Global signal peptide profiling reveals principles of selective Sec61 inhibition.

Cotransins target the Sec61 translocon and inhibit the biogenesis of an undefined subset of secretory and membrane proteins. Remarkably, cotransin inhibition depends on the unique signal peptide (SP) of each Sec61 client, which is required for cotranslational translocation into the endoplasmic reticulum. It remains unknown how an SP's amino acid sequence and biophysical properties confer sensitivity to structurally distinct cotransins. Here we describe a fluorescence-based, pooled-cell screening platform to interrogate nearly all human SPs in parallel. We profiled two cotransins with distinct effects on cancer cells and discovered a small subset of SPs, including the oncoprotein human epidermal growth factor receptor 3 (HER3), with increased sensitivity to the more selective cotransin, KZR-9873. By comparing divergent mouse and human orthologs, we unveiled a position-dependent effect of arginine on SP sensitivity. Our multiplexed profiling platform reveals how cotransins can exploit subtle sequence differences to achieve SP discrimination.

Zetomipzomib (KZR-616) attenuates lupus in mice via modulation of innate and adaptive immune responses.

Zetomipzomib (KZR-616) is a selective inhibitor of the immunoproteasome currently undergoing clinical investigation in autoimmune disorders. Here, we characterized KZR-616 in vitro and in vivo using multiplexed cytokine analysis, lymphocyte activation and differentiation, and differential gene expression analysis. KZR-616 blocked production of >30 pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs), polarization of T helper (Th) cells, and formation of plasmablasts. In the NZB/W F1 mouse model of lupus nephritis (LN), KZR-616 treatment resulted in complete resolution of proteinuria that was maintained at least 8 weeks after the cessation of dosing and was mediated in part by alterations in T and B cell activation, including reduced numbers of short and long-lived plasma cells. Gene expression analysis of human PBMCs and tissues from diseased mice revealed a consistent and broad response focused on inhibition of T, B, and plasma cell function and the Type I interferon pathway and promotion of hematopoietic cell lineages and tissue remodeling. In healthy volunteers, KZR-616 administration resulted in selective inhibition of the immunoproteasome and blockade of cytokine production following ex vivo stimulation. These data support the ongoing development of KZR-616 in autoimmune disorders such as systemic lupus erythematosus (SLE)/LN.

Inhibition of the Sec61 translocon overcomes cytokine-induced glucocorticoid resistance in T-cell acute lymphoblastic leukaemia.

Glucocorticoid (GC) resistance is a poor prognostic factor in T-cell acute lymphoblastic leukaemia (T-ALL). Interleukin-7 (IL-7) mediates GC resistance via GC-induced upregulation of IL-7 receptor (IL-7R) expression, leading to increased pro-survival signalling. IL-7R reaches the cell surface via the secretory pathway, so we hypothesized that inhibiting the translocation of IL-7R into the secretory pathway would overcome GC resistance. Sec61 is an endoplasmic reticulum (ER) channel that is required for insertion of polypeptides into the ER. Here, we demonstrate that KZR-445, a novel inhibitor of Sec61, potently attenuates the dexamethasone (DEX)-induced increase in cell surface IL-7R and overcomes IL-7-induced DEX resistance.

Proteasome Inhibitors and Their Pharmacokinetics, Pharmacodynamics, and Metabolism.

The proteasome is responsible for mediating intracellular protein degradation and regulating cellular function with impact on tumor and immune effector cell biology. The proteasome is found predominantly in two forms, the constitutive proteasome and the immunoproteasome. It has been validated as a therapeutic drug target through regulatory approval with 2 distinct chemical classes of small molecular inhibitors (boronic acid derivatives and peptide epoxyketones), including 3 compounds, bortezomib (VELCADE), carfilzomib (KYPROLIS), and ixazomib (NINLARO), for use in the treatment of the plasma cell neoplasm, multiple myeloma. Additionally, a selective inhibitor of immunoproteasome (KZR-616) is being developed for the treatment of autoimmune diseases. Here, we compare and contrast the pharmacokinetics (PK), pharmacodynamics (PD), and metabolism of these 2 classes of compounds in preclinical models and clinical studies. The distinct metabolism of peptide epoxyketones, which is primarily mediated by microsomal epoxide hydrolase, is highlighted and postulated as a favorable property for the development of this class of compound in chronic conditions.

