Dual PI3K/mTOR inhibition shows antileukemic activity in MLL rearranged acute. Supplemental method S1: Confirmation of RAS mutations

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1 Supplementary information Dual PI3K/mTOR inhibition shows antileukemic activity in MLL rearranged acute myeloid leukemia Sandhöfer N. et al. Supplemental methods S1-S4: Supplemental method S1: Confirmation of RAS mutations Supplemental method S2: Generation of stable murine MLL-ENL transduced and immortalized cells Supplemental method S3: Microarray analysis Supplemental method S4: quantitative real-time PCR (qrt-pcr) Supplemental tables S1-S4 Supplemental table S1: Characteristics of patient samples used for in vitro treatment experiments (n=18). Supplemental table S2: Genetic background of human cell lines. Supplemental table S3: Characteristics of patient samples used for gene expression analysis. Supplemental table S4: Frequencies of specific codon mutations in cytogenetically normal (CN) AML (n=125) and MLL rearranged AML patients (n=38) Supplemental figures S1-S9 Supplemental figure S1: Pathway inhibition in patient-derived xenografted cells. Supplemental figure S2: Spontaneous apoptosis and Ki67 expression.

2 Supplemental figure S3: Pathway inhibition in murine MLL-ENL immortalized (MLL-ENL ICs) bone marrow progenitor cells. Supplemental figure S4: Inhibition of PI3K/AKT/mTOR signaling in cell lines. Supplemental figure S5: MK-226 and Rapamycin inhibit PI3K/AKT/mTOR signaling but do not induce apoptosis. Supplemental figure S6: Tumor load and body weight during the treatment with BEZ-235 and AC22. Supplemental figure S7: EVI1 and PTEN expression in AML patient samples and cell lines. Supplemental figure S8: Mutational analysis in AML patient samples. Supplemental figure S9: Combination treatment with AZD-6244 in human AML cell lines and PDX cells.

3 Supplemental method S1: Confirmation of RAS mutations KRAS mutations were independently verified using PCR followed by 454 Titanium amplicon chemistry sequencing (Roche Applied Science, Mannheim, Germany), and NRAS mutations by PCR and melting curve analysis. For 454 Titanium amplicon chemistry, a working dilution of 1 6 molecules/l was used as starting concentration for the GS Junior empcr Kit (Lib-A) (Roche Applied Science). Following the empcr amplification, clonally amplified beads were enriched for 454 next-generation sequencing according to the manufacturer s recommendations. The 454 sequencing data were generated on a GS Junior using the GS Junior Titanium Sequencing Kit (Roche Applied Science). Data analysis was performed utilizing JSI Sequence Pilot, SEQNext (JSI medical systems GmbH, Kippenheim, Germany). Supplemental method S2: Generation of stable murine MLL-ENL transduced and immortalized cells The retroviral vectors were purchased from Addgene (pmscv neo empty vector and pmscv neo -MLL-ENL). The production of retroviral supernatants by Phoenix eco cells was conducted as described previously (1). Four to six weeks old C57BL/6 mice were injected intravenously with 15 mg/kg 5-FU in PBS. Two days later bone marrow cells were harvested and cultured overnight in RPMI Glutamax (Life Technologies, Carlsbad, CA, USA) supplemented with 2% fetal calf serum (FCS; Pan Biotech, Aidenbach, Germany), interleukin-3 (IL-3; 1 ng/ml), interleukin-6 (IL-6; 1 ng/ml), stem cell factor (SCF; 1 ng/ml; all from Immunotools, Friesoythe, Germany). The next day cells were infected by spinoculation (2 hour at 25 g at 33 C in 2 ml retroviral supernatant in the presence of 8 µg/ml polybrene) which was

4 repeated on the next day. After the second spinoculation cells were again cultured overnight in RPMI supplemented with 2% FCS, IL-3, IL-6 and SCF. 4, cells were seeded per 1.1 ml of Methocult M3534 (Stem Cell Technologies Inc., Vancouver, Canada) with or without.75 mg/ml G-418 (Life Technologies). Cells transduced with the empty vector could not be replated more than three rounds. After four rounds of serial replating in Methocult3534 MLL-ENL transduced cells were cultured in RPMI Glutamax supplemented with 2% FCS, 1 ng/ml IL-3, 1 ng/ml IL-6 and 5 ng/ml SCF. Supplemental method S3: Microarray data Total RNA was extracted from 562 bone marrow samples as described previously (2) and analyzed using Affymetrix HG-U133 A/B and 2. plus oligonucleotide microarrays (Affymetrix, Santa Clara, CA). Hybridization and image acquisition followed official Affymetrix protocols. No cell sorting was performed. For probes to probe set annotation we used custom chip definition files (CDFs) based on GeneAnnot version 2., synchronized with GeneCards Version 3.4 (available at (3). These CDFs decrease the total number of probe sets (one probe set per gene), and potentially increases the specificity of the analyses by eliminating cross-hybridizing probes (probes are restricted by sequence specificity). Data normalization was performed using the Robust Multichip Average (RMA) method as described by Irizarry (4). Only the probe sets present both on the A, B chips and the 2. plus chips were included in the analysis. Some probe sets on the A, B chips tend to have lower mean signal levels and higher standard deviations than the corresponding probe sets on the Plus 2. chips. To eliminate this

