Introduction

Glioblastoma multiforme (GBM) is a form of brain cancer that forms in astrocytes. GBM is the most aggressive and lethal form of brain cancer with a standard life expectancy of 12 to 18 months post diagnosis¹ and five-year survival rate of 6.8%². With most cancers, current treatment options have increased the standard life expectancy and five-year survival rates. However, GBM has evaded most current treatment options with no increase in survival statistics in decades².

The etiology of GBM is still mostly a mystery. There are no current environmental factors or carcinogens that have been associated with the onset of GBM aside from high level radiation³. However, genetic mutations are observed in some forms of GBM. There are many possible genetic mutations that could account for tumorigenesis, but this review will focus on mutations in two specific genes, ROS1 and NTRK1. 

ROS1 is a proto-oncogene responsible for the coding of type I integral membrane receptor tyrosine kinase proteins⁴. Under normal conditions receptor tyrosine kinases (RTK) bind to ligands, driving cell growth and differentiation. Improper functioning of these receptors may result in uncontrolled and unregulated cell growth and differentiation, ultimately forming certain cancers such as GBM or non-small cell lung cancer¹².

NTRK1 is another RTK gene that works slightly different than that of ROS1. NTRK1 binds neurotrophin and phosphorylates the MAPK pathway which, among other functions, plays a role in cell proliferation and differentiation⁵. Disruptions in the functioning of both RTKs can be caused by genetic mutations that force the formation of ROS1 or NTRK1 fusion proteins resulting in permanent autophosphorylation and activation of cell proliferation^6,7.

The presence of ROS1 and NTRK1 fusions also provide potential GBM treatment options by means of tropomyosin receptor kinase (TRK) inhibitors. There are currently TRK inhibitors in clinical trials like entrecitinib and dovitinib⁸. A single patient case study from 2021 highlights the use of a specific TRK inhibitor, lorlatinib, as a treatment for RTK fusion induced GBM and provided interesting results in terms of recurrance⁹.

Understanding the genetic factors that lead to the onset of GBM generates a stronger grasp on its etiology. It also begs the question, what other protein fusions could be at play. Finding more genetic mutations like ROS1 and NTRK1 could be the missing component for breakthrough treatments and creating a greater overall impact on survival statistics.

ROS1 Genomic Microdeletions Form Fusion Proteins

The ROS1 gene has been a target in various types of cancer research for around 35 years. The first indication of ROS1 involvement in GBM etiology was in 1987 when a team from Cold Springs Harbor Laboratory found an ROS1 abnormality in the GBM U118MG cell line¹⁰. This cell line was used in further research to see ROS1’s role in GBM.

In 2018 a study was performed that used The Cancer Genome Atlas (TCGA) to look for ROS1 mutations in GBM datasets. ROS1 fusions were present in 0.55% of the analyzed GBM tissue⁶. Two specific fusions were analyzed, CEP85L-ROS1 and GOPC-ROS1, through sanger sequencing⁶. The sequence showed that intrachromosomal microdeletions were responsible for the fusions of exons and therefore fused proteins⁶. These two fusions took place at 6q22⁶ which differs from other types of ROS1 fusions that were observed at 6q21¹¹. Knowing that all ROS1 fusions are not homologous provides an element of specificity to potential treatment options. These ROS1 fusions result in continued autophosphorylation¹² which drives uncontrolled cell proliferation¹³.

The same 2018 study introduced GBM into a mouse model. This was done through xenograft of the U118MG fusion cell line. The xenograft was performed by introducing 1x10⁶ U118MG cells by way of lentivirus to 8-week-old male mice. The mice were confirmed to have GBM using bioluminescent imaging reaffirming the role of these ROS1 fusions in GBM⁶. But ROS1 fusions are not the only fusion proteins that can lead to GBM. Other rare fusions like NTRK fusions appear to play a role as well.

NTRK Fusions with Neurofascin are Rarely Observed

Unlike ROS1 fusions, NTRK fusions do not directly stimulate cell proliferation. NTRK fusions activate the MAPK pathway which has a downstream effect on cell proliferation⁵.

