Combinatorial Inhibition of Focal Adhesion Kinase and BCL-2 Enhances Antileukemia Activity of Venetoclax in Acute Myeloid Leukemia

Focal adhesion kinase (FAK) is frequently activated or overexpressed in cancer cells (1). FAK activation is initiated through autophosphorylation at Y397, resulting in SRC phosphorylation and formation of the FAK/SRC complex, which leads to phosphorylation of other tyrosine sites and full activation of FAK (2). Activated FAK modulates multiple signaling pathways, including PI3K/AKT and MAPK, to promote cell growth, survival, migration, and tumor metastasis (1). Tumor FAK also suppresses antitumor immunity and plays important roles in forming the tumor microenvironment that conveys therapeutic resistance (3), suggesting that FAK may be a target for cancer therapy.

We previously reported that high FAK expression in patients with acute myeloid leukemia (AML) was associated with unfavorable cytogenetics and higher rates of disease relapse following therapy (4), indicating its importance in therapy resistance. Several small-molecule FAK inhibitors have been tested in clinical trials against solid tumors (5–7). VS-4718 (also known as PND-1186; ref. 8), an oral small-molecule FAK inhibitor, shows antiproliferative effects in numerous cancer cell lines, and targets cancer stem cells in solid tumors (9, 10). We demonstrated that inhibition of FAK by VS-4718 markedly inhibited cell growth and induced apoptosis in AML cell lines even when they were cocultured with mesenchymal stem cells (MSC; ref. 4). VS-4718 also suppressed migration and adhesion of leukemia cells to MSCs in vitro supporting a role for FAK in leukemia-microenvironment interactions. VS-4718 treatment reduced the leukemia burden and statistically significantly prolonged the survival in NSGS (NOD-SCID IL2Rgnull-3/GM/SF) mice xenografted with Molm14 AML cells.

The BCL-2 family proteins are key regulators of mitochondria-mediated apoptosis. Among them, BCL-2, an antiapoptotic protein (11), is a novel target for cancer including AML (12, 13). Preclinical and clinical studies have demonstrated that the selective and highly potent BCL-2 inhibitor ABT-199 (venetoclax), has antileukemia activities against various hematologic malignancies, including MDS, CLL, and AML (14–17). ABT-199 is FDA approved for a subset of patients with CLL and AML (18, 19). Although ABT-199 demonstrated strong antileukemia activity preclinically (17), as a single agent it has shown limited clinical efficacy in AML (14). The FDA-approved combination of ABT-199 with hypomethylating agents is resulting in CR/CRi rates of 70%–95% and good tolerability in elderly patients with AML, but patients invariably relapse (20, 21). MCL-1 was identified as a key resistance factor (17, 22). Recent studies showed that low-dose ABT-199 can induce drug resistance in hematologic malignancies by upregulating MCL-1 and BCL-XL, two other antiapoptotic BCL-2 family members, indicating that MCL-1 and BCL-XL are potential targets for combination therapy with ABT-199 to reduce drug-induced resistance (23, 24).

Many signaling pathways regulate MCL-1 and BCL-XL levels. In FLT3-ITD–mutated AML, phosphorylated STAT5 (p-STAT5) plays an important role in maintaining the expression of MCL-1 and BCL-XL (25). MCL-1 and BCL-XL are also downstream of MAPK and PI3K/AKT pathway, which are frequently constitutively activated in AML (26, 27). Chatterjee and colleagues demonstrated that constitutive activation of FAK modulates the nuclear translocation of p-STAT5 in FLT3 and KIT mutated leukemia cells (28). Targeting p21-activated kinase, a downstream effector of FAK and RAC1, inhibited the nuclear translocation of p-STAT5, suppressed the growth of FLT3-ITD AML cells, and prolonged mouse survival (28). Furthermore, the FAK-activated SRC signaling can activate the RAS pathway, which activates MAPK and PI3K/AKT signaling cascades (29), leading to increased MCL-1 and BCL-XL expression.

