The research team, working at Rice University with collaborators at Vanderbilt University Medical Center, exposed two well-known prostate cancer cell lines to repeated pulses of high-intensity fluid shear stress reaching 3950 dyn/cm2, a level far beyond anything previously tested in sustained experiments. For context, venous blood flow typically imposes forces of roughly 0.5 to 4 dyn/cm2, arterial flow ranges from 4 to 30 dyn/cm2, and turbulent regions near arterial bifurcations or inside the heart can briefly generate pulses on the order of 1000 dyn/cm2. The researchers used a syringe pump to perfuse cells through a fine 30-gauge needle at high speed, producing a brief pulse lasting just over a millisecond, with each pulse delivered two minutes apart. Over four to five months, the cells endured approximately sixty of these treatments, with the number of pulses per session gradually increased.

The two cell lines told strikingly different stories. LNCaP cells, originally derived from a lymph node metastasis, showed limited innate resistance and could only tolerate up to ten shear pulses per treatment. PC3 cells, which came from a bone metastasis and therefore plausibly experienced circulatory forces in the patient from whom they were isolated, proved innately tough, maintaining viability above eighty percent even after twenty pulses. By repeatedly selecting the survivors, the team generated what they call mechanoresistant, or MR, populations. After ten pulses, the mechanoresistant LNCaP cells retained nearly seventy percent viability, while unmodified parental cells dropped to under thirty-eight percent. The PC3 cells, already robust, showed that innate resistance alone does not capture the full picture of how metastatic cells adapt.

Under the microscope, the mechanoresistant PC3 cells revealed a fascinating physical transformation. They became significantly smaller, less granular, and less spread out when attached to a surface, adopting features consistent with an amoeboid morphology, a rounded, highly deformable state that has been linked to enhanced metastatic capacity in several cancer types. Analysis of epithelial and mesenchymal markers showed that these cells occupied a hybrid state, expressing both E-cadherin and vimentin, rather than committing fully to either program. This plasticity may be exactly what allows circulating tumor cells to survive the mechanical gauntlet of the bloodstream and then squeeze back out of vessels to seed new tumors, a process during which many cells are known to undergo partial transitions between epithelial and mesenchymal states.

The cell cycle findings were equally unexpected. Previous studies had generally shown that fluid shear stress drives tumor cells into cell cycle arrest and promotes stress-induced death. Here, the mechanoresistant cells did the opposite. Twenty-four hours after shear exposure, the PC3 mechanoresistant cells showed a significantly elevated fraction of cells in S phase, the DNA replication stage, and a corresponding reduction in the G1 phase, indicating accelerated progression through the cycle rather than arrest. Both mechanoresistant lines also displayed elevated expression of Piezo1, a calcium-permeable mechanosensitive ion channel that has previously been implicated in prostate cancer progression, migration, and proliferation. The researchers found that Piezo1 levels correlated positively with proliferative activity, suggesting a mechanistic link between mechanical sensing and the growth behavior of these adapted cells.

Bulk RNA sequencing, performed after roughly sixty shear treatments, confirmed that the mechanoresistant cells had acquired genuinely distinct molecular identities, clustering separately from both parental cells and higher-passage controls designed to rule out artifacts of long-term culture. Among the significantly upregulated genes in the PC3 mechanoresistant cells, one stood out: CALB2, which encodes the calcium-binding protein calretinin. Calretinin buffers intracellular calcium levels, placing it in a prime position to modulate signaling downstream of mechanosensitive channels like Piezo1. Quantitative PCR and Western blotting confirmed the elevated mRNA and protein expression, and the finding carried particular weight because calretinin had never before been examined in the context of fluid shear stress in any cancer.

To probe calretinin’s role in human disease, the team measured the protein in prostate tissue microarrays. Calretinin expression was highest in adenocarcinoma and metastatic samples compared with normal adjacent tissue and samples from patients with hyperplasia or prostatitis, and in paired comparisons, four of six patients showed elevated calretinin in tumor tissue relative to their healthy neighbors. Mining large clinical datasets, including the TCGA prostate adenocarcinoma cohort, revealed that high CALB2 expression correlated with reduced overall survival, although the association did not reach statistical significance in that particular analysis. Intriguingly, CALB2-high outlier tumors appeared across all Gleason grades, suggesting that calretinin elevation may reflect a patient-specific transcriptional state rather than a simple marker of tumor aggressiveness. The team also found that CALB2 expression correlated with DGK1, a lipid signaling gene, and with EPB41L3, a tumor suppressor, hinting at a broader co-expression network tied to membrane and cytoskeletal biology.

