The work, led by Deeksha Singh, Premachandran Yadukrishnan, Puthan Valappil Rahul, and Sourav Datta, focuses on three proteins with long histories in plant biology. ABI5, the ABSCISIC ACID INSENSITIVE 5 transcription factor, is the canonical executor of ABA’s decision to halt seedling growth. HY5, ELONGATED HYPOCOTYL 5, is a light-activated transcription factor that promotes photomorphogenesis, the program by which seedlings open green cotyledons and begin photosynthesis. COP1, CONSTITUTIVELY PHOTOMORPHOGENIC1, is an E3 ubiquitin ligase best known as HY5’s executioner: it tags HY5 for destruction in the dark, and light inactivates it, allowing HY5 to accumulate. What the IISER team shows is that these three proteins form a regulatory module whose behavior during early seedling development is far more intricate than the textbook picture suggests.

The researchers began by tracking ABI5 and HY5 protein levels across the entire arc of early development in Arabidopsis thaliana, from dry siliques and dry seeds through imbibition, stratification, germination, and seedling establishment. The two proteins traced mirror-image trajectories. ABI5 was abundant in mature and dry seeds, stayed high through imbibition and stratification, and then declined as germination proceeded. HY5 did the opposite: it was barely detectable in dry and imbibed seeds and rose steadily during germination and post-germination growth. This inverse pattern makes intuitive sense. High ABA in the seed keeps ABI5 in command and the embryo dormant; as ABA levels fall and light takes over, HY5 rises and the seedling commits to growth.

But the story deepened when the team applied exogenous ABA to germinated seeds. Within three hours, HY5 protein levels dropped measurably at both germinated-seed and post-germination stages, a result confirmed independently with a fluorescent HY5-YFP reporter line. Meanwhile, ABA treatment caused ABI5, which normally fades after germination, to climb back up over a four-day window. The two master regulators were being pushed in opposite directions by the very hormone that governs the checkpoint, raising the question of whether they were also pushing on each other.

To answer that, the researchers exploited loss-of-function mutants. In hy5-215 plants lacking functional HY5, ABI5 protein accumulated more strongly than in wild-type seedlings from day two onward under ABA treatment, even though HY5 is known to bind the ABI5 promoter and activate its transcription. Conversely, in abi5-8 mutants lacking ABI5, HY5 protein built up to higher levels than in wild type across all stages examined. The interpretation is striking: each transcription factor restrains the other’s protein abundance, even as HY5 positively regulates ABI5 at the level of DNA. This arrangement, in which two factors each suppress the other’s accumulation, constitutes a double-negative feedback loop, a circuit motif that can sharpen transitions and create a bistable switch between the quiescent and growth states.

Chromatin immunoprecipitation followed by quantitative PCR added a layer of nuance. When ABA was present, HY5 enrichment at the ABI5 promoter fell, consistent with the protein’s degradation. In four-day-old seedlings, ABI5 bound its target promoters more strongly in the hy5 mutant background, while HY5 bound the RBCS and HY5 promoters more strongly in the abi5 mutant background. In other words, neither factor needs the other to occupy DNA; their mutual antagonism operates at the protein level rather than through competition for binding sites. The authors suggest that the previously documented physical interaction between HY5 and ABI5 proteins may underlie this mutual suppression, though the precise mechanism remains to be worked out.

Genetic epistasis experiments pinned down the hierarchy. The team generated a hy5-215 abi5-8 double mutant and scored seedling establishment, defined by open, expanded green cotyledons, with and without 1 micromolar ABA. Without the hormone, all genotypes established fully by day three. With ABA, hy5-215 was hypersensitive, meaning it arrested more readily, while abi5-8 was hyposensitive, resisting arrest. The double mutant behaved like abi5-8, not like hy5-215. That result places ABI5 genetically downstream of HY5: whatever HY5 does to influence ABA response, it must act through ABI5. It also reconciles an apparent paradox in the literature, since earlier studies had variously described HY5 as a positive regulator of ABA signaling and as a suppressor of ABA-mediated arrest. The resolution is that HY5’s transcriptional activation of ABI5 and its protein-level suppression of ABI5 are two faces of the same module, with the balance between them determining the outcome.

