migration and induction of Pyk2 expression in K562 cells following imatinib exposure

Leukemia Research 37 (2013) 1729–1736

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Enhanced adhesion/migration and induction of Pyk2 expression in K562 cells following imatinib exposure Adelina Ovcharenko a,1 , Galit Granot a,∗,1 , Oshrat Hershkovitz Rokah a , Jennifer Park a,b , Ofer Shpilberg a,c , Pia Raanani a,c a

Felsenstein Medical Research Center, Beilinson Hospital, Sackler School of Medicine, Tel Aviv University, Israel Massachusetts Institute of Technology, Cambridge, MA, USA c Institute of Hematology, Davidoff Cancer Center, Rabin Medical Center, Beilinson Hospital, Israel b

a r t i c l e

i n f o

Article history: Received 28 May 2013 Received in revised form 16 September 2013 Accepted 4 October 2013 Available online 14 October 2013 Keywords: Chronic myeloid leukemia Extramedullary disease Imatinib Pyk2 Adhesion Migration

a b s t r a c t Concern about extramedullary relapse (EMR) despite favorable response in the bone marrow has been raised with the use of imatinib for treatment of chronic myeloid leukemia (CML). In the present study we show an increase in adhesion, migration and invasion capabilities of the CML cell line K562 following imatinib administration. Imatinib induced upregulation of Pyk2 mRNA and protein levels. Pyk2 inhibition resulted in a reduction of K562 cells’ adhesion and migration subsequent to imatinib treatment. This effect was similar to that shown by us previously with all trans retinoic acid (ATRA) in the acute promyelocytic leukemia (APL) cell line NB4. Our data pinpoint Pyk2 as a shared key mediator of targeted-therapy induced adhesion and migration and may implicate that targeting Pyk2 may serve as an effective therapeutic strategy to reduce EMR in APL and CML. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Chronic myeloid leukemia (CML) is a clonal hematopoietic disorder characterized by the Philadelphia chromosome (Ph), ensuing a translocation between chromosomes 9 and 22. This results in the formation of the BCR–ABL fusion gene encoding for an aberrant tyrosine kinase with an oncogenic activity. Since the BCR–ABL protein is not expressed in normal cells but is expressed and active in >90% of the CML cells, it was possible to design an effective targeted molecular therapy that inhibits BCR–ABL kinase activity in leukemic cells with little effect on the normal cell population [1,2]. Imatinib was the first targeted therapy used for the treatment of CML patients. Since its introduction, it has rapidly become firstline therapy for CML, with the attainment of complete hematologic remission in 95% of patients and very high rates of cytogenetic and molecular response [3–5]. However, this success story is not without any detrimental effects such as the development of resistance to imatinib treatment, most often due to point mutations in the

∗ Corresponding author at: Felsenstein Medical Research Center, Rabin Medical Center, Beilinson Hospital, 39 Jabotinski Street, Petah-Tikva 49100, Israel. Tel.: +972 3 9376795; fax: +972 3 9376744. E-mail address: [email protected] (G. Granot). 1 These authors contributed equally to this work. 0145-2126/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2013.10.005

kinase domain of the ABL gene [6]. Furthermore, although very rare in CML in the past, when patients were treated with chemotherapy and/or interferon, cases of extramedullary relapse (EMR) have been increasingly reported in the imatinib era during the past few years [1,2,7]. It is difficult to quantify these cases in terms of incidence of this phenomenon since they are presented as case reports and not reported in large series, thus publication bias might be introduced. Yet, the number of these cases is impressive and increasing compared to the pre-imatinib era [8,9]. EMR in CML usually suggests an accelerated phase or blast crisis [10–13] as well as being the first manifestation of post allogeneic transplant relapse [14–16]. Breccia et al. [17] were first to report the occurrence of extramedullary blast crisis manifested by pleural effusion containing Ph(+) cells in a patient treated with imatinib in major cytogenetic response, followed by numerous other reports of EMR in CML patients. The most common extramedullary sites reported are the lymph nodes (10–61% of cases), followed by bone (around 37% of cases) and skin or soft tissues (29%) [10–12,18]. Several mechanisms could contribute to the development of EMR in CML patients treated with imatinib: prolonged survival of patients receiving imatinib-containing regimens may allow for disease progression arising from dormant cells in sanctuary sites despite response in the bone marrow; another possible mechanism is poor penetration of this drug to sanctuary sites. Petzer et al. documented a 2-log reduction of imatinib levels in the cerebrospinal

