<p> 1Supplementary Materials & Methods</p><p>2</p><p>3Cell lines, reagents and patient samples</p><p>4Multiple myeloma cell lines (U266, H929, MM.1S) were obtained from ATCC and</p><p>5grown in RPMI 1640 supplemented with 10% heat inactivated FCS (Sigma), 60</p><p>6ug/mL penicillin, and 100 ug/mL streptomycin. PI-103 (Calbiochem), Bafilomycin A1</p><p>7and Rapamycin (Sigma Aldrich), Bortezomib (Cambridge Bioscience) and BEZ235</p><p>8(Synkinase) were reconstituted in DMSO while Chloroquine (Sigma Aldrich) was</p><p>9reconstituted in PBS. Following informed consent, myeloma cells were obtained from</p><p>10bone marrow aspirates using Ficoll-PaqueTM separation gradient (Invitrogen)</p><p>11followed by CD138 selection by magnetic-assisted cell sorting (Miltenyi Biotec). Cells</p><p>12were cultured for 24 hours as above. Similarly bone marrow stromal cells (BMSCs)</p><p>13were obtained from aspirates using a Ficoll-PaqueTM separation gradient (Invitrogen)</p><p>14and cells were cultured in RPMI 1640 supplemented with 20% heat inactivated FCS</p><p>15for 24-72 hours before any non-adherent cells were removed. Adherent cells were</p><p>16grown for up to 8 weeks with the media changed every 3-4 days. </p><p>17</p><p>18Detection and quantification of acidic acridine orange-positive vesicles</p><p>19Acidic vesicles were detected by staining cells with acridine orange (Sigma Aldrich)</p><p>20at a final concentration of 1.5 µg/ml for 15 minutes at 37 °C. Vesicles were visualised</p><p>21under a Leica LSM700 confocal microscope at 63 times magnification (Carl Zeiss</p><p>22Inc.) using the spectral unmixing function of the Zen 2009 software. Alternatively,</p><p>23cells were harvested, washed and analysed by flow cytometry using the ratio of red</p><p>24(660nm) to green (530nm) fluorescence.</p><p>25</p><p>1 1 2 26Immunoblotting </p><p>27Protein was extracted as previously described1 and the concentration measured</p><p>28using a BCA protein assay (Pierce). For preparation of insoluble fractions, cell pellets</p><p>29were resuspended in lysis buffer and subjected to sonication. Primary antibodies</p><p>30were purchased from Cell Signalling Technology (ubiquitin, phospho (Ser2448) and</p><p>31total mTOR, phospho (Ser473) and total AKT), Novus Biologicals (LC3), Sigma</p><p>32Alridch (p62, actin). Secondary antibodies (anti-mouse and anti-rabbit) conjugated to</p><p>33horseradish peroxidase (GE Healthcare and Cell Signalling Technology) were used</p><p>34and ECL-Plus (GE Healthcare) was used for detection.</p><p>35</p><p>36Proteasome assay</p><p>37Cells were lysed in buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM</p><p>38EGTA, 1% Triton) containing 1x protease inhibitor cocktail (Roche) and 1mM Na3VO4</p><p>39(Sigma Aldrich). Proteasome chymotrypsin-like activity was determined using</p><p>40proteasome assay buffer and SucLLVY-AMC as the substrate (Enzo Life Sciences)</p><p>41and 5 µg cell lysate per well. Plates were read at one minute intervals on a Mithras</p><p>42LB 940 plate reader (Berthold Technologies) using 355 nM excitation and 460 nM</p><p>43emission wavelengths. Kinetic data were obtained from the linear part of curves</p><p>44representing increased fluorescence due to substrate cleavage and expressed as a</p><p>45percentage of the control. P values were calculated using a two-tailed t-test.