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Solid tumor growth is dependent on angiogenesis, the formation of neovasculature from existing vessels. Endothelial activation of the extracellular signal–regulated kinase 1/2, c-jun NH2-terminal kinase, and p38 mitogen-activated protein kinase pathways is central to this process, and thus presents an attractive target for the development of angiogenesis inhibitors. Anthrax lethal toxin (LeTx) has potent catalytic mitogen-activated protein kinase inhibition activity. Preclinical studies showed that LeTx induced potent tumor growth inhibition via the inhibition of xenograft vascularization. However, LeTx receptors and the essential furin-like activating proteases are expressed in many normal tissues, potentially limiting the specificity of LeTx as an antitumor agent. To circumvent nonspecific LeTx activation and simultaneously enhance tumor vascular targeting, a substrate preferably cleaved by the gelatinases class of matrix metalloproteinases (MMP) was substituted for the furin LeTx activation site. In vivo efficacy studies showed that this MMP-activated LeTx inhibited tumor xenografts growth via the reduced migration of endothelial cells into the tumor parenchyma. Here we have expanded on these initial findings by showing that this MMP-activated LeTx reduces endothelial proangiogenic MMP expression, thus causing a diminished proteolytic capacity for extracellular matrix remodeling and endothelial differentiation into capillary networks. Additionally, our data suggest that inhibition of the c-jun NH2-terminal kinase and p38, but not extracellular signal–regulated kinase-1/2, pathways is significant in the antiangiogenic activity of the MMP-activated LeTx. Collectively, these results support the clinical development of the MMP-activated LeTx for the treatment of solid tumors. (Mol Cancer Res 2009;7(4):452–61)

Supplementary Data from Matrix Metalloproteinase–Activated Anthrax Lethal Toxin Inhibits Endothelial Invasion and Neovasculature Formation during In vitro Morphogenesis

Supplementary Figures S1-S3 from Matrix Metalloproteinase–Activated Anthrax Lethal Toxin Inhibits Endothelial Invasion and Neovasculature Formation during In vitro Morphogenesis

Anthrax lethal toxin (LeTx) shows potent mitogen-activated protein kinase pathway inhibition and apoptosis in melanoma cells that harbor the activating V600E B-RAF mutation. LeTx is composed of two proteins, protective antigen and lethal factor. Uptake of the toxin into cells is dependent on proteolytic activation of protective antigen by the ubiquitously expressed furin or furin-like proteases. To circumvent nonspecific LeTx activation, a substrate preferably cleaved by gelatinases was substituted for the furin LeTx activation site. Here, we have shown that the toxicity of this matrix metalloproteinase (MMP)–activated LeTx is dependent on host cell surface MMP-2 and MMP-9 activity as well as the presence of the activating V600E B-RAF mutation, making this toxin dual specific. This additional layer of tumor cell specificity would potentially decrease systemic toxicity from the reduction of nonspecific toxin activation while retaining antitumor efficacy in patients with V600E B-RAF melanomas. Moreover, our results indicate that cell surface-associated gelatinase expression can be used to predict sensitivity among V600E B-RAF melanomas. This finding will aid in the better selection of patients that will potentially respond to MMP-activated LeTx therapy. [Mol Cancer Ther 2008;7(5):1218–26]

Purpose:The heterodimeric transcription factor HIF-2 is arguably the most important driver of clear cell renal cell carcinoma (ccRCC). Although considered undruggable, structural analyses at the University of Texas Southwestern Medical Center (UTSW, Dallas, TX) identified a vulnerability in the α subunit, which heterodimerizes with HIF1β, ultimately leading to the development of PT2385, a first-in-class inhibitor. PT2385 was safe and active in a first-in-human phase I clinical trial of patients with extensively pretreated ccRCC at UTSW and elsewhere. There were no dose-limiting toxicities, and disease control ≥4 months was achieved in 42% of patients.Patients and Methods:We conducted a prospective companion substudy involving a subset of patients enrolled in the phase I clinical trial at UTSW (n = 10), who were treated at the phase II dose or above, involving multiparametric MRI, blood draws, and serial biopsies for biochemical, whole exome, and RNA-sequencing studies.Results:PT2385 inhibited HIF-2 in nontumor tissues, as determined by a reduction in erythropoietin levels (a pharmacodynamic marker), in all but one patient, who had the lowest drug concentrations. PT2385 dissociated HIF-2 complexes in ccRCC metastases, and inhibited HIF-2 target gene expression. In contrast, HIF-1 complexes were unaffected. Prolonged PT2385 treatment resulted in the acquisition of resistance, and we identified a gatekeeper mutation (G323E) in HIF2α, which interferes with drug binding and precluded HIF-2 complex dissociation. In addition, we identified an acquired TP53 mutation elsewhere, suggesting a possible alternate mechanism of resistance.Conclusions:These findings demonstrate a core dependency on HIF-2 in metastatic ccRCC and establish PT2385 as a highly specific HIF-2 inhibitor in humans. New approaches will be required to target mutant HIF-2 beyond PT2385 or the closely related PT2977 (MK-6482).

