Veethika Pandey

Natural killer cells stimulated with PM21 particles expand and biodistribute in vivo: Clinical implications for cancer treatment

BACKGROUND AIMS Natural killer (NK) cell immunotherapy for treatment of cancer is promising, but requires methods that expand cytotoxic NK cells that persist in circulation and home to disease site. METHODS We developed a particle-based method that is simple, effective and specifically expands cytotoxic NK cells from peripheral blood mononuclear cells (PBMCs) both ex vivo and in vivo. This method uses particles prepared from plasma membranes of K562-mb21-41BBL cells, expressing 41BBL and membrane bound interleukin-21 (PM21 particles). RESULTS Ex vivo, PM21 particles caused specific NK-cell expansion from PBMCs from healthy donors (mean 825-fold, range 163-2216, n = 13 in 14 days) and acute myeloid leukemia patients. The PM21 particles also stimulated in vivo NK cell expansion in NSG mice. Ex vivo pre-activation of PBMCs with PM21 particles (PM21-PBMC) before intraperitoneal (i.p.) injection resulted in 66-fold higher amounts of hNK cells in peripheral blood (PB) of mice compared with unactivated PBMCs on day 12 after injection. In vivo administration of PM21 particles resulted in a dose-dependent increase of PB hNK cells in mice injected i.p. with 2.0 × 10(6) PM21-PBMCs (11% NK cells). Optimal dose of 800 µg/injection of PM21 particles (twice weekly) with low-dose interleukin 2 (1000 U/thrice weekly) resulted in 470 ± 40 hNK/µL and 95 ± 2% of total hCD45(+) cells by day 12 in PB. Furthermore, hNK cells were found in marrow, spleen, lung, liver and brain (day 16 after i.p. PM21/PBMC injection), and mice injected with PM21 particles had higher amounts. CONCLUSIONS The extent of NK cells observed in PB, their persistence and the biodistribution would be relevant for cancer treatment.

Constitutively Active Akt1 Cooperates with KRasG12D to Accelerate In Vivo Pancreatic Tumor Onset and Progression

BACKGROUND AND AIMS: Pancreatic adenocarcinoma is a deadly disease characterized by metastatic progression and resistance to conventional therapeutics. Mutation of KRAS is the most frequent early event in pancreatic tumor progression. AKT isoforms are frequently activated in pancreatic cancer, and reports have implicated hyperactivation of AKT1, as well as AKT2, in pancreatic tumor formation. The objective here is to delineate the role of AKT in facilitating in vivo pancreatic tumor progression in the context of KRAS mutation and predisposition to pancreatic cancer. METHODS: Mice with Akt1 and KRas mutant alleles expressed using the pancreas Pdx promoter were mated to characterize the incidence and frequency of histologic and genetic alterations known to occur commonly in human pancreatic ductal adenocarcinoma. RESULTS: Active Akt1 (Akt1Myr, containing a myristoylation sequence) cooperated with active mutant KRasG12D to accelerate pancreatic carcinoma onset and progression and increase phosphorylation of downstream effectors in the Akt pathway. Mucin and smooth muscle actin expression was found in and around pancreatic intraepithelial neoplasms (PanINs), and accelerated time to metastasis was found in Akt1Myr/KRasG12D mice. CONCLUSIONS: In contrast to prior reports of pancreatic KRas mutant mice mated with mice deficient for various tumor suppressor genes, which resulted in aggressive disease within a few months of age, Akt1Myr/KRasG12D mice enabled the study of PanINs and spontaneous pancreatic transformation more characteristic of human pancreatic progression in elderly individuals. The Akt1Myr/KRasG12D model holds promise for delineating the tumor biology and biomarkers critical for understanding their cooperation in cancer oncogenesis and future targeting in therapeutic strategies.

Synthesis and biological evaluation of antimetastatic agents predicated upon dihydromotuporamine C and its carbocyclic derivatives.

