AbstractMantle cell lymphoma and other lymphoma subtypes often spread to the bone marrow, and stromal interactions mediated by focal adhesion kinase frequently enhance survival and drug resistance of the lymphoma cells. To study the role of focal adhesion kinase in mantle cell lymphoma, immunohistochemistry of primary cases and functional analysis of mantle cell lymphoma cell lines and primary mantle cell lymphoma cells co-cultured with bone marrow stromal cells (BMSC) using small molecule inhibitors and RNAi-based focal adhesion kinase silencing was performed. We showed that focal adhesion kinase is highly expressed in bone marrow infiltrates of mantle cell lymphoma and in mantle cell lymphoma cell lines. Stroma-mediated activation of focal adhesion kinase led to activation of multiple kinases (AKT, p42/44 and NF-κB), that are important for prosurvival and proliferation signaling. Interestingly, RNAi-based focal adhesion kinase silencing or inhibition with small molecule inhibitors (FAKi) resulted in blockage of targeted cell invasion and induced apoptosis by inactivation of multiple signaling cascades, including the classic and alternative NF-κB pathway. In addition, the combined treatment of ibrutinib and FAKi was highly synergistic, and ibrutinib resistance of mantle cell lymphoma could be overcome. These data demonstrate that focal adhesion kinase is important for stroma-mediated survival and drug resistance in mantle cell lymphoma, providing indications for a targeted therapeutic strategy.
Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with a poor prognosis, and a significant number of patients relapse after treatment.1 Promising results can be achieved in relapsed or refractory MCL with ibrutinib, a small molecule inhibitor of Bruton tyrosine kinase (BTK), with a significant improvement in progression-free survival. However, despite this, primary resistance to ibrutinib occurs in one-third of all patients. Acquired secondary resistance has also been described.42 Although some mechanisms of resistance, such as activation of the alternative NF-αB signaling pathway,5 mutations in the BTK binding site and others6 have been identified, most mechanisms of ibrutinib resistance remain unclear, and multiple mechanisms are likely to be involved.
In several B-cell malignancies, stromal interactions support cell survival, and it has been shown that in MCLs bone marrow (BM) stromal interaction can increase drug resistance.7 Over 90% of MCL patients have extranodal manifestations, and especially the aggressive blastoid variant of MCL is characterized by bone marrow involvement. Homing to the BM requires the expression of adhesion molecules on the lymphoma cells and intact intracellular signaling, with the classic and alternative NF-κB signaling pathway being some of the major components.7
Recently, focal adhesion kinase (FAK), a major signaling molecule that functions downstream of integrins and that translates signals from the extracellular matrix,98 has gained attention as a drug target in the treatment of solid tumors. Several studies have demonstrated that FAK can enhance cell proliferation, survival and migration in response to stromal interaction.1110
Therefore, we chose to study the role of FAK in BM stroma-mediated enhancement of MCL proliferation and survival. We identified FAK inhibition as a possible mechanism of restoring the ibrutinib response, which makes it an attractive target for combination treatment, especially in patients who present with BM involvement.
Primary cases and cell lines
Thirty primary MCL cases [10 typical MCLs, 10 MCLs of the blastoid variant, and 10 paired typical MCL samples of BM infiltrates and extramedullary infiltrates (lymph node or gastro-intestinal tract)] were selected from the files of the Institute of Pathology, University of Wuerzburg, Germany. The cases were classified according to the World Health Organization (WHO) classification as typical MCL or as blastoid variant. All human specimens were processed after informed consent in compliance with the institutional review board of the Faculty of Medicine of the University of Wuerzburg, Germany, and conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.
Nine well-characterized and widely used MCL cell lines were used in this study: Granta 519, Z138C, HBL-2, REC-1, JEKO, MINO, MAVER, JVM-2 and UPN-1. BM stromal cells (BMSC) were isolated from BM samples from patients as previously described.12 For co-culture experiments, BMSC were plated overnight, and after confirming the confluence of the stroma layer, medium was replaced by 5×10 MCL cells in RPMI-1640. Drugs were added after 4 hours (h) of incubation and ibrutinib was pre-incubated for 30 minutes (min) before addition of VS-6063.
Immunoreagents and inhibitors
The following antibodies were used for immunoblotting and immunohistochemistry: FAK, pFAK (Tyr397), pPaxillin (Tyr118), pAKT (Ser473), actin, p-p42/44 (Tyr202/204), pGSK3β (Ser9), pIκBα (Ser32/36), IKKα, pIKKα/β (Ser176/180), p52, cleaved caspase-3, anti-mouse and anti-rabbit IgG horseradish peroxidase (HRP)-linked from Cell Signaling (Beverly, MA, USA). Cyclin D1 was obtained from Thermo Scientific (Waltham, MA, USA); c-Myc was from Abcam (Cambridge, UK). Immunodetection was performed with the DAKO REAL detection kit (DAKO GmbH, Hamburg, Germany).
