“Suzanne George, MD is Assistant Professor of Medicine, Harvard Medical School, and Clinical Director, Center for Sarcoma and Bone Oncology, Dana-Farber Cancer Institute, Boston. In her capacity as medical oncologist, she is part of a multidisciplinary team that treats and provides consultation to sarcoma patients throughout the New England area, as well as elsewhere in United States and abroad. She is also extremely active in clinical research — developing and executing clinical trials focused on new avenues of therapy for GIST and other soft tissue sarcomas.”

GSI posed questions about the evolution and management of GIST resistance mutations to Suzanne George, MD, Assistant Professor of Medicine, Harvard Medical School, and Clinical Director, Center for Sarcoma and Bone Oncology, Dana-Farber Cancer Institute, Boston.

1. What role do mutations in KIT and PDGFRα proteins have in GIST?

KIT and PDGFRα belong to the family of proteins called Class III receptor tyrosine kinases, which act as cellular “on/off” switches, promoting cell growth and survival when in the “on” state. Under normal conditions this activity is tightly regulated, so that KIT and PDGFRα are only active when bound to specific signaling molecules (ligands), such as hormones or growth factors. However, the KIT or PDGFRa proteins found in GISTs are often defective, containing mutations that favor the “on” state and result in deregulated cell growth and tumor formation.

2. How do these mutations affect GIST treatments?

Standard kinase inhibitor drugs for GIST (imatinib, sunitinib, regorafenib) bind to KIT and PDGFRα proteins in a manner that stabilizes the “off” state, silencing the abnormal growth signals. However, research has shown that a few specific mutations can alter the structure of KIT or PDGFRα in ways that are incompatible with the binding of a given drug. When the drug’s ability to bind to its target protein is impaired, signaling proceeds unchecked and tumor growth occurs.
Such “resistance mutations” are uncommon in treatment-naïve primary GISTs — although one primary mutation, PDGFRα D842V, does notably confer resistance to standard kinase inhibitors. In the majority of cases, though, resistance mutations emerge only after prolonged drug exposure. Some resistance mutations can be addressed by switching to a different kinase inhibitor, but mutations in a critical region of the KIT and PDGFRα protein structure called the activation loop (A loop) have shown a high degree of resistance to all GIST treatments to date.

3. What is the activation loop (or A loop)? Why are mutations located in the A loop referred to as “Exon 17” for KIT and “Exon 18” for PDGFRa, and what is their clinical significance?

The DNA of a gene is very long, and specific locations along that stretch are indicated by a system of numbered “exons.” Exons are analogous to the mile markers along a highway that tell you where you are. They indicate which segment of the gene or its related part of the protein is being discussed. The individual structural components of a protein correlate to specific exon segments (Figure 1).

Figure 1: The molecular architectures of KIT protein and KIT gene. A gene is divided into functional segments called exons. The individual structural components of a protein correlate to specific exon segments. Exon 17 encodes the activation loop in the kinase domain of KIT. The activation loop is a structural feature that guides the “on/off” state of the kinase.

Instructions for the A loop – the flexible region in protein kinases that governs their “on/off” switch – are encoded via exon 17 of KIT and exon 18 of PDGFRa. When the A-loop is switched “on” (open conformation) the result is an active kinase. When the A loop is switched “off “ (closed conformation) the kinase is incapable of signaling and remains in an inactive state.

The structural status of the A loop is critical for the binding of kinase inhibitors such as imatinib and sunitinib, and mutations distorting it can cause drug resistance. Resistance mutations in KIT exon 17 have been identified at positions 816, 820, 822 and 823. The The PDGFRα exon 18, D842V, mutation structurally analogous to the D816V mutation in KIT.

4. Drugs that inhibit KIT and PDGFRα have been categorized as Type 1 or Type 2 kinase inhibitors based on their binding characteristics. How do mutations in KIT Exon 17 and PDGFRα D842 affect a Type 1 inhibitors vs Type 2 inhibitors?

Type 2 kinase inhibitors like imatinib fit neatly into “binding pockets” that are created when the A loop adopts a closed conformation (Figure 2). However, they are incapable of binding to KIT mutants with the A loop in an open conformation. GIST primary mutations such as those in KIT exon 11, while activating, do not limit the A loop to an open conformation. Therefore, Type 2 inhibitors can effectively bind to and inhibit oncogenic KIT activated by exon 11 mutations. In contrast, exon 17/18 mutations stabilize the A loop in an open conformation, which obstructs binding of Type 2 inhibitors (Figure 3A).
Type 1 inhibitors bind to and inhibit kinases more potently when the A loop is in the open conformation (Figure 3B). The investigational drug BLU-285 is an example of a selective Type 1 kinase inhibitor that binds potently to KIT and PDGFRα having exon 17/18 activating mutations.

Figure 2:

Figure 2 shows KIT bound to imatinib (yellow) with the A loop in a closed conformation (purple). When in this conformation, imatinib fits nicely into the binding pocket of KIT. Exon 17/18 mutations in KIT or PDGFRα stabilize the A-loop in an open conformation (green), which obstructs binding of imatinib and other type 2 inhibitors.