Focal Adhesion Kinase as a Potential Target in AML and MDS

Focal adhesion kinase (FAK) coordinates a signaling network that orchestrates a diverse range of cellular processes through both kinase-dependent and independent mechanisms (1). Once activated through integrin (ITG) ligation (2) or growth factor receptor–mediated (3) signals within the tumor microenvironment, FAK cooperates with SRC, leading to SRC phosphorylation and subsequent FAK/SRC phosphorylation at multiple sites, which relays the external signal into cells by activating multiple cell proliferating/survival pathways, such as PI3K/AKT and MAPK, and by regulating the expression of various genes (4). Most recently, FAK was found to be able to generate a tumor-suppressive microenvironment by regulating chemokine transcription and promoting regulatory T-cell (Treg) recruitment and retention in squamous cell carcinoma (5) and inhibition of FAK renders pancreatic cancers responsive to checkpoint immunotherapy (6).

FAK is overexpressed and/or constitutively activated in many solid tumors, and both increased FAK expression and activity are associated with poor clinical outcomes (7–9), suggesting that FAK is a potentially critical target for cancer therapy. Indeed, FAK inhibition has been investigated extensively in solid tumors (10, 11). A recent study in a patient-derived xenograft model of mesothelioma showed that FAK inhibitor VS-4718 preferentially eliminated the cancer stem cells that were enriched following treatment with chemotherapeutic agents (11). VS-4718 treatment in combination with dasatinib prolonged survival in a model of B-cell acute lymphoblastic leukemia (B-ALL; ref. 12). Several FAK inhibitors, such as VS-4718, have entered clinical trials (NCT01849744, NCT02651727) in solid tumors. However, the proposed trial in acute myeloid leukemia (AML; NCT02215629) was withdrawn largely due to lack of preclinical studies of the compound in acute leukemia.

The critical role of the bone marrow microenvironment in leukemia progression and drug resistance has only been recognized and investigated in the past decade (13). Although it is extensively studied in solid tumors, limited work was done with regard to the expression and function of FAK in myeloid leukemia. Recher and colleagues demonstrated that FAK is expressed in approximately 40% of AML patient samples and that high expression of FAK in AML was associated with enhanced blast migration, increased cellularity, and poor prognosis (14). Subsequently, Tavernier-Tardy and colleagues showed that FAK expression negatively associated with overall survival (OS) in AML, and patients overexpressing two to three factors of FAK, CXCR4, and VLA4 had a significantly shorter OS (15). Given the importance of the bone marrow microenvironment in leukemogenesis, progression, and drug resistance, it is not surprising that leukemic cell adhesion molecules play critical roles in regulating the interactions with mesenchymal stromal cells (MSC), an important bone marrow component affecting patient outcome. Although FAK as a therapeutic strategy has not been explored in AML, other agents potentially impacting the bone marrow microenvironment have been investigated. Pharmacologic disruption of the CXCR4–CXCL12 interaction has demonstrated preclinically and in clinical trials that it is capable of mobilizing leukemia cells from the protective bone marrow microenvironment and sensitizes to chemotherapy (16–19). Targeting VLA4/VCAM-1 signaling has also shown the potential of overcoming stroma-mediated chemoresistance in bone marrow–resident leukemia cells (20), indicating that disruption of adhesion pathways in leukemic cells may have a number of clinical utilities.

Utilizing improved proteomic technologies, we examined the expression of FAK in a large cohort of newly diagnosed AML (n = 511) patient samples by reverse-phase protein array (RPPA) and correlated its expression with patient clinical characteristics. As approximately 30% of MDS patients will progress to AML and these patients with secondary AML carry poor prognosis, we also examined the expression of FAK and its clinical correlation in a large cohort of newly diagnosed MDS (n = 133) patient samples. We determined a role of FAK in AML cell lines in vitro and in vivo AML models by knocking down FAK with shRNA and/or using a FAK inhibitor VS-4718. These experiments offer a compelling rationale for the use of FAK inhibitors clinically in the AML setting.

