Otto Phanstiel - Academia.edu (original) (raw)

Papers by Otto Phanstiel

Research paper thumbnail of Loss of Anti-Tumor Efficacy by Polyamine Blocking Therapy in GCN2 Null Mice

Biomedicines

GCN2 is one of the main sensors of amino acid starvation stress, and its activation in the stress... more GCN2 is one of the main sensors of amino acid starvation stress, and its activation in the stressful tumor microenvironment plays a crucial role in tumor survival and progression. We hypothesized that elevated polyamine biosynthesis and subsequent depletion of precursor arginine activates GCN2, thus rewiring metabolism to support tumor cell survival and drive myeloid immunosuppressive function. We sought to determine if the anti-tumor efficacy of a polyamine blocking therapy (PBT) may be mediated by its effect on GCN2. Unlike wild-type mice, PBT treatment in GCN2 knockout mice bearing syngeneic B16.F10 or EG7 tumors resulted in no tumor growth inhibition and no changes in the profile of infiltrating tumor immune cells. Studies with murine bone marrow cell cultures showed that increased polyamine metabolism and subsequent arginine depletion and GCN2 activation played an essential role in the generation and cytoprotective autophagy of myeloid derived suppressor cells (MDSCs) as well a...

Research paper thumbnail of Abstract 4503: Polyamine blockade therapy: A strategy to block immunosuppression in pancreatic cancer

Cancer Research, Apr 4, 2023

Research paper thumbnail of Abstract 3546: DFMO based improvement in survival of pancreatic cancer-bearing mice is associated with modulation of immune suppression in the tumor microenvironment

Cancer Research, Jun 15, 2022

Existing therapeutics have failed to improve pancreatic ductal adenocarcinoma (PDAC) patient outc... more Existing therapeutics have failed to improve pancreatic ductal adenocarcinoma (PDAC) patient outcomes, in part due to tumor associated immune suppression and upregulation of compensatory mechanisms. KRAS and c-MYC are important oncogenes in PDAC linked to tumor immune suppression. We describe the use of difluoromethylornithine (DFMO) and a c-RAF inhibitor (GW5074) to indirectly target c-MYC and KRAS, respectively. DFMO in combination with GW5074 was tested for ability to improve pancreatic cancer outcomes. Pancreatic cancer cells showed decreased cell viability and increased apoptosis in response to a combination of DFMO + GW5074. In vivo orthotopic models of pancreatic tumors had decreased tumor weight in DFMO + GW5074 treated mice, when compared to control group. However, only treatment with single agent DFMO resulted in improved survival of immunocompetent pancreatic tumor bearing mice with respect to control, in contrast to treatment with GW5074 alone or DFMO + GW5074. To understand why the in vivo data was contrary to in vitro results, immunohistochemical analysis of immune cells in the tumor microenvironment was used to reveal increased expression of markers associated with anti-tumor effects such as CD86, CD3, CD4 and CD8 in DFMO treated tumors. Tumors treated with DFMO also displayed decreased expression of MYC, suggesting that DFMO-associated MYC suppression could be linked to decreased immune suppression in the tumor microenvironment and improved survival. In contrast, GW5074 treatment maintained MYC expression in tumors. Overall, the present study points to DFMO being an immunomodulatory agent, and a need for further understanding of DFMO-based therapeutic strategies in PDAC. Citation Format: Sai Preethi Nakkina, Sarah B. Gitto, Jordan M. Beardsley, Veethika Pandey, Michael M. Rohr, Jignesh Parikh, Otto Phanstiel, Deborah A. Altomare. DFMO based improvement in survival of pancreatic cancer-bearing mice is associated with modulation of immune suppression in the tumor microenvironment [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 3546.

Research paper thumbnail of A <i>Drosophila</i> Model To Identify Polyamine−Drug Conjugates That Target the Polyamine Transporter in an Intact Epithelium

Journal of Medicinal Chemistry, Dec 29, 2007

Polyamine transport is elevated in many tumor types, suggesting that toxic polyamine-drug conjuga... more Polyamine transport is elevated in many tumor types, suggesting that toxic polyamine-drug conjugates could be targeted to cancer cells via the polyamine transporter (PAT). We have previously reported the use of Chinese hamster ovary (CHO) cells and its PAT-deficient mutant cell line, CHO-MG, to screen anthracene-polyamine conjugates for their PAT-selective targeting ability. We report here a novel Drosophila-based model for screening anthracene-polyamine conjugates in a developing and intact epithelium ( Drosophila imaginal discs), wherein cell-cell adhesion properties are maintained. Data from the Drosophila assay are consistent with previous results in CHO cells, indicating that the Drosophila epithelium has a PAT with vertebrate-like characteristics. This assay will be of use to medicinal chemists interested in screening drugs that use PAT for cellular entry, and it offers the possibility of genetic dissection of the polyamine transport process, including identification of a Drosophila PAT.

