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The widespread notion of a unique national humour involves an impulse to apply the commonplace assumptions of national identity that demand uniqueness of identity, history, language and culture for a political society. What is deemed true and distinctive of the nation must be also be true and distinctive of its national humour, goes the thinking. However, such cultural exclusivity has not been reconciled with cultural exchanges between nations. Paradoxically, conceptions of national humour have been formulated in dynamic tension with such exchanges during the various phases of globalization that have taken place since the 19th century. The Americanisation of humour, in particular, has been an important component of such transmissions and resulted from the commercial popular culture dominated by America since the nineteenth century. Australia is a prime example examined here along with examples from Britain. To complicate matters of transmission, Americanisation sometimes arrived in Australia via Britain as well as directly from America itself. Australians and Britons periodically reacted against American culture, including humour, as a threat to national identity. But this was part of a dynamic tension played out between modern and traditional, imported and local in their selections and adaptations of humour imports from America. There is a huge and historic complexity of cultural anxiety and cultural transfer lying behind the apparent cultural comforts of belonging to a nation-state. Moreover, humour has played its part in the continual discursive recreation of the nation in the form of constant searches for the unique national humour of a people.

Supplementary Methods, Figures, and Tables

Thalidomide analogues exert their therapeutic effects by binding to the CRL4CRBN E3 ubiquitin ligase, promoting ubiquitination and subsequent proteasomal degradation of specific protein substrates. Drug-induced degradation of IKZF1 and IKZF3 in B-cell malignancies demonstrates the clinical utility of targeting disease-relevant transcription factors for degradation. Here, we found that avadomide (CC-122) induces CRBN-dependent ubiquitination and proteasomal degradation of ZMYM2 (ZNF198), a transcription factor involved in balanced chromosomal rearrangements with FGFR1 and FLT3 in aggressive forms of hematologic malignancies. The minimal drug-responsive element of ZMYM2 is a zinc-chelating MYM domain and is contained in the N-terminal portion of ZMYM2 that is universally included in the derived fusion proteins. We demonstrate that avadomide has the ability to induce proteasomal degradation of ZMYM2–FGFR1 and ZMYM2–FLT3 chimeric oncoproteins, both in vitro and in vivo. Our findings suggest that patients with hematologic malignancies harboring these ZMYM2 fusion proteins may benefit from avadomide treatment.Significance:We extend the potential clinical scope of thalidomide analogues by the identification of a novel avadomide-dependent CRL4CRBN substrate, ZMYM2. Avadomide induces ubiquitination and degradation of ZMYM2–FGFR1 and ZMYM2–FLT3, two chimeric oncoproteins involved in hematologic malignancies, providing a proof of concept for drug-induced degradation of transcription factor fusion proteins by thalidomide analogues.

Supplementary Table 1. Proteomic data

Supplementary Figure S1:Effect of CB-5083 on cell growth and survival in multiple myeloma and solid tumor cell lines; Supplementary Figure S2:CB-5083 activates UPR;Supplementary Figure S3:CB-5083 transcriptional response is unique in comparison to proteasome inhibitors;Supplementary Figure S4:CB-5083 enhances the antitumor activity of proteasome inhibitors; Supplementary Figure S5: Nrf1 upregulation induced by bortezomib is inhibited by CB-5083; Supplementary Figure S6:CB-5083 demonstrates a broad activity in multiple myeloma relevant in vivo models; Supplementary Table S1: List of antibodies used in the manuscript.

Inhibition of the AAA ATPase, p97, was recently shown to be a novel method for targeting the ubiquitin proteasome system, and CB-5083, a first-in-class inhibitor of p97, has demonstrated broad antitumor activity in a range of both hematologic and solid tumor models. Here, we show that CB-5083 has robust activity against multiple myeloma cell lines and a number of in vivo multiple myeloma models. Treatment with CB-5083 is associated with accumulation of ubiquitinated proteins, induction of the unfolded protein response, and apoptosis. CB-5083 decreases viability in multiple myeloma cell lines and patient-derived multiple myeloma cells, including those with background proteasome inhibitor (PI) resistance. CB-5083 has a unique mechanism of action that combines well with PIs, which is likely owing to the p97-dependent retro-translocation of the transcription factor, Nrf1, which transcribes proteasome subunit genes following exposure to a PI. In vivo studies using clinically relevant multiple myeloma models demonstrate that single-agent CB-5083 inhibits tumor growth and combines well with multiple myeloma standard-of-care agents. Our preclinical data demonstrate the efficacy of CB-5083 in several multiple myeloma disease models and provide the rationale for clinical evaluation as monotherapy and in combination in multiple myeloma. Mol Cancer Ther; 16(11); 2375–86. ©2017 AACR.

