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The bone marrow (BM) microenvironment actively promotes multiple myeloma pathogenesis, and therapies targeting both cancer cells and the niche are highly effective. We were interested in identifying novel signaling pathways supporting multiple myeloma–BM cross-talk. Mutations in the transmembrane receptor Roundabout 1 (ROBO1) were recently identified in patients with multiple myeloma; however, their functional consequences are uncertain. Through protein structure–function studies, we discovered that ROBO1 is necessary for multiple myeloma adhesion to BM stromal and endothelial cells and that ROBO1 knockout (KO) compromises BM homing and engraftment in a disseminated mouse model. ROBO1 KO significantly decreases multiple myeloma proliferation in vitro and intra- and extramedullary tumor growth in vivo. Mechanistically, the ROBO1 C-terminus is cleaved in a ligand-independent fashion and is sufficient to promote multiple myeloma proliferation. Vice versa, mutants lacking the cytoplasmic domain, including the human-derived G674* truncation, act dominantly negative. Interactomic and RNA-sequencing studies suggest that ROBO1 may be involved in RNA processing, supporting further studies.Significance:ROBO1 is highly expressed in t(4;14) multiple myeloma and supports homing and dissemination to the BM niche. ROBO1 knockout causes reduced tumor growth in intramedullary and extramedullary myeloma animal models, while the ROBO1 C-terminus is cleaved in multiple fragments and it is necessary and sufficient to sustain myeloma proliferation.

Supplementary Figure 1 showing ROBO1 expression in primary MM cells versus normal donor bone marrow plasma cells via gene expression profiling and immunohistochemistry. Supplementary Figure 2 showing distribution of ROBO1 expression in the CoMMpass database; ROBO1 expression in CoMMpass database according to cytogenetics; ROBO1 expression in IFM170 database according to cytogenetics; ROBO1 expression in t(4;14) cell lines WT or KO for MMSET. Supplementary Figure 3 showing ROBO1 protein down regulation after ROBO1 knock down via shRNA. Supplementary Figure 4 showing effects of shRNA4- and shRNA-5 mediated ROBO1 KD on cell viability across a panel of MM cell lines with western confirming proper protein down regulation. Supplementary Figure 5 showing successful ROBO1 KO across several KMS11 monoclones, but not H929 monoclones. Supplementary Figure 6 showing expression of ROBO1 FL construct in polyclonally and monoclonally-transduced OPM2 cells. Supplementary Figure 7 showing expression of FL ROBO1 addback in distinct KMS11 ROBO1 KO monoclones. Supplementary Figure 8 showing radiographic images of WT versus KO ROBO1 mu-SCID mice. Supplementary Figure 9 showing adhesion defect in ROBO1 KO KMS11 cells that is fully rescued by FL ROBO1 addback. Supplementary Figure 10 showing Kaplan-Meier survival curve of disseminated mouse model injected with ROBO1 KO versus FL OPM2 cells. Supplementary Figure 11 showing cleaved cytosolic ROBO1 fragments in 293T cells transduced with FL ROBO1 tagged with C-LAP tag and in WT MM.1S cells. Supplementary Figure 12 showing interacting partners of ROBO1 cytosolic domain as deconvoluted via STRING database. Supplementary Figure 13 showing differentially expressed genes in ROBO1 WT versus KO plasmacytoma represented via Volcano plot. Supplementary Figure 14 showing gene set enrichment analysis of differentially expressed genes in ROBO1 WT versus KO plasmacytoma. Supplementary Figure 15 showing schema of patient-derived ROBO1 mutation and expression of G674 truncation in WT and KO ROBO1 OPM2 cells. Supplementary Table 1 showing clinical annotation of newly diagnosed MM parents used in Supplementary figure 1.

