The microenvironment of pancreatic cancer adenocarcinoma (PDAC) is highly desmoplastic with distinct tumor‐restraining and tumor‐promoting fibroblast subpopulations. Re‐education rather than indiscriminate elimination of these fibroblasts has emerged as a new strategy for combination therapy. Here, we studied the effects of global loss of profibrotic noncoding regulatory microRNA‐21 (miR‐21) in K‐Ras‐driven p53‐deleted genetically engineered mouse models of PDAC. Strikingly, loss of miR‐21 accelerated tumor initiation via mucinous cystic neoplastic lesions and progression to locally advanced invasive carcinoma from which animals precipitously succumbed at an early age. The absence of tumor‐restraining myofibroblasts and a massive infiltrate of immune cells were salient phenotypic features of global miR‐21 loss. Stromal miR‐21 activity was required for induction of tumor‐restraining myofibroblasts in in vivo isograft transplantation experiments. Low miR‐21 expression negatively correlated with a fibroblast gene expression signature and positively with an immune cell gene expression signature in The Cancer Genome Atlas PDAC data set (n = 156) mirroring findings in the mouse models. Our results exposed an overall tumor‐suppressive function of miR‐21 in in vivo PDAC models. These results have important clinical implications for anti‐miR‐21‐based inhibitory therapeutic approaches under consideration for PDAC and other cancer types. Mechanistic dissection of the cell‐intrinsic role of miR‐21 in cancer‐associated fibroblasts and other cell types will be needed to inform best strategies for pharmacological modulation of miR‐21 activity to remodel the tumor microenvironment and enhance treatment response in PDAC.

Malignant peripheral nerve sheath tumors (MPNSTs) are chemotherapy resistant sarcomas that are a leading cause of death in neurofibromatosis type 1 (NF1). Although NF1-related MPNSTs derive from neural crest cell origin, they also exhibit intratumoral heterogeneity. TP53 mutations are associated with significantly decreased survival in MPNSTs, however the mechanisms underlying TP53-mediated therapy responses are unclear in the context of NF1-deficiency. We evaluated the role of two commonly altered genes, MET and TP53, in kinome reprograming and cellular differentiation in preclinical MPNST mouse models. We previously showed that MET amplification occurs early in human MPNST progression and that Trp53 loss abrogated MET-addiction resulting in MET inhibitor resistance. Here we demonstrate a novel mechanism of therapy resistance whereby p53 alters MET stability, localization, and downstream signaling leading to kinome reprogramming and lineage plasticity. Trp53 loss also resulted in a shift from RAS/ERK to AKT signaling and enhanced sensitivity to MEK and mTOR inhibition. In response to MET, MEK and mTOR inhibition, we observed broad and heterogeneous activation of key differentiation genes in Trp53-deficient lines suggesting Trp53 loss also impacts lineage plasticity in MPNSTs. These results demonstrate the mechanisms by which p53 loss alters MET dependency and therapy resistance in MPNSTS through kinome reprogramming and phenotypic flexibility.

Malignant peripheral nerve sheath tumors (MPNSTs) are chemotherapy resistant sarcomas that are a leading cause of death in neurofibromatosis type 1 (NF1). Although NF1-related MPNSTs derive from neural crest cell origin, they also exhibit intratumoral heterogeneity. TP53 mutations are associated with significantly decreased survival in MPNSTs, however the mechanisms underlying TP53-mediated therapy responses are unclear in the context of NF1-deficiency. We evaluated the role of two commonly altered genes, MET and TP53, in kinome reprograming and cellular differentiation in preclinical MPNST mouse models. We previously showed that MET amplification occurs early in human MPNST progression and that Trp53 loss abrogated MET-addiction resulting in MET inhibitor resistance. Here we demonstrate a novel mechanism of therapy resistance whereby p53 alters MET stability, localization, and downstream signaling leading to kinome reprogramming and lineage plasticity. Trp53 loss also resulted in a shift from RAS/ERK to AKT signaling and enhanced sensitivity to MEK and mTOR inhibition. In response to MET, MEK and mTOR inhibition, we observed broad and heterogeneous activation of key differentiation genes in Trp53-deficient lines suggesting Trp53 loss also impacts lineage plasticity in MPNSTs. These results demonstrate the mechanisms by which p53 loss alters MET dependency and therapy resistance in MPNSTS through kinome reprogramming and phenotypic flexibility.

