Search

Metastasis continues to be the primary cause of cancer-related death in women with breast cancer, needing targeted therapies to inhibit this process. Therefore, we developed inhibitors targeting the homologous Rho GTPases Rac1 and Cdc42, which direct the actin cytoskeletal changes required for cell migration/invasion; and thus, metastasis. We characterized the potent inhibitor, MBQ-167 that blocks both Rac1 and Cdc42 at IC50s 103 nM and 78 nM, respectively, in human HER2 (+) and triple-negative breast cancer (TNBC) cells. Consequently, MBQ-167 inhibits tumor growth and metastasis by ~90% in pre-clinical mouse models of breast cancer (Humphries-Bickley, et al., 2017). To improve the activity and solubility of MBQ-167, we synthesized a panel of derivatives, maintaining the 9-ethyl-3-(1H-1,2,3-triazol-1-yl)-9H-carbazole as the core for each derivative. Of the derivatives tested, MBQ-168 demonstrated improved solubility and comparable efficacy. Similar to MBQ-167, MBQ-168 induced actin cytoskeletal disintegration, cell rounding, and detachment to ultimately undergo anoikis. In 120hrs, MBQ-168 significantly inhibited the viability of MDA-MB-231 at a GI50 of 228nM and HER2+ MDA-MB-435 cells with a GI50 of 137 nM, without affecting the viability of Human Mammary Epithelial Cells (HMEC). In apoptosis assays, MBQ-168 treatment, at 500nM for 48 hrs, demonstrated a similar response to MBQ-167 by >4-fold increase in Caspase 3/7 activity in HER2 (+), and MDA-MB-231 and MDA-MB-468 TNBC cells. In wound healing assays, MBQ-168 also responded similar to MBQ-167 by a ~80% inhibition of breast cancer cell migration at 250nM and 500nM for 24hrs. Moreover, as quantified from Rac1.GTP pulldown assays, following 250nM treatment for 24hrs, MBQ-168 inhibited Rac1 activation in the attached cell population by ~70%, and in the detached cell population by ~100%, demonstrating a potent inhibition of Rac1 activation. In a preliminary study for relative efficacy in mice, MBQ-168 significantly reduced HER2+ MDA-MB-435 mammary tumor growth (~90%) at 5mg/kg BW via IP administration. In conclusion, MBQ-168 is an effective Rac inhibitor that reduces breast cancer cell viability, induces apoptosis, and inhibits breast cancer cell migration, actin cytoskeletal extensions, and Rac1 activation to result in a drastic reduction in mammary tumor growth in mice. We predict that the increased solubility of MBQ-168 will make it more bioavailable than MBQ-167. Thus, we have demonstrated that small structural modifications of MBQ-167 can affect the cytotoxic activity of carbazole derivatives with potential as anti-cancer drugs, and as tools to block Rac/Cdc42 activities in biological systems. Citation Format: Julia Medina, Tatiana Matos, Luis Velazquez, Michael Rivera, Ailed Cruz-Collazo, Eliud Hernandez, Cornelis Vlaar, Suranganie Dharmawardhane. Characterization of the Rac/Cdc42 inhibitor MBQ-168 as an anti-cancer compound [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-09.

Intensified production of smart drug modafinil under continuous flow and sustainable conditions.

