Search

AIMS: High-dose methotrexate (HDMTX) is an essential part of the treatment of several adult and paediatric malignancies. Despite meticulous supportive care during HDMTX administration, severe toxicities, including acute kidney injury (AKI), may occur contributing to patient morbidity. Population pharmacokinetics provide a powerful tool to predict time to clear HDMTX and adjust subsequent doses. We sought to develop and validate pharmacokinetic models for HDMTX in adults with diverse malignancies and to relate systemic exposure with the occurrence of severe toxicity.METHODS: Anonymized, de-identified data were provided from 101 US oncology practices that participate in the Guardian Research Network, a non-profit clinical research consortium. Modelled variables included clinical, laboratory, demographic and pharmacological data. Population pharmacokinetic analysis was performed by means of nonlinear mixed effects modelling using MonolixSuite.RESULTS: A total of 693 HDMTX courses from 243 adults were analysed, of which 62 courses (8.8%) were associated with stage 2/3 acute kidney injury (43 stage 2, 19 stage 3). A three-compartment model adequately fitted the data. Time-dependent serum creatinine, baseline serum albumin and allometrically scaled bodyweight were clinically significant covariates related to methotrexate clearance. External evaluation confirmed a satisfactory predictive performance of the model in adults receiving HDMTX. Dose-normalized methotrexate concentration at 24 and 48 hours correlated with AKI incidence.CONCLUSION: We developed a population pharmacometric model that considers weight, albumin and time-dependent creatinine that can be used to guide supportive care in adult patients with delayed HDMTX elimination.

During development, extracellular cues guiding cell fate determination are provided by morphogens. One mechanism by which morphogens are proposed to traverse extracellular space is by traveling along specialized filopodia called cytonemes. These cellular highways extend between signal-producing and -receiving cells to enable direct morphogen delivery. Although genetic studies support cytoneme involvement in morphogen transport, mechanistic insight into how they are regulated is limited owing to technical challenges associated with performing cell biological analysis of the delicate filopodial structures. Here, we introduce a fixation method whereby cultured cell cytonemes can be preserved for imaging studies, allowing investigation of cytoneme regulation using standard cell biological techniques. Using this method, we examined Hedgehog-containing cytonemes and identified a role for the Hedgehog deployment protein Dispatched in cytoneme stabilization. We demonstrate that Hedgehog and Dispatched colocalize in cytonemes, and that cholesterol-modified Hedgehog acts through Dispatched to increase cytoneme occurrence. Live imaging suggests that this occurs through Dispatched-mediated slowing of cytoneme retraction rates. Dispatched-induced cytoneme modulation was recapitulated in wing imaginal discs of transgenic Drosophila, providing evidence that cultured cell cytoneme analysis is predictive of in vivo functionality., Summary: A new fixation method for preserving cultured cell cytonemes, used in combination with live cell imaging, reveals that the Hedgehog deployment protein Dispatched promotes cytoneme occurrence by slowing retraction rates.

This data article includes supporting information for the research article entitled “The Small GTPase Rap1 is a Modulator of Hedgehog Signaling” [1]. Drosophila wing phenotypes induced by expression of a dominant negative Smoothened (Smo) mutant were cataloged into five distinct classes. Class distributions observed following expression of dominant negative Smo in control and sensitized backgrounds were quantified to serve as references for strength of phenotypic modification. Shifts in class distribution of Hedgehog (Hh) wing phenotypes resulting from introduction of loss-of-function alleles of select Ras family G protein genes and the Hh pathway regulators Fused and Suppressor of Fused are shown.