The degradation machinery emerged as the pivot of the whole system. When germinated seeds were treated with MG132, an inhibitor of the 26S proteasome, the ABA-induced decline in HY5 was abolished, while cycloheximide, a translation inhibitor, had no effect. ABA therefore does not stop HY5 from being made; it accelerates its destruction. That destruction depends on COP1, because in cop1-4 mutants ABA failed to reduce HY5 levels. Curiously, total COP1 protein abundance itself did not change with ABA treatment in a YFP-COP1 reporter line. Instead, the hormone changed where COP1 resides. Confocal microscopy of germinated seeds expressing YFP-COP1 showed that a three-hour ABA treatment enhanced the nuclear localization of the ligase. Since COP1 only ubiquitinates HY5 in the nucleus, this relocalization is the mechanistic link between the ABA signal and HY5 destruction.

This finding adds a twist to a complicated picture of COP1 regulation. Darkness is classically known to drive COP1 into the nucleus, degrading light-responsive factors, while light expels it to the cytoplasm. Recent studies had shown that ABA can promote COP1’s cytoplasmic translocation during seed germination in some contexts and enhance its nuclear localization in dark-grown seedlings. The new data show that under normal light-dark cycling, ABA pushes COP1 into the nucleus of germinated seeds, tipping the module toward ABI5 accumulation and growth arrest. The authors also note that endoplasmic reticulum stress and high temperature have been reported to reduce HY5 by enhancing COP1 nuclear localization, suggesting that the COP1-HY5 pair functions as a general stress-responsive rheostat whose output depends on the specific environmental input.

The model that emerges is elegant in its economy. ABA recruits COP1 to the nucleus, COP1 degrades HY5, and with HY5 out of the way, ABI5 protein accumulates unchecked, imposing the developmental arrest that conserves the seedling’s resources until conditions improve. When ABA falls, COP1 retreats from the nucleus, HY5 rebounds, and HY5’s suppression of ABI5 protein helps extinguish the arrest program, allowing growth to resume. The double-negative feedback loop between HY5 and ABI5 likely sharpens this transition, ensuring that the checkpoint flips decisively rather than drifting. For agriculture, the implications are tantalizing: seedling establishment is a vulnerable window for crops facing drought, salinity, and temperature extremes, and understanding the COP1-HY5-ABI5 module at this level of molecular detail may eventually inform strategies for tuning how aggressively seedlings pause or proceed when the environment turns against them. The authors caution that key questions remain, particularly how ABA relocalizes COP1 and whether disrupting the physical HY5-ABI5 interaction alters ABI5 stability, but the framework they have built turns a once-linear signaling pathway into a dynamic switch with a built-in memory of stress.

Subject of Research: Regulation of ABA-mediated post-germination developmental arrest in Arabidopsis by the COP1-HY5-ABI5 module

Article Title: A COP1-HY5-ABI5 module regulates ABA-mediated post-germination developmental arrest

Article References: Singh, D., Yadukrishnan, P., Rahul, P. V., & Datta, S. (2025). A COP1-HY5-ABI5 module regulates ABA-mediated post-germination developmental arrest. Frontiers in Plant Physiology, 3, Article 1675932. https://doi.org/10.3389/fphgy.2025.1675932

Image Credits: AI Generated

DOI: 10.3389/fphgy.2025.1675932

Keywords: abscisic acid, ABI5, HY5, COP1, post-germination arrest, seedling development, Arabidopsis thaliana, ubiquitin ligase, proteasome, light signaling, plant hormone, stress response

The study, led by Prasanth Viswanathan and colleagues, focused on a protein called myeloid cell leukemia-1, or MCL1. MCL1 belongs to a family of proteins that act as molecular bodyguards, blocking the self-destruct program known as apoptosis. It is an oncogene, meaning that when it is amplified or overactive it drives cancer, and tumors with high MCL1 levels are notoriously resistant to chemotherapy. Because of its clinical importance, MCL1 inhibitors are already being developed and tested in trials, making it an attractive target for drug designers. Previous work had shown that PEL cell lines, the laboratory models of this lymphoma, cannot survive without MCL1, but the details of how MCL1 protects these virus-transformed cells remained murky.