Among all cancers worldwide, breast cancer is the first cause of death in women. One of the most aggressive subtypes of breast cancer is the triple negative form, which account for the 10-15% of breast cancer cases. Due to its complex heterogeneity triple negative breast cancer available treatments are limited, being chemotherapy in the first line of use. Despite advances in the search for new treatments, there is still a lack of effective therapies, being the metastatic disease the principal cause of breast cancer mortality. Therefore, it is critical to develop a new and effective strategies to inhibit metastasis. The Rho GTPase Rac has been identified as a promising target for anti-metastatic cancer therapy as it has been shown to play key roles in metastatic cancer cells dynamics as: cellular adhesion, migration, proliferation, survival and invasion. To this extent, in an effort to find a compound with increased Rac inhibitory capacity our group developed Ehop-016 derivatives HV-107 and HV-118. Previously we have reported HV-107 and HV-118 to affect cell viability promoting a G2-M cell cycle arrest with a sub-G1 population indicative of cell death in MDA-MB-231. Also, an increase of caspases activity validating apoptosis as a mechanism of cell death was shown. In addition, a decrease in tumor growth and metastasis by ~35 to 40% in an in vivo study using HV-118 at 1 mg/kg body weightwas also reported. Currently, through pulldown assays we have showed HV-107 and HV-118 to inhibit Rac activity at~500-2000 nM and 10 nM respectively in MDA-MB-231 and MDA-MB-468 breast cancer cells. Using trypan blue excision assay HV-107(>500 nM) and HV-118(>50 nM) were shown to affect cell viability promoting a G2-M cell cycle arrest in MDA-MB-468. We also demonstrated HV-107 and HV-118 to inhibit the direct downstream effector of Rac, PAK at >500 nM(HV-107) and >50 nM(HV-118) in MDA-MB-231 cells. In addition, HV-107 was also shown to increase Rho activity at concentrations significantly higher than the effective for Rac inhibition. Rho activity up-regulation has been reported to negatively affect migration of cancerous cells. Finally, we tested HV-107 in a mouse model of metastatic breast cancer. A decrease of ~ 40% in liver metastasis was shown for mice treated with 5 mg/kg BW HV-107. Taken together, our results indicate HV-107 and HV-118 are approximately 4-100 times more efficient than the parent compound Ehop-016 and have potential as anti-breast cancer metastasis therapeutics. This study is supported by NIH Grant 1SC1GM122691, and PRINBRE P20GM103475 from NIGMS of the NIH to GVC Citation Format: Grace Enid Velez Crespo, Eliud Hernandez, Suranganie Dharmawardhane, Cornelis Vlaar, Linnette Castillo, Lilia Kucheryavykh. Rac inhibitors for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2973.

Metastasis continues to be the most difficult phase for cancer treatment due to few targeted therapy options. Therefore, we focus on targeting the Rho GTPases Rac1 and Cdc42, key drivers of the primary steps of metastasis: cell migration and invasion. We developed MBQ-167 as a small molecule dual inhibitor for Rac1 and Cdc42 activation with IC50s of 103 and 78 nM, respectively. In metastatic human breast cancer cells, MBQ-167 inhibits the Rac/Cdc42 downstream effector p21-activated kinase (PAK)/LIM kinase/Cofilin signaling, lamellipodia extension, and thus, cell polarity, and migration. In addition, in metastatic breast cancer cells, MBQ-167 inhibits cell proliferation and cell cycle progression to ultimately induce apoptosis via anoikis. MBQ-167 inhibits tumor growth and metastasis in experimental and spontaneous HER-positive and Triple negative breast cancer (TNBC) mouse models. MBQ-167 has 35% bioavailability and is not toxic in rodent or canine models up to 1000 mg/kg. Therefore, we are further developing MBQ-167 as a lead anti metastatic cancer compound. The objective in this study is to elucidate potential activity of MBQ-167 metabolites. Prior work has identified MBQ-167 metabolites via LC MS/MS from a mammalian liver microsome (rats and human) assay. Four of the MBQ-167 metabolites with the highest MS peak area, were analyzed in metastatic breast cancer cell lines, for cell viability via MTT assays and apoptosis via caspase 3/7 assays. We report that the metabolites M6, M7, M8, and M9 did not inhibit cell viability or induce apoptosis at 250 or 500 nM in HER2-type MDA-MB-435, and TNBC MDA-MB-231 and MDA-MB-468 breast cancer cell lines, compared to MBQ-167. We next analyzed whether these metabolites could inhibit the activation of Rac and Cdc42 by analyzing the phosphorylation status of its downstream effector p-21 activated kinase isoforms 1/2/3 (PAK1/2/3). When MBQ-167 and metabolites at 250nM were incubated for 24h in HER2-type MDA-MB-435 metastatic cancer cells, and the lysates western blotted with antibodies to total PAK1/2/3 or phospho-Pak1/2/3, we found a decrease in the phosphorylation levels of PAK in response to MBQ-167 and metabolite M6. However, since the peak area of M6 in the microsome assay is only 6% compared to MBQ-167, we conclude that MBQ-167 is the major active compound with anti-metastatic cancer properties. Citation Format: Julia I. Medina, Eliud Hernández, Cornelis Vlaar, Suranganie Dharmawardhane. Comparative activity of MBQ-167 metabolites in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 347.