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fluid compared to plasma levels [19]. We support an alternative concept proposing enhancement of leukemic cells’ adhesion and migration abilities via exposure to this drug [20]. Hematopoietic cell migration and adhesion are complex, highly regulated processes mediated in part by members of the integrin superfamily [21,22] and controlled by cues from microenvironmental niches or from external signals triggered by drugs [23]. Despite the importance of these processes, the mechanisms regulating adhesion and migration remain largely unknown. Focal adhesion complexes linking the extracellular matrix (ECM) and the cell cytoskeleton play an important role in these processes [23,24]. Several known signaling enzymes, including protein kinase C (PKC), Src, focal adhesion kinase (FAK), proline-rich tyrosine kinase 2 (Pyk2) and adaptor proteins such as paxillin, talin and vinculin are localized to focal adhesion sites through interactions with the intracellular domains of integrins, linking them to the actin-containing cytoskeleton [23,25,26]. We have recently shown an increase in the migration, adhesion and invasion capabilities of NB4 cells (APL cell line) following treatment with the targeted therapy all trans retinoic acid (ATRA), routinely used for the treatment of APL patients and identified Pyk2 as one of the major mediators of these processes [27]. In agreement with our previous report, our current studies reveal that CML cell lines and patient cells also demonstrate elevated Pyk2 expression when exposed to the targeted therapy imatinib. This increase in Pyk2 expression subsequently led to enhanced adhesion, migration and invasion capabilities of K562 cells. Clinical observation proposes that EMR is a broad phenomenon in the targeted therapy era. Our data raise the possibility that the origin of EMR occurrence following targeted therapy exposure may be common to a substantial portion of the targeted agents administered today. The results presented here together with our previous publication [27] put a spotlight on Pyk2 as a key mediator of targeted therapydependent migration and adhesion, implicating that targeting this kinase might serve as an effective therapeutic strategy to reduce EMR in APL and CML patients. 2. Materials and methods 2.1. Cell lines K562 cells (CML cell line, ATCC) and NB4 cells (kindly provided by Dr. Ben-Zion Katz, Tel-Aviv Sourasky Medical Center, Israel), were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 1% penicillin and streptomycin and cultured at 37 ◦ C in a humidified incubator with 5% CO2 . 5 × 104 cells/ml were used for all experiments.