Membrane‐less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher‐order assemblies influences the recruitment of the speckle‐type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin‐3‐RING ubiquitin ligase (CRL3) substrate adaptor, self‐associates into higher‐order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild‐type SPOP localizes to liquid nuclear speckles, self‐association‐deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB‐mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration‐dependent populations of the resulting oligomeric species. Higher‐order oligomerization of SPOP stimulates CRL3 SPOP ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher‐order protein self‐association may be a general mechanism to concentrate functional components in membrane‐less cellular bodies.

e20525 Background: Despite new treatments, Relapsed-Refractory Multiple Myeloma (RRMM) remains an incurable disease1. In a recent study by Moreau et al, there was a demonstrated survival benefit to treatment with pomalidomide in RRMM patients2. To better understand how the relationships between initial depth of response and long-term prognosis can inform clinical decision making and guide new compound development, we performed an analysis of standardized clinical trial patient pool data compared with Real-World Data (RWD), across multiple studies. Methods: Pooled clinical trial data was obtained from a Study Data Tabulation Model (SDTM) dataset (n=1,815) from the Medidata Enterprise Data Store. De-identified Oncology EMR data was sourced from the Guardian Research Network™ (GRN)5 of integrated delivery systems from 2010-2018, with robust clinical endpoint extraction and curation to enable outcomes comparisons (n=962). Subject selection was refined based on the inclusion/exclusion criteria from the NIMBUS trial2. Response, Progression-free survival (PFS), and Overall Survival (OS) were extracted. Patients were stratified by age, gender, and prior regimens. Log-rank tests were conducted to compare PFS and OS in patient sub-populations at 90, 180, and 240 days from most recent treatment start. Cox proportional hazard models assessed predictors of survival. Rates were estimated for common adverse events, including leukopenia, neutropenia, and thrombocytopenia. Factors associated with neutropenia were assessed using logistic regression. Results: Within pooled trial and RWD patients, the majority were on regimens with proteasome inhibitors, followed by immunomodulators, and approximately one third on monoclonal antibodies. Within the pooled trial analysis, survival rates were consistent with published literature rates, at ~4 months and ~12 months, respectively. EMR data analysis showed overall longer times to progression with an increased depth of response, with similar associations. Frequency-Factored Time-to-Progression also decreased. Conclusions: The use of SDTM for pooled clinical trial analyses, together with real-world data, can overcome individual trial sample size limitations and biases. Together this approach can expand the range of populations, relative treatment comparisons, and clinical events that can be studied to more comprehensively understand the complexity of the oncology treatment landscape.

Hedgehog ligands activate an evolutionarily conserved signaling pathway that provides instructional cues during tissue morphogenesis, and when corrupted, contributes to developmental disorders and cancer. The transmembrane protein Dispatched is an essential component of the machinery that deploys Hedgehog family ligands from producing cells, and is absolutely required for signaling to long-range targets. Despite this crucial role, regulatory mechanisms controlling Dispatched activity remain largely undefined. Herein, we reveal vertebrate Dispatched is activated by proprotein convertase-mediated cleavage at a conserved processing site in its first extracellular loop. Dispatched processing occurs at the cell surface to instruct its membrane re-localization in polarized epithelial cells. Cleavage site mutation alters Dispatched membrane trafficking and reduces ligand release, leading to compromised pathway activity in vivo. As such, convertase-mediated cleavage is required for Dispatched maturation and functional competency in Hedgehog ligand-producing cells., eLife digest As an embryo develops, its cells divide many times until they specialize to become distinct cell types that make up the tissues and organs. To do so, the cells need to communicate, and some send signals by making and releasing proteins that travel to more distant cells. One such signaling pathway is called Hedgehog signaling. This pathway is necessary so that the tissue and organs develop properly. If faulty, it can stop the embryo from developing properly and even lead to diseases such as cancer. Hedgehog signaling is initiated by the Hedgehog protein, which needs to be released from the cells that produce the message to transport the signal to the target cells. A protein called Dispatched helps Hedgehog to get free and travel to its destination. Without Dispatched, Hedgehog cannot be released and the embryos will not develop. Now, Stewart, Marada et al. wanted to find out if and how Dispatched itself is controlled by studying embryo cells of mice. The results showed that a protein called Furin activates Dispatched by splitting it at a specific point. When the break-point on Dispatch was genetically modified, Furin could no longer cleave the protein. As a consequence, Dispatched did not reach the correct location within cells to help Hedgehog move away from signal-releasing cells. This suggests that Furin is an essential protein of the Hedgehog pathway. A next step will be to see if this is also the case in humans. Some cancer cells can produce large amounts of Hedgehog protein, which makes tumors grow faster. A better understanding of Hedgehog signaling may help to find new cancer therapies that can block this pathway in cancer cells.

