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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.

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.

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.

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.

Lobar torsion is well documented after pneumonectomy, but is very rare after lung transplant. To the best of our knowledge, this is the twelfth reported case of lobar torsion after lung transplant. In our case, bronchoscopies and chest radiographs were inconclusive; however, CT scan clearly demonstrated findings consistent with right middle lobe torsion. We review the literature and discuss the epidemiology, clinical presentation, imaging features, and treatment considerations for this condition. We also propose that if a clinical picture could be secondary to torsion and bronchoscopies and chest x ray are inconclusive that a CT scan should be obtained as soon as possible since early recognition increases the likelihood of being able to successfully detorse the lung and avoid lobectomy.

The proteasome inhibitors Carfilzomib (Cfz) and Bortezomib (Btz) are used successfully to treat MM, but have not shown clinical efficacy in solid tumors. Here we show that clinically achievable inhibition of the β5 site of the proteasome by Cfz and Btz does not result in loss of viability of triple-negative breast cancer cell lines. We use site-specific inhibitors and CRISPR-mediated genetic inactivation of β1 and β2 to demonstrate that inhibiting a second site of the proteasome, particularly the β2 site, sensitizes cell lines to Btz and Cfz in vitro and in vivo. Inhibiting both β5 and β2 suppresses production of the soluble, active form of the transcription factor Nrf1 and prevents the recovery of proteasome activity through induction of new proteasomes. These findings provide a strong rationale for the development of dual β5 and β2 inhibitors for the treatment of solid tumors.

MLN4924 is a potent and selective small molecule NEDD8-activating enzyme (NAE) inhibitor. In most cancer cells tested, inhibition of NAE leads to induction of DNA rereplication, resulting in DNA damage and cell death. However, in preclinical models of activated B cell–like (ABC) diffuse large B-cell lymphoma (DLBCL), we show that MLN4924 induces an alternative mechanism of action. Treatment of ABC DLBCL cells with MLN4924 resulted in rapid accumulation of pIκBα, decrease in nuclear p65 content, reduction of nuclear factor-κB (NF-κB) transcriptional activity, and G1 arrest, ultimately resulting in apoptosis induction, events consistent with potent NF-κB pathway inhibition. Treatment of germinal-center B cell–like (GCB) DLBCL cells resulted in an increase in cellular Cdt-1 and accumulation of cells in S-phase, consistent with cells undergoing DNA rereplication. In vivo administration of MLN4924 to mice bearing human xenograft tumors of ABC- and GCB-DLBCL blocked NAE pathway biomarkers and resulted in complete tumor growth inhibition. In primary human tumor models of ABC-DLBCL, MLN4924 treatment resulted in NF-κB pathway inhibition accompanied by tumor regressions. This work describes a novel mechanism of targeted NF-κB pathway modulation in DLBCL and provides strong rationale for clinical development of MLN4924 against NF-κB–dependent lymphomas.

Inhibition of the renin-angiotensin-aldosterone system (RAAS) is a key strategy in treating hypertension and cardiovascular and renal diseases. However, RAAS inhibitors (angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, aldosterone receptor antagonists, and direct renin inhibitors) increase the risk of hyperkalemia (serum potassium >5.5 mmol/L). This review evaluates the effects on serum potassium levels of RAAS inhibitors. Using PubMed, we searched for clinical trials published up to December 2008 assessing the effects on serum potassium levels of angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, aldosterone receptor antagonists, and direct renin inhibitors, alone and in combination, in patients with hypertension, heart failure (HF), or chronic kidney disease (CKD); 39 studies were identified. In patients with hypertension without risk factors for hyperkalemia, the incidence of hyperkalemia with RAAS inhibitor monotherapy is low (< or =2%), whereas rates are higher with dual RAAS inhibition ( approximately 5%). The incidence of hyperkalemia is also increased in patients with HF or CKD (5% to 10%). However, increases in serum potassium levels are small ( approximately 0.1 to 0.3 mmol/L), and rates of study discontinuation due to hyperkalemia are low, even in high-risk patient groups (1% to 5%). Patients with HF or CKD are at greater risk of hyperkalemia with RAAS inhibitors than those without these conditions. However, the absolute changes in serum potassium are generally small and unlikely to be clinically significant. Moreover, these patients are likely to derive benefit from RAAS inhibition. Rather than denying them an effective treatment, electrolyte levels should be closely monitored in these patients.