FAK expression in large cohorts of AML and MDS patient samples and normal controls was determined by RPPA as described previously (21, 22). The AML patient population was the same as published previously (23, 24). Briefly, peripheral blood and bone marrow specimens were collected from 511 newly diagnosed AML patients evaluated at The University of Texas MD Anderson Cancer Center (MDACC; Houston, TX) from September 1999 to July 2010. A paired relapse sample was available for 47 patients. Of the 511 AML patients, 415 were treated at MDACC and were evaluable for outcome. For the MDS population, bone marrow or peripheral blood specimens were collected from 133 newly diagnosed MDS patients who were evaluated at MDACC between 1999 and 2007. The 133 newly diagnosed MDS patients include 7 refractory anemia, 8 refractory cytopenia with multilineage dysplasia (RCMD), 3 RCMD and ring sideroblasts, 67 refractory anemia with excess blast, 3 refractory anemia with ringed sideroblasts, 1 myelodysplastic syndrome unclassified, 31 chronic myelomonocytic leukemia, and 13 others. The median age at diagnosis was 68.3 years (range, 26.5–89.4). By International Prognostic Scoring System, 13 were low risk, 48 intermediate-1, 37 intermediate-2, 18 high risk, and 17 unknown. Cytogenetics of the population includes 72 diploid, 31 isolated 5q/7q, 4 isolated 20q, and 26 others. CD34⁺ cells (n = 191, 116 diagnosis and 75 sequential including 73 relapse) and CD34⁺CD38⁻ cells (n = 88, 47 diagnosis and 41 sequential including 40 relapse) were separated as reported previously (21). Sample collection and RPPA analysis were done according to the protocols approved by the MDACC Investigational Review Board. As controls, FAK expression level was also determined in CD34⁺ cells from 16, peripheral blood mononuclear cells from 9, and CD133⁺ cells from 5 normal donors (Table 1). FAK antibody was purchased from Cell Signaling Technology (cat #3285).

OCI-AML3 was provided in 2004 by Dr. M. Minden (Ontario Cancer Institute, Toronto, ON, Canada). Molm13 and Molm14 were obtained in 2005 and 2014, respectively, from the German Collection of Microorganisms and Cell Cultures. MV4-11, purchased in 2006, and KG-1, KG-1a, HL-60, and THP1, all purchased in 1998, were obtained from the ATCC. Cell lines were validated by STR DNA fingerprinting using the AmpF_STR Identifier Kit according to the manufacturer's instructions (Applied Biosystems, cat #4322288). The STR profiles were compared with known ATCC fingerprints and with the Cell Line Integrated Molecular Authentication database (CLIMA) version 0.1.200808 (http://archive.is/http://bioinformatics.istge.it/clima/; ref. 25). The STR profiles matched known DNA fingerprints or were identified as unique (OCI-AML3). Authenticated cells are stored under liquid nitrogen and are never kept in culture for more than 4 months. Cell lines were cultured in RPMI1640 medium supplemented with 10% heat-inactivated FCS, 2 mmol/L l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Primary leukemic samples were acquired from AML patients with high blast counts, after informed consent following the institution-approved protocol. Mononuclear cells were isolated from these samples by density gradient centrifugation using a Lymphocyte Separation Medium (Corning) and cultured in α-MEM medium supplemented with 10% heat-inactivated FCS. Cells were kept at 37°C in a humidified atmosphere of 5% CO₂. Human MSCs isolated from bone marrow samples obtained from healthy subjects as described previously (26, 27) and mouse MSC cell line MS5 were cultured in the same medium as AML patient samples. For coculture experiments, leukemia cells were added to MSCs (AML cells: MSCs = 4:1 ratio) that were plated the night before and cultured as above. Leukemia cells cultured alone or cocultured with MSCs were collected at 24 hours for measuring FAK expression or treated with FAK inhibitor VS-4718 for further assessment. VS-4718 was purchased from Chemietek. The chemical structure for VS-4718 is presented in Supplementary Fig. S1.

Migration of leukemia cells toward and adhesion to MSCs were determined as reported previously (28). Migration was determined at 6 hours and adhesion at 24 hours.