Supplementary Figure 2 from Inhibition of Tumor Angiogenesis by the Matrix Metalloproteinase–Activated Anthrax Lethal Toxin in an Orthotopic Model of Anaplastic Thyroid Carcinoma

Supplementary Figure S1. Pharmacodynamic analyses and illustrative mpMRI studies.

Supplementary Fig. S6 from Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma

Supplementary Fig. S3 from Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma

Supplementary Tables S2-S9.

Supplementary Data Legends

Patients with anaplastic thyroid carcinoma (ATC) typically succumb to their disease months after diagnosis despite aggressive therapy. A large percentage of ATCs have been shown to harbor the V600E B-Raf point mutation, leading to the constitutive activation of the mitogen-activated protein kinase pathway. ATC invasion, metastasis, and angiogenesis are in part dependent on the gelatinase class of matrix metalloproteinases (MMP). The explicit targeting of these two tumor markers may provide a novel therapeutic strategy for the treatment of ATC. The MMP-activated anthrax lethal toxin (LeTx), a novel recombinant protein toxin combination, shows potent mitogen-activated protein kinase pathway inhibition in gelatinase-expressing V600E B-Raf tumor cells in vitro. However, preliminary in vivo studies showed that the MMP-activated LeTx also exhibited dramatic antitumor activity against xenografts that did not show significant antiproliferative responses to the LeTx in vitro. Here, we show that the MMP-activated LeTx inhibits orthotopic ATC xenograft progression in both toxin-sensitive and toxin-resistant ATC cells via reduced endothelial cell recruitment and subsequent tumor vascularization. This in turn translates to an improved long-term survival that is comparable with that produced by the multikinase inhibitor sorafenib. Our results also indicate that therapy with the MMP-activated LeTx is extremely effective against advanced tumors with well-established vascular networks. Taken together, these results suggest that the MMP-activated LeTx-mediated endothelial cell targeting is the primary in vivo antitumor mechanism of this novel toxin. Therefore, the MMP-activated LeTx could be used not only in the clinical management of V600E B-Raf ATC but potentially in any solid tumor. Mol Cancer Ther; 9(1); 190–201

Supplementary Figure Legends from Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma

Supplementary Table S1 from Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma

Supplementary Fig. S4 from Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma

Supplementary Fig. S1 from Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma

Supplementary Fig. S5 from Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma

Supplementary Fig. S2 from Cytotoxicity of the matrix metalloproteinase–activated anthrax lethal toxin is dependent on gelatinase expression and B-RAF status in human melanoma cells

Supplementary Figure 1 from Inhibition of Tumor Angiogenesis by the Matrix Metalloproteinase–Activated Anthrax Lethal Toxin in an Orthotopic Model of Anaplastic Thyroid Carcinoma

Supplementary Figure S4. p53 mutation in Pt27 metastasis progressing on PT2385.

Supplementary Table S1. Multiparametric magnetic resonance imaging (mpMRI) acquisition parameters.

Supplementary Fig. S1 from Cytotoxicity of the matrix metalloproteinase–activated anthrax lethal toxin is dependent on gelatinase expression and B-RAF status in human melanoma cells

Supplementary Fig. S2 from Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma

Supplementary Figure S2. PT2385 does not affect HIF-1 complexes.

Supplementary Figure S3. HIF-2a resistance mutation in Pt35 metastasis progressing on PT2385.

The novel recombinant anthrax toxin, PrAgU2/FP59, composed of the urokinase-activated protective antigen and a fusion protein of Pseudomonas exotoxin and lethal factor was tested for anti–lung cancer efficacy in an in vivo human tumor model. Male athymic nude mice (age 4–6 weeks) were inoculated s.c. with 10 million H1299 non–small cell lung cancer (NSCLC) cells in the left flank. When tumor volumes reached 200 mm3 (6–8 days), i.p. injection of 100 μL saline or different ratios and doses of PrAgU2/FP59 in 100 μL saline were given every 3 days for four doses and an additional dose at day 29. Animals were monitored twice daily and tumor measurements were made by calipers. The maximum tolerated doses of PrAgU2/FP59 differed dependent on the ratios of PrAgU2 to FP59 over the range of 3:1 to 25:1, respectively. At tolerated doses, tumor regressions were seen in all animals. Complete histologic remission lasting 60 days occurred in 30% of animals. PrAgU2/FP59 showed dramatic anti-NSCLC efficacy and warrants further clinical development for therapy of patients with advanced NSCLC. [Cancer Res 2007;67(7):3329–36]

