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Pet dogs with naturally occurring cancers play an important role in studies of cancer biology and drug development. We assessed tolerability, efficacy, and pharmacokinetic/pharmacodynamic relationships with a first-in-class small molecule inhibitor of valosin-containing protein (VCP/p97), CB-5339, administered to 24 tumor-bearing pet dogs. Tumor types assessed included solid malignancies, lymphomas, and multiple myeloma. Through a stepwise dose and schedule escalation schema, we determined the maximum tolerated dose to be 7.5 mg/kg when administered orally on a 4 days on, 3 days off schedule per week for 3 consecutive weeks. Adverse events were minimal and mainly related to the gastrointestinal system. Pharmacokinetic/pharmacodynamic data suggest a relationship between exposure and modulation of targets related to induction of the unfolded protein response, but not to tolerability of the agent. An efficacy signal was detected in 33% (2/6) of dogs with multiple myeloma, consistent with a mechanism of action relating to induction of proteotoxic stress in a tumor type with abundant protein production. Clinical trials of CB-5339 in humans with acute myelogenous leukemia and multiple myeloma are ongoing.

Supplementary Data from Comparative Oncology Assessment of a Novel Inhibitor of Valosin-Containing Protein in Tumor-Bearing Dogs

CB-5083 is an inhibitor of p97/valosin-containing protein (VCP), for which phase I trials for cancer were terminated because of adverse effects on vision, such as photophobia and dyschromatopsia. Lower dose CB-5083 could combat inclusion body myopathy with early-onset Paget disease and frontotemporal dementia or multisystem proteinopathy caused by gain-of-function mutations in VCP. We hypothesized that the visual impairment in the cancer trial was due to CB-5083's inhibition of phosphodiesterase (PDE)-6, which mediates signal transduction in photoreceptors. To test our hypothesis, we used in vivo and ex vivo electroretinography (ERG) in mice and a PDE6 activity assay of bovine rod outer segment (ROS) extracts. Additionally, histology and optical coherence tomography were used to assess CB-5083's long-term ocular toxicity. A single administration of CB-5083 led to robust ERG signal deterioration, specifically in photoresponse kinetics. Similar recordings with known PDE inhibitors sildenafil, tadalafil, vardenafil, and zaprinast showed that only vardenafil had as strong an effect on the ERG signal in vivo as did CB-5083. In the biochemical assay, CB-5083 inhibited PDE6 activity with a potency higher than sildenafil but lower than that of vardenafil. Ex vivo ERG revealed a PDE6 inhibition constant of 80 nM for CB-5083, which is 7-fold smaller than that for sildenafil. Finally, we showed that the inhibitory effect of CB-5083 on visual function is reversible, and its chronic administration does not cause permanent retinal anomalies in aged VCP-disease model mice. Our results warrant re-evaluation of CB-5083 as a clinical therapeutic agent. We recommend preclinical ERG recordings as a routine drug safety screen. SIGNIFICANCE STATEMENT: This report supports the use of a valosin-containing protein (VCP) inhibitor drug, CB-5083, for the treatment of neuromuscular VCP disease despite CB-5083's initial clinical failure for cancer treatment due to side effects on vision. The data show that CB-5083 displays a dose-dependent but reversible inhibitory action on phosphodiesterase-6, an essential enzyme in retinal photoreceptor function, but no long-term consequences on retinal function or structure.

The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry-based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9-driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.

RUVBL1 and RUVBL2 are ATPases associated with diverse cellular activities (AAAs) that form a complex involved in a variety of cellular processes, including chromatin remodeling and regulation of gene expression. RUVBLs have a strong link to oncogenesis, where overexpression is correlated with tumor growth and poor prognosis in several cancer types. CB-6644, an allosteric small-molecule inhibitor of the ATPase activity of the RUVBL1/2 complex, interacts specifically with RUVBL1/2 in cancer cells, leading to cell death. Importantly, drug-acquired-resistant cell clones have amino acid mutations in either RUVBL1 or RUVBL2, suggesting that cell killing is an on-target consequence of RUVBL1/2 engagement. In xenograft models of acute myeloid leukemia and multiple myeloma, CB-6644 significantly reduced tumor growth without obvious toxicity. This work demonstrates the therapeutic potential of targeting RUVBLs in the treatment of cancer and establishes a chemical entity for probing the many facets of RUVBL biology.

