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The spindle assembly checkpoint (SAC) is critical for sensing defective microtubule-kinetochore attachments and tension across the kinetochore and functions to arrest cells in prometaphase to allow time to repair any errors before proceeding into anaphase. Dysregulation of the SAC leads to chromosome segregation errors that have been linked to human diseases like cancer. Although much has been learned about the composition of the SAC and the factors that regulate its activity, the proximity associations of core SAC components have not been explored in a systematic manner. Here, we have taken a BioID2-proximity-labeling proteomic approach to define the proximity protein environment for each of the five core SAC proteins BUB1, BUB3, BUBR1, MAD1L1, and MAD2L1 in mitotic-enriched populations of cells where the SAC is active. These five protein association maps were integrated to generate a SAC proximity protein network that contains multiple layers of information related to core SAC protein complexes, protein-protein interactions, and proximity associations. Our analysis validated many known SAC complexes and protein-protein interactions. Additionally, it uncovered new protein associations, including the ELYS-MAD1L1 interaction that we have validated, which lend insight into the functioning of core SAC proteins and highlight future areas of investigation to better understand the SAC.

BackgroundIn a metastatic setting, systemically-administered therapies that overcome the immunosuppressive tumor microenvironment to promote T-cell recruitment and T-cell cytolytic function will be required to elicit durable anti-tumor immunity. To accomplish this, the STACT (S. Typhimurium-Attenuated Cancer Therapy) platform was developed. STACT is a live bacterial product that has been highly modified using precision genome editing for the following properties: (1) enhanced tolerability after IV dosing, (2) tumor-specific enrichment, (3) phagocytosis by tumor-resident antigen-presenting cells (APCs) with a lack of epithelial cell infectivity, (4) multiplexed genetic cargo delivery, and (5) attenuation of bacterial pathways that impair CD8+ T-cell function. An extensive screening campaign was performed to identify ideal encoded immunomodulatory payload combinations delivered by STACT for efficacy against T-cell excluded tumors.MethodsChromosomal edits to the STACT platform strain were made using PCR. A panel of immunomodulatory proteins, including cytokines, type I interferon (IFN)-inducing factors, co-stimulatory receptors, checkpoint antibodies and TGFβR-Fc decoys were tested for combinatorial potency using STACT. An engineered STING (eSTING) was designed through an extensive protein engineering campaign to identify optimal variants. Combinations were evaluated in primary human APCs using in vitro functional assays, where STACT IL-15Rα-IL-15 (IL-15) + eSTING (ACTM-838) emerged as a lead candidate. ACTM-838 was then evaluated in multiple murine tumor models for therapeutic efficacy and mechanism, as well as tolerability in rodents and primates after systemic administration.ResultsCombinatorial target profiling led to the discovery of ACTM-838, a STACT encoding IL-15 + eSTING. In vitro, ACTM-838 payloads synergistically produced high levels of type I IFN and T-cell recruitment and activation factors from primary human APCs. In vivo, ACTM-838 demonstrated a high degree of complete tumor responses that were entirely CD8+ T-cell dependent. In an autochthonous breast cancer model that lacks any significant lymphocyte infiltrate, ACTM-838 was able to uniformly enrich in each spontaneous lesion to high levels after IV dosing and resulted in significant CD8+ T-cell infiltration. In primates, ACTM-838 was well-tolerated, rapidly cleared, and elicited minimal cytokine response after IV dosing.ConclusionsACTM-838 is a highly attenuated, precision genome-engineered bacterial immunotherapy that delivers IL-15 + eSTING to phagocytic APCs of the solid tumor microenvironment after systemic administration. In preclinical studies, ACTM-838 promotes CD8+ T-cell mediated tumor clearance in T-cell excluded tumors and elicits durable anti-tumor immunity, and is well tolerated in primates. Based on these data, ACTM-838 was nominated for clinical development and has entered cGMP manufacturing and IND-enabling studies.Ethics ApprovalAll animals were used according to protocols approved by an Institutional Animal Care and Use Committee and maintained in specific pathogen-free conditions in a barrier facility.