Supplementary Figure from Systematic Urokinase-Activated Anthrax Toxin Therapy Produces Regressions of Subcutaneous Human Non–Small Cell Lung Tumor in Athymic Nude Mice

Purpose: The heterodimeric transcription factor HIF-2 is arguably the most important driver of clear cell renal cell carcinoma (ccRCC). Although considered undruggable, structural analyses at the University of Texas Southwestern Medical Center (UTSW, Dallas, TX) identified a vulnerability in the α subunit, which heterodimerizes with HIF1β, ultimately leading to the development of PT2385, a first-in-class inhibitor. PT2385 was safe and active in a first-in-human phase I clinical trial of patients with extensively pretreated ccRCC at UTSW and elsewhere. There were no dose-limiting toxicities, and disease control ≥4 months was achieved in 42% of patients. Patients and Methods: We conducted a prospective companion substudy involving a subset of patients enrolled in the phase I clinical trial at UTSW (n = 10), who were treated at the phase II dose or above, involving multiparametric MRI, blood draws, and serial biopsies for biochemical, whole exome, and RNA-sequencing studies. Results: PT2385 inhibited HIF-2 in nontumor tissues, as determined by a reduction in erythropoietin levels (a pharmacodynamic marker), in all but one patient, who had the lowest drug concentrations. PT2385 dissociated HIF-2 complexes in ccRCC metastases, and inhibited HIF-2 target gene expression. In contrast, HIF-1 complexes were unaffected. Prolonged PT2385 treatment resulted in the acquisition of resistance, and we identified a gatekeeper mutation (G323E) in HIF2α, which interferes with drug binding and precluded HIF-2 complex dissociation. In addition, we identified an acquired TP53 mutation elsewhere, suggesting a possible alternate mechanism of resistance. Conclusions: These findings demonstrate a core dependency on HIF-2 in metastatic ccRCC and establish PT2385 as a highly specific HIF-2 inhibitor in humans. New approaches will be required to target mutant HIF-2 beyond PT2385 or the closely related PT2977 (MK-6482).

Introduction FASN inhibition is a novel approach to cancer treatment involving selective disruption of palmitate biosynthesis that, in tumor cells, leads to apoptosis. TVB-2640 is an oral, first-in-class, small molecule reversible inhibitor of FASN that demonstrates in vivo antitumor effects. We previously reported the results of dose escalation and now present evidence of preliminary activity in breast cancer patients treated in the dose expansion cohort. Methods This ongoing international, multicenter Phase I trial enrolls patients (pts) with advanced solid tumors with adequate organ function. TVB-2640 is given orally once daily at the MTD (100 mg/m2) as monotherapy (mono) or in combination (combo) with weekly IV paclitaxel (80 mg/m2). Results The most common AE's observed [mono, N=53; combo, N=47] were: alopecia (57%), palmar-plantar erythrodysesthesia (PPE) (36%), dry eye (13%) and increased lacrimation (11%). Gr. 3 toxicities include corneal edema (3%) and PPE (10%). Other toxicities were ≤ Gr. 2 and only minor GI symptoms occurred. All toxicities were reversible on dose interruption and no enhancement of common paclitaxel toxicity was observed when given with TVB-2640. Rare cases of pneumonitis in combination have been observed but the contribution of TVB-2640 to this effect is uncertain. 14 breast cancer patients were enrolled and treated in combination with weekly paclitaxel while 3 breast patients were given monotherapy (during the dose escalation phase). Among patients treated in combination, three (3) confirmed RECIST partial responses (cPR) were seen and multiple cases of prolonged stable disease (SD) (≥16 wks) despite heavy pre-treatment and taxane resistance in all but 2 cases. One ER+, PR+, Her2+ patient achieved a cPR and was on study for 26 weeks. One ER-/PR-/Her2+ patient whose previous best response to paclitaxel treatment was SD for 24 weeks, reached a cPR at week 12, discontinued paclitaxel at week 21 and remains on monotherapy TVB-2640 with a sustained cPR at week 29. The third responder with cPR is an ER+, PR+, HER2- patient whose previous taxane treatment lasted 15 weeks (response unknown) and she remains on study at week 24. Of the remaining 11 patients, 10 achieved SD > 12 weeks and 8 of the 10 maintained that response for 16-45 weeks. The ongoing SD patient at week 45, discontinued paclitaxel at week 35 and remains on monotherapy TVB-2640. Summary TVB-2640 demonstrated multiple cPRs and prolonged SD when combined with weekly paclitaxel in 93% of patients treated. Further exploration of response in patients recently progressed on a taxane (progression within the prior 6 months) and safety with TVB-2640 in combination with docetaxel is being explored. Citation Format: Brenner AJ, Falchook G, Patel M, Infante JR, Arkenau H-T, Dean EM, Borazanci E, Lopez JS, Moore K, Schmid P, Frankel AE, Jones S, McCulloch W, Kemble G, Grimmer K, Burris H. Heavily pre-treated breast cancer patients show promising responses in the first in human study of the first-In-class fatty acid synthase (FASN) inhibitor, TVB-2640 in combination with paclitaxel [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-11-09.

