For knockdown of individual genes, the following TRC library clones were used: Gene Species Library TRC number Designation in manuscript

Transcription

1 Supplemental Methods shrna constructs for validation experiments For knockdown of MLL-AF9, a custom shrna with the following stem sequence directed against the fusion breakpoint (underlined) was designed using shrna Explorer ( and cloned into the plko.1 vector: 5 -AAGTCTGAACAACCCAGTCCT-3 For knockdown of individual genes, the following TRC library clones were used: Gene Species Library TRC number Designation in manuscript AF9 human TRC-Hs 1.0 (Human) TRCN shaf9_5 TRC-Hs 1.0 (Human) TRCN shaf9_3 CAMK1 human TRC-Hs 1.0 (Human) TRCN shcamk1-679 TRC-Hs 1.0 (Human) TRCN shcamk CDK4 human TRC-Hs 1.0 (Human) TRCN shcdk4_1 TRC-Hs 1.0 (Human) TRCN shcdk4_2 CDK6 human TRC-Hs 1.0 (Human) TRCN shcdk4_3 TRC-Hs 1.0 (Human) TRCN shcdk6_1 TRC-Hs 1.0 (Human) TRCN shcdk6_2 TRC-Hs 1.0 (Human) TRCN shcdk6_3 DGKH human TRC-Hs 1.0 (Human) TRCN shcdk6_4 TRC-Hs 1.0 (Human) TRCN shdgkh-1357 TRC-Hs 1.0 (Human) TRCN shdgkh-1360 MAP3K12 human TRC-Hs 1.0 (Human) TRCN shmap3k TRC-Hs 1.0 (Human) TRCN shmap3k SRPK2 human TRC-Hs 1.0 (Human) TRCN shsrpk TRC-Hs 1.0 (Human) TRCN shsrpk THOC4 human TRC-Hs 1.0 (Human) TRCN shthoc4-351 TRC-Hs 1.0 (Human) TRCN shthoc4-352 UNC13B human TRC-Hs 1.0 (Human) TRCN shunc13b-2356 TRC-Hs 1.0 (Human) TRCN shunc13b-2359 Cdk6 mouse TRC-Mm 1.0 (Mouse) TRCN shcdk6_m1 TRC-Mm 1.0 (Mouse) TRCN shcdk6_m2 1

2 RNA isolation, cdna synthesis, and qrt-pcr Total RNA was isolated using the RNeasy Mini or Micro Kit (Qiagen) and reversetranscribed (2 µg in a total volume of 20 µl) using the High Capacity cdna Reverse Transcription Kit (Applied Biosystems). Real-time PCR was used to quantify gene expression relative to endogenous PBGD or HPRT1. Primers were combined with LightCycler 480 SYBR Green I Master Mix (Roche) or TaqMan SYBR Green PCR Master Mix (Applied Biosystems) reagents, and cdna was used as template. Reactions were run on a LightCycler 480 (Roche) or an ABI PRISM 7900HT Sequence Detection System at default thermal cycling conditions. Results were analyzed using the standard curve method. Primer sequences for qrt-pcr Gene Species Forward primer Reverse primer CAMK1 human GGGCAAGGAAGGCAGCAT GGTAGAGGTGGCCCCCACT CDK4 human CTGGTGTTTGAGCATGTAGACC AAACTGGCGCATCAGATCCTT CDK6 human TGCACAGTGTCACGAACAGA ACCTCGGAGAAGCTGAAACA CEBPA human AACATCGCGGTGCGCAAGAG TTCGCGGCTCAGCTGTTCCA DGKH human AAACCTTCCTCCCAGAAAGC GGTTCACAGGGGTGAACTGT HOXA9 human CCGAGAGGCAGGTCAAGATC AAATAAGCCCAAATGGCATCA HPRT1 human TTGCTGACCTGCTGGATTAC TCTCCACCAATTACTTTTATGTCC IRF8 human CCGAGCCATACAAAGTTTACCG CAGAGCGACCGCACTCCA MAP3K12 human GAACTACCTGCACCTGCACA TCCTTGGAAGTGCCAAAATC MLL-AF9 8A a human GTCCAGAGCAGAGCAAACAGAAA TTGTTGCCTGGTCTGGGATG MLL-AF9 9A b human GCAGATGGAGTCCACAGGAT ACCTCAAAGGACCTTGTTGC MLL-AF9 plasmid human GCAGATGGAGTCCACAGGAT ATGCCTTGTCACATTCACCAT PBGD human GGAGCCATGTCTGGTAACGGCA GGTACCCACGCGAATCACTCTCA SPI1 human TGTTACAGGCGTGCAAAATGG TGCGTTTGGCGTTGGTATAGA SRPK2 human CTTTGCCAGACCCCACAC GTCTCCAATTTTCACTGGATGAT THOC4 human GGAAACTGCTGGTGTCCAAT CGACCAGAGCGATCATAGTG UNC13B human CTGAAGACTATTCGTCAGTCGGA TGAGGAGTTGGGTTTCTAGTTCC Cdk4 mouse AACTGATCGGGACATCAAGG CAGGCCGCTTAGAAACTGAC Cdk6 mouse AGAAGTCCTGCTCCAGTCCA CACGTCTGAACTTCCACGAA Hoxa9 mouse CCCTGACTGACTATGCTTGTG GCATCGCTTCTTCCGAGTG Hprt1 mouse CTTTGCTGACCTGCTGGATT TATGTCCCCCGTTGACTGAT a Fusion between MLL exon 8 and AF9 exon 6 (site A) b Fusion between MLL exon 9 and AF9 exon 6 (site A) 2