Research paper thumbnail of Abstract PO-119: DFMO mediated improvement in survival of an orthotopic model of pancreatic cancer is associated with modulating immune suppression in the tumor microenvironment

There remains an urgent need to target pancreatic tumor cells using innovative strategies. KRAS a... more There remains an urgent need to target pancreatic tumor cells using innovative strategies. KRAS and MYC, are important oncogenes in pancreatic ductal adenocarcinoma (PDAC) which pose a challenge to successful treatment of PDAC. Our previous studies have shown that inhibition of ornithine decarboxylase 1 (ODC1) using difluoromethylornithine (DFMO) decreases MYC expression and tumorigenesis. GW5074 can modulate RAF1, a downstream effector of KRAS. Here we test the responsiveness of pancreatic tumor cells treated with DFMO alone and in combination with GW5074. We used an orthotopic animal model of pancreatic tumor using KRas-driven murine pancreatic cancer cells (PanO2) to test the effects of these compounds on tumor microenvironment and overall survival. Cellular and molecular changes in the tumor microenvironment were assessed using immunohistochemistry. The results showed an inhibition of pancreatic cancer cell viability in DFMO and DFMO+GW5074 treatment groups in vitro, with a significant decrease in tumor weight compared to control treatment group in vivo. However, in terms of overall survival, DFMO alone resulted in a dramatic increase in survival compared to control treatment group. Interestingly, GW5074 alone or DFMO in combination with GW5074 did not result in a detectable effect on survival. Further investigation of immune cells in the tumor microenvironment revealed that standalone DFMO treatment was associated with an increase in infiltration of macrophages, T cell costimulatory marker CD86 and T cell markers (CD3, CD4 and CD8) compared to control and GW5074 treated groups. Additionally, DFMO is associated with decreased MYC expression compared to control and GW5074 treated groups. In conclusion, DFMO decreased MYC expression and associated immune suppression in the PDAC microenvironment. In contrast standalone GW5074 treatment resulted in maintenance of MYC expression and worse survival. In conclusion, the present study highlights the success of DFMO in PDAC treatment in part through downregulation of MYC and a decrease in associated immune suppression. Citation Format: Sai Preethi Nakkina, Sarah B. Gitto, Veethika Pandey, Jordan M. Beardsley, Michael W. Rohr, Jignesh G. Parikh, Otto Phanstiel, Deborah A. Altomare. DFMO mediated improvement in survival of an orthotopic model of pancreatic cancer is associated with modulating immune suppression in the tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-119.

Research paper thumbnail of Synthesis and Biological Evaluation of <i>N</i>-(Anthracen-9-ylmethyl)triamines as Molecular Recognition Elements for the Polyamine Transporter

Journal of Medicinal Chemistry, May 20, 2003

An efficient modular synthesis of N(1)-substituted triamines containing different tether lengths ... more An efficient modular synthesis of N(1)-substituted triamines containing different tether lengths between nitrogen centers was developed. A series of N(1)-(9-anthracenylmethyl)triamines were evaluated for biological activity in L1210 (murine leukemia), alpha-difluoromethylornithine (DFMO)-treated L1210, Chinese hamster ovary (CHO), and CHO-MG cell lines. All triamines 8 had increased potency in DFMO-treated L1210 cells. The 4,4- and 5,4-triamine systems had the highest affinity for the polyamine transporter (PAT) with L1210 K(i) values of 1.8 and 1.7 microM, respectively. This trend was also reflected in the CHO studies. Surprisingly, the respective 4,4- and 5,4-triamine systems had 150-fold and 38-fold higher cytotoxicity in CHO cells containing active polyamine transporters. Initial microscopy studies revealed the rapid formation of vesicular structures within A375 melanoma cells treated with the N(1)-(9-anthracenylmethyl)homospermidine (4,4-triamine) conjugate. In summary, the 4,4- and 5,4-triamines were identified as selective vector motifs to ferry anthracene into cells via the PAT.

Research paper thumbnail of The Effect of Polyamine Homologation on the Transport and Cytotoxicity Properties of Polyamine−(DNA-Intercalator) Conjugates

Journal of Organic Chemistry, Oct 12, 2000

Research paper thumbnail of Kinetic and stereochemical effect of a fluorine substituent on the Cope and the homodienyl 1,5-hydrogen shift rearrangements

Journal of the American Chemical Society, May 1, 1987

The kinetics and stereochemical outcome for the thermal rearrangements of 3-fluoro-1,5-hexadiene ... more The kinetics and stereochemical outcome for the thermal rearrangements of 3-fluoro-1,5-hexadiene and 1-(flueromethyl)-2-vinylcyclopropane have been investigated. The relative proportions of Z and E products in each case reflected their relative stability, thus indicating the lack of any dramatic kinetic effects due to the presence of the fluorine substituents in contrast to the very dramatic effects of this nature observed in cyclobutene-butadiene interconvetsions. The recently reported dramatic kinetic effect of substituents, particularly fluorine substituents, upon electrocyclic cyclo-butene-butadiene interconversions,'v2 combined with the subsequent theoretical explanation for these effect^,^.^ induced US to

Research paper thumbnail of Abstract 1483: Development of FUBP1 inhibitors to reprogram pancreatic cancer cells into an anti-cancer phenotype

Cancer Research

The purpose of this project was to identify small molecules which reprogram tumor cells into an a... more The purpose of this project was to identify small molecules which reprogram tumor cells into an anticancer phenotype. Far upstream binding protein 1 (FUBP1) has been identified as a master regulator of a panel of genes involved in tumor survival including c-Myc and p21. FUBP1 controls gene expression by binding to specific far upstream element (FUSE) DNA sequences of the promoters of key tumor survival genes to regulate their transcription to provide a pro-tumor phenotype (high c-Myc, low p21). This makes FUBP1 a novel therapeutic target as its inhibition reprograms tumors to an anti-cancer phenotype (low c-Myc, high p21). This report describes the development of novel anthranilic acid derivatives which bind to FUBP1 (as shown by ChIP and gel shift assays) and modulate the mRNA and protein expression (qRT-PCR and Western) of FUSE-controlled genes (e.g., c-Myc and p21). The lead compound UCF699 was shown to reprogram pancreatic ductal adenocarcinoma (PDAC) cells into an anti-cancer p...