As a master regulator of chromatin function, the lysine methyltransferase EZH2 orchestrates transcriptional silencing of developmental gene networks. Overexpression of EZH2 is commonly observed in human epithelial cancers, such as non-small cell lung carcinoma (NSCLC), yet definitive demonstration of malignant transformation by deregulated EZH2 remains elusive. Here, we demonstrate the causal role of EZH2 overexpression in NSCLC with new genetically-engineered mouse models of lung adenocarcinoma. Deregulated EZH2 silences normal developmental pathways leading to epigenetic transformation independent from canonical growth factor pathway activation. As such, tumors feature a transcriptional program distinct from KRAS- and EGFR-mutant mouse lung cancers, but shared with human lung adenocarcinomas exhibiting high EZH2 expression. To target EZH2-dependent cancers, we developed a novel and potent EZH2 inhibitor JQEZ5 that promoted the regression of EZH2-driven tumors in vivo, confirming oncogenic addiction to EZH2 in established tumors and providing the rationale for epigenetic therapy in a subset of lung cancer.

Purpose: The novel bispecific ligand-directed toxin (BLT) DT2219 consists of a recombinant fusion between the catalytic and translocation enhancing domain of diphtheria toxin (DT) and bispecific single-chain variable fragments (scFV) of antibodies targeting human CD19 and CD22. We conducted a phase I dose-escalation study to assess the safety, maximum tolerated dose, and preliminary efficacy of DT2219 in patients with relapsed/refractory B-cell lymphoma or leukemia. Experimental Design: DT2219 was administered intravenously over 2 hours every other day for 4 total doses. Dose was escalated from 0.5 μg/kg/day to 80 μg/kg/day in nine dose cohorts until a dose-limiting toxicity (DLT) was observed. Results: Twenty-five patients with mature or precursor B-cell lymphoid malignancies expressing CD19 and/or CD22 enrolled to the study. Patients received median 3 prior lines of chemotherapy and 8 failed hematopoietic transplantation. All patients received a single course of DT2219; one patient was retreated. The most common adverse events, including weight gain, low albumin, transaminitis, and fever were transient grade 1–2 and occurred in patients in higher dose cohorts (≥40 μg/kg/day). Two subjects experienced DLT at dose levels 40 and 60 μg/kg. Durable objective responses occurred in 2 patients; one was complete remission after 2 cycles. Correlative studies showed a surprisingly low incidence of neutralizing antibody (30%). Conclusions: We have determined the safety of a novel immunotoxin DT2219 and established its biologically active dose between 40 and 80 μg/kg/day ×4. A phase II study exploring repetitive courses of DT2219 is planned. Clin Cancer Res; 21(6); 1267–72. ©2015 AACR.

We hypothesized that signaling through multiple mitogen-activated protein kinase (MAPK) kinase (MKK) pathways is essential for the growth and vascularization of soft-tissue sarcomas, which are malignant tumors derived from mesenchymal tissues. We tested this using HT-1080, NCI, and Shac fibrosarcoma-derived cell lines and anthrax lethal toxin (LeTx), a bacterial toxin that inactivates MKKs. Western blots confirmed that LeTx treatment reduced the levels of phosphorylated extracellular signal-regulated kinase and p38 MAPK in vitro. Although short treatments with LeTx only modestly affected cell proliferation, sustained treatment markedly reduced cell numbers. LeTx also substantially inhibited the extracellular release of angioproliferative factors including vascular endothelial growth factor, interleukin-8, and basic fibroblast growth factor. Similar results were obtained with cell lines derived from malignant fibrous histiocytomas, leiomyosarcomas, and liposarcomas. In vivo, LeTx decreased MAPK activity and blocked fibrosarcoma growth. Growth inhibition correlated with decreased cellular proliferation and extensive necrosis, and it was accompanied by a decrease in tumor mean vessel density as well as a reduction in serum expression of angioproliferative cytokines. Vital imaging using high-resolution ultrasound enhanced with contrast microbubbles revealed that the effects of LeTx on tumor perfusion were remarkably rapid (

