William E. Fogler

ENMD-2076 Is an Orally-Active Kinase Inhibitor with Antiangiogenic and Antiproliferative Mechanisms of Action

ENMD-2076 is a novel orally active, small molecule kinase inhibitor with a mechanism of action involving several pathways key to tumor growth and survival: angiogenesis, proliferation, and the cell cycle. ENMD-2076 has selective activity against the mitotic kinase Aurora A, as well as kinases involved in angiogenesis (VEGFRs, FGFRs). ENMD-2076 inhibited the growth in vitro of a wide range of human solid tumor and hematopoietic cancer cell lines with IC50 values ranging from 0.025 to 0.7 μmol/L. ENMD-2076 was also shown to induce regression or complete inhibition of tumor growth in vivo at well-tolerated doses in tumor xenograft models derived from breast, colon, melanoma, leukemia, and multiple myeloma cell lines. Pharmacodynamic experiments in vivo showed that in addition to inhibiting Aurora A, single doses of ENMD-2076 had sustained inhibitory effects on the activation of Flt3 as well as the angiogenic tyrosine kinases, VEGFR2/KDR and FGFR1 and 2. ENMD-2076 was shown to prevent the formation of new blood vessels and regress formed vessels in vivo at doses equivalent to those that gave substantial activity in tumor xenograft models. These results indicate that ENMD-2076 is a well-tolerated, orally active multitarget kinase inhibitor with a unique antiangiogenic/antiproliferative profile and provides strong preclinical support for use as a therapeutic for human cancers. Several phase 1 studies involving ENMD-2076 have been recently completed, and the compound is currently being evaluated in a phase 2 clinical trial in patients with platinum-resistant ovarian cancer. Mol Cancer Ther; 10(1); 126–37. ©2010 AACR.

Significant antitumor activity in vivo following treatment with the microtubule agent ENMD-1198

Clinical studies using the microtubule-targeting agent 2-methoxyestradiol (2ME2; Panzem) in cancer patients show that treatment is associated with clinical benefit, including prolonged stable disease, complete and partial responses, and an excellent safety profile. Studies have shown that 2ME2 is metabolized by conjugation at positions 3 and 17 and oxidation at position 17. To define structure-activity relationships for these positions of 2ME2 and to generate metabolically stable analogues with improved anti-tubulin properties, a series of analogues was generated and three lead analogues were selected, ENMD-1198, ENMD-1200, and ENMD-1237. These molecules showed improved metabolic stability with >65% remaining after 2-h incubation with hepatocytes. Pharmacokinetic studies showed that oral administration of the compounds resulted in increased plasma levels compared with 2ME2. All three analogues bind the colchicine binding site of tubulin, induce G2-M cell cycle arrest and apoptosis, and reduce hypoxia-inducible factor-1α levels. ENMD-1198 and ENMD-1200 showed improved in vitro antiproliferative activities. Significant reductions in tumor volumes compared with vehicle-treated mice were observed in an orthotopic breast carcinoma (MDA-MB-231) xenograft model following daily oral treatment with all compounds (ANOVA, P < 0.05). Significantly improved median survival time was observed with ENMD-1198 and ENMD-1237 (200 mg/kg/d) in a Lewis lung carcinoma metastatic model (P < 0.05). In both tumor models, the high-dose group of ENMD-1198 showed antitumor activity equivalent to that of cyclophosphamide. ENMD-1198 was selected as the lead molecule in this analogue series and is currently in a phase I clinical trial in patients with refractory solid tumors. [Mol Cancer Ther 2008;7(6):1472–82]

Disease modifying and antiangiogenic activity of 2-Methoxyestradiol in a murine model of rheumatoid arthritis