Background High-dose methotrexate (HDMTX, doses higher than 500 mg/m2) is an essential component of therapy for several hematologic cancers but requires meticulous supportive care and therapeutic drug monitoring to deliver safely. Community oncologists manage 85% of adults with cancer in the USA but may lack infrastructure to support HDMTX administration and patient outcomes can be compromised by avoidance of HDMTX when it would have been appropriate, toxicities, and early discontinuation after toxicity occurs. Methods We evaluated HDMTX use and outcomes in the Guardian Research Network, (www.GuardianResearch.org), a non-profit USA clinical trials consortium of 110 community oncology centers with a database containing patients' entire medical, including all demographics, diagnoses, labs, medications, procedures, encounters, and notes of all kinds (clinical, radiology reports). Anonymized, de-identified data about HDMTX doses, administration, supportive care, and outcomes was analyzed to determine its safety and efficacy in the community setting. Results Of 529784 cancer patients diagnosed from 2011 through 2019, 230 received at 1 to 18 courses of HDMTX (median 3) for acute lymphoblastic leukemia (n=41), NHL (n=142), or other cancers (n=47). Patient characteristics are shown in the Table. In 106 cases (46%), patients received only 1 or 2 doses of HDMTX, usually due to delayed methotrexate clearance and acute kidney injury (AKI). Only 2 patients (1%) received dialysis, but 23 (10%) died within 28 days of a HDMTX course. Survival at 3 years for patients who received only 1 or 2 doses of HDMTX was 50% (Standard error, SE 6%), compared to 71% for those who received 3 or 4 doses (SE 6%) and 64% for patients who received 5 or more doses (SE 7%), p=0.002. Age older than 80 years predicted survival (p=0.02), but no other patient features, including: race (p=0.40), sex (p=0.72), cancer type (p=0.82). In multivariable Cox proportional hazards analysis, receiving 2 or less HDMTX courses was associated with a hazard ratio (HR) of 2.0 (95% CI 1.3 to 3.3, p=0.004) and age increased the risk of death for each decade older than 50 years (HR 4.8 for patients older than 80 years, 95% CI 1.6 to 14, p=0.004. Conclusions HDMTX can be safely delivered in community oncology practices, but requires meticulous supportive care to allow completion of all doses and maximize survival Figure Disclosures Howard: BTG: Consultancy, Research Funding; Servier: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; EUSA: Consultancy; Resonance: Equity Ownership.

To define the mutation spectrum in non-Down syndrome acute megkaryoblastic leukemia (non-DS-AMKL), we performed transcriptome sequencing on diagnostic blasts from 14 pediatric patients and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult AMKL leukemia samples. Our analysis identified a cryptic chromosome 16 inversion [inv(16)(p13.3q24.3)] in 27% of pediatric cases, which encodes a CBFA2T3-GLIS2 fusion protein. Expression of CBFA2T3-GLIS2 in Drosophila and murine hematopoietic cells induced bone morphogenic protein (BMP) signaling, and resulted in a marked increase in the self-renewal capacity of hematopoietic progenitors. These data suggest that expression of CBFA2T3-GLIS2 directly contributes to leukemogenesis.

Summary The conserved Hippo signaling pathway acts in growth control and is fundamental to animal development and oncogenesis [1–3]. Hippo signaling has also been implicated in adult midgut homeostasis in Drosophila . Regulated divisions of intestinal stem cells (ISCs), giving rise to an ISC and an enteroblast (EB) that differentiates into an enterocyte (EC) or an enteroendocrine (EE) cell [4–6], enable rapid tissue turnover in response to intestinal stress [7–15]. The damage-related increase in ISC proliferation requires deactivation of the Hippo pathway and consequential activation of the transcriptional coactivator Yorkie (Yki) [16–18] in both ECs and ISCs [19–22]. Here, we identify Pez, an evolutionarily conserved FERM domain protein containing a protein tyrosine phosphatase (PTP) domain, as a novel binding partner of the upstream Hippo signaling component Kibra [23–26]. Pez function—but not its PTP domain—is essential for Hippo pathway activity specifically in the fly midgut epithelium. Thus, Pez displays a tissue-specific requirement and functions as a negative upstream regulator of Yki in the regulation of ISC proliferation.

