Mapping the IMiD-dependent cereblon interactome using BioID-proximity labelling.

Immunomodulatory imide drugs (IMiDs) are central components of therapy for multiple myeloma (MM). IMiDs bind cereblon (CRBN), an adaptor for the CUL4-DDB1-RBX1 E3 ligase to change its substrate specificity and induce degradation of 'neosubstrate' transcription factors that are essential to MM cells. Mechanistic studies to date have largely focussed on mediators of therapeutic activity and insight into clinical IMiD toxicities is less developed. We adopted BioID2-dependent proximity labelling (BioID2-CRBN) to characterise the CRBN interactome in the presence and absence of various IMiDs and the proteasome inhibitor, bortezomib. We aimed to leverage this technology to further map CRBN interactions beyond what has been achieved by conventional proteomic techniques. In support of this approach, analysis of cells expressing BioID2-CRBN following IMiD treatment displayed biotinylation of known CRBN interactors and neosubstrates. We observed that bortezomib alone significantly modifies the CRBN interactome. Proximity labelling also suggested that IMiDs augment the interaction between CRBN and proteins that are not degraded, thus designating 'neointeractors' distinct from previously disclosed 'neosubstrates'. Here we identify Non-Muscle Myosin Heavy Chain IIA (MYH9) as a putative CRBN neointeractor that may contribute to the haematological toxicity of IMiDs. These studies provide proof of concept for proximity labelling technologies in the mechanistic profiling of IMiDs and related E3-ligase-modulating drugs.

The Drosophila ZNRF1/2 homologue, detour, interacts with HOPS complex and regulates autophagy.

Autophagy, the process of elimination of cellular components by lysosomal degradation, is essential for animal development and homeostasis. Using the autophagy-dependent Drosophila larval midgut degradation model we identified an autophagy regulator, the RING domain ubiquitin ligase CG14435 (detour). Depletion of detour resulted in increased early-stage autophagic vesicles, premature tissue contraction, and overexpression of detour or mammalian homologues, ZNRF1 and ZNRF2, increased autophagic vesicle size. The ablation of ZNRF1 or ZNRF2 in mammalian cells increased basal autophagy. We identified detour interacting proteins including HOPS subunits, deep orange (dor/VPS18), Vacuolar protein sorting 16A (VPS16A), and light (lt/VPS41) and found that detour promotes their ubiquitination. The detour mutant accumulated autophagy-related proteins in young adults, displayed premature ageing, impaired motor function, and activation of innate immunity. Collectively, our findings suggest a role for detour in autophagy, likely through regulation of HOPS complex, with implications for healthy aging.

Autophagy captures the retromer-TBC1D5 complex to inhibit receptor recycling.

Retromer prevents the destruction of numerous receptors by recycling them from endosomes to the trans-Golgi network or plasma membrane. This enables retromer to fine-tune the activity of many signaling pathways in parallel. However, the mechanism(s) by which retromer function adapts to environmental fluctuations such as nutrient withdrawal and how this affects the fate of its cargoes remains incompletely understood. Here, we reveal that macroautophagy/autophagy inhibition by MTORC1 controls the abundance of retromer+ endosomes under nutrient-replete conditions. Autophagy activation by chemical inhibition of MTOR or nutrient withdrawal does not affect retromer assembly or its interaction with the RAB7 GAP protein TBC1D5, but rather targets these endosomes for bulk destruction following their capture by phagophores. This process appears to be distinct from amphisome formation. TBC1D5 and its ability to bind to retromer, but not its C-terminal LC3-interacting region (LIR) or nutrient-regulated dephosphorylation, is critical for retromer to be captured by autophagosomes following MTOR inhibition. Consequently, endosomal recycling of its cargoes to the plasma membrane and trans-Golgi network is impaired, leading to their lysosomal turnover. These findings demonstrate a mechanistic link connecting nutrient abundance to receptor homeostasis.Abbreviations: AMPK, 5'-AMP-activated protein kinase; APP, amyloid beta precursor protein; ATG, autophagy related; BafA, bafilomycin A1; CQ, chloroquine; DMEM, Dulbecco's minimum essential medium; DPBS, Dulbecco's phosphate-buffered saline; EBSS, Earle's balanced salt solution; FBS, fetal bovine serum; GAP, GTPase-activating protein; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; LIR, LC3-interacting region; LANDO, LC3-associated endocytosis; LP, leupeptin and pepstatin; MTOR, mechanistic target of rapamycin kinase; MTORC1, MTOR complex 1; nutrient stress, withdrawal of amino acids and serum; PDZ, DLG4/PSD95, DLG1, and TJP1/zo-1; RPS6, ribosomal protein S6; RPS6KB1/S6K1, ribosomal protein S6 kinase B1; SLC2A1/GLUT1, solute carrier family 2 member 1; SORL1, sortillin related receptor 1; SORT1, sortillin 1; SNX, sorting nexin; TBC1D5, TBC1 domain family member 5; ULK1, unc-51 like autophagy activating kinase 1; WASH, WASH complex subunit.

