Isolation of newly synthesised RNA can be achieved by treatment of cells in culture with 6-thioguanosine or 4-thiouridine followed by separation of thiol-containing RNA by affinity chromatography on mercurated cellulose columns. After short periods of treatment with 6-thioguanosine the proportion of RNA retained on mercurated cellulose is the same for both poly(A)-containing and poly(A)-free RNA, indicating similar incorporation of the drug into mRNA and rRNA. However, after longer periods of exposure, the cytotoxic effect of 6-thioguanosine results in diminished incorporation of radioactive uridine into RNA and of radioactive leucine into protein ; this suggests that synthesis of both RNA and protein are impaired. On the other hand, even after long exposure to high concentrations of 4-thiouridine, the syntheses of RNA and protein are not significantly affected. Proteins synthesised after treatment of cells with 6-thioguanosine are less stable than proteins synthesised after treatment of cells with 4-thiouridine.Following treatment of cells with thiol-containing analogues of nucleic acid precursors, RNA containing thiol groups can be physically separated from nonthiol RNA by affinity chromatography on columns of mercurated cellulose or agarose [I, 21. The possible application of this technique to studies of RNA metabolism and gene expression has been pointed out An alternative technique involving the use of mercury-substituted UTP and CTP as precursors for RNA biosynthesis in isolated chromatin preparations and subsequent isolation of mercurated RNA on thiol-Sepharose columns has already been applied to studies such as the fidelity of transcription of chromatin by Escherichiu coli RNA polymerase [3,4]. However, it appears that mercurated RNA may be subject to aggregation [5,6] and, in addition, the technique is limited to studies on isolated nuclei or chromatin [3]. Aggregation of mercurated RNA molecules with normal RNA leads to contamination of the RNA newly synthesised in vitvo with endogenous RNA carried over in the nuclear preparation Thiol-containing RNA can be isolated from intact cells and does not appear to be subject to aggregation. Physical isolation of RNA newly synthesised in vivo is therefore possible and provides a technique with which such questions as the extent of reutilisation of RNA precursors and the real half-lives of nuclear RNA and mRNA molecules can be tackled. In this paper we present further information on the use of [I, 21. PI. different precursors as regards incorporation into thiol-containing RNA, and their influence on macromolecular metabolism. MATERIALS AND METHODS Mutevials6-Thioguanosine, 6-mercaptopurine ribonucleoside, 8-thioguanosine, 2-mercaptopyrimidine, 2-thiocytosine and 4-thiouridine were obtained from Sigma

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor-positive breast cancer and are currently in clinical development in melanoma, a tumor that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib and demonstrate that the activity of PRMT5, a protein arginine methyltransferase and indirect target of CDK4, is essential for CDK4/6 inhibitor sensitivity. By indirectly suppressing PRMT5 activity, palbociclib alters the pre-mRNA splicing of MDM4, a negative regulator of p53, leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 induces p21, leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib. Loss of the ability of palbociclib to regulate the PRMT5–MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treating naive and resistant models and also delays the emergence of resistance. Our studies have uncovered a mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5–MDM4 axis is essential for robust melanoma cell sensitivity and provide preclinical evidence that coinhibition of CDK4/6 and PRMT5 is an effective and well-tolerated therapeutic strategy. Overall, our data provide a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors, not only in melanoma but other tumor types, including breast, pancreatic, and esophageal carcinoma.

Multidrug resistant prostate cancer cell lines DU 0.03 and PC 0.03 were established from the parental prostate cancer cell lines DU145 and PC-3 respectively by stepwise selection in doxorubicin (DOX) from 0.001 to 0.03 m mg/ml. As cells adapted to each concentration of DOX. the drug concentration was increased by 0.001 m mg/ml. The chemosensitivity of each line was determined by growth inhibition assay. The DU 0.03 and PC 0.03 lines exhibit a 5 ± 10-fold and 1.3 ± 2.8-fold increase in resistance to anthracyclines, vinblastine (VLB) and mitozantrone (Mito), respectively. Verapamil (5 m mM) partially reversed the resistance to the anthracycline and completely reversed the resistance to VLB and Mito. Drug kinetic studies measured by intracellular accumulation of 3 H-daunorubicin demonstrated a 3 fold decrease in the level of intracellular 3 H-daunorubicin in the PC 0.03 and DU 0.03 resistant lines compared with their respective parental line. This effect was partially reversed by 5 m mM verapamil. The expression of MDR1 and MRP genes was analysed by Northern blotting and RT-PCR. P-glycoprotein (Pgp) and MRP protein were tested by immunocytochemistry staining using the monoclonal antibodies J-SB1. C219 and MRK16 (Pgp) and MRPm6 and MRPr1 (MRP). Neither Northern blot analysis nor the more sensitive RT-PCR demonstrated detectable MDR1 transcripts in any of the prostate cancer cell lines and the three Pgp monoclonal antibodies failed to reveal expression of Pgp.A 2 ± 4-fold increase in MRP1 mRNA levels in the drug resistant DU 0.03 and PC 0.03 lines were demonstrated by both Northern blotting and RT-PCR consistent with the ®ndings observed after staining by the two speci®c monoclonal antibodies, MRPm6 and MRPr1. Southern blot analysis demonstrated a 2-fold increase in the MRP1 gene copy number in the PC 0.03 line but not in the DU 0.03 line, suggesting that the overexpression of the MRP gene was regulated at the level of transcription in the latter line. We conclude that MRP1 not MDR1 overexpression. contributes to acquired drug resistance in these two prostate cancer cell lines. Prostate Cancer and Prostatic Diseases (2000) 3, 66±75.