Supplementary Fig. S2 from Comparison of biochemical and biological effects of ML858 (salinosporamide A) and bortezomib

Supplementary Table S1 from Comparison of biochemical and biological effects of ML858 (salinosporamide A) and bortezomib

Strains within the genus Salinospora have been shown to produce complex natural products having antibiotic and antiproliferative activities. The biochemical basis for the cytotoxic effects of salinosporamide A has been linked to its ability to inhibit the proteasome. Synthetically accessible salinosporamide A (ML858) was used to determine its biochemical and biological activities and to compare its effects with those of bortezomib. ML858 and bortezomib show time- and concentration-dependent inhibition of the proteasome in vitro. However, unlike bortezomib, which is a reversible inhibitor, ML858 covalently binds to the proteasome, resulting in the irreversible inhibition of 20S proteasome activity. ML858 was equipotent to bortezomib in cell-based reporter stabilization assays, but due to intramolecular instability is less potent in long-term assays. ML858 failed to maintain levels of proteasome inhibition necessary to achieve efficacy in tumor models responsive to bortezomib. Our results show that ML858 and bortezomib exhibit different kinetic and pharmacologic profiles and suggest that additional characterization of ML858 is warranted before its therapeutic potential can be fully appreciated. [Mol Cancer Ther 2006;5(12):3052–61]

The proteasome was validated as an oncology target following the clinical success of VELCADE (bortezomib) for injection for the treatment of multiple myeloma and recurring mantle cell lymphoma. Consequently, severalgroups are pursuing the development of additional small-molecule proteasome inhibitors for both hematologic and solid tumor indications. Here, we describe MLN9708, a selective, orally bioavailable, second-generation proteasome inhibitor that is in phase I clinical development. MLN9708 has a shorter proteasome dissociation half-life and improved pharmacokinetics, pharmacodynamics, and antitumor activity compared with bortezomib. MLN9708 has a larger blood volume distribution at steady state, and analysis of 20S proteasome inhibition and markers of the unfolded protein response confirmed that MLN9708 has greater pharmacodynamic effects in tissues than bortezomib. MLN9708 showed activity in both solid tumor and hematologic preclinical xenograft models, and we found a correlation between greater pharmacodynamic responses and improved antitumor activity. Moreover, antitumor activity was shown via multiple dosing routes, including oral gavage. Taken together, these data support the clinical development of MLN9708 for both hematologic and solid tumor indications. Cancer Res; 70(5); 1970–80

Supplementary Data, Figure Legends 1-3 from Evaluation of the Proteasome Inhibitor MLN9708 in Preclinical Models of Human Cancer

Supplementary Figure 3 from Evaluation of the Proteasome Inhibitor MLN9708 in Preclinical Models of Human Cancer

Supplementary Figure 1 from Evaluation of the Proteasome Inhibitor MLN9708 in Preclinical Models of Human Cancer

Supplementary Figure 2 from Evaluation of the Proteasome Inhibitor MLN9708 in Preclinical Models of Human Cancer

The multiplexed cancer cell line screening platform PRISM demonstrated its utility in testing hundreds of cell lines in a single run, possessing the potential to speed up anti-cancer drug discovery, validation and optimization. Here we described the development and implementation of a next-generation PRISM platform combining Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing, cell line DNA barcoding and next-generation sequencing to enable genetic and/or pharmacological assessment of target addiction in hundreds of cell lines simultaneously. Both compound and CRISPR-knockout PRISM screens well recapitulated the results from individual assays and showed high consistency with a public database., Xia et al. report the development and optimization of a high-throughput screening platform to systematically determine cancer cell sensitivity to pharmacological and genetic perturbations, BMS-PRISM, based on PRISM and high-throughput CRISPR/Cas9 loss-of-function screen technologies using cell line barcoding.