Purpose: In this study, we tested the hypothesis that inhibition of mitogen-activated protein kinase kinases (MKK) inhibits tumor growth by acting on angiogenic signaling and by extension may form the basis of an effective strategy for treatment of Kaposi's sarcoma.Experimental Design: Murine endothelial cells expressing the human herpes virus 8 G protein–coupled receptor (vGPCR-SVEC) were treated with anthrax lethal toxin (LeTx). LeTx is a binary toxin ordinarily secreted by Bacillus anthracis and is composed of two proteins: protective antigen (the binding moiety) and lethal factor (the active moiety). Lethal factor is a protease that cleaves and inactivates MKKs.Results: In vitro, treatment of vGPCR-SVEC with LeTx inhibited MKK signaling, moderately inhibited cell proliferation, and blocked the ability of these cells to form colonies in soft agar. Treatment with LeTx also blocked the ability of these cells to release several angioproliferative cytokines, notably vascular endothelial growth factor (VEGF). In contrast, inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 with U0126 caused a substantial inhibition of proliferation but only modestly inhibited VEGF release. In xenograft models, i.v. injection of LeTx caused reduced tumor growth characterized immunohistochemically by inhibition of MKK signaling, decreased rates of proliferation, and reduced levels of VEGF and VEGF receptor 2, with a corresponding decrease in vascular density.Conclusions: These data support a role for MKK signaling in tumor growth and vascularization and are consistent with the hypothesis that inhibition of MKK signaling by LeTx or a similar agent may be an effective strategy for the treatment of Kaposi's sarcoma as well as other vascular tumors.

The novel recombinant anthrax toxin, PrAgU2/FP59, composed of the urokinase-activated protective antigen and a fusion protein of Pseudomonas exotoxin and lethal factor was tested for anti–lung cancer efficacy in an in vivo human tumor model. Male athymic nude mice (age 4–6 weeks) were inoculated s.c. with 10 million H1299 non–small cell lung cancer (NSCLC) cells in the left flank. When tumor volumes reached 200 mm3 (6–8 days), i.p. injection of 100 μL saline or different ratios and doses of PrAgU2/FP59 in 100 μL saline were given every 3 days for four doses and an additional dose at day 29. Animals were monitored twice daily and tumor measurements were made by calipers. The maximum tolerated doses of PrAgU2/FP59 differed dependent on the ratios of PrAgU2 to FP59 over the range of 3:1 to 25:1, respectively. At tolerated doses, tumor regressions were seen in all animals. Complete histologic remission lasting 60 days occurred in 30% of animals. PrAgU2/FP59 showed dramatic anti-NSCLC efficacy and warrants further clinical development for therapy of patients with advanced NSCLC. [Cancer Res 2007;67(7):3329–36]

Purpose: Anthrax Lethal Toxin (LeTx), composed of protective antigen and lethal factor, catalytically cleaves mitogen-activated protein kinase (MAPK) kinases and inhibits the MAPK signaling pathways. The majority of metastatic melanomas possess the V599E BRAF mutation, which constitutively activates MAPK1/2 signaling. LeTx is cytotoxic to BRAF mutant melanoma cell lines in vitro, whereas most normal cells are resistant to this toxin. In this study, we determine the in vivo potency and safety of systemically administered LeTx. Experimental Design: A s.c. xenograft melanoma model in athymic nude mice was treated with different i.p. doses of LeTx. Results: In this study, we show that in vivo systemic LeTx treatment of s.c. xenograft melanoma tumors in athymic nude mice yields partial and complete tumor regressions with minor toxicity to mice. When animal toxicity was observed, we did not find any histologic evidence of tissue damage. Conclusions: LeTx is one of the rare targeted agents to produce complete remissions of human melanomas in an animal model and thus warrants further preclinical development.

Urokinase plasminogen activator (uPA) is a tumor-specific protease highly expressed in several types of solid tumors and rarely present on normal cells under physiologic conditions. Due to its high expression on metastatic tumors, several different strategies have been used to target the urokinase system. These have mostly led to tumor growth inhibition rather than tumor regression. A different approach was adopted by replacing the furin activation site on a recombinant anthrax toxin with a urokinase activation site. The resulting toxin, PrAgU2/FP59, was highly potent against tumors both in vitro and in vivo. In this study, we show that PrAgU2/FP59 is toxic to a wide range of tumor cell lines, including non–small cell lung cancer, pancreatic cancer, and basal-like breast cancer cell lines. Of the few cell lines found to be resistant to PrAgU2/FP59, most became sensitive upon addition of exogenous pro-uPA. PrAgU2/FP59 was much less toxic to normal human cells. The potency of PrAgU2/FP59 was dependent on anthrax toxin receptor, uPA receptor, and uPA levels but not on total plasminogen activator inhibitor-1 levels. In this study, we show that PrAgU2/FP59 is a wide-range, highly potent, and highly selective toxin that is capable of specifically targeting uPA-expressing tumor cells, independently of the tissue of origin of these cells. Furthermore, we identify three molecular markers, anthrax toxin receptor, uPA, and uPA receptor, which can be used as predictors of tumor cell sensitivity to PrAgU2/FP59. [Mol Cancer Ther 2006;5(10):2556–62]