BackgroundA critical component of disease progression in rheumatoid arthritis (RA) involves neovascularization associated with pannus formation. 2-methoxyestradiol (2ME2) is a naturally occurring molecule with no known physiologic function, although at pharmacologic concentrations it has antiproliferative and antiangiogenic activities. We investigated the impact of orally administered 2ME2 on the initiation and development of proliferative synovitis using the anti-collagen monoclonal antibodies (CAIA) model.MethodsSevere polyarticular arthritis was induced in Balb/c female mice by administration of 2 mg of a monoclonal antibody cocktail intravenously into the tail vein of mice. Twenty-four hours following monoclonal antibody administration, mice were injected with 25 μg of LPS (E. coli strain 0111:B4) via the intraperitoneal route. Treatment with 2ME2 (100, 75, 50, 25, 10, 1 mg/kg, p.o., daily), or vehicle control began 24 hrs following LPS challenge and continued to day 21. Hind limbs were harvested, sectioned and evaluated for DMARD activity and general histopathology by histomorphometric analysis and immunohistochemistry (vWF staining). In a separate study, different dosing regimens of 2ME2 (100 mg/kg; q.d. vs q.w. vs q.w. × 2) were evaluated. The effect of treatment with 2ME2 on gene expression of inflammatory cytokines and angiogenic growth factors in the joint space was evaluated 5 and 14 days after the induction of arthritis.ResultsMice treated with 2ME2 beginning 24 hours post anti-collagen monoclonal antibody injection, showed a dose-dependent inhibition in mean arthritic scores. At study termination (day 21), blinded histomorphometric assessments of sectioned hind limbs demonstrated decreases in synovial inflammation, articular cartilage degradation, pannus formation, osteoclast activity and bone resorption. At the maximal efficacious dosing regimen (100 mg/kg/day), administration of 2ME2 resulted in total inhibition of the study parameters and prevented neovascularization into the joint. Examination of gene expression on dissected hind limbs from mice treated for 5 or 14 days with 2ME2 showed inhibition of inflammatory cytokine message for IL-1β, TNF-α, IL-6 and IL-17, as well as the angiogenic cytokines, VEGF and FGF-2.ConclusionThese data demonstrate that in the CAIA mouse model of RA, 2ME2 has disease modifying activity that is at least partially attributable to the inhibition of neovascular development. Further, the data suggests new mechanistic points of intervention for 2ME2 in RA, specifically inhibition of inflammatory mediators and osteoclast activity.

Stereoselective synthesis of 3,3-diarylacrylonitriles as tubulin polymerization inhibitors.

A series of 3,3-diarylacrylonitriles were synthesized stereoselectively as tubulin polymerization inhibitors for potential use in cancer chemotherapy. This synthetic route features stannylcupration and palladium-catalyzed Stille cross-coupling chemistry, allowing both E and Z isomers of 3,3-diarylacrylonitriles to be prepared in a very short sequence of reactions.

Biological characterization of a chimeric mouse-human IgM antibody directed against the 17-1A antigen

SummaryA chimeric antibody was constructed in which the murine H- and L-chain variable regions of mAb 17-1A, raised against human colorectal cancer cells, were joined with the human constant μ and κ regions. Transfection of these constructs into the murine myeloma Sp2/0 resulted in the expression and secretion of a pentameric Ig, designated chimeric 17-1A IgM. The chimeric 17-1A IgM was subsequently compared to a previously described chimeric 17-1A IgG1 for biological activities. Both chimeric mAbs were equally effective (weight basis) in competing against the binding of murine125I-17-1A to cultures of HT-29 colon carcinoma cells. The calculated association constants for the chimeric 17-1A IgM and IgG1 were 1.63 × 108 1/mol and 3.41 × 107 1/mol, respectively. Unlike chimeric 17-1A IgG1, the chimeric 17-1A IgM was able to render colon carcinoma target cells susceptible to lysis by both xenogeneic (rabbit) and human complement. The extent of complement-mediated lysis dependent upon chimeric 17-1A IgM was correlated to 17-1A antigen expression on target cells. HT-29 colon carcinoma cells treated with chimeric 17-1A IgM did not directly result in antibody-dependent cellular cytotoxicity by human peripheral blood monocytes. However, chimeric 17-1A IgM greatly enhanced the deposition of C3 on complementtreated HT-29 cells, and concomitant incubation with monocytes resulted in heightened lysis of the tumor cells. The feasibility of enhancing host defense against gastrointestinal malignancies by the administration of this chimeric 17-1A IgM may have certain clinical advantages.

Abstract 425: A small molecule glycomimetic antagonist of E-selectin and CXCR4 (GMI-1359) prevents pancreatic tumor metastasis and improves chemotherapy

