NOTCH1 promotes T cell leukemia-initiating activity by RUNXmediated. regulation of PKC-θ and reactive oxygen species

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1 NOTCH1 promotes T cell leukemia-initiating activity by RUNXmediated regulation of PKC-θ and reactive oxygen species Vincenzo Giambra, Christopher R. Jenkins, Hongfang Wang, Sonya Lam, Olena O. Shevchuk, Oksana Nemirovsky, Carol Wai, Sam Gusscott, Mark Y. Chiang, Jon C. Aster, R. Keith Humphries, Connie Eaves & Andrew P. Weng Supplementary Information including Supplementary Figures 1-15 Supplementary Table 1 Nature Medicine doi:1.138/nm.296 SI-1

2 CD44 DCFDA *FITC *FITC *FITC CD44 DCFDA Survival (%) a 1 8 NOTCH1-ΔE in NSG CD44 1 (n = 3) 1 (n = 4) 1 (n = 12) Time after transplant (d) CD44 + ROS high 1 (n = 4) 1 (n = 4) 1 (n = 1) CD44 + ROS low 1 (n = 4) 1 (n = 4) 1 (n = 11) b CD all cells Presort K 2K 3K K 2K 3K *FSC K 1K 15K 2K 25K FSC FSC Transplanted recipient CD44 + ROS low K 2K 3K K 2K 3K 92.9 *FSC C57BL/6 Postsort CD44 + ROS high K 2K 3K K 2K 3K *FSC FSC CD K 2K 3K FSC Supplementary Figure 1. LICs are asymmetrically distributed in the CD44 + ROS low fraction. (a) Survival of immunodeficient NSG recipient mice after injection in the tail vein of primary mouse NOTCH1-ΔE leukemia cells, with the number of injected cells from each FACS-sorted subset indicated. Throughout the figure, n indicates the number of mice in each group. Raw survival data are provided in Supplementary Table 1. (b) Flow cytometric phenotyping of NOTCH1-ΔE leukemia cells before FACS sorting, after FACS sorting, and after transplantation of FACS-sorted subsets into secondary recipients. Data depicted are representative of several similar experiments. Nature Medicine doi:1.138/nm.296 SI-2

3 Survival (%) Survival (%) a NOTCH1-ΔE CD44 (1 4 ; n = 4) CD44 + (1 4 ; n = 4) Time after transplant (d) b NOTCH1-L161P ΔPEST Time after transplant (d) Total (1 6 ; n = 4) Total (1 5 ; n = 5) CD44 (1 4 ; n = 8) CD44 (1 3 ; n = 4) CD44 + (1 4 ; n = 8) CD44 + (1 3 ; n = 4) Supplementary Figure 2. LICs are asymmetrically distributed in the CD44 + fraction. Survival of syngeneic (C57BL/6) recipient mice after transplantation with primary mouse NOTCH1 ΔE (a) and L161P-ΔPEST (b) leukemia cells, with the number of transplanted cells from each FACS-sorted subset indicated. Throughout the figure, n indicates the number of mice in each group. Raw survival data are provided in Supplementary Table 1. Nature Medicine doi:1.138/nm.296 SI-3

4 % of Max Events % of Max CD4 PKCθ null spleen (non-leukemic) PKCθ WT spleen (non-leukemic) CD8 1 8 SP4 1 8 SP8 PKCθ null 6 6 PKCθ WT 4 4 Unstained DCFDA Supplementary Figure 3. Normal splenic T cells from PKCθ null and PKCθ wild-type mice show similar levels of ROS. Flow cytometric analysis of freshly explanted splenocytes from normal, age-matched animals. WT, wild-type; SP4, CD4 + CD8 ; SP8, CD4 CD8 +. Nature Medicine doi:1.138/nm.296 SI-4

5 a HPBALL b RPMI PKCθ β-actin PKCθ β-actin Supplementary Figure 4. PKCθ overexpression and knock-down in human T-ALL cell lines. Western blot analysis of PKCθ protein expression in human T-ALL cell lines after retroviral overexpression of PKCθ (a) and lentiviral shrna-mediated knock-down of PKCθ (b). Sample labels are as in Fig. 2e,f. Numbers below each blot indicate the fold change over control cells after normalization to the loading control, β-actin. Results depicted are representative of at least two replicates. Nature Medicine doi:1.138/nm.296 SI-5