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

The NF kappa B family is composed by five subunits (p65/RelA, c-Rel, RelB, p105-p50/NF kappa B(1), p100-p52/NF kappa B(2)) and controls the expression of many genes that participate in cell cycle, apoptosis, and other key cellular processes. In a canonical pathway, NF kappa B activation depends on the IKK complex activity, which is formed by three subunits (IKKalpha and IKKbeta and IKKgamma/NEMO). There is an alternative NF kappa B activation pathway that does not require IKKbeta or IKKgamma/NEMO, in which RelB is a major player. We report in a panel of human breast cancer cells that the IKK/NF kappa B system is generally overexpressed in breast cancer cells and there is heterogeneity in expression levels of individual members between different cell lines. Doxorubicin, an anticancer agent used in patients with breast cancer, activated NF kappa B and appeared to be less effective in cells expressing predominantly members of the canonical IKK/NF kappa B. Two NF kappa B inhibitors, bortezomib and NEMO-Binding Domain Inhibitory Peptide, prevented doxorubicin-induced NF kappa B activation and increased doxorubicin antitumor effects in BT-474 cells. Transient down-regulation of members of the canonical pathway (p65, p52, c-Rel and IKKgamma/NEMO) by siRNA in HeLa cells increased doxorubicin cytotoxicity. In contrast, silencing of RelB, a key subunit of the alternative pathway, had no evident effects on doxorubicin cytotoxicity. To conclude, NF kappa B inhibition sensitized cells to doxorubicin, implying directly p65, p52, c-Rel and IKKgamma/NEMO subunits in chemoresistance, but not RelB. These findings suggest that selective inhibition of the canonical NF kappa B pathway is sufficient to improve doxorubicin antitumor effects.

Purpose: To determine whether the combination of the proteasome inhibitor bortezomib and the bcl-2 antisense molecule oblimersen can sensitize human lymphoma to cyclophosphamide. Experimental Design: Cytotoxicity assays were conducted to determine if there was any additive or synergistic interaction between the combinations of bortezomib, oblimersen, and cyclophosphamide using a standard trypan blue exclusion assay. Based on these experiments, in vivo experiments in severe combined immunodeficiency beige mice were done using human lymphoma xenografts in which different schedules were explored. Bcl-2 and oblimersen levels were determined in treated tumors, some of which were resected at the end of the in vivo experiment and evaluated pathologically. Results: The results suggest that the combination of bortezomib and oblimersen seem to interact in at least an additive fashion, and that the addition of cyclophosphamide to this drug combination can markedly improve tumor cell kill. In addition, it seems that these drug combinations may be schedule-dependent, with a requirement for oblimersen pretreatment. Animals treated with the triplet drug combination in a schedule-dependent manner experienced pathologic complete regression of disease, which was not observed in other treatment cohorts. The addition of bortezomib also seemed to increase the levels of intracellular oblimersen, which resulted in a marked reduction in Bcl-2. Histologic studies confirmed marked necrosis and caspase-3 activation only in the cohort receiving all three drugs. Conclusion: The use of Bcl-2-directed therapy and a proteasome inhibitor sensitizes human lymphoma cells to cytotoxic drugs like cyclophosphamide. This combination may offer new opportunities for integrating novel targeted therapies with conventional chemotherapy.

The regulatory agency approvals in the United States and Europe of imatinib mesylate (Gleevec) for patients with bcr/abl-positive chronic myelogenous leukemia, cetuximab (Erbitux) for patients with epidermal growth factor receptor overexpressing metastatic colorectal cancer, the antiangiogenesis agent bevacizumab (Avastin), and the proteasome inhibitor bortezomib (Velcade)—and the considerable public interest in new anticancer drugs that take advantage of specific genetic defects that render the malignant cells more likely to respond to specific treatment—are driving a new era of integrated diagnostics and therapeutics. The recent discovery of a drug response predicting activating mutation in the epidermal growth factor receptor gene for patients with non–small cell lung cancer treated with gefitinib (Iressa) has intensified this interest. In this review, the history of targeted anticancer therapies is highlighted, with focus on the development of molecular diagnostics for hematologic malignancies and the emergence of trastuzumab (Herceptin), an antibody-based targeted therapy for HER-2/neu overexpressing metastatic breast cancer. The potential of pharmacogenomic strategies and the use of high-density genomic microarrays to classify and select therapy for cancer are briefly considered. This review also considers the widely held view that, in the next 5 to 10 years, the clinical application of molecular diagnostics will further revolutionize the drug discovery and development process; customize the selection, dosing, route of administration of existing and new therapeutic agents; and truly personalize medical care for cancer patients. The regulatory approvals of trastuzumab (Herceptin), imatinib mesylate (Gleevec), and cetuximab (Erbitux) for patients with HER-2/neu overexpressing breast cancer, bcr/abl translocation–positive chronic myelogenous leukemia (CML), and epidermal growth factor receptor (EGFR)-overexpressing colorectal cancer, respectively, have headlined the considerable public interest in new targeted anticancer drugs. 1-3 In addition, a large number of new targeted therapies for patients with cancer, including additional small-molecule tyrosine kinase inhibitors, antisense messenger RNA inhibitors, and antibodies are in both early and late stages of clinical development. Many scientists, clinicians, and pharmaceutical company executives believe that during the next 5 to 10 years, the integration of molecular oncology and molecular diagnostics will further revolutionize oncology drug discovery and development; customize the selection, dosing, route of administration of previously approved traditional agents and new therapeutics in clinical trials; and truly personalize medical care for patients with cancer. 4-7