Research paper thumbnail of Discovery of Anthranilic Acid Derivatives as Difluoromethylornithine Adjunct Agents That Inhibit Far Upstream Element Binding Protein 1 (FUBP1) Function

Research paper thumbnail of Polyamine-Based Nanostructures Share Polyamine Transport Mechanisms with Native Polyamines and Their Analogues: Significance for Polyamine-Targeted Therapy

Medical Sciences

Polyamines are small polycationic alkylamines involved in many fundamental cellular processes, in... more Polyamines are small polycationic alkylamines involved in many fundamental cellular processes, including cell proliferation, survival, and protection from oxidative stress. Polyamine homeostasis is tightly regulated through coordinated biosynthesis, catabolism, and transport. Due to their continual proliferation, cancer cells maintain elevated intracellular polyamine pools. Both polyamine metabolism and transport are commonly dysregulated in cancer, and as such, polyamine analogues are a promising strategy for exploiting the increased polyamine requirement of cancer cells. One potential polyamine analogue resistance mechanism is the downregulation of the poorly defined polyamine transport system. Recent advances in nanomedicine have produced nanostructures with polyamine analogue-based backbones (nanopolyamines). Similar nanostructures with non-polyamine backbones have been shown to be transported by endocytosis. As these polyamine-based nanoparticles could be a method for polyamine...

Research paper thumbnail of Abstract 235: Differential polyamine expression in human pancreatic cancer supports the use of polyamine blockade therapy to modulate the in vivo pancreatic tumor microenvironment

Cancer Research

Pancreatic cancer has a poor five-year survival rate of less than 8% and is projected to be the s... more Pancreatic cancer has a poor five-year survival rate of less than 8% and is projected to be the second leading cause of cancer related deaths in the US by the year 2030. Polyamine metabolism is an underexplored therapeutic target in pancreatic ductal adenocarcinoma (PDAC). The current investigation profiled RNA expression of genes associated with polyamine metabolism, transport and homeostasis in clinical pancreatic cancer samples to reveal evidence for dysregulation of polyamine-based pathways in PDAC. Increased expression of select pro-polyamine genes was associated with poorer patient prognosis in data obtained from the TCGA dataset. Preclinical experimental data supported the use of polyamine blockade therapy (PBT) in pancreatic tumor bearing mice, and revealed that PBT increased survival and decreased tumor weights in comparison to control treatment. PBT therapeutic effects were associated with increased expression of CD86 T cell co-stimulatory marker present on antigen present...

Research paper thumbnail of Harnessing the polyamine transport system to treat BRAF inhibitor-resistant melanoma

Cancer Biology & Therapy

ABSTRACT BRAF mutations are present in over half of all melanoma tumors. Although BRAF inhibitors... more ABSTRACT BRAF mutations are present in over half of all melanoma tumors. Although BRAF inhibitors significantly improve survival of patients with metastatic melanoma, recurrences occur within several months. We previously reported that BRAF mutant melanoma cells are more sensitive to a novel arylmethyl-polyamine ( AP ) compound that exploits their increased polyamine uptake compared to that of BRAF wildtype cells. Using an animal model of BRAF inhibitor-resistant melanoma, we show that co-treatment with the BRAF inhibitor, PLX4720, and AP significantly delays the recurrence of PLX4720-resistant melanoma tumors and decreases tumor-promoting macrophages. Development of BRAF inhibitor-resistance enriches for metastatic cancer stem cells (CSC) and increases tumor-promoting macrophages. In vitro studies demonstrated that CD304+, CXCR4+ spheroid cultures of BRAF mutant melanoma cells are resistant to PLX4720 but are more sensitive to AP compared to monolayer cultures of the same cells. AP significantly inhibited YUMM1.7 melanoma cell invasiveness across a Matrigel-coated filter using the CXCR4 ligand, SDF-1α, as the chemoattractant. AP also blocked the chemotactic effect of SDF-1α on CXCR4+ macrophages and inhibited M2 polarization of macrophages. In melanoma-macrophage co-cultures, AP prevented the PLX4720-induced release of pro-tumorigenic growth factors, such as VEGF, from macrophages and prevented the macrophage rescue of BRAF mutant melanoma cells treated with PLX4720. Our study offers a novel therapy ( AP ) to treat chemo-resistant melanoma. AP is unique because it targets the polyamine transport system in BRAF inhibitor-resistant CSCs and also blocks CXCR4 signaling in invasive melanoma cells and pro-tumorigenic macrophages.

Research paper thumbnail of The discovery of indolone GW5074 during a comprehensive search for non-polyamine-based polyamine transport inhibitors

The International Journal of Biochemistry & Cell Biology, 2021

The native polyamines putrescine, spermidine, and spermine are essential for cell development and... more The native polyamines putrescine, spermidine, and spermine are essential for cell development and proliferation. Polyamine levels are often increased in cancer tissues and polyamine depletion is a validated anticancer strategy. Cancer cell growth can be inhibited by the polyamine biosynthesis inhibitor difluoromethylornithine (DFMO) which inhibits ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine biosynthesis pathway. Unfortunately, cells treated with DFMO often replenish their polyamine pools by importing polyamines from their environment. Several polyamine-based molecules have been developed to work as polyamine transport inhibitors (PTIs) and have been successfully used in combination with DFMO in several cancer models. Here, we present the first comprehensive search for potential non-polyamine based PTIs that work in human pancreatic cancer cells in vitro. After identifying and testing five different categories of compounds, we have identified the c-RAF inhibitor, GW5074, as a novel non-polyamine based PTI. GW5074 inhibited the uptake of all three native polyamines and a fluorescent-polyamine probe into human pancreatic cancer cells. GW5074 significantly reduced pancreatic cancer cell growth in vitro when treated in combination with DFMO and a rescuing dose of spermidine. Moreover, GW5074 alone reduced tumor growth when tested in a murine pancreatic cancer mouse model in vivo. In summary, GW5074 is a novel non-polyamine-based PTI that potentiates the anticancer activity of DFMO in pancreatic cancers.

