Efficacy of Rac and Cdc42 Inhibitor MBQ-167 in Triple-negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer, with a high predisposition for locally invasive and metastatic cancer. With the objective to reduce cancer metastasis, we developed small molecule inhibitors to target the drivers of metastasis, the Rho GTPases Rac and Cdc42. Of these, MBQ-167 inhibits both Rac and Cdc42 with IC50s of 103 and 78 nmol/L, respectively; and consequently, inhibits p21-activated kinase (PAK) signaling, metastatic cancer cell proliferation, migration, and mammosphere growth; induces cell-cycle arrest and apoptosis; and decreases HER2-type mammary fatpad tumor growth and metastasis (Humphries-Bickley and colleagues, 2017). Herein, we used nuclear magnetic resonance to show that MBQ-167 directly interacts with Rac1 to displace specific amino acids, and consequently inhibits Rac.GTP loading and viability in TNBC cell lines. Phosphokinome arrays in the MDA-MB-231 human TNBC cells show that phosphorylation status of kinases independent of the Rac/Cdc42/PAK pathway are not significantly changed following 200 nmol/L MBQ-167 treatment. Western blotting shows that initial increases in phospho-c-Jun and phospho-CREB in response to MBQ-167 are not sustained with prolonged exposure, as also confirmed by a decrease in their transcriptional targets. MBQ-167 inhibits tumor growth, and spontaneous and experimental metastasis in immunocompromised (human TNBC) and immunocompetent (mouse TNBC) models. Moreover, per oral administration of MBQ-167 at 100 mg/kg body weight is not toxic to immunocompetent BALB/c mice and has a half-life of 4.6 hours in plasma. These results highlight the specificity, potency, and bioavailability of MBQ-167, and support its clinical potential as a TNBC therapeutic.

Conflict of interest statement

Conflict of interest: LBG is a current, and HP is a former, employee of MBQ Pharma, Inc. SD, LCP, EH, and CPV and own stock at MBQ Pharma, Inc., which has licensed patents US 9,981,980, US10,392,396 and international patents related to PCT/US2017/029921 relevant to this work from the University of Puerto Rico.

©2021 American Association for Cancer Research.

Figures

Figure 1.

Effect of MBQ-167 on TNBC cells. A, Bright field images (40X) of MDA-MB-231 human metastatic breast cancer cells following vehicle (0) or MBQ-167 (200 or 500 nM) for 24 h. B,C, Clonogenic assay. MDA-MB-231 cells were treated for 24 h with 0 (vehicle= 0.04% DMSO), or varying concentrations of MBQ-167 (250, or 500 nM). The attached (A) and detached (D) cell populations were recovered and equal numbers of cells plated for 7 days. B, Representative fixed and Trypan-blue stained colonies for each treatment. C, Relative colony number for vehicle (0) or attached or detached cell populations following MBQ-167 treatment. N=4; * = P < 0.05, ** = P < 0.01 **** = P < 0.0001 in a one-way ANOVA compared to Vehicle (0). Error bars represent ± SEM. D, E, MDA-MB-468 (D) or 4T-1 (E) cells were treated for 48 h with 0, 250, or 500 nM MBQ-167 and cell viability measured by a MTT assay. N=3±SEM; *, P<0.05, **, P<0.01, **, P<0.001. F, G, Effect of MBQ-167 on Rac and Cdc42 activation in TNBC cells. F, MDA-MB-468 EGFR ++ human TNBC cells; G, 4T-1 mouse TNBC cells were treated for 24 h with 0, 250, or 500 nM MBQ-167. Both attached and detached cell populations were collected, pooled, and subjected to a pulldown assay, which identifies the fraction of Rac or Cdc42 bound to GTP, using a GST-fusion construct of the Cdc42 and Rac interactive binding (CRIB) domain of the Rac/Cdc42 downstream effector p21- activated kinase (PAK). Representative western blots probed with specific pan Rac antibodies are shown (N=3).

Figure 2.

Kinome array following MBQ-167 treatment from pooled attached and detached cells. MDA-MB-231 cells were treated with 200 nM MBQ-167 for 24 hours. Both attached and detached cells were pooled and total cell lysates were subjected to a Proteome Profiler Human Phospho-Kinase Array Kit (ARY003B) from R&D Systems for 45 phospho proteins. N=3±SEM. A, Fold change of phospho protein levels in reference to vehicle controls are shown. N=4, P<0.05. B, C, Western blot confirmation of Jun pathway changes in response to MBQ-167. MDA-MB-231 cells were treated with 200 nM MBQ-167 for 24 hours. Cell lysates were western blotted using specific antibodies for phospho (P)-c-Jun, c-Jun, P-Jun kinase (JNK), and JNK. B, Representative western blot; C, Relative integrated density of positive bands from MBQ-167 treated compared to vehicle treated cells. N=3±SEM; *, P<0.05.

