Supplementary Figure 6 - PDF file 74K, Synergistic interaction of YM155 and CTLs. In A, the effect of YM155 on survivin and MCL-1 protein levels of MM cells is shown. The cells were cultured with YM155 during 24h and the western blot assays were carried out as indicated in material methods. Band intensities were quantified using image J software, calculated as relative values to the control protein tubuline and plotted as relative to the expression levels of cells cultured without YM155. In B, the type of interaction between YM155 and CTLs (see figure 5C-D) in the presence of accessory cells was analyzed using Compusyn software (version 1.0, 2004 ). In the plots (A) the CI values corresponding to the doses in figure 5 are shown. A CI value of <1, 1 or >1 indicates synergy, additive effects or antagonism, respectively. In C the supernatants of assays as described in Fig 5C-D granzyme B release was measured using ELISA (B). Results are expressed as the mean values of the triplicate cultures. Error bars represent the SEM. Results are representative of 3 independent assays

PDF file - 111K, GVHD and GvT regulation in the LME-1 MM tumor model. (A) GvHD scores at weeks 3, 4,and 5. The total score is the sum of the scores for weight loss ( 0=0-10 %; 1=10-20%; 2=>20%. ) , mobility (0= mobile; 1= diminished mobility;2 = immobile) and fur appearance (0=normal; 1= ruffled fur; 2= ruffled fur + red swollen skin; 3= ruffled fur + red swollen skin + patchy alopecia) (B) CD4 and CD8 T cell counts in peripheral blood, BM and spleen at week 3. (C) BLI imaging results of three representative animals per group at week 3. The depicted areas indicate the regions of extramedullar vs medullar tumors . The BLI counts in the corresponding table are in thousands. For each mouse total, extramedullary, and medullary ( total-extramedullary) tumor load are indicated separately. The mean values of the ratio of extramedullary vs medullary tumor load are also depicted.

Transfer of surface membrane from UM9 cells to monocytes.

Supplementary Figure 3 - PDF file 55K, RGDw reduces adhesion of MM cells to accessory cells. Adherent HS-5 en HUVEC accessory cells were incubated with RGDw or an irrelevant peptide for 1hour. After washing away the unbound peptide, UM9 (A) or U266 (B) cells were added and incubated overnight. Plates were reversely centrifuged to determine adhesion to accessory cells. Results represent the mean values of triplicate cultures (+/- SEM). Differences in adhesion were tested by unpaired two tailed student's t test. *= p<0.05; **= p<0.01, ***= p<0.001

Supplementary Figure Legends - PDF file 73K, Legends for supplementary figures

Supplementary Figure 1 - PDF file 53K, Antigen specific, HLA-restricted and dose-dependent recognition of the MM cell lines U266 and UM9, but not of accessory cells by CD8+ and CD4+ mHag-specific CTLs. The CD4+ (A) and CD8+ (B) CTLs were co-cultured with the luc+ MM cell lines UM9 and U266 in different effector to target ratios. The HLA- and mHag typing of these MM cell lines are indicated. MM cell survival was determined 24 h after addition of T cells by CS-BLI. Results represent the mean values of triplicate cultures (+/- SEM). Results are representative of 3 independent assays. In (C and D) CD4+ (C) and CD8+ (D) CTLs were co-cultured with accessory cells. Accessory cell survival was determined 24h after addition of T cells by FACS annexine V/ propidium iodide staining of CD4 and CD8 negative cells in the co-cultures. Results are depicted as % surviving cells, measured as relative number annexin V/propidium iodide negative cells, compared to no T cells control

Supplementary Figure 1. The LB-TTK-1D-specific (clone 10-5) and USP11-specific T cell clones have a similar affinity for their cognate peptide. Synthetic peptides RLHDGRVFV (peptideTTK) or FTWEGLYNV (peptideUSP11) were loaded on T2 cells in a serial dilution. The depicted T cell clones were stimulated using these target cells followed by determination of IFN-γ secretion in the supernatant (ELISA). Calculated EC50 values are depicted.

Supplementary Figure 5 - PDF file 41K, Bortezomib does not improve the CTL mediated lysis of Fas negative L363 cellsL3L3 reactive CD8+CTLs were incubated at the indicated effector to target ratios with luc-transduced L3L3 cells alone or in the presence of various dilutions of bortezomib. The CTL mediated and bortezomib mediated lysis of L3L3 cells was determined after 48 hours by BLI. Results are depicted as % surviving cells using the triplicate cultures without T cells and bortezomib as the 100% survival control

Supplementary Figure 2 - PDF file 50K, Accessory cells protect MM cells from WT-1 specific CTLs. U266 cells were co-cultured with WT-1 specific T cells in presence and absence of different accessory cells. MM cell viability was determined 24h after addition of T cells by CS-BLI (A). Granzyme B production in the supernatant was determined by ELISA (B). Significance of inhibition of MM cell survival in the presence of accessory cells was tested by unpaired two tailed student's t test. *= p<0.05; **= p<0.01

Daratumumab-mediated CD38 reduction of MM cells in the presence of PBMCs as shown by Western blot analysis.

Supplementary Figure 2. The generation of the LB-TTK-1D peptide is proteasome-dependent. LB-TTK-1D antigen positive EBV-LCLs were treated with increasing concentrations of the proteasome inhibitor bortezomib for 48h, washed and exposed to LB-TTK-1D-specific T cells (clone 10-5). T cell-produced IFN-γ was determined in the supernatant of the cultures by ELISA. Depicted are OD450 values.