Research paper thumbnail of Development of Non‐Polyamine based Polyamine Transport Inhibitors for the Treatment of Pancreatic Ductal Adenocarcinoma

Research paper thumbnail of Sustained Polyamine Depletion for the Treatment of Pancreatic Ductal Adenocarcinoma

The FASEB Journal, 2019

Of all human tissues, the human pancreas has the highest level of the polyamine spermidine. Pancr... more Of all human tissues, the human pancreas has the highest level of the polyamine spermidine. Pancreatic cancers use these high levels to drive their growth. Indeed, polyamine metabolism plays critic...

Research paper thumbnail of Abstract PO-120: Differential expression of polyamine pathways in human pancreatic tumor progression and effects of polyamine blockade therapy on the in vivo pancreatic tumor microenvironment

Cancer Research, 2021

Pancreatic cancer is the fourth leading cause of cancer death in the United States, with a five-y... more Pancreatic cancer is the fourth leading cause of cancer death in the United States, with a five-year survival rate of less than 8%. Existing therapies have failed to improve pancreatic ductal adenocarcinoma (PDAC) patient prognosis. The dense desmoplastic reaction which occurs in PDAC makes it challenging for drugs and immune cells to infiltrate the fibrotic barrier. There is a need to exploit lesser explored targets in PDAC that can influence both the tumor and its microenvironment. One such avenue could be via targeting polyamine metabolism which is upregulated in pancreatic tumors. Though aberrant polyamine upregulation in pancreatic tumors has been known for decades, there has been little progress in translating this information into a PDAC therapeutic strategy. Additionally, there is a dearth of information regarding the dysregulation of polyamine metabolism in human PDAC and its association with clinical outcomes. Thus far, preclinical studies targeting polyamines using polyam...

Research paper thumbnail of Correction to Antibiotic Adjuvants: Make Antibiotics Great Again!

Journal of Medicinal Chemistry, 2020

Research paper thumbnail of Serendipitous Discovery of Leucine and Methionine Depletion Agents during the Search for Polyamine Transport Inhibitors

Journal of Medicinal Chemistry, 2020

Targeting polyamine metabolism is a proven anticancer strategy. Cancers often escape the polyamin... more Targeting polyamine metabolism is a proven anticancer strategy. Cancers often escape the polyamine biosynthesis inhibitors by increased polyamine import. Therefore, there is much interest in identifying polyamine transport inhibitors (PTIs) to be used in combination therapies. In a search for new PTIs, we serendipitously discovered a LAT-1 efflux agonist, which induces intracellular depletion of methionine, leucine, spermidine, and spermine, but not putrescine. Since S-adenosylmethioninamine is made from methionine, a loss of intracellular methionine leads to an inability to biosynthesize spermidine and spermine. Importantly, we found that this methioninedepletion approach to polyamine depletion could not be rescued by exogenous polyamines, thereby obviating the need for a PTI. Using 3 H-leucine (the gold standard for LAT-1 transport studies) and JPH-203 (a specific LAT-1 inhibitor), we showed that the efflux agonist did not inhibit the uptake of extracellular leucine, but instead facilitated the efflux of intracellular leucine pools.

Research paper thumbnail of FP028CALCIUM Sensor Receptor Modulation is a Mechanism for the Medicinal Effects of Plant-Derived Polyphenols

Nephrology Dialysis Transplantation, 2019

INTRODUCTION: The present study investigated the role of upstream and downstream components of Ye... more INTRODUCTION: The present study investigated the role of upstream and downstream components of Yes-associated protein (YAP) signaling pathway in the pathogenesis of kidney fibrosis. METHODS: Unilateral ureteral obstruction (UUO) was induced in C57BL/6J mice for 2 weeks. We determined the changes of angiotensin II type 1 receptor (AT1R), epidermal growth factor receptor (EGFR), Akt, CREB, YAP and pro-fibrotic proteins in the obstructed kidney of mice with UUO. We also investigated the protein expression of the upstream and downstream components of YAP signaling pathway after human renal proximal tubular (HK-2) cells were cultured with angiotensin II (Ang II) in the absence or presence of verteporfin, a YAP inhibitor. RESULTS: The protein expression of AT1R, transforming growth factor (TGF)-b1, and a-smooth muscle actin (SMA) and connective tissue growth factor (CTGF) was increased in the ureteral obstructed kidney of mice with UUO. The phosphorylation of EGFR, Akt and CREB, and the expression of YAP were increased. In HK-2 cells, the treatment with Ang II increased the protein expression of TGF-b1, a-SMA, CTGF and fibronectin. These changes were counteracted by the pretreatment with verteporfin. In addition, the treatment with Ang II increased the phosphorylation of EGFR, which was also attenuated by the inhibition of YAP. CONCLUSIONS: The expression of AT1R, EGFR, YAP and pro-fibrotic proteins is increased in the ureteral obstructed kidney. In HK-2 cells, the inhibition of YAP attenuates the activation of pro-fibrotic proteins and EGFR by the Ang II treatment. Our results suggests that YAP may regulate EGFR, an upstream component of YAP, as well as pro-fibrotic proteins.

