Gene Fusions Yield Increasingly Broad Spectrum of Targeted Therapies

We have come a long way since German biologist Theodore Boveri first proposed more than 100 years ago that tumor growth results from chromosomal abnormalities. The identification of chromosomal rearrangements that result in oncogenic gene fusions ushered in the era of molecularly targeted therapies in oncology.

Next-generation sequencing (NGS) technologies have expanded the number of known fusions from several hundred to nearly 10,000 across the spectrum of cancer types, according to recent updates. The highest frequency of gene fusions (90%) is found in hematologic malignancies, particularly lymphomas. Among solid tumors, the highest number of gene fusions have been found in bladder cancer, with thyroid cancer on the opposite end of the spectrum. Although some types of gene fusions are found in only a single tumor type, others are more prolific.

Thus far, only a small portion of known gene fusions have been tested with functional assays in an effort to understand if, and how, they drive cancer. Several dominant types of gene fusion have emerged. Newly identified and well-established gene fusions alike continue to provide promising therapeutic targets and broaden our understanding of cancer development (Table).

Rearranging the Genome

The genomic instability of cancer cells can lead to structural defects in chromosomes as they break apart and join back together in different configurations. This can take different forms: translocations involve whole segments of 1 chromosome transferred to another; inversions occur when a piece of chromosome flips 180 degrees before reinserting; insertions describe the reinsertion of a piece of chromosome back into the same chromosome at a different location; and deletions involve the loss of a portion of a chromosome.

As researchers have been able to examine the genes contained within the breakpoints of rearranged chromosomes, gene fusions emerged as one of the molecular consequences. These occur when 2 previously separate regulatory and/or protein-coding regions from 2 genes are put next to each other in a manner that means they can be “read” by the transcription machinery as a single gene.

Most Common Fusion Targets

Gene fusions can drive cancer in several different ways: through abnormal transcription of otherwise normal cellular genes, resulting in changes in the expression level of that gene; by generating fusion proteins with altered regulation, activity or structure; or via creation of shortened, inactivated tumor suppressor genes.BCR-ABL1

Among the first chromosomal alterations linked to cancer was the Philadelphia chromosome, a translocation between chromosomes 9 and 22 found in chronic myeloid leukemia (CML) and acute myeloid leukemia. Identification of the BCR-ABL1 fusion gene as the molecular driver behind the Philadelphia chromosome and the tyrosine kinase inhibitors (TKIs) that were developed to target the resulting fusion protein ushered in the era of molecularly targeted therapy in oncology (Figure 1).

This gene fusion results from the reciprocal exchange of genetic material between 2 chromosomes that causes the Abelson kinase (ABL1) gene, which encodes a receptor tyrosine kinase (RTK), to become fused to the breakpoint cluster region (BCR) gene. The ABL1 gene is then “read” as part of the BCR gene, creating a hybrid BCR-ABL protein. Because the BCR protein contains a dimerization domain, it permits activation of the ABL kinase without the need for ligand binding, resulting in a kinase that is always switched on. This general mechanism is thought to be shared by all oncogenic tyrosine kinase fusions identified to date.

Imatinib (Gleevec), a TKI that includes the BCR-ABL protein among its targets, was approved by the FDA for the treatment of CML in 2001. Because the BCR-ABL protein is present in more than 95% of CML cases, imatinib became known as the “magic bullet” that revolutionized the treatment of this cancer.

EML4-ALK

Drugs targeting the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion provide another prime example of rapid bench-to-bedside translation (Figure 2). The ALK gene encodes an RTK, and all ALK fusions identified to date drive cancer through constitutive activation of its kinase activity. Although first identified in hematologic malignancies, this fusion is best known for its role in a subset of patients with non—small cell lung cancer (NSCLC). The EML4-ALK fusion is found in only 3% to 7% of patients with NSCLC, but because of the prevalence of this cancer, that translates into thousands of cases in the United States each year. Within 5 years of the discovery of ALK fusions in NSCLC, the FDA approved ALK inhibitor crizotinib (Xalkori) for the treatment of ALK fusion-positive patients with NSCLC.