Viable cell numbers were determined by flow cytometry using counting beads (Life Technologies). Apoptosis was estimated via flow cytometry measurement of phosphatidylserine externalization with Annexin V staining (BD Biosciences). Cell membrane integrity was simultaneously assessed by 7-aminoactinomycin D (7AAD) exclusion in the Annexin V–stained cells. To assess cell numbers and apoptosis in leukemia cells cocultured with MSCs, CD45⁺ cells were counted and apoptotic cells were defined as Annexin V⁺/AAD⁺ CD45⁺ cells.

FAK was knocked down by lentiviral transduction using gene-specific shRNA transfer vectors (clones TRCN0000001620: shRNA1-targeting residues 3053-3073 and TRCN0000001621: shRNA2-targeting residues 2739-2759 on RefSeq NM_005607.4; GE Dharmacon). Lentivirus was prepared by cotransfecting HEK293T cells (ATCC) with an equimolar mix of transfer vector and packaging plasmids (psPAX2 and pMD2.G, gifts from Didier Trono (School of Life Sciences at the Swiss Institutes of Technology, Lausanne, Switzerland), plasmids #12260 and 12259, Addgene) using JetPrime transfection reagent as directed by the manufacturer (Polyplus). Fresh lentiviral supernatants were passed through 0.45-μm pore surfactant-free cellulose acetate membranes and then used immediately to infect leukemic cells by incubation overnight at 37°C under 5% CO₂. Infected cells were selected with puromycin (InvivoGen) starting at 0.5 μg/mL. In parallel, cells were transduced using lentivirus delivering a nonspecific control [pLKO.1-TRC control, a gift from David Root (Board Institute of MIT and Harvard, Cambridge, MA), plasmid 10879, Addgene]. Knockdown was verified by Western blot and real time RT-PCR analyses.

Western blot analysis was carried out as described previously (28). Antibodies against FLT3, p-FLT3, and FAK were purchased from Cell Signaling Technology and p-FAK^y397 from Abcam. β-Actin was used as a loading control. Signals were detected using the Odyssey Infrared Imaging System (LI-COR Biosciences) and quantified using the Odyssey software (version 3.0, LI-COR Biosciences).

Mononuclear cells from AML primary patient samples were stained with antibodies for cell surface and intracellular proteins (Table 2) and subjected to CyTOF mass cytometry as described previously (29, 30). Data were exported as FCS for subsequent analysis in SPADE (v3.0, http://pengqiu.gatech.edu/software/SPADE/).

Animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee at MDACC. Molm14 cells (0.6 × 10⁶) stably expressing a dual luciferase-GFP reporter (Molm14-GFP/Luc) were injected via the tail vein into NOD/SCID IL2Rγ Null-3/GM/SF (NSGS) mice (The Jackson Laboratory). Once engraftment was confirmed by the IVIS-200 noninvasive bioluminescence in vivo imaging system (Xenogen), mice were either untreated or treated with VS-4718 twice a day at 75 mg/kg via oral gavage (n = 10/group) for 16 days. Leukemia burden was monitored by IVIS in vivo imaging, flow cytometric measurement of human CD45 cells (anti-human CD45 antibody, BD Biosciences) in mouse peripheral blood, and IHC staining for human CD45⁺ cells in mouse tissues (stained with anti-human CD45 antibody and visualized by Biotin-free Tyramide Signal Amplification System, both from Dako). Mouse survival was recorded.

Protein expressions determined by RPPA, correlations of FAK expression with other proteins in patient samples and with patient clinical outcomes in AML were analyzed as described previously (23, 24). For RPPA analysis of MDS patient samples, Pearson and Spearman correlation coefficient of the protein level and continuous variables were calculated. A log-rank test was used to evaluate the survival difference among the groups of subjects with different FAK protein level. All in vitro experiments were conducted in triplicate. Correlation coefficient for two sets of values was determined by Pearson (Microsoft Excel 2010). Mouse survival was analyzed using log-rank test. Statistical differences between groups were determined using paired Student t test with P ≤ 0.05 being considered statistically significant. Results are expressed as mean ± SEs.

