Acute BRCAness Induction and AR Signaling Blockage through CDK12/7/9 Degradation Enhances PARP Inhibitor Sensitivity in Prostate Cancer.

Current treatments for advanced prostate cancer (PCa) primarily target androgen receptor (AR)-pathways. However, the emergence of castration-resistant prostate cancer (CRPC) and resistance to AR signaling inhibitors (ARSI) remains a significant clinical challenge. This study introduces BSJ-5-63, a novel triple degrader targeting cyclin-dependent kinases (CDKs) CDK12, CDK7, and CDK9, with potential to transform CRPC therapy. BSJ-5-63 effectively downregulates homologous recombination repair (HRR) genes, including BRCA1 and BRCA2, through CDK12 degradation, and attenuates AR signaling through CDK7 and CDK9 degradation, further enhancing its therapeutic impact. Importantly, BSJ-5-63 induces a "BRCAness" state that persists for a significant duration, enabling sequential combination therapy with PARP inhibitors (PARPis) while potentially minimizing drug-related toxicity and resistance. In both in vitro and in vivo studies, BSJ-5-63 exhibited potent antiproliferative effects in both AR-positive and AR-negative CRPC models. This study presents a promising multi-pronged approach for CRPC treatment, addressing both DNA repair mechanisms and AR signaling, with the potential to benefit a wide range of patients regardless of their BRCA1/2 mutational status. SIGNIFICANCE: This study introduces BSJ-5-63, a triple degrader designed to target CDK12, CDK7, and CDK9, making a significant advancement in CRPC therapy. The distinctive mechanism of BSJ-5-63 involves downregulating HRR genes and inhibiting AR signaling, thereby inducing a BRCAness state. This enhances sensitivity to PARP inhibition, effectively addressing ARSI resistance and improving the overall efficacy of treatment. The development of BSJ-5-63 represents a promising therapeutic approach, with the potential to benefit a broad spectrum of CRPC patients.

A spatiotemporal map of co-receptor signaling networks underlying B cell activation.

The B cell receptor (BCR) signals together with a multi-component co-receptor complex to initiate B cell activation in response to antigen binding. Here, we take advantage of peroxidase-catalyzed proximity labeling combined with quantitative mass spectrometry to track co-receptor signaling dynamics in Raji cells from 10 s to 2 h after BCR stimulation. This approach enables tracking of 2,814 proximity-labeled proteins and 1,394 phosphosites and provides an unbiased and quantitative molecular map of proteins recruited to the vicinity of CD19, the signaling subunit of the co-receptor complex. We detail the recruitment kinetics of signaling effectors to CD19 and identify previously uncharacterized mediators of B cell activation. We show that the glutamate transporter SLC1A1 is responsible for mediating rapid metabolic reprogramming and for maintaining redox homeostasis during B cell activation. This study provides a comprehensive map of BCR signaling and a rich resource for uncovering the complex signaling networks that regulate activation.

TYK2 as a novel therapeutic target in Alzheimer's Disease with TDP-43 inclusions.

Neuroinflammation is a pathological feature of many neurodegenerative diseases, including Alzheimer's disease (AD)1,2 and amyotrophic lateral sclerosis (ALS)3, raising the possibility of common therapeutic targets. We previously established that cytoplasmic double-stranded RNA (cdsRNA) is spatially coincident with cytoplasmic pTDP-43 inclusions in neurons of patients with C9ORF72-mediated ALS4. CdsRNA triggers a type-I interferon (IFN-I)-based innate immune response in human neural cells, resulting in their death4. Here, we report that cdsRNA is also spatially coincident with pTDP-43 cytoplasmic inclusions in brain cells of patients with AD pathology and that type-I interferon response genes are significantly upregulated in brain regions affected by AD. We updated our machine-learning pipeline DRIAD-SP (Drug Repurposing In Alzheimer's Disease with Systems Pharmacology) to incorporate cryptic exon (CE) detection as a proxy of pTDP-43 inclusions and demonstrated that the FDA-approved JAK inhibitors baricitinib and ruxolitinib that block interferon signaling show a protective signal only in cortical brain regions expressing multiple CEs. Furthermore, the JAK family member TYK2 was a top hit in a CRISPR screen of cdsRNA-mediated death in differentiated human neural cells. The selective TYK2 inhibitor deucravacitinib, an FDA-approved drug for psoriasis, rescued toxicity elicited by cdsRNA. Finally, we identified CCL2, CXCL10, and IL-6 as candidate predictive biomarkers for cdsRNA-related neurodegenerative diseases. Together, we find parallel neuroinflammatory mechanisms between TDP-43 associated-AD and ALS and nominate TYK2 as a possible disease-modifying target of these incurable neurodegenerative diseases.

