Targeting TRK family proteins in cancer

Yekaterina B. Khotskaya, Vijaykumar R. Holla, Anna F. Farago, Kenna
R. Mills Shaw, Funda Meric-Bernstam, David S. Hong
PII: S0163-7258(17)30020-7
DOI: doi:10.1016/j.pharmthera.2017.02.006
Reference: JPT 7014

To appear in: Pharmacology and Therapeutics

Please cite this article as: Khotskaya, Y.B., Holla, V.R., Farago, A.F., Mills Shaw, K.R., Meric-Bernstam, F. & Hong, D.S., Targeting TRK family proteins in cancer, Pharmacol- ogy and Therapeutics (2017), doi:10.1016/j.pharmthera.2017.02.006

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P&T 23100
Targeting TRK family proteins in cancer

Yekaterina B. Khotskaya1, Vijaykumar R. Holla1, Anna F. Farago2, Kenna R. Mills Shaw1, Funda Meric-Bernstam1,3,4, David S. Hong3,*

1Sheikh Khalifa Bin Zayed Al Nahyan Institute for Personalized Cancer Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX; Department of Medicine, Massachusetts General Hospital, Boston, MA2; and Departments of Investigational Cancer Therapeutics3 and Surgical Oncology4, The University of Texas MD Anderson Cancer Center, Houston, TX.

*Corresponding author:
David S. Hong, MD
Department of Investigational Cancer Therapeutics The University of Texas MD Anderson Cancer Center 1515 Holcombe Boulevard, Unit# 455
Houston, TX 77030
Tel#: 713-563-5844
E-mail address: [email protected]

ACKNOWLEDGMENTS:
This work was supported in part by The Cancer Prevention and Research Institute of Texas (RP1100584), the Sheikh Khalifa Bin Zayed Al Nahyan Institute for Personalized Cancer Therapy, the MD Anderson Cancer Center Support Grant (P30 CA016672), and the NCATS Grant UL1 TR000371 (Center for Clinical and Translational Sciences).

Conflict of interest:
Dr. David S. Hong has research support from LOXO oncology and from Ignyta. All other author have no conflict of interest.

Keywords: NTRK1; NTRK2; NTRK3; cancer; gene fusions; central nervous system

Abstract

The tropomyosin receptor kinase (TRK) family includes TRKA, TRKB, and TRKC proteins, which are encoded by NTRK1, NTRK2 and NTRK3 genes, respectively. Binding of neurotrophins to TRK proteins induces receptor dimerization, phosphorylation, and activation of the downstream signaling cascades via PI3K, RAS/MAPK/ERK, and PLC-gamma. TRK pathway aberrations, including gene fusions, protein overexpression, and single nucleotide alterations, have been implicated in the pathogenesis of many cancer types, with NTRK gene fusions being the most well validated oncogenic events to date. Although the NTRK gene fusions are infrequent in most cancer types, certain rare tumor types are predominately driven by these events. Conversely, in more common histologies, such as lung and colorectal cancers, prevalence of the NTRK fusions is well below 5%. Selective inhibition of TRK signaling may therefore be beneficial among patients whose tumors vary in histologies, but share underlying oncogenic NTRK gene alterations. Currently, several TRK-targeting compounds are in clinical development. The ongoing Phase 2 trials with entrectinib and LOXO- 101, two of the leading TRK inhibitors, are designed as ‘basket trials’, inclusive of patients whose tumors harbor NTRK gene fusions, independent of histology. Additional Phase 1 studies of other TRK inhibitors, including MGCD516, PLX7486, DS-6051b, and TSR-011, are
underway. Interim data examining NTRK-rearranged tumors treated with entrectinib or LOXO-

101 demonstrate encouraging activity, with patients achieving rapid and durable responses. Consequently, both drugs have achieved orphan designation from regulatory agencies, and efforts are underway to further expedite their development.

Abbreviations

AKT, protein kinase B; ALK, anaplastic lymphoma kinase; ATP, adenosine triphosphate; BBB, blood-brain barrier; BDNF, brain-derived neurotrophic factor; CIPA, congenital insensitivity to pain with anhidrosis syndrome; cMET, hepatocyte growth factor receptor; CNS, central nervous system; DIPG, diffuse intrinsic pontine glioma; ERK, extracellular signal-regulated kinase; FISH, fluorescence in situ hybridization; FLT3, FMS-like tyrosine kinase 3; JAK1, Janus-associated kinase 1; JAK2, Janus-associated kinase 2; LRM, leucine-rich motif; MAPK, mitogen-activated protein kinase; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; NTRK1, neurotrophic tyrosine receptor kinase 1; NTRK2, neurotrophic tyrosine receptor kinase 2; NTRK3, neurotrophic tyrosine receptor kinase 3; PI3K, phosphatidylinositol 3-kinase; PLC-g, phospholipase C-gamma; RAS, rat sarcoma oncogene family; ROS1, ROS proto-oncogene 1; STAT, signal transducer and activator of transcription; TRKA, tropomyosin receptor kinase A; TRKB, tropomyosin receptor kinase B; TRKC, tropomyosin receptor kinase C; TPM3, tropomyosin 3.