2.4. Real-time PCR Total RNA was extracted using Ambion RNAqueous-4PCR kit (Ambion) following the manufacturer’s instructions. RNA was reverse transcribed into cDNA using high capacity cDNA RT kit (Ambion). Real-time PCR was performed using the SDS 7000 machine (Applied Biosystems) using FAM dye-labeled TaqMan probes (Ambion). GusB was used as control. Relative expression was calculated using the Ct method. 2.5. Immunoblot analysis Cells were lysed using lysis buffer (50 mM Tris–HCL pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitor cocktail). Equal amounts of protein were separated by SDS-PAGE. After protein transfer, membranes were incubated with primary antibodies and secondary horseradish peroxidase-conjugated antibodies (Jackson Laboratories). Detection was performed with a chemiluminescent peroxidase substrate (Sigma–Aldrich). Primary antibodies: ␣-Pyk2(N-19) (Santa Cruz Biotechnology), ␣-Tubulin (Sigma–Aldrich), ␣-GAPDH (Santa Cruz Biotechnology). 2.6. Adhesion assay 40 mg/ml FN was added to 6-well plates and incubated overnight at 4 ◦ C. Next, treated or untreated cells were added to the FN-covered wells and allowed to adhere at 37 ◦ C for varying time lengths. Fractions of floating and adherent cells were separated and counted under a light microscope. 2.7. Migration and invasion assays Cells were plated on the upper side of a polycarbonate membrane of a Boyden chamber in medium without serum. Uncoated polycarbonate membranes were used for transwell migration assays (Calbiochem) and ECM-coated membranes were used for invasion assays (BD Cell BioLabs) (96-well insert; pore size, 5 ␮m). In both assays, serum was used as a chemoattractant in the lower chamber. The cells were incubated for 3–4 h at 37 ◦ C. Cells that had migrated to or invaded the lower chamber were transferred to a black 96-well plate and labeled with calcein-AM or with CyQuant GR fluorescent dye, respectively. The number of migrating or invading cells was detected by a fluorescent measure (excitation wavelength: ∼485 nm; emission wavelength: ∼520 nm). 2.8. ShRNA-mediated knockdown of pyk2 K562 cells were transduced with 2 different pyk2 specific shRNApGIPZ lentiviral clones (clone IDs: V2LHS 210405, V2LHS 172999, Open Biosystems) according to the manufacturer’s instructions. All clones were infected at a concentration of MOI = 15Tu/cell. The cells were seeded at a concentration of 1.5 × 105 cells/well, and positive clones were selected after 48 h and maintained on puromycin (0.5 ␮g/mL). A nonsilencing shRNA (RHS4348, Open Biosystems) with no homology to known mammalian genes was used as a non-targeting negative control. 2.9. Statistics Two tailed, unpaired student’s t-test was used. Statistical significance was given at p < 0.05.

3. Results 2.2. Leukocyte isolation According to the local policy of our institute we initiate treatment to newly diagnosed CML patients with escalating doses of imatinib, thus a dose of 100 mg/day is administered on days 1–7, a dose of 200 mg/day is used on days 8–14, a dose of 300 mg/day is given on days 15–21, and a dose of 400 mg/day is delivered from day 22 and thereafter. This research was approved by the Rabin Medical Center Institutional Review Board (IRB) and Ethics Committee. All healthy volunteers were members of the Experimental Hematology Lab in the Felsenstein Medical Research Center at the Rabin Medical Center and verbal consent was documented from them according to IRB and ethics committee procedures. Peripheral blood samples were drawn, after informed consent had been obtained, from newly diagnosed patients before and on days 5 and 30 of treatment with imatinib as well as from healthy volunteers. Only blood samples with 67–70% of white blood cells being granulocytes (according to routine blood counts) were used. Blood samples, meeting this criterion, were treated with erythrocyte lysis buffer (Qiagen). 2.3. Reagents Imatinib (LC Laboratories) was dissolved in sterile double distilled water and added to the cells at a final concentration of 1 ␮M. Fibronectin (FN) (Biological Industries Ltd.) was used at a concentration of 40 mg/ml. Recombinant human interferon gamma (ProSpec-Tany Techno Gene Ltd.) was dissolved in sterile double distilled water and added to the cells at a final concentration of 100 ng/ml.

3.1. Imatinib induces Pyk2 mRNA and protein expression in K562 cells Cell adhesion and migration requires organized changes in cytoskeletal structures and in focal adhesion sites. We have previously shown that the mRNA and protein expression of the focal adhesion protein Pyk2 is elevated in NB4 cells exposed to ATRA [27]. We therefore sought to explore whether the targeted therapy, imatinib, has a similar effect on Pyk2 expression in K562 cells. To this end, K562 cells were exposed to 1 ␮M imatinib for 24 h and Pyk2 expression was determined by real-time PCR and Western blot. Imatinib increased Pyk2 mRNA levels by 2.5-fold (p < 0.004) and protein levels by 2-fold (p < 0.002) (Fig. 1A and B). Notably, Pyk2 mRNA expression remained unchanged following imatinib treatment in leukocytes derived from healthy donors (Fig. 1C). In order to assess whether this is a BCR–ABL dependent manifestation, we studied Pyk2 mRNA expression in a BCR–ABL negative hematopoietic cell line; NB4 following exposure to 1 ␮M imatinib. Since we did not expect these BCR–ABL negative cells to be sensitive to imatinib,

