Letters to the Editor 1163 13 Kuruvilla J, Gutierrez M, Shah BD, Gabrail NY, de Nully Brown P, 14 Yu L, Mohamed AJ, Simonson OE, Vargas L, Blomberg KE, Bjorkstrand B et al. Stone RM et al. Preliminary evidence of anti tumor activity of selinexor Proteasome-dependent autoregulation of Bruton tyrosine kinase (Btk) promoter (KPT-330) in a phase I trial of a first-in-class oral selective inhibitor via NF-kappaB. Blood 2008; 111: 4617–4626. of nuclear export (SINE) in patients (pts) with relapsed/refractory non 15BurgerJA,BurgerM,KippsTJ.Chronic lymphocytic B cells Hodgkin’s (NHL) and chronic lymphocytic leukemia (CLL). Blood 2013; express functional CXCR4 chemokine receptors that mediate spontaneous 122: 90. migration beneath bone marrow stromal cells. Blood 1999; 94: 3658–3667.

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

B-cell receptor pathway inhibitors affect CD20 levels and impair antitumor activity of anti-CD20 monoclonal

Leukemia (2014) 28, 1163–1167; doi:10.1038/leu.2014.12 also tested a primary MCL sample and upon treatment with BCR inhibitors observed a significant downregulation of surface CD20 levels and a trend towards impaired R-CDC and O-CDC (Supplementary Figure 1b). Moreover, we determined the Signaling via the aberrantly activated B-cell receptor (BCR) has a influence of BCR inhibitors on CD20 surface levels in a critical role in the pathogenesis of B-cell tumors by promoting series of 15 tumor cell lines, including Burkitt’s lymphoma (Ramos, survival and clonal expansion of malignant B cells.1,2 Multiple Daudi and BJAB), ALL (NALM-6), diffuse large B-cell lymphoma preclinical studies indicate that blocking various components of (BCR-dependent Ly-1, Ly-7, Ly-10, DHL-6, HBL-1, U2932 and the BCR signaling pathway holds a great therapeutic potential in BCR-independent Ly-4, Ly-19, Pfeiffer) and CLL (EHEB and MEC-1). the treatment of B-cell and .3,4 This is further We observed a significantly decreased surface CD20 levels in most supported by clinical data from recent and ongoing clinical trials, CD20-positive tumor cells irrespectively of the tumor type/subtype which demonstrate considerable activity of SYK inhibitors (Figure 1e). Surprisingly, as opposed to the primary CLL samples we (fostamatinib and GS-9973),5 BTK inhibitors (, AVL-292, did not observe a significant CD20 downregulation in either of CC-292 and ONO-4059)6,7 and PI3Kd inhibitor (CAL-101)8 as single the two tested CLL cell lines (Figure 1e). Since anti-CD20 mAbs are agents or in combination with other therapies in patients with known to also mediate -dependent cell-mediated cytotoxi- 10 B-cell tumors, for whom -based regimens have become a city (ADCC), we carried out additional analyses to measure the standard of care. However, tumor relapses occur in a significant effects of R406, ibrutinib or CAL-101 on ADCC in various settings. percentage of patients treated even with the most effective Pre-incubation of Raji cells with the increasing concentrations of modalities. Therefore, combinations of anti-CD20 monoclonal inhibitors, followed by a coculture with rituximab and NK effector antibodies (mAbs) with targeted therapeutics inhibiting BCR cells in the absence of tested drugs, failed to influence R-ADCC, as signaling pathways have recently become