To dig deeper, the team turned to a systems biology approach, mapping the web of genetic interactions surrounding MCL1 in PEL cells. This unbiased strategy surfaced a surprise: MCL1 appeared to function in the same pathway as a protein called MARCHF5, a membrane-anchored E3 ubiquitin ligase residing in the mitochondria. MARCHF5 was not an obvious suspect. It is best known for maintaining mitochondrial homeostasis and for tuning antiviral signaling pathways, not for regulating apoptosis. The genetic interaction hinted, however, that the two proteins cooperate in keeping PEL cells alive, and the researchers set out to test that idea directly.

The first experiment was decisive. Using CRISPR gene editing, the team knocked out the MARCHF5 gene in PEL cell lines. Even though MCL1 was still present and functional, the loss of MARCHF5 triggered a significant increase in apoptosis. In other words, MCL1 alone was not enough to keep the cells alive; they also needed MARCHF5. This was a striking result because it established that a mitochondrial ubiquitin ligase, a protein class usually discussed in the context of mitochondrial quality control, plays an essential anti-apoptotic role in these virus-transformed tumor cells.

MARCHF5 is an enzyme, and enzymes can often be disabled by breaking their catalytic machinery. The researchers probed which activities of MARCHF5 were required for its survival function. Two features proved essential: its E3 ligase activity, the enzymatic ability to attach ubiquitin tags to target proteins, and its capacity to dimerize, or pair up with another MARCHF5 molecule. When either of these activities was disrupted, the protein could no longer protect PEL cells from apoptosis. This mechanistic detail matters because it suggests the survival function of MARCHF5 is not a passive structural role but an active enzymatic process, one that could in principle be targeted with drugs.

What was MARCHF5 actually doing? The clue came from an unexpected direction. When the researchers removed MARCHF5, or when they treated the cells with inhibitors of the 26S proteasome, the cellular machine that destroys ubiquitin-tagged proteins, one particular protein accumulated: NOXA. NOXA is a pro-apoptotic member of the same protein family as MCL1, and its job is essentially to antagonize MCL1 and push the cell toward self-destruction. Intriguingly, the levels of MCL1 itself did not change under these conditions. Instead, MARCHF5 appeared to be keeping NOXA in check, and without MARCHF5 the lethal signal built up unchecked.

The team then asked whether NOXA was truly the pro-apoptotic signal that MCL1 and MARCHF5 exist to neutralize. Several lines of evidence converged on that conclusion. Endogenous reciprocal co-immunoprecipitation experiments, which pull protein complexes out of cells without artificial tagging, showed that MARCHF5 and NOXA are found together in the same protein complex, consistent with a direct physical relationship. Even more telling, when the researchers knocked out the NOXA gene on its own, the PEL cells gained a fitness advantage, suggesting that NOXA exerts a constant, damaging pressure on these tumor cells that must be continuously suppressed for survival.

The most convincing test came from a genetic rescue experiment. If NOXA accumulation is the reason that MARCHF5-deficient cells die, then deleting NOXA should save them. That is exactly what the researchers observed: knocking out NOXA restored cell viability in PEL lines that had lost MARCHF5. Together, these experiments provide the mechanistic link that underlies the original genetic interaction between MCL1 and MARCHF5. The model that emerges is elegant: MARCHF5, using its ubiquitin ligase activity, induces the degradation of NOXA, the natural enemy of MCL1, thereby reinforcing MCL1’s pro-survival function and allowing the KSHV-transformed cells to evade apoptosis.

This finding adds a new layer to the biology of KSHV-associated malignancies. Primary effusion lymphoma arises when the virus reprograms B cells into rapidly proliferating, apoptosis-resistant tumor cells that typically grow as lymphomas in body cavities. The virus achieves this in part by manipulating the host cell’s apoptotic machinery, and the MCL1-MARCHF5 axis now appears to be a central node in that manipulation. The fact that both proteins are required, and that they act through a defined substrate, NOXA, gives researchers a clearer picture of the survival circuitry that these tumors depend on. It also raises the possibility that similar cooperation between MCL1 and mitochondrial ubiquitin ligases operates in other cancers where MCL1 is amplified.