The recent clinical and commercial success of anticancer antibodies, such as rituximab (Rituxan) and trastuzumab (Herceptin) has created great interest in antibody-based therapeutics for hematopoietic malignancies and solid tumors. Given the likely lower toxicity for antibodies versus small molecules, the potential increase in efficacy by conjugation to radioisotopes and other cellular toxins and the ability to characterize the target with clinical laboratory diagnostics to improve the drug's clinical performance, it is anticipated that current and future antibody therapeutics will find substantial roles alone and in combination therapy strategies for the treatment of patients with cancer. It is also likely that conjugation strategies will add new radiolabeled and toxin-linked products to the market to complement the recent approvals of ibritumomab tiuxetan (Zevalin) and gemtuzumab ozogamycin (Mylotarg). However, although there are a large number of agents in both early and later stages of clinical development, only a handful will make it through regulatory approval and become successful products. This review considers the structure of anticancer therapeutic antibodies, the techniques used to reduce their antigenicity, factors that influence efficacy and toxicity, conjugation with isotopes and toxins and antibody target validation.

Anomalous pulmonary vein anatomy is infrequently encountered during reconstructive lung surgery, especially lung transplantation. Complications of pulmonary venous anastomosis carry high morbidity and mortality. We report a case of anomalous pulmonary vein reconstruction with a decellularized porcine small intestinal submucosa-derived extracellular matrix during bilateral lung transplantation in an 18-year-old woman with cystic fibrosis.

Invasive cervical cancer is very highly correlated with the presence of high-risk human papillomavirus (HPV) types 16 and 18. Two viral proteins, E6 and E7, act in concert to subvert growth control of infected cells by inactivating the tumor suppressor proteins, p53 and Rb, respectively. E6 is thought to abrogate p53 function by stimulating its degradation via ubiquitin-mediated proteolysis in a reaction requiring E6AP (E6-Associated Protein). Here we evaluate the in vivo role of E6AP in p53 degradation in normal and HPV-infected cell types using antisense phosphorothioate oligodeoxynucleotides (S-ODNs). This study shows that reduction of E6AP in vivo in high-risk HPV-infected cells leads to an elevation of p53, confirming the function of E6AP predicted by in vitro experiments. Further, we demonstrate that reduction of E6AP in normal cells has no effect on p53 levels, indicative of an E6AP-indpendent mechanism for p53 degradation. These experiments show that inhibition of intermediate proteins in the ubiquitin-mediated proteolysis pathway (ubiquitin-conjugating enzymes or associated recognition proteins) can result in specific inhibition of substrate degradation. We propose that modulation of p53 levels by elimination of E6AP function may have therapeutic potential for cervical cancer.

The ubiquitin proteasome system (UPS) is a process present in all mammalian cells where proteins that are no longer needed or have been misfolded are labeled by ubiquitin which targets them for destruction. A cascade of ubiquitination enzymes is involved in the specific labeling of target proteins with ubiquitin. The labeled proteins are recognized by chaperone proteins that direct them to the 26S proteasome—a large multiprotein complex which systematically degrades the unneeded/misfolded proteins into smaller peptide fragments. Cancer cells heavily rely on this pathway to maintain protein homeostasis and selective inhibition of UPS enzymes causes a buildup of proteins inducing apoptotic cell death. The proteasome inhibitors bortezomib and carfilzomib have been clinically approved to treat hematological malignancies. Other compounds targeting various enzymes in this pathway are in earlier stages of development and will represent an important new class of cancer treatments.

The p27 mammalian cell cycle protein is an inhibitor of cyclin-dependent kinases. Both in vivo and in vitro, p27 was found to be degraded by the ubiquitin-proteasome pathway. The human ubiquitin-conjugating enzymes Ubc2 and Ubc3 were specifically involved in the ubiquitination of p27. Compared with proliferating cells, quiescent cells exhibited a smaller amount of p27 ubiquitinating activity, which accounted for the marked increase of p27 half-life measured in these cells. Thus, the abundance of p27 in cells is regulated by degradation. The specific proteolysis of p27 may represent a mechanism for regulating the activity of cyclin-dependent kinases.

