Curr Hematol Malig Rep (2014) 9:17–23 DOI 10.1007/s11899-013-0190-1

CHRONIC LEUKEMIAS (J GOLDMAN, SECTION EDITOR)

Immunology and Immunotherapy of Chronic Myeloid Leukemia Mette Ilander & Can Hekim & Satu Mustjoki

Published online: 5 January 2014 # Springer Science+Business Media New York 2014

Abstract Chronic myeloid leukemia (CML) is a clonal bone marrow stem cell neoplasia known to be responsive to immunotherapy. Despite the success of tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 oncokinase, patients are not considered to be cured with the current therapy modalities. However, there have been recent advancements in understanding the immunobiology of the disease (such as tumor specific antigens and immunostimulatory agents), and this may lead to the development of novel, curative treatment strategies. Already there are promising results showing that a small proportion of CML patients are able to discontinue the therapy although they have a minimal amount of residual leukemia cells left. This implies that the immune system is able to restrain the tumor cell expansion. In this review, we aim to give a brief update of the novel aspects of the immune system in CML patients and of the developing strategies for controlling CML by the means of immunotherapy.

Keywords CML . Immunology . Tyrosine Kinase Inhibitors . Tumor Antigens . Vaccine . Stem Cell . NK-Cell

M. Ilander : C. Hekim : S. Mustjoki Hematology Research Unit Helsinki, Department of Medicine, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland M. Ilander e-mail: [email protected] C. Hekim e-mail: [email protected] S. Mustjoki (*) Helsinki Hematology Research Unit, Biomedicum Helsinki, University of Helsinki and Helsinki University Central Hospital, P.O. Box 700, FIN-00029 Helsinki, Finland e-mail: [email protected]

Introduction During the last decades, chronic myeloid leukemia (CML) has been a model disease in targeted cancer therapy. By understanding the underlying molecular mechanism (Philadelphia chromosome and BCR-ABL1 fusion gene) the invention of several different tyrosine kinase inhibitors (TKIs) has been possible. The first generation TKI imatinib (Glivec®) has been in clinical use already over ten years, and more potent second generation TKIs, dasatinib (Sprycel™), nilotinib (Tasigna®), and bosutinib (Bosulif®) are all currently approved for the treatment of CML [1–5]. TKI therapy has improved the prognosis significantly [6], and deaths due to CML are nowadays rare. However, some patients may develop resistance due to the ABL kinase domain mutations, and, therefore, third generation TKIs such as ponatinib (Iclusig®) have been developed [7]. Although treatment results in CML are far better than in most other cancers, the treatment is still not considered to be curative [8]. Most likely, the quiescent leukemic stem cells are resistant to TKI therapy [9–12], and therefore, relapses occur shortly if treatment is discontinued. Although TKIs are usually well tolerated, some long-lasting side effects such as nausea, cramps and peripheral edema may hinder adherence to life-long therapy. In addition, the cost of the drugs is significant which may cause unbearable burden to the health economy in the future [13]. Therefore, there is a significant need to find novel effective treatment options in order to cure the patients. Most definitive long-term eradication of CML has been achieved by allogeneic hematopoietic stem cell transplantation (HSCT). However, the morbidity (such as graft versus host disease) and mortality related to HSCT hampers its use as a first-line treatment in chronic phase CML patients. Some old drugs, such as IFN-α, may experience a comeback in CML due to their beneficial immunological effects. Before the discovery of TKIs, IFN-α was used as the first-line treatment

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in CML, and in some patients it induced complete cytogenetic remissions. Due to the superior treatment responses achieved with TKI therapy, the use of IFN-α almost vanished from CML, but currently it is being tested in several clinical trials in combination with TKI therapy. In addition to IFN-α, there are other promising new candidate drugs, which could stimulate the immune system in CML. Thus, this review summarizes the recent advancements in the field of CML immunology and focuses on novel immunotherapeutic options.