Discovery and Early Clinical Development of Selective Immunoproteasome Inhibitors.

Inhibitors of the proteolytic activity of the 20S proteasome have transformed the treatment of multiple B-cell malignancies. These agents have also been employed with success in the treatment of patients with autoimmune diseases and immune-mediated disorders. However, new agents are needed to fully unlock the potential of proteasome inhibitors as immunomodulatory drugs. The discovery that selective inhibitors of the immunoproteasome possess broad anti-inflammatory activity in preclinical models has led to the progression of multiple compounds to clinical trials. This review focuses on the anti-inflammatory potential of immunoproteasome inhibition and the early development of KZR-616, the first selective inhibitor of the immunoproteasome to reach clinical testing.

Co-inhibition of immunoproteasome subunits LMP2 and LMP7 is required to block autoimmunity.

Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine-inducible proteasome variant comprising the proteolytic subunits LMP2 (ß1i), MECL-1 (ß2i), and LMP7 (ß5i). Targeting the immunoproteasome in pre-clinical models of autoimmune diseases with the epoxyketone inhibitor ONX 0914 has proven to be effective. ONX 0914 was previously described as a selective LMP7 inhibitor. Here, we show that PRN1126, developed as an exclusively LMP7-specific inhibitor, has limited effects on IL-6 secretion, experimental colitis, and experimental autoimmune encephalomyelitis (EAE). We demonstrate that prolonged exposure of cells with ONX 0914 leads to inhibition of both LMP7 and LMP2. Co-inhibition of LMP7 and LMP2 with PRN1126 and LMP2 inhibitors LU-001i or ML604440 impairs MHC class I cell surface expression, IL-6 secretion, and differentiation of naïve T helper cells to T helper 17 cells, and strongly ameliorates disease in experimental colitis and EAE. Hence, co-inhibition of LMP2 and LMP7 appears to be synergistic and advantageous for the treatment of autoimmune diseases.

Clinical activity of carfilzomib correlates with inhibition of multiple proteasome subunits: application of a novel pharmacodynamic assay.

While proteasome inhibition is a validated therapeutic approach for multiple myeloma (MM), inhibition of individual constitutive proteasome (c20S) and immunoproteasome (i20S) subunits has not been fully explored owing to a lack of effective tools. We utilized the novel proteasome constitutive/immunoproteasome subunit enzyme-linked immunosorbent (ProCISE) assay to quantify proteasome subunit occupancy in samples from five phase I/II and II trials before and after treatment with the proteasome inhibitor carfilzomib. Following the first carfilzomib dose (15-56 mg/m(2) ), dose-dependent inhibition of c20S and i20S chymotrypsin-like active sites was observed [whole blood: ≥67%; peripheral blood mononuclear cells (PBMCs): ≥75%]. A similar inhibition profile was observed in bone marrow-derived CD138(+) tumour cells. Carfilzomib-induced proteasome inhibition was durable, with minimal recovery in PBMCs after 24 h but near-complete recovery between cycles. Importantly, the ProCISE assay can be used to quantify occupancy of individual c20S and i20S subunits. We observed a relationship between MM patient response (n = 29), carfilzomib dose and occupancy of multiple i20S subunits, where greater occupancy was associated with an increased likelihood of achieving a clinical response at higher doses. ProCISE represents a new tool for measuring proteasome inhibitor activity in clinical trials and relating drug action to patient outcomes.

Inhibition of the Immunoproteasome Subunit LMP7 with ONX 0914 Ameliorates Graft-versus-Host Disease in an MHC-Matched Minor Histocompatibility Antigen-Disparate Murine Model.