Table of Contents:

Overview of NTRK Gene Family

In 1982, high-molecular weight DNA from two colon carcinoma patients showed transformation capabilities when transfected into the NIH-3T3 cells (Pulciani, Santos et al. 1982). Subsequently, cloning and sequencing of the same DNA samples in 1986 identified a fusion of the non-muscle tropomyosin to at that time unknown protein kinase, later identified as TRKA (Martin-Zanca, Hughes et al. 1986). This was the first published report that identified a member of the tropomyosin receptor kinase (TRK) family, which includes TRKA, TRKB, and TRKC proteins, encoded by NTRK1, NTRK2, and NTRK3 genes, respectively. Structurally, TRK proteins contain extracellular and intracellular regions separated by a single transmembrane domain. All three TRK proteins share a high degree of structural homology, including the three leucine-rich motifs (LRM) that are flanked by two cysteine clusters and two immunoglobulin (Ig)-like C2 type domains located within the extracellular region of the protein.
Although initially identified in cancer, under normal physiologic conditions TRK proteins serve as the high-affinity receptors for the nerve growth factors (NGFs), a type of neurotrophins (Kaplan, Martin-Zanca et al. 1991). During organogenesis, TRK proteins are expressed in the neuronal tissues where they play a critical role in the development of central and peripheral nervous systems (Nakagawara 2001, Arevalo and Wu 2006). Binding of neurotrophins [(brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4)] to their cognate receptors elicits the formation of TRK homo-dimers and

phosphorylation of their tyrosine residues (Figure 1). In turn, this triggers TRK kinase activation that induces cell proliferation, differentiation, apoptosis, and survival of neurons and other cells types where this signaling may be activated via the downstream PI3K, RAS/MAPK/ERK and phospholipase C-gamma (PLC-g) signaling transduction pathways (Kaplan, Martin-Zanca et al. 1991, Klein, Jing et al. 1991, Loeb, Stephens et al. 1994, Kawamura, Kawamura et al. 2007, Kawamura, Kawamura et al. 2009, Kawamura, Kawamura et al. 2011).

NTRK Gene Alterations

The TRK pathway has been implicated in the pathogenesis of many cancer types.

Chromosomal rearrangements resulting in oncogenic gene fusions, protein overexpression, and single nucleotide variants are the most commonly described alterations associated with the NTRK family.
NTRK Fusions

Currently, NTRK gene fusions are the best-characterized aberrations among all NTRK alterations occurring in cancers. These gene fusions are known to be oncogenic, as they promote tumorigenesis through the constitutive activation of downstream cell growth and proliferative pathways (Rubin and Segal 2003). In 1986, TPM3 was identified as the first NTRK1 fusion partner, resulting from an intrachromosomal rearrangement at 1q22-23 (inv(1) (q21-22q22-23) in a colorectal cancer patient (Martin-Zanca, Hughes et al. 1986). In general, NTRK gene fusions promote oncogenesis when the 3’ portion of the NTRK gene, containing the catalytic tyrosine kinase domain, achieves an in-frame fusion to the 5’ portion of a partner gene, driving gene expression and facilitating protein dimerization. This is analogous to the oncogenic fusions observed with the ALK and ROS1 genes, among others (Shaw, Hsu et al.

2013). Although the wild-type TRK proteins are usually activated upon the neurotrophin ligand binding to the receptor, structural studies of other oncogenic receptor tyrosine kinase fusions show that a fusion with a 5’ partner can lead to ligand-independent kinase activation and oncogenic signaling (Fujimoto, Shiota et al. 1996, Wang, Li et al. 2015). To date, multiple fusion partners have been identified in NTRK1, 2, and 3-rearranged tumors (Table 1).
NTRK Copy Number Variations and Gene Overexpression

NTRK copy number aberrations have been observed in a variety of malignancies, and may have clinical implications. NTRK1 copy number gain has been reported in 13% of liver cancers, 12% of invasive breast cancers, 10% of lung adenocarcinomas, and 9% of uterine cancers, according to the cBioPortal (Gao, Aksoy et al. 2013). Moreover, a recent paper identifies NTRK1 amplification in 4 of 10 breast cancer brain metastasis, suggesting that TRK signaling may be important for mediating metastatic dissemination to the central nervous system (Bollig-Fischer, Michelhaugh et al. 2015). Furthermore, changes in TRK mRNA and protein levels have been evaluated and may possess prognostic and/or diagnostic values in several human malignancies. One study described TRKA mRNA overexpression, possibly indicative of gene amplification, in some adrenal, pancreatic, ovarian, esophageal, urinary bladder, and endometrial cancers, as well as in 100% of pheochromocytomas (Narayanan, Yepuru et al. 2013). Additionally, in patients with Wilms’ tumors, high TRKB mRNA levels are associated with poor relapse-free survival (Eggert, Grotzer et al. 2001). Finally, NTRK deletions are also reported in the literature. For example, pancreatic and prostate cancers are characterized by NTRK1 deletions in approximately 2% of patient samples documented in the cBioPortal (Gao, Aksoy et al. 2013), although their functional significance is somewhat unclear.