Supplementary Methods

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China is facing dual pressures to reduce both PM

Nitrous acid (HONO) is a key precursor of the hydroxyl radicals (OH) and has a significant impact on air quality. Nowadays, the source of HONO is still controversial due to its complex formation mechanisms, which is widely explored in extensive field and laboratory studies. In this study, the pollution characteristics and source contribution of HONO under different air quality conditions in summer in Beijing were analyzed. The observation periods were classified as three typical periods: clean, ozone pollution, and double high pollution (co-occurrence of high PM

Nitrates formed on mineral dust through heterogeneous reactions in high NOx areas can undergo photolysis to regenerate NOx and potentially interfere in the photochemistry in the downwind low NOx areas. However, little is known about such renoxification processes. In this study, photolysis of various nitrates on different mineral oxides was comprehensively investigated in a flow reactor and in situ diffuse reflectance Fourier-transform infrared spectroscopy (in situ DRIFTS). TiO2 was found much more reactive than Al2O3 and SiO2 with both NO2 and HONO as the two major photolysis products. The yields of NO2 and HONO depend on the cation basicity of the nitrate salts or the acidity of particles. As such, NH4NO3 is much more productive than other nitrates like Fe(NO3)3, Ca(NO3)2, and KNO3. SO2 and water vapor promote the photodegradation by increasing the surface acidity due to the photoinduced formation of H2SO4/sulfate and H+, respectively. O2 enables the photo-oxidation of NOx to regenerate nitrate and thus inhibits the NOx yield. Overall, our results demonstrated that the photolysis of nitrate can be accelerated under complex air pollution conditions, which are helpful for understanding the transformation of nitrate and the nitrogen cycle in the atmosphere.

High values of ozone (O3) occur frequently in the dry spring season; thus, understanding the evolution characteristics of volatile organic compounds (VOCs) in spring is of great significance for preventing O3 pollution. In this study, a total of 101 VOCs from April 16 to May 21, 2019, were quantified using an online gas chromatography mass spectrometer/flame ionization detector (GCMS/FID). The results indicated that the observed concentration of total VOCs (TVOCs) was 30.4 ± 17.0 ppbv, and it was dominated by alkanes (44.3%), followed by oxygenated VOCs (OVOCs) (17.4%), halocarbons (12.7%), aromatics (9.5%), alkenes (8.2%), acetylene (5.3%) and carbon disulfide (2.5%). The average mixing ratio of VOCs showed obvious diurnal variation (high at night, low during daytime). We conducted a source apportionment study based on 32 major VOCs using positive matrix factorization (PMF), and coal + biomass burning (25.2%), diesel exhaust (16.0%), gasoline exhaust + evaporation (17.4%), secondary + long-lived species (16.7%), biogenic sources (4.3%), industrial emissions (9.3%) and solvent use (11.2%) were identified as major sources of VOCs. In addition to local emissions, most of the atmospheric VOCs were derived from long-distance air masses (65.7%), and the average mixing ratio of VOCs in the northwest direction was 29.4 ppbv. Combined with the results of the potential source contribution function (PSCF) indicate that research should focus on the local emissions of combustion, transportation sources and solvents usage to control atmospheric VOCs. Additionally, transmission of the northwest air mass is an important component that cannot be ignored during spring in Beijing.

Stressed plants and polymer production can emit many unsaturated volatile organic esters (UVOEs). However, secondary organic aerosol (SOA) formation of UVOEs remains unclear, especially under complex ambient conditions. In this study, we mainly investigated ethyl methacrylate (EM) ozonolysis. Results showed that a substantial increase in secondary H2SO4 particles promoted SOA formation with increasing SO2. An important reason was that the homogeneous nucleation of more H2SO4 at high SO2 level provided greater surface area and volume for SOA condensation. However, increased primary H2SO4 with seed acidity enhanced EM uptake but reduced SOA formation. This was ascribed to the fact that the ozonolysis of more adsorbed EM was hampered with the formation of surface H2SO4 at higher particle acidity. Moreover, the increase in secondary H2SO4 particle via homogeneous nucleation favored to the oligomerization of oxidation products, whereas the increasing of primary H2SO4 with acidity in the presence of seed tended to promote the functionalization conversion products. This study indicated that the role of increased H2SO4 to EM-derived SOA may not be the same under different ambient conditions, which helps to advance our understanding of the complicated roles of H2SO4 in the formation of EM-derived SOA.

Secondary organic aerosol (SOA) plays a critical role in sustained haze pollution in megacities. Traditional observation of atmospheric aerosols usually analyzes the ambient organic aerosol (OA) but neglects the SOA formation potential (SOAFP) of precursors remaining in ambient air. Knowledge on SOAFP is still limited, especially in megacities suffering from frequent haze. In this study, the SOAFP of ambient air in urban Beijing was characterized at different pollution levels based on a two-year field observation using an oxidation flow reactor (OFR) system. Both OA and SOAFP increased as a function of ambient pollution level, in which increasing concentrations of precursor volatile organic compounds (VOCs) and decreasing atmospheric oxidation capacity were found to be the two main influencing factors. To address the role of the atmospheric oxidation capacity in SOAFP, a relative OA enhancement ratio (EROA = 1 + SOAFP/OA) and the elemental composition of the OA were investigated in this study. The results indicated that the atmospheric oxidation capacity was weakened and resulted in higher SOAFP on more polluted days. The relationship found between SOAFP and the atmospheric oxidation capacity could be helpful in understanding changes in SOA pollution with improving air quality in the megacities of developing countries.