As with other TKIs, resistance rapidly became an issue that limited the effectiveness of crizotinib. Second- and third-generation ALK inhibitors have since been developed to overcome key molecular mechanisms of resistance. In 2014, ceritinib (Zykadia) was awarded regulatory approval for the treatment of ALK fusion-positive NSCLC, followed by alectinib (Alecensa) in 2015. Although both drugs are approved for the second-line treatment of patients who have progressed on crizotinib, they are also being compared with crizotinib in the frontline setting in late-stage clinical trials. Alectinib received a breakthrough therapy designation for this indication in late 2016 based on the findings of the phase III J-ALEX study in which the drug reduced the risk of disease progression or death by more than 60% compared with crizotinib.

On April 28, the FDA approved a third-generation inhibitor, brigatinib (Alunbrig), in the second-line setting for ALK-positive patients with NSCLC. In the phase II ALTA trial, the overall response rate (ORR) by independent review was 53% among patients (n = 110) who received the recommended dosing regimen of 90 mg daily for the first 7 days followed by 180 mg daily if tolerable, with a median response duration of 13.8 months (95% CI, 9.3-not estimable).

ROS1

ROS1, encoded by ROS proto-oncogene 1, is another RTK that frequently undergoes fusion events in a variety of tumor types. The FIG-ROS1 fusion was first identified in patients with glioblastoma but, like ALK, the discovery of these fusions (most commonly CD74-ROS1) in NSCLC fueled development of targeted therapy.

Chromosomal rearrangements involving the ROS1 gene occur in approximately 2% of patients with NSCLC. Since the kinase domains of ROS1 and ALK have similar structures, ALK inhibitors have been rapidly repurposed for use in ROS1 fusion-positive patients.

In March 2016, crizotinib’s approval was expanded to include patients with ROS1-positive NSCLC, and second- and third-generation inhibitors are also being tested in this setting. The results of a phase I/II study of lorlatinib were presented at the 2016 ASCO Annual Meeting. The ORR was 46% among 41 patients with ALK-positive tumors and 50% among 12 patients with ROS1-positive disease. Responses were seen in treatment-naïve and refractory patients, including those with crizotinib-resistance mutations, and in patients with brain metastases.

RET

Approximately 1% of patients with NSCLC display fusions in the RET proto-oncogene, which also results in activation of its RTK activity. Although individually ALK, ROS, and RET fusions are present in only small percentages of patients with NSCLC, they are often mutually exclusive with each other and with other known drivers, such as EGFR mutations; thus, they significantly expand the targetable patient pool.

In addition to NSCLC, RET fusions are found in patients with thyroid cancer—up to half of all patients with medullary thyroid cancer and 10% to 20% of those with sporadic papillary thyroid cancers—and in other tumor types. Although there are not yet any TKIs approved for use specifically in RET fusion-positive patients, various multitargeted drugs include RET among their targets and several of these are already approved for use in patients with medullary thyroid cancer. Trials of these and other RET-targeting drugs are ongoing, including the novel selective RET inhibitor BLU-667 in patients with RET-positive NSCLC and other advanced solid tumors.

FGFR

The fibroblast growth factor receptors (FGFRs) constitute a family of RTKs that, upon binding of their ligands, initiate signaling pathways that regulate important processes during embryonic development and in adults, including wound healing and angiogenesis.

A variety of FGFR aberrations have been identified as potential cancer drivers and FGFR inhibitors have been developed to target them. Thus far, interest in these drugs has focused on their potential antiangiogenic properties and they have been tested across a broad range of tumor types, either in unselected patient populations or in those with FGFR amplifications or mutations.

In the 1990s, the first FGFR gene fusions were described in patients with high-grade glioma (FGFR3-TACC3). Since then, FGFR fusions have been observed across a variety of other tumors. In solid tumors, FGFR fusions tend to have the FGFR gene in the 3’ position, while the reverse is true in hematologic malignancies. However, both types are oncogenic for similar reasons, either allowing for ligand-independent activation of FGFR kinase activity or recruitment of downstream signaling proteins. The results of preclinical studies suggest that patients with FGFR fusions may experience benefit from FGFR inhibitors, prompting early-stage clinical trials across tumor types. NTRK

The tropomycin receptor kinase (TRK) family of RTKs plays an essential role in the neuronal signaling pathways involved in the development and function of the nervous system. They are encoded by the NTRK1, 2, and 3 genes, and fusions involving them were among the first reported gene fusions in solid tumors.