NTRK Single Nucleotide Variants

Many single nucleotide alterations in the activating and catalytic loops of the NTRK genes have been detected, but very few of these are known to be oncogenic. For example, approximately 10% (10 of 95) of pulmonary neuroendocrine tumors are associated with mutations in NTRK2 and NTRK3 genes, although the function of these mutations is yet to be investigated (Marchetti, Felicioni et al. 2008). Conversely, several inactivating mutations in different regions of the NTRK genes are associated with various non-malignant syndromes and are thought to be largely germline, rather than somatic. Patients with congenital insensitivity to pain with anhidrosis syndrome (CIPA) carry several inactivating NTRK1 mutations located in the leucine-rich motif 1 (L93P), first Ig-like domain (L213P), and the kinase domain (G516R, F521L, G571R, R643W, R648C, D664Y, D668Y, R686H, G708S, and
R774P), all resulting in a protein that cannot be phosphorylated, thus reducing cell growth and survival signals in neurons (Mardy, Miura et al. 1999, Greco, Villa et al. 2000, Miura, Mardy et al. 2000, Indo, Mardy et al. 2001, Mardy, Miura et al. 2001, Gao, Guo et al. 2013). Several other NTRK1 mutations identified in CIPA patients, including R85S (LRM region), H598Y and G607V (kinase domain), show no change in the functional activity when compared to the wild- type TRKA protein, suggesting that they are likely inconsequential (Mardy, Miura et al. 2001). Functional characterization of NTRK2 mutations identified in patients with severe early-onset of obesity revealed that the Y722C mutation results in an impaired MAPK, PLC-g, and AKT signaling, and is inactivating in nature (Gray, Yeo et al. 2007). Lastly, mutations in NTRK3 are associated with susceptibility to congenital heart defects and at least one of the mutations, T93M, shows decreased TRKC autophosphorylation in response to ligand (Werner, Paluru et al. 2014).

Other NTRK Alterations

Less common somatic alterations detected in the NTRK genes include an in-frame deletion of NTRK1, which promotes constitutive activation of the downstream pathways in acute myeloid leukemia (Tomasson, Xiang et al. 2008), and an alternative splicing of NTRK1 that produces a transcript lacking exons 6-7, which is constitutively active and tumor promoting in neuroblastoma (Tacconelli, Farina et al. 2004). In addition, the extracellular portion of TRK proteins plays a critical regulatory role, as seen in studies that show that deletions, rearrangements, or splice variants resulting in a loss of the entire or a portion of the TRK extracellular region lead to constitutive receptor activation and promote tumor growth (Arevalo, Conde et al. 2000, Vaishnavi, Le et al. 2015).

TRK as a Therapeutic Target

To date, the most promising clinical activity of TRK inhibitors has been observed among patients whose tumors harbor NTRK gene fusions. Therefore, we will focus our discussion of TRK inhibitors on these targets. Detection of NTRK gene fusions in clinical samples can occur via targeted next-generation sequencing-based approaches, using either an RNA or DNA template, or fluorescence in situ hybridization (FISH) (Vaishnavi, Capelletti et al. 2013, Farago, Le et al. 2015, Barr 2016). However, a more recent integration of multiplexed genomic sequencing into routine patient care has allowed for the detection of multiple potential gene fusions from a single nucleic acid preparation, enabling a broader, ‘shotgun’ approach to screening (Zheng, Liebers et al. 2014, Farago, Le et al. 2015).
Although oncogenic driver aberrations in NTRK genes, such as NTRK fusions, are relatively infrequent events in cancer, certain rare tumor types are predominately driven by these rearrangements (Hong 2016). For example, in pediatric patients with diffuse intrinsic

pontine glioma (DIPG) and pediatric non-brainstem glioblastoma, NTRK gene fusions account for up to 47% of driver events (Wu, Diaz et al. 2014). Although the overall number of the affected patients in this specific population is relatively small, TRK-directed therapy may provide a tremendous advancement to the currently available and not highly effective therapeutic options that include steroids and whole-brain radiation (Warren 2012). Conversely, in more common histologies, such as lung, colorectal, and head and neck cancers, prevalence of the NTRK gene fusions is well below 5% (Hong 2016). Thus, screening for these alterations broadly across a variety of cancer subtypes is critical for identification of patients who may benefit from TRK-directed therapy. The low frequency of these alterations can also lead to challenges in clinical trial accrual if studies are limited to specific histologies.
There are several TRK inhibitor compounds in clinical development. The furthest along to date are entrectinib and LOXO-101, which are being studied in ongoing Phase 2 ‘basket trials’ that recruit patients with specific genomic or molecular aberrations independent of histology. Additionally, several multi-kinase inhibitors, including MGCD516, PLX7486, DS- 6051b, and TSR-011, are being studied in Phase 1 trials (direct gene target are shown in Table 3A, and clinical trial information is summarized in Table 4). Other clinically available drugs with mild to modest activity against at least one member of the TRK family, including crizotinib (NTRK1, (Vaishnavi, Capelletti et al. 2013)), BMS-754807 (NTRK1, NTRK2, (Schroeder, An et al. 2009)), and midostaurin (ETV6-NTRK3, (Chi, Ly et al. 2012)), are summarized in Table 3B. Currently the best clinically characterized compounds are entrectinib and LOXO-101, which will be discussed in greater detail below.
TRK Inhibitors in Solid Tumor Malignancies

