A potent tumor-selective ERK pathway inactivator with high therapeutic index

Abstract

FDA-approved BRAF and MEK small molecule inhibitors have demonstrated some level of efficacy in patients with metastatic melanomas. However, these "targeted" therapeutics have a very low therapeutic index, since these agents affect normal cells, causing undesirable, even fatal, side effects. To address these significant drawbacks, here, we have reengineered the anthrax toxin-based protein delivery system to develop a potent, tumor-selective MEK inactivator. This toxin-based MEK inactivator exhibits potent activity against a wide range of solid tumors, with the highest activity seen when directed toward tumors containing the BRAF^V600E mutation. We demonstrate that this reengineered MEK inactivator also exhibits an extremely high therapeutic index (>15), due to its in vitro and in vivo activity being strictly dependent on the expression of multiple tumor-associated factors including tumor-associated proteases matrix metalloproteinase, urokinase plasminogen activator, and anthrax toxin receptor capillary morphogenesis protein-2. Furthermore, we have improved the specificity of this MEK inactivator, restricting its enzymatic activity to only target the ERK pathway, thereby greatly diminishing off-target toxicity. Together, these data suggest that engineered bacterial toxins can be modified to have significant in vitro and in vivo therapeutic effects with high therapeutic index.

Keywords: CMG2; ERK signaling; anthrax lethal toxin; intermolecular complementation; tumor targeting.

Figure 1.

IC3-PA requires simultaneous presence of the three tumor markers MMPs, urokinase, and CMG2 for its cytotoxic action. (A) Mode of action of the reengineered anthrax toxin-based MEK inactivator. IC3-PA consists of PA-L1-I207R/I656Q and PA-U2-R200A/I656Q, which are activated by MMPs and uPA, respectively. Upon proteolytic activation, PA-L1-I207R/I656Q and PA-U2-R200A/I656Q form oligomers and gain the capacity to bind LF or LF variants. Each active LF-binding site is formed by two subsites (I207 and R200) on two adjacent hetero PA protomers PA-L1-I207R/I656Q and PA-U2-R200A/I656Q, each proving one subsite, i.e. R200 and I207, respectively. High tumor specificity of IC3-PA is derived by its selectively binding to CMG2 receptor and relying on concurrent presence of the two distinct tumor-associated proteases (MMPs and uPA) for activation. Thus, LF-derived effector proteins (such as LF-W271A) can be selectively delivered into tumor cells to target the ERK pathway. (B) IC3-PA requires intermolecular complementation of PA-L1-I207R/I656Q and PA-U2-R200A/I656Q for its cytotoxic action. Lewis lung carcinoma (LLC) cells were incubated with various concentrations of PA proteins in the presence of FP59 (100 ng/mL) for 48 h, followed by an MTT assay for assessing cell viability. IC3-PA (1 μg) = PA-L1-I207R/I656Q (0.5 μg) + PA-U2-R200A/I656Q (0.5 μg). Means ± SD. (C) IC3-PA needs binding to CMG2 receptor for its cytotoxic action. While LLC–WT cells were sensitive to IC3-PA/FP59 (in B), LLC(CMG2-KO) cells were completely resistant to the toxin. (D) and (E) IC3-PA relies on protease activities of MMPs and uPA for its cytotoxic action. LLC–WT cells were incubated with/without 1 or 10 μM GM6001 or UK371804HCL and various concentrations of PA proteins in the presence of FP59 (100 ng/mL) for 2 h. Then the cells were replaced with fresh medium without the toxin and protease inhibitors and cultured for 48 h, followed by an MMT assay for assessing cell viability (D). We also included PA-U2 and PA-L1 as additional controls, verifying that GM6001 could only block PA-L1’s cytotoxicity, and UK371804HCL could only inhibit PA-U2’s activity (E). Of note, neither inhibitor affected WT-PA's cytotoxicity. In (E), PA variants (10 ng/mL) plus FP59 (100 ng/mL) were used as in (D) in the presence of the protease inhibitors as indicated. Means ± SD.

Figure 2.

Selective inhibitory activity of LF-W271A to the ERK but not the p38 and JNK pathways. (A) MEK1/2-selectivity of LF-W271A. LLC cells were incubated with various concentrations of LF/PA or LF-W271A/PA for 3 h, followed by western blotting using anti-MEK1, -MEK2, -MEK3, -MEK4, -MEK6, and -MEK7 antibodies for assessing proteolytic cleavage of the MEKs, or using antiphospho-ERK (T202/Y204), -phospho-p38 (T100/Y182), and -phospho-JNK (T183/Y185) antibodies to evaluate activation status of these pathways. Compared to WT LT, LT-W271A had similar proteolytic activity toward MEK1/2, but lost activity to other MEKs. Consistently, by restricting inhibitory activity to the ERK pathway, LT-W271A could not disrupt the p38 and JNK pathways. Of note, MEK7 was not a target of LT. (B) LF-W271A/PA and LF/PA are equally cytotoxic to LLC cells. LLC cells were incubated with various concentrations of LF-W271A/PA or LF/PA for 72 h, followed by an MTT assay to measure cell viability. PA only and PA plus LF-E687C (LF catalytically inactive variant) were used as additional controls. Means ± SD. (C) HT144, Colo205, and HT29 cells that are dependent on the ERK pathway for proliferation are equally susceptible to LF-W271A/PA and LF/PA. The cells were incubated with various concentrations of LF-W271A or LF in the presence of 500 ng/mL PA for 72 h, followed by an MTT assay to measure cell viability. Means ± SD. (D) LF-W271A/IC3-PA and LF/IC3-PA exhibit comparable antitumor activity. LLC tumor-bearing C57BL/6 J mice were treated intraperitoneally (IP) with LF-W271A/IC3-PA (6.7 µg/20 µg) or LF/IC3-PA (6.7 µg/20 µg) as indicated by the arrows. Means ± SE.