Acute myeloid leukemia (AML) therapy has remained relatively unchanged for more than 40 years with the majority of patients not achieving long-term remission when treated with currently available agents. Novel strategies are urgently needed to improve outcomes. The constitutive dysregulation of protein synthesis/turnover contributes to disease progression and drug resistance in many forms of cancer including AML. p97 (VCP) is a master regulator of protein turnover that has been implicated in oncogenesis and malignant pathogenesis. CB-5083 is a first-in-class selective and potent orally available inhibitor of p97 that in currently being evaluated in phase I clinical trials in patients with multiple myeloma and advanced solid tumors. To assess the potential benefit of p97 inhibition as a novel approach for AML therapy, we investigated the efficacy, pharmacodynamics (PD), and pharmacokinetics (PK) of CB-5083 in a panel of human AML cell lines with diverse genetic backgrounds, primary AML specimens from both newly diagnosed and relapsed/refractory patients, and xenograft mouse models of AML. In vitro treatment with CB-5083 potently diminished the viability of AML cell lines (n = 7) and primary CD34+ blasts obtained from patients (n = 10) with IC50s significantly below 1 µM (range 200 - 700 nM) in all lines and specimens evaluated to date. Diminished viability was associated with reduced clonogenic survival and increased apoptosis in AML cell lines and primary blasts. In contrast to many conventional and experimental drugs that are less active against primary AML cells than established AML cell lines, primary cells exhibited sensitivity to CB-5083 that was similar to cell lines. Additionally, CB-5083 was highly active in 3 different cell line models of cytarabine resistance and primary cells from refractory AML patients. This suggests that CB-5083 may be effective for patients who are relapsed/refractory to conventional therapy. In vitro PD analyses demonstrated that CB-5083 rapidly triggered the accumulation of ubiquitin-conjugated proteins, activated the unfolded protein response (UPR), disrupted STAT5 signaling, reduced levels of key STAT5 targets including BCL-xL and PIM-2, and induced apoptosis. The pro-apoptotic effects of CB-5083 were associated with activation of the endoplasmic reticulum (ER) resident initiator caspase-4 and induction of the BH3-only protein NOXA, which has been previously demonstrated to be an important mediator of cell death induced by other agents that disrupt protein homeostasis. RNA sequencing (RNASeq) gene ontology (GO) analyses of MV4-11 and MOLM-13 AML cells following treatment with CB-5083 demonstrated that short-term treatment (6h) caused significant increases in multiple regulators of the unfolded protein response, protein biosynthesis, and other ubiquitin-related pathways (p Disclosures LE Moigne: Cleave Biosciences: Employment. Rolfe:Cleave Biosciences: Employment. Djakovic:Cleave Biosciences: Employment. Anderson:Cleave Biosciences: Employment. Wustrow:Cleave Biosciences: Employment. Zhou:Cleave Biosciences: Employment. Wong:Cleave Biosciences: Employment. Sekeres:TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Carew:Boehringer Ingelheim: Research Funding.

Treatment of Syrian hamster kidney cells with the demethylating agent 5-aza-2'-deoxycytidine (azadC) increased both the frequency and the rate of gene amplification appreciably. AzadC caused substantial DNA demethylation, which is likely to be responsible. The magnitude of the increases depended on the concentrations of both azadC and the drug used for selection. A transient stress response is not responsible since the increases were not dependent on cytotoxicity and were still apparent after several weeks. We discuss mechanisms by which azadC treatment may induce amplification by rendering DNA more prone to this process or by increasing the transcription of genes whose protein products stimulate amplification.

E3 ubiquitin ligases regulate many dynamic cellular processes important for cancer cell survival. Together with ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s), E3s catalyze the ubiquitination of numerous protein substrates that are subsequently targeted to the 26S proteasome for degradation. The clinical success of the proteasome inhibitor bortezomib has encouraged the evaluation of other components of the ubiquitin proteasome system for pharmaceutical intervention. Targeting specific E3s is particularly attractive because there is the potential to selectively block the degradation of certain cellular proteins and possibly avoid unwanted effects on other proteins. The cullin-RING ubiquitin E3 ligases (CRLs) represent the largest subfamily of E3s. The requirement that CRLs be activated by NEDD8 modification on the cullin protein offers an “achilles heel” for modulating this entire subfamily. NEDD8-activating enzyme (NAE) catalyzes the first step in the NEDD8 pathway and as such controls the activity of CRLs. In this article, we describe the role of the NEDD8 pathway in activating CRLs and discuss the preclinical findings with a first-in-class NAE inhibitor that is currently in phase I clinical trials for both solid tumor and hematological malignancies. In addition, we speculate where NAE inhibitors may find clinical utility.

