Checkpoint Activation Research Articles

LILRB3 supports acute myeloid leukemia development and regulates T-cell antitumor immune responses through the TRAF2-cFLIP-NF-κB signaling axis.

Leukocyte immunoglobulin-like receptor B (LILRB), a family of immune checkpoint receptors, contribute to acute myeloid leukemia (AML) development, but the specific mechanisms triggered by activation or inhibition of these immune checkpoints in cancer is largely unknown. Here we demonstrated that the intracellular domain of LILRB3 is constitutively associated with the adaptor protein TRAF2. Activated LILRB3 in AML cells leads to recruitment of cFLIP and subsequent NF-κB upregulation, resulting in enhanced leukemic cell survival and inhibition of T cell-mediated anti-tumor activity. Hyperactivation of NF-κB induces a negative regulatory feedback loop mediated by A20, which disrupts the interaction of LILRB3 and TRAF2; consequently the SHP-1/2-mediated inhibitory activity of LILRB3 becomes dominant. Finally, we show that blockade of LILRB3 signaling with antagonizing antibodies hampers AML progression. LILRB3 thus exerts context-dependent activating and inhibitory functions, and targeting LILRB3 may become a potential therapeutic strategy for AML treatment.

Phosphorylation and Pin1 binding to the LIC1 subunit selectively regulate mitotic dynein functions.

The dynein motor performs multiple functions in mitosis by engaging with a wide cargo spectrum. One way to regulate dynein's cargo-binding selectivity is through the C-terminal domain (CTD) of its light intermediate chain 1 subunit (LIC1), which binds directly with cargo adaptors. Here we show that mitotic phosphorylation of LIC1-CTD at its three cdk1 sites is required for proper mitotic progression, for dynein loading onto prometaphase kinetochores, and for spindle assembly checkpoint inactivation in human cells. Mitotic LIC1-CTD phosphorylation also engages the prolyl isomerase Pin1 predominantly to Hook2-dynein-Nde1-Lis1 complexes, but not to dynein-spindly-dynactin complexes. LIC1-CTD dephosphorylation abrogates dynein-Pin1 binding, promotes prophase centrosome-nuclear envelope detachment, and impairs metaphase chromosome congression and mitotic Golgi fragmentation, without affecting interphase membrane transport. Phosphomutation of a conserved LIC1-CTD SP site in zebrafish leads to early developmental defects. Our work reveals that LIC1-CTD phosphorylation differentially regulates distinct mitotic dynein pools and suggests the evolutionary conservation of this phosphoregulation.

DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks.

In acute myeloid leukemia (AML), recurrent DNA methyltransferase 3A (DNMT3A) mutations are associated with chemoresistance and poor prognosis, especially in advanced-age patients. Gene-expression studies in DNMT3A-mutated cells identified signatures implicated in deregulated DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here, we tested whether pharmacologically induced replication fork stalling, such as with cytarabine, creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. Leukemia cell lines, genetic mouse models, and isogenic cells with and without DNMT3A(mut) were used to evaluate sensitivity to nucleoside analogues such as cytarabine in vitro and in vivo, followed by analysis of DNA damage and signaling, replication restart, and cell-cycle progression on treatment and after drug removal. Transcriptome profiling identified pathways deregulated by DNMT3A(mut) expression. We found increased sensitivity to pharmacologically induced replication stress in cells expressing DNMT3A(R882)-mutant, with persistent intra-S-phase checkpoint activation, impaired PARP1 recruitment, and elevated DNA damage, which was incompletely resolved after drug removal and carried through mitosis. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine washout demonstrated a higher rate of fork collapse in DNMT3A(mut)-expressing cells. RNA-seq studies supported deregulated cell-cycle progression and p53 activation, along with splicing, ribosome biogenesis, and metabolism. Together, our studies show that DNMT3A mutations underlie a defect in recovery from replication fork arrest with subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during replication stress. See related commentary by Viny, p. 573.