PM2.5 concentrations have decreased remarkably in China in recent years, coinciding with a more rapid decrease in SO2 concentrations and a slower decrease in NO2 concentrations, while O3 concentrat...

To clarify how coexisting atmospheric pollutants affect SOA formation, we investigated the effects of mixed gaseous pollutants (CO and SO2) and mixed organic-inorganic (MOI) particles on SOA format...

Multiple myeloma (MM) is a cancer of terminally differentiated plasma cells. MM remains incurable, but overall survival of patients has progressively increased over the past two decades largely due to novel agents such as proteasome inhibitors (PI) and the immunomodulatory agents. While these therapies are highly effective, MM patients can be de novo resistant and acquired resistance with prolonged treatment is inevitable. There is growing interest in early, accurate identification of responsive versus non-responsive patients, however limited sample availability and need for rapid assays are limiting factors. Here, we test dry mass and volume as label-free biomarkers to monitor early response of MM cells to treatment with bortezomib, doxorubicin, and ultraviolet light. For the dry mass measurement, we use two types of phase-sensitive optical microscopy techniques: digital holographic tomography and computationally-enhanced quantitative phase microscopy. We show that human MM cell lines (RPMI8226, MM.1S, KMS20, and AMO1) increase dry mass upon bortezomib treatment. This dry mass increase after bortezomib treatment occurs as early as 1 hour for sensitive cells and 4 hours for all tested cells. We further confirm this observation using primary multiple myeloma cells derived from patients and show that a correlation exists between increase in dry mass and sensitivity to bortezomib, supporting the use of dry mass as a biomarker. The volume measurement using Coulter counter shows a more complex behavior; RPMI8226 cells increase the volume at an early stage of apoptosis, but MM.1S cells show the volume decrease typically observed with apoptotic cells. Altogether, this cell study presents complex kinetics of dry mass and volume at an early stage of apoptosis, which may serve as a basis for the detection and treatment of MM cells.

Secondary organic aerosol (SOA) formation originating from the emission of anthropogenic volatile organic compounds (VOCs) makes a significant contribution to fine particulate matter (PM

Publisher Copyright: © Since the 1960s, many indoor and outdoor smog chambers have been developed worldwide. However, most of them are made of Teflon films, which have relatively high background contaminations due to the wall effect. We developed the world's first medium-size quartz chamber (10 m(3)), which is jointed with 32 pieces of 5 mm thick polished quartz glasses and a stainless-steel frame. Characterizations show that this chamber exhibits excellent performance in terms of relative humidity (RH) (2-80%) and temperature (15-30 +/- 1 degrees C) control, mixing efficiency of the reactants (6-8 min), light transmittance (>90% above 290 nm), and wall loss of pollutants. The wall loss rates of the gas-phase pollutants are on the order of 10(-4) min(-1) at 298 K under dry conditions. It is 0.08 h(-1) for 100-500 nm particles, significantly lower than those of Teflon chambers. The photolysis rate of NO2 (J(NO2)) is automatically adjustable to simulate the diurnal variation of solar irradiation from 0 to 0.40 min(-1). The inner surface of the chamber can be repeatedly washed with deionized water, resulting in low background contaminations. Both experiments (toluene-NOx and alpha-pinene-ozone systems) and box model demonstrate that this new quartz chamber can provide high-quality data for investigating SOA and O-3 formation in the atmosphere.

