Cancer-Associated Fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through pro-tumor signalling and the generation of fibrosis, the latter of which creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumor stroma and its prognostic significance. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poorer overall survival. Orthogonal approaches showed that PDACderived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis and that SLC7A11 inhibition significantly decreases CAF proliferation, reduces their resistance to oxidative stress, and inhibits their ability to remodel collagen and support PDAC cell growth.Importantly, specific ablation of SLC7A11 from the tumor compartment of transgenic mouse PDAC tumors did not affect tumor growth, suggesting the stroma can substantially influence PDAC tumor response to SLC7A11 inhibition. In a mouse orthotopic PDAC model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle genesilencing drug against SLC7A11, developed by our laboratory, reduced PDAC tumor growth, incidence of metastases, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth. Statement of Significance:This study demonstrates that SLC7A11 in PDAC stromal cells is important for the tumor-promoting activity of CAF and validates a clinically translatable nanomedicine for therapeutic SLC7A11 inhibition in PDAC.

Cancer is the second leading cause of death globally, with therapeutic resistance being a major cause of treatment failure in the clinic. The dynamic signaling that occurs between tumor cells and the diverse cells of the surrounding tumor microenvironment actively promotes disease progression and therapeutic resistance. Improving the understanding of how tumors evolve following therapy and the molecular mechanisms underpinning de novo or acquired resistance is thus critical for the identification of new targets and for the subsequent development of more effective combination regimens. Simultaneously targeting multiple hallmark capabilities of cancer to circumvent adaptive or evasive resistance may lead to significantly improved treatment response in the clinic. Here, the latest applications of functional genomics tools, such as clustered regularly interspaced short palindromic repeats (CRISPR) editing, to characterize the dynamic cancer resistance mechanisms, from improving the understanding of resistance to classical chemotherapeutics, to deciphering unique mechanisms that regulate tumor responses to new targeted agents and immunotherapies, are discussed. Potential avenues of future research in combating therapeutic resistance, the contribution of tumor-stroma signaling in this setting, and how advanced functional genomics tools can help streamline the identification of key molecular determinants of drug response are explored.

Background Pancreatic ductal adenocarcinoma (PDA) has a 5year survival of only 10% and persists as the 3rd most common cause of cancer-related death in Western societies. New treatment options are urgently needed. We have previously defined specific molecular subgroups of PDA associated with pre-clinical and clinical response to select tailored treatment strategies. [1][2] One such molecular-guided therapy, RXC004, a potent and selective inhibitor of the Wnt/b-Catenin pathway regulator porcupine, is being investigated in a Ph2 study in patients with pancreatic cancer (NCT04907851). We have previously demonstrated interesting effects of tumour-cell targeted therapies on the environment of PDA. [2][3][4] Methods We determined the preclinical efficacy and detailed antistromal effects of RXC004 and selective ROCK2 inhibitors in a range of patient derived and genetically-defined PDA models, including clinically relevant combinations with standard of care (SoC) chemotherapy and immunotherapy. Mechanistic assessment of alterations in tumour cell-stromal cell cross-talk was performed using comprehensive transcriptomics and immunofluorescence approaches. Results In addition to reducing tumour growth and improving overall survival in patient-derived models of aggressive PDA, RXC004 demonstrated striking antifibrotic effects in vivo, with changes in cancer-associated fibroblast phenotype, accompanied by decreased levels of extracellular matrix components (fibronectin, periostin) and their organisation (collagen). Moreover, treatment with RXC004 as part of 'priming' combination therapy or 'maintenance' regimen significantly improved in vivo chemosensitivity. We also demonstrate that titrated modulation of fibrotic elements in vivo via ROCK2 targeting and as part of clinically-applicable therapeutic regimens, can lead to improved outcomes in diverse highly fibrotic and chemoresistant in vivo settings. Importantly, selective modulation of ROCK2 or Wnt signalling within the microenvironment of the immunocompetent LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) model of metastatic PDA revealed significant positive modulation of distinct immune components. These alterations include decreased level of immunosuppressive regulatory T cells, improved CD8+ and CD4+ T cell infiltration and increased presence of M1 pro-inflammatory macrophages in KPC tumours post-treatment, evident both within the tumour body and the invasive edge. Conclusions These data demonstrate that therapeutic efficacy of RXC004 and select anti-fibrotics in preclinical development may be the result of targeting both tumour cells and key aspects of the fibrotic and immune PDA microenvironment and in addition provide scientific rationale for the design of future SoC chemotherapy as well as immunotherapy-based combinations in pancreatic cancer.