Based on the efficacy of EHop-016 as an inhibitor of migration and Rac1 activation, a new series of carbazole derivatives has been synthesized. Cytotoxic and anti-migratory effects of these compounds were evaluated in MCF-7 and MDA-MB-231 breast cancer cell lines. Preliminary investigations of their anticancer activity demonstrated that several compounds have moderate antiproliferative effects on cancer cell lines with GI50 values in the range of 13–50 µM. Furthermore, compounds 3b and 11b inhibit migration activity of metastatic cell line MDA-MB-231 by 32% and 34%, respectively. Compound 11b was shown to inhibit activation of the Rho GTPase Rac1 by 55% at 250 nM in both MDA-MB-231 and MDA-MB-435 cell lines. Compared with the IC50 of Rac1 inhibition by lead compound EHop-016 of 1.1 µM, compound 11b demonstrates 4X improved in vitro efficacy.

It is well established that glucotoxicity (caused by high glucose concentrations; HG) underlies pathogenesis of islet dysfunction in diabetes. We have recently demonstrated that Nox2 plays a requisite role in the generation of reactive oxygen species (ROS) under HG conditions, resulting in mitochondrial dysregulation and loss of islet β-cell function. Herein, we investigated roles of Nox2 in the regulation of downstream stress kinase (p38MAPK) activation under HG conditions (20 mM; 24 h) in normal rodent islets and INS-1 832/13 cells. We observed that gp91-ds-tat, a specific inhibitor of Nox2, but not its inactive analog, significantly attenuated HG-induced Nox2 activation, ROS generation and p38MAPK activation, thus suggesting that Nox2 activation couples with p38MAPK activation. Since Rac1, is an integral member of the Nox2 holoenzyme, we also assessed the effects of Rac1 inhibitors (EHT 1864, NSC23766 and Ehop-016) on HG-induced p38MAPK activation in isolated β-cells. We report a significant inhibition of p38MAPK phosphorylation by Rac1 inhibitors, implying a regulatory role for Rac1 in promoting the Nox2-p38MAPK signaling axis in the β-cell under the duress of HG. 2-Bromopalmitate, a known inhibitor of protein (Rac1) palmitoylation, significantly reduced HG-induced p38MAPK phosphorylation. However, GGTI-2147, a specific inhibitor of geranylgeranylation of Rac1, failed to exert any significant effects on HG-induced p38MAPK activation. In conclusion, we present the first evidence that the Rac1-Nox2 signaling module plays novel regulatory roles in HG-induced p38MAPK activation and loss in glucose-stimulated insulin secretion (GSIS) culminating in metabolic dysfunction and the onset of diabetes.