The decisive experiment came with genetic deletion. Using lentiviral CRISPR constructs, the researchers knocked out CALB2 in their PC3 lines and re-exposed the cells to twenty shear pulses. The result was dramatic and highly specific: while parental and mechanonaive knockout cells barely noticed the treatment, the mechanoresistant cells lost their hard-won protection, with viability plummeting to roughly thirty-six percent after twenty pulses. The knockout cells also showed reduced proliferative activity, with a larger fraction displaying low Ki67 expression. Because parental cells never depended on CALB2 for shear survival, the gene appears to be a genuine acquired mechanoresistance factor, a molecular prop that the adapted cells came to rely upon. This makes CALB2 the first gene to be functionally validated as a mediator of resistance to high-intensity fluid shear stress through repeated exposure experiments.

In vivo work reinforced the picture. In a pilot subcutaneous study, mechanoresistant cells produced higher tumor burden than parental controls, showing that their adapted traits persisted outside the dish. The team then performed an orthotopic model, surgically implanting luciferase-labeled PC3 cells into the prostate of immunodeficient mice and tracking tumor growth by bioluminescence for twelve weeks. Mechanoresistant tumors grew slightly faster during the first nine weeks, and calretinin levels in the resected tumors were highest in the mechanoresistant group, mirroring the in vitro findings, though the magnitude of the difference was more modest, likely reflecting the complexity of the tumor microenvironment. Few metastases were detected in any group, a limitation the authors attribute to the Matrigel used for engraftment and the severe immunodeficiency of the mouse strain.

The implications reach beyond prostate cancer. Circulating tumor cell survival remains the least studied stage of metastasis, partly because these cells spend an estimated twenty-five to thirty minutes in the bloodstream and are extraordinarily difficult to capture and study. By building the first stable mechanoresistant cancer cell lines, the researchers have created a durable experimental platform for dissecting how tumor cells withstand hemodynamic forces, a question that touches on cytoskeletal remodeling, membrane repair, calcium signaling, and ion channel biology all at once. Calretinin itself lacks well-defined drug-binding pockets, so direct pharmacological targeting may be difficult, and compensatory calcium buffers such as calmodulin could blunt any single-gene strategy. But the discovery that a calcium-buffering protein underpins mechanical survival opens a concrete therapeutic avenue: combining mechanosensitive channel inhibitors, such as the Piezo1 blocker GsMTx4, with existing apoptosis-triggering agents like TRAIL could exploit the very adaptations that make metastatic cells lethal. For the roughly seventy percent drop in five-year survival that accompanies distant prostate cancer metastasis, understanding and disrupting this mechanical armor may prove to be a critical step forward.

Subject of Research: Mechanoresistance and the role of CALB2 in metastatic prostate cancer cell survival under fluid shear stress

Article Title: CALB2 is a Mechanoresistance Gene in Metastatic Prostate Cancer

Article References: Fabiano, A. R., Luo, A. C., Taufalele, P., Dombroski, J. A., Aalaei, E., Rowland, S. J., Cantú, M. S., Carter, A. T., Knoblauch, S. V., Reinhart‐King, C. A., & King, M. R. (2026). CALB2 is a Mechanoresistance Gene in Metastatic Prostate Cancer. Advanced Science, 13(55), Article e76535. https://doi.org/10.1002/advs.76535

Image Credits: AI Generated

DOI: 10.1002/advs.76535

Keywords: prostate cancer, metastasis, circulating tumor cells, fluid shear stress, CALB2, calretinin, mechanotransduction, Piezo1, CRISPR knockout, RNA sequencing, orthotopic mouse model, Advanced Science

The core problem the researchers set out to solve is a bottleneck familiar to anyone working in targeted protein degradation. Proteomics-based approaches can, in principle, reveal every protein whose abundance changes when a compound is applied, but they are expensive and slow, making them impractical as a first-pass screen for large chemical libraries. Target-based biochemical assays such as AlphaLISA or TR-FRET require purified proteins and, crucially, prior knowledge of which target-E3 pair to test—an assumption that defeats the purpose of hunting for glues against unknown or classically undruggable substrates like transcription factors and scaffold proteins. Phenotypic screens, meanwhile, are biologically rich but cannot easily distinguish a true degrader from an ordinary inhibitor, forcing laborious downstream mechanistic deconvolution. What the field needed was a cheap, fast, and mechanistically informative primary readout.