Given the potential relationship between FAK activation and MCL-1/BCL-XL expression, we hypothesized that FAK inhibition could suppress the expression of MCL-1 and BCL-XL, which should potentiate the activity of ABT-199. We here report that inhibition of FAK with VS-4718 enhances ABT-199 activity in AML cells by suppressing MCL-1 and BCL-XL through several pathways, and the combinatorial inhibition of FAK and BCL-2 exhibits antileukemia activity in vitro and in vivo in patient-derived xenograft (PDX) murine models of AML.

OCI-AML3 was provided by Dr. M. Minden (Ontario Cancer Institute). Molm14 was obtained from the German Collection of Microorganisms and Cell Cultures. Cell lines were validated by short tandem repeat (STR) DNA fingerprinting using the AmpF_STR Identifier kit according to the manufacturer's instructions (Applied Biosystems). The STR profiles were compared with known ATCC fingerprints, and to the Cell Line Integrated Molecular Authentication database (CLIMA) version 0.1.200808 (http://bioinformatics.hsanmartino.it/clima2/; ref. 30). The STR profiles matched known DNA fingerprints or were identified as unique. Authenticated cells are stored under liquid nitrogen and are kept in culture for no more than 3 months. Mycoplasma was tested with Detection Kit from Applied Biological Materials as per the manufacturer's instructions. 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 samples were obtained after acquiring written informed consent from patients following MD Anderson Cancer Center IRB-approved protocols and the studies were conducted in accordance with recognized guidelines described in the Declaration of Helsinki. Mononuclear cells were isolated from these samples using Lymphocyte Separation Medium (Corning Cellgro) by density-gradient centrifugation and cultured in α-MEM supplemented with 10% FCS. MSCs were isolated from normal human bone marrow (BM) samples as described previously (31). For coculture experiments, leukemia cells were added to MSCs (4:1) that had been plated overnight in α-MEM supplemented with 20% FCS. Cells were treated with VS-4718 (provided by Verastem, Inc.), ABT-199 (Active Biochem), or the combination in the presence or absence of MSCs or pretreated with proteasome inhibitor MG132 (Sigma-Aldrich) or caspase inhibitor z-VAD-FMK (Cayman Chemical) for 1 hour then treated with VS-4718.

Viable cell numbers were determined by flow cytometry using counting beads (Life Technologies). Apoptosis was estimated via flow cytometry measurement of cells after Annexin V staining (BD Biosciences) in the presence of 7-aminoactinomycin D (7AAD). For AML and MSC coculture experiments, CD45⁺ cells were counted and apoptotic cells were defined as Annexin V⁺/7AAD⁺CD45⁺. For primary samples, apoptosis was assessed in both CD45⁺ bulk and CD45⁺CD34⁺ stem/progenitor cells and expressed as specific apoptosis:

Cells were transfected with 1 μmol/L ON-TARGET plus human PTK2 siRNA smart pool (catalog no. L-003164-00-0010) or nontargeting pool (catalog no. D-001810-10-05; both from Dharmacon, Inc.) in Amaxa Nucleofector using Nucleofector Kit V (Program O-017; Lonza) and then were cultured in RPMI1640 medium supplemented with 10% FCS for 24 hours. AML cell lines with MCL-1 overexpression or knockdown were generated as described previously (32). Control and BCL-XL–overexpressing HL-60 cells were kindly provided by Dr. K. Bhalla (MD Anderson Cancer Center, Houston, TX).

Western blot analysis was performed as described previously (4). Antibodies against FAK (catalog no. 13009s), p-STAT5 (catalog no. 9351s), STAT5 (catalog no. 9363s), p-AKT (catalog no. 4060s), AKT (catalog no. 2920s), BCL-XL (catalog no. 2764s), p-ERK (catalog no. 4370s), 4EBP1 (catalog no. 9644), and p-4EBP1 (catalog no. 2855) were purchased from Cell Signaling Technology. Antibodies against MCL-1 (catalog no. sc-819) and BCL-2 (catalog no. M0887) were purchased from Santa Cruz Biotechnology and DAKO, respectively. β-Actin or α-tubulin was used as a loading control. Signals were detected using the Odyssey Infrared Imaging System (LI-COR Biosciences) and quantified using Odyssey software (version 3.0, LI-COR Biosciences).

Animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee at MD Anderson Cancer Center (Houston, TX). PDX cells (2 × 10⁶) were injected via tail vein into 40 male (16–20 or 5–9 weeks) NSGS mice (Jackson Laboratory). Engraftment was confirmed by flow cytometry measuring human CD45⁺ cells (anti-human CD45 antibody, BD Biosciences; average 1%) in mouse peripheral blood (PB). The mice were treated with vehicle only, VS-4718 twice daily (75 mg/kg; ref. 8), ABT-199 daily (100 or 50 mg/kg), or the combination via oral gavage (n = 10/group) for 29 or 27 days. VS-4718 was suspended in 5% sucrose. ABT-199 was dissolved in 60% PHOSAL 50PG (LIPOID), 30% polyethylene glycol 400 (Spectrum Chemical), and 10% 200-proof ethyl alcohol (Pharmco-Aaper).

The leukemia progression was monitored weekly by flow cytometric measurement of human CD45⁺ cells in mouse PB. At the end of the treatments, BM cells were collected from one of the PDX models (PDX1; Supplementary Table S1, #14; n = 3/treatment group) for RNA-sequencing analysis and spleen were harvested from both PDX models (n = 3/group). Mice were monitored daily and survival was recorded.

RNA was extracted using TRIzol reagent (Invitrogen) and purified. RNA-sequencing was performed by the Sequencing and Microarray Facility (Department of Genetics, MD Anderson Cancer Center, Houston, TX) using stranded mRNA-seq.

cDNA was generated using SuperScript III Reverse Transcriptase Kit (Invitrogen). TaqMan Fast Universal PCR Master Mix was purchased from Thermo Fisher Scientific (Applied Biosystems). Human XAF1 (TaqMan, Assay ID Hs01550142_m1), human IL1β (TaqMan, Assay ID Hs01555410_m1), MCL-1 (TaqMan, Assay ID Hs03043899_m1), and BCL2L1 (TaqMan, Assay ID Hs00236329_m1) primers were purchased from Thermo Fisher Scientific. The abundance of each transcript relative to RPL13A or ABL was calculated using the 2^−ΔC_t method, where ΔC_t is the mean C_t of the transcript of interest minus the mean C_t of the transcript for RPL13A or ABL housekeeping gene.

All in vitro experiments were conducted in triplicate. The combination index (CI), based on the Chou–Talalay method (33) and determined by CalcuSyn software (BIOSOFT), was expressed as the mean of the CI values obtained at the effective doses of 50%, 75%, and 90% in the population exposed to the different agents (expressed as mean ± SD). CI < 1 was considered synergistic, CI = 1 additive, and CI > 1 antagonistic. The correlation coefficient was determined by Pearson (Microsoft Excel 2013). Statistical differences between groups were determined using a Student t test, with P ≤ 0.05 being considered statistically significant. The results are expressed as mean ± SE. Mouse survival was estimated by the Kaplan–Meier method and analyzed using the log-rank test. RNA-sequencing data were analyzed by the Department of Bioinformatics and Computational Biology (MD Anderson Cancer Center, Houston, TX). Paired end reads from PDX cells were aligned against the hg19 build of human transcriptome using Kallisto (34), and the mapped reads were analyzed using DESeq2 (35) in R. Genes differentially expressed under different treatment conditions were identified by using an Benjamini–Hochberg–corrected P-value threshold of 0.05 and absolute log₂ fold-change threshold of 1. Heatmaps of identified genes were plotted using the ComplexHeatmap (36) package in R, after scaling the estimated count abundances. Pathway enrichment analysis was performed using IPA (QIAGEN Inc., https://digitalinsights.qiagen.com/ingenuity-pathway-analysis-resources/).

We treated Molm14 cells with VS-4718 and examined the effect on FAK signaling and the levels of antiapoptotic BCL-2 proteins. Western blot analysis showed that VS-4718 treatment (24 hours) decreased total FAK (T-FAK) as well as MCL-1 and BCL-XL levels, with lesser effect on BCL-2 (Fig. 1A). We also treated the cells with ABT-199 and the combination of ABT-199 and VS-4718 (24 hours). ABT-199 increased MCL-1 and BCL-XL levels, consistent with the reported literature (23), while these increases were antagonized when combined with VS-4718 (Fig. 1A).

