Search

Proteasome inhibitors (PI) have evolved as the central backbone of treatment for multiple myeloma (MM), with first-in-class bortezomib and second- and third-generation PI, carfilzomib and ixazomib, having been approved for this indication. Proteasome inhibition disrupts the unfolded protein response to resolve excessive endoplasmic reticulum stress, but also leads to massive metabolic changes manifested by the induction of amino acid biosynthesis, an anti-oxidant response, lipogenesis and an increase in protein folding. In this sense, PI represent a unique class of drugs targeting cancer cell metabolism by affecting the balance between protein biosynthesis, folding and destruction. However, the adaptive changes in MM cell metabolism may provide the basis of resistance to PI, as high glycolytic activity or rewiring glucose metabolism is associated with bortezomib resistance in MM.

Downregulation of the unfolded protein response mediates proteasome inhibitor resistance in multiple myeloma. The Human Immunodeficieny Virus protease inhibitor nelfinavir activates the unfolded protein response in vitro. We determined dose-limiting toxicity and recommended dose for phase II of nelfinavir in combination with the proteasome inhibitor bortezomib. Twelve patients with advanced hematologic malignancies were treated with nelfinavir (2500-5000 mg/day p.o., days 1-14, 3+3 dose escalation) and bortezomib (1.3 mg/m(2), days 1, 4, 8, 11; 21-day cycles). A run in phase with nelfinavir monotherapy allowed pharmakokinetic/pharmakodynamic assessment of nelfinavir in the presence or absence of concomittant bortezomib. End points included dose-limiting toxicity, activation of the unfolded protein response, proteasome activity, toxicity and response to trial treatment. Nelfinavir 2×2500 mg was the recommended phase II dose identified. Nelfinavir alone significantly up-regulated expression of proteins related to the unfolded protein response in peripheral blood mononuclear cells and inhibited proteasome activity. Of 10 evaluable patients in the dose escalation cohort, 3 achieved a partial response, 4 stable disease for 2 cycles or more, while 3 had progressive disease as best response. In an exploratory extension cohort with 6 relapsed, bortezomib-refractory, lenalidomide-resistant myeloma patients treated at the recommended phase II dose, 3 reached a partial response, 2 a minor response, and one progressive disease. The combination of nelfinavir with bortezomib is safe and shows promising activity in advanced, bortezomib-refractory multiple myeloma. Induction of the unfolded protein response by nelfinavir may overcome the biological features of proteasome inhibitor resistance. (clinicaltrials.gov identifier: 01164709).

Proteasome inhibitors (PIs) are a backbone of multiple myeloma (MM) therapy. The proteasome harbors six proteolytically active subunits (β1, β2, β5), while β5 was identified as rate-limiting and is a primary target of clinically available PIs. The most effective pattern of subunit inhibition provided by these PIs for cytotoxic activity in MM is unknown. A head-to-head comparison of clinically available PIs shows that in the clinically relevant setting only the co-inhibition of β1 or β2 with β5 activity achieves meaningful functional proteasome inhibition and cytotoxicity, while the selective β2/β5 inhibition of both constitutive and immunoproteasome is the most cytotoxic. In the long-term setting, selective inhibition of β5 subunit is sufficient to induce cytotoxicity in PI-sensitive, but not in PI-resistant MM, and the β5/β2 co-inhibition is the most cytotoxic in PI-resistant MM. These results give a rational basis for selecting individual PIs for the treatment of MM.

Proteasome inhibitor (PI) carfilzomib (CFZ) has activity superior to bortezomib (BTZ) and is increasingly incorporated in multiple myeloma (MM) frontline therapy and relapsed settings. Most MM patients ultimately experience PI-refractory disease, an unmet medical need with poorly understood biology and dismal outcome. Pharmacologic targeting of ABCB1 improved patient outcomes, including MM, but suffered from adverse drug effects and insufficient plasma concentrations. Proteomics analysis identified ABCB1 overexpression as the most significant change in CFZ-resistant MM cells. We addressed the functional role of ABCB1 overexpression in MM and observed significantly upregulated ABCB1 in peripheral blood malignant plasma cells (PCs) vs untreated patients' bone marrow PC. ABCB1 overexpression reduces the proteasome-inhibiting activity of CFZ due to drug efflux, in contrast to BTZ. Likewise, the cytotoxicity of established anti-MM drugs was significantly reduced in ABCB1-expressing MM cells. In search for potential drugs targeting ABCB1 in clinical trials, we identified the HIV protease inhibitors nelfinavir (NFV) and lopinavir (LPV) as potent functional modulators of ABCB1-mediated drug export, most likely via modulation of mitochondria permeability transition pore. NFV and LPV restored CFZ activity at therapeutically relevant drug levels and thus represent ready-to-use drugs to be tested in clinical trials to target ABCB1 and to re-sensitize PC to established myeloma drugs, in particular CFZ.