S_ummary Studies were carried out in a variant human multidrug-resistant (MDR) cell line CEM/A7R, which expresses very low levels of mdrl mRNA and P-glycoprotein (P-gp). The induction of mdrl RNA expression by three anthracycines, (doxorubicin, daunorubicin, epirubicin), VP-16 and two vinca alkaloids (vincristine, vinblastine) was semiquantitatively assessed by scanning Northern blots on a phosphorimager. The relative level of mdrl expression was expressed as ratio of mdrl to the internal RNA (actin). A significant increase (P < 0.02) in expression of mdrl was noted within 4 hrs of exposure to 1.5 ig ml-' daunorubicin or epirubicin. Neither vinblastine nor vincristine had any effect on mdrl levels after an 8 h exposure. With increasing concentrations of daunorubicin or epirubicin in a fixed 24 h time period, mdrl expression increased, although a biphasic response was seen. Based on MRK 16 binding, an increase in P-gp levels was seen in the CEM/A7R line after a 24 h exposure to 1 Zig ml1 l daunorubicin or epirubicin. The rapid increase in mdurl expression after a short period of exposure to doxorubicin, daunorubicin or epirubicin suggests that induction of mdrl expression may have an important role in the development of drug-resistant tumours.

Both targeted therapy and immunotherapy have been used successfully to treat melanoma, but the development of resistance and poor response rates to the individual therapies has limited their success. Designing rational combinations of targeted therapy and immunotherapy may overcome these obstacles, but requires assessment in preclinical models with the capacity to respond to both therapeutic classes. Herein, we describe the development and characterization of a novel, immunogenic variant of the BrafV600ECdkn2a−/−Pten−/− YUMM1.1 tumor model that expresses the immunogen, ovalbumin (YOVAL1.1). We demonstrate that, unlike parental tumors, YOVAL1.1 tumors are immunogenic in vivo and can be controlled by immunotherapy. Importantly, YOVAL1.1 tumors are sensitive to targeted inhibitors of BRAFV600E and MEK, responding in a manner consistent with human BRAFV600E melanoma. The YOVAL1.1 melanoma model is transplantable, immunogenic and sensitive to clinical therapies, making it a valuable platform to guide strategic development of combined targeted therapy and immunotherapy approaches in BRAFV600E melanoma.

Combined inhibition of BRAF, MEK, and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a BRAFV600 mutation. While this triple therapy has potent tumor-intrinsic effects, the impact of this combination on antitumor immunity remains unexplored. Here, using a syngeneic BrafV600ECdkn2a−/−Pten−/− melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA sequencing analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted proinflammatory macrophages and cross-priming CD103+ dendritic cells, the absence of which correlated with poor overall survival and clinical responses to ICB in patients with melanoma. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses ex vivo. While combined BRAF, MEK, and CDK4/6 inhibition demonstrates favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact antitumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK, and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma.

SummaryThe effects of 4-demethoxydaunorubicin (idarubicin, IDA) and MX2, a new morpholino-anthracycline, on up-regulation of the MDR1 gene in the low-level multidrug resistant (MDR) cell line CEM/A7R were compared at similar concentrations (IC 10 , IC 50 and IC 90 ) over a short time exposure (4 and 24 h). The chemosensitivity of each drug was determined by a 3-day cell growth inhibition assay. Compared with epirubicin (EPI), IDA and MX2 were 17-and eightfold more effective in the CEM/A7R line respectively. No cross-resistance to 5-FU was seen in the CEM/A7R line. Verapamil (5 µM) and PSC 833 (1 µM), which dramatically reversed resistance to EPI in the CEM/A7R line, had no sensitizing effect on the resistance of this line to MX2, but slightly decreased resistance to IDA. The sensitivity to 5-FU was unchanged by these modulators. The induction of MDR1 mRNA expression by IDA, MX2 and 5-FU was analysed by Northern blotting and semiquantitatively assessed by scanning Northern blots on a phosphorimager. The relative level of MDR1 expression was expressed as a ratio of MDR1 mRNA to the internal RNA control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). IDA, MX2 and 5-FU differentially up-regulated MDR1 mRNA in the CEM/A7R line in a dose-dependent manner. Both IDA and MX2 induced MDR1 expression within 4 h. 5-FU up-regulated MDR1 expression only when drug exposure was prolonged to 24 h. Based on MRK 16 binding, flow cytometric analysis of P-glycoprotein (Pgp) expression paralleled the increase in MDR1 mRNA levels. For the three anthracyclines, the increase in MDR1 expression was stable in cells grown in the absence of drug for more than 3 weeks after drug treatment. The induction of MDR1 expression by 5-FU was transient, associated with a rapid decrease in the increased Pgp levels which returned to baseline 72 h after the removal of 5-FU. This study demonstrates that MDR1 expression can be induced by analogues of anthracyclies not pumped by Pgp, and that this induction appears to be stable despite a 3-week drug-free period.

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