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 minutes. (B-D) MOLT-4 cells were treated with either vehicle, whole extract or fractions 1-4 (3 μg/mL) and (B) apoptosis was analyzed after 24 h (N=3) via Annexin V/PI, (C-D) NICD and cleaved caspase-3 (C. Cas. 3) expressions were evaluated (N=3) after 3 h with β-tubulin as the loading control. A representative blot is shown. (E) UHPLC/UV chromatogram of fraction 2, the specific compounds constituting the fraction are marked (C1-C5). (F) Chemical structure and peak assignment of CBD and 331-18A according to 1H and 13C NMR. Atom numbering is according to the monoterpene numbering system. (G-I) MOLT-4 cells were treated with the whole extract (3 μg/mL) or with 0.06 µg/mL 331-18A, 0.06 µg/mL CBDV and 1.5 µg/mL CBD, their corresponding concentrations in the extract, and their different combinations. Cells were assessed for (G) apoptosis after 24 h (N=3) via Annexin V/PI assay and (H-I) NICD expression after 3 h (N=3) with GAPDH as the loading control. Results are presents as mean ± SEM and statistically analyzed with One-way ANOVA (*p<0.05, **p<0.01, ***p<0.001).

331-18A, CBDV and CBD mediate their effect through CB2 and TRPV1.

(A) The mRNA levels of receptors CNR1 (CB1), CNR2 (CB2), GPR55, TRPV1, TRPA1 and TRPM8 were evaluated by qRT-PCR. Gene expression levels were calculated as ΔCT normalized to GUSB housekeeping gene. Results are presented as mean expression of three biological replicates. High ΔCT values indicate low receptor expression. (B) MOLT-4 cells were pretreated with 50 µM antagonist to CB2 (AM630) or TRPV1 (AMG9810) for 30 min, then treated with the combination of the three cannabinoids for 3 h. NICD expression was evaluated by western blot (N=3) with GAPDH as the loading control and statistically analyzed with an unpaired student’s t-test (**p<0.01, ***p<0.001). (C) Representative blots of B. (D) Calcium release by MOLT-4 cells was measured with the Fluo-4 calcium probe immediately following treatment with either vehicle, whole extract, a combination of 331-18A, CBDV and CBD, and each cannabinoid separately. The calcium curves represent an average of three independent experiments. (E) Reduction of each phytocannabinoid-induced calcium release by pretreatment with 50 µM of antagonist to CB2 (AM630) or TRPV1 (AMG9810) for 30 min. The calcium curve for each compound is given again in this graph to compare the effects with the antagonists (N=3, two-way ANOVA, *p<0.05, **p<0.01). (F) MOLT-4 cells were pretreated with 50 µM antagonist to CB2 (SR-144,528) or TRPV1 (AMG9810) or both for 30 min, then treated with the combination of the three cannabinoids or left untreated. Apoptosis was analyzed after 24 h (N=3) by Annexin V/PI. Results are presented as mean ± SEM and statistically analyzed with one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001).

Notch1 downregulation by 331-18A, CBDV and CBD combination is mediated via ATF4-CHOP-CHAC1 signaling pathway.

(A) MOLT-4 cells were treated for 3 h with either vehicle or the whole Extract (3 µg/mL) and an RNA-seq (affymetrix) scatter plot presents the differential expression; the increased abundance genes CHAC1, CHOP and ATF4 are marked (N=3). (B-D) MOLT-4 cells were treated for 3 h with 331-18A, CBDV and CBD combination (N=3), the mRNA expression of ATF4, CHOP and CHAC1 genes was evaluated with qRT-PCR at different time points (0-60 min). Differences are presented as fold-change ± SEM and statistically analyzed with one-way ANOVA (***p<0.001, ****p<0.0001). (E-G) MOLT-4 cells were treated as in B-D and the protein expression of ATF4, CHOP and CHAC1 were evaluated with β-tubulin as the loading control. Intensity analysis was performed on three independent experiments (N=3) and statistically analyzed with an unpaired Student’s t-test (**p<0.01). (H) Representative blots. (I-K) Analysis of ATF4, CHOP and CHAC1 mRNA expression at different time points following pretreatment with CB2 and TRPV1 antagonists for 30 min and then treatment with the cannabinoid combination (N=3), analyzed with two-way ANOVA (***p<0.001, ****p<0.0001).

331-18A, CBDV and CBD combination activates eIF2α.