The following inhibitors and immunoreagents were used: VS-6063 (Selleckchem, Muenchen, Germany), ibrutinib (Selleckchem, Muenchen, Germany), and rhCXCL-12 (R&D Systems, Wiesbaden, Germany).
Western blot analysis, immunoprecipitation and immunohistochemistry
Western blot analysis, immunoprecipitation and immunohistochemistry were performed as previously described.13
microRNA sequences, plasmid constructs and lentiviral transduction
A short hairpin oligonucleotide directed against FAK was designed. The sense strand of the FAK-microRNA sequence was TGCTGTTGACAGTGAGCGAAGCGATTATGTTAGAGATAT AGTGAAGCCACAAGATGTATATCTAACATATAATCGCTCT GCCTACTGCCTCGGA. FAK oligonucleotide and a control microRNA, which lacks complementary sequences in the human genome, was cloned into lentiviral Tet-regulated expression vector (pRRLT3GmiR-E, a kind gift from J. Zuber13 to MTR).
Molecular targets for therapy test and combination treatment
The molecular targets for therapy (MTT) test was performed as previously described.13 Synergism was assessed by the Chou-Talalay method,14 calculating combination index (CI) values using CalcuSyn software (Biosoft, Cambridge, UK).
Matrigel-coated nucleopore filter inserts in a 24-well transwell chamber (Corning Biocoat, New York, USA) were used for the invasion assays. Cells (treated or untreated with VS-6063) were seeded at a density of 40,000 cells/well into the upper part of the Matrigel-coated filter, and RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum and rhCXCL-12 (R&D Systems, Wiesbaden, Germany) (1 ng/mL) was added to the lower part. After 24 h, the cells that had migrated through the Matrigel and the 8-μm pore-size membrane were fixed, stained, and counted under a light microscope.
Continuous variables and categorical variables were compared by t-test or Fisher exact test. All reported P-values were two-sided; P<0.05 was considered statistically significant.
FAK is highly expressed in bone marrow infiltrates of MCL and in MCL cell lines
We first examined the expression of FAK in primary MCL samples by immunohistochemistry. Cases selected were lymph node infiltrates of MCL that were classified according to the WHO as 10 typical MCLs and 10 MCLs of the blastoid variant. They showed only mild (n=14) or no (n=5) expression of FAK, with only one typical case displaying high FAK expression. FAK expression did not correlate with the WHO classification (typical or blastoid) (Figure 1A).
As FAK can be induced and activated by integrin and extracellular matrix signaling,158 we next compared FAK expression in BM infiltrates versus an extramedullary (lymph node or gastro-intestinal tract) manifestation of MCL. We performed immunohistochemistry of 10 paired samples (medullary and extramedullary infiltrates) of typical MCLs. All BM infiltrates were characterized by high FAK expression, whereas only one infiltrate in the colon showed high expression of FAK. All other extramedullary infiltrates displayed no or only weak staining of FAK (Figure 1C). Thus, high FAK expression correlated with infiltration of the BM (P=0.0001).
We then evaluated FAK expression in MCL cell lines. We performed western blot analysis of 9 well-characterized MCL cell lines (Granta 519, Z138C, REC-1, HBL-2, JEKO, MAVER, MINO, JVM-2, UPN-1). All cell lines showed clear FAK expression with the highest levels of FAK expression in MINO and HBL-2. They also showed high expression of pFAK (Tyr397), which is the autophosphorylation and binding site for Src family kinase members (PI3K, PLCα) (Figure 1D).
FAK expression and activation in MCL can be induced by CXCL12
Lymphoma migration and homing requires the co-operation between chemokines, adhesion molecules, ligands and their receptors expressed by stromal and lymphoma cells. MCLs express G-protein-coupled chemokine receptors such as CXCR4 and CXCR5 that bind CXCL12.16 Hence, we determined whether FAK expression and activation could be induced by CXCL12. We cultured two different MCL cell lines, JEKO and Z138C, in the presence of 1 ng/mL rhCXCL12. Western blot analysis was performed after 15, 30 and 60 min. Expression of total FAK and pFAK (Tyr397) increased after incubation with rhCXCL12. In addition, rhCXCL12 induced increased phosphorylation of the direct downstream target of FAK, Paxillin, confirming FAK activation (Figure 2A).