A number of clinically validated drugs have been developed by repurposing the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex with molecular glue degraders to eliminate disease-driving proteins. Here, we present the identification of a first-in-class GSPT1-selective cereblon E3 ligase modulator, CC-90009. Biochemical, structural, and molecular characterization demonstrates that CC-90009 coopts the CRL4CRBN to selectively target GSPT1 for ubiquitination and proteasomal degradation. Depletion of GSPT1 by CC-90009 rapidly induces acute myeloid leukemia (AML) apoptosis, reducing leukemia engraftment and leukemia stem cells (LSCs) in large-scale primary patient xenografting of 35 independent AML samples, including those with adverse risk features. Using a genome-wide CRISPR-Cas9 screen for effectors of CC-90009 response, we uncovered the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreases the production of full-length cereblon protein via modulating CRBN messenger RNA alternative splicing, leading to diminished response to CC-90009. The screen also revealed that the mTOR signaling and the integrated stress response specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 by reducing CC-90009-induced binding of GSPT1 to cereblon and subsequent GSPT1 degradation. On the other hand, GSPT1 degradation promoted the activation of the GCN1/GCN2/ATF4 pathway and subsequent apoptosis in AML cells. Collectively, CC-90009 activity is mediated by multiple layers of signaling networks and pathways within AML blasts and LSCs, whose elucidation gives insight into further assessment of CC-90009s clinical utility. These trials were registered at www.clinicaltrials.gov as #NCT02848001 and #NCT04336982).

Thalidomide-dependent degradation of the embryonic transcription factor SALL4 by the CRL4CRBN E3 ubiquitin ligase is a plausible major driver of thalidomide teratogenicity. The structure of the second zinc finger of SALL4 in complex with pomalidomide, cereblon and DDB1 reveals the molecular details of recruitment. Sequence differences and a shifted binding position relative to Ikaros offer a path to the rational design of cereblon-binding drugs with reduced teratogenic risk.

Supplemental Digital Content is available in the text., Objectives Management of pain is suboptimal in many prehospital and emergency department settings, and European guidelines are lacking. We carried out the Consensus On Management of PAin Caused by Trauma (COMPACT) Delphi initiative to gain insights into the factors physicians consider important when selecting analgesics for trauma pain. Patients and methods A pan-European panel of experts in emergency medicine or pain (N = 31) was recruited to participate in the COMPACT Delphi initiative. In round 1, panelists supplied free-text responses to an open question about the attributes of analgesics for emergency pain relief favored by physicians. Common themes were consolidated into factors. In round 2, factors rated important by more than 75% of the panel were taken forward into round 3. In round 3, the point at which the consensus was achieved was defined a priori as at least 75% of panelists agreeing or strongly agreeing that a factor was important. Results Twenty-nine experts participated, representing 12 European countries and with a mean (SD) of 20 (8.6) years of clinical experience. Most worked in an emergency department (79.3%). The consensus was achieved for 10 factors that were important to consider when selecting analgesics for trauma pain relief. The highest level of consensus was achieved for ‘efficacy’ (100%), followed by ‘safety and tolerability’ (96.6%), and ‘ease of use’ (93.1%). Conclusion These findings may facilitate the development of evidence-based guidelines supporting the provision of pain management in prehospital, emergency department, and critical care settings.

When John Clarke died in 2016, the Australian media rushed for the opinions of politicians on his passing. Among the many cliches about political satire, former prime ministers intoned that he ‘spo...