In the current study we evaluated the effects of immunoproteasome inhibition using ONX 0914 (formerly PR-957) to ameliorate graft-versus-host disease (GVHD). ONX 0914, an LMP7-selective epoxyketone inhibitor of the immunoproteasome, has been shown to reduce cytokine production in activated monocytes and T cells and attenuate disease progression in mouse models of rheumatoid arthritis, colitis, systemic lupus erythematosus, and, more recently, encephalomyelitis. Inhibition of LMP7 with ONX 0914 in the B10.BR→CBA MHC-matched/minor histocompatibility antigen (miHA)-disparate murine blood and marrow transplant (BMT) model caused a modest but significant improvement in the survival of mice experiencing GVHD. Concomitant with these results, in vitro mixed lymphocyte cultures revealed that stimulator splenocytes, but not responder T cells, treated with ONX 0914 resulted in decreased IFN-γ production by allogeneic T cells in both MHC-disparate (B10.BR anti-B6) and miHA-mismatched (B10.BR anti-CBA) settings. In addition, a reduction in the expression of the MHC class I-restricted SIINFEKL peptide was observed in splenocytes from transgenic C57BL/6-Tg(CAG-OVA)916Jen/J mice exposed to ONX 0914. Taken together, these data support that LMP7 inhibition in the context of BMT modulates allogeneic responses by decreasing endogenous miHA presentation and that the consequential reduction in allogeneic stimulation and cytokine production reduces GVHD development.

Assessment of cytokine-modulated proteasome activity.

This chapter presents two methods for assessment of proteasome function. The first is a modification of the standard fluorogenic peptide cleavage assay which takes into account the effect of ATP on proteasome activity. This method is described in both its macro and high throughput micro-assay forms. The second is the Proteasome Constitutive Immuno-Subunit (active site) ELISA or ProCISE method. ProCISE is a modification of active site directed probe analysis and allows for convenient differentiation between active constitutive and immuno-subunits. While the utility of measuring proteasome activity and its relationship to cytokine action and inflammation are clear, the assessment and interpretation is not always straightforward. Therefore, we also discuss the pitfalls of the standard fluorogenic assay, particularly in the interpretation of results obtained, and the advantages of the newer, ProCISE assay.

Carfilzomib and oprozomib synergize with histone deacetylase inhibitors in head and neck squamous cell carcinoma models of acquired resistance to proteasome inhibitors.

Acquired resistance to proteasome inhibitors represents a considerable impediment to their effective clinical application. Carfilzomib and its orally bioavailable structural analog oprozomib are second-generation, highly-selective, proteasome inhibitors. However, the mechanisms of acquired resistance to carfilzomib and oprozomib are incompletely understood, and effective strategies for overcoming this resistance are needed. Here, we developed models of acquired resistance to carfilzomib in two head and neck squamous cell carcinoma cell lines, UMSCC-1 and Cal33, through gradual exposure to increasing drug concentrations. The resistant lines R-UMSCC-1 and R-Cal33 demonstrated 205- and 64-fold resistance, respectively, relative to the parental lines. Similarly, a high level of cross-resistance to oprozomib, as well as paclitaxel, was observed, whereas only moderate resistance to bortezomib (8- to 29-fold), and low level resistance to cisplatin (1.5- to 5-fold) was seen. Synergistic induction of apoptosis signaling and cell death, and inhibition of colony formation followed co-treatment of acquired resistance models with carfilzomib and the histone deacetylase inhibitor (HDACi) vorinostat. Synergism was also seen with other combinations, including oprozomib plus vorinostat, or carfilzomib plus the HDACi entinostat. Synergism was accompanied by upregulation of proapoptotic Bik, and suppression of Bik attenuated the synergy. The acquired resistance models also exhibited elevated levels of MDR-1/P-gp. Inhibition of MDR-1/P-gp with reversin 121 partially overcame carfilzomib resistance in R-UMSCC-1 and R-Cal33 cells. Collectively, these studies indicate that combining carfilzomib or oprozomib with HDAC or MDR-1/P-gp inhibitors may be a useful strategy for overcoming acquired resistance to these proteasome inhibitors.

Anti-leukemic activity and mechanisms underlying resistance to the novel immunoproteasome inhibitor PR-924.

