Niki Zacharias Millward

Long Noncoding RNA Ceruloplasmin Promotes Cancer Growth by Altering Glycolysis

Long noncoding RNAs (lncRNAs) significantly influence the development and regulation of genome expression in cells. Here, we demonstrate the role of lncRNA ceruloplasmin (NRCP) in cancer metabolism and elucidate functional effects leading to increased tumor progression. NRCP was highly upregulated in ovarian tumors, and knockdown of NRCP resulted in significantly increased apoptosis, decreased cell proliferation, and decreased glycolysis compared with control cancer cells. In an orthotopic mouse model of ovarian cancer, siNRCP delivered via a liposomal carrier significantly reduced tumor growth compared with control treatment. We identified NRCP as an intermediate binding partner between STAT1 and RNA polymerase II, leading to increased expression of downstream target genes such as glucose-6-phosphate isomerase. Collectively, we report a previously unrecognized role of the lncRNA NRCP in modulating cancer metabolism. As demonstrated, DOPC nanoparticle-incorporated siRNA-mediated silencing of this lncRNA in vivo provides therapeutic avenue toward modulating lncRNAs in cancer.

Hypoxia-Activated Prodrug TH-302 Targets Hypoxic Bone Marrow Niches in Preclinical Leukemia Models

Purpose: To characterize the prevalence of hypoxia in the leukemic bone marrow, its association with metabolic and transcriptional changes in the leukemic blasts and the utility of hypoxia-activated prodrug TH-302 in leukemia models. Experimental Design: Hyperpolarized magnetic resonance spectroscopy was utilized to interrogate the pyruvate metabolism of the bone marrow in the murine acute myeloid leukemia (AML) model. Nanostring technology was used to evaluate a gene set defining a hypoxia signature in leukemic blasts and normal donors. The efficacy of the hypoxia-activated prodrug TH-302 was examined in the in vitro and in vivo leukemia models. Results: Metabolic imaging has demonstrated increased glycolysis in the femur of leukemic mice compared with healthy control mice, suggesting metabolic reprogramming of hypoxic bone marrow niches. Primary leukemic blasts in samples from AML patients overexpressed genes defining a “hypoxia index” compared with samples from normal donors. TH-302 depleted hypoxic cells, prolonged survival of xenograft leukemia models, and reduced the leukemia stem cell pool in vivo. In the aggressive FLT3/ITD MOLM-13 model, combination of TH-302 with tyrosine kinase inhibitor sorafenib had greater antileukemia effects than either drug alone. Importantly, residual leukemic bone marrow cells in a syngeneic AML model remain hypoxic after chemotherapy. In turn, administration of TH-302 following chemotherapy treatment to mice with residual disease prolonged survival, suggesting that this approach may be suitable for eliminating chemotherapy-resistant leukemia cells. Conclusions: These findings implicate a pathogenic role of hypoxia in leukemia maintenance and chemoresistance and demonstrate the feasibility of targeting hypoxic cells by hypoxia cytotoxins. Clin Cancer Res; 22(7); 1687–98. ©2015 AACR.

Real-Time MRI-Guided Catheter Tracking Using Hyperpolarized Silicon Particles

Visualizing the movement of angiocatheters during endovascular interventions is typically accomplished using x-ray fluoroscopy. There are many potential advantages to developing magnetic resonance imaging-based approaches that will allow three-dimensional imaging of the tissue/vasculature interface while monitoring other physiologically-relevant criteria, without exposing the patient or clinician team to ionizing radiation. Here we introduce a proof-of-concept development of a magnetic resonance imaging-guided catheter tracking method that utilizes hyperpolarized silicon particles. The increased signal of the silicon particles is generated via low-temperature, solid-state dynamic nuclear polarization, and the particles retain their enhanced signal for ≥40 minutes—allowing imaging experiments over extended time durations. The particles are affixed to the tip of standard medical-grade catheters and are used to track passage under set distal and temporal points in phantoms and live mouse models. With continued development, this method has the potential to supplement x-ray fluoroscopy and other MRI-guided catheter tracking methods as a zero-background, positive contrast agent that does not require ionizing radiation.

Photoacoustic-based SO 2 assessment of femoral bone marrow in a murine model of leukemia

Leukemia commonly leads to hypoxia in the bone marrow, which can then result in increased resistance to chemotherapy. However, the relationship between local hypoxia and disease progression is not well understood, and it is unclear whether hypoxia in the bone marrow is diffuse or focal in presentation. Spectroscopic photoacoustic (PA) imaging-based estimation of blood oxygen saturation (SO2) can be used as a biomarker for tissue hypoxia. In this study, we investigate the longitudinal repeatability of PA-based SO2 estimates in the femoral bone marrow of a murine model of leukemia.