5 batch effect resulting from the different chip designs, we performed a second normalization using an empirical Bayesian method. Supplemental method S4: quantitative real-time PCR (qrt-pcr) Total RNA was extracted from eleven human myeloid cell lines (RNeasy RNA isolation kit (Qiagen,Hilden Germany) and 1 µg RNA was reverse transcribed into copydna (RevertAid First Strand cdna Synthesis Kit; Thermo Scientific, Waltham, MA, USA). The expression levels of the targets (EVI1, PTEN) and a housekeeping gene (GAPDH) were determined using the TaqMan Gene Expression assay kit (Applied Biosystems, Darmstadt, Germany). Amplification and quantification was performed using the 75 Fast Real-Time PCR System (Applied Biosystems). At least three independent experiments were performed with each reaction in duplicates. Supplemental references 1. Polzer H, Janke H, Schmid D, Hiddemann W, Spiekermann K. Casitas B- lineage lymphoma mutants activate AKT to induce transformation in cooperation with class III receptor tyrosine kinases. Experimental hematology. 213;41(3):271-8 e4. 2. Haferlach T, Kern W, Schnittger S, Schoch C. Modern diagnostics in acute leukemias. Critical reviews in oncology/hematology. 25;56(2): Ferrari F, Bortoluzzi S, Coppe A, Sirota A, Safran M, Shmoish M, et al. Novel definition files for human GeneChips based on GeneAnnot. BMC bioinformatics. 27;8:446.

6 4. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics. 23;4(2):

7 Table S1: Characteristics of patient samples used for in vitro treatment experiments (n=18). # Age [y] Gender Blast cells FAB MLL FLT3 RAS NPM1 CEBPA Type 1 68 M 8% M1 WT WT NA WT WT BM 2 45 F 83% M4 MLL-PTD ITD NA WT NA BM 3 53 F 88% M WT ITD WT NPM1c WT PB 4 2 F 95% M5a MLL-t WT WT WT NA BM 5 77 F 76% M2 WT WT NRAS G12D NPM1c NA BM 6 6 F 8% M2 WT ITD WT NPM1c NA BM 7 58 F 77% M1 MLL-PTD ITD WT WT WT BM 8 65 F 87% M1 WT TKD NRAS G12C NPM1c WT BM 9 79 F 83% M1 WT WT NRAS Q61R KRAS G12D WT WT BM 1 73 F 8% M5 MLL-t WT WT WT NA BM F 85% NA WT ITD WT WT WT BM M 88% M1 WT TKD NRAS G12S WT NA BM F 84% M4 WT TKD NA NPM1c WT BM F 71% M2 WT ITD WT WT WT PB M 1% M4 MLL-t WT KRAS G13D KRAS Q61H WT WT BM F 8% M1 WT WT WT WT WT BM M 74% M2 MLL-PTD ITD NA WT WT BM M 77% M3 WT ITD NA WT WT BM M: male, F: female, FAB: French-American-British Classification, WT: wildtype, MLL-t: MLL-translocation, MLL-PTD: MLLpartial tandem duplication, FLT3-ITD: internal tandem duplication, FLT3-TKD: tyrosine kinase domain, NA: not analyzed, BM: bone marrow, PB: peripheral blood Table S2: Genetic background of human cell lines. FAB MLL FLT3 CBL NRAS KRAS p53 NPM1 DNMT3A MOLM-13 M5 MLL-AF9 ITD 49del WT WT WT WT WT MV4-11 M5 MLL-AF4 ITD WT WT WT R248W WT WT THP-1 M5 MLL-AF9 WT WT G12D WT R174fs*3 WT WT OCI-AML3 M4 WT WT WT WT WT WT NPM1c R882C K-562 CML WT WT WT WT WT WT WT WT HL-6 M2 WT WT WT Q61L WT 1182del WT WT CML: chronic myeloid leukemia, ITD: internal tandem duplication, WT: wildtype