Entrectinib and LOXO-101 are orally bioavailable tyrosine kinase inhibitors that both inhibit TRK catalytic activity with low nano-molar potency (Table 3A). LOXO-101 preferentially

blocks the ATP binding site of TRKA, TRKB, and TRKC proteins. In assays with cells expressing NTRK-family rearrangements, LOXO-101 is characterized by IC50 values in a low nano-molar range (Doebele, Davis et al. 2015). Similarly, entrectinib also inhibits TRKA, TRKB, and TRKC, with IC50 values ranging from 0.1 to 1.7 nM. In addition, it also potently inhibits the kinase activities of ROS1 and ALK (Rolfo, Ruiz et al. 2015).
Both entrectinib and LOXO-101 have demonstrated potent clinical activity among patients with metastatic or unresectable solid tumors that harbor an NTRK gene fusion (summarized in Table 2). Based on the most recent results of the Phase 1 study of entrectinib, presented in April 2016 at the Annual Meeting of the American Association for Cancer Research, 3 of 3 patients with rearrangements in NTRK1 or NTRK3 achieved confirmed partial response, as of a data cutoff of March 7, 2016 (Drilon 2016). Data specifically highlighted a patient with LMNA-NTRK1-rearranged colorectal cancer who achieved a 30% tumor burden reduction and remained on study for 4 months, and a patient with ETV6-NTRK3-rearranged mammary analogue secretory carcinoma who achieved a 89% tumor reduction, with overall on-study time of 10 months (Drilon 2016). The most robust response, however, was observed in a patient with SQSTM1-NTRK1-rearranged non-small cell lung cancer, who achieved a tumor burden reduction of 80% and had remained on the study for 12 months with ongoing response (Drilon 2016). Importantly, in all three patients, documented time to response was within 1 month of drug initiation.
Similar to entrectinib, early clinical data indicate that treatment with LOXO-101 results in rapid and sustained clinical responses among patients with solid tumors harboring NTRK gene fusions. As of a March 25, 2016 data cutoff, 7 patients with NTRK-rearranged tumors (three with soft tissue sarcoma, LMNA-NTRK1; and one each with mammary analogue secretory carcinoma of the salivary gland, ETV6-NTRK3; thyroid, ETV6-NTRK3; and non-small cell lung carcinoma, TPR-NTRK1) had responded to treatment, and none had discontinued treatment

due to toxicity or drug resistance (Hong 2016). Among 6 NTRK-rearranged cases whose clinical data were available for analysis (one was enrolled less than 8 weeks before the data cutoff), 5 achieved confirmed partial response, and one patient had confirmed stable disease (overall response rate 83%). Importantly, these responses were durable, with treatment duration ranging from 7 cycles (3 patients) to 14 cycles (1 patient) (Hong 2016). Specifically, a patient with LMNA-NTRK1-rearranged undifferentiated soft tissue sarcoma widely metastatic to the lungs exhibited a rapid 80% tumor burden reduction, accompanied by the resolution of dyspnea and hypoxemia, and continued on therapy. In another case, a patient with mammary analogue secretory carcinoma of the salivary gland achieved 50% tumor reduction for 10 cycles on treatment (Hong 2016).
Emergence of Acquired Resistance to TRK Inhibitors

As is the case with other tyrosine kinase inhibitors directed against oncogenic gene fusions in solid tumors, TRK inhibitors are likely not curative. Indeed, emergence of acquired resistance is beginning to be described, and this will likely pose both challenges and opportunities for development of further TRK-directed therapies in the future. In spite of rapid, durable responses to entrectinib among patients with several NTRK-rearranged tumor types, two cases of acquired drug resistance mediated by the emergence of secondary mutations in NTRK genes have now been described. Specifically, the patient with LMNA-NTRK1- rearranged colorectal cancer developed two novel, treatment-associated G595R and G667C mutations in the kinase domain of NTRK1 (Russo, Misale et al. 2016). Interestingly, preclinical studies showed that the emergence of G595R and G667C mutations occurred in a dose- dependent manner: G595R was induced by the high-dose, and G667C – by the low-dose entrectinib treatment. Thus, this entrectinib dose-dependence may need to be considered in the design of the future clinical dosing schemas for entrectinib administration, although at this

time, there is not enough evidence to predict what type of clinical drug dosing may induce resistance. Additionally, it should be considered that while the resistance associated with G667C could be overcome with a subsequent LOXO-101 treatment, cells expressing NTRK1 G595R-mutant protein were resistant to all tested TRK inhibitors, indicative of a highly resistant phenotype and necessitating a complete change in the therapeutic approach (Russo, Misale et al. 2016). Likewise, the ETV6-NTRK3-rearranged mammary analogue secretory carcinoma patient also developed an acquired resistance to entrectinib, albeit after a significantly longer exposure to entrectinib (Drilon, Li et al. 2016). Pre- and post-treatment biopsies taken from this patient revealed an emergence of NTRK3 G623R mutation in the kinase ATP binding pocket.
Functionally, mutation of the highly evolutionally conserved glycine 623 codon to arginine resulted in the reduced affinity of ETV6-NTRK3-expressing cells to entrectinib by over 250-fold (Drilon, Li et al. 2016). Moreover, homology alignment showed that NTRK3 G623 residue was a paralog of NTRK1 G595, ALK 1202, and ROS1 G2032 codons, all of which have previously been implicated as drivers of acquired drug resistance (Awad, Katayama et al. 2013, Friboulet, Li et al. 2014, Russo, Misale et al. 2016). Thus, based on its homology with NTRK1 G595R, it is likely that NTRK3 G623R could also be resistant to other clinically available TRK inhibitors. Taken together, although entrectinib achieves remarkable clinical efficacy, its current use is associated with the emergence of drug resistance that in some instances cannot be overcome by subsequent treatment with other clinically available TRK inhibitors.
Acquired resistance to LOXO-101 in the clinic has not yet been reported, though is anticipated based on the experience with other therapies targeting oncogenic gene fusions. Therefore, a second-generation TRK inhibitor, LOXO-195, is slated to enter Phase 1 testing in 2017. This highly selective pan-TRK inhibitor targets TRKA, TRKB, and TRKC with IC50 values in the nano-molar range. Experimental assays utilizing NTRK1 G595R-mutant cells also exhibit high compound activity in this setting (Hong 2016).