Sublethal concentrations of high glucose prolong mitotic arrest in a spindle assembly checkpoint activity dependent manner in budding yeast

Sublethal concentrations of high glucose prolong mitotic arrest in a spindle assembly checkpoint activity dependent manner in budding yeast

Pharmacologic Ascorbate Enhances the Therapeutic Index of ATM-Inhibitor Based Chemoradiation for Colorectal Cancer

Ataxia telangiectasia mutated protein (ATM) is one of the key sensors of DNA damage and specific inhibitors of ATM are potent radiosensitizers. However, their clinical utility with radiation (RT) is limited because they lack tissue specificity and increase normal tissue injury. Pharmacologic (high dose) ascorbate (P-AscH-) selectively increases oxidative stress in tumors while functioning as a donor antioxidant and reducing RT damage in normal tissues. We hypothesized that P-AscH- could enhance the therapeutic index of ATM-inhibitor based chemoradiation (CRT) for colorectal cancer (CRC) by simultaneously enhancing efficacy and reducing RT bowel injury.Human HCT116, SW480, and HT29 and murine CT26 and MC38 CRC models were used. Clonogenic survival was assessed following single-fraction RT (2-8 Gy) +/- P-AscH- (5 pM/cell) +/- veliparib (PARP), VE821 (ATR), or KU60019 (ATM). Catalase expression was induced using HCT116 cells expressing a doxycycline inducible catalase transgene. DNA double strand breaks (DSBs) were quantified using neutral comet assays 0-24 hours post RT. Cell cycle phases were assessed using flow cytometry. ATM and pATM localization were assessed using IF. Jejunal toxicity was assessed using IHC in fixed tissues following single fraction (10 Gy) whole abdominal RT in c57bl/6 mice. Tumor growth delay was assessed following RT (5 Gy x 3) +/- drug treatment in unilateral flank tumors.Veliparib, VE821, and KU60019 were potent radiosensitizers in HCT116, SW480, HT29, MC38, and CT26 CRC tumor models and P-AscH- further reduced clonogenic survival with DRIs in all lines except for HT29. In contrast, P-AscH- enhanced survival of cultured HUVEC and FHs-74 cells exposed to RT. Enhanced cell kill with P-AscH- is H202 mediated as it is completely attenuated by inducible catalase expression. P-AscH- significantly increased the number of DNA DSBs in tumors after RT in vitro. Despite the increase in DNA DSBs, P-AscH-significantly decreased nuclear localization of activated pATM after RT and significantly decreased the fraction of cells in G2/M phases of the cell cycle. In vivo, RT + P-AscH- + KU60019 induced more tumor growth delay/clearance than all other combinations in unilateral MC38 or HCT116 flank tumors. Finally, P-AscH- significantly reduced loss of jejunal crypt cell density, epithelial architecture, and markers of lipid and protein oxidation following whole abdominal RT.P-AscH- selectively enhances the efficacy of ATM-based CRT in CRC tumor models while simultaneously decreasing RT-mediated small bowel toxicity. In tumors, P-AscH- enhances DNA DSBs by stimulating an H202 flux and prevents activation of DNA repair pathways and cell cycle checkpoints by inhibiting RT-induced activation of ATM. Selective radioprotectors like P-AscH- could facilitate the clinical translation ATM inhibitors as radiosensitizers.