Adaptive resistance of myeloma to proteasome inhibition represents a clinical challenge, whose biology is poorly understood. Proteasome mutations were implicated as underlying mechanism, while an alternative hypothesis based on low activation status of the unfolded protein response was recently suggested (IRE1/XBP1-low model). We generated bortezomib- and carfilzomib-adapted, highly resistant multiple myeloma cell clones (AMO-BTZ, AMO-CFZ), which we analyzed in a combined quantitative and functional proteomic approach. We demonstrate that proteasome inhibitor-adapted myeloma cells tolerate subtotal proteasome inhibition, irrespective of a proteasome mutation, and uniformly show an 'IRE1/XBP1-low' signature. Adaptation of myeloma cells to proteasome inhibitors involved quantitative changes in >600 protein species with similar patterns in AMO-BTZ and AMO-CFZ cells: proteins involved in metabolic regulation, redox homeostasis, and protein folding and destruction were upregulated, while apoptosis and transcription/translation were downregulated. The quantitatively most upregulated protein in AMO-CFZ cells was the multidrug resistance protein (MDR1) protein ABCB1, and carfilzomib resistance could be overcome by MDR1 inhibition. We propose a model where proteasome inhibitor-adapted myeloma cells tolerate subtotal proteasome inhibition owing to metabolic adaptations that favor the generation of reducing equivalents, such as NADPH, which is supported by oxidative glycolysis. Proteasome inhibitor resistance may thus be targeted by manipulating the energy and redox metabolism.

Purpose Proteasome-inhibiting drugs (PI) are gaining importance in hematologic oncology. The proteasome carries three proteolytically active subunits (β1, β2, β5). All established PI (bortezomib and carfilzomib), as well as experimental drugs in the field (dalanzomib, oprozomib, and ixazomib), by design target the rate-limiting β5 subunit. It is unknown whether β2-selective proteasome inhibition can also be exploited toward anticancer treatment. Combining PI with the pan B-cell-directed Bruton tyrosine kinase inhibitor ibrutinib appears a natural option for future improved treatment of multiple myeloma (MM) and B-cell lymphomas. However, bortezomib induces phosphorylation of IκB and activation of NF-κB in MM cells, while ibrutinib inhibits the IκB/NF-κB axis, suggesting antagonistic signaling. A β2-selective proteasome inhibitor may lack such antagonistic signaling effects. Methods We recently introduced LU-102, the first β2-selective PI available for preclinical testing. We here compare bortezomib with carfilzomib and LU-102 in MM and MCL in vitro with regard to their effects on pIκB/NF-κB signaling and their cytotoxic activity in combination with ibrutinib. Results LU-102 reduced phosphorylation of IκB, in contrast to bortezomib and carfilzomib, and was a superior inhibitor of NF-κB activation in MM cells. This translated into highly synergistic cytotoxicity between LU-102 and ibrutinib, which was able to overcome BTZ resistance and CFZ resistance. By contrast, BTZ lacked consistent synergistic cytotoxicity with ibrutinib. Conclusion Ibrutinib is highly synergistic with β2-selective proteasome inhibition against MM and MCL in vitro. Novel β2-selective proteasome inhibitors may be exploited to overcome bortezomib/carfilzomib resistance and boost the activity of BTK inhibitors against B-cell-derived malignancies.