Focal adhesion kinase can mediate cell proliferation and survival in many types of solid and non-solid tumors17 and, therefore, we analyzed downstream targets affected by FAK signaling in MCL. Indeed, FAK expression and activation led to phosphorylation and activation of several downstream targets. An increase in the phosphorylation of p42/44 and AKT, as well as a constant upregulation of c-MYC and Cyclin D1, could be observed (Figure 2B).
Co-culture with BMSC activates FAK signaling in MCL cell lines and primary MCL-cells
We could observe a high FAK expression in BM infiltrates, and incubation of MCL cell lines with rhCXCL12 led to FAK upregulation and activation. As CXCL12 is highly expressed by BMSCs,1918 we determined whether co-culture of MCL with BMSCs could activate FAK signaling. Co-culture of the MCL cell lines Z138C and JEKO with BMSC resulted in FAK upregulation. Western blot analysis revealed a steady increase of FAK and phospho-Paxillin after 8, 24 and 48 h. FAK activation led to the phosphorylation and activation of multiple downstream targets, such as p42/44 and AKT, and to the upregulation of Cyclin D1 (Figure 3A). Interestingly, we could observe interactions of FAK with the NF-κB signaling pathway. FAK activation resulted in an increase in the phosphorylation of IKKα as shown by the immunoprecipitation of IKKα and detection with pIKKα/β. (To our knowledge, there is no phospho-specific IKKα antibody commercially available). IKKα is an important component of the canonical and non-canonical NF-κB pathway.2120 Indeed, phosphorylation of IkBα led to the phosphorylation of IkBα and to an increase in p52 in MCL cell lines (Figure 3C). To rule out cell culture artefacts due to the use of MCL cell lines, we repeated the experiment with primary MCL cells. Western blot analysis showed similar results with an upregulation and activation of FAK after 72 h accompanied by an increase in p52 and increase in the phosphorylation of IkBα and AKT (Figure 3B).
FAK silencing suppresses multiple signaling pathways and FAK is essential for CXCL12 induced activation of several downstream targets
Our results indicate that incubation with rhCXCL12 or co-culture with BMSCs can activate FAK and several downstream targets in MCLs. Although the downstream targets studied are well-established FAK targets, it is possible that these proteins could have been activated through alternative kinases. Therefore, we studied the effect of FAK silencing on the activation of downstream targets.
We used a Tet-inducible microRNA system in the two MCL cell lines HBL-2 and MINO with high constitutive FAK expression and activation. Western blot analysis showed that, after 72 and 96 h of incubation with doxicyclin in both cell lines, FAK expression was completely abolished, whereas expression of the kinase PYK2 with high homology to FAK did not change. As expected, FAK downregulation dramatically decreased or resulted in a complete loss of activation or phosphorylation of the downstream targets Paxillin, p42/44, IkBα, p52, AKT and GSK3β, as well as in downregulation of Cyclin D1 (Figure 4A).
Interestingly, incubation with doxycyclin and silencing of FAK also reduced the upregulation of phosphorylation of AKT, IkBα and p42/44 and upregulation of p52 expression after rhCXCL12 treatment, as shown by western blot analysis for HBL-2 (Figure 4B). This result clearly demonstrates that FAK participates in CXCL12-mediated activation of the AKT, p42/44 and NF-κB signaling pathways.
FAK inhibition leads to inhibition of the alternative and classical NF-κB signaling pathway
Silencing of FAK resulted in the inhibition of multiple downstream targets. There are several FAK-inhibitors available that are currently being evaluated for use in clinical trials for the treatment of solid tumors.2322 Therefore, we determined whether we could achieve a blockage of the downstream targets of FAK in MCL with VS-6063. VS-6063 is a 2-generation inhibitor that is in clinical trials for solid tumors and that targets the FAK/PYK2 kinase domain in an (ATP)-competitive, reversible way.24 We were especially interested in the regulation of the NF-κB signaling pathway, as this pathway was highly activated by co-culture with BMSCs. We performed experiments with different MCL cell lines with activation of the classic or alternative NF-κB signaling pathway. We chose the MINO and HBL-2 cell lines, which are characterized by the activation of the classic NF-κB signaling pathway, and which showed high expression levels of FAK in our western blot analysis. In addition, we chose the cell line Z138C, with a TRAF2 mutation. The TRAF2 mutation leads to an activation of the alternative NF-κB pathway, so Z138C is characterized by the activation of the classic and alternative NF-κB signaling pathway. The cell lines were co-cultured with BMSCs and treated with VS-6063 (100 nM). After 15 min, 8 h, and 24 h, the cells were harvested and western blots were performed. VS-6063 treatment inhibited the phosphorylation of Paxillin, p42/44, AKT and GSK3β. We also observed a downregulation of the total protein levels of Cyclin D1 and c-MYC after 15 min and an increase in cleaved caspase-3 after 8 h (Figure 5A). In each cell line, even in Z138C (carrier of a TRAF2 mutation), an inhibition of the activation of the classic and alternative NF-κB signaling pathway could be observed (Figure 5B and C). There was a clear inhibition of the phosphorylation of IKKα (shown by immunoprecipitation) and IkBα as components of the classic NF-κB signaling pathway. In addition, downregulation of p52 indicated the inhibition of the alternative NF-κB signaling pathway. We confirmed the western blot results with immunofluorescence microscopy. HBL-2 and MINO were treated with VS-6063 (100 nM) for 8 h, the cells were fixed and immunofluorescence staining for p65 was performed. After VS-6063 treatment, the p65 subunit accumulated in the cytoplasma and decreased in the cell nucleus in both cell lines (Figure 5D).