6 Events Events Events Events a HPBALL RV-Empty RV-PKCθ Unstained DCFDA DHE Mitosox RPMI 842 shscramble shpkc -92 shpkc -91 Unstained DCFDA DHE Mitosox Primary mouse leukemias PKCθ high PKCθ low PKCθ null Unstained DCFDA DHE Mitosox b Mouse PKCθ high leukemia CD44 + CD44 Total Unstained DCFDA DHE Mitosox Supplementary Figure 5. Alternate ROS detection dyes recapitulate results with DCFDA. Flow cytometric analysis of intracellular ROS amounts as measured by DCFDA, dihydroethidine (DHE), and Mitosox staining in human and mouse T-ALL cells with naturally varying or engineered amounts of PKCθ expression (a) and within gated CD44 + and CD44 subsets (b). N.B. Some DCFDA panels are reproduced from the main Figures to facilitate comparison. Nature Medicine doi:1.138/nm.296 SI-6

7 PKC (FITC) per cell (normalized to DAPI signal) PKC (FITC) per cell (normalized to DAPI signal) a PKCθ null PKCθ low PKCθ high 6 μm DAPI PKCθ b Primary NOTCH1-ΔE mouse leukemias CD44 + CD μM DAPI PKCθ CD44 + CD44 c NSG-expanded human T-lymphoblasts ROS low ROS high μM DAPI PKCθ ROS low ROS high Supplementary Figure 6. CD44 + and ROS low fractions of mouse and human T-ALLs, respectively, exhibit reduced PKCθ protein expression. (a) PKCθ immunofluorescence by leukemia cells with high, low, or no PKCθ expression (as confirmed by western blot; see Fig. 2a). Representative cells are depicted. (b,c) PKCθ immunofluorescence by FACS-sorted subsets of primary mouse NOTCH1-ΔE leukemia cells (b) and NSGexpanded human T-ALL lymphoblasts (c). Immunofluorescence quantitation, given as the PKCθ (FITC) signal after normalization to the DAPI signal for each imaged cell, is shown to the right of each panel. Error bars indicate standard deviation., P<.1 (Student's t-test). Nature Medicine doi:1.138/nm.296 SI-7

8 Live fraction (%) PKCθ WT cells (GFP) a Donors PKCθ low 1 5 Wild-type cells with low PKCθ PKCθ null cells Recipients e e PKCθ null PKCθ null cells (NGFR) PKCθ null cells (NGFR) PKCθ high 1 4 PKCθ null cells Wild-type cells with high PKCθ Recipients e b Subject F LV-Empty LV-PKCθ-CA PKCθ null cells (NGFR) Time cultured in vitro (d) Supplementary Figure 7. (a) PKCθ null leukemia cells outperform PKCθ wild-type cells in competitive transplant assay. Flow cytometric analysis of C57BL/6 recipient mice co-injected with PKCθ null (NGFR + ) plus PKCθ wild-type (GFP + ) NOTCH1-ΔE leukemia cells, the latter in ten-fold excess and with either high (PKCθ high ) or low (PKCθ low ) endogenous PKCθ expression (n = 4 for each group). Data depicted are representative of two replicate experiments. WT, wild-type. (b) Enforced expression of constitutively activated PKCθ is not toxic. Viability of NSG-expanded human T-ALL cells cultured in vitro after lentiviral transduction with a constitutively activated form of PKCθ (LV-PKCθ-CA) or empty virus control (LV-Empty). Nature Medicine doi:1.138/nm.296 SI-8

