Cannabinoid combination targets NOTCH1-mutated T-cell acute lymphoblastic leukemia through the integrated stress response pathway.

In T-cell acute lymphoblastic leukemia (T-ALL), more than 50% of cases display autoactivation of Notch1 signaling, leading to oncogenic transformation. We have previously identified a specific chemovar of Cannabis that induces apoptosis by preventing Notch1 maturation in leukemia cells. Here, we isolated three cannabinoids from this chemovar that synergistically mimic the effects of the whole extract. Two were previously known, cannabidiol (CBD) and cannabidivarin (CBDV), whereas the third cannabinoid, which we termed 331-18A, was identified and fully characterized in this study. We demonstrated that these cannabinoids act through cannabinoid receptor type 2 and TRPV1 to activate the integrated stress response pathway by depleting intracellular Ca2+. This is followed by increased mRNA and protein expression of ATF4, CHOP, and CHAC1, which is hindered by inhibiting the upstream initiation factor eIF2α. The increased abundance of CHAC1 prevents Notch1 maturation, thereby reducing the levels of the active Notch1 intracellular domain, and consequently decreasing cell viability and increasing apoptosis. Treatment with the three isolated molecules resulted in reduced tumor size and weight in vivo and slowed leukemia progression in mice models. Altogether, this study elucidated the mechanism of action of three distinct cannabinoids in modulating the Notch1 pathway, and constitutes an important step in the establishment of a new therapy for treating NOTCH1-mutated diseases and cancers such as T-ALL.

The biosynthetic pathway of the hallucinogen mescaline and its heterologous reconstruction.

Mescaline, among the earliest identified natural hallucinogens, holds great potential in psychotherapy treatment. Nonetheless, despite the existence of a postulated biosynthetic pathway for more than half a century, the specific enzymes involved in this process are yet to be identified. In this study, we investigated the cactus Lophophora williamsii (Peyote), the largest known natural producer of the phenethylamine mescaline. We employed a multi-faceted approach, combining de novo whole-genome and transcriptome sequencing with comprehensive chemical profiling, enzymatic assays, molecular modeling, and pathway engineering for pathway elucidation. We identified four groups of enzymes responsible for the six catalytic steps in the mescaline biosynthetic pathway, and an N-methyltransferase enzyme that N-methylates all phenethylamine intermediates, likely modulating mescaline levels in Peyote. Finally, we reconstructed the mescaline biosynthetic pathway in both Nicotiana benthamiana plants and yeast cells, providing novel insights into several challenges hindering complete heterologous mescaline production. Taken together, our study opens up avenues for exploration of sustainable production approaches and responsible utilization of mescaline, safeguarding this valuable natural resource for future generations.

Parallel evolution of cannabinoid biosynthesis.

Modulation of the endocannabinoid system is projected to have therapeutic potential in almost all human diseases. Accordingly, the high demand for novel cannabinoids stimulates the discovery of untapped sources and efficient manufacturing technologies. Here we explored Helichrysum umbraculigerum, an Asteraceae species unrelated to Cannabis sativa that produces Cannabis-type cannabinoids (for example, 4.3% cannabigerolic acid). In contrast to Cannabis, cannabinoids in H. umbraculigerum accumulate in leaves' glandular trichomes rather than in flowers. The integration of de novo whole-genome sequencing data with unambiguous chemical structure annotation, enzymatic assays and pathway reconstitution in Nicotiana benthamiana and in Saccharomyces cerevisiae has uncovered the molecular and chemical features of this plant. Apart from core biosynthetic enzymes, we reveal tailoring ones producing previously unknown cannabinoid metabolites. Orthology analyses demonstrate that cannabinoid synthesis evolved in parallel in H. umbraculigerum and Cannabis. Our discovery provides a currently unexploited source of cannabinoids and tools for engineering in heterologous hosts.

Correction: Bar-Sela et al. Cannabis Consumption Used by Cancer Patients during Immunotherapy Correlates with Poor Clinical Outcome. Cancers 2020, 12, 2447.

In the original article [...].

Prolonged Medical Cannabis Treatment is Associated With Quality of Life Improvement and Reduction of Analgesic Medication Consumption in Chronic Pain Patients.

Introduction: Chronic non-cancer pain (CNCP) is one of the most prevalent indications for medical cannabis (MC) treatment globally. In this study, we investigated CNCP parameters in patients during prolonged MC treatment, and assessed the interrelation between CNCP parameters and the chemical composition of MC chemovar used. Methods: A cross-sectional questionnaire-based study was performed in one-month intervals for the duration of six months. Subjects were adult patients licensed for MC treatment who also reported a diagnosis of CNCP by a physician. Data included self-reported questionnaires. MC treatment features included administration route, cultivator, cultivar name and monthly dose. Comparison statistics were used to evaluate differences between the abovementioned parameters and the monthly MC chemovar doses at each time point. Results: 429, 150, 98, 71, 77 and 82 patients reported fully on their MC treatment regimens at six one-month intervals, respectively. Although pain intensities did not change during the study period, analgesic medication consumption rates decreased from 46 to 28% (p < 0.005) and good Quality of Life (QoL) rates increased from 49 to 62% (p < 0.05). These changes overlapped with increase in rates of (-)-Δ9-trans-tetrahydrocannabinol (THC) and α-pinene high dose consumption. Conclusion: Even though we observed that pain intensities did not improve during the study, QoL did improve and the rate of analgesic medication consumption decreased alongside with increasing rates of high dose THC and α-pinene consumption. Understanding MC treatment composition may shed light on its long-term effects.