an active area of determined by CD107a mobilization (Figure 1f). However, in a investigation. For example, ibrutinib and CAL-101 are currently clinically more relevant co-incubation model, where both target Raji being investigated in combination with chemotherapy and/or cells and effector NK cells were cocultured for 4 h in presence of BCR anti-CD20 mAbs (rituximab and ) in several phase I, II inhibitors, rituximab-induced degranulation of NK cells was severely and III clinical trials (summarized in Kharfan-Dabaja et al.9). impaired at 1 mM of R406 or ibrutinib (Figure 1f). Similarly, in a Conspicuously, the antitumor effects of these combinations have co-incubation model R406 and ibrutinib inhibited NK cytotoxicity not been studied in the preclinical setting. Therefore, we decided against rituximab- (Figure 1g) and ofatumumab-opsonized to investigate the molecular interactions between anti-CD20 (Supplementary Figure 1c) carboxyfluorescein succinimidyl ester mAbs and R406 (active metabolite of fostamatinib), ibrutinib (CFSE)-labeled Raji cells as assessed using flow cytometry. Moreover, and CAL-101. the release of tumor factor (TNF) and interferon (IFN)-g from Startlingly, we observed a significant impairment of ritux- NK cells incubated with either R406 or ibrutinib was severely imab-(R-CDC, Figure 1a) and ofatumumab-induced comple- impaired (Figure 1h). Noteworthy, even the highest concentrations of ment-dependent cytotoxicity (O-CDC) (Supplementary Figure 1a) in all tested BCR inhibitors did not affect the viability of NK cells in a 48- Raji cells pre-incubated for 48 h with increasing concentrations of h culture (Supplementary Figure 1d). Conspicuously, CAL-101 exerted R406, ibrutinib or CAL-101, when compared with controls. The no antagonistic effect on NK cell cytotoxicity, CD107a mobilization highest tested concentration (1 mM) of R406 and ibrutinib almost and cytokine secretion in a co-incubation model. This is in 11 completely abrogated both R-CDC and O-CDC, whereas CAL-101 accordance with a study by Herman et al., where CAL-101 was affected anti-CD20 mAb-mediated CDC to a lesser extent. Flow reported not to interfere with rituximab-mediated or - cytometry studies further revealed that pre-incubation of Raji cells mediated ADCC, although it diminished IFN-g production by NK cells. with BCR pathway inhibitors severely impaired the binding of The levels of complement regulatory (CD46 and CD55) various anti-CD20 mAbs (fluorescein isothiocyanate-conjugated L27 as well as other B-cell surface antigens (CD38 and CD19) remained clone (Figure 1b), rituximab and ofatumumab (Figure 1c)). All three roughly unchanged upon treatment with BCR inhibitors compounds induced a downregulation of surface CD20 levels in a (Supplementary Figure 2). However, we observed a significant panel of 26 primary chronic lymphocytic leukemia (CLL) samples downregulation of CD21 and CD22, of which CD22 is currently pre-incubated for 48 h with 1 mM of each BCR signaling inhibitor used as a target for toxin-conjugated anti-CD22 mAbs (inotuzu- (Figure 1d). Since ibrutinib has been recently approved as a second- mab ozogamicin). Thus, our results may suggest that CD20 line monotherapy for patients with (MCL), we downregulation is not an isolated phenomenon and BCR