PR-924 is a novel prototypic immunoproteasome inhibitor bearing markedly enhanced specificity for the ß5i immunoproteasome subunit, compared to the classical proteasome inhibitor bortezomib. Here, we assessed the growth inhibitory potential of PR-924 in three human hematologic malignancy cell lines (CCRF-CEM, THP1, and 8226) and their bortezomib-resistant sublines. Parental cells displayed equal sensitivity to PR-924 (IC50: 1.5-2.8 µM), whereas their bortezomib-resistant tumor lines displayed a 10-12 fold cross-resistance to PR-924. However, PR-924 cross-resistance factors for bortezomib-resistant sublines were markedly lower compared to the resistance factors to bortezomib. Proteasome inhibition experiments confirmed that PR-924 specifically inhibited ß5i activity, even far below concentrations that exerted anti-proliferative activity. We further determined whether PR-924 activity might be compromised by acquisition of drug resistance phenomena. Indeed, CEM cells rendered stepwise resistant to 20 µM PR-924 (CEM/PR20) displayed 13-fold PR-924-resistance and 10-fold cross-resistance to bortezomib. CEM/PR20 cells were devoid of mutations in the PSMB8 gene (encoding ß5i), but acquired Met45Ile mutation in the PSMB5 gene (encoding constitutive ß5), consistent with ß5 mutations observed in bortezomib-resistant cells. Furthermore, compared to parental CEM cells, CEM/PR20 cells exhibited 2.5-fold upregulation of constitutive proteasome subunit expression, whereas immunoproteasome subunit expression was 2-fold decreased. In conclusion, PR-924 displayed potent anti-leukemic activity including toward bortezomib-resistant leukemia cells. Despite the specificity of PR-924 to the ß5i immunoproteasome subunit, its anti-leukemic effect required concentrations that blocked both ß5 and ß5i subunits. This is underscored by the emergence of mutations in PSMB5 rather than in PSMB8.

Interferon-γ-induced upregulation of immunoproteasome subunit assembly overcomes bortezomib resistance in human hematological cell lines.

BACKGROUND: Despite encouraging results with the proteasome inhibitor bortezomib in the treatment of hematologic malignancies, emergence of resistance can limit its efficacy, hence calling for novel strategies to overcome bortezomib-resistance. We previously showed that bortezomib-resistant human leukemia cell lines expressed significantly lower levels of immunoproteasome at the expense of constitutive proteasomes, which harbored point mutations in exon 2 of the PSMB5 gene encoding the ß5 subunit. Here we investigated whether up-regulation of immunoproteasomes by exposure to interferon-γ restores sensitivity to bortezomib in myeloma and leukemia cell lines with acquired resistance to bortezomib. METHODS: RPMI-8226 myeloma, THP1 monocytic/macrophage and CCRF-CEM (T) parental cells and sub lines with acquired resistance to bortezomib were exposed to Interferon-γ for 24-48 h where after the effects on proteasome subunit expression and activity were measured, next to sensitivity measurements to proteasome inhibitors bortezomib, carfilzomib, and the immunoproteasome selective inhibitor ONX 0914. At last, siRNA knockdown experiments of ß5i and ß1i were performed to identify the contribution of these subunits to sensitivity to proteasome inhibition. Statistical significance of the differences were determined using the Mann-Whitney U test. RESULTS: Interferon-γ exposure markedly increased immunoproteasome subunit mRNA to a significantly higher level in bortezomib-resistant cells (up to 30-fold, 10-fold, and 6-fold, in ß1i, ß5i, and ß2i, respectively) than in parental cells. These increases were paralleled by elevated immunoproteasome protein levels and catalytic activity, as well as HLA class-I. Moreover, interferon-γ exposure reinforced sensitization of bortezomib-resistant tumor cells to bortezomib and carfilzomib, but most prominently to ONX 0914, as confirmed by cell growth inhibition studies, proteasome inhibitor-induced apoptosis, activation of PARP cleavage and accumulation of polyubiquitinated proteins. This sensitization was abrogated by siRNA silencing of ß5i but not by ß1i silencing, prior to pulse exposure to interferon-γ. CONCLUSION: Downregulation of ß5i subunit expression is a major determinant in acquisition of bortezomib-resistance and enhancement of its proteasomal assembly after induction by interferon-γ facilitates restoration of sensitivity in bortezomib-resistant leukemia cells towards bortezomib and next generation (immuno) proteasome inhibitors.