The dynamic TRPV2 ion channel proximity proteome reveals functional links of calcium flux with cellular adhesion factors NCAM and L1CAM in neurite outgrowth.

TRPV2 voltage-insensitive, calcium-permeable ion channels play important roles in cancer progression, immune response, and neuronal development. Despite TRPV2's physiological impact, underlying endogenous proteins mediating TRPV2 responses and affected signaling pathways remain elusive. Using quantitative peroxidase-catalyzed (APEX2) proximity proteomics we uncover dynamic changes in the TRPV2-proximal proteome and identify calcium signaling and cell adhesion factors recruited to the molecular channel neighborhood in response to activation. Quantitative TRPV2 proximity proteomics further revealed activation-induced enrichment of protein clusters with biological functions in neural and cellular projection. We demonstrate a functional connection between TRPV2 and the neural immunoglobulin cell adhesion molecules NCAM and L1CAM. NCAM and L1CAM stimulation robustly induces TRPV2 [Ca2+]I flux in neuronal PC12 cells and this TRPV2-specific [Ca2+]I flux requires activation of the protein kinase PKCα. TRPV2 expression directly impacts neurite lengths that are modulated by NCAM or L1CAM stimulation. Hence, TRPV2's calcium signaling plays a previously undescribed, yet vital role in cell adhesion, and TRPV2 calcium flux and neurite development are intricately linked via NCAM and L1CAM cell adhesion proteins.

STMND1 is a phylogenetically ancient stathmin which localizes to motile cilia and exhibits nuclear translocation that is inhibited when soluble tubulin concentration increases.

Stathmins are small, unstructured proteins that bind tubulin dimers and are implicated in several human diseases, but whose function remains unknown. We characterized a new stathmin, STMND1 (Stathmin Domain Containing 1) as the human representative of an ancient subfamily. STMND1 features a N-terminal myristoylated and palmitoylated motif which directs it to membranes and a tubulin-binding stathmin-like domain (SLD) that contains an internal nuclear localization signal. Biochemistry and proximity labeling showed that STMND1 binds tubulin, and live imaging showed that tubulin binding inhibits translocation from cellular membranes to the nucleus. STMND1 is highly expressed in multiciliated epithelial cells, where it localizes to motile cilia. Overexpression in a model system increased the length of primary cilia. Our study suggests that the most ancient stathmins have cilium-related functions that involve sensing soluble tubulin.

Proteomic profiling across breast cancer cell lines and models.

We performed quantitative proteomics on 60 human-derived breast cancer cell line models to a depth of ~13,000 proteins. The resulting high-throughput datasets were assessed for quality and reproducibility. We used the datasets to identify and characterize the subtypes of breast cancer and showed that they conform to known transcriptional subtypes, revealing that molecular subtypes are preserved even in under-sampled protein feature sets. All datasets are freely available as public resources on the LINCS portal. We anticipate that these datasets, either in isolation or in combination with complimentary measurements such as genomics, transcriptomics and phosphoproteomics, can be mined for the purpose of predicting drug response, informing cell line specific context in models of signalling pathways, and identifying markers of sensitivity or resistance to therapeutics.

A spatiotemporal Notch interaction map from plasma membrane to nucleus.

Notch signaling relies on ligand-induced proteolysis of the transmembrane receptor Notch to liberate a nuclear effector that drives cell fate decisions. Upon ligand binding, sequential cleavage of Notch by the transmembrane protease ADAM10 and the intracellular protease γ-secretase releases the Notch intracellular domain (NICD), which translocates to the nucleus and forms a complex that induces target gene transcription. To map the location and timing of the individual steps required for the proteolysis and movement of Notch from the plasma membrane to the nucleus, we used proximity labeling with quantitative, multiplexed mass spectrometry to monitor the interaction partners of endogenous NOTCH2 after ligand stimulation in the presence of a γ-secretase inhibitor and as a function of time after inhibitor removal. Our studies showed that γ-secretase-mediated cleavage of NOTCH2 occurred in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurred within 45 min of inhibitor washout. These findings provide a detailed spatiotemporal map tracking the path of Notch from the plasma membrane to the nucleus and identify signaling events that are potential targets for modulating Notch activity.

HUWE1 Amplifies Ubiquitin Modifications to Broadly Stimulate Clearance of Proteins and Aggregates.