The Rho GTPase Rac is an important regulator of cancer cell migration and invasion; processes required for metastatic progression. We previously characterized the small molecule EHop-016 as a novel Rac inhibitor in metastatic breast cancer cells and recently found that EHop-016 was effective at reducing tumor growth in nude mice at 25 mg/kg bodyweight (BW). The purpose of this study was to compare the pharmacokinetics and bioavailability of EHop-016 at different dosages in a single dose input scheme (10, 20 and 40 mg/kg BW) following intraperitoneal (IP) and oral gavage (PO) administration to nude mice. We developed and validated a rapid and sensitive method for the quantitation of EHop-016 in mouse plasma by ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC/MS/MS). Separation was carried out on an Agilent Poroshell 120 EC-C18 column (3.0 × 50 mm) using organic and aqueous mobile phases. EHop-016 was identified from its accurate mass and retention time from the acquired full-scan chromatogram and quantified by its peak area. The validated method was linear (R2> 0.995) over the range of 5 – 1000 ng/mL (1/x2 weighting). Pharmacokinetic parameters were obtained by non-compartmental analysis using WinNonlin®. The area under the curve (AUC0-∞) ranged from 328 – 1869 ng·hr/mL and 133 – 487 ng·hr/mL for IP and PO dosing respectively. The elimination half-life (t1/2) ranged from 3.8 – 5.7 hours and 3.4 – 26.8 hours for IP and PO dosing respectively. For both IP and PO administration, the AUC0-∞values were proportional to the tested doses demonstrating linear PK profiles. The relative bioavailability of EHop-016 after oral gavage administration ranged from 26% - 40%. These results support further preclinical evaluation of EHop-016 as a new anti-cancer therapy.

Breast cancer is the first cause of death in women globally. Metastatic breast cancer is stimated to affect more than a quarter of million of women in the US. Due to the lack of effective treatment, metastasis remains the main cause of breast cancer mortality. Therefore, it is imperative to develop novel effective strategies against metastasis. A promising target for anti-metastatic therapy is the Rho GTPase Rac because it plays a key role in metastatic cancer progression by regulating cellular processes such as adhesion, migration, proliferation and survival. Our group developed Ehop-016, a small molecule that inhibits Rac in metastatic breast cancer cells with an IC50=1µM and significantly reduces tumor growth, angiogenesis, and metastasis in a mouse model of metastatic breast cancer. However, its relative bioavailability is moderate and should be improved. Therefore, to find a compound with increased potency and bioavailability, we tested several Ehop-016 derivatives. Using Rac pulldown assays we show that HV-107 and HV-118 inhibit Rac activation by 60% in MDA-MB-231 and MDA-MB-435 metastatic breast cancer cells at 100 and 250nM, respectively. MTT assays show HV-107 (at ≥500nM) and HV-118 (at ≥50nM) significantly inhibit metastatic breast cancer cell viability, while showing minimal toxicity towards non-cancerous cells. Cell cycle analysis by flow cytometry demonstrates a G2-M arrest and a prominent sub-G1 population, indicative of cell death, in metastatic breast cancer cells treated with HV-107 (1000 nM) and HV-118 (100 nM). To evaluate apoptosis as a potential cell death mechanism, we measured caspase 3 activity. Our results show HV-107 and HV-118 significantly induce caspase 3 activity by approximately 1.6-fold at 1000 and 100nM, respectively in metastatic breast cancer cells. Therefore, these Rac inhibitors affect cell viability by inhibiting cell cycle progression and inducing apoptosis. Finally, we tested HV-118 (at 1mg/kg BW) in a mouse model of metastatic breast cancer and found a 30% reduction in tumor growth and a 90% inhibition in metastasis. Taken together, our results indicate HV-107 and HV-118 have potential as anti-breast cancer metastasis therapeutics. This study was supported by awards from the Susan Komen for the Cure, NIH/NIMHHD U54MD008149, and the Puerto Rico Science and Technology Trust to SD; NIH/NCRR R25GM061838 to UPR MSC; NIH/NIMHHD RCMI 8G12MD007583RCMI, Title V PPOHA 031M10505 and Title V Cooperative P031S130068 from U.S. Department of Education to UCC; and and PRINBRE (NIH/NIGMS P20GM103475-13) Sub-Award to LCP. Citation Format: Grace E. Velez, Cornelis Vlaar, Eliud Hernandez, Anibal Valentin, Linette Castillo-Pichardo, Suranganie Dharmawardhane. The Rac inhibitors HV-107 and HV-118 as potential therapeutics for metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1997.