We determined FAK expression by RPPA in peripheral blood or bone marrow samples obtained from a large cohort of newly diagnosed AML (n = 511). High FAK expression was associated with the unfavorable cytogenetic group (P = 2 × 10⁻⁴; Fig. 1A). The lowest FAK expression was seen in patients with favorable cytogenetic group, including inv16 (n = 19), t(8;21) (n = 15), and t(15;17) (n = 20) chromosome translocations (green circles, Fig. 1B). Patients with −5, −7, and/or +8 (n = 100) expressed relatively higher FAK (red box, Fig. 1B). Localization of FAK on chromosome 8 may explain high FAK levels in samples from AML patients with trisomy of chromosome 8. FAK expression was higher in relapsed compared with paired newly diagnosed samples (n = 47, P = 0.02; Fig. 1C). Interestingly, we found that FAK expression was significantly lower in patients with FLT3-ITD (n = 83, P = 0.0024) or RAS (n = 64, P = 0.05) mutations and tended to be lower in patients with FLT3-D835 mutation (n = 24, P = 0.06; Fig. 1D), suggesting functional compensation of these signaling pathways. Perhaps the hyperleukocytosis characteristic of FLT3-ITD AML is related to the decreased FAK levels observed.

Figure 1E shows the distribution of FAK expression in samples with newly diagnosed AML (n = 511) and CD34⁺ cells from normal controls (n = 21). Among the 415 evaluable patients, we did not observe significant differences in OS (P = 0.23) among patients whose FAK expression was lower, equal, or higher compared with that of normal controls, although patients with lower FAK tended to do worse short term and patients with higher FAK did worse long term (Fig. 1F). Interestingly, FAK expression levels significantly impact remission duration of the patients (P = 0.02). Patients with FAK levels the same as normal controls had the longest remission duration, followed by patients with lower FAK levels, the shortest being the patients with FAK levels higher than normal controls (Fig. 1G). Thus, by multiple criteria, increased FAK expression is a poor prognostic marker in AML and associates with unfavorable cytogenetic profiles.

RPPA was performed on 279 bone marrow or peripheral blood samples collected from 133 newly diagnosed MDS patients and their follow-up (Table 1). Elevated or decreased expression was defined as expression levels above or below 90% confidence interval of CD34⁺ normal specimen mean expression, respectively. Overexpression of FAK was seen in 90.7% of all samples compared with CD34⁺ normal specimens. Furthermore, overexpression of FAK was observed in 91.6% of CD34⁺ samples, including 91.4% in newly diagnosed samples and 91.8% in samples from relapsed patients. Overexpression of FAK was observed in 88.6% of CD34⁺CD38⁻ samples, including 85.1% in newly diagnosed samples and 92.5% in samples from relapsed patients (Table 1). We then compared the distribution of FAK level in the 116 newly diagnosed CD34⁺ samples and 47 newly diagnosed CD34⁺CD38⁻ samples with 16 normal CD34⁺ controls. FAK expression levels were significantly higher in both CD34⁺ (n = 116, P = 5.42 × 10⁻²⁰) and CD34⁺CD38⁻ (n = 47, P = 7.62 × 10⁻⁹) cells from MDS patient samples compared with CD34⁺ cells from normal controls (n = 16; Fig. 2A), suggesting their involvement in MDS pathogenesis. Patients with higher FAK expression in CD34⁺ cells tended to have a better OS (P = 0.05) in newly diagnosed MDS (Fig. 2B). This result was not found for FAK levels in CD34⁺CD38⁻ cells (not shown).

We next compared FAK levels in MDS patients not transformed (n = 83) with those later transformed to AML (n = 26) and found significantly higher FAK in the latter group (P = 0.013 or 0.022 by F or Kruskal-Wallis H test, respectively; Fig. 2C). We then compared FAK levels between de novo (n = 262) and MDS-transformed (n = 127) AML and found significantly higher FAK (P < 10⁻⁶ by either F or Kruskal-Wallis H test) in MDS-transformed AML patients (Fig. 2C).