Selective breakdown of proteins and aggregates is crucial for maintaining normal cellular activities and is involved in the pathogenesis of diverse diseases. How the cell recognizes and tags these targets in different structural states for degradation by the proteasome and autophagy pathways has not been well understood. Here, we discovered that a HECT-family ubiquitin ligase HUWE1 is broadly required for the efficient degradation of soluble factors and for the clearance of protein aggregates/condensates. Underlying this capacity of HUWE1 is a novel Ubiquitin-Directed ubiquitin Ligase (UDL) activity which recognizes both soluble substrates and aggregates that carry a high density of ubiquitin chains and rapidly expand the ubiquitin modifications on these targets. Ubiquitin signal amplification by HUWE1 recruits the ubiquitin-dependent segregase p97/VCP to process these targets for subsequent degradation or clearance. HUWE1 controls the cytotoxicity of protein aggregates, mediates Targeted Protein Degradation and regulates cell-cycle transitions with its UDL activity.

A Spatiotemporal Map of Co-Receptor Signaling Networks Underlying B Cell Activation.

The B cell receptor (BCR) signals together with a multi-component co-receptor complex to initiate B cell activation in response to antigen binding. This process underlies nearly every aspect of proper B cell function. Here, we take advantage of peroxidase-catalyzed proximity labeling combined with quantitative mass spectrometry to track B cell co-receptor signaling dynamics from 10 seconds to 2 hours after BCR stimulation. This approach enables tracking of 2,814 proximity-labeled proteins and 1,394 quantified phosphosites and provides an unbiased and quantitative molecular map of proteins recruited to the vicinity of CD19, the key signaling subunit of the co-receptor complex. We detail the recruitment kinetics of essential signaling effectors to CD19 following activation, and then identify new mediators of B cell activation. In particular, we show that the glutamate transporter SLC1A1 is responsible for mediating rapid metabolic reprogramming immediately downstream of BCR stimulation and for maintaining redox homeostasis during B cell activation. This study provides a comprehensive map of the BCR signaling pathway and a rich resource for uncovering the complex signaling networks that regulate B cell activation.

Rad regulation of CaV1.2 channels controls cardiac fight-or-flight response.

Fight-or-flight responses involve ß-adrenergic-induced increases in heart rate and contractile force. In the present study, we uncover the primary mechanism underlying the heart's innate contractile reserve. We show that four protein kinase A (PKA)-phosphorylated residues in Rad, a calcium channel inhibitor, are crucial for controlling basal calcium current and essential for ß-adrenergic augmentation of calcium influx in cardiomyocytes. Even with intact PKA signaling to other proteins modulating calcium handling, preventing adrenergic activation of calcium channels in Rad-phosphosite-mutant mice (4SA-Rad) has profound physiological effects: reduced heart rate with increased pauses, reduced basal contractility, near-complete attenuation of ß-adrenergic contractile response and diminished exercise capacity. Conversely, expression of mutant calcium-channel ß-subunits that cannot bind 4SA-Rad is sufficient to enhance basal calcium influx and contractility to adrenergically augmented levels of wild-type mice, rescuing the failing heart phenotype of 4SA-Rad mice. Hence, disruption of interactions between Rad and calcium channels constitutes the foundation toward next-generation therapeutics specifically enhancing cardiac contractility.

Exploiting endogenous and therapy-induced apoptotic vulnerabilities in immunoglobulin light chain amyloidosis with BH3 mimetics.

Immunoglobulin light chain (AL) amyloidosis is an incurable hematologic disorder typically characterized by the production of amyloidogenic light chains by clonal plasma cells. These light chains misfold and aggregate in healthy tissues as amyloid fibrils, leading to life-threatening multi-organ dysfunction. Here we show that the clonal plasma cells in AL amyloidosis are highly primed to undergo apoptosis and dependent on pro-survival proteins MCL-1 and BCL-2. Notably, this MCL-1 dependency is indirectly targeted by the proteasome inhibitor bortezomib, currently the standard of care for this disease and the related plasma cell disorder multiple myeloma, due to upregulation of pro-apoptotic Noxa and its inhibitory binding to MCL-1. BCL-2 inhibitors sensitize clonal plasma cells to multiple front-line therapies including bortezomib, dexamethasone and lenalidomide. Strikingly, in mice bearing AL amyloidosis cell line xenografts, single agent treatment with the BCL-2 inhibitor ABT-199 (venetoclax) produces deeper remissions than bortezomib and triples median survival. Mass spectrometry-based proteomic analysis reveals rewiring of signaling pathways regulating apoptosis, proliferation and mitochondrial metabolism between isogenic AL amyloidosis and multiple myeloma cells that divergently alter their sensitivity to therapies. These findings provide a roadmap for the use of BH3 mimetics to exploit endogenous and induced apoptotic vulnerabilities in AL amyloidosis.