Thalidomide analogues exert their therapeutic effects by binding to the CRL4CRBN E3 ubiquitin ligase, promoting ubiquitination and subsequent proteasomal degradation of specific protein substrates. Drug-induced degradation of IKZF1 and IKZF3 in B-cell malignancies demonstrates the clinical utility of targeting disease-relevant transcription factors for degradation. Here, we found that avadomide (CC-122) induces CRBN-dependent ubiquitination and proteasomal degradation of ZMYM2 (ZNF198), a transcription factor involved in balanced chromosomal rearrangements with FGFR1 and FLT3 in aggressive forms of hematologic malignancies. The minimal drug-responsive element of ZMYM2 is a zinc-chelating MYM domain and is contained in the N-terminal portion of ZMYM2 that is universally included in the derived fusion proteins. We demonstrate that avadomide has the ability to induce proteasomal degradation of ZMYM2–FGFR1 and ZMYM2–FLT3 chimeric oncoproteins, both in vitro and in vivo. Our findings suggest that patients with hematologic malignancies harboring these ZMYM2 fusion proteins may benefit from avadomide treatment. Significance: We extend the potential clinical scope of thalidomide analogues by the identification of a novel avadomide-dependent CRL4CRBN substrate, ZMYM2. Avadomide induces ubiquitination and degradation of ZMYM2–FGFR1 and ZMYM2–FLT3, two chimeric oncoproteins involved in hematologic malignancies, providing a proof of concept for drug-induced degradation of transcription factor fusion proteins by thalidomide analogues.

CC-122 is a next-generation cereblon E3 ligase–modulating agent that has demonstrated promising clinical efficacy in patients with relapsed or refractory diffuse large B-cell lymphoma (R/R DLBCL). Mechanistically, CC-122 induces the degradation of IKZF1/3, leading to T-cell activation and robust cell-autonomous killing in DLBCL. We report a genome-wide CRISPR/Cas9 screening for CC-122 in a DLBCL cell line SU-DHL-4 with follow-up mechanistic characterization in 6 DLBCL cell lines to identify genes regulating the response to CC-122. Top-ranked CC-122 resistance genes encode, not only well-defined members or regulators of the CUL4/DDB1/RBX1/CRBN E3 ubiquitin ligase complex, but also key components of signaling and transcriptional networks that have not been shown to modulate the response to cereblon modulators. Ablation of CYLD, NFKBIA, TRAF2, or TRAF3 induces hyperactivation of the canonical and/or noncanonical NF-κB pathways and subsequently diminishes CC-122–induced apoptosis in 5 of 6 DLBCL cell lines. Depletion of KCTD5, the substrate adaptor of the CUL3/RBX1/KCTD5 ubiquitin ligase complex, promotes the stabilization of its cognate substrate, GNG5, resulting in CC-122 resistance in HT, SU-DHL-4, and WSU-DLCL2. Furthermore, knockout of AMBRA1 renders resistance to CC-122 in SU-DHL-4 and U-2932, whereas knockout of RFX7 leads to resistance specifically in SU-DHL-4. The ubiquitous and cell line–specific mechanisms of CC-122 resistance in DLBCL cell lines revealed in this work pinpoint genetic alternations that are potentially associated with clinical resistance in patients and facilitate the development of biomarker strategies for patient stratification, which may improve clinical outcomes of patients with R/R DLBCL.

Targeted protein degradation via small-molecule modulation of cereblon offers vast potential for the development of new therapeutics. Cereblon-binding therapeutics carry the safety risks of thalidomide, which caused an epidemic of severe birth defects characterized by forelimb shortening or phocomelia. Here we show that thalidomide is not teratogenic in transgenic mice expressing human cereblon, indicating that binding to cereblon is not sufficient to cause birth defects. Instead, we identify SALL4 as a thalidomide-dependent cereblon neosubstrate. Human mutations in SALL4 cause Duane-radial ray, IVIC, and acro-renal-ocular syndromes with overlapping clinical presentations to thalidomide embryopathy, including phocomelia. SALL4 is degraded in rabbits but not in resistant organisms such as mice because of SALL4 sequence variations. This work expands the scope of cereblon neosubstrate activity within the formerly 'undruggable' C2H2 zinc finger family and offers a path toward safer therapeutics through an improved understanding of the molecular basis of thalidomide-induced teratogenicity.