The motuporamines isolated from the sea sponge Xestospongia exigua are of biological interest because of their unique antimigration and antiangiogenic properties. Key bioactive features were found to be a saturated 15-membered heterocycle and a norspermidine motif. This paper describes new analogues that modulate the cytotoxicity of this compound class and have enhanced antimigration properties. By movement of the polyamine chain outside the ring, new carbocycles were discovered that doubled the antimigration potency and reduced compound toxicity by 133-fold. Mice injected with metastatic human L3.6pl pancreatic cancer cells demonstrated significant reduction in liver metastases when treated with N(1)-(3-aminopropyl)-N(3)-(cyclopentadecylmethyl)propane-1,3-diamine compared with dihydromotuporamine C. Significant changes in specific ceramide populations (N16:0 and N22:1) were noted in L3.6pl cells treated with dihydromotuporamine C but not for the cyclopentadecylmethylnorspermidine derivative, which had lower toxicity. Both compounds gave increased levels of specific low molecular weight sphingomyelins, suggesting that they may act upon sphingomyelin processing enzymes.

Anti-ovarian tumor response of donor peripheral blood mononuclear cells is due to infiltrating cytotoxic NK cells.

Treatment of ovarian cancer, a leading cause of gynecological malignancy, has good initial efficacy with surgery and platinum/taxane-based chemotherapy, but poor long-term survival in patients. Inferior long-term prognosis is attributed to intraperitoneal spreading, relapse and ineffective alternate therapies. Adoptive cell therapy is promising for tumor remission, although logistical concerns impede widespread implementation. In this study, healthy PBMCs were used to examine the immune response in a mouse model with human ovarian cancer, where natural killer (NK) cells were found to be the effector cells that elicited an anti-tumor response. Presence of tumor was found to stimulate NK cell expansion in mice treated intraperitoneally with PBMC+Interleukin-2 (IL-2), as compared to no expansion in non-tumor-bearing mice given the same treatment. PBMC+IL-2 treated mice exhibiting NK cell expansion had complete tumor remission. To validate NK cell mediated anti-tumor response, the intratumoral presence of NK cells and their cytotoxicity was confirmed by immunohistochemistry and granzyme activity of NK cells recovered from the tumor. Collectively, this study highlights the significance of NK cell-cytotoxic response to tumor, which may be attributed to interacting immune cell types in the PBMC population, as opposed to clinically used isolated NK cells showing lack of anti-tumor efficacy in ovarian cancer patients.

Difluoromethylornithine Combined with a Polyamine Transport Inhibitor Is Effective against Gemcitabine Resistant Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is highly chemo-resistant and has an extremely poor patient prognosis, with a survival rate at five years of <8%. There remains an urgent need for innovative treatments. Targeting polyamine biosynthesis through inhibition of ornithine decarboxylase with difluoromethylornithine (DFMO) has had mixed clinical success due to tumor escape via an undefined transport system, which imports exogenous polyamines and sustains intracellular polyamine pools. Here, we tested DFMO in combination with a polyamine transport inhibitor (PTI), Trimer44NMe, against Gemcitabine-resistant PDAC cells. DFMO alone and with Trimer44NMe significantly reduced PDAC cell viability by inducing apoptosis or diminishing proliferation. DFMO alone and with Trimer44NMe also inhibited in vivo orthotopic PDAC growth and resulted in decreased c-Myc expression, a readout of polyamine pathway dysfunction. Moreover, dual inhibition significantly prolonged survival of tumor-bearing mice. Collectively, these studies demonstrate that targeting polyamine biosynthesis and import pathways in PDAC can lead to increased survival in pancreatic cancer.

Prediabetes linked to excess glucagon in transgenic mice with pancreatic active AKT1

Protein kinase B/AKT has three isoforms (AKT1-3) and is renowned for its central role in the regulation of cell growth and proliferation, due to its constitutive activation in various cancers. AKT2, which is highly expressed in insulin-responsive tissues, has been identified as a primary regulator of glucose metabolism as Akt2 knockout mice (Akt2(-/-)) are glucose-intolerant and insulin-resistant. However, the role of AKT1 in glucose metabolism is not as clearly defined. We previously showed that mice with myristoylated Akt1 (AKT1(Myr)) expressed through a bicistronic Pdx1-TetA and TetO-MyrAkt1 system were susceptible to islet cell carcinomas, and in this study we characterized an early onset, prediabetic phenotype. Beginning at weaning (3 weeks of age), the glucose-intolerant AKT1(Myr) mice exhibited non-fasted hyperglycemia, which progressed to fasted hyperglycemia by 5 months of age. The glucose intolerance was attributed to a fasted hyperglucagonemia, and hepatic insulin resistance detectable by reduced phosphorylation of the insulin receptor following insulin injection into the inferior vena cava. In contrast, treatment with doxycycline diet to turn off the transgene caused attenuation of the non-fasted and fasted hyperglycemia, thus affirming AKT1 hyperactivation as the trigger. Collectively, this model highlights a novel glucagon-mediated mechanism by which AKT1 hyperactivation affects glucose homeostasis and provides an avenue to better delineate the molecular mechanisms responsible for diabetes mellitus and the potential association with pancreatic cancer.

