenic, CD8+T cells independent, tumours.Ethics ApprovalMouse Experiments were approved by the animal ethics committee at KU Leuven (project P114/2019 and p195/2020) following the European directive 2010/63/EU as amended by the Regulation (EU) 2019/1010 and the Flemish government decree of 17 February 2017.
Infection with HIV ultimately leads to advanced immunodeficiency resulting in an increased incidence of cancer. For example primary effusion lymphoma (PEL) is an aggressive non-Hodgkin lymphoma with ...very poor prognosis that typically affects HIV infected individuals in advanced stages of immunodeficiency. Here we report on the dual anti-HIV and anti-PEL effect of targeting a single process common in both diseases. Inhibition of the exportin-1 (XPO1) mediated nuclear transport by clinical stage orally bioavailable small molecule inhibitors (SINE) prevented the nuclear export of the late intron-containing HIV RNA species and consequently potently suppressed viral replication. In contrast, in CRISPR-Cas9 genome edited cells expressing mutant C528S XPO1, viral replication was unaffected upon treatment, clearly demonstrating the anti-XPO1 mechanism of action. At the same time, SINE caused the nuclear accumulation of p53 tumor suppressor protein as well as inhibition of NF-κB activity in PEL cells resulting in cell cycle arrest and effective apoptosis induction. In vivo, oral administration arrested PEL tumor growth in engrafted mice. Our findings provide strong rationale for inhibiting XPO1 as an innovative strategy for the combined anti-retroviral and anti-neoplastic treatment of HIV and PEL and offer perspectives for the treatment of other AIDS-associated cancers and potentially other virus-related malignancies.
•Selective inhibition of XPO1 suppresses the replication of HIV and induces apoptosis in PEL cells.•Clinical stage orally bioavailable XPO1 inhibitors display potent anti-HIV and anti-PEL activity.•This study validates XPO1 inhibition as a treatment strategy for PEL, especially in the setting of HIV-infection.
Infection with human immunodeficiency virus (HIV) compromises the body's immune system leaving infected individuals vulnerable to other pathologies including cancer. Some forms of cancer typically develop in AIDS patients, as for example the very aggressive and most often deadly primary effusion lymphoma (PEL). There is currently no standard treatment for PEL but the use of anti-HIV drugs is associated with better prognosis. Here we show in preclinical tests that inhibitors of nuclear export suppress both HIV replication as well as PEL progression. These findings provide a rationale for further evaluating these inhibitors as treatment strategy for dual HIV/lymphoma therapy.
An enzymatic method has been successfully established enabling the generation of partially base-modified RNA (previously named RZA) constructs, in which all G residues were replaced by isomorphic ...fluorescent thienoguanosine (thG) analogs, as well as fully modified RZA featuring thG, 5-bromocytosine, 7-deazaadenine and 5-chlorouracil. The transcriptional efficiency of emissive fully modified RZA was found to benefit from the use of various T7 RNA polymerase variants. Moreover, dthG could be incorporated into PCR products by Taq DNA polymerase together with the other three base-modified nucleotides. Notably, the obtained RNA products containing thG as well as thG together with 5-bromocytosine could function as effectively as natural sgRNAs in an in vitro CRISPR-Cas9 cleavage assay. N1-Methylpseudouridine was also demonstrated to be a faithful non-canonical substitute of uridine to direct Cas9 nuclease cleavage when incorporated in sgRNA. The Cas9 inactivation by 7-deazapurines indicated the importance of the 7-nitrogen atom of purines in both sgRNA and PAM site for achieving efficient Cas9 cleavage. Additional aspects of this study are discussed in relation to the significance of sgRNA-protein and PAM--protein interactions that were not highlighted by the Cas9-sgRNA-DNA complex crystal structure. These findings could expand the impact and therapeutic value of CRISPR-Cas9 and other RNA-based technologies.
The ongoing COVID-19 pandemic has caused a global economic and health crisis. To identify host factors essential for coronavirus infection, we performed genome-wide functional genetic screens with ...severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human coronavirus 229E. These screens uncovered virus-specific as well as shared host factors, including TMEM41B and PI3K type 3. We discovered that SARS-CoV-2 requires the lysosomal protein TMEM106B to infect human cell lines and primary lung cells. TMEM106B overexpression enhanced SARS-CoV-2 infection as well as pseudovirus infection, suggesting a role in viral entry. Furthermore, single-cell RNA-sequencing of airway cells from patients with COVID-19 demonstrated that TMEM106B expression correlates with SARS-CoV-2 infection. The present study uncovered a collection of coronavirus host factors that may be exploited to develop drugs against SARS-CoV-2 infection or future zoonotic coronavirus outbreaks.
While drug resistance mutations provide the gold standard proof for drug target engagement, target deconvolution of inhibitors identified from a phenotypic screen remains challenging. Genetic ...screening for functional in-frame drug resistance mutations by tiling CRISPR-Cas nucleases across protein coding sequences is a method for identifying a drug’s target and binding site. However, the applicability of this approach is constrained by the availability of nuclease target sites across genetic regions that mediate drug resistance upon mutation. In this study, we show that an enhanced AsCas12a variant (enAsCas12a), which harbors an expanded targeting range, facilitates screening for drug resistance mutations with increased activity and resolution in regions that are not accessible to other CRISPR nucleases, including the prototypical SpCas9. Utilizing enAsCas12a, we uncover new drug resistance mutations against inhibitors of NAMPT and KIF11. These findings demonstrate that enAsCas12a is a promising new addition to the CRISPR screening toolbox and allows targeting sites not readily accessible to SpCas9.
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CRISPR-Cas mutagenesis screening across protein coding sequences allows for drug target deconvolution. However, the applicability of this approach can be severely restricted by the absence of CRISPR target sites. In this study, we show that the enhanced AsCas12a (enAsCas12a) CRISPR endonuclease, which harbors an expanded targeting range, facilitates efficient screening for drug resistance mutations.
SARS-CoV-2 is associated with broad tissue tropism, a characteristic often determined by the availability of entry receptors on host cells. Here, we show that TMEM106B, a lysosomal transmembrane ...protein, can serve as an alternative receptor for SARS-CoV-2 entry into angiotensin-converting enzyme 2 (ACE2)-negative cells. Spike substitution E484D increased TMEM106B binding, thereby enhancing TMEM106B-mediated entry. TMEM106B-specific monoclonal antibodies blocked SARS-CoV-2 infection, demonstrating a role of TMEM106B in viral entry. Using X-ray crystallography, cryogenic electron microscopy (cryo-EM), and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we show that the luminal domain (LD) of TMEM106B engages the receptor-binding motif of SARS-CoV-2 spike. Finally, we show that TMEM106B promotes spike-mediated syncytium formation, suggesting a role of TMEM106B in viral fusion. Together, our findings identify an ACE2-independent SARS-CoV-2 infection mechanism that involves cooperative interactions with the receptors heparan sulfate and TMEM106B.
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•TMEM106B directly engages the receptor-binding domain of SARS-CoV-2 spike•Substitution E484D increases TMEM106B binding, enhancing TMEM106B-mediated entry•TMEM106B-specific antibodies neutralize SARS-CoV-2 infection•TMEM106B promotes spike-mediated syncytium formation
The lysosomal transmembrane protein TMEM106B can serve as an alternative receptor for SARS-CoV-2 entry into ACE2-negative cells. Spike substitution E484D improves spike binding to TMEM106B, enhancing TMEM106B-mediated SARS-CoV-2 infection.
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