A combined immunopeptidomics, proteomics, and cell surface proteomics approach to identify immunotherapy targets for diffuse intrinsic pontine glioma.

Introduction: Diffuse intrinsic pontine glioma (DIPG), recently reclassified as a subtype of diffuse midline glioma, is a highly aggressive brainstem tumor affecting children and young adults, with no cure and a median survival of only 9 months. Conventional treatments are ineffective, highlighting the need for alternative therapeutic strategies such as cellular immunotherapy. However, identifying unique and tumor-specific cell surface antigens to target with chimeric antigen receptor (CAR) or T-cell receptor (TCR) therapies is challenging. Methods: In this study, a multi-omics approach was used to interrogate patient-derived DIPG cell lines and to identify potential targets for immunotherapy. Results: Through immunopeptidomics, a range of targetable peptide antigens from cancer testis and tumor-associated antigens as well as peptides derived from human endogenous retroviral elements were identified. Proteomics analysis also revealed upregulation of potential drug targets and cell surface proteins such as Cluster of differentiation 27 (CD276) B7 homolog 3 protein (B7H3), Interleukin 13 alpha receptor 2 (IL-13Rα2), Human Epidermal Growth Factor Receptor 3 (HER2), Ephrin Type-A Receptor 2 (EphA2), and Ephrin Type-A Receptor 3 (EphA3). Discussion: The results of this study provide a valuable resource for the scientific community to accelerate immunotherapeutic approaches for DIPG. Identifying potential targets for CAR and TCR therapies could open up new avenues for treating this devastating disease.

CD98 defines a metabolically flexible, proinflammatory subset of low-density neutrophils in systemic lupus erythematosus.

BACKGROUND: Low-density neutrophils (LDN) are a distinct subset of neutrophils rarely detected in healthy people but appear in the blood of patients with autoimmune diseases, including systemic lupus erythematosus (SLE), and are mobilised in response to granulocyte colony-stimulating factor (G-CSF). The aim of this study was to identify novel mechanisms responsible for the pathogenic capacity of LDN in SLE. METHODS: Neutrophils were isolated from donors treated with G-CSF, and whole-cell proteomic analysis was performed on LDN and normal-density neutrophils. RESULTS: CD98 is significantly upregulated in LDN from G-CSF donors and defines a subset of LDN within the blood of SLE patients. CD98 is a transmembrane protein that dimerises with L-type amino acid transporters. We show that CD98 is responsible for the increased bioenergetic capacity of LDN. CD98 on LDN mediates the uptake of essential amino acids that are used by mitochondria to produce adenosine triphosphate, especially in the absence of glucose. Inhibition of CD98 reduces the metabolic flexibility of this population, which may limit their pathogenic capacity. CD98+ LDN produce more proinflammatory cytokines and chemokines than their normal density counterparts and are resistant to apoptosis, which may also contribute to tissue inflammation and end organ damage in SLE. CONCLUSIONS: CD98 provides a phenotypic marker for LDN that facilitates identification of this population without density-gradient separation and represents a novel therapeutic target to limit its pathogenic capacity.

Inflammasome sensor NLRP1 disease variant M1184V promotes autoproteolysis and DPP9 complex formation by stabilizing the FIIND domain.