</p><p>46</p><p>47RNA extraction, amplification and Real-Time Polymerase Chain Reaction </p><p>48RNA was extracted and amplified as previously described. Details of CHOP, ATF4,</p><p>49XBP1/XBP1s and ACTNB primer sequences and thermal cycling conditions are also</p><p>50described. </p><p>3 2 4 51</p><p>52Competitive binding assay</p><p>53Cells were treated for 24 hours with either PI-103 or Bortezomib, harvested, washed</p><p>54and replated in medium containing 2μmol/l proteasome activity probe (Me4BodipyFL-</p><p>3 55Ahx3Leu3VS; R&D Systems) for 2 hours. Protein was then extracted and quantified</p><p>56as above. Lysates were separated by SDS-PAGE and fluorescent images were</p><p>57captured on a LAS 4000 imager (Fuji).</p><p>58</p><p>59RNA extraction for microarrays, cDNA synthesis and array hybridization </p><p>60Triplicate RNA was extracted from MM.1S cells treated for 0, 6 or 12 hours with</p><p>611μmol/l PI-103 using RNeasy Plus mini kit (Qiagen). RNA was quantified on a</p><p>62Nanodrop spectrophotometer and purity and integrity evaluated using the small RNA</p><p>63chips on the Bioanalyser 2100 (Agilent) according to manufacturer’s instructions.</p><p>64First cycle cDNA was synthesized from 100ng total RNA and 10μg cRNA was used</p><p>65to synthesize second cycle cDNA using the Ambion WT Expression kit (Life</p><p>66Technologies). Single sense strand DNA (5.5μg) was fragmented and labelled using</p><p>67the GeneChip WT Terminal Labeling kit (Affymetrix) and hybridized to Human Gene</p><p>68ST 2.0 arrays (Afymetrix) using the GeneChip Hybridiztion, Wash and Stain kit</p><p>69(Affymetrix) as per the protocol. Arrays were washed on the Affymetrix Fluidics</p><p>70Station 450 and scanned on a GeneChip Scanner 3000.</p><p>71</p><p>72Microarray analysis</p><p>73Background correction, normalization and transcript cluster summaries were</p><p>74performed at the gene level in Affymetrix Expression Console using robust multi-</p><p>75array (RMA) sketch.4 Transcript cluster log expression values were used to detect</p><p>5 3 6 76differentially expressed genes with at least a 1.5 fold change and p value with false</p><p>77discovery rate of <0.05 using 1 way analysis of variance in the Genomics Suite</p><p>78(Partek). Heatmaps were generated from these genes by hierarchical clustering. The</p><p>79list of proteasome and autophagy genes were taken from public databases</p><p>80(http://www.genenames.org/; http://autophagy.lu/index.html) and research papers.5-8</p><p>81</p><p>82Proliferation, survival and cell cycle assays</p><p>83Proliferation was measured by MTT (Sigma-Aldrich) or Wst-1 (Roche) assays</p><p>84according to manufacturer’s instructions. P values were calculated using a two-tailed</p><p>85t-test. For co-culture experiments, BMSC were grown in phenol-free medium for 4</p><p>86days, the medium collected and used in an MTT assay. Plates were read on a</p><p>87Dynatech Laboratories (Billingshurst) MRX plate reader. Cell death was verified in</p><p>88treated cells using trypan blue exclusion and Annexin V/propidium iodide binding (BD</p><p>89Biosciences) as described previously. </p><p>90</p><p>91References</p><p>921. Davenport, EL, Moore, HE, Dunlop, AS, Sharp, SY, Workman, P, Morgan, GJ, et al. Heat shock protein 93inhibition is associated with activation of the unfolded protein response pathway in myeloma plasma cells. 94Blood 2007; 110: 2641-2649. 952. Moore, HE, Davenport, EL, Smith, EM, Muralikrishnan, S, Dunlop, AS, Walker, BA, et al. 96Aminopeptidase inhibition as a targeted treatment strategy in myeloma. Mol Cancer Ther 2009; 8: 762-770. 973. Berkers, CR, van Leeuwen, FWB, Groothuis, TA, Peperzak, V, van Tilburg, EW, Borst, J, et al. 98Profiling Proteasome Activity in Tissue with Fluorescent Probes. Molecular Pharmaceutics 2007; 4: 739-748. 994. Irizarry, RA, Bolstad, BM, Collin, F, Cope, LM, Hobbs, B, Speed, TP. Summaries of Affymetrix 100GeneChip probe level data. Nucleic Acids Research 2003; 31: e15. 1015. Behrends, C, Sowa, ME, Gygi, SP, Harper, JW. Network organization of the human autophagy system. 