The team’s answer was to make the transcriptome itself the universal readout. Gene expression signatures are high-dimensional proxies for cellular state, a principle demonstrated at scale by the L1000 platform and made affordable by DRUG-seq, which skips RNA purification and uses direct cell lysis with multiplexed library preparation. The researchers employed an optimized version, DRUG-seq2, which they report offers superior sensitivity for low-abundance regulatory transcripts—a critical feature when the signals of interest come from immature, weak molecular glues. In their workflow, Hep3B liver cancer cells were seeded in 96-well plates, treated with compounds at a uniform concentration of 1 micromolar for 24 hours, and processed through the miniaturized sequencing protocol. The library comprised 117 compounds: 11 commercial molecular glues serving as validated benchmarks and 106 in-house candidates synthesized by two collaborative research groups, all rationally designed to modulate the cereblon (CRBN) E3 ligase.

A deceptively simple but essential innovation lies in how the baseline is defined. Because DRUG-seq2 relies on direct lysis, technical noise can swamp the subtle transcriptional ripples produced by nascent glues. Before any analysis, the team performed systematic pairwise Pearson correlation analysis across all DMSO vehicle wells on each plate, excluding any replicate with a correlation coefficient below 0.95. By constructing the reference transcriptome solely from high-consensus control wells, they established a robust, self-consistent zero-point that maximizes statistical power to resolve low-magnitude perturbations. Downstream, differential expression analysis with DESeq2—flagging genes with absolute log2 fold change greater than 1 and adjusted p-value below 0.05—was paired with gene set enrichment analysis (GSEA) built from six curated collections, including Hallmark, Reactome, KEGG, Gene Ontology Biological Process, C6, and the C3 transcription factor target set. This two-layer design means even compounds with few or zero individual differentially expressed genes can still betray coordinated pathway-level activity.

The mechanistic heart of the platform is a genetic benchmark: an isogenic pair of Hep3B cell lines, one wild-type and one in which CRBN has been knocked out via CRISPR-Cas9. The researchers established a stable Cas9-expressing parent line, introduced single-guide RNAs targeting CRBN, and isolated single-cell clones. Clone 3 emerged as the definitive knockout after rigorous validation. Treatment with CC-90009, a clinical molecular glue that degrades the translation termination factor GSPT1, provided the proof: in wild-type cells the drug rapidly and almost completely eliminated GSPT1 protein, while in the knockout the effect vanished entirely, along with the drug’s cytotoxic potency, which showed a pronounced right-shift in the IC50 curve. Any compound whose transcriptional signature collapses in the knockout is therefore flagged as CRBN-dependent—a hallmark of a genuine glue—whereas signals that persist likely reflect off-target toxicity, conventional inhibition, or hijacking of a different E3 ligase such as VHL or DCAF16.

Screening the full library revealed a strikingly polarized activity spectrum. Roughly 20 percent of the compounds were transcriptionally inert, producing zero differentially expressed genes under the test conditions. At the other extreme, the high-activity cluster was dominated by known glues: SJ3149, which targets CK1α, elicited the most profound response, followed by lenalidomide, BMS-986397, and a MYC degrader. For most of these, transcriptional activity was almost completely abolished in the CRBN knockout, exactly as expected for classic glues. Encouragingly, one in-house compound, LC-02-047-P1, landed among the potent performers, marking it as a promising novel candidate. Conversely, compounds such as LC-02-105 and LC-02-033 displayed a paradoxical knockout-amplified phenotype, indicating CRBN-independent mechanisms and allowing the team to filter out molecules whose intrinsic scaffold toxicity overwhelmed any degradation function.