Accepted article preview online 10 January 2014; advance online publication, 4 February 2014

& 2014 Macmillan Publishers Limited Leukemia (2014) 1129 – 1174 Letters to the Editor 1164

Leukemia (2014) 1129 – 1174 & 2014 Macmillan Publishers Limited Letters to the Editor 1165 inhibitors could potentially reduce the activity of other mAbs- incubation with R406, ibrutinib or CAL-101, we observed a based modalities. significant decrease in luciferase activity (Figure 2e, black bars). The selectivity of small-molecule signaling inhibitors is rather Since stimulation of BCR signaling is followed by recruitment and limited, and most inhibitors target various kinases. Therefore, to activation of SYK, we co-transfected Raji cells with the CD20 investigate whether modulation of CD20 levels results from promoter reporter and the TEL–SYK fusion ensuring inhibition of selected components of BCR signaling, we efficiently constitutive autophosphorylation and activation of SYK. knocked down SYK and BTK with short hairpin RNA (shRNA) Expression of TEL–SYK protected Raji cells from R406- or (Figure 2a) and using flow cytometry observed significantly ibrutinib-mediated CD20 promoter downregulation but had no decreased surface CD20 levels when compared with controls influence on CAL-101 activity. Furthermore, given the role of AKT modified with vector-encoding non-silencing shRNA (Figure 2a). in propagation of signals from BCR,13 we co-transfected Raji cells These results indicate that the effects of R406 and ibrutinib are with the CD20 promoter reporter plasmid and a constitutively likely caused by inhibition of SYK and BTK, respectively. active AKT1 expression vector (pMIG-myrAKT). We observed that Immunoblotting revealed a significant, dose-dependent down- myrAKT1 reversed downregulatory effects of BCR inhibitors regulation of total CD20 at the level in Raji cells pre- on CD20 promoter activity (Figure 2e, gray bars). The rescue incubated with increasing concentrations of all tested BCR effect was most pronounced for CAL-101 but was also inhibitors (Figure 2b). Accordingly, quantitative reverse transcrip- significant for both ibrutinib and R406. Similarly, Raji cells tion polymerase chain reaction (qRT-PCR) analyses revealed that retrovirally modified to express myrAKT1 (Raji pMIG-myrAKT) CD20 is downregulated at the mRNA level in a dose-dependent were protected from BCR inhibitor-induced surface CD20 decrease manner for all tested drugs (Figure 2c). Noteworthy, we to a higher extent than cells modified with a control plasmid reanalyzed the microarray data originally published by Chen (Raji pMIG-con) (Figure 2g). et al.12 and concluded that the MS4A1 (CD20)genewas Collectively, these results indicate that BCR signaling inhibitors significantly downregulated upon treatment with R406 in a downregulate CD20 levels through an AKT-dependent pathway. panel of five DLBCL cell lines. MS4A1 downregulation was Remarkably, flow cytometry study of Raji cells incubated for 48 h ranked especially high, among the first 15 most significantly with BCR inhibitors followed by washout periods of 0–96 h regulated probe sets after 6-h incubation with R406 (not revealed that downregulation of CD20 levels is largely reversible shown). After 24 h, the global differences in (Figure 2h). This finding might be relevant in terms of designing between R406 and the vehicle DMSO-treated cells were much clinical combination studies, where treatment with BCR signaling more pronounced and downregulation of the MS4A1 gene inhibitors and anti-CD20 mAbs should be applied in alternative, remained strong and statistically significant (Figure 2d). To sequential schedules to fully unleash independent mechanisms of further confirm that downregulation of CD20 expression is antitumor effects of these therapies and to avoid direct negative caused by transcriptional mechanism, a Firefly luciferase reporter interactions. plasmid containing the CD20 promoter ( À 431/ þ 52 bp) was Altogether, our preclinical investigation reveals for the first time introduced via nucleofection into Raji cells. Upon 48-h that inhibition of BCR signaling with SYK inhibitor (R406), BTK