9 Disease latency (d) Proliferative fraction (% BrdU + ) Proliferative fraction (% BrdU + ) Survival (%) Survival (%) a Primary transplants b Secondary transplants Time after transplant (d) PKCθ wild-type (n = 4) PKCθ null (n = 4) Time after transplant (d) PKCθ null 1 5 (n = 4) 1 4 (n = 4) 1 3 (n = 4) c Secondary transplants d e ns wild-type null ns -Trolox ns +Trolox HPBALL RV-Empty RV-PKCθ RPMI 842 ns PKC genotype Supplementary Figure 8. (a,b) LIC frequency in PKCθ null leukemia is comparable to PKCθ wild-type leukemia. (a) Survival of mice transplanted with NOTCH1-ΔE transduced bone marrow from PKCθ null or wild-type backgrounds (primary recipients). (b) Survival of secondary recipient mice transplanted at limiting dilution with total PKCθ null leukemia cells generated in a. The calculated PKCθ null LIC frequency is 1 in ~3, cells (95% confidence interval of 1 in 8 8,9 cells). Compare with the PKCθ wild-type LIC frequency of 1 in ~6,1 cells (95% confidence interval of 1 in 2,8 13,2 cells) (see Fig. 1a). Throughout the figure, n indicates the number of mice in each group. (c e) PKCθ does not confer a growth advantage or disadvantage. (c) Latency of disease in mice developing leukemia after transplantation with primary PKCθ wild-type or PKCθ null leukemia cells. Data are replotted from secondary transplant survival curves for PKCθ wild-type and PKCθ null genotypes in Fig. 1a and Supplementary Fig. 8b, respectively. (d) Proliferation of the human T-ALL cell line HPBALL (low endogenous PKCθ expression) after transduction with PKCθ retrovirus (RV-PKCθ) or empty virus control (RV-Empty). Cells were also treated with the antioxidant Trolox (5 μm final) for 24 h prior to assay in order to counteract increased ROS levels (see Fig. 2e). (e) Proliferation of the human T-ALL cell line RPMI 842 (high endogenous PKCθ expression) after transduction with lentiviral shrnas against PKCθ (shpkcθ) or scrambled shrna control (shscramble). Error bars indicate s.d. for assays performed in triplicate. ns, not significant. Nature Medicine doi:1.138/nm.296 SI-9

10 Cell viability (% of untreated control) Cell viability (% of untreated control) Cell viability (% of untreated control) Cell viability (% of untreated control) Cell viability (% of untreated control) Cell viability (% of untreated control) a Mouse PKCθ high Mouse PKCθ low Mouse PKCθ null b ns * Mouse PKCθ high PKCθ inhibitor + PKCθ inhibitor c * * Irrad PKCθ inhibitor, then irrad Mouse PKCθ high Untreated Doxorubicin Irradiation d Doxorubicin Untreated ** Untreated Dexamethasone ** HPBALL RV-Empty RV-PKCθ 2 2 Trolox +Trolox Untreated Doxo Doxo Irrad +Trolox Irrad +Trolox e Untreated ** ** Doxo ** Irrad RPMI 842 shscramble shpkcθ-91 shpkcθ-92 f * ** Irrad ns PKCθ inhibitor, then irrad NSG-human PKCθ high (M71) NSG-human PKCθ low (F1313) Supplementary Figure 9. PKCθ expression and activity correlate with sensitivity to chemotherapy and radiation in a manner partially dependent on ROS. (a f) Cell viability h after treatment with doxorubicin, dexamethasone, or irradiation in vitro. Some samples were pretreated with either PKCθ inhibitor or the antioxidant Trolox 24 h prior to exposure to chemotherapy or irradiation. Error bars indicate s.d. for assays performed in triplicate. ns, not significant; *, P<.5; **, P<.1;, P<.1 (Student's t-test). Nature Medicine doi:1.138/nm.296 SI-1

11 Events % γh2ax + cells % γh2ax + cells g 1 ** HPBALL h 1 * RPMI ** Parental RV-PKCθ ns LV-Empty shpkcθ-92 Nonirrad Nonirrad Irrad Irrad i H2AX Mouse PKCθ null RV-PKCθ + PKCθ inhibitor, then irradiated RV-PKCθ, then irradiated Irradiated Non-irradiated Supplementary Figure 9 (continued). (g i) Cellular response to DNA damage as measured by γh2ax expression 1 h after irradiation in vitro. Some samples were pretreated with PKCθ inhibitor 24 h prior to irradiation. Error bars indicate s.d. for assays performed in triplicate. ns, not significant; *, P<.5; **, P<.1 (Student's t-test). Nature Medicine doi:1.138/nm.296 SI-11