Amines are frequently detected in atmospheric particles and are internally mixed with other particle-phase components. However, research on the further reactions of amine with reactive species after entering the particle phase is still limited. This study investigated the nitration reaction process of particulate dimethylamine (DMA), formed via a substitution reaction between DMA and (NH

Current air quality models usually underestimate the concentration of ambient air sulfate, but the cause of this underestimation remains unclear. One reason for the underestimation is that the sulfate formation mechanism in the models is incomplete, and does not adequately consider the impact of the synergistic effects of high concentrations of multiple pollutants on sulfate formation. In this work, the roles of gaseous NO2, NH3 and solution ionic strength in the formation of sulfate in the aqueous phase were quantitatively investigated using a glass reactor and a 30 m3 smog chamber, separately. The results showed that sulfate formation was enhanced to different degrees in the presence of gas-phase NO2, NH3 and their coexistence as solutes in both liquid solution and aerosol water. NH3 enhances the aqueous oxidation of SO2 by NO2 mainly by accelerating the uptake of SO2 through increased solubility. More importantly, we found that high ionic strength in aerosol water could significantly accelerate the aqueous oxidation of SO2, resulting in unexpectedly high S(VI) formation rates. We estimate that under severe haze conditions, heterogeneous oxidation of SO2 by NO2 on aerosols may be much shorter than that through gas phase oxidation by OH, aided by high ionic strengths in aerosols. Considering the existence of complex air pollution conditions with high concentrations of NO2, NH3 and aerosol water, as expected in typical urban and suburban settings, the sulfate formation mechanisms revealed in the present work should be incorporated into air quality models to improve the prediction of sulfate concentrations.

Alkyl vinyl ethers are widely used as fuel additives. Despite this, their atmospheric chemistry and secondary organic aerosol (SOA) formation potentials are still not well-known under complex pollu...

Gasoline evaporative emissions have become an important anthropogenic source of urban atmospheric volatile organic compounds (VOCs) and secondary organic aerosol (SOA). These emissions have a significant impact on regional air quality, especially in China where car ownership is growing rapidly. However, the contribution of evaporative emissions to secondary aerosol (SA) is not clear in an air pollution complex in which a high concentration of SO2 and NH3 was present. In this study, the effects of SO2 and NH3 on SA formation from unburned gasoline vapor were investigated in a 30 m3 indoor smog chamber. It was found that an increase in SO2 and NH3 concentrations (0–151 and 0–200 ppb, respectively) could linearly promote the formation of SA, which could be enhanced by a factor of 1.6–2.6 and 2.0–2.5, respectively. Sulfate was most sensitive to the SO2 concentration, followed by organic aerosol, which was due not only to the acid catalytic effect, but was also related to the formation of organic sulfur-containing compounds. In the case of an increasing NH3 concentration, ammonium nitrate increased more significantly than organic aerosol, and nitrogen-containing organics were also enhanced, as revealed by the results of positive matrix factorization (PMF) analysis. New particle formation (NPF) and particle size growth were also significantly enhanced in the presence of SO2 and NH3. This work indicates that gasoline evaporative emissions will be a significant source of SA, especially in the presence of high concentrations of SO2 and NH3. Meanwhile, these emissions might also be a potential source of sulfur- and nitrogen-containing organics. Our work provides a scientific basis for the synergistic emission reduction of secondary aerosol precursors, including NOx, SO2, NH3, and particularly VOCs, to mitigate particulate matter (PM) pollution in China.

Guaiacol (2-methoxyphenol) has a high emission rate from wood burning and mainly exists in the gas phase, but the formation potential of secondary organic aerosol (SOA) from the atmospheric oxidation of guaiacol has not been well determined yet. In this work, SOA formation from the gas-phase reaction of guaiacol with OH radicals was investigated using an oxidation flow reactor (OFR) under different experimental conditions. The results showed that SOA yield was dependent on guaiacol concentration, OH exposure, and the presence of SO2 and NO2. SOA yield firstly increased and then decreased as a function of OH exposure. The maximum SOA yield (0.28–0.54) obtained at different guaiacol concentrations could be well-expressed by a one-product model. The SOA oxidation degree was represented by the carbon oxidation state (OSC) and f44/f43 (the ratio of organic mass fractions of m/z 44 to m/z 43), which both increased linearly and significantly with the increase of OH exposure. In addition, SO2 and NO2 promoted SOA formation, for which the maximum yield enhancements were 13.38% and 10.69%, respectively. The N/C ratio (0.034–0.045) indicated that NO2 participated in the OH-initiated reaction of guaiacol, consequently resulting in the formation of organic nitrates. The experimental results would be helpful to further the understanding of SOA formation from the atmospheric oxidation of guaiacol and its subsequent impacts on air quality and climate.

