Immunomodulatory Effects of Tyrosine Kinase Inhibitors

INTERNATIONAL TRENDS IN IMMUNITY VOL.1 NO.3 JULY 2013 ISSN

ISSN 2326-3121 (Print) ISSN 2326-313X (Online) http://www.researchpub.org/journal/iti/iti.html

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1

Abstract— Tyrosine kinase inhibitors (TKIs) have dramatically

improved the outcome of chronic myeloid leukemia (CML). Even

though TKI therapy does not necessarily eliminate all leukemic

cells, a small proportion of CML patients is able to discontinue the

treatment successfully and be considered cured. Besides inhibiting

the actual target BCR-ABL1 kinase in leukemic cells, TKIs inhibit

several off-target kinases in normal healthy cells, such as

lymphocytes. To date, little is known about the long-term in vivo

effects of TKI treatment on the function of the immune system.

However, constantly growing evidence suggests that some TKIs

have a dual-mode of action; the direct cytotoxic effects induced by

oncokinase inhibition are accompanied by the activation of

immune system2, which is potentially relevant for the long-term

control of CML.

In this review, we have summarized the reported

immunomodulatory effects of the three TKIs, which currently are

accepted for first-line use in CML (imatinib, dasatinib and

nilotinib). Understanding the mechanisms of TKI-induced

functional cure and the role of immune system in this process will

hopefully not only benefit CML patients, but also patients with

other malignant diseases in the future.

Index Terms— Chronic myeloid leukemia, dasatinib, imatinib,

immunomodulation, nilotinib, tyrosine kinase inhibitors.

I. INTRODUCTION

he treatment of CML has significantly improved during the

past 10-20 years after the tyrosine kinase inhibitors (TKIs)

entered the clinics.[1] Even though the TKI therapy has

Received April 25., 2013 and accepted after minor revision May 15, 2013.

This work was supported in part by the Finnish special governmental subsidy

for health sciences, research and training, and grants from the Finnish Cancer

Societies, the Emil Aaltonen Foundation, the Academy of Finland, the Biomedicum Helsinki Foundation, the Finnish Medical Foundation, the Blood

Disease Foundation, the Finnish Association of Hematology, the Sigrid

Jusélius Foundation and the KA Johansson Foundation. A, Kreutzman was with Hematology Research Unit Helsinki, Helsinki

University Central Hospital, Finland. She is now with the Department of

Immunology, Princes University Hospital, Madrid, Spain (e-mail: [email protected]).

K. Porkka is with the Hematology Research Unit Helsinki, Department of

Medicine, Helsinki University Central Hospital, P.B. 700, 00029 HUCH, Finland. (e-mail: [email protected]).

S. Mustjoki is with the Hematology Research Unit Helsinki, Department of

Medicine, Helsinki University Central Hospital, P.B. 700, 00029 HUCH, Finland. (e-mail: [email protected]).

2 Figure

changed the course of the disease, these drugs do not eradicate

all tumor cells as it has been shown in vitro that leukemic stem

cells are resistant to TKI therapy.[2-4] This means that the

majority of patients will relapse if the treatment is stopped,[5-7]

but recent results suggest that in some patients therapy

discontinuation is feasible although residual leukemic cells

exist.[8-10] It would be important to understand the

mechanisms of cure and ways to achieve it in a larger

proportion of patients in order to avoid life-long treatment with

expensive TKI drugs.

One curative approach could be the modulation of the

immune system with the single agents or the combination

regimes including TKIs in order to better target the malignant

cells. This idea is supported by several arguments. First, the

chronic nature of the disease and the slow progression rate in

most cases is a favorable setting for immunotherapy.

Furthermore, in most patients TKI therapy induces a state of

minimal residual disease, which is often needed for successful

immunotherapy.[11] Second, the malignant CML cells carry a

well-defined and specific tumor antigen and the peptides

derived from the fusion zone of the p210bcr-abl protein are able to

produce an immune response.[12-20] In accordance with this,

leukemia antigen-specific T-cells such as anti-PR1 (the

HLA-A2-restricted 9-mer peptide from proteinase 3), and

anti-WT1 (Wilms tumor 1 protein) T-cells are shown to be

present in CML patients, although they may be anergic.[21, 22]

However, it is believed that the immune system is not

permanently compromised, as CML patients can successfully

be treated with immunomodulatory agents such as IFN-.[21,

23-28] It is also thought that the high efficiency of allogeneic

stem cell transplantation is another evidence of the immune

system controlling CML.[29, 30] Furthermore, donor

lymphocyte infusions in the case of relapse have been reported

to induce complete remissions.[31-34] In addition, as already

mentioned above, a small proportion of CML patients are able

to discontinue the TKI treatment successfully [8] although the

disease is clearly measurable with the sensitive BCR-ABL1

RQ-PCR technique.[9] This proves that the immune system

plays a role in the disease control of CML and can eliminate

malignant cells

II. LYMPHOCYTE ANERGY IN CML

In the majority of cancer patients, the immune system is

dysfunctional and unable to produce an efficient immune

response against the malignant cells.[35] Indeed, immune cell

Immunomodulatory Effects of Tyrosine Kinase

Inhibitors

A. Kreutzman, K. Porkka, and S. Mustjoki

T



18

anergy has been described in several types of cancers and it is

thought to be one of the main hinderances in successful

immunotherapy.[36, 37] Similarly in CML, the observed

leukemia-specific T-cells have shown to be functionally

exhausted [38] or anergic.[39] One contributing factor is the

activation of programmed death receptor 1 (PD-1)

