Intratumoral spatial heterogeneity of BTK kinomic activity dictates distinct therapeutic response within a single glioblastoma tumor

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  • 1 Departments of Neurosurgery and
  • | 2 Radiation Oncology, and
  • | 4 Comprehensive Cancer Center, University of Alabama at Birmingham, Alabama; and
  • | 3 Brain Tumor Translational Targets, German Cancer Research Center, Heidelberg, Germany
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OBJECTIVE

Despite significant recent efforts applied toward the development of efficacious therapies for glioblastoma (GBM) through exploration of GBM’s genome and transcriptome, curative therapeutic strategies remain highly elusive. As such, novel and effective therapeutics are urgently required. In this study, the authors sought to explore the kinomic landscape of GBM from a previously underutilized approach (i.e., spatial heterogeneity), followed by validation of Bruton’s tyrosine kinase (BTK) targeting according to this stepwise kinomic-based novel approach.

METHODS

Twelve GBM tumor samples were obtained and characterized histopathologically from 2 patients with GBM. PamStation peptide-array analysis of these tissues was performed to measure the kinomic activity of each sample. The Ivy GBM database was then utilized to determine the intratumoral spatial localization of BTK activity by investigating the expression of BTK-related transcription factors (TFs) within tumors. Genetic inhibition of BTK family members through lentiviral short hairpin RNA (shRNA) knockdown was performed to determine their function in the core-like and edge-like GBM neurosphere models. Finally, the small-molecule inhibitor of BTK, ONO/GS-4059, which is currently under clinical investigation in nonbrain cancers, was applied for pharmacological inhibition of regionally specified newly established GBM edge and core neurosphere models.

RESULTS

Kinomic investigation identified two major subclusters of GBM tissues from both patients exhibiting distinct profiles of kinase activity. Comparatively, in these spatially defined subgroups, BTK was the centric kinase differentially expressed. According to the Ivy GBM database, BTK-related TFs were highly expressed in the tumor core, but not in edge counterparts. Short hairpin RNA–mediated gene silencing of BTK in previously established edge- and core-like GBM neurospheres demonstrated increased apoptotic activity with predominance of the sub-G1 phase of core-like neurospheres compared to edge-like neurospheres. Lastly, pharmacological inhibition of BTK by ONO/GS-4059 resulted in growth inhibition of regionally derived GBM core cells and, to a lesser extent, their edge counterparts.

CONCLUSIONS

This study identifies significant heterogeneity in kinase activity both within and across distinct GBM tumors. The study findings indicate that BTK activity is elevated in the classically therapy-resistant GBM tumor core. Given these findings, targeting GBM’s resistant core through BTK may potentially provide therapeutic benefit for patients with GBM.

ABBREVIATIONS

bFGF = basic fibroblast growth factor; BTK = Bruton’s tyrosine kinase; CLL = chronic lymphocytic leukemia; EGF = epidermal growth factor; EGFR = endothelial growth factor receptor; FITC = fluorescein isothiocyanate; GAP = Glioblastoma Atlas Project; GBM = glioblastoma; GSC = glioma stemlike cell; IDH = isocitrate dehydrogenase; MCL = mantle cell lymphoma; PCA = principal component analysis; PDGFR = platelet-derived endothelial growth factor; PI = propidium iodide; PTK = protein tyrosine kinase; shRNA = short hairpin RNA; TF = transcription factor; UAB = University of Alabama at Birmingham.

Supplementary Materials

    • Figs S1-S5 and Tables S1 and S2 (PDF 2.71 MB)

The Neurosurgery Research & Education Foundation (NREF) is celebrating its 40th anniversary this month. Since its inception, the NREF has invested nearly $30 million in the future of neurosurgery through its support of basic science and clinical research, as well as life-long education, to foster improved outcomes for our patients with neurosurgical diseases. See the article by Agarwal et al. (pp 1905–1912).

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