Disruption of cotranscriptional splicing suggests that RBM39 is a therapeutic target in acute lymphoblastic leukemia.

There are few options for patients with relapse/refractory B-cell acute lymphoblastic leukemia (B-ALL), thus this is a major area of unmet medical need. Here, we reveal that inclusion of a poison exon in RBM39, which could be induced both by CDK9 or CDK9 independent CMGC (cyclin-dependent kinases, mitogen-activated protein kinases, glycogen synthase kinases, CDC-like kinases) kinase inhibition, is recognized by the nonsense-mediated mRNA decay (NMD) pathway for degradation. Targeting this poison exon in RBM39 with CMGC inhibitors lead to protein downregulation and inhibition of ALL growth, particularly in relapse/refractory B-ALL. Mechanistically, disruption of co-transcriptional splicing by inhibition of CMGC kinases including DYRK1A, or inhibition of CDK9, which phosphorylate the C-terminal domain of RNA polymerase II (Pol II), results in alteration of SF3B1 and Pol II association. Disruption of SF3B1 and transcriptional elongation complex alters Pol II pausing, which promotes the inclusion of a poison exon in RBM39. Moreover, RBM39 ablation suppresses the growth of human B-ALL, and targeting RBM39 with sulfonamides, which degrade RBM39 protein, showed strong anti-tumor activity in preclinical models. Our data reveal that relapse/refractory B-ALL is susceptible to pharmacologic and genetic inhibition of RBM39 and provide two potential strategies to target this axis.

Hyper-active RAS/MAPK introduces cancer-specific mitotic vulnerabilities.

Aneuploidy, the incorrect number of whole chromosomes, is a common feature of tumors that contributes to their initiation and evolution. Preventing aneuploidy requires properly functioning kinetochores, which are large protein complexes assembled on centromeric DNA that link mitotic chromosomes to dynamic spindle microtubules and facilitate chromosome segregation. The kinetochore leverages at least two mechanisms to prevent aneuploidy: error correction and the spindle assembly checkpoint (SAC). BubR1, a factor involved in both processes, was identified as a cancer dependency and therapeutic target in multiple tumor types; however, it remains unclear what specific oncogenic pressures drive this enhanced dependency on BubR1 and whether it arises from BubR1's regulation of the SAC or error-correction pathways. Here, we use a genetically controlled transformation model and glioblastoma tumor isolates to show that constitutive signaling by RAS or MAPK is necessary for cancer-specific BubR1 vulnerability. The MAPK pathway enzymatically hyperstimulates a network of kinetochore kinases that compromises chromosome segregation, rendering cells more dependent on two BubR1 activities: counteracting excessive kinetochore-microtubule turnover for error correction and maintaining the SAC. This work expands our understanding of how chromosome segregation adapts to different cellular states and reveals an oncogenic trigger of a cancer-specific defect.

BuGZ facilitates loading of spindle assembly checkpoint proteins to kinetochores in early mitosis.

BuGZ is a kinetochore component that binds to and stabilizes Bub3, a key player in mitotic spindle assembly checkpoint signaling. Bub3 is required for kinetochore recruitment of Bub1 and BubR1, two proteins that have essential and distinct roles in the checkpoint. Both Bub1 and BubR1 localize to kinetochores through interactions with Bub3, which are mediated through conserved GLEBS domains in both Bub1 and BubR1. BuGZ also has a GLEBS domain, which is required for its kinetochore localization as well, presumably mediated through Bub3 binding. Although much is understood about the requirements for Bub1 and BubR1 interaction with Bub3 and kinetochores, much less is known regarding BuGZ's requirements. Here, we used a series of mutants to demonstrate that BuGZ kinetochore localization requires only its core GLEBS domain, which is distinct from the requirements for both Bub1 and BubR1. Furthermore, we found that the kinetics of Bub1, BubR1, and BuGZ loading to kinetochores differ, with BuGZ localizing prior to BubR1 and Bub1. To better understand how complexes containing Bub3 and its binding partners are loaded to kinetochores, we carried out size-exclusion chromatography and analyzed Bub3-containing complexes from cells under different spindle assembly checkpoint signaling conditions. We found that prior to kinetochore formation, Bub3 is complexed with BuGZ but not Bub1 or BubR1. Our results point to a model in which BuGZ stabilizes Bub3 and promotes Bub3 loading onto kinetochores in early mitosis, which, in turn, facilitates Bub1 and BubR1 kinetochore recruitment and spindle assembly checkpoint signaling.