Research paper thumbnail of Loss of Anti-Tumor Efficacy by Polyamine Blocking Therapy in GCN2 Null Mice

Biomedicines

GCN2 is one of the main sensors of amino acid starvation stress, and its activation in the stress... more GCN2 is one of the main sensors of amino acid starvation stress, and its activation in the stressful tumor microenvironment plays a crucial role in tumor survival and progression. We hypothesized that elevated polyamine biosynthesis and subsequent depletion of precursor arginine activates GCN2, thus rewiring metabolism to support tumor cell survival and drive myeloid immunosuppressive function. We sought to determine if the anti-tumor efficacy of a polyamine blocking therapy (PBT) may be mediated by its effect on GCN2. Unlike wild-type mice, PBT treatment in GCN2 knockout mice bearing syngeneic B16.F10 or EG7 tumors resulted in no tumor growth inhibition and no changes in the profile of infiltrating tumor immune cells. Studies with murine bone marrow cell cultures showed that increased polyamine metabolism and subsequent arginine depletion and GCN2 activation played an essential role in the generation and cytoprotective autophagy of myeloid derived suppressor cells (MDSCs) as well a...

Research paper thumbnail of Abstract 4503: Polyamine blockade therapy: A strategy to block immunosuppression in pancreatic cancer

Cancer Research, Apr 4, 2023

Research paper thumbnail of Abstract 3546: DFMO based improvement in survival of pancreatic cancer-bearing mice is associated with modulation of immune suppression in the tumor microenvironment

Cancer Research, Jun 15, 2022

Existing therapeutics have failed to improve pancreatic ductal adenocarcinoma (PDAC) patient outc... more Existing therapeutics have failed to improve pancreatic ductal adenocarcinoma (PDAC) patient outcomes, in part due to tumor associated immune suppression and upregulation of compensatory mechanisms. KRAS and c-MYC are important oncogenes in PDAC linked to tumor immune suppression. We describe the use of difluoromethylornithine (DFMO) and a c-RAF inhibitor (GW5074) to indirectly target c-MYC and KRAS, respectively. DFMO in combination with GW5074 was tested for ability to improve pancreatic cancer outcomes. Pancreatic cancer cells showed decreased cell viability and increased apoptosis in response to a combination of DFMO + GW5074. In vivo orthotopic models of pancreatic tumors had decreased tumor weight in DFMO + GW5074 treated mice, when compared to control group. However, only treatment with single agent DFMO resulted in improved survival of immunocompetent pancreatic tumor bearing mice with respect to control, in contrast to treatment with GW5074 alone or DFMO + GW5074. To understand why the in vivo data was contrary to in vitro results, immunohistochemical analysis of immune cells in the tumor microenvironment was used to reveal increased expression of markers associated with anti-tumor effects such as CD86, CD3, CD4 and CD8 in DFMO treated tumors. Tumors treated with DFMO also displayed decreased expression of MYC, suggesting that DFMO-associated MYC suppression could be linked to decreased immune suppression in the tumor microenvironment and improved survival. In contrast, GW5074 treatment maintained MYC expression in tumors. Overall, the present study points to DFMO being an immunomodulatory agent, and a need for further understanding of DFMO-based therapeutic strategies in PDAC. Citation Format: Sai Preethi Nakkina, Sarah B. Gitto, Jordan M. Beardsley, Veethika Pandey, Michael M. Rohr, Jignesh Parikh, Otto Phanstiel, Deborah A. Altomare. DFMO based improvement in survival of pancreatic cancer-bearing mice is associated with modulation of immune suppression in the tumor microenvironment [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 3546.

Research paper thumbnail of A <i>Drosophila</i> Model To Identify Polyamine−Drug Conjugates That Target the Polyamine Transporter in an Intact Epithelium

Journal of Medicinal Chemistry, Dec 29, 2007

Polyamine transport is elevated in many tumor types, suggesting that toxic polyamine-drug conjuga... more Polyamine transport is elevated in many tumor types, suggesting that toxic polyamine-drug conjugates could be targeted to cancer cells via the polyamine transporter (PAT). We have previously reported the use of Chinese hamster ovary (CHO) cells and its PAT-deficient mutant cell line, CHO-MG, to screen anthracene-polyamine conjugates for their PAT-selective targeting ability. We report here a novel Drosophila-based model for screening anthracene-polyamine conjugates in a developing and intact epithelium ( Drosophila imaginal discs), wherein cell-cell adhesion properties are maintained. Data from the Drosophila assay are consistent with previous results in CHO cells, indicating that the Drosophila epithelium has a PAT with vertebrate-like characteristics. This assay will be of use to medicinal chemists interested in screening drugs that use PAT for cellular entry, and it offers the possibility of genetic dissection of the polyamine transport process, including identification of a Drosophila PAT.

