Age-stratified comorbid and pharmacologic analysis of patients with glioblastoma.

Background: Increased age is a strong and unfavorable prognostic factor for patients with glioblastoma (GBM). However, the relationships between stratified patient age, comorbidities, and medications have yet to be explored in GBM patient survival analyses. Objective: To evaluate co-morbid conditions, tumor-related symptoms, medication prescriptions, and subject age for patients with GBM and to establish potential targets for prospective studies. Methods: Electronic health records for 565 patients with IDHwt GBM were evaluated at a single center between January 1, 2000 and August 9, 2021 were retrospectively assessed. Data were stratified by MGMT promoter methylation status when available and were used to construct multivariable time-dependent cox models and intra-cohort hazards. Results: Younger (<65 years of age) but not older (≥65 years) GBM patients demonstrated a worse prognosis with movement related disabilities (P < 0.0001), gait/balance difficulty (P = 0.04) and weakness (P = 0.007), as well as psychiatric conditions, mental health disorders (P = 0.002) and anxiety (P = 0.001). In contrast, older but not younger GBM patients demonstrated a worse prognosis with epilepsy (P = 0.039). Both groups had worse survival with confusion/altered mental status (P = 0.023 vs < 0.000) and an improved survival with a Temozolomide prescription. Older but not younger GBM patients experienced an improved hazard with a prescription of ace-inhibitor medications (P = 0.048). Conclusion: Age-dependent novel associations between clinical symptoms and medications prescribed for co-morbid conditions were demonstrated in patients with GBM. The results of the current work support future mechanistic studies that investigate the negative relationship(s) between increased age, comorbidities, and drug therapies for differential clinical decision-making across the lifespan of patients with GBM.

Advanced Age in Humans and Mouse Models of Glioblastoma Show Decreased Survival from Extratumoral Influence.

PURPOSE: Glioblastoma (GBM) is the most common aggressive primary malignant brain tumor in adults with a median age of onset of 68 to 70 years old. Although advanced age is often associated with poorer GBM patient survival, the predominant source(s) of maladaptive aging effects remains to be established. Here, we studied intratumoral and extratumoral relationships between adult patients with GBM and mice with brain tumors across the lifespan. EXPERIMENTAL DESIGN: Electronic health records at Northwestern Medicine and the NCI SEER databases were evaluated for GBM patient age and overall survival. The commercial Tempus and Caris databases, as well as The Cancer Genome Atlas were profiled for gene expression, DNA methylation, and mutational changes with varying GBM patient age. In addition, gene expression analysis was performed on the extratumoral brain of younger and older adult mice with or without a brain tumor. The survival of young and old wild-type or transgenic (INK-ATTAC) mice with a brain tumor was evaluated after treatment with or without senolytics and/or immunotherapy. RESULTS: Human patients with GBM ≥65 years of age had a significantly decreased survival compared with their younger counterparts. While the intra-GBM molecular profiles were similar between younger and older patients with GBM, non-tumor brain tissue had a significantly different gene expression profile between young and old mice with a brain tumor and the eradication of senescent cells improved immunotherapy-dependent survival of old but not young mice. CONCLUSIONS: This work suggests a potential benefit for combining senolytics with immunotherapy in older patients with GBM.

Positive Allosteric Modulator of SERCA Pump NDC-1173 Exerts Beneficial Effects in Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is an irreversible neurodegenerative disease that affects millions of people worldwide. AD does not have a cure and most drug development efforts in the AD field have been focused on targeting the amyloid pathway based on the "amyloid cascade hypothesis". However, in addition to the amyloid pathway, substantial evidence also points to dysregulated neuronal calcium (Ca2+) signaling as one of the key pathogenic events in AD, and it has been proposed that pharmacological agents that stabilize neuronal Ca2+ signaling may act as disease-modifying agents in AD. In previous studies, we demonstrated that positive allosteric regulators (PAMs) of the Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump might act as such Ca2+ stabilizing agents. In the present study, we report the development of a novel SERCA PAM agent, compound NDC-1173. To test the effectiveness of this compound, we performed behavioral studies with the APP/PS1 transgenic AD mouse model. We also evaluated effects of this compound on expression of endoplasmic reticulum (ER) stress genes in the hippocampus of APP/PS1 mice. The results of this study support the hypothesis that the SERCA pump is a potential novel therapeutic drug target and that NDC-1173 is a promising lead molecule for developing disease-modifying agents in AD.

