Validation and development of MTH1 inhibitors for treatment of cancer.

BACKGROUND: Previously, we showed cancer cells rely on the MTH1 protein to prevent incorporation of otherwise deadly oxidised nucleotides into DNA and we developed MTH1 inhibitors which selectively kill cancer cells. Recently, several new and potent inhibitors of MTH1 were demonstrated to be non-toxic to cancer cells, challenging the utility of MTH1 inhibition as a target for cancer treatment. MATERIAL AND METHODS: Human cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA or shRNA. 8-oxodG was measured by immunostaining and modified comet assay. Thermal Proteome profiling, proteomics, cellular thermal shift assays, kinase and CEREP panel were used for target engagement, mode of action and selectivity investigations of MTH1 inhibitors. Effect of MTH1 inhibition on tumour growth was explored in BRAF V600E-mutated malignant melanoma patient derived xenograft and human colon cancer SW480 and HCT116 xenograft models. RESULTS: Here, we demonstrate that recently described MTH1 inhibitors, which fail to kill cancer cells, also fail to introduce the toxic oxidized nucleotides into DNA. We also describe a new MTH1 inhibitor TH1579, (Karonudib), an analogue of TH588, which is a potent, selective MTH1 inhibitor with good oral availability and demonstrates excellent pharmacokinetic and anti-cancer properties in vivo. CONCLUSION: We demonstrate that in order to kill cancer cells MTH1 inhibitors must also introduce oxidized nucleotides into DNA. Furthermore, we describe TH1579 as a best-in-class MTH1 inhibitor, which we expect to be useful in order to further validate the MTH1 inhibitor concept.

Conditioned media from cultured blood cells of atopic individuals can induce differentiation in the human basophilic leukemia cell line KU812.

The basophilic propagating activity was determined in conditioned media obtained from cell cultures of mononuclear cells from atopic and healthy individuals. The activity was analyzed as the capacity to induce differentiation in the human basophilic cell line KU812. The KU812 cells responded primarily to cultured media with histamine production and secondarily with granulation. Eight atopic individuals, 3 of whom having mild symptoms, with known birch allergies were selected together with 3 control individuals. Total and specific IgE were analyzed in sera, and basophil and eosinophil counts were determined from blood smears after Wright's staining. Significant differences between the symptomatic atopic individuals and the control group were obtained for eosinophil counts (p less than 0.05) and in the KU812 basophilic differentiation activity assay (p less than 0.01). No single test could significantly discriminate between symptomatic and nonsymptomatic individuals, but the KU812 cell assay in combination with the eosinophil count clearly distinguished the symptomatic individuals from the control group. Increased levels of IgE specific to birch allergen were only obtained in the atopic individuals (p less than 0.001). No difference in basophilic propagating activity was observed with supernatants from cells incubated with birch pollen allergen. Serum tested for basophilic propagating activity showed no or decreased values of inducing histamine production in the KU812 cell line. In conclusion, the KU812 assay for basophilic propagating activity was the most useful discriminating test to select symptomatic atopic individuals from nonsymptomatic atopic and control individuals.

Induction of basophilic differentiation in the human basophilic cell line KU812.

The potential for differentiation of the human basophilic leukaemia cell line KU812 was examined by means of a panel of physiologic and non-physiologic substances used as inducers. The phenotypic characteristics of non-induced KU812 cells included an immature morphology with scanty cytoplasmic granulation, expression of a low amount of high affinity, but no low affinity receptors (CD 23) for IgE, and a capacity for low-rate histamine synthesis. The differentiation process was characterized by a rapid (24 h) increase in histamine production a slower morphological maturation with the development of Alcian blue stainable granula demonstrable after 72 h. Concomitant with the phenotypic alterations, cell growth was inhibited. Differentiation in KU812 cells was inducible by Ara-C and to some extent by sodium butyrate, but not by dimethyl sulphoxide, retinoic acid, or gamma-interferon. Conditioned medium (CM) from cultured peripheral blood cells from atopic individuals and 18 out of 22 analysed glioma cell lines induced differentiation of the KU812 cells, whereas supernatant from only 1 out of 21 other cell lines, including carcinoma, melanoma, sarcoma, leukaemia, and normal fibroblasts had this activity. CM from the T-leukaemic cell line, Mo, also induced KU812 differentiation. A primary fractionation of the active substance from this cell line by reversed phase chromatography eluted the active substance at a concentration of 42-44% acetonitrile. Our present study has shown that the KU812 may serve as an appropriate model to study differentiation of basophils. In addition, its fast and specific response to biological factors makes it suitable as a biological assay for determination of active factor produced by atopic individuals.