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Fig. 1. Imatinib induces Pyk2 mRNA and protein expression in K562 cells. K562 cells were left untreated or treated with 1 ␮M imatinib for 24 h. Pyk2 mRNA (A) and protein (B) expression were determined by real-time PCR using Pyk2 specific primers as well as GusB control primers and by Western blot using Pyk2 specific antibodies and GAPDH antibody as a standard control. (C) Freshly isolated leukocytes from 4 healthy volunteers were treated or not with 1 ␮M imatinib for 24 h. RNA samples were prepared and Pyk2 mRNA expression was measured by real-time PCR. The real-time PCR histograms show the percentage of Pyk2 expression normalized to GusB ± SEM from at least 3 experiments. The Western blot histograms show the percentage of Pyk2 expression normalized to GAPDH ± SEM from at least 3 experiments. (D) NB4 cells were left untreated or treated with 1 ␮M and 10 ␮M imatinib for 24 h. Pyk2 mRNA expression was determined by real-time PCR using Pyk2 specific primers as well as GusB primers as a standard control. IM; imatinib. *p < 0.004, **p < 0.002.

we also exposed them to very high concentrations (10 ␮M) of imatinib. As seen in Fig. 1D, Pyk2 mRNA expression remained unchanged following exposure to imatinib, even following incubation with the high 10 ␮M concentration. 3.2. Imatinib induces K562 cell adhesion, migration and invasion Pyk2 has been implicated in mediating cell adhesion and motility [28]. In our previous work we also showed that increased Pyk2 expression in NB4 cells exposed to ATRA paralleled the adhesion, migration, and invasion capabilities of these cells [27]. We therefore evaluated the effect of imatinib on the adhesion ability of the K562 cell line to FN. Untreated K562 cells demonstrated very low adhesion potential when seeded on FN (∼6% of the cells). However, a 24 h exposure to 1 ␮M imatinib prior to FN seeding resulted in a 5-fold increase in K562 cells adhering to FN (p < 0.0001) (Fig. 2A). While performing these experiments we noticed that when the adherent and floating cell populations were incubated separately for 24 h in the presence of 1 ␮M imatinib, the floating cells were 50% more sensitive to the treatment (data not shown). These data suggest that the acquired adhesive phenotype of K562 cells following exposure to imatinib may grant the cells with a certain level of resistance to this treatment. In contrast to our previous observation that NB4 cells adhere to FN even 5 days after ATRA withdrawal [27], upon withdrawal of imatinib K562 cells quickly (as soon as 3 h post imatinib

withdrawal) stop adhering, demonstrating that the enhanced adhesion of these cells is strictly dependent on the presence of imatinib in the culture medium (data not shown). In comparison, the ability of K562 cells to adhere to FN following treatment with interferon, which was considered the “gold standard” treatment for CML before imatinib, was significantly lower (6% of the cells adhered) than their ability to adhere following imatinib treatment (32% of the cells adhered) (Fig. 2B). Similarly, the adhesion ability of the BCR–ABL negative hematopoietic cell line; NB4 was unchanged following exposure to 1 ␮M imatinib (Fig. 2F). The adhesion ability of these cells was also not enhanced and even reduced following exposure to high concentrations (10 ␮M) of imatinib (Fig. 2F). These data, along with the fact that Pyk2 expression in NB4 cells was not influenced by imatinib treatment (see Fig. 1D), support our hypothesis that the effect of imatinib on Pyk2 expression and on cellular adhesion is probably BCR–ABL dependent. Untreated K562 cells exhibited little basal cell migration, whereas exposure to imatinib increased their migration capabilities by over 7-fold (p < 0.0001) (Fig. 2C). Similarly, the invasion rate of imatinib-treated K562 cells through an ECM-coated membrane in response to FBS as a chemoattractant was significantly higher (p < 0.001) than that of the untreated cells (2.4% compared to 1.27%, respectively in the presence of 30% FBS and 1.8% compared to 1.2%, respectively, in the presence of 10% FBS) (Fig. 2D). Importantly, leukocytes derived from healthy volunteers demonstrated

