EGFR blockade in GBM brain tumor stem cells synergizes with JAK2/STAT3 pathway inhibition to abrogate compensatory mechanisms in vitro and in vivo

Abstract

Background The EGFR pathway is frequently mutated in glioblastoma (GBM). However, to date, EGFR therapies have not demonstrated efficacy in clinical trials. Poor brain penetration of conventional inhibitors, lack of patient stratification for EGFR status, and mechanisms of resistance are likely responsible for the failure of EGFR-targeted therapy. We aimed to address these elements in a large panel of molecularly diverse patient-derived GBM brain tumor stem cells (BTSCs). Methods In vitro growth inhibition and on-target efficacy of afatinib, pacritinib, or a combination were assessed by cell viability, neurosphere formation, cytotoxicity, limiting dilution assays, and western blotting. In vivo efficacy was assessed with mass spectrometry, immunohistochemistry, magnetic resonance imaging, and intracranial xenograft models. Results We show that afatinib and pacritinib decreased BTSC growth and sphere-forming capacity in vitro. Combinations of the 2 drugs were synergistic and abrogated the activation of STAT3 signaling observed upon EGFR inhibition in vitro and in vivo. We further demonstrate that the brain-penetrant EGFR inhibitor, afatinib, improved survival in EGFRvIII mt orthotopic xenograft models. However, upregulation of the oncogenic STAT3 signaling pathway was observed following afatinib treatment. Combined inhibition with 2 clinically relevant drugs, afatinib and pacritinib, synergistically decreased BTSC viability and abrogated this compensatory mechanism of resistance to EGFR inhibition. A significant decrease in tumor burden in vivo was observed with the combinatorial treatment. Conclusions These data demonstrate that brain-penetrant combinatorial therapies targeting the EGFR and STAT3 signaling pathways hold therapeutic promise for GBM.

The Role and Therapeutic Targeting of JAK/STAT Signaling in Glioblastoma

“…The reliance on several dysregulated—and often redundant—signaling pathways, including EGFR, PIK3CA, PDGF, and NF-κB, has been well-characterized in glioblastoma, and cross-talk between these have likely contributed in large part to the historical failures of targeted therapy [ 53 , 145 , 146 ]. Indeed, compensatory STAT3 upregulation has been demonstrated in numerous studies of other receptor tyrosine kinases (e.g., EGFR, MEK, HER2) in other solid tumors [ 147 , 148 , 149 ]. Treatment of GBM with anti-VEGF monoclonal antibody bevacizumab for instance—currently the only FDA-approved targeted treatment for glioblastoma and one that has not been shown to have survival benefit—eventually induces STAT3 activation likely via the hypoxia response, leading to the expression of stemness and invasive markers such as nestin and ICAM-1, respectively [ 150 ].…”

Targeting the JAK/STAT pathway in solid tumors

“…Pacritinib is a selective JAK2 inhibitor currently being studied in a Phase III clinical trial for treatment of myelofibrosis (NCT02055781) [125,126] . In GBM cell lines, pacritinib, alone or in combination with afatinib, inhibited STAT3 activation, cell viability, and spheroid formation [127][128][129] . Pacritinib plus afatinib was also shown to decrease tumor burden in mice with GBM tumors [129] .…”

“…In GBM cell lines, pacritinib, alone or in combination with afatinib, inhibited STAT3 activation, cell viability, and spheroid formation [127][128][129] . Pacritinib plus afatinib was also shown to decrease tumor burden in mice with GBM tumors [129] . Similar to momelotinib, pacritinib reduced resistance to temozolomide in GBM in vivo models [127,128] .…”

Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

“…EGFRvIII/STAT3 signalling plays crucial roles in cell growth and proliferation 36,37 . Identification of LGALS1 as a direct downstream target gene of EGFRvIII/STAT3 oncogenic pathway in human BTSCs led us next to examine the role of LGALS1 in the regulation of BTSC growth.…”