102Nature 2010; 466: 68-76. 1036. Mizushima, N, Yoshimori, T, Ohsumi, Y. The role of Atg proteins in autophagosome formation. Annual 104review of cell and developmental biology 2011; 27: 107-132. 1057. Schulman, BA, Wade Harper, J. Ubiquitin-like protein activation by E1 enzymes: the apex for 106downstream signalling pathways. Nat Rev Mol Cell Biol 2009; 10: 319-331. 1078. Ye, Y, Rape, M. Building ubiquitin chains: E2 enzymes at work. Nat Rev Mol Cell Biol 2009; 10: 755- 108764. 109 110</p><p>111Supplementary Table 1. Relative expression of proteasome, ubiquitin E1 and E2</p><p>7 4 8 112genes that are significantly changed over 6 and 12 hour treatment with PI-103</p><p>Affymetrix Fold Change p-value (12 and Transcript Gene symbol 0 hr 6 hr 12 hr (12 and 6 vs. 0) 6 vs. 0) Cluster ID 16925674 PSMG1 1.309133 -0.54637 -0.76277 -2.67895 6.52E-06 16886919 PSMD14 1.205157 -0.21343 -0.99173 -2.1312 0.000154895 16791059 PSME2 1.139209 -0.06066 -1.07855 -2.00231 0.00026903 16694429 UBQLN4 1.299657 -0.63952 -0.66015 -1.92519 3.93E-05 16892039 PSMD1 1.204643 -0.14977 -1.05488 -1.89868 1.43E-05 16837029 PSMC5 1.123883 -0.00418 -1.11971 -1.86176 0.000126451 16698023 UBE2T 1.221935 -0.34811 -0.87383 -1.81504 0.000487049 16848032 PSMD12 1.297567 -0.53918 -0.75838 -1.81294 2.77E-05 16833139 PSMD11 1.234783 -0.27384 -0.96094 -1.77358 6.59E-05 16834486 PSME3 1.32088 -0.69279 -0.62809 -1.76133 1.78E-06 16784642 PSMA3 1.208756 -0.52851 -0.68024 -1.73708 0.0017845 17054460 PSMG3 1.096645 0.030252 -1.1269 -1.6529 0.000293898 16926412 UBE2G2 1.211882 -0.40207 -0.80981 -1.63959 0.00105934 16738023 PSMC3 1.103181 0.060993 -1.16418 -1.61702 3.37E-05 16862145 PSMC4 1.197533 -0.12149 -1.07605 -1.61328 6.29E-06 17115118 HAUS7 1.102149 -0.12759 -0.97456 -1.59844 0.00318017 17104176 UBQLN2 1.249344 -0.89239 -0.35696 -1.59844 0.000120645 16803540 PSMA4 1.246477 -0.72165 -0.52483 -1.58557 0.000561097 16827170 NAE1 1.234633 -0.3228 -0.91183 -1.5351 0.000183953 16945777 UBA5 1.172803 -0.65481 -0.51799 -1.51572 0.00383206 17117724 PSMD3 1.013678 -0.42189 -0.59178 -1.51222 0.027867 16989243 UBE2B -1.21934 0.969223 0.250122 1.50004 0.000129665 17062811 UBE2H -1.25551 0.852185 0.403324 1.56193 0.000155958 113</p><p>114</p><p>115</p><p>116</p><p>117</p><p>118</p><p>119</p><p>120</p><p>121</p><p>122Supplementary Table 2. Relative expression of autophagy genes that are</p><p>123significantly changed over 6 and 12 hour treatment with PI-103</p><p>9 5 10 Affymetrix Gene Fold Change p-value (12 Transcript 0 hr 6 hr 12 hr symbol (12 and 6 vs. 0) and 6 vs. 0) Cluster ID 16748304 GABARAPL1 -1.31456 0.500743 0.813818 5.65032 2.92E-07 17086496 DAPK1 -1.32238 0.671534 0.650851 3.02793 1.36E-06 16834578 NBR1 -1.32007 0.737052 0.583016 2.51112 1.15E-06 17110771 WDR45 -1.27674 0.331134 0.94561 2.37293 7.27E-07 16794441 ZFYVE1 -1.30658 0.721058 0.585521 2.30537 1.53E-05 16740161 ATG2A -1.24233 0.769153 0.473171 2.03967 0.000529 16759350 ULK1 -1.31717 0.671454 0.645711 2.03261 4.35E-06 17077135 RB1CC1 -1.32301 0.616107 0.706902 2.02792 8.44E-07 16752322 RAB5B -1.28266 0.645171 0.637492 1.78386 0.000133 16724358 ATG13 -1.29637 0.668318 0.628058 1.74715 5.13E-05 16964764 TBC1D14 -1.30059 0.739341 0.561252 1.74513 2.49E-05 16796479 ATG2B -1.1945 0.449237 0.745266 1.70133 0.002098 16848079 WIPI1 -1.28889 0.48 0.808888 1.6529 3.14E-05 17043230 WIPI2 -1.24397 0.908716 0.335254 1.63014 0.000122 16793263 ATG14 -1.24878 0.424714 0.824071 1.57644 0.000285 16908656 ATG9A -1.19929 0.946145 0.253149 1.54935 0.000445 17084866 GLIPR2 1.15395 -0.10535 -1.04859 -2.24752 0.000335</p><p>124 125 126 127 128</p><p>129</p><p>130</p><p>131</p><p>132</p><p>133</p><p>134 135 136</p><p>11 6 12</p>