The pathway-level analysis added mechanistic texture that raw gene counts alone could not provide. Potent GSPT1 degraders such as CC-90009 upregulated regulators of the integrated stress response—DDIT3 (CHOP), GDF15, TRIB3, DDIT4, and PPP1R15A—consistent with the biology of translation termination failure: ribosomes stall at stop codons, collide, and trigger the ZAKα-mediated ribotoxic stress response, which in turn activates ATF4 and suppresses the translational apparatus to mitigate proteotoxic stress. GSEA of CC-90009 confirmed enrichment of integrated stress response, proteasome, and apoptosis pathways alongside downregulation of translation machinery. LC-02-047-P1, by contrast, showed metabolic rather than stress-related regulatory characteristics, hinting that its degraded target might be an upstream switch governing multiple metabolic pathways. The enrichment layer also rescued latent hits: LC-02-051 performed modestly in the differential expression analysis but ranked near the top in pathway enrichment, and its signal was completely blocked by CRBN knockout—suggesting a genuine but weak glue worth optimizing. Even CC-92480 yielded an instructive anomaly, showing slightly expanded enrichment in knockout cells, which the authors attribute to the absence of its high-affinity target IKZF1 in Hep3B, pushing the compound toward non-canonical substrates or generic xenobiotic stress.

To translate these multidimensional data into decisions, the researchers built a scatterplot integrating transcriptional potency (differential gene counts) with functional breadth (enriched pathway counts) across both wild-type and knockout conditions, then stratified the library into four tiers. Tier one comprises potent glues with high wild-type activity that nearly vanishes upon CRBN loss—the highest priorities for optimization. Tier two captures intermediate and latent leads whose coordinated functional signals suggest seed scaffolds needing structural refinement. Tier three contains active but E3-independent perturbators, likely conventional inhibitors, and tier four collects inert compounds. The spatial logic is intuitive: strong glues such as SJ3149, lenalidomide, CC-90009, and LC-02-047-P1 occupy the upper-right quadrant and collapse toward the origin in the knockout, while pharmacologically inert molecules cluster near the origin in both conditions and target-independent agents stubbornly refuse to move. The thresholds, the authors note, can be customized for different libraries.

Proteomic cross-validation demonstrated that the transcriptomic tiers predict real degradation events. Tandem mass tag quantitative proteomics of LC-02-047-P1-treated Hep3B cells revealed marked downregulation of CSNK1A1 (CK1α), and western blotting across Hep3B, Huh7, and HepG2 hepatocellular carcinoma lines confirmed near-complete, selective CK1α degradation with no effect on GSPT1. A decisive control sealed the case: cells engineered to express a non-degradable GSPT1 mutant abolished CC-90009’s activity but left LC-02-047-P1’s CK1α degradation untouched, proving the candidate acts entirely independently of GSPT1. The weaker tier-two compound LC-02-051 produced only minor proteomic perturbation, with TPM3 changes failing statistical validation—consistent with its latent classification. On scalability, the economics are compelling: the platform costs less than 100 RMB (roughly 14 US dollars) per sample, about 2 percent of standard TMT proteomics, with an end-to-end turnaround of three to four weeks even beyond 1,000 compounds.

The authors are candid about limitations. The single 24-hour window captures an integrated cellular state—primary degradation consequences blended with secondary cascades and stress responses—rather than the immediate transcriptional footprints of neosubstrate depletion, and future iterations will incorporate time-resolved profiling. Transcriptomic signatures also remain an indirect proxy for protein-level events, so candidate leads must ultimately be confirmed by quantitative proteomics and structural biology demonstrating direct ternary complex formation. The choice of screening cell line matters too, since E3 and substrate abundance dictate sensitivity; the authors suggest abundance-matched models, such as VHL-high or DCAF15-high backgrounds, for targeted campaigns. Even so, the GPS platform fills a genuine gap, bridging high-volume chemical screening and low-throughput mechanistic validation, and offering the field a practical roadmap for converting molecular glue discovery from a lucky accident into an engineering discipline.

Subject of Research: High-throughput transcriptomic screening for the discovery of CRBN-dependent molecular glue degraders

Article Title: A scalable, high-throughput glue perturbation screening platform for molecular glue discovery

Article References: Huang, D., Shuang, Z., Chen, L., Ouyang, H., Yang, P., Huang, L., Lu, W., Shi, M., Ding, X., Jiang, B., & Wu, W. (2026). A scalable, high-throughput glue perturbation screening platform for molecular glue discovery. iScience, 29(10), Article 117600. https://doi.org/10.1016/j.isci.2026.117600

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117600

Keywords: molecular glue degraders, targeted protein degradation, CRBN, DRUG-seq2, transcriptomics, gene set enrichment analysis, CRISPR knockout, E3 ubiquitin ligase, proteomics, drug discovery, GSPT1, CK1α