12 PKCθ corr (r) p-value RUNX E-13 ZNF75A E-1 NBR E-1 NBAS E-1 SMAD E-1 ZNF354A E-9 SYNRG E-9 ZNRF E-9 RCSD E-9 LOC E-9 CEP E-9 CEP E-9 C11orf E-9 THYN E-9 FAM48A E-9 CEP E-9 CD E-8 DLG E-8 ETS E-8 FAM48A E-8 5 T-ALL patient samples (GSE14615) PKCθ corr (r) p-value TOX E-1 AGTPBP E-8 ARID5B E-8 FRYL E-8 SH2D1A E-8 SLC25A E-8 LAT E-8 AKAP E-7 CALM E-7 ELOVL E-7 RUNX E-7 DLG E-7 ERG E-7 BCL11B E-7 TNFAIP E-7 PSMD E-7 GNAQ E-7 EVI2A E-7 TNFAIP E-7 TOX E-6 42 T-ALL patient samples (GSE14613) PKCθ corr (r) p-value AKAP E-15 RUNX E-1 GNAQ E-9 AKAP E-9 MECP E-8 RHOH E-8 CPT1A E-8 FAM89B E-8 ATP2B E-7 DDAH E-7 ARHGAP E-7 ATP2B E-7 LCK E-7 CYFIP E-7 CD3E E-7 SYNRG E-7 SYNRG E-7 ATP2C E-6 DDAH E-6 VAT E-6 55 ETP-ALL patient samples (GSE8879) Supplementary Figure 1. Expression of PKCθ and RUNX1 are highly correlated in patient T-ALL samples. Microarray expression profile data from three independent patient cohorts. Patient samples (columns) are ordered left to right by PKCθ expression. The top 2 genes (rows) most correlated with PKCθ are ordered from top to bottom. RUNX1 is highlighted in each data series by arrowheads. Pearson correlation coefficients (r) and correlation P-values are indicated for each gene. Expression values are normalized for each probeset across samples in each dataset with mean of and an s.d. of 1. Nature Medicine doi:1.138/nm.296 SI-12

13 a HPBALL RPMI 842 HPBALL Jurkat RUNX1 RUNX ERK RUNX RUNX3 β-actin ERK2 b Jurkat CUTLL1 KOPTK1 RPMI 842 TALL GSI x 4 d RUNX3 β-actin c PF382 Jurkat CUTLL1 TALL-1 RPMI 842 HPBALL GSI x 6 7 d + + GSI x 4 d PKCθ PKCθ β-actin tubulin d Primary mouse NOTCH1-ΔE leukemias NSG-expanded human T-lymphoblasts GSI x 3 4 d PKCθ β-actin Supplementary Figure 11. (a) Knock-down of RUNX3 induces RUNX1 in T-ALL cells. Western blot analyses of RUNX1 and RUNX3 protein expression in human T-ALL cell lines transduced with lentiviral shrnas against RUNX3 (shrunx3) or scrambled negative control (shscramble). Numbers below each blot indicate the fold change after normalization to the loading control, β-actin or ERK2. Data depicted are representative of at least two replicates. (b d) NOTCH inhibition decreases RUNX3 and increases PKCθ in T-ALL cells. Western blot analyses of RUNX3 (b) and PKCθ (c,d) protein expression in T-ALL cells treated in vitro with γ-secretase inhibitor (GSI) to block NOTCH signaling or DMSO vehicle control. Numbers below each blot indicate fold change after normalization to the loading control, β-actin or tubulin. Data depicted are representative of at least two replicates. Nature Medicine doi:1.138/nm.296 SI-13

14 Events e CUTLL1 RV-Empty DN-MAML Secondary Ab only Unstained RUNX3 (MFI) (Normalized) RUNX3 f RPMI 842 TetOn DN-MAML Jurkat TetOn DN-MAML + + Dox RUNX3 β-actin Supplementary Figure 11 (continued). (e,f) Dominant negative Mastermind-like-1 (DN-MAML) confirms that effects of GSI on RUNX3 are NOTCHspecific. (e) Flow cytometric analysis of RUNX3 protein expression in the human T-ALL cell line CUTLL1 3 d after retroviral transduction with DN-MAML or empty virus control. MFI, mean fluorescence intensity. (f) Western blot analysis of RUNX3 protein expression in human T-ALL cell lines RPMI 842 and Jurkat in which DN-MAML expression is under control of a doxycycline-inducible promoter. Numbers below the blot indicate fold change after normalization to the loading control, β-actin. Data depicted are representative of two replicates. Nature Medicine doi:1.138/nm.296 SI-14