We then examined the protein levels of STAT5/p-STAT5, AKT/p-AKT, and p-ERK, which are FAK targets and regulators of MCL-1 and BCL-XL. VS-4718, and its combination with ABT-199, decreased the levels of p-AKT and p-STAT5, but had a minimal effect on p-ERK (24 hours; Fig. 1A and B). We next treated OCI-AML3, an AML cell line with wild-type FLT3 and low p-STAT5 expression, with VS-4718, ABT-199, and the combination. VS-4718 also decreased T-FAK, MCL-1, and BCL-XL levels, and the combination antagonized the ABT-199–induced MCL-1 and BCL-XL expression (24 hour; Fig. 1C) in these cells. However, VS-4718 had minimal effect on p-ERK, p-AKT, and T-AKT levels in OCI-AML3 cells 24 or 48 hours after treatment (Fig. 1D). Similarly, VS-4718 also had minimal effect on BCL-2 levels in OCI-AML3 cells (Fig. 1D).

To further understand the regulation of MCL-1 and BCL-XL by FAK, we treated Molm14 and OCI-AML3 cells with VS-4718 (24 hours) and determined MCL-1 and BCL-XL RNA and protein levels in the absence or presence of the proteasome inhibitor MG132 or pan-caspase inhibitor z-VAD-FMK. Inhibition of FAK with VS-4718 decreased MCL-1 significantly and there was minor decrease in BCL-XL RNA levels in Molm14 cells (Fig. 1E, top). While although statistically significant, it decreased MCL-1 to a much lesser degree and intriguingly increased BCL-XL RNA levels in OCI-AML3 cells (Fig. 1F, top). The decrease of MCL-1 RNA in Molm14 cells is consistent with the report of its regulation by STAT5 in FLT3-ITD cells (25). The reduction of MCL-1 and BCL-XL protein levels was inhibited by MG132, but not by z-VAD-FMK, in both cell lines (Fig. 1E and F, bottom) indicating that FAK also potentially regulates MCL-1/BCL-XL posttranslationally through proteasome degradation and their decreases are not due to caspase cleavage. It is not surprising that MCL-1 and BCL-XL protein levels were not greatly affected by caspase inhibition because cell viability was largely unaffected (Fig. 1E and F).

VS-4718 decreased FAK as well as the ABT-199 resistance factors MCL-1 and BCL-XL, and antagonized the ABT-199–induced increases of MCL-1 and BCL-XL, suggesting that the combined inhibition of FAK and BCL-2 could have enhanced antileukemic activity. We treated Molm14 and OCL-AML3 cells with VS-4718, ABT-199, or both. Each single agent induced low levels of apoptosis and the combination was highly synergistic in both Molm14 (CI = 0.293 ± 0.025) and OCI-AML3 (CI = 0.008 ± 0.002) cells (48 hours; Fig. 2A). This synergy was also observed when AML cells were treated under MSC coculture conditions that mimic the BM microenvironment known to protect leukemia cells from drug-induced cell death (Fig. 2A). The CI was 0.164 ± 0.039 and 0.157 ± 0.072 for Molm14 and OCI-AML3 cells under the coculture conditions, respectively. We previously showed that VS-4718 suppressed AML cell growth (4). As shown in Fig. 2B, its combination with ABT-199 was more effective in decreasing viable cell numbers in both Molm14 and OCI-AML3 even under coculture conditions.

We also observed that p-4EBP1, the downstream target of mTOR, decreased in Molm14 cells treated with VS-4718 or in combination with ABT-199 (24 hours; Fig. 2C), suggesting that in addition to the effect on MCL-1 and BCL-XL, targeting of PI3K/AKT/mTOR/4EBP1 pathway contributed, at least in part, to VS-4718 activity.