Figure 1. Inhibitors of BCR signaling affect surface CD20 and impair antitumor activity of anti-CD20 monoclonal antibodies. (a)Rajicells pre-incubated for 48 h with increasing concentrations of R406, ibrutinib or CAL-101 were washed and incubated for 1 h with rituximab (1–100 mg/ml) and 10% human AB serum. Cell viability in the CDC assay was measured with propidium iodide (PI) staining using BD FACScan flow cytometer (BD Biosciences, La Jolla, CA, USA). The survival of cells is calculated as a percentage of corresponding control cultures incubated with medium and serum without antibody and expressed as a relative viability of cells±s.d. (b) Raji cells pre-incubated for 48 h with increasing concentrations of R406, ibrutinib or CAL-101 were incubated with saturating amounts of fluorescein isothiocyanate-conjugated anti-CD20 mAb (L27; Becton Dickinson, San Jose, CA, USA) or IgG1 isotype control for 30 min at room temperature (RT) in the dark, washed with phosphate-buffered saline (PBS) and resuspended in PBS supplemented with 4 mg/ml of PI. Results are presented as a percentage of control mean fluorescence intensity (MFI) of PI-negative cells±s.d. from three independent experiments. (c) To determine the binding of clinically available anti-CD20 mAbs, Raji cells pre-incubated for 48 h with 1 mM R406, ibrutinib or CAL-101 were blocked with 1% bovine serum albumin/PBS for 30 min, washed in PBS and incubated with 100 mg/ml rituximab or 0 ofatumumab for 30 min at RT. After washing, cells were incubated with saturating amounts of Alexa Fluor 488-conjugated AffiniPure F(ab )2 Fragment Goat anti-Human IgG (H þ L) (Jackson Immunoresearch, West Grove, PA, USA). Before flow cytometry analysis, cells were washed and resuspended in PBS supplemented with PI (final concentration 4 mg/ml). Bars show the means of MFI±s.d. from three independent experiments. (d) Primary CLL samples (n ¼ 26) were pre-incubated for 48 h with 1 mM R406, ibrutinib or CAL-101. CD20 levels were determined with flow cytometry as described earlier. The differences in CD20 MFI between control and drug-treated groups were statistically significant (*Po0.0001) as measured using Wilcoxon-signed test. (e)Burkitt’slymphoma(Ramos,Daudi,BJAB),chronic lymphocytic leukemia (EHEB, MEC-1), acute lymphoblastic leukemia (NALM-6) pre-incubated for 48 h and DLBCL (Ly-1, Ly-4, Ly-7, Ly-10, Ly-19, DHL-6, Pfeiffer, HBL-1, U2932) pre-incubated for 24 h with 1 mM R406, ibrutinib or CAL-101 were analyzed for CD20 levels with flow cytometry as described earlier. (f) Degranulation assays were performed using NK cells negatively isolated from peripheral blood mononuclear cells (PBMC) with EasySep system (STEMCELL Technologies, Vancouver, BC, Canada) and activated with 100 IU/ml of human rIL-2 (Proleukin; Chiron, Emeryville, CA, USA) and 10 ng/ml human rIL-15 (R&D Systems, Minneapolis, MN, USA) overnight. In a pre- incubation model Raji cells or NK cells pre-incubated for 24 h with 1 mM R406, ibrutinib or CAL-101 were washed before degranulation assay and incubated at effector:target (E:T) ratio 1:1 for 4 h in the presence of 100 mg/ml rituximab, GolgiStop and anti-CD107a antibody. In a co-incubation model, Raji cells were incubated for 4 h with NK cells in presence of 100 mg/ml rituximab, GolgiStop, anti-CD107a antibody and 1 mM R406, ibrutinib or CAL-101. Before analysis, NK cells were stained with PE-Vio770-conjugated anti-CD56, PerCP-Cy5.5-conjugated anti-CD3 and Fixable Viability Dye. Degranulation of NK cells is presented from three independent experiments as a percentage of CD107a-positive cells±s.d. within the whole NK cells population. (g) ADCC was evaluated in the CFSE/PI flow cytometry assay. Fluorescently labeled with CFSE, Raji cells were incubated at E:T ratio 6:1 with rituximab (100 mg/ml) and NK cells isolated from PBMC (Human CD56-Positive Selection Kit, STEMCELL Technologies) in the presence of R406, ibrutinib or CAL-101 for 4 h at 37 1C. The percentage of cellular cytotoxicity±s.d. was calculated in the CFSE-positive population from three independent experiments. (h) Cytokine production was determined using NK cells isolated as described above. NK cells and Raji cells were incubated with rituximab and 1 mM R406, ibrutinib or CAL-101 for 4 h at 37 1C. Cells were washed, stained with anti-CD56 and anti-CD3 antibodies and Fixable Viability Dye. Upon washing, cells were permeabilized with Cytoperm/Cytofix (BD Biosciences), stained with Alexa Fluor 700-conjugated anti-IFN-g antibody or eFluor 450-conjugated anti-TNF and analyzed using flow cytometry. Results are presented as a percentage of TNF- or IFN-g-positive NK cells±s.d. within the whole NK cell population. CFSE, carboxyfluorescein succinimidyl ester.