Role of TRK Inhibitors in the Central Nervous System

The role of TRKA, TRKB, and TRKC in normal physiology lends itself to the study of the TRK inhibitor activity in the central nervous system (CNS). Specifically, germline mutations in NTRK1, which normally functions in the development and appropriate differentiation of the sensory neurons, are associated with the development of CIPA syndrome that can predispose patients to mental retardation (Indo 2012). Similarly, TRKB knockout mice are characterized by the loss of ~32% neurons, including a significant reduction in the number of sensory neurons (Perez-Pinera, Garcia-Suarez et al. 2008). Another model of TRKB inactivation revealed that NTRK2 expression was required for the formation of synaptic connections and loss of its normal expression pattern resulted in an anxiety-like behavior in the affected mice (Bergami, Rimondini et al. 2008). Similarly, rare inactivating NTRK2 mutations detected in humans are associated with phenotypic changes that include memory impairment and developmental delay (Yeo, Connie Hung et al. 2004). Moreover, TRKB levels are also reduced at the RNA and protein levels in patients with depression (Tripp, Oh et al. 2012), as are NTRK2 transcription level and TRKB phosphorylation in the brains of suicide victims (Pandey, Ren et al. 2008).
Subsequent studies show that mRNA levels of TRKA and TRKC are also reduced in the hippocampal regions of the suicide victims’ brains, and that the phosphorylation of all three TRK family members was significantly reduced, indicative of these proteins’ crucial role in depression and suicide (Dwivedi, Rizavi et al. 2009). Importantly, in a very recent study utilizing an animal model of depression-like behavior, a pan-TRK inhibitor K252a blocked an antidepressant-stimulated TRKB phosphorylation and hampered the improvement of behavioral symptoms (Fukuda, Takatori et al. 2016). Thus, TRK family inhibitors that possess the ability to cross the blood-brain barrier (BBB) must be carefully tested to ensure lack of adverse neuropsychiatric effects, especially as these inhibitors undergo clinical testing in the
pediatric population whose nervous system would not complete its development until well into

the adolescence, a population that has one of the highest rates of depression and suicide (Tau and Peterson 2010, Devenish, Berk et al. 2016).
One of the earlier generation multi-tyrosine kinase inhibitors that included members of the TRK family, AZD1480, was ultimately withdrawn from further clinical development due to the severe neurotoxicity in a high number of patients, which included dizziness, aphasia, ataxia, dysarthria, memory loss, hallucinations, anxiety, and behavioral changes (Plimack, Lorusso et al. 2013, Verstovsek, Hoffman et al. 2015). AZD1480 was originally developed as a selective inhibitor of Janus-associated kinases 1 and 2 (JAK1, JAK2), but in preclinical enzymatic testing it exhibited equal potency towards TRKA, TRKB, and TRKC (Plimack, Lorusso et al. 2013). In preclinical animal testing, certain concentrations of AZD1480 also resulted in ataxia, mirroring results of the Phase 1 study (Verstovsek, Hoffman et al. 2015).
Clinical use of other JAK1/2 inhibitors had also been associated with adverse neurological effects, hence it is plausible that the adverse side effects observed with AZD1480 may be related to the modulation of the JAK/STAT signaling pathway (Pardanani, Gotlib et al. 2013, Pardanani, Laborde et al. 2013, Verstovsek, Tam et al. 2014). Another potential explanation may lie in this compound’s relatively high blood brain barrier (BBB) penetration and its effect on the TRK-related signaling in the brain, which would correlate with the effects of TRK dysregulation observed in animals and humans described above.
Some of the other clinically tested inhibitors that show affinity towards TRK proteins, such as crizotinib, lestaurtinib, midostaurin, and PHA-848125AC (direct targets are listed in Table 3B) have shown varying degrees of neurological side effects. This interesting phenomenon could be due to the fact that the first three of these compounds have limited affinity towards the TRK family: crizotinib predominately targets ALK, ROS1, and cMET, and lestaurtinib and midostaurin are potent inhibitors of FLT3 (Mathias, Natarajan et al. 2015).
Moreover, in preclinical testing these drugs have shown limited BBB penetration, likely also