The inflammasome sensor NLRP1 (nucleotide-binding oligomerization domain-like receptor containing a pyrin domain 1) detects a variety of pathogen-derived molecular patterns to induce an inflammatory immune response by triggering pyroptosis and cytokine release. A number of mutations and polymorphisms of NLRP1 are known to cause autoinflammatory diseases, the functional characterization of which contributes to a better understanding of NLRP1 regulation. Here, we assessed the effect of the common NLRP1 variant M1184V, associated with asthma, inflammatory bowel disease, and diabetes, on the protein level. Our size-exclusion chromatography experiments show that M1184V stabilizes the "function-to-find" domain (FIIND) in a monomeric conformation. This effect is independent of autoproteolysis. In addition, molecular dynamics simulations reveal that the methionine residue increases flexibility within the ZU5 domain, whereas valine decreases flexibility, potentially indirectly stabilizing the catalytic triad responsible for autocleavage. By keeping the FIIND domain monomeric, formation of a multimer of full-length NLRP1 is promoted. We found that the stabilizing effect of the valine further leads to improved dipeptidyl peptidase 9 (DPP9)-binding capacities for the FIIND domain as well as the full-length protein as determined by surface plasmon resonance. Moreover, our immunoprecipitation experiments confirmed increased DPP9 binding for the M1184V protein in cells, consistent with improved formation of an autoinhibited complex with DPP9 in activity assays. Collectively, our study establishes a molecular rationale for the dichotomous involvement of the NLRP1 variant M1184V in autoimmune syndromes.

Ubiquitylation of RIPK3 beyond-the-RHIM can limit RIPK3 activity and cell death.

Pathogen recognition and TNF receptors signal via receptor interacting serine/threonine kinase-3 (RIPK3) to cause cell death, including MLKL-mediated necroptosis and caspase-8-dependent apoptosis. However, the post-translational control of RIPK3 is not fully understood. Using mass-spectrometry, we identified that RIPK3 is ubiquitylated on K469. The expression of mutant RIPK3 K469R demonstrated that RIPK3 ubiquitylation can limit both RIPK3-mediated apoptosis and necroptosis. The enhanced cell death of overexpressed RIPK3 K469R and activated endogenous RIPK3 correlated with an overall increase in RIPK3 ubiquitylation. Ripk3 K469R/K469R mice challenged with Salmonella displayed enhanced bacterial loads and reduced serum IFNγ. However, Ripk3 K469R/K469R macrophages and dermal fibroblasts were not sensitized to RIPK3-mediated apoptotic or necroptotic signaling suggesting that, in these cells, there is functional redundancy with alternate RIPK3 ubiquitin-modified sites. Consistent with this idea, the mutation of other ubiquitylated RIPK3 residues also increased RIPK3 hyper-ubiquitylation and cell death. Therefore, the targeted ubiquitylation of RIPK3 may act as either a brake or accelerator of RIPK3-dependent killing.

Human RIPK3 C-lobe phosphorylation is essential for necroptotic signaling.

Necroptosis is a caspase-independent, pro-inflammatory mode of programmed cell death which relies on the activation of the terminal effector, MLKL, by the upstream protein kinase RIPK3. To mediate necroptosis, RIPK3 must stably interact with, and phosphorylate the pseudokinase domain of MLKL, although the precise molecular cues that provoke RIPK3 necroptotic signaling are incompletely understood. The recent finding that RIPK3 S227 phosphorylation and the occurrence of a stable RIPK3:MLKL complex in human cells prior to exposure to a necroptosis stimulus raises the possibility that additional, as-yet-unidentified phosphorylation events activate RIPK3 upon initiation of necroptosis signaling. Here, we sought to identify phosphorylation sites of RIPK3 and dissect their regulatory functions. Phosphoproteomics identified 21 phosphorylation sites in HT29 cells overexpressing human RIPK3. By comparing cells expressing wild-type and kinase-inactive D142N RIPK3, autophosphorylation sites and substrates of other cellular kinases were distinguished. Of these 21 phosphosites, mutational analyses identified only pT224 and pS227 as crucial, synergistic sites for stable interaction with MLKL to promote necroptosis, while the recently reported activation loop phosphorylation at S164/T165 negatively regulate the kinase activity of RIPK3. Despite being able to phosphorylate MLKL to a similar or higher extent than wild-type RIPK3, mutation of T224, S227, or the RHIM in RIPK3 attenuated necroptosis. This finding highlights the stable recruitment of human MLKL by RIPK3 to the necrosome as an essential checkpoint in necroptosis signaling, which is independent from and precedes the phosphorylation of MLKL.

Tankyrase-mediated ADP-ribosylation is a regulator of TNF-induced death.