From a therapeutic standpoint, the implications are considerable. MCL1 inhibitors are an active area of drug development precisely because MCL1 amplification drives chemoresistance in many cancers. The new work suggests that combination therapies targeting both MCL1 and MARCHF5, or approaches that prevent MARCHF5 from degrading NOXA, could be more effective than hitting either protein alone, since the two oncogenes reinforce each other’s function. Disabling MARCHF5’s ligase activity would strip away one of the defenses that keeps NOXA suppressed, potentially sensitizing tumor cells to MCL1 inhibition or to existing chemotherapy regimens. As the authors note, this newly appreciated interaction between the MCL1 and MARCHF5 oncogenes may prove useful in improving the design of combination therapies for KSHV malignancies, offering a rational strategy against a cancer that has so far proven difficult to treat.

Subject of Research: How the mitochondrial ubiquitin ligase MARCHF5 cooperates with MCL1 to inhibit apoptosis in KSHV-transformed primary effusion lymphoma cells

Article Title: The Mitochondrial ubiquitin ligase MARCHF5 cooperates with MCL1 to inhibit Apoptosis in KSHV-transformed primary effusion lymphoma cell lines

Article References: Viswanathan, P., Bersonda, J. R., Gill, J., Navarro, A., Farrar, A. C., Caldarera, T., Murphy, A., Dunham, D., Boehme, K. W., & Manzano, M. (2026). The mitochondrial ubiquitin ligase MARCHF5 cooperates with MCL1 to inhibit apoptosis in KSHV-transformed primary effusion lymphoma cell lines. PLOS Pathogens, 22(10), e1014665. https://doi.org/10.1371/journal.ppat.1014665

Image Credits: AI Generated

DOI: 10.1371/journal.ppat.1014665

Keywords: KSHV, primary effusion lymphoma, MCL1, MARCHF5, NOXA, apoptosis, ubiquitin ligase, mitochondria, CRISPR, oncogene, viral oncology, combination therapy

The research team began with a systematic question about a large and underexplored molecular family. Ubiquitination, the process by which the small protein ubiquitin is attached to target proteins, governs countless cellular activities in eukaryotes, from protein degradation to DNA repair and cell differentiation. The process requires a cascade of enzymes: E1 activating enzymes priming ubiquitin, E2 conjugating enzymes transferring it, and E3 ligases conferring substrate specificity. While ubiquitination was known to operate in Plasmodium parasites, whether any particular ubiquitination machinery specifically controlled the sexual conversion step was unknown. To find out, the researchers deployed CRISPR–Cas9 mutagenesis screens against the complete repertoire of E2 ubiquitin-conjugating enzyme genes in the rodent malaria parasite Plasmodium yoelii, systematically deleting each of the fourteen E2 genes and assessing the consequences for parasite development across the life cycle.

The screen produced a clear winner. An E2 enzyme the authors designate Rad6B emerged as essential for gametocytogenesis, the process by which asexual blood-stage parasites convert into gametocytes. When Rad6B was deleted, gametocyte production collapsed to severe defects, and the mutant parasites proved dramatically impaired in their ability to form oocysts in the mosquito midgut, the critical developmental stage that follows ingestion of gametocytes during a blood meal. Parasitemia and host survival in mice infected with the Rad6B deletion mutant remained comparable to wild-type infections, indicating that the enzyme’s essential function was not in general blood-stage proliferation but specifically in the developmental switch that enables transmission. Whole genome sequencing and Sanger sequencing confirmed that the observed phenotypes stemmed cleanly from the targeted gene disruptions rather than off-target alterations.

Rad6B did not act alone. Through interaction analyses, the team identified its partner: a RING-type E3 ubiquitin ligase called Tex1. The pairing follows a well-established logic in ubiquitin biology, in which the E2 enzyme carries activated ubiquitin while the RING-domain E3 ligase recognizes the substrate and catalyzes the transfer. Deleting Tex1 phenocopied the loss of Rad6B, producing severe defects in gametocyte production and oocyst formation, and the two proteins were shown to function as a complex. To verify that the enzymatic activities of both partners were required, the researchers engineered point-mutant parasites: a Rad6B variant in which the conserved catalytic cysteine in the ubiquitin-conjugating domain was substituted, and Tex1 variants in which conserved arginine and lysine residues of the RING domain were mutated or the domain deleted outright. In every case, the disruption of catalytic function abolished the pathway’s effect on gametocytogenesis, confirming that genuine ubiquitin transfer—not a scaffolding role—lay behind the phenotype.