3 Immunoblotting Immunoblotting was performed using standard procedures with the following antibodies: anti-ß-actin (Sigma-Aldrich); anti-ß-tubulin, anti-cdk4, anti-cdk6 (Cell Signaling); anti- MLL1 (Bethyl). Whole-cell protein extracts were prepared using lysis buffer containing 20 mm Tris, 1% Triton X-100, 150 mm NaCl, 1 mm EDTA, and 10% glycerol supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Pierce), and µg of protein were subjected to SDS-PAGE and immunoblotting. Determination of viable cell numbers Viable cell numbers in suspension culture were determined using the CellTiter96AQ ueous One Solution Proliferation Assay (Promega). Cell cycle, apoptosis, and differentiation analysis For cell cycle analysis, cells were either stained for 15 minutes on ice with Nicoletti buffer (0.1% sodium citrate, 0.1% Triton-X 100, 50 µg/ml propidium iodide, ph 7.4) or pulsed with BrdU for 2 hours and processed according to the FITC BrdU Flow Kit staining protocol (BD Pharmingen). For apoptosis analysis, cells were stained with Annexin V-PE or Annexin V-FITC and 7-AAD in Annexin V Binding Buffer (BD Pharmingen). To determine the expression of surface antigens associated with myelomonocytic differentiation, cells were stained with human FITC-conjugated anti- CD14 and PE-conjugated anti-cd11b (BD Biosciences), mouse Pacific Blue-conjugated anti-f4/80 and APC-conjugated anti-cd115 (ebioscience), or isotype control. Flow cytometric analyses were performed on a BD LSR II flow cytometer (BD Biosciences) 6 days after lentiviral transduction of human cells, 5 days after PD treatment, and 4 days after retroviral transduction of murine cells, respectively. Colony formation assays Cell lines derived from mouse HSPC expressing MLL-AF9 or MOZ-TIF2 (1x10 4 to 3x10 4 cells) were transduced with shrna constructs and plated in methylcellulose medium (MethoCult GF M3434; StemCell Technologies), and colonies were counted after 5 days. Human AML cell lines (1x10 3 to 1x10 4 cells) were plated in methylcellulose medium (MethoCult H4236; StemCell Technologies) with or without PD , and colonies were counted after 10 days. For colony formation assays with mononuclear cells from AML patients, 1x10 6 to 2x10 6 cells were plated in methylcellulose medium 3

4 (MethoCult H4435 Enriched; StemCell Technologies) with or without PD After 5 to 8 days, colonies were counted and cell numbers determined. Images were taken on a Cell Observer system (Zeiss). Primary human hematopoietic cells BM or blood samples from AML patients were obtained at diagnosis prior to therapy. Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation. The diagnosis of AML was made according to the World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues or French-American-British Cooperative Group criteria. All specimens were karyotyped by chromosome banding. In selected cases, the presence of an MLL fusion gene was confirmed by RT-PCR or fluorescence in situ hybridization. Normal CD34 pos hematopoietic progenitor cells were isolated from cryopreserved leukapheresis products. Mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation. Enrichment of CD34 pos cells to a purity of greater than 95% was performed by positive selection using immunomagnetic microbeads (Miltenyi Biotec) according to the manufacturer's instructions. 4

