Cannabinoid combination targets NOTCH1-mutated T-cell acute lymphoblastic leukemia through the integrated stress response pathway
- The Laboratory of Cancer Biology and Cannabinoid Research, Faculty of Biology, Technion – Israel Institute of Technology, Israel
Figures
Figure 1 with 3 supplements
Identifying specific phytocannabinoids responsible for the cytotoxic effect of the whole extract.
(A) A semi-preparative HPLC-UV chromatogram of the whole extract showing the four fractions collected every 10 min. (B–D) MOLT-4 cells were treated with either vehicle, whole extract, or fractions …
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Figure 1—source data 1
Original file for the western blot presented in Figure 1D.
- https://cdn.elifesciences.org/articles/90854/elife-90854-fig1-data1-v1.zip
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Figure 1—source data 2
Original file for the western blot presented in Figure 1I.
- https://cdn.elifesciences.org/articles/90854/elife-90854-fig1-data2-v1.zip
Figure 1—figure supplement 1
Fraction 2 is as effective as the whole extract in reducing the viability of MOLT-4 cells.
MOLT-4 cells were treated with either whole extract or each of the four fractions and 24 hr later the viability of the cells was assessed with XTT.
Figure 1—figure supplement 2
Specific identification of phytocannabinoids with anti-cancer properties by spectral matching and peak purity.
(A) MS/MS spectral matching of C1, C2, and C5 versus our spectral library and analytical standards of cannabidivarin (CBDV) and cannabidiol (CBD), respectively. (B) UHPLC/UV chromatogram of extract …
Figure 1—figure supplement 3
Cannabidiol (CBD) and 331-18A nuclear magnetic resonance (NMR) spectra.
(A) 1H NMR spectrum of CBD. (B) 13C NMR spectrum of CBD. (C) 1H NMR spectrum of 331-18A. (D) 13C NMR spectrum of 331-18A.
Figure 2 with 1 supplement
331-18A, cannabidivarin (CBDV), and cannabidiol (CBD) mediate their effect through CB2 and TRPV1.
(A) The mRNA levels of receptors CNR1 (cannabinoid receptor type 1 [CB1]), CNR2 (CB2), GPR55, TRPV1, TRPA1, and TRPM8 were evaluated by qRT-PCR. Gene expression levels were calculated as ΔCT …
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Figure 2—source data 1
Original file for the western blot presented in Figure 2C.
- https://cdn.elifesciences.org/articles/90854/elife-90854-fig2-data1-v1.zip
Figure 2—figure supplement 1
Notch1 downregulation by extract 12 is mediated through CB2 and TRPV1 followed by ATF4-CHOP-CHAC1 signaling pathway.
(A) MOLT-4 cells were pretreated for 30 min with either of the following antagonists: CID (10 µM) for GPR55, BIM (10 µM) for general Gαq GPCR, AM630 (50 µM) for CB2 and AMG9810 (50 µM) for TRPV1; …
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Figure 2—figure supplement 1—source data 1
Original file for the western blot presented in Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/90854/elife-90854-fig2-figsupp1-data1-v1.zip
Figure 3 with 1 supplement
Notch1 downregulation by 331-18A, cannabidivarin (CBDV), and cannabidiol (CBD) combination is mediated via ATF4-CHOP-CHAC1 signaling pathway.
(A) MOLT-4 cells were treated for 3 hr with either vehicle or the whole extract (3 µg/mL) and an Affymetrix scatter plot presents the differential expression; the increased-abundance genes CHAC1, DDI…
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Figure 3—source data 1
Original file for the western blot presented in Figure 3H.
- https://cdn.elifesciences.org/articles/90854/elife-90854-fig3-data1-v1.zip
Figure 3—figure supplement 1
Notch1 downregulation by extract 12 is mediated through the ATF4-CHOP-CHAC1 signaling pathway.
(A–C) Time course (n=3) qRT-PCR of ATF4, DDIT3, and CHAC1 genes at different time points (0-60 min) following treatment with extract 12 (3 µg/mL) and statistically analyzed with one-way ANOVA (*p<0.0…
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Figure 3—figure supplement 1—source data 1
Original file for the western blot presented in Figure 3—figure supplement 1E.
- https://cdn.elifesciences.org/articles/90854/elife-90854-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Original file for the western blot presented in Figure 3—figure supplement 1I.
- https://cdn.elifesciences.org/articles/90854/elife-90854-fig3-figsupp1-data2-v1.zip
Figure 4 with 1 supplement
331-18A, cannabidivarin (CBDV), and cannabidiol (CBD) combination activates eIF2α.