Tumor necrosis factor (TNF) is a key component of the innate immune response. Upon binding to its receptor, TNFR1, it promotes production of other cytokines via a membrane-bound complex 1 or induces cell death via a cytosolic complex 2. To understand how TNF-induced cell death is regulated, we performed mass spectrometry of complex 2 and identified tankyrase-1 as a native component that, upon a death stimulus, mediates complex 2 poly-ADP-ribosylation (PARylation). PARylation promotes recruitment of the E3 ligase RNF146, resulting in proteasomal degradation of complex 2, thereby limiting cell death. Expression of the ADP-ribose-binding/hydrolyzing severe acute respiratory syndrome coronavirus 2 macrodomain sensitizes cells to TNF-induced death via abolishing complex 2 PARylation. This suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

K-29 linked ubiquitination of Arrdc4 regulates its function in extracellular vesicle biogenesis.

Extracellular vesicles (EVs) are important mediators of intercellular communication. However, EV biogenesis remains poorly understood. We previously defined a role for Arrdc4 (Arrestin domain containing protein 4), an adaptor for Nedd4 family ubiquitin ligases, in the biogenesis of EVs. Here we report that ubiquitination of Arrdc4 is critical for its role in EV secretion. We identified five potential ubiquitinated lysine residues in Arrdc4 using mass spectrometry. By analysing Arrdc4 lysine mutants we discovered that lysine 270 (K270) is critical for Arrdc4 function in EV biogenesis. Arrdc4K270R mutation caused a decrease in the number of EVs released by cells compared to Arrdc4WT , and a reduction in trafficking of divalent metal transporter (DMT1) into EVs. Furthermore, we also observed a decrease in DMT1 activity and an increase in its intracellular degradation in the presence of Arrdc4K270R . K270 was found to be ubiquitinated with K-29 polyubiquitin chains by the ubiquitin ligase Nedd4-2. Thus, our results uncover a novel role of K-29 polyubiquitin chains in Arrdc4-mediated EV biogenesis and protein trafficking.

SFPQ-ABL1 and BCR-ABL1 use different signaling networks to drive B-cell acute lymphoblastic leukemia.

Philadelphia-like (Ph-like) acute lymphoblastic leukemia (ALL) is a high-risk subtype of B-cell ALL characterized by a gene expression profile resembling Philadelphia chromosome-positive ALL (Ph+ ALL) in the absence of BCR-ABL1. Tyrosine kinase-activating fusions, some involving ABL1, are recurrent drivers of Ph-like ALL and are targetable with tyrosine kinase inhibitors (TKIs). We identified a rare instance of SFPQ-ABL1 in a child with Ph-like ALL. SFPQ-ABL1 expressed in cytokine-dependent cell lines was sufficient to transform cells and these cells were sensitive to ABL1-targeting TKIs. In contrast to BCR-ABL1, SFPQ-ABL1 localized to the nuclear compartment and was a weaker driver of cellular proliferation. Phosphoproteomics analysis showed upregulation of cell cycle, DNA replication, and spliceosome pathways, and downregulation of signal transduction pathways, including ErbB, NF-κB, vascular endothelial growth factor (VEGF), and MAPK signaling in SFPQ-ABL1-expressing cells compared with BCR-ABL1-expressing cells. SFPQ-ABL1 expression did not activate phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling and was associated with phosphorylation of G2/M cell cycle proteins. SFPQ-ABL1 was sensitive to navitoclax and S-63845 and promotes cell survival by maintaining expression of Mcl-1 and Bcl-xL. SFPQ-ABL1 has functionally distinct mechanisms by which it drives ALL, including subcellular localization, proliferative capacity, and activation of cellular pathways. These findings highlight the role that fusion partners have in mediating the function of ABL1 fusions.

Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis.

The ancestral origins of the lytic cell death mode, necroptosis, lie in host defense. However, the dysregulation of necroptosis in inflammatory diseases has led to widespread interest in targeting the pathway therapeutically. This mode of cell death is executed by the terminal effector, the MLKL pseudokinase, which is licensed to kill following phosphorylation by its upstream regulator, RIPK3 kinase. The precise molecular details underlying MLKL activation are still emerging and, intriguingly, appear to mechanistically-diverge between species. Here, we report the structure of the human RIPK3 kinase domain alone and in complex with the MLKL pseudokinase. These structures reveal how human RIPK3 structurally differs from its mouse counterpart, and how human RIPK3 maintains MLKL in an inactive conformation prior to induction of necroptosis. Residues within the RIPK3:MLKL C-lobe interface are crucial to complex assembly and necroptotic signaling in human cells, thereby rationalizing the strict species specificity governing RIPK3 activation of MLKL.

