Abstract A067: βIII-tubulin is a dual cell therapeutic target in pancreatic cancer and regulates sensitivity to TRAIL through a DR5-dependent mechanism

Background: Pancreatic ductal adenocarcinoma (PDAC) urgently requires novel, effective therapies as its 5-year survival rate remains at just 13%. Our group has previously shown that silencing βIII-tubulin induced chemosensitisation and apoptosis in vitro, and reduced metastasis and tumour growth in vivo (McCarroll et al, Oncotarget, 2015). Recently, we have demonstrated that βIII-tubulin regulates the extrinsic (caspase-8) apoptosis pathway in PDAC cells, where silencing βIII-tubulin increased PDAC cell sensitivity to TNF-related apoptosis-inducing ligand (TRAIL). Extrinsic apoptosis is activated when TRAIL binds to its receptors, death receptor 4 (DR4) or 5 (DR5). This study expanded on these findings to assess whether these effects (i) are dependent on DR4 or DR5 signaling; (ii) occur in PDAC cancer-associated fibroblasts (CAFs); (iii) are therapeutically relevant in an in vivo setting. Aims: 1) Determine the effect of (a) DR5 knockout and (b) DR4 and DR5 agonists on βIII-tubulin knockdown-mediated sensitization of PDAC cells to TRAIL in vitro; 2) Evaluate the effect of βIII-tubulin knockdown on CAF proliferation and sensitivity to TRAIL-induced apoptosis in vitro; 3) Assess the therapeutic potential of silencing βIII-tubulin in combination with TRAIL in a xenograft PDAC mouse model. Methods: 1) MiaPaCa2 PDAC cells were treated with (a) CRISPR to knockout DR5 ± control or βIII-tubulin-siRNA ± TRAIL, or (b) control- or βIII-tubulin-siRNA ± TRAIL, DR4 or DR5 agonist, and apoptosis measured (Annexin V + DAPI via flow cytometry). 2) Patient-derived PDAC CAFs were transfected with control- or βIII-tubulin ± TRAIL and proliferation (IncuCyte S3), apoptosis (Annexin V + DAPI via flow cytometry) and senescence (β-galactosidase senescence stain) measured. 3) Subcutaneous PDAC tumours were treated with control- or βIII-tubulin-siRNA complexed to STAR 3 nanoparticles twice weekly (intravenous) ± TRAIL once weekly (intratumoural) for 4 weeks. Results: 1a) Knockout of DR5 in PDAC inhibited βIII-tubulin knockdown-mediated sensitization to TRAIL. 1b) βIII-tubulin knockdown combined with DR5 agonists induced apoptosis to a greater extent than DR4 agonists in PDAC. 2) Knockdown of βIII-tubulin reduced CAF proliferation and viability, and induced senescence. However, it did not induce apoptosis ± TRAIL. 3) Knockdown of βIII-tubulin in combination with TRAIL induced a significant increase in the frequency of responders, in comparison to either treatment alone. Conclusions: 1) Sensitization of PDAC cells to TRAIL mediated by βIII-tubulin knockdown is DR5-dependent; 2) βIII-tubulin knockdown inhibits both CAF and PDAC proliferation and sensitises PDAC cells to TRAIL-induced apoptosis. Citation Format: Grace Schulstad, John Kokkinos, Janet Youkhana, Aparna S. Raina, Meagan E. Davis, Oliver S.M. Arkell, Keilah Garcia Netto, Cyrille Boyer, David Goldstein, Koroush S. Haghighi, George Sharbeen, Joshua A. McCarroll, Phoebe A. Phillips. βIII-tubulin is a dual cell therapeutic target in pancreatic cancer and regulates sensitivity to TRAIL through a DR5-dependent mechanism [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3): nr A067.