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EGFR inhibitor resistance in glioblastoma: deciphering the role of FGFR-mediated PTEN phosphorylation

Malignant gliomas are the most common primary brain tumors and their highly invasive and neurologically destructive nature makes them among the deadliest of adult human cancers. Glioblastoma multiforme (GBM) is the most aggressive manifestation of glioma, with roughly 15,000 new cases diagnosed annually in the United States. The median survival for GBM ranges between 12-15 months despite maximum treatment efforts -- a devastating statistic that has changed little over several decades of technological advances in neurosurgery, radiation therapy and clinical trials of conventional and novel therapeutics. It is perplexing that therapies used effectively in the treatment of other solid tumors, such as breast, lung and prostate, are overwhelmingly ineffective in the treatment of GBM. Thus, there is a desperate need for more effective, novel therapeutic approaches to improve the clinical outcome of patients with this aggressive disease.

On the basis of clinical presentation, GBMs can be subdivided into two subtypes: primary or secondary. Primary GBMs account for the great majority of cases in older patients, while secondary GBMs are more rare, and tend to occur in patients below the age of 45 years. Primary GBM presents in an acute manner with no evidence of prior symptoms or precursor lower grade pathology. In contrast, secondary GBM derives consistently from lower grade disease which progresses over the course of 5-10 years from the time of diagnosis. Remarkably, despite these distinct clinical histories, primary and secondary GBMs are clinically indistinguishable after diagnosis as reflected by their equally poor prognosis.

Like all cancers, GBMs of both the primary and secondary type are caused by mutations or alterations in genes that control the normal growth of the cells from which they arise. These affected genes can be classified into two broad categories, oncogenes and tumor suppressor genes. These two genes types can best be described by comparison to the accelerator and brakes of a car, where oncogenes typically act as the gas or accelerator of tumor cell growth and survival, and tumor suppressor genes act as the brake on tumor cell growth and survival. The combination of mutations in both types of genes within the same cell has the greatest effect on promoting aggressive tumor growth, as acceleration of tumor growth is left undaunted by the brakes to slow it down. For example, the gene that encodes a molecule known as epidermal growth factor receptor (EGFR) is commonly expressed at very high levels (amplified) or mutated in GBM and thus promotes tumor growth acceleration through activation of a complex network of molecules, the chief of which is an enzyme, phosphatidylinositol-3 kinase (PI3-K). In contrast, PI3-K is kept under tight control by a tumor suppressor gene that codes for the phosphatase and tensin (PTEN) protein. If EGFR is mutated and becomes highly active (gas is applied), it can accelerate tumor cell growth. If the tumor cell also becomes mutated for PTEN (removing the brake), this combination of elevated EGFR activity and defective PTEN cooperate to dramatically enhance tumor aggressiveness.

It is this EGFR/PTEN accelerator/brake circuit that has received considerable attention in the field of GBM therapeutics. A range of potential therapies that target EGFR or a mutant, highly oncogenic form of EGFR specific to GBM, have been developed. Examples of therapies that are currently in development or in clinical trials for the treatment of this disease are inhibitors that block the activity of the receptor, in essence removing the accelerator (tyrosine kinase inhibitors; TKIs); antibodies that bind to the receptor; and vaccines that prompt an immunological response targeting GBM cells expressing the receptor. Data from experimental studies evaluating these therapies, in particular, TKIs, have been very promising; however, their efficacy in the clinic has so far been limited.

Some of the documented mechanisms that tumors use to cause resistance to EGFR TKIs include the acquisition of changes in the EGFR protein that blocks drug binding, compensatory elevated expression of other growth factor receptors that function similar to EGFR but are different enough proteins that the EGFRspecific drug doesn't bind to them, activation of molecules that pump the TKI out of the tumor cell, or the existence of minor populations of cancer cells that are inherently resistant to a broad spectrum of therapeutics. Several of these mechanisms have been firmly established for cancers such as non-small cell lung cancer and breast cancer. In GBM, several labs have shown that expression of mutated EGFR coupled with mutation of PTEN is a major mechanism that confers poor clinical responses to EGFR TKIs, in essence because the brake that blocks EGFR-mediated activation of PI3-K has been compromised. Unfortunately, tumors with activated EGFR and intact, non-mutated PTEN tend to show only modest or short-lived treatment responses, pointing to the existence of additional mechanisms that promote drug resistance.

This raises the question-- are there mechanisms that an EGFR-expressing tumor cell might use that mimic the mutation of PTEN? In other words, PTEN is retained in the tumor cell, but fails to exert its normal functions. In an effort toward understanding this phenomenon of PTEN retention coupled with EGFR TKI resistance, my laboratory has sought to determine if the PTEN brake has been compromised through a mutation-independent mechanism. By analyzing the PTEN protein in cell lines derived from GBM patient samples, we have in fact identified a unique mechanism whereby PTEN is modified by phosphorylation, an alteration found on many proteins that regulates diverse features, such as its activity, its localization in the cell, and its rate of degradation. In the case of PTEN, phosphorylation occurred on a specific amino acid of the protein, a tyrosine located at position 240 in the chain of 403 amino acids that make up the PTEN protein. Using an antibody that we developed as a tool to detect PTEN tyrosine 240 phosphorylation, we determined that in addition to loss or mutation of PTEN, its phosphorylation at tyrosine 240 is linked to very poor prognosis and EGFR TKI resistance in GBM patients. Further analysis of these patient samples revealed that an additional growth factor receptor known as fibroblast growth factor receptor (FGFR) was expressed in EGFR TKI resistant, PTEN-expressing tumor cells and subsequent experimentation showed this receptor to be directly responsible for causing the phosphorylation of PTEN and promoting EGFR TKI resistance. These findings show that this specific FGFR-prompted modification of PTEN causes this tumor suppressor to malfunction, similar to having grease on a brake pad.

Given the above findings, we predict that restoration of PTEN's normal function, which would be akin the cleaning of grease from the brake pads, should be possible in GBMs where PTEN is tyrosine phosphorylated by FGFR. This could be achieved by using inhibitors targeting FGFR, which would block phosphorylation of PTEN's tyrosine 240, and in conjunction with co-administration of an EGFR TKI, we should be able to overcome drug resistance in a GBM patient that fits this profile.

Targeted therapies are an important new class of agents for the treatment of GBM patients. Based on the previously defined PTEN deletion mechanism for promoting EGFR TKI resistance in GBM and our finding of the involvement of FGFR-mediated PTEN tyrosine 240 phosphorylation, it is clear that single targeted agents will not suffice as a curative approach for treating this disease. The studies in this proposal are designed to understand how FGFR is able to negate PTEN function through the identified phosphorylation event and with this knowledge, test for the restoration of EGFR TKI sensitivity by blocking FGFR activity or associated mechanisms that lead to PTEN tyrosine 240 phosphorylation. It is hoped that information gained here can be applied to patients in the long run for more effective treatment, and even cure, of this universally fatal brain cancer.