Discovery and Characterization of PROTACs Targeting Tissue Transglutaminase (TG2).

Tissue transglutaminase (TG2) is a multifunctional enzyme involved in the cross-linking of extracellular matrix proteins, formation of complexes with fibronectin (FN) and integrins, and GTP hydrolysis. TG2 is activated in several pathological conditions, including cancer. We recently described a novel series of ligands that bind to TG2 and inhibit its interaction with FN. Because TG2 acts via multiple mechanisms, we set out to pursue a targeted protein degradation strategy to abolish TG2's myriad functions. Here, we report the synthesis and characterization of a series of VHL-based degraders that reduce TG2 in ovarian cancer cells in a proteasome-dependent manner. Degradation of TG2 resulted in significantly reduced cancer cell adhesion and migration in vitro in scratch-wound and migration assays. These results strongly indicate that further development of more potent and in vivo efficient TG2 degraders could be a new strategy for reducing the dissemination of ovarian and other cancers.

Chemokine receptor CXCR7 activates Aurora Kinase A and promotes neuroendocrine prostate cancer growth.

CXCR7 is an atypical chemokine receptor that recruits ß-arrestin (ARRB2) and internalizes into clathrin-coated intracellular vesicles where the complex acts as a scaffold for cytoplasmic kinase assembly and signal transduction. Here, we report that CXCR7 was elevated in the majority of prostate cancer (PCa) cases with neuroendocrine features (NEPC). CXCR7 markedly induced mitotic spindle and cell cycle gene expression. Mechanistically, we identified Aurora Kinase A (AURKA), a key regulator of mitosis, as a novel target that was bound and activated by the CXCR7-ARRB2 complex. CXCR7 interacted with proteins associated with microtubules and golgi, and, as such, the CXCR7-ARRB2-containing vesicles trafficked along the microtubules to the pericentrosomal golgi apparatus, where the complex interacted with AURKA. Accordingly, CXCR7 promoted PCa cell proliferation and tumor growth, which was mitigated by AURKA inhibition. In summary, our study reveals a critical role of CXCR7-ARRB2 in interacting and activating AURKA, which can be targeted by AURKA inhibitors to benefit a subset of patients with NEPC.

Medicinal chemistry approaches to target the MNK-eIF4E axis in cancer.

Aberrant translation of proteins that promote cell proliferation is an essential factor that defines oncogenic processes and cancer. The process for ribosomal translation of proteins from mRNA requires an essential initiation step which is controlled by the protein eIF4E, which binds the RNA 5'-cap and forms the eIF4F complex that subsequently translates protein. Typically, eIF4E is activated by phosphorylation on Ser209 by MNK1 and MNK2 kinases. Substantial work has shown that eIF4E and MNK1/2 are dysregulated in many cancers and this axis has therefore become an active area of interest for developing new cancer therapeutics. This review summarizes and discusses recent work to develop small molecules that target different steps in the MNK-eIF4E axis as potential cancer therapeutics. The aim of this review is to cover the breadth of different molecular approaches being taken and the medicinal chemistry basis for their optimization and testing as new cancer therapeutics.

Targeting pleckstrin-2/Akt signaling reduces proliferation in myeloproliferative neoplasm models.