signaling.[38] PD-1 is a member of the immunoglobulin

superfamily, which expression can be induced on T- and

B-cells and activated monocytes.[40] PD-1 mediates inhibitory

signals through the binding of its ligand PD-L1 and PD-L2. In

chronic virus infections, the PD-1 signaling has been identified

as an important mechanism for antigen-specific T-cell

dysfunction.[41, 42] Similarly, many tumor cells highly

express PD-L1 and in this manner suppress tumor

immunity.[43, 44] Mumbrecht et al. have demonstrated that

also in CML, the CD8+ T-cells derived from patients express

higher levels of PD-1 than healthy CD8+ T-cells, and in the

mouse model the PD-L1 blockade on tumor cells leads to

prolonged survival.[38] Similarly recent paper by

Christiansson et al. described that cytotoxic CD8+ T-cells in

CML patients express high levels of PD-1. Furthermore, the

PD-L1 expression on myeloid tumor cells was higher in

patients than in healthy controls, and interestingly, patients

with high Sokal risk score had increased levels of PD-L1

expressing CD34+ stem cells.[45] In addition to the

PD-1/PD-L1 pathway, also other anergy mechanisms (such as

regulatory T-cells, myeloid derived suppressor cells) may

operate in CML patients.[45]

III. TYROSINE KINASE INHIBITORS

CML is caused by an oncogenic translocation forming the

characteristic Philadelphia chromosome (Ph) in the

hematopoietic stem cells. The resulting fusion gene produces

BCR-ABL1 protein with constant tyrosine kinase activity,

which in turn leads to uncontrolled cell proliferation and

reduced apoptosis.[46] After the discovery of BCR-ABL1, the

development of targeted therapies has emerged rapidly and the

first trials with a selective inhibition against the BCR-ABL1

tyrosine kinase began the revolution in the treatment of

CML.[47-50]

The first-generation TKI imatinib quickly replaced the

previous standard therapies for CML as it became soon evident

that imatinib treatment resulted in superior treatment

responses.[47-49, 51] However, long-term follow-up has

shown that a proportion of imatinib-treated patients do not

respond well to the treatment and eventually progress to

advanced phases of CML.[52] In addition, some patients are

unable to continue imatinib treatment because of

intolerance.[53] Since the emerging cases of resistance and

intolerance, several pharmaceutical companies have developed

second-generation TKIs such as dasatinib, nilotinib and

bosutinib. In addition to inhibiting BCR-ABL1 more

effectively, they are more flexible in binding to different

BCR-ABL1 conformations [54-57] and they are able to inhibit

several imatinib-resistant mutations.[57-61] Therefore, in

many countries these drugs have been approved for the

treatment of imatinib-resistant CML patients.[62-64] Moreover,

in the first-line setting, both dasatinib and nilotinib are shown to

result in deeper therapy responses in a shorter time than

imatinib treatment.[65, 66]

The key challenges today in treating CML are the evolving

mutations against TKIs. For example, several dasatinib

(T315I/A, F317L/V/I/C, and V299L)[67, 68], nilotinib (T315I,

E255K/V, Y253H, F359V/C/I)[68] and bosutinib-resistant

mutations (T315I and V299L) have been observed.[69] Of

these, the most important is the T315I mutation, which is

resistant to all first- and second-generation drugs. For these

patients, there are several third-generation TKIs in phase I/II

trials. These new candidate molecules are potential inhibitors of

the T315I mutation and include compounds such as XL228,

PHA-739358, AT9283, and DCC-2036.[70] However, the most

promising at the moment is ponatinib (AP24534), which is

highly active against the T315I mutation and has already

entered in clinical trials.[71]

IV. IMMUNOMODULATORY EFFECTS OF TYROSINE KINASE

INHIBITORS

The first TKI that entered to the clinics was imatinib, which

is therefore also the most studied. Numerous groups have

shown that imatinib has inhibitory effects on several kinases

including ABL, ARG, BCR-ABL1, c-KIT, platelet derived

growth factor receptor (PDGFR), DDR1 and NQO2.[72]