CC-99282 is a novel, oral CELMoD ® agent currently under investigation in phase 1 clinical studies in patients with relapsed or refractory (R/R) non-Hodgkin lymphomas (NHL) and chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Mechanistically, CC-99282 interacts with the CRL4 CRBN E3 ubiquitin ligase substrate receptor CRBN to induce recruitment and ubiquitin-mediated proteasomal degradation of transcription factors Ikaros and Aiolos. The design intent for CC-99282 included efficient absorption, deep tissue distribution, and prolonged exposure to optimize activity in bulky lymphoma lesions. Recently, we reported that CC-99282 shows potent antitumor activity in different preclinical models of diffuse large B cell lymphoma (DLBCL; Lopez-Girona, et al. Hematol Oncol. 2021). Here, we provide an expanded analysis of CC-99282 activity as a monotherapy, as well as examine its synergistic activity with anti-CD20 antibody treatment, in preclinical models of NHL including DLBCL and follicular lymphoma (FL). Compared with existing agents targeting Ikaros/Aiolos that show activity in hematologic malignancies, such as lenalidomide, avadomide, and iberdomide (CC-220), CC-99282 induced a more rapid, deep, and sustained degradation of Ikaros/Aiolos, causing derepression of cyclin-dependent kinase (CDK) inhibitors and interferon-stimulated genes (IRF7, IFIT3, and DDX58), and the reduction of the highly critical oncogenic factors c-Myc and IRF4. These molecular changes were followed by potent, 10- to 100-fold enhanced, autonomous cell killing and induction of apoptosis (Figure). Our results show that these effects were independent of the cell of origin (activated B cell [ABC; TMD8 cell line], germinal center B cell [GCB; WSU-DLCL2 cell line], or primary mediastinal B cell lymphoma [PMBL] subtypes of DLBCL) or presence of high-risk chromosomal translocations (MYC, BCL2, and/or BCL6), as observed in a panel of 36 lymphoma cell lines that included DLBCL and FL cell lines. In vivo, CC-99282 demonstrated robust tissue distribution that favored target tissues and exhibited antitumor activity resulting in improved tumor regression and tumor-free animals in several lymphoma xenograft models, including an intracranial xenograft model. This strong antitumor activity was observed using various continuous and intermittent dosing paradigms. The potent, direct autonomous cell-killing activity of CC-99282 was augmented when CC-99282 was combined with the anti-CD20 antibody rituximab. In vitro combination studies of CC-99282 with rituximab in lymphoma cell lines demonstrated enhanced cell killing by human natural killer (NK) cells, macrophage-mediated phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). In FL and DLBCL cell lines, we showed that the combination of CC-99282 with rituximab resulted in increases in both NK-mediated ADCC and macrophage-mediated ADCP of up to 20% compared with rituximab treatment alone. In vivo, combination treatment with CC-99282 and rituximab induced dose-dependent tumor growth inhibition in WSU-DLCL2 and RL (FL) xenograft models. In the WSU-DLCL2 model, CC-99282 (1 mg/kg) or rituximab (10 mg/kg) monotherapy resulted in modest tumor growth inhibition, whereas the combination of CC-99282 (1 mg/kg) and rituximab (10 mg/kg) resulted in tumor regression in 100% of animals. Similar results were obtained in FL xenograft models using the RL cell line, where combinations of CC-99282 (1 mg/kg) with rituximab (25 mg/kg) induced complete tumor regression in 100% of animals. In conclusion, CC-99282 is a novel CELMoD agent with an improved substrate degradation profile compared with existing Ikaros/Aiolos-degrading agents. CC-99282 demonstrated enhanced antiproliferative and apoptotic activities across a broad range of lymphoma cells and a robust distribution profile that favors target tissues such as lymphoid organs. In addition, CC-99282 acts synergistically in combination with anti-CD20 monoclonal antibody treatment. Collectively, these data support the clinical investigation of CC-99282 as monotherapy and in combination with rituximab in patients with R/R NHL. Figure 1 Figure 1. Disclosures Carrancio: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Groocock: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Janardhanan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Jankeel: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Galasso: Ryan Galasso: Current Employment, Current equity holder in publicly-traded company. Guarinos: Bristol Myers Squibb: Current Employment. Narla: Bristol Myers Squibb: Current Employment. Groza: Bristol Myers Squibb: Current Employment. Leisten: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Pierce: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Rolfe: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Lopez-Girona: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