Purpose: The novel fusion protein, DAB389EGF, composed of the catalytic and translocation domains of diphtheria toxin (DAB389) fused with a His-Ala linker to human epidermal growth factor (EGF) was tested for antiglioma efficacy in an in vivo model of human glioma. Experimental Design: Female athymic nude mice (ages 4-6 weeks) were inoculated s.c. with 10 million U87MG human glioma cells in the right flank. When tumor volumes reached ∼100 mm3 (∼6-8 days), i.t. injections of saline, DAB389IL2, or DAB389EGF 1, 3, 5 or 10 μg in 50 μL were given every other day for three to six doses. Animals were monitored twice daily and tumor measurements were made by calipers. Results: The maximal tolerated dose (MTD) of DAB389EGF was 3 μg every other day. Above the MTD, animals experienced loss of activity, reduced oral intake, and dehydration. Blood chemistries confirmed elevated blood urea nitrogen, creatinine, aspartate transaminase, and alanine transaminase. Histopathology revealed renal tubular necrosis. At the MTD, tumor regression was seen in all animals. Relapses occurred in 4 of 16 (25%) of animals after 1 month. These tumors contained EGF receptor, were sensitive in vitro to DAB389EGF, and responded to a second course of i.t. DAB389EGF. Conclusions: DAB389EGF fusion protein shows in vivo antiglioma efficacy in a s.c. tumor model and warrants further preclinical testing in an i.c. tumor model for eventual treatment of patients with recurrent or refractory EGF receptor–positive glioblastoma multiforme.

DT388GMCSF, a fusion toxin composed of the NH2-terminal region of diphtheria toxin (DT) fused to human granulocyte-macrophage colony-stimulating factor (GMCSF) has shown efficacy in the treatment of acute myeloid leukemia. However, the primary dose-limiting side effect is liver toxicity. We have reproduced liver toxicity in rats using the rodent cell-tropic DT-murine GMCSF (DT390mGMCSF). Serum aspartate aminotransferase and alanine aminotransferase were elevated 15- and 4-fold, respectively, in DT390mGMCSF-treated rats relative to controls. Histologic analysis revealed hepatocyte swelling; however, this did not lead to hepatic necrosis or overt histopathologic changes in the liver. Immunohistochemical staining showed apoptotic cells in the sinusoids, and depletion of cells expressing the monocyte/macrophage markers, ED1 and ED2, indicating that Kupffer cells (KC) are targets of DT390mGMCSF. In contrast, sinusoidal endothelial cells seemed intact. In vitro, DT390mGMCSF was directly cytotoxic to primary KC but not hepatocytes. Two related fusion toxins, DT388GMCSF, which targets the human GMCSF receptor, and DT390mIL-3, which targets the rodent IL-3 receptor, induced a less than 2-fold elevation in serum transaminases and did not deplete KC in vivo. In addition, DTU2mGMCSF, a modified form of DT390mGMCSF with enhanced tumor cell specificity, was not hepatotoxic and was significantly less toxic to KC in vivo and in vitro. These results show that DT390mGMCSF causes liver toxicity by targeting KC, and establish a model for studying how this leads to hepatocyte injury. Furthermore, alternative fusion toxins with potentially reduced hepatotoxicity are presented.