Research paper thumbnail of Abstract PO-119: DFMO mediated improvement in survival of an orthotopic model of pancreatic cancer is associated with modulating immune suppression in the tumor microenvironment

There remains an urgent need to target pancreatic tumor cells using innovative strategies. KRAS a... more There remains an urgent need to target pancreatic tumor cells using innovative strategies. KRAS and MYC, are important oncogenes in pancreatic ductal adenocarcinoma (PDAC) which pose a challenge to successful treatment of PDAC. Our previous studies have shown that inhibition of ornithine decarboxylase 1 (ODC1) using difluoromethylornithine (DFMO) decreases MYC expression and tumorigenesis. GW5074 can modulate RAF1, a downstream effector of KRAS. Here we test the responsiveness of pancreatic tumor cells treated with DFMO alone and in combination with GW5074. We used an orthotopic animal model of pancreatic tumor using KRas-driven murine pancreatic cancer cells (PanO2) to test the effects of these compounds on tumor microenvironment and overall survival. Cellular and molecular changes in the tumor microenvironment were assessed using immunohistochemistry. The results showed an inhibition of pancreatic cancer cell viability in DFMO and DFMO+GW5074 treatment groups in vitro, with a significant decrease in tumor weight compared to control treatment group in vivo. However, in terms of overall survival, DFMO alone resulted in a dramatic increase in survival compared to control treatment group. Interestingly, GW5074 alone or DFMO in combination with GW5074 did not result in a detectable effect on survival. Further investigation of immune cells in the tumor microenvironment revealed that standalone DFMO treatment was associated with an increase in infiltration of macrophages, T cell costimulatory marker CD86 and T cell markers (CD3, CD4 and CD8) compared to control and GW5074 treated groups. Additionally, DFMO is associated with decreased MYC expression compared to control and GW5074 treated groups. In conclusion, DFMO decreased MYC expression and associated immune suppression in the PDAC microenvironment. In contrast standalone GW5074 treatment resulted in maintenance of MYC expression and worse survival. In conclusion, the present study highlights the success of DFMO in PDAC treatment in part through downregulation of MYC and a decrease in associated immune suppression. Citation Format: Sai Preethi Nakkina, Sarah B. Gitto, Veethika Pandey, Jordan M. Beardsley, Michael W. Rohr, Jignesh G. Parikh, Otto Phanstiel, Deborah A. Altomare. DFMO mediated improvement in survival of an orthotopic model of pancreatic cancer is associated with modulating immune suppression in the tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-119.

Research paper thumbnail of Synthesis and Biological Evaluation of <i>N</i>-(Anthracen-9-ylmethyl)triamines as Molecular Recognition Elements for the Polyamine Transporter

Journal of Medicinal Chemistry, May 20, 2003

An efficient modular synthesis of N(1)-substituted triamines containing different tether lengths ... more An efficient modular synthesis of N(1)-substituted triamines containing different tether lengths between nitrogen centers was developed. A series of N(1)-(9-anthracenylmethyl)triamines were evaluated for biological activity in L1210 (murine leukemia), alpha-difluoromethylornithine (DFMO)-treated L1210, Chinese hamster ovary (CHO), and CHO-MG cell lines. All triamines 8 had increased potency in DFMO-treated L1210 cells. The 4,4- and 5,4-triamine systems had the highest affinity for the polyamine transporter (PAT) with L1210 K(i) values of 1.8 and 1.7 microM, respectively. This trend was also reflected in the CHO studies. Surprisingly, the respective 4,4- and 5,4-triamine systems had 150-fold and 38-fold higher cytotoxicity in CHO cells containing active polyamine transporters. Initial microscopy studies revealed the rapid formation of vesicular structures within A375 melanoma cells treated with the N(1)-(9-anthracenylmethyl)homospermidine (4,4-triamine) conjugate. In summary, the 4,4- and 5,4-triamines were identified as selective vector motifs to ferry anthracene into cells via the PAT.

Research paper thumbnail of The Effect of Polyamine Homologation on the Transport and Cytotoxicity Properties of Polyamine−(DNA-Intercalator) Conjugates

Journal of Organic Chemistry, Oct 12, 2000

Research paper thumbnail of Kinetic and stereochemical effect of a fluorine substituent on the Cope and the homodienyl 1,5-hydrogen shift rearrangements

Journal of the American Chemical Society, May 1, 1987

The kinetics and stereochemical outcome for the thermal rearrangements of 3-fluoro-1,5-hexadiene ... more The kinetics and stereochemical outcome for the thermal rearrangements of 3-fluoro-1,5-hexadiene and 1-(flueromethyl)-2-vinylcyclopropane have been investigated. The relative proportions of Z and E products in each case reflected their relative stability, thus indicating the lack of any dramatic kinetic effects due to the presence of the fluorine substituents in contrast to the very dramatic effects of this nature observed in cyclobutene-butadiene interconvetsions. The recently reported dramatic kinetic effect of substituents, particularly fluorine substituents, upon electrocyclic cyclo-butene-butadiene interconversions,'v2 combined with the subsequent theoretical explanation for these effect^,^.^ induced US to

Research paper thumbnail of Abstract 1483: Development of FUBP1 inhibitors to reprogram pancreatic cancer cells into an anti-cancer phenotype

Cancer Research

The purpose of this project was to identify small molecules which reprogram tumor cells into an a... more The purpose of this project was to identify small molecules which reprogram tumor cells into an anticancer phenotype. Far upstream binding protein 1 (FUBP1) has been identified as a master regulator of a panel of genes involved in tumor survival including c-Myc and p21. FUBP1 controls gene expression by binding to specific far upstream element (FUSE) DNA sequences of the promoters of key tumor survival genes to regulate their transcription to provide a pro-tumor phenotype (high c-Myc, low p21). This makes FUBP1 a novel therapeutic target as its inhibition reprograms tumors to an anti-cancer phenotype (low c-Myc, high p21). This report describes the development of novel anthranilic acid derivatives which bind to FUBP1 (as shown by ChIP and gel shift assays) and modulate the mRNA and protein expression (qRT-PCR and Western) of FUSE-controlled genes (e.g., c-Myc and p21). The lead compound UCF699 was shown to reprogram pancreatic ductal adenocarcinoma (PDAC) cells into an anti-cancer p...