Figure 3.

Extremely high therapeutic index of the tumor-selective MEK inactivator LF-W271A/IC3-PA. (A) Reduced in vivo toxicity of LF-W271A compared to WT LF. C57BL/6 J mice were injected (IP) with three doses of LF-W271A/PA (20 µg/20 µg) or LF/PA (20 µg/20 µg) as indicated (red arrows), with survival monitored for 2 weeks. (B) Dose-escalation studies to measure maximum tolerated doses of LF-W271A/IC3-PA. C57BL/6 J mice were injected (IP) with six doses of LF-W271A/IC3-PA as indicated (red arrows). Of note, mice tolerated six doses of 50 µg/150 µg LF-W271A/IC3-PA without any signs of malaise. Only one mouse succumbed to 100 µg/300 µg LF-W271A/IC3-PA. (C) Antitumor activity of LF-W271A/IC3-PA in syngeneic B16F10 melanomas. B16F10 melanoma-bearing mice were injected IP with various doses of LF-W271A/IC3-PA as indicated (red arrows). Survival of each treatment group was monitored (right panel). Of note, significant antitumor activity was observed for doses as low as 3.3 µg/10 µg of LF-W271A/IC3-PA. Thus, the therapeutic index of LF-W271A/IC3-PA is ≥ 15 (50 µg/150 µg ÷ 3.3 µg/10 µg) in B16F10 syngeneic tumors. Means ± SE.

Figure 4.

Succeptibility of human cancer cells with oncogenic BRAF or KRAS mutation to the MEK inhibition by LF-W271A. (A) Cancer cells with the BRAF^V600E mutation but not the oncogenic RAS mutations are more sensitive to the MEK inhibition by LF-W271A/PA. Cancer cells with the indicated mutations were incubated with various concentrations of LF-W271A in the presence of 500 ng/mL PA for 72 h, followed by an MTT assay for assessing cell viability. Means ± SD. (B) Signal transduction pathways driven by the oncogenic RAS and BRAF. Oncogenic RAS proteins can drive both the MEK–ERK and PI3K effector pathways for cell proliferation. Therefore, under the MEK–ERK inhibition, the cells with oncogenic RAS mutations may still survive via the PI3K pathway signaling. (C) Cells were incubated with various concentrations of LF/PA or LF-W271A/PA for 3 h, followed by western blotting using anti-MEK2 or anti-Phospho-ERK (T202/Y204) antibody to evaluate activation status of ERK pathway. (D) Synergistic or additive cytotoxic effect of LF-W271A/PA and BEZ235 to a set of human cancer cells. Cells were treated with LF-W271A/PA/(1 nM = 85 ng/mL each of LF-W271A and PA), BEZ235 (30 nM), or their combination for 48 h, followed by an MTT assay to assess cell viability. Unpaired two-tailed Student's t test.

Figure 5.

Antitumor activity of our reengineered toxin in long-term experimental cancer therapies. (A)–(C). HT29 (BRAF^V600E) (A), HT144 (BRAF^V600E) (B), or HCT116 (KRAS^G13D) (C) tumor-bearing mice were treated (IP) with PBS or LF-W271A/IC3-PA twice a week as indicated by the red arrows. Tumor weights, mean ± SE. Right panels: Body weights, mean ± SD.

Figure 6.

Long-term antitumor activity of LF-W271A/IC3-PA in immunocompetent mice. (A) B16F10 melanoma-bearing immunocompetent C57BL/6 J mice were treated (IP) with PBS, LF-W271A/IC3-PA (10 µg/30 µg), LF-W271A/IC3-PA combined with PC regimen (20 µg pentostatin plus 1 mg cyclophosphamide). PBS vs. all other groups, P < 0.00001; LF-W271A/IC3-PA + PC vs. LF-W271A/IC3-PA, P < 0.001. Tumor weights, mean ± SE. (B) Body weights of tumor-bearing mice during the course of treatment in (A). Due to tumor burden, the PBS-treated group was euthanized at day 10 after the first treatment. Mean ± SD. (C) Neutralizing activities of antibodies against the toxin. RAW264.7 cells were incubated with PA/LF (100 ng/mL each) for 5 h in the presence of various dilutions of sera obtained from representative mice in (A) (each line represents serum from each mouse) after 2 weeks of therapy (at day 10 for the PBS group). Cell viabilities were determined by an MTT assay as described in Methods. Of note, toxin neutralizing activity could be detected only in sera of the toxin only group.