The ubiquitin-proteasome system (UPS) sustains cancer cell viability by alleviating proteotoxic stress caused by an imbalance of protein synthesis and degradation. While proteasome inhibitors and immunomodulators (IMiDs) are approved for the treatment of hematological malignancies, relapse following treatment is common. Additionally, these agents have poor efficacy in solid tumors, prompting the need for discovery of novel drug targets within the UPS which might provide a more profound anti-tumor effect. The homo-hexameric AAA-ATPase VCP/p97 functions by converting chemical energy into mechanical force to extract proteins from cell membranes and protein complexes. p97 is directly involved in several facets of protein homeostasis, including ubiquitin-dependent protein degradation, endoplasmic reticulum-associated degradation (ERAD), and autophagy. Therefore p97 inhibition could provide a novel and more potent approach than proteasome inhibitors to exploit cancer cell addiction to protein homeostatic mechanisms. Through a targeted medicinal chemistry effort we have discovered CB-5083, a novel small molecule ATP-competitive inhibitor of p97 ATPase activity with nanomolar enzymatic and cellular potency. Treatment of cancer cells with CB-5083 causes a dramatic increase in poly-ubiquitinated proteins and an accumulation of substrates of the UPS and ERAD. The blockade of ERAD causes induction of an irresolvable unfolded protein response (UPR), leading to caspase cleavage and apoptosis in a manner that is distinct from proteasome inhibitors. Indeed, ER-related stress and ERAD genes were the most significantly upregulated gene ontologies uncovered from transcriptome profiling. Additionally, knockdown of DR5 protects against CB-5083 mediated cell death, suggesting that death receptor signaling plays a key role in the apoptotic response caused by inhibition of p97 function. In animal models, our p97 inhibitor is orally bio-available and causes rapid and sustained accumulation of poly-ubiquitin and markers of the UPR and apoptosis. Furthermore, strong anti-cancer effects were observed in solid tumor and hematological murine models. We have developed a clinical-grade multiparametric flow cytometry assay to measure the accumulation of lysine-48 linked poly-ubiquitin and cPARP from human whole blood and circulating multiple myeloma cells, providing a way to monitor changes to the UPS and biological activity in real time. CB-5083 is currently being tested in ongoing phase I clinical trials for relapsed/refractory multiple myeloma and solid tumor patients. Taken together, these results indicate that CB-5083 has great promise as a novel therapeutic agent for the treatment of cancer. Citation Format: Daniel J. Anderson, Ronan Le Moigne, Stevan Djakovic, Brajesh Kumar, Julie Rice, Steve Wong, Jinhai Wang, Bing Yao, Eduardo Valle, MK Menon, Szerenke Kiss von Soly, Antonett Madriaga, Ferdie Sorlano, Mike Longhi, Alessandra Cesano, Han-Jie Zhou, David Wustrow, Mark Rolfe. Inhibition of the AAA-ATPase p97 with the first in class inhibitor CB-5083 as a novel approach to treat cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr DDT02-01. doi:10.1158/1538-7445.AM2015-DDT02-01

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 aim of this study was to validate quantitative metabolic response of tumors to a treatment measured by longitudinal 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) micro positron emission tomography (microPET) as a robust tool for preclinical evaluation of new anticancer agents.Severe combined immunodeficiency mice with CWR22 xenografts were intravenously treated with bortezomib (Velcade) at 0.8 mg/kg on days 0, 3, 7, 10, and 14 and imaged with FDG microPET before, during and after treatment. Quantitative indices of tumor FDG uptake were developed.FDG microPET images successfully revealed the gradual reduction of tumor FDG uptake on day 4 onward despite no absolute tumor shrinkage. The standardized uptake values of FDG in tumors was reduced to 43% of the baseline values. Using the total tumor FDG uptake as the viable tumor burden, we found 86% tumor inhibition, compared to a 55% tumor growth inhibition in tumor volume measurement.FDG microPET imaging can provide an additional dimension of the efficacy of anticancer therapies that may otherwise be underestimated by tumor volume measurement.

The cellular and molecular effects of the proteasome inhibitor bortezomib on breast cancer cells are as yet poorly characterized. Here, in a panel of six breast cancer cell lines, bortezomib reduced viability in a concentration-dependent, time-dependent, and cell line–dependent manner. Proteasome activity was relatively high in two of the three more resistant cell lines. No relationship was observed between bortezomib effects on cell viability and expression/phosphorylation of HER-2, epidermal growth factor receptor (EGFR), AKT, or extracellular signal-regulated kinase 1/2 (ERK1/2). Molecular effects of bortezomib were further studied in SK-BR-3 and BT-474 cells because they share expression of EGFR and overexpression of HER-2 while, in contrast, SK-BR-3 cells were 200-fold more sensitive to this agent. Proteasome activity was inhibited to a similar extent in the two cell lines, and known proteasome substrates accumulated similarly. In SK-BR-3 cells, a marked inhibition of EGFR, HER-2, and AKT phosphorylation was observed at a clinically relevant concentration of bortezomib. In contrast, phosphorylation of Raf/mitogen-activated protein kinase kinase 1/2 (MEK 1/2)/ERK1/2 increased by bortezomib. In BT-474 cells, the effects were much less pronounced. Treatment of SK-BR-3 cells with bortezomib combined with pharmacologic inhibitors of EGFR, phosphatidylinositol 3′-kinase, or MEK resulted in modest or no enhancement of the effects on cell viability. Collectively, these results show that bortezomib has differential cellular and molecular effects in human breast cancer cells. The bortezomib-observed effects on signaling transduction molecules might be relevant to help to design mechanistic-based combination treatments. [Mol Cancer Ther 2006;5(3):665–75]

The recent clinical and commercial success of anti-cancer antibodies such as rituximab, trastuzumab, cetuximab and bevacizumab has continued to foster great interest in antibody-based therapeutics for the treatment of both hematopoietic malignancies and solid tumors. Given the likely lower toxicity for antibodies which, in contrast with traditional cytotoxic small molecule drugs, target tumor cells and have a lower impact on non-malignant by-stander organs, the potential increases in efficacy associated with conjugation to radioisotopes and other cellular toxins and the ability to characterize the target with clinical laboratory diagnostics to improve the drugs clinical performance, it is anticipated that current and future antibody therapeutics will find substantial roles alone and in combination therapy strategies for the treatment of patients with cancer. A significant number of cell surface proteins, glycoproteins, receptors, enzymes and peptides have been discovered that have become targets for the treatment of advanced hormone-refractory prostate cancer. A variety of naked antibodies and antibody conjugates have currently progressed through preclinical development and are in early or more advanced stages of clinical development. Clinicians, scientists and prostate cancer patients are all keenly interested to learn whether these agents when administered alone or in combination with other hormonal-based and cytotoxic therapies will show lasting benefit for sufferers of this common disease.

