Tumor Heterogeneity

A central issue that confounds successful treatment of GBM patients is the heterogeneous nature of this aggressive tumor. As a result, multiple and spatially distinct heterotypic populations exist within a single GBM, making any lesion- or pathway-specific therapy less effective. While considerable effort has been placed on understanding cell intrinsic mechanisms conferring therapeutic resistance, much less is known about the interactions between heterogeneous tumor cells within these neoplasms that contribute to the recalcitrant nature of this cancer.

Inter-clonal cross talk and receptor activation: Leaders and followers

The most common genetic alteration found in high-grade astrocytic gliomas is amplification of the EGFR gene, which encodes a transmembrane glycoprotein receptor and is a member of the ERBB receptor tyrosine kinase family. EGFR amplification occurs in approximately 60% of GBMs and is typically present as double-minute extrachromosomal DNA elements (ecDNA). From a clinical perspective the presence of amplification of this receptor has been shown to correlate with a shorter interval to relapse and poorer survival. In about half the cases with wild type EGFR (wtEGFR) amplification, the event is coupled with gene rearrangement, and the majority result in a truncated, constitutively active mutant lacking exons 2-7, known as EGFRvIII. Paradoxically, despite a potent ability to enhance tumorigenicity, EGFRvIII expression is typically observed only in a subpopulation of cells and almost never in the entirety of the tumor. Our data support the possibility that the minority EGFRvIII population of cells not only enhance their own intrinsic tumorigenic abilities, but also actively potentiate the proliferation of the neighboring majority of tumor cells expressing amplified wtEGFR through inflammatory paracrine activity of interleukin 6 (IL-6). These results support the concept that heterogeneity could be maintained through interclonal cooperativity of genetically distinct tumor cells. More importantly, it demonstrates that even a small minority of phenotypically distinct cells can have a profound impact on behavior of the rest of the population by, for example, conferring an enhanced growth rate.

EGFR/EGFRvIII crosstalk converges on NF-κB to attenuate EGFR inhibitor sensitivity: When heterogeneity presents an achilles heel:

Heterogeneous expression of amplified and mutated EGFR presents a substantial challenge for the effective use of EGFR-directed therapeutics. To address this, we used human and murine GBM cell lines, patient derived xenograft (PDX) models and clinical specimens from GBM patients, to describe an active attenuation in sensitivity to EGFR tyrosine kinase inhibitors (TKIs) mediated by EGFRvIII-driven production of IL-6. Mechanistically, activation of the IL-6 common receptor, gp130, expressed on wtEGFR cells, promotes NF-κB activation and subsequent expression of a pro-survival gene target, survivin (BIRC5), through deposition of NF-κB in complex with BRD4 at its promoter. This signaling axis is efficiently uncoupled by the BET inhibitor JQ1, leading to inhibition of survivin expression and restored sensitivity to anti-EGFR therapy. These results underscore that in addition to cell intrinsic mechanisms, attenuation to therapy can also occur through extrinsic cues fostered by interclonal tumor cell communication within the tumor ecosystem. Understanding heterotypic tumor cell interactions, as exemplified by the EGFR mutational landscape found in GBM, will better inform strategies to overcome therapy resistance commonly encountered for this tumor.

Generation of EGFRvIII: When less is more

EGFRvIII, which is the product of a truncation encompassing exons two to seven, is the most common EGFR mutant in glioblastoma (GBM) and is associated with its aggressiveness. Our detailed analyses of EGFRvIII breakpoint sequences showed traces of different varieties of DNA repair mechanisms accompanied by alterations of chromosome seven, suggesting that complex genomic rearrangements are involved in generation of EGFRvIII as an underlying mechanism.

Molecular determinants of glioma invasion: Not all EGFR mutations are alike

Malignant gliomas are the most common, infiltrative, and lethal primary brain tumors affecting the adult population. The poor prognosis for this disease is due to a combination of the presence of highly invasive tumor cells that escape surgical resection and extreme cellular, genetic and epigenetic heterogeneity making any single therapeutic approach likely to fail. Understanding the invasion mechanisms governing how cells adhere to their environment is thus critical to devise new therapeutic strategies. We explored the clinical and pathological impact of EGFR extracellular domain missense mutations. Retrospective assessment of de novo glioblastoma (GBM) patients revealed a significant reduction in overall survival in patients with EGFR mutations at alanine 289 (EGFR-A289D/T/V). Quantitative multi-parametric MRI analyses indicated increased tumor invasion for EGFR-A289D/T/V mutants, a finding corroborated in mice bearing intracranial tumors expressing EGFR-A289V and dependent on ERK-mediated expression of matrix metalloproteinase-1 (MMP1). These findings indicate a highly invasive and prognostic indicator for the EGFR-A289V missense mutation in GBM.