Background: Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are clonal hematopoietic disorders that predominantly occur in older adults. For a limited number of fit patients, induction followed by consolidation chemotherapy and/or allogeneic stem-cell transplant (SCT) leads to cure. However, many AML patients are ineligible for aggressive therapy; up to 30% have primary refractory disease and up to 50% will relapse after front-line therapy, requiring alternative approaches. For these patients, durable long-term remission rate is low, with 5-year overall survival rates lower than 10%. Therefore, there remains an urgent unmet need for novel therapies for such patients. CB-5339 is a second generation, potent and selective, orally bioavailable small molecule inhibitor of valosin containing protein (VCP)/p97. VCP/p97 is a key cellular enzyme involved in cellular stress response pathways critical to cancer cell growth and survival such as protein homeostasis and the response to DNA damage. Inhibition of VCP/p97 in a panel of 131 cancer cell lines representing 16 cancer types revealed AML as the most exquisitely dependent disease (p=0.004) on VCP/p97 function. Further, CB-5339 demonstrated antiproliferative potency in a panel of 16 AML cell lines (IC50:100nM - 500nM). To better characterize the effects of CB-5339 on human leukemia, viability assays were performed on a set of 30 genetically diverse primary AML patient samples. Cellular viability was impacted with a similar potency across samples, irrespective of underlying genetic abnormalities (mean IC50: 423nM). In vivo, CB-5339 treatment resulted in decreased circulating leukemic cells and significantly prolonged survival in an MLL-AF9 syngeneic AML mouse model (p=0.02). In addition, evidence of synergy was exhibited with standard of care AML therapy, a combination of an anthracycline and cytarabine. This triple combination regimen resulted in a 96% mean relative decrease in leukemic burden compared to control mice and significantly prolonged mice survival compared to each regimen alone (p Here, we present a phase 1 study to evaluate CB-5339 in patients with relapsed/refractory (R/R) AML and intermediate or higher-risk MDS. Study design: This is a single arm, open-label, multi-center phase 1 clinical trial. The study includes two parts: 1) a Dose Escalation phase using a single-participant cohort accelerated titration schema in R/R AML or intermediate/higher-risk MDS (IPSS-R) participants and 2) a Dose Expansion phase using a 2 stage design for further assessment of disease specific cohorts and potential combination strategies at the recommended phase 2 dose (RP2D). CB-5339 will be administered orally once-daily (QD) for 4 days, followed by a 3-day treatment-free period weekly, in successive 28-day cycles until progressive disease or intolerable toxicity. Intraparticipant dose escalation will be permitted after Cycle 1. Twice-daily (BID) dosing may also be tested depending on initial PK assessments. The primary objective of this trial is to determine the safety, tolerability, and MTD and/or RP2D of CB-5339. The secondary objectives are to characterize PK properties and antitumor activity of CB-5339. Exploratory objectives will evaluate potential pharmacodynamic (PD) biomarkers and characterize the PK/PD