Genetic and phenotypic attributes of splenic marginal zone lymphoma

AbstractSplenic marginal zone B-cell lymphoma (SMZL) is a heterogeneous clinico-biological entity. The clinical course is variable, multiple genes are mutated with no unifying mechanism, and essential regulatory pathways and surrounding microenvironments are diverse. We sought to clarify the heterogeneity of SMZL by resolving different subgroups and their underlying genomic abnormalities, pathway signatures, and microenvironment compositions to uncover biomarkers and therapeutic vulnerabilities. We studied 303 SMZL spleen samples collected through the IELSG46 multicenter international study (NCT02945319) by using a multiplatform approach. We carried out genetic and phenotypic analyses, defined self-organized signatures, validated the findings in independent primary tumor metadata and determined correlations with outcome data. We identified 2 prominent genetic clusters in SMZL, termed NNK (58% of cases, harboring NF-κB, NOTCH, and KLF2 modules) and DMT (32% of cases, with DNA-damage response, MAPK, and TLR modules). Genetic aberrations in multiple genes as well as cytogenetic and immunogenetic features distinguished NNK- from DMT-SMZLs. These genetic clusters not only have distinct underpinning biology, as judged by differences in gene-expression signatures, but also different outcomes, with inferior survival in NNK-SMZLs. Digital cytometry and in situ profiling segregated 2 basic types of SMZL immune microenvironments termed immune-suppressive SMZL (50% of cases, associated with inflammatory cells and immune checkpoint activation) and immune-silent SMZL (50% of cases, associated with an immune-excluded phenotype) with distinct mutational and clinical connotations. In summary, we propose a nosology of SMZL that can implement its classification and also aid in the development of rationally targeted treatments.

THSC/TREX-2 deficiency causes replication stress and genome instability in Caenorhabditis elegans.

Transcription is an essential process of DNA metabolism, yet it makes DNA more susceptible to DNA damage. THSC/TREX-2 is a conserved eukaryotic protein complex with a key role in mRNP biogenesis and maturation that prevents genome instability. One source of such instability is linked to transcription, as shown in yeast and human cells, but the underlying mechanism and whether this link is universal is still unclear. To obtain further insight into the putative role of the THSC/TREX-2 complex in genome integrity, we have used Caenorhabditis elegans mutants of the thp-1 and dss-1 components of THSC/TREX-2. These mutants show similar defective meiosis, DNA damage accumulation and activation of the DNA damage checkpoint. However, they differ from each other regarding replication defects, as determined by measuring dUTP incorporation in the germline. Interestingly, this specific thp-1 mutant phenotype can be partially rescued by overexpression of RNase H. Furthermore, both mutants show a mild increase in phosphorylation of histone H3 at Ser10 (H3S10P), a mark previously shown to be linked to DNA-RNA hybrid-mediated genome instability. These data support the view that both THSC/TREX-2 factors prevent transcription-associated DNA damage derived from DNA-RNA hybrid accumulation by separate means.

Resolvin D1 attenuates acid-induced DNA damage in esophageal epithelial cells and rat models of acid reflux

The role of resolvin D1 (RvD1) in gastroesophageal reflux disease (GERD) remains largely unknown. Here, we investigated the potential role of RvD1 in acid-induced DNA damage in esophageal epithelial cells, patients with refractory GERD and a rat model of acid reflux. Weak acid exposure induced longer comet tails, reactive oxygen species (ROS) generation, oxidative DNA damage and DNA double-strand breaks (DSBs) in cells and RvD1 (0.1 μM) blocked all these effects. Mechanistic analyses showed that apart from ROS-reducing effects, RvD1 possessed a strong capacity to promote DNA damage repair, augmenting cell cycle checkpoint activity and DSB repair by modulating phosphatase and tensin homolog (PTEN) in cells. We also detected the surface expression of formyl peptide receptor 2 (FPR2), a receptor for RvD1, in the esophageal epithelial cells, and inhibition of FPR2 abrogated the protective effects of RvD1 on cells. Furthermore, a positive correlation between RvD1 and PTEN was observed predominantly in the esophageal epithelium from patients with refractory GERD (r = 0.67, P < 0.05). Additionally, RvD1 administration upregulated PTEN, suppressed DNA DSBs and alleviated microscopic damage in the rat model of gastric reflux. FPR2 gene silencing abolished the therapeutic effects of RvD1 on the rat model. Taken together, RvD1 binding to FPR2 protects the esophageal epithelium from acid reflux-induced DNA damage via a mechanism involving the inhibition of ROS production and facilitation of DSB repair. These findings support RvD1 as a promising approach that may be valuable for the treatment of GERD.