Metastatic disease lacks effective treatments, and remains the primary cause of mortality from epithelial cancers. The Rho family GTPases Rac and Cdc42 and their downstream effector p21-activated kinase (PAK) are pivotal regulators of metastatic cancer cell migration and invasion; thus, overexpression of Rac/Cdc42/PAK has been correlated with reduced patient survival in breast, gastric, and lung cancer. Our studies on the efficacy and pharmacokinetics of the Rac inhibitor EHop-016 (US patents # 8,884,006 B2; 9,278,956B1) validated the development of Rac and Cdc42 inhibitors as anti-metastatic cancer therapeutics. To improve the pharmacologic utility of EHop-016, structural derivatives were screened for enhanced efficacy and bioavailability. We recently identified a dual Rac and Cdc42 inhibitor, MetaBloq(MBQ)-167, which inhibits Rac and Cdc42 activation with IC50s of 103 nM for Rac inhibition and 78 nM for Cdc42 inhibition. Moreover, MBQ-167 is specific to cancer cells that have undergone epithelial-to-mesenchymal transition (EMT), but not epithelial cancer cells or noncancer cells. In metastatic cancer cells, MBQ-167 induces a unique phenotype of loss in cell polarity, cell surface actin extensions, and cell-substrate attachments to undergo anoikis (apoptosis due to inadequate cell-matrix interactions). In vivo, MBQ-167 inhibits HER2 type mammary tumor growth and metastasis in immunocompromised mice by ~90-100% (Humphries-Bickley et al., Mol Cancer Ther 2017; patent applications US 15/499532, PCT/US17/29921). In this study, we have further tested the pharmacokinetics and utility of MBQ-167 in additional models of cancer and screened MBQ-167 derivatives for their utility as Rac/Cdc42 inhibitors. Similar to the results with HER2 type breast cancer, in severe combined immunodeficiency (SCID) mice with mammary fat pad tumors from the triple-negative MDA-MB-231 human breast cancer cell line, the effect of MBQ-167 saturated at 1 mg/kg BW with a 80% reduction in tumor growth and a 100% reduction in metastasis. In gastric cancer, MBQ-167 reduced the viability of metastatic NCI-N87 gastric cancer cells, induced anoikis without affecting the AGS nonmetastatic gastric cancer cells, and inhibited tumor growth in SCID mice. We have screened a range of MBQ-167 derivatives for cell viability and the “anoikis” phenotype and isolated 4 compounds that reduced breast cancer cell viability with GI50s in the nM range. From this screen, MBQ-14 acts similar to MBQ-167, and thus is considered to be a viable inhibitor for further investigation. We have developed a quantitative method for identifying MBQ-167 and MBQ-14 from mouse plasma using ultra-performance convergence chromatography (UPC2)/MS/MS, and find that MBQ-167 is bioavailable in mouse plasma with a half-life of ~4h following oral or intraperitoneal administration. Overall our data demonstrate the utility of further developing MBQ-167 and derivatives as anti-metastatic cancer therapeutics. Citation Format: Linette Castillo-PIchardo, Maria Del Mar Maldonado, Jean Ruiz-Calderon, Jose F. Rodriguez-Orengo, Eliud Hernandez, Cornelis Vlaar, Surangani F. Dharmawardhane Flanagan. The dual Rac/Cdc42 inhibitor MBQ-167 and derivatives as anticancer compounds [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B094.

A convenient method to prepare a series of benzodithiine derivatives was developed, via the synthesis of cyclic disulfide building blocks containing an amino-group linker. Some of the novel cyclic disulfide compounds are shown to modulate the activity of the redox-enzyme glutathione reductase.