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Fig. 2. Imatinib induces K562 cell adhesion, migration and invasion. (A) K562 cells were left untreated or treated with 1 ␮M imatinib for 24 h and then were allowed to adhere to FN-coated wells. Shown is the adherent K562 cell population as photographed by light microscope and the percentage of adherent K562 cells. (B) K562 cells were left untreated or treated with 100 ng/ml interferon gamma for 24 h. The cells were allowed to adhere to FN-coated wells. Migration (C) and invasion (D) were tested using a modified Boyden Chamber assay. K562 cells treated or not treated with imatinib were seeded in the upper chamber while the lower chamber was filled with medium with or without serum as a chemoattractant. Migrated or invaded cells recovered from the lower chamber were labeled with fluorescent dye and counted as described in Materials and Methods. (E) Freshly isolated leukocytes from 4 healthy volunteers were treated or not with 1 ␮M imatinib for 24 h and (F) NB4 cells were treated or not with 1 ␮M or 10 ␮M imatinib for 24 h. The cells were plated on FN-coated wells. The adherent cell fraction was counted under a light microscope. Shown is the percentage of adherent leukocytes ± SEM from at least 3 experiments. All results are presented as the mean ± SEM from 3 separate experiments. The adhesion experiments were performed in duplicates, the migration experiments were performed in triplicates. IM; imatinib. *p < 0.0001.

no increase, and even a slight decrease, in adhesion abilities following exposure to imatinib (Fig. 2E).

3.4. Pyk2 expression and induction of adhesion in CML patient’s leukocytes following imatinib treatment

3.3. Pyk2 knockdown inhibits imatinib-induced adhesion and migration of K562 cells

Finally, to extend these observations we assessed the in vivo effect of imatinib on Pyk2 mRNA expression in leukocytes from 4 CML patients before and during imatinib treatment. In leukocytes derived from 3 patients, imatinib was proven to increase Pyk2 mRNA levels. This was evident as soon as 5 days after the initiation of imatinib treatment, when patients were treated with 100 mg imatinib, where Pyk2 mRNA expression was enhanced 1.5fold compared to baseline. Pyk2 mRNA expression continued to increase and reached an increment of 3-fold on day 30 of imatinib treatment, when patients were treated with 400 mg imatinib (p < 0.006) (Fig. 4A). In one patient tested, Pyk2 mRNA expression remained unchanged even after one month of imatinib treatment (Fig. 4A). Interestingly, this patient experienced a suboptimal response to imatinib during follow-up according to the updated European LeukemiaNet recommendations for the management of CML and treatment was switched later to nilotinib [29]. In order to confirm our results, the adhesion ability of CML patient leukocytes was studied in vitro. Thus, freshly isolated leukocytes from one of the above mentioned 3 newly diagnosed CML patients were either

In order to assess whether Pyk2 is essential for imatinibdependent adhesion and migration of K562 cells, these cells were infected with increasing concentrations of 2 different Pyk2 specific shRNAs cloned into the pGIPZ lentiviral expression vector. Our optimization studies indicated that infection with clone #V2LHS 172999, at a concentration of 1.63 × 108 Tu/ml (MOI = 15 Tu/cell), significantly decreased Pyk2 expression by ∼80% at the mRNA level and by ∼60% at the protein level, in comparison with non-targeting shRNA infected cells (Fig. 3A). When exposed to imatinib, the Pyk2-shRNA-infected K562 cells showed a statistically significant (p < 0.02) 2-fold reduction in adhesion as compared to parental K562 cells and to cells infected with non-targeting shRNA control (Fig. 3B). Additionally, these cells exhibited over 5-fold reduction in migration potential when exposed to imatinib (p < 0.002) (Fig. 3C). These data support our hypothesis that Pyk2 is required for imatinib-mediated adhesion and migration.