Myeloproliferative neoplasms (MPNs) are characterized by the activated JAK2/STAT pathway. Pleckstrin-2 (Plek2) is a downstream target of the JAK2/STAT5 pathway and is overexpressed in patients with MPNs. We previously revealed that Plek2 plays critical roles in the pathogenesis of JAK2-mutated MPNs. The nonessential roles of Plek2 under physiologic conditions make it an ideal target for MPN therapy. Here, we identified first-in-class Plek2 inhibitors through an in silico high-throughput screening approach and cell-based assays, followed by the synthesis of analogs. Plek2-specific small-molecule inhibitors showed potent inhibitory effects on cell proliferation. Mechanistically, Plek2 interacts with and enhances the activity of Akt through the recruitment of downstream effector proteins. The Plek2-signaling complex also includes Hsp72, which protects Akt from degradation. These functions were blocked by Plek2 inhibitors via their direct binding to the Plek2 dishevelled, Egl-10 and pleckstrin (DEP) domain. The role of Plek2 in activating Akt signaling was further confirmed in vivo using a hematopoietic-specific Pten-knockout mouse model. We next tested Plek2 inhibitors alone or in combination with an Akt inhibitor in various MPN mouse models, which showed significant therapeutic efficacies similar to that seen with the genetic depletion of Plek2. The Plek2 inhibitor was also effective in reducing proliferation of CD34-positive cells from MPN patients. Our studies reveal a Plek2/Akt complex that drives cell proliferation and can be targeted by a class of antiproliferative compounds for MPN therapy.

Identification and Characterization of a Novel Indoleamine 2,3-Dioxygenase 1 Protein Degrader for Glioblastoma.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a potent immunosuppressive enzyme that inhibits the antitumor immune response through both tryptophan metabolism and non-enzymatic functions. To date, most IDO1-targeted approaches have focused on inhibiting tryptophan metabolism. However, this class of drugs has failed to improve the overall survival of patients with cancer. Here, we developed and characterized proteolysis targeting chimeras (PROTACs) that degrade the IDO1 protein. IDO1-PROTACs were tested for their effects on IDO1 enzyme and non-enzyme activities. After screening a library of IDO1-PROTAC derivatives, a compound was identified that potently degraded the IDO1 protein through cereblon-mediated proteasomal degradation. The IDO1-PROTAC: (i) inhibited IDO1 enzyme activity and IDO1-mediated NF-κB phosphorylation in cultured human glioblastoma (GBM) cells, (ii) degraded the IDO1 protein within intracranial brain tumors in vivo, and (iii) mediated a survival benefit in mice with well-established brain tumors. This study identified and characterized a new IDO1 protein degrader with therapeutic potential for patients with glioblastoma.

Effects of Small Molecule Ligands on ACKR3 Receptors.

Chemokines such as stromal derived factor 1 and their G protein coupled receptors are well-known regulators of the development and functions of numerous tissues. C-X-C motif chemokine ligand 12 (CXCL12) has two receptors: C-X-C chemokine motif receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3). ACKR3 has been described as an atypical "biased" receptor because it does not appear to signal through G proteins and, instead, signals solely through the ß-arrestin pathway. In support of this conclusion, we have shown that ACKR3 is unable to signal through any of the known mammalian G α isoforms and have generated a comprehensive map of the G α activation by CXCL12/CXCR4. We also synthesized a series of small molecule ligands which acted as selective agonists for ACKR3 as assessed by their ability to recruit ß-arrestin to the receptor. Using select point mutations, we studied the molecular characteristics that determine the ability of small molecules to activate ACKR3 receptors, revealing a key role for the deeper binding pocket composed of residues in the transmembrane domains of ACKR3. The development of more selective ACKR3 ligands should allow us to better appreciate the unique roles of ACKR3 in the CXCL12/CXCR4/ACKR3-signaling axis and better understand the structural determinants for ACKR3 activation. SIGNIFICANCE STATEMENT: We are interested in the signaling produced by the G protein coupled receptor atypical chemokine receptor 3 (ACKR3), which signals atypically. In this study, novel selective ligands for ACKR3 were discovered and the site of interactions between these small molecules and ACKR3 was defined. This work will help to better understand the unique signaling roles of ACKR3.