& 2014 Macmillan Publishers Limited Leukemia (2014) 1129 – 1174 Letters to the Editor 1166

Figure 2. Inhibitors of BCR signaling modulate CD20 levels at a transcriptional level in a manner dependent on AKT. (a) Raji cells were stably transduced with lentiviral vectors to silence BTK or SYK kinases. For lentivirus production, HEK-293T cells were co-transfected with 2 mgofgeneof interest (GOI)-containing vector and components of second generation of lentiviral system using GeneJuice transfection reagent, according to the manufacturer’s protocol. After 72 h, lentivirus-containing medium was added to target cells. GOI-expressing cells were selected using 2 mg/ml puromycin. The degree of silencing of GOI was confirmed using qRT-PCR amplification with LightCycler Fast Start DNA Master PLUS SYBRGreen I (Roche Diagnostics, Basel, Switzerland). Thereafter, cells were analyzed using flow cytometry for surface CD20 levels. Results are presented as a percentage of control cells transduced with non-targeting shRNA pLKO.1 vector±s.d. (b) Raji cells pre-incubated for 48 h with increasing concentrations of R406, ibrutinib or CAL-101 were lysed in lysis buffer (50 mM Hepes, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10% glycerol, pH 7.4) supplemented with Complete protease inhibitor cocktail (Roche Diagnostics) and phosphatase inhibitor cocktail 3 (Sigma-Aldrich, St Louis, MO, USA). Protein concentration was measured using Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). Equal amounts of whole-cell proteins (10 mg) were separated in 10% polyacrylamide gel, transferred onto Protran nitrocellulose membranes, blocked with TBST (Tris-buffered saline (pH 7.4) and 0.05% Tween 20) supplemented with 5% nonfat dry milk. Anti-CD20 (polyclonal, Abcam, Cambridge, UK) and anti-b-actin (clone AC-15, Sigma-Aldrich) antibodies were used to detect corresponding proteins. (c) Raji cells pre-incubated for 48 h with increasing concentrations of R406, ibrutinib or CAL-101 were washed with PBS, pelleted and resuspended in 0.5 ml of TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) to extract total RNA according to the manufacturer’s protocol. RNA concentration was measured with NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). One microgram of total RNA was primed with oligo(dT) and used for the first- strand complementary DNA (cDNA) synthesis using AMV-reverse transcriptase (EURx). Real-time PCR was performed as described earlier. The amplification of cDNA was performed using a LightCycler 480 II device (Roche Diagnostics) according to the manufacturer’s recommendations and primers for CD20, b-actin and RPL29 in a final volume of 20 ml. In each PCR run, the samples were measured in duplicates in order to estimate their reproducibility. (d) The raw Affymetrix U133 Plus 2.0 Array intensity values available under GSE43510 accession number from Gene Expression Omnibus (GEO) were background-corrected, log2-transformed and quantile-normalized using the Robust Multi-array Average (RMA) algorithm. The differences in MS4A1 expression between control and drug-treated groups were statistically significant (Po0.0003) as measured using Wilcoxon-signed rank test. (e, f) Raji cells were nucleofected with the plasmid mix consisting of Firefly luciferase reporter (wild-type CD20 promoter, 1.5 mg) and Renilla luciferase reporter (pRL-TK, 0.5 mg) using the Cell Line Nucleofector Kit V (Lonza, Basel, Switzerland) and Amaxa Nucleofector Technology (Basel, Switzerland). To introduce TEL–SYK fusion protein, 2 mg of pCDNA3– TEL–SYK or control pCDNA3 was additionally used for nucleofection. To introduce constitutively active AKT1 isoform, 2 mg of either pMIG- myrAKT-IRES-GFP or control pMIG-GFP vectors was additionally used for nucleofection. Two hours after transfection, cells were incubated with 1 mM R406, ibrutinib or CAL-101. After 24 h, cells were lysed with Passive Lysis Buffer (Promega, Madison, WI, USA) and the relative luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega) and a multilabel reader (Victor X4, PerkinElmer, Waltham, MA, USA). (g) Raji cells stably transduced as described above with retroviral vectors encoding constitutively active myrAKT1 isoform and corresponding pMIG control plasmid were pre-incubated for 48 h with 1 mM R406, ibrutinib or CAL-101 and were analyzed for CD20 levels with flow cytometry as described earlier. Bars show the means of MFI±s.d. from three independent experiments. (h) Raji cells pre-incubated with 1 mM R406, ibrutinib or CAL-101 for 48 h were washed and subsequently incubated without drugs for indicated times. CD20 levels were determined with flow cytometry as described earlier. Bars show the means of MFI±s.d. from three independent experiments.