As FAK is activated by ITG ligation or growth factors from the microenvironment and is known to relay the extracellular signal intracellularly through the FAK/SRC signaling cascade, we compared FAK expression with the expression of SRC and ITGs in the same AML RPPA samples. FAK expression was highly positively correlated with p-SRC^Y416 and ITGβ3 expression in a 3D surface blot (Fig. 3A), suggesting a role for ITG/FAK/SRC signaling in AML cells.

To better define growth factor and cell–cell interaction determinants of FAK signaling in AML, we first investigated whether FAK in AML cells is regulated by myeloid growth factors. GM-CSF induced FAK expression in OCI-AML3 cells (Fig. 3B). MSCs secrete multiple growth factors/cytokines and are an important component of the bone marrow microenvironment that is critical for the homing and survival of leukemia cells. We next examined whether FAK expression in AML cells is increased by MSC coculture. We cultured leukemic mononuclear cells obtained from patients with AML with or without a murine MSC cell line (MS5) for 24 hours and determined FAK expression by Western blot analysis. In an evaluation of 7 AML patient samples, leukemic cells cocultured with MSCs expressed significantly more FAK than without (P = 0.016; Fig. 3C), suggesting that the microenvironment modulates leukemia cell function in part through activating FAK signaling.

To determine FAK signaling, we treated primary AML cells from 2 patients, one with wild-type FLT3 and one with FLT3-ITD with FAK inhibitor VS-4718 and measured cell signaling in blasts and CD34⁺ subset by CyTOF mass cytometry, aided by SPADE analysis. We found that VS-4718 decreased p-FAK, FAK, and p-SRC as well as FLT3 downstream targets p-AKT and p-STAT5, but not p-FLT3 and FLT3 in bulk and CD34⁺ cells of both samples (Fig. 3D), suggesting that VS-4718 inhibits cell survival signaling through FAK, not FLT3 inhibition. We also treated OCI-AML3 and Molm14 cells with VS-4718 and found that VS-4718 reduced FAK in both AML cell lines and did not affect p-FLT3 and FLT3 levels in Molm14 cells with FLT3-ITD (Fig. 3D).

To determine whether FAK has a role in leukemia–microenvironment interactions, we conducted adhesion and migration assays. Inhibition of FAK by VS-4718 decreased the adhesion (Fig. 4A) and migration (Fig. 4B) of OCI-AML3 cells to bone marrow–derived MSCs, suggesting that inhibition of FAK blocks leukemia–stroma interactions. Note that reduced adhesion and migration of AML cells to MSCs were not caused by FAK inhibition–induced cell death, as at the doses and time points for the assay, no marked decreases in cell viability were observed (Fig. 4A and B). Furthermore, inhibition of FAK expression by shRNA in OCI-AML3 cells decreased the adhesion and migration of these cells in a dose-dependent manner to human bone marrow–derived MSCs (Fig. 4C), supporting that FAK mediates the interactions.

To determine the role of FAK in leukemia cell growth and survival, we first determined the expression of FAK and p-FAK^y397 in AML cell lines (Fig. 5A). We then treated these cell lines with FAK inhibitor VS-4718. VS-4718 potently decreased viable cell numbers in all the leukemia cell lines tested (IC₅₀ range from 90 nmol/L to <2 μmol/L; Fig. 5B). Six of eight cell lines tested had IC₅₀ at nmol/L levels, and VS-4718 was active even under MSC coculture conditions with most of these cell lines (Fig. 5B, top). IC₅₀ did not correlate with the levels of p-FAK (R = 0.26, P = 0.54). However, when AML cells were cocultured with MSCs, IC₅₀ was significantly correlated with the level of p-FAK (R = 0.73, P = 0.04; Fig. 5B, bottom right), suggesting a role of FAK in mediating bone marrow microenvironment signals to leukemia cells. VS-4718 induced cell death in leukemia cell lines with variable potencies, even in cells cocultured with MSCs (Fig. 5C).

