In this paper, we have extended our previous findings demonstrating ectopic expression of MerTK in pediatric T-cell ALL.21
We detected MerTK expression in 5 out of 12 (41.7%) pediatric patient samples at the time of diagnosis, and in 6 out of 11 (54.5%) T-ALL cell lines analyzed. These findings are consistent with the data previously reported (MerTK expression detected in 50–55.8% of banked pediatric T-ALL patient samples),21
and also demonstrate the relative ease with which MerTK expression analysis can be incorporated into current flow cytometry diagnostic protocols.
To better understand the mechanism by which MerTK activation provides an advantage in T-cell leukemia cells, we screened a human phospho-kinase array for changes in phosphorylation status of proteins in response to Gas6-mediated activation of MerTK. Of the 38 different phospho-proteins present on the array, six proteins exhibited a change in phosphorylation status, including the ERK1/2 and AKT kinases and the STAT5 protein. Activation of the STAT pathway has been observed in AML and ALL, and is known to occur through a variety of mechanisms, including expression of Jak kinase fusion proteins (for example, TEL-JAK2).32
Although there are a few reports of STAT activation downstream of the TAM (Tyro3/Axl/Mer) receptors,33
MerTK-dependent STAT phosphorylation has only been observed in COS cells overexpressing a constitutively active chimeric MerTK receptor.34
In this report, we show that Stat5 is phosphorylated in response to Gas6 treatment and that MerTK inhibition reduces this response. Our data are the first to demonstrate a ligand-dependent role for endogenous MerTK protein in STAT signaling. In addition, STAT signaling as a consequence of MerTK activation has not been previously reported in leukemia. The mechanism of MerTK-dependent Stat5 phosphorylation will require further study, as we were not able to detect changes in Jak kinase activation (data not shown). It is possible that Stat5 phosphorylation could be mediated by another kinase, such as Src or MAP kinases,35
which have also been shown to be activated in response to MerTK stimulation.
Both the ERK1/2 and AKT kinases are well known modulators of antiapoptotic signals in cancer cells and have been shown to be downstream of MerTK stimulation. Both pathways can be activated by a variety of inputs and mechanisms. For example, in Jurkat cells AKT is constitutively active due to a mutation in PTEN
, a phosphatase that regulates AKT activity.36
Thus, AKT activity in Jurkat cells was not significantly affected by MerTK stimulation or inhibition (data not shown). ERK1/2 activation, however, was responsive to Gas6 treatment in the Jurkat and HSB2 cells, and was blunted in the shRNA derived Mer knockdown cell lines.
The results of our signaling analysis suggests that targeted inhibition of MerTK would reduce prosurvival signaling in cells and result in a more effective outcome in response to cytotoxic treatment. Accordingly, both Jurkat and HSB2 MerTK knockdown cells were more sensitive to treatment with chemotherapeutic agents currently used for the treatment of leukemia.31
The increased sensitivity was mediated by a higher rate of apoptosis, which was detected by increased levels of caspase 3 and PARP cleavage. Interestingly, MerTK inhibition also reduced the clonogenic potential of the Jurkat cell line, both in an in vitro
assay and in a mouse model of leukemia. Importantly, we were able to rescue the increased chemosensitivity and reduced clonogenic potential in the MerTK knockdown cell lines by exogenous expression of MerTK, indicating that the results presented here are unlikely to be due to off-target effects of the shRNA construct.
In conclusion, in this report we have confirmed that MerTK is ectopically expressed in ~50% of patients with T-cell ALL, and we demonstrated that MerTK expression analysis can be incorporated into current flow cytometry diagnostic protocols. We have shown that MerTK activation leads to upregulation of prosurvival signaling pathways in T-cell lines, and identified novel downstream signaling via Stat5. Furthermore, we have shown that inhibition of MerTK increases the sensitivity of leukemia cells to treatment with cytotoxic agents, decreases their colony-forming potential, and MerTK inhibition also decreased the leukemogenic potential of T-cell lines in a mouse model. We believe these results offer compelling evidence that MerTK is a viable target for the development of targeted inhibitors that can be used in combination with cytotoxic therapies for T-cell leukemia, and that these inhibitors will increase the benefit of current treatment protocols while allowing for dose reduction and a decrease in the severity of the side effects that are now observed.