15 Events % of Max Primary leukemias Mouse NOTCH1-ΔE NSG-expanded human T-lymphoblasts Human T-ALL cell lines RPMI 842 Jurkat KOPTK DMSO GSI Unstained DCFDA Supplementary Figure 12. NOTCH inhibition increases ROS in T-ALL cells. Flow cytometric analysis of intracellular ROS amounts by DCFDA staining in human and mouse T-ALL cells after treatment in vitro with γ-secretase inhibitor (GSI) to block NOTCH signaling or DMSO vehicle control for 3 d. Data depicted are representative of at least two replicates. Nature Medicine doi:1.138/nm.296 SI-15

16 Cell proliferation (normalized to mock) Mock GSI.4.2 PKCθ null leukemia PKCθ WT leukemia Supplementary Figure 13. PKCθ null and PKCθ wild-type leukemias are similarly dependent on NOTCH signaling. Cell proliferation as measured by BrdU incorporation in freshly explanted primary NOTCH1-ΔE leukemia cells that were cultured briefly in vitro, then treated with γ-secretase inhibitor (GSI) or DMSO vehicle control (Mock) for 1 3 d. Nature Medicine doi:1.138/nm.296 SI-16

17 Events CD44 mrna level (normalized to β-actin) a * Mock GSI.5.1 Primary mouse NOTCH1-ΔE leukemia NSG-expanded human T-lymphoblasts b Primary mouse NOTCH1-ΔE leukemias NSG-expanded human T-lymphoblasts Mock GSI Unstained CD Supplementary Figure 14. NOTCH inhibition decreases CD44 expression in T-ALL cells. (a) Quantitative RT-PCR analysis of CD44 mrna expression in freshly explanted mouse and human T-ALL cells cultured in vitro with γ-secretase inhibitor (GSI) to block NOTCH signaling or DMSO vehicle control (Mock) for 3 d. Error bars indicate s.d. of assays performed in triplicate. *, P<.5;, P<.1 (Student's t-test). (b) Flow cytometric analysis of CD44 protein expression in freshly explanted mouse and human T-ALL cells cultured in vitro with γ-secretase inhibitor (GSI) to block NOTCH signaling or DMSO vehicle control (Mock) for 3 d. Nature Medicine doi:1.138/nm.296 SI-17

18 CD44 DCFDA CD4 CD44 ROS high ROS low CD44 + CD K 1K 15K 2K 25K 5K 1K 15K 2K 25K K 1K 15K 2K 25K 5K 1K 15K 2K 25K K 1K 15K 2K 25K 5K 1K 15K 2K 25K K 1K 15K 2K 25K K 1K 15K 2K 25K e FSC FSC CD8 CD8 CD8 CD8 Supplementary Figure 15. CD44 + ROS low cells variably express CD4 and CD8. Flow cytometric analysis of freshly explanted primary NOTCH1-ΔE leukemia cells. Each row depicts cells from a different leukemic animal. Live, NGFR + leukemia cells are shown. Gated analyses of CD44, CD44 + ROS high, and CD44 + ROS low subsets are indicated. Depicted examples were selected to demonstrate the range of observed CD4/CD8 immunophenotypes among total cells. Nature Medicine doi:1.138/nm.296 SI-18