relationship. Key inclusion criteria include participants with pathologically confirmed AML or intermediate/higher-risk MDS relapsed and/or refractory to standard therapies, ECOG ≤2, and adequate kidney and liver functions. Key exclusion criteria include pathologically confirmed acute promyelocytic leukemia, central nervous system involvement, immediately life-threatening/severe complications of AML/MDS such as uncontrolled bleeding, uncontrolled active infection, pneumonia with hypoxia or shock, and/or disseminated intravascular coagulation, SCT within 100 days of first dose, active GVHD, or post-SCT systemic immunosuppressive therapy, concomitant active malignancy requiring chemotherapy and adverse cardiac history. Up to 60 participants (20 in dose escalation and 40 in dose expansion) will be enrolled at approximately 10 sites in the U.S and Australia. Recruitment is ongoing and this trial is registered with clinicaltrials.gov: NCT04402541. Disclosures Benajiba: Gilead Foundation: Research Funding. Carraway:BMS: Consultancy, Other: Research support, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; Stemline: Consultancy, Speakers Bureau; Takeda: Other: Independent Advisory Committe (IRC); ASTEX: Other: Independent Advisory Committe (IRC); Abbvie: Other: Independent Advisory Committe (IRC). Hamad:Novartis: Honoraria; Abbvie: Honoraria. Stein:Biotheryx: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Seattle Genetics: Consultancy; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy; Astellas Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy. Burroughs:Chiasma: Current equity holder in publicly-traded company; Synthetic Genomics: Current equity holder in private company; Cleave Therapeutics: Current Employment, Current equity holder in private company; Crinetics Pharmaceuticals: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Harris:Cleave Therapeutics: Current Employment, Current equity holder in private company. Lane:Cleave Therapeutics: Consultancy; Regimmune Corporation: Consultancy; Star Therapeutics: Consultancy; Nanoscope Therapeutics: Consultancy; Viewpoint Therapeutics: Ended employment in the past 24 months; Valitor: Consultancy; Revolution Medicines: Consultancy. Nguyen:Cleave Therapeutics, Inc: Current Employment, Current equity holder in private company. Stuart:Cleave Therapeutics: Consultancy; Triphase Accelerator U.S Corporation: Consultancy; Revolution Medicines Inc: Consultancy; RegImmune Corp: Consultancy; IgM Biosciences: Consultancy; Portola Pharmaceuticals Inc: Consultancy; Artiva Biotherapeutics: Consultancy; Gilead Sciences Inc: Consultancy; Theravance Biopharma: Consultancy; Integral Medicines Inc: Consultancy. Vargas:Cytomx Therapeutics, Inc.: Current equity holder in publicly-traded company; Sangamo Therapeutics: Current equity holder in publicly-traded company; Cleave Therapeutics, Inc.: Current Employment, Current equity holder in private company. Stegmaier:Novartis: Research Funding; Auron Therapeutics: Consultancy. DiNardo:ImmuneOnc: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Notable Labs: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Research Funding; MedImmune: Honoraria; Agios: Consultancy, Honoraria, Research Funding; Jazz: Honoraria; Novartis: Consultancy; Calithera: Research Funding; Syros: Honoraria.