Inhibition of the immunoproteasome ameliorates experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a chronic demyelinating immune mediated disease of the central nervous system. The immunoproteasome is a distinct class of proteasomes found predominantly in monocytes and lymphocytes. Recently, we demonstrated a novel function of immunoproteasomes in cytokine production and T cell differentiation. In this study, we investigated the therapeutic efficacy of an inhibitor of the immunoproteasome (ONX 0914) in two different mouse models of MS. ONX 0914 attenuated disease progression after active and passive induction of experimental autoimmune encephalomyelitis (EAE), both in MOG35-55 and PLP139₋151-induced EAE. Isolation of lymphocytes from the brain or spinal cord revealed a strong reduction of cytokine-producing CD4(+) cells in ONX 0914 treated mice. Additionally, ONX 0914 treatment prevented disease exacerbation in a relapsing-remitting model. An analysis of draining lymph nodes after induction of EAE revealed that the differentiation to Th17 or Th1 cells was strongly impaired in ONX 0914 treated mice. These results implicate the immunoproteasome in the development of EAE and suggest that immunoproteasome inhibitors are promising drugs for the treatment of MS.

Higher ratio immune versus constitutive proteasome level as novel indicator of sensitivity of pediatric acute leukemia cells to proteasome inhibitors.

The ex vivo sensitivity of pediatric leukemia cells to the proteasome inhibitor bortezomib was compared to 3 next generation proteasome inhibitors: the epoxyketone-based irreversible proteasome inhibitors carfilzomib, its orally bio-available analog ONX 0912, and the immunoproteasome inhibitor ONX 0914. LC50 values were determined by MTT cytotoxicity assays for 29 childhood acute lymphoblastic leukemia and 12 acute myeloid leukemia patient samples and correlated with protein expression levels of the constitutive proteasome subunits (ß5, ß1, ß2) and their immunoproteasome counterparts (ß5i, ß1i, ß2i). Acute lymphoblastic leukemia cells were up to 5.5-fold more sensitive to proteasome inhibitors than acute myeloid leukemia cells (P<0.001) and the combination of bortezomib and dexamethasone proved additive/synergistic in the majority of patient specimens. Although total proteasome levels in acute lymphoblastic leukemia and acute myeloid leukemia cells did not differ significantly, the ratio of immuno/constitutive proteasome was markedly higher in acute lymphoblastic leukemia cells over acute myeloid leukemia cells. In both acute lymphoblastic leukemia and acute myeloid leukemia, increased ratios of ß5i/ß5, ß1i/ß1 and ß2i/ß2 correlated with increased sensitivity to proteasome inhibitors. Together, differential expression levels of constitutive and immunoproteasomes in pediatric acute lymphoblastic leukemia and acute myeloid leukemia constitute an underlying mechanism of sensitivity to bortezomib and new generation proteasome inhibitors, which may further benefit from synergistic combination therapy with drugs including glucocorticoids.

Reactive glia show increased immunoproteasome activity in Alzheimer's disease.

The proteasome is the major protein degradation system within the cell, comprised of different proteolytic subunits; amyloid-ß is thought to impair its activity in Alzheimer's disease. Neuroinflammation is a prominent hallmark of Alzheimer's disease, which may implicate an activation of the immunoproteasome, a specific proteasome variant induced by immune signalling that holds slightly different proteolytic properties than the constitutive proteasome. Using a novel cell-permeable proteasome activity probe, we found that amyloid-ß enhances proteasome activity in glial and neuronal cultures. Additionally, using a subunit-specific proteasome activity assay we showed that in the cortex of the APPswePS1dE9 plaque pathology mouse model, immunoproteasome activities were strongly increased together with increased messenger RNA and protein expression in reactive glia surrounding plaques. Importantly, this elevated activity was confirmed in human post-mortem tissue from donors with Alzheimer's disease. These findings are in contrast with earlier studies, which reported impairment of proteasome activity in human Alzheimer's disease tissue and mouse models. Targeting the increased immunoproteasome activity with a specific inhibitor resulted in a decreased expression of inflammatory markers in ex vivo microglia. This may serve as a potential novel approach to modulate sustained neuroinflammation and glial dysfunction associated with Alzheimer's disease.

