Poster Presentation NSW State Cancer Conference 2023

Novel drug (CRO-67) in commercial pipeline have both anti-tumour and stromal reprogramming capacity (#108)

Shannon Chiang 1 , Keilah G Netto 1 , John Kokkinos 1 , Omali Pitiyarachchi 1 , Aparna Raina 1 , Janet Youkhana 1 , Quach Truong 2 , Daniel Wenholz 2 , Xiang Li 2 , Olivier Laczka 2 , Naresh Kumar 3 , John Wilkinson 2 , George Sharbeen 1 , Phoebe Phillips 1
  1. School of Biomedical Sciences, University of New South Wales, Sydney, NSW, Australia
  2. Noxopharm Limited, Sydney, NSW, Australia
  3. School of Chemistry, University of New South Wales, Sydney, NSW, Australia

Objective/rationale:
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy that urgently needs more effective therapies. Stromal cancer-associated fibroblasts (CAFs) play a major role in promoting tumour progression and chemoresistance, by creating a fibrotic tumour microenvironment that physically impedes drug access, and feeds PDAC cells nutrients and pro-tumour signals. Thus, in partnership with Noxopharm Limited we have developed a drug (CRO-67) designed to target both PDAC cells and CAFs. Aims: 1) Investigate the therapeutic potential of CRO-67 in patient-derived PDAC tumour explant model, mimicking the heterogeneity and multicellular architecture of human disease. 2) Assess the effect of CRO-67 on PDAC cell and CAF proliferation, apoptosis, and cell cycle progression to elucidate the mode of action.

Methods:
1) PDAC tumour samples were collected from 7 patients undergoing pancreatic resection. Tumour explants (1 – 2 mm diameter) were cultured on gelatin sponges and treated with CRO-67 (0 – 50  μg/mL) every 3 days and fixed on day 12. Therapeutic response was assessed by immunohistochemistry for markers of tumour cells (cytokeratin), CAFs (α-Smooth Muscle Actin), cell proliferation (Bromodeoxyuridine), and cell death (TUNEL).
2) PDAC cells and patient-derived CAFs were treated with CRO-67 for 24 h before cell proliferation (IncuCYTE S3) and apoptosis (Annexin V/DAPi staining and flow cytometry) analyses, or for 4 h before cell cycle analysis (flow cytometry).

Results:
1) CRO-67 treatment decreased tumour and CAF cell frequency in 5/7 and 6/7 patient PDAC tumour explants, respectively. It also decreased cell proliferation, and increased cell death relative to controls. 2) CRO-67 reduced proliferation of CAFs (IC50 = 346.9 nM) and PDAC cells (IC50 = 251.5 nM), increased apoptosis by 219 ± 6.5% (p<0.0001) and increased the fraction of cells in the G2/M cell cycle phase by 37 ± 3.6% (p<0.0001) relative to controls.

Conclusions:
Our findings demonstrate that CRO-67: 1) reduced PDAC cells and CAFs in human tumour explants; 2) reduced proliferation of PDAC cells and CAFs, increased apoptosis, and disrupted cell cycle progression through G2/M phase. Implication: CRO-67 is an ideal candidate for further preclinical investigation in vivo. We predict that this drug could lead to a more effective therapy for pancreatic cancer, able to directly kill PDAC cells and alleviate physical barriers to drug delivery by targeting CAFs, allowing sensitisation to chemotherapy.