Immune System in Untreated CML Patients In CML, like in other malignancies, the immune response against cancer is impaired resulting in immune escape of the malignant cells, which is required for the cancer to develop. In patients diagnosed with CML, the cells from the innate immune system are dysfunctional. For example, Chen et al. [14] observed that in newly diagnosed CML patients the proportion of NK-cells is decreased, and their function is impaired when studied by degranulation and expansion assays. Thereafter the administration of imatinib did not succeed in elevating the NK-cell numbers or improving their function [14]. In addition, invariant NKT-cells (iNKT) have been reported to be dysfunctional [15], but their function improved in patients

who achieved complete cytogenetic remission (CCyR) with IFN-α or TKI treatment. The regulation of immune activation has also been found to be altered in CML. Bachy et al. [16] showed that regulatory Tcells (Tregs) are significantly increased in CML patients with intermediate or high-risk Sokal scores compared to the lowrisk patients. Furthermore, myeloid derived suppressor cell (MDSC) associated arginase 1, which is able to inhibit T-cell function, has been found to be increased in the high Sokal risk patients [17]. The same study showed that at diagnosis CML cells (including also CD34+ stem cells) expressed programmed death receptor ligand 1 (PD-L1), and its receptor PD-1 was expressed on T-cells. By binding to the inhibitory receptor PD-1 on T-cells, PD-L1 is able to suppress the T-cell effector functions (Fig. 1).

Tumor Specific Antigens in CML In CML, a clear antigen target for immunotherapy is the oncoprotein Bcr-Abl1, as the fusion of the two proteins Bcr and Abl1 creates a junction peptide sequence unique to leukemic cells. Most of the CML cells have been shown to present endogenous p210/e13a2 or p210/e14a2 Bcr/Abl1 derived junctional peptides in the context of class I HLA-

Clonal Expansion of CD8+T-cells

CD8+T-cell clone

Dasatinib

Regulatory T-cells Function of NK-cells

NK cells

IFN-α Dasatinib

Arginase1

anti-WT-1/MHC-I ADCC by T-cells PD1-L PD-1 PD-1L

anti-PD1 + PD1

Myeloid derived suppressor cells

T-cell recognition and lysis anti-IL1RAP IL1RAP T-cells ADCC by NK-cells

At Diagnosis

Leukemic cells

Fig. 1 At diagnosis the function of the immune system is impaired in CML. T-cells are anergic and NK-cells dysfunctional. Immunoinhibitory mechanisms such as regulatory T-cells and myeloid derived suppressor cells are increased and tumor cells display ligands for the PD-1 inhibitory receptor. Some therapeutic options (such as IFN-α and TKIs) may reverse the immune cell anergy and activate the immune cell function. In addition

During Treatment to treatments already in use, there are several promising agents such as anti-PD1, anti-IL1RAP and anti-WT1 antibodies, which could enhance the immune system and may enter in clinical use in the future. Harnessing the immune system to battle against the leukemic cells in synergy with the direct oncokinase inhibiting drugs could result in a disease-free situation where the immune system is in control