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this principle, the purification of critical proteins required for the functioning of these pathways along with their native interacting partners has not only allowed the mapping of the protein constituents of these pathways, but has also provided a deeper understanding of how these proteins coordinate to regulate these pathways. Within this context, understanding a protein's spatiotemporal localization and its protein-protein interaction network can aid in defining its role within a pathway, as well as how its misregulation may lead to disease pathogenesis. To address this need, several approaches for protein purification such as tandem affinity purification (TAP) and localization and affinity purification (LAP) have been designed and used successfully. Nevertheless, in order to apply these approaches to pathway-scale proteomic analyses, these strategies must be supplemented with modern technological developments in cloning and mammalian stable cell line generation. Here, we describe a method for generating LAP-tagged human inducible stable cell lines for investigating protein subcellular localization and protein-protein interaction networks. This approach has been successfully applied to the dissection of multiple cellular pathways including cell division and is compatible with high-throughput proteomic analyses.

The sterol regulatory element-binding protein (SREBP) transcription factors have become attractive targets for pharmacological inhibition in the treatment of metabolic diseases and cancer. SREBPs are critical for the production and metabolism of lipids and cholesterol, which are essential for cellular homeostasis and cell proliferation. Fatostatin was recently discovered as a specific inhibitor of SREBP cleavage-activating protein (SCAP), which is required for SREBP activation. Fatostatin possesses antitumor properties including the inhibition of cancer cell proliferation, invasion, and migration, and it arrests cancer cells in G2/M phase. Although Fatostatin has been viewed as an antitumor agent due to its inhibition of SREBP and its effect on lipid metabolism, we show that Fatostatin's anticancer properties can also be attributed to its inhibition of cell division. We analyzed the effect of SREBP activity inhibitors including Fatostatin, PF-429242, and Betulin on the cell cycle and determined that only Fatostatin possessed antimitotic properties. Fatostatin inhibited tubulin polymerization, arrested cells in mitosis, activated the spindle assembly checkpoint, and triggered mitotic catastrophe and reduced cell viability. Thus Fatostatin's ability to inhibit SREBP activity and cell division could prove beneficial in treating aggressive types of cancers such as glioblastomas that have elevated lipid metabolism and fast proliferation rates and often develop resistance to current anticancer therapies.

The Katanin family of microtubule-severing enzymes is critical for remodeling microtubule-based structures that influence cell division, motility, morphogenesis and signaling. Katanin is composed of a catalytic p60 subunit (A subunit, KATNA1) and a regulatory p80 subunit (B subunit, KATNB1). The mammalian genome also encodes two additional A-like subunits (KATNAL1 and KATNAL2) and one additional B-like subunit (KATNBL1) that have remained poorly characterized. To better understand the factors and mechanisms controlling mammalian microtubule-severing, we have taken a mass proteomic approach to define the protein interaction module for each mammalian Katanin subunit and to generate the mammalian Katanin family interaction network (Katan-ome). Further, we have analyzed the function of the KATNBL1 subunit and determined that it associates with KATNA1 and KATNAL1, it localizes to the spindle poles only during mitosis and it regulates Katanin A subunit microtubule-severing activity in vitro. Interestingly, during interphase, KATNBL1 is sequestered in the nucleus through an N-terminal nuclear localization signal. Finally KATNB1 was able to compete the interaction of KATNBL1 with KATNA1 and KATNAL1. These data indicate that KATNBL1 functions as a regulator of Katanin A subunit microtubule-severing activity during mitosis and that it likely coordinates with KATNB1 to perform this function.

A unique insertion in STARD9's motor domain is phosphorylated by mitotic kinases, including Plk1, which regulate its levels through an SCFb-TrCP ubiquitin ligase and proteasome-dependent process. These results imply that in vivo, full-length STARD9 could be regulated by Plk1 and SCFβ-TrCP to promote proper mitotic spindle assembly., STARD9 is a largely uncharacterized mitotic kinesin and putative cancer target that is critical for regulating pericentriolar material cohesion during bipolar spindle assembly. To begin to understand the mechanisms regulating STARD9 function and their importance to cell division, we took a multidisciplinary approach to define the cis and trans factors that regulate the stability of the STARD9 motor domain. We show that, unlike the other ∼50 mammalian kinesins, STARD9 contains an insertion in loop 12 of its motor domain (MD). Working with the STARD9-MD, we show that it is phosphorylated in mitosis by mitotic kinases that include Plk1. These phosphorylation events are important for targeting a pool of STARD9-MD for ubiquitination by the SCFβ-TrCP ubiquitin ligase and proteasome-dependent degradation. Of interest, overexpression of nonphosphorylatable/nondegradable STARD9-MD mutants leads to spindle assembly defects. Our results with STARD9-MD imply that in vivo the protein levels of full-length STARD9 could be regulated by Plk1 and SCFβ-TrCP to promote proper mitotic spindle assembly.