To demonstrate that VS-4718–mediated FAK inhibition potentiates ABT-199 activity in AML, we genetically silenced FAK expression using siRNA in Molm14 cells. As shown in Fig. 2D, decreasing FAK by siRNA also decreased MCL-1 and BCL-XL. At 24 hours posttransfection, cells were treated with ABT-199 for an additional 24 hours. The FAK knockdown Molm14 cells were more sensitive to ABT-199 compared with controls. These results further support that FAK regulates MCL-1 and BCL-XL, and its inhibition sensitizes AML cells to ABT-199, at least in part through decreasing MCL-1 and BCL-XL levels.

To further validate that FAK inhibition–mediated MCL-1 and BCL-XL decreases contributes to the synergism with ABT-199, we treated MCL-1 knockdown OCI-AML3, MCL-1–overexpressing Molm14, and BCL-XL–overexpressing HL-60 cells with VS-4718, ABT-199, and the combination (48 hours). MCL-1 knockdown OCI-AML3 cells were more sensitive to ABT-199 than the controls and combined inhibition of FAK and BCL-2 synergistically induced cell death in the controls, and more so in the MCL-1 knockdown cells (Fig. 3A). Conversely, MCL-1–overexpressing Molm14 cells were more resistant to ABT-199 than the controls, and combined inhibition of FAK and BCL-2 synergistically induced cell death in the controls and less so in the MCL-1–overexpressing cells (Fig. 3B). Similarly, HL-60 cells highly overexpressing BCL-XL were extremely resistant to ABT-199 and combined inhibition of FAK and BCL-2 synergistically induced cell death in the control, but although synergistic, the combination did not markedly induce apoptosis in the BCL-XL–overexpressing HL-60 cells because BCL-XL levels were exceedingly high in this cell (Fig. 3C).

To examine the effects of the combined FAK and BCL-2 inhibition on primary AML cells, we treated BM cells from patients with AML (n = 18) with VS-4718, ABT-199, or their combination. The patient characteristics are listed in Supplementary Table S1. Available CI values of the combination treatment for each sample are shown in Table 1 and apoptosis induction in samples with CI < 1 is plotted (Fig. 4A and B). Each single agent induced apoptosis in AML blasts (CD45⁺ cells) and the combination treatment was statistically significantly more effective (Fig. 4A). As shown in Fig. 4A and Table 1, synergy was observed in most samples (CI < 1), independent of the patient's cytogenetics, mutational status, previous treatments, or response (Supplementary Table S1). Notably, the VS-4718 and ABT-199 combination statistically significantly increased apoptosis in these samples even with MSC coculture (Fig. 4A). Apoptosis was also assessed in CD34⁺ AML progenitor cells in 16 of 18 BM samples. Synergy was observed in 14 of 16 samples (CI < 1; Fig. 4B; Table 1), indicating that combined inhibition of FAK and BCL-2 also enhanced apoptosis in primary AML stem/progenitor cells.

Among the 18 samples, 14 had enough cells for measuring the basal BCL-2, MCL-1, and BCL-XL levels. Interestingly, we found the samples with higher synergy (n = 7; CI < 0.6) for the combination treatment had statistically significantly higher BCL-XL (P = 0.0024) and MCL-1 (P < 0.0001) compared with those with lower synergy (n = 7; CI > 0.6). There was no statistically significant difference in BCL-2 levels (P = 0.56) observed between the two groups (Fig. 4C). These results support the notion that inhibition of FAK with VS-4718 decreases MCL-1, BCL-XL, or both, and antagonizes the ABT-199–induced increase of MCL-1 and BCL-XL. This could contribute to the synergistic antileukemic effect. We also treated normal BM samples with VS-4718, ABT-199, or the combination at the same dose used in primary AML cells and minimal cytotoxicity was observed in CD45⁺ cells (n = 5) or CD34⁺ progenitor cells (n = 3) (Fig. 4D).