A Tale of Two Checkpoints: ATR Inhibition and PD-(L)1 Blockade

Innate immunity and the DNA damage response (DDR) pathway are inextricably linked. Within the DDR, ataxia telangiectasia and Rad3-related (ATR) is a key kinase responsible for sensing replication stress and facilitating DNA repair through checkpoint activation, cell cycle arrest, and promotion of fork recovery. Recent studies have shed light on the immunomodulatory role of the ATR-CHK1 pathway in the tumor microenvironment and the specific effects of ATR inhibition in stimulating an innate immune response. With several potent and selective ATR inhibitors in developmental pipelines, the combination of dual ATR and PD-(L)1 blockade has attracted increasing interest in cancer therapy. In this review, we summarize the clinical and preclinical data supporting the combined inhibition of ATR and PD-(L)1, discuss the potential challenges surrounding this approach, and highlight biomarkers relevant for selected patients who are most likely to benefit from the blockade of these two checkpoints.

DRTEL1 is essential for the maintenance of Drosophila male germline stem cells.

Stem cells have the potential to maintain undifferentiated state and differentiate into specialized cell types. Despite numerous progress has been achieved in understanding stem cell self-renewal and differentiation, many fundamental questions remain unanswered. In this study, we identify dRTEL1, the Drosophila homolog of Regulator of Telomere Elongation Helicase 1, as a novel regulator of male germline stem cells (GSCs). Our genome-wide transcriptome analysis and ChIP-Seq results suggest that dRTEL1 affects a set of candidate genes required for GSC maintenance, likely independent of its role in DNA repair. Furthermore, dRTEL1 prevents DNA damage-induced checkpoint activation in GSCs. Finally, dRTEL1 functions to sustain Stat92E protein levels, the key player in GSC maintenance. Together, our findings reveal an intrinsic role of the DNA helicase dRTEL1 in maintaining male GSC and provide insight into the function of dRTEL1.

DDK/Hsk1 phosphorylates and targets fission yeast histone deacetylase Hst4 for degradation to stabilize stalled DNA replication forks.

In eukaryotes, paused replication forks are prone to collapse, which leads to genomic instability, a hallmark of cancer. Dbf4-dependent kinase (DDK)/Hsk1Cdc7 is a conserved replication initiator kinase with conflicting roles in replication stress response. Here, we show that fission yeast DDK/Hsk1 phosphorylates sirtuin, Hst4 upon replication stress at C-terminal serine residues. Phosphorylation of Hst4 by DDK marks it for degradation via the ubiquitin ligase SCFpof3. Phosphorylation-defective hst4 mutant (4SA-hst4) displays defective recovery from replication stress, faulty fork restart, slow S-phase progression and decreased viability. The highly conserved fork protection complex (FPC) stabilizes stalled replication forks. We found that the recruitment of FPC components, Swi1 and Mcl1 to the chromatin is compromised in the 4SA-hst4 mutant, although whole cell levels increased. These defects are dependent upon H3K56ac and independent of intra S-phase checkpoint activation. Finally, we show conservation of H3K56ac-dependent regulation of Timeless, Tipin, and And-1 in human cells. We propose that degradation of Hst4 via DDK increases H3K56ac, changing the chromatin state in the vicinity of stalled forks facilitating recruitment and function of FPC. Overall, this study identified a crucial role of DDK and FPC in the regulation of replication stress response with implications in cancer therapeutics.

Epistasis, aneuploidy, and functional mutations underlie evolution of resistance to induced microtubule depolymerization.