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restricted T-cell cytotoxicity [18, 19]. Other Bcr-Abl1 translocations, such as e1a2, have also been identified, but these are shown to be poorly immunogenic [20, 21]. Nevertheless, the immunogenicity has been improved by modifying the peptides and increasing their binding to HLA class I molecules [22]. In addition to the unique Bcr-Abl fusion peptide, selectively expressed or overexpressed leukemia-specific antigens are other targets for CML immunotherapy. Among these, a zinc finger transcription factor Wilms tumor antigen 1 (WT1) has been an attractive target as it is overexpressed in leukemic hematopoietic stem cells and also in a wide range of solid tumors (Fig. 1) [23]. As WT1 is localized in the nucleus, it is not possible to target the oncoprotein via classical antibody targeting methods. However, WT1-specific cytotoxic T-cells responses can be generated by presenting the peptides on the cell surface by MHC class I molecules. A WT1-derived peptide RMFPNAPYL presented by HLA-A0201 has been extensively studied and shown to induce cytotoxic CD8 T-cells that are capable of killing WT1+ tumor cells in human clinical vaccine trials [24–26]. Very recently, Dao et al., have described a human monoclonal antibody specific for the RMF/ HLA-A0201 complex that mediates antibody-dependent cellmediate cytotoxicity in human leukemia xenografts [27•]. The authors confirmed that the cytotoxicity effect is restricted to WT1+ and HLA-A0201+ tumor cells and this antibody could be a promising novel immunotherapeutic agent. In addition to WT1, a number of other leukemia-associated antigens have been described in CML including hyaluronan acid-mediated motility (RHAMM), telomerase, PR1, PR3, PPP2R5C, ELA2, PRAME and a novel epitope derived from the M-phase phosphoprotein 11 protein (MPP11) [28–31]. IFN-α treatment in CML has been shown to increase PR3 expression in peripheral blood and induce the increase of PR1-specific CTLs, which may contribute to sustained remission after imatinib discontinuation [32]. PR1 is a part of both PR3 and ELA2 and can induce specific CTL responses [33, 34]. Furthermore, a 9-mer Aurora-A kinase derived peptide has been shown to be capable of inducing anti-leukemic cytotoxic T-cell activation in the context of HLA-A0201 [35]. Aurora-A kinase is a serine/threonine kinase that regulates cell division and plays a role in tumorigenesis. Interestingly, a recent in vitro study involving computational prediction methods to identify and generate neoantigens for imatinib-resistant BCR-ABL mutations was able to describe a novel mutation derived peptide, which bound to HLA-A3 in the imatinib-resistant CML patients [36]. This finding demonstrates an intriguing possibility of immunizing relapsed CML patients, who carry ABL mutations against newly acquired antigens. Although the tumor antigen specific immunotherapy holds promising potential in CML, it is noteworthy to recognize that persistent vaccination with stable peptide emulsions have also

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shown to cause sequestering and dysfunction of the cytotoxic CD8+ T-cells [37].

Leukemic Stem Cells and the Immune System TKI therapy eradicates very efficiently the majority of leukemic cells, but it seems that they are not able to kill the most primitive quiescent leukemic stem cells (LSCs) [9–12], although, recent clinical trials have shown that most of the leukemic progenitors and LSCs are also very sensitive to TKIs [38]. Therefore, there has been a considerable interest in finding LSC targeting novel therapies. One option to target LSCs is immunotherapy, but to be specific for LSCs and not to target healthy hematopoietic stem cells for example; it is necessary to understand which antigens are selectively expressed or overexpressed by LSCs. Recently, it was shown that IL-1 receptor accessory protein (IL1RAP) distinguishes LSCs from normal HSCs [39•]. In addition to CML, it has been shown to be expressed by AML LSCs [40•]. A monoclonal antibody against IL1RAP has already been developed [39•, 40•], and the antibody bound IL1RAP enables the immune system to recognize the LSCs and expose them to antibody dependent cellular cytotoxicity (ADCC). However, this antibody therapy has not yet entered clinical trials. One of the widely accepted methods to eliminate the residual LSCs is hematopoietic stem cell transplantation (HSCT), where the grafted donor cells mediate the graft versus leukemia (GvL) effect. The main effectors of GvL are thought to be the alloreactive T-cells in the unrelated HLA-matched and partially-HLA mismatched transplants, which are recognizing minor histocompatibility antigens and HLA molecules on the residual leukemic cells. The potency of T-cells mediating GvL effects have been demonstrated in clinical studies, which have shown that the relapse rate is significantly higher among CML patients who have received T-cell depleted transplants [41]. However, the same factors are able to trigger the graft versus host disease (GvHD), which may have a fatal outcome. Boosting solely the GvL effect has been difficult; but it has been shown recently in the xenograft model that the stimulation of HLA-haploidentical donor T lymphocytes with leukemic antigen-presenting cells can lead to the expansion of leukemia reactive T cell populations [42]. Yet, NK-cells are also capable of recognizing partiallyHLA mismatched transplants, and they do not mediate GvHD effect. Alloreactive NK-cells have been suggested to prevent relapse after HSCT; the reason for this is claimed to be the absence of the killer immunoglobulin receptors (KIRs) on the recipient cells [43]. In a CML mouse transplant model it was recently described that NK-cells were effective in killing primary CML cells, and the NK-mediated protective effect was based on the missing-self recognition [44].

