(T1030-07-46) Sustained Release of a Pan-RAF–MEK Molecular-Glue (NST-628): A New Strategy for Long-Acting Cancer Therapy

Purpose: Despite decades of research, pancreatic cancer is characterized by aggressive progression, late diagnosis, and chemoresistance to standard therapies. The aberrant signaling of the mitogen-activated protein kinase (MAPK) pathway poses to be the central driver in pancreatic cancer, primarily due to KRAS mutation, that occur in 95% of the cases. Considering the current landscape of therapies that offer limited survival benefit and rapid emergence of drug resistance, a paradigm-shifting approach that utilizes a novel molecular glue is essential to transcend the limitations of existing chemotherapeutic agents. NST-628 (NST) is a potent pan-RAF–MEK molecular glue that provide dual blockade by preventing the activation of MEK and by blocking the RAF proteins, thus enabling prolonged inhibition of MAPK oncogenic signaling pathway (Fig. 1). Given the physicochemical characteristics of NST, oral administration will prove to be suboptimal, prompting for the development of a long-acting, sustained-release injectable formulation of NST to ensure therapeutic efficacy. Recognizing the potential of NST to target and modulate intracellular signaling pathways, we aim (a) to optimize and characterize a self-injectable, biodegradable, sustained-release depot formulation for NST (DepNeST); and (b) to determine anticancer efficacy in 2D and 3D pancreatic cancer cell culture models. This study represents the first investigation of a novel molecular glue as an extended drug release formulation in cancer therapy.

Methods: DepNeST was fabricated by dissolving biodegradable polymers in a biocompatible solvent, N-methyl pyrrolidone (NMP). Two different biodegradable polymers, poly(lactide-co-glycolide) (PLGA) and the triblock polymer of polycaprolactone (PCL) and polyethylene glycol (PEG), specifically PCL-PEG-PCL, were utilized for formulating DepNeST at concentrations of 15% w/v and 30% w/v. Subsequently, NST was dissolved into a polymer-NMP solution as illustrated in Fig. 2A.  The in vitro release profile of NST from DepNeST was performed. DepNeST was evaluated for formulations containing PLGA and PCL-PEG-PCL at 15%w/v and 30% w/v polymer concentrations. To determine the clinical efficacy of NST, cellular studies were performed was determined in MIA PaCa-2 cells. The impact of NST on cellular morphology and cytoskeletal integrity was examined by immunofluorescence staining of F-actin and were confirmed with Giemsa staining. Induction of apoptosis following NST treatment was analyzed using acridine orange staining to determine nuclear integrity and cell viability. The cytotoxic potential of NST was determined in MIA PaCa-2 cells using the MTT assay. A multicellular 3D spheroid model of MIA PaCa-2 cells was developed to mimic in vivo tumor conditions and investigated acute and chronic treatment regimens. In vitro Live/Dead cell assay was performed to assess the cell apoptotic effect of the treatment groups on 3D spheroids of MIA PaCa-2 cells.

Results: Four different batches were prepared using two biodegradable polymers at concentration 15% w/v and 30% w/v in NMP. Fig. 2B illustrates visual appearance of blank polymeric depot of PLGA and PCL-PEG-PCL at these varying polymer concentrations. The in vitro release study demonstrated an initial burst release followed by a sustained release of NST from DepNeST over a period of 24 h (Fig. 2C). Specifically, DepNeST formulation containing 15% w/v polymer concentration exhibited nearly 12% cumulative drug release within 24 h compared to 30% w/v polymer concentration showing nearly 8%, indicating that lower polymer concentrations facilitated faster NST release. NST treatment led enhanced formation of lamellipodia and filopodia in MIA PaCa-2 cells as shown in Fig. 3A. MIA PaCa-2 cells when stained with immunofluorescent F-actin and DAPI, revealed substantial accumulation of actin filaments and actin-rich protrusions, suggesting that NST negatively impacted the cytoskeletal integrity of the cells in a concentration-dependent manner. Cell staining with acridine orange revealed condensed chromatin fragments post NST treatment. Giemsa staining further confirmed that treatment with NST led to morphological changes and inhibited cell growth. We found very promising in vitro cytotoxicity in nanomolar concentrations where the half-maximal inhibitory concentration (IC₅₀) of NST in MIA PaCa-2 cells was found to be 183.8 ± 10.5 nM (Fig. 3B). The acute treatment regimen in 3D spheroids showed a non-significant decrease in cell growth compared to chronic treatment exposure (Fig. 3C, D). From the in vitro Live/Dead Cell assay, higher red fluorescence was found in treatment groups as compared to control confirming large number of apoptotic cells in both acute and chronic exposure to NST (Fig. 3E).

Conclusion: This is the very first study to investigate the molecular glue, NST-628, in pancreatic cancer, highlighting its sustained and potent antitumor activity as a highly promising therapeutic strategy. The engineering of DepNeST, in situ depot system for sustained release of NST, offers a novel, groundbreaking approach to enhance the therapeutic efficacy while reducing the dose. This strategy could potentially revolutionize the therapeutic landscape for treating RAS/RAF-driven oncogenic tumors.