An AML PDX model (PDX1) was generated using cells from a patient with NPM1, FLT3-ITD, TET2, DNMT3A, and WT1 mutations and complex karyotype who had relapsed following multiple chemotherapies (Supplementary Table S1, #14). The combination of VS-4718 and ABT-199 synergistically killed the human CD45⁺ PDX cells in vitro, even with MSC coculture (Supplementary Fig. S1). The PDX cells were transplanted into NSGS mice. Once engrafted, the mice were treated with vehicle, VS-4718 (75 mg/kg, twice daily), ABT-199 (100 mg/kg, daily), or their combination via oral gavage for 29 days (Fig. 5A). Both single agents, and the combination, showed antileukemia activity. At the end of treatments, the VS-4718–treated group had significantly reduced circulating human CD45⁺ cells (P = 0.0003), while ABT-199, alone (P = 5.90E-07) and in combination with VS-4718 (P = 5.38E-07), was even more effective (Fig. 5B). VS-4718 alone, and in combination with ABT-199, reduced the leukemia burden in the spleen, while ABT-199 alone did not (Fig. 5C), indicating that ABT-199 had a stronger antileukemic effect on PB AML cells and VS-4718 was more effective at reducing AML cells in tissues. The combination was effective in reducing both. VS-4718–treated mice survived statistically significantly longer than did the untreated controls (median survival, 72 vs. 36 days; P = 0.0002). ABT-199 did not prolong survival (median survival, 35 days). The combination of VS-4718 and ABT-199 statistically significantly prolonged survival (median survival, 65.5 days; P = 0.011 vs. control and P = 0.0072 vs. ABT-199). No statistically significant difference in survival was observed between VS-4718 and the combination treatment groups (P = 0.33) (Fig. 5D). Although demonstrating stronger antileukemia activities in PB and was at least equally effective on tissue compared with VS-4718 alone, the combination did not further extend the survival compared with VS-4718 alone. We noticed that all of the mice had splenomegaly at death except those animals in the combination treated group that died earlier during treatment (Supplementary Fig. S2), suggesting that other factors, such as drug toxicity, contributed to the early death of the first four mice in the combination treatment group, resulting in a shortened median survival.

We then treated the second PDX mouse model (PDX2) using a lower dose of ABT-199. The PDX cells were generated from an AML patient sample with IDH1 and TP53 mutations and complex karyotype (Supplementary Table S1, #19). After engrafted, the mice were treated with vehicle, VS-4718 (75 mg/kg, twice daily), ABT-199 (50 mg/kg, daily), or their combination via oral gavage for 27 days. Due to the aggressiveness of this model, the controls started to die 11 days into treatment. The combination treatment reduced spleen size (13 days after treatment; Fig. 5E). VS-4718 (19 days) or ABT-199 (17 days) alone did not significantly extended survival compared with controls of 14.5 days (P = 0.07 for VS-4718 vs. control, and P = 0.3 for ABT-199 vs. control), while the combination statistically significantly prolonged the survival (25 days, P = 0.03 for the combination vs. control). Even though the combination did not show significantly longer survival time compared with VS-4718 or ABT-199 alone, >70%, >45%, and 30% survival extension of the combination versus the control, ABT-199–treated, or VS-4718–treated mice, respectively suggested the benefit of VS-4718 and ABT-199 combination even in a TP53-mutated, highly aggressive AML model (Fig. 5F).

To further understand the mechanisms of action, we isolated RNA from BM cells at the end of the treatment from the PDX1 mouse model (n = 3) and performed RNA-sequencing. The RNA-sequencing analysis revealed that genes associated with NFκB signaling, which affects proliferation and survival of AML cells, were inhibited in the VS-4718–treated compared with the control group (Fig. 6A). Consistent with this finding, we found that IL1β, downstream of NFκB signaling, was noticeably decreased in the VS-4718–treated group (Fig. 6A). The results also showed that genes associated with IFN signaling were markedly activated, and correspondingly genes that are regulated by the IFN family proteins were markedly upregulated in the combination treatment group, among them the proapoptotic protein XAF1 (Fig. 6B). To validate these findings, we measured the mRNA levels of IL1β and XAF1 in BM cells (n = 3) by RT-PCR, and found that IL1β mRNA expression significantly decreased in VS-4718–treated compared with the control groups (P = 0.02; Fig. 6C). XAF1 mRNA expression markedly increased in the VS-4718 treatment group (P = 0.048 vs. control) and even more so in the combinatorial treatment group (P = 0.006 vs. control) (Fig. 6D).