Pancreatic ductal adenocarcinoma (PDAC) is a recalcitrant and lethal disease with an overall 5-year survival rate of less than 8%. The abundance and heterogeneity of cancer-associated fibroblasts (CAFs) in a highly desmoplastic tumor microenvironment are unique features of PDAC. Therapeutic strategies aimed at indiscriminately eliminating CAFs have failed in clinical trials. The opposing tumor-restraining or tumor-promoting role of distinct CAFs subpopulations of has emerged as a new paradigm in PDAC basic and translational research. Understanding how the composition of these distinct CAF subpopulations is affected by and interferes with current treatments is a key question to solve in order to obtain more effective and durable clinical responses. Genetic studies in K-Ras-driven genetically engineered mouse models (GEMMs) indicate that α-smooth muscle actin-expressing (SMA+) CAFs have a predominant tumor-restraining role. Here, we studied global loss of pro-fibrotic non-coding regulatory microRNA-21 (miR-21) in PDAC GEMMs. We generated mouse strains carrying wild type or knockout alleles of Mir-21 in a well-characterized K-Ras-driven, p53-deleted PDAC model (LSL-KrasG12D; p53lox/+;Pdx1-Cre, KPC). Even though miR-21 expression was upregulated in cancer cells and cancer-associated fibroblasts in KPC tumors, global loss of miR-21 activity did not inhibit tumor growth. Strikingly, loss of miR-21 accelerated tumor initiation via mucinous cystic neoplastic lesions and progression to locally advanced invasive carcinoma from which animals precipitously succumbed at an early age. We uncovered a cell-autonomous requirement of miR-21 activity for induction of tumor-restraining SMA+ CAFs, whose absence led to a profound remodeling of the stroma and massive infiltrate of tumor-promoting immune cells. Low miR-21 expression negatively correlated with a fibroblast gene expression signature and positively with an immune cell gene expression signature in TCGA PDAC data set (n = 156) mirroring findings in GEMMs. In vitro and in vivo isograft experiments showed that miR-21 activity was dispensable for cancer cell growth, but required for complete execution of TGF-β-mediated programs. Our results suggest that modulation of miR-21 activity may provide a novel therapeutic opportunity to affect the composition of CAF subpopulations. We will present our genetic and pharmacological approaches to modulate stromal activity of miR-21 in established disease in order to enhance current chemotherapy and/or immunotherapy treatments. Citation Format: Josh Schipper, Brooke Jackson, Ian Beddows, Elizabeth Kenyon, Katie Powell, Neil Robertson, Galen Hostetter, Matti Kiupel, Anna Moore, Jose Conejo-Garcia, Lorenzo sempere. Tumor-suppressive stromal activity of pro-fibrogenic microRNA-21 in initiation and progression of K-Ras-driven mouse models of pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5719.

The tumor microenvironment of pancreatic cancer adenocarcinoma (PDAC) is highly desmoplastic with distinct tumor-restraining and tumor-promoting fibroblast subpopulations. Re-education rather than indiscriminate elimination of these fibroblasts has emerged as a new strategy for combination therapy. Here, we studied global loss of profibrotic noncoding regulatory microRNA-21 (miR-21) in K-Ras-driven genetically engineered mouse models of PDAC. We generated mouse strains carrying wild-type or knockout alleles of Mir-21 in a well-characterized K-Ras-driven, p53-deleted PDAC model (LSL-KrasG12D; p53lox/+;Pdx1-Cre, KPC). Even though miR-21 expression was upregulated in cancer cells and cancer-associated fibroblasts in KPC tumors, global loss of miR-21 activity did not inhibit tumor growth. Strikingly, loss of miR-21 accelerated tumor initiation via mucinous cystic neoplastic lesions and progression to locally advanced invasive carcinoma from which animals precipitously succumbed at an early age. We uncovered a cell-autonomous requirement of miR-21 activity for induction of tumor-restraining myofibroblasts, whose absence led to a profound remodeling of the stroma and massive infiltrate of tumor-permissive immune cells. Low miR-21 expression negatively correlated with a fibroblast gene expression signature and positively with an immune cell gene expression signature in TCGA PDAC data set (n = 156) mirroring findings in genetic models. In vitro and in vivo isograft experiments showed that miR-21 activity was dispensable for cancer cell growth but required for complete execution of TGF-b-mediated programs. Our results suggest that modulation of miR-21 activity may provide a novel therapeutic opportunity to affect the composition of fibroblast subpopulations and enhance current chemotherapy and/or immunotherapy treatments. Citation Format: Josh Schipper, Ian Beddows, Matti Kiupel, Jose Conejo-Garcia, Lorenzo Sempere. Genetic ablation of microRNA-21 profoundly remodels stroma and shortens survival of K-Ras-driven pancreatic cancer mouse models [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A48.