Research paper thumbnail of Discovery of Anthranilic Acid Derivatives as Difluoromethylornithine Adjunct Agents That Inhibit Far Upstream Element Binding Protein 1 (FUBP1) Function

Research paper thumbnail of Polyamine-Based Nanostructures Share Polyamine Transport Mechanisms with Native Polyamines and Their Analogues: Significance for Polyamine-Targeted Therapy

Medical Sciences

Polyamines are small polycationic alkylamines involved in many fundamental cellular processes, in... more Polyamines are small polycationic alkylamines involved in many fundamental cellular processes, including cell proliferation, survival, and protection from oxidative stress. Polyamine homeostasis is tightly regulated through coordinated biosynthesis, catabolism, and transport. Due to their continual proliferation, cancer cells maintain elevated intracellular polyamine pools. Both polyamine metabolism and transport are commonly dysregulated in cancer, and as such, polyamine analogues are a promising strategy for exploiting the increased polyamine requirement of cancer cells. One potential polyamine analogue resistance mechanism is the downregulation of the poorly defined polyamine transport system. Recent advances in nanomedicine have produced nanostructures with polyamine analogue-based backbones (nanopolyamines). Similar nanostructures with non-polyamine backbones have been shown to be transported by endocytosis. As these polyamine-based nanoparticles could be a method for polyamine...

Research paper thumbnail of Abstract 235: Differential polyamine expression in human pancreatic cancer supports the use of polyamine blockade therapy to modulate the in vivo pancreatic tumor microenvironment

Cancer Research

Pancreatic cancer has a poor five-year survival rate of less than 8% and is projected to be the s... more Pancreatic cancer has a poor five-year survival rate of less than 8% and is projected to be the second leading cause of cancer related deaths in the US by the year 2030. Polyamine metabolism is an underexplored therapeutic target in pancreatic ductal adenocarcinoma (PDAC). The current investigation profiled RNA expression of genes associated with polyamine metabolism, transport and homeostasis in clinical pancreatic cancer samples to reveal evidence for dysregulation of polyamine-based pathways in PDAC. Increased expression of select pro-polyamine genes was associated with poorer patient prognosis in data obtained from the TCGA dataset. Preclinical experimental data supported the use of polyamine blockade therapy (PBT) in pancreatic tumor bearing mice, and revealed that PBT increased survival and decreased tumor weights in comparison to control treatment. PBT therapeutic effects were associated with increased expression of CD86 T cell co-stimulatory marker present on antigen present...

Research paper thumbnail of Harnessing the polyamine transport system to treat BRAF inhibitor-resistant melanoma

Cancer Biology & Therapy

ABSTRACT BRAF mutations are present in over half of all melanoma tumors. Although BRAF inhibitors... more ABSTRACT BRAF mutations are present in over half of all melanoma tumors. Although BRAF inhibitors significantly improve survival of patients with metastatic melanoma, recurrences occur within several months. We previously reported that BRAF mutant melanoma cells are more sensitive to a novel arylmethyl-polyamine ( AP ) compound that exploits their increased polyamine uptake compared to that of BRAF wildtype cells. Using an animal model of BRAF inhibitor-resistant melanoma, we show that co-treatment with the BRAF inhibitor, PLX4720, and AP significantly delays the recurrence of PLX4720-resistant melanoma tumors and decreases tumor-promoting macrophages. Development of BRAF inhibitor-resistance enriches for metastatic cancer stem cells (CSC) and increases tumor-promoting macrophages. In vitro studies demonstrated that CD304+, CXCR4+ spheroid cultures of BRAF mutant melanoma cells are resistant to PLX4720 but are more sensitive to AP compared to monolayer cultures of the same cells. AP significantly inhibited YUMM1.7 melanoma cell invasiveness across a Matrigel-coated filter using the CXCR4 ligand, SDF-1α, as the chemoattractant. AP also blocked the chemotactic effect of SDF-1α on CXCR4+ macrophages and inhibited M2 polarization of macrophages. In melanoma-macrophage co-cultures, AP prevented the PLX4720-induced release of pro-tumorigenic growth factors, such as VEGF, from macrophages and prevented the macrophage rescue of BRAF mutant melanoma cells treated with PLX4720. Our study offers a novel therapy ( AP ) to treat chemo-resistant melanoma. AP is unique because it targets the polyamine transport system in BRAF inhibitor-resistant CSCs and also blocks CXCR4 signaling in invasive melanoma cells and pro-tumorigenic macrophages.

Research paper thumbnail of The discovery of indolone GW5074 during a comprehensive search for non-polyamine-based polyamine transport inhibitors

The International Journal of Biochemistry & Cell Biology, 2021

The native polyamines putrescine, spermidine, and spermine are essential for cell development and... more The native polyamines putrescine, spermidine, and spermine are essential for cell development and proliferation. Polyamine levels are often increased in cancer tissues and polyamine depletion is a validated anticancer strategy. Cancer cell growth can be inhibited by the polyamine biosynthesis inhibitor difluoromethylornithine (DFMO) which inhibits ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine biosynthesis pathway. Unfortunately, cells treated with DFMO often replenish their polyamine pools by importing polyamines from their environment. Several polyamine-based molecules have been developed to work as polyamine transport inhibitors (PTIs) and have been successfully used in combination with DFMO in several cancer models. Here, we present the first comprehensive search for potential non-polyamine based PTIs that work in human pancreatic cancer cells in vitro. After identifying and testing five different categories of compounds, we have identified the c-RAF inhibitor, GW5074, as a novel non-polyamine based PTI. GW5074 inhibited the uptake of all three native polyamines and a fluorescent-polyamine probe into human pancreatic cancer cells. GW5074 significantly reduced pancreatic cancer cell growth in vitro when treated in combination with DFMO and a rescuing dose of spermidine. Moreover, GW5074 alone reduced tumor growth when tested in a murine pancreatic cancer mouse model in vivo. In summary, GW5074 is a novel non-polyamine-based PTI that potentiates the anticancer activity of DFMO in pancreatic cancers.