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Fig. 3. Pyk2 knockdown inhibits imatinib-induced adhesion and migration of K562 cells. K562 cells were infected with 2 Pyk2-specific shRNA and with a non-targeting shRNA (for specific clone number see Materials and Methods). (A) mRNA and protein expression of Pyk2 in Pyk2-knockdown cells analyzed by real-time PCR and Western blot analysis, respectively. Cells infected with the Pyk2-specific shRNA and with the non-targeting shRNA were treated or not with imatinib. These cells were seeded in transwells and their adhesion (B) and migration (C) abilities were determined. K562 cell adhesion was tested on FN-coated wells. Cells were treated with 1 ␮M imatinib and allowed to adhere. The adherent cell fraction was counted under a light microscope. Migration was tested using a modified Boyden Chamber assay. K562 cells treated or not treated with 1 ␮M imatinib for 24 h were seeded in the upper chamber while the lower chamber was filled with medium with or without serum as a chemoattractant. Migrated cells recovered from the lower chamber were labeled with fluorescent dye and counted as described in “Materials and Methods”. All results are presented as the mean ± SEM from 3 separate experiments. IM; imatinib. *p < 0.02, **p < 0.002.

left untreated or treated in vitro with 1 ␮M imatinib for 24 h. As is evident in Fig. 4B, untreated CML leukocytes had virtually no adhesion potential (0.24% adhesion) whereas exposure to imatinib increased their adhesion capabilities considerably to 24%. 4. Discussion Extramedullary progression despite bone marrow remission is a rare phenomenon in CML patients. Nevertheless, with the introduction of imatinib, there has been increasing concern about this complication. A possible explanation for the association of EMR and imatinib treatment could be life prolongation by this sophisticated treatment allowing sufficient time for disease progression in extramedullary sites. Alternatively, extramedullary progression despite remission in the bone marrow may arise from cells’ propagation in sanctuary sites, where imatinib is less accessible. We support an alternative concept, suggesting that imatinib exposure might result in the acquisition of enhanced adhesion and migration virtues of the leukemic cells via overexpression of Pyk2. In the present study we showed an increase of adhesion, migration and invasion features of imatinib-treated K562 cells in a Pyk2-dependent manner. This information is consistent with the existing literature showing increased cell adhesion and migration of these cells with imatinib treatment [30,31].

We have previously shown upregulated Pyk2 expression in an additional hematological malignancy, APL treated with ATRA [27]. Collectively our data state that ATRA and imatinib induce Pyk2 mRNA expression. The C/EBPb transcription factor has been shown to be important for Pyk2 transcription [32]. It is possible that the molecular mechanism accountable for Pyk2 upregulation following ATRA or imatinib exposure is through enhanced C/EBPb transcription factor expression. Supporting this notion is the fact that ATRA, for example, has been shown to induce an increase in protein level and binding activity of C/EBPb [33]. In contrast to ATRA, imatinib has not been shown to affect C/EBPb expression to date. Taken together, these data clarify the reason for the upregulation in Pyk2 mRNA transcript in cells exposed to ATRA and may provide a reasonable explanation for the upregulation in Pyk2 mRNA transcript in cells exposed to imatinib. Several studies have identified Pyk2 as a key element in controlling cellular processes such as cellular polarization, adhesion and migration [34]. In agreement, we identified Pyk2 as a major factor in regulating ATRA- and imatinib-induced adhesion. Recent studies have highlighted that within a tumor exists a small subpopulation of cancer cells that are thought to be the driving force involved in carcinogenesis, local invasion and metastasis [35,36]. Moreover, accumulating data support the idea that these cells could play an important role in chemo-resistance [37]. Therefore, while this is a