Specific phytocannabinoid compositions are associated with analgesic response and adverse effects in chronic pain patients treated with medical cannabis.

Medical cannabis (MC) treatment for chronic pain is increasing, but evidence regarding short- and long-term efficacy and associated adverse effects (AEs) of the different cannabis plant components is limited. Most reports focus on two phytocannabinoids, (-)-Δ9-trans-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). This study, aimed to identify patterns of phytocannabinoid compositions associated with MC treatment response and with related AEs. Participants in this multicenter prospective cohort were patients with chronic non-cancer pain that were prescribed MC by physicians. Data was collected before MC treatment, at one month (short-term) and at 12 months (long-term). Simultaneously, liquid chromatography mass spectrometry identification and quantification of phytocannabinoids from the cultivars were performed. The monthly dose of each phytocannabinoid for each patient was z-scaled and clustered into ten groups to assess the difference in analgesic treatment response (≥30%/50% pain intensity reduction) and AEs rates. We identified ten clusters that had similar analgesic treatment response rates. However, there were significant differences in AEs rates both at short- and long-term. We identified specific phytocannabinoid compositions that were associated with overall AEs rates (5% compared to 53% at short-term and 44% at long-term) and with specific AEs rates such as MC related central nervous system, gastrointestinal and psychological AEs. To conclude, Evaluating only Δ9-THC or CBD is insufficient to find associations with MC related AEs. Therefore, comprehensive profiling of phytocannabinoids is needed to discover associations to related AEs and help physicians prescribe safer cannabis with less AEs while still relieving pain.

Cannabis Consumption Used by Cancer Patients during Immunotherapy Correlates with Poor Clinical Outcome.

Cannabis or its derivatives are widely used by patients with cancer to help with cancer symptoms and treatment side effects. However, cannabis has potent immunomodulatory properties. To determine if cannabis consumption during immunotherapy affects therapy outcomes, we conducted a prospective observatory study including 102 (68 immunotherapy and 34 immunotherapy plus cannabis) consecutive patients with advanced cancers who initiated immunotherapy. Cannabis consumption correlated with a significant decrease in time to tumor progression and overall survival. On the other hand, the use of cannabis reduced therapy-related immune-related adverse events. We also tested the possibility that cannabis may affect the immune system or the tumor microenvironment through the alteration of the endocannabinoid system. We analyzed a panel of serum endocannabinoids (eCBs) and eCB-like lipids, measuring their levels before and after immunotherapy in both groups. Levels of serum eCBs and eCB-like lipids, before immunotherapy, showed no significant differences between cannabis users to nonusers. Nevertheless, the levels of four eCB and eCB-like compounds were associated with patients' overall survival time. Collectively, cannabis consumption has considerable immunomodulatory effects, and its use among cancer patients needs to be carefully considered due to its potential effects on the immune system, especially during treatment with immunotherapy.

The heterogeneity and complexity of Cannabis extracts as antitumor agents.

The Cannabis plant contains over 100 phytocannabinoids and hundreds of other components. The biological effects and interplay of these Cannabis compounds are not fully understood and yet influence the plant's therapeutic effects. Here we assessed the antitumor effects of whole Cannabis extracts, which contained significant amounts of differing phytocannabinoids, on different cancer lines from various tumor origins. We first utilized our novel electrospray ionization liquid chromatography mass spectrometry method to analyze the phytocannabinoid contents of 124 Cannabis extracts. We then monitored the effects of 12 chosen different Cannabis extracts on 12 cancer cell lines. Our results show that specific Cannabis extracts impaired the survival and proliferation of cancer cell lines as well as induced apoptosis. Our findings showed that pure (-)-Δ9-trans-tetrahydrocannabinol (Δ9-THC) did not produce the same effects on these cell lines as the whole Cannabis extracts. Furthermore, Cannabis extracts with similar amounts of Δ9-THC produced significantly different effects on the survival of specific cancer cells. In addition, we demonstrated that specific Cannabis extracts may selectively and differentially affect cancer cells and differing cancer cell lines from the same organ origin. We also found that cannabimimetic receptors were differentially expressed among various cancer cell lines and suggest that this receptor diversity may contribute to the heterogeneous effects produced by the differing Cannabis extracts on each cell line. Our overall findings indicate that the effect of a Cannabis extract on a specific cancer cell line relies on the extract's composition as well as on certain characteristics of the targeted cells.