Dissolving Fusion Oncoprotein Condensates to Reverse Aberrant Gene Expression.

In a recent study, Wang and colleagues reported that a significant fraction of cancer-associated fusion proteins display a common structural topology, including an N-terminal phase separation-prone region (PS) from one parent protein and a C-terminal DNA-binding domain (DBD) from the other. This is reminiscent of the structural topology of transcription factors and led to the hypothesis that the PS-DBD fusions form aberrant transcriptional condensates through phase separation, which was supported through transcriptomic data analysis and cellular condensate assays. The authors developed a high-throughput screen based upon time-lapse, high-content imaging to identify 114 compounds that dissolved condensates formed by a chromatin-dissociated mutant of FUS::ERG (FUS::ERGmut). One of these compounds, LY2835219, was shown to dissolve FUS::ERGmut condensates by promoting lysosome formation and was also active against condensates formed by other PS-DBD fusions, including EWS::FLI1. Finally, condensate dissolution by LY2835219 was shown to reverse aberrant gene expression driven by EWS::FLI1, although how this compound specifically marshals lysosomes to target some PS-DBD fusions and not other condensate-forming proteins remains elusive. This work not only highlights likely roles for aberrant condensate formation in the oncogenic function of PS-DBD fusions, but also provides proof of principle for mechanistically unbiased screening to identify compounds that modulate fusion protein-driven condensates and their oncogenic functions.

Defining the condensate landscape of fusion oncoproteins.

Fusion oncoproteins (FOs) arise from chromosomal translocations in ~17% of cancers and are often oncogenic drivers. Although some FOs can promote oncogenesis by undergoing liquid-liquid phase separation (LLPS) to form aberrant biomolecular condensates, the generality of this phenomenon is unknown. We explored this question by testing 166 FOs in HeLa cells and found that 58% formed condensates. The condensate-forming FOs displayed physicochemical features distinct from those of condensate-negative FOs and segregated into distinct feature-based groups that aligned with their sub-cellular localization and biological function. Using Machine Learning, we developed a predictor of FO condensation behavior, and discovered that 67% of ~3000 additional FOs likely form condensates, with 35% of those predicted to function by altering gene expression. 47% of the predicted condensate-negative FOs were associated with cell signaling functions, suggesting a functional dichotomy between condensate-positive and -negative FOs. Our Datasets and reagents are rich resources to interrogate FO condensation in the future.

An Image Analysis Pipeline for Quantifying the Features of Fluorescently-Labeled Biomolecular Condensates in Cells.

