Abstract Zelasudil (RXC007) is an orally available, highly selective small molecule inhibitor of Rho-associated coiled-coil containing protein kinase 2 (ROCK2), which has demonstrated pre-clinical anti-fibrotic efficacy and is currently being tested in a Phase 2a clinical trial for the treatment of idiopathic pulmonary fibrosis (IPF) (NCT05570058). Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and therapeutically challenging disease with a dense and highly fibrotic tumor microenvironment which may limit efficacy of conventional therapies. Here, we investigate the benefit of combining an anti-fibrotic, RXC007, with either standard of care chemotherapy or immunotherapy in mouse models of PDAC.In a fibrotic patient-derived metastatic PDAC orthotopic model, RXC007 (10mg/kg, 50mg/kg and 100mg/kg BID 5d on/2d off PO) in combination with SoC chemotherapy Gemcitabine (100mg/kg IP QW)/Abraxane (30 mg/kg IP QW) improved survival in a dose-dependent manner (all P<0.0001) compared to Gemcitabine/Abraxane alone.In the immunocompetent orthotopic LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) model of metastatic PDAC, survival was significantly increased (P<0.0001) upon treatment with a combination of RXC007 (50 mg/kg BID 5d on/2d off PO) and anti-PD1 (200 ug BIW) compared to anti-PD1 alone. ROCK2 inhibition elicited positive immunomodulatory effects in KPC pancreatic tumors, with increase of CD8+ (P=0.0031) and CD4+ (P=0.008) T cell infiltrate into the tumor cortex and reduction in immunosuppressive FOXP3+ regulatory T cells at the tumor border. Furthermore, RXC007 monotherapy exhibited anti-fibrotic effects, via a decrease in collagen content and organization, effects associated with CAF reprogramming and reduced intra-tumoral aSMA+ PDPN+ myofibroblast-like CAFs (myCAFs; P=0.0406).This data highlights the potential for treating highly fibrotic tumours with RXC007 in combination with SoC chemotherapy or immunotherapy in order to improve patient outcomes in this aggressive and treatment-refractory cancer. Citation Format: Marina Pajic, Benjamin McLean, Diego Chacon Fajardo, Sean Porazinski, Dannielle H. Upton, Diana Schuhmacher, Aji Istadi, Thomas Cox, Paul Timpson, Daniel J. Wilcock, Katie J. Anderson, Helen Mason, Nicolas E. Guisot, Clifford D. Jones, Caroline Phillips, Richard Armer. Combining zelasudil, a small molecule ROCK2 inhibitor, with chemotherapy or immunotherapy improves response in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 720.

Abstract Pancreatic ductal adenocarcinoma (PDA) has a 5-year survival of less than 10% and remains the 3rd leading cause of cancer-related death in Western societies. New treatment options are urgently needed. TGF-β promotes stromal cell reprogramming, immunosuppression, and fibrinogenesis in cancers, including PDA. Integrins αVβ8 and αVβ1 are important activators of TGF-β signalling. Selective integrin blockade has recently emerged as a promising therapeutic approach to address TGF-β-mediated immunotherapy resistance and improve anti-tumour response across cancer models. The purpose of this study was to assess the in vivo efficacy of small molecule inhibitor PLN-104, a selective inhibitor of αVβ1, and PLN-101095, a dual inhibitor of αVβ8 and αVβ1, in well-annotated models of advanced PDA. We determined the preclinical efficacy and detailed anti-stromal effect of PLN-101095 inhibitor in genetically defined and patient-derived PDA models, including clinically relevant combinations with standard of care (SoC) chemotherapy and anti-programmed death receptor-1 antibody (anti-mPD-1). Mechanistic assessment of alterations in tumour cell-stromal cell crosstalk was performed using comprehensive transcriptomics and immunofluorescence approaches. Dual targeting of αVβ8 and αVβ1 with PLN-101095 in the syngeneic LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx1-Cre (KPC) model effectively reduced tumour growth by 45% in comparison to the vehicle, and significantly delayed disease progression in vivo. Single-cell analysis of PLN-101095-treated KPC pancreatic tumours revealed a positive reprogramming of malignant cells from a mesenchymal to a more epithelial-like state. This effect was further associated with an upregulation of MHC type I/II markers and corresponding downregulation of pro-metastatic factors across diverse cancer sub-populations. Additionally, combining PLN-101095 with anti-mPD-1 antibody further improved survival in this aggressive model of metastatic PDA. In a second syngeneic model, Pan02, PLN-101095 in combination with anti-mPD-1 antibody significantly reduced tumour growth, while increasing CD8+ lymphocyte infiltration. The combination of PLN-101095 with Anti-PD1 increased MHC and IFN gene expression. Finally, utilizing patient-derived models of metastatic PDA revealed that both PLN-104 and PLN-101095 significantly blocked tumour growth, improved the response to SoC chemotherapy Gemcitabine/Abraxane, and reduced metastatic spread to distant sites. In conclusion, these data provide scientific rationale for the design of future PLN-101095 and SoC chemotherapy as well as immunotherapy combinations in pancreatic cancer, with Phase I first-in-human oncology studies with PLN-101095 plus ICB already underway. Citation Format: Dannielle Upton, Diego Chacon Fajardo, Sofia Omari, Sean Porazinski, Benjamin McLean, Diana Schuhmacher, Aji Istadi, Australian Pancreatic Cancer Genome Initiative, Vishal Kothari, Darren Finkelstein, Tim Machajewski, Fernando Rock, Scott Turner, Marina Pajic. Selective targeting of integrins αVβ8 and αVβ1 within the dynamic ecosystem of pancreatic cancer to improve the overall anti-tumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6575.