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Fig. 4. Pyk2 expression is upregulated in CML patients receiving imatinib treatment. (A) RNA samples were prepared from freshly isolated leukocytes of newly diagnosed CML patient at days 0, 5, 30 imatinib treatment (for specific treatment course, see Materials and Methods). Pyk2 expression was measured by real-time PCR. Three patients were shown to have increased expression levels of Pyk2 mRNA during the course of their treatment, while in one patient the expression of Pyk2 mRNA remained unaffected by imatinib exposure. *p < 0.005, **p < 0.0025. (B) Freshly isolated leukocytes from a newly diagnosed CML patient were left untreated or treated in vitro with 1 ␮M imatinib for 24 h and were then allowed to adhere to FN-coated wells. Shown is the percentage of adherent leukocytes.

relatively small population, since these cells are considered to be chemo-resistant, it is quite possible that these cells may be the origin of EMR after an initial favorable response to treatment. It has long been known that interactions between environmental factors such as extracellular matrix (ECM) and cellular counter-receptors may contribute to tumor cell survival during exposure to cytotoxic stress such as radiation [38]. Teicher et al. showed that mammary tumors made resistant to alkylating agents in vivo were sensitized to cytotoxic drugs once removed from their ECM surroundings [39]. Over a decade ago, it was realized that cell–cell or cell–ECM adhesion can regulate apoptosis and cell survival in a wide variety of cell types [40–42]. For instance, adhesion has been shown to parallel upregulated p27kip1 expression and acquired resistance to chemotherapeutic agents in several cell lines [43]. In a different paper, adhesion to ECM has been reported to induce P-glycoprotein expression and confer doxorubicin resistance in rat hepatocytes [44]. This observation was termed “cell adhesion-mediated drug resistance” (CAM-DR). It is possible that in the case of CML, the cells that acquire the adhesive phenotype following exposure to imatinib are the core of a resistant subpopulation. The emergence of such an adhesion-dependent chemo-resistant population and the occurrence of CAM-DR have been recognized as important mechanisms of resistance to therapy in CML as well. Puissant et al. have observed that continuous imatinib treatment selects for a subpopulation of imatinib-resistant and highly adherent CML cells [30]. Damiano et al. have shown that the CAM-DR phenotype in CML cells is not the result of decreased drug accumulation [45]. A possible explanation for the CAM-DR phenomenon in K562 cells treated with imatinib is the fact that integrins, which are responsible for the interaction with the ECM, are known to mimic the activation of signal transduction cascades activated by BCR–ABL, effectively reconstituting the survival pathway inhibited by imatinib [46]. This is consistent with our findings that the adherent K562 cells were less responsive to imatinib treatment. Strikingly, Puissant et al. continue to show that upon withdrawal of imatinib, the adherent cells quickly