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Immunostimulatory Treatments (TKIs, IFN-α and Novel Checkpoint Inhibitors) In general, in vitro studies have suggested that TKIs are immunosuppressive inhibiting T-cell and NK-cell activation [45–49]. This is thought to be mediated by their inhibition of off-target kinases (such as c-KIT, TEC, and SRC), and especially second generation TKIs have broader inhibition profiles than imatinib. In vivo, the immunological effects of TKIs seem to differ, and there are a few publications describing the immune profiles of CML patients treated with imatinib, nilotinib and dasatinib [50, 51]. During imatinib therapy the immune system seems to recover and the immune cell profile resembles closely that seen in healthy controls. In contrast, in a proportion of dasatinib treated patients immunostimulation can be observed with increased numbers of CD8+ T-cells, NKcells, NKT-like cells and decreased numbers of Tregs [50, 52]. Furthermore, clonal expansion of CD8+ T-cells has been observed during dasatinib treatment [53], which has been linked to cytomegalovirus reactivation [54] in some patients. These immunostimulatory effects seem to be contrast with the profound immunoinhibitory effects seen in vitro [45, 46], but this could be due to very short half-life (only 3-4 hours) of dasatinib in vivo. Actually there are already a couple of recent publications suggesting that short-term exposure of dasatinib in vitro is immunostimulatory and enhances NK-cell function [55, 56]. Interestingly, dasatinib has also been reported to exert immediate effects on immune cells in vivo. After a single dose of dasatinib, there is a marked mobilization of lymphocytes leading to 2-8 fold increase in the absolute lymphocyte counts [57•, 58]. Furthermore, mobilization is associated with the enhanced NK-cell cytotoxicity and increased transmigration. These effects seem to be dasatinib-specific as they were not observed with imatinib, nilotinib or bosutinib treatment. The effects of TKI treatment on B-cell maturation and activation have not been studied in detail, although the same kinases are also involved in these processes. However, there is one recent publication showing that TKI treatment (either imatinib, nilotinib or dasatinib) impairs B-cell responses in CML patients [59]. This can be observed as weakened humoral responses after vaccination attempts. Further studies with a larger number of patients are warranted to understand whether different TKIs differ in their effects on B-cells. Already in the 1990s there were several studies published with clear evidence of immune activation followed by IFN-α treatment. The NK-cell cytotoxicity against K562 cells and autologous CML cells was found to increase in the beginning of IFN-α therapy [60, 61]. Due to the advent of TKIs, IFN-α treatment was almost totally abandoned in CML, but recent clinical trials have shown promising results confirming that the combination of IFN-α with imatinib leads to higher response rates [62•, 63•]. In addition, the likelihood of stopping the TKI therapy without subsequent relapse increases when

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IFN-α is used in combination with imatinib as compared with TKI monotherapy [32]. Currently, there are many on-going clinical trials combining different TKIs with pegylated IFN-α. Two have combined nilotinib with pegylated IFN-α2b (NCT01657604, NCT01866553), one imatinib with pegylated IFN-α2b (NCT00573378) and one dasatinib with pegylated IFN-α2b (NCT01725204). It will be interesting to follow whether these combinations lead to deeper molecular responses and whether that translates to increased likelihood of stopping the therapy in the future. IFN-α therapy could also have a role in treating TKI resistant patients, such as patients with the multi-TKI resistant T315I mutation. There are a few recent case reports suggesting that T315I patients may benefit from the IFN-α monotherapy or from the combination treatment with TKIs [64–66]. Notably, one of the cases showed a significant immune activation in conjunction with the disappearance of the T315I mutated clone, and the patient was even able to discontinue the therapy without disease relapse [64]. However, basic immunological studies are necessary to understand the detailed mechanisms of action behind the effects of IFN-α. The immune checkpoint inhibitors such as α-CTLA4 (ipilimumab) and α-PD1 (nivolumab), which take the breaks off the immune system, are promising new immunotherapeutic agents and are currently being tested in various solid tumors such as melanoma and kidney cancer [67]. The first results are encouraging and they have been shown to improve significantly the survival of advanced phase cancer patients [68, 69•]. These drugs would be interesting novel candidate drugs in hematological malignancies as well, but the first clinical phase 1 trials are just commencing (NCT01592370), and it remains to be seen whether their side-effect profiles allow their use in chronic phase CML patients.