Specifically, fusions of NTRK1 and the neurofascin (NFASC) gene showed roughly a 50% increase of downstream cellular activity of nerve growth factor/tyrosine kinase A (NGF/TrkA) when analyzed using RNA sequencing of human tissue samples from TCGA⁷. This points towards an increase in overall cell proliferation in the presence of these fusions. Not only was there an increase in downstream activity, there was also roughly a ninefold increase in overall NTRK1 expression within the samples⁷. This would indicate that NFASC-NTRK1 fusions have a higher rate of transcription than the wild type genes, adding to the potential for higher rates of cell proliferation.

While discovering some etiology is an exciting prospect, these NTRK fusion tumors are very rare. Less than 1% of GBM samples (2 out of 162) in TCGA contain NTRK fusions⁷. Another data set of 248 GBM samples did not show any NTRK fusions further adding to their rarity¹⁴. However, just like with ROS1 fusions, understanding the specific etiology of the tumor provides greater insight for potential treatments.

TRK Inhibitors as Potential Treatment

Knowing the etiology of a tumor can provide avenues for treatment. Both NTRK and ROS1 genes code for RTK proteins. The fusions that cause GBM result in increased cell proliferation from upregulated protein activity. Therefore, an easy target would be to test the inhibition of these fusion proteins using small molecule TRK inhibitors. These inhibitors act by selectively inhibiting activity of the fusion proteins¹⁵. As each fusion protein binds different ligands, specific treatments based on fusion type could result in increased survival time.

In ROS1 fusions, the TRK inhibitor lorlatinib showed promising results in the previously mentioned U118MG xenograft mice models⁶. Lorlatinib was administered orally once a day for four weeks. The size of tumors decreased in all subjects and the mice showed no disease symptoms or weight loss for 36 days after treatment was stopped⁶.

However, there are some concerns with reoccurrence. Lorlatinib was given to a three-year-old child with an anaplastic lymphoma kinase (ALK) fusion induced GBM⁹. The initial results were promising as the tumor regressed. It was then fully removed via surgical resection. Another tumor showed up six months later and treatment started again. Once again, the tumor shrunk, and the patient has continued lorlatinib treatment as no significant side effects were observed⁹. While lorlatinib may not fully eradicate RTK fusion GBM it appears to be an option for slowing cell proliferation and shrinking tumor size.

There are many TRK inhibitor treatment options available for NTRK fusions. Larotrectinib is now approved by the FDA for treatment of solid tumors with NTRK fusions⁸. An 18-month-old presented with an NTRK fusion GBM. Three months post tumor resection a small tumor continued to grow at the resection site. She was then treated with oral larotrectinib. Eight weeks after treatment an MRI showed significant tumor regression¹⁶.

While both lorlatinib and larotrectinib appear to have promising results, both have shown recurring tumors after stopping the drug. Also, no long-term data are available. It is unknown whether TRK inhibitors can be used as a first line treatment option, in conjunction with chemotherapeutics and surgical resection, or should be a second line treatment option. It is still too soon to know if TRK inhibitors will increase survival statistics.

Conclusion

GBM is the most aggressive and fatal form of brain cancer and has many different etiologies. It is known that mutations cause ROS1 and NTRK fusions. However, there is still a large subset of GBM where the genetic component is unknown and needs to be examined. These two fusion types are only a small subset of all GBM tumors, leaving the door open for multiple other fusion types to be discovered. It is also unknown how these fusions occur. It is apparent that microdeletions can cause fusions, but what underlying factors are causing the microdeletions?

While ROS1 and NTRK fusions make a small subsection of GBM etiology, understanding these rare forms allows for a more precise treatment plan. TRK inhibitors are showing promise but lack the long-term data to prove their effectiveness. To improve survival rates, long term TRK inhibitor studies need to be performed in ROS1 and NTRK fusion GBM models. It is vital to know the recurrence rate of GBM tumors when using TRK inhibitors over various timelines. Understanding the long-term effects of TRK inhibitors will guide medical providers to more precise usage of this tool and more positive outcomes for patients.

As it currently appears, TRK inhibitors limit uncontrolled growth and do not fully remove the cancerous genes. Full removal could be done through gene therapeutics such as CRISPR by removing the fused gene sequence and replacing with the correct sequence. While CRISPR technology is still being refined and ethical implications are up for debate, this provides another point to continue research and potentially cure a subset of this aggressive, lethal disease.

[+] References

[+] Other Work By David Stull