(W1230-03-18) Harnessing Albumin-Anchored BRD4 PROTAC Nanoliposomes for Selective and Targeted Degradation in Pancreatic Ductal Adenocarcinoma

Purpose: Pancreatic ductal adenocarcinoma (PDAC) remains a highly aggressive malignancy in solid tumors having a 5-year survival rate of less than 8%. Despite the standard single-agent therapies like gemcitabine, irinotecan and combination treatment approaches like FOLFIRINOX, PDAC has a less favorable prognosis and poor outcomes, usually characterized by silent progression. PROteolysis TArgeting Chimera (PROTAC) is an emerging technology that offers a paradigm shift for degrading the proteins that were traditionally deemed to be ‘undruggable’ by utilizing the cell’s own ubiquitin-proteasome mechanism. Unlike the conventional small-molecule inhibitors, PROTACs regulate the protein function by degradation of the target protein rather than mere inhibition. The highly expressed Bromodomain protein 4 (BRD4) significantly amplifies the oncogenic activity of c-MYC and KRAS, thus driving tumorigenesis in PDAC. For addressing this, a BRD4 PROTAC, ARV-825 (ARV), was potentially explored for targeted protein degradation by formulating non-targeted ARV-loaded liposomes (ARL) and human serum albumin-conjugated nanoliposomes of ARV (HARL) for targeted delivery. This innovative approach leverages the active uptake mechanism of albuminosomes via gp60 and/or secreted protein acidic and rich in cysteine (SPARC), a glycoprotein that is overexpressed in PDAC stroma as well as the tumor cells (Fig. 1). We aim to (a) develop and characterize ARV encapsulated nanoliposomes, ARL and HARL; and (b) to investigate efficacy of ARL and HARL in 2D and 3D pancreatic cancer cell lines.

Methods: ARV-loaded nanoliposomes were developed using modified hydration method, as illustrated in Fig. 2(a). HARL were prepared by incorporating ARV, DOPC, cholesterol, DSPE-PEG-2000, and DGS-NTA-Ni in a weight ratio of 1:60:10:2.5:2.5. Alternatively, ARL was formulated using the same composition, excluding DGS-NTA-Ni. HARL was formulated by incubating DGS-NTA-Ni containing liposomes with Histidine-tagged human serum albumin (HSA). Subsequent analyses were conducted using the particle size and surface charge potential to evaluate whether the conjugation has efficiently occurred. The particle sizes of ARL and HARL formulation were performed using dynamic light scattering (DLS) to measure the hydrodynamic diameter. The polydispersity index (PDI) were assessed to evaluate homogeneity and zeta potential was performed to determine the surface charge. Antiproliferative activity of ARV was evaluated using MTT assay in pancreatic cancer cell lines and in non-tumorigenic cell lines including HEK-293 and RAW 264.7. The selectivity index of ARV was performed in both pancreatic cancer cell lines and in non-cancerous cell lines to evaluate the selective toxicity and safety profile. In-vitro2D assays including migration assay, clonogenic assay, apoptosis and 3D spheroid assay was performed for ARV, ARL and HARL to understand the anticancer efficacy.

Results: Human serum albumin-conjugated ARV nanoliposomes were formulated by incubating DGS-NTA-Ni and HSA in 1:0.5 molar ratio.  ARV-loaded nanoliposomes containing DGS-NTA-Ni (before conjugation) yielded a monomodal size distribution of average particle size 144.8 ± 0.707 nm and polydispersity index of 0.231 ± 0.001, while the zeta potential was determined to be -32.75 ± 3.323 mV> (Fig. 3b) <. HARL conjugation with His-tagged HSA demonstrated a significant increase in particle size from 144.8 ± 0.707 nm to 174.95 ± 1.344 nm as mentioned in Fig. 2(c). A notable increase in the zeta potential of HARL from –32.75 ± 3.323 mV to –9.31 ± 0.863 mV was observed, justifying successful conjugation of HSA to the targeted ARVL. ARV revealed comparable IC₅₀ values in cancerous as well as the non-cancerous cell lines. The antiproliferation activity was comparable in the pancreatic cancer cell lines and higher in healthy cell lines as shown in Table 1 (a). The selectivity indices, derived from comparing the IC₅₀ values of ARV between non-tumorigenic cell lines HEK-293 and RAW 264.7 and cancer cells, were significantly higher than 1, demonstrating enhanced toxicity of ARV towards cancerous cells and not the healthy cells. As shown in Table 1 (b), the differential selectivity observed in non-cancerous cell lines underscores ARV’s potency and cytotoxicity towards pancreatic cancer cell lines.  In-vitro cytotoxic evaluation including migration assay, clonogenic assay, apoptosis and 3D spheroid assay for ARV, ARL and HARL are currently under investigation.

Conclusion: In summary, ARV-loaded nanoliposomes were developed using modified hydration method which exhibited successful conjugation with HSA, reinforcing the potential of HARL as a targeted and therapeutic drug delivery strategy. The targeted approach allows the BRD4 degrader to exhibit pronounced selectivity towards the tumor cells, resulting in maximal therapeutic action under potential safety index. Overall, this study reports on a novel strategy for elucidating anti tumor efficacy of PROTACs against pancreatic ductal adenocarcinoma.

Acknowledgements: This project was funded by NIH R16 (1R16GM145556-01A1)