Current atmospheric quality models usually underestimate the level of ambient secondary organic aerosol (SOA), one of the possible reasons is that the precursors at different concentrations may undergo different oxidation processes and further affect SOA formation. Therefore, there is a need to perform more chamber studies to disclose the influence. In this work, SOA formation over a wide range of initial precursor concentrations (tens of ppb to hundreds of ppb levels) was investigated in a 30 m3 indoor smog chamber, and mainly through the analysis of multiple generations of VOCs detected from HR-ToF-PTRMS to expound the difference in the oxidation process between low and high precursor concentrations. Compared to high initial concentrations, gas-phase intermediates formed at low concentrations had a higher intensity by about one order of magnitude, and the low-volatility compounds also had a higher formation potential due to the competition between semi-volatile intermediates and precursors with oxidants. In addition, the formed SOA was more oxidized with higher f44 value (0.14 ± 0.02) and more relevant to real atmosphere than that formed at high concentrations. This work should help to deeply understand SOA formation and improve the performance of air quality models for SOA simulation.

Gasoline emissions are the largest source of urban atmospheric VOCs, which are critical precursors of ozone (O3) and secondary organic aerosol (SOA). Besides vehicle exhaust, emissions from gasoline evaporation also have a potentially significant effect on SOA formation. However, there are still few studies on the relationship between gasoline vapor composition and SOA formation, especially for gasoline in China. In this study, SOA formation from three unburned gasoline vapors were investigated in a 30 m3 indoor smog chamber. The experimental results showed that with the increase of aromatic content (especially toluene and C2 benzenes) in gasoline from 23% to 50%, the SOA yield was significantly enhanced by a factor of 4.0–6.7. This phenomenon might be related to the higher amounts of intermediate volatility organic compounds (IVOCs) and semi-volatile organic compounds (SVOCs) formed, which promoted the gas-particle partitioning and SOA formation. Additionally, the synergistic effects between precursors in the mixtures might also be a key factor, which could be supported by the higher SOA yield accompanied by a higher ratio of toluene/benzene. Meanwhile, there were more oxygenated organic aerosols (OOA) observed when using high-aromatic gasoline. This work will help in understanding the effect of aromatic content or gasoline quality on the SOA formation from gasoline evaporation emissions, and in providing the scientific basis for taking corresponding control measures to relieve haze events in China.

The bone marrow (BM) microenvironment actively promotes multiple myeloma pathogenesis, and therapies targeting both cancer cells and the niche are highly effective. We were interested in identifying novel signaling pathways supporting multiple myeloma–BM cross-talk. Mutations in the transmembrane receptor Roundabout 1 (ROBO1) were recently identified in patients with multiple myeloma; however, their functional consequences are uncertain. Through protein structure–function studies, we discovered that ROBO1 is necessary for multiple myeloma adhesion to BM stromal and endothelial cells and that ROBO1 knockout (KO) compromises BM homing and engraftment in a disseminated mouse model. ROBO1 KO significantly decreases multiple myeloma proliferation in vitro and intra- and extramedullary tumor growth in vivo. Mechanistically, the ROBO1 C-terminus is cleaved in a ligand-independent fashion and is sufficient to promote multiple myeloma proliferation. Vice versa, mutants lacking the cytoplasmic domain, including the human-derived G674* truncation, act dominantly negative. Interactomic and RNA-sequencing studies suggest that ROBO1 may be involved in RNA processing, supporting further studies. Significance: ROBO1 is highly expressed in t(4;14) multiple myeloma and supports homing and dissemination to the BM niche. ROBO1 knockout causes reduced tumor growth in intramedullary and extramedullary myeloma animal models, while the ROBO1 C-terminus is cleaved in multiple fragments and it is necessary and sufficient to sustain myeloma proliferation.

Smog chamber experimental systems, which have been widely used in laboratory simulation for studying atmospheric processes, are comprehensively reviewed in this paper. The components, development history, main research topics and main achievements of smog chambers are introduced. Typical smog chambers in the world, including their volumes, wall materials, light sources and features, are summarized and compared. Key factors of smog chambers and their influences on the simulation of the atmospheric environment are discussed, including wall loss, wall emission and background pollutants. The features of next-generation smog chambers and their application prospect in future studies of the atmospheric environment are also outlined in this paper.