FAK inhibition results in suppression of targeted cell invasion
One major escape mechanism of lymphoma cells treated with chemotherapy is homing to the BM niche. For this reason, we analyzed whether treatment with VS-6063 could inhibit the targeted migration of MCL cells. VS-6063 treatment (100 nM) inhibited the invasion of the MCL cell line MINO, HBL-2, JEKO and Z138C towards medium supplemented with rhCXCL12. After 24 h almost no invasion could be observed in the VS-6063-treated group, whereas untreated cells showed a high invasion towards rhCXCL12 (P=0.0001) (Figure 6).
FAK inhibition acts highly synergistically with ibrutinib treatment
In the treatment of refractory MCL, promising results can be achieved with ibrutinib. However, activation of the alternative NF-κB signaling pathway can lead to primary or secondary resistance to ibrutinib. As FAK inhibition resulted in the suppression of the classic and alternative NF-κB signaling pathway, we questioned whether resistance to ibrutinib could be overcome with VS-6063 treatment, and whether ibrutinib and VS-6063 treatment are synergistic. We chose four MCL cell lines whose resistance to ibrutinib is known: JEKO and MINO (which are responsive to ibrutinib), Z138C (with a TRAF2 mutation), and HBL-2 (which are resistant to ibrutinib).5 Cells co-cultured with BMSCs were treated with escalating doses of ibrutinib and VS-6063 (0–1 μM) for 48 h and MTT-assays were performed. We observed a highly synergistic effect in all four MCL cell lines with CI values less than 0.6 (Figure 7A) (ED50, ED75, ED90) and resistance to ibrutinib could be overcome by combination treatment.
Combination of VS-6063 and ibrutinib leads to complete abrogation of the NF-κB signaling pathway
We observed a high synergistic effect by combination treatment (ibrutinib and VS-6063) of different MCL cell lines. As ibrutinib and VS-6063 can both inhibit the classic and alternative NF-κB signaling pathway, we performed western blot analysis to study the effect on this pathway by the various treatments (VS-6063 or ibrutinib alone (100 nM) or in combination (10 nM). After 48 h of single or combination treatment, western blot assays were performed (Figure 7B). As expected, single treatment with ibrutinib or VS-6063 resulted in the downregulation of the phosphorylation of IkBα, AKT and p42/44, whereas combination treatment resulted in complete abrogation of the phosphorylation. In addition, treatment with VS-6063 alone or in combination with ibrutinib led to the downregulation or complete loss of p52 expression. Therefore, combination treatment resulted in the complete inhibition of the classic and alternative NF-κB signaling pathways.
Mantle cell lymphoma and other lymphoma subtypes frequently spread to the BM, and stromal interactions often lead to enhanced survival and drug resistance.272516 Therefore, targeting deregulated kinases activated by the microenvironment has emerged as a promising strategy for the treatment of lymphomas. It is well known that integrin-mediated signaling cascades play important roles in cell adhesion and interaction with the microenvironment.3028 FAK kinase is activated in response to chemokines, cytokines or ligand-binding, and plays a key role in integrin signaling.311198 Here, we show that FAK was highly expressed in BM infiltrates of MCLs with only weak to moderate expression in lymph node infiltrates and moderate to high expression in MCL cell lines, consistent with previous studies demonstrating weak or moderate expression of FAK in the majority (63%) of lymph node infiltrates of MCLs.32 Earlier studies of MCLs showed that FAK could be activated by BCR or Hedgehog-signaling.3433 A recently published paper35 demonstrated that FAK expression in MCL is regulated by SOX11, and FAK expression leads to invasion of MCL and homing to the BM. As we observed high FAK expression, especially in BM infiltrates, we investigated whether FAK could also be activated by stromal interaction and integrin signaling in MCL. Indeed, FAK activation and expression could be induced in MCL by incubation with rhCXCL12 or co-culture with BMSC. Incubation with rhCXCL12 or co-culture with BMSCs resulted in a significant increase in FAK, and in an increase in the phosphorylation of the autophosphorylation site of FAK and an increase in the phosphorylation of Paxillin, a direct downstream target of FAK.