Discovery of a small-molecule inhibitor of the TRIP8b-HCN interaction with efficacy in neurons.

Major depressive disorder is a critical public health problem with a lifetime prevalence of nearly 17% in the United States. One potential therapeutic target is the interaction between hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and an auxiliary subunit of the channel named tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). HCN channels regulate neuronal excitability in the mammalian hippocampus, and recent work has established that antagonizing HCN function rescues cognitive impairment caused by chronic stress. Here, we utilize a high-throughput virtual screen to find small molecules capable of disrupting the TRIP8b-HCN interaction. We found that the hit compound NUCC-0200590 disrupts the TRIP8b-HCN interaction in vitro and in vivo. These results provide a compelling strategy for developing new small molecules capable of disrupting the TRIP8b-HCN interaction.

Regulation of IFNα-induced expression of the short ACE2 isoform by ULK1.

The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been shown to hijack angiotensin converting enzyme 2 (ACE2) for entry into mammalian cells. A short isoform of ACE2, termed deltaACE2 (dACE2), has recently been identified. In contrast to ACE2, the short dACE2 isoform lacks the ability to bind the spike protein of SARS-CoV-2. Several studies have proposed that expression of ACE2 and/or dACE2 is induced by interferons (IFNs). Here, we report that drug-targeted inhibition or silencing of Unc51-like kinase 1 (ULK1) results in repression of type I IFN-induced expression of the dACE2 isoform. Notably, dACE2 is expressed in various squamous tumors. In efforts to identify pharmacological agents that target this pathway, we found that fisetin, a natural flavonoid, is an ULK1 inhibitor that decreases type I IFN-induced dACE2 expression. Taken together, our results establish a requirement for ULK1 in the regulation of type I IFN-induced transcription of dACE2 and raise the possibility of clinical translational applications of fisetin as a novel ULK1 inhibitor.

A MYC inhibitor selectively alters the MYC and MAX cistromes and modulates the epigenomic landscape to regulate target gene expression.

MYC regulates multiple gene programs, raising questions about the potential selectivity and downstream transcriptional consequences of MYC inhibitors as cancer therapeutics. Here, we examined the effect of a small-molecule MYC inhibitor, MYCi975, on the MYC/MAX cistromes, epigenome, transcriptome, and tumorigenesis. Integrating these data revealed three major classes of MYCi975-modulated gene targets: type 1 (down-regulated), type 2 (up-regulated), and type 3 (unaltered). While cell cycle and signal transduction pathways were heavily targeted by MYCi, RNA biogenesis and core transcriptional pathway genes were spared. MYCi975 altered chromatin binding of MYC and the MYC network family proteins, and chromatin accessibility and H3K27 acetylation alterations revealed MYCi975 suppression of MYC-regulated lineage factors AR/ARv7, FOXA1, and FOXM1. Consequently, MYCi975 synergistically sensitized resistant prostate cancer cells to enzalutamide and estrogen receptor-positive breast cancer cells to 4-hydroxytamoxifen. Our results demonstrate that MYCi975 selectively inhibits MYC target gene expression and provide a mechanistic rationale for potential combination therapies.

Tumor Cell IDO Enhances Immune Suppression and Decreases Survival Independent of Tryptophan Metabolism in Glioblastoma.

