NOTCH pathway inactivation reprograms stem-like oral cancer cells to JAK-STAT dependent state and provides the opportunity of synthetic lethality

Background: Recent evidence demonstrates that stem-like oral cancer cells (oral-SLCCs) exhibit interconvertible cellular states, contributing to non-genetic heterogeneity. This study investigates the role of NOTCH pathway activity as a potential mechanism underlying this stochastic plasticity.
Methods: Oral-SLCCs were enriched through 3D spheroid culture. The NOTCH pathway was modulated to achieve constitutively active or inactive states using genetic and pharmacological approaches. Gene expression profiling was conducted via RNA sequencing and real-time PCR. Cytotoxicity assays were performed using AlamarBlue, and in vivo effects were assessed through zebrafish embryo xenograft models.
Results: Oral-SLCCs demonstrated stochastic plasticity, maintaining coexisting NOTCH-active and NOTCH-inactive states. Cisplatin resistance was linked to a post-treatment shift toward the NOTCH-active state. In contrast, NOTCH-inactive oral-SLCCs exhibited aggressive tumor growth and poor prognosis. RNA sequencing revealed upregulation of the JAK-STAT pathway in NOTCH-inactive cells. Spheroids with lower NOTCH activity displayed heightened sensitivity to JAK inhibitors, such as Ruxolitinib and Tofacitinib, or STAT3/4 knockdown via siRNA. Programming oral-SLCCs toward the NOTCH-inactive state with γ-secretase inhibitors (LY411575 or RO4929097) followed by JAK inhibition significantly reduced 3D spheroid viability and suppressed zebrafish xenograft initiation.
Conclusion: This study identifies for the first time that the NOTCH-inactive state activates the JAK-STAT pathway, forming a synthetic lethal interaction. Combined inhibition of these pathways offers a promising therapeutic strategy for combating aggressive oral cancer.