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the signal transducer of the developmentally and therapeutically relevant Hedgehog (Hh) pathway. Although recent structural analyses have advanced our understanding of Smo biology, several questions remain. Chief among them are the identity of its natural ligand, the regulatory processes controlling its activation, and the mechanisms by which it signals to downstream effectors. In this review, we discuss recent discoveries from multiple model systems that have set the stage for solving these mysteries. We focus on the roles of distinct Smo functional domains, post-translational modifications, and trafficking, and conclude by discussing their contributions to signal output.

Cellular “bodies”, i.e. organelles in cells that are not enclosed by membranes, have been characterized as large protein assemblies with liquid-like properties, though the biophysical basis for their formation is currently unclear. Recent work demonstrated that weak, multivalent protein interactions can result in the formation of large higher-order complexes, can undergo liquid demixing phase separation in vitro and may enable the formation of cellular bodies. The inherent size heterogeneity of higher-order complexes renders them difficult to characterize biophysically and structurally. As a result, their size distributions remain largely unquantified, limiting molecular insight into their biological functions. We report novel mechanisms governing the formation of nuclear SPOP bodies and stress granules in which the self-association of folded domains into large homo-oligomers and of long disordered regions play key roles. We have used biophysical, biochemical and cell biological approaches to explore the self-association of proteins in vitro, to quantify the size distribution of the resulting higher-order oligomers, and to relate these to the function of cellular bodies in cells. We further explore how changes in self-association properties of constituent proteins, i.e. the destabilization or rigidification of cellular bodies, can lead to cancer and neurodegenerative diseases. We propose that dynamic, higher-order protein self-association is a general mechanism underlying the formation of cellular bodies. These may serve as hotspots of enzymatic or signaling activity, which can be dynamically turned on or off through the regulated assembly and disassembly of the organelle bodies, respectively.

The Drosophila melanogaster genome has been extensively characterized, but there remains a pressing need to associate gene products with phenotypes, subcellular localizations, and interaction partners. A multifunctional, Minos transposon-based protein trapping system called Hostile takeover (Hto) was developed to facilitate in vivo analyses of endogenous genes, including live imaging, purification of protein complexes, and mutagenesis. The Hto transposon features a UAS enhancer with a basal promoter, followed by an artificial exon 1 and a standard 5′ splice site. Upon GAL4 induction, exon 1 can splice to the next exon downstream in the flanking genomic DNA, belonging to a random target gene. Exon 1 encodes a dual tag (FLAG epitope and mCherry red fluorescent protein), which becomes fused to the target protein. Hto was mobilized throughout the genome and then activated by eye-specific GAL4; an F1 screen for abnormal eye phenotypes was used to identify inserts that express disruptive fusion proteins. Approximately 1.7% of new inserts cause eye phenotypes. Of the first 23 verified target genes, 21 can be described as regulators of cell biology and development. Most are transcription factor genes, including AP-2, CG17181, cut, klu, mamo, Sox102F, and sv. Other target genes [l(1)G0232, nuf, pum, and Syt4] make cytoplasmic proteins, and these lines produce diverse fluorescence localization patterns. Hto permits the expression of stable carboxy-terminal subfragments of proteins, which are rarely tested in conventional genetic screens. Some of these may disrupt specific cell pathways, as exemplified by truncated forms of Mastermind and Nuf.

Smoothened (Smo) is a member of the Frizzled (FzD) class of G-protein-coupled receptors (GPCRs), and functions as the key transducer in the Hedgehog (Hh) signalling pathway. Smo has an extracellular cysteine-rich domain (CRD), indispensable for its function and downstream Hh signalling. Despite its essential role, the functional contribution of the CRD to Smo signalling has not been clearly elucidated. However, given that the FzD CRD binds to the endogenous Wnt ligand, it has been proposed that the Smo CRD may bind its own endogenous ligand. Here we present the NMR solution structure of the Drosophila Smo CRD, and describe interactions between the glucocorticoid budesonide (Bud) and the Smo CRDs from both Drosophila and human. Our results highlight a function of the Smo CRD, demonstrating its role in binding to small-molecule modulators.