Whereas nilotinib has significantly similar inhibition profile as

imatinib, dasatinib inhibits a broader range of kinases including

the SRC family (FGR, FYN, SRC, YES, BLK, HCK, LCK,

LYN), TEC family (BMX, BTK, ITK, TEC, TXK), and SYK

families (SYK and ZAP-70)(Table 1).[55, 73-76] Of these,

several are known to be important in the function of the

immune system. For example LCK, FYN, and ITK are essential

in T-cell signaling, whereas LYN, SYK, and BTK are involved

in B-cell signaling.[77] Further, the GCK-activated signaling

pathways participate in the modulation of cell adhesion, cell

migration and immune cell responses.[78] PTK6 (BRK) has

been described to mediate signaling pathways that are involved

in the regulation of cell cycle and migration.[79] A unique

feature of bosutinib is that it does not inhibit c-KIT or PDGFR,

and it targets the STE family of kinases and in particular the

STE20 subfamily.[80] These kinases are known regulators of

apoptosis, cell cycle, migration, lymphocyte polarization and

adhesion, and B-cell class switch.[81] Also CAMK2G, a

Ca2+

/calmodulin-dependent protein kinase shown to be

involved in regulating signaling networks controlling the

proliferation of myeloid leukemia cells,[82] is inhibited by

bosutinib but not by dasatinib.[80] The third-generation TKI

ponatinib is a potential inhibitor of a discrete subset of protein

tyrosine kinases including members of the class III/IV

subfamily of tyrosine kinases (RTK), FLT3, c-KIT, fibroblast

growth factor receptor 1 (FGFR1) and PDGFR.[83, 84]

In the following paragraphs, the immunomodulatory

effects of different TKIs will be presented in detail by the

immune cell type (figure 1).



19

A. T-cells

T-cells have several well-characterized functions in tumor

immunology. They circulate through tissues and scan major

histocompatibility complex (MHC)-peptide complexes

presented on antigen-presenting cells (APCs), which can

specifically activate T-cells through the T cell receptor (TCR).

In addition, T-cells are capable of sensing a variety of signals,

such as potential danger signals associated with cancer.

Activated T-cells are then able to recognize antigens directly on

the tumor cell surfaces (reviewed by Restifo et al).[85]

Tumor-infiltrating lymphocytes (TILs) are a well-known

example of the antitumor activity of T-cells and the presence of

TILs have been correlated with clinical outcome.[86] Several

vaccine studies have also indicated the usefulness of cytotoxic

T-cell (CTL) responses, especially in treating minimal residual

disease.[87] In CML, the importance of T cell-mediated

immune responses was observed already in the 1980 s when

CML patients were transplanted with T cell-depleted grafts to

avoid graft-versus-host disease (GVHD). Even though the

depletion of T-cells successfully reduced the mortality from

GVHD, it also significantly increased the risk of relapse.[88]

Further proof of the beneficial role of antileukemic T-cells was

obtained when transplanted patients were given donor

lymphocyte infusions, which alone induced complete

remissions in patients who had relapsed.[31-34]

To date, several in vitro studies have shown that imatinib,

nilotinib, and dasatinib have inhibitory effects on T-cells.

Imatinib was reported to impair T-cell function first time in

2004 when Dietz et al. showed that the presence of imatinib

inhibited T-cell proliferation when cells were stimulated with

dendritic cells (DCs) or phytohaemagglutinin (PHA). Washout

of imatinib restored the proliferative capacity of T-cells. These

results were confirmed in a mouse in vivo-model.[89] The

following reports supported these first findings as they showed

that imatinib inhibits the proliferation of activated T-cells, and

reduce antigen-triggered expansion of CD8+ T-cells in

response to cytomegalovirus (CMV) and Epstein-Barr virus

(EBV) peptides.[90] Further, imatinib was shown to inhibit

PHA-induced proliferation of normal peripheral blood

mononuclear cells (PBMCs) in vitro.[91] The effects of

imatinib on the expression of T-cell activation markers (CD25

and CD69) have been controversial; both suppressive and

neutral effects have been reported [89-91].

The cytokine production of CD4+ and CD8+ T-cells has

also been reported to be reduced by imatinib treatment.[92] For

example the activation of whole blood with Staphylococcus

enterotoxin B resulted in lower production of Th1-type of

cytokines such as IL-1, IFN- and TNF- in imatinib treated

patients.[93] Similar inhibitory effects of imatinib on the

production of IL-2 and IFN- by T-cells have also been

reported in other studies.[90, 91]

The suppressive effects of imatinib therapy on leukemia

specific T-cells (such as the inhibition of IFN- and granzyme

B (GrB) release) have also shown to affect the lysis of CML

progenitor cells.[94] Further, imatinib seemed to inhibit the

antigen-specific IFN- secretion of both CD4+ and CD8+ T

effector cells. The same study showed that the cytolytic

functions of CD8+ CTLs were not altered.[92] Ex vivo studies

using autologous blood samples as stimulators to detect

antileukemic T-cell responses have shown that CML patients in

remission on imatinib were able to respond to stimulation by

cytokine secretion.[95]