The tumor microenvironment9s cellular and molecular composition includes a complex matrix that encases tumor cells and presents a conundrum in respect to pancreatic tumor therapy. On one hand, the dense desmoplastic reaction that accompanies growth of well differentiated pancreatic tumors reduces blood flow to tumors and creates an environment in which it is difficult to deliver therapies. On the other hand, elimination of certain stromal elements enhances the aggressiveness and metastatic potential of pancreatic cancer cells. Several current studies are attempting to target stroma in an effort to enhance delivery and efficacy of therapeutic agents and to block metastasis. We have investigated the activity of GMI-1359, a potent dual antagonist that targets both E-selectin and CXCR4. Adhesion protein E-selectin plays an important role in the tumor microenvironment by regulating cell contacts, including tumor cell binding to vascular and lymphatic endothelial cells during extravasation. Chemokine receptor CXCR49s role in the chemoattraction of tumor cells toward endothelial cells (ECs) contributes to tumor microenvironment remodeling by influencing lymphangiogenesis/angiogenesis, tumor cell survival/proliferation, and tumor stem cell mobilization. Our in vitro studies show that pancreatic ductal adenocarcinoma (PDAC) cells do not attract growth of lymphatic nor vascular ECs toward themselves. However, tumor associated fibroblasts, a major component of the PDAC tumor microenvironment, significantly increase EC directional migration. GMI-1359 completely blocked lymphatic and vascular EC migration toward fibroblast cells and disrupted these cell-cell interactions. In addition, GMI-1359 inhibited the capacity of invasive PDAC cell lines S2.013 and Colo357 to bind and migrate across EC barriers. The dual antagonist was more effective than an independent small molecule E-selectin inhibitor. We evaluated the capacity of GMI-1359 to inhibit growth and metastasis of orthotopically implanted S2.013 cells with and without administration of Gemcitabine. Two weeks post implantation, mice were treated by intraperitoneal injection for 4 weeks with either PBS once daily; 40 mg/kg GMI-1359 once daily; 100 mg/kg Gemcitabine every 4 days; or a combination of GMI-1359 and Gemcitabine. GMI-1359 treatment slightly, but not significantly, decreased primary tumor size as compared to the vehicle control. However, GMI-1359 in combination with Gemcitabine significantly reduced metastasis of this tumor to liver and diaphragm as compared to mice that received only Gemcitabine. Further studies of GMI-1359 are warranted to understand its potential for disrupting cellular contacts and blocking pancreatic tumor dissemination through the vascular and lymphatic systems, and for enhancing the efficacy of chemotherapeutic approaches to pancreatic cancer. Citation Format: Maria M. Steele, William E. Fogler, John L. Magnani, Michael A. Hollingsworth. A small molecule glycomimetic antagonist of E-selectin and CXCR4 (GMI-1359) prevents pancreatic tumor metastasis and improves chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 425. doi:10.1158/1538-7445.AM2015-425

Abstract 428: Dual E-selectin and CXCR4 inhibition reduces tumor growth and increases the sensitivity to docetaxel in experimental bone metastases of prostate cancer

Prostate cancers preferentially metastasize to the skeleton where the bone microenvironment can stimulate excessive tumor cell growth and spread, and promote the emergence of clinically-resistant disease. An improved understanding of the complex relationship between prostate carcinoma (PCa) cells and the bone microenvironment has created a powerful opportunity to develop novel therapies. PCa cells preferentially roll and adhere on bone marrow vascular endothelial cells, where constitutive E-selectin expression and abundant stromal cell-derived factor-1α (SDF-1α) are expressed and interact with E-selectin ligands and CXCR4 present on PCa cells. These molecular interactions initiate a cascade of activation events that lead to the development of treatment resistant metastases. This suggests that agents able to antagonize these molecular interactions may be used as pharmacological treatments of bone metastatic disease. In the current studies we investigate if a dual E-selectin/CXCR4 inhibitor (GMI-1359) could impact the intraosseous growth of the metastatic, androgen-independent PC3M cell line and affect chemosensitivity to docetaxel. PCa cells, including PC3M, selected for increased visceral and bone metastatic potential express high levels of E-selectin ligands and CXCR4 as compared to nonmetastatic PCa cell lines. We evaluated the ability of GMI-1359 administered alone or in combination with docetaxel to inhibit the growth and metastasis of intratibially implanted luciferase-transfected PC-3M cells. Approximately two weeks post tumor cell implantation, mice were treated by intraperitoneal injection for 2 weeks with either saline twice daily; 40 mg/kg GMI-1359 twice daily, 5 mg/kg docetaxel once weekly or a combination of GMI-1359 and docetaxel. Thirty-five days after initiation of treatment, the percentage of tibiae positive by X-ray and the size of osteolytic lesions was impacted by treatment with GMI-1359 alone or in combination with docetaxel. Docetaxel alone had only a modest impact on intraosseous lesions. Lytic units were reduced by 38%, 78% and 88% in mice treated with docetaxel alone, GMI-1359 alone, or GMI-1359 in combination with docetaxel, respectively. The significantly reduced intraosseous growth of PC3M cells correlated with decreased serum levels of both mTRAP and type I collagen fragments. Our data provides a clear biologic rationale for the use of a dual E-selectin/CXCR4 inhibitor as an adjuvant to taxane-based chemotherapy in men with high-risk prostate cancer to prevent bone metastases. Given its complementary mechanism of action to traditional chemotherapy, GMI-1359 warrants further development not only in prostate carcinoma, but also in other malignancies where tumor cells are likely to spread to bone. Citation Format: Giovanni L. Gravina, Andrea Mancini, Alessandro Colapietro, Simona D. Monache, Adriano Angelucci, Alessia Calgani, William E. Fogler, John L. Magnani, Claudio Festuccia. Dual E-selectin and CXCR4 inhibition reduces tumor growth and increases the sensitivity to docetaxel in experimental bone metastases of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 428. doi:10.1158/1538-7445.AM2015-428