To explore the role of FAK in an in vivo model of AML, NSGS mice were injected with Molm14-GFP/Luc cells and treated with 75 mg/kg VS-4718 twice a day via oral gavage for 16 days following the experimental scheme shown in Fig. 6A. VS-4718–treated mice had decreased leukemia burden by in vivo imaging, lower human CD45 positivity in peripheral blood by flow cytometry analysis, and less tissue infiltration of leukemia cells by IHC staining of human CD45⁺ cells (Fig. 6B). VS-4718–treated mice survived significantly longer than the untreated controls (medium survival 27 vs. 20 days, P = 0.0003; Fig. 6C). No weight loss or other treatment-related toxicities were observed. One mouse in the treatment group died of causes unrelated to the experiment.

We demonstrate in this study that FAK is expressed in AML patient samples and that high expression is associated with unfavorable cytogenetics. FAK, activated in AML cells by the bone marrow microenvironment, promotes leukemia/stroma interaction and supports the survival of leukemia cells. FAK inhibition decreases viability of leukemia cells in vitro and prolongs mouse survival in a human AML xenograft model.

There were significantly higher FAK levels in relapsed versus paired newly diagnosed AML samples, suggesting that high FAK expression contributes to drug-acquired or intrinsic resistance in AML. FAK was reported by others to predict poor prognosis in patients with AML (14, 15). Although we observed a significant difference in remission duration in AML patients with different FAK levels (the shortest in patients with FAK levels higher than normal controls), we did not find significant differences in OS among these patients based on their FAK levels. When the analyses were performed in subsets of patients with intermediate cytogenetics, intermediate cytogenetics with or without FLT3-ITD mutations, or unfavorable cytogenetics, we did not find an FAK level impact OS either. These findings may in part be due to the highly heterogeneous genetic and epigenetic backgrounds of AML patients and to the fact that leukemia cell signaling is regulated and compensated by multiple interconnected signaling pathways. For example, we found that FAK expression was significantly lower in patients with FLT3-ITD or RAS mutations, suggesting that FAK signaling and FLT3/RAS signaling may compensate each other. Although FAK plays multiple roles to support cancer cell survival, it, by itself, may not be sufficient to predict AML patient OS. Reports from Tavernier-Tardy and colleagues (15) demonstrated that AML patients with combined overexpression of two or three adhesion proteins, including CXCR4, VLA4, and FAK, had a significantly shorter OS. In that study, flow cytometry was used to determine functional cell surface CXCR4 and VLA4 levels. RPPA analysis cannot detect cell localization, and CXCR4 and VLA4 were not included in our RPPA panels.

The ITG/FAK/SRC signaling cascade is a well-established pathway that translates environmental signaling and activates multiple intracellular signaling pathways to support cell growth and survival. ITGβ3 was identified by in vivo RNAi screening to be essential for leukemia cells but not for normal hematopoietic stem/progenitor cells (31). We observed that AML samples expressing high ITGβ3 simultaneously expressed high levels of FAK and p-SRC^Y416. The data suggest a functional role of ITG/FAK/SRC signaling in AML cells. We demonstrate that FAK in AML cells is induced by cytokines and MSCs and that FAK regulates AML–MSC interactions. Interestingly, it was previously reported that FAK in AML cells modulates the function of MSCs (32), suggesting that FAK can facilitate the bidirectional cross-talk between leukemic cells and the bone marrow microenvironment.

Although VS-4718 reduced cell viability in all tested AML cell lines, Molm14, Molm13, and MV4-11, all harboring the FLT3-ITD mutation, were the most sensitive. It was previously shown that in addition to FAK, VS-4718 has activities against other kinases, particularly FLT3 by in vitro kinase profiling (33). We do not exclude that VS-4718 can inhibit FLT3 and other kinases in a biochemical assay. However, at the doses used, VS-4718 was able to decrease FAK but not p-FLT3 or FLT3 in AML cell lines or patient samples by either Western blot or CyTOF analysis. Interestingly, VS-4718 decreased FLT3 downstream signaling proteins p-AKT and p-STAT5, independent of FLT3 mutation status. FLT3-mutated cells may depend more than wild-type cells on these signaling proteins for growth and survival.