PURPOSE: Glioblastoma (GBM) is an incurable primary brain tumor that has not benefited from immunotherapy to date. More than 90% of GBM expresses the tryptophan (Trp) metabolic enzyme, indoleamine 2,3-dioxygenase 1 (IDO). This observation supported the historical hypothesis that IDO suppresses the antitumor immune response solely through a mechanism that requires intratumoral Trp depletion. However, recent findings led us to investigate the alternative hypothesis that IDO suppresses the anti-GBM immune response independent of its association with Trp metabolism. EXPERIMENTAL DESIGN: IDO-deficient GBM cell lines reconstituted with IDO wild-type or IDO enzyme-null cDNA were created and validated in vitro and in vivo. Microarray analysis was conducted to search for genes that IDO regulates, followed by the analysis of human GBM cell lines, patient GBM and plasma, and The Cancer Genome Atlas (TCGA) database. Ex vivo cell coculture assays, syngeneic and humanized mouse GBM models, were used to test the alternative hypothesis. RESULTS: Nonenzymic tumor cell IDO activity decreased the survival of experimental animals and increased the expression of complement factor H (CFH) and its isoform, factor H like protein 1 (FHL-1) in human GBM. Tumor cell IDO increased CFH and FHL-1 expression independent of Trp metabolism. Increased intratumoral CFH and FHL-1 levels were associated with poorer survival among patients with glioma. Similar to IDO effects, GBM cell FHL-1 expression increased intratumoral regulatory T cells (Treg) and myeloid-derived suppressor cells while it decreased overall survival in mice with GBM. CONCLUSIONS: Our study reveals a nonmetabolic IDO-mediated enhancement of CFH expression and provides a new therapeutic target for patients with GBM.

Going beyond Polycomb: EZH2 functions in prostate cancer.

The Polycomb group (PcG) protein Enhancer of Zeste Homolog 2 (EZH2) is one of the three core subunits of the Polycomb Repressive Complex 2 (PRC2). It harbors histone methyltransferase activity (MTase) that specifically catalyze histone 3 lysine 27 (H3K27) methylation on target gene promoters. As such, PRC2 are epigenetic silencers that play important roles in cellular identity and embryonic stem cell maintenance. In the past two decades, mounting evidence supports EZH2 mutations and/or over-expression in a wide array of hematological cancers and solid tumors, including prostate cancer. Further, EZH2 is among the most upregulated genes in neuroendocrine prostate cancers, which become abundant due to the clinical use of high-affinity androgen receptor pathway inhibitors. While numerous studies have reported epigenetic functions of EZH2 that inhibit tumor suppressor genes and promote tumorigenesis, discordance between EZH2 and H3K27 methylation has been reported. Further, enzymatic EZH2 inhibitors have shown limited efficacy in prostate cancer, warranting a more comprehensive understanding of EZH2 functions. Here we first review how canonical functions of EZH2 as a histone MTase are regulated and describe the various mechanisms of PRC2 recruitment to the chromatin. We further outline non-histone substrates of EZH2 and discuss post-translational modifications to EZH2 itself that may affect substrate preference. Lastly, we summarize non-canonical functions of EZH2, beyond its MTase activity and/or PRC2, as a transcriptional cofactor and discuss prospects of its therapeutic targeting in prostate cancer.

Identification of a druggable protein-protein interaction site between mutant p53 and its stabilizing chaperone DNAJA1.

The TP53 gene is the most frequently mutated gene in human cancers, and the majority of TP53 mutations are missense mutations. As a result, these mutant p53 (mutp53) either directly lose wildtype p53 (wtp53) tumor suppressor function or exhibit a dominant negative effect over wtp53. In addition, some mutp53 have acquired new oncogenic function (gain of function). Therefore, targeting mutp53 for its degradation may serve as a promising strategy for cancer prevention and therapy. Based on our previous finding that farnesylated DNAJA1 is a crucial chaperone in maintaining mutp53 stabilization, and by using an in silico approach, we built 3D homology models of human DNAJA1 and mutp53R175H proteins, identified the interacting pocket in the DNAJA1-mutp53R175H complex, and found one critical druggable small molecule binding site in the DNAJA1 glycine/phenylalanine-rich region. We confirmed that the interacting pocket in the DNAJA1-mutp53R175H complex was crucial for stabilizing mutp53R175H using a site-directed mutagenesis approach. We further screened a drug-like library to identify a promising small molecule hit (GY1-22) against the interacting pocket in the DNAJA1-mutp53R175H complex. The GY1-22 compound displayed an effective activity against the DNAJA1-mutp53R175H complex. Treatment with GY1-22 significantly reduced mutp53 protein levels, enhanced Waf1p21 expression, suppressed cyclin D1 expression, and inhibited mutp53-driven pancreatic cancer growth both in vitro and in vivo. Together, our results indicate that the interacting pocket in the DNAJA1-mutp53R175H complex is critical for mutp53's stability and oncogenic function, and DNAJA1 is a robust therapeutic target for developing the efficient small molecule inhibitors against oncogenic mutp53.