19 Species Clone Recipient Cell Population Cell Dose Leukemic / Injected Mice Mouse ΔE (C3-1/3-2) C57BL/6 Total 1,, 4 / Mouse ΔE (C3-1/3-2) C57BL/6 Total 1, 8 / Mouse ΔE (C3-1/3-2) C57BL/6 Total 1, 6 / Mouse ΔE (C3-1/3-2) C57BL/6 Total 1, 2 / 7 4 Mouse ΔE (C3-1/3-2) C57BL/6 Total 1 / 4 NR Mouse ΔE (H14-4) C57BL/6 CD44 1, 2 / Mouse ΔE (H14-4) C57BL/6 CD44 + 1, 4 / 4 32 Mouse L161PΔPEST (H3-7;1) C57BL/6 Total 1,, 3 / Mouse L161PΔPEST (H3-7;1) C57BL/6 Total 1, 1 / 5 NR Mouse L161PΔPEST (H3-7;1) C57BL/6 CD44 1, 1 / 8 NR Mouse L161PΔPEST (H3-7;1) C57BL/6 CD44 1, / 4 NR Mouse L161PΔPEST (H3-7;1) C57BL/6 CD44 + 1, 8 / 8 4 Mouse L161PΔPEST (H3-7;1) C57BL/6 CD44 + 1, 1 / 4 NR Mouse ΔE (H14-2) C57BL/6 CD44 1, 1 / 8 NR Mouse ΔE (H14-2) C57BL/6 CD44 1 / 4 NR Mouse ΔE (H14-2) C57BL/6 CD44 + ROS high 1, 2 / 4 65 Mouse ΔE (H14-2) C57BL/6 CD44 + ROS high 1 / 4 NR Mouse ΔE (H14-2) C57BL/6 CD44 + ROS low 1, 8 / Mouse ΔE (H14-2) C57BL/6 CD44 + ROS low 1 4 / 4 42 Mouse ΔE (H14-2) NSG CD / 3 28 Mouse ΔE (H14-2) NSG CD44 1 / 4 NR Mouse ΔE (H14-2) NSG CD / 12 NR Mouse ΔE (H14-2) NSG CD44 + ROS high 1 / 4 NR Mouse ΔE (H14-2) NSG CD44 + ROS high 1 / 4 NR Mouse ΔE (H14-2) NSG CD44 + ROS high 1 / 1 NR Mouse ΔE (H14-2) NSG CD44 + ROS low 1 4 / 4 15 Mouse ΔE (H14-2) NSG CD44 + ROS low 1 4 / 4 27 Mouse ΔE (H14-2) NSG CD44 + ROS low 1 2 / 11 NR Mouse K-ras G12D (LCR 1985) C57BL/6 CD44 1, / 4 NR Mouse K-ras G12D (LCR 1985) C57BL/6 CD44 1, / 4 NR Mouse K-ras G12D (LCR 1985) C57BL/6 CD44 1 / 4 NR Mouse K-ras G12D (LCR 1985) C57BL/6 CD44 + ROS high 1, 2 / Mouse K-ras G12D (LCR 1985) C57BL/6 CD44 + ROS high 1, / 4 NR Mouse K-ras G12D (LCR 1985) C57BL/6 CD44 + ROS high 1 / 4 NR Mouse K-ras G12D (LCR 1985) C57BL/6 CD44 + ROS low 1, 6 / 6 21 Mouse K-ras G12D (LCR 1985) C57BL/6 CD44 + ROS low 1, 1 / 4 NR Mouse K-ras G12D (LCR 1985) C57BL/6 CD44 + ROS low 1 / 4 NR Mouse PKCθ null ΔE (V14-4) C57BL/6 Total 1, 4 / 4 3 Mouse PKCθ null ΔE (V14-4) C57BL/6 Total 1, 4 / 4 43 Mouse PKCθ null ΔE (V14-4) C57BL/6 Total 1, 1 / 4 NR Median Survival (Days) Nature Medicine doi:1.138/nm.296 SI-19

20 Human M71-1;14 NSG ROS low 1, 4 / 4 72 Human M71-1;14 NSG ROS high 1, 4 / Human M71-1;14 NSG Total 1, 4 / Human D115-1;2 NSG ROS low 1, 4 / 4 31 Human D115-1;2 NSG ROS high 1, 4 / Human D115-1;2 NSG Total 1, 4 / 4 44 Human F (Empty vector) NSG Total 1, 4 / 4 56 Human F (Lenti PKCθ-CA) NSG Total 1, / 4 NR Supplementary Table 1. Summary of leukemia-initiating cell (LIC) transplant assays. All donor mouse leukemias were C57BL/6 background. Donor human leukemia cells were diagnostic T-ALL biopsy specimens previously expanded up to 2 passages in immunodeficient NOD/Scid/Il2rg / (NSG) mice. Transplant recipients were either non-irradiated C57BL/6 mice or sub-lethally irradiated NSG mice as indicated. All transplants were performed by tail vein injection. NR = not reached. Nature Medicine doi:1.138/nm.296 SI-2