The effects of imatinib treatment on antiviral immune

responses in vivo was studied by Mumprecht et al. Primary

CTL responses were not impaired in imatinib-treated mice, but

the secondary expansion of specific memory CTLs was

reduced both in vitro and in vivo, which resulted in impaired

protection against reinfection.[96] Imatinib treatment in vivo

selectively inhibited the expansion of antigen-experienced

memory CTLs without affecting primary T- or B-cell

responses.[96] The in vivo effect of imatinib has also been

studied by analyzing the response of CD8+ T-cells to Listeria

moncytogenes.[97] In vitro, imatinib had no effect on

antigen-specific expansion, cell division, cell cycle progression

or IFN- expression of naïve or memory T-cells. However,

imatinib induced apoptosis in these specific cells. Again, the

primary response was not altered whereas imatinib treatment

resulted in diminished memory response, suggesting that

imatinib treatment affects the ability of the CD8 memory pool

TABLE 1

TARGETS OF IMATINIB, NILOTINIB, AND DASATINIB

Modified from Hantschel et al., 2008.[55]

Imatinib Nilotinib Dasatinib

ABL ABL ABL HCK TXK EPHA3 LIMK2 SLK

ARG ARG ARG LCK DDR1 EPHA4 MYT1 STK36

BCR-ABL1 BCR-ABL1 BCR-ABL1 FGR DDR2 EPHA5 NLK SYK

c-KIT c-KIT c-KIT BLK ACK FAK PTK6/BRK TAO3

PDGFR PDGFR PDGFR FRK ACTR2B GAK QIK TESK2

DDR1 DDR1 SRC CSK ACVR2 GCK QSK TYK2

NQO2 NQO2 YES BTK BRAF HH498 RAF1 ZAK

FYN

LYN

TEC

BMX

EGFR

EPHA2

ILK

LIMK1

RET

RIPK2

Fig. 1. Immunomodulatory effects of imatinib and dasatinib on lymphocytes.

CML

Treg

Dasatinib

Mobilization of cytotoxic lymphocytes and expansion of LGLs, sensitized for

Th-1 type immune responses

NK-cells

LGL

Imatinib

Downregulation of

Tregs

Expansion of NK-cells and enhanced

cytotoxicity

NK-cells Restored NK-function

Treg Downregulation of Tregs

NK-cells

Treg T-cells

T-cells

T-cells

B-cells

B-cells

LGL

?

Anti-

leukemic activity

through antibody

production

? Inhibition

Expansion of differentiated memory

T-cells, decreased

expression of PD-1

Anergic T-cells (eg. expression of PD-1)

B-cells ? Quantitative and functional defects

Significant

increase in the percentage of

Tregs

Fig. 1. Immunomodulatory effects of imatinib and dasatinib on lymphocytes in CML patients.



20

to respond to antigen and has the potential to increase

susceptibility to infection.[97]

Similarly to imatinib, dasatinib blocks the function of

normal human T-cells in vitro at clinically relevant

concentrations. Several studies have reported that dasatinib,

when constantly present, inhibits the proliferation of both

CD4+ and CD8+ T-cells.[39, 98-101] Dasatinib has also shown

to inhibit the activation of T-cells[39, 98-100] and

antigen-specific proliferation of murine CD4+ and CD8+

transgenic T-cells in vitro and in vivo. The induction of

T-cell-mediated cytotoxicity following immunization with a

non-replicating recombinant virus was also inhibited.[102]

Furthermore, the presence of dasatinib hindered T-cell cytokine

production and degranulation in both CD4+ and CD8

+ T-cells in

a dose-dependent manner.[39, 98-100] It has been suggested

that CD4+ T-cells are more sensitive to the inhibitory effects of

dasatinib than CD8+ T-cells, and that naïve T-cells are more

sensitive than memory T-cell subsets.[99] In addition, the

inhibition of the proliferation of CD8+ T-cells has been

associated with lower secretion of IFN- and GrB.[101] Several

studies have also shown that dasatinib inhibits TCR-mediated

signal transduction.[100] The impaired TCR signaling led to

decreased proliferation and function of CD8+ T-cells.[101]

This seemed to be mediated through the SRC-family kinase

LCK, a candidate kinase for suppressed T-cell function.[98]

Furthermore, signal transduction and proliferative responses

via IL-2 remained unaffected, suggesting that the inhibitory

effect of dasatinib is specific for TCR mediated signaling.[100]

Dasatinib has also been reported to arrest CD8+ T-cells in the

G0/G1 phase of cell cycle. These effects were mediated by the

downregulation of the TCR phosphorylation and the NF-B

signaling transduction cascade.[101] Despite of these

inhibitory effects, dasatinib does not seem to induce apoptosis

in T-cells.[100, 101]

To date, only a few studies have reported

immunomodulatory effects of nilotinib. However, the available

data is similar to those of imatinib and dasatinib. It has been

shown that nilotinib inhibits the proliferation of CD8+ T-cells

in vitro at therapeutically relevant concentrations.[103] The

inhibition of CD8+ T-cells specific for leukemia or viral

antigens was associated with a reduced expansion of antigen

peptide specific CD8+ T-cells and with a decreased release of

IFN- and GrB. In addition, the inhibitory effect caused by

nilotinib was reported to be two times stronger than by imatinib.

