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.

Correction: Blal et al. The Effect of Cannabis Plant Extracts on Head and Neck Squamous Cell Carcinoma and the Quest for Cannabis-Based Personalized Therapy. Cancers 2023, 15, 497.

In the original publication [...].

The Effect of Cannabis Plant Extracts on Head and Neck Squamous Cell Carcinoma and the Quest for Cannabis-Based Personalized Therapy.

Cannabis sativa plants have a wide diversity in their metabolite composition among their different chemovars, facilitating diverse anti-tumoral effects on cancer cells. This research examined the anti-tumoral effects of 24 cannabis extracts representative of three primary types of chemovars on head and neck squamous cell carcinoma (HNSCC). The chemical composition of the extracts was determined using High-Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS). The most potent anti-tumoral extracts were type III decarboxylated extracts, with high levels of Cannabidiol (CBD). We identified extract 296 (CAN296) as the most potent in inducing HNSCC cell death via proapoptotic and anti-proliferative effects. Using chemical fractionation of CAN296, we identified the CBD fraction as the primary inducer of the anti-tumoral activity. We succeeded in defining the combination of CBD with cannabichromene (CBC) or tetrahydrocannabinol (THC) present in minute concentrations in the extract, yielding a synergic impact that mimics the extract's full effect. The cytotoxic effect could be maximized by combining CBD with either CBC or THC in a ratio of 2:1. This research suggests using decarboxylated CBD-type extracts enriched with CBC for future preclinical trials aimed at HNSCC treatment.

Glycine decarboxylase maintains mitochondrial protein lipoylation to support tumor growth.

The folic acid cycle mediates the transfer of one-carbon (1C) units to support nucleotide biosynthesis. While the importance of serine as a mitochondrial and cytosolic donor of folate-mediated 1C units in cancer cells has been thoroughly investigated, a potential role of glycine oxidation remains unclear. We developed an approach for quantifying mitochondrial glycine cleavage system (GCS) flux by combining stable and radioactive isotope tracing with computational flux decomposition. We find high GCS flux in hepatocellular carcinoma (HCC), supporting nucleotide biosynthesis. Surprisingly, other than supplying 1C units, we found that GCS is important for maintaining protein lipoylation and mitochondrial activity. Genetic silencing of glycine decarboxylase inhibits the lipoylation and activity of pyruvate dehydrogenase and impairs tumor growth, suggesting a novel drug target for HCC. Considering the physiological role of liver glycine cleavage, our results support the notion that tissue of origin plays an important role in tumor-specific metabolic rewiring.

Tumor Reliance on Cytosolic versus Mitochondrial One-Carbon Flux Depends on Folate Availability.

Folate metabolism supplies one-carbon (1C) units for biosynthesis and methylation and has long been a target for cancer chemotherapy. Mitochondrial serine catabolism is considered the sole contributor of folate-mediated 1C units in proliferating cancer cells. Here, we show that under physiological folate levels in the cell environment, cytosolic serine-hydroxymethyltransferase (SHMT1) is the predominant source of 1C units in a variety of cancers, while mitochondrial 1C flux is overly repressed. Tumor-specific reliance on cytosolic 1C flux is associated with poor capacity to retain intracellular folates, which is determined by the expression of SLC19A1, which encodes the reduced folate carrier (RFC). We show that silencing SHMT1 in cells with low RFC expression impairs pyrimidine biosynthesis and tumor growth in vivo. Overall, our findings reveal major diversity in cancer cell utilization of the cytosolic versus mitochondrial folate cycle across tumors and SLC19A1 expression as a marker for increased reliance on SHMT1.

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.

Combined shRNA over CRISPR/cas9 as a methodology to detect off-target effects and a potential compensatory mechanism.

Inhibition of genes is a powerful approach to study their function. While RNA interference is a widely used method to achieve this goal, mounting evidence indicates that such an approach is prone to off-target effects. An alternative approach to gene function inhibition is genetic mutation, such as the CRISPR/cas9 method. A recent report, however, demonstrated that genetic mutation and inhibition of gene expression do not always give corresponding results. This can be explained by off-target effects, but it was recently shown, at least in one case, that these differences are the result of a compensatory mechanism induced only by genetic mutation. We present here a combination of RNA inhibition and CRISPR/cas9 methods to identify possible off targets as well as potential compensatory effects. This approach is demonstrated by testing a possible role for Sema4B in glioma biology, in which our results implicate Sema4B as having a critical function. In stark contrast, by using shRNA over CRISPR/cas9 combined methodology, we clearly demonstrate that the Sema4B targeted shRNA effects on cell proliferation is the result of off-target effects. Nevertheless, it also revealed that certain splice variants of Sema4B are important for the ability of glioma cells to grow as individual clones.

Astrogliosis Induced by Brain Injury Is Regulated by Sema4B Phosphorylation

Injury to the CNS induces astrogliosis, an astrocyte-mediated response that has both beneficial and detrimental impacts on surrounding neural and non-neural cells. The precise signaling events underlying astrogliosis are not fully characterized. Here, we show that astrocyte activation was altered and proliferation was reduced in Semaphorin 4B (Sema4B)-deficient mice following injury. Proliferation of cultured Sema4B(-/-) astrocytes was also significantly reduced. In contrast to its expected role as a ligand, the Sema4B ectodomain was not able to rescue Sema4B(-/-) astrocyte proliferation but instead acted as an antagonist against Sema4B(+/-) astrocytes. Furthermore, the effects of Sema4B on astrocyte proliferation were dependent on phosphorylation of the intracellular domain at Ser825. Our results suggest that Sema4B functions as an astrocyte receptor, defining a novel signaling pathway that regulates astrogliosis after CNS injury.

A new impedance based approach to test the activity of recombinant protein--Semaphorins as a test case.

The biological activity of a recombinant protein is routinely measured using a bioassay such as an enzyme assay. However, many proteins have no enzymatic activity and in many cases it is difficult to devise a simple and reliable approach to test their activity. Semaphorins, Ephrins, Slits, Netrins or amylin-assisted proteins have numerous activities affecting many systems and cell types in the human body. Most of them are also able to induce rapid cytoskeleton changes at least in some cell types. We assumed therefore, that such proteins might be tested based on their ability to modulate the cytoskeleton. Here we tested a number of semaphorins in an impedance based label-free platform that allows for dynamic monitoring of subtle morphological and adhesive changes. This system has proved to be a very fast, sensitive and effective way to monitor and determine the activity of such proteins. Furthermore we showed that it is possible to customize a cell-protein system by transfecting the cells with specific receptors and test the cell response following the addition of the recombinant ligand protein. Since other protein families such as Ephrins and Netrins can also influence the cytoskeleton of some cells, this approach may be applicable to a large number of proteins.