Dynamic reconfiguration of pro-apoptotic BAK on membranes.

BAK and BAX, the effectors of intrinsic apoptosis, each undergo major reconfiguration to an activated conformer that self-associates to damage mitochondria and cause cell death. However, the dynamic structural mechanisms of this reconfiguration in the presence of a membrane have yet to be fully elucidated. To explore the metamorphosis of membrane-bound BAK, we employed hydrogen-deuterium exchange mass spectrometry (HDX-MS). The HDX-MS profile of BAK on liposomes comprising mitochondrial lipids was consistent with known solution structures of inactive BAK. Following activation, HDX-MS resolved major reconfigurations in BAK. Mutagenesis guided by our HDX-MS profiling revealed that the BCL-2 homology (BH) 4 domain maintains the inactive conformation of BAK, and disrupting this domain is sufficient for constitutive BAK activation. Moreover, the entire N-terminal region preceding the BAK oligomerisation domains became disordered post-activation and remained disordered in the activated oligomer. Removal of the disordered N-terminus did not impair, but rather slightly potentiated, BAK-mediated membrane permeabilisation of liposomes and mitochondria. Together, our HDX-MS analyses reveal new insights into the dynamic nature of BAK activation on a membrane, which may provide new opportunities for therapeutic targeting.

The intracellular domains of the EphB6 and EphA10 receptor tyrosine pseudokinases function as dynamic signalling hubs.

EphB6 and EphA10 are two poorly characterised pseudokinase members of the Eph receptor family, which collectively serves as mediators of contact-dependent cell-cell communication to transmit extracellular cues into intracellular signals. As per their active counterparts, EphB6 and EphA10 deregulation is strongly linked to proliferative diseases. However, unlike active Eph receptors, whose catalytic activities are thought to initiate an intracellular signalling cascade, EphB6 and EphA10 are classified as catalytically dead, raising the question of how non-catalytic functions contribute to Eph receptor signalling homeostasis. In this study, we have characterised the biochemical properties and topology of the EphB6 and EphA10 intracellular regions comprising the juxtamembrane (JM) region, pseudokinase and SAM domains. Using small-angle X-ray scattering and cross-linking-mass spectrometry, we observed high flexibility within their intracellular regions in solution and a propensity for interaction between the component domains. We identified tyrosine residues in the JM region of EphB6 as EphB4 substrates, which can bind the SH2 domains of signalling effectors, including Abl, Src and Vav3, consistent with cellular roles in recruiting these proteins for downstream signalling. Furthermore, our finding that EphB6 and EphA10 can bind ATP and ATP-competitive small molecules raises the prospect that these pseudokinase domains could be pharmacologically targeted to counter oncogenic signalling.

Multimodal regulation of encystation in Giardia duodenalis revealed by deep proteomics.

Cyst formation in the parasitic protist Giardia duodenalis is critical to its transmission. Existing proteomic data quantifies only 17% of coding genes transcribed during encystation and does not cover the complete process from trophozoite to mature cyst. Using high-resolution mass spectrometry, we have quantified proteomic changes across encystation and compared this with published transcriptomic data. We reproducibly identified 3863 (64.5% of Giardia proteins) and quantified 3382 proteins (56.5% of Giardia proteins) over standard trophozoite growth (TY), during low-bile encystation priming (LB), 16 h into encystation (EC), and at cyst maturation (C). This work provides the first known expanded observation of encystation at the proteomic level and triples the coverage of previous encystation proteomes. One-third (1169 proteins) of the quantified proteome is differentially expressed in the mature cyst relative to the trophozoite, including proteasomal machinery, metabolic pathways, and secretory proteins. Changes in lipid metabolism indicated a shift in lipid species dependency during encystation. Consistent with this, we identified the first, putative lipid transporters in this species, representing the steroidogenic acute regulatory protein-related lipid transfer (StARkin), oxysterol binding protein related protein (ORP/Osh) and glycosphingolipid transfer protein (GLTP) families, and follow their differential expression over cyst formation. Lastly, we undertook correlation analyses of the transcriptome and proteome of trophozoites and cysts, and found evidence of post-transcriptional regulation of key protein classes (RNA binding proteins) and stage-specific genes (encystation markers) implicating translation-repression in encystation. We provide the most extensive proteomic analysis of encystation in Giardia to date and the first known exploration across its complete duration. This work identifies encystation as highly coordinated, involving major changes in proteostasis, metabolism and membrane dynamics, and indicates a potential role for post-transcriptional regulation, mediated through RNA-binding proteins. Together our work provides a valuable resource for Giardia research and the development of transmission-blocking anti-giardials.