contributing to their lack of associated neuropsychological adverse effects (Tang, Nguyen et al. 2014, Mathias, Natarajan et al. 2015). On the other hand, PHA-848125AC, a dual cyclin- dependent kinase (CDK) and TRKA inhibitor, is characterized by high BBB penetration and has exhibited neurotoxicity in all tested animal models (Weiss, Hidalgo et al. 2012). Moreover, in a Phase 1 study, treatment-related tremors were observed in approximately 50% patients (all treatment doses combined), and 3 patients experienced Grade 3, and 1 patient – Grade 4 ataxia that were resolved upon treatment discontinuation (Weiss, Hidalgo et al. 2012).
The newer generation of TRK inhibitors currently in clinical development (listed in Table 3A and Table 4) have exhibited limited neurotoxicity to date, likely due to their enhanced target specificity and more selective preclinical testing. Specifically, preliminary results of a Phase 1/2a study of TSR-011, a dual, potent inhibitor of ALK and TRKA, TRKB, and TRKC have recently been reported. Although the reported patient population consisted entirely of patients with ALK rearrangements (data from NTRK-rearranged patients are yet to be published), the most significant neurological side effects reported in the study were fatigue and headaches (Arkenau, Sachdev et al. 2015). In a Phase 1 study of entrectinib, most patients only experienced Grade 1-2 neurological side effects: fatigue/asthenia (47%), dizziness (21%), and parasthesia (21%) (Drilon (2016). Two Grade 3 cognitive disturbances were reported, and these resolved upon treatment discontinuation (Drilon 2016). Importantly, however, entrectinib shows a promising clinical activity in both primary and metastatic tumors occurring in the central nervous system (data summarized in Table 2). In a patient with SQSTM1-NTRK1- rearranged non-small cell lung carcinoma with previously untreated CNS metastases, treatment with entrectinib resulted in complete response of all brain metastases, and the patient remains on study progression-free at over 12 months without Grade 3-4 adverse effects (Farago, Le et al. 2015, Drilon 2016). Another patient with BCAN-NTRK1-rearranged, unresectable low-grade astrocytoma achieved stable disease by RECIST 1.1 measurements

and a 45% reduction in tumor burden by volumetric assessment, as well as improvement in clinical symptoms for 7 months following treatment with entrectinib (Drilon 2016). Finally, a pediatric patient with recurrent infantile fibrosarcoma metastatic to the brain with leptomeningeal involvement and characterized by ETV6-NTRK3 rearrangement treated with entrectinib showed radiographic tumor response, as well as an overall improvement in symptoms, including increased alertness and improved appetite; no adverse neurological side effects were reported for this patient (Drilon 2016).
LOXO-101 may also have clinical activity in the CNS, as demonstrated by a case study response in a patient with a non-small cell lung carcinoma characterized by the TPR-NTRK1 rearrangement and metastatic to the brain (Table 2). The patient achieved a radiographic response in his brain lesion, an overall stable disease, and has remained on the study progression-free for over 7 cycles with no reported neuropsychological side effects related to treatment (Hong 2016). In the recently reported interim results of the Phase 1 study, adverse neuropsychological effects associated with LOXO-101 exposure were fatigue (33%), dizziness (23%), anxiety (12%), and delirium (5%), with 5% patients experiencing Grade 3-4 fatigue and
delirium (Hong 2016).

Taken together, the newer generation of TRK inhibitors is well tolerated with few neuropsychiatric side effects. This is likely due to the more stringent preclinical testing that aims to identify compounds that have minimally sufficient BBB penetration and low neurotoxicity in animal models for further clinical testing. Moreover, these inhibitors, especially entrectinib and LOXO-101, are characterized by their ability to penetrate the BBB and achieve durable responses in patients with tumors in the central nervous system.

Conclusions

Clinical data obtained with entrectinib and LOXO-101 are encouraging (Drilon 2016, Hong 2016). Building upon this early success, entrectinib received an orphan drug designation from the U.S. Food and Drug Administration for the treatment of TRKA-, TRKB-, TRKC-, ROS1-, and ALK-positive non-small cell lung cancer in 2015. Likewise, LOXO-101 recently received a first-ever breakthrough indication based on the molecular indication alone (June 13, 2016). Collectively, TRK inhibitors provide an exciting treatment option for patients with malignancies harboring oncogenic NTRK gene fusions.

Figure Legends:

Figure 1. TRK signaling pathways. Binding of neurotrophins to TRK proteins induces receptor dimerization, phosphorylation, and activation of the downstream signaling cascades via PI3K, RAS/MAPK/ERK, and PLC-gamma.

Table 1. NTRK gene family fusion variants known or predicted to activate TRK kinase domain.

Table 2. NTRK gene family-relevant therapeutic implications.

Table 3A. TRK protein-targeting drugs currently in clinical trials selecting for patients with

NTRK aberrations.

Table 3B. Clinically available TRK protein-targeting drugs that are currently not being investigated in NTRK-selected clinical trials.

Table 4. Open clinical trials recruiting patients with NTRK alterations.