These effects were mediated through the inhibition of the

phosphorylation of ZAP-70, LCK and ERK 1/2 and the NF-B

signaling transduction pathway.[103]

As a conclusion, it seems that when assessed in vitro, all the

studied TKIs possess more or less inhibitory effect on various

T-cell functions by blocking different kinases. However, it

needs to be taken into account that in these studies TKIs are

usually present throughout the experiment, which is not true in

the in vivo setting. For example, the maximum plasma

concentration for dasatinib is achieved already within one hour

after the drug intake and the half-life of only 3-5 hours,[104,

105] which obviously can affect the in vivo effects of the TKI.

B. Dendritic cells

Dendritic cells (DCs) are specialized APCs found in all

tissues. DCs ingest samples from the environment and present

the collected information to T- and B-cells in the adaptive

immune system. By presenting antigens, the DCs are capable of

initiating an immune response. Compared with other APCs

such as macrophages, DCs are very effective and can elicit very

low number of T-cells to respond. Once the DCs have captured

the antigen in the peripheral tissues the cells migrate into lymph

nodes where they present the antigen to lymphocytes.[106]

Distinct deficiencies have been reported to occur in CML

derived DCs (CML-DCs), such as reduced migration,

endocytosis, phagocytosis, antigen processing, DC maturation

and cytokine production as reviewed by Eisendle et al.[107]

Imatinib has been shown to restore the beneficial DC-related

immune functions in CML.[108] In addition, Sato et al. showed

that adding imatinib to DCs derived from CML patients

resulted in better allogeneic antigen presentation by mixed

leukocyte culture compared with the control cells without

imatinib.[109] Furthermore, imatinib enhanced antitumor

immune responses to DC-based immunization against an

imatinib-resistant BCR-ABL negative lymphoma.[110] In vitro

treatment of APCs with imatinib has also shown to enhance the

activation of naive antigen-specific T-cells and restore the

responsiveness of tolerant T-cells from tumor-bearing hosts.

Furthermore, in vivo treatment with imatinib not only prevented

the induction of tolerance of tumor-specific CD4+ T-cells

preserving their responsiveness to a subsequent immunization,

but also resulted in enhanced vaccine efficacy.[111] Although

imatinib seems to have beneficial effects on the function of

DCs, there are no reports of the effects of second generation

TKIs.

C. B-cells

B-cells are responsible for antibody production and errors in

B-cell development can give rise to immunodeficiency,

leukemia and lymphomas, and autoimmune diseases.[112] To

date, very few studies have explored the effects of the different

TKIs on B-cells. In the context of CML, B-cells have been

proposed to have beneficial anti-leukemic activity in patients

treated with imatinib as the treatment induces CD5+ B-cells

and IgM natural antibodies with anti-leukemic reactivity.[113]

However, in imatinib-treated patients the phenotype of plasma

cells was reported to be abnormal and it correlated with the

reduced gamma-globulin levels.[114] Imatinib treatment has

also been reported to decrease Ig levels and cause

hypogammaglobulinemia.[115-117] In addition to CML,

similar observations have also been reported in gastrointestinal

stromal tumor (GIST) patients who have been treated with

imatinib.[117]

D. NK-cells

NK-cells are a crucial part of the innate immunity and they

have a well-defined ability to recognize stressed cells (for

example transformed or infected cells) and destroy them.

Further, NK-cells can also provide help to the adaptive immune



21

system for example by producing IFN-.[118] Besides

cytotoxic T-cells, also NK-cells may have a role in the immune

control of cancer.

A recent report by Chen et al. concluded that CML is

associated with quantitative and functional defects within the

NK-cell compartment.[119] Several groups have reported

effects of the different TKIs on NK-cells and the results have

been controversial. Some studies have reported that imatinib

has no direct influence on NK-cell reactivity in vitro.[120] In

contrast, an earlier study by Cebo et al. showed that imatinib

treatment inhibits the NKG2D activating pathway and that the

modulation of NKG2D by imatinib interferes with NK-cell

recognition and cytolysis.[121] Accordingly, Chen et al.

reported that imatinib treatment cause a limited in vitro

expansion of NK-cells from CML patients and a reduced

degranulation response to K562 target cells.[119]

Similarly dasatinib has been shown to reduce NK-cell

cytotoxicity and cytokine production both using human cells

and an in vivo mouse model.[39, 102, 120] In contrast to these

studies, one publication reported that NK-cells in

dasatinib-treated patients have increased capability to kill K562

cells.[122] This finding is supported by reports showing that

NK-cell cytotoxicity is improved in dasatinib-treated CML

patients who have achieved a good therapy response.[122, 123]

Analysis of NK-cell signaling has revealed that dasatinib

inhibits proximal signaling events leading to decreased

phosphorylation of PI3K and ERK that are crucial for NK-cell

reactivity. Imatinib and nilotinib showed no relevant effect on

NK-cell PI3K or ERK activity.[120]