MOLT-4 cells were pretreated for 30 min with the eIF2α inhibitor ISRIB (150 µM) or left untreated, then treated with vehicle or the combination of 331-18A, CBDV and CBD for 3 h. (A) Viability after 24 hrs was assessed with XTT (N=3). (B) NICD protein expression was assessed (N=3) with β-Tubulin as the loading control (unpaired Student’s t-test, *p<0.05). (C) A representative image also showing the protein levels of phosphorylated eIF2α (Ser51) and total eIF2α. (D-F) ATF4, CHOP and CHAC1 gene expression was assessed via qRT-PCR 4 hrs after treatment (N=3). Results are presented as mean ± SEM and statistically analyzed with one-way ANOVA (****p<0.0001).

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 separatly was plotted. (B-E) Synergy distribution for different ratios of cannabinoids was calculated by HSA model with SynergyFinder web application (v 2.0), representative dose-response matrices are presented for CBD at (B) 0, (C) 0.1, (D) 0.25 and (E) 0.5 µg/ml. (F-G) Best synergy scores according to HSA model presented for the triple combination relative to the two-cannabinoid combinations.

Inhibition of cancer progression in-vivo by a combination of 331-18A, CBDV and CBD.

(A) Female NOD/Scid mice (N=4-5/group, two independent experiments) were engrafted subcutaneously with 1×106 MOLT-4 cells. After seven days, the mice were randomly divided into two groups and alternate-day treated intraperitoneally with either vehicle or a combination of 331-18A (1.11 mg/kg), CBDV (1.11 mg/kg) and CBD (17.77 mg/kg). Ectopic tumor volume was measured using a vernier caliper and calculated according to the formula (length × width2) × 0.5. The average difference between groups was statistically analyzed by Bonferroni’s multiple comparisons test (*p<0.05, ***p<0.001). (B) After 34 days of treatment, the mice were sacrificed and tumors were excised and weighed (N=9). The difference in the weights of the tumors between treatment groups was statistically analyzed by unpaired Student’s t-test (****p<0.0001). (C) Representative photograph of excised tumors. (D) A representative blot (top) and fold change (bottom) of NICD protein expression in excised tumors at day 34 after treatment with either vehicle (C) or a combination of the three cannabinoids (T), with GAPDH as the loading control. Data are presented as mean ± SEM (N=3) and statistically analyzed by unpaired Student’s t-test (**p<0.01). (E) Body weight (grams) in vehicle- and cannabinoids-treated mice. (F) Female NSG mice were intravenously injected with 1×106 human CCRF-CEM cells. Mice were randomly divided into three groups (N=5/group) and treated intraperitoneally daily for five days, then left untreated for two days before starting another cycle, for a period of three weeks. Treatment groups were vehicle, pure CBD (30 mg/kg), and a combination of CBD (26.67 mg/kg), CBDV (1.67 mg/kg) and 331-18A (1.67 mg/kg) at the same overall concentration as pure CBD. After 24 days, the percentage of hCD45+ cells in the bone marrow was measured with flow cytometry and statistically analyzed by unpaired student’s t-test (*p<0.05). (G) Body weight (grams) in vehicle- and cannabinoids-treated mice. (H) Survival analyses after CCRF-CEM injection followed by treatment with either vehicle or the cannabinoid combination (N=10/group), statistical differences were calculated with the Log-rank (Mantel-Cox) test (*p<0.05).

Schematic representation.

(A) In WT cells, the Notch1 receptor is only activated upon ligand binding. In T-ALL cells that have a Notch1 mutation, the receptor is constitutively active regardless of ligand binding, leading to a positive feedback loop in which the mature receptor is cleaved by a series of sequential cleavage events, causing the release of NICD, which translocates to the nucleus where it promotes transcription of target genes involved in cell growth. The premature Notch1 is cleaved by a furin-like convertase in the trans-Golgi apparatus, resulting in a mature receptor that is transported to the membrane. (B) The three phytocannabinoids CBD, CBDV and 331-18A in the whole extract stimulate CB2 and TRPV1 leading to an increase in cytosolic Ca2+ and ER stress induced activation of eIF2α followed by ATF4-CHOP-CHAC1 signaling. CHAC1 inhibits the furin-like cleavage of Notch1, preventing its maturation and resulting in reduced NICD formation, reduced cell viability and increased apoptosis.