5 Supplemental Table 1 Cell line Disease MLL fusion Additional mutations (selection) NOMO-1 AML MLL-AF9 KRAS G13D ML-2 AML MLL-AF6 KRAS A146T MOLM-14 AML MLL-AF9 FLT3 ITD Mono-Mac-6 AML MLL-AF9 FLT3 V592A MV4-11 AML MLL-AF4 FLT3 ITD THP-1 AML MLL-AF9 NRAS G12D HL-60 AML NRAS Q61L K562 CML-BC BCR-ABL1 NB4 APL PML-RARA, KRAS A18D OCI-AML3 AML NPM1 mut, NRAS Q61L U937 AML PTPN11 G60R CML-BC, chronic myeloid leukemia blast crisis; APL, acute promyelocytic leuekmia; ITD, internal tandem duplication. 5

6 Supplemental Figure 1 Supplemental Figure 1. Requirement for CDK6 in MLL-rearranged AML cell lines. (A) CDK6 mrna and protein expression of AML cell lines transduced with a nontargeting control shrna or shrnas targeting CDK6. MM6, Mono-Mac-6. (B) CDK6 mrna and protein expression of MV4-11 and ML-2 cells transduced with a nontargeting control shrna or shrnas targeting CDK6. (C) Cdk6 protein expression of cells used in Figure 2E and F. 6

7 Supplemental Figure 2 7

8 Supplemental Figure 2 continued Supplemental Figure 2. Effects of CDK6 suppression in AML cells. (A) Flow cytometric analysis of cell cycle progression using propidium iodide. Numbers indicate percentages of cells in G0/G1. (B, C) Flow cytometric analysis of cell cycle progression using BrdU/7AAD. Shown are the mean ± SEM of 3 independent experiments (panel B) and representative plots (panel C). Numbers in panel C indicate percentages of cells. (D) Flow cytometric analysis of apoptosis. Numbers indicate percentages of cells. (E) CDK6, IRF8, CEBPA, and SPI1 mrna expression of MLL-AF9 pos THP-1 cells following suppression of CDK6 with 3 different shrnas. (F, G) Flow cytometric analysis of myeloid differentiation in THP-1 (panel F) and NOMO-1 (panel G) cells transduced with lentiviral vectors expressing GFP and the coding sequences of CDK6, catalytically deficient CDK6 (CDK6 K43M), and CDK4, respectively, or an empty control vector (EV). Cells were GFP-sorted, transduced with an shrna targeting the CDK6 3 UTR, and analyzed for CD11b expression after 6 days. Shown are the percentages of GFP pos /CD11b pos cells (left panels) and counts relative to CD11b fluorescence intensity (right panels). (H) AF9 mrna expression of various AML cell lines. 8

9 Supplemental Figure 3 9

10 Supplemental Figure 3 continued Supplemental Figure 3. Pharmacologic inhibition of CDK6 in human AML cells. (A) Colony formation in methylcellulose of MLL-AF9 pos (NOMO-1, THP-1) and MLL-AF9 neg (HL-60, K562) AML cell lines treated with varying concentrations of PD Original magnification, x4.7. (B) CDK4 mrna and protein expression of AML cell lines transduced with a non-targeting control shrna or shrnas targeting CDK4. (C) Characteristics of primary human MLL-rearranged leukemia samples. AUL, acute undifferentiated leukemia. (D) Absolute colony numbers of primary human MLLrearranged leukemia samples cultured in methylcellulose in the presence of varying concentrations of PD (E) IRF8, CEBPA, and SPI1 mrna expression of MLL- AF9 pos and MLL-AF9 neg AML cell lines treated with varying concentrations of PD NE, not expressed. 10

11 Supplemental Figure 4 Supplemental Figure 4. Gating strategy for GFP pos /mcherry pos leukemic cells used for in vitro and in vivo experiments shown in Figure 6. Flow cytometric analysis of GFP (marker for MLL-AF9) and mcherry (marker for shrna) expression of leukemic cells from mice with secondary MLL-AF9-induced AML transduced with a LeGO-C2 lentiviral gene ontology vector encoding either an shrna against Cdk6 or a non-targeting control sequence. Gates for sorting of cells with strong mcherry expression are indicated. 11