8 Table S3: Characteristics of patient samples used for gene expression analysis (n=562). Variable Number of patients No. of patients 562 Median age, years (range) 58 (18-85) Female sex, no. (%) 282 (5.2) Bone marrow blasts, %, median(range) 8 (1-1) de novo AML 437 (83.1) taml 27 (5.1) MDS 14 (2.7) saml 48 (9.1) FAB M, no. (%) 22 (3.9) M1, no. (%) 113 (2.1) M2, no. (%) 164 (29.2) M3, no. (%) 15 (2.7) M3v, no. (%) 12 (2.1) M4, no. (%) 121 (21.5) M5, no. (%) 66 (11.7) M6, no. (%) 22 (3.9) M7, no. (%) 3 (.1) NA, no. (%) 24 (4.3) Cytogenetic CN-AML (total), no. (%) 199 (35.4) Complex Cytogenetics, no. (%) 74 (13.2) inv16/t(16;16), no. (%) 38 (6.7) t(8;21), no. (%) 3 (5.3) t(15;17), no. (%) 24 (4.2) (v;11)(v;q23), no. (%) 19 (3.3) t(9;11), no. (%) 19 (3.3) del(7q)/monosomy 7, no. (%) 16 (2.8) inv3/t(3/3), no. (%) 15 (2.6) del(5q)/monosomie 5, no. (%) 6 (1.1) t(6;9), no. (%) 4 (.7)

9 del(9q) soley 7 (1.2) Isolated trisomy 8 14 (2.5) Isolated trisomy 13 9 (1.6) Isolated trisomy 11 7 (1.2) Various (non-complex), no. (%) 51 (9.1) NA, no. (%) 3 (5.3) ELN classification Favorable, no. (%) 141 (25.1) Intermediate-I, no. (%) 111 (19.8) Intermediate-II, no. (%) 17 (19) Adverse, no. (%) 134 (23.8) NA 69 (12.3) CN: cytogentically normal, del: deletion, inv: inversion, MDS: myelodysplastic syndrome, NA: not analyzed, No: number, saml: secondary AML, taml: therapy-related AML, t: translocation Table S4: Frequencies of specific codon mutations in cytogenetically normal (CN) AML (n=125) and MLL rearranged AML patients (n=38) NRAS KRAS codon 12 codon 13 codon 61 others codon 12 codon 13 codon 61 others CN- AML MLL-t AML 43.6% 2.5% 2.5% 15.4% 36.4% 27.3% 9.% 27.3% 7.% 2.% - 1.% 31.6% 26.3% 31.6% 1.5%

10 Supplemental figure S1 untreated BEZ-235 MK-226 Rapamycin isotype PS6rp 62.5 nm 25 nm 1 nm untreated treated 25 nm 1 nm 1 nm counts 1 nm 4 nm 1, nm P-S6rp Supplemental figure S1: Pathway inhibition in patient-derived xenografted cells: Patient-derived xenograft cells were treated with BEZ- 235, MK-226 and Rapamycin, respectively. Phosphorylation of ribosomal protein S6 (S6rp) was measured by FACS staining after three hours. The isotype control is shown in grey, untreated cells are shown in black, treated cells are shown in blue.

11 Supplemental figure S2 A B spontaneous apoptosis [%] spontaneous apoptosis [%] BEZ-235 resistent MLL WT p=.78 BEZ-235 sensitiv p=.64 MLL-t / MLL-PTD C log2 intensities Ki67 expression MLL wt MLL mu Supplemental figure S2: Spontaneous apoptosis and Ki67 expression. Human AML blast cells were freshly isolated and cultured for hours with or without inhibitors. The spontaneous apoptosis of untreated cells was measured by FACS analysis and compared between BEZ-235 sensitive and non-sensitive (A) and between MLL wildtype and MLL mutated samples (B). There was no significant difference. By using microarray analysis Ki67 expression levels were determined and compared between MLL wildtype and MLL rearranged samples (C).

12 Supplemental figure S3 untreated isotype PS6rp BEZ-235 untreated treated counts 125 nm 25 nm 1 nm P-S6rp Supplemental figure S3: Pathway inhibition in murine MLL-ENL immortalized (MLL-ENL ICs) bone marrow progenitor cells Cells were treated with BEZ-235. Phosphorylation of ribosomal protein S6 (S6rp) was measured by FACS staining after three hours. The isotype control is shown in grey, untreated cells are shown in black, treated cells are shown in blue.

13 Supplemental figure S4 A MOLM-13 BEZ-235 MK226 Rapamycin ps6k T389 HL-6 PC -,6, S6K BEZ-235 MK226 Rapamycin PC -,6, ps6k T389 S6K pakt S473 pakt S473 AKT AKT perk T22/Y24 perk T22/Y24 ERK ERK Actin Actin B P-AKT/AKT ratio 1.2 * MDA-MB-453 THP-1 MOLM-13 MV4-11 HL-6 K-562 OCI-AML3.2 Supplemental figure S4: Inhibition of PI3K/AKT/mTOR signaling in cell lines. The human myeloid cell lines were cultured for six hours with or without BEZ-235, MK-226, and Rapamycin. As a positive control (PC) lysates from the human breast cancer cell line MDA-MB-453 were used. Activation of signaling pathways was analyzed by western blot (A) and quantification of P-AKT/AKT ratios was done using Image J software (B; * p<.5). Shown are the mean values +/- standard error of at least three quantifications.