The substrate that the Rad6B–Tex1 complex targets turned out to be histone H2B, one of the core proteins around which DNA is wound. The complex catalyzes monoubiquitination of H2B, the attachment of a single ubiquitin molecule to a specific lysine residue on the histone. This modification has an illustrious history in biology: it was first characterized in yeast, where the Rad6 ortholog and its RING partner Bre1 mark H2B, and it was subsequently shown to regulate gene silencing, H3 methylation and differentiation of multipotent stem cells in mammals. The new study demonstrates that the same modification operates in Plasmodium yoelii and, crucially, in Plasmodium falciparum, the parasite responsible for the most deadly form of human malaria. Using ChIP-seq and CUT&Tag-seq, the researchers mapped H2B monoubiquitination across the parasite genome and found it dramatically reduced at specific loci in both the Rad6B and Tex1 deletion mutants.

The mechanistic chain then became clear through a series of elegant chromatin analyses. In wild-type parasites, H2B monoubiquitination at the ap2-g promoter region promotes the occupancy of tri-methylated histone H3 lysine 4, or H3K4me3, an activating histone mark classically associated with open, transcriptionally permissive chromatin. When the Rad6B–Tex1 complex was absent, this H3K4me3 enrichment at the ap2-g promoter was lost, ap2-g expression failed to rise, and the parasites remained locked in the asexual program. CUT&Tag-seq in the deletion mutants showed parallel losses of both H2Bub and H3K4me3 signals at the ap2-g locus, with other chromatin marks such as H3K79me3 and heterochromatin protein 1 distributions assessed for comparison. The effect was spatially specific: other AP2 family transcription factors and regulatory genes, including ap2-g2, ap2-g3, ap2-g5, hda2 and hp1, showed no comparable loss of H2Bub, indicating that the pathway does not broadly remodel the chromatin landscape but instead focuses on the master regulator of sexual commitment.

To prove that the histone mark itself, rather than some other function of Rad6B, was the causal agent, the team performed knock-in experiments that border on molecular surgery. They generated parasite lines in which the endogenous histone H2B was replaced with versions carrying substitutions at the ubiquitination site, and similarly modified H3 at the lysines subject to methylation. Parasites carrying an H2B version that cannot be monoubiquitinated failed to activate ap2-g and could not commit to gametocytogenesis, while restoring the ubiquitin-acceptable residue restored the pathway. Complementary experiments showed that altering the H3 lysine targeted for tri-methylation likewise disrupted sexual commitment, placing H2B monoubiquitination upstream of H3K4me3 in a coherent signaling relay. Transcriptomic comparisons reinforced the picture: genes downregulated in the Rad6B and Tex1 mutants overlapped substantially, and their orthologs in P. falciparum and P. berghei were enriched for genes whose expression peaks during gametocyte development, indicating that the two enzymes converge on the sexual differentiation program.

Perhaps the most consequential finding is the conservation of this mechanism in the human parasite. In Plasmodium falciparum, disruption of either Rad6B or Tex1 produced the same chromatin consequences—loss of H2Bub and H3K4me3 at the ap2-g locus—and the same failure to produce gametocytes, while asexual blood-stage growth of the mutants proceeded normally across three erythrocytic cycles. Using a parasite line in which ap2-g expression is tagged with green fluorescent protein, the researchers sorted individual trophozoites that had committed to sexual development from those that had not. The committed, AP2-G-positive parasites displayed significantly enriched H2Bub and H3K4me3 at the ap2-g upstream region compared with their AP2-G-negative siblings. The team also manipulated culture conditions known to influence sexual conversion, growing parasites under commitment-inducing conditions of high parasitemia with reduced albumin supplementation, and observed that these inducing conditions were accompanied by increased H2Bub enrichment at the ap2-g promoter, linking the epigenetic mark to the environmental cues that naturally trigger gametocyte production.

