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NOTCH1-driven UBR7 stimulates nucleotide biosynthesis to promote T cell acute lymphoblastic leukemia

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Science Advances  27 Jan 2021:
Vol. 7, no. 5, eabc9781
DOI: 10.1126/sciadv.abc9781
  • Fig. 1 Identification of UBR7-interacting proteins.

    (A) UBR7-FLAG expression in stable HEK293T cells and silver stain showing the elutes from FLAG immunoprecipitation (IP) in parental and UBR7-FLAG–expressing HEK293T cells. (B) Venn diagram representing the number of common and exclusive FLAG-UBR7–associated proteins between two independent experiments E1 and E2. (C) Scatter plot from two independent MS runs (E1 and E2) showing the UBR7-FLAG–associated proteins. The axes represent the log of spec counts of UBR7-FLAG normalized to parental. A curated list of UBR7-interacting proteins can be found in table S1. (D) KEGG pathway analysis representing top 10 significantly enriched pathways associated with UBR7-interacting partners from E1.

  • Fig. 2 UBR7 interacts with PRPS1 and PRPS2.

    (A and B) Co-IP from the whole-cell extracts of HEK293T parental or stably expressing UBR7-FLAG showing the interaction of UBR7-FLAG with endogenous PRPS1 (A) or PRPS2 (B). (C and D) FLAG-PRPS1 (C) or FLAG-PRPS2 (D) interaction with endogenous UBR7 in HEK293T cells transfected with either empty vector, FLAG-PRPS1, or FLAG-PRPS2. (E and F) Co-IP showing the interaction of endogenous UBR7 with PRPS1 (E) or PRPS2 (F) in HEK293T cells. (G) Schematic representing N-terminally FLAG-tagged UBR7 full length and various truncation mutants. (H to J) A Co-IP assay from the whole-cell extracts of HEK293T cells transiently transfected with FLAG-UBR7 wild-type, various truncation (H and I), or point mutants (J), with endogenous PRPS1 and PRPS2. The numbers below the IP blots in (J) represent the band intensities of PRPS relative to FLAG-UBR7.

  • Fig. 3 UBR7 stabilizes PRPS enzymes through the degradation of PRPSAP.

    (A and B) Immunoblot showing the PRPS1 (A) and PRPS2 (B) levels in HEK293T cells transduced with control (Ctrl.) or UBR7 shRNA. (C) UBR7, PRPS1, and PRPS2 mRNA fold change in UBR7 knockdown cells normalized to control as measured from qPCR. (D and E) PRPS1 and PRPS2 levels in doxycycline-inducible UBR7 shRNA stable HEK293T cells treated with doxycycline (1 μg/ml) or DMSO for 48 hours. (F) UBR7, PRPS1, and PRPS2 mRNA fold change in doxycycline-inducible UBR7 knockdown cells normalized to control as measured from qPCR. (G and H) Co-IP demonstrating the interaction of FLAG-tagged PRPS1 (G) or UBR7 (H) with endogenous PRPSAP1 in HEK293T cells 1 and 2, represented by arrows, indicate two isoforms of PRPSAP1. (I) PRPS1 and PRPSAP1 levels in control or UBR7 shRNA HEK293T cells. (J) Ubiquitination of transiently transfected vector or FLAG-PRPSAP1 in control or UBR7 shRNA–transduced HEK293T cells treated with 10 μM MG132 for 6 hours before anti-FLAG IP. (K) PRPS1 levels in control or PRPSAP1 shRNA transduced HEK293T cells. Data points in (C) and (F) represent independent biological replicates. Each biological replicate is the mean of at least three technical replicates. Error bars are means ± SD, and P values were computed from Student’s t test and shown with respect to control shRNA (n.s.P > 0.05, *P ≤ 0.05, **P ≤ 0.01). Numbers below the immunoblots represent the relative band intensities.

  • Fig. 4 UBR7 is highly expressed in T-ALL and is regulated by NOTCH1.

    (A) UBR7 mRNA expression from CCLE dataset. (B) Tracks showing NOTCH1 ChIP-seq signal enrichment at the UBR7 locus in CUTLL1 cells treated with GSI (+) or GSI washed off (−). These data are accessible through GSE51800 (41). (C) Immunoblot showing UBR7, PRPS1, and PRPS2 levels in two NICD1-negative cell lines, T-ALL1 and Loucy, and various NICD1-positive cell lines. The band intensities (arbitrary unit) of UBR7, PRPS1, and PRPS2 normalized to β-actin and correlation curve with 95% confidence interval and Pearson correlation value (R) are shown below immunoblot. (D to G) UBR7, PRPS1, and PRPS2 levels in DND41 or ALL-SIL cells treated with increasing doses of GSI, DAPT, for 24 hours. The table below immunoblots shows the relative levels of UBR7, PRPS1, and PRPS2 normalized to β-actin.

