Illumina HiSeq 4000 paired end sequencing; Inhibition of PARP14 restores response to α-PD1 immune checkpoint inhibition in tumours with resistance driven by IFNγ. Mouse tumour samples
Inhibition of PARP14 restores response to α-PD1 immune checkpoint inhibition in tumours with resistance driven by IFNγ. Mouse tumour samples
Female, 8–12-week-old C57BL/6 mice were purchased from ENVIGO and housed in the Biological Services Facility of The University of Manchester on a 12/12-hour light/dark cycle. Mice were given unlimited access to food (Bekay, B&K Universal, Hull, UK) and water. All procedures were approved by the institution's review board and performed under relevant Home Office licenses according to the UK Animals (Scientific Procedures) Act, 1986. YUMM2.1 cells (7 × 106 cells in 100 µL) were pre-treated with IFNγ cell culture for 2 weeks (maintained in RPMI-1640 (Sigma-Aldrich) medium supplemented with 10% fetal bovine serum (FBS; Life Technologies), 1% penicillin-streptomycin (P/S; Sigma-Aldrich) and with 50 IU/ml, replacing medium and IFNγ every 72 h with passaging of cells as necessary). These pretreated cells were subcutaneously injected into the left flank of mice under isoflurane anaesthesia. Tumour size (determined by calliper measurements) and mouse weight were monitored 3 times per week (every 2-3 days).  When tumours reached approximately 150 mm3, mice were randomised into either α-PD-1 group or α-PD-1 + PARP14i group. Both groups of mice were administered with 2 doses of 300 µg of α-PD-1 antibody (BioXCell) via intraperitoneal (i.p.) injection (1 dose every 3 days). Mice were administered concurrently 14 doses vehicle or 500 mg/kg of RBN012759 by oral gavage (BID) (2 doses daily). RBN012759 was dissolved in the vehicle 0.5% methylcellulose (Sigma-Aldrich) + 0.2% Tween 80 (Sigma-Aldrich). Each dose was delivered in a volume of 0.2 mL/20 g mouse (10 mL/kg) and adjusted for the last recorded weight of individual animals. 24 h following the last dose of RBN012759, mice were euthanized and tumours dissected. Female, 8–12-week-old C57BL/6 mice were purchased from ENVIGO and housed in the Biological Services Facility of The University of Manchester on a 12/12-hour light/dark cycle. Mice were given unlimited access to food (Bekay, B&K Universal, Hull, UK) and water. All procedures were approved by the institution's review board and performed under relevant Home Office licenses according to the UK Animals (Scientific Procedures) Act, 1986. YUMM2.1 cells (7 × 106 cells in 100 µL) were pre-treated with IFNγ cell culture for 2 weeks (maintained in RPMI-1640 (Sigma-Aldrich) medium supplemented with 10% fetal bovine serum (FBS; Life Technologies), 1% penicillin-streptomycin (P/S; Sigma-Aldrich) and with 50 IU/ml, replacing medium and IFNγ every 72 h with passaging of cells as necessary). These pretreated cells were subcutaneously injected into the left flank of mice under isoflurane anaesthesia. Tumour size (determined by calliper measurements) and mouse weight were monitored 3 times per week (every 2-3 days).  When tumours reached approximately 150 mm3, mice were randomised into either α-PD-1 group or α-PD-1 + PARP14i group. Both groups of mice were administered with 2 doses of 300 µg of α-PD-1 antibody (BioXCell) via intraperitoneal (i.p.) injection (1 dose every 3 days). Mice were administered concurrently 14 doses vehicle or 500 mg/kg of RBN012759 by oral gavage (BID) (2 doses daily). RBN012759 was dissolved in the vehicle 0.5% methylcellulose (Sigma-Aldrich) + 0.2% Tween 80 (Sigma-Aldrich). Each dose was delivered in a volume of 0.2 mL/20 g mouse (10 mL/kg) and adjusted for the last recorded weight of individual animals. 24 h following the last dose of RBN012759, mice were euthanized and tumours dissected. Female, 8–12-week-old C57BL/6 mice were purchased from ENVIGO and housed in the Biological Services Facility of The University of Manchester on a 12/12-hour light/dark cycle. Mice were given unlimited access to food (Bekay, B&K Universal, Hull, UK) and water. All procedures were approved by the institution's review board and performed under relevant Home Office licenses according to the UK Animals (Scientific Procedures) Act, 1986. YUMM2.1 cells (7 × 106 cells in 100 µL) were pre-treated with IFNγ cell culture for 2 weeks (maintained in RPMI-1640 (Sigma-Aldrich) medium supplemented with 10% fetal bovine serum (FBS; Life Technologies), 1% penicillin-streptomycin (P/S; Sigma-Aldrich) and with 50 IU/ml, replacing medium and IFNγ every 72 h with passaging of cells as necessary). These pretreated cells were subcutaneously injected into the left flank of mice under isoflurane anaesthesia. Tumour size (determined by calliper measurements) and mouse weight were monitored 3 times per week (every 2-3 days).  When tumours reached approximately 150 mm3, mice were randomised into either α-PD-1 group or α-PD-1 + PARP14i group. Both groups of mice were administered with 2 doses of 300 µg of α-PD-1 antibody (BioXCell) via intraperitoneal (i.p.) injection (1 dose every 3 days). Mice were administered concurrently 14 doses vehicle or 500 mg/kg of RBN012759 by oral gavage (BID) (2 doses daily). RBN012759 was dissolved in the vehicle 0.5% methylcellulose (Sigma-Aldrich) + 0.2% Tween 80 (Sigma-Aldrich). Each dose was delivered in a volume of 0.2 mL/20 g mouse (10 mL/kg) and adjusted for the last recorded weight of individual animals. 24 h following the last dose of RBN012759, mice were euthanized and tumours dissected. Total RNA was isolated using the RNAeasy mini kit. RNA integrity was assessed on the Agilent 2200 TapeStation (Agilent Technologies). TruSeq Stranded mRNA assay (Illumina)