14 Supplemental figure S5 A -ps6k -S6K -pakt -AKT 6 h 24 h -ps6k -S6K -pakt -AKT -Aktin -Aktin BEZ-235 [μm] MK-226 [μm] Rapamycin [μm], ,1,1-1 1, ,1,1-1 1 B specific apoptosis [%] BEZ-235 [μm] MK-226 [μm] Rapamycin [μm] viable cells [%] BEZ-235 [μm] MK-226 [μm] Rapamycin [μm] * ** Supplemental figure S5: MK-226 and Rapamycin inhibit PI3K/AKT/mTOR signaling but do not induce apoptosis HL-6 cells were cultured for 6 or 24 hours (h) with or without inhibitors. Activation of signaling pathways was analyzed by Western Blot (A). Similar concentrations were used to culture cells in the absence or presence of those inhibitors. Apoptosis was determined after 72 hours by FACS analysis and is depicted as specific apoptosis in comparison to untreated controls. Shown are mean values +/- standard error of at least three independent experiments. Cells were seeded in the absence or presence of inhibitors and after 72 hours viable cells were counted by Trypan Blue exclusion. Results show the relative cell number compared to untreated controls and represent mean +/- standard error of at least three independent experiments (B). *p<.5; **p<.1

15 Supplemental figure S6 A dpi AC B Vehicle; n=2 BEZ-235; n=2 AC22; n=2 CD45-APC C Body weight [g] GFP 26 Vehicle dpi Body weight [g] BEZ dpi Body weightt [g] AC dpi Supplemental figure S6: Tumor load and body weight during the treatment with BEZ-235 and AC22. Tumor progression of AC22-treated mice. Shown are imaging files of one representative animal of this group at different time points (A). At day 15 two animals out of each group were sacrificed and their tumor load in the bone marrow was determined by measuring human CD45 positive cells in total mouse bone marrow. Shown are the FACS blots of all six animals (B). The body weight of all mice was determined during the treatment period of 21 days (C).

16 Supplemental figure S7 A log2 Intensity EVI1 expression *** *** *** B log2 intensities EVI1 PTEN ns 1st 2nd 3d 4th quartiles CN-AML inv(3) t(3;3) t(8;21) t(15;17) inv(16) del(5q) t(9;11) t(v;11)(v;q23) t(16;16) C EVI1 PTEN MOLM-13 MV4-11 THP-1 MM6 # # MOLM-13 MV4-11 THP-1 MM6 HL-6 OCI-AML3 U937 K562 # HL-6 OCI-AML3 U937 K562 K562 EV K562 MLL-AF9 K562 EV K562 MLL-AF9 HEL HEL Delta Ct-value Delta Ct-value Supplemental figure S7: EVI1 and PTEN expression in in AML patient samples and cell lines. EVI1 expression was analyzed in 35 AML patient samples and compared between different cytogenetic subgroups. MLL-rearranged patient samples are indicated in blue (A). EVI1 expression quartiles were correlated with PTEN expression in 38 MLLrearranged patient samples (B). EVI1 and PTEN expression levels were determined via qpcr in 11 human cell lines. Cell lines expressing an MLL-rearrangement are indicated in blue. Shown are mean delta Ct-values from three independent experiments. +/- standard errors. Gapdh was used as house-keeping gene (C) EV: empty vector, #: not detectable, ns: not significant, ***p<.1.

17 Supplemental figure S8 CN-AML MLL FLT3 NRAS KRAS CBL 37% 19% 9% 5% Supplemental figure S8: Mutational analysis in AML patient samples. 125 cytogenetically normal (CN)-AML patient samples were analyzed for mutations in FLT3, RAS and CBL. Mutations are indicated in red and frequency rates are shown for each gene.

18 Figure for reviewer only specific apoptosis [%] Daunorubicine [.1 μm] n=17 ns; p=.4 48 h 72 h incubation time specific apoptosis [%] BEZ-235 [4 μm] n=16 ns; p=.8 48 h 72 h incubation time Comparison between primary patient samples treated for 48 hours and 72 hours, respectively. Freshly isolated blast cells from AML samples were cultured in the presence of Daunorubicine or BEZ-235 in duplicates. Apoptosis was determined by FACS analysis after 48 hours (white bars) - 72 hours (green bars) and is depicted as specific apoptosis in comparison to untreated controls. The average of two values is shown. There were no significant differences between 48 hours and 72 hours. ns: not significant