MOLT-4 cells were pretreated for 30 min with the eIF2α inhibitor integrated stress response inhibitor (ISRIB) (150 µM) or left untreated, then treated with vehicle or the combination of 331-18A, …
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Figure 4—source data 1
Original file for the western blot presented in Figure 4C.
- https://cdn.elifesciences.org/articles/90854/elife-90854-fig4-data1-v1.zip
Figure 4—figure supplement 1
Whole extract treatment activates eIF2α.
(A) MOLT-4 cells were with extract 12 and the phosphorylation of eIF2α was assessed at different times up to 2 hr after treatment. (B) Representative blots of phosphorylated eIF2α (Ser51), total …
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Figure 4—figure supplement 1—source data 1
Original file for the western blot presented in Figure 4—figure supplement 1B.
- https://cdn.elifesciences.org/articles/90854/elife-90854-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Original file for the western blot presented in Figure 4—figure supplement 1E.
- https://cdn.elifesciences.org/articles/90854/elife-90854-fig4-figsupp1-data2-v1.zip
Figure 5
All three cannabinoids must be combined for a synergistic effect.
(A) MOLT-4 cell death (N=3) was assessed by XTT following treatments with concentrations ranging 0–2 µg/mL and a dose-response curve for each cannabinoid separately was plotted. (B–E) Synergy …
Figure 6 with 2 supplements
Inhibition of cancer progression in vivo by a combination of 331-18A, cannabidivarin (CBDV), and cannabidiol (CBD).
(A) Female nonobese diabetic-severe combined immunodeficiency (NOD/Scid) mice (N=4–5/group, two independent experiments) were engrafted subcutaneously with 1×106 MOLT-4 cells. After 7 days, the mice …
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Figure 6—source data 1
Original file for the western blot presented in Figure 6D.
- https://cdn.elifesciences.org/articles/90854/elife-90854-fig6-data1-v1.zip
Figure 6—figure supplement 1
Inhibition of tumor growth in vivo by whole extract.
(A) Female NSG mice were intravenously injected with 1×106 human CCRF-CEM cells. Mice were randomly divided into two groups (n=19) and alternate-day treated intraperitoneally with either vehicle or …
Figure 6—figure supplement 2
Treatment with extract containing the three cannabinoids inhibits leukemic expansion in an in vivo patient-derived xenografts (PDX) model.
NSG mice were engrafted with 1×106 primary Notch1-mutated cells expanded from a T-cell acute lymphoblastic leukemia (T-ALL) patient. After 35 days, the mice were randomly divided into two groups …
Figure 7
Schematic representation.
(A) In WT cells, the Notch1 receptor is only activated upon ligand binding. In T-cell acute lymphoblastic leukemia (T-ALL) cells that have a Notch1 mutation, the receptor is constitutively active …
Author response image 1
Partial knockdown of CHAC1 hinders extract-induced cell death.
(A) MOLT-4 cells were treated with either an empty vector or shRNA for Chac1, 369 and 739 represent two different areas of Chac1, for 48 hrs. Then, the gene expression of CHAC1 was assessed via …
Author response image 2
Partial knockdown of CHAC1 hinders extract-induced cell death.
(A) MOLT-4 cells were treated with either an empty vector or shRNA for Chac1, 369 and 739 represent two different areas of Chac1, for 48 hrs. Then, the gene expression of CHAC1 was assessed via …
Author response image 3
Dose-response of CB2 and TRPV1 inhibitors in MOLT-4 cells.
MOLT-4 cells were treated with increasing concentrations (µM) of (A) CB2 inhibitor AM630 or (B) TRPV1 inhibitor AMG9810; and 24 hrs later the viability of the cells was assessed with XTT.
Additional files
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Supplementary file 1
Phytocannabinoid concentrations by UHPLC/LC-MS of fractions relative to the whole extract.
- https://cdn.elifesciences.org/articles/90854/elife-90854-supp1-v1.docx
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Supplementary file 2
Phytocannabinoid concentrations by UHPLC/LC-MS of Cannabis fraction 2 peaks.
- https://cdn.elifesciences.org/articles/90854/elife-90854-supp2-v1.docx
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Supplementary file 3
1H and 13C peak assignments and chemical shifts of 331-18A.
- https://cdn.elifesciences.org/articles/90854/elife-90854-supp3-v1.docx
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Supplementary file 4
Ten most increased- and decreased-abundance genes following treatment of MOLT-4 cells with the whole extract according to Affymetrix.
- https://cdn.elifesciences.org/articles/90854/elife-90854-supp4-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/90854/elife-90854-mdarchecklist1-v1.docx