Loss of CD38 from the MM cell membrane is associated with reduced CDC and ADCC

Monocytes take up AF488-labeled daratumumab-CD38 complexes from PKH-26-stained UM9 cells.

Supplementary Figure 4 - PDF file 219K, FasL upregulation on CTLs upon MM cell recognition is not hampered by presence of accessory cells. CD4+ (A) and CD8+ (B) CTLs were cultured alone (unstimulated) or in the presence of tumor cells and in presence or absence of accessory cells as indicated. After 3 hours of co-incubation CTLs were evaluated for CD178 (Fas ligand) expression. Percentage of FasL expressing cells is indicated

Daratumumab is transferred from UM9 cells to monocytes.

Supplemental Methods, Supplementary Tables, Supplementary Figure Legends

Cancer patients benefit from early tumor detection since treatment outcomes are more favorable for less advanced cancers. Platelets are involved in cancer progression and are considered a promising biosource for cancer detection, as they alter their RNA content upon local and systemic cues. We show that tumor-educated platelet (TEP) RNA-based blood tests enable the detection of 18 cancer types. With 99% specificity in asymptomatic controls, thromboSeq correctly detected the presence of cancer in two-thirds of 1,096 blood samples from stage I-IV cancer patients and in half of 352 stage I-III tumors. Symptomatic controls, including inflammatory and cardiovascular diseases, and benign tumors had increased false-positive test results with an average specificity of 78%. Moreover, thromboSeq determined the tumor site of origin in five different tumor types correctly in over 80% of the cancer patients. These results highlight the potential properties of TEP-derived RNA panels to supplement current approaches for blood-based cancer screening.

Lenalidomide (LEN) maintenance (MT) post autologous stem cell transplantation (ASCT) is standard of care in newly diagnosed multiple myeloma (MM) but has not been compared to other agents in clinical trials. We retrospectively compared bortezomib (BTZ; n = 138) or LEN ( n = 183) MT from two subsequent GMMG phase III trials. All patients received three cycles of BTZ-based triplet induction and post-ASCT MT. BTZ MT (1.3 mg/m 2 i.v.) was administered every 2 weeks for 2 years. LEN MT included two consolidation cycles (25 mg p.o., days 1–21 of 28 day cycles) followed by 10–15 mg/day for 2 years. The BTZ cohort more frequently received tandem ASCT (91% vs. 33%) due to different tandem ASCT strategies. In the LEN and BTZ cohort, 43% and 46% of patients completed 2 years of MT as intended ( p = 0.57). Progression-free survival (PFS; HR = 0.83, p = 0.18) and overall survival (OS; HR = 0.70, p = 0.15) did not differ significantly with LEN vs. BTZ MT. Patients with <nCR after first ASCT were assigned tandem ASCT in both trials. In patients with <nCR and tandem ASCT (LEN: n = 54 vs. BTZ: n = 84), LEN MT significantly improved PFS (HR = 0.61, p = 0.04) but not OS (HR = 0.46, p = 0.09). In conclusion, the significant PFS benefit after eliminating the impact of different tandem ASCT rates supports the current standard of LEN MT after ASCT.

Over the past decades, immunomodulatory drugs, proteasome inhibitors, and targeted antibodies have significantly improved the overall survival of multiple myeloma (MM) patients. Nonetheless, the disease remains incurable as patients ultimately develop resistance to all available modalities. One of the well-known mechanisms of therapy resistance in MM is evasion of apoptosis, characterized by an up-regulation of anti-apoptotic proteins of the Bcl-2 family and/or inhibitor of apoptosis (IAP) proteins including Survivin, whose expression is associated with a worse clinical outcome. Apoptosis resistance can be MM cell intrinsic, but can also be induced by their interactions with accessory cells and the extracellular matrix in the bone marrow (BM). Inside-out signaling via integrins, signaling via NOTCH, or soluble factors like IL6, can significantly contribute to this microenvironmentmediated drug resistance (EM-DR). To overcome apoptosis resistance, Li et al. have recently screened for compounds that could alter the expression of Survivin in tumor cells and described a new small molecule, FL118. Although FL118 was identified as a camptothecin analog, it appeared to inhibit the promoter activity of Survivin and its gene expression with significantly greater efficiency than it could inhibit DNA topoisomerase-I. In addition, FL118 was shown to selectively and independently inhibit additional anti-apoptotic genes including Mcl-1, XIAP and cIAP2. In preclinical settings, FL118 showed a favorable toxicity profile in experimental animals, and an effective antitumor activity against human colon and head-and-neck tumors in a topoisomerase Iand P53 status-independent manner. The latter is of importance for MM, since mutations or the loss of the P53 encoding gene, through the deletion of chromosome 17p, are characteristics of high-risk MM with poor prognosis. Prompted by these encouraging results, we have addressed here for the first time the anti-MM activity of FL118 in vitro and in vivo. We first evaluated FL118 against a panel of six MM cell lines with a different P53 status (Online Supplementary Table S1). Since stromal cells can induce EM-DR, the assays were executed in the presence or absence of MM patient-derived BM mesenchymal stromal cells (BMMSC). While FL118 showed only minimal toxicity against BMMSC (Online Supplementary Figure S1), it exhibited a clear dose-dependent anti-MM activity in the MM cell lines, independent of the P53 status, with the half maximal effective concentration (EC50) values ranging from 7.4 nmol/L to 344.8 nmol/L (Figure 1A). Importantly, lysis of MM cell lines by FL118 was not reduced, or even increased, in the presence of BMMSC for five of six MM cell lines, except for a minimal reduc-