Abstract 1757: Mobilization of tumor-primed, marrow-infiltrating lymphocytes into peripheral blood with inhibitors of E-selectin or E-selectin and CXCR4

Marrow-infiltrating lymphocytes (MILs) primed to tumor antigens have been described in patients with hematologic malignancies and in metastatic disease arising from carcinomas. The presence of tumor-reactive MILs in these patients has suggested the possibility of their utilization in T-cell immunotherapeutic approaches. Inherent in this approach are considerations that mediate MIL interactions with the microenvironment and how these may be governed for adoptive or active immunotherapy. Both E-selectin and CXCR4 are known to regulate the homing and retention of T cells to the bone marrow. GMI-1271 and GMI-1359 are potent, small-molecule glycomimetic antagonists of E-selectin and E-selectin/CXCR4, respectively. GMI-1359 is a potent small-molecule glycomimetic dual antagonist targeting E-selectin and CXCR4. In the present studies tumor-specific MILs were established in BALB/c mice that had been induced to reject the syngeneic CT26 colon carcinoma via treatment with anti-CTLA-4 T cell checkpoint antibody, and the subsequent effects of antagonizing E-selectin and/or CXCR4 with GMI-1271 or GMI-1359 on the mobilization and distribution of these bone marrow-derived tumor-specific CD8+ T cells were determined. CT26-immune mice were treated for three days with saline, GMI-1271 (40 mg/kg), or GMI-1359 (40 mg/kg) and 12 hours following the last injection, the phenotype and functional activity of CD8+ T cells were determined in bone marrow and peripheral blood. Additional controls included CT26-immune mice treated with G-CSF (0.125 mg/kg) and tumor-naive mice treated with saline. Treatment of mice with GMI-1271 and to a greater extent with GMI-1359 led to an approximate 3-4 fold increase in CD8+CD62L+CD44- naive and CD8+CD62L+CD44+ central memory T cells in peripheral blood. This was not observed following treatment of tumor-immune mice with G-CSF. Treatment of mice with GMI-1271 or GMI-1359 did not affect distribution of CD8+CD62L-CD44+ effector memory T cells in peripheral blood. The increase in percentages of CD8+ naive and central memory T cells in peripheral blood following treatment with GMI-1271 or GMI-1357 functionally correlated with increased production of IFN-γ ex vivo in response to irradiated CT26 tumor cells or the immunodominant CT26 peptide, AH-1. Collectively these results demonstrate the mobilization or redistribution of marrow-infiltrating tumor-specific CD8+ T cells into peripheral blood as a consequence of E-selectin and/or E-selectin and CXCR4 antagonism. Once in the periphery, these MILs could (1) be collected for adoptive immunotherapy approaches or (2) serve as a systemic augmentation of T cells for combination with immune stimulants as a foundation to boost active immunotherapy. Citation Format: William E. Fogler, Theodore A. Smith, Rachel K. King, John L. Magnani. Mobilization of tumor-primed, marrow-infiltrating lymphocytes into peripheral blood with inhibitors of E-selectin or E-selectin and CXCR4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1757.

Abstract 902: A small molecule glycomimetic antagonist of E-selectin and CXCR4 (GMI-1359) delays pancreatic tumor metastasis and significantly alters the pancreatic tumor microenvironment