Introduction FASN inhibition causes selective disruption of palmitate biosynthesis that, in tumor cells, leads to apoptosis. TVB-2640 is an oral, first-in-class, small molecule reversible inhibitor of FASN that demonstrated in vivo antitumor effects. We previously reported the results of dose escalation and now present evidence of activity in patients (pts) treated in the dose expansion cohorts. Methods This fully enrolled multicenter phase I trial included pts with advanced solid tumors. TVB-2640 was given PO once daily at the MTD (100 mg/m2) as monotherapy (mono) or in combination (combo) with weekly IV paclitaxel (80 mg/m2). Results The most common related AEs observed in both groups (mono, N=44, combo, N=43) included alopecia (41%), palmar-plantar erythrodysesthesia (PPE) (47%), and decreased appetite (13%). Additional common AEs in the mono group included dry skin (19%) and in the combo group included nausea (28%). Gr3 related AEs included decreased appetite (8%) and PPE (9%); all other related AEs were ≤ Gr2. All related AEs were reversible on dose interruption. No enhancement of paclitaxel toxicity was observed with TVB-2640. Pneumonitis in the combo arm was observed uncommonly (9%), but the contribution of TVB-2640 to this effect is uncertain. Pharmacokinetic analyses showed that TVB-2640 had a similar half-life whether given alone or in combo with paclitaxel. Clearance rates were similar on days 1 and 8 (or day 15 for combo). Multiple pharmacodynamic markers demonstrated potent inhibition of FASN and lipogenesis in pts. With respect to clinical activity, overall, 5 confirmed RECIST partial responses (cPR) were seen. Among pts with NSCLC, 18 of 31 were KRASmut, and KRASmut pts achieved longer progression-free survival on TVB-2640 monotherapy, with 60% of KRASmut pts vs. 0% of KRASwt pts on study > 12 wks. Among 18 KRASmut pts, 11 achieved prolonged SD (≥16 wks), including 6 mono pts (SD=19-46 wks) and 5 combo pts (SD=23-54 wks), whereas no KRASwt pts achieved prolonged SD. One NSCLC combo pt achieved cPR at wk 12 and remained on study for 39 wks. Of 14 breast cancer pts, 3 pts achieved cPR and 8 pts achieved prolonged SD (≥16 wks), despite extensive previous treatment, including taxane resistance. One ongoing breast cancer pt with SD, entering her 78th wk of treatment, discontinued paclitaxel at wk 35 and remains on monotherapy . One pt with peritoneal carcinoma (combo) achieved cPR and a 58% decrease in CA125. Reductions in CA125 were seen in 5 out of 12 ovarian cancer pts, who were typically heavily pretreated and taxane-resistant. Summary TVB-2640 demonstrated antitumor activity, including objective responses when combined with weekly paclitaxel, as well as prolonged SD as monotherapy or in combination with paclitaxel. Further studies are planned to evaluate the efficacy of TVB-2640 in NSCLC and breast cancer pts. Citation Format: Gerald Falchook, Manish Patel, Jeffrey Infante, Hendrik-Tobias Arkenau, Emma Dean, Andrew Brenner, Erkut Borazanci, Juanita Lopez, Kathleen Moore, Peter Schmid, Arthur Frankel, Suzanne Jones, William McCulloch, George Kemble, Howard Burris. First in human study of the first-in-class fatty acid synthase (FASN) inhibitor TVB-2640 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT153. doi:10.1158/1538-7445.AM2017-CT153

Immunotoxins are hybrid proteins composed of peptide cytotoxins covalently linked to target cell-selective peptide ligands such as monoclonal antibodies, antibody fragments, growth factors, or cytokines. Immunotoxins have been synthesized that kill cells selectively in tissue culture with great

Tumor Endothelial Marker 8 (TEM8) is an integrin-like cell-surface receptor originally identified being specific to tumor endothelial cells in colorectal cancer (CRC) blood vessels. Further investigation showed upregulated TEM8 expression in tumor vessels of various tumor types, as well as in certain tumor cells. We observe an increase in TEM8 expression in metastatic cancer cell lines compared to their non-metastatic counterpart in both colon and breast cancer cell lines. In order to investigate whether TEM8 is required for tumor growth and metastasis we have developed a humanized anti-TEM8-toxin-conjugated antibody. The antibody shows preclinical effectiveness in a CRC model in vivo. We have also utilized the CRISPR-Cas9 technology to create TEM8 knockout cell lines, which show impaired proliferation and invasion in vitro and reduced tumor growth in vivo compared to control cell lines. Intriguingly, microarray analysis showed a significant increase in TEM8 mRNA levels in breast cancer cells seeded on collagen modified by the crosslinking enzyme lysyl oxidase (LOX). We have previously shown that LOX plays a role in tumor progression, metastasis, and pre-metastatic niche formation through its ability to modulate matrix stiffness. Importantly, both high TEM8 and LOX expression is correlated with decreased survival in ER- breast cancer patients. Collagen VI, a TEM8 ligand, and their interaction have been implicated in being important for migration and for stimulating Wnt-signaling in breast cancer stem cells. Using global quantitative mass spectrometry we observe an increase in collagen VI in the extracellular matrix of CRC tumors overexpressing catalytically active, but not catalytically inactive, LOX compared to low-LOX expressing tumors. Furthermore, using publicly available databases TEM8 and collagen VI are found to be co-expressed in CRC patient datasets. Thus, we speculate that another possible mechanism of LOX mediated tumor and metastasis progression is through TEM8. Further, that combined targeting of TEM8 and LOX may inhibit primary and metastatic tumor growth. Citation Format: Anette M. Høye, Sofie Tolstrup, Thomas R. Cox, Jeremy P. Mauldin, Arthur E. Frankel, Janine T. Erler. Disrupting tumor growth through targeting TEM8. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B17.