Stress-response transcription factors such as NFκB turn on hundreds of genes and must have a mechanism for rapid cessation of transcriptional activation. We recently showed that the inhibitor of NFκB signaling, IκBα, dramatically accelerates the dissociation of NFκB from transcription sites, a process we have called "stripping." To test the role of the IκBα C-terminal PEST (rich in proline, glutamic acid, serine, and threonine residues) sequence in NFκB stripping, a mutant IκBα was generated in which five acidic PEST residues were mutated to their neutral analogs. This IκBα(5xPEST) mutant was impaired in stripping NFκB from DNA and formed a more stable intermediate ternary complex than that formed from IκBα(WT) because DNA dissociated more slowly. NMR and amide hydrogen-deuterium exchange mass spectrometry showed that the IκBα(5xPEST) appears to be "caught in the act of stripping" because it is not yet completely in the folded and NFκB-bound state. When the mutant was introduced into cells, the rate of postinduction IκBα-mediated export of NFκB from the nucleus decreased markedly.

Formation of the nuclear envelope (NE) around segregated chromosomes occurs by the reshaping of the endoplasmic reticulum (ER), a reservoir for disassembled nuclear membrane components during mitosis. In this study, we show that inner nuclear membrane proteins such as lamin B receptor (LBR), MAN1, Lap2β, and the trans-membrane nucleoporins Ndc1 and POM121 drive the spreading of ER membranes into the emerging NE via their capacity to bind chromatin in a collaborative manner. Despite their redundant functions, decreasing the levels of any of these trans-membrane proteins by RNAi-mediated knockdown delayed NE formation, whereas increasing the levels of any of them had the opposite effect. Furthermore, acceleration of NE formation interferes with chromosome separation during mitosis, indicating that the time frame over which chromatin becomes membrane enclosed is physiologically relevant and regulated. These data suggest that functionally distinct classes of chromatin-interacting membrane proteins, which are present at nonsaturating levels, collaborate to rapidly reestablish the nuclear compartment at the end of mitosis.

The nuclear factor kappa B (NFκB) family of transcription factors is a key regulator of immune development, immune responses, inflammation, and cancer. The NFκB signaling system (defined by the interactions between NFκB dimers, IκB regulators, and IKK complexes) is responsive to a number of stimuli, and upon ligand-receptor engagement, distinct cellular outcomes, appropriate to the specific signal received, are set into motion. After almost three decades of study, many signaling mechanisms are well understood, rendering them amenable to mathematical modeling, which can reveal deeper insights about the regulatory design principles. While other reviews have focused on upstream, receptor proximal signaling (Hayden MS, Ghosh S. Signaling to NF-κB. Genes Dev 2004, 18:2195-2224; Verstrepen L, Bekaert T, Chau TL, Tavernier J, Chariot A, Beyaert R. TLR-4, IL-1R and TNF-R signaling to NF-κB: variations on a common theme. Cell Mol Life Sci 2008, 65:2964-2978), and advances through computational modeling (Basak S, Behar M, Hoffmann A. Lessons from mathematically modeling the NF-κB pathway. Immunol Rev 2012, 246:221-238; Williams R, Timmis J, Qwarnstrom E. Computational models of the NF-KB signalling pathway. Computation 2014, 2:131), in this review we aim to summarize the current understanding of the NFκB signaling system itself, the molecular mechanisms, and systems properties that are key to its diverse biological functions, and we discuss remaining questions in the field. WIREs Syst Biol Med 2016, 8:227-241. doi: 10.1002/wsbm.1331 For further resources related to this article, please visit the WIREs website.

The magnitude, duration and oscillation of cellular signalling pathway responses are often limited by negative feedback loops, defined as an ‘activator-induced inhibitor’ regulatory motif. Within the NF κ B signalling pathway, a key negative feedback regulator is I κ B α . We show here that, contrary to current understanding, NF κ B-inducible expression is not sufficient for providing effective negative feedback. We then employ computational simulations of NF κ B signalling to identify I κ B α molecular properties that are critical for proper negative feedback control and test the resulting predictions in biochemical and single-cell live-imaging studies. We identified nuclear import and nuclear export of I κ B α and the I κ B α –NF κ B complex, as well as the free I κ B α half-life, as key determinants of post-induction repression of NF κ B and the potential for subsequent reactivation. Our work emphasizes that negative feedback is an emergent systems property determined by multiple molecular and biophysical properties in addition to the required ‘activator-induced inhibitor’ relationship.

The innate immune response is largely initiated by pathogen-responsive activation of the transcription factor IRF3. Among other target genes, IRF3 controls the expression of IFN-β, which triggers the activation of the transcription factor ISGF3 via the IFNAR. IRF3 and ISGF3 have been reported to control many of the same target genes and together, control the antimicrobial innate-immune program; however, their respective contributions and specificities remain unclear. Here, we used genomic technologies to characterize their specificity in terms of their physical DNA-binding and genetic function. With the use of ChiP-seq and transcriptomic measurements in WT versus ifnar−/− versus ifnar−/−irf3−/− macrophages responding to intracellular dsRNA, we confirmed the known ISGF3 DNA-binding motif and further specified a distinct IRF3 consensus sequence. The functional specificity of IRF3 is particularly pronounced in cytokine/chemokine regulation; yet, even in the control of IFN-β, that specificity is not absolute. By mathematically modeling IFN-β production within an abstracted tissue layer, we find that IRF3 versus ISGF3 specificity may be critical to limiting IFN-β production and ISGF3 activation, temporally and spatially, but that partial overlap in their specificity is tolerable and may enhance the effectiveness of the innate-immune response.

