[“Molecule Background

DDR targeting significantly increased the total level of PDL protein in all cell lines tested with prexasertib inducing the greatest PDL fold change as detected by RPPA. The RPPA result was validated by immunoblot analysis, which further demonstrated PDL upregulation upon DDR targeting in a timedependent manner. As PDL must be expressed on the cell surface for successful targeting, we then assayed cell surface PDL expression by FACS.Cell surface PDL levels significantly increased following treatment with either prexasertib or olaparib in a timedependent manner in both human. To confirm that PDL upregulation is specifically due to inhibition of CHK or PARP and not an offtarget effect of the inhibitors, we knocked down CHEK or PARP in multiple SCLC cell lines.Consistent with pharmacologic inhibition, PDL expression was substantially higher in CHEK knockdown cells compared with the scrambled control.PDL upregulation upon CHK targeting was further confirmed by treating cells with a second CHK inhibitor. To test this possibility, we compared the effect of a low dose of prexasertib, {|buy {Endurobol|Amiodarone previously shown to cause growth delay but not tumor regression mice.However, a greater degree of PDL upregulation was seen in the IC model. The enhancement of PDL expression in the IC model was further confirmed by immunoblot. As predicted, prexasertib treatment increased PDL protein expression in this model in a timedependent manner. The induction of PDL expression, rapid tumor regression, and intratumoral immune infi ltration following CHK targeting support a direct role for DDR modulation in regulating the immune microenvironment in these SCLC models.Mice treated with antiPDL alone showed no antitumor response in this model and were sacrificed due to excessive tumor burden within weeks.However, within week, remarkable tumor regression was observed in the combinationtreated group. Of the mice treated with the combination of prexasertib and antiPDL, had a complete response and analyzed by multicolor flow cytometry for the changes in tumorinfi ltrating lymphocytes. AD, DDR inhibition by targeting with smallmolecule inhibitors of CHK and immunoblot analysis SCLC cell lines.Prexasertib showed enhanced antitumor efficacy in IC model. H were harvested at day, the immune profiling was analyzed by FACS at the endpoint, and the representative plots and cumulative data for all the tumors are shown.CD and CD IHC staining and scoring were performed on the resected tumors. The IHC data confirm the flow cytometry observations with higher CD staining intensity and percentage of CD cells in the prexasertibtreated group as compared with vehicle or antiPDL alone and further enhancement of this population in the anti PDL prexasertib treatment tumors. CD depletion slightly enhanced tumor growth in the vehicle and PDL antibody treated groups. In tumors treated with singleagent prexasertib or combined treatment of prexasertib and anti PDL, we observed that depletion of CD cytotoxic T cells significantly decreased the degree of tumor shrinkage relative to the control arms. FACS analysis of CD CD total T cells from the endpoint primary tumors.We have previously shown that PARP is overexpressed in SCLC tumors, and recent clinical trials have demonstrated clinical benefit from PARP inhibitors in some patients with SCLC.

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