Volatile organic compounds (VOCs) are easily degraded by oxidants during atmospheric transport. Therefore, the contribution of VOCs to ozone (O3) and secondary organic aerosol (SOA) formation at a receptor site is different from that in a source area. In this study, hourly concentrations of VOCs and other pollutants, such as O3, NOx, HONO, CO, and PM2.5, were measured in the suburbs (Daxing district) of Beijing in August 2019. The photochemical initial concentrations (PICs), in which the photochemical losses of VOCs were accounted for, were calculated to evaluate the contribution of the VOCs to O3 and SOA formation. The mean (±standard deviation) measured VOC concentrations and the PICs were 11.2 ± 5.7 and 14.6 ± 8.4 ppbv, respectively, which correspond to O3 formation potentials (OFP) of 57.8 ± 26.3 and 103.9 ± 109.4 ppbv and SOA formation potentials (SOAP) of 8.4 ± 4.1 and 10.3 ± 7.4 μg m−3, respectively. Alkenes contributed 80.5% of the consumed VOCs, followed by aromatics (13.3%) and alkanes (6.2%). The contributions of the alkenes and aromatics to the OFPPICs were 56.8% and 30.3%, respectively; while their corresponding contributions to the SOAPPICs were 1.9% and 97.3%, respectively. The OFPPICs was linearly correlated with the observed O3 concentrations (OFPPICs = 41.5 + 1.40 × cO3, R2 = 0.87). The O3 formation was associated with a VOC-limited regime at the receptor site based on the measured VOCs and changed to a transition regime and a NOx sensitive regime based on the PIC. Our results suggest that more attention should be paid to biogenic VOCs when studying O3 formation in summer in Beijing, while the control of anthropogenic aromatic compounds should be given priority in terms of SOA formation.

AL amyloidosis (AL) is an incurable plasma cell disorder. The solely pathogenic mechanism in AL is deposition of immunoglobulin free light chains (FLC) organized in fibrils in target organs. Surprisingly, therapeutic strategies directly targeting FLC secretion are not available. SNARE proteins, which are the specific target of botulinum neurotoxin (BoNT), are involved in the docking and fusion of secretory vesicles. We hypothesized that certain BoNT serotypes may block FLC secretion and trigger cytotoxicity triggering a terminal UPR. Gene expression profiling in a cohort of 170 newly diagnosed multiple myeloma patients (IFM170) was used to interrogate SNAREs expression in malignant plasma cells. We developed tetracycline inducible, bicistronic vectors expressing distinct BoNT serotypes (BoNT/A-F), T2A and GFP. Lentivirally transduced cells would express BoNT in a 1:1 stochiometric ratio with GFP, upon doxycycline (dox) administration. We transduced AL cell lines stably expressing rRTA with Tet-On lentivirus expressing 7 distinct BoNTs and performed two sets of experiments. First, we performed time-course viability assays on polyclonally transduced cells and compared relative proportion of GFP+ cells over time. Then, we single-cell sorted transduced cells, triggered BoNT expression and assessed GFP kinetic and apoptosis at 24, 48 and 72 hours post dox. SNAREs cleavage following induction of BoNT expression was evaluated via WB in GFP+ clones. To assess if BoNT cytotoxicity correlated with cessation of FLC secretion, we performed a secretion assay in monoclones expressing distinct BoNTs. IFM170 GEP and AL/MM cell lines analysis showed VAMP2, VAMP3 and SNAP23 as the top expressed SNAREs. By using polyclonally transduced cells, we show that GFP+ (transduced) cells are rapidly depleted over time after dox, across all serotypes, except BoNT/B, consistent with cytotoxic effect. We noted an association between SNAP23 and VAMP3 cleavage and BoNT toxicity, suggesting that dual targeting of SNAP23/VAMP3 may be necessary to mediate BoNT cytotoxicity. We next show that only BoNTs causing early cytotoxicity (A, D, E and F) significantly inhibited FLC secretion and induced UPR activation, presumably through FLC retention. Indeed, cytotoxic BoNTs, activated PERK pathway with eIF2a phosphorylation (p-eIF2a); ATF4, CHOP and GADD34 upregulation. Importantly preliminary data in CHOP KO AL cells show rescue of BoNT_induced death, confirming that a terminal UPR is the mechanism of cytotoxicity of BoNT in AL. We show that in all seven BoNT serotypes examined, except for BoNT/B, simultaneous cleavage of SNAP23 and VAMP3 predicts BoNT cytotoxicity and correlates with cessation of FLC secretion and terminal UPR. Overall, our preliminary data provide proof of concept that targeting FLC secretion is feasible and of therapeutic efficacy in preclinical AL models, suggesting potential clinical translatability of this innovative approach. Citation Format: Maria Moscvin, Peter Gregor Czarnecki, Tianzeng Chen, Annamaria Gullá, Kenneth Carl Anderson, Giada Bianchi. Targeting protein secretion as a novel therapeutic strategy in AL amyloidosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 141.