We demonstrate in this study that genetic or pharmacologic inhibition of FAK decreases MCL-1 and BCL-XL, two resistance factors for the BCL-2 inhibitor ABT-199, and that combinatorial inhibition of FAK and BCL-2 enhances antileukemia activity of ABT-199 in AML, in an MCL-1– and BCL-XL–dependent manner.

We previously reported that inhibition of FAK by VS-4718 suppresses growth and induces apoptosis in AML cell lines and prolonged survival in an AML xenograft mouse model (4). While ABT-199 has shown limited efficacy as a single agent (14, 37), mechanism-based combinatorial therapies could improve AML treatment outcomes. Targeting MCL-1 and BCL-XL is a potential means of rescuing leukemia cells from resistance and maximizing apoptosis (22, 38, 39). To extend our findings, we investigated the mechanisms of action of FAK inhibition in AML cells. We found that inhibition of FAK by VS-4718, or by silencing with siRNA, decreased MCL-1 and BCL-XL levels. We demonstrated that FAK inhibition–mediated MCL-1 and BCL-XL decreases were regulated by transcriptional and posttranslational mechanisms. Furthermore, we identified FAK-regulated signaling pathways as regulators of MCL-1 and BCL-XL expression. We further demonstrated that FAK inhibitor VS-4718 suppressed the ABT-199–mediated induction of MCL-1 and BCL-XL and potentiated the antileukemia activity of ABT-199 in AML cell lines and primary AML cells including CD34⁺ AML stem/progenitor cells in vitro and in vivo in two AML PDX mouse models.

High FAK expression in AML was found to be associated with unfavorable cytogenetics and higher rate of disease relapse following therapy (4). VS-4718 is a selective FAK kinase inhibitor and decreases p-FAK, not FAK, levels in solid tumors (8, 10). p-FAK levels are low in AML cells and often are difficult to detect by Western blot analysis. However, we found that VS-4718 decreased T-FAK in both AML cell lines (Figs. 1 and 2) and primary samples (4). Inhibition of FAK decreases multiple signaling pathways in AML cells. Whether FAK decrease is mediated through the feedback mechanisms of signaling pathway inhibitions or other mechanisms are currently unclear. Nevertheless, VS-478–mediated FAK inhibition exerts antileukemia activity and potentiates ABT-199 in AML in vitro and in vivo.

The level of p-STAT5 is strongly correlated with FLT3-ITD mutation (40), and FLT3-ITD constitutively activates STAT5 (28, 41, 42). We previously showed that VS-4718 did not affect FLT3 in Molm14 cells (4). We found that FAK inhibition with VS-4718 decreased p-STAT5 and p-AKT in FLT3-ITD–mutant cells. In FLT3 wild-type OCI-AML3 cells, which have low p-STAT5, MCL-1, and BCL-XL, these levels also decreased after a 24-hour treatment with VS-4718, but there was no change in PI3K/AKT signaling. These results indicated that while MCL-1 and BCL-XL were decreased in both FLT3-ITD–mutated and wild-type cells by the FAK inhibitor, they were regulated through different pathways. We speculate that in the FLT3-ITD–mutated cells, this was mediated through inhibition of p-STAT5, in agreement with the results of transcriptional regulation of MCL-1 by FAK, and PI3K/AKT, and in the FLT3 wild-type cells this was mediated by suppressing other pathways that are not clear at present. Nevertheless, in both cell lines, MCL-1 and BCL-XL levels are regulated posttranslationally through proteasome degradation.

We and others have shown that VS-4718 decreases viability of AML cell lines and inhibit the growth of cancer stem cells (4, 10, 43). In this study, we demonstrated that combined inhibition of FAK and BCL-2 effectively and synergistically induced apoptosis not only in bulk primary AML cells, but also in CD34⁺ AML stem/progenitor cells even when they were cocultured with MSCs. Importantly, the synergy of the combination treatment in primary AML cells was affected by the expression levels of MCL-1 or BCL-XL. This combinatorial approach should benefit most patients with AML, especially those with high MCL-1/BCL-XL expression, suggesting that high expression of MCL-1 and BCL-XL in patients with AML may predict enhanced synergy of the VS-4718 and ABT-199 combination therapy.