Overcoming bortezomib resistance in human B cells by anti-CD20/rituximab-mediated complement-dependent cytotoxicity and epoxyketone-based irreversible proteasome inhibitors.

BACKGROUND: In clinical and experimental settings, antibody-based anti-CD20/rituximab and small molecule proteasome inhibitor (PI) bortezomib (BTZ) treatment proved effective modalities for B cell depletion in lymphoproliferative disorders as well as autoimmune diseases. However, the chronic nature of these diseases requires either prolonged or re-treatment, often with acquired resistance as a consequence. METHODS: Here we studied the molecular basis of acquired resistance to BTZ in JY human B lymphoblastic cells following prolonged exposure to this drug and examined possibilities to overcome resistance by next generation PIs and anti-CD20/rituximab-mediated complement-dependent cytotoxicity (CDC). RESULTS: Characterization of BTZ-resistant JY/BTZ cells compared to parental JY/WT cells revealed the following features: (a) 10-12 fold resistance to BTZ associated with the acquisition of a mutation in the PSMB5 gene (encoding the constitutive ß5 proteasome subunit) introducing an amino acid substitution (Met45Ile) in the BTZ-binding pocket, (b) a significant 2-4 fold increase in the mRNA and protein levels of the constitutive ß5 proteasome subunit along with unaltered immunoproteasome expression, (c) full sensitivity to the irreversible epoxyketone-based PIs carfilzomib and (to a lesser extent) the immunoproteasome inhibitor ONX 0914. Finally, in association with impaired ubiquitination and attenuated breakdown of CD20, JY/BTZ cells harbored a net 3-fold increase in CD20 cell surface expression, which was functionally implicated in conferring a significantly increased anti-CD20/rituximab-mediated CDC. CONCLUSIONS: These results demonstrate that acquired resistance to BTZ in B cells can be overcome by next generation PIs and by anti-CD20/rituximab-induced CDC, thereby paving the way for salvage therapy in BTZ-resistant disease.

The next generation proteasome inhibitors carfilzomib and oprozomib activate prosurvival autophagy via induction of the unfolded protein response and ATF4.

The proteasome inhibitor bortezomib has shown remarkable clinical success in the treatment of multiple myeloma. However, the efficacy and mechanism of action of bortezomib in solid tumor malignancies is less well understood. In addition, the use of this first-in-class proteasome inhibitor is limited by several factors, including off-target effects that lead to adverse toxicities. We recently reported the impact and mechanisms of carfilzomib and oprozomib, second-in-class proteasome inhibitors with higher specificities and reduced toxicities, against head and neck squamous cell carcinoma (HNSCC). Carfilzomib and oprozomib potently inhibit HNSCC cell survival and the growth of HNSCC tumors. Both compounds promote upregulation of proapoptotic BIK and antiapoptotic MCL1, which serves to mediate and attenuate, respectively, the killing activities of these proteasome inhibitors. Both compounds also induce complete autophagic flux that is partially dependent on activation of the unfolded protein response (UPR) and upregulation of ATF4. Carfilzomib- and oprozomib-induced autophagy acts to promote HNSCC cell survival. Our study indicates that the therapeutic benefit of these promising proteasome inhibitors may be improved by inhibiting MCL1 expression or autophagy.

Immunoproteasome subunit LMP7 deficiency and inhibition suppresses Th1 and Th17 but enhances regulatory T cell differentiation.