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Figure 1

Table 1. NTRK gene family fusion variants that are known or predicted to activate TRK kinase domain

NTRK Fusion Partners Location of
fusion partners Tumor Type References

NTRK1 (1q21- q22) ARHGEF2 1q21-q22 Glioblastoma (Zheng et al. 2014)
CD74* 5q32 Non-small cell lung cancer (Vaishnavi et al. 2013)*
CHTOP 1q21.3 Glioblastoma (Zheng et al. 2014)

LMNA*
1q22 Spitzoid tumors (Wiesner et al. 2014)
Colorectal cancer (Sartore-Bianchi et al. 2016)
Soft tissue sarcoma (Doebele et al. 2015)
MPRIP* 17p11.2 Non-small cell lung cancer (Vaishnavi et al. 2013)
NFASC* 1q32.1 Glioblastoma multiforme (Frattini et al. 2013; Kim et al. 2014)
SQSTM1* 5q35 Non-small cell lung cancer (Farago et al. 2015)

TFG* 3q12.2
Papillary thyroid carcinoma (Greco et al. 1997; Hernandez et al. 2002; Edel et al. 2004)
TP53 17p13.1 Spitzoid tumors (Wiesner et al. 2014)

TPM3*
1q21.2
Colorectal cancer (Martin-Zanca et al. 1986; Ardini et al. 2014)
Papillary thyroid carcinoma (Beimfohr et al. 1999)

TPR* 1q25 Papillary thyroid carcinoma (Greco et al. 1992; Greco et al. 1997)
Colorectal cancer (Creancier et al. 2015)

NTRK2
(9q22.1) AFAP1 4p16 Low-grade glioma (Stransky et al. 2014)
NACC2 9q34.3 Pilocytic astrocytoma (Jones et al. 2013)
PAN3 13q12.2 Head and neck squamous cell carcinoma (Stransky et al. 2014)
QKI 6q26 Pilocytic astrocytoma (Jones et al. 2013)
TRIM24 7q32-q34 Non-small cell lung cancer (Stransky et al. 2014)

NTRK3 (15q25)

ETV6

12p13 Congenital fibrosarcoma (Knezevich et al. 1998b)
Congenital mesoblastic nephroma (Knezevich et al. 1998a; Rubin et al.
1998)
Secretory breast carcinoma (Tognon et al. 2002)
Acute myeloid leukemia (Eguchi et al. 1999)
Mammary analogue secretory carcinoma (Skalova et al. 2016)

Table 2. NTRK gene family-relevant therapeutic implications

Drug Name Alteration Type
Alteration
Tumor Type Sensitivity Vs.
Resistance
References

LOXO-101

Fusion LMNA- NTRK1 Soft tissue sarcoma
Sensitive
(Doebele, et al., 2015)
TPR- NTRK1 Non-small cell lung cancer Sensitive (Hong, 2016)

ETV6- NTRK3 Gastrointestinal stromal tumor, mammary analogue secretory carcinoma, papillary thyroid cancer, infantile
fibrosarcoma

Sensitive

(Hong, 2016; Nagasubramanian, et al., 2016)

Entrectinib Fusion SQSTM1- NTRK1 Non-small cell lung cancer Sensitive (Farago, et al., 2015)

Fusion

BCAN- NTRK1

Astrocytoma

Sensitive (A. Drilon, De Braud, F. G., Siena, S.,
Ou, S-H., Patel, M., Ahn, M-J., Lee, J.,
Bauer, T. M., Farago, A. F., Liu, S. V.,
Reddinger, N., Patel, R., Luo, D., Maneval, E. C., Multani, P. S., Doebele,
R. C., Shaw., A. T., 2016)
Fusion LMNA- NTRK1 Colorectal cancer Sensitive (Sartore-Bianchi, et al., 2016)

Secondary mutation LMNA- NTRK1 G595R;
G667C
Colorectal cancer
Resistant
(Russo, et al., 2016)

Fusion

ETV6- NTRK3 Mammary analogue secretory carcinoma, infantile fibrosarcoma

Sensitive (A. Drilon, De Braud, F. G., Siena, S.,
Ou, S-H., Patel, M., Ahn, M-J., Lee, J.,
Bauer, T. M., Farago, A. F., Liu, S. V.,
Reddinger, N., Patel, R., Luo, D., Maneval, E. C., Multani, P. S., Doebele,
R. C., Shaw., A. T., 2016; A. Drilon, et al., 2016)

Secondary mutation ETV6- NTRK3 G623R Mammary analogue secretory carcinoma
Resistant
(A. Drilon, et al., 2016)

Table 3A. TRK protein-targeting drugs currently in clinical trials selecting for patients with
NTRK aberrations

Drug Name Direct Gene Targets Development Phase Company
LOXO-101 NTRK1, NTRK2, NTRK3 Phase 2 Loxo Oncology
Entrectinib NTRK1, NTRK2, NTRK3, ALK, ROS1 Phase 2 Ignyta
Cabozantinib* NTRK2, KDR, MET, RET, KIT, FLT1, FLT3, FLT4, AXL Phase 2 Exelixis

Merestinib NTRK1, NTRK2, NTRK3, MET, AXL, ROS1, MKNK1, MKNK2, FLT3, TEK,
DDR1, DDR2
Phase 2
Eli Lilly and Company
TSR-011 NTRK1, NTRK2, NTRK3, ALK Phase 1/2 Tesaro
DS-6051b NTRK1, NTRK2, NTRK3, ROS1 Phase 1 Daiichi-Sankyo
PLX7486 NTRK1, NTRK2, NTRK3, CSF1R Phase 1 Plexxicon
MGCD516 NTRK1, NTRK2, NTRK3, DDR2, MET, KIT, KDR, PDGFR, Phase 1 Mirati Therapeutics
*Cabozantinib is FDA approved for other indications

