ALBERT

Description: Phytoplankton have attracted increasing attention in climate science due to their impacts on climate systems. A new generation of climate models can now provide estimates of future climate change, considering the biological feedbacks through the development of the coupled physical–ecosystem model. Here we present the geophysical impact of phytoplankton, which is often overlooked in future climate projections. A suite of future warming experiments using a fully coupled ocean−atmosphere model that interacts with a marine ecosystem model reveals that the future phytoplankton change influenced by greenhouse warming can amplify Arctic surface warming considerably. The warming-induced sea ice melting and the corresponding increase in shortwave radiation penetrating into the ocean both result in a longer phytoplankton growing season in the Arctic. In turn, the increase in Arctic phytoplankton warms the ocean surface layer through direct biological heating, triggering additional positive feedbacks in the Arctic, and consequently intensifying the Arctic warming further. Our results establish the presence of marine phytoplankton as an important potential driver of the future Arctic climate changes.

Description: Adaptive resistance of myeloma cells to proteasome inhibition is poorly understood. It is suggested to base on point mutations in PSMB5 and/or downmodulation of the activation state of the unfolded protein response (UPR) via reduced activity of its major regulatory axis IRE-1/XBP-1. We have generated subclones of the AMO-1 myeloma cell line resistant to bortezomib 〉 1000 nM (AMO-BTZ), or carfilzomib 〉 1000 nM (AMO-CFZ), that do or do not carry the PSMB5 A310G mutation in the β5 substrate pocket. We combine this model with a global quantitative proteomics approach, the analysis of the activation status of the IRE-1/XBP-1 pathway, and with an advanced set of proteasome activity-specific fluorescent affinity probes that allow direct, selective, simultaneous visualization of the activity of all six active β-subunits of the constitutive and the immunoproteasome. Our results demonstrate that the A310G mutation has a modest impact on β5c proteasome inhibition by bortezomib (increasing the IC50 from 25nM to 80 nM), and likewise by carfilzomib (IC50 increase from 10 nM to 50 nM). Strikingly, when AMO-CFZ or AMO-BTZ were exposed to the same functional level of proteasome inhibition (〉 90% inhibition of β5c/5i, 20% inhibition of β1/1i, β2/2i) that resulted in 70-90% cytotoxicity in AMO-1 cells, no cytotoxicity was observed in AMO-BTZ and AMO-CFZ cells. Likewise, AMO-BTZ and AMO-CFZ cells were resistant to the next generation proteasome inhibitors ixazomib, oprozomib and dalanzomib, irrespective of the presence or absence of the PSMB5 mutation. Analysis of the UPR and its major regulators on protein and mRNA levels revealed that all clones of AMO-BTZ and AMO-CFZ showed significantly lower expression of IRE-1 and its product, spliced XBP-1, compared to AMO-1 cells, in contrast to all other major regulators of the UPR (ATF6, PERK, elF2a). Proteasome inhibitor treatment induced phosphorylation of IRE-1 and the induction of sXBP1 similarly in AMO-1, AMO-BTZ and AMO-CFZ cells, however, the induction of downstream proteins of the UPR (ATF4, PDI) was exclusively found in AMO-1 cells. Mass spectrometry-based quantitative global proteomic analysis was performed to compare AMO-1 cells with AMO-BTZ and AMO-CFZ with a cut off of at least a 50% change in abundance of differentially expressed proteins in at least 2 out of triplicate experiments. This yielded 〉 3500 identified individual proteins in proteasome inhibitor adapted cells, of which 〉 600 were differentially expressed and subsequently subjected to a protein-protein interaction (PPI) search and a Gene Ontology (GO) analysis, resulting in an average of 30 GO terms for the overexpressed proteins and 10 for downregulated species in AMO-BTZ and AMO-CFZ. Manual grouping of GO into functionally related clusters resulted in 5-6 groups that were largely concordant between AMO-BTZ and AMO-CFZ. The clusters found overexpressed in AMO-BTZ and AMO-CFZ were proteins involved in protein catabolism, redox control and protein folding. Uniform downregulation was observed for protein clusters involved in transcription/translation, differentiation, apoptosis and structural/cytoskeletal functions. The quantitatively largest group of proteins with significantly altered expression levels in AMO-BTZ/AMO-CFZ vs. AMO-1 control cells consisted of proteins involved in metabolic regulation. This big cluster comprised close to 50 % of all polypeptides with significant quantitative changes, suggesting a key role for metabolic homeostasis. The quantitatively most significantly upregulated protein in both AMO-CFZ and AMO-BTZ was NADPH dehydrogenase, the most important reducing enzyme in eukaryotic cells (4-6 x upregulated). The top individual upregulated protein in AMO-CFZ was the p-glycoprotein 1 (Pgp, 12 x upregulated), while the transcription factor IKZF3 was among the top downregulated proteins in AMO-BTZ cells (0.2 x). Our data indicate that proteasome gene mutations are not required for proteasome inhibitor resistance of myeloma cells, that proteasome inhibitor adapted myeloma cells can compensate subtotal proteasome inhibition irrespective of the type of inhibitor used, and that they have undergone complex adaptive changes in particular in proteins that regulate metabolic functions. Thus we suggest that the metabolic machinery rather than the proteasome should be explored for drug targets in myeloma cells with acquired proteasome inhibitor resistance. Disclosures No relevant conflicts of interest to declare.