Leukemia (2014) 1129 – 1174 & 2014 Macmillan Publishers Limited Letters to the Editor 1167 inhibitor (ibrutinib) or PI3Kd inhibitor (CAL-101) results in REFERENCES increased resistance to antitumor activity of anti-CD20 mAbs. 1 Davis RE, Ngo VN, Lenz G, Tolar P, Young RM, Romesser PB et al. Chronic active Current study demonstrates that BCR inhibitors strongly down- B-cell-receptor signalling in diffuse large B-cell lymphoma. Nature 2010; 463:88–92. regulate CD20 expression in tumor cells, leading to decreased 2 Duhren-von Minden M, Ubelhart R, Schneider D, Wossning T, Bach MP, Buchner M binding of anti-CD20 mAbs to the surface of tumor cells and et al. Chronic lymphocytic leukaemia is driven by antigen-independent cell- impairment of CDC and ADCC mechanisms that mediate autonomous signalling. Nature 2012; 489: 309–312. antitumor effects of anti-CD20 mAbs in vivo. Our observations 3 Gobessi S, Laurenti L, Longo PG, Carsetti L, Berno V, Sica S et al. Inhibition of strongly imply that before investigating novel therapeutic constitutive and BCR-induced Syk activation downregulates Mcl-1 and induces apoptosis in chronic lymphocytic leukemia B cells. Leukemia 2009; 23: combinations in cancer patients, extensive preclinical studies 686–697. should be carried out to evaluate possible interactions between 4 Buchner M, Baer C, Prinz G, Dierks C, Burger M, Zenz T et al. Spleen tyrosine kinase drugs at the molecular level. inhibition prevents chemokine- and integrin-mediated stromal protective effects in chronic lymphocytic leukemia. Blood 2010; 115: 4497–4506. 5 Herman SE, Barr PM, McAuley EM, Liu D, Wiestner A, Friedberg JW. Fostamatinib CONFLICT OF INTEREST inhibits B-cell receptor signaling, cellular activation and tumor proliferation in patients with relapsed and refractory chronic lymphocytic leukemia. Leukemia The authors declare no conflict of interest. 2013; 27: 1769–1773. 6 Byrd JC, Furman RR, Coutre SE, Flinn IW, Burger JA, Blum KA et al. Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia. N Engl J Med 2013; 369: ACKNOWLEDGEMENTS 32–42. This work was supported by the National Science Center grant 2012/07/B/NZ6/03498 7 Cheng S, Ma J, Guo A, Lu P, Leonard JP, Coleman M et al. BTK inhibition targets (MW), the Polish Ministry of Science and Higher Education grant IP2011 060271 (MW), in vivo CLL proliferation through its effects on B-cell receptor signaling activity. the European Commission 7th Framework Programme: FP7-REGPOT-2012- Leukemia 2013; e-pub ahead of print 25 November 2013; doi:10.1038/leu. CT2012-316254-BASTION (JG), and the Medical University of Warsaw grant 2013.358. 1M19/PM12D/13 (KB). 8 Hoellenriegel J, Meadows SA, Sivina M, Wierda WG, Kantarjian H, Keating MJ et al. The phosphoinositide 30-kinase delta inhibitor, CAL-101, inhibits B-cell receptor K Bojarczuk1, M Siernicka1, M Dwojak1, M Bobrowicz1, signaling and chemokine networks in chronic lymphocytic leukemia. Blood 2011; B Pyrzynska1, P Gaj1, M Karp2, K Giannopoulos2, DG Efremov3, 118: 3603–3612. C Fauriat4, J Golab1,5 and M Winiarska1 9 Kharfan-Dabaja MA, Wierda WG, Cooper LJN. Immunotherapy for chronic 1 lymphocytic leukemia in the era of BTK inhibitors. Leukemia 2013; e-pub ahead of Department of Immunology, Center for Biostructure Research, print 25 October 2013; doi:10.1038/leu.2013.311. Medical University of Warsaw, Warsaw, Poland; 10 Winiarska M, Glodkowska-Mrowka E, Bil J, Golab J. Molecular mechanisms of the 2 Department of Experimental Hematooncology, Medical University of antitumor effects of anti-CD20 antibodies. Front Biosci (Landmark Ed) 2011; 16: Lublin, Lublin, Poland; 277–306. 3International Centre for Genetic Engineering and Biotechnology, 11 Herman SE, Gordon AL, Wagner AJ, Heerema NA, Zhao W, Flynn JM et al. Molecular Hematology Group, Campus A. Buzzati-Traverso, Phosphatidylinositol 3-kinase-delta inhibitor CAL-101 shows promising preclinical Rome, Italy; activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic 4IBiSA Cancer Immunomonitoring Platform, Institut Paoli Calmettes, cellular survival signals. Blood 2010; 116: 2078–2088. UM 105, Aix-Marseille Universite´, Marseille, France and 12 Chen L, Monti S, Juszczynski P, Ouyang J, Chapuy B, Neuberg D et al. SYK 5 inhibition modulates distinct PI3K/AKT- dependent survival pathways and choles- Department 3, Institute of Physical Chemistry, Polish Academy of terol biosynthesis in diffuse large lymphomas. Cancer Cell 2013; 23: 826–838. Sciences, Warsaw, Poland 13 Srinivasan L, Sasaki Y, Calado DP, Zhang B, Paik JH, DePinho RA et al. PI3 kinase E-mail: [email protected] or [email protected] signals BCR-dependent mature B cell survival. Cell 2009; 139: 573–586.