The immunoproteasome generates peptides presented on MHC class I molecules to cytotoxic T cells. ONX 0914 (formerly called PR-957) is a selective inhibitor of the immunoproteasome subunit low molecular mass polypeptide (LMP) 7 (ß5i) that attenuates disease progression in mouse models of diabetes, colitis, and arthritis. The aim of this study was to investigate the effect of LMP7-specific inhibition on major Th cell differentiation pathways involved in the progression of autoimmune diseases in vitro and in vivo. We used ONX 0914-treated wild-type CD4(+) T cells and also LMP7(-/-) CD4(+) T cells under different Th cell-polarizing conditions, focusing on the effector cytokines and transcription factors involved, and compared them with wild-type CD4(+) T cells. Mouse models of dextran sodium sulfate-induced colitis and a T cell transfer model of colitis were used for in vivo assessment. Deletion or inhibition of LMP7 suppressed generation of Th17 but promoted regulatory T cell (Treg) development. In developing Th17 cells, immunoproteasome inhibition blocked phosphorylation of STAT3, whereas in Tregs, SMAD phosphorylation was enhanced. Additionally, LMP7 inhibition led to reduced STAT1 phosphorylation and Th1 differentiation. These findings were confirmed in vivo as LMP7 inhibition or deficiency resulted in reduced Th1 and Th17 expansion while promoting Treg development in dextran sodium sulfate-induced colitis. Also, in a T cell-dependent transfer model of colitis, LMP7-specific inhibition led to reduced Th1 and Th17 differentiation in vivo. LMP7 governs Th cell lineage determination by affecting the balance of receptor proximal signals during differentiation. These data render LMP7 a promising drug target for the treatment of autoimmune diseases.

Carfilzomib and ONX 0912 inhibit cell survival and tumor growth of head and neck cancer and their activities are enhanced by suppression of Mcl-1 or autophagy.

PURPOSE: Carfilzomib is a selective, irreversible inhibitor of the chymotrypsin-like activity of the proteasome and is undergoing clinical evaluation in myeloma. ONX 0912 (oprozomib) is an orally bioavailable derivative. The activities of carfilzomib and ONX 0912 against solid tumor malignancies are less well understood. We investigated the impact and mechanisms of action of carfilzomib and ONX 0912 in preclinical models of head and neck squamous cell carcinoma (HNSCC). EXPERIMENTAL DESIGN: The effects of carfilzomib and ONX 0912 on HNSCC cell survival and xenograft tumor growth were evaluated. The impact and mechanisms of both agents on apoptosis and autophagy induction were also investigated. The contribution of the unfolded protein response (UPR) to autophagy induction and the role of autophagy in attenuating HNSCC cell death were determined. RESULTS: Carfilzomib and ONX 0912 potently induced apoptosis in HNSCC cell lines via upregulation of pro-apoptotic Bik. Upregulation of Mcl-1 by these agents served to dampen their efficacies. Carfilzomib and ONX 0912 also induced autophagy, mediated, in part, by activation of the UPR pathway involving upregulation of ATF4 transcription factor. Autophagy induction served a prosurvival role. Oral administration of ONX 0912 inhibited the growth of HNSCC xenograft tumors in a dose-dependent manner. CONCLUSIONS: These results show that carfilzomib and ONX 0912 are potently active against HNSCC cells, and the activities of these agents can be enhanced via suppression of Mcl-1 or inhibition of autophagy. Oral ONX 0912 exhibits in vivo activity against HNSCC tumors and may represent a useful therapeutic agent for this malignancy.

Discovery and development of second-generation proteasome inhibitors.

Proteasome inhibition is a validated therapeutic strategy for the treatment of B-cell neoplasms. The peptide boronate based inhibitor bortezomib has become an important tool in the armamentarium for the treatment of multiple myeloma (MM) and has spurred the development of new agents that target the catalytic activities of the proteasome. Five of these agents, representing three distinct chemical classes, have reached clinical testing. These compounds have properties similar to and distinct from bortezomib. Here, the preclinical activity and clinical development of these agents are reviewed with special attention given to comparisons with bortezomib.