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  • br To visualize whether RORg blockade impacts

    2019-10-14


    To visualize whether RORg blockade impacts tumor progres-sion by targeting stem cells, SR2211 was delivered in REM2-KPf/fC mice with late-stage autochthonous tumors and re-sponses tracked via live imaging. In vehicle-treated mice, large stem cell clusters could be readily identiÞed throughout the tumor based on GFP expression driven by the Msi reporter (Fig-ures 5K and 5L). SR2211 led to a marked depletion of the major-ity of large stem cell clusters within 1 week of treatment (Figures 5K and 5L), with no increased necrosis observed in surrounding tissues. This unique spatio-temporal analysis suggests that stem cell depletion is an early consequence of RORg blockade and highlights the REM2-KPf/fC model as an effective platform to assess the impact of new agents on therapy-resistant cells.
    Figure 4. The Immuno-regulatory Gene RORg Is a Critical Dependency of Pancreatic Cancer
    (A) Rorc expression in stem and non-stem REM2-KPf/fC tumor cells; representative of three biological replicates.
    (B) Representative images of RORg expression in normal adjacent human pancreas (left), PanINs (middle), and PDAC (right). RORg (green), E-cadherin (red), DAPI (blue), scale bars represent 50 mm.
    (C) Frequency of RORg+ Oxidopamine hydrochloride within E-cadherin+ epithelial fraction in patient samples quantiÞed by immunoßuorescence; Normal adjacent, n = 3; pancreatitis,
    (D and E) Impact of shRNA-mediated RORg inhibition on 3D growth of KPR172H/+C (D) and KPf/fC (E) cells, n = 3 per shRNA. (F) Impact of shRNA-mediated RORg inhibition on Msi2-GFP stem cell content in KPf/fC cells in 3D culture (H), n = 3. (G) Impact of shRNA-mediated RORg inhibition on ßank tumor growth of KPf/fC cells, n = 4. (H and I) Reduced tumor burden in Rorc / KP f/f C mice. Age-matched wild-type (WT) KP f/f C and Rorc / KP f/f C mice displayed reduced tumor cell number (H) and reduced adenocarcinoma content (I); low-grade PanIN indicated with red arrow, PDAC indicated with black arrow, scale bars represent 100 mm; n = 3 mice from 8Ð10 weeks of age; representative plots and images from matched mice are displayed.
    (J and K) Relative RNA expression of stem cell programs (J) and pro-tumorigenic factors (K) in KPf/fC cells transduced with shCtrl or shRorc. Red, over-rep-resented; blue, under-represented; color denotes fold change.
    (L) Venn diagram of genes downregulated with RORg loss (q < 0.05, purple). Stem-speciÞc super-enhancer-associated genes (green) and genes associated with H3K27ac peaks with RORg consensus binding sites (orange) are shown.
    (M) Number of RORg, CBFB, and Sox9 binding sites found in stem cell super-enhancers relative to random genomic background of equivalent base-pair coverage (p < 0.05).
    (N) Relative RNA expression of super-enhancer-associated oncogenes in KPf/fC cells transduced with shCtrl or shRorc. Red, over-represented; blue, under-represented; color denotes fold change from median values.
    (O) H3K27ac ChIP-seq reads for genes marked by stem cell super-enhancers and downregulated in RORg-depleted KPf/fC cells.
    Because treatment with the inhibitor in immunocompetent mice or in patients in vivo could have an impact on both cancer cells and immune cells, we tested the effect of SR2211 in the context of an immunocompromised environment. SR2211 
    signiÞcantly impacted growth of KPf/fC tumors in an immunode-Þcient background (Figures 6A and 6B), suggesting that inßam-matory T cells were not necessary for its effect. Further, in chimeric mice where wild-type tumors were transplanted into
    Figure 5. Pharmacologic Targeting of RORg Impairs Progression and Improves Survival in Mouse Models of Pancreatic Cancer