Small Molecule Approaches for Targeting the Polycomb Repressive Complex 2 (PRC2) in Cancer.

The polycomb repressive complex 2 (PRC2) is composed of three core subunits, enhancer of zeste 2 (EZH2), embryonic ectoderm development (EED), and suppressor of zeste 12 (SUZ12), along with a number of accessory proteins. It is the key enzymatic protein complex that catalyzes histone H3 lysine 27 (H3K27) methylation to mediate epigenetic silencing of target genes. PRC2 thus plays essential roles in maintaining embryonic stem cell identity and in controlling cellular differentiation. Studies in the past decade have reported frequent overexpression or mutation of PRC2 in various cancers including prostate cancer and lymphoma. Aberrant PRC2 function has been extensively studied and proven to contribute to a large number of abnormal cellular processes, including those that lead to uncontrolled proliferation and tumorigenesis. Significant efforts have recently been made to develop small molecules targeting PRC2 function for potential use as anticancer therapeutics. In this review, we describe recent approaches to identify and develop small molecules that target PRC2. These various strategies include the inhibition of the function of individual PRC2 core proteins, the disruption of PRC2 complex formation, and the degradation of its subunits.

Heterologous Expression of the Unusual Terreazepine Biosynthetic Gene Cluster Reveals a Promising Approach for Identifying New Chemical Scaffolds.

Advances in genome sequencing have revitalized natural product discovery efforts, revealing the untapped biosynthetic potential of fungi. While the volume of genomic data continues to expand, discovery efforts are slowed due to the time-consuming nature of experiments required to characterize new molecules. To direct efforts toward uncharacterized biosynthetic gene clusters most likely to encode novel chemical scaffolds, we took advantage of comparative metabolomics and heterologous gene expression using fungal artificial chromosomes (FACs). By linking mass spectral profiles with structural clues provided by FAC-encoded gene clusters, we targeted a compound originating from an unusual gene cluster containing an indoleamine 2,3-dioxygenase (IDO). With this approach, we isolate and characterize R and S forms of the new molecule terreazepine, which contains a novel chemical scaffold resulting from cyclization of the IDO-supplied kynurenine. The discovery of terreazepine illustrates that FAC-based approaches targeting unusual biosynthetic machinery provide a promising avenue forward for targeted discovery of novel scaffolds and their biosynthetic enzymes, and it also represents another example of a biosynthetic gene cluster "repurposing" a primary metabolic enzyme to diversify its secondary metabolite arsenal.IMPORTANCE Here, we provide evidence that Aspergillus terreus encodes a biosynthetic gene cluster containing a repurposed indoleamine 2,3-dioxygenase (IDO) dedicated to secondary metabolite synthesis. The discovery of this neofunctionalized IDO not only enabled discovery of a new compound with an unusual chemical scaffold but also provided insight into the numerous strategies fungi employ for diversifying and protecting themselves against secondary metabolites. The observations in this study set the stage for further in-depth studies into the function of duplicated IDOs present in fungal biosynthetic gene clusters and presents a strategy for accessing the biosynthetic potential of gene clusters containing duplicated primary metabolic genes.

Immunosuppressive IDO in Cancer: Mechanisms of Action, Animal Models, and Targeting Strategies.