Only recently, as the list of tumor types in which they have been observed has continued to grow, have drugs directed against these fusions been explored as potential targets for anticancer therapy. Several inhibitors that target the TRK receptors are in clinical development. NTRK fusions appear to be drivers of NSCLC in up to 3% of cases; thus, several of these drugs are designed to target TRK in addition to ALK and ROS1.

Entrectinib (RXDX-101) is currently being evaluated in a phase II basket trial across a range of tumor types with NTRK fusions (STARTRK-2; NCT02568267). Combined data from 2 phase I studies were presented at the 2016 American Association of Cancer Research Annual Meeting. Among 24 evaluable patients, the ORR was 79%, including responses in patients with primary brain tumors and brain metastases.

Transcription Factors Are a Tougher Foe

Larotrectinib (LOXO-101), a more selective inhibitor of TRK receptors, is also in development. In results from an ongoing phase I study, 7 of 41 patients with advanced solid tumors were subsequently found to have NTRK fusions. Responses were observed in 5 of 6 evaluable patients, for an ORR of 83%, while the drug showed no activity in patients without NTRK fusions. The FDA has granted larotrectinib a breakthrough therapy designation in patients with unresectable or metastatic NTRK fusion-positive solid tumors.Other prevalent types of gene fusion involve genes encoding transcription factors. This type of alteration dominates the genomic landscape in sarcomas and prostate cancers.

The discovery of gene fusions involving the E-twenty-six (ETS) transcription factor family in prostate cancer demonstrated for the first time that gene fusions could characterize a major subset of patients with solid tumors. A dozen or more ETS transcription factor fusion partners have now been described in patients with prostate cancer. Most commonly, the ETS transcription factor gene ERG is fused to the constitutively activated promoter of an androgen-regulated gene, TMPRSS2.

In patients with Ewing sarcoma, the ETS transcription factor genes are most often fused to genes encoding members of the TET family of RNA-binding proteins. In 85% to 90% of cases, the EWSR1 gene is fused to the FLI1 gene.

Given their prevalence in these cancer types, these gene fusions offer a rational drug target, but the problem is that transcription factors are not readily “druggable.” Efforts to date have focused on targeting proteins that are activated downstream of the transcription factors as a result of their oncogenic activity.

In both prostate cancer and Ewing sarcoma, studies demonstrated an association between ETS gene fusions and components of the DNA damage response pathways, including the poly (ADP-ribose) polymerase 1 (PARP-1) enzyme. Inhibitors of PARP are already available and clinical trials are ongoing in both cancer types. Studies in patients with ETS gene fusions, however, are limited to 1 ongoing study of veliparib in patients with prostate cancer that is evaluating the correlation between ETS fusions and response.

The fact that the TMPRSS2 gene contains an androgen-responsive promoter suggests that prostate cancers with this fusion might bene t preferentially from treatment with antiandrogen therapies. An ongoing study is attempting to address if the presence of ETS fusions can dictate improved response to enzalutamide (Xtandi).

In Ewing sarcoma, insulin-like growth factor 1 receptor (IGF1R) was identified as a downstream target of the EWSR1-FLI1 fusion, sparking significant interest in inhibitors of this kinase. Unfortunately, clinical trials demonstrated limited success, potentially due to the development of resistance. The activation of the mammalian target of rapamycin (mTOR) pathway was found to be a potential mechanism of resistance to IGF1R inhibition; dual IGF1R-mTOR inhibitors have also been tested. Clinical success was limited to a few exceptional responders, but researchers are currently trying to dissect the molecular mechanisms of these responses to find additional therapeutic clues.

The paucity of other targetable alterations in patients with Ewing sarcoma means that the EWSR1-FLI1 fusion is still a central focus of drug development. This fusion has been shown to be associated with dysregulation of the epigenetic machinery and inhibitors of the bromodomain and extra-terminal domain (BET) proteins, which are an important family of epigenetic “readers” that recognize specific tags on histone tails and help to induce an open chromatin structure that is conducive to gene expression. BET-targeting drugs have shown preclinical promise.

Finally, a first-in-class small molecule that binds to the EWSR1-FLI1 fusion has been developed. The FDA recently granted TK-216 an orphan drug designation and a phase I trial has been initiated.

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