became round with a very slight adhesion phenotype, demonstrating, similar to what we have witnessed, that the enhanced adhesion of the cells is strictly dependent on the presence of imatinib in the medium. Since CAM-DR has been acknowledged as a significant mechanism of resistance to chemotherapy in hematopoietic malignancies and solid cancers, anti-adhesion strategies aimed at evading such resistances have received increasing attention. These anti-adhesion strategies include approaches such as targeting of surface antigens or inhibition of cell adhesion-associated proteins such as the focal adhesion kinase, Fak. Our demonstration of the existence of a subpopulation of CML cells that overexpresses the Pyk2 kinase and possess an acquired adhesive phenotype may represent the starting point for the use of anti-adhesion therapies in hematopoietic malignancies where CAM-DR may be activated. In this respect, an original therapeutic approach could be combining TKIs with anti-adhesion molecules such as Pyk2 antagonists to prevent the occurrence of a rare cell population with high invasive potential culminating with the relapse in extramedullary sites. An interesting observation of this study was that while the expression of Pyk2 mRNA in leukocytes derived from patients responding to imatinib increased, it remained unchanged, even after one month of treatment, in the single patient with suboptimal response. The variability in patients’ sensitivity and response to imatinib is mainly due to differences in the efficiency of imatinib uptake and retention [47]. These might be induced by variability in the function of hOCT-1 which is an influx transporter, responsible for the uptake of imatinib into CML cells [48–50]. The suboptimal response of this specific patient could be due to low intracellular concentration of imatinib and these low concentrations can be the reason for the lack of increase in Pyk2 expression in this patient. Relying on the data presented here and in a previous paper published by us recently [27], we would like to propose that extramedullary progression despite good response in the bone

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marrow may be a more global phenomenon in the era of sophisticated targeted therapies, requiring our attention. In this paper we have suggested that a conceivable explanation for the increment of EMR in CML may be enhancement of leukemic cells’ adhesion and migration abilities via imatinib. In our previous paper we showed an ATRA-dependent increase in adhesion, migration and invasion capabilities of the APL cell line, NB4 [27]. In both cases, Pyk2 was found to be a key regulator of these processes. Intuitively, one may contemplate a particular and individual mechanism for EMR for each targeted therapy. For example, poor penetration of imatinib (but not of ATRA in APL) to sanctuary sites such as the cerebrospinal fluid may enable disease progression arising from CML cells ‘hidden’ in this sanctuary site, not accessible to the treatment [19]. However, further to these separate mechanisms, we suggest a common denominator for the tendency of some of the new targeted agents to be associated with extramedullary disease despite a good response in the bone marrow, mediated through overexpression of Pyk2 kinase. Thus, we suggest a more active role of these agents in the occurrence of EMR. Agents such as ATRA and imatinib could induce expression of adhesion molecules that promote migration and extravasation of the leukemic cells and eventually encourage adhesion to extramedullary sites, forming a reservoir of viable cells. The latter can later proliferate and expand, thus resulting in an extramedullary relapse. Since the use of sophisticated therapies such as ATRA and TKIs is broadening, we should be aware that alongside their therapeutic effect they may potentially obtain a detrimental nature with respect to extramedullary manifestation in hematological malignancies. Further studies are needed to see whether this phenomenon is common also to other targeted therapies used for other hematological malignancies and if so is there a role for Pyk2 in these instances as well. Funding source The source of funding for this work was the Van Bits Scholarship donated by the Cancer Research Center, Tel-Aviv University, Israel and the Boaz Adar foundation from the Israel Cancer Association. Conflict of interest The authors have no conflicts of interest to declare. Acknowledgements Contributors: A.O., G.G., O.H.R. and J.P. acquisition of data, analysis and interpretation of data. G.G. and P.R. conception and design of the study. G.G. drafting the article, final approval of the version to be submitted. O.S. and P.R. revising the article, final approval of the version to be submitted. A.O. and G.G. contributed equally to the work. References [1] Melo JV, Barnes DJ. Chronic myeloid leukaemia as a model of disease evolution in human cancer. Nat Rev Cancer 2007;7:441–53. [2] Druker BJ. Translation of the Philadelphia chromosome into therapy for CML. Blood 2008;112:4808–17. [3] Hughes T, Deininger M, Hochhaus A, Branford S, Radich J, Kaeda J, et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR–ABL transcripts and kinase domain mutations and for expressing results. Blood 2006;108:28–37. [4] O’Brien SG, Guilhot F, Larson RA, Gathmann I, Baccarani M, Cervantes F, et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2003;348:994–1004. [5] Druker BJ, Guilhot F, O’Brien SG, Gathmann I, Kantarjian H, Gattermann N, et al. Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med 2006;355:2408–17.

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