  • Fig. 5 UBR7 regulates nucleotide metabolism in T-ALL cells.

    (A and B) Co-IP demonstrating the interaction of endogenous UBR7 with PRPS1 or PRPS2 in CUTLL1 (A) and Jurkat (B) cells. (C and D) Immunoblot showing the PRPS1 and PRPS2 levels in CUTLL1 (C) and Jurkat (D) cells transduced with control or UBR7 shRNA. (E and F) Fold change in UBR7, PRPS1, and PRPS2 mRNA in UBR7 shRNA CUTLL1 (E) or Jurkat (F) normalized to their control counterparts as measured from qPCR. (G) Schematic representing the involvement of PRPP in nucleotide synthesis by de novo and salvage pathway. 14C-glycine and 3H-hypoxanthine are highlighted as they are used in (J) and (K) to show their involvement in de novo and salvage purine synthesis pathway. Dashed lines represent the multiple steps. PPP, pentose phosphate pathway. (H and I) PRPP levels per unit total protein in control or UBR7 shRNA–treated CUTLL1 (H) and Jurkat (I) cells as measured from LC-MS. (J and K) De novo and salvage purine synthesis measured from 14C-glycine or 3H-hypoxanthine incorporation, respectively, in total RNA in CUTLL1 (J) and Jurkat (K) vector or UBR7-FLAG–expressing cells transduced with control or UBR7 shRNA. Data points represent independent biological replicate. Error bars are means ± SD, and P values were computed from Student’s t test and shown only with respect to control or vector + control shRNA as indicated (n.s.P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001).

  • Fig. 6 UBR7 knockdown attenuates T-ALL cell proliferation and oncogenic potential.

    (A and B) Cell proliferation curves of CUTLL1 (A) and Jurkat (B), vector and UBR7-FLAG cells transduced with a constitutive UBR7 shRNA targeting 3′UTR as measured by relative fluorescence unit (RFU) of reduced alamarBlue. Data points represent mean of five technical replicates. Error bars are mean normalized to day 0 ± SD. P values are computed from Student’s t test and shown only with respect to vector + control shRNA (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001). Western blot analysis below the proliferation curve shows the knockdown of endogenous UBR7 and the expression of exogenous UBR7 from the CUTLL1 (A) and Jurkat (B) lysates of day 4. (C and D) Representative images and colony counts from colony formation assay of control or UBR7 shRNA–infected CUTLL1 (C) or Jurkat (D) cells cultured in methylcellulose-based medium for 3 weeks. Data points represent the independent biological replicate. P values are computed from Student’s t test (**P ≤ 0.01, and ****P ≤ 0.0001). (E and F) Representative IVIS images at days 12 and 25 after intravenous transplantation of mice with control or UBR7 shRNA–transduced CUTLL1-luciferase (E) and Jurkat-luciferase cells (F). The plots below represent the tumor progression measured from mean normalized IVIS intensity from all the mice in the group (CUTLL1-luciferase control shRNA, n = 5; CUTLL1-luciferase UBR7 shRNA, n = 7; Jurkat-luciferase control shRNA, n = 7; Jurkat-luciferase UBR7 shRNA, n = 7). (G and H) Mouse survival curves of CUTLL1 (G) or Jurkat (H) control and UBR7 shRNA groups. (I) Model showing the upstream regulation of UBR7 by NOTCH1 signaling and T-ALL promotion through UBR7-PRPS–mediated nucleotide synthesis. PRPSAP1 polyubiquitination–mediated degradation by UBR7 and inhibition of PRPS activity are also depicted.

  • Table 1 List of primers used in the study.

    qPCR primers for gene expression
    UBR75′-GAACAGGGAAAGGATGATGTCCG-3′ (forward)
    5′-AGCTCCTGAAGTTTGCAGCCAG-3′ (reverse)
    PRPS15′-GGCTGACACTTGTGGCACAATC-3′ (forward)
    5′-GATGCGAGAAATAGCAGGACCG-3′ (reverse)
    PRPS25′-GGTCACGAAGAAGTTCAGCAACC-3′ (forward)
    5′-GAGGAGTTCCATCAGGTTGTCG-3′ (reverse)
    GAPDH5′-TTCAACAGCGACACCCACTC-3′ (forward)
    5′-TGACAAAGTGGTCGTTGAGGG-3′ (reverse)
    qPCR primers for ChIP
    HES15′-AAGTTTCACACGAGCCGTTC-3′ (forward)
    5′-GCTGTTATCAGCACCAGCTC-3′ (reverse)
    UBR75′- AGCTTCCAGAACACGACACC-3′ (forward)
    5′-TCCGACAAACGGATGTCACT-3′ (reverse)
    UBR7 distal site5′-GGCTGACACTTGTGGCACAATC-3′ (forward)
    5′-GATGCGAGAAATAGCAGGACCG-3′ (reverse)

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