The first-in-class alkylating HDAC inhibitor EDO-S101 is highly synergistic with proteasome inhibition against multiple myeloma through activation of multiple pathways

Background Protein metabolism is an innovative potential therapeutic target for AML. Proteotoxic stress (PS) sensitizes malignant cells for proteasome inhibitor treatment. Some HIV protease inhibitors (HIV-PI) induce PS and may therefore be combined with proteasome inhibitors to achieve PS-targeted therapy of AML. Methods We investigated the effects of all nine approved HIV-PI alone and in combination with proteasome inhibitors on AML cell lines and primary cells in vitro. Results Ritonavir induced cytotoxicity and PS at clinically achievable concentrations, and induced synergistic PS-triggered apoptosis with bortezomib. Saquinavir, nelfinavir and lopinavir were likewise cytotoxic against primary AML cells, triggered PS-induced apoptosis, inhibited AKT-phosphorylation and showed synergistic cytotoxicity with bortezomib and carfilzomib at low micromolar concentrations. Exclusively nelfinavir inhibited intracellular proteasome activity, including the β2 proteasome activity that is not targeted by bortezomib/carfilzomib. Conclusions Of the nine currently approved HIV-PI, ritonavir, saquinavir, nelfinavir and lopinavir can sensitize AML primary cells for proteasome inhibitor treatment at low micromolar concentrations and may therefore be tested clinically toward a proteotoxic stress targeted therapy of AML.

Malignant plasma cells drive massive overproduction of monoclonal immunoglobulin in multiple myeloma (MM). Hence MM cells critically rely on a proper balance between protein biosynthesis and folding, and degradation of unfolded/ misfolded protein orchestrated by the unfolded protein response (UPR). Inhibitors of the proteasome, the terminal protease along the UPR pathway, are highly effective against MM. Adaptive resistance to current proteasome inhibitors (PI) (Bortezomib-Btz, Carfilzomib-Cfz) is a major cause of treatment failure. Btz is a peptide boronate-type reversible inhibitor, whereas Cfz is of epoxyketone-type and binds irreversibly to the same proteasomal B5 active site target. Next generation PI are likewise B5-directed and either of the peptide boronate (ixazomib, dalanzomib), or the epoxyketone type (oprozomib). PI resistance is currently understood via two alternative concepts, the load/capacity concept characterized by proteasome upregulation in conjunction with downregulation of the UPR via reduced IRE/XBP-1 signaling, and the mutation concept basing on B5 active site mutations that prevent PI binding. Only IRE1/XBP1 downregulation have been confirmed in patient-derived material. We generated an in vitro model where AMO-1 myeloma cells (IC50: 3.1nM for Btz, 4.9nM Cfz) were adapted to Btz (AMOaBtz; IC50: 298.7nM Btz, 43.2nM Cfz) or to Cfz, (AMOaCfz; IC50: 27.9nM Btz, 411.5nM Cfz). AMOaBtz cells showed preferential resistance against ixazomib and dalanzomib, while AMOaCfz cells were preferentially resistant to oprozomib and other epoxyketone-type experimental inhibitors. AMOaBtz and AMOaCfz likewise downregulated IRE/XBP-1, but only AMOaBtz contained an acquired proteasome mutation (A310G in PSMB5), however this had only a modest influence on Btz binding and proteasome inhibition. Global gene expression analysis revealed 1430 differentially expressed genes in AMOaBtz/ AMOaCfz (FC>2, p Citation Format: Andrej Besse, Lenka Besse, Marianne Kraus, Jurgen Bader, Mario Novkovic, Fedor Kryukov, Pavel Nemec, Tobias Silzle, Christoph Driessen. Inside of multiple mechanism of adaption towards the proteasome inhibitors in multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1243.

