Aberrant activation of various RTKs has long been associated with tumorigenesis. Point mutations in kinase domains of RTKs such as EGFR, HER2, MET, KIT, and FLT3 (among others) have been implicated as driver mutations in various cancers such as lung, breast, renal, liver, intestinal, and leukemia (reviewed in 
). Such mutations tend to result in constitutive activation of the kinase domain, which ultimately contributes to escape from normal cellular growth controls. The gene for LTK, an RTK highly similar to ALK, is located within a chromosomal region implicated as a major breakpoint cluster domain in mouse models of radiation-induced AML 
. Further evidence for the involvement of LTK in malignancies emerged when the gene was found to be overexpressed in a subset of AML patients 
and overexpression of LTK was found to confer an increased risk of metastasis in NSCLCs 
. Despite this knowledge, uncovering the specifics of LTK function has been hampered by the fact that the ligand(s) for this receptor is not known. Studies using chimeras constructed from the extracellular portion of the EGF receptor with the transmembrane and cytoplasmic domains of LTK have provided evidence that activation of the LTK kinase domain leads to signaling through the Ras pathway via Grb2 and the adaptor protein Shc 
. Along with cell growth, LTK appears to be involved in anti-apoptotic signaling 
. Thus, disruption of normal LTK function may carry important consequences for neoplastic cell growth. For these reasons, we undertook the current study to investigate likely ways in which LTK could become constitutively activated and to observe the implications of such changes.
We utilized the high degree of conservation of kinase domain residues between LTK and ALK to inform our choice of candidate residues most likely to confer transforming properties when mutated. Two ALK residues in particular—F1174 and R1275—result in constitutive kinase domain activation when mutated in neuroblastomas 
. As with many kinase domain mutations implicated in cancer, the F1174 and R1275 mutations in ALK leads to phosphorylation of downstream targets and result in heightened cell proliferation, invasion, and survival 
. We report here for the first time the consequences of the expression of LTK proteins containing mutations at the analogous sites to these ALK residues. Our analyses revealed that, in many ways, LTK-F568L and LTK-R669Q behave similarly to the F1174L and R1275Q mutants of ALK. Overall, the F568L mutation was a stronger activator of LTK signaling than the R669Q mutation (, , , and ). While R669Q mutant cells showed evidence of being able to escape normal growth controls (losing contact inhibition and exhibiting anchorage independent growth), this activity was considerably weaker than that of LTK-F568L ( and ). Additionally, while the F568L mutant of LTK was able to transform hematopoietic cells to IL-3 independence, LTK-R669Q was not ( and ). Such findings are consistent with research of the corresponding ALK mutations, wherein ALK-F1174L is considered more highly transforming than the R1275Q mutation 
The F568L mutation of LTK results in constitutive tyrosine phosphorylation of the receptor and expression of this LTK mutant leads to phosphorylation of several key signaling proteins that appear to act downstream of LTK (, , , and ). LTK has three phosphotyrosine sites that have previously been reported to be key in mitogenic and survival signaling: Y485, Y753, and Y862 
. Tyrosine 753 of LTK is located within a kinase domain YXXM motif and appears to be involved in survival signaling via PI3K activation 
. Tyrosine 485 of LTK is part of a NPXY motif located within the juxtamembrane domain which is highly conserved among the insulin receptor family (and which corresponds to Y1096 of ALK) 
. Once phosphorylated, both Y485 and Y862 have been reported to associate with downstream signaling molecules, with Y862 being the major site of association with Shc resulting in the recruitment of Grb2/Sos and Ras activation 
. We found evidence of this LTK/Shc relationship, as numerous cell types expressing LTK-F568L revealed a marked increase in the phosphorylation of Shc tyrosines 239, 240, and 317 (known to be GRB2-binding sites) 
, compared to cells expressing wildtype LTK (, , , and ). We also found evidence that activated LTK leads to phosphorylation of various proteins within the JAK/STAT pathway, including JAK1, JAK2, STAT3, and STAT5 (, , , and ), and that survival of hematopoietic cells transformed to cytokine independence by LTK-F568L expression requires JAK signaling. When hematopoietic cells transformed by LTK-F568L were treated with a pan JAK inhibitor, we found a decrease in or complete loss of the phosphorylated form of JAK1 and JAK2 as well as their downstream targets STAT3 and STAT5, as would be expected (). Tyrosine phosphorylation of LTK remained unchanged during JAK inhibitor treatment. However, we observed a decrease in phosphorylated Shc and a complete disappearance of phosphorylated ERK in these cells. These data suggest, but do not prove, that activated JAK signaling contributes to Shc tyrosine phosphorylation and ERK activation downstream of activated LTK.