Cells with blocked microtubule polymerization are delayed in mitosis, but eventually manage to proliferate despite substantial chromosome missegregation. While several studies have analyzed the first cell division after microtubule depolymerization, we have asked how cells cope long-term with microtubule impairment. We allowed 24 clonal populations of yeast cells with beta-tubulin mutations preventing proper microtubule polymerization, to evolve for ˜150 generations. At the end of the laboratory evolution experiment, cells had regained the ability to form microtubules and were less sensitive to microtubule-depolymerizing drugs. Whole-genome sequencing identified recurrently mutated genes, in particular for tubulins and kinesins, as well as pervasive duplication of chromosome VIII. Recreating these mutations and chromosome VIII disomy prior to evolution confirmed that they allow cells to compensate for the original mutation in beta-tubulin. Most of the identified mutations did not abolish function, but rather restored microtubule functionality. Analysis of the temporal order of resistance development in independent populations repeatedly revealed the same series of events: disomy of chromosome VIII followed by a single additional adaptive mutation in either tubulins or kinesins. Since tubulins are highly conserved among eukaryotes, our results have implications for understanding resistance to microtubule-targeting drugs widely used in cancer therapy.

USP7 and VCPFAF1 define the SUMO/Ubiquitin landscape at the DNA replication fork.

SummaryThe AAA+ ATPase VCP regulates the extraction of SUMO and ubiquitin-modified DNA replication factors from chromatin. We have previously described that active DNA synthesis is associated with a SUMO-high/ubiquitin-low environment governed by the deubiquitylase USP7. Here, we unveil a functional cooperation between USP7 and VCP in DNA replication, which is conserved from Caenorhabditis elegans to mammals. The role of VCP in chromatin is defined by its cofactor FAF1, which facilitates the extraction of SUMOylated and ubiquitylated proteins that accumulate after the block of DNA replication in the absence of USP7. The inactivation of USP7 and FAF1 is synthetically lethal both in C. elegans and mammalian cells. In addition, USP7 and VCP inhibitors display synergistic toxicity supporting a functional link between deubiquitylation and extraction of chromatin-bound proteins. Our results suggest that USP7 and VCPFAF1 facilitate DNA replication by controlling the balance of SUMO/Ubiquitin-modified DNA replication factors on chromatin.

Relationship between IGF-1 and body weight in inflammatory bowel diseases: Cellular and molecular mechanisms involved

Inflammatory bowel diseases (IBD), represented by ulcerative colitis (UC) and Crohn's disease (CD), are characterized by chronic inflammation of the gastrointestinal tract, what leads to diarrhea, malnutrition, and weight loss. Depression of the growth hormone-insulin-like growth factor-1 axis (GH-IGF-1 axis) could be responsible of these symptoms. We demonstrate that long-term treatment (54 weeks) of adult CD patients with adalimumab (ADA) results in a decrease in serum IGF-1 without changes in serum IGF-1 binding protein (IGF1BP4). These results prompted us to conduct a preclinical study to test the efficiency of IGF-1 in the medication for experimental colitis. IGF-1 treatment of rats with DSS-induced colitis has a beneficial effect on the following circulating biochemical parameters: glucose, albumin, and total protein levels. In this experimental group we also observed healthy maintenance of colon size, body weight, and lean mass in comparison with the DSS-only group. Histological analysis revealed restoration of the mucosal barrier with the IGF-1 treatment, which was characterized by healthy quantities of mucin production, structural maintenance of adherers junctions (AJs), recuperation of E-cadherin and β-catenin levels and decrease in infiltrating immune cells and in metalloproteinase-2 levels. The experimentally induced colitis caused activation of apoptosis markers, including cleaved caspase 3, caspase 8, and PARP and decreases cell-cycle checkpoint activators including phosphorylated Rb, cyclin E, and E2F1. The IGF-1 treatment inhibited cyclin E depletion and partially protects PARP levels. The beneficial effects of IGF-1 in experimental colitis could be explained by a re-sensitization of the IGF-1/IRS-1/AKT cascade to exogenous IGF-1. Given these results, we postulate that IGF-1 treatment of IBD patients could prove to be successful in reducing disease pathology.

Ubc1 turnover contributes to the spindle assembly checkpoint in Saccharomyces cerevisiae.