Abstract A108: Targeting sphingolipid metabolism and metastasis with motuporamine derivatives

The purpose of this research is to develop non-toxic antimetastatic agents for use in the treatment of metastatic pancreatic cancers. Dihydromotuporamine C ( 1 ) and related motuporamines were previously isolated from the sea sponge Xestospongia exigua found off the coast of Motupore island, New Guinea. The motuporamine structure contains a large heterocycle with an appended polyamine chain. These are of biological interest due to their potent anti-migration and anti-angiogenic properties. Previous work demonstrated key structural features (i.e., saturated 15-membered heterocycle and norspermidine) necessary for their antimetastatic activity. Genetic studies in yeast suggested that these compounds target sphingolipid metabolism. In this report, a series of carbocycle-polyamine conjugates were explored to find compounds with improved properties. Of this panel, (N 1 -(3-aminopropyl)-N 3 -(cyclopentadecylmethyl)propane-1,3-diamine 2 ) was discovered which had a two-fold increase in anti-migration potency and was 133-fold less toxic than 1 . Several experimental procedures were used to investigate perturbations in sphingolipid metabolism. Significant changes in specific ceramide populations (N16:0 and N22:1) were noted in human L3.6pl metastatic pancreatic cancer cells treated with 1 , but not with derivative 2 (which was similar to the untreated control). Both 1 and 2 gave increased levels of low molecular weight sphingomyelins and inhibited cell migration in vitro. A mouse model using metastatic human L3.6pl pancreatic cancer cells demonstrated significant reduction in L3.6pl liver metastases for mice treated with the new derivative 2 compared with the parent compound 1 . In summary, alterations in the N16:0/N22:1 ceramide ratio provided a potential explanation for the toxicity of 1 . Significantly increased levels of low molecular weight sphingomyelins in cells treated with either 1 or 2 suggested that they potentially target sphingomyelin synthase (as agonists) or sphingomyelinase enzymes (as inhibitors). We speculate that these increased levels of sphingomyelin may inhibit metastatic pathways associated with CXCR4 signaling; a pathway with known sensitivity to sphingomyelin. In conclusion, this report describes for the first time how compounds of this type alter chain-length specific ceramide and sphingomyelin populations in metastatic pancreatic cancer cells and suggests a novel mechanism for how they inhibit pancreatic cell metastasis. References: 1. Synthesis and Biological Evaluation of Dihydromotuporamine Derivatives in Cells Containing Active Polyamine Transporters. Kaur, N,; Delcros, J-G.; Martin, B.; Phanstiel, IV, O. J. Med. Chem. 2005, 48 , 3832-3839. 2. Modeling the Preferred Shapes of Polyamine Transporter Ligands and Dihydromotuporamine-C Mimics: Shovel versus Hoe. Breitbeil III, F. Kaur, N.; Delcros, J-G.; Martin,B.; Abboud, K.A.; Phanstiel, IV, O. J. Med. Chem. 2006, 49 , 2407-2416. 3. Motuporamine Mimic Agents. Otto Phanstiel IV, U.S. Patent : 7,728,041, June 1, 2010. 4. Antimetastatic Agents Predicated upon Polyamine-macrocyclic conjugates. Otto Phanstiel IV and Aaron Muth. World Patent A1, WO 2013148219, October 3, 2013. Citation Format: Aaron Muth, Veethika Pandey, Xianlin Han, Deborah Altomare, Otto Phanstiel, IV. Targeting sphingolipid metabolism and metastasis with motuporamine derivatives. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A108.