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

Subhashis Ghosh; Paromita Mitra; Uday Saha; Rimpa Nandi; Subhashree Jena; Arnab Ghosh; Shantanu Saha Roy; Moulinath Acharya; Nidhan Kumar Biswas; Sandeep Singh

doi:10.1016/j.tranon.2023.101669

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

Transl Oncol. 2023 Jun:32:101669. doi: 10.1016/j.tranon.2023.101669. Epub 2023 Apr 11.

Affiliations

¹ National Institute of Biomedical Genomics, Kalyani, West Bengal 741251, India.
² National Institute of Biomedical Genomics, Kalyani, West Bengal 741251, India. Electronic address: ss5@nibmg.ac.in.

Abstract

Background: We have recently provided the evidence of interconvertible cellular states, driving non-genetic heterogeneity among stem-like oral cancer cells (oral-SLCCs). Here, NOTCH pathway-activity status is explored as one of the possible mechanisms behind this stochastic plasticity.

Methods: Oral-SLCCs were enriched in 3D-spheroids. Constitutively-active and inactive status of NOTCH pathway was achieved by genetic or pharmacological approaches. RNA sequencing and real-time PCR was performed for gene expression studies. in vitro cytotoxicity assessments were performed by AlamarBlue assay and in vivo effects were studied by xenograft growth in zebrafish embryo.

Results: We have observed stochastic plasticity in oral-SLCCs, spontaneously maintaining both NOTCH-active and inactive states. While cisplatin refraction was associated with post-treatment adaptation to the active-state of NOTCH pathway, oral-SLCCs with inactive NOTCH pathway status showed aggressive tumor growth and poor prognosis. RNAseq analysis clearly suggested the upregulation of JAK-STAT pathway in NOTCH pathway-inactive subset. The 3D-spheroids with lower NOTCH-activity status displayed significantly higher sensitivity to JAK-selective drugs, Ruxolitinib or Tofacitinib or siRNA mediated downregulation of tested partners STAT3/4. Oral-SLCCs were programmed to adapt the inactive status of NOTCH pathway by exposing to γ-secretase inhibitors, LY411575 or RO4929097, followed by targeting with JAK-inhibitors, Ruxolitinib or Tofacitinib. This approach resulted in a very significant inhibition in viability of 3D-spheroids as well as xenograft initiation in Zebrafish embryos.

Conclusion: Study revealed for the first time that NOTCH pathway-inactive state exhibit activation of JAK-STAT pathways, as synthetic lethal pair. Therefore, co-inhibition of these pathway may serve as novel therapeutic strategy against aggressive oral cancer.

Keywords: Cellular plasticity; JAK-STAT; NOTCH; Oral cancer; Stemness; Synthetic lethal pair.