Furthermore, we demonstrated that VS-4718, both as a single agent and in combination with ABT-199, has remarkable antileukemia activity using the first AML PDX model. Although effectively decreasing circulating blasts, ABT-199 did not reduce tissue leukemia burden or prolonged survival in vivo. Although less effective than ABT-199 on circulating blasts, VS-4718, as a single agent, remarkably reduced tissue leukemia burden as well as prolonged survival, supporting a pivotal role of FAK in the leukemia microenvironment. The combination showed antileukemia activity both in circulation and in tissues, and statistically significantly prolonged survival. However, mice in the ABT-199 and combination treatment groups started losing weight and became weak during treatment with pale spleens, suggesting toxicity. We therefore tested the combination in the second PDX model (PDX2) using a reduced dose of ABT-199. In this model, all the control mice quickly died because of the aggressiveness of TP53-mutated cells. Neither VS-4718 nor ABT-199 treatment reduced spleen size and only slightly prolonged survival. However, the combination demonstrated remarkable antileukemia activity, and statistically significantly prolonged survival (72% longer than controls). This is of great potential clinical significance because patients with AML with TP53 mutations and complex karyotype have dismal outcomes.

NFκB signaling is known to regulate AML cell growth and survival (44). IL1β, an important mediator of the inflammatory response to infection and injury, is regulated by NFκB, and it in turn can activate NFκB and JNK signaling (45, 46). We found that inhibition of FAK attenuated NFκB–related gene expression including IL1β in the PDX mouse model.

The IFN family members are important immune-modulators. Their downregulation has been shown to play roles in the immunotherapy resistance of cancer cells (47). We found that the combined inhibition of FAK and BCL-2 activated genes associated with the IFN pathway in vivo, such as proapoptotic XAF1 (48, 49), indicating that the combination of VS-4718 and ABT-199 may potentiate immunotherapy.

In conclusion, we demonstrate that inhibition of FAK has antileukemia activity in AML, at least in part, by suppressing MCL-1 and BCL-XL, which can be synergistically enhanced via combined BCL-2 inhibition. Importantly, the combined inhibition of FAK and BCL-2 extended survival in two PDX models derived from patients with complex karyotype AML carrying either TP53 mutation or multiple mutations suggesting that targeting FAK and BCL-2 may be agnostic to the presence of TP53 and other mutations, and thus benefit patients with high-risk AML with poor outcome. Our results warrant characterization of this combinatorial approach in the clinic, perhaps in combination with immunotherapy.

A. Rao is a member of Voxel Analytics, LLC and reports receiving a commercial research grant from Agilent Technologies. J.A. Pachter is a chief scientific officer for Verastem. D.T. Weaver is VP, Translation Medicine, at and has ownership interest (including patents) in Verastem Oncology, and is an advisory board member for Hillstream Biopharmaceuticals and FemtoDX. No potential conflicts of interest were disclosed by the other authors.

Conception and design: M. Andreeff, B. Xu, B.Z. Carter

Development of methodology: P.Y. Mak, H. Mu, A. Rao, M. Andreeff

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): X. Wang, P.Y. Mak, H. Mu, V. Ruvolo

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): X. Wang, P.Y. Mak, H. Mu, W. Tao, A. Rao, R. Visweswaran, J.A. Pachter, M. Andreeff, B.Z. Carter

Writing, review, and/or revision of the manuscript: X. Wang, A. Rao, J.A. Pachter, D.T. Weaver, M. Andreeff, B.Z. Carter

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): X. Wang, H. Mu

Study supervision: M. Andreeff, B. Xu, B.Z. Carter

We thank Ann Sutton at the Department of Scientific Publications and Numsen Hail for editing this manuscript. This work was supported in part by the University Cancer Foundation via the Institutional Support: Research Grant program at MD Anderson (to B.Z. Carter), the Paul and Mary Haas Chair in Genetics (to M. Andreeff), and MD Anderson's Cancer Center Support Grant CA016672 (MD Anderson North Campus Flow Cytometry and Cellular Imaging Core Facility, Characterized Cell Line core, and Sequencing and Microarray Facility).

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