Time-series transcriptomics and proteomics reveal alternative modes to decode p53 oscillations.

The cell stress-responsive transcription factor p53 influences the expression of its target genes and subsequent cellular responses based in part on its dynamics (changes in level over time). The mechanisms decoding p53 dynamics into subsequent target mRNA and protein dynamics remain unclear. We systematically quantified p53 target mRNA and protein expression over time under two p53 dynamical regimes, oscillatory and rising, using RNA-sequencing and TMT mass spectrometry. Oscillatory dynamics allowed for a greater variety of dynamical patterns for both mRNAs and proteins. Mathematical modeling of empirical data revealed three distinct mechanisms that decode p53 dynamics. Specific combinations of these mechanisms at the transcriptional and post-transcriptional levels enabled exclusive induction of proteins under particular dynamics. In addition, rising induction of p53 led to higher induction of proteins regardless of their functional class, including proteins promoting arrest of proliferation, the primary cellular outcome under rising p53. Our results highlight the diverse mechanisms cells employ to distinguish complex transcription factor dynamics to regulate gene expression.

Rixosomal RNA degradation contributes to silencing of Polycomb target genes.

Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) are histone-modifying and -binding complexes that mediate the formation of facultative heterochromatin and are required for silencing of developmental genes and maintenance of cell fate1-3. Multiple pathways of RNA decay work together to establish and maintain heterochromatin in fission yeast, including a recently identified role for a conserved RNA-degradation complex known as the rixosome or RIX1 complex4-6. Whether RNA degradation also has a role in the stability of mammalian heterochromatin remains unknown. Here we show that the rixosome contributes to silencing of many Polycomb targets in human cells. The rixosome associates with human PRC complexes and is enriched at promoters of Polycomb target genes. Depletion of either the rixosome or Polycomb results in accumulation of paused and elongating RNA polymerase at Polycomb target genes. We identify point mutations in the RING1B subunit of PRC1 that disrupt the interaction between PRC1 and the rixosome and result in diminished silencing, suggesting that direct recruitment of the rixosome to chromatin is required for silencing. Finally, we show that the RNA endonuclease and kinase activities of the rixosome and the downstream XRN2 exoribonuclease, which degrades RNAs with 5' monophosphate groups generated by the rixosome, are required for silencing. Our findings suggest that rixosomal degradation of nascent RNA is conserved from fission yeast to human, with a primary role in RNA degradation at facultative heterochromatin in human cells.

Detecting Cardiovascular Protein-Protein Interactions by Proximity Proteomics.

Rapidly changing and transient protein-protein interactions regulate dynamic cellular processes in the cardiovascular system. Traditional methods, including affinity purification and mass spectrometry, have revealed many macromolecular complexes in cardiomyocytes and the vasculature. Yet these methods often fail to identify in vivo or transient protein-protein interactions. To capture these interactions in living cells and animals with subsequent mass spectrometry identification, enzyme-catalyzed proximity labeling techniques have been developed in the past decade. Although the application of this methodology to cardiovascular research is still in its infancy, the field is developing rapidly, and the promise is substantial. In this review, we outline important concepts and discuss how proximity proteomics has been applied to study physiological and pathophysiological processes relevant to the cardiovascular system.

Orphan nuclear receptor COUP-TFII enhances myofibroblast glycolysis leading to kidney fibrosis.

Recent studies demonstrate that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in human fibrotic kidneys, lungs, kidney organoids, and mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts also induced production of alpha-smooth muscle actin (αSMA) and collagen 1. Knockout of COUP-TFII decreased glycolysis and collagen 1 levels in fibroblasts. Chip-qPCR revealed the binding of COUP-TFII on the promoter of PGC1α. Overexpression of COUP-TFII reduced the cellular level of PGC1α. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potentially fibrosis in other organs.

Time-resolved proteomics profiling of the ciliary Hedgehog response.

The primary cilium is a signaling compartment that interprets Hedgehog signals through changes of its protein, lipid, and second messenger compositions. Here, we combine proximity labeling of cilia with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary proteome in response to Hedgehog. This approach correctly identifies the three factors known to undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together with the G protein-coupled receptor GPR161 in response to Hedgehog, and we propose that the GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, we identify the phosphatase Paladin as a cell type-specific regulator of Hedgehog signaling that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling malfunctions.

Discovery and resistance mechanism of a selective CDK12 degrader.