Biomolecular condensates are cellular organelles formed through liquid-liquid phase separation (LLPS) that play critical roles in cellular functions including signaling, transcription, translation, and stress response. Importantly, condensate misregulation is associated with human diseases, including neurodegeneration and cancer among others. When condensate-forming biomolecules are fluorescently-labeled and examined with fluorescence microscopy they appear as illuminated foci, or puncta, in cells. Puncta features such as number, volume, shape, location, and concentration of biomolecular species within them are influenced by the thermodynamics of biomolecular interactions that underlie LLPS. Quantification of puncta features enables evaluation of the thermodynamic driving force for LLPS and facilitates quantitative comparisons of puncta formed under different cellular conditions or by different biomolecules. Our work on nucleoporin 98 (NUP98) fusion oncoproteins (FOs) associated with pediatric leukemia inspired us to develop an objective and reliable computational approach for such analyses. The NUP98-HOXA9 FO forms hundreds of punctate transcriptional condensates in cells, leading to hematopoietic cell transformation and leukemogenesis. To quantify the features of these puncta and derive the associated thermodynamic parameters, we developed a live-cell fluorescence microscopy image processing pipeline based on existing methodologies and open-source tools. The pipeline quantifies the numbers and volumes of puncta and fluorescence intensities of the fluorescently-labeled biomolecule(s) within them and generates reports of their features for hundreds of cells. Using a standard curve of fluorescence intensity versus protein concentration, the pipeline determines the apparent molar concentration of fluorescently-labeled biomolecules within and outside of puncta and calculates the partition coefficient (Kp) and Gibbs free energy of transfer (ΔGTr), which quantify the favorability of a labeled biomolecule partitioning into puncta. In addition, we provide a library of R functions for statistical analysis of the extracted measurements for certain experimental designs. The source code, analysis notebooks, and test data for the Punctatools pipeline are available on GitHub: https://github.com/stjude/punctatools. Here, we provide a protocol for applying our Punctatools pipeline to extract puncta features from fluorescence microscopy images of cells.

Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation.

NUP98 fusion oncoproteins (FO) are drivers in pediatric leukemias and many transform hematopoietic cells. Most NUP98 FOs harbor an intrinsically disordered region from NUP98 that is prone to liquid-liquid phase separation (LLPS) in vitro. A predominant class of NUP98 FOs, including NUP98-HOXA9 (NHA9), retains a DNA-binding homeodomain, whereas others harbor other types of DNA- or chromatin-binding domains. NUP98 FOs have long been known to form puncta, but long-standing questions are how nuclear puncta form and how they drive leukemogenesis. Here we studied NHA9 condensates and show that homotypic interactions and different types of heterotypic interactions are required to form nuclear puncta, which are associated with aberrant transcriptional activity and transformation of hematopoietic stem and progenitor cells. We also show that three additional leukemia-associated NUP98 FOs (NUP98-PRRX1, NUP98-KDM5A, and NUP98-LNP1) form nuclear puncta and transform hematopoietic cells. These findings indicate that LLPS is critical for leukemogenesis by NUP98 FOs. SIGNIFICANCE: We show that homotypic and heterotypic mechanisms of LLPS control NUP98-HOXA9 puncta formation, modulating transcriptional activity and transforming hematopoietic cells. Importantly, these mechanisms are generalizable to other NUP98 FOs that share similar domain structures. These findings address long-standing questions on how nuclear puncta form and their link to leukemogenesis. This article is highlighted in the In This Issue feature, p. 873.

Spindle assembly checkpoint signaling and sister chromatid cohesion are disrupted by HPV E6-mediated transformation.

Aneuploidy, a condition that results from unequal partitioning of chromosomes during mitosis, is a hallmark of many cancers, including those caused by human papillomaviruses (HPVs). E6 and E7 are the primary transforming proteins in HPV that drive tumor progression. In this study, we stably expressed E6 and E7 in noncancerous RPE1 cells and analyzed the specific mitotic defects that contribute to aneuploidy in each cell line. We find that E6 expression results in multiple chromosomes associated with one or both spindle poles, causing a significant mitotic delay. In most cells, the misaligned chromosomes eventually migrated to the spindle equator, leading to mitotic exit. In some cells, however, mitotic exit occurred in the presence of pole-associated chromosomes. We determined that this premature mitotic exit is due to defects in spindle assembly checkpoint (SAC) signaling, such that cells are unable to maintain a prolonged mitotic arrest in the presence of unaligned chromosomes. This SAC defect is caused in part by a loss of kinetochore-associated Mad2 in E6-expressing cells. Our results demonstrate that E6-expressing cells exhibit previously unappreciated mitotic defects that likely contribute to HPV-mediated cancer progression.