There are only a few papers reporting the effects of nilotinib

on NK-cells. In one publication by Salih et al., nilotinib did not

alter the cytotoxicity of NK-cells, but at high levels it impaired

the cytokine production in vitro.[120] This could be explained

by the finding that nilotinib increased the cell death within the

CD56bright

CD16neg

NK-cell subset, which is known to secrete

cytokines.[120] In contrast, Hayashi et al reported that nilotinib

decreased NK-cell cytotoxicity.[122]

In summary, the effects of TKIs on NK-cell function are

still controversial and additional research is needed. As with

T-cells, it should be kept in mind that the in vivo

pharmacokinetics of the TKIs differs from in vitro situation. In

addition, also the in vitro results have shown that the inhibitory

effects of TKIs on the NK cell function are reversible, as the

washing of the dasatinib-treated NK-cells restored their

cytotoxicity.[39, 120, 124]

E. Immunosuppressive cells

Tumors possess several mechanisms how they can disrupt

the anti-tumor immunity. This can be done for example by high

jacking the parts of the immune system such as by recruiting

myeloid-derived suppressor cells (MDSC) or regulatory T-cells

(Tregs) to provide protection against the immune attack.

Normally these cells contribute to peripheral tolerance, but in

cancer patients they have been shown to enhance tumor

survival and growth as reviewed by Whiteside [125] and

Lindau et al.[126]

MDCS are a group of immature CD11b+CD14-CD33+

cells (including precursors of macrophages, granulocytes, DCs

and myeloid cells) that are produced in response to various

tumor-derived cytokines. These inhibitory myeloid cells are

able to suppress both CD4+ and CD8+ T-cell mediated

immunity.[85] Interestingly, Christiansson et al. reported that

untreated CML patients have higher MDSC levels compared to

healthy individuals.[45]

In addition to MDSCs, the presence of Tregs has shown to

be correlated with poor prognosis in a number of cancers and

therefore several studies are now exploring the possibility to

eliminate Tregs to improve therapy responses.[127] However,

the elimination of Tregs can lead to autoimmune side effects in

addition to improved anti-tumor immunity.[128, 129] Imatinib

is known to inhibit the proliferation of Tregs and their

production of IL-10, TGF-1 and GrB in a dose-dependent

manner. In addition, the expression of CD69, GITR, FoxP3,

and CTLA-4 of activated Tregs was inhibited by imatinib.[110,

130] In contrast, another study showed that when used at

concentrations achieved in clinical use, imatinib did not impair

the production of IL-10 and TGF- by Tregs.[110] Analysis of

Treg TCR-induced signaling cascade indicated that imatinib

inhibited phosphorylation of ZAP70 and LAT.[110] Imatinib

treatment of mice decreased the frequency of Tregs and

impaired their immunosuppressive function in vivo.[110]

Dasatinib has been shown to profoundly prevent the

proliferation of Tregs in a dose-dependent manner, and to

decrease the production of corresponding cytokines. Treatment

of Tregs with dasatinib also inhibited the suppressive capacity

of Tregs. The mechanisms of this inhibition included the arrest

of cells in the G0/G1 phase of cell cycle, down-regulation of the

transcription factor FoxP3, glucocorticoid-induced tumor

necrosis factor receptor and CTLA-4 as well as inhibition of

signaling events through SRC and NF-B.[131] Similarly

nilotinib has been shown to inhibit and impair the function of

Tregs in a dose-dependent manner.[132]

In summary, TKIs also affect the immune regulatory cells

such as Tregs and this may lead to beneficial effects by

reversion of the suppressive mechanisms.

V. CLINICAL OBSERVATIONS

As described above, TKIs seem to possess mostly

immunosuppressive effects in vitro. Therefore, it has been of

interest to see whether similar effects are observed in vivo in

patients, as such strong inhibition of the immune system would

most likely lead to serious infections and other adverse-effects.

However, to date no increased rate of infections, secondary

malignancies or autoimmune disorders in TKI-treated patients

have been observed in large clinical studies.[66] A few cases of

panniculitis has been reported in imatinib-resistant CML

patients who were later treated with dasatinib.[133] Moreover,

Duman et al. reviewed eight cases of CML patients who had

developed secondary tumors after they had been treated with

imatinib.[134-136] Further, a few case reports suggesting that

in some circumstances dasatinib treatment may be associated

with abnormal viral infections. For example, parvovirus B19,

herpes virus 6 and reactivation of CMV infections have been



22

described in dasatinib-treated patients.[137] Our group has also

observed that in a selected group of patients experiencing large

granular lymphocyte (LGL) lymphocytosis during dasatinib

treatment, 40% had CMV reactivation.[138] Also, a high

number of CMV-specific T-cells were detected in these

patients.[39, 138] In contrast, in a recent report including only

first-line dasatinib-treated CML patients no increased incidence

of CMV reactivation was observed.[139]

