Single-Cell Proteomic Profiling Identifies Combined AXL and JAK1 Inhibition as a Novel Therapeutic Strategy for Lung Cancer

Abstract

Cytometry by time-of-flight (CyTOF) simultaneously measures multiple cellular proteins at the single-cell level and is used to assess intertumor and intratumor heterogeneity. This approach may be used to investigate the variability of individual tumor responses to treatments. Herein, we stratified lung tumor subpopulations based on AXL signaling as a potential targeting strategy. Integrative transcriptome analyses were used to investigate how TP-0903, an AXL kinase inhibitor, influences redundant oncogenic pathways in metastatic lung cancer cells. CyTOF profiling revealed that AXL inhibition suppressed SMAD4/TGFb signaling and induced JAK1-STAT3 signaling to compensate for the loss of AXL. Interestingly, high JAK1-STAT3 was associated with increased levels of AXL in treatment-na€ ve tumors. Tumors with high AXL, TGFb, and JAK1 signaling concomitantly displayed CD133-mediated cancer stemness and hybrid epithelialto-mesenchymal transition features in advanced-stage patients, suggesting greater potential for distant dissemination. Diffusion pseudotime analysis revealed cell-fate trajectories among four different categories that were linked to clinicopathologic features for each patient. Patient-derived organoids (PDO) obtained from tumors with high AXL and JAK1 were sensitive to TP-0903 and ruxolitinib (JAK inhibitor) treatments, supporting the CyTOF findings. This study shows that single-cell proteomic profiling of treatment-na€ ve lung tumors, coupled with ex vivo testing of PDOs, identifies continuous AXL, TGFb, and JAK1-STAT3 signal activation in select tumors that may be targeted by combined AXL-JAK1 inhibition.Significance: Single-cell proteomic profiling of clinical samples may facilitate the optimal selection of novel drug targets, interpretation of early-phase clinical trial data, and development of predictive biomarkers valuable for patient stratification.

Targeting the JAK/STAT pathway in solid tumors

“…The JAK1/2-selective inhibitor ruxolitinib is FDA approved for the treatment of polycythemia vera, myelofibrosis, and graft versus host disease, and it has been shown to decrease STAT3 activation in preclinical models of several solid tumors [18,22,65] . Ruxolitinib inhibited STAT3 activation and decreased cell growth in breast cancer [66,67] , NSCLC [68] , HNC [69] , esophageal cancer [70] , bladder cancer [71] , HCC [72] , cervical cancer [73] , and colorectal cancer [74,75] cell lines. In pancreatic cancer cells, ruxolitinib treatment was also shown to decrease expression of pro-angiogenic genes and impede epithelial-to-mesenchymal transition [76,77] .…”

Markers and Reporters to Reveal the Hierarchy in Heterogeneous Cancer Stem Cells

“…There was another study conducted in parallel using single-cell proteomic profiling with lung cancer samples, where a CyTOF panel of 21 antibodies was designed to probe lung cancer cells. They observed a higher level of CD133 in comparison to other stemness markers (OCT3/4, NANOG, CD44, and ALDH1A1) in a subpopulation that exhibited a hybrid nature of epithelial/mesenchymal plasticity (Taverna et al, 2020). Recently it is postulated that epithelial-mesenchymal plasticity is the driving force of CSCs during metastasis (Celia-Terrassa and Jolly, 2020).…”

“…Additionally, this dormant population can acquire proliferative capacity to facilitate differentiation. Recent advances in the single-cell-based technologies, such as single-cell DNA/RNA-Sequencing, mass cytometry (CyTOF), next-generation fluorescence flow cytometry, and “imaging mass cytometry” platform, help us to understand how functional heterogeneity is reflected by phenotypic heterogeneity ( Akrap et al, 2016 ; Puram et al, 2017 ; Colacino et al, 2018 ; Sharma et al, 2018 ; Prieto-Vila et al, 2019 ; Taverna et al, 2020 ; Frank et al, 2021 ; Gonzalez Castro et al, 2021 ).…”

“…The recent evidences suggest that the proliferative non-CSCs can acquire resistance and aid in metastasis after stemness induction depending on the CSC niche, probably explaining why the ITGA6 + cells showed metastatic ability. In a proteomic analysis, CD133 was observed in hybrid EMT phenotype, which is shown to be important for metastatic cells ( Taverna et al, 2020 ). Whether the CD133 expression is exclusive to the Progenitor-like population is not yet evident.…”

“…The significance of ALDH Hi population expressing ALDH1A1 at the single-cell level was investigated in the cancer context also. In lung cancer, there is a subpopulation of ALDH1A1 cells, the population size of which increases progressively with the stage of the disease ( Taverna et al, 2020 ). But this increase was not associated with acquisition of drug resistance ( Taverna et al, 2020 ).…”