Immunogenetics in CML Recent publications in CML suggest that inherent features such as genes involved in the activation and inhibition of the immune system may play a role in treatment responses. The killer-cell immunoglobulin-like receptors (KIRs) are inhibiting and activating receptors of NK-cells. In a large group of imatinib treated patients it was shown that patients who carried the activating KIR2DS1 gene had a significantly lower probability of achieving CCyR [70]. The KIR2DS1 positive genotype was also linked to shorter overall survival. In a follow-up paper from the same group, the authors suggested that dasatinib may overcome this adverse prognostic factor and no differences were found in the achievement of CCyR between the KIR2DS1 positive and negative groups [71]. However, in the other study, a trend between KIR2DS1 genotype and molecular response was still observed in firstline dasatinib treated patients, and overall, the absence of

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several KIR receptors was related to better treatment responses [72]. Similar results have also been obtained by an Italian group showing that the low number of inhibitory KIR genes is associated with the achievement of complete molecular remission [73]. Immunogenetics in CML warrants further studies as it may play a role in future discontinuation attempts as well.

Immune System in Successful Treatment Discontinuation Recent reports suggest that nearly half of CML patients treated with imatinib who have reached durable complete molecular response are able to stop the treatment without relapse [74]. This is especially interesting as it has been shown that patients still have BCR-ABL positive cells left when measured with sensitive DNA based assays [75•]. Although there is a lack of specific prognostic factors, which could determine which patients are able to discontinue the therapy, there is increasing evidence suggesting that NK-cells are important in controlling the leukemic growth. In animal experiments, it has been shown that NK-cells are able to control the leukemic cells after implanting them in irradiated bone marrow of the recipient mice [44]. In CML patients, increased NK-cell counts seem to correlate with the successful imatinib discontinuation [76, 77]. Similarly, in IFN-α monotherapy treated patients increased NK-cell counts are found in patients who have been able to stop the treatment without disease relapse [78]. The function and the genetic determinants of the NK-cell expansion need further study, but it is tempting to speculate that successful CML therapy in the future should aim to increase the number and activity of NK-cells.

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The dual role of TKI therapy- both direct kinase mediated cytotoxicity and immunomodulatory effects- has gained significant interest during the last few years. Patients who are able to discontinue the TKI monotherapy without disease relapse still have residual leukemia cells left. This implies that the TKI therapy has at least restored the function of the normal immune system or probably in some cases further strengthened it. The immunomodulatory effects of small molecule kinase inhibitors warrant further studies and these findings may lead to the discovery of previously unidentified mechanisms of immune activation, and thus probably, identification of novel targets for immunotherapy. In conclusion, it seems likely that the immune system will play a significant role in the future therapy of CML as the current treatment aim should be a cure. Whether this will be achieved with the combination of old immunomodulatory drugs such as IFN-a with TKIs or with some novel agents such as WT1 targeting antibodies, immune checkpoint inhibitors or vaccine treatments need still further studies, but still there is already hope that CML could be completely eradicated. Compliance with Ethics Guidelines Conflict of Interest MSc. Mette Ilander and Dr. Can Hekim declare no potential conflicts of interest relevant to this article. Dr. Satu Mustjoki has received honoraria and research funding from Novartis and Bristol-Myers Squibb. Human and Animal Rights and Informed Consent This article does not describe any studies with human or animal subjects performed by any of the authors.

References Conclusions The current standard treatment for CML patients is targeted therapy using TKIs, which are generally well tolerated but do not completely cure the disease. Thus, there is still a need for novel medical interventions. The recent advances in understanding the CML immunobiology have significantly improved the possibility of developing novel immunotherapeutic strategies. For example, identification of several leukemiaspecific antigens is one of the promising steps forward in CML immunotherapy. Among these tumor-specific antigens, WT1 stands out as an attractive target as it is expressed in many leukemia subtypes and also in leukemic stem or progenitor cells. WT1 targeting immunotherapies are currently in clinical trials for several types of cancers. In addition to WT1, the potential targeting of the other recognized and emerging CML-specific antigens are under investigation by many study groups.

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