The implications for malaria control are considerable. Transmission-blocking interventions—drugs, vaccines or other strategies that prevent parasites from completing their development in the mosquito—remain a critical but underserved frontier in malaria elimination, because a treatment that kills asexual parasites while leaving gametocyte production intact can still leave the treated patient infectious to mosquitoes. By identifying the Rad6B–Tex1 ubiquitin ligase complex as the enzymatic gatekeeper of sexual commitment, the study supplies a pair of concrete molecular targets whose inhibition would suppress gametocyte formation at its epigenetic source. Because the pathway is conserved between rodent and human malaria parasites, the P. yoelii system can serve as a tractable model for screening inhibitors against the human equivalents. Beyond the translational angle, the work adds malaria parasites to the growing list of eukaryotes in which the ancient Rad6–Bre1 module of H2B monoubiquitination, first described in yeast more than two decades ago, serves as a master regulator of cell differentiation—here repurposed by a single-celled pathogen to choreograph one of the most consequential decisions in its life cycle, and in doing so, to choreograph the spread of a disease that still claims hundreds of thousands of lives each year.

Subject of Research: Epigenetic control of sexual commitment in malaria parasites via histone H2B monoubiquitination

Article Title: Histone H2B monoubiquitination drives sexual commitment in malaria parasites

Article References: Jiao, Z., Lin, L., Tang, R., Zhong, C.-Q., Wu, C., Li, W., Xu, R., Gao, Y., He, P., Wang, C., Zhang, Q.-Q., Liu, S., Yuan, J., Zhang, Q., & Li, J. (2026). Histone H2B monoubiquitination drives sexual commitment in malaria parasites. Nature Microbiology. https://doi.org/10.1038/s41564-026-02475-4

Image Credits: AI Generated

DOI: 10.1038/s41564-026-02475-4

Keywords: malaria, Plasmodium, gametocytogenesis, histone H2B monoubiquitination, Rad6B, Tex1, AP2-G, epigenetics, ubiquitin ligase, H3K4me3, sexual commitment, transmission blocking

The study, led by Maja Louise Arendt of the University of Copenhagen and Jennifer R. S. Meadows of Uppsala University and SciLifeLab, focused on cereblon, the protein that thalidomide binds. Cereblon was only identified as the drug’s primary target in 2010, when researchers showed that thalidomide attaches to this substrate receptor of the cullin-4 E3 ubiquitin ligase complex, known as CUL4-RBX1-DDB1. When thalidomide occupies cereblon, the degradation machinery’s specificity changes: it begins recruiting, ubiquitinating and destroying an unnatural set of proteins called neo-substrates, including transcription factors such as SALL4, p63, Ikaros and Aiolos. These proteins govern critical developmental and immunological processes, which helps explain both the drug’s teratogenic tragedy and its therapeutic benefits. At the same time, native substrates such as MEIS2 are no longer degraded, adding further biological consequences. To date, cereblon remains the only described mammalian target of thalidomide.

The crux of the new findings lies in a single amino acid position within the cereblon protein. The C-terminal region of human cereblon, spanning roughly amino acids 318 to 426, forms the thalidomide-binding CULT domain, and variation within this 109-amino-acid stretch is known to determine how different species respond to the drug. In humans, the amino acid at position 388 is a valine, abbreviated V388. In mice, the equivalent position holds an isoleucine instead. This seemingly minor substitution does not prevent thalidomide from binding to cereblon, but it creates steric hindrance that blocks neo-substrates from entering the binding pocket, preventing their ubiquitination and degradation. The result is that thalidomide and its derivatives are pharmacologically ineffective in mice, a fact that contributed to the drug’s original licensing as a supposedly safe sedative, since rodent tests failed to predict its devastating effects in humans.

When the researchers mined the Dog10K consortium datasets, which include single nucleotide and structural variant calls from 1,929 and 1,879 canids respectively, they found that every single dog examined encodes an isoleucine at the position equivalent to human V388. Across the roughly 30-kilobase CRBN gene region, 384 variable sites were detected, but only three had any potential to alter the protein’s coding sequence, and each of these was extraordinarily rare, with allele frequencies below 0.2 percent. Two structural deletions overlapping the gene were also identified, both at frequencies below 0.5 percent. In other words, the canine cereblon protein is essentially uniform across the species, and it uniformly carries the mouse-like isoleucine that has been shown to abolish thalidomide’s neo-substrate degradation activity.