B acute lymphoblastic leukemia (B-ALL) cells are distinctively vulnerable to endoplasmic reticulum (ER) stress. Recently, inhibition of p97 was shown to induce ER stress and subsequently cell death in solid tumors and in multiple myeloma. We investigated the role of a novel, orally available, p97 inhibitor (CB-5083; Cleave Biosciences) in B-ALL. CB-5083 induced a significant reduction in viability in 10 human B-ALL cell lines, harboring the most common fusion-genes involved in pediatric and adult B-ALL, with IC50s ranging from 0.34 to 0.76 μM. Moreover, CB-5083 significantly reduced the colony formation of OP1 and NALM6 cells. Early and strong induction of apoptosis was demonstrated in BALL1 and OP1 cells, together with a robust cleavage of PARP. CB-5083 induced ER stress, as documented through: 1) prominent expression of chaperones (GRP78, GRP94, PDI, DNAJC3, and DNAJB9); 2) increased activation of IRE1-alpha, as demonstrated by the splicing of XBP1; and 3) activation of PERK, which resulted in a significant overexpression of CHOP, and its downstream genes. CB-5083 reduced the viability also in GRP78−/−, GRP94−/−, and XBP1−/− cells, suggesting that none of these proteins alone was strictly required for CB-5083 activity. Moreover, we showed that the absence of XBP1 (XBP1−/−) increased the sensitivity to CB-5083, leading to the hypothesis that XBP1 splicing counteracts the activity of CB-5083, probably mitigating ER stress. Finally, vincristine was synergistic with CB-5083 in both BALL1 and OP1 cells. In summary, the targeting of p97 with CB-5083 is a novel promising therapeutic approach that should be further evaluated in B-ALL.

Thalidomide-dependent degradation of the embryonic transcription factor SALL4 by the CRL4

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.

The AAA-ATPase p97 plays vital roles in mechanisms of protein homeostasis, including ubiquitin-proteasome system (UPS) mediated protein degradation, endoplasmic reticulum-associated degradation (ERAD), and autophagy. Herein we describe our lead optimization efforts focused on in vitro potency, ADME, and pharmaceutical properties that led to the discovery of a potent, ATP-competitive, D2-selective, and orally bioavailable p97 inhibitor 71, CB-5083. Treatment of tumor cells with 71 leads to significant accumulation of markers associated with inhibition of UPS and ERAD functions, which induces irresolvable proteotoxic stress and cell death. In tumor bearing mice, oral administration of 71 causes rapid accumulation of markers of the unfolded protein response (UPR) and subsequently induces apoptosis leading to sustained antitumor activity in in vivo xenograft models of both solid and hematological tumors. 71 has been taken into phase 1 clinical trials in patients with multiple myeloma and solid tumors.

Glioblastoma (GBM) is the most prevalent and aggressive malignant tumor of the central nervous system in adults. Patients with GBM have a poor prognosis, with a median survival of 15 months with current standard-of-care treatment, consisting of surgery followed by radiotherapy with concomitant and adjuvant temozolomide. Marizomib is an irreversible, pan-proteasome inhibitor that crosses the blood–brain barrier, currently under investigation in a phase 3 trial for newly diagnosed GBM. This preclinical study investigated the mechanism of action of marizomib in patient-derived GBM spheres and explored a series of combination treatments to further enhance its antitumor potency. Marizomib inhibited the 3 proteolytic subunits of the 20S proteasome in a dose-dependent manner, and this correlated with reduced cell proliferation compared with vehicle control (dimethyl sulfoxide). Marizomib induced transient expression of unfolded protein response (UPR) pathway genes, with a sustained upregulation of the SQSTMI gene. Combining marizomib with the histone deacetylase (HDAC) inhibitors panobinostat and vorinostat demonstrated cooperative upregulation of UPR genes and enhanced inhibition of cell proliferation. Furthermore, marizomib treatment upregulated survivin, an inhibitor of apoptosis protein (IAP). Compared with marizomib alone, addition of the IAP inhibitors AZD5582 and LCL161 increased marizomib antiproliferative activity in GBM spheres. In conclusion, these findings indicate that combining marizomib with HDAC or IAP inhibitors can enhance the overall antitumor activity of marizomib in patient-derived GBM spheres. Additional studies in preclinical models of GBM are needed to corroborate these findings.