Background: EDO-S101 is a first-in-class alkylating histone-deacetylase inhibitor (HDACi) fusion molecule that combines the strong DNA damaging effect of bendamustine, with a fully functional pan-HDAC inhibitor, vorinostat. Bendamustine has substantial clinical activity against B-cell malignancies, lacks cross resistance with many other anticancer drugs, has superior antimyeloma activity compared to melphalan, and can safely be combined with proteasome inhibitors. The Histone deacetylase inhibitor (HDACi) vorinostat has a broad spectrum of epigenetic activities and sensitizes lymphoma and myeloma cells for a variety of cytotoxic drugs. Vorinostat in particular has a strong synergy with proteasome inhibitors, presumably due to the inhibition of HDAC6. HDAC6 allows polyubiquitinated substrate protein to bypass the inhibited proteasome towards degradation via the autophagy pathway. The EDO-S101 molecule was designed to create a very potent cytotoxic agent for systemic use upon exploiting the synergies of a bi-functional mode of action. Methods: The aim of the in vitro study was to compare the cytotoxicity of EDO-S101 against Multiple Myeloma (MM), leukemia and lymphoma cells with established alkylating agents and to investigate its cellular and molecular effects in combination with proteasome inhibitors. Results: The IC50 of EDO-S101 ranged between 5-13 μM in 8 myeloma cell lines and thus one order of magnitude lower than the IC50 for bendamustine (70 - > 200 μM). Myeloma cell lines with adaptive resistance against bortezomib or carfilzomib did not differ from non-adapted cells in their IC50 for EDO-S101. Likewise, the IC50 for 3 ABC type DLBCL cell lines ranged between 3-8 μM for EDO-S101, compared to bendamustine > 50 μM. EDO-S101 had significant synergistic cytotoxicity with the proteasome inhibitors bortezomib and carfilzomib across all cell types tested, in contrast to melphalan and bendamustine. In a panel of 6 MM cell lines, the combination of EDO-S101 yielded a mean combination index for synergistic cytotoxicity of 0.12 (± 0.06) and 0.08 (± 0.06) for bortezomib or carfilzomib combinations, respectively (with values < 0.8 indicating significant synergism), in contrast to bendamustine 1.35 (± 0.87) and 1.29 (± 0.86), and melphalan 1.09 (± 0.66) and 1.20 (± 1.44). Likewise, EDO-S101 showed synergistic cytotoxicity with bortezomib and carfilzomib against mantle cell lymphoma cells (mean CIs 0.6 and 0.2), in contrast to bendamustine (CIs 1.72, 1.22) and melphalan (CIs 1.16 and 1.17), as well as ABC type DLBCL (CIs 0.32 and 0.28 for EDO S-101, compared to 15 and 34 for bendamustine and 0.87 and 0.78 for melphalan). To dissect the molecular mechanism for the unique synergistic cytotoxicity of EDO-S101 with proteasome inhibition, which contrasted to the established alkylating drugs, we analysed proteasome activity, protein acetylation status, accumulation of polyubiquitinated proteins as well as regulatory and effector proteins of the unfolded protein response (UPR) in RPMI8226 myeloma cells by western blot. EDO-S101 induced strong protein and histone acetylation, confirming its HDACi-like activity. Interestingly, and in contrast to bendamustine, melphalan and vorinostat, EDO-S101 was a strong inducer of pIRE-1, the key activator protein of the UPR in MM cells. IRE1 activation and induction of the UPR have recently been shown to be the major determinants of proteasome inhibitor sensitivity in human MM. Conclusions: We conclude that EDO-S101, an alkylating HDAC inhibitor fusion molecule, displays bi-functional activity. Compared to bendamustine and melphalan, it has superior monoactivity in vitro against hematologic malignancies including MM, mantle cell lymphoma and ABC type DLBCL. Of particular interest is the strong synergy of EDO-S101 with proteasome inhibitors which also stands out in comparison to the established alkylating agents. The latter is associated with induction of pIRE1, the key regulator of the UPR by EDO- S101. Both, the superior monoactivity of EDO-S101 and its mechanism-based synergy with proteasome inhibitors warrant further development of the compound towards clinical testing. Disclosures Driessen: Mundipharma: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Rationale: Overcoming proteasome inhibitor (PI) resistance is a challenge in multiple myeloma (MM) therapy since most MM patients ultimately develop PI resistance. Induction of excessive activation of the unfolded protein response (UPR) is the major mechanism of PI-induced cytotoxicity in MM. The UPR