Indoleamine 2, 3-dioxygenase 1 (IDO; IDO1; INDO) is a rate-limiting enzyme that metabolizes the essential amino acid, tryptophan, into downstream kynurenines. Canonically, the metabolic depletion of tryptophan and/or the accumulation of kynurenine is the mechanism that defines how immunosuppressive IDO inhibits immune cell effector functions and/or facilitates T cell death. Non-canonically, IDO also suppresses immunity through non-enzymic effects. Since IDO targeting compounds predominantly aim to inhibit metabolic activity as evidenced across the numerous clinical trials currently evaluating safety/efficacy in patients with cancer, in addition to the recent disappointment of IDO enzyme inhibitor therapy during the phase III ECHO-301 trial, the issue of IDO non-enzyme effects have come to the forefront of mechanistic and therapeutic consideration(s). Here, we review enzyme-dependent and -independent IDO-mediated immunosuppression as it primarily relates to glioblastoma (GBM); the most common and aggressive primary brain tumor in adults. Our group's recent discovery that IDO levels increase in the brain parenchyma during advanced age and regardless of whether GBM is present, highlights an immunosuppressive synergy between aging-increased IDO activity in cells of the central nervous system that reside outside of the brain tumor but collaborate with GBM cell IDO activity inside of the tumor. Because of their potential value for the in vivo study of IDO, we also review current transgenic animal modeling systems while highlighting three new constructs recently created by our group. This work converges on the central premise that maximal immunotherapeutic efficacy in subjects with advanced cancer requires both IDO enzyme- and non-enzyme-neutralization, which is not adequately addressed by available IDO-targeting pharmacologic approaches at this time.

A novel strategy to block mitotic progression for targeted therapy.

BACKGROUND: Blockade of mitotic progression is an ideal approach to induce mitotic catastrophe that suppresses cancer cell expansion. Cdc20 is a critical mitotic factor governing anaphase initiation and the exit from mitosis through recruiting substrates to APC/C for degradation. Results from recent TCGA (The Cancer Genome Atlas) and pathological studies have demonstrated a pivotal oncogenic role for Cdc20-APC/C in tumor progression as well as drug resistance. Thus, deprivation of the mitotic role for Cdc20-APC/C by either inhibition of Cdc20-APC/C activity or elimination of Cdc20 protein via induced protein degradation emerges as an effective therapeutic strategy to control cancer. METHODS: We designed a proteolysis targeting chimera, called CP5V, which comprises a Cdc20 ligand and VHL binding moiety bridged by a PEG5 linker that induces Cdc20 degradation. We characterized the effect of CP5V in destroying Cdc20, arresting mitosis, and inhibiting tumor progression by measuring protein degradation, 3D structure dynamics, cell cycle control, tumor cell killing and tumor inhibition using human breast cancer xenograft mouse model. FINDINGS: Results from our study demonstrate that CP5V can specifically degrade Cdc20 by linking Cdc20 to the VHL/VBC complex for ubiquitination followed by proteasomal degradation. Induced degradation of Cdc20 by CP5V leads to significant inhibition of breast cancer cell proliferation and resensitization of Taxol-resistant cell lines. Results based on a human breast cancer xenograft mouse model show a significant role for CP5V in suppressing breast tumor progression. INTERPRETATION: CP5V-mediated degradation of Cdc20 could be an effective therapeutic strategy for anti-mitotic therapy.

Small-Molecule MYC Inhibitors Suppress Tumor Growth and Enhance Immunotherapy.

Small molecules that directly target MYC and are also well tolerated in vivo will provide invaluable chemical probes and potential anti-cancer therapeutic agents. We developed a series of small-molecule MYC inhibitors that engage MYC inside cells, disrupt MYC/MAX dimers, and impair MYC-driven gene expression. The compounds enhance MYC phosphorylation on threonine-58, consequently increasing proteasome-mediated MYC degradation. The initial lead, MYC inhibitor 361 (MYCi361), suppressed in vivo tumor growth in mice, increased tumor immune cell infiltration, upregulated PD-L1 on tumors, and sensitized tumors to anti-PD1 immunotherapy. However, 361 demonstrated a narrow therapeutic index. An improved analog, MYCi975 showed better tolerability. These findings suggest the potential of small-molecule MYC inhibitors as chemical probes and possible anti-cancer therapeutic agents.