Rho GTPases (Ras-related C3 botulinum toxin substrate 1 [Rac1] and cell division cycle 42 [Cdc42]) have been shown to regulate glucose-stimulated insulin secretion (GSIS) via cytoskeletal remodelling, trafficking and fusion of insulin-secretory granules with the plasma membrane. GTP loading of these G proteins, which is facilitated by GDP/GTP exchange factors, is a requisite step in the regulation of downstream effector proteins. Guanine nucleotide exchange factor VAV2 (VAV2), a member of the Dbl family of proteins, has been identified as one of the GDP/GTP exchange factors for Rac1. Despite recent evidence on the regulatory roles of VAV2 in different cell types, roles of this guanine nucleotide exchange factor in the signalling events leading to GSIS remain undefined. Using immunological, short interfering RNA (siRNA), pharmacological and microscopic approaches we investigated the role of VAV2 in GSIS from islet beta cells.Co-localisation of Rac1 and VAV2 was determined by Triton X-114 phase partition and confocal microscopy. Glucose-induced actin remodelling was quantified by live cell imaging using the LifeAct-GFP fluorescent biosensor. Rac1 activation was determined by G protein linked immunosorbent assay (G-LISA).Western blotting indicated that VAV2 is expressed in INS-1 832/13 beta cells, normal rat islets and human islets. Vav2 siRNA markedly attenuated GSIS in INS-1 832/13 cells. Ehop-016, a newly discovered small molecule inhibitor of the VAV2-Rac1 interaction, or siRNA-mediated knockdown of VAV2 markedly attenuated glucose-induced Rac1 activation and GSIS in INS-1 832/13 cells. Pharmacological findings were recapitulated in primary rat islets. A high glucose concentration promoted co-localisation of Rac1 and VAV2. Real-time imaging in live cells indicated a significant inhibition of glucose-induced cortical actin remodelling by Ehop-016.Our data provide the first evidence to implicate VAV2 in glucose-induced Rac1 activation, actin remodelling and GSIS in pancreatic beta cells.

The expression and activities of the Rho GTPases Rac and Cdc42, and their downstream effector P21-activated kinase (PAK), have been correlated with metastatic cancer. Our published studies with the Rac/PAK inhibitor EHop-016 demonstrate the validity of using Rac inhibitors as anti metastatic cancer therapeutics. However, the relatively high effective concentrations (Rac activity IC50 1μM, tumor inhibition at 25 mg/kg body weight (BW)), and the moderate bioavailability (∼30%, t1/2 4.5h) of EHop-016 need improvement. Therefore, we developed EHop-016 derivatives and identified EHop-167 as a Rac inhibitor at nM concentrations. Unlike EHop-016, which does not substantially change breast cancer cell shape, but only reduces cell surface actin based invadopodia, EHop-167 induced a marked decrease in breast cancer cell polarity, cell surface extensions, and cell-extracellular matrix (ECM) attachments (focal adhesions). This phenotype of cell rounding and detachment in response to EHop-167 was demonstrated only by breast cancer cell lines that have undergone epithelial to mesenchymal transition, but not by epithelial breast cancer cells, or MCF-10A mammary epithelial cells. As assessed by pulldown assays and western blotting, Rac and PAK activities were reduced by 80-90% in response to 250 nM EHop-167, in the detached cells. As demonstrated by caspase assays, the cell rounding and detachment from the ECM ultimately resulted in anoikis (cell death due to loss of focal adhesions). Accordingly, Transwell assays of mesenchymal breast cancer cells following 250 nM Ehop-167 showed a ∼90% reduction in cell migration in the detached breast cancer cells, and a ∼60% inhibition in the attached cells. EHop-167 also reduced the mammosphere formation efficiency of metastatic cancer cells by 50%, indicating an inhibitory effect on cancer stem cells. To determine the in vivo efficacy of EHop-167, athymic nude mice, bearing mammary fatpad tumors of MDA-MB-435 metastatic cancer cells, were treated 3X a week with 0, 1, or 10 mg/kg BW EHop-167 for 50 days. Treatment with 1.0 mg/kg BW EHop-167 resulted in a 50% reduction in tumor growth, while 10.0 mg/kg BW EHop-167 induced an ∼95% reduction in tumor growth, compared to controls. Additionally, these mice did not show gross signs of toxicity or significant weight loss. Since the parental compound EHop-016 has no anticancer effects at similar concentrations, we conclude that EHop-167 is an improved Rac/PAK inhibitor that holds promise as an anti metastatic breast cancer therapeutic. Citation Format: Linette Castillo-Pichardo, Tessa Humphries-Bickley, Ingrid Forrestier-Roman, Luis Borrero-Garcia, Fabiola Pagan Melendez, Eliud Hernandez, Cornelis Vlaar, Luis A. Cubano, Surangani Dharmawardhane. Characterization of an improved derivative of the Rac/PAK inhibitor EHop-016 in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3076.