The cereblon modulating agents (CMs) including lenalidomide, pomalidomide and CC-220 repurpose the Cul4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase complex to induce the degradation of specific neomorphic substrates via polyubiquitination in conjunction with E2 ubiquitin-conjugating enzymes, which have until now remained elusive. Here we show that the ubiquitin-conjugating enzymes UBE2G1 and UBE2D3 cooperatively promote the K48-linked polyubiquitination of CRL4CRBN neomorphic substrates via a sequential ubiquitination mechanism. Blockade of UBE2G1 diminishes the ubiquitination and degradation of neomorphic substrates, and consequent antitumor activities elicited by all tested CMs. For example, UBE2G1 inactivation significantly attenuated the degradation of myeloma survival factors IKZF1 and IKZF3 induced by lenalidomide and pomalidomide, hence conferring drug resistance. UBE2G1-deficient myeloma cells, however, remained sensitive to a more potent IKZF1/3 degrader CC-220. Collectively, it will be of fundamental interest to explore if loss of UBE2G1 activity is linked to clinical resistance to drugs that hijack the CRL4CRBN to eliminate disease-driving proteins., eLife digest Cells routinely breakdown damaged or unwanted proteins to recycle their building blocks. In humans, most of these unwanted proteins are first tagged with a chain of smaller proteins called ubiquitin, in a process known as ubiquitination. Three kinds of enzymes – named E1, E2 and E3 – act one after the other to recruit and transfer ubiquitin onto the protein. Any problem with this protein-disposal system may cause diseases including cancers. Several drugs such as thalidomide are known to hijack the ubiquitination process by binding to the E3 enzyme. Instead of targeting unwanted proteins, the E3 enzyme-drug complex targets proteins that are driving a disease. These drugs are particularly useful for treating blood cancers. The problem is patients often become resistant to these drugs, and not always because the activity of the E3 enzyme is impaired. An alternative suspect would be an E2 enzyme, but the role of these enzymes remains unclear. Lu et al. have now asked whether a faulty E2 enzyme can lead to drug resistance in a form of blood cancer called multiple myeloma. The experiments tested how proteins relevant for the growth of cancerous myeloma cells were degraded in the presence of different drugs. Genes for the E2 enzymes were inactivated one at a time using a gene editing approach to see which ones would affect the degradation of the proteins and result in drug resistance. Two E2 enzymes, UBE2G1 and UBE2D3, were found to be critical. UBE2D3 first links the disease-driving proteins with one ubiquitin before UBE2G1 can subsequently assemble a chain of ubiquitin proteins. If either of these E2 enzymes was missing from myeloma cells treated with drugs, the disease-driving proteins could not be properly tagged with ubiquitin. This interfered with the degradation of the proteins and allowed the myeloma cells to continue to grow. Yet, myeloma cells that did not have UBE2G1 and were resistant to certain drugs could still respond to other more potent drugs. This suggests that the success of the drugs depends on UBE2G1. Therefore, a better understanding of the activity of this E2 enzyme may be useful for the development of future anticancer drugs.

Inhibition of the AAA ATPase, p97, was recently shown to be a novel method for targeting the ubiquitin proteasome system, and CB-5083, a first-in-class inhibitor of p97, has demonstrated broad antitumor activity in a range of both hematologic and solid tumor models. Here, we show that CB-5083 has robust activity against multiple myeloma cell lines and a number of in vivo multiple myeloma models. Treatment with CB-5083 is associated with accumulation of ubiquitinated proteins, induction of the unfolded protein response, and apoptosis. CB-5083 decreases viability in multiple myeloma cell lines and patient-derived multiple myeloma cells, including those with background proteasome inhibitor (PI) resistance. CB-5083 has a unique mechanism of action that combines well with PIs, which is likely owing to the p97-dependent retro-translocation of the transcription factor, Nrf1, which transcribes proteasome subunit genes following exposure to a PI. In vivo studies using clinically relevant multiple myeloma models demonstrate that single-agent CB-5083 inhibits tumor growth and combines well with multiple myeloma standard-of-care agents. Our preclinical data demonstrate the efficacy of CB-5083 in several multiple myeloma disease models and provide the rationale for clinical evaluation as monotherapy and in combination in multiple myeloma. Mol Cancer Ther; 16(11); 2375–86. ©2017 AACR.