To place this finding in evolutionary context, the team turned to two massive comparative genomics resources: the Zoonomia alignment of 240 mammalian species and the TOGA alignment covering 344 eutherian mammals. At the nucleotide level, the codon encoding position 388 showed little evolutionary constraint, with phyloP conservation scores of 0.40, 4.66 and -1.74 across its three bases; only the second position showed purifying selection. This wobbling tolerance is mirrored by amino acid variability across the mammalian tree. Among 344 species, 72 percent encode an isoleucine at the equivalent position, including all 55 available species of the order Carnivora, to which dogs belong. Only bats, rodents and rabbits, and the order Primates, which includes humans, were found to encode both valine and isoleucine at this site. Notably, rabbits and guinea pigs carry a valine, consistent with decades of toxicology data showing that rabbits, unlike mice, do develop thalidomide-induced birth defects similar to those seen in humans.

The researchers also considered whether cancer itself might change the equation. Tumors accumulate mutations that can alter drug sensitivity, so the team searched for non-silent, protein-altering mutations in the CRBN gene within two published canine tumor-normal sequencing datasets, comprising 55 mammary tumors and 43 diffuse large B-cell lymphoma cases, as well as 723 samples from the cBioportal Canine Cancer Genome Atlas. The search yielded almost nothing: a single non-synonymous mutation, p.CRBN A10T, was found in one lymphoma sample, affecting the N-terminal region far from the thalidomide-binding CULT domain. No mutations were identified that would plausibly make canine cancer cells more susceptible to thalidomide than normal cells. In human multiple myeloma, somatic CRBN mutations are likewise reported to be neutral or to confer resistance, never enhanced sensitivity.

These genetic findings cast a revealing light on the veterinary literature. Thalidomide has been tested in dogs with splenic hemangiosarcoma, mammary carcinoma, lung carcinoma, lymphoma and multiple myeloma, usually in combination with other therapies, making efficacy difficult to assess. One recent multicentre retrospective study reported that 71 percent of seven dogs with relapsed multiple myeloma maintained or achieved complete remission on thalidomide as a single-agent rescue therapy, with moderate lethargy in two dogs as the only recorded side effect. Strikingly, a 53-week toxicology study in beagles using doses up to 1,000 milligrams per kilogram daily, roughly fifty times the human clinical dose for a ten-kilogram dog, found no clinically significant adverse effects, no hematological or biochemical changes and no evidence of the peripheral neuropathy that plagues human patients. The authors argue that this remarkable tolerance is itself a warning sign: a drug with no side effects at massive doses may simply have no pharmacological activity.

There are caveats. The sedative effect of thalidomide appears to be independent of cereblon, and one canine study reported reduced vascular endothelial growth factor staining in metastatic tissue from treated dogs, hinting that anti-angiogenic mechanisms independent of the CRBN-CUL4-RBX1-DDB1 complex might operate in dogs. A pharmacokinetic study in tumor-bearing dogs also reported a range of clinical signs, though the authors of the new analysis note these could reflect advanced cancer rather than drug toxicity. The researchers are careful not to claim that thalidomide is definitively useless in dogs; rather, they conclude that the molecular function of the drug in this species must be directly investigated before its therapeutic role in canine cancer can be understood or justified.

Beyond its immediate veterinary implications, the study is a compelling demonstration of how publicly available genomic data can inform clinical pharmacology. Because DNA-damaging chemotherapy agents tend to work comparably across humans and dogs, veterinary oncologists have reasonably borrowed human anticancer drugs. But for targeted agents such as thalidomide, whose action depends on the precise structure of a single protein, species differences in that target can silently nullify the drug’s effect. Given thalidomide’s tragic history and its strict regulation in human medicine, the authors advocate that molecular evidence of function should be established before the drug is used therapeutically in dogs, a lesson that applies equally to the growing arsenal of cereblon-targeting protein degraders now entering human clinical development.

Subject of Research: Genetic variation in the canine cereblon protein and its implications for thalidomide therapy in dogs with cancer

Article Title: Genetic evidence supports that thalidomide should not be used therapeutically in dogs

Article References: Arendt, M. L., & Meadows, J. R. S. (2026). Genetic evidence supports that thalidomide should not be used therapeutically in dogs. Veterinary Oncology, 3(1), Article 14. https://doi.org/10.1186/s44356-026-00067-2

Image Credits: AI Generated

DOI: 10.1186/s44356-026-00067-2

Keywords: thalidomide, cereblon, CRBN, dogs, canine cancer, veterinary oncology, comparative genomics, neo-substrates, ubiquitin ligase, drug repurposing, teratogenicity, Dog10K