Figure 3.

Western blot validation of phosphokinome array results in detached and attached cells for P-CREB and P-c-Jun and their transcriptional targets following MBQ-167. MDA-MB-231 cells were treated with 200 nM MBQ-167 for 48 hours. Attached and detached cells were recovered separately and the lysates subjected to western blotting. A, Representative western blots for CREB and p-CREB (left) and ZEB1, Survivin, Cyclin D, p-c-Jun, and c-Jun (Right). Actin was used as a loading control. B-D, Western blot analysis. Average fold change relative to vehicle controls are shown. B, ZEB1, Survivin, Cyclin D, P-c-Jun, and c-Jun levels in attached and detached cells after 48 h. MDA-MB-231 cells were treated for 24, 48, and 96 hours with 200 nM MBQ-167. Attached (Att) and detached (Det) cells were isolated and lysed. Lysates were western blotted for transcriptional targets of CREB and c-Jun: ZEB1, Survivin, Cyclin D1, P-c-Jun, and c-Jun. B, Representative western blot at 48 h following vehicle or MBQ-167. C. Relative (relative to actin and vehicle) Integrated density of positive bands from western blots. N=3±SEM; *, P<0.05; **, P<0.01, ***, P<0.001. D, Relative integrated density of P-c-Jun, c-Jun, Zeb1, Survivin, and Cyclin D levels from western blots following time in MBQ-167 (0, 24, 48, 96 h).

Figure 4.

Effect of MBQ-167 (IP treatment) on TNBC tumor growth, metastasis, and liver enzymes in mouse models. A, B, C, SCID mice were inoculated at the mammary fat pad with GFP-MDA-MB-231 TNBC cells. One week following tumor establishment, mice were treated with vehicle control (12.5% EtOH, 12.5% Cremophor, 75% PBS) or 1, 5, or 10 mg/kg BW MBQ-167 3X a week by IP for 108 days. A, Relative tumor growth was quantitated by fluorescence image analysis using Image J, and percentage tumor growth was calculated as the integrated density (ID) for each tumor on each day of imaging (1X a week 2 months) divided by its ID on Day 1. N=10±SEM, Asterisk=pC, Representative fluorescence micrograph of excised lungs from vehicle (0) or MBQ-167 (1 mg/kg BW) treated mice with MDA-MB-231 primary mammary tumors, i.e. spontaneous metastasis. D, Experimental metastasis assay in BALB/c mice. GFP-4T-1 mouse breast cancer cells were inoculated at the tail vein of BALB/c mice. 24 hours following inoculation, mice were treated with vehicle control (12.5% EtOH, 12.5% Cremophor, 75% PBS) or 5 mg/kg BW MBQ-167 3X a week by IP for 21 days. Top panel, Representative fluorescent micrographs from lungs. Bottom panel, Average integrated density of fluorescent metastatic foci per treatment group. N=8, Asterisk=P<0.05.

Figure 5.

Effect of MBQ-167 (PO) on TNBC tumor growth and metastasis. Balb/C mice were inoculated at the mammary fat pad with GFP-4T-1 mouse TNBC cells. One week following inoculation, when ~100 mm3 tumors were established, mice received 0 (0.5% methyl cellulose, 0.1% Tween 80), 5, 25, 50, or 100 mg/kg BW MBQ-167 by oral gavage 5X a week for 28 days. A, Tumor growth was quantitated by fluorescence image analysis using Image J, and relative tumor growth was calculated as the integrated density (ID) for each tumor on each day of imaging (1X a week for 4 weeks) divided by its ID on Day 1, relative to vehicle (1). N=10±SEM. B, The change in integrated density is shown for each treatment from fluorescent images acquired on day 28 calculated as the average integrated density (ID) on day 28 divided by its ID on Day 1. C, Average integrated density of fluorescent metastatic foci from lungs harvested from vehicle or MBQ-167 treated mice.

Figure 6.

Safety and bioavailability of P.O. administration of MBQ-167 in Balb/c mice in formulation B (0.5% methyl cellulose, 0.1% Tween 80). A, Average mouse weight following vehicle or MBQ-167 treatment by PO 5X a week for 28 days. B, mouse Liver proteins from plasma of Balb/C mice following 4 weeks of 100 mg/kg MBQ-167 3X a week by IP. N=5±SEM. C, Plasma availability of MBQ-167 in Balb/C mice following administration of 100 mg/kg BW MBQ-167 at 0–24 hrs.