Contemporary satire and cartooning in Australia and the USA share a populist strain of discourse that encourages a stereotypical view of politicians as participants in a dirty, slippery game involving spin and dubious language. This rhetoric is often mistaken for speaking truth to power (parrhesia), but it is an historic legacy of anti-politics. Populism is not an aberration of democracy but a result of tensions inherent in representative democracy from its beginnings. The anti-political rhetoric is traced to early eighteenth-century Britain and the Augustan polemical writers who established what became common attitudes towards politicians. The literature passed to America as standard reading for the Founding Fathers and for many educated Americans before the 1830s. It became the basis of not only US populism but a permanent dissatisfaction with politicians actively cultivated by the political system itself and the media. From the 1820s to the 1830s, the strain passed to Australia.

Medicinal chemistry efforts leading to the discovery of inhibitors of the AAA + ATPase p97 are described. Special focus is given to the discovery of CB-5083, a first-in-class agent currently in clinical trials against various cancers.

Summary Many calls have been made since 2001 for a ‘new public diplomacy’ of the information age that utilizes the internet to reach public opinion. They have been especially forthcoming from the Obama administration, although they have been just as popular with the political classes in the United States and elsewhere. However, such recent calls form only the latest instalment of a rhetorical tradition of public diplomacy that stretches back to Woodrow Wilson and beyond to the 1790s. There is a thematic recurrence in the rhetoric of public diplomacy, as there is in the rhetoric of democracy, and for the same reason: representative democracy has always involved a complex tension between, on the one hand, the political class of politicians and diplomats and, on the other, public opinion, which needs to be appeased since it confers legitimacy on representatives. This results in a recurring pattern of language involving suspicions of the political class, declarations of a new era of diplomacy and claims to credibility. There are hence frequent bouts of anti-politics politics and anti-diplomacy politics, sometimes utilizing a discourse of technological optimism, which politicians and diplomats attempt to assuage with similar calls for new political dawns.

CC-90009 is a novel cereblon E3 ligase modulator (CELMoD) currently under investigation in a phase I clinical study in relapsed or refractory acute myeloid leukemia (R/R AML) (CC-90009-AML-001; NCT02848001). CC-90009 coopts the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex to target the translation termination factor G1 to S phase transition 1 (GSPT1) for ubiquitination and proteasomal degradation, resulting in rapid induction of apoptosis and growth inhibition in AML cell lines and primary patient blasts. To further elucidate the mechanism of action of CC-90009 in AML, we performed a genome-wide CRISPR/Cas9 screen to identify gene(s) whose knockout abrogate(s) the response to CC-90009 in a sensitive AML cell line. In addition to well-established key regulatory proteins required for the activity of all known cereblon modulators, which include components of the CRL4CRBN complex, E2 ubiquitin conjugating enzymes UBE2G1 and UBE2D3, and members of the neddylation and deneddylation machinery, interestingly, the screen identified the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreased the production of full-length CRBN transcript via modulating alternative splicing of CRBN mRNA, leading to significant downregulation of cereblon expression and hence diminished response to CC-90009. The screen also revealed that mTOR signaling and the integrated stress response (ISR) specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Since CC-90009 inhibits protein translation, it is reasonable to expect interactions with regulators of this pathway. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 , at least in part by reducing CC-90009 induced binding of GSPT1 to cereblon and subsequent GSPT1 degradation. On the other hand, GSPT1 degradation promoted the activation of the GCN1/GCN2/ATF4 pathway and subsequent apoptosis in AML cells. Loss of GCN2 significantly attenuated the growth inhibitory effect of CC-90009, and this effect can be rescued with GCN2 wild-type but not enzymatically-dead mutants. Collectively, the antitumor activity of CC-90009, a first-in-class GSPT1 degrader, in AML cell lines is mediated by multiple layers of signaling networks and machinery, the elucidation of which reveals the underlying mechanism by which CC-90009 exerts its anti-AML activity and informs on the pathways for further study of CC-90009's clinical utility. Disclosures Lu: Celgene Corporation: Employment, Equity Ownership. Surka:Celgene: Employment, Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Jang:Celgene: Employment, Equity Ownership. Wang:Celgene: Employment, Equity Ownership. Rolfe:Celgene: Employment, Equity Ownership.