Hematological malignancies such as multiple myeloma (MM) have an increased reliance on the ubiquitin proteasome system (UPS) presumably as a consequence of their high protein synthetic and secretory burden. Chemical agents that target the proteasome, such as bortezomib and carfilzomib, have been successful in treating multiple myeloma; however patients treated with these drugs ultimately relapse. The AAA-ATPase p97/VCP (p97) facilitates ATP-dependent extraction and degradation of ubiquitinated proteins destined for proteasomal elimination. In addition to ubiquitin-dependent protein degradation, p97 is also closely involved in other aspects of protein homeostasis, including endoplasmic reticulum-associated degradation (ERAD) and autophagy. Pharmacologic inhibition of p97 provides a compelling therapeutic approach for hematological malignancies that rely on tight regulation of protein homeostasis as a component of their survival. CB-5083 is a novel small molecule inhibitor of p97 ATPase activity with nanomolar enzymatic and cellular potency. Treatment of cancer cells with CB-5083 causes a dramatic increase in poly-ubiquitinated proteins as well as an accumulation of substrates of the UPS and ERAD. CB-5083 causes a profound induction of the unfolded protein response (UPR) with consequent activation of the DR5 death receptor, caspase 8, caspase 3/7 and ultimately cell death. Induction of the UPR occurs to a greater magnitude with CB-5083 when compared to the proteasome inhibitor, bortezomib, suggesting the potential for increased efficacy in cancers with sensitivity to UPR-mediated cell death. In addition, activation of apoptosis and cell death occur more rapidly with CB-5083 than with bortezomib. Sequencing of cell lines made resistant to CB-5083 reveals missense mutations mapping to the D2 ATPase site in p97, supporting on-target association with cytotoxicity. In an expanded panel of MM cell lines there is no correlation between the cytotoxic sensitivity to CB-5083 and the cytotoxic sensitivity to proteasome inhibitors, suggesting differential mechanisms of cytotoxicity and potential activity of CB-5083 in proteasome inhibitor resistant settings. Compared to myeloma cell lines, CB-5083 has reduced cytotoxic potency in immortalized stromal cell lines and in patient-derived CD138-negative bone marrow mononuclear cells. Furthermore, unlike the reduced potency demonstrated by carfilzomib in the context of MM cell-bone marrow stromal cell (BMSC) interactions, the cyto-reductive potential of CB-5083 is unaffected in co-cultures of MM cells with patient-derived BMSCs or immortalized BMSCs from healthy donors. In vivo, CB-5083 is orally bioavailable, shows a pharmacodynamic effect in tumor tissue (as measured by poly-ubiquitin accumulation) and demonstrates robust anti-tumor activity across several MM models. CB-5083 treatment of mice bearing subcutaneous xenografts leads to tumor stasis and regression in RPMI8226 and AMO1 MM models, respectively. In advanced models of disseminated, ortho-metastatic disease, intermittent oral administration of CB-5083 demonstrates significant inhibition of myeloma burden and improves survival, with an overall efficacy profile that compares favorably to that of clinically approved proteasome inhibitors. Furthermore, in the Vk*Myc genetically engineered mouse model of MM, treatment with CB-5083 results in a significant reduction in M-spike by 55%. Combination treatment of mice bearing the RPMI8226 subcutaneous xenograft model with CB-5083, dexamethasone and lenalidomide results in tumor regression. Taken together, these data demonstrate that CB-5083 is a potent and selective inhibitor of the p97 ATPase with robust activity in vitro and in vivo in numerous MM models and strongly support clinical evaluation. Based on these observations, a phase 1 dose-escalation trial has recently been initiated and is currently underway in patients with relapsed/refractory multiple myeloma. Disclosures Anderson: Cleave Biosciences: Employment. Le Moigne:Cleave Biosciences: Employment. Djakovic:Cleave Biosciences: Employment. Rice:Cleave Biosciences: Employment. Wong:Cleave Biosciences: Employment. Kumar:Cleave Biosciences: Employment. Valle:Cleave Biosciences: Employment. Menon:Cleave Biosciences: Employment. Kiss von Soly:Cleave Biosciences: Employment. Wang:Cleave Biosciences: Employment. Yao:Cleave Biosciences: Employment. Soriano:Cleave Biosciences: Employment. Bergsagel:ONYX: Consultancy; Janssen: Consultancy; BMS: Consultancy; Novartis: Research Funding. Yakes:Cleave Biosciences: Employment. Zhou:Cleave Biosciences: Employment. Wustrow:Cleave Biosciences: Employment. Rolfe:Cleave Biosciences: Employment.