STAT3 activation and AKT phosphorylation have been reported following ALK-F1174L expression 
. Consistent with this, we also found evidence of STAT3 activation following the transformation of two hematopoietic cell lines by LTK-F568L as well as upon expression of this LTK mutant in epithelial cells (, , and ). When we examined mutant LTK cells for AKT activation, we found that in 32D cells only LTK-F568L expression increased AKT phosphorylation (). In BAF3 cells the expression of LTK-F568L resulted in a slight increase in phosphorylated AKT (compared to vector control), while expression of LTK-R669Q exhibited a more marked increase in phosphorylated AKT in these cells (). The opposite was true in epithelial cells, where LTK-F568L activated AKT to a greater extent than LTK-R669Q did (). However, 293T cells failed to show any changes in AKT phosphorylation with expression of either mutation (). Expression of ALK-R1275Q has been shown to lead to ERK1/2 activation 
, while results are conflicting as to whether ALK-F1174L does 
or does not 
result in similar activation of ERK 1/2. In our experiments, we observed that LTK-F568L is as good and in some cell types a stronger activator of ERK than LTK-R669Q. Such findings suggest, not surprisingly, that cell type may play a role in determining which downstream signaling pathways become activated when a LTK mutation confers gain of function signaling activity.
In addition to holding important implications for hematopoietic cells, we found that mutant LTK confers important changes in cells of other types. In epithelial cells, both mutations were able to confer the ability to escape normal growth controls, including exhibiting anchorage independent growth (). Additionally, our findings reveal that the F568L mutation of LTK is sufficient to induce differentiation of PC12 cells as measured by neuronal outgrowth (). This provides additional evidence that LTK-F568L is a constitutively activated receptor tyrosine kinase. These observations are consistent with previous work utilizing a CSF1R/LTK chimera in PC12 cells, which suggests that LTK activation can signal through pathways resulting in neuronal differentiation 
. Importantly, expression of LTK-R669Q also induced differentiation of PC12 cells, albeit to a significantly less extent than LTK-F568L (). Nonetheless, this indicates LTK-R669Q is capable of inducing differentiation signals in PC12 cells, suggesting this mutant LTK does exhibit a level of increased signaling. In support of this, we observed that BAF3 cells expressing LTK-R669Q show an increase in phosphorylation of certain signaling proteins such as STAT5 and AKT, compared to wildtype LTK (). Taken together, our data suggest that while LTK-R669Q does not readily exhibit potent transforming and cell signaling-inducing activity, expression of this LTK mutant does suggest it is a weakly activating mutation.
It remains to be determined whether or not activating LTK mutations are present in human cancer. Our work suggests that certain LTK mutations may have the ability to contribute to neoplastic cell growth, as has been demonstrated for ALK, whose kinase domain is nearly 80% identical to the kinase domain of LTK (). Mutations of the corresponding residues of ALK have proved important in understanding the pathology of neuroblastomas that carry these genetic changes 
. Moreover, the F1174 mutation of ALK occurs in a region of the kinase domain that is often mutated in EGFR and HER2 
. The R1275Q mutation of ALK is correspondingly adjacent to the most common lung cancer-associated mutation (L858R) in EGFR 
. The similarity in the location of these ALK mutations, and thus the corresponding LTK mutations investigated in our study, to other activating tyrosine kinase domain mutations in cancer underscores the important consequences of mutation of this region of tyrosine kinases. Mutationally activated ALK is found in NSCLC and, interestingly, examination of LTK expression in patients with NSCLC revealed that patients with LTK overexpression had a three-fold higher risk of metastasis 
. While our work shows that mutationally activated LTK can induce transformation of various cell types including epithelial cells, overexpression of wildtype LTK does not. However, overexpression of wildtype LTK does lead to activation of some downstream signaling proteins, such as ERK, in certain cell types (, , and ). Thus, it is possible that overexpression of LTK may contribute in some manner to enhanced signaling of distinct intracellular pathways, which if not significant on its own, may sensitize cells to additional genomic insults. Also, constitutively activated ALK is known to carry prognostic value in cancers such as lung cancer 
and ALCL 
, thus providing further evidence that mutations in LTK that induce constitutive signaling may provide clinically important information.
Importantly, we found that cells transformed by LTK-F568L are susceptible to the ALK inhibitor PF-2341066 (Crizotinib) ( and ). While little is known about the normal role of LTK, it is worth noting that treatment of cells with PF-2341066 to target mutationally-activated ALK may produce off-target effects through inhibition of LTK. Our work suggests that the similarities between ALK and LTK may be exploited for treatment options if LTK is found to have a role in driving certain cohorts of cancer patients. Having a potential therapeutic agent available makes the identification of potential activating LTK mutations in cancer all the more intriguing. While the size of the patient population with cancers containing activating LTK mutations, if any, is not yet known, advances in genomic sequencing, which will provide data for the personalization of therapeutic treatments for patients, makes the identification of such a population significant. This is especially true if these cancers can be effectively targeted by drugs already being used in patients. While further research is needed to elucidate the role of LTK in human cancer, the potential for improved prognosis is significant if LTK-driven neoplasms can be identified and met with targeted treatments. Future whole genome sequencing approaches will rely heavily on studies such as ours presented here to determine the significance of identified mutations.
In conclusion, we demonstrate that expression of LTK mutations homologous to known activating mutations of ALK leads to elevated activation and cell signaling compared to wildtype LTK. LTK-F568L is a stronger transforming mutation than LTK-R669Q in multiple cell types. Signaling and transforming activity of mutated LTK proteins are evident in cells of hematopoietic and epithelial origin, as well as in cells used to model neuronal differentiation, suggesting aberrant activation of LTK may play a role in neoplastic disease of multiple cell types.