The Hedgehog signaling pathway, an essential regulator of developmental patterning, has been implicated in playing causative and survival roles in a range of human cancers. The signal-transducing component of the pathway, Smoothened, has revealed itself to be an efficacious therapeutic target in combating oncogenic signaling. However, therapeutic challenges remain in cases where tumors acquire resistance to Smoothened antagonists, and also in cases where signaling is driven by active Smoothened mutants that exhibit reduced sensitivity to these compounds. We previously demonstrated that active Smoothened mutants are subjected to prolonged endoplasmic reticulum (ER) retention, likely due to their mutations triggering conformation shifts that are detected by ER quality control. We attempted to exploit this biology and demonstrate that deregulated Hedgehog signaling driven by active Smoothened mutants is specifically attenuated by ER stressors that induce the unfolded protein response (UPR). Upon UPR induction, active Smoothened mutants are targeted by ER-associated degradation, resulting in attenuation of inappropriate pathway activity. Accordingly, we found that the UPR agonist thapsigargin attenuated mutant Smoothened-induced phenotypes in vivo in Drosophila melanogaster. Wild-type Smoothened and physiological Hedgehog patterning were not affected, suggesting that UPR modulation may provide a novel therapeutic window to be evaluated for targeting active Smoothened mutants in disease.

Acute Megakaryoblastic Leukemia (AMKL) accounts for ∼10% of childhood acute myeloid leukemia (AML). Although AMKL patients with Down syndrome (DS-AMKL) have an excellent 5 year event-free survival (EFS), non-DS-AMKL patients have an extremely poor outcome with a 3 year EFS < 40%. To define the landscape of mutations that occur in non-DS-AMKL, we performed transcriptome sequencing on diagnostic blasts from 14 cases. Our results identified chromosomal rearrangements resulting in the expression of novel fusion transcripts in 12/14 cases. Remarkably, in 7/14 cases, we detected an inversion on chromosome 16 [inv(16)(p13.3;q24.3)] that resulted in the juxtaposition of CBFA2T3, a member of the ETO family of transcription factors, next to GLIS2 resulting in a CBFA2T3-GLIS2 chimeric gene encoding an in frame fusion protein. GLIS2 is a member of the GLI family of transcription factors that mediate sonic hedgehog (SHH) signaling and has been demonstrated to play a role in regulating expression of GLI target genes. Evaluation of a recurrency cohort of 52 samples including 24 additional pediatric cases and 28 adult cases revealed 6 additional pediatric samples carrying the fusion for an overall frequency of 34% in pediatric AMKL. To gain insight into the mechanism whereby CBFA2T3-GLIS2 promotes leukemogenesis, we introduced the fusion into murine hematopoietic cells and assessed its effect on in vitro colony replating as a surrogate measure of self-renewal. Cells transduced with a mCherry expressing retrovirus failed to form colonies after the 2nd replating. By contrast, expression of either wild-type GLIS2 or CBFA2T3-GLIS2 resulted in a marked increase in the self-renewal capacity, with colony formation persisting through 12 replatings. Immunophenotypic analysis of the CBFA2T3-GLIS2 expressing colonies revealed evidence of megakaryocytic differentiation. GLI transcription factors modulate expression of multiple downstream targets including components of BMP, WNT, and SHH pathways. To interrogate these pathways as potential contributors to the enhanced self-renewal capacity, we conducted luciferase reporter assays and found that CBFA2T3-GLIS2 functioned as a strong activator of the BMP responsive element. Furthermore, expression of CBFA2T3-GLIS2 in Drosophila resulted in ectopic expression of endogenous dpp, the fly homolog of BMP4, and conferred a dpp gain of function phenotype. Taken together these data identify a novel inv(16)-encoded CBFA2T3-GLIS2 fusion protein as a recurrent driver mutation in ∼35% of non-infant pediatric non-DS-AMKLs. The alteration of a key transcriptional regulator within the SHH signaling pathways in a substantial percentage of pediatric AMKL raises the possibility that inhibition of this pathway or downstream activated pathways may have a therapeutic benefit in this aggressive form of AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4867. doi:1538-7445.AM2012-4867