Abstract PL07-01: Altered metabolism in leukemic microenvironment

Interactions of leukemia cells and their bone marrow (BM) microenvironment are known to play a key role in the survival and growth of leukemic cells. It has been postulated that specific niches provide a sanctuary where subpopulations of leukemic cells evade chemotherapy-induced death and acquire a drug-resistant phenotype. Understanding the cellular and molecular biology of the leukemia stem cell (LSC) niche and of microenvironment/leukemia interactions may provide new targets that allow destruction of LSCs without adversely affecting normal stem cell self-renewal. Key emerging therapeutic targets include chemokine receptors such as CXCR4 and hypoxia-related proteins, as well as the metabolic abnormalities of the leukemia-associated stroma. We have recently reported that CXCR4 inhibition causes leukemia cell dislodgement from CXCL12-producing marrow niches, reduced proliferation and induction of differentiation of AML cells in an in vivo model of AML, translating into pronounced anti-leukemia effects. Studies in murine leukemia models using the hypoxia probe pimonidazole demonstrated extensive areas of hypoxia within leukemic, but not healthy normal, bone marrow. Time-course analysis of bone marrow spaces within calvaria and femurs by multiphoton intravital microscopy (MP-IVM) demonstrated lodging of p190-Bcr/Abl tdTomato cells in close proximity to blood vessels, followed by accumulation of leukemia cells localized within the sinusoidal and marrow spaces resulting in the demise of the animals within 3 weeks. In this model, pimonidazole detected hypoxic areas despite abundant vascular supply in the marrow cavities. In vivo magnetic resonance imaging with hyperpolarized pyruvate showed higher pyruvate-lactate conversion (high glycolytic flux) in leukemic marrows. These findings were supported by significant pimonidazole uptake by the diseased bone marrow in patients with acute leukemia, causing stabilization of HIF-1α in 55% (76/138) of primary AML patients and of its target CA9. Paradoxically, AML cells become highly dependent on mitochondrial oxidative phosphorylation (OXPHOS) for their survival, and inhibition of OXPHOS with the novel small molecule complex I inhibitor IACS-10759 inhibits oxygen consumption, eliminates hypoxia in vivo and inhibits AML growth. These findings suggest that altered tumor metabolism underlies the hypoxia observed in leukemias. We postulate that the altered tumor microenvironment within the hypoxic niche cells will influence leukemia development and response to therapy. Hence, targeting key metabolic alterations should be considered in the armamentarium of anti-AML therapies. IACS-10759 is presently completing IND enabling safety/toxicity studies with first in human studies targeting relapsed/refractory AML planned for 2016. Citation Format: Marina Y. Konopleva, Tomasz Zal, Niki M. Zacharias Millward, Byoung-Sik Cho, Karine Harutyunyan, Anna Zal, Hong Mu, Sergej Konoplev, Juliana Benito, Juliana Velez, Carlos Bueso-Ramso, Jennifer Molina, Pratip K. Bhattacharya, Maria Emilia Di Francesco, Joseph Marszalek, Michael Andreeff. Altered metabolism in leukemic microenvironment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL07-01.

Abstract 210: In vivo metabolic imaging to differentiate aggressive versus indolent prostate cancer

Purpose: Many prostate cancers (PCa) detected by screening are indolent (will not leave the prostate) however, 90% of patients will receive immediate treatment such as surgery or radiation therapy. There is a pressing need in the clinic for determining the aggressiveness of PCa that allows the whole prostate to be examined and is directly correlated to the metastatic behavior of the tumor. We are using a multi-prong approach to understand the metabolic changes that occur in the progression of PCa. (1) We are metabolic profiling tumor and normal prostate tissue. (2) We are metabolic profiling both the intracellular and secretion/consumption of metabolites of four human prostate cancer cell lines with different aggressiveness profiles: RWPE-1 (non-tumorigenic, considered benign), RWPE-2 (non-metastatic), PC3 (aggressive, castration resistant), most aggressive cell line PC3M (castration resistant). (3) We are following the progression of PCa with hyperpolarized magnetic resonance (MR) agents in PCa animal models. Hyperpolarization allows for over >10,000 fold sensitivity enhancement using conventional MRI and MRS. The polarization (signal enhancement) can be retained on the metabolites of the hyperpolarized molecule. Hyperpolarization allows for in vivo real time metabolic profiling. Results: Based on our metabolic profiling of PCa tissue and cell culture, we observe significant differences in uptake of glutamine and the amount of intracellular glutamine, differences in phosphocholine and glycerophosphocholine, and differences in intracellular succinate levels between aggressive versus non-aggressive cell lines and in PCa tissue versus normal prostate tissue. In addition, in our hands we see no significant difference in the glycolytic rate (production of lactate) between the indolent and aggressive PCa cell lines in culture. Using dynamic nuclear polarization, we are designing new in vivo methods for interrogating metabolic pathways. Using our chemical reaction-induced multi-molecular polarization (CRIMP) technique, we can fully transfer the high polarization levels of pyruvate in an irreversible reaction to 1-13C acetate and 13C-carbon dioxide without substantial signal loses. Because hyperpolarized carbon dioxide nearly instantly equilibrates with bicarbonate in the aqueous environment, the pH of the media can be simply calculated from the signal intensity ratio of the two compounds using the Henderson Hasselbalch equation. Discussion: As an alternative to hyperpolarized pyruvate for analysis of the aggressiveness of prostate cancer, compounds that more comprehensively interrogate glutaminolysis, choline metabolism, and the Krebs cycle in combination may provide better metabolic imaging information. Using the CRIMP technique and single component hyperpolarization of 15N-choline, we are currently using MR to metabolically image PCa animal models. Citation Format: Niki Zacharias Millward, Christopher McCullough, Youngbok Lee, Jingzhe Hu, Prasanta Dutta, David Piwnica-Worms, Pratip Bhattacharya. In vivo metabolic imaging to differentiate aggressive versus indolent prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 210. doi:10.1158/1538-7445.AM2015-210