Research paper thumbnail of Development of Non‐Polyamine based Polyamine Transport Inhibitors for the Treatment of Pancreatic Ductal Adenocarcinoma

Research paper thumbnail of Sustained Polyamine Depletion for the Treatment of Pancreatic Ductal Adenocarcinoma

The FASEB Journal, 2019

Of all human tissues, the human pancreas has the highest level of the polyamine spermidine. Pancr... more Of all human tissues, the human pancreas has the highest level of the polyamine spermidine. Pancreatic cancers use these high levels to drive their growth. Indeed, polyamine metabolism plays critic...

Research paper thumbnail of Abstract PO-120: Differential expression of polyamine pathways in human pancreatic tumor progression and effects of polyamine blockade therapy on the in vivo pancreatic tumor microenvironment

Cancer Research, 2021

Pancreatic cancer is the fourth leading cause of cancer death in the United States, with a five-y... more Pancreatic cancer is the fourth leading cause of cancer death in the United States, with a five-year survival rate of less than 8%. Existing therapies have failed to improve pancreatic ductal adenocarcinoma (PDAC) patient prognosis. The dense desmoplastic reaction which occurs in PDAC makes it challenging for drugs and immune cells to infiltrate the fibrotic barrier. There is a need to exploit lesser explored targets in PDAC that can influence both the tumor and its microenvironment. One such avenue could be via targeting polyamine metabolism which is upregulated in pancreatic tumors. Though aberrant polyamine upregulation in pancreatic tumors has been known for decades, there has been little progress in translating this information into a PDAC therapeutic strategy. Additionally, there is a dearth of information regarding the dysregulation of polyamine metabolism in human PDAC and its association with clinical outcomes. Thus far, preclinical studies targeting polyamines using polyam...

Research paper thumbnail of Correction to Antibiotic Adjuvants: Make Antibiotics Great Again!

Journal of Medicinal Chemistry, 2020

Research paper thumbnail of Serendipitous Discovery of Leucine and Methionine Depletion Agents during the Search for Polyamine Transport Inhibitors

Journal of Medicinal Chemistry, 2020

Targeting polyamine metabolism is a proven anticancer strategy. Cancers often escape the polyamin... more Targeting polyamine metabolism is a proven anticancer strategy. Cancers often escape the polyamine biosynthesis inhibitors by increased polyamine import. Therefore, there is much interest in identifying polyamine transport inhibitors (PTIs) to be used in combination therapies. In a search for new PTIs, we serendipitously discovered a LAT-1 efflux agonist, which induces intracellular depletion of methionine, leucine, spermidine, and spermine, but not putrescine. Since S-adenosylmethioninamine is made from methionine, a loss of intracellular methionine leads to an inability to biosynthesize spermidine and spermine. Importantly, we found that this methioninedepletion approach to polyamine depletion could not be rescued by exogenous polyamines, thereby obviating the need for a PTI. Using 3 H-leucine (the gold standard for LAT-1 transport studies) and JPH-203 (a specific LAT-1 inhibitor), we showed that the efflux agonist did not inhibit the uptake of extracellular leucine, but instead facilitated the efflux of intracellular leucine pools.

Research paper thumbnail of FP028CALCIUM Sensor Receptor Modulation is a Mechanism for the Medicinal Effects of Plant-Derived Polyphenols

Nephrology Dialysis Transplantation, 2019

INTRODUCTION: The present study investigated the role of upstream and downstream components of Ye... more INTRODUCTION: The present study investigated the role of upstream and downstream components of Yes-associated protein (YAP) signaling pathway in the pathogenesis of kidney fibrosis. METHODS: Unilateral ureteral obstruction (UUO) was induced in C57BL/6J mice for 2 weeks. We determined the changes of angiotensin II type 1 receptor (AT1R), epidermal growth factor receptor (EGFR), Akt, CREB, YAP and pro-fibrotic proteins in the obstructed kidney of mice with UUO. We also investigated the protein expression of the upstream and downstream components of YAP signaling pathway after human renal proximal tubular (HK-2) cells were cultured with angiotensin II (Ang II) in the absence or presence of verteporfin, a YAP inhibitor. RESULTS: The protein expression of AT1R, transforming growth factor (TGF)-b1, and a-smooth muscle actin (SMA) and connective tissue growth factor (CTGF) was increased in the ureteral obstructed kidney of mice with UUO. The phosphorylation of EGFR, Akt and CREB, and the expression of YAP were increased. In HK-2 cells, the treatment with Ang II increased the protein expression of TGF-b1, a-SMA, CTGF and fibronectin. These changes were counteracted by the pretreatment with verteporfin. In addition, the treatment with Ang II increased the phosphorylation of EGFR, which was also attenuated by the inhibition of YAP. CONCLUSIONS: The expression of AT1R, EGFR, YAP and pro-fibrotic proteins is increased in the ureteral obstructed kidney. In HK-2 cells, the inhibition of YAP attenuates the activation of pro-fibrotic proteins and EGFR by the Ang II treatment. Our results suggests that YAP may regulate EGFR, an upstream component of YAP, as well as pro-fibrotic proteins.