Hayashi et al. recently reported that imatinib, nilotinib and

dasatinib-treated patients have similar numbers of CD8+ and

CD4+ T-cells during the treatment.[122] In contrast, Rohon et

al. reported that dasatinib-treated patients have a lower

proportion of T-cells in the blood,[140] and consequently an

increased number of NK-cells.[122, 140] The percentage of

immunosuppressive regulatory T-cells (CD4+CD25high

FoxP3+)

has been reported to be significantly higher in untreated CML

patients compared to healthy controls.[141] We and others have

reported that the number of Tregs in the blood is decreased

during dasatinib treatment.[138, 142] However, in other TKI

treated patients the number of Tregs has been found to be

similar as in healthy volunteers.[122, 141]

Mumprecht et al. reported that in imatinib-treated patients,

the PD-1 expression in CD8+ T-cells is increased up to 60%

whereas it is less than 10% in healthy controls.[38]

Interestingly, in dasatinib-treated patients CD8+ T-cells are

shown to express less than 20% of PD-1.[39] This finding

indicates that unlike imatinib, dasatinib may decrease PD-1

expression close to the levels of healthy controls, which could

be of importance as the lack of PD-1 signaling pathway is

associated with an improved survival and might restore the

function of CML-specific CTLs as shown in a CML mouse

model.[38]

The role of immunoregulatory genes has also been studied

in association with the response to TKI therapy. Especially

killer-immunoglobulin-like receptors (KIRs) are of special

interest as they are able to regulate both the function of

NK-cells and CD8+ T-cells. The presence of a stimulatory

KIR2DS1 gene was found to be a negative prognostic factor

and a lower proportion of KIR2DS1+ patients achieved

complete cytogenetic remission (CCgR) during imatinib

therapy.[143] Similar association with the rate of CCgR was

not found among dasatinib treated patients,[144] but the

absence of the inhibitory KIR2DL5A, 2DL5B, and 2DL5all

genes were associated with improved molecular response.[143,

145, 146]

In summary, despite the highly immunoinhibitory effects of

TKIs in vitro, these drugs do not seem to cause increased risks

of infections. In contrast, especially dasatinib, as described in

more detail below, seems to modify the immune system to a

more cytotoxic direction and decrease the number of

suppressive Tregs, which might have a positive impact on the

therapy outcome.

A. Dasatinib-induced lymphocytosis

In 2007, we noticed that imatinib treatment is associated

with the increased number of lymphocytes in the bone marrow,

and this correlated with the better therapy response.[147] The

lymphocyte infiltration was a mixed population including

B-cells and their precursors, T-cells and NK-cells. A few years

later, our group observed that dasatinib-treated patients had an

increased number of LGLs in the blood, which was also

associated with an improved therapy response.[138] This

observation was later confirmed by several other groups.[123,

139, 148, 149] The dasatinib-associated lymphocytosis is

characterized by an increased absolute blood lymphocyte count

(> 3.6 x 109/L) and it typically occurs 3-4 months after the

initiation of dasatinib treatment. It has been reported that nearly

30% of dasatinib-treated patients develop lymphocytosis.[148,

150] Importantly, there is an increasing evidence indicating that

lymphocytosis is associated with excellent therapy

responses.[123, 138, 148, 150] Such an expansion of T-LGLs

(CD3+CD8+CD56neg

) has also been described after autologous

and allogeneic bone marrow transplantation, and also in these

patient groups it has been associated with better treatment

responses.[151-155]

The role of cytomegalovirus (CMV) infection in the

development of LGL lymphocytosis has also been considered.

In our original reports we observed that 40% of the patients

who developed LGL lymphocytosis during dasatinib therapy

had also modest CMV reactivation and some patients even

suffered from symptomatic CMV reactivation such as from

CMV colitis.[39, 138] Further, all patients who had LGL

expansion during dasatinib therapy were cytomegalovirus

(CMV) seropositive[142] and most patients also had an

elevated number of CMV-specific CD8+ T-cells.[39] However,

in these studies most patients were treated with dasatinib

second-line and in other publications studying first-line CML

patients, no CMV reactivations have been observed.[139] The

understanding of the causative role of CMV in the development

of LGL lymphocytosis needs further studies, but it could be that

previous CMV infection increases the pool of specific

lymphocytes which are more prone to expand and mobilize

during dasatinib therapy.[156]

The exact mechanism causing LGL lymphocytosis during

dasatinib-therapy is still unknown. However, our group

recently demonstrated that dasatinib is able to induce a rapid

and dose-dependent mobilization of non-leukemic

lymphocytes and monocytes in blood peaking 1-2 h after the

oral intake of the drug. Importantly, the blood counts closely

mirrored drug plasma concentration. The mobilization was

coupled with a more effective transmigration of leukocytes

through an endothelial cell layer. Similar effects on blood cell

dynamics and function were not observed with any other TKI

(imatinib, nilotinib and bosutinib).[156] Furthermore, NK-cell

cytotoxicity was shown to be improved 1 hour after the intake

of dasatinib.[156] The wide kinase inhibition profile of

dasatinib seems to affect signaling pathways in leukocytes or

surrounding tissues causing rapid leukocyte increase in blood,

but the exact kinase involved in this process is still unknown.