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

OPEN Expression of putative targets of immunotherapy in acute myeloid leukemia and healthy tissues

Leukemia (2014) 28, 1167–1170; doi:10.1038/leu.2014.14 comprehensive evidence-based list of AML antigen targets has not yet been established. As a first step toward this goal, we therefore analyzed, using quantitative real-time PCR, the gene expression of 65 potential LAAs (Supplementary Table S1) in de-identified, The ability to target myeloid malignancies using immunotherapy clinically annotated samples from 48 newly diagnosed untreated through means other than allogeneic transplantation depends on AML patients that were collected under institutional review board- the capability to target leukemic clones while sparing normal approved protocols from three NCCN cancer centers. tissues. It is now possible to generate clinical grade ex-vivo A total of 52 samples (30 peripheral blood (PB) and 22 bone expanded T cells specific for leukemia-associated antigens (LAAs) marrow aspirate (BM) samples) from 48 AML patients were analyzed, for use in adoptive cell therapy.1 Although a variety of putative which included 4 patients for whom both PB and BM samples were LAAs in acute myeloid leukemia (AML) have been identified for available. The average age of the patients was 52 years (range use as potential targets for immunotherapy2–8 and consensus 24–86); 52% of the patients were women. A total of 7 patients had panels have attempted to prioritize generic cancer antigens,9 a favorable cytogenetics, whereas 11 were classified as adverse,

Accepted article preview online 10 January 2014; advance online publication, 28 January 2014

& 2014 Macmillan Publishers Limited Leukemia (2014) 1129 – 1174