Table 3B. Clinically available TRK protein-targeting drugs that are currently not being investigated in NTRK-selected clinical trials

Drug Name Direct Gene Targets Company References
Crizotinib* NTRK1, ALK, MET, ROS1 Pfizer (Vaishnavi et al., 2013)
Regorafenib* NTRK1, BRAF, KDR, KIT, PDGFRA, PDGFRB, RAF1, RET, FGFR1, FGFR2 Bayer (Regorafenib FDA label)
Dovitinib NTRK1, FGFR1, FGFR, FLT1, FLT3, FLT4, KDR, KIT, RET Novartis Pharmaceuticals (Sarker et al., 2008)
Lestaurtinib NTRK1, NTRK2, NTRK3, JAK2, FLT3 Teva Pharmaceutical (Festuccia et al., 2007a; Festuccia et al., 2007b)
BMS-754807 NTRK1, NTRK2, IGF1R, MET Bristol-Myers Squibb (Carboni et al., 2009)
Danusertib NTRK1, ABL1, AURKA, AURKB, FGFR1, RET Nerviano Medical Sciences (Zhang et al., 2014)
ENMD-2076 NTRK1, AURKA, AURKB, FLT1, FLT3,
FLT4, KDR, FGFR, RET CASI
Pharmeceuticals (Fletcher et al., 2011)
Midostaurin ETV6-NTRK3, FLT1, FLT3, PDGFRA, PDGFRB, KDR, KIT Novartis Pharmaceuticals (Chi et al., 2012)
PHA-848125 AC NTRK1, CDK1, CDK2, CDK4, CDK5,
CDK7 Nerviano
Medical Sciences (Weiss et al., 2012)
BMS-777607 NTRK1, NTRK2, MET, FLT3, KDR Aslan Pharmaceuticals (Schroeder et al., 2009)
Altiratinib NTRK1, NTRK2, NTRK3, MET, KDR Deciphera Pharmaceuticals (Smith et al., 2015)
AZD7451 NTRK1, NTRK2, NTRK3 AstraZeneca (Tatematsu et al., 2014)
MK5108 NTRK1, NTRK2, AURKA, AURKB, AURKC Merck (Shimomura et al., 2010)
PF-03814735 NTRK1, AURKA, AURKB, FLT1, MET, FGFR1, PTK2 Pfizer (Jani et al., 2010)
SNS-314 NTRK1, NTRK2, AURKA, AURKB, FLT4, CSF1R, DDR2, RAF1 Sunesis Pharmaceuticals (Arbitrario et al., 2010)
*Crizotinib and regorafenib are FDA approved for other indications

Table 4. Open clinical trials recruiting patients with NTRK alterations

Drugs Phase Title NCTID
LOXO-101 Phase 2 A Phase II Basket Study of the Oral TRK Inhibitor LOXO-101 in Subjects With NTRK Fusion-Positive Tumors NCT02576431

Entrectinib
Phase 2 An Open-Label, Multicenter, Global Phase 2 Basket Study of Entrectinib for the Treatment of Patients With Locally Advanced or Metastatic Solid Tumors That Harbor NTRK1/2/3, ROS1, or ALK Gene Rearrangements
NCT02568267

Cabozantinib Phase 2 A Phase II Study of Cabozantinib in Patients With RET Fusion-Positive Advanced Non-Small Cell Lung Cancer and Those With Other Genotypes: ROS1 or NTRK
Fusions or Increased MET or AXL Activity NCT01639508

Merestinib
Phase 2
Merestinib In Non-Small Cell Lung Cancer And Solid Tumors
NCT02920996

Entrectinib
Phase 1 A Phase 1/2a, Multicenter, Open-Label Study of Oral Entrectinib (RXDX-101) in Adult Patients With Locally Advanced or Metastatic Cancer Confirmed to be Positive for NTRK1, NTRK2, NTRK3, ROS1, or ALK Molecular Alterations
NCT02097810
LOXO-101 Phase 1 A Phase 1 Study of the Oral TRK Inhibitor LOXO-101 in Adult Patients With Solid Tumors NCT02122913

LOXO-101
Phase 1 A Phase 1 Study of the Oral TRK Inhibitor LOXO-101 in Pediatric Patients With Advanced Solid or Primary Central Nervous System Tumors
NCT02637687

Entrectinib
Phase 1 A Phase 1/1b, Open-Label, Dose-Escalation and Expansion Study of Entrectinib (RXDX 101) in Children and Adolescents With Recurrent or Refractory Solid Tumors and Primary CNS Tumors
NCT02650401
MGCD516 Phase 1 A Phase 1/1b Study of MGCD516 in Patients With Advanced Solid Tumor Malignancies NCT02219711

DS6051b
Phase 1 A Phase 1, Two-Part, Multi-Center, Non Randomized, Open-Label, Multiple Dose First-In-Human Study Of DS-6051b, An Oral ROS1 And NTRK Inhibitor, In Subjects With Advanced Solid Tumors
NCT02279433
DS-6051b Phase 1 Phase 1 Study of DS-6051b in Japanese Subjects With Advanced Solid Malignant Tumors Harboring Either a ROS1 or NTRK Fusion Gene NCT02675491