The spindle assembly checkpoint protects the integrity of the genome by ensuring that chromosomes are properly attached to the mitotic spindle before they are segregated during anaphase. Activation of the spindle checkpoint results in inhibition of the Anaphase-Promoting Complex (APC), an E3 ubiquitin ligase that triggers the metaphase–anaphase transition. Here, we show that levels of Ubc1, an E2 enzyme that functions in complex with the APC, modulate the response to spindle checkpoint activation in Saccharomyces cerevisiae. Overexpression of Ubc1 increased resistance to microtubule poisons, whereas Ubc1 shut-off sensitized cells. We also found that Ubc1 levels are regulated by the spindle checkpoint. Checkpoint activation or direct APC inhibition led to a decrease in Ubc1 levels, charging, and half-life. Additionally, stabilization of Ubc1 prevented its down-regulation by the spindle checkpoint and increased resistance to checkpoint-activating drugs. These results suggest that down-regulation of Ubc1 in response to spindle checkpoint signaling is necessary for a robust cell cycle arrest.

The N-Terminal Region of the Polo Kinase Cdc5 Is Required for Downregulation of the Meiotic Recombination Checkpoint.

During meiosis, the budding yeast polo-like kinase Cdc5 is a crucial driver of the prophase I to meiosis I (G2/M) transition. The meiotic recombination checkpoint restrains cell cycle progression in response to defective recombination to ensure proper distribution of intact chromosomes to the gametes. This checkpoint detects unrepaired DSBs and initiates a signaling cascade that ultimately inhibits Ndt80, a transcription factor required for CDC5 gene expression. Previous work revealed that overexpression of CDC5 partially alleviates the checkpoint-imposed meiotic delay in the synaptonemal complex-defective zip1Δ mutant. Here, we show that overproduction of a Cdc5 version (Cdc5-ΔN70), lacking the N-terminal region required for targeted degradation of the protein by the APC/C complex, fails to relieve the zip1Δ-induced meiotic delay, despite being more stable and reaching increased protein levels. However, precise mutation of the consensus motifs for APC/C recognition (D-boxes and KEN) has no effect on Cdc5 stability or function during meiosis. Compared to the zip1Δ single mutant, the zip1Δ cdc5-ΔN70 double mutant exhibits an exacerbated meiotic block and reduced levels of Ndt80 consistent with persistent checkpoint activity. Finally, using a CDC5-inducible system, we demonstrate that the N-terminal region of Cdc5 is essential for its checkpoint erasing function. Thus, our results unveil an additional layer of regulation of polo-like kinase function in meiotic cell cycle control.

PIGN spatiotemporally regulates the spindle assembly checkpoint proteins in leukemia transformation and progression

Phosphatidylinositol glycan anchor biosynthesis class N (PIGN) has been linked to the suppression of chromosomal instability. The spindle assembly checkpoint complex is responsible for proper chromosome segregation during mitosis to prevent chromosomal instability. In this study, the novel role of PIGN as a regulator of the spindle assembly checkpoint was unveiled in leukemic patient cells and cell lines. Transient downregulation or ablation of PIGN resulted in impaired mitotic checkpoint activation due to the dysregulated expression of spindle assembly checkpoint-related proteins including MAD1, MAD2, BUBR1, and MPS1. Moreover, ectopic overexpression of PIGN restored the expression of MAD2. PIGN regulated the spindle assembly checkpoint by forming a complex with the spindle assembly checkpoint proteins MAD1, MAD2, and the mitotic kinase MPS1. Thus, PIGN could play a vital role in the spindle assembly checkpoint to suppress chromosomal instability associated with leukemic transformation and progression.

