Cutaneous T-cell lymphoma (CTCL) is the most frequent primary lymphoma of the skin.
Patients diagnosed in early stages often experience an indolent disease course and
have a favorable prognosis. Yet, the disease follows an aggressive course in a substantial
fraction (15–20%) of patients and despite recent progress in novel therapies, advanced
disease remains a major challenge as relapses are common and cure is rare.
1
Recently, it was discovered,
2
and independently confirmed in a meta-analysis study,
3
that malignant T cells in the majority of patients display ectopic expression of the
B-lymphoid tyrosine kinase (Blk), a member of the Src kinase family. Importantly,
gene knockdown experiments showed that Blk promoted the proliferation of malignant
T cells from CTCL patients,
2
suggesting that Blk—in analogy with other Src family members—may function as an oncogene.
In support, Montero-Ruiz et al.
4
provided evidence that Blk is implicated in childhood acute lymphoblastic leukemia.
However, studies in mice suggested that murine Blk also has tumor-suppressive functions
depending on the specific cellular context.
5
To study the oncogenic potential of human Blk, we therefore transfected a cytokine
(IL-3)-dependent lymphoid cell line (Ba/F3) with plasmids expressing either wild-type
(wt) Blk or a constitutively active form of Blk lacking the kinase-inhibitory site
due to a tyrosine-to-phenylalanine substitution at amino-acid position 501 (Y501F).
Stable transfectants were established by selecting for the plasmid-encoded blasticidin
resistance gene, and before experimentation, transformed cells were maintained in
blasticidin- and IL-3-supplemented growth media. As shown in Figure 1a, the constitutively
active form of Blk (Y501F) was fully able to transform growth factor (IL-3)-dependent
Ba/F3 cells into IL-3-independent cells, whereas non-transfected and Blk-wt-transfected
Ba/F3 cells remained dependent on exogenous IL-3 to survive and proliferate. In accordance,
IL-3 deprivation induced massive apoptosis in non-transfected and Blk-wt-transfected
Ba/F3 cells, whereas no increase in apoptosis was observed in Blk(Y501F)-transfected
Ba/F3 cells following IL-3 withdrawal (Figure 1b). As expected, Blk(Y501F) was phosphorylated
on the activating tyrosine (Y388) and not on the inhibitory tyrosine phosphorylation
site (Y501), whereas Blk-wt was heavily phosphorylated on the kinase-inhibitory site
(Y501) (Figures 1c and d). The well-characterized Src family kinase inhibitor, Lck
inhibitor (LckI, Calbiochem, San Diego, CA, USA), selectively inhibited the proliferation
of Blk(Y501F)-transfected Ba/F3 cells, whereas an inhibitor of mitogen-activated protein
kinase p38 (SB203580, Selleck Chemicals, Houston, TX, USA) did not (Figure 1e). Likewise,
the dual-specificity inhibitor of Bcr-Abl and the Src family kinases, dasatinib (Sprycel,
Selleck Chemicals), inhibited Y388 phosphorylation and proliferation of the Blk(Y501F)-transfected
Ba/F3 cells (Figures 1f and g). Taken together, these results indicate that the active
form of human Blk is able and sufficient to transform cytokine-dependent lymphoid
cells into cytokine-independent cells. These findings support the previous observation
made by others
6
that murine Blk(Y495F) is lymphomagenic in mice. In keeping, enzymatic inhibition
by LckI and other Src family kinase inhibitors, as well as siRNA-mediated knockdown
of Blk, inhibits proliferation of Blk-positive malignant T cells including MyLa2059
and MyLa2000 (ref. 2 and data not shown). As dasatinib profoundly inhibited Blk(Y501F)-transformed
Ba/F3 cells and tyrosine phosphorylation of Blk in the MyLa2059 cells (Figure 2a),
we hypothesized that dasatinib—which is used for treatment of chronic myelogenous
leukemia and other malignancies
7
—has a potential for treatment of CTCL. To address this hypothesis, we initially studied
the effect of dasatinib on malignant proliferation in vitro. As shown in Figure 2b,
dasatinib inhibited the spontaneous proliferation of the malignant CTCL T-cell line
MyLa2059 in a concentration-dependent manner. Likewise, the Blk-positive CTCL cell
lines MyLa2000 and PB2B
2
were also inhibited by dasatinib, whereas the Blk-negative Sezary Syndrome cell line
(SeAx) was resistant (Supplementary Figure S1). As malignant T cells, including some
cell lines obtained from CTCL
2
and peripheral T-cell lymphoma patients,
8
often express Fyn (another Src kinase), we cannot exclude the possibility that the
effect of dasatinib was partially mediated through an inhibition of both Blk and Fyn.
However, Fyn is not tyrosine phosphorylated in the malignant MyLa cells suggesting
that Fyn may not be functionally active in these cells (data not shown). The observation
that dasatinib inhibited Blk-positive tumor cells prompted us to examine the effect
of dasatinib on tumor growth in a xenograft transplantation model of CTCL.
9, 10
In a preliminary experiment, mice were inoculated subcutaneously (s.c.) with MyLa2059
cells and treated orally with different concentrations of dasatinib (or vehicle as
a control) to evaluate the effect on tumor formation in vivo. The average time of
tumor onset was significantly (P<0.05) delayed from day 13 in the control group (N=5)
to day 20 in animals (N=6) treated with dasatinib (data not shown). Next, we addressed
whether dasatinib inhibited growth of already established tumors. Accordingly, eight
mice were inoculated s.c. with MyLa2059 cells and following detection of palpable
tumors (day 1) the mice were treated with either dasatinib (Sprycel) (40 mg/kg) or
vehicle as control. Tumor dimensions were measured in each group on days 3, 5, 8 and
10. As shown in Figure 2c, dasatinib significantly inhibited tumor growth. Likewise,
volume of the resected tumors harvested on day 10 was significantly lower in the dasatinib-treated
mice when compared with the control mice, confirming that dasatinib does inhibit CTCL
tumor growth in vivo (Figure 2d). As the malignant T cells do not express the Bcr-Abl
oncogene (data not shown), the present finding suggests that Blk functions as an oncogene
in the CTCL cells. As NF-κB is active in CTCL
2
and supports growth of dasatinib/imatinib-resistant cells,
11, 12
we tested dasatinib in combination with NF-κB inhibitors. Interestingly, dasatinib
and NF-κB inhibitors had an additive effect on malignant proliferation in vitro (data
not shown), which might explain why Blk(Y501F)-transformed Ba/F3 cells were more sensitive
to dasatinib than the malignant MyLa cells. In summary, (i) Blk is enzymatically active
in malignant CTCL cells and expressed in situ,
2
(ii) its constitutively active form confers cytokine independence (Figure 1) and (iii)
it promotes tumor growth in vivo as indicated by the effect of dasatinib on tumor
growth in mice (Figure 2). Taken together, these findings strongly suggest that Blk
is a potential therapeutic target in CTCL for dasatinib and other clinical-grade dual-specificity
Bcr-Abl and Src family kinase inhibitors. As dasatinib and other dual-specific inhibitors
are already used in treatment of other hematological malignancies with a high efficacy,
tolerability and compliance,
7
these drugs are attractive novel candidates for the treatment of CTCL expressing Blk.
In conclusion, our study provides novel evidence that human Blk—in its active form—is
an oncogene with the potential to support growth of lymphoid cells in vitro and to
promote tumor growth in vivo. Thus, Blk is a potential novel therapeutic target in
CTCL.