Confirmation of SLC7A11 knockdown in KPC tumour cells and CAFs and collagen fibril analysis in tumour sections at therapeutic model endpoint.

Background Pancreatic ductal adenocarcinoma (PDA) has a 5-year survival of less than 10% and remains the 3rd leading cause of cancer-related death in Western societies. New treatment options are urgently needed. We previously characterized molecular subsets of PDA, including fibrotic elements of the disease, associated with pre-clinical and clinical response to select tailored treatment strategies.1–4 TGF-β promotes stromal cell reprogramming, immunosuppression, and fibrinogenesis in cancers, including PDA.5 6 Integrins αVβ8 and αVβ1 are important activators of TGF-β signaling. Selective integrin blockade has recently emerged as a promising therapeutic approach to address TGF-β-mediated immunotherapy resistance, and improve anti-tumor response across cancer models.7–9 Here, we assessed the in vivo efficacy of PLN-101095, a dual selective small molecule inhibitor of αVβ8 and αVβ1, in well-annotated models of advanced PDA. Methods We determined the pre-clinical efficacy of PLN-101095 in genetically-defined (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC), and Pan02) and genomically diverse patient-derived PDA xenograft models, testing clinically relevant combinations with standard of care (SoC) chemotherapy and anti-programmed death receptor-1 antibody (anti mPD-1), by monitoring tumor growth, metastasis, and animal survival. Mechanistic assessment of alterations in the tumor microenvironment (TME) was performed using comprehensive transcriptomic, immunohistochemical, and immunofluorescence approaches. Results Single cell analysis of KPC pancreatic tumors revealed restricted expression of integrin αVβ8 (ITGB8) within the T-reg and NK cell subsets, while components of TGF-β signaling were more widely represented across cancer and stromal cell subsets. Dual targeting of αVβ8 and αVβ1 with PLN-101095 in this setting effectively reduced tumor growth (45% reduction in tumor weight compared with Vehicle; P=0.003) and significantly delayed disease progression in vivo (median OS Vehicle 29.5 days vs PLN-101095 45 days, P<0.0001). Of note, combining PLN-101095 with anti mPD-1 antibody further improved survival in this aggressive model of metastatic PDA (median OS anti mPD-1 33 days vs PLN-101095 + anti mPD-1 51 days, 22% CR; P<0.0001). In a second syngeneic model of PDA (Pan02), PLN-101095 in combination with immune checkpoint blockade (ICB) significantly reduced tumor growth, TGF-β signaling, and fibrosis, while increasing CD8+ lymphocyte infiltration. Finally, utilizing patient-derived models of metastatic PDA revealed that PLN-101095 significantly blocked tumor growth, improved the response to SoC chemotherapy Gemcitabine/Abraxane, and reduced the number and size of lung metastases. Conclusions These data demonstrate that PLN-101095 significantly enhances ICB or SoC chemotherapy response in advanced PDA models and provide scientific rationale for future combination studies testing PLN-101095 in pancreatic cancer. References Waddell N, et al Nature 2015;518(7540):495 Chou A, et al. Gut 2018;67(12):2142 Vennin C, et al. Science Translational Medicine 2017;pii:eaai8504 Murphy KJ, et al. Science Advances 2021;7(40):eabh0363 Biffi G, et al. Cancer Discovery 2019;9(2):282 Krishnamurty AT, et al. Nature 2022;611(7934):148 Takasaka N, et al. JCI Insight 2018;3(20):e122591 Dodagatta-Marri E, et al. Cell Reports 2021;36(1):109309 Qiang L, et al. Gastroenterology 2023:S0016–5085(23)00809–0 [Online ahead of print] Ethics Approval In vivo experiments were approved by the Garvan Institute of Medical Research/St Vincent's Hospital Animal Ethics Committee (Animal Research Authority 21_30, 22_26). Patient samples and data were acquired through the Australian Pancreatic Genome Initiative (APGI)/International Cancer Genome Consortium (n=200), with ethics approval for the analyses of data and biological material under HREC X11–0220 (GHRP 1150).

Australian Pancreatic Cancer Genome Initiative International Cancer Genome Cohort patient cohort characteristics.

SLC7A11 multivariate survival analysis parameters.

SLC7A11 knockdown in PDAC CAFs does not affect glutamate secretion and is maintained in the presence of oxidative stress.

Validation of SLC7A11 antibodies and SLC7A11 knockdown in CAFs and PDAC cells in vitro.

Anti-proliferative effect of SLC7A11 knockdown in CAFs and the effect of SLC7A11 inhibition in MiaPaCa-2 PDAC cells and normal human pancreatic ductal epithelial (HPDE) cells.

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 5-year 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/β-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.2-4 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. References Waddell N, Pajic M, et al. Whole genomes redefine the mutational landscape of pancreatic cancer. Nature 2015;518(7540):495. Chou A, et al. Tailored First- and Second-Line CDK4-Targeting Treatment Combinations in Mouse Models of Pancreatic Cancer. Gut 2018;67(12):2142. Vennin C, et al. Transient tissue 'priming' via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy and the onset of the metastatic niche. Science Translational Medicine (2017) pii: eaai8504 Murphy KJ, et al. Merlin status guides epithelial versus stromal targeting of FAK in pancreatic cancer: improving outcome in personalized medicine. Science Advances 2021;7(40):eabh0363. Ethics Approval This study has been approved by the Garvan Institute of Medical Research/St Vincent's Hospital Animal Ethics Committee.

Abstract Cancer-associated fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through protumor signaling 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 PDAC-derived 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 gene-silencing 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. Significance: This study demonstrates that SLC7A11 in PDAC stromal cells is important for the tumor-promoting activity of CAFs and validates a clinically translatable nanomedicine for therapeutic SLC7A11 inhibition in PDAC.