is a complex transcriptional response that balances biosynthesis, folding and proteasomal destruction of cellular protein. UPR inactivation results in PI resistance in vitro, and MM cells with low UPR activation accumulate and drive the relapse in PI-resistant MM patients. Pharmacologic activation of the UPR overcomes PI-resistance in preclinical models of MM and provides an option for clinical testing. The HIV protease inhibitor nelfinavir (NFV) has UPR-inducing activity via an unknown mechanism that may involve interference with regulatory proteases in the UPR and/or proteasome activity. NFV has single agent activity in MM and sensitizes MM and AML cells for PI treatment in vitro and in vivo. Methods: We performed a multicenter phase I dose escalation study to assess safety and recommended dose for phase II of NFV in combination with bortezomib (BTZ) in patients with advanced hematologic malignancies, and to detect signals for activity. NFV was given d 1-14 twice daily p.o. at the dose levels 1250 mg (DL0), 1875 mg (DL1) and 2500 mg (DL2), BTZ was dosed 1.3 mg/m2 d 1, 4, 8, 11 i.v. in 21 day cycles. The first treatment cycle was preceded by one week of NFV monotherapy for assessment of pharmacokinetic/pharmacodynamic parameters (NFV plasma concentrations, proteasome activity and expression of UPR-related proteins in peripheral blood mononuclear cells (PBMC)). Patients were treated for 3 cycles per protocol with the option to receive up to a total of 7 cycles. Results: 12 patients were treated in the dose escalation cohort (median age 58 years; 8 patients with MM, 1 each with ALL, AML, DLBCL, MCL) for an average of 2.6 cycles. All MM patients had received prior BTZ. DLT was determined in cycle 1 in which 93 % of planned dose was delivered. One DLT was observed (G4 ALT elevation at DL2 that spontaneously resolved). Toxicity was mostly mild, could be handled symptomatically, and did not lead to study drug discontinuation except for one case of thrombocytopenia. Diarrhoea G1-2 was the most frequent toxicity observed. Ten patients were evaluable for best response while on trial therapy after having received at least one full cycle. Of these, three patients achieved a PR (1 MCL, 2 MM), 4 remained in SD for at least 2 cycles (2 MM, 1 AML, 1 ALL), while 3 progressed (2 MM, 1 DLBCL). Peak NFV plasma concentrations during monotherapy were in the dose range putatively required for UPR activation, tended to be higher in patients treated at DL1, compared to DL2 (means 13.3 vs. 8.9 mM, p=0.08) and were significantly higher during NFV monotherapy than during combination therapy with BTZ (means 9.24 vs. 6.60 mM, p=0.04), suggesting induction of NFV clearance either by autoinduction, concomitant BTZ application, or both. Pharmacodynamic analysis revealed upregulation of proteins related to UPR-induced apoptosis by NFV monotherapy in PBMC (CHOP +56%, p=0.008; PARP +57%, p=0.04, n=10). Activity of the BTZ-insensitive proteasome b2 subunit in PBMC decreased (-16%, p=0.01) during NFV monotherapy, compared to baseline, as did the BTZ-sensitive b1/b5 subunit (-17%, p=0.001). To detect additional signals for activity, an extension cohort of 6 heavily pretreated MM patients that had shown BTZ-resistance during the past 12 months and were in addition lenalidomide-resistant was treated at the recommended dose (DL2). Three of these patients achieved a PR and 2 a MR, while 1 showed PD with a mean of 4.3 cycles administered. Overall, 12 MM patients could be evaluated for best response while on therapy with BTZ + NFV in this study, of which 5 achieved a paraprotein reduction of > 50% compared to baseline (figure 1). Conclusion: Nelfinavir 2500 mg p.o. twice daily induces UPR activation and proteasome inhibition. It can safely be combined with bortezomib (1.3 mg/m2 d 1, 4, 8, 11) to potentially increase bortezomib sensitivity of hematologic malignancies. The combination yields promising clinical activity signals in patients with bortezomib-resistant myeloma. Figure 1: Best paraprotein response, relative to baseline, of evaluable patients with relapsed-refractory myeloma treated with bortezomib + nelfinavir at any dose level for at least one full cycle. Figure 1:. Best paraprotein response, relative to baseline, of evaluable patients with relapsed-refractory myeloma treated with bortezomib + nelfinavir at any dose level for at least one full cycle. Disclosures Off Label Use: the presentation will include off label use of nelfinavir as investigational medicinal product (IMP). Hitz:Celgene: Research Funding.