The Hedgehog (Hh) signaling pathway plays an instructional role during development, and is frequently activated in cancer. Ligand-induced pathway activation requires signaling by the transmembrane protein Smoothened (Smo), a member of the G-protein-coupled receptor (GPCR) superfamily. The extracellular (EC) loops of canonical GPCRs harbor cysteine residues that engage in disulfide bonds, affecting active and inactive signaling states through regulating receptor conformation, dimerization and/or ligand binding. Although a functional importance for cysteines localized to the N-terminal extracellular cysteine-rich domain has been described, a functional role for a set of conserved cysteines in the EC loops of Smo has not yet been established. In this study, we mutated each of the conserved EC cysteines, and tested for effects on Hh signal transduction. Cysteine mutagenesis reveals that previously uncharacterized functional roles exist for Smo EC1 and EC2. We provide in vitro and in vivo evidence that EC1 cysteine mutation induces significant Hh-independent Smo signaling, triggering a level of pathway activation similar to that of a maximal Hh response in Drosophila and mammalian systems. Furthermore, we show that a single amino acid change in EC2 attenuates Hh-induced Smo signaling, whereas deletion of the central region of EC2 renders Smo fully active, suggesting that the conformation of EC2 is crucial for regulated Smo activity. Taken together, these findings are consistent with loop cysteines engaging in disulfide bonds that facilitate a Smo conformation that is silent in the absence of Hh, but can transition to a fully active state in response to ligand.

During development, the evolutionarily conserved Hedgehog (Hh) morphogen provides instructional cues that influence cell fate, cell affinity and tissue morphogenesis. To do so, the Hh signaling cascade must coordinate its activity with other morphogenetic signals. This can occur through engagement of or response to effectors that do not typically function as core Hh pathway components. Given the ability of small G proteins of the Ras family to impact cell survival, differentiation, growth and adhesion, we wanted to determine whether Hh and Ras signaling might intersect during development. We performed genetic modifier tests in Drosophila to examine the ability of select Ras family members to influence Hh signal output, and identified Rap1 as a positive modulator of Hh pathway activity. Our results suggest that Rap1 is activated to its GTP-bound form in response to Hh ligand, and that the GTPase exchange factor C3G likely contributes to this activation. The Rap1 effector Canoe (Cno) also impacts Hh signal output, suggesting that a C3G-Rap1-Cno axis intersects the Hh pathway during tissue morphogenesis.

Light microscopically detectable, non-membrane bound cellular “bodies” are large protein assemblies with liquid-like properties, but the biophysical basis of their formation is unclear. Weak, multivalent protein interactions can result in higher-order complexes and can enable the formation of cellular bodies. The inherent size heterogeneity of higher-order complexes renders them difficult to characterize biophysically. As a result, their size distributions remain largely unquantified, limiting molecular insight into their biological functions. We report a novel mechanism governing cellular body formation of the Speckle-type POZ protein (SPOP), which was recently identified as tumor suppressor, is a ubiquitin ligase substrate adaptor that localizes to nuclear puncta. We demonstrate that its cellular localization is dependent upon the ability of SPOP to form higher-order homo-oligomers through indefinite self-association, mediated by two distinct oligomerization domains. Furthermore, in vitro ubiquitination of substrates is enhanced through higher-order self-association of SPOP, suggesting that SPOP puncta are hotspots of substrate ubiquitination. One of SPOP's domains dimerizes with nanomolar affinity yielding stable SPOP dimers as “building blocks” for indefinite self-association, while the other domain dimerizes with micromolar affinity, rendering SPOP oligomers highly dynamic. Together, this results in isodesmic self-association, in which each addition of a dimer occurs with the same affinity, independent of the oligomer size. From this model, we describe the size distribution of SPOP oligomers, providing for the first time a quantitative analysis of protein assemblies participating in the formation of cellular bodies. Mutations within both oligomerization domains have been observed in a variety of cancers, supporting our conclusion that SPOP self-association is important for its biological function. We propose that dynamic, higher-order protein self-association is a general mechanism underlying the formation of cellular bodies, which may serve as switches to fine-tune signaling cascades.