Perfluorooctane sulfonate affects mouse oocyte maturation in vitro by promoting oxidative stress and apoptosis induced bymitochondrial dysfunction

Perfluorooctane sulphonate (PFOS), as a surfactant, is widely applied in the agricultural production activities and has become a potential menace to human health. The mechanism of its effect on the maturation of mammalian oocytes is unclear. This study explored the toxic effect of PFOS on mouse oocyte maturation in vitro. The results revealed that PFOS under a concentration of 600 μM could significantly reduce the polar body extrusion rate (PBE) of mouse oocytes and cause symmetrical cell division. Further experiments showed that PFOS resulted in the abnormal cytoskeleton of the oocytes, causing the abnormal spindles and misplaced chromosomes, as well as the impaired dynamics of actin. Moreover, PFOS exposure inhibited the process of oocyte meiosis, which reflected in the slower spindle migration and continuous activation of spindle assembly checkpoint (SAC), then ultimately increased the probability of aneuploidy. Most importantly, PFOS exposure reduced the quality of oocytes, specifically by disrupting the function of mitochondria, inducing cell oxidative stress, and triggering early apoptosis. Furthermore, the level of methylation of histones is additionally influenced. In summary, our findings showed that PFOS exposure interfered with the maturation of mouse oocytes through affecting cytoskeletal dynamics, meiotic progression, oocyte quality, and histone modifications.

Treating ICB-resistant glioma with anti-CD40 and mitotic spindle checkpoint controller BAL101553 (lisavanbulin).

Glioblastoma is a highly malignant brain tumor with no curative treatment options, and immune checkpoint blockade has not yet shown major impact. We hypothesized that drugs targeting mitosis might affect the tumor microenvironment and sensitize cancer cells to immunotherapy. We used 2 glioblastoma mouse models with different immunogenicity profiles, GL261 and SB28, to test the efficacy of antineoplastic and immunotherapy combinations. The spindle assembly checkpoint activator BAL101553 (lisavanbulin), agonistic anti-CD40 antibody, and double immune checkpoint blockade (anti–programmed cell death 1 and anti–cytotoxic T lymphocyte–associated protein 4; anti–PD-1 and anti–CTLA-4) were evaluated individually or in combination for treating orthotopic GL261 and SB28 tumors. Genomic and immunological analyses were used to predict and interpret therapy responsiveness. BAL101553 monotherapy increased survival in immune checkpoint blockade–resistant SB28 glioblastoma tumors and synergized with anti-CD40 antibody, in a T cell–independent manner. In contrast, the more immunogenic and highly mutated GL261 model responded best to anti–PD-1 and anti–CTLA-4 therapy and more modestly to BAL101553 and anti-CD40 combination. Our results show that BAL101553 is a promising therapeutic agent for glioblastoma and could synergize with innate immune stimulation. Overall, these data strongly support immune profiling of glioblastoma patients and preclinical testing of combination therapies with appropriate models for particular patient groups.

Targeting Mps1 in combination with paclitaxel inhibits osteosarcoma progression by modulating spindle assembly checkpoint and Akt/mTOR signaling.

Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents and is characterized by early metastasis and frequent recurrence, which greatly affects patient prognosis and survival rates. However, the treatment of OS, its recurrence and subsequent metastasis is now at a clinical bottleneck. To explore new OS chemotherapeutic targets, investigate new therapeutic strategies and improve patient prognosis and survival rates, the roles of paclitaxel (PTX) and monopolar spindle kinase 1 (Mps1) in OS were investigated using in vivo and in vitro models. Mps1 expression was upregulated in OS samples and associated with patient survival times. Moreover, spindle assembly checkpoint (SAC) activation and upregulation of Akt/mTOR signaling were both positively associated with OS progression. PTX treatment significantly inhibited Mps1 expression, as well as migration of OS cells both in vitro. In addition, the combination of Mps1 knockdown and PTX treatment inhibited OS progression in vivo. Mps1 overexpression inhibited the expression of SAC markers and upregulated Akt and mTOR expression, while Mps1 knockdown had the opposite effect. Cells subjected to combined Mps1 knockdown and PTX treatment exhibited activation of SAC and inhibition of Akt/mTOR signaling compared with Mps1 knockdown or PTX treatment alone. Based on these observations, Mps1 inhibition combined with PTX treatment may represent a potentially effective strategy for the treatment of OS.