Inhibition of FAK by VS-4718 for only 16 days significantly prolongs survival of leukemia-engrafted mice, which may possibly be extended by prolonged inhibition. Although inhibition of FAK significantly prolonged survival of leukemia-engrafted mice and greatly suppressed cell growth in all the cell lines tested (IC₅₀ < 2 μmol/L) and was also effective under MSC coculture at a clinically achievable dose (about 3 μmol/L) in vitro, it was less potent in inducing cell death, suggesting combined strategies are needed for enhancing cytotoxic efficacy, which is currently under investigation. In addition, a recent study demonstrated that FAK splice variants are overexpressed in stem/progenitor cells of AML patients with poor prognosis and maintain primitive AML cells, suggesting that targeting FAK has the potential to eliminate AML stem/progenitor cells (34). Inhibition of FAK in combination with agents that potently eliminate bulk leukemia cells may be more effective in the eradication of various leukemic subpopulations supported by a recent study in Ph⁺ B-ALL showing synergism of combined inhibition of FAK and Bcr-Abl tyrosine kinase (12).

Although FAK expression tended to positively impact survival in MDS, overexpression of FAK in CD34⁺ cells from MDS patients may also suggest that FAK signaling is involved in the pathogenesis of the disease. It was reported that the 5′-flanking region of FAK promoter contains several potential transcription factor–binding sites, including NFκB-binding sites (35). Inhibition of NFκB decreased and activation of NFκB by TNFα induced FAK transcription. The presence of abnormal levels of cytokines and chemokines, such as TNFα, in MDS patients have been extensively documented (36). Elevated NFκB signaling has been correlated with the progression of MDS (37). It is possible that aberrantly high levels of chemokines and cytokines and upregulated NFκB signaling induce overexpression of FAK in MDS, but the association with disease severity and progression has yet to be determined. Interestingly, we found FAK expression was significantly higher in MDS patients who later transformed to compared with those who did not transform to AML and in AML patients who transformed from MDS compared with those with de novo AML, suggesting functional importance of FAK expression in AML and MDS patients.

Collectively, our data suggest that FAK regulates leukemia–stromal interactions and supports leukemia cell survival and hence is a potential therapeutic target in myeloid leukemia. Combination strategies may improve the efficacy of FAK inhibition in AML, which is currently under investigation.

No potential conflicts of interest were disclosed.

Conception and design: B.Z. Carter, H. Yang, G. Garcia-Manero, J.A. Pachter, S. Kornblau

Development of methodology: P.Y. Mak, G. Garcia-Manero, Y. Qiu, S. Kornblau

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): P.Y. Mak, X. Wang, H. Yang, G. Garcia-Manero, D.H. Mak, H. Mu, V.R. Ruvolo, S. Kornblau

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): B.Z. Carter, P.Y. Mak, X. Wang, H. Yang, G. Garcia-Manero, D.H. Mak, K. Coombes, N. Zhang, B. Ragon, S. Kornblau

Writing, review, and/or revision of the manuscript: B.Z. Carter, X. Wang, H. Yang, G. Garcia-Manero, K. Coombes, B. Ragon, D.T. Weaver, J.A. Pachter, M. Andreeff

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): P.Y. Mak, G. Garcia-Manero, V.R. Ruvolo

Study supervision: B.Z. Carter

Other (partially supported the study): B.Z. Carter

We thank Numsen Hail for editorial support and assistance with the preparation of the manuscript.

This work was supported in part by the University Cancer Foundation via the Institutional Research Grant program at the University of Texas MD Anderson Cancer Center (to B.Z. Carter), grants from the NIH (P01CA055164), Cancer Prevention Research Institute of Texas (CPRIT, RP121010), the Paul and Mary Haas Chair in Genetics (to M. Andreeff), and MD Anderson's Cancer Center Support Grant CA016672 (Flow Cytometry and Cellular Image Facility and Characterized Cell Line Core).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.