Background AL amyloidosis (AL) is an incurable plasma cell (PC) disorder. The solely pathogenic mechanism in AL is deposition of immunoglobulin free light chains (FLC) organized in fibrils in target organs. Surprisingly, therapeutic strategies directly targeting FLC secretion are not available. SNARE proteins, which are the specific target of botulinum neurotoxin (BoNT), are involved in the docking and fusion of secretory vesicles. We hypothesized that certain BoNT serotypes may block FLC exocytosis, causing retention of FLC-loaded vesicles and triggering a terminal unfolded protein response (UPR). Methods Gene expression profiling in a cohort of 170 newly diagnosed multiple myeloma (MM) patients (IFM170) was used to interrogate SNAREs expression in malignant plasma cells. Western blotting (WB) was used to assess SNARE expression across MM and AL cell lines. We developed tetracycline inducible, lentiviral vectors expressing distinct BoNT serotypes (BoNT/A-F), T2A and GFP. Lentivirally transduced cells would express BoNT in a 1:1 stochiometric ratio with GFP, upon doxycycline (dox) administration, allowing for flow cytometry-based analysis. A vector comprising solely T2A and GFP was used as negative control. We transduced AL cell lines with Tet-On lentivirus expressing 7 distinct BoNTs and performed two sets of experiments. First, we performed time-course viability assays on polyclonally transduced cells and compared relative proportion of GFP+ cells over time. Then, we single-cell sorted transduced cells, triggered BoNT expression and assessed GFP kinetic and apoptosis via AnnexinV/DAPI flow cytometry at 24, 48 and 72 hours post dox. SNAREs cleavage following induction of BoNT expression was evaluated via WB in GFP+ clones. To assess if BoNT cytotoxicity correlated with cessation of FLC secretion, we performed a secretion assay in monoclones expressing distinct BoNTs. Results IFM170 GEP analysis showed VAMP2, VAMP3 and SNAP23 as the top expressed SNAREs. This was further confirmed in AL/MM cell lines. By using polyclonally transduced cells, we show that GFP+ cells are rapidly depleted over time after dox, across all serotypes, except BoNT/B, consistent with cytotoxic effect. Similarly, we observed rapid apoptosis in monoclones expressing any BoNT serotypes, except BoNT/B. We noted an association between SNAP23 and VAMP3 cleavage and BoNT toxicity, suggesting that dual targeting of SNAP23/VAMP3 may be necessary to mediate BoNT cytotoxicity. We next show that only BoNTs causing early cytotoxicity significantly inhibited FLC secretion. Cytotoxic BoNTs, activated PERK pathway with eIF2a phosphorylation (p-eIF2a); CHOP and GADD34 upregulation, presumably through FLC retention. Conclusions We show that cytotoxic BoNTs block FLC secretion, trigger a terminal UPR and induce apoptosis in AL and MM models. We provide proof of concept that targeting FLC secretion has a potential clinical translatability.

Intense and frequent new particle formation (NPF) events have been observed in polluted urban environments, yet the dominant mechanisms are still under debate. To understand the key species and governing processes of NPF in polluted urban environments, we conducted comprehensive measurements in downtown Beijing during January–March, 2018. We performed detailed analyses on sulfuric acid cluster composition and budget, as well as the chemical and physical properties of oxidized organic molecules (OOMs). Our results demonstrate that the fast clustering of sulfuric acid (H2SO4) and base molecules triggered the NPF events, and OOMs further helped grow the newly formed particles toward climate- and health-relevant sizes. This synergistic role of H2SO4, base species, and OOMs in NPF is likely representative of polluted urban environments where abundant H2SO4 and base species usually co-exist, and OOMs are with moderately low volatility when produced under high NOx concentrations. publishedVersion