Arrdc4-dependent extracellular vesicle biogenesis is required for sperm maturation.

Extracellular vesicles (EVs) are important players in cell to cell communication in reproductive systems. Notably, EVs have been found and characterized in the male reproductive tract, however, direct functional evidence for their importance in mediating sperm function is lacking. We have previously demonstrated that Arrdc4, a member of the α-arrestin protein family, is involved in extracellular vesicle biogenesis and release. Here we show that Arrdc4-mediated extracellular vesicle biogenesis is required for proper sperm function. Sperm from Arrdc4-/- mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as observed by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and two-cell embryo production. We found a significant reduction in extracellular vesicle production by Arrdc4-/- epididymal epithelial cells, and further, supplementation of Arrdc4-/- sperm with additional vesicles dampened the acrosome reaction defect and restored zona pellucida binding. These results indicate that Arrdc4 is important for proper sperm maturation through the control of extracellular vesicle biogenesis.

The PP2A-Integrator-CDK9 axis fine-tunes transcription and can be targeted therapeutically in cancer.

Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.

Targeting histone acetylation dynamics and oncogenic transcription by catalytic P300/CBP inhibition.

To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.

CDK4/6 Inhibition Promotes Antitumor Immunity through the Induction of T-cell Memory.

Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) are an approved treatment for hormone receptor-positive breast cancer and are currently under evaluation across hundreds of clinical trials for other cancer types. The clinical success of these inhibitors is largely attributed to well-defined tumor-intrinsic cytostatic mechanisms, whereas their emerging role as immunomodulatory agents is less understood. Using integrated epigenomic, transcriptomic, and proteomic analyses, we demonstrated a novel action of CDK4/6 inhibitors in promoting the phenotypic and functional acquisition of immunologic T-cell memory. Short-term priming with a CDK4/6 inhibitor promoted long-term endogenous antitumor T-cell immunity in mice, enhanced the persistence and therapeutic efficacy of chimeric antigen receptor T cells, and induced a retinoblastoma-dependent T-cell phenotype supportive of favorable responses to immune checkpoint blockade in patients with melanoma. Together, these mechanistic insights significantly broaden the prospective utility of CDK4/6 inhibitors as clinical tools to boost antitumor T-cell immunity. SIGNIFICANCE: Immunologic memory is critical for sustained antitumor immunity. Our discovery that CDK4/6 inhibitors drive T-cell memory fate commitment sheds new light on their clinical activity, which is essential for the design of clinical trial protocols incorporating these agents, particularly in combination with immunotherapy, for the treatment of cancer.This article is highlighted in the In This Issue feature, p. 2355.

Proteomic analyses reveal that immune integrins are major targets for regulation by Membrane-Associated Ring-CH (MARCH) proteins MARCH2, 3, 4 and 9.

MARCH proteins are membrane-associated Ring-CH E3 ubiquitin ligases that dampen immune responses by downregulating cell surface expression of major histocompatibility complexes I and II as well as immune co-stimulatory receptors. We recently showed that MARCH2,3,4 and 9 also downregulate cell surface expression of the inflammatory cytokine receptor for interleukin-6 (IL6Rα). Here we use over-expression of these MARCH proteins in the M1 myeloid leukaemia cell line and cell surface proteomic analyses to globally analyse other potential targets of these proteins. A large range of cell surface proteins regulated by more than one MARCH protein in addition to several MARCH protein-specific cell surface targets were identified most of which were downregulated by MARCH expression. Prominent among these were several integrin complexes associated with immune cell homing, adhesion and migration. Integrin α4ß1 (VLA4 or VCAM-1 receptor) was downregulated only by MARCH2 and we showed that in MARCH2 knockout mice, Integrin α4 was upregulated specifically in mature B-lymphocytes and this was accompanied by decreased numbers of B-cells in the spleen.