Cancer pain is a great fear, at times greater than even death, in the progression of the disease. 10-15% of cancer patients have pain that is refractory to opioid treatment. There is a common fear among terminal patients that pain treatments will leave them unable to function normally at a time when that is extremely important for them. Substance P-Saporin (SP-SAP), targets with Substance P (SP), a neuromodulator that binds to receptors on laminae I and X of the dorsal horn of NK1r-bearing neurons. These neurons are critical components of the high level, intense pain pathway. The toxic portion of the drug is Saporin (SAP), a ribosome-inactivating protein that, once assisted inside a cell by Substance P, ablates neurons that express NK1r. A single intrathecal infusion of SP-SAP alleviated pathologic pain perception without affecting other sensory signal pathways in preclinical studies conducted in rats. Studies published in the journal Science show pathological pain relief is permanent with no long-term side effects such as central sensitization. A veterinary single-blind clinical trial for the treatment of pain associated with bone tumors in canine companion animals showed that after one injection of SP-SAP, dogs had a much higher quality of life throughout the cancer. Patients in the ongoing SP-SAP phase I clinical trial (NCT02036281) are treated at University of Texas Southwestern. Eligibility: 18 years or older, signed consent, able to verbally report pain, able to complete pain surveys, perform motor/sensory tests, able to undergo intrathecal catheter placement, other therapeutics and palliative options have been exhausted, and have been diagnosed with advanced cancer leading to intractable pain with minimum expected survival of one month. Treatment: a percutaneous intraspinal catheter is placed at the L5-S1 interspace and the advanced 4-5 cm into the intrathecal space under fluoroscopic guidance. 1 mL SP-SAP is mixed with 1 mL patient CSF fluid and administered via the catheter. Subjects are monitored for four hours in the recovery room before catheter is removed, monitored in hospital 24 hours, and discharged. The starting dose of SP-SAP was 1 mcg in 1mL, 1/10th the dog no observable adverse effect level. Dose: Single subject cohorts are at doses of 1, 2, 4, 8, 16, 32, 64, and 90 mcg. If a drug-related dose-limiting toxicity is observed in two or more patients in a cohort, the maximal tolerated dose (MTD) has been exceeded, and the next lower dose level will be the MTD. An additional 15 patients will be treated at the MTD to confirm safety. Endpoints: Response measurements include pain level measurements, Brief Pain Inventory (BPI), VAS “pain bothersomeness,” VAS “pain intensity,” ODI, SF-36, EQ-5D, BDI, and Medication Use logs in patient diary. Primary endpoints for the trial are either a 20% reduction in pain scores or a 20% reduction in pain medications taken by the patient. Citation Format: Arthur E. Frankel, Douglas A. Lappi, Carl Noe, Denise Higgins, Brian J. Russell. Preliminary results from a phase I study of Substance P-Saporin in advanced cancer patients with intractable pain. [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 CT030.

Acute myeloid leukemia (AML) is the most common leukemia in adults and the second most common leukemia in children, accounting for a significant share of health care costs with ten thousand cases diagnosed per annum in the US alone. Enzyme-based therapy in the form of asparaginase has revolutionized the treatment of acute lymphoblastic leukemia and there is increasing interest in exploiting analogous tractable metabolic defects in AML. Interestingly, arginine auxotrophic tumors due to deficiency of the rate-limiting enzyme for arginine production, argininosuccinate synthetase (ASS1) are susceptible to arginine-degrading enzymes. Here, we studied whether ASS1 negativity would predict for the efficacy of pegylated arginine deiminase (ADI-PEG20) using AML cell lines and primary AML samples. A lack of ASS1 protein was identified in three of seven leukemic cell lines (K562, Kasumi and KG-1) and in all nine samples from patients with cytogenetically normal and abnormal karotype AML. Methylation of the ASS1 promoter correlated with reduced levels of ASS1 mRNA and absence of ASS1 expression. Bone marrow trephines from patients with AML revealed absence of ASS1 protein in 87% (46/53) of samples by immunohistochemistry, indicating that ASS1 expression may be a biomarker of response to ADI-PEG20 in vivo. Increased levels of ASS1 mRNA and detectable ASS1 protein expression were noted in acute promyelocytic leukemia with the translocation t(15;17). Significantly, ADI-PEG20 reduced the viability of ASS1-negative AML lines whereas the ASS1-positive control lines, Fujioka and U937, were resistant to drug-induced arginine deprivation. Recently, we have identified primary ASS1-negative AML samples with good engraftment in NOD/SCID mice and are proceeding to test the efficacy of ADI-PEG20 using this primary AML xenograft model. Based on our preliminary data and the potential efficacy with low toxicity of arginine deprivation in humans, a phase II trial of ADI-PEG20 is planned in patients with relapsed AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4067. doi:10.1158/1538-7445.AM2011-4067