This rapid mobilization is thought to account partially for the

observed LGL lymphocytosis during dasatinib treatment, but

additional mechanisms such as the decreased amount of Tregs,

genetic factors and primary lymphocyte pool may play a role.



23

VI. IMMUNOMODULATORY EFFECTS OF TKIS IN OTHER

CANCERS

The most famous success story of using TKIs in other

cancers than CML is the treatment of gastrointestinal stromal

tumors (GIST) with imatinib. GIST is a common type of

soft-tissue sarcoma mainly caused by a mutation in the KIT

gene, which results in the activation of the receptor in the

absence of its ligand and finally to uncontrolled cell growth and

tumor formation.[157] The discovery of the molecular

background of the disease led to successful clinical trials with

imatinib, which is also able to inhibit KIT. The

second-generation TKIs dasatinib and nilotinib are also able to

induce beneficial therapeutic responses in GIST patients.[158,

159] Interestingly, recent reports indicate that the superior

therapy responses seen in imatinib-treated GIST patients might

not only be due to the effective inhibition of the KIT

oncoprotein but also due to the immunomodulatory effects.

Balachandran et al. reported that imatinib potentiates antitumor

T-cell responses in a mouse model of GIST. Within the tumors,

imatinib was shown to increase the number of activated CD8+

T-cells, but to decrease the frequency of Tregs, consequently

significantly increasing the CD8+ T-cell to Treg ratio.

Interestingly, imatinib-treatment significantly decreased the

intratumoral IDO-expression. IDO is a protein, which promotes

the development, stabilization and activation of Tregs while

suppressing effector T-cells. These results were confirmed in

patient samples; imatinib-sensitive tumors contained a higher

frequency of CD8+ T-cells, but lower frequency of Tregs when

compared to imatinib-resistant tumors.[160]

Another disease associated with an activating mutation in

the KIT oncoprotein is systemic mastocytosis (SM). Already in

2006, Shah et al. demonstrated a significant inhibitory activity

of dasatinib against both wild-type and mutated c-KIT.

Dasatinib was shown to selectively kill primary neoplastic bone

marrow mast cells from patients with SM.[161] Interestingly,

in a recent paper by Yang et al., the in vivo antitumor effects of

dasatinib on the c-KIT mutant mastocytoma tumor was

demonstrated to be dependent on T cell-mediated immunity.

They showed that dasatinib treatment significantly decreased

the levels of Tregs while improving the antigen-specific T-cell

responses against tumors. The combination of dasatinib and

agonistic anti-OX40 (a potent co-stimulatory signal to T-cells)

antibody resulted in better therapeutic efficacy compared with

either drug alone, which was associated with increased number

of tumor antigen-specific T-cells in the tumor

microenvironment. Moreover, the combination of dasatinib and

anti-OX40 inhibited the function of Tregs and resulted in a

significant up-regulation of the IFN--induced chemokines

CXCL9, 10, and 11, which attracts CTL to the tumor

microenvironment.[162]

These two examples (GIST and SM) in addition to effects

observed in CML patients suggest that TKI treatment may have

beneficial immunomodulatory effects in vivo which need to be

studied in more detail in order to be able to utilize them in

future treatment regimens.

VII. FUTURE PROSPECTS

The current experience with TKIs in CML suggests that

only a small proportion of patients can achieve a state of

complete molecular remission (CMR) where therapy

discontinuation can be considered.[163] The CMR rates with

the 2nd

generation inhibitors dasatinib and nilotinib are better

and it could be that these drugs are also more effective against

the leukemic stem cells.[164] However, it seems that the total

eradication of leukemic cells is not needed in order to be able to

discontinue the treatment successfully and therefore better

ways to boost the immune system may increase the rate of

successful discontinuations.

There are already data showing that the combination of

immunomodulatory agents such as IFN- with the imatinib

treatment may increase the rate of superior molecular

responses.[165, 166] In addition, IFN- monotherapy after

imatinib treatment increases the rate of successful therapy

discontinuations.[166, 167] Ongoing clinical trials have

combined IFN- with 2nd

generation TKIs and their effects

remains to be seen. Further, the combination of imatinib with

anti-CML vaccines (mostly against p210bcr-abl) seems to

improve therapy responses.[168]

In the future it would be of interest to combine TKI

treatment with different types of immunomodulatory agents

such as with immune checkpoint inhibitors (for example PD1,

PD-L1, CTLA-4 and OX40 antibodies), which have now

entered in the first clinical trials.[169, 170] They could have

synergistic beneficial effects with TKIs, although also the risk

of side effects may increase. However, such combination

regimens may not only turn out to be useful in CML, but also in

other cancers, as was already shown in mastocytoma mouse

model.[162]

ACKNOWLEDGMENT

Personnel at the Hematology Research Unit Helsinki are

acknowledged for their expert technical assistance.

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Immunomodulatory Effects of Tyrosine Kinase Inhibitors

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