The Rho GTPase Rac is a pivotal regulator of cancer cell migration and invasion; processes required for metastatic progression. We previously characterized the small molecule EHop-016 as a novel Rac inhibitor in metastatic breast cancer cells and recently reported that EHop-016 was effective at reducing tumor growth, metastasis, and angiogenesis in nude mice at 25 mg/kg bodyweight (BW) (Castillo-Pichardo, et al. 2014). We also determined the pharmacokinetics and bioavailability of EHop-016, and reported that EHop-016 is rapidly cleared from mouse plasma with a half-life of ∼5 hrs and ∼30% bioavailability (Humphries-Bickley, et al., 2015). To improve the bioavailability and efficacy of EHop-016, we synthesized and screened a number of derivatives, from which EHop-016A was identified as a potent Rac inhibitor at nM concentrations. Moreover, as determined from immunofluorescence and brightfield microscopy of several breast cancer cell lines, EHop-016A has a dramatic effect on cell morphology by inducing a loss of cell polarity, and inhibiting cell surface actin-based extensions and focal adhesions, to ultimately result in the detachment of cells from the extracellular matrix (ECM). In addition, EHop-016A reduces breast cancer cell migration in a transwell assay. EHop-016A also decreases mammosphere formation, indicating an inhibitory effect on breast cancer stem cell-like properties. The effect of EHop-016A on metastatic cancer cell viability was determined via MTT assays. EHop-016A decreases cell viability with a GI50 of 150 nM and 110 nM in MDA-MB-435 and MDA-MB-231 human metastatic cancer cell lines respectively. Western blotting demonstrated that EHop-016A decreases anti-apoptotic proteins BCL-2 and BCL-xL without affecting their gene expression, as quantified by qPCR. Consequently, EHop-016A increases pro-apoptotic caspase 3/7 activity. These results indicate that the EHop-016A induced cell rounding and detachment from the substratum results in anoikis (apoptosis due to dissolution of integrin-mediated cell to ECM attachments). Therefore, this new small molecule compound has potential as an inhibitor of metastatic breast cancer progression, and warrants further investigation as an anticancer agent. Citation Format: Tessa Humphries-Bickley, Linette Castillo-Pichardo, Luis Borrero-Garcia, Ingrid Forestier-Roman, Luis Cubano, Eliud Hernandez-O'Farrill, Cornelis Vlaar, Suranganie Dharmawardhane. A novel inhibitor of malignant signaling in metastatic breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A141.