The splicing factor SPF45 (RBM17) is frequently overexpressed in many solid tumors, and stable expression in HeLa cells confers resistance to doxorubicin and vincristine. In this study, we characterized stable transfectants of A2780 ovarian carcinoma cells. In a 3-day cytotoxicity assay, human SPF45 overexpression conferred 3- to 21-fold resistance to carboplatin, vinorelbine, doxorubicin, etoposide, mitoxantrone, and vincristine. In addition, resistance to gemcitabine and pemetrexed was observed at the highest drug concentrations tested. Knockdown of SPF45 in parental A2780 cells using a hammerhead ribozyme sensitized A2780 cells to etoposide by ∼5-fold relative to a catalytically inactive ribozyme control and untransfected cells, suggesting a role for SPF45 in intrinsic resistance to some drugs. A2780-SPF45 cells accumulated similar levels of doxorubicin as vector-transfected and parental A2780 cells, indicating that drug resistance is not due to differences in drug accumulation. Efforts to identify small molecules that could block SPF45-mediated drug resistance revealed that the selective estrogen receptor (ER) modulators tamoxifen and LY117018 (a raloxifene analogue) partially reversed SPF45-mediated drug resistance to mitoxantrone in A2780-SPF45 cells from 21-fold to 8- and 5-fold, respectively, but did not significantly affect the mitoxantrone sensitivity of vector control cells. Quantitative PCR showed that ERβ but not ERα was expressed in A2780 transfectants. Coimmunoprecipitation experiments suggest that SPF45 and ERβ physically interact in vivo. Thus, SPF45-mediated drug resistance in A2780 cells may result in part from effects of SPF45 on the transcription or alternate splicing of ERβ-regulated genes.

Ubiquitin-mediated proteolysis is involved in the turnover of many short-lived regulatory proteins. This pathway leads to the covalent attachment of one or more multiubiquitin chains to target substrates which are then degraded by the 26S multicatalytic proteasome complex. Multiple classes of regulatory enzymes have been identified that mediate either ubiquitin conjugation or ubiquitin deconjugation from target substrates. Timed destruction of cellular regulators by the ubiquitin-proteasome pathway plays a critical role in ensuring normal cellular processes. This review provides multiple examples of key growth regulatory proteins whose levels are regulated by ubiquitin-mediated proteolysis. Pharmacological intervention which alters the half-lives of these cellular proteins may have wide therapeutic potential. Specifically, prevention of p53 ubiquitination (and subsequent degradation) in human papilloma virus positive tumors, and perhaps all tumors retaining wild-type p53 but lacking the retinoblastoma gene function, should lead to programmed cell death. Specific inhibitors of p27 and cyclin B ubiquitination are predicted to be potent antiproliferative agents. Inhibitors of IkappaB ubiquitination should prevent NFkappaB activation and may have utility in a variety of autoimmune and inflammatory conditions. Finally, we present a case for deubiquitination enzymes as novel, potential drug targets.

Background: The ubiquitin-proteasome system (UPS) sustains cancer cell viability by alleviating proteotoxic stress caused by an imbalance of protein synthesis and degradation. Bortezomib and carfilzomib are 26S proteasome inhibitors approved for the treatment of multiple myeloma. Unfortunately, these agents have poor efficacy in solid tumors, prompting the need for discovery of novel drugs targeting other enzymes within the UPS. The AAA-ATPase p97/VCP functions by converting chemical energy into mechanical force and is closely involved in several facets of protein homeostasis. These include ubiquitin-dependent protein degradation, endoplasmic reticulum-associated degradation (ERAD), and autophagy. p97 inhibition will provide a novel approach to exploit cancer cell addiction to protein homeostatic mechanisms. Results: Through a targeted medicinal chemistry effort, we have discovered novel small molecule inhibitors of p97 ATPase activity with nanomolar enzymatic and cellular potency. Sequencing of human cancer cell lines that have developed resistance to our p97 inhibitors has revealed mutations in p97, strongly suggesting on-target cellular activity. Treatment of normal and cancer cells with this class of p97 inhibitors causes a dramatic increase in poly-ubiquitinated proteins and an accumulation of substrates of the UPS and ERAD. These inhibitors also disrupt other specific p97 cellular functions including macroautophagy and receptor endocytosis. Inhibitor treatment ultimately leads to a blockade of NF-kB signaling and a decrease in downstream survival factors followed by the induction of caspase cleavage and apoptosis. In animal models, our p97 inhibitors are orally bio-available and cause rapid accumulation of poly-ubiquitin in tumor xenografts at levels that exceed the accumulation seen with bortezomib. Furthermore, significant tumor growth inhibition was observed in a number of solid tumor and hematological models. Conclusion: Together, these data provide novel insights into the role of p97 in cancer cell growth and the mechanism of death due to p97 inhibition. Animal model data suggests promise for our inhibitors as therapeutic agents for patients with hematological and solid tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C188. Citation Format: Stevan N. Djakovic, Daniel J. Anderson, Szerenke Kiss von Soly, Ronan Le Moigne, Julie Rice, Mark Rolfe, Ferdie Soriano, Eduardo Valle, Jinhai Wang, Steve Wong, David Wustrow, F. Michael Yakes, Bing Yao, Han-Jie Zhou. Novel small molecule inhibitors of p97 disrupt cellular protein homeostasis and demonstrate anti-tumor activity in solid and hematological models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C188.