Description: Background. EDO-S101 is a first-in-class alkylating, histone-deacetylase inhibitor (HDACi) fusion molecule with dual activity that is currently in Phase I. It structurally combines the strong DNA damaging effect of bendamustine with a fully functional pan-HDAC inhibitor, vorinostat. Bendamustine has substantial activity against B-cell malignancies; vorinostat sensitizes the same type of cancers against alkylators or proteasome inhibitors (PI). Bendamustine combined with the PI bortezomib (BTZ) is active against multiple myeloma (MM). Cytotoxicity of PI in MM relies on excess induction of proteotoxic stress and triggering of the unfolded protein response (UPR). Upon proteasome inhibition, HDACi synergize with PI by interfering with the a-tubulin-mediated transport of poly-ubiquitinated proteasome substrates to lysosomal destruction. Indeed, EDO-S101 has strong synergistic cytotoxicity with PI in vitro against hematological malignancies, including MM, mantle cell lymphoma and ABC type diffuse large B-cell lymphoma. The aim of this work is to characterize the molecular mechanism of action of the synergy of EDO-S101 with PI in comparison to its established structurally related drugs, bendamustine and vorinostat. Methods. The cytotoxic and molecular activity of EDO-S101 in combination with BTZ and other types of PI was assessed in vitro using the RPMI-8226 and several other MM cell lines. HDAC-inhibiting activity, accumulation of poly-ubiquitinated proteins and induction of ER stress, apoptotic signaling and autophagy induction were assessed by quantitative PCR and western blotting. Proteasome activity was measured with activity based probes (ABP). Apoptosis was assessed by AnnexinV/FITC staining with flow cytometry. Cell viability was evaluated by MTS assay. Results. EDO-S101 showed substantially stronger cytotoxicity in combination with PI than melphalan, bendamustine, cyclophosphamide or PI combined with equimolar vorinostat. EDO-S101 had higher HDACi-type of activity, compared to vorinostat, as demonstrated judged in particular by increased a-tubulin acetylation, providing a potential mechanistic basis for its superior synergy with PI. Consistent with this, EDO-S101 alone induced moderate cellular accumulation of poly-ubiquitinated proteins already in the absence of proteasome inhibition, which was potentiated when EDO-S101 was combined with BTZ. EDO-S101 induced activation of the UPR-regulators XBP1 and IRE1 known to control BTZ sensitivity of MM, in contrast to vorinostat or bendamustine alone. Co-treatment with BTZ and EDO-S101 or vorinostat resulted in highly synergistic triggering of the UPR (ATF4, CHOP, BIP). Interestingly, EDO-S101 in addition induced the pro-apoptotic machinery via upregulation of NOXA, downregulation of BCL2 and an increase of the BAX/BCL2 ratio, and also activated autophagy, as evidenced by upregulation of LC3A and LC3B. While this pro-apoptotic signaling of EDO-S101 was highly synergistic with BTZ-induced apoptotic signals, co-treatment with BTZ and vorinostat reduced apoptotic signaling compared to BTZ alone. EDO-S101 reduced c-Myc expression by 60%, while vorinostat had no effect on c-Myc levels. The combination BTZ+EDO-S101 decreased c-Myc levels by approx. 90%, while these levels remained unchanged during treatment with BTZ+vorinostat. Conclusion. EDO-S101 is a first-in-class, dual-mechanism, alkylator-HDAC-inhibitor fusion molecule that combines key structural features of bendamustine and vorinostat. The molecular mode of action of EDO-S101 differs from that of its structurally related drugs by a more effective interaction with a-tubulin, which may in part explain superior synergy with PI. Most importantly, EDO-S101 has a direct pro-apoptotic activity via downregulation of c-Myc and BCL2 while upregulating NOXA, features not observed with vorinostat. This results in highly synergistic signaling with the PI-induced pro-apoptotic effects. EDO-S101 is a promising advancement of bendamustine with molecular features clearly different from and superior to a combination of bendamustine with vorinostat. EDO-S101 should be explored in combination with proteasome inhibitors in particular in poor risk B cell neoplasms with c-Myc overexpression such as aggressive MM, Burkitt lymphoma or "double hit" aggressive B cell lymphoma. Disclosures Besse: Mundipharma-EDO: Other: travel support. Mehrling:Mundipharma-EDO: Employment. Driessen:Mundipharma-EDO: Honoraria, Membership on an entity's Board of Directors or advisory committees; celgene: Consultancy; janssen: Consultancy.

Description: 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.

Description: 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.

Description: The tropical easterly jet (TEJ) is a characteristic upper-level feature of the West African monsoon (WAM) circulation. Moreover, the TEJ over West Africa is significantly correlated with summer Sahel rainfall on interannual and decadal time scales. In contrast, the relationship between Sahel rainfall and the regional TEJ on synoptic to intraseasonal time scales is unclear. Therefore, this relationship is investigated by means of multiple statistical analyses using temporally highly resolved measurement and reanalysis data. It is shown that average correlations between convective activity and regional TEJ intensity remain below 0.3 for all synoptic to intraseasonal time scales. Especially on the synoptic time scale, the TEJ significantly lags anomalies in convective activity by one or two days, which indicates that convection anomalies are more likely to drive changes in the regional TEJ than vice versa. To further shed light on the role of the TEJ for rainfall over West Africa, a previously proposed effect of TEJ-induced upper-level divergence on the development of mesoscale convective systems (MCSs) is examined more closely. An analysis of nearly 300 Sahelian MCSs shows that their initiation is generally not associated with significant TEJ anomalies or jet-induced upper-level divergence. Furthermore, no statistically significant evidence is found that preexisting TEJ-related upper-level divergence anomalies affect intensity, size, and lifetime of MCSs. A limiting factor of this study is the focus on TEJ-induced upper-level divergence. Therefore, a possible effect of the TEJ on Sahel rainfall via other mechanisms cannot be ruled out and should be subject to future studies.