Understanding the functions of multi-cellular organs in terms of the molecular networks within each cell is an important step in the quest to predict phenotype from genotype. B-lymphocyte population dynamics, which are predictive of immune response and vaccine effectiveness, are determined by individual cells undergoing division or death seemingly stochastically. Based on tracking single-cell time-lapse trajectories of hundreds of B cells, single-cell transcriptome, and immunofluorescence analyses, we constructed an agent-based multi-modular computational model to simulate lymphocyte population dynamics in terms of the molecular networks that control NF-κB signaling, the cell cycle, and apoptosis. Combining modeling and experimentation, we found that NF-κBcRel enforces the execution of a cellular decision between mutually exclusive fates by promoting survival in growing cells. But as cRel deficiency causes growing B cells to die at similar rates to non-growing cells, our analysis reveals that the phenomenological decision model of wild-type cells is rooted in a biased race of cell fates. We show that a multi-scale modeling approach allows for the prediction of dynamic organ-level physiology in terms of intra-cellular molecular networks.

DEAF1 is a transcriptional regulator associated with autoimmune and neurological disorders and is known to bind TTCG motifs. To further ascertain preferred DEAF1 DNA ligands, we screened a random oligonucleotide library containing an “anchored” CpG motif. We identified a binding consensus that generally conformed to a repeated TTCGGG motif, with the two invariant CpG dinucleotides separated by 6–11 nucleotides. Alteration of the consensus surrounding the dual CpG dinucleotides, or cytosine methylation of a single CpG half-site, eliminated DEAF1 binding. A sequence within the Htr1a promoter that resembles the binding consensus but contains a single CpG motif was confirmed to have low affinity binding with DEAF1. A DEAF1 binding consensus was identified in the EIF4G3 promoter and ChIP assay showed endogenous DEAF1 was bound to the region. We conclude that DEAF1 preferentially binds variably spaced and unmethylated CpG-containing half-sites when they occur within an appropriate consensus.

Neoplastic cells are often characterized by specific morphological abnormalities of the nuclear envelope (NE), which have been used for cancer diagnosis for more than a century. The NE is a double phospholipid bilayer that encapsulates the nuclear genome, regulates all nuclear trafficking of RNAs and proteins and prevents the passive diffusion of macromolecules between the nucleoplasm and the cytoplasm. Whether there is a consequence to the proper functioning of the cell and loss of structural integrity of the nucleus remains unclear. Using live cell imaging, we characterize a phenomenon wherein nuclei of several proliferating human cancer cell lines become temporarily ruptured during interphase. Strikingly, NE rupturing was associated with the mislocalization of nucleoplasmic and cytoplasmic proteins and, in the most extreme cases, the entrapment of cytoplasmic organelles in the nuclear interior. In addition, we observed the formation of micronuclei-like structures during interphase and the movement of chromatin out of the nuclear space. The frequency of these NE rupturing events was higher in cells in which the nuclear lamina, a network of intermediate filaments providing mechanical support to the NE, was not properly formed. Our data uncover the existence of a NE instability that has the potential to change the genomic landscape of cancer cells.

[reaction--see text] The type 2 intramolecular Diels-Alder cycloaddition utilizing N-acylnitroso dienophiles provides an efficient entry into bridged oxazinolactams. In contrast to the bimolecular counterpart, the reaction is completely regioselective. Structural characterization of the cycloadducts allows for evaluation of the olefin distortion and the degree of pyramidalization of the bridgehead oxazinolactam.