In addition to cell adhesion and cytoskeletal reorganization, FAK mediates proliferation and survival signals.37361198 Here, we show, that FAK activation by rhCXCL12 or co-culture with BMSCs leads to the activation of AKT and p42/44, two kinases that are important for cell proliferation and survival. In addition, the classic and alternative NF-κB pathway was activated, as shown by the phosphorylation of pIkBα, IKKα and by an increase in p52 expression. Previous studies in MCL, CLL and solid tumors emphasize the importance of the NF-κB signaling pathway in stromal-lymphoma interactions, supporting long-term expansion and drug resistance.40387 In our experiments, FAK was essential for the activation of multiple downstream targets, including the classic and alternative NF-κB signaling cascade, as FAK silencing resulted in a nearly complete abrogation of the activation of AKT, p44/42, pIkBα, and IKKα or in downregulation of p52. This is in line with Balsas et al.35 who were also able to demonstrate a blockage of the PI3K/AKT pathway after FAK inhibition.
These data are clinically highly relevant, given that there are several FAK inhibitors2422 currently being tested in patients. We chose the 2-generation inhibitor VS-6063 to perform the inhibition experiments, as this targets the FAK kinase domain with a better pharmacodynamics profile than first-line inhibitors. In MCL, enhanced activation of several downstream targets (AKT, c-Myc and p42/44) by co-culture with BMSCs could be blocked by the inhibition of FAK with VS-6063. In addition, the downregulation of Cyclin D1, which is up-regulated in MCLs by the t(11;14)(q13;q32) translocation could be achieved after 8–24 h. This effect could be due to reactivation of GSK3β kinase, which targets Cyclin D1 for degradation4341 After 24 h, we observed an increase in cleaved caspase-3 as an indicator that cells underwent apoptosis, which makes FAK an attractive target in MCLs. As previously shown in combination with sorafenib treatment,33 inhibition of FAK also resulted in a dramatic decrease in targeted cell invasion, underlining its key role in tissue microenvironmental regulation of lymphoma dissemination to the BM.
Mesenchymal stromal cells protect MCL from apoptosis through the activation of the classic and alternative NF-κB signaling pathways.7 These pathways have gained great attention as promising new therapeutic targets in lymphoma with activated BCR/NF-κB pathway,4744 and with available BTK-inhibitors such as ibrutinib, a significant efficacy could be achieved in the treatment of refractory or relapsed MCL.4832 However, somatic mutations in NF-κB regulatory genes can confer resistance to ibrutinib treatment in MCLs.495 Interestingly, FAK inhibition resulted in the inhibition of the classic and alternative NF-κB signaling pathways not only in the MCL cell lines MINO and HBL2, but also in Z138C, which has a known TRAF2 mutation and is characterized by the upregulation of p52.5 This result could be achieved by abrogation of the phosphorylation of IKKα, which has been previously described as a substrate of FAK.50 IKKα is a component of both the classic and the alternative NF-κB pathway by functioning within an IkB kinase complex to directly phosphorylate the negative regulator IkB and by facilitating the cleavage of p100 to p52/Rel B.2120 Combined treatment with ibrutinib and FAK inhibition turned out to be highly synergistic, and ibrutinib resistance in HBL2 and Z138C could be overcome by complete inhibition of the alternative and classic NF-κB signaling pathway. This supports previously published data35 demonstrating that FAK confers cell-adhesion-mediated drug resistance and contributes to a more aggressive phenotype.
In conclusion, our results provide evidence that FAK modulates the migratory and prosurvival signals mediated by the microenvironment in MCL. Furthermore, FAK inhibition, especially in combination with ibrutinib, may represent a promising approach to treat patients with advanced MCL presenting with BM involvement.
The excellent technical assistance of Sabine Roth and Barbara Leibbrandt is gratefully acknowledged.
- Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/103/1/116
- Funding This work was supported in part through a grant to M.T.R. (DFG-FOR2314) from the German Research Foundation (DFG).
- Received July 29, 2017.
- Accepted October 16, 2017.
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