Key Points MM cells suffer from proteotoxicity and depend on NRF1, the master regulator of proteasome stress response, for survival.KO of NRF1 or its activating protease DDI2 is cytotoxic in myeloma and overcomes de novo and acquired PI resistance., Visual Abstract, Multiple myeloma (MM) cells suffer from baseline proteotoxicity as the result of an imbalance between the load of misfolded proteins awaiting proteolysis and the capacity of the ubiquitin-proteasome system to degrade them. This intrinsic vulnerability is at the base of MM sensitivity to agents that perturb proteostasis, such as proteasome inhibitors (PIs), the mainstay of modern-day myeloma therapy. De novo and acquired PI resistance are important clinical limitations that adversely affect prognosis. The molecular mechanisms underpinning PI resistance are only partially understood, limiting the development of drugs that can overcome it. The transcription factor NRF1 is activated by the aspartic protease DNA damage inducible 1 homolog 2 (DDI2) upon proteasome insufficiency and governs proteasome biogenesis. In this article, we show that MM cells exhibit baseline NRF1 activation and are dependent upon DDI2 for survival. DDI2 knockout (KO) is cytotoxic for MM cells, both in vitro and in vivo. Protein structure-function studies show that DDI2 KO blocks NRF1 cleavage and nuclear translocation, causing impaired proteasome activity recovery upon irreversible proteasome inhibition and, thereby, increasing sensitivity to PIs. Add-back of wild-type, but not of catalytically dead DDI2, fully rescues these phenotypes. We propose that DDI2 is an unexplored promising molecular target in MM by disrupting the proteasome stress response and exacerbating proteotoxicity.

Multiple myeloma (MM), the second most common hematological malignancy, is an incurable cancer of plasma cells. MM cells diffusely involves the bone marrow (BM) and establish a close interaction with the BM niche that in turn supports MM survival, proliferation, dissemination and drug resistance. In spite of remarkable progress in understanding MM biology and developing drugs targeting MM in the context of the BM niche, acquisition of multi-class drug resistance is almost universally inevitable. Exosomes are small, secreted vesicles that have been shown to mediate bidirectional transfer of proteins, lipids, and nucleic acids between BM microenvironment and MM, supporting MM pathogenesis by promoting angiogenesis, osteolysis, and drug resistance. Exosome content has been shown to differ between MM patients and healthy donors and could potentially serve as both cancer biomarker and target for novel therapies. Furthermore, the natural nanostructure and modifiable surface properties of exosomes make them good candidates for drug delivery or novel immunomodulatory therapy. In this review we will discuss the current knowledge regarding exosome’s role in MM pathogenesis and its potential role as a novel biomarker and therapeutic tool in MM.

Stressed plants and polymer production can emit many unsaturated volatile organic esters (UVOEs). However, secondary organic aerosol (SOA) formation of UVOEs remain unclear, especially under complex ambient conditions. In this study, we mainly investigated ethyl methacrylate (EM) ozonolysis. Results showed that a substantial increase in secondary H2SO4 particles promoted SOA formation with increasing SO2. An important reason was that the homogeneous nucleation of more H2SO4 at high SO2 level provided greater surface area and volume for SOA condensation. However, increased primary H2SO4 with seed acidity enhanced EM uptake, but reduced SOA formation. This was ascribed to the fact that the ozonolysis of more adsorbed EM was hampered with the formation of surface H2SO4 at higher particle acidity. Moreover, the increase in secondary H2SO4 particle via homogeneous nucleation favored to the oligomerization of oxidation products, whereas the increasing of primary H2SO4 with acidity in the presence of seed tended to promote the functionalization conversion products. This study indicated that the role of increased H2SO4 to EM-derived SOA maybe not the same under different ambient conditions, which helps to advance our understanding of the complicated roles of H2SO4 in the formation of EM-derived SOA.

Secondary organic aerosol (SOA) is an important component of atmospheric fine particles (PM2.5), while the key factors controlling SOA formation in ambient air remain poorly understood. In this work, the SOA formation in Beijing urban ambient air was investigated using an oxidation flow reactor (OFR) with high concentrations of OH radicals. The SOA formation potential increased significantly with the increase of ambient PM2.5 concentration during the observation. The optimum ambient exposure time, which is the aging time equivalent to atmospheric oxidation (with similar OH exposure) associated with the peak SOA formation, varied between 2 and 4 days in this study. The OA enhancement in this study was much higher than that of developed countries under different environmental conditions. The higher OA enhancement is probably due to the higher concentrations of volatile organic compounds (VOCs) in the urban air of Beijing. This might also have occurred because fragmentation did not dominate in the oxidation ...