Abstract 810 Gain-of-function mutations in the KIT receptor tyrosine kinase have been associated with highly malignant human neoplasms. In particular, an acquired somatic mutation at codon 816 in KIT involving an aspartic acid to valine substitution is found in ∼90% of patients with systemic mastocytosis (SM) and in ∼40% of core binding factor acute myeloid leukemia (AML). The presence of this mutation in SM and AML is associated with poor prognosis and overall survival. In mice, presence of this mutation is sufficient to recapitulate many cardinal features of human SM. This mutation changes the conformation of KIT receptor resulting in altered substrate recognition and constitutive tyrosine autophosphorylation leading to constitutive ligand independent growth, which is resistant to imatinib and shows little therapeutic efficacy in response to Dasatinib in most SM patients. As there are currently no efficacious therapeutic agents against this mutation, we sought to define novel therapeutic targets that contribute to aberrant signaling downstream from KITD816V that promote transformation of primary hematopoietic stem/progenitor cells (HSC/Ps) in diseases such as AML and SM. Previously, we and others have demonstrated that the regulatory subunit of PI3K, p85α, is required for KITD814V (murine homolog) induced transformation. Although difficult to target, we hypothesized that perhaps the downstream effectors of the PI3K signaling pathway, in particular p21 activated kinase (PAK1) and its upstream effectors including guanine exchange factors (GEF) such as Tiam1, Trio and Vav as well as the Rho family of GTPases (Rac) contribute to gain-of-function mutant-mediated transformation. We show that KITD814V (mouse) and KITD816V (human) bearing leukemic cells exhibit constitutive activation of PAK, Rac GTPases, and GEF Vav. Importantly, treatment of KITD814V bearing murine cells or mastocytosis patient derived cells bearing the KITD816V mutation with an allosteric inhibitor of PAK1 (i.e. IPA-3) results in significant inhibition in growth due to enhanced apoptosis. Consistently, expression of a dominant negative form of PAK1 (K299R) in KITD814V bearing cells profoundly inhibited their growth but not the growth of normal cells. Upstream of PAK, we show that suppression of Rac GTPases by expression of a dominant negative form of Rac (RacN17) abrogates activating KIT-induced hyperproliferation, and activity of downstream effector, PAK1. Although both Rac1 and Rac2 are activated due to the presence of KITD814V in primary HSC/Ps; loss of Rac1 only modestly corrects the growth of KITD814V bearing cells and loss of Rac2 contributes to only 50% correction. In contrast, loss of both Rac1 and Rac2 in HSC/Ps resulted in 75% correction in KITD814V induced ligand independent growth in vitro. In vivo, Rac repression significantly delayed the onset of KITD814V induced myeloproliferative neoplasms (MPN). Although, all KITD814V bearing mice died around 20 days of transplantation due to splenomegaly, increased white cell counts and massive lung infiltration by leukemic cells; KITD814V bearing mice in which Rac was repressed showed prolonged survival, significantly reducted spleen size, white cell counts and myeloid cell infiltration in the lungs. Prior studies have shown that Rac GTPases can be activated by GEFs such as Tiam1, Trio and Vav. To assess the specific role of these GEFs in KITD814V induced transformation, we utilized small molecule inhibitors that uniquely target different GEFs. We synthesized and utilized a novel inhibitor of Rac, EHop-016, which is based on the structure of an existing GEF inhibitor, NSC23766. While NSC23766 targets Tiam1 and Trio, EHop-016 targets Vav. The IC50 of EHop-016 is ∼50 fold lower than that of NSC23766. Using these two drugs, we demonstrate that EHop-016 is 50-fold more potent in inhibiting the growth of both murine and human patient derived leukemic cells compared to NSC23766. These observations were confirmed utilizing mice and bone marrow cells deficient in the expression of Vav1 engineered to express the KITD814V mutation. Taken together, a series of experiments using knockout mouse models, mouse models of MPN, dominant negative approaches, and a novel allosteric inhibitor of PAK1 and a novel small molecule inhibitor of GEF Vav provide a mechanism of KITD816V induced transformation and provide potential novel therapeutic targets for treating oncogenic KIT bearing neoplasms. Disclosures: No relevant conflicts of interest to declare.