HIV protease inhibitors (HIV-PI) are oral drugs for HIV treatment. HIV-PI have antitumor activity via induction of ER-stress, inhibition of phospho-AKT (p-AKT) and the proteasome, suggesting antimyeloma activity. We characterize the effects of all approved HIV-PI on myeloma cells. HIV-PI were compared regarding cytotoxicity, proteasome activity, ER-stress induction and AKT phosphorylation using myeloma cells in vitro. Nelfinavir is the HIV-PI with highest cytotoxic activity against primary myeloma cells and with an IC50 near therapeutic drug blood levels (8–14 μM), irrespective of bortezomib sensitivity. Only nelfinavir inhibited intracellular proteasome activity in situ at drug concentrations

Abstract 4069 Background: HIV protease inhibitors (HIV-PI: Ritonavir, Lopinavir, Saquinavir, Nelfinavir, Amprenavir, Indinavir, Atazanavir, Tipranavir and Darunavir) are oral drugs approved for HIV treatment. Although designed to inhibit the HIV protease, HIV-PI likely have additional, yet unidentified, mammalian targets. Individual HIV-PI have been reported to inhibit the proteasome as well as AKT phosphorylation, induce ER stress and exert synergistic cytotoxicity with bortezomib in solid tumors, suggesting a therapeutic potential of HIV-PI especially in myeloma and AML. However, testing and comparing the effects of the different HIV-PI in AML or myloma cells, a prerequisite to identify the most promising HIV-PI to enter clinical testing, have not yet been performed. Methods: We systematically compared and analysed the effects of all currently approved HIV-PI on proteasome inhibition, cytotoxicity, induction or ER-stress, inhibition of p-AKT, and synergism with bortezomib in human myeloma and AML cell lines and primary cells in vitro. Results: HIV-PI can be divided into two subclasses, based on their activity on myeloma and AML cells: Lopinavir, Nelfinavir, Ritonavir and Saquinavir (LNRS-PI) showed biological and molecular activity at concentrations within or near to therapeutic drug levels (10-20 μ M). All remaining HIV-PI were inactive in this concentration range. The LNRS-PI induced ER stress in a very similar fashion (concentration-dependent increase in expression of the chaperones BIP and PDI at 10 μ M). This translated into a uniform pattern of ER stress-induced apoptosis, as deferred from increased expression of CHOP and cleaved PARP, leading to cell death. Likewise, the concentration-dependent inhibition of AKT-phosphorylation was similarly observed for all LNRS-PI, starting at 10 μ M. The LNRS-PI also showed a uniform synergistic cytotoxic activity with bortezomib at therapeutic drug levels, as observed with myeloma cell lines, as well as AML cell lines and primary cells in vitro. By contrast, all remaining HIV-PI did not show a synergistic effect with bortezomib. To assess the effects of the different HIV-PI on proteasome activity, we used activity-based proteasome-specific probes that visualize the activities of the proteasome β2 and β1/β5 active sites in intact, viable cells. Nelfinavir stood out as the only HIV-PI with proteasome-inhibiting activity at therapeutic drug levels. Nelfinavir led to a dose-dependent decrease in active proteasome β1/β5 as well as β2 species in the concentration range of 10–40 μ M. By contrast, low doses of Lopinavir, Ritonavir or Saquinavir induced proteasome β2 and β1/β5 activity, which then decreased to baseline activity levels upon higher drug concentrations (up to 80 μ M). All remaining HIV-PI had no effect on proteasome activity. When cells were pre-treated with bortezomib, which preferentially inhibits the proteasome β1/β5 subunits, and were then exposed to the different HIV-PI, only Nelfinavir led to an additional inhibition of the proteasomal β1/5 and also β2-activity, while all other HIV-PI did not have such an effect. Strikingly, primary myeloma cells from a patient who was refractory to bortezomib-lenalidomide combination therapy, and which were refractory to Bortezomib 10 nM also in vitro, showed robust (> 90%) cytotoxicity when bortezomib was combined with therapeutic drug levels of Nelfinavir (10 μ M). Conclusions: Nelfinavir is a unique drug in the class of HIV-PI, which leads not only to the induction of ER stress and inhibition of AKT-phosphorylation, but also to proteasome inhibition of all active subunits in intact cells at therapeutic drug levels. It re-sensitizes bortezomib-refractory myeloma cells towards bortezomib treatment. Nelfinavir may therefore be an active drug warranting clinical testing in hematologic malignancies, such as myeloma, mantle cell lymphoma or AML. Disclosures: No relevant conflicts of interest to declare.