Abstract 2709 Poster Board II-685 Introduction: The ubiquitin-proteasome system processes the majority of cellular proteins and is the principal manner by which cells regulate protein homeostasis. The successful development of bortezomib for multiple myeloma and previously treated mantle cell lymphoma has validated the proteasome as a therapeutic target for hematological malignancies. MLN9708 was identified in screens for a proteasome inhibitor with greater antitumor activity than bortezomib in preclinical xenograft models. MLN9708 immediately hydrolyzes to MLN2238, the biologically active form, upon exposure to aqueous solutions or plasma. MLN2238 was used for all preclinical studies described below. Results: MLN2238 inhibited the 20S proteasome b5 site (IC50 of 3.4 nM), an engineered proteasome substrate (4x ubiquitin-luciferase reporter) and blocked TNFa-induced activation of the NFkB pathway. Cell viability studies confirmed that MLN2238 has potent activity against both myeloma and lymphoma cell lines. The proteasome dissociation half life of MLN2238 was determined to be approximately 6-fold faster than bortezomib, consistent with data generated from Proteasome-Glo wash out experiments where proteasome activity recovered more quickly in MLN2238-treated cells compared to bortezomib. In immunocompromised mice MLN2238 achieved exposures that resulted in significant blood and tumor proteasome inhibition and had increased plasma and tumor exposure compared to bortezomib (when dosed at MTD). In Sprague-Dawley rats, MLN2238 had improved plasma exposure, lower plasma clearance, higher blood Vdss, better oral F% and higher plasma protein binding than bortezomib. In WSU-DLCL2 xenografts, a model of diffuse large B cell lymphoma, greater antitumor activity was seen in mice treated with IV or SC doses of MLN2238 compared to bortezomib. For example, MLN2238 dosed SC QD at its MTD resulted in a T/C of 0.29 compared to bortezomib dosed SC QD at its MTD which resulted in a T/C of 0.79. Pharmacodynamic (PD) responses (in mice) were assessed by evaluating tumor 20S b5 site-specific activity and expression levels of GADD34, an unfolded protein response (UPR) pathway gene shown to be upregulated in response to proteasome inhibition. Consistent with the efficacy difference between MLN2238 and bortezomib in WSU-DLCL2 xenografts, MLN2238 demonstrated an improved PD response compared to bortezomib, showing higher levels of tumor proteasome inhibition and pathway marker elevation. MLN2238 and bortezomib were then evaluated for their ability to reduce tumor burden and improve overall survival in a disseminated model of lymphoma. Tumor burden was tracked over time via bioluminescent scans in NOD-SCID mice inoculated with OCI-Ly7-luciferase cells. The strongest antitumor response was seen in mice treated with MLN2238 SC QD at its MTD, and this dosing regimen also significantly prolonged overall survival compared to vehicle treated controls (median survival was 54 vs. 33 days). Weaker antitumor responses were seen following treatment with bortezomib (either at its SC QD MTD or weekly IV MTD) and these dose regimens did not significantly prolong survival in this study. Conclusions: MLN2238 is a potent, reversible and orally bioavailable proteasome inhibitor with improved pharmacokinetics, pharmacodynamics and antitumor activity in preclinical xenograft models compared to bortezomib. MLN9708 is currently in clinical development for a variety of oncology indications. Disclosures: Kupperman: Milllennium: Employment. Lee:Milllennium: Employment, Equity Ownership. Cao:Milllennium: Employment, Equity Ownership. Bannerman:Milllennium: Employment. Fitzgerald:Millennium Pharmaceuticals: Employment. Berger:Millennium Pharmaceuticals: Employment. Yu:Millennium Pharmaceuticals: Employment. Zhang:Millennium Pharmaceuticals: Employment. Hales:Millennium Pharmaceuticals: Employment. Bruzzese:Millennium Pharmaceuticals: Employment. Liu:Millennium Pharmaceuticals: Employment. Blank:Millennium Pharmaceuticals: Employment. Garcia:Millennium Pharmaceuticals: Employment. Tsu:Millennium Pharmaceuticals: Employment. Dick:Millennium Pharmaceuticals: Employment. Fleming:Millennium Pharmaceuticals: Employment. Yu:Millennium Pharmaceuticals: Employment. Manfredi:Millennium Pharmaceuticals: Employment. Rolfe:Millennium Pharmaceuticals: Employment. Bolen:Millennium Pharmaceuticals: Employment.

Two UV-inducible proteins have been detected in the yeast, Saccharomyces cerevisiae. The proteins have molecular weights of 78,000 Daltons and 23,000 Daltons. This induction is specific for UV-irradiation as exposure to X-rays, mitomycin C and heat shock does not result in the synthesis of the proteins. The larger (78 kD) protein is induced in various rad strains and in a rho degree cir degree strain. Attempts are being made to isolate the genes coding for these inducible proteins.