Cell Lines, Reagents and Patient Samples

1Supplementary Materials & Methods

3Cell lines, reagents and patient samples

4Multiple myeloma cell lines (U266, H929, MM.1S) were obtained from ATCC and

5grown in RPMI 1640 supplemented with 10% heat inactivated FCS (Sigma), 60

6ug/mL penicillin, and 100 ug/mL streptomycin. PI-103 (Calbiochem), Bafilomycin A1

7and Rapamycin (Sigma Aldrich), Bortezomib (Cambridge Bioscience) and BEZ235

8(Synkinase) were reconstituted in DMSO while Chloroquine (Sigma Aldrich) was

9reconstituted in PBS. Following informed consent, myeloma cells were obtained from

10bone marrow aspirates using Ficoll-PaqueTM separation gradient (Invitrogen)

11followed by CD138 selection by magnetic-assisted cell sorting (Miltenyi Biotec). Cells

12were cultured for 24 hours as above. Similarly bone marrow stromal cells (BMSCs)

13were obtained from aspirates using a Ficoll-PaqueTM separation gradient (Invitrogen)

14and cells were cultured in RPMI 1640 supplemented with 20% heat inactivated FCS

15for 24-72 hours before any non-adherent cells were removed. Adherent cells were

16grown for up to 8 weeks with the media changed every 3-4 days.

18Detection and quantification of acidic acridine orange-positive vesicles

19Acidic vesicles were detected by staining cells with acridine orange (Sigma Aldrich)

20at a final concentration of 1.5 µg/ml for 15 minutes at 37 °C. Vesicles were visualised

21under a Leica LSM700 confocal microscope at 63 times magnification (Carl Zeiss

22Inc.) using the spectral unmixing function of the Zen 2009 software. Alternatively,

23cells were harvested, washed and analysed by flow cytometry using the ratio of red

24(660nm) to green (530nm) fluorescence.

1 1 2 26Immunoblotting

27Protein was extracted as previously described1 and the concentration measured

28using a BCA protein assay (Pierce). For preparation of insoluble fractions, cell pellets

29were resuspended in lysis buffer and subjected to sonication. Primary antibodies

30were purchased from Cell Signalling Technology (ubiquitin, phospho (Ser2448) and

31total mTOR, phospho (Ser473) and total AKT), Novus Biologicals (LC3), Sigma

32Alridch (p62, actin). Secondary antibodies (anti-mouse and anti-rabbit) conjugated to

33horseradish peroxidase (GE Healthcare and Cell Signalling Technology) were used

34and ECL-Plus (GE Healthcare) was used for detection.

36Proteasome assay

37Cells were lysed in buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM

38EGTA, 1% Triton) containing 1x protease inhibitor cocktail (Roche) and 1mM Na3VO4

39(Sigma Aldrich). Proteasome chymotrypsin-like activity was determined using

40proteasome assay buffer and SucLLVY-AMC as the substrate (Enzo Life Sciences)

41and 5 µg cell lysate per well. Plates were read at one minute intervals on a Mithras

42LB 940 plate reader (Berthold Technologies) using 355 nM excitation and 460 nM

43emission wavelengths. Kinetic data were obtained from the linear part of curves

44representing increased fluorescence due to substrate cleavage and expressed as a

45percentage of the control. P values were calculated using a two-tailed t-test.

47RNA extraction, amplification and Real-Time Polymerase Chain Reaction

48RNA was extracted and amplified as previously described. Details of CHOP, ATF4,

49XBP1/XBP1s and ACTNB primer sequences and thermal cycling conditions are also

50described.

3 2 4 51

52Competitive binding assay

53Cells were treated for 24 hours with either PI-103 or Bortezomib, harvested, washed

54and replated in medium containing 2μmol/l proteasome activity probe (Me4BodipyFL-

3 55Ahx3Leu3VS; R&D Systems) for 2 hours. Protein was then extracted and quantified

56as above. Lysates were separated by SDS-PAGE and fluorescent images were

57captured on a LAS 4000 imager (Fuji).

59RNA extraction for microarrays, cDNA synthesis and array hybridization

60Triplicate RNA was extracted from MM.1S cells treated for 0, 6 or 12 hours with

611μmol/l PI-103 using RNeasy Plus mini kit (Qiagen). RNA was quantified on a

62Nanodrop spectrophotometer and purity and integrity evaluated using the small RNA

63chips on the Bioanalyser 2100 (Agilent) according to manufacturer’s instructions.

64First cycle cDNA was synthesized from 100ng total RNA and 10μg cRNA was used

65to synthesize second cycle cDNA using the Ambion WT Expression kit (Life

66Technologies). Single sense strand DNA (5.5μg) was fragmented and labelled using

67the GeneChip WT Terminal Labeling kit (Affymetrix) and hybridized to Human Gene

68ST 2.0 arrays (Afymetrix) using the GeneChip Hybridiztion, Wash and Stain kit

69(Affymetrix) as per the protocol. Arrays were washed on the Affymetrix Fluidics

70Station 450 and scanned on a GeneChip Scanner 3000.

72Microarray analysis

73Background correction, normalization and transcript cluster summaries were

74performed at the gene level in Affymetrix Expression Console using robust multi-

75array (RMA) sketch.4 Transcript cluster log expression values were used to detect

5 3 6 76differentially expressed genes with at least a 1.5 fold change and p value with false

77discovery rate of <0.05 using 1 way analysis of variance in the Genomics Suite

78(Partek). Heatmaps were generated from these genes by hierarchical clustering. The

79list of proteasome and autophagy genes were taken from public databases

80(http://www.genenames.org/; http://autophagy.lu/index.html) and research papers.5-8

82Proliferation, survival and cell cycle assays

83Proliferation was measured by MTT (Sigma-Aldrich) or Wst-1 (Roche) assays

84according to manufacturer’s instructions. P values were calculated using a two-tailed

85t-test. For co-culture experiments, BMSC were grown in phenol-free medium for 4

86days, the medium collected and used in an MTT assay. Plates were read on a

87Dynatech Laboratories (Billingshurst) MRX plate reader. Cell death was verified in

88treated cells using trypan blue exclusion and Annexin V/propidium iodide binding (BD

89Biosciences) as described previously.

91References

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

111Supplementary Table 1. Relative expression of proteasome, ubiquitin E1 and E2

7 4 8 112genes that are significantly changed over 6 and 12 hour treatment with PI-103

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

122Supplementary Table 2. Relative expression of autophagy genes that are

123significantly changed over 6 and 12 hour treatment with PI-103

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

124 125 126 127 128

134 135 136

11 6 12