The cellular and molecular composition of the pancreatic tumor microenvironment includes a complex matrix that encases tumor cells and presents a conundrum in respect to therapy. The dense desmoplasia that accompanies tumor growth reduces blood flow to tumors and creates an environment that compromises delivery of therapeutics. As well, elimination of certain stromal elements enhances the aggressiveness and metastatic potential of pancreatic cancer cells. Recent studies attempt to target stroma in an effort to enhance delivery and efficacy of therapeutic agents and to block metastasis. In this context we have investigated the activity of GMI-1359, a potent dual antagonist that targets both E-selectin and CXCR4. Adhesion protein E-selectin is crucial for regulating vascular and lymphatic endothelial cell (EC) interactions with tumor cells during transmigration. The CXCL12-CXCR4 chemokine axis contributes to the formation of the tumor microenvironment including fibroblast and immune cell recruitment, lymph- and angiogenesis, tumor cell proliferation/survival, and tumor stem cell mobilization. Our in vitro results demonstrate that pancreatic fibroblasts secrete significant amounts of CXCL12 which promotes tumor cell and lymphatic and vascular EC directional migration; this migration toward CXCL12-secreting fibroblasts was completely blocked by GMI-1359. Additionally, CXCL12-stimulated ECs facilitated increased transendothelial migration (TEM) by pancreatic tumor cells. Dual antagonist GMI-1359 inhibited this CXCL12-dependent increase in pancreatic tumor cell TEM and was more effective than an independent small molecule E-selectin only inhibitor. Using an in vivo orthotopic model of pancreatic cancer in athymic mice, we evaluated the ability of GMI-1359 (with and without co-administration Gemcitabine (Gem)) to suppress tumor progression, modulate tumor microenvironment composition, and prolong survival. Our work demonstrated GMI-1359 slightly inhibited tumor growth when used alone or in combination with Gem but did not prolong survival in this immune compromised model. However, GMI-1359 inhibited tumor metastasis to the spleen, liver, and lungs. Interestingly, GMI-1359 significantly modulated the cellular composition of the tumor microenvironment. Immunohistochemical analysis revealed mice treated with GMI-1359 (with or without Gem administration) had drastically reduced desmoplasia and reduced lymphatic and blood vessel densities compared to mice treated with vehicle control or Gem alone. The E-selectin only inhibitor had no effect on tumor microenvironment composition. Further studies of GMI-1359, particularly in immune competent models, are warranted to understand its potential for disrupting the pancreatic tumor microenvironment, inhibiting dissemination, and enhancing anti-tumor immune responses. Citation Format: Maria M. Steele, William E. Fogler, John L. Magnani, Michael A. Hollingsworth. A small molecule glycomimetic antagonist of E-selectin and CXCR4 (GMI-1359) delays pancreatic tumor metastasis and significantly alters the pancreatic tumor microenvironment. [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 902.

Abstract 3284: Targeting E-selectin/CXCR4 with GMI-1359 effectively mobilizes bone marrow leukemia cells and enhances FLT3 inhibitor-induced anti-leukemia efficacy in a murine acute myeloid leukemia model

FLT3 internal tandem duplication (ITD) mutations are the most common alterations in acute myeloid leukemia (AML) and are associated with poor disease prognosis. Targeted therapy using FLT3-ITD inhibitors showed limited effect in reducing leukemia blasts in bone marrow (BM) than that in peripheral blood. The BM microenvironment is enriched with cytokines and adhesion molecules, such as CXCR4 and E-selectin, which are believed to provide AML cells protection against chemotherapeutic agents. Therefore, blocking CXCR4 and E-selectin concomitantly with FLT3 inhibition could theoretically eliminate the protection in FLT3-mutant AML patients. We recently reported that targeting CXCR4/E-selectin with the dual inhibitor GMI-1359 showed significant prolongation of survival of mice engrafted with FLT3-ITD mutant MV4-11 leukemia cells. In the present study, we further investigated anti-leukemia effects of dual CXCR4/E-selectin inhibition. Results indicate efficient mobilization of leukemia cells into the circulation by GMI-1359 in a MOLM14-engrafted murine model 2h after drug treatment, which was 3.3-fold (± 0.3) higher compared with the CXCR4 antagonist plerixafor, and 7.4-fold (± 2.7) higher compared with controls. In addition, GMI-1359 also mobilized normal murine leukocytes from the BM, suggesting that GMI-1359 may block interactions of leukemia cells with various BM components. Combination therapy of GMI-1359 and sorafenib significantly reduced leukemia burden (1.9e7 vs. 2.3e9, 1.0e9 and 8.5e7 photons/sec in the combination group versus control, GMI-1359 and sorafenib groups, respectively, at day 20 as determined by Xenogen IVIS bioluminescence Imaging; p Citation Format: Weiguo Zhang, Hong Mu, Qi Zhang, Nalini B. Patel, William E. Fogler, John L. Magnani, Michael Andreeff. Targeting E-selectin/CXCR4 with GMI-1359 effectively mobilizes bone marrow leukemia cells and enhances FLT3 inhibitor-induced anti-leukemia efficacy in a murine acute myeloid leukemia model. [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 3284.