Cyclin-dependent kinase 12 (CDK12) is an emerging therapeutic target due to its role in regulating transcription of DNA-damage response (DDR) genes. However, development of selective small molecules targeting CDK12 has been challenging due to the high degree of homology between kinase domains of CDK12 and other transcriptional CDKs, most notably CDK13. In the present study, we report the rational design and characterization of a CDK12-specific degrader, BSJ-4-116. BSJ-4-116 selectively degraded CDK12 as assessed through quantitative proteomics. Selective degradation of CDK12 resulted in premature cleavage and poly(adenylation) of DDR genes. Moreover, BSJ-4-116 exhibited potent antiproliferative effects, alone and in combination with the poly(ADP-ribose) polymerase inhibitor olaparib, as well as when used as a single agent against cell lines resistant to covalent CDK12 inhibitors. Two point mutations in CDK12 were identified that confer resistance to BSJ-4-116, demonstrating a potential mechanism that tumor cells can use to evade bivalent degrader molecules.

Targeting immunosuppressive macrophages overcomes PARP inhibitor resistance in BRCA1-associated triple-negative breast cancer.

Despite objective responses to PARP inhibition and improvements in progression-free survival compared to standard chemotherapy in patients with BRCA-associated triple-negative breast cancer (TNBC), benefits are transitory. Using high dimensional single-cell profiling of human TNBC, here we demonstrate that macrophages are the predominant infiltrating immune cell type in BRCA-associated TNBC. Through multi-omics profiling we show that PARP inhibitors enhance both anti- and pro-tumor features of macrophages through glucose and lipid metabolic reprogramming driven by the sterol regulatory element-binding protein 1 (SREBP-1) pathway. Combined PARP inhibitor therapy with CSF-1R blocking antibodies significantly enhanced innate and adaptive anti-tumor immunity and extends survival in BRCA-deficient tumors in vivo and is mediated by CD8+ T-cells. Collectively, our results uncover macrophage-mediated immune suppression as a liability of PARP inhibitor treatment and demonstrate combined PARP inhibition and macrophage targeting therapy induces a durable reprogramming of the tumor microenvironment, thus constituting a promising therapeutic strategy for TNBC.

Impact of Zika virus on the human type I interferon osteoimmune response.

BACKGROUND: The developing field of osteoimmunology supports importance of an interferon (IFN) response pathway in osteoblasts. Clarifying osteoblast-IFN interactions is important because IFN is used as salvage anti-tumor therapy but systemic toxicity is high with variable clinical results. In addition, osteoblast response to systemic bursts and disruptions of IFN pathways induced by viral infection may influence bone remodeling. ZIKA virus (ZIKV) infection impacts bone development in humans and IFN response in vitro. Consistently, initial evidence of permissivity to ZIKV has been reported in human osteoblasts. HYPOTHESIS: Osteoblast-like Saos-2 cells are permissive to ZIKV and responsive to IFN. METHODS: Multiple approaches were used to assess whether Saos-2 cells are permissive to ZIKV infection and exhibit IFN-mediated ZIKV suppression. Proteomic methods were used to evaluate impact of ZIKV and IFN on Saos-2 cells. RESULTS: Evidence is presented confirming Saos-2 cells are permissive to ZIKV and support IFN-mediated suppression of ZIKV. ZIKV and IFN differentially impact the Saos-2 proteome, exemplified by HELZ2 protein which is upregulated by IFN but non responsive to ZIKV. Both ZIKV and IFN suppress proteins associated with microcephaly/pseudo-TORCH syndrome (BI1, KI20A and UBP18), and ZIKV induces potential entry factor PLVAP. CONCLUSIONS: Transient ZIKV infection influences osteoimmune state, and IFN and ZIKV activate distinct proteomes in Saos-2 cells, which could inform therapeutic, engineered, disruptions.

A conserved RNA degradation complex required for spreading and epigenetic inheritance of heterochromatin.

Heterochromatic domains containing histone H3 lysine 9 methylation (H3K9me) can be epigenetically inherited independently of underlying DNA sequence. To gain insight into the mechanisms that mediate epigenetic inheritance, we used a Schizosaccharomyces pombe inducible heterochromatin formation system to perform a genetic screen for mutations that abolish heterochromatin inheritance without affecting its establishment. We identified mutations in several pathways, including the conserved and essential Rix1-associated complex (henceforth the rixosome), which contains RNA endonuclease and polynucleotide kinase activities with known roles in ribosomal RNA processing. We show that the rixosome is required for spreading and epigenetic inheritance of heterochromatin in fission yeast. Viable rixosome mutations that disrupt its association with Swi6/HP1 fail to localize to heterochromatin, lead to accumulation of heterochromatic RNAs, and block spreading of H3K9me and silencing into actively transcribed regions. These findings reveal a new pathway for degradation of heterochromatic RNAs with essential roles in heterochromatin spreading and inheritance.