Stoker, A. | Search bioRxiv

bioRxiv2026

DUSP6 is transcriptionally upregulated by activated ALK and cooperates with ALK signaling to reduce lorlatinib sensitivity in neuroblastoma cells

Neuroblastoma is a pediatric, sympathoadrenal tumour accounting for 7-10% of childhood malignancies. Some neuroblastomas are driven by activating mutations in ALK kinase and inhibitors show promise in clinical trials. Nevertheless, with resistance an ever-present concern, it remains important to understand better the effectors and modulators of ALK signaling. Wild type ALK promotes ERK activation, raising expression of negative regulators such as the dual-specificity phosphatase (DUSP) DUSP6. DUSP6 though can be pro- or anti-oncogenic in different cancers and its role in neuroblastoma cells remains unclear. We sought to understand its role in cells with either wild type or mutated ALK. Mutated ALK strongly promotes DUSP6 transcription, but apparently not through the ERK pathway. DUSP6 also appears to promote neuroblastoma cell proliferation without affecting ERK. Additionally, when DUSP6 is lost, the cells become more sensitive to ALK inhibitors lorlatinib and crizotinib. Phosphoproteomic analysis of such cells demonstrates that mutated ALK cooperates with DUSP6 to maximise signaling through several potential pathways, but again not through ERK or AKT. Their cooperation may also maintain optimal levels of N-Myc in MYCN-amplified neuroblastoma cells. While key substrates of DUSP6 remain to be determined in neuroblastoma cells, our study defines a novel role for this phosphatase in supporting the action of oncogenic ALK.

Cancer Biology↗

bioRxiv2026

Pharmaceutical assessment of low global warming potential alternatives to HFA-134a in a budesonide, glycopyrrolate, and formoterol fumarate pressurized metered dose inhaler

Manufacturers are adopting propellants for use in pressurized metered-dose inhalers (pMDIs) that have lower global warming potentials (GWPs) than the propellants traditionally used in pMDIs. Hydrofluoroalkane (HFA)-134a has been used as the propellant in the pMDI used to deliver the fixed-dose triple combination of budesonide, glycopyrrolate and formoterol fumarate (BGF); following successful clinical evaluation, the BGF pMDI is now being transitioned to the next generation propellant hydrofluoroolefin (HFO)-1234ze(E), which has near-zero GWP. We describe formulation development efforts that led to selection of HFO-1234ze(E) over another propellant, HFA-152a, for reformulation. Propellant-specific studies evaluated active pharmaceutical ingredient (API) stability and aerodynamic particle size distribution (aPSD). Those analyses have been complemented by in silico regional lung deposition modeling conducted after the clinical evaluation of the reformulated BGF pMDI. HFO-1234ze(E) supported favorable stability and aPSD characteristics for BGF pMDI reformulation, compared with HFA-152a, and modeling predicted regional deposition consistent with therapeutic intent. Given that each pMDI is a unique combination of APIs, device, propellant, and excipients, propellant substitution requires product-specific evidence and regulatory approval, and typically takes several years. Targeted analyses, such as those described here, helped to identify the most suitable candidate propellant for successful substitution in the BGF pMDI. HighlightsO_LIFormulation development efforts that led to evaluation of a budesonide-glycopyrrolate-formoterol fumarate pressurized metered-dose inhaler (BGF pMDI) reformulated with the next generation propellant HFO-1234ze(E) in a clinical trial program are described; the suitability of another propellant, HFA-152a, was also assessed C_LIO_LIOver 6 months under accelerated storage conditions (40{degrees}C/75% relative humidity [RH]), the HFA-152a formulation approached and, in one replicate, fell below the 90% of formulation label claim threshold of evaluation, whereas the original HFA-134a product and the HFO-1234ze(E) formulation remained above that threshold C_LIO_LIOver 6 months under accelerated storage conditions (40{degrees}C/75% RH) and 18 months under long-term stability storage conditions (25{degrees}C/60% RH), the fine particle mass and fine particle fraction for all active pharmaceutical ingredients (APIs) showed that the HFO-1234ze(E) formulation tracked more closely than the HFA-152a formulation to the original HFA-134a product C_LIO_LILater in silico modeling, conducted after clinical testing, predicted a trend for greater deposition of APIs in early airway generations with HFA-152a, whereas HFO-1234ze(E) was predicted to more closely match HFA-134a, indicating a greater likelihood of achieving equivalence to the original HFA-134a product with HFO-1234ze(E) than with HFA-152a C_LIO_LIBased on these analyses and other formulation development efforts, HFO-1234ze(E) was identified as the most suitable propellant for reformulation of the BGF pMDI; for HFA-152a, analyses